Human monoclonal antibody production and characterization

Transcription

UNIVERSITY OF COPENHAGEN
Master's thesis
Anine Engholm Jeppesen, BTK08027
Human monoclonal anti-malaria antibody
production and characterization
Academic supervisors:
Post Doc Michael Dalgaard
Post Doc Lea Barfod
Professor Lars Hviid
Centre for Medical Parasitology
Faculty of Health Sciences
University of Copenhagen
Submitted:01/03/2011
Associate professor Søren Skov
Department of Veterinary Disease Biology
Faculty of Life Sciences
University of Copenhagen
PREFACE
This study is a master thesis (60 ECTS) finalizing my studies of Biology-Biotechnology at Faculty of
Life Sciences, University of Copenhagen. The work has primarily been conducted at Centre for
Medical Parasitology at Faculty of Health Science, University of Copenhagen and to some extent at
the Noguchi Memorial Institute for Medical Research, University of Ghana.
I would like to thank my supervisors at Centre for Medical Parasitology Michael Back Dalgaard, Lea
Barfod and Lars Hviid as well as Søren Skov at Faculty of Life Sciences. In particular thanks to my
daily supervisors Michael and Lea for always finding time to answer my questions and discuss my
work - and for believing in me.
Thanks to Michael Ofori and his colleagues at the Noguchi Memorial Institute for Medical
Research for letting us use their laboratory facilities and for their great help during B cell
collection. A thank to Justina Ansah, Mavis Okyere and their colleagues at the Blood Bank at Korle
Bu Teaching Hospital, Accra, Ghana for their help with donor blood collection.
I would like to thank everybody at Centre for Medical Parasitology for keeping up a great work
environment, for answering numerous questions and for fun times in and outside the lab.
Especially thanks to Kirsten Pihl, Katrine Vegener, Maiken Visti and Lisbeth Stolpe for great help in
the laboratory, to Louise Turner, Gerald Cham, and Mafalda Resende for assistance with the
luminex assay, Anja Jensen and Anja Bengtsson for providing binding data and to Louise Jørgensen
for parasite supply. A special thank to Mette Agerbæk and Kamilla Jensen for sharing my
enthusiasm when experiments were successful and for their cake-based support when
experiments did not go as planned.
A special thank to my family for always being supportive and for their efforts to understand my
molecular world.
Thanks to the Novo foundation for financial support by awarding me their scholarship.
The thesis was submitted 1st of March 2011
______________________________
Anine Engholm Jeppesen, BTK08027
Front page: Animation of antibodies (http://lifesciencedigest.com/2010/07/11/monoclonal-antibody-companiescommand-premiums/), rosette of infected erythrocyte binding to uninfected erythrocytes (Photo by David Ferguson),
structure model of PFD1235w with predicted epitopes of the antibody AB01.15 marked in yellow.
I
ABSTRACT
Malaria remains one of the most important human health problems in the developing world killing
nearly one million people each year while causing disease in more than 200 millions. Development
of a malaria-vaccine is troublesome as the parasite causing malaria is able to switch between
expression of different antigens and because the antigen repertoire is vast. However, it has been
shown that the antigens expressed by parasites causing severe and potentially fatal malaria are
more conserved. Discovering conserved epitopes could lead to a morbidity reducing vaccine –
protecting from severe and fatal malaria. Monoclonal antibodies derived from individuals with
acquired protective immunity to malaria are a powerful tool to define and characterize conserved
epitopes.
At Centre for Medical Parasitology, Epstein-Barr virus-immortalized memory B cells from donors
who are clinically immune to malaria have been used to produce human monoclonal anti-malaria
antibodies. However, the Epstein-Barr technology gives relatively low-yield, is susceptible to cell
culture contamination and cultures often die out. Symplex™ is a technology for the isolation of
antibody genes from single B cells and their expression as monoclonal recombinant antibodies. It
is advantageous compared to production by the Epstein-Barr method as production of high
quantities is convenient, the production is stable and engineering of the antibody is possible. It
would thus be beneficial to use the Symplex™ technology to make recombinant versions of
Epstein-Barr virus-immortalized culture-antibodies.
The aim of this thesis has been to establish the Symplex™ technology at Centre for Medical
Parasitology and generate and characterize recombinant versions of antibodies produced by
Epstein-Barr virus-immortalized B cells.
The Symplex™ technology was successfully established at Centre for Medical Parasitology together
with methods for sequence verification, detection of antibody production, and optimization of
antibody production. Contamination impeded the establishment, but various implemented
initiatives have reduced the risk of contamination.
Antibody genes from tree different Epstein-Barr virus-immortalized B cell cultures were
successfully cloned by the Symplex™ technology and all recombinant antibodies were shown to
retain the activity of their counterpart produced by Epstein-Barr virus-immortalized cells. One of
the antibodies showed cross-reactivity to several antigen domains, which suggests that conserved
epitopes among antigens involved in severe malaria exist and adds further hope to the possibility
of developing a morbidity-reducing vaccines.
The Symplex™ technology has yet broader perspectives. The future goal is to apply it directly to
memory B cells from malaria exposed donors to generate anti-malaria antibody libraries.
II
RESUMÉ
Malaria er fortsat et af de vigtigste sundhedsproblemer i udviklingslande og dræber næsten en
million mennesker årligt samt forårsager sygdom hos flere end 200 millioner. Udvikling af en
malaria vaccine er vanskelig, idet parasitten kan skifte mellem hvilke antigener, der udtrykkes, og
fordi antigen repertoiret er enormt. Det er dog blevet vist, at antigener udtrykt i parasitter, der
forårsager alvorlig og potentiel dødelig malaria, er mere konserverede. Opdagelsen af
konserverede epitoper ville kunne føre til en morbiditetsreducerende vaccine, der beskytter mod
alvorlig og dødelig malaria. Monoklonale antistoffer dannet i personer, der har udviklet
beskyttende immunitet mod malaria, er et vigtigt redskab til bestemmelse og karakterisering af
konserverede epitoper.
På Center for Medicinsk Parasitologi har Epstein-Barr virus immortaliserede hukommelses B celler
fra klinisk immune donorer været brugt til at producere humane monoklonale anti-malaria
antistoffer. Epstein-Barr metoden giver dog et relativt lavt udbytte, er modtagelig for forurening af
kulturer og kulturer dør ofte ud. Symplex™ er en metode til at isolere antistofgener fra enkelte
celler og udtrykke dem som rekombinante monoklonale antistoffer. Sammenlignet med EpsteinBarr metoden har Symplex™ metoden de fordele at produktionen af større mængder antistoffer er
nem, produktionen er stabil og genetisk manipulation af antistoffet er muligt. Det vil derfor være
fordelagtigt at bruge Symplex™ metoden til at lave rekombinante udgaver af antistoffer
produceret af Epstein-Barr virus immortaliserede kulturer.
Formålet med dette speciale har været at etablere Symplex™ metoden på Center for Medicinsk
Parasitologi samt producere og karakterisere rekombinante udgaver af antistoffer produceret af
Epstein-Barr virus immortaliserede B celler.
Det lykkedes at etablere af Symplex™ metoden på Center for Medicinsk Parasitologi samt metoder
til verificering af sekvenser, påvisning af antistof produktion og optimering af antistof
produktionen. Forurening besværliggjorde etableringen, men forskellige tiltag har reduceret
risikoen for forurening.
Antistof gener fra tre forskellige Epstein-Barr virus immortaliserede B celle kulturer blev klonet
med Symplex™ metoden og det blev vist at alle tre bibeholdt reaktiviteten af samme antistof
produceret af Epstein-Barr virus immortaliserede B celler. Et af disse antistoffer var kryds-reaktivt
med adskillige antigen domæner, hvilket tyder på at der findes konserverede epitoper i antigener
involverede i alvorlig malaria og giver håb om muligheden for at udvikle en
morbiditetsreducerende vaccine.
Symplex™ metoden har videre muligheder. Målet er for fremtiden at bruge metoden direkte på
hukommelses B celler fra malaria eksponerede donorer og dermed lave anti-malaria antistof
biblioteker.
III
TABLE OF CONTENTS
PREFACE
I
ABSTRACT
II
RESUMÉ
III
TABLE OF CONTENTS
IV
LIST OF ABBREVIATIONS
VII
INTRODUCTION
1
MALARIA DISEASE
PLASMODIUM FALCIPARUM
LIFE CYCLE
PLASMODIUM FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1
CYTOADHESION
IMMUNITY TO MALARIA
VSAS AS VACCINE CANDIDATES
MONOCLONAL ANTIBODY PRODUCTION
ANTIBODIES
IMMORTALIZATION OF B CELLS
THE HYBRIDOMA TECHNOLOGY
RECOMBINANT PRODUCTION OF MONOCLONAL ANTIBODIES
SYMPLEX™ TECHNOLOGY
1
1
2
2
3
5
6
7
7
8
9
10
11
OUTLINE FOR EXPERIMENTAL WORK
14
MATERIALS AND METHODS
16
B CELL COLLECTION
ISOLATION OF B CELLS
MALARIA EXPOSURE DETERMINING ELISA
EPSTEIN BARR VIRUS IMMORTALIZED B CELLS
SINGLE CELL SORTING
SINGLE CELL SORTING BY LIMITING DILUTION
SINGLE CELL SORTING BY FACSARIA
CELL SORTING USING MICRO MANIPULATOR
CLONING OF VH-VCL FROM EBV CELLS
ISOLATION OF VH-VCL FROM EBV-IMMORTALIZED B CELLS
16
16
16
17
17
17
18
18
18
20
IV
INSERTION OF VH-VCL INTO IGG1 EXPRESSION VECTOR
SEQUENCING
SEQUENCE ANALYSIS
ANTIBODY PRODUCTION, PURIFICATION AND VERIFICATION
HEK293 TRANSFECTION
OPTIMIZATION OF ANTIBODY PRODUCTION
ANTIBODY PURIFICATION
SDS-PAGE
WESTERN BLOT
DETECTION OF CONTAMINATION
PCR ON REAGENTS
PLASMODIUM FALCIPARUM PARASITES
MAINTENANCE
SELECTION
MACS-PURIFICATION
ANTIBODY CHARACTERIZATION
ANTIBODY DETECTION ELISA
FLOW CYTOMETRY
LUMINEX
ROSETTE REVERSAL ASSAY
EPITOPE PREDICTIONS
22
23
24
26
26
26
26
27
27
28
28
28
28
28
29
29
29
30
30
31
31
RESULTS
33
B CELL COLLECTION
ISOLATION OF ANTIBODY ENCODING GENES
ANTIBODY SEQUENCE ANALYSIS
EXPRESSION AND PURIFICATION OF RECOMBINANT ANTIBODIES
DETECTION OF ANTIBODIES
OPTIMIZING ANTIBODY-DETECTION ELISA
PURIFICATION OF ANTIBODIES
PROTEIN VERIFICATION
DETERMINATION AND PREVENTION OF CONTAMINATION
OPTIMIZING CELL SORTING BY FACSARIA
RECOMBINANT AB01.15 RETAINS ORIGINAL SPECIFICITY
RECOMBINANT AR03.1 RETAINS ACTIVITY
RECOMBINANT AR04.1 RETAINS ITS ACTIVITY
EPSTEIN-BARR VIRUS-IMMORTALIZED B CELL CULTURE AR05.2 PRODUCE IGM
33
35
37
40
40
41
42
43
43
44
47
47
47
54
55
56
DISCUSSION
57
V
B CELL COLLECTION
ESTABLISHMENT OF THE SYMPLEX™ TECHNOLOGY
CELL SORTING
ISOLATION OF ANTIBODY-ENCODING GENES
SEQUENCING
DETECTION OF ANTIBODY PRODUCTION
OPTIMIZING PRODUCTION
VERIFICATION OF FUNCTION
DETERMINATION AND ELIMINATION OF CONTAMINATION
RECOMBINANT ANTIBODY PRODUCTION VS. EBV-ANTIBODIES
LARGER SCALE MONOCLONAL ANTIBODY PRODUCTION
PERSPECTIVES OF APPLYING THE SYMPLEX™ TECHNOLOGY FOR LARGER SCALE ANTIBODY GENERATION
LARGER SCALE ANTIBODY GENERATION BY ALTERNATIVE METHODS
AR03.1
AR04.1
AB01.15
57
58
58
59
59
60
60
61
61
63
64
64
65
67
67
68
69
CONCLUSION
71
PERSPECTIVE
72
REFERENCES
73
APPENDIX
A
DNA LADDERS
VECTOR 00VP002
PLEX 5
A
B
C
VI
LIST OF ABBREVIATIONS
ASC
Antibody secreting cell
CDR
Complementarity-determining region
CH
Heavy chain constant domain
CIDR
Cysteine-rich interdomain region
CL
Light chain constant domain
CMP
Centre for Medical Parasitology
CSA
Chondroitin sulfate A
CR1
Complement receptor 1
DBL
Duffy binding-like
EBV cell
Epstein-Barr virus immortalized B cell
Fab
Antigen binding fragment
Fc
Constant fragment
FACS
fluorescence-activated cell sorting
FW
Framework region
ICAM-1
Intercellular adhesion molecule 1
IE
Infected erythrocyte
Ig
Immunoglobulin
ON
over night
P.falciparum Plasmodium falciparum
PfEMP1
Plasmodium falciparum erythrocyte membrane protein 1
rt
Room temperature
RT-PCR
Reverse-transcription PCR
SM
Severe malaria
SvFv
Single chain variable fragment
TM
Transmembrane domain
UM
Uncomplicated malaria
VH
Heavy chain variable domain
VL
Light chain variable domain
VSA
Variant surface antigen
VII
INTRODUCTION
Malaria is one of the most common life-threatening diseases in the tropics and adds one million to
the death records every year. These are mainly children from sub-Saharan countries and in fact
every fifth childhood death in Africa is due to malaria. A total of 200-300 million people suffer
from the disease each year and around half the world’s population is at risk of malaria - mainly
people living in poverty. The disease burden also adds an economic burden to these already
challenged countries. Malaria is estimated to decrease the gross domestic product with 1.3% in
countries with high levels of transmission and accounting for 40% of public health care expenses
(World Health Organisation 2010).
Although malaria is preventable and treatable many of the people affected by malaria cannot
afford treatment or live in remote areas without access to regular health care. This makes a
malaria vaccine highly desirable (World Health Organisation 2010).
MALARIA DISEASE
Malaria is seen as an acute febrile illness with symptoms such as headache, chills, vomiting and
periodic fevers. Although the disease is agonizing most patients survive uncomplicated malaria
(UM) even without treatment. However, in 1-2% of cases the disease develops into severe,
potentially fatal malaria (SM). This is seen as further complications such as cerebral malaria,
severe anemia or metabolic acidosis. Another variant of severe disease is pregnancy malaria.
Other complications such as organ failure are also seen in countries with low transmission. The
focus of this thesis will however be on malaria in high endemicity countries. Severe malaria has a
mortality rate of 15-20%. Parasitaemia can also occur without the development of any disease
symptoms, known as asymptomatic malaria (Mackintosh et al. 2004; Rowe et al. 2009; Taylor &
Molyneux 2002).
PLASMODIUM FALCIPARUM
Malaria is caused by eukaryotic single-celled parasites of the genus Plasmodium. The species
infecting humans are P. vivax, P. malariae, P. ovale, P. knowlesi and P. falciparum. Focus of this
thesis will be on P. falciparum as it is responsible for approximately 40% of all malaria cases and
nearly all malaria-caused deaths (Wellems et al. 2009).
1
LIFE CYCLE
P. falciparum has a complex life cycle which involves two different hosts; humans and mosquitoes
of the Anopheles genus. When an infected mosquito bites it injects parasites in the sporozoite
stage into the human host (figure 1). Quickly these will make their way to the liver and infect
hepatocytes. Inside the hepatocytes the parasites undergo asexual replication and each sporozoite
gives rise to tens of thousands of merozoites. The hepatocytes will eventually burst and release
merozoites into the bloodstream, where erythrocytes rapidly are invaded. Inside the infected
erythrocytes (IE) the parasites go through additional rounds of asexual reproduction developing
through the ring, trophozoite and schizont stage leading to around 20 new merozoites per
erythrocyte (Miller et al. 2002). The
blood stage cycle takes 48 hours
and ends by synchronous rupture
of the IEs and release of
merozoites. This rupture is
associated with the periodic fevers
seen in P. falciparum infected
patients. Each of the merozoites
will invade a new erythrocyte and
the cycle will be repeated. A subset
of the merozoites develops into
male and female gametocytes that
are taken up when a mosquito
takes a blood meal. Inside the
mosquito the parasite undergo
sexual
replication
producing
thousands of progeny sporozoites
that are ready to initiate another
life cycle when injected into a new
human host (Rowe et al. 2009).
Figure 1. Plasmodium falciparum life cycle. See text for
description. Figure from Rowe et al. (2009).
PLASMODIUM FALCIPARUM ERYTHROCYTE MEMBRANE PROTEIN 1
During the blood stage parasites live in a protected area in that erythrocytes lack a nucleus and
are thus not able to present foreign proteins to cells of the immune system. The parasite has even
come up with a way to avoid splenic removal by adhering to cells of the vascular system. This is
mediated by parasite encoded proteins that are exported and displayed at the erythrocyte
surface. Several parasite proteins are displayed at the erythrocyte surface and are known as
variant surface antigens (VSA) due to their vast number of variants. VSAs consist of different
protein families: RIFINs, STEVORs, SURFINs and Plasmodium falciparum erythrocyte membrane
protein 1 (PfEMP1). The latter has been shown to play a key role in pathogenesis, immunity and
immune evasion and will thus be the focus here (Rowe et al. 2009).
2
PfEMP1s are encoded by the highly variable var gene family and every haploid genome contains
around 60 genes each encoding a distinct protein with particular antigenic and adhesive
properties. Due to frequent recombination var genes also differ between parasites and the total
repertoire within the parasite population is thus immense. The expression of PfEMP1s is tightly
regulated and the general agreement is that only a single variant is expressed at the erythrocyte
surface at any given time. However, recent evidence has shown that two PfEMP1s can be
expressed at the surface simultaneously (Joergensen et al. 2010). The variant being expressed can
be switched at each blood stage cycle with a frequency of around 1-2% per generation. PfEMP1s
are high molecular weight proteins and consist of a conserved cytoplasmic and transmembrane
(TM) region and a variable extracellular region (figure 2). The extracellular regions are build up of
duffy-binding-like (DBL) domains, cysteine-rich interdomain regions (CIDR) and C2 domains. Based
on sequence signatures these can further be classified into DBLα-ε and CIDRα-γ although extensive
variation is seen even within groups. The number, organization, and type of DBL and CIDR domains
among PfEMP1 vary, however, the first domain from the N-terminal is a DBL1α usually followed by
a CIDR1α or γ. var genes can be subgrouped into A, B, C or intermediate A/B or B/C groups based
on their chromosomal location, transcriptional orientation, and upstream sequence (Lavstsen et
al. 2003; Mercereau-Puijalon et al. 2008; Rowe et al. 2009).
Figure 2. Schematic representation of Plasmodium
falciparum erythrocytes membrane protein 1
(PfEMP1). PfEMP1s are expressed by the parasite and
exported to the infected erythrocyte surface where they
participate in binding to receptors on e.g. the human
vascular endothelium. NTS: N-terminal sequence, DBL:
Duffy binding-like domain, CIDR: Cysteine-rich
interdomain region, TM: Transmembrane region. Figure
from Rowe et al. (2009).
CYTOADHESION
PfEMP1s are known to bind to different receptors at the host cell surface making the IEs adhere to
host cells. There are four different types of adhesion; adhesion to endothelial cells, adhesion to
uninfected erythrocytes, adhesion to platelets and adhesion to cells of the immune system (figure
3) (Mercereau-Puijalon et al. 2008; Rowe et al. 2009).
3
Figure 3 Adhesion types of IEs. By adhering to endothelial cells IEs are able to accumulate in the placenta, brain or
microvasculature. Other adhesion types are adhesion to erythrocytes, which causes the IE to form rosettes, to platelets,
which is responsible for platelet mediated clumping of the IEs, or to cells of the immune system. Figure modified from
Miller et al. (2002)
Adhesion to endothelial cells make the IEs accumulate in the post-capillary venules. This is thought
to be an advantage to the parasite as it avoids the host’s normal splenic clearance of aged or
damaged erythrocytes. The accumulation leads to microvascular obstruction and inflammation
causing disease in the host. This is the case in cerebral and pregnancy malaria where parasites
accumulate in the brain and placenta respectively causing severe disease. A number of host
molecules have been shown to be adhesion receptors. Among these are intercellular adhesion
molecule 1 (ICAM-1), to which adhesion in some, but not all studies has been linked to cerebral
malaria (Rogerson et al. 1999; Smith et al. 2000) and chondroitin sulfate A (CSA) in the placenta to
which IEs causing pregnancy malaria adhere. Several PfEMP1 variants have been shown to be able
to mediate adhesion to cell surface receptors. Among these are the PFD1235w variant that
adheres to ICAM-1 and the VAR2CSA variant adhering to CSA (Joergensen et al. 2010; Rowe et al.
2009; Salanti et al. 2004).
Adhesion of an IE to uninfected erythrocytes is called rosetting. It is not clear what advantage
rosetting gives parasites, but the phenomenon enhance microvascular obstruction and has been
linked to severe disease as it is more abundant in parasites isolated from children with severe
disease. Adherence to erythrocytes has been shown to occur through various receptors. Among
these complement receptor 1 (CR1), heparan sulfate like molecules and blood group antigens A
and B. Furthermore immunoglobulin M (IgM) has been suggested to play a role in rosette
formation. Adherence to erythrocytes has been mapped to the DBL1α of several different PfEMP1
variants among these IT4VAR60 (Albrecht et al. 2011; Mercereau-Puijalon et al. 2008; ViganWomas et al. 2008).
Less is known about the last two adhesion types. IEs are able to adhere to platelets resulting in
platelet-mediated clumping, which is thought to contribute to microvascular obstruction. The
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implication of platelet-mediated clumping in severe malaria is though being debated (Wassmer et
al. 2008). IEs are also able to adhere to cells of the immune system, but whether this adhesion aids
in immune evasion or parasite clearance is unclear. For both adhesion types the PfEMP1s involved
are unknown (Rowe et al. 2009).
Furthermore some PfEMP1s have been shown to bind to natural IgM in addition to cell adhesion,
possibly conferring immune evasion (Ghumra et al. (2008), Barfod et al. submitted for
publication).
Extensive research is being done on PfEMP1. Both to study its role in adhesion, but also their role
as target in naturally acquired immunity.
IMMUNITY TO MALARIA
In malaria endemic areas protection to malaria disease is acquired gradually after repeated
infections. This immunity is referred to as clinical immunity. Severe disease predominates in early
childhood, and immunity to SM has been shown to be acquired after only a few episodes (Gupta
et al. 1999). On the contrary susceptibility to uncomplicated disease continues into adolescence or
even longer. Sterile immunity is probably never achieved and although no disease symptoms are
seen low-density parasitaemia can still occur.
Several factors of the innate and adaptive immune systems play together in combating malaria.
However, in this thesis only the most studied antibody-mediated immunity will be described.
Studies have shown that IgG antibodies are central to malaria immunity in that immunity can be
transferred passively (Cohen et al. 1961). Antibodies normally act by neutralizing the pathogen or
targeting it for opsonization or complement activation (Murphy et al. 2008). Protection against
malaria has been linked to antibodies targeting VSAs and are thought to protect by blocking
adhesion and hence promoting splenic removal or targeting IEs for opsonization or complement
activation (Beeson et al. 2008).
The maintenance of immunity to malaria is being debated. While clinical immunity persists in
people living in malaria endemic areas, people leaving these areas, become susceptible to disease
when they revisit after a period abroad. However, they have decreased risk of experiencing severe
complication compared to non-immune travelers (Struik & Riley 2004). It thus seems like immune
memory to severe disease is longer lasting, but memory in general is inefficient. In a study on
Madagascans, where Malaria has been eliminated, they showed that 8 years after the last malaria
outbreak, memory B cells towards malaria parasites were still found. This suggest that at least
some Plasmodium falciparum-specific memory B cells are long lived (Migot et al. 1995).
VSAs have proven to be the major target of protective antibodies as level of VSA-specific
antibodies correlates with protection. Studies have shown that parasites isolated from malaria
5
patients express VSAs that are not recognized by the patients’ plasma prior to infection, although
other unrelated VSAs are recognized. However, when examining serum after infection, high level
of IgG specific for the disease-associated VSA has been acquired (Bull et al. 1998; Ofori et al.
2002). Susceptibility thus reflects ‘holes’ in the antibody repertoire and a disease episode results
in closing the hole to the particular VSA. The great variation in VSAs and thus many holes to close
explains the slow acquisition of protective immunity.
VSAs expressed by parasites isolated from children with severe disease (VSASM) are more
frequently recognized by plasma from healthy children than are VSAs expressed in parasites
isolated from patients with uncomplicated disease (VSAUM) (Nielsen et al. 2002). This suggests that
the repertoire of VSASM is relatively conserved and that VSASM seem to predominate early in life.
Furthermore it indicates that VSAs are expressed in a certain order. This fits with the observation
of severe malaria occurring early in life and the quick achievement of immunity to severe disease
mentioned above.
Consistent with this, the transcription of PfEMP1s belonging to group A were found to be upregulated in VSASM expressing parasites (Jensen et al. 2004). Group A consists of large PfEMP1s
that resemble each other more than var genes do in general. This is further supported by
(Warimwe et al. 2009) who linked young host age and severe disease to expression of PfEMP1s of
group A and A/B in field isolates. That VSASM seem to be relatively conserved opens the possibility
that a vaccine targeting VSA and thereby reducing malaria morbidity and mortality can be
developed.
VSAS AS VACCINE CANDIDATES
Extensive research has been done and is being done in order to develop vaccines against malaria,
but still no effective vaccine exists. Different approaches aim at targeting different stages in the
Plasmodium falciparum life cycle. Some aim at targeting the sporozoite and liver stages in order to
prevent infection, some aim at the blood stage in order to prevent disease and some seek to
prevent transmission by targeting gametocytes (Wipasa et al. 2002). Blood stage vaccines are the
focus of research at Centre for Medical Parasitology and as VSAs seem to be a central target in
naturally acquired immunity, they are the main focus. Furthermore, due to PfEMP1s known
implication in adhesion these are the VSAs that are currently considered for vaccine-development
(Hviid 2010).
VSAs have been considered inconvenient vaccine candidates due to their substantial variability.
However, the increasing evidence of a limited repertoire of VSAs causing severe disease, gives
hope to the possibility of developing a morbidity reducing vaccine. If a vaccine eliciting antibodies
targeting VSASM could be developed it would prevent severe disease and thus reduce morbidity
6
and mortality. However, uncomplicated disease would not be prevented, but this might only aid in
boosting the vaccine (Hviid 2010).
The challenge now is to find out, which VSAs are involved in severe malaria and where the VSASM
are conserved and thus what could be a vaccine-candidate. As group A PfEMP1s and in particular
the PFD1235w variant have been shown to be up-regulated in VSASM expressing parasites (Jensen
et al. 2004), it would be obvious to search for conserved parts between these PfEMP1s. Rosetting,
which has been associated with severe malaria, is another subject of research. Although different
PfEMP1s have been found to be implicated in rosetting it is still possible that these have
similarities since they mediate the same function. An approach for finding conserved parts of
VSASM is to study monoclonal antibodies from malaria exposed donors. Finding antibodies that are
cross-reactive to different VSASM would reveal conserved epitopes on these proteins and thus
point to possible vaccine-candidates.
MONOCLONAL ANTIBODY PRODUCTION
ANTIBODIES
Human antibodies are composed of light and heavy chains held together by disulfide bonds. The
light chain can be either of the lambda or kappa type and consists of a variable domain (VL) and a
constant domain (CL). Heavy chains also consist of a variable domain (VH) followed by different
numbers of constant domains (CH) depending on its isotype (IgG, IgM, IgD, IgA, and IgE). An
example of the IgG structure is given
in figure 4. The epitope binding site
is located at the VL and VH interface
and
consists
of
three
complementarity-determining
regions (CDR) on both domains. The
regions around the CDRs are more
conserved and are called frame work
regions (FW). Between the CH1 and
CH2 of the IgG molecule is a hinge
region that divides the antibody into
a constant fragment (Fc) and two Figure 4. Structure of antibody of the IgG isotype. See text for
antigen binding fragments (Fab) explanation. Figure from Penichet & Morrison (2004).
when cleaved (Murphy et al. 2008).
The human antibody repertoire is vast due to numerous different rearrangements of V, D and J
gene segments which make up the variable domains. Furthermore these are affinity matured in
activated B cells by somatic hypermutation. B cells carrying antigen specific membrane bound
antibodies are stimulated on the encounter with antigen (and helper T cell stimulation) that makes
7
them proliferate and differentiate into plasma cells and memory B cells. Plasma cells secrete
antibodies, while memory B cells protect in future exposures as it upon antigen stimulation quickly
proliferates and differentiates into antibody secreting plasma cells (Murphy et al. 2008).
Monoclonal antibodies are identical antibodies specific for only one epitope and are a powerful
tool to find conserved epitopes among VSASM. They can be generated by various techniques with
basically two different approaches to producing monoclonal antibodies: Either by establishing
cultures of immortalized B cells or by generation of recombinant antibodies. An overview of
features of different methods is given in table 1, but each of the different techniques will be
described below. Intense research is currently done in developing antibodies for therapeutic use in
e.g. infection, cancers, and autoimmune diseases. Although therapeutic antibodies would be
useful regarding cure of severe malaria disease these will not be discussed in this thesis as the aim
is to produce antibodies to aid in vaccine discovery.
Table 1. Features of antibody-production methods
The methods vary in which cells are used for immortalization or antibody gene isolation, which isotypes can be
produced, whether heavy and light chains are paired as they were in the B cell they originate from, to which antigens
antibodies can be produced and what assays can be used to select antibodies with desired specificity.
IMMORTALIZATION OF B CELLS
One approach to produce monoclonal antibodies is by establishing a culture of B cells originating
from the same B cell thus producing the same antibody. The repertoire of antibodies generated by
this approach has the advantage of having no bias in which antibody isotype can be generated.
8
Two different methods exist for production of monoclonal antibodies by immortalized B cells:
Immortalization by Epstein-Barr virus and hybridoma formation.
E PSTEIN -B ARR VIRUS - IMMORTALIZATION OF B CELLS
The Epstein-Barr method was developed in 1977 by Steinitz et al. (1977) and relies on infecting
memory B cells with Epstein-Barr virus, which transforms the cells into continuously dividing
antibody-secreting cells. Epstein-Barr is a herpes virus known to be the etiologic agent of acute
infectious mononucleosis. Memory B cells are purified from peripheral blood and mixed with
supernatant from the Epstein-Barr virus-infected cell line B95-8 (overview in figure 5.a). To
improve efficiency of immortalization a CpG oligonucleotide is added. This is a TLR9 agonist and a
polyclonal activator of memory B cells. To improve growth the immortalized cells are cultured with
irradiated mononuclear cells that do not divide. After two weeks the supernatants of the
immortalized B cell cultures can be screened for antigen specific antibodies. Positive cultures are
subcloned by limiting dilution and seeded onto growth-impaired irradiated mononuclear cells to
obtain a pure cell line producing the monoclonal antibodies of interest. A great advantage of the
Epstein-Barr method is that it uses memory B cells. Memory B cells are readily available from
peripheral blood and can be collected long time after pathogen exposure. This makes it easier to
obtain antibodies developed as a response to natural infection (Fraussen et al. 2010; Lanzavecchia
et al. 2007; Traggiai et al. 2004). A drawback of the Epstein-Barr method is that it often results in
the isolation of relatively few antibodies due to inefficient immortalization, culture death or
outgrow of non-specific B cells (Smith et al. 2009), Lea Barfod, personal communications)
THE HYBRIDOMA TECHNOLOGY
The hybridoma technology was developed by Kohler & Milstein (1975) and relies on immortalizing
B lymphocytes by fusion to cancer plasma cells called myeloma cells (overview in figure 5.b). Fused
cells will then possess the ability to grow indefinite from the cancer cell and produce the specific
antibody of the lymphocyte. When hybridomas have been formed their supernatants are screened
for desired antibody specificities and positive cultures are cloned to obtain a pure cell line
producing the monoclonal antibodies of interest. Hybridomas are commonly made from murine B
cells isolated from spleens. Prior to sampling mice are immunized with the antigen of interest to
increase the number of antigen-specific B cells. The hybridoma technology is well-established for
murine cells, but hybridoma formation with human cells has been inefficient and is thus rarely
used. Alternatively, human antibodies can be produced by the hybridoma technology by using
transgenic mice. These are mouse lines that have had human antibody genes inserted and their
own antibody genes knocked out. In this way human monoclonal antibodies can be made by the
hybridoma technology (Chiarella & Fazio 2008; Green 1999; Penichet & Morrison 2004).
9
Figure 5. Technologies for human monoclonal antibody production. A) Epstein-Barr virus-immortalization. B)
Hybridoma formation. C) Phage display of antibody libraries. Figure modified from Marasco & Sui (2007)
When producing monoclonal antibodies by mouse hybridomas, mice need to be immunized with
the antigen of interest or infected if the pathogen is shared between mice and humans.
Immunization with antigen demands knowledge and production of the antigen of interest and the
antibodies being produced are not necessarily targeting the antigen in its natural conformation
during infection. Antibodies developed in mice might also target different epitopes due to
differences between mice and human antibody-responses (Hviid & Barfod 2008; Smith et al.
2009).
RECOMBINANT PRODUCTION OF MONOCLONAL ANTIBODIES
An alternative to producing monoclonal antibodies by immortalized cells is producing recombinant
antibodies. This can either be done by making combinatorial libraries of antibody variable domain
genes and screen for antigen-specific binding by e.g. phage display or by copying antibody genes
from individual B cells by single cell PCR e.g. with the Symplex™ technology.
10
A NTIBODY P HAGE DISPLAY
Generation of large libraries of antibodies displayed on phages is a widely used method for
antibody production and was first described by Mccafferty et al. (1990). Antibody libraries are
formed by selecting B cells from immune (immune library) or non-immune donors (naïve library)
and amplify cDNA of heavy and light chain variable genes (overview of method in figure 5.c).
Synthetic libraries have also been made by introducing variation in the CDR regions of variable
domains in vitro. The heavy and light chains are then randomly combined into a phagemid vector
and coupled to a phage coat protein. By transformation into Escherichia coli, virus particles are
formed with antibodies displayed at the surface. Antibodies are either displayed as Fab fragments
or Single chain-Fv (ScFv), which are a heavy and a light chain variable domain linked together. The
display system gives the advantages of coupling of phenotype and genotype facilitating selection.
The antibodies of desired reactivity are selected by panning the phages onto an antigen-coated
surface. The non-binders are washed away and the binding phages can be collected and amplified
in Escherichia coli. The panning is repeated to increase over all affinity of selected antibodies. This
practical selection method however also limits the technique in that functional selection assays
cannot be carried out. After selection the variable domain encoding-genes from the selected
phages are then isolated and inserted into vectors for Fab or full-length antibody secretion. In
addition to phages, techniques exist for antibody library display by mRNA, ribosome, bacteria,
yeast or mammalian cells. A disadvantage of library display techniques is that the heavy and light
chains are randomly combined. In this way information of epitopes developed by natural immune
response is lost (de Haard et al. 1999; Hoogenboom et al. 1998; Hviid & Barfod 2008; Marasco &
Sui 2007).
SYMPLEX™ TECHNOLOGY
Another way to generate human monoclonal antibodies is by isolating antibody-encoding genes
from single cells, express the recombinant antibodies in bacteria or mammalian cells and screen
supernatants for the desired reactivity. In contrast to library display techniques antibodies
generated by single cell PCR retain the pairing of heavy and light chains from the original B cell and
the antibodies are thus identical to the ones developed in the donor’s natural immune response.
Single cell PCR techniques are however biased in that only antibody genes of a certain subtype can
be amplified in one PCR reaction, thus neglecting cells of any other isotype.
A technology relying on single cell PCR is the high throughput technology Symplex™ developed by
Meijer et al. (2006) at the company Symphogen. Antibody secreting cells (ASC) are isolated after
donor booster immunization by specific staining followed by fluorescence-activated cell sorting
(FACS) into PCR-plates. In the first PCR reaction (referred to as 1st PCR) Reverse transcription-PCR
(RT-PCR) followed by regular PCR amplification is performed in one step (Overview in figure 6).
The primers used are a cocktail of primers amplifying the variable and constant domain 1 of the
IgG heavy chains and kappa light chains separately. As mentioned above antibody variable
domains are made up from recombination of different V, D, and J segments. These segments differ
11
Figure 6. The Symplex™ technology. A
multiplex 1st PCR is performed on single B
cells with variable domain primers with
overlapping 5’ tails. A 2nd PCR is performed
on the product and amplifies the heavy
chain variable domain and the light chain
variable and constant domains as one
fragment due to the overlapping sequence
from the 1st PCR. The fragments from all
nd
positive 2 PCRs are pooled together and
bulk cloned into an antibody-expression
vector followed by exchange of the linker
sequence with a double promoter. The
constructs are then transformed into an
expression organism and antibodies can be
harvested from supernatant. CH1:
Constant domain 1 of heavy chain. VH:
Variable domain of heavy chain. VL:
Variable domain of light chain. CL:
Constant domain of light chain. Linker:
sequence
generated
by
the
complementary 5’end tails. Figure
modified from Meijer et al. (2006)
in sequence and therefore multiple primers are needed to cover many different antibody variants.
The tails of the 1st PCR primers annealing at the variable domain of the heavy chain are
complementary to the tails of the primers annealing at the variable domain of the light chain. The
product from the 1st PCR is subjected to a second nested PCR (referred to as 2nd PCR), where
primers are annealing at the J segment of the heavy chain variable domain and the constant
domain of the light chain. The complementary sequence of the primers of the 1st PCR results in the
linking of the light and heavy chain DNA. Heavy and light chains are thus amplified as one
fragment in the 2nd PCR. The positive wells (verified by gel-analysis) are pooled together for bulk
cloning. Bulk cloning limits the work associated with cloning. However, it also necessitates
isolation of large amounts of colonies to make sure a substantial proportion of the different
antibodies are represented.
The primers used in the 2nd PCR contain restriction sites in their tails facilitating ligation into an
antibody-expression vector. This could either be a Fab-expression vector as described in Meijer et
12
al. (2006) containing the C1 domain of the IgG heavy chain or a full-length expression vector
containing the C1, C2, and C3 domains of the IgG heavy chain. Expression vectors conferring the
expression of other isotypes could all so be utilized. The linker sequence formed by the
complementary primers contain restriction sites that facilitates the exchange of the linker
sequence with a bi-directional double promoter with leader cassettes for the expression of heavy
and light chain. The leader cassettes contain start codons and restriction sites to ensure in frame
placement of antibody genes. The final construct is used to transform Escherichia coli cells or to
transfect mammalian cells. Culture supernatants are then tested for the desired activity in either
binding or functional assays. Production of positive antibodies are easily scaled up for further
characterization (Meijer et al. 2006). A drawback of the Symplex™ method is that it relies on ASCs.
These are only available at a narrow time window after immunization or infection and are
therefore difficult to collect after natural infection. ASCs cannot be selected for antigen-specificity
prior to cloning, meaning expensive cloning of cells that generate uninteresting antibodies must
be done as well (Lanzavecchia et al. 2007).
Other technologies for single cell PCR have been developed recently by (Smith et al. 2009; Tiller et
al. 2008). While the method developed by Smith et al. (2009) resembles the Symplex™ technology,
the method developed by Tiller et al. (2008) differs in that it can be performed on memory B cells.
However, none of the methods rely on linking the heavy and light chain fragments together thus
allowing bulk cloning as Symplex™ does. Instead each light chain and heavy chain 2nd PCR product
is cloned separately and expression is performed by co-transfecting with light and heavy chain
constructs which adds considerable work to the methods.
13
OUTLINE FOR EXPERIMENTAL WORK
Monoclonal antibodies developed in protective immune responses to natural infections by
Plasmodium falciparum are an invaluable tool to discover relevant, conserved epitopes of VSASM.
The original aim of this thesis was to generate human monoclonal antibodies with specificity for
clinically important PfEMP1 antigens using the Symplex™ technology. The intended approach was
to collect B cells from malaria exposed donors, purify memory B cells and select these for binding
to recombinant Plasmodium falciparum proteins, stimulate memory B cells to become plasma cells
and copy the antibody genes by the Symplex™ method. Thereafter the recombinant antibodies
would be produced and anti-malaria antibodies would be characterized.
Unfortunately, as is described in the results’ ‘B cell collection’ section, the collected B cells showed
too low a viability to continue in the intended direction. Therefore the project took a new turn and
the aim was revised.
At Centre for Medical Parasitology (CMP), Epstein-Barr virus-immortalized memory B cells from
donors who are clinically immune to malaria have been used to produce monoclonal anti-malaria
antibodies. Some of the monoclonal antibodies generated at CMP by the Epstein-Barr method
have been shown to have interesting activities such as binding to the PfEMP1 PFD1235w which is
associated with severe childhood malaria, binding to the PfEMP1 VAR2CSA which is implicated in
pregnancy malaria, or inhibiting rosette-formation, which also has been linked to severe malaria.
However, the Epstein-Barr technology gives relatively low-yield, is susceptible to cell culture
contamination and cultures often die out. Recombinant production would give advantages to
further studies of the antibodies, as production of high quantities is convenient, the production is
stable and engineering of the antibody is possible. Therefore I wanted to establish the Symplex™
technology at CMP to produce recombinant versions of Epstein-Barr virus-immortalized cultureantibodies. The establishment of the Symplex™ technology for small-scale antibody generation
would also prepare the laboratory for larger scale antibody library generation in future.
Aim:
To clone antibody genes from Epstein-Barr virus-immortalized B cell cultures using the
Symplex™ technology, express them as recombinant antibodies and characterize these
recombinant antibodies.
To pursue this aim, I need to establish the Symplex™ technology at CMP. To accomplish
this, I must be able to:
•
•
•
•
•
Isolate antibody genes from single cells producing antibodies
Verify correct antibody gene insertion into expression vector
Verify and optimize antibody production
Purify antibodies
Verify that recombinant antibodies retain activity of natural antibody
14
•
Solve problems that may arise (contamination, cell sorting)
Finally based on experience from present experiments and relevant literature I want to evaluate
the establishment of the Symplex™ method and compare it to the presently used Epstein-Barr
method and to other methods for larger scale monoclonal antibody generation.
15
MATERIALS AND METHODS
B CELL COLLECTION
ISOLATION OF B CELLS
B cells were collected in the malaria endemic country Ghana (World Health Organization 2009).
Blood samples were a kind gift from Dr. Justina Ansah at the Korle-Bu Teaching Hospitals blood
bank in Accra, Ghana, who obtained necessary ethical clearance. Approximately 250 ml blood
samples were taken from healthy donors with life-long exposure to P. falciparum parasites and a
small aliquot of approximately 200 µl was taken out for ELISA. ELISA was done to test for anti-P.
falciparum antibodies and thus a greater probability of having memory B cells specific for P.
falciparum. (see procedure below). The highest reacting donors were selected for B cell isolation.
B cells were isolated using the RosetteSep® technique following the manufacturer’s Buffy Coat
Procedure (StemCell Technologies). The blood samples were centrifuged at 200 x g for 10 min at
room temperature (rt) with the brakes off. The leukocyte band and a small fraction of plasma and
erythrocytes were collected and added to the RosetteSep® antibody cocktail. RosetteSep is a
negative selection method that cross-links unwanted cells to red blood cells and leaves B cell
unbound. After 20 min incubation the solution was diluted three fold in dilution buffer (DPBS
(Lonza, #BE17-512F) + 2% Fetal Bovine Serum (FBS) (Sigma-Aldrich, #F6178)). This was layered on
top of the density medium RosetteSep DM-L (StemCell Technologies) and centrifuged at 1200 x g
for 20 min at rt with the brakes off. Afterwards the B cell layer between the plasma and the DM-L
could be removed and cells were washed twice with dilution buffer. Cells were counted, their
viability verified using trypan blue solution (1% w/W trypan blue diluted 1:10 in DPBS) and diluted
in freezing media (RPMI 1640 medium (Invitrogen, #31870-025), 10% Dimethylsulphoxide (DMSO)
(Sigma-Aldrich, #D5879), 12.5% FBS (Sigma-Aldrich, #F6178)). Finally the B cells were aliquoted
into 1.8 ml Cryotubes (Nunc, #115511) with 0.5·106-1.7·106 per tube, cryopreserved in liquid
nitrogen on a gradient-controlled device as described in Hviid et al. (1993) and stored in liquid
nitrogen until use. The cells were transported to Denmark in a cryogenic dry shipper.
MALARIA EXPOSURE DETERMINING ELISA
ELISA was performed on plasma from the blood donors in order to test for anti-P. falciparum
antibodies. At least one day prior to the experiment ELISA plates (Nunc, #456437) were coated
with 100 µl antigen solution. These were either 10 ng/ml GLURP R0 (protein prepared as in
(Theisen et al. 1995)), 1:100 or 1:10 crude P. falciparum extract (described under the MACSpurification section), Ghanaian P. falciparum extract with 50.000 or 500.000 parasites/well (kind
gift from Michael Ofori, Noguchi Memorial Institute for Medical Research, University of Ghana),
controls with crude extract of uninfected erythrocytes (uninfected erythrocytes for parasite
cultivation, spun down and sonicated) or coating buffer (0.1M Glycin (Sigma-Aldrich, #G7403),
adjusted to pH 2.75 with HCl (VWR, #30018.298))). The plates were stored at 4°C until use and all
following steps were performed at rt. Wells were emptied and blocked with 200 µl blocking buffer
16
(1% Bovine Serum Albumin (BSA) (Sigma-Aldrich, #A3059), 0.1% Tween®20 (Sigma-Aldrich, #
P7949) in DPBS (Lonza, #BE17-512F)) for 15 min under rocking conditions. After emptying the
wells 100 µl of the plasma samples diluted 1:100 in blocking buffer was added to each well and
incubated 15 min under rocking conditions. Blocking buffer without plasma was used as negative
controls. The wells were then washed 3 times with washing buffer (0.1% Tween®20 (SigmaAldrich, # P7949) in DPBS (Lonza, #BE17-512F)) and 100 µl 1:3000 Polyclonal rabbit anti-human
IgG-HRP (DakoCytomation, #P0214) was added and the plate incubated 15 min under rocking
conditions. After additional 3 washes in washing buffer, 100 µl of OPD solution (4 OPD-tablets
(Dako, #S2045), 12 ml ultrapure water, 10 µl 30% wt. H2O2 (Sigma-Aldrich, #H3410)) was added
and incubated in the dark until color developed (approximately 10 min). To stop the reaction 100
µl 2.5M H2SO4 (Merck, #4803641000) was added and the plate was read on an ELISA reader at
492nm. For the final ELISA on day 4 DPBS (Lonza, #BE17-512F) was used as coating buffer and the
amount of the Ghanaian P. falciparum extract was increased to 500.000 parasites/well.
EPSTEIN BARR VIRUS IMMORTALIZED B CELLS
Epstein Barr Virus-immortalized B cell cultures (EBV cells) were provided by Lea Barfod. Peripheral
Blood Mononuclear cells were obtained from malaria-exposed donors in malaria-endemic Ghana
and the immortalization was done as in Barfod et al. (2007). The cultures had been subcloned
once and expanded. The cultures had been selected due to their rosette-inhibiting activity, binding
to PFD1235w or IEs expressing VAR2CSA.
SINGLE CELL SORTING
SINGLE CELL SORTING BY LIMITING DILUTION
EBV cells were passed through MACS® Pre-separation filters (Miltenyi Biotec, #130-041-407) to
avoid cell clumps, counted using a haemocytometer and diluted in DPBS (Lonza, #BE17-512F) to
0.5 cell/µl or 10 cells/µl. 1 µl of 0.5 cell/µl was added to each well on a 96 well plate (Bioplastics,
#AB70651) containing 9 µl reaction mix (described in ‘Isolation of VH-VCL from EBV cells’). 1 µl
10cells/µl was added to positive semi-control wells and 1 µl DPBS was added to negative control
wells. The 0.5 cell/µl dilution was used in order to reduce the risk of multiple cells per well and
thus secure the natural pairing of the heavy and light chain genes. The 10 cells/well is not a true
positive control as it will not always be positive. Instead it is a control where template should be
present and will be positive if the mRNA can be amplified by the specific primer set (and cells in
fact produce mRNA). Real positive controls were not included due to risk of them contaminating
the rest of the wells.
17
SINGLE CELL SORTING BY FACSARIA
Single cell sorting was performed on a FACSAria™ cell sorter from Beckton Dickenson by core
facilities at Department of International Health, Immunology and Microbiology, University of
Copenhagen. At the day of sorting 96 well plates were prepared with 10 µl reaction mix (described
in ‘Isolation of VH-VCL from EBV cells’) and stored on dry ice until sorting. EBV cells were
suspended in media and passed through MACS® Pre-separation filters (Miltenyi Biotec, #130-041407) to obtain single cells. The cells were then applied to the FACSAria and sorted into the 96-well
plates. The P1 gate (see figure 7) was chosen to avoid cell debris (assumedly in the P2 area) or cell
clusters (assumedly in the P3 area). The FACSAria
was set to apply one cell pr well or 10 cells into
positive semi-control wells. Nothing was added to
the negative control wells. Three different kinds of
96 well plates were used: ‘regular’ PCR-plates from
Bioplastics, (#AB70651), skirted, ‘stabile’ plates
from Eppendorf (#951020401) and plates identical
to the ones used by (Meijer et al. 2006) namely
plates from ABgene (#AB0800). In the initial
experiments a dummy plate was used to calibrate
cell-containing droplets to the wells and for the rest
of the experiments the FACSAria was calibrated on
the reaction-mix containing plate while covering it
with a lid.
Figure 7. Forward and side scatter data of EBV
cells. P1 gate was selected for cell sorting.
CELL SORTING USING MICRO MANIPULATOR
A Nikon Eclipse TE2000-E microscope and a Nikon Narishige NT-88-V3 micro manipulator
containing a 11 µm, 30° angle needle (VitroLife, #14324) were used. EBV B cells were placed under
the microscope and a single, healthy looking cell was sucked into the needle and transferred to 10
µl reaction mix (described in ‘Isolation of VH-VCL from EBV-immortalized B cells’), which was kept
on ice until use. Immediately thereafter the solution was mixed and transferred to a well on a 96
well plate (Bioplastics, #AB70651) on ice. For the positive semi-control 10 cells were sucked into
the needle and transferred to the reaction mix. The negative control was made by sucking up
media from the cell culture or simply not adding anything to the reaction mix.
CLONING OF VH-VCL FROM EBV CELLS
An overview of the different steps involved in the cloning procedure is given in figure 8.
18
Figure 8. Overview of the cloning process. Flow-chart from EBV cell to recombinant antibody production.
19
ISOLATION OF VH-VCL FROM EBV-IMMORTALIZED B CELLS
Cloning of the heavy chain variable domain (VH) and the light chain constant and variable domains
(VCL) genes was essentially done as in Meijer et al. (2006), but with some modifications. Instead of
only using primer sets amplifying IgGκ antibodies, primer sets for the amplification of IgM and λlight chain antibodies had been modified from de Haard et al. (1999) and included (provided by
Michael Dalgaard). A 96 well plate was prepared for multiplex overlap-extension RT-PCR by adding
10 Units of RNasin (Promega, #N2515), 2 µl One-Step Buffer, 400 µM dNTP mix, 0.4 µl OneStep RT
PCR Enzyme Mix (Qiagen OneStep RT PCR kit, #210212) 1 µl of either the 1st PCR G+κ, G+λ2, G+λ7,
M+κ, M+λ2 or M+λ7 primer sets (table 2, individual primers are listed in table 3) and RNase free
H2O to a final volume of 9 µl or 10 µl. The Quiagen OneStep Enzyme Mix contains enzymes both
for reverse transcription and PCR amplification. All work was performed in a DNA clean-bench in
order to avoid DNA and RNA contamination. EBV cells were applied to the wells by either limiting
dilution, FACSAria cell sorting or micro manipulation. When using the micro manipulator OneStep
RT PCR Enzyme mix was not added until after the cell sort. A volume of 0.8 µl of enzyme mix
diluted 1:1 was then added. After cell sorting the plates were given a short spin (e.g. 20 sec, 200 x
g) and placed at -80 °C for approximately 15 min to release mRNA by rupturing the cells.
Subsequently reverse transcription was performed by incubating 30 min. at 55 °C. The following
15 min at 95 °C inactivates the reverse transcriptases and activates the amplification PCR enzyme.
Products were then amplified by 35 cycles of 94 °C, 30 s, 60 °C, 30 s, 72 °C, 5 min, followed by an
extension step of 10 min at 72 °C (run on a Duocycler, VWR). This PCR reaction will further on be
referred to as 1st PCR. 1 µl of the 1st PCR product was added to a new plate with 4 µl 5x Phusion®
HF Reaction Buffer (Finnzymes, #F-518), 0.2 µl Phusion® DNA polymerase (Finnzymes, #F-530L),
200µM dNTP mix (Invitrogen, # 10297018), 2 µl of the primer set 2nd PCR G/M+κ, G/M+λ2 or
G/M+λ7 to a final volume of 20 µl. A 2nd PCR was then performed by a 30 s step at 98 °C followed
by 30 cycles of 98 °C, 10 s, 60 °C, 10 s, 72 °C, 30 s and ending with an extension step of 5 min at 72
°C. To check for positive product, 3 µl was run on a 1% agarose gel and wells with products around
1100 bp were selected for further amplification. GeneRuler™ 100 bp Plus (Fermentas, #SM0321)
was used as DNA ladder. To obtain greater amounts of the positive product a new 2nd PCR (2nd PCR
amplification) was set up with 1st PCR product from positive wells. All product from the positive
wells of the 1st PCR was used to set up as many 2nd PCRs wells as possible. 2µl 1st PCR product was
used per well together with 8 µl 5x Phusion® HF Reaction Buffer (Finnzymes, #F-518), 0.4 µl
Phusion® DNA polymerase (Finnzymes, #F-530L), 200 µM dNTP mix (Invitrogen, # 10297018) and 4
µl of the relevant 2nd PCR primer set to a final volume of 40 µl/well. The 2nd PCR amplification
products stemming from the same 1st PCR well were pooled and run on a 1% agarose gel.
Fragments around 1100 bp (referred to as VH-VCL) were cut out using a clean scalpel and purified
with E.Z.N.A.® Gel Extraction kit (Omega, #D2500-02) following the manufacturer’s instructions.
GeneRuler™ 100 bp Plus (Fermentas, #SM0321) was used as DNA ladder. All PCR-work was
performed using gloves and filter tips. After discovering problems with contamination a
sequencing step was inserted after fragment purification. Sequencing procedure is described in a
separate section below.
20
Table 2. List of primer sets
Primer set
1st PCR G+κ
1st PCR M+κ
1st PCR G+λ2
1st PCR M+λ2
1st PCR G+λ7
1st PCR M+λ7
2nd PCR G/M+κ
2nd PCR G/M+λ2
2nd PCR G/M+λ7
Primer name
S7, S8, S9, S10, S11, S12, S13, S14, S15a, S16, S17,
S18, S19, S20, S21, S22a
S7, S8, S9, S10, S11, S12, S13, S14, S16, S17, S18,
S19, S20, S21, S22a, S28a
S7, S8, S9, S10, S11, S12, S13, S14, S15a, S29, S30,
S31, S32, S33, S34, S35, S36, S37, S38, S39, S40a
S7, S8, S9, S10, S11, S12, S13, S14, S28a, S29, S30,
S31, S32, S33, S34, S35, S36, S37, S38, S39, S40a
S7, S8, S9, S10, S11, S12, S13, S14, S15a, S29, S30,
S31, S32, S33, S34, S35, S36, S37, S38, S39, S41a
S7, S8, S9, S10, S11, S12, S13, S14, S28a, S29, S30,
S31, S32, S33, S34, S35, S36, S37, S38, S39, S41a
S23a, S24a, S25a, S26a, S27a
S23a, S24a, S25a, S26a, S42a
S23a, S24a, S25a, S26a, S43a
Final primer concentration Is 40nM or 200nM if marked with an a.
Table 3. List of primers
Primer name
Primer sequence
S1
GGGCCCTTGGTGGAGGC
S2
CCTCTACAAATGTGGTATGGCTG
S3
CTTCTGTGTTCTCTCCACAGGAG
S4
GTCTCCACTCCTAGGTACGC
S7
tattcccatggcgcgccCAGRTGCAGCTGGTGCART
S8
tattcccatggcgcgccSAGGTCCAGCTGGTRCAGT
S9
tattcccatggcgcgccCAGRTCACCTTGAAGGAGT
S10
tattcccatggcgcgccSAGGTGCAGCTGGTGGAG
S11
tattcccatggcgcgccCAGGTGCAGCTACAGCAGT
S12
tattcccatggcgcgccCAGSTGCAGCTGCAGGAGT
S13
tattcccatggcgcgccGARGTGCAGCTGGTGCAGT
S14
tattcccatggcgcgccCAGGTACAGCTGCAGCAGTC
S15
GACSGATGGGCCCTTGGTGG
S16
ggcgcgccatgggaatagctagccGACATCCAGWTGACCCAGTCT
S17
ggcgcgccatgggaatagctagccGATGTTGTGATGACTCAGTCT
S18
ggcgcgccatgggaatagctagccGAAATTGTGWTGACRCAGTCT
S19
ggcgcgccatgggaatagctagccGATATTGTGATGACCCACACT
S20
ggcgcgccatgggaatagctagccGAAACGACACTCACGCAGT
S21
ggcgcgccatgggaatagctagccGAAATTGTGCTGACTCAGTCT
S22
atatatatgcggccgcACACTCTCCCCTGTTGAA
S23
ggaggcgctcGAGACGGTGACCAGGGTGCC
S24
ggaggcgctcGAGACGGTGACCATTGTCCC
S25
ggaggcgctcGAGACGGTGACCAGGGTTCC
S26
ggaggcgctcGAGACGGTGACCGTGGTCCC
S27
accgcctccaccggcggccgcACACTCTCCCCTGTTGAAGCTCTT
S28
TGGAAGAGGCACGTTCTTTTCTTT
S29
ggcgcgccatgggaatagctagccCAGTCTGTGCTGACTCAGCCA
Reference
Michael Dalgaard, unpublished
RT-PCR Meijer et al 2006
Nested PCR Meijer et al 2006
RT.PCR de Haard et al 1999
21
S30
S31
S32
S33
S34
S35
S36
S37
S38
S39
S40
S41
S42
ggcgcgccatgggaatagctagccCAGTCTGTGYTGACGCAGCCG
ggcgcgccatgggaatagctagccCAGTCTGTCGTGACGCAGCCG
ggcgcgccatgggaatagctagccCARTCTGCCCTGACTCAGCCT
ggcgcgccatgggaatagctagccTCCTATGWGCTGACTCAGCCA
ggcgcgccatgggaatagctagccTCTTCTGAGCTGACTCAGGAC
ggcgcgccatgggaatagctagccCACGTTATACTGACTCAACCG
ggcgcgccatgggaatagctagccCAGGCTGTGCTGACTCAGCCG
ggcgcgccatgggaatagctagccAATTTTATGCTGACTCAGCCC
ggcgcgccatgggaatagctagccCAGRCTGTGGTGACYCAGGAG
ggcgcgccatgggaatagctagccCWGCCTGTGCTGACTCAGCCM
atatatatgcggccgcTGAACATTCTGTAGGGGCCACTG
atatatatgcggccgcAGAGCATTCTGCAGGGGCCACTG
accgcctccaccggcggccgcTGAACATTCTGTAGGGGCCACTG
S43
accgcctccaccggcggccgcAGAGCATTCTGCAGGGGCCACTG
RT-PCR de Haard et al 1999
RT-PCR de Haard et al 1999
Michael Dalgaard, unpublished,
modified from de Haard et al 1999
Michael Dalgaard, unpublished,
modified from de Haard et al 1999
Table continued from previous page.
All primers were obtained from eurofins MWG Operon. S = C or G, W = A or T, R = A or G.
INSERTION OF VH-VCL INTO IGG1 EXPRESSION VECTOR
15 µl of each of the purified VH-VCL products were digested with 1.5 µl NotI (New England Biolabs
(NEB), #R0189L) and 1.5 µl XhoI (NEB, #R0146M) in a final volume of 50 µl using Buffer 3 (NEB,
#B7003S) at a final dilution of 1:10 and BSA (NEB, #B9001S) of 100 µg/ml. Following one hour
incubation at 37 °C the digested fragments were purified with E.Z.N.A.® MicroElute® DNA Clean up
kit (Omega, #D6296-02) following the manufacturer’s instructions. The IgG1-expression vector
00VP002 (Symphogen) was also digested with NotI and XhoI (as described above). This digestion
generates two bands of similar size when run on an agarose gel. Therefore, prior to digestion with
NotI and XhoI, 00VP002 was digested with BsmBI (NEB, #R0580S) one hour at 55 °C using the same
reagents as described above. NotI and XhoI were then added and incubated at 37 °C for two
hours. To prevent re-circulation the 5’ phosphate groups were removed by one hour incubation at
37 °C with 0.5U/mgDNA Alkaline Phosphatase, Calf Intestinal (CIP)(NEB, #M0290S)). The digested
vector was then isolated by running the mixture on an 0.7% agarose gel, cutting out the band of
approximately 5400 bp and purifying them with E.Z.N.A.® Gel Extraction kit (Omega, #D2500-02)
following the manufacturer’s instructions. GeneRuler™ 1 kb Plus (Fermentas, #SM1331) was
used as DNA ladder. The digested 00VP002 and VH-CVL fragments were then ligated in a 1:5 mole
ratio using 1 µl 1:10 T4 ligase, 1 µl T4 DNA ligase buffer (NEB, # M0202) and a final volume of 10 µl.
After incubation either two hours at rt or ON at 14 °C the ligation mix was transformed into One
Shot® TOP10 Chemically Competent Escherichia coli (Invitrogen, #C4040-10). 3 µl ligation mix was
added to 25 µl TOP10 cells and incubated on ice for 10 min. Then plasmid-uptake was facilitated
by heat shocking the cells in a 42 °C water bath for 30 sec followed by 1 min on ice. Next 250 µl
S.O.C. medium (Invitrogen, #15544-034) was added and the cells were incubated at 37 °C for one
hour and plated onto LB agar (10g Bacto-tryptone (BD, #211705), 5g Yeast-extract (Sigma-Aldrich,
22
#70161), 10 NaCl (Sigma-Aldrich, #S9625), 15g agar (Merck, #1.01614) in 1l deionized water)
containing 100 µg/ml ampicillin (Sigma-Aldrich, #A9518). Up to four colonies from each VH-CVL
fragments were selected and verified for VH-CVL fragments insertion by 1st colony PCR. This was
performed in a 96 well plate with 10 µl TEMPase Hot Start Master Mix (Amplicon, #230303), 1 µl
20 µM S1 and 1 µl 20 µM S2 primers (annealing in 00VP002) (see figure 9) in a final volume of 20
µl. Pipette tips were used to transfer a bit of the colonies to the wells and were afterwards left in
the wells. To release the cells the plate with the pipette tips was left shaking at app. 200rpm for
5min. For negative controls colonies from transformation with 00VP002 without insert were
included as well as wells without template. Hereafter the 1st colony PCR was run on a VWR
Duocycler (95 °C, 15 min, 35 cycles of 95 °C, 30 s, 55 °C, 30 s, 72 °C, 1 min and a final extention
step of 72 °C, 10 min). 5 µl of the product was run on a 1% agarose gel. GeneRuler™ 100 bp Plus
(Fermentas, #SM0321) was used as DNA ladder. Positive transformants (seen as bands around
1100 bp) were cultured in LB medium (10g Bacto-tryptone (BD, #211705), 5g Yeast-extract (SigmaAldrich, #70161), 10 NaCl (Sigma-Aldrich, #S9625) in 1l deionized water) containing 100 µg/ml
ampicillin (Sigma-Aldrich, #A9518) over night (ON) and plasmids were purified with E.Z.N.A.™
Plasmid Mini Kit I (Omega, #D6943-02) following the manufacturer’s instructions. Subsequently
DNA concentration was measured on Nanodrop-2000 (Thermo Scientific). To further verify
insertion of the VH-CVL fragments in the plasmids, the insertion was sequenced (described in a
separate section below)
The verified sequences were then ready for insertion of the bi-directional promoter. The bidirectional promotor was cut out of 00VP002. This was done by incubation with 1 µl of each of the
restriction enzymes NheI and AscI in 2 µl 1:10 BSA and 2 µl Buffer 4 (NEB, #R0131, #R0558L,
#B9001S, #B7004S) in a total volume of 20 µl and gel purified as above - this time cutting out the
band around 3.9 kb. The VH-CVL containing plasmids were digested with NheI and AscI, the same
way as 00VP002, cutting out the LINK fragment. A fragment of 6.5 kb was isolated by gel
purification as above. The digested plasmid and promoter were ligated and transformed into One
Shot® TOP10 Chemically Competent Escherichia coli (Invitrogen, #C4040-10) as described above.
2nd Colony PCR was run as 1st PCR - only now using the primers S1 (00VP002 annealing) and S3
(promoter annealing) (see figure 9) and selecting positive transformants with bands around 400
bp. Plasmids were amplified and purified as above and sequenced (described below).
SEQUENCING
Sequencing was done by adding template DNA to wells on a MicroAmp™ optical 96 well research
plate (Applied Biosystems, #N801-0560) containing 1.75 µl BigDye® Terminator v3.1 5X
Sequencing Buffer, 0.5 µl BigDye® Terminator v3.1 Enzyme mix v3.1 (BigDye® Terminator v3.1
Cycle Sequencing Kit 100rxn, Applied Biosystems, #4337455) and 2 µl 0.8 µM primer in a final
volume of 10 µl. Sequencing-PCR was performed on a 2730 Thermal Cycler (Applied Biosystems)
(96 °C, 1 min, 25 cycles of 96 °C, 10 s, 50 °C, 5 s, 60 °C, 4 min and a final step of 60 °C for 1 s. 100ng
or 200ng template and primers S23 or S27 were used for the sequencing the VH-VCL fragment (1st
23
sequencing). 300ng or 600ng template was used for sequencing after ligation into 00VP002 either
with primers S1 or S2 (2nd sequencing) before promoter insertion or with S3 or S4 (3rd sequencing)
after promoter insertion. Location of the primer annealing sites is shown in figure 9. After the PCR
2.5 µl 125 mM EDTA (Invitrogen, #15576-028 dissolved in ultrapure water) and 32 µl ice cold 96%
Ethanol (Solveco AB, #200-578) was mixed and added to each well to precipitate DNA. Hereafter
the plate was left at -20 °C for at least 30 min, spun down at 2169 x g, -10 °C for at least 30 min,
inverted and emptied with a gentle flick followed by spinning up site down at 180 x g, -10 °C, 30,
washed with 100 µl 70% ethanol (96% Ethanol (Solveco AB, #200-578) diluted in ultrapure water)
repeating the spinning down and emptying but this time spinning for 1 min instead of 30 s. Finally
10 µl Hi-Di™ formamide (Applied Biosystems, #4311320) was added and mixed and after a quick
spin to avoid air bubbles the plate was run on a sequenator 3130 Genetic Analyzer (Applied
Biosystems).
Figure 9. Overview of sequencing primer annealing-sites.
SEQUENCE ANALYSIS
The sequences were analyzed using the program CLC Main Workbench (CLC Bio) and its default
settings. The VH-CVL fragment sequences from the 1st sequencing were assembled using the
‘assemble’ function. With this function sequence of quality below a certain threshold is cut off and
sequences are aligned with each other. Conflicts between sequences were verified manually and a
consensus sequence was made based on an assessment of which sequence had better quality at
the given point. The consensus sequence was then divided into light chain sequence until the NheI
restriction site at the linker sequence (GCTAGCTATTCCCATGGCGCGCC) and heavy chain sequence
from the AscI restriction site in the LINK sequence. To verify for contamination a multiple
alignment was made with other previously cloned antibody heavy and light chain sequences
separately. If the antibody was not identical with any previous sequences (no apparent
contamination) its sequence was blasted against the nucleotide collection (nr/nt) database using
the blastn program at NCBI (http://blast.ncbi.nlm.nih.gov/). If the highest scoring alignment was a
24
human antibody sequence, the antibody gene isolation was assumed to be successful and the
fragment chosen for continuation to plasmid insertion.
Sequences from the 2nd sequencing after ligation into 00VP002 were assembled and verified as
above and a consensus light and heavy chain were generated. A Heavy and a light chain leader
sequence is situated in the double promotor and contains a start codon as well as a short part of
the variable domain genes. To obtain a correct Open Reading Frame (ORF) this sequence must be
added. The sequence from the start codon inside the promoter until the AscI
(ATGAGAAAGAGATTGAGTCCAGTCCAGGGAGATCTCATCCACTTCTGTGTTCTCTCCACAGGAGGGCGCGC
C) or NheI (ATGTCTCCACTCCTAGGTACGCTAGC) restriction sites were added to the heavy and light
chains
respectively
and
submitted
to
the
ORF
finder
tool
at
NCBI
(http://www.ncbi.nlm.nih.gov/projects/gorf/) using default settings. Sequences disrupted by a
stop codon in the middle of the sequence were discarded. Cloning of a culture of EBV cells might
produce several sequences with few base pair differences between consensus sequences either
due to PCR errors, sequencing errors or mutations. These differences were found when running a
multiple alignment. Heavy and light chain sequences were then run separately in the V-QUEST
program from IMGT (http://imgt.cines.fr/IMGT_vquest/vquest) using default settings. This
immunoglobulin alignment tool predicts where in the sequence the Complementary Determining
Regions (CDRs) are situated. If the base pair differences between sequences were in one of the
CDRs – increasing the chances of different binding properties - both varieties were chosen for
continuation to promoter insertion.
After the 3rd sequencing on the final plasmid with promoter, sequences from 2nd and 3rd
sequencing were assembled as above. The sequence between the NheI restriction site in S2 and
the NotI restriction site in S4 plus the leader sequence represent the entire light chain (CVL)
sequence and between the S1 AscI restriction site and the S3 XhoI restriction site plus leader
sequence represent the entire heavy chain variable domain (VH) sequence. Once again the
sequences were verified in ORF finder for correct stop codons (however, the heavy chain should
not have a stop codon as it is supposed to continue transcription into the vectors IGHG1). Multiple
alignments would tell the base pair differences between the sequences and the sequence
resembling the most probable consensus sequence was chosen for expression. If differences were
inside the CDR’s (verified by V-QUEST) the different variants were chosen for expression.
To show the entire amino acid sequences the exons of constant domain 1, hinge region, and
constant domain 2 and 3 inside the IgG1 expression vector were inserted after the variable
domain sequence. The heavy and light chains were translated in the +1 frame in the CLC
workbench. The molecular weight of each chain was calculated using the
http://www.biopeptide.com/PepCalc/PepMWCalc2.dll/Calculate website.
Amino acid sequences were analyzed using the blastp program at NCBI with default settings
(http://blast.ncbi.nlm.nih.gov/).
25
ANTIBODY PRODUCTION, PURIFICATION AND VERIFICATION
HEK293 TRANSFECTION
FreeStyle™ 293-F (Invitrogen, #R790-07) which are Human Embryonic Kidney cells (HEK293) that
have been adapted to high density, serum-free suspension culture were kept in liquid nitrogen,
thawed and grown in suspension in serum-free FreeStyle™ 293 Expression media (Invitrogen,
#12338-018) following the manufacturer’s instructions. The transfection was performed
essentially according to the manufacturer’s instructions. The day prior to transfection the HEK293
cells were passed to 0.7·106 cells/ml. On the transfection day cells were diluted in media to 1·106
cells/ml and 30 or 130 ml were added to 125 ml or 500 ml Erlenmeyer flasks. 1 µg plasmid DNA/ml
media was diluted in OptiPro™ Serum-Free Medium (SFM) (Invitrogen, #12309-050) (warmed to
rt) in polycarbonate tubes (Thermo Scientific, #31180010) to a final volume of 20 µl/ml media. 1 µl
Freestyle™ MAX reagent (Invitrogen, #16447-100)/ml media was diluted in OptiPro™ SFM in
polycarbonate tubes to a final volume of 20 µl/ml media. Immediately after addition the two tubes
were mixed gently. The solution was incubated between 10-20 min at rt and added drop-wise to
the HEK293 cells while slowly swirling the flask. The flasks were then incubated at 37 °C, 5% CO2
for 7 days. Antibodies were harvested by centrifuging the cell suspension at 800g for 5 min, rt and
collecting the supernatant. This fraction will further on be referred to as supernatant.
To determine optimal plasmid DNA amount and day of harvest 6 125 ml Erlenmeyer flasks with 30
ml HEK293 cell solution were set up as described for HEK293 transfection with plasmid DNA
amounts of 25 µg, 30 µg, 35 µg, 40µg, 50 µg and a negative control with 35 µg 00VP002. A 0.5 ml
sample was harvested each day until the 9th day after transfection. Samples were stored at -20 °C.
ANTIBODY PURIFICATION
To up-concentrate larger proteins (Antibodies included) the HEK293 supernatants were passed
through Vivaspin columns (Sartorius Stedim, #VS2022) by centrifugation at 3893 x g filtering out
anything with molecular weight less than 30 kDa. Prior to the concentration, supernatants had
been passed through a 0.80 µm filter (Sartorius Stedim, #16592) to avoid bigger components
clotting the Vivaspin filter. This fraction will further on be referred to as concentrated supernatant.
Antibodies were purified from the concentrated supernatant by use of κ light chain binding
protein L columns (Thermo Scientific, # 89959). The purification was done following the
manufacturers Spin Purification Protocol except from a moderation of the sample application step.
In short Protein L columns were prepared by washing twice with 2 ml binding buffer (0.1 M
sodium phosphate (Sigma-Aldrich, #S0751), 0.15 M NaCl (Sigma-Aldrich, #S9625), adjusted to pH
7.2 with HCl (VWR, #30018.298)). The protein L resins were transferred from the column to
samples diluted 1:1 in binding buffer, after 10 min incubation the sample/resin mix was applied to
the column 3 ml at a time. The columns were then washed three times in 2 ml binding buffer
(wash 1) before Antibodies were eluted by three steps of 1 ml elution buffer (0.1 M glycine
26
(Sigma-Aldrich, #G7403), adjusted to pH 2 with HCl (VWR, #30018.298)) collecting each eluate
separately. To prepare the column for re-use it was washed twice with 3 ml elution buffer (wash
2), once in 3 ml DPBS (Lonza, #BE17-512F) and stored at 4 °C with 3 ml storage solution (DPBS
(Lonza, #BE17-512F), 0.02% sodium azide). Each passage through the column was performed by
centrifuging 1 min at 1000 g. Protein concentration of the eluates were measured on a NanoDrop2000 (Thermo Scientific). This fraction will further on be referred to as purified antibodies.
SDS-PAGE
10 µl sample were incubated with 2 µl of SDS loading buffer (6 ml Trizma base (Sigma-Aldrich,
#T1503) adjusted to pH 6.8 with HCl (VWR, #30018.298), 2.4g SDS (Sigma-Aldrich, #L4390), 12 ml
glycerol (Sigma-Aldrich, #G7757), 0.12g Bromphenol blue (Sigma-Aldrich, #B5525), 2 ml ultrapure
water) with or without 0.92g DTT (Sigma-Aldrich, #D0632)) at 87 °C for 10 min and applied to a
Nu-PAGE 4-12% Bis-Tris Gel (Invitrogen, #NP0323Box). For the positive control ChromPure Human
IgG, Fab fragment (Jackson Immunoresearch, #009-000-007) diluted in ultrapure water was
loaded. 6 µl ProSieve Color protein marker 10-190 kDa (Lonza, #50552) was used for ladder. The
gel was washed three times in deionized water for 5 min under rocking conditions before coloring
for 20 min in coomassie blue solution (in 1 liter: 2g coomassie blue (Sigma-Aldrich, #B0149), 500
ml 96% EtOH (Solveco AB, #200-578), 80 ml acidic acid (Merck, 1.01830), 420 ml deionized water)
and leaving ON in decoloring (in 5 liters: 350 ml acidic acid (Merck, 1.01830) and 650 ml 96%
Ethanol (Solveco AB, #200-578)).
WESTERN BLOT
A Nu-PAGE 4-12% Bis-Tris Gel (Invitrogen, #NP0323Box) was run as above, but with samples
diluted 1:10 in ultrapure water. Separated proteins were blotted onto a nitrocellulose membrane
(GE-Healthcare, # RPN203E) for 1.5 hours and left in blocking buffer (5% skimmed milk powder
(ISIS foods, #500101) in TBST (6g Tris-base (Sigma-Aldrich, #T6066), 29.2g NaCl (Sigma-Aldrich,
#S9625), 500 µl Tween®20 (Sigma-Aldrich, # P7949) adjusted to pH 7.4 with HCl (VWR,
#30018.298)) one hour on a rotational shaker. The membrane was then washed 3 x 5 min with
TBST and incubated one hour with Polyclonal rabbit anti-human IgG/HRP (DakoCytomation,
P0214) diluted 1:1000 in TBST. After washing 3 x 5 min with TBST, Pierce® ECL Western Blotting
Substrate (Thermo Scientific, #32209) was added to the membrane and photos were taken as
bands became visible.
An additional western blot was made and developed as above. Only this time the membrane was
incubated one hour with 1:3000 Goat Anti-Human IgG, F(ab')2 Fragment Specific (Jackson
Immunoresearch, #109-005-097) followed by 1 hour incubation with Polyclonal Rabbit Anti-Goat
Immunoglobulins/HRP (DakoCytomation, #P0449) with a washing step in between. Purified IgG
from plasma and EBV cultures (kindly provided by Mette Andersen and Katrine Vegener) were
included as positive controls.
27
DETECTION OF CONTAMINATION
PCR ON REAGENTS
Reagents were tested for contamination by 2nd PCR. The PCR was set up as described for a 40 µl
total volume 2nd PCR in the ‘Isolation of VH-VCL from EBV-immortalized B cells’ section. The 2nd PCR
G/M + κ primer set was used and 2 µl of each reagent. Instead of 30 cycles, 40 cycles were run, 10
µl was taken out for gel-analysis, and additional 30 cycles were run. 10 µl of each well was gelanalyzed on a 1% agarose gel with 10 µl GeneRuler™ 100 bp Plus (Fermentas, #SM0321) as DNA
ladder.
PLASMODIUM FALCIPARUM PARASITES
MAINTENANCE
Parasites of the long-term in vitro cultured line 3D7 were maintained as described in (Cranmer et
al. 1997) and was performed by laboratory technicians. In short parasites were grown in blood
type 0 Rh+ erythrocytes (obtained by washing buffy coats provided by the blood bank at
Copenhagen University hospital) at 4% hematochrit and albumax media (500 ml RPMI 1640
(Lonza, #BE12-115F), 10 ml glutamin (14.6g glutamine (Sigma-Aldrich, #G3126) in 500 ml 0.9%
NaCl solution (Sigma-Aldrich, #S9625)), 50 ml Albumax (0.2g Hypoxantine (Sigma-Aldrich, #H9377),
50 g Albumax (Invitrogen, #11021037), 1 l RPMI 1640 (Lonza, #BE12-115F)), 2.5 ml Gentamycin
(Lonza, #BE02-012E)) in 25 cm2 cultureflasks (Nunc™, #156340). Following application of new
media the parasite culture was gassed with CO2 for 30 sec. The culture was incubated at 37 °C.
Parasitaemia was held below 10%. When counting parasitaemia a smear with erythrocytes was
made on a glass slide, fixed in methanol (AppliChem, #A1833.1000) and stained with Giemsa
(Merck, #1.09204.0500) for 10 min. The percentage of IEs of total erythrocytes was counted using
a light microscope and corresponds to parasitaemia.
The long-term in vitro cultured rosetting parasite line PAR+ was grown as 3D7, but in NHS media
instead of Albumax media containing 10% Normal human sera from the Blood Bank at
Copenhagen University Hospital instead of albumax.
SELECTION
To synchronize the parasites, the PAR+ cultures were selected for ring stages by sorbitol treatment
and for rosetting with percoll-treatment when in the trophozoite stage. Sorbitol will only
penetrate and lyse cells more than 20 hours post-invasion thus leaving early ring stages and
uninfected erythrocytes intact (Lambros & Vanderberg 1979). Sorbitol treatment was performed
when most parasites were at the ring stage (verified by light microscopy of giemsa stained
cultures). The culture was spun down, supernatant removed and pellet resuspended in 5% sorbitol
(Sigma-Aldrich, #S3889) diluted in ultrapure water. After incubation at 37 °C for 15 min the culture
was spun down, supernatant removed and cells were washed twice with RBC wash (500 ml RPMI
28
1640 (Lonza, #BE12-115F), 2.5 gentamycin (Lonza, #BE02-012E)) before incubation in media as
above.
Percoll treatment was performed by spinning parasites down, resuspending them in around 2 ml
media and layering them on top of 7 ml percoll solution (30 ml Percoll (GE Healthcare, #17-089101), 16.7 ml RBC wash media, 3.3 ml 10x RPMI (Invitrogen, #51800019)). After centrifugation at
567 x g for 10 min supernatant was removed and the pellet containing rosetting IEs, uninfected
erythrocytes and IEs at the ring stage was washed in RBC wash media and returned to culture.
Parasites of the 3D7 line had been selected for PFD1235w expression by antibody-selection using
dynabeads as in (Joergensen et al. 2010) and were kindly provided by Louise Jørgensen, CMP.
MACS-PURIFICATION
IEs with parasites in the trophozoite stage can be purified using magnetic cell sorting. This is due
to a magnetic waste product produced at this stage (Paul et al. 1981). A MACS CS-column (Miltenyi
Biotec, # 130-041-305) was placed on a Vario-MACS magnet (Miltenyi Biotec) and prepared by
addition of buffer (PBS (VWR, #10001633) with 2% FBS (Sigma-Aldrich, #F6178)). Parasite cultures
were suspended in buffer and added to the column followed by washing with 50 ml buffer. Finally
IEs were eluted by disconnecting the column from the magnet and eluting the bound material and
flushing with 50 ml buffer.
For flow cytometry the eluate was centrifuged and resuspended to the desired dilution. For use as
crude parasite mix the eluate was spun down and sonicated to release parasite proteins.
ANTIBODY DETECTION ELISA
In the Antibody detection ELISA 100 µl of supernatant - if required diluted in DPBS (Lonza, #BE17512F) - was added to each well on a 96 well ELISA plate (Nunc, #456437) and incubated at 37 °C
for one hour. 100 µl plasma sample diluted 1:100 and 100 µl DPBS or media were used as positive
and negative controls. The plate was emptied and blocked with 200 µl dilution buffer (146.1g NaCl
(Sigma-Aldrich, #71380), 1,0g KCl, 1.0g KH2PO4, 5.75g Na2HPO4·2H2O (Merck, #49369, #4783,
#6580), 50 ml Triton® X-100 (VWR, #306324N), 50g BSA (Applichem, #A1391), Phenol red (Merck,
#7241) in 5L ultrapure water) for 30 min on a rotational shaker at rt followed by addition of 100 µl
1:600 Goat Anti-Human IgG, F(ab')2 Fragment Specific (Jackson Immunoresearch, #109-005-097)
diluted in dilution buffer and 1 hour shaking at rt. Afterwards the plate was washed 4 times in
washing buffer (1% Triton X-100 (VWR, #306324N), adjusted to pH 7.2 with HCl (VWR,
#30018.298)), 100 µl 1:6000 Polyclonal Rabbit Anti-Goat Immunoglobulins/HRP (DakoCytomation,
#P0449) was added and the plate was left shaking at rt for 1 hour. Finally the plate was washed 4
times and developed with the addition of 100 µl OPD solution (4 OPD tablets (Dako, #S2045), 10 µl
29
H2O2 (Sigma-Aldrich, #H3410) dissolved in 12 ml ultrapure water) and left until color formation
was seen clearly (between 5-10 min). The reaction was stopped with 100 µl 2.5 M H2SO4 (Merck,
#4803641000) and read at 490nm on a VersaMax microplate reader (Molecular Devices).
Alternatively Polyclonal rabbit anti-human IgG/HRP (DakoCytomation, P0214) diluted 1:1000 in
dilution buffer was used for detection, skipping one labeling and washing step.
IgM-detection was identical to the anti-human IgG/HRP set-up, except from the detecting
antibody being Polyclonal Rabbit anti-human IgM/HRP (DakoCytomation, #P0215) and samples
stemming from the supernatant of EBV culture.
In the antigen-specific ELISA plates were coated with 100 µl 1 µg/ml protein diluted in DPBS
(Lonza, #BE17-512F) and left at 4 °C ON. The plates were emptied, blocked as above and 100 µl 1:1
supernatant diluted in PBS was added to each well and left shaking at rt for 1 h. Immune plasma
and DPBS or media was used as positive and negative controls as above. Afterwards the plate was
washed 4 times and the protocol from above was followed starting from the addition of Goat-αhuman Fab antibodies. The coating proteins had been made as in Victor et al. (2010) for the
PFD1235w proteins and as in Khunrae et al. (2010) for the VAR2CSA protein.
FLOW CYTOMETRY
Flow cytometry was performed to verify whether the recombinant antibodies were able to bind to
the surface of IEs. 25 µl supernatant or 5 µl human immune plasma (positive control) was added to
each well of a round bottomed 96 well plate (Nunc™, #163320). Nothing was added to wells of
negative controls. IEs were MACS-purified as described above and adjusted to 2·106/ml. 100 µl IE
mixture was added to each well and the plate was incubated 30 min at 4 °C. The plate was washed
three times with washing buffer (PBS (VWR, #10001633) + 2% FBS (Sigma-Aldrich, #F6178)) (once
added 100 µl, twice adding 200 µl). This was done by adding washing buffer, spinning the plate 4
min at 567 x g, 4 °C, and flicking it upside down to empty it. Next, 100 µl 1:600 Goat Anti-Human
IgG, F(ab')2 Fragment Specific (Jackson Immunoresearch, #109-005-097) was added, incubated 30
min, 4 °C and washed as above. Then 100 µl FITC conjugated rabbit-anti goat (Sigma-Aldrich,
#F1016) diluted 1:100 + 20 µl/ml 0.1 mg/ml Ethidium Bromide (Invitrogen, #15585011) was added
and the plate incubated for another 30 min at 4 °C followed by washing twice. Finally 100 µl
washing buffer was added and 5000 Ethidium Bromide positive IEs were read from each well on a
Cytomics FC500 MPL from Beckmann Coulter. Data was analyzed by WinList 5.0 software
(www.vsh.com).
LUMINEX
Luminex is a multiplex assay where coupling of different proteins to beads with different emission
wave lengths makes it possible to measure binding of antibodies to multiple proteins in one assay.
Beads were coated with proteins as in Cham et al. (2008) and mixed to obtain plexes containing
various different proteins. Beads were provided by Mafalda Resende, Gerald Cham and Louise
30
Turner who also performed the luminex assay. Filter bottom 96 well plates (Millipore,
#MSBVS1210) were pre-wetted with 50 µl SP buffer (PBS (VWR, #10001633), 0.5% BSA (SigmaAldrich, #A3059), 0.02% Tween®20 (Sigma-Aldrich, # P7949), adjusted to pH 7.4 with HCl (VWR,
#30018.298)), left at rt for 30 min and aspirated. 50 µl beads (app. 1250 beads) were added to
each well and the wells were washed three times with 100 µl SP buffer. Prior to addition the beads
had been sonicated for 30 sec, diluted in SP buffer and vortexed for 10 sec. 50 µl antibody sample
diluted 1:10 in SP buffer was added to the wells and the plate was incubated on a shaker in the
dark for 30 min. Immune plasma was used as positive controls while SP buffer was used for
negative controls. After three washes with 100 µl SP buffer, 50 µl 1:400 R-phycoerythrinconjugated F(ab’)2 Goat Anti-human IgG, Fc fragment specific (Jackson Immunoresearch, #109116-098) was added and the plate was incubated in the dark on a shaker for another 30 min. Wells
were aspirated and washed three times with 100 µl SP buffer. Beads were resuspended by fast
shaking for 4 min with 50 µl/well SP buffer. After addition of another 50 µl, the plate was shaken
for 5 min and 100 beads/well read on a Luminex®100™ IS Total System.
ROSETTE REVERSAL ASSAY
To test whether recombinant antibodies retained anti-rosetting properties of their EBV cellproduced counterpart, a rosette reversal assay was performed on the supernatants of AR03.1 and
AR04.1. Parasites mainly in the trophozoite stage were spun down and resuspended in NHS media
to a hematocrit of 4%. 50 µl IE suspension was added to each well of a round bottomed 96 well
plate (Nunc™, #163320) together with either 17.5 µl sample. Samples were either undiluted
supernatant, 17.5 µl 20 U/ml Heparin (Sigma-Aldrich, #H4784) (positive control), 17.5 µl DPBS
(Lonza, #BE17-512F) (negative control) 2.2 µg or 0.5 µg of EBV culture-produced antibodies or 2.5
µl immune plasma (positive control) diluted in DPBS. After gassing with CO2 for 3 min followed by
incubation at 37 °C for 30 min 5 µl 0.1 mg/ml Ethidium Bromide (Invitrogen, #15585011) was
added to each well and the percentage of rosetting IEs of total IEs were counted on a Leica DM LB
fluorescence microscope. An IE were classified as rosetting if at least two erythrocytes were bound
to it.
EPITOPE PREDICTIONS
AB01.15 antibodies from Epstein-Barr virus immortalized B cells had been tested in ELISA on DBLγ
domains and in luminex on various DBLγ and non-DBLγ domains.
Alignments of sequences recognized and non-recognized in ELISA by AB01.15 were made
separately by Anja Jensen. Alignments of sequences recognized and non-recognized in luminex
were made separately using the ClustalW2 program for multiple alignments
(http://www.ebi.ac.uk/Tools/msa/clustalw2/). Epitopes were predicted by manual verification of
differences between alignments of recognized and non-recognized DBLγ domains. Differences
were only considered different if the amino acids had different properties (e.g. hydrophobichydrophilic). A 3D model of PFD1235w DBLγ had been made by Pernille Andersen modeled as the
VAR2CSA domains in (Andersen et al. 2008). To visualize the location of the predicted epitopes
31
they were marked in
(http://www.pymol.org/).
the
model
with
the
protein
structure
program
PyMol
32
RESULTS
B CELL COLLECTION
Blood samples from a total of 24 healthy Ghanaian donors with life-long exposure to P. falciparum
were collected.
Due to limited capacity a maximum of 3 blood samples could be processed per day. However, on
day 1 only one donor was selected in order to test the set-up. Each day the samples to be
processed were selected out of 6 donors based on amount of anti-P. falciparum antibodies in
plasma samples tested in ELISA. If anti-P. falciparum antibodies are present it indicates recent
malaria incident and increases the chances of presence of anti-P. falciparum memory B cells.
Samples were selected based on their reactivity to sonicated erythrocytes infected with a mix of
parasite (crude parasite mix) and recombinant GLURP R0 and no reactivity to non-infected
erythrocytes or coating buffer.
Compared to regular ELISAs this assay had been time-reduced in order to reduce B cell processing
time and thus enhance B cell viability. The ELISA assay had been optimized and quality tested in
Denmark prior to B cell collection. At this time reactivity of positive controls to crude parasite mix
was above OD490nm 3 when incubated with OPD solution for 10 min (data not shown). Still the first
three days in Ghana the assay showed inconsistency (figure 10). No pronounced reactivity was
seen with crude parasite mix and the negative controls were relatively high. A new crude parasite
mix was included but did not improve reactivity. Donors were therefore selected based on
reactivity to GLURP R0 and low reactivity to the negative controls. Donor 1.4 (MBD01) was
selected on day 1, donor 2.1, 2.5 and 2.6 (MBD02, MBD03, and MBD04) on day 2, and donor 3.4,
3.5, and 3.6 (MBD05, MBD06, and MBD07) on day 3. Great variation in reactivity between
duplicate wells was seen. An example is the results of the negative control at day 1, where the
reactivity of OD490nm 0.22 to GLURP R0 is the average of two wells showing reactivity of 0.35 and
0.09. Furthermore, in some of the wells it seemed like color only developed in one side of the well.
To avoid this, the OPD solution tablets were more thoroughly dissolved on day 2 and 3. While it
seemed to improve the results of day 2, day 3 gave very unreliable results. Thus at day 4 the batch
of the OPD was changed and PBS was used as coating buffer. Finally the assay showed the
expected reactivity and donor 4.2, 4.3, and 4.5 (MBD08, MBD09, and MBD10) were selected based
on their reactivity with GLURP R0 and crude parasite mix while showing low reactivity to the
negative controls.
33
Figure 10. ELISA-analysis of donor plasma samples. Determination of relative amount of anti-malaria antibodies in
plasma samples of 24 donors distributed on 4 days with 6 donors/day.*indicate the donors selected for B cell
purification the given day. The figure shows mean reactivity of duplicates.
B cells were negatively selected using the rosettesep® technology and between 3.5·106 and
22.5·106 cells were recovered from each donor (table 4). Light microscopy examination of cells
colored with trypan blue showed viability of >99% and a uniform cell population devoid of red
blood cells and bigger cells like neutrophils. The processing time from blood sampling to
cryopreservation of the B cells was between 6 and 8 hours. Cells were transported in a dryshipper
held cold by liquid nitrogen and immediately transferred to liquid nitrogen when in Denmark. Four
weeks after selection random samples were investigated. Unexpectedly the fluid level in the tubes
was no longer horizontal as if the vials had been thawed and re-frozen. Coloring with trypan blue
confirmed this suspicion as it revealed a cell viability less than 1%. As the samples still were frozen
at arrival to Denmark, it is possible that customs have emptied the container and let the samples
thaw before returning them to the cold container. Most deplorable this made it impossible to
continue with the samples in further experiments.
Table 4. Amount of B cells purified from each donor
Donor
MBD01
MBD02
MBD03
MBD04
MBD05
MBD06
MBD07
MBD08
MBD09
MBD10
Amount
of B cells
3,5·106
3,5·106
10·106
10·106
22,5·106
7,5·106
9,45·106
13,2·106
20,4·106
8,4·106
34
ISOLATION OF ANTIBODY ENCODING GENES
At CMP anti-P. falciparum antibodies have already been generated by the Epstein-Barr method.
However, recombinant production would give advantages to further studies of these antibodies,
as production of high amounts is convenient, the production is stable and genetic engineering of
the antibody is possible. The conversion from production by EBV cell to recombinant production
was therefore desirable. This was pursued while establishing the Symplex™ technology.
The present approach differs from the Symplex™ technology described in the introduction in two
ways. 1: The aim of the original method is to clone antibody genes from as many single cells as
possible, while the present aim is to clone antibody genes from a particular cell line. To be able to
clone antibody-genes from cultures expressing IgM and/or the lambda light chain new primer
pairs were included (made by Michael Dalgaard by modifying primers from de Haard et al. (1999)
the same way the Symplex™ authors did for the IgG/κ primer set). 2: Instead of bulk cloning which
is advantageous when generating many different antibodies, fragments from each positive well
were cloned separately.
EBV cell cultures had been selected for their reactivity to the PfEMP1s PFD1235w or VAR2CSA or
for anti-rosetting activity and are thus highly interesting regarding severe malaria. After
subculturing and extensive expansion the cultures are assumed to be monoclonal.
Onwards, EBV cultures are designated their name followed by –EBV (e.g. AB01.15-EBV), the
antibodies they produce are designated EBV- followed by their name (e.g. EBV-AB01.15), the
constructs containing their antibody genes are designated their name followed by –construct (e.g.
AB01.15-construct), and the antibodies expressed from these constructs are designated only their
name (e.g. AB01.15).
All together antibody genes were isolated from 8 different cultures. In the following section
examples from the isolation from one culture are shown. Single cells from EBV cells obtained
either by limiting dilution, single cell sorting or micro-manipulation were subjected to 1st and 2nd
PCR. The PCR-products were verified by gel-analysis where positive wells were seen as a band
around 1100 bp (figure 11, lane 1, 2, 9, 12, and 14). Figure 11 only shows gel-analysis of products
from amplification with the primer set 1st PCR G+κ as all other primer sets yielded no positive
bands on this particular culture. Each EBV cell culture was subjected to each of the primer sets but
as expected only one of the primer sets yielded positive wells. An exception is however the 1st PCR
G+κ set that was seen to cross-anneal to IgM mRNA. The set-up had been tested on cells
producing antibodies of IgG and κ/λ2/λ7 and IgM and κ/ λ2/λ7. These true positive controls were
amplified by the respective primer set (data not shown). These were not included in the actual
experiments due to risk of contamination.
35
Figure 11. Representative gel-analysis of PCR-products.
Agarose gel (1%) analysis of PCR products from the
amplification of AR03.1-EBV antibody genes with the G+κ
primer set. Size of positive bands: 1100 bp. The PCR was
performed on 0.5 cell/well, 10 cells/well (positive control)
or PBS (negative control). 3 µl PCR product was loaded in
each well and 5 µl Generuler 100 bp plus ladder
(fermentas, shown in appendix A).
As other groups have reported previously (Meijer et al. 2006; Smith et al. 2009), single cell PCR is
very sensitive to contamination and the Symplex™ technology is no exception. Negative controls
randomly appeared positive, as is seen in figure 11, and contamination with foreign antibody
DNA/RNA was seen in sample wells. To eliminate the risk of contamination, only one cell type was
cloned at a time, the 1st PCR was set up in a DNA clean-bench, lab-coats were changed when
entering the DNA clean-bench room, and gloves and filter pipettes were used when setting up 1st
and 2nd PCRs. The contamination problems are described more thoroughly in a later section.
The purified VH-CVL fragments were inserted into the IgG1 expression vector 00VP002 (appendix
B). The expression vector contains the C1, hinge, C2, and C3 domains of IgG1 for full length IgG1expression and an ampicillin resistance gene for the selection in Escherichia coli. The constructs
were amplified in Escherichia coli and VH-CVL fragments insertion was verified by gel-analysis of
colony-PCR products (figure 12.a). Plasmids were purified from positive colonies yielding bands
around 1100 bp and their linker sequence was replaced by a fragment with bi-directional
Figure 12. Representative agarose (1%) gel-analysis of colony PCR products. 5 µl PCR product is loaded and 10 µl
Generuler 100 bp plus (Fermentas, shown in appendix A). Each lane represents one colony. Neg: Negative control
without template A) Products from 1st Colony PCR to verify VH-CVL insertion. Positive band size: 1100 bp. B) Products
from 2nd colony PCR to verify promotor insertion. Positive band size: 400 bp.
36
promoter for eukaryotic expression and heavy and light chain leader sequences. The expression
constructs were amplified in Escherichia coli and verified for promoter insertion by colony-PCR and
gel-analysis. Colonies with correct promoter insertion yielded a band around 400 bp (figure 12.b).
ANTIBODY SEQUENCE ANALYSIS
Absence of contamination, insertion of antibody genes and insertion of promotor were verified by
DNA sequencing. Three sequencing steps were performed: on the purified VH-CVL fragments
before vector insertion (1st sequencing), before (2nd sequencing) and after (3rd sequencing)
promotor insertion.
The 1st sequencing was performed on the PCR product before vector insertion and yielded part of
the sequence of the VH-CVL fragment. The sequence was divided into light and heavy chain
sequences and a multiple alignment was performed with all previous sequences to secure no
presence of contamination (data not shown). An example of a sequence of a VH-CVL fragment is
given in figure 13. To confirm that the insert indeed was human antibody genes, the heavy and
light chain sequences were blasted against NCBIs nucleotide collection database. All PCR products
Figure 13. Sequence of VH-CVL fragment of AR04.1. The linker sequence generated by overlapping 5’ tail primers is
high-lighted and restriction sites used in cloning are marked.
37
that yielded good quality sequence information showed alignment with human antibody genes
(example given in table 5).
Table 5. Nucleotide Blast-search of AR04.1 light chain sequence
Accession
Description
Homo sapiens IGK mRNA for immunoglobulin kappa light
AB064103.1
chain VLJ region, partial cds, clone:K62
Homo sapiens IGK mRNA for immunoglobulin kappa light
AB064111.1
chain VLJ region, partial cds, clone:K70
BC017870.1 Homo sapiens cDNA clone IMAGE:4274551
E-value
0.0
Max Identity
0.0
96%
0.0
96%
97%
Open reading frames of sequences from the 2nd and 3rd sequencings were verified and the few
clones that showed disruption by stop codons were discarded. Open reading frames of sequences
from the 1st sequencing were not verified as 1st sequences often were of lower quality, which
would increase the risk of seeing false stop codons.
Multiple alignments comparing sequences from the 2nd and 3rd sequencing respectively showed
few base pair differences (example given in figure 14) between different clones. Examination of
the location of these differences in the V-QUEST program of IMGT showed that they rarely were
located inside the CDR regions (data not shown). It should though be noted that the CDRpredictions are uncertain and CDR3 is particularly difficult to predict due to its variable length. The
clone with the sequence most closely resembling the consensus sequence was chosen for
expression. When analyzing the sequences in the V-QUEST program, base pair differences
between the input sequence and the aligning IMGT sequences were often seen inside the CDR
Figure 14. Segment of multiple alignment of light chain sequences from different clones of AR04.1. Sequences
were obtained from the 2nd sequencing.
38
regions as expected (data not shown). Analysis of ORFs in NCBIs blastp program indicated absence
of frame shifts as sequences aligned to human heavy or light chains (data not shown). In figure 15
amino acid sequences, calculated molecular weights and CDR regions are shown for the selected
clones of the successfully cloned AB01.15, AR03.1, and AR04.1. In total antibody genes were
cloned from 8 different EBV cell cultures, but due to contamination the sequences of the
remaining 5 are not shown.
AB01.15
>light chain
>---------FW1------------<>CDR1<>----FW2-------<>CDR2<>---------------FW3--------------<>-MSPLLGTLAEIVLTQSPSSLSASIGDRVTITCRASEDIFAFLNWYQKKPGTAPKLLIYDRTNLQAGVPSRFSGSGSGTEFTLTINNLQPEDIGTYYCQQY
CDR3-<
>---------CD-----------------------------------------------------------------------DEFPLTFGGGTKVEIKGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSRESVTEQDSKDSTYSLSSTLTLSKADYEKHKL
----------------------<
YACEVTHQGLSSPVTKSFNRGEC**
>heavy chain
>---------FW1-----------<>-CDR1-<>------FW2------<>--CDR2<>------------FW3-MRKRLSPVQGDLIHFCVLSTGGRAEVQLVESGAEVKRPGSSVSVSCEASGGTFSGYAVSWVRQAPGQGLEWMGGIIPILETTEYTQKFQDRVTIIADEST
-------------------<>---CDR3-------<
>-----------------------------------C1--------------STVYMELRSLTSEDTGVYYCARGYYGSNSYYLALDLWGKGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
--------------------------------------------<>----H--------<>--------------------------------------PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
-----------C2--------------------------------------------------------<>----------------------------PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
--------C3------------------------------------------------------------------<
GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
Mw light chain = 24.3 kDa
Mw heavy chain = 52.1 kDa
Mw antibody = 152.8 kDa
AR03.1
>light chain
>----------FW1-----------<>---CDR1---<>-----FW2------<>CDR2<>---------FW3-----------------MSPLLGTLADIVMTQSPDSLAVSLGERATIKCKSSQSVLYSSNNKHYLGWYQQKPGQPPKLLIHWASTRESGVPDRFIGSGSGTDFTLTISSLQAEDVAV
--<>--CDR3-<
>------------------------------------------CD--------------------------------YYCQQYYSIPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD
----------------------------<
YEKHKVYACEVTHQGLSSPVTKSFNRGEC**
>heavy chain
>-------FW1-------------<>-CDR1-<>---FW2---------<>--CDR2<>-------------FW3MRKRLSPVQGDLIHFCVLSTGGRAEVQLVQSGGGLVQPGGSLRLSCAASGFTFSDYWMHWVRQAPGKGLVWVSRINGDGSSTNYADSMKGRIDTSRDNTT
-------------------<>--CDR3---<
>------------------------------------C1-------------------NTLHLHINSLRAEDTAVYYCVRSRYYRMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
--------------------------------------<>-----H-------<>-----------------------------------C2-------SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN
---------------------------------------------------------------<>----------------------------------WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD
-----------------C3---------------------------------------------------<
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
39
AR04.1
>light chain
>---------FW1------------<>CDR1<>-----FW2------<>CDR2<>---------------FW3--------------<>-MSPLLGTLADVVMTQSPSSLSASLGDRVTITCRASQDISHYLGWFQQTPGKAPKSLISAATNLQSGVPSRFSGSGSGADFTLTISSLQPEDFATYYCQQY
CDR3-<
>--------------------------------------------------CD------------------------------TTYPRNFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKL
----------------------<
YACEVTHQGLSSPVTKSFNRGEC**
>heavy chain
>--------FW1------------<>-CDR1-<>------FW2------<>- CDR2<>----------------MRKRLSPVQGDLIHFCVLSTGGRAQITLKEWGAGLLKPSETLSLTCAVYGGSFSDYYWTWIRQPPGKGLEWIGEITHTGSTNYNPSLKSRITMSIDTSKN
--FW3-------------<>-----CDR3----------<
>------------------------------C1---------------QFSLKLTSVTAADTAMYFCARGPSVSVTPPIIATTTTEGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG
------------------------------------------------<>-----H-------<>----------------------------------VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
---------C2--------------------------------------------------------------<>------------------------SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
-------------------C3-----------------------------------------------------------<
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
Figure 15. Amino acid sequence and calculated molecular weights of AB01.15, AR03.1, and AR04.1. Framework
regions (FW), complementarity determining regions (CDR), light chain constant domain (CD), heavy chain constant
domain 1 (C1), hinge region (H), constant domain 2 (C2) and constant domain 3 (C3) are shown. Frame work regions
and complementarity determining regions have been determined by the V-QUEST program from IMGT. The calculated
molecular weights of light chain, heavy chain and the entire antibody is shown below the sequences.
EXPRESSION AND PURIFICATION OF RECOMBINANT ANTIBODIES
DETECTION OF ANTIBODIES
Antibodies were expressed by Human embryonic kidney cells (HEK293) after transient transfection
with the AB01.15-, AR03.1-, or AR04.1-construct. Antibodies were harvested by centrifuging
cultures and collecting the supernatant. To confirm that antibodies indeed were expressed from
the constructs, supernatants were tested for presence of antibodies in ELISA. An optimization of
this assay is described below. Figure 16 shows that supernatants from cultures transfected with all
three constructs contained antibodies. Negative control with media resulted in absorbance similar
to PBS. Results stem from three different assays and amounts are thus not comparable. It was a
general observation that antibodies were produced whenever prior sequence analysis had shown
antibody genes with undisrupted open reading frames.
40
Figure 16. Detection of production of recombinant antibodies. Verification of presence of antibodies in
supernatants determined by ELISA. Presence of antibodies in AR03.1, AR04.1 and AB01.15 supernatants was
verified in three separate assays. Supernatants were diluted 1:160.
OPTIMIZING ANTIBODY-DETECTION ELISA
Antibodies were detected by coating ELISA plates with culture supernatants, and detecting the
bound antibodies with anti-Fab or anti-IgG antibodies. This was not straight forward as the
reactivity seen with undiluted supernatants was almost as low as the negative controls with media
or PBS. Extra washes before applying secondary antibody did not improve the assay. However,
when samples were diluted 1:10 in PBS the reactivity increased and even more when diluted 1:100
(data not shown). When a two-fold dilution series was made it revealed highest reactivity at 1:160
dilution, and that dilution in media reduced reactivity (figure 17). On this background a dilution of
1:150 or a two-fold dilution series was chosen for antibody-detection. This reverse correlation
Figure 17. Detection of antibodies in supernatants of
different dilutions. Detection of antibodies in AR04.1
supernatant by ELISA to determine optimal dilution
for antibody-detection assay. Final dilutions are
shown to the right. 1:10 in media was diluted in
FreeStyle media (Invitrogen).
41
between dilution and absorbance was not seen when coating plates with antigen, letting
antibodies in supernatant bind to the antigens and then detecting antibodies (data not shown).
To maximize the antibody amount produced, optimal construct-DNA concentration and day of
harvest was determined. Six cultures with 30 ml HEK293 cells were set up with 25, 30, 35, 40, and
50 µg AR04.1-construct-DNA respectively and one culture with 35 µg expression vector 00VP002
without VH-CVL insertion. Samples were taken each day for nine days and the antibody amount in
the supernatant was measured in ELISA. All samples were examined in one assay and determined
in duplicates. Negative control samples with either PBS or media were included and yielded
absorbance less than OD490nm 0.04, while a positive control with plasma diluted 1:100 gave
absorbance around OD490nm 3.6. As is seen in figure 18 transfection with 30 µg construct DNA gives
higher antibody yield than any of the other concentrations. All construct-DNA transfected cultures
showed a high increase of antibody level from day 6 to day 7 and then the antibody level seemed
to stabilize. It should be noted that all duplicates showed very little variation except from 30 µg
construct DNA, day 7, which is the average of reactivity of OD490nm 0.94 and 1.36. No antibodies
were detected from the culture transfected with 00VP002 as reactivity was as low as the PBS and
media controls. Based on these studies the following HEK293 cultures were transfected with 1 µg
construct-DNA/ml culture and antibodies were harvested on day 7.
Figure 18. Relative antibody-concentration of samples of variable construct-DNA concentration and day of harvest.
Reactivities from antibody-detection ELISA on AR04.1 supernatants from cultures transfected with different amount of
construct-DNA and harvested at different time points to determine optimal conditions for antibody production. Each
sample was diluted 1:150. Each data-point is the average of duplicate determinations.
42
PURIFICATION OF ANTIBODIES
AB01.15 and AR03.1 antibodies were up-concentrated from the supernatant by Vivaspin columns
(Sartorius Stedim). Afterwards antibodies were purified from concentrated supernatant using
protein L columns (Thermo Scientific). Due to time limitation AR04.1 was never purified. The
purification of 30 ml AR03.1 supernatant yielded 147 µg IgG (distributed on 89 µg in eluate 1, 43
µg in eluate 2, and 15 µg in eluate 3) giving a yield of 4.9 µg/ml. SDS-PAGE-analysis of the different
fractions from the purification indicated that the eluates are pure as they do not contain other
protein-bands than the antibody-bands around 180 kDa (figure 19.a). Furthermore the main part
of the antibodies was present in eluate 1 and 2 whereas the flow-through and wash fraction
before and after elution seem to be devoid of IgG as no bands were present. An IgG-detecting
Western Blot (figure 19.b) confirmed that all IgG was washed out in the three elution fractions as
the wash 2 after elution did not contain any IgG. However, IgG was present in both the flowthrough and wash 1 fraction, revealing inefficient binding of IgG to the column.
Figure 19. Protein verification of recombinant antibodies and fractions from AR03.1 purification. A) SDS-PAGE.analysis. Amount loaded: 6 ProSieve Color Protein Marker 10-190 kDa (Lonza), 10 µl concentrated AR03.1
supernatant, 10 µl flow through, 10 µl Wash 1, 10 µl (890 ng) eluate 1, 10 µl (430 ng) eluate 2, 10 µl (150 ng) eluate 3,
10 µl wash 2, 10 µl AR04.1 supernatant, 10 µl (3 µg) purified AB01.15, 3 µg standard Fab fragments. B) Western blotanalysis with anti-IgG detecting antibody. Amount loaded: 1:10 of the amount loaded in the SDS-PAGE, 6 µl ProSieve
Color Protein Marker 10-190 kDa (Lonza). C) Western blot analysis with anti-Fab detecting antibody. Amount loaded: 6
µl ProSieve Color Protein Marker 10-190 kDa (Lonza), 89 ng purified AR03.1, 10 µl AR04.1 supernatant, 300 ng purified
AB01.15, 300 ng purified EBV-AB01.15, 10 µl purified plasma IgG. Red = samples have been reduced by incubation with
DTT. The predicted IgG, heavy chain (HC) and light chain (LC) bands are pointed out.
PROTEIN VERIFICATION
IgG molecules were seen as bands around 180 kDa in SDS-PAGE and Western Blot (figure 19.a). As
this was somewhat bigger than the expected IgG sizes around 150 kDa (figure 15), control
43
antibodies were included in a Western Blot to see if this was general of IgG antibodies (e.g. due to
glycosylations) or an artifact of recombinant production. Both EBV antibodies and IgG purified
from human plasma revealed bands of the same size (figure 19.c). When reducing the samples
with DTT, heavy (around 50 kDa, detected with anti-IgG antibody in figure 19.b) and light chains
(around 25 kDa, detected with anti-Fab antibody in figure 19.c) were separated as expected. The
Fab-detecting Western Blot with un-reduced samples showed additional bands that might be due
to different degradation products (figure 19.c).
DETERMINATION AND PREVENTION OF CONTAMINATION
After having cloned and expressed antibody genes from 8 different EBV cell cultures
contamination was discovered. A multiple alignment of light and heavy chain sequences
respectively showed that some of the sequences were identical (light chain variable domain
alignment shown in figure 20, heavy chain alignment not shown), and contamination thus was
Figure 20. Multiple alignment of light chain variable domain sequences. 8 different EBV cell cultures were cloned and
their light chain variable domain sequences aligned.
44
present. The contaminants appeared to be previously cloned sequences. The sequences of the
contaminants were identical to the AB01.15 sequences or clone No. 89. The identity of AB01.15
was certain as it had been characterized and showed to retain the specificity of its EBVcounterpart. However, which EBV-culture clone no. 89 belongs to is unknown. The sequence
appeared for the first time when cloning PAM4.07-EBV, which antibodies bind to the protein
VAR2CSA. But this binding was not shown when testing the recombinant antibody on full length
VAR2CSA in ELISA (data not shown). From figure 20 clone 230 seems to be unique, but alignment
of heavy chains revealed contamination with AB01.15. In general pairing of heavy and light chain
genes in contaminated sequences was random. The light chain could be unique but paired with an
AB01.15 heavy chain, the light chain could be of AB01.15 origin but paired with a heavy chain of
clone no. 89, or both chains could derive from one of the contaminants.
Before starting over with cloning of antibody genes from EBV cell cultures, the source of
contamination had to be identified and eliminated. A 2nd PCR was performed on all reagents and
gel-analyzed. If any contaminating DNA was present it would be amplified by the 2nd PCR G/M + κ
primer set and yield a band around 1100 bp as the contaminating sequences are of IgGκ origin.
Two of the vials with NotI and XhoI digested vector appeared as positive (figure 21, lane 33 and
35). The two contaminated vials were thrown out and cloning of AR03.1-EBV antibody genes was
repeated. Sadly, when 2nd sequencing the construct, the AB01.15 sequences appeared again.
Figure 21. Gel-analysis of 2nd PCR on reagents. Agarose gel (1%) analysis of products from 40 cycles of 2nd PCR on
reagents. 10 µl Generuler 100 bp plus loaded (Fermentas, shown in appendix A). 10 µl sample was loaded. 1: Qiagen
One Step buffer. 2: RNAsin. 3-8: 1st PCR primer sets. 9+22+27: dNTP. 10+25: Quiagen One Step Enzyme mix. 11: RNAse
free H2O. 12+19: AB01.15-construct. 13-16+28-29: H2O. 17: 00VP002 cut by NheI and AscI. 18: 00VP002. 20: PBS.
21+26: 5x HF Phusion buffer. 23: Phusion Polymerase. 24+30-35: 00VP002 cut by NotI and XhoI batch 1-7. Results from
70 cycles PCR were identical.
When inserting the VH-CVL fragment into 00VP002, few colonies were sometimes seen on control
plates with Escherichia coli cells that had been transformed only with NotI and XhoI digested
vector. To test whether these were colonies with re-ligated vector or contaminating constructs
45
that had not been detected in the PCR, these were sequenced with the S1 and S2 primers. The
sequences showed only vector sequence and thus no contamination of the vector (data not
shown).
Until now sequencing had only been performed on constructs (2nd and 3rd sequencing). To test
whether contamination already was introduced prior to insertion into the vector, a new
sequencing step was added; the 1st sequencing of the VH-CVL fragment. Sequencing of the VH-CVL
fragment revealed that contamination already was present in the PCR product. When sequencing
PCR-products from negative control wells that came up as positive (e.g. in figure 11) the
contaminating sequences were found, hence contamination was not specific to cell-containing
wells.
To eliminate contaminants all reagents were exchanged. New reagents were aliquoted in smaller
volumes and kept in separate freezer boxes on a separate freezer shelf dedicated to PCR-work
only. DNA-hood and freezer blocks were washed down with a chlorine and NaOH solution, filter
tips and PCR plates were kept separately and not used for any other lab-work.
Reactions were set up for AR03.1-EBV - One where cells were sorted by micro manipulation and
one sorted by limiting dilution. A 1st sequencing of the PCR-products showed that no
contamination was present in wells where cells were applied by micro-manipulation and later
characterization of the final recombinant antibody, showed retained activity of EBV-AR03.1 (as
described in ‘Recombinant AR03.1 retains activity’).
However, contamination still appeared in reactions where cells had been applied by limiting
dilution. In the gel-analysis a wrong positive/negative pattern was seen (figure 22.b). Although
around 10 cells should have been applied to the semi-positive controls, only one appeared with a
positive band. Sequences from the one positive control well also showed to be identical to
contaminating sequences. It was a general observation that whenever positive controls were not
nd
Figure 22. Example of right and wrong positive/negative patterns. Agarose (1%) gel-analysis of 2 PCR products
from AR03.1-EBV amplified with G+κ primer set. 3 µl was loaded in each well and 5 µl Generuler 100 bp plus ladder
(fermentas, see appendix A) was loaded. Expected size of positive bands is 1100 bp. A) Right positive/negative pattern.
Positive controls were positive and negative controls were negative. Cells were applied by micro-manipulation. B)
Wrong positive/negative pattern. All positive controls were not positive. Cells were applied by limiting dilution.
46
positive and/or negative controls were not negative (designated wrong positive/negative pattern),
contamination was seen in the products. When correct patterns were seen, no contamination was
found (figure 22.a).
Later on contamination-free products were obtained from AR04.1 with both use of micromanipulation and limiting dilution. Positive/negative patterns were right here as well (data not
shown).
OPTIMIZING CELL SORTING BY FACSARIA
When viable B cells from malaria-exposed donors are obtained, the Symplex™ technology will be
applied to a greater number of cells. Therefore cell sorting by micromanipulation is not feasible as
it is too laborious. Limiting dilution is not optimal either, as the many empty wells will make it
expensive. Furthermore dilutions are not precise as the concentration needed for counting the
cells is much higher than the concentration needed in limiting dilution. Both to prepare for future
Symplex™ experiments and to make sure the correct number of cells was applied to the wells,
cells were sorted by flow cytometry using a FACSAria cell sorter. As is seen in figure 7, EBV cells are
quite heterogeneous in size. The gate was set as P1, as we assumed P2 would contain cell debris
and P3 might be cell clusters. Later light microscopy examination of cells from the P2 gate
revealed cell debris, while cells from the P1 and P3 gate were single cells. From experiments using
the micro manipulator to sort cells, antibody-encoding genes were isolated regardless of the cell
being large or small. In initial experiments with FACSAria, regular PCR-plates (Bioplastics) were
used. The following gel-analysis revealed a wrong positive/negative control pattern. This might be
because no cells reached the reaction mixture, either due to incorrect adjustment of the
FACSAria/plate, instability of the plate, or the cell-squirt bending off leaving the cell on the side of
the wells. Therefore a more stabile skirted plate from Eppendorf was used, the FACSAria was
adjusted directly on the plates and the plates were spun down after cells sorting. However this
was no improvement as the wrong positive/negative control pattern still was seen and sequencing
of products from positive wells showed presence of contamination (data not shown). Even when
using the same plates (ABgene) as the inventors of the Symplex™ technology, the wrong
positive/negative control pattern appeared (data not shown). In cooperation with the FACSAria
core facility this work is in progress.
RECOMBINANT AB01.15 RETAINS ORIGINAL SPECIFICITY
EBV-AB01.15 has previously been characterized. It has been shown that EBV-AB01.15 is of the IgG
isotype, it binds to the DBL4 domain (a DBLγ domain) of PFD1235w (Lea Barfod, personal
47
communication), it is cross-reactive to DBLγ domains from other parasite proteins (Luminex assays
and ELISA, Michael Dalgaard, Lea Barfod, Anja Jensen and Anja Bengtsson, personal
communication), it binds to the surface of 3D7-PFD1235w IEs (flow cytometry, figure 24.c), and
the epitope it recognizes is conformational (western blot of reduced and non-reduced PFD1235w
DBL4, Anja Bengtsson, personal communication).
The antibody genes of AB01.15-EBV were successfully cloned by the Symplex™ technology. The
primers that amplified the genes were from the 1st PCR G + κ primer set. To determine whether
the correct antibody genes had been cloned and the recombinant antibody retained the specificity
of EBV-AB01.15, different characterization experiments were performed.
In ELISA AB01.15 reacted with recombinant PFD1235w DBL4 domain. It did not show any
unspecific binding to full length VAR2CSA (figure 23). Identical binding properties had been seen
for EBV-AB01.15 (Lea Barfod, personal communication).
Figure 23. Reactivity of AB01.15. AB01.15 reactivity to
PFD1235w DBL4 determined by antigen-specific ELISA.
Absorbance of PBS samples were too low to be seen in
the figure. 0.1 µg protein was coated to each well.
To test whether AB01.15 also binds to surface exposed PFD1235w in native conformation, a flow
cytometry assay on 3D7-PFD1235w parasite IEs was performed. As is seen in figure 24.a
recombinant AB01.15 reacted with the surface of the IEs, as EBV-AB01.15 had been shown to do
previously (figure 24.c). It should be noted that the two assays can not be compared for reactivity
as different antibody concentrations have been used and different secondary antibodies. AB01.15
did not bind to erythrocytes infected with control PAR+ that express IT4VAR60, while immune
plasma did (figure 24.b).
48
Figure 24. Surface-reactivity of AB01.15. A) Reactivity of recombinant AB01.15 supernatant (black curve), buffer
(grey area), and immune plasma (red curve) with the surface of 3D7-PFD1235w IEs. B) Reactivity of recombinant
AB01.15 (black curve), buffer (grey area) and immune plasma (red curve) with the surface of PAR+ IEs. C)
Reactivity of purified EBV-AB01.15 (black curve) or buffer (grey area) with the surface of 3D7-PFD1235 IEs.
Both recombinant AB01.15 and EBV-AB01.15 were included in the multiplexed ELISA assay
Luminex. The included plex consisted of various domains of different PfEMP1 from different
parasite isolates. Both AB01.15 and EBV-AB01.15 showed to react specifically with three DBLγ
domains from three different PfEMP1s (Pf11_008, PFD1235w, and PF08_0141) (figure 25). The
reactivity of the antibodies cannot be compared as different concentrations have been used.
Figure 25. Reactivity of EBV-AB01.15, recombinant AB01.15 and AR04.1 to recombinant PfEMP1 domains. Reactivity
of antibodies to Plasmodium falciparum proteins of plex 5 by luminex assay. DBLγ domains are written fully, the
identity of the rest of the domains can be found in appendix C. MFI: Median fluorescent intensity.
49
Multiple alignments were made of DBLγ domains being recognized or not recognized by EBVAB01.15 in ELISA (figure 26) and luminex (figure 27 and 28) (results provided by Michael Dalgaard,
Lea Barfod, Anja Bengtsson and Anja Jensen). From these alignments differences in sequences
between the recognized and non-recognized domains were verified manually and are marked in
the figure. A predicted 3D model structure is shown in figure 29 with the location of the epitopes
predicted by ELISA data (figure 29.a) and luminex data (figure 29.b and c).
Figure 26. Alignment of DBLγ domains. A) Alignment of domains recognized in ELISA assays. B) Alignment of
domains not recognized in ELISA. Areas where recognized domains differ from unrecognized domains are marked.
50
Figure 27. Alignment of DBLγ domains recognized by AB01.15 in luminex. Areas where recognized domains differ
from unrecognized domains are marked.
51
Figure continues on next page
52
Figure 28. Alignment of DBLγ domains not recognized by AB01.15 in luminex. Areas where recognized domains differ
from unrecognized domains are marked.
53
Figure 29. Location of predicted epitopes on model of PFD1235w. Amino acids of the predicted epitopes are
highlighted in yellow. A) Prediction based on binding pattern in ELISA assays. B) Prediction based on binding pattern in
luminex assays. C) As in B, turned 135° to the right.
RECOMBINANT AR03.1 RETAINS ACTIVITY
EBV-AR03.1 has been shown to be of the IgG3 isotype, reverse rosettes and inhibit rosette
formation around 90%. Any specific epitope has not been found, but EBV-AR03.1 has been shown
to bind to the DBL2γ domain of IT4VAR60, CIDR domains, MSP3 and various serum proteins. The
binding to CIDR domains and MSP3 could however not be confirmed in antigen-specific ELISA.
EBV-AR03.1 did not to bind to the surface of PAR+ IEs in flow cytometry assays (Luminex, western
blot, immunoprecipitation, ELISA, flow cytometry data, Lea Barfod, personal communication).
The antibody genes of AR03.1-EBV were successfully cloned by the Symplex™ technology. The
primers that amplified the genes were from the 1st PCR G + κ primer set. Due to time limitations
recombinant AR03.1 was only tested for ability to reverse rosettes and on a limited number of
parasite proteins in luminex. The luminex assay was performed on a plex containing mainly
domains from VAR2CSA but also the EBV-AR03.1-reacting MSP3. Recombinant AR03.1 reacted
slightly with MSP3 as is seen in figure 30. Positive and negative controls reacted as expected.
Recombinant AR03.1 and EBV-AR03.1 were tested in a rosette reversal assay. Both showed
reduction of percentage of rosettes compared to treatment with only PBS (figure 31). The activity
of EBV-AR03.1 seems to be concentration-dependent. The reversal-efficiencies of EBV-AR03.1 and
recombinant AR03.1 cannot be compared as different concentrations have been used.
54
Figure 30. Reactivity of
recombinant AR03.1 and
AR04.1 to Plasmodium
falciparum protein domains.
Reactivity of antibodies to
Plasmodium falciparum
proteins on STOPPAM plex by
luminex assay. Concentrated
supernatant of AR03.1 was
diluted 1:100, while AR04.1
supernatant was diluted 1:10.
Figure 31. Par+ rosette reversal activity of EBVAR03.1, AR03.1 and AR04.1. Percentage of IEs
forming rosettes after incubation with recombinant
AR03.1
supernatant,
recombinant
AR04.1
supernatant, purified EBV-AR03.1, heparin, immune
plasma or PBS. Results for PBS is the average of two
samples.
RECOMBINANT AR04.1 RETAINS ITS ACTIVITY
EBV-AR04.1 has been shown to be of the IgM isotype, reverse rosettes and inhibit rosette
formation around 50%. The antigen of EBV-AR04.1 has not been found (Lea Barfod, personal
communication).
Antibody genes from AR04.1-EBV were cloned successfully with the Symplex™ technology. The
primers that amplified the antibody genes were from the 1st PCR M + κ set, although some positive
wells also were seen with the primer set 1st PCR G + κ (data not shown). Recombinant AR04.1 was
shown to retain the activity of EBV-AR04.1 as it reduced the percentage of rosettes in the rosette
reversal assay (figure 31).
55
The parasite line PAR+ has previously been found to mediate rosetting through the DBL1-α domain
of IT4VAR60 (Albrecht et al. 2011). To test whether the rosette-reversal activity of AR04.1 was due
to binding to this domain, AR04.1 was tested against the domain in ELISA. No reactivity to
IT4VAR60 DBL1-α was detected (figure 32.b). Neither did AR04.1 show any reactivity to the
proteins included in either of the luminex assay (figure 25 and 30). When tested in flow cytometry
AR04.1 did not show any surface reactivity to PAR+ IEs (figure 32.a).
Figure 32 Reactivity of recombinant AR04.1. A) Reactivity of recombinant AR04.1 (black curve), buffer (grey
area) and immune plasma (red curve) to the surface of PAR+ IEs. B) Reactivity of recombinant AR04.1
supernatant, immune plasma and buffer to recombinant IT4VAR60 DBL1α by antigen specific ELISA.
EPSTEIN-BARR VIRUS-IMMORTALIZED B CELL CULTURE AR05.2 PRODUCE IGM
Cloning of antibody genes from AR05.2-EBV was attempted several times, but without getting any
positive bands after the 1st and 2nd PCR – or only isolating contaminants. AR05.2 has previously
shown to be of the IgM isotype and to inhibit rosette formation up to 50% (Lea Barfod, personal
communication). To test whether the unsuccessful cloning was due to loss of ability to produce
antibodies of the culture, a µ-chain-specific ELISA was set up. As is seen in figure 33, AR05.2 does
produce IgM and lack of IgM mRNA cannot be the reason for the unsuccessful cloning.
Figure 33. Detection of IgM
production.
IgM-detection
of
supernatant from EBV-AR05.2,
immune plasma (positive control)
and PBS (negative control) by ELISA.
56
DISCUSSION
B CELL COLLECTION
According to the World Health Organization’s World Malaria Report (World Health Organization
2009), the incidence of malaria in most of Ghana is more than 100 cases per 1000 inhabitants per
year. The adult B cell donors have thus most probably had malaria at some point and thus most
probably possess memory B cells towards malaria parasites. However, as there are differences in
how often individual donors are infected and whether they are treated with anti-malarial drugs,
the abundance of memory B cells will vary accordingly. The ELISA was included to give a hint of P.
falciparum exposure and thus increase the probability of obtaining circulating anti-P. falciparum
memory B cells.
10 out of 24 donors were selected for B cell purification based on GLURP R0 reactivity in ELISA.
The intention was to base selection on reactivity to IE mix and recombinant GLURP R0 while
showing no reactivity to uninfected erythrocytes or coating buffer. As described in ‘results’ the
ELISA set-up was inconsistent as problems with high variation between duplicate wells and high
negative controls were seen. However, on day 4 when the batch of OPD was changed and the
coating buffer was changed to PBS, less variation was seen and reactivity to crude parasite mix
appeared. As the assay showed fine reactivity patterns when tested in Denmark prior to sampling
and at day 4 in Ghana, the set-up does work. Because it showed consistency at day 4 the problems
must have been due to the OPD tablets or the coating buffer. Although these reagents were
brought to Ghana in polystyrene boxes with freezer packs, it is possible that they were damaged
during transport.
Despite the variability, the ELISA assay was not repeated. The assay takes around one hour, and if
it was to be repeated the total B cell processing time would increase and viability decrease. Due to
time limitations it was not possible to postpone the B cell collection until the ELISA assay was up
running again. If the B cell collection is to be repeated, the set-up should be tested on a plasma
sample known to react with crude parasite mix and recombinant GLURPR0 and not with
uninfected erythrocytes or coating buffer in Ghana prior to sampling. This positive control should
also be included in the following donor selection assays.
In spite of the variability of the assay and the lack of reactivity to crude parasite mix, it seems safe
to state that the selected donors on day 1 and 2 – and certainly on day 4 – have anti-Plasmodium
falciparum antibodies. Especially when keeping in mind that the high reactivity to GLURP R0 of the
negative control on day 1 was due to uneven color development in one of the wells. Whether the
donors selected on day 3 really possess anti-P. falciparum antibodies is less certain due to the high
reactivity of the negative controls. Still this might not mean that these donors are devoid of
memory B cells to P. falciparum.
57
Pre-selection of donors would have been optimal for B cell collection. If small blood samples had
been taken prior to collection the best donors could be selected without being restricted by time.
Then the ELISA assay could have been repeated if showing inconsistency, but more interestingly
there would have been time to perform other assays such as ELISPOT or multiplexed luminex
assays. Luminex would detect antibodies to multiple P. falciparum protein domains simultaneously
while ELISPOT would give a direct count of P. falciparum specific memory B cell (Crotty et al.
2004). When obtaining blood samples from the blood bank it is not possible to obtain any presamples as most donors only come to the hospital the day of sampling. However, the plan was to
test the B cells in ELISPOT after purification to determine which of the selected donors to apply to
the Symplex™ technology.
With the rough estimates of 1.25·106 PBMC/ml blood and 10% of these being B cells (Crotty et al.
2004), the expected B cell amount from 250 ml blood would be around 31.25·106. In this study
between 3.5·106-22.5·106 cells were collected per donor, which is close to the estimate. Some B
cells might have been lost from the purification, which by investigation under a light microscope,
revealed a seemingly uniform cell population corresponding to the size of B cells. No further
examinations of B cell purity was necessary as memory B cells anyway were to be sorted by FACS
analysis or specific magnetic assisted cell sorting. B cell viability verified in light microscopy was
>99%, so the cells did not seem to have been affected by the 6-8 hours processing time. Most
deplorable the viability was less than 1% when cells were brought to Denmark. This loss of viability
is likely to be due to thawing of the samples during transport. It was therefore impossible to
continue with the collected cells. At the time of writing new B cell collections are being planned.
This time transport will be carried out by a well-established courier.
ESTABLISHMENT OF THE SYMPLEX™ TECHNOLOGY
The Symplex™ technology was established at CMP to be able to produce recombinant versions of
antibodies generated by EBV cells. Furthermore, future larger scale antibody generations would
benefit from this establishment. The different experiments will be discussed in the order they
appear in the cloning protocol ending out with a discussion of the contamination problem.
CELL SORTING
Due to ease of the procedure, limiting dilution was initially used to sort single cells into reaction
wells. Because of the risk of incorrect dilutions and thus uncertainty of whether cells actually were
applied to the wells, micro-manipulation and FACS single cell sorting were introduced. Sorting by
micro-manipulation gives certainty that the correct number of cells is applied to each well.
However, this is extremely laborious and applying cells to a 96 well plate would probably take an
entire day - even for a skilled person. It would thus be an inconvenient method to use for larger
scale antibody generation. Additionally, handling by the micro-manipulator might kill the cell.
Although one cell has been applied to each of the wells (except from positive and negative
58
controls) many wells do not give positive bands (see figure 22.a). This could also be due to some of
the EBV cells not producing antibodies. The FACS single cell sorting would be the optimal way of
applying cells, but unfortunately we could not make it work. It should be possible as the machine
and plates used are identical to the ones used in the paper by Meijer et al. (2006). Either the
FACSAria is not handled optimally or we simply chose a bad gating on the cell population. As is
seen in figure 7 EBV cell cultures consist of a heterogeneous population when it comes to size and
granulation. It is possible that only the bigger and more granulated cells are producing antibodies
and the p3 gates should be chosen (figure 7). Work on optimizing FACS cell sorting is ongoing as
the method is essential for future larger scale set-ups. However, when using the Symplex™
technology on few cells, micro-manipulation proved to be a fine way to sort cells.
ISOLATION OF ANTIBODY-ENCODING GENES
The isolation of antibody encoding genes was relatively easy and both the Meijer et al. (2006)- and
the in-house-developed primer sets were working. Cross-annealing of the G+κ set developed by
Meijer et al. (2006) was observed with cells of the IgMκ isotype. However, I do suspect that the
cross-annealing was caused by an IgGκ contaminant. Although several primer sets have been
developed these are not likely to cover all possible antibodies of the IgM and IgG type. This can be
the reason why the antibody genes from the IgM-producing AR05.2-EBV could not be isolated.
SEQUENCING
Though colony-PCR could confirm fragment insertion of the correct length and insertion of
promoter (S3 annealing site is inside the promoter/leader fragment), sequence analysis was the
ultimate verification of correct insert into vector, promoter insertion and presence of
contamination. Sequence verification was also used to decide which clones should be selected for
promoter insertion or antibody expression. Base-pair differences between different clones were
often seen. These could be due to cell to cell variation in the EBV cell culture caused by random
mutations, errors introduced in 1st or 2nd PCR or could be an artifact introduced in the sequencing
PCR or analysis. The clones most resembling the consensus sequence were selected. Despite basepair differences between clones, expression of the selected clones did end up in production of
antibodies with retained specificity. Altogether this suggests that the sequence verification and
selection steps applied were sufficient to ascertain correct antibody production.
Sequence analysis was also used to determine presence of contamination. Implementation of the
1st sequencing was a great improvement in order to detect contamination on an earlier stage and
thus avoid fruitless work. This 1st sequencing can further be improved by optimizing primers used.
It would be obvious to design primers annealing in the linker region instead. As this sequence is
conserved in any VH-CVL fragment and is located inside the fragment, the quality of the sequence
would probably increase. Improved sequence quality would make it possible to exclude sequences
with interrupting stop codons already after the 1st sequencing. In future work on converting EBVproduced antibodies into recombinant antibodies it might be possible to exclude the 2nd
59
sequencing and rely on 1st colony-PCR alone to determine fragment insertion. This would cut out
substantial work and time from the protocol.
DETECTION OF ANTIBODY PRODUCTION
A simple antibody-detection assay was developed, and proved that the optimal dilution for
antibody-detection is around 1:150. It is unclear why the reverse correlation between antibody
dilution and reactivity was seen. The low reactivity when supernatant was diluted in media (figure
17) suggests that a protein in the media binds better to the ELISA plate thus outcompeting
antibodies. This would continue until the supernatant is diluted to an extent where not all binding
sites can be occupied by this protein and antibodies would get to bind. Instead of diluting the
sample, sandwich ELISA could be employed by coating with anti-IgG, apply supernatant and detect
with another anti-immunoglobulin as is done by Tiller et al. (2008). Dot blot would be another
time-saving solution. The present assay was though sufficient to determine whether antibodies
were produced or not.
OPTIMIZING PRODUCTION
To maximize antibody production the optimal day of harvest and the optimal construct-DNA
transfection concentration were determined. A construct-DNA transfection concentration of 1
µg/ml and an incubation period of 7 days were found to be optimal for antibody production. It is
clear from figure 18 that 1 µg/ml (30 µg in 30 ml) construct-DNA is the optimal DNAconcentration. Harvest day 9 was not chosen despite showing highest antibody titer. This was due
to the general pattern at this day being a decline in antibody production and due to increased risk
of antibody degradation. If production is to be optimized further it would be interesting to include
more construct-DNA transfection concentrations around 1 µg/ml. By the current antibodydetection assay it could not be determined whether the antibodies were functional or not. To be
able to detect only functional antibodies the assay could be changed into an antigen-specific
assay. However, the first antibody being produced – and thus the one to be subjected to
optimization – was the AR04.1. The antigen of AR04.1 is not known and an antigen-specific assay
was thus not applicable.
Western blot of the recombinant antibodies in figure 19.c revealed presence of degradation
products. It would be useful to run samples from earlier days of harvest as well to test whether
sooner harvest would reduce the amount of degradation products and the optimal day of harvest
thus should be changed.
The additional five bands seen in figure 19.c have sizes around what is expected for two heavy
chains (H) and one light chain (L), two H, one H and one L, one H, and one L. The degradation
products may thus reflect incorrect linkage of heavy and light chains. This is a common problem
for recombinant production in E. coli where disulphide bonds are not easily formed (Reilly &
Yansura 2010), but to my knowledge normally not associated with recombinant production in
human cells. As the purification column is specific for kappa light chain it is unlikely that products
60
not containing light chains will be purified. Instead, the reduction of the disulfide bonds might be
caused by prolonged storage in low pH elution buffer. However, AR04.1 also contains degradation
products albeit being supernatant and not stored in elution buffer. The degradation products seen
could also be due to too long incubation at 87 °C when preparing the gel. The same amount of
degradation products is though not seen for the EBV-AB01.15 that has been treated the same way
(comparing AB01.15 and EBV-AB01.15 in figure 19.c). Although the sizes of recombinant IgG is
bigger than expected (around 180kDa versus around 150kDa) the sizes are comparable with
plasma IgG and could be detected to consist of heavy and light chains when reduced (figure 19.b
and c). The larger size could be due to glycosylation of the antibodies or the simple explanation
that the band sizes of the ladder being incorrect. To investigate degradation products further it
would be preferable to repeat the western blot with a polyclonal detecting antibody that is
specific to the whole IgG and not as here one specific for the heavy chain (figure 19.b) and one
seemingly specific for the light chain (not mentioned in the manufactures’ specification sheet)
(figure 19.c).
When comparing to other studies with antibody concentration of 20-40 µg/ml or 10-80 µg/ml
(Geisse & Fux 2009; Tiller et al. 2008) the present yield of 4.9 µg/ml is not high. As is seen in figure
19 some antibodies are lost during purification despite the antibody amount not exceeding the
capacity of the column (5-10mg). As the purification protocol has been altered compared with the
manufacturer’s guidelines, it is likely that optimizing the protocol would increase antibody
amount. As all recombinant antibodies are produced as IgGs the purification column could be
changed to protein G or A that binds the Fc region. The reason why protein L was used for
purification was that the intention was to produce Fab fragments that are not bound by protein A
or G. However, well into the project it was discovered that IgG and not Fab was produced. The
expression vector was thus for IgG and not - as assured - Fab. Therefore many of the
characterization experiments have been designed for Fabs.
VERIFICATION OF FUNCTION
The ultimate proof that correct antibody genes are being expressed and are functional were the
characterization experiments. AB01.15 showed identical specificities as its EBV cell-produced
counterpart recognizing the same protein domains and AR03.1 and AR04.1 both retained the
rosette-reversal activities of their EBV cell-produced counterparts. This also means that the
recombinant production did not affect the specificity of the antibodies. The characterization
experiments will be discussed later.
DETERMINATION AND ELIMINATION OF CONTAMINATION
Extensive work has been put into trying to sort out where contamination stems from and how it
could be eliminated.
61
At first, PCR on reagents pointed to contamination of digested vectors, but further sequencing of
re-ligated vector did not confirm this. Instead 1st sequencing revealed that contamination
appeared already in the PCR product. The presence of contamination in wells without any added
cells left only the possibility of contamination being introduced in either 1st PCR or in the 2nd PCR.
As no cell, no DNA, and no RNA (except for the researchers’ own!) is introduced into the DNA
clean-bench or the room where it is located and plates are sealed before leaving the hood, it is
hard to believe that contamination is introduced already in the 1st PCR. The 2nd PCR was
performed in a common laboratory where further work with constructs also took place. Although
contamination was sought to be avoided by using gloves, filter tips, plates and seals separated
from other lab-work, and separate freezer and shelf spaces, contamination could be introduced
here - maybe in construct-containing dust in the air or on pipettes. A thing contradicting this is the
fact that sometimes combinations of heavy and light chains were seen, which is more probable if
both have been amplified in 1st PCR. Also, less amount of the contaminating DNA is required if it is
present in the 1st PCR as more cycles of amplifications are performed then. Contamination often
appeared when cells had been applied by limiting dilution or by FACS cell sorting, but never when
cells were applied by micro manipulation. Although cells are applied in a separate (but nonprotected) room when using micromanipulation, enzyme mix is added in the same hood, where
cells from limiting dilution are applied. It is thus not likely that the contamination is introduced
when adding cells. The only difference of using the micro-manipulator instead of FACS cell sorting
or limiting dilution is the certainty that the correct amount of cells reaches the wells.
Although it was not possible to determine what is causing the contamination and exactly where it
is introduced, it seems to dominate in set-ups where template is lacking. The wrong
positive/negative pattern that was seen in connection with contamination adds to this theory. If
cells are applied to the well, template will probably be present in larger amounts than the
contaminating DNA and the many amplification cycles of the PCRs will make the proportion of the
right fragment greater. However if cells are not applied to the well (or cells do not produce
sufficient mRNA) no bands are seen in the positive wells (wrong positive/negative patter) and only
the contaminant will be amplified.
To look further into where contamination is introduced, additional negative controls should be
included in the 2nd PCR. If water samples turn out to give positive results when subjected to 2nd
PCR it would confirm introduction of contamination in this step. However, such negative controls
have been included previously and never turned out positive.
Apparently contamination is a common problem when working with single cell PCR. To cite Smith
and co-authors (2009): “Extreme caution must be used; even talking over the plate can cause
contamination”. At Symphogen – the inventor-company of Symplex™ – separate rooms are used
for different steps to avoid contamination (Allan Jensen, Symphogen, personal communication).
62
Unfortunately it has not been possible to eliminate the risk of contamination. My suggestions for
precautions to avoid contamination would be to start setting up the 2nd PCR in a separate room
where no construct-work is taking place, using separate pipettes and changing lab-coat when
entering the room. Ideally the 2nd PCR could be set-up in another DNA clean-bench, but this would
be a somewhat pricey solution.
Until (and if!) the contamination puzzle has been solved the best way to deal with contamination
is to perform extensive verifications like the 1st sequencing of PCR products and aligning them to
known sequences. The contamination experienced in this study was caused by antibody genes
previously cloned. Therefore aligning 1st sequences to sequences of previously cloned cells, was
successful in discovering contamination. There is of cause the possibility that an unknown
contaminant will appear and not being recognized as contamination. As long as the method is
applied to already characterized EBV cells the contamination can be discovered as lack of function
of the recombinant antibody. However, when generating novel antibodies for antigen discovery
this is not possible.
Taken together the Symplex™ method has now been established for human monoclonal antibody
production at CMP. Three sets of antibody encoding genes were isolated from three different EBV
cell cultures and expressed as recombinant antibodies. Although antibodies were produced, there
is still room for optimizing the production further. Unfortunately, it was not possible to eliminate
the risk of contamination. However, the initiatives taken have reduced the risk of contamination
to a somewhat acceptable level.
RECOMBINANT ANTIBODY PRODUCTION VS. EBV-ANTIBODIES
Monoclonal antibodies have so far been produced by EBV cells at CMP. Now Symplex™ has been
established to clone the antibody genes from these cells and express them in a recombinant
version.
The recombinant antibodies give the advantage of a stable production. Although the yield
obtained in the present study is not as high as expected (4.9 µg/ml), it does compare with
antibody production of EBV cells (3-20 µg/ml, (Traggiai et al. 2004)) and certainly exceeds the
amount produced by EBV cells at CMP (0.02-0.3 µg/ml, Lea Barfod, personal communication). It
did seem like more degradation products were seen in recombinant antibody production
compared to EBV-production (figure 19.c). However, the results are not comparable as the EBVproduced antibodies were purified differently (protein A column). Production is a lot faster in
transiently transfected HEK293 cells, easy to set up in larger culture volumes and does not require
any work between transfection and harvest. The antibody production by EBV cells often declines
63
over time and some cultures grow very slowly, get contaminated, or die out ((Steinitz 2009) and
our laboratory observations). There might even be a decrease in antibody affinity in long term
cultures due to mutations (Steinitz 2009). On the contrary recombinant antibody production relies
on expression from antibody encoding constructs leading to stable and uniform production.
Antibody-encoding constructs also make it possible to engineer the antibody further. When
expressed from the constructs, all antibodies are of the IgG1 isotype making characterization
experiments such as luminex and flow cytometry easier to perform.
EBV cells do have the advantage of being able to express any kind of antibody. The generation of
recombinant antibodies is only possible if the encoding genes can be amplified by primers used.
This could for example be what limited the cloning of AR05.2-EBV.
Generation of antibody-constructs is quite laborious when only cloning one cell type at a time and
especially when contamination delays the work. Still recombinant antibody production is stable,
fast and easy making the Symplex™ technology advantageous. Conversion to recombinant
production is thus valuable if high antibody amounts are wanted or if the EBV culture is growing
slowly or dying out.
LARGER SCALE MONOCLONAL ANTIBODY PRODUCTION
PERSPECTIVES OF APPLYING THE SYMPLEX™ TECHNOLOGY FOR LARGER SCALE ANTIBODY GENERATION
The Symplex™ technology was established at CMP for small throughput recombinant antibody
generation. To be able to exploit the technology for higher throughput production of many
different antibodies at the same time some adjustments need to be made. Instead of cloning
fragments from each positive well separately, bulk cloning as described in Meijer et al. (2006)
would decrease cloning work. However, introducing bulk cloning would make it difficult to detect
contamination by 1st sequencing as done in this study as fragments are not purified separately.
Instead other initiatives could be taken. Positive wells could be randomly selected for separate
purification and 1st sequencing. These sequences should be aligned to all previous sequences to
check whether any known contaminant is present. Presence of novel contaminants would be
indicated if several of the 1st sequences are identical. Various negative controls in both 1st PCR and
2nd PCR should be included on each plate in order to monitor contamination. Finally, initiatives
should be taken to reduce the risk of contamination. The PCR steps should be performed in
separate rooms isolated from any construct-work and separate lab coats should be used when
working in construct-free rooms. Unfortunately, total elimination of the risk of contamination is
probably impossible.
The Symplex™ technology uses ASCs as mRNA source. This can be an advantage, when analyzing
antibody responses to a vaccine, as the antibody secreting plasma cells are readily available in
64
peripheral blood at a narrow time window after vaccination. It would also give the advantage of a
high proportion of the cells being antigen-specific (Lanzavecchia et al. 2007). However, when
working with natural infections such as malaria the narrow time window is troublesome to
determine. Instead of collecting blood from a blood bank as was done in this study, malaria
patients would have to be monitored to determine the peak of ASCs. Another drawback of ASCs is
that they cannot be selected for antigen-specificity prior to cloning i.e. all uninteresting cells will
be cloned as well.
Memory B cells on the other hand are readily available in peripheral blood long after infection. In
addition they carry B cell receptors on their surface making it possible to select antigen-specific
cells prior to cloning. In malaria research this could be done by selecting cells adhering to relevant
recombinant proteins or to the surface of IEs. However, the feasibility of these approaches still
needs to be proven.
The method developed by Tiller et al. (2008) performs single cell PCR on memory B cells. As this
method does not possess the convenience of bulk cloning, it would be advantageous if Symplex™
could be modified to isolate genes from memory B cells instead. However, this would probably
require an increased number of cycles in the PCRs. The Tiller method uses a total of 100 cycles to
amplify genes from memory B cells, which I think, would increase risk of contamination
substantially. Another possibility of applying the Symplex™ technology to memory B cells is to
stimulate the cells to become ASCs and then subject them to cloning. Memory B cell can be
stimulated to differentiate into ASCs by stimuli such as IL-21 and CD40 (Ettinger et al. 2005). This
was the intended approach in this project if the collected B cells had been viable. A similar
approach has previously been taken by (Weitkamp et al. 2003). Single B cells binding the antigen
of interest were sorted, stimulated for differentiation, and supernatants were tested after
expansion. Positive cells were afterwards subjected to RT-PCR and further cloning. The drawback
of stimulating cells prior to cloning would be that some cells will die and thus be lost.
LARGER SCALE ANTIBODY GENERATION BY ALTERNATIVE METHODS
As described in the introduction other methods are available for monoclonal antibody production.
Phage display libraries have been used to isolate malaria antigen-specific monoclonal antibodies
previously (e.g. Lundquist et al. (2006) and McIntosh et al. (2007)). What makes phage display
stand out from the other methods is its ability to select antigen-specific antibodies from huge
libraries. However, this selection is also what limits the technology regarding antigen or conserved
epitope discovery. In phage display antibodies are coupled to phage particles during selection,
whereas antibodies in other methods are in suspension. This gives some drawbacks. Functional
assays cannot be performed, thus selecting antibodies based on their anti-rosetting activity would
not be possible. Further obstructing antigen discovery are the difficulties of selection on infected
erythrocytes (Hviid & Barfod 2008) although possible in theory (Hoogenboom 2005). Due to
difficulties of purifying VSAs from infected erythrocytes, selection of VSA-specific antibodies relies
65
on recombinant antibodies. A priori knowledge of the antigen is thus necessary (Hviid & Barfod
2008). I question how antibodies can be selected for cross-reactivity using phage display. Because
of the particles not being in a monoclonal suspension, they cannot be tested against various
proteins simultaneously. Cross-reactive antibodies produced by the other methods could on the
other hand be found using luminex or flow cytometry on different IE isolates and thus assist in
conserved epitope discovery. To be used in malaria antigen or conserved epitope discovery phage
displayed antibodies first have to be selected on recombinant protein and then expressed in
suspension where they can be tested in functional assays or for cross-reactivity. Another possible
drawback is the combination of random V gene pairing and selection on recombinant antigen,
which means that the epitopes of the monoclonal antibodies produced might not reflect epitope
specificities that human generate in vivo and not necessarily be present in the intact native protein
(Hviid & Barfod 2008; Smith et al. 2009).
The hybridoma technology has also been used in malaria research (e.g. (Kobayashi et al. 2007)). In
contrast to phage display functional assays and cross-reactivity screenings can be carried out with
the hybridoma technology. The major drawback of the hybridoma technology is that it is best
suited for murine cells. The generation of anti-PfEMP1 antibodies by the hybridoma technology
thus relies on immunizing mice with recombinant antigens. Compared to production of antibodies
developed during natural infection antibodies will be restricted to target the antigen used for
immunization thus antigen discovery would not be feasible. Furthermore there are considerations
of whether the epitope targeted by mice reflect the ones important in human immune responses
(Smith et al. 2009). Also, as recombinant antigens are needed for immunization this raises the risk
of incorrect protein folding leading to epitopes not necessarily being present in the native protein
(Hviid & Barfod 2008). Finally, if a protective cross-reacting antibody should be found it cannot be
used as a therapeutic antibody due to risk of the human immune system reacting to the foreign
murine antibody (Penichet & Morrison 2004).
The Epstein-Barr virus-immortalization method has been used for monoclonal antibody
production at CMP and has among other things been used to show that different VAR2CSA
variants have shared epitopes and that these epitopes are involved in the adhesion of IEs to CSAs
(Barfod et al. 2010). As described above, there are drawbacks of the Epstein-Barr method: the
production is relatively slow, risk of decreasing antibody production and affinity over time and risk
of culture contamination. Most importantly there is a risk of cells dying out or being overgrown by
irrelevant B cells. This often results in relatively few different specific antibodies being isolated
from one immortalization (Lea Barfod, personal communication). The Epstein-Barr method and
the hybridoma technology share the advantage of a relatively early initial screening. 2-3 weeks
after immortalization, antibodies can be screened in functional assays or for cross-reactivity.
Interesting antibodies are thus relative quickly found and focused on. Furthermore the EpsteinBarr method has the advantage of generating an unbiased fully human repertoire, where
antibodies of any isotype can be generated.
66
The Epstein-Barr method and Symplex™ technology stand out from the other methods when it
comes to production of monoclonal antibodies for P. falciparum antigen and conserved epitope
discovery purposes. They are both able to generate fully human antibodies developed as a
response to native proteins in natural infection. Furthermore it is possible to screen antibodies on
native proteins on the surface of IEs using flow cytometry or in functional assays such as the
rosette reversal assay used in the present study. Together this increases the chances of obtaining
antibodies relevant in protection.
While the Epstein-Barr method suffers from relatively few antibodies being isolated from each
immortalization as cells die out or are overgrown it enjoys the possibility of screening for
antibody-specificity relatively quickly. The Symplex™ technology on the other hand, enjoys the
advantage of faster and stable production of recombinant antibodies, but work is wasted on
cloning uninteresting antibodies as the method lacks the possibility of screening cells prior to
cloning. The advantages of both methods could be achieved by combining the methods. The setup would be identical to EBV-immortalization only instead of subcloning cells from positive wells,
cells are single cell sorted and cloned by the Symplex™ method. This would give the advantage of
selecting which cells produce interesting antibodies and facilitate cloning only of the antibodies
with desired activity. The recombinant antibody production would be stable, making it possible to
isolate and characterize numerous antibodies.
Despite unresolved problems with contamination it seem to me that the Symplex™ technology
possibly combined with the Epstein-Barr method or other ASC-inducing stimulation would be the
best suited method for monoclonal antibody production for use in antigen and conserved epitope
discovery.
Three different recombinant antibodies were successfully made by the Symplex™ method:
AB01.15, AR03.1, and AR04.1. Unfortunately, the contamination problems took up a great amount
of time which limited the work on characterizing the recombinant antibodies.
AR03.1
EBV-AR03.1 has previously been characterized and shown to bind to various proteins (listed in
‘results’). Luminex data of AR03.1 showed that the recombinant antibody has retained ability to
bind at least one of the proteins EBV-AR03.1 binds to. The absorbance is not very high, but this
could be due to diluting the antibody too much. Unfortunately AR03.1 could not be tested on
other plex’es in the luminex assay. Plex’es are expensive and laborious to produce and preferably
many samples are run at a time, so I had to rely on colleagues to fit my samples into their larger
67
set-ups. AR03.1 was though confirmed to retain EBV-AR03.1s ability to reverse rosettes and this
strongly indicates that AR03.1 is identical to EBV-AR03.1. However the reversal assay has been
performed on supernatants and without a negative media control without antibodies. So in theory
the rosette reversal could be caused by a component of the media. Unfortunately, the PAR+
parasites have been troublesome to cultivate, so the planned repeat of the experiment with
purified antibodies of known concentration has not been performed. When future reversal assays
are set up to characterize the recombinant antibodies further they should be performed with
purified antibodies, so the concentration is known and influence of media can be ruled out.
It was not possible to confirm the binding of AR03.1 to the DBL2γ domain of IT4VAR60 as there
was not any recombinant IT4VAR60 DBL2 available. Although EBV-AR03.1 binds to this domain it is
not capable of binding to the surface of PAR+ IEs expressing IT4VAR60. As this could be due to the
antibodies ability to bind to different components of the serum used in flow cytometry assays, an
assay without serum was set up. Still no surface binding was seen (Lea Barfod, personal
communication). The many different target proteins of EBV-AR03.1 must mean that the epitope of
EBV-AR03.1 is fairly common. It is obvious to speculate that something else than binding to the
PfEMP1 is crucial for its ability to reverse rosettes. What this component is, is still interesting in
order to achieve more knowledge about rosetting - especially as rosetting has been associated
with severe malaria (Rowe et al. 2009). However, regarding direct VSA-antigen discovery this
antibody does not seem to be a helpful tool.
AR04.1
The only thing that previously had been determined about EBV-AR04.1 was that it was of the IgM
subtype and it was capable of inhibiting and reversing rosettes of PAR+ IEs. Flow cytometry and
luminex assays have been impossible to perform as IEs bind IgM unspecifically and the luminex
assays have been developed for IgG. The recombinant IgG AR04.1 did not show reactivity to the
surface of PAR+ infected erythrocytes indicating a similar non-VSA binding mediated ability to
reverse rosettes as seen with AR03.1. However, the flow cytometry assay has only been
performed once and as PAR+ seems to lose ability to form rosettes every now and then it should
be repeated when PAR+ is certain to form rosettes. Although AR04.1 did not show any reactivity in
the luminex assay or to the DBL1α domain of IT4VAR60 in ELISA it does not mean that the
antibody is non-functional. It is quite possible that its epitope is not included in the luminex plexes
and that it is another domain of IT4VAR60 than the DBL1α that it binds to. The AR04.1 supernatant
was able to reverse rosette formation which indicates that AR04.1 is identical to EBV-AR04.1 and
that the conversion of IgM to IgG did not influence this activity. As for AR03.1 these data need to
be repeated with purified AR04.1 and/or media as negative control to finally confirm this activity.
At the time of writing recombinant domains of IT4VAR60 are being produced. AR04.1 will be
tested against these and immunoprecipitation on IEs will be carried out to find the antigen of
AR04.1.
68
AB01.15
EBV-AB01.15 has been characterized previously, and the recombinant counterpart showed to bind
the same protein domains in ELISA, flow cytometry and luminex. From luminex and flow
cytometry at first there seems to be differences in strength of binding, but as supernatants have
been used for AB01.15 and purified antibodies used for EBV-AB01.15 it is not possible to compare
affinity. The luminex data of AB01.15 and EBV-AB01.15 presented in this study and data from
previous studies on EBV-AB01.15 show that this antibody is able to recognize DBLγ domains from
various group A PfEMP1s from different parasite isolates. By aligning DBLγ sequences that EBVAB01.15 recognizes and does not recognize and compare these to each other 5 different regions
were found where the recognized domains differed from the unrecognized (2 found in alignments
based on ELISA data and 3 found in alignments based on luminex data). These differences have
been verified manually and it should be underpinned that they are speculative. The AB01.15
epitope could e.g. consist of amino acids conserved in all DBLγ, but additional amino acids in nonrecognized domains alleviate this binding. The analysis is further biased in that correct
conformation of the recombinant domains is pivotal and incorrect folding would classify an
otherwise recognized domain as non-recognized. In a structure-model of a DBLγ domain (figure
29) the predicted epitopes were found to be situated at the surface of the protein and could be an
antibody target. Two of the residues found in luminex data alignments are even located close to
each other, so the epitope of AB01.15 could consist of both these regions. This also fits to the
observation of AB01.15 being conformation-dependent. To test the hypothesis of the AB01.15
epitope, DBLγ domains with sequences with and without the predicted epitopes should be
produced. If we are able to predict whether a domain will be recognized or not, it suggests
discovery of the correct epitope. This could further be verified by subjecting a binding domain to
point-mutations inside the predicted epitope and test if AB01.15 loses or decreases its ability to
bind.
Further characterization of AB01.15 would also involve testing whether the antibody is
functionally important and blocks PfEMP1 binding to human receptors e.g. PFD1235w adhesion to
ICAM-1. It would also be interesting to test AB01.15 on field isolates collected from children with
severe malaria by flow cytometry. If it recognizes the surface of many different field isolates it will
be highly interesting regarding vaccine development. The epitope would then be obvious to
include in a morbidity-reducing vaccine. However, it is not plausible that only one epitope will
induce protection from all severe malaria variants. But as only relatively few malaria-exposures
are needed in order to acquire natural protection to severe malaria it is possible that a collection
of few epitopes in a vaccine will induce protection. More conserved epitope discovery is thus
needed.
PfEMP1s of the relatively conserved group A variants have been shown to be up-regulated in
parasites expressing VSASM (Jensen et al. 2004). Although extensive sequence analysis has been
carried out at CMP it has not been possible to identify any conserved epitopes on the PfEMP1s
69
involved in severe malaria. The study of AB01.15 underpin the advantages of studying antibodies
developed from human immune responses as they might reveal conserved epitopes that due to
their conformation-dependency will not be discovered by sequence analysis.
70
CONCLUSION
During this thesis I succeeded in establishing the Symplex™ technology for small scale monoclonal
antibody production at Centre for Medical Parasitology. Single cell sorting by micro manipulation
was found to be most successful, but in future set-ups it will be time saving to establish single cell
sorting by FACS. A sequence verification procedure was established to confirm antibody gene and
promotor insertion as well as to select which clone should proceed to promotor insertion or
expression. Furthermore, the antibody production was optimized. It was found that a transfection
construct-DNA concentration of 1µg/ml and an incubation time of 7 days were optimal. The
antibody yield of 4.9µg/ml was lower than expected and western blot analysis suggests that
production and purification could be optimized further. The establishment of the Symplex™
technology was set back by contamination caused by previously cloned antibody genes.
Elimination of contamination, or determination of where it was introduced, was not accomplished.
However, several initiatives limited the risk of contamination and improved its detection. Despite
contamination, the establishment of the Symplex™ technology was successful and cloning of three
different Epstein-Barr virus immortalized cell cultures resulted in production of recombinant
antibodies with retained reactivity.
At Centre for Medical Parasitology monoclonal antibodies have previously been produced by
Epstein-Barr virus immortalized B cells. The conversion into recombinant antibody production by
the Symplex™ technology resulted in a more convenient and stable production.
The Symplex™ would also be applicable for larger scale production of anti-malaria antibodies,
though risk of contamination should be kept in mind and its presence should be strictly monitored.
Other methods are also applicable to larger scale monoclonal antibody generation. However,
when the purpose is to isolate antibodies for antigen or conserved epitope discovery, the
Symplex™ technology seems the most suitable as functional assays can be performed and
antibodies developed during natural infection can be generated. To further improve the Symplex™
technology, it could be combined with the Epstein-Barr method making selection of interesting
antibodies prior to cloning possible.
Stable recombinant production of the antibodies AR03.1, AR04.1 and AB01.15 has been achieved
and the recombinant antibodies have been shown to retain the activity of their Epstein-Barr virusimmortalized B cell counterparts. Further characterization of AR03.1 and AR04.1 was halted
primarily by difficulties in culturing malaria parasites with the right phenotype. However, this work
will proceed.
The cross reactivity of AB01.15 to a subset of DBLγ domains is exciting as this suggests that
conserved epitopes among VSASM exist further adding hope to the possibility of developing a
morbidity-reducing vaccines.
71
PERSPECTIVE
With the establishment of the Symplex™ method for small scale antibody-production, the method
is now ready to be established for larger set-ups to generate many different antibodies directly
from memory B cells.
As shown for AB01.15, cross-reactive antibodies towards malaria antigens do exist and provides us
knowledge that cannot be achieved by sequence studies. The Symplex™ technology may aid in
isolating more cross-reactive antibodies. When the Symplex™ technology has been established for
larger scale antibody generation more extensive experiments could be set up to find conserved
epitopes of VSASM: Blood samples from children with severe malaria could be collected to obtain
VSASM-expressing parasites and after recovery another blood sample with memory B-cells or ASCs
could be collected for antibody generation. Antibodies could then be selected according to crossreactivity with the surface of various VSASM expressing parasites by flow cytometry, which would
increase the probability of retrieving antibodies important in protection against VSASM. Finding the
epitopes of these antibodies could provide us with conserved epitopes of different VSASM and
might reveal new antigens that have not been found previously. Also it would be useful to obtain
more antibodies being able to inhibit rosette-formation in field isolates. Finding their epitopes
would reveal crucial antigens implemented in the rosetting phenomenon associated with severe
malaria.
It is the hope that finding various conserved epitopes among VSASM will make it possible to
generate a vaccine to prevent VSASM expression in infection. Although not eliminating malaria, it
could prevent disease that otherwise would cause extensive suffering and deaths of children in
sub-Saharan countries.
72
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78
APPENDIX
DNA LADDERS
http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm032generuler-100bp-plus
http://www.fermentas.com/en/products/all/dna-electrophoresis/generuler-dna-ladders/sm133generuler-1kb-plus
A
VECTOR 00VP002
B
PLEX 5
C

Human monoclonal antibody production and characterization

Transcription

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