Citation
Inherently immunogenic co-arrays of defined size and valence bearing T cell epitopes and haptenic B cell epitopes induce a switch of the anti-hapten response from IgM to IgG

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Title:
Inherently immunogenic co-arrays of defined size and valence bearing T cell epitopes and haptenic B cell epitopes induce a switch of the anti-hapten response from IgM to IgG
Creator:
Cook, Cheryl Ann
Publication Date:
Language:
English
Physical Description:
xvi, 205 leaves : illustrations ; 29 cm

Thesis/Dissertation Information

Degree Divisions:
Department of Chemistry, CU Denver
Degree Disciplines:
Chemistry

Subjects

Subjects / Keywords:
Vaccines -- Biotechnology ( lcsh )
Immunologic memory ( lcsh )
Peptides ( lcsh )
Immunologic memory ( fast )
Peptides ( fast )
Vaccines -- Biotechnology ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 193-205).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Basic Science, Department of Chemistry
Statement of Responsibility:
by Cheryl Ann Cook.

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Source Institution:
|University of Colorado Denver
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Auraria Library
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
35326335 ( OCLC )
ocm35326335
Classification:
LD1190.L46 1995m .C66 ( lcc )

Full Text
INHERENTLY IMMUNOGENIC CO-ARRAYS OF DEFINED SIZE AND
VALENCE BEARING T CELL EPITOPES AND HAPTENIC B CELL EPITOPES
INDUCE A SWITCH OF THE ANTI-HAPTEN RESPONSE FROM IgM to IgG
by
Cheryl Ann Cook
B.S., Indiana University 1982
A Thesis submitted to the
Faculty of the Graduate School of the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Basic Science
Department of Chemistry
1995


This thesis for the Master of Basic Science
degree by
Cheryl Ann Cook
has been approved for the
John Cambier
Date


Cook, Cheryl Ann (M.B.S., Masters of Basic Science)
Inherently Immunogenic Co-arrays of Defined Size and Valence Bearing T Cell
Epitopes and Haptenic or Peptide B Cell Epitopes Switch the Response from IgM
to IgG
Thesis directed by Douglas Dyckes and James Blodgett
ABSTRACT
In an effort to prevent infectious disease, the immune system of the host
attempts to destroy the pathogen and neutralize its products. This strategy underlies
the field of vaccine development which is based on two key elements of adaptive
immunity: specificity and immunological memory. Specificity increases
effectiveness of a vaccine whereas memory allows the immune system to mount a
stronger response in reaction to a second encounter with antigen. This secondary
response is both faster to appear and more effective than the primary response.
The aim of this thesis has been to develop a general method for constructing
vaccines containing relevant peptides from pathogens linked to a carrier molecule of
optimized chemistry and geometry. Although the model peptides used in these
studies have been derived from the pathogens Plasmodium falciparum, Plasmodium
berghei and human immunodeficiency virus I (HIV), it is hoped that the findings
m


might be generally applicable to the generation of vaccines against other pathogens.
Importantly, it is anticipated that such vaccines will be inherently immunogenic and
will produce an immune response without the aid of adjuvant.
This abstract accurately represents the content of the candidate's thesis. I recommend
its publication.
Signed
Signed
Douglas Dyckes
IV


DEDICATION AND ACKNOWLEDGEMENTS
Thank you to Cortech, Inc. for supporting my education and allowing me to
complete this work. Thank you to the Immunochemistry group for supplying the
conjugates used in these studies and to Sherman Ross, in the Biochemistry Group for
performing the in vitro peptide stability studies. Thanks to my advisors for agreeing
to be a part of this process. Particular thanks to Corrine Campbell for encouraging
me to enroll in the program and for being the best teacher Ive ever had. Thanks to
Jim Blodgett for putting up with all of my chemistry questions for being patient
enough to hear those questions over and over and over again. If I had known Dr.
Campbell and Dr. Blodgett as an undergraduate, this thesis would have been in
biochemistry or peptide chemistry. These individuals have a gift for teaching. The
unsung heroines of this endeavor have been the members of the Immunology group
at Cortech particularly Claire Coeshott and Catherine McCall. They have been
unofficial members of my committee and have supplied all of the immunological
advice as well as personal encouragement. Completion of this work would not have
been possible without them. Thanks to Chris Ohnemus, Anna Gallegos and Jane
Kehl also, for help with those unbearably huge ELISAs. Thanks for the extra-
hands as well as the unselfish spirit for helping me to get the assays completed.
Thank you dear for putting up with the classes, studying and the extra hours away
from the mountains that it took to get this done.
v


ABBREVIATIONS
Ac........................................................Acetylated
ACA...............................................Amino Caproic Acid
ASIM.................................Antigen Specific Immunomodulation
Al(OH)3 ....................................Aluminum Hydroxide/Alum
BSA............................................ Bovine Serum Albumin
CD4 ..............................................Cluster Determinant 4
CFA......................................... Complete Freunds Adjuvant
cOA .............................................. Chicken Ovalbumin
CTL ................................. cytotoxic T lymphocyte dextrandex
dex500 .......................dextran of molecular weight 500 kilodaltons
dex70.........................dextran of molecular weight 70 kilodaltons
ELISA ............................ Enzyme-Linked Immunosorbent Assay
HIV.................................... Human Immunodeficiency Virus
FI ....................................................... Flourescein
Ig .....................................................Immunoglobulin
IgM, IgG, IgA, IgE....Immunoglobulin of the heavy chain isotype M,
G, A or E
IL-2..................................................... Interleukin 2
i.p....................................................intraperitoneal
Kd..........................................................Kilodalton
LN .......................................................lymph node
MAPS...................................Multiple Antigen Peptide System
MHC .................................Major Histocompatability Complex
mlg./slg ................................... membrane immunoglobulin
MW ...................................................molecular weight
O. D.....................................................optical density
P. berghei........................................Plasmodium berghei
P. falciparum.................................. Plasmodium falciparum
PND.................................. Principle Neutralizing Determinant
s.c.......................................................subcutaneous
TD ................................................... T cell dependent
Th....................................................... T helper cell
TI ...................................................T cell independent
vi


CONTENTS
Abstract ........................................................... iii
Dedication and Acknowledgments .........................................v
Abbreviations..........................................................vi
Table of Contents.................................................... vii
List of Tables ...................................................... xii
List of Legends...................................................... xii
List of Figures......................................................xiii
1. Introduction.....................................................1
1.1 Current V accine Strategies.....................................10
1.2 The Immunon Hypothesis .........................................17
1.3 Scope...........................................................20
2. Materials and Methods...........................................22
2.1 Chemistry.......................................................22
2.1.1 Preparation of Peptides ........................................22
2.2 Epitopes used in these Studies..................................23
2.2.1 Descriptive Information.........................................23
2.2.2 Modifications of the Native Sequence............................24
2.2.3 The Epitopes ...................................................24
2.3 Preparation and Maleimidation of High Molecular Weight Dexamine
Vll


and Subsequent Peptide Conjugation...............................29
2.4 Fluoresceination and Maleimidation of High Molecular Weight
Dexamine and Subsequent Peptide Conjugation......................33
2.5 Amino Acid Analysis and Substitution Density
Determinations...................................................34
2.6 Animals ........................................................35
2.7 Preparation of Adjuvant ........................................36
2.8 Immunizations for Antibody Production...........................36
2.9 Measurement of Antibody Titers..................................38
2.9.1 Solid-Phase ELISA ..............................................38
2.9.2 Competition ELISA...............................................40
2.10 In Vitro Peptide Stability Studies..............................41
2.11 Pyrogenicity Testing ...........................................42
2.12 In vitro Infectiity Testing of P. falciparum ...................43
2.13 In vitro T cell Proliferation Assays (Lymph Node Proliferation
Assay)...........................................................43
3. Presentation and Discussion of Results..........................45
3.1 Hapten B Epitope (FI), Peptide T Epitope Co-arrays .............46
3.1.1 Antibody Response to the B cell (FI) Epitope....................47
3.1.2 Antibody Response to the T cell cOA(323.339)Epitope.............49
3.1.3 IgG Antibody Response to Conjugates Lacking the T cell Epitope .. 59
Vlll


3.1.4
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.2.7
3.2.8
3.3
3.3.1
3.3.2
3.3.3
3.3.4
3.4
Summary of Section 3.1..........................................63
Peptide B Epitope, (NANP) 3, Peptide T Epitope (cOA323.339) Co-array:
Part I..........................................................64
(NANP)3 Antibody Response in H-2d mice..........................65
(NANP)3 Antibody Response in H-2b mice..........................66
cOA(323.339)Antibody Response...................................67
(NANP)3 as a T helper Epitope for IgG Antibody
Production .....................................................69
(NANP)3 as a T helper Epitope via a Lymph Node Proliferation
Assay...........................................................74
Summary of Antibody Response and Lymph Node Proliferation Data
(sections 3.2-3.2.5)............................................77
In vitro Protective Ability of anti-(NANP)3 Antibodies .........80
Anti-Dextran Antibody Response .................................81
Peptide B Epitope, (NANP) 3, Peptide T Epitope (cOA323.339) Co-array:
Part II.........................................................84
Antibody Response to (NANP)3 ...................................85
Antibody Response to the cOA Epitope .......................... 86
Antibody Response to Dextran ...................................88
Summary of Sections 3.3-3.3.4 ..................................99
Peptide B Epitope, (NANP) 3, Peptide T Epitope (cOA323_339) Co-arrays
with Varied T cell Epitope Substitution Density........................100
IX


3.4.1 Overview Section 3.4
100
3.4.2 Effect of T cell Epitope Substitution Density of the Antibody
Response to (NANP)3 ........................................104
3.4.3 Antibody Response to the cOA Epitope ........................105
3.4.4 Antibody Response to Dextran ................................110
3.5 Peptide B Epitope, P. berghei, Peptide T Epitope, cOA, Co-array 122
3.5.1 Primary Antibody Response to the P. berghei Epitope..........124
3.5.2 Primary Antibody Response to the cOA T cell Epitope .........126
3.5.3 Secondary Antibody Response to the P. berghei Epitope........127
3.5.4 Secondary Antibody Response to the cOA T cell Epitope .......128
3.5.5 Repeat Experiment............................................141
3.5.5.1 Primary Antibody Response to the B cell and T cell
Epitopes...................................................142
3.5.5.2 Secondary Antibody Response to the B cell and T cell
Epitopes...................................................143
3.5.5.3 Tertiary Antibody Response to the P. berghei Epitope.......150
3.5.6 Differences Between the Original and Repeat Experiments......153
3.5.7 Anomolous IgG Titers to the cOA Epitope......................162
3.5.8 Pyrogenicity Testing ........................................170
3.5.9 Summary of Sections 3.5-3.5.8 ...............................171
3.5.10 Importance of the Stability of Epitopes.....................172
x


3.6 Subthreshold Co-arrays of the Hapten FI and the T Epitope cOA:
Implications for Vaccine Design...............................175
4. Summary and Future Directions ................................180
References...........................................................193
xi


TABLES, LEGENDS AND FIGURES
Table
1 Conjugates......................................................26
2 Anti-Dextran IgG Titres........................................ 83
Legend
Figure 3 ..............................................................31
Figures 4-10...........................................................51
Figure 11 .............................................................61
Figures 12-15..........................................................69
Figure 16 .............................................................75
Figures 17-25..........................................................89
Figures 26-33 ...................................................... Ill
Figure 34 ............................................................120
Figures 35 -44 ...................................................... 130
Figures 45 -48 ...................................................... 145
Figure 49 ............................................................151
Figures 50 53 .................................................... 157
Figure 54 ............................................................166
xii


Figure 55 .............................................................168
Figure 56 .............................................................178
Figure
1 B Cell-T Cell Collaboration.......................................5
2 Antigen Specific Immunomodulation................................16
3 Preparation and Anlysis of Conjugates ...........................32
4 IgM Antibody Response to Fl: Primary Immunization Fl-dex500.... 52
5 IgG Antibody Response to Fl: Primary Immunization Fl-dex500 .... 53
6 IgG Antibody Response to Fl: Primary Immunization [Fl + peptide]-
dex5oo ..........................................................54
7 IgM Antibody Response to Fl: Primary Immunization [Fl + peptide]-
dexjoo ..........................................................55
8 IgM Antibody Response to the cOA(323.339) Epitope................56
9 IgG Antibody Response to the cOA(323.339) Epitope................57
10 IgG Antibody Response to Fl High Substitution Density Readout
Antigen..........................................................58
11 Isotypes ........................................................62
12 IgG Antibody Response to the (NANP)3 Epitope: H-2d ..............70
13 IgG Antibody Response to the (NANP)3 Epitope: H-2bmice ..........71
14 IgG Antibody Response to the cOA(323.339) Epitope, H-2d mice....72
xm


15 IgG Antibody Response to the cOA(323.339) Epitope, H-2b mice..73
16 Lymph node Proliferation Assay.................................76
17 IgG Antibody Response to the (NANP)3 Epitope ..................90
18 IgG Tertiary Antibody Response to (NANP)3 Epitope .............91
19 (NANP)3-dex500 Immunized groups only:IgM Antibody Response to
(NANP)3........................................................92
20 Co-array Immunized groups only: IgM Antibody Response to
(NANP)3 Epitope................................................93
21 (NANP)3-dex500 Immunized groups only: IgM Antibody Response to
the cOA(323.339) Epitope.......................................94
22 All groups IgG Antibody Response to the cOA(323.339) Epitope..95
23 Co-array Immunized groups only IgM: Antibody Response to the
cOA(323-339) Epitope...........................................96
24 All groups IgG Antibody Response to Dextran....................97
25 All groups IgM Antibody Response to Dextran ...................98
26 IgG Antibody Response to (NANP)3: 14 days POST-BOOST .... 112
27 Primary IgM Antibody Response to (NANP)3 Epitope..............113
28 IgM Antibody Response to (NANP)3: POST-BOOST .................114
29 Primary IgG Antibody Response to (NANP)3 Epitope .............115
30 Primary IgM Antibody Response to cOA(323-339) Epitope.........116
31 IgM Antibody Response to cOA{323_339) Epitope: 14 days POST-
BOOST ...............................................................117
xiv


32 Primary IgG Antibody Response to cOA^^) Epitope..................118
33 IgG Antibody Response to cOA(323_339) Epitope: 14 days POST-
BOOST ..................................................................119
34 Effect of Substitution Density on Antibody Response .............121
35 Primary IgG Antibody Response to P. berghei Epitope:
H-2d mice.........................................................131
36 Primary IgG Antibody Response to P. berghei Epitope:
H-2k mice.........................................................132
37 Primary IgM Antibody Response to P. berghei Epitope:
H-2d mice.........................................................133
38 Primary IgM Antibody Response to P. berghei Epitope:
H-2k mice.........................................................134
39 Primary IgG Antibody Response to the cOA(323.339) Epitope: H-2d
mice..............................................................135
40 Primary IgG antibody response to the cOA(323.339) epitope: H-2k
mice..............................................................136
41 Primary IgM Antibody Response to the cOA(323.339) Epitope: H-2d
mice..............................................................137
42 Primary IgM Antibody Response to the cOA(323.339) Epitope: H-2k
mice .............................................................138
43 IgG Antibody Response to P. berghei Epitope: 7 days Post-
Boost ........................................................... 139
44 IgG Antibody Response to the cOA(323_339) Epitope: 7 days Post-
Boost ........................................................... 140
xv


45 Repeat Experiment: Primary IgG Antibody Response to the P. Berghei
Epitope.........................................................146
46 Primary IgG Antibody Response to the cOA(323.339) T Epitope .... 147
47 Repeat Experiment: Secondary IgG Antibody Response to P. berghei
Epitope.........................................................148
48 Repeat Experiment: Secondary IgG Antibody Response to the cOA(323.
339) Epitope....................................................149
49 Repeat Experiment: Tertiary IgG Antibody Response to the P. berghei
Epitope.........................................................152
50 Original Experiment: IgG Antibody Response to the P. berghei
Epitope.........................................................158
51 Original Experiment: IgG Antibody Response to the cOA(323.339)
Epitope.........................................................159
52 Repeat Experiment: IgG antibody response to the P. berghei
Epitope.........................................................160
53 Repeat Experiment: IgG Antibody Response to the cOA
epitope.........................................................161
54 Competition.....................................................167
55 IgG Antibody Response to cOA(323.339) Epitope: GMB or MBS
Linker..........................................................169
56 Inherent Immunogenicity.........................................179
xvi


1.
Introduction
The immune system is the primary biological defense of the host against
potentially lethal agents. These agents may be pathogens such as bacteria or viruses
as well as modified-self cells including virus-infected cells, tumor cells or other
abnormal cells of the host which may otherwise cause disease. The immune system
can recognize a vast array of these molecules, termed antigens, and the recognition of
antigen by the immune system rapidly mobilizes immune mechanisms to ensure the
sanctity of the host's environment. It is well known that upon repeated exposure to
the same antigen, the immune response that occurs is significantly superior to the
primary exposure1. This memory of earlier exposures is the fundamental concept
underlying programmes of vaccination to prevent infectious diseases.
The selective force of the antigen operates on two types of cells, the B
lymphocyte and the T lymphocyte, each capable of discriminating between those
antigens that are foreign and those present on the body's own tissues, which are
termed self- or auto-antigens. Each of these cells expresses a unique receptor which
recognizes antigen. Membrane immunoglobulins (mlg) constitute the B cell antigen
receptors whose sites recognize native antigen. The structurally related T cell antigen
receptors differ in their binding of antigen. They recognize complexes of antigenic
1


determinants in conjunction with molecules encoded by the Major Histocompatibility
Complex (MHC). The uniqueness of the specificity of both B cells and T cells is
primarily determined by diversity in the antigen binding portion of the receptor2,3,4.
The capacity to respond precisely to the enormous possibilities of antigenic
structures, both natural and synthetic, is impressive and is accounted for by the clonal
selection theory which estimates that the precursors of the antibody-producing cells
of the immune system, the B lymphocytes, are capable of recognizing up to 1010
different possible specificities5. The theory states that those cells expressing
receptors most complementary to an antigen will be specifically selected6,7 and it
predicts that B cell variants with the "best fit" antigen receptors will be preferred in
subsequent selection by antigen, a process termed affinity maturation. Repeated
exposure to the same antigen can therefore result in an immune response that is
significantly superior to that which occurs after a primary exposure, a process termed
immunological memory1. The ability of an individual to survive depends directly on
this inherent capability to mount adequate memory responses to pathogens leading to
their removal, destruction or neutralization. We utilize these concepts in the design
of vaccines to prevent infectious diseases.
2


Both normal and pathological immune responses are the result of a complex
interplay among B cells, T cells and other cells of the immune system, such as
antigen-presenting cells. The ability of the immune system to recognize and eliminate
foreign antigens is governed by these cell populations. However, the specific
interactions that occur depend in large part on the type of response most useful for
eliminating the pathogen, i.e., whether the antigen is an intracellular agent such as a
virus or an extracellular one such as a bacterium. Whereas B cells recognize antigen
directly and, as a consequence, become activated to differentiate and produce specific
antibodies that bind to antigen and cause its clearance from the body, T cells
recognize antigen only in association with MHC molecules on the surface of other
cells. T cells can be divided into several subsets which respond to contact with their
appropriate antigen/MHC molecules with a variety of effector functions. For
purposes of this thesis, the subset of interest is the T helper cell (TH) population
which provide help for the production of antibodies. Their role in antibody
production is a result of a direct interaction with the B cells which initiates a cascade
of events that influences the amount, type and affinity of antibody produced (Figure
!)
3


Legend for Figure 1
B Cell /T Cell Collaboration
Simplified diagram of B cell and T cell collaboration. B cells recognize
discrete epitopes on the surface of the antigen via its membrane immunoglobulin
(mlg). Antigen is internalized and processed to epitopes which, in conjunction with
the Class II Major Histocompatability Complex (MHCII), are presented on the
surface of the B cell to T helper (Th) cells. The recognition occurs via the T cell
receptor complex (TCR) and other surface receptors. Once the TCR is engaged, the
T cell is activated to secrete lymphokines such as interleukin 2 (IL-2) and interleukin
4 (IL-4) which further acitvate the B cell resulting in immunoglobulin production.
4


Antigen Presentation
Encounter and
Activation
Antibody
Production
T-B
Collaboration
t


It is believed that antigen entering a host interacts initially with T and B cells
along two separate but parallel pathways. The B cell recognizes a defined portion of
the antigen molecule known as a B epitope via mlg without the need for prior
processing of the antigen. The B cell can become activated as a consequence of this
recognition. It is postulated that two types of signal are required to activate a B cell
resulting in the production of antibody. Recognition of antigen that initiates the
cross-linking of surface Ig receptors provides the first of these signals. For example,
efficient cross-linking of mlg is essential for intracellular calcium flux, one of the
best characterized events among several biochemical signaling pathways to the B
cell8,9. Once activated, the B cell internalizes the antigen and enzymatically processes
it to smaller peptides which can be presented by the B cell to other cells of the
immune system. In contrast, T cell epitopes must be processed by the antigen-
presenting cell and presented to the appropriate T cell in conjunction with another
cell surface molecule, Class IIMHC. These MHC molecules are those preferentially
recognized by the T cell population that subsequently helps the B cells differentiate
into antibody producing cells. At the cell surface, these complexes of peptide and
MHC class II are recognized by specific receptors on the appropriate TH cell that
recognize this particular epitope/MHC combination. As a consequence, the TH cell
then also becomes activated and a complex series of signaling events occur including
release of many soluble factors with multiple intermolecular interactions4 and
6


costimulatory signals10. One of the best studied factors is IL-2 which is secreted from
antigen specific CD4+ T cells activated by B cell presentation of processed antigen
fragments in conjunction with MHC Class II molecules. The close physical contact
of an antigen specific TH cell with an antigen presenting B cell allows the precise
delivery of this and other helper functions11. Thus, to produce antibody, the B cell
must receive one signal when antigen interacts and crosslinks mlg receptors, and a
second stimulating signal or signals from T cells. The premise is that an optimal
stimulatory response to any antigen involves both interactions.
After the B cell is activated upon a first encounter with antigen, it becomes
>
stimulated to proliferate and differentiate12,13 giving rise to populations of cells known
as plasma cells, the terminally differentiated B cell that secretes antibody of the same
specificity as that of the original B cell. For the primary encounter with antigen,
these antibodies are of the IgM isotype. In addition to producing plasma cells, B cells
can be stimulated to proliferate and differentiate along a different pathway to become
memory B cells which bear Ig receptors of higher affinity than those of their
predecessors. These long-lived memory B cells are responsible for responding to a
subsequent contact with the same antigen with a faster and stronger antibody
response, generally of the IgG, IgA or IgE isotype. This type of response leading to
immunologic memory is termed a T-dependent (TD) response and as the name
suggests, requires the helper function provided by T cells as described above.
7


Some B cells can be triggered by certain types of antigens in the relative
absence of T cell help. These antigens such as bacterial cell wall polysaccharides are
referred to as T-independent (TI)14 antigens and tend to have simple repetitive
structures. The immune response to T-dependent and T-independent antigens is
qualitatively different; TI antigens induce a response consisting of antibodies
primarily of the IgM isotype whether this is upon a primary or secondary encounter
with antigen. The level of the secondary response to these antigens resembles the
primary encounter with antigen in that it does not exceed it in magnitude or kinetics
and is short-lived. In contrast, the secondary response to a T-dependent antigen is far
stronger, appears earlier, more rapidly and to a larger extent than the primary
response to T independent antigens. Thus, it seems that T-dependent but not T-
independent antigens induce memory and maturation of the responses as
/
characterized by class switching to IgG, IgA or IgE antibodies with an increase in
affinity of antibodies for antigen. Therefore, vaccines that do not elicit T cell help for
B cells are of limited value since they will not result in a memory response.
Whereas the ability to elicit a memory response requires T cell helper
functions, the ability to be a strong and effective immunogen depends on physical and
chemical characteristics. Antigenicity depends on the state of aggregation of the
immunogen. It is well known that experimentally induced aggregation of protein
molecules by physical methods such as heat, adsorption to alum and emulsification
8


with Freund's adjuvant or by chemical methods (cross-linking with glutaraldehyde) or
alum15 greatly enhances their antigenicity. Presumably, one of the reasons for this
increased antigenicity is the increased molecular weight of the antigen. Protein
molecules made free from aggregates via centrifugation have been shown to be not
only non-immunogenic but also tolerogenic, whereas aggregated material with
presumed multiple antigenic sites per molecule produces an immune response16,17.
However, not all polymeric molecules of high MW with repeating arrays of identical
epitopes are able to elicit a memory response since for this to occur T cell helper
involvement is required.
The vast majority of soluble T-dependent antigens elicit only low level
antibody responses and no memory responses unless they are administered with an
adjuvant, a substance which non-specifically enhances the immune response to an
antigen. Adjuvants act by mechanisms such as I) formation of depots at the site of
inoculation which then slowly release antigen, ii) the focused presentation of antigen
to immunocompetent cells or iii) the stimulation of the production of different
lymphokines such as various interleukins to aid in immunostimulation. Adjuvants
also act to aggregate the antigen and form larger molecular weight, multivalent
constructs18. However, the use of adjuvants may be associated with several
disadvantages; they are often toxic, they may skew the antibody response to certain
isotypes which may have different effector functions from those produced in the
9


absence of adjuvants and they may alter the nature of the antigen so that epitopes
other than those recognized in the native antigen become immunodominant18'2'.
1.1 Current Vaccine Strategies
Despite the impressive success of vaccination in preventing diseases, there are
still a substantial number of diseases for which effective vaccines do not exist or for
which vaccination fails to offer adequate protection, especially for certain groups at
high risk. Pathogens often implicated in these types of diseases include Neisseria
meningitidis, Streptococcus pneumoniae, Salmonella typhi, Pseudomonas
aeruginosa, Escherichia coli, Klebsiella, Vibrio cholerae22. One clear characteristic
that distinguishes these pathogen groups is their ability to produce specific and
unique surface carbohydrate compounds. The surface polysaccharides, which include
capsular polysaccharides, may enhance the virulence of these organisms by acting as
protective surface molecules, though many of these capsular polysaccharides can be
used in vaccines and elicit protective antibodies in healthy adults. However, children
under 18 months and some elderly and immunocompromised individuals either fail to
produce antibodies after vaccination or produce antibodies in levels too low to be
protective. Furthermore, booster injections fail to elicit an enhanced immune
response in these groups of people. As the vaccines are composed of mainly
10


polysaccharides with repeating subunits, they are considered T-independent antigens
and do not produce a memory response. It would be desirable, however, to convert
these T-independent antigens to T-dependent antigens. Furthermore, malaria,
bacterial meningitis and infections from Pseudomonas aeruginosa and Neisseria
gonarrhoeae are especially dangerous due to increasing resistance of the pathogens to
chemotherapeutic agents and/or antibiotics. The need for vaccines to these pathogens
is also a high priority. Therefore, a clear need exists for numerous vaccines capable
of inducing the production of memory B cells and eliciting a specific antibody
response to many pathogenic invaders (reviewed by multiple authors in Science
1994)23.
In addition, though many diseases such as polio and tetanus can be
successfully prevented or treated with vaccines capable of producing protective
antibodies, these vaccines could be improved. Most vaccines currently in use consist
of attenuated or killed pathogens or complex mixtures of components derived from
them. More recent work on vaccine development carried out by numerous companies
and academic laboratories has been directed at producing simpler, better-defined and
more efficacious prophylactic and therapeutic vaccines. One widely used approach is
to define the relevant B and T epitopes of protein antigens of the pathogen of interest
and use peptides or small recombinant proteins comprising these epitopes as the
immunizing agent. Unfortunately, such molecules make poor immunogens and their
11


use has met with limited success.
One strategy designed to improve the efficacy of vaccines combined peptide
B and T cell epitopes into structures termed MAPS (multiple antigenic peptide
systems)24'26. MAPS have been used for linking of B cell and T cell epitopes in
various configurations in efforts to produce synthetic vaccines with multiple epitopes.
Through the use of MAPS, protection against a malaria sporozoite challenge has been
reported in murine models25 This has promoted the development of MAPS constructs
for human malaria26. Although MAPS constructs appear to have enhanced
immunogenicity over monomeric forms of the antigen, administration of these
constructs is always carried out in strong adjuvants such as Freunds adjuvant and in
many cases, only low level production of antibodies was elicited, multiple
immunizations were required and infants failed to mount a secondary response. Their
ability to stimulate responses when alum is used as an adjuvant depends on the
ability of the material to bind alum (personal communication Elisabeth Nardin,
NYU), suggesting that the MAPS-type construct is not inherently immunogenic. In
view of the Immunon Hypothesis (discussed in the following section 1.2) MAPS
constructs should be inherently suppressive.
Another approach used the non-immunogenic B cell epitope conjugated to
immunogenic carrier proteins such as tetanus, cholera toxin or diphtheria toxoid27'31.
Some of the problems with this type of approach include the reaction used in
12


coupling which may modify or destroy the epitope32,33 or alter its conformation34,35
Furthermore, suppression rather than stimulation of the immune response to the B cell
epitope36'38 has been documented using this approach. There is a potential problem of
hypersensitivity to the carrier protein and poor batch to batch reproducibility of the
conjugates. Also, depending on the construct, a considerable portion of the immune
response may be directed at the carrier molecule rather than the hapten target epitope
itself resulting in an ineffective vaccine26.
Some have tried conjugation of a hapten or peptide B epitopes to small
peptide T epitopes. This allows conjugation of non-immunogenic hapten or peptides
to small immunogenic carrier peptides thus creating artificial B-T epitope chimeras.
The chimeras have been made in various ways such as co-synthesis40, conjugation of
separate epitopes41, or co-polymerization42. The major problem reported with this
approach has been the production of altered/overlapping epitopes with changes in
specificity of the antibody response41,42.
An entirely different strategy examines the use of delivery vehicles such as
adjuvants or liposomes to enhance immunogenicity. These agents generally impart
immunogenicity as a result of their particulate nature and their ability to multivalently
array antigen on their surface. However, many of these systems require complex
preparative schemes and some have been found to be toxic43.
These are only a sampling of the strategies employed by vaccinologists to
13


improve the design of vaccines. The approach on which this work is based is a novel
interpretation of the work established by H.M. Dintzis and R. Z. Dintzis. Whereas
their work established the criteria necessary to establish a T-independent antibody
response to an antigen, this thesis has extended this work to a T-dependent, memory
response. A review of the Dintzis' work follows.
14


Legend for Figure 2
Antigen Specific Immunomodulation
It was proposed that a minimum number of surface immunoglobulin receptors
must be cross-linked in a cluster termed an IMMUNON, in order to trigger the B
cell. In order to elicit an antibody response, conjugates whould have both a minimun
threshold molecular weight of approximately 100,000-15000 Da and a minimun
substitution density of approximately 20. Conjugaes of subthreshold size or density
are considered not only non-immunogenic but suppressive. Subthreshold arrays are
postulated to act by competing for binding to surface immunglobulin receptors,
preventing suprathreshold arrays from cross-linking sufficient receptors to trigger the
cell.
15


mw m mv


1.2 The Immunon Hypothesis
Native antigens activate B cells by cross-linking mlg receptors on their
surface. While this concept is not a new one, it is chiefly quantitative rather than
quantitative. The Dintzis' sought to quantify the response by defining the parameters
necessary to make an ideal model antigen44
Their model proposed that when polymer molecules containing properly
spaced multiple copies of an epitope encounter the surface of a B lymphocyte which
bears many receptor molecules having sufficient specific affinity for that epitope,
multiple surface receptor molecules can simultaneously bind to epitopes on a single
polymer molecule. Further, the receptor molecules slgM or slgD are spatially
concentrated and brought closer together on the surface of the lymphocyte than they
could otherwise be. The model postulated that if enough receptors were concentrated
sufficiently for a long enough period of time, the cooperative formation of a complex
multi-protein structure, the IMMUNON, would occur (Figure 2). The formation of a
sufficient number of immunons on the cell surface was assumed to trigger a B
lymphocyte to begin the complex process of cellular division and differentiation
which would lead to the formation of antibody producing plasma cells.
Their model is consistent with the three experimentally based generalizations:
a) Soluble polymer molecules of molecular mass <100Kd cannot stimulate an
17


immune response to their epitopes. Since each cell surface receptor is a globular
protein of relatively large size, a stimulatory, soluble, polymer molecule able to
physically link a sufficient number of receptors simultaneously must have substantial
spatial characteristics of spread. In order to achieve this spatial spread, a stimulatory
molecule can be expected to need considerable mass which their experiments indicate
is in the range of lOOKd. Molecules with molecular mass < lOOKd are not large
enough to span and bind to a sufficient number of membrane receptors, b) Small
non-immunogenic polymers with molecular mass< lOOKd are competitive inhibitors
of large stimulatory polymers of the same chemical composition. This observation is
to be expected if such small molecules which cannot bind enough receptors to form a
signaling structure can nevertheless bind smaller numbers of receptors. They would
then be capable of "stealing" receptors from stimulatory polymer molecules thereby
interfering with the ability of stimulatory polymers to form immunons. These small,
non-immunogenic molecules can be viewed as competitive inhibitors which are
intrinsically inhibitory for the epitopes which they contain in multiple copies, c) At
the high end of the dose response curve, immunogenic soluble polymers are self
inhibitory. This observation is to be expected if immunogenic polymer molecules can
"steal" receptors from each other, i.e., equilibrate among themselves for bound
receptors. At sufficiently high polymer concentration, the ability of any one polymer
molecule to gather a stimulatory number of receptors is reduced by opposing
18


tendencies of other polymer molecules to bind receptors. This amounts to
competitive inhibition by like molecules. Therefore, a particular kind of
immunogenic polymer molecule can be either immunogenic or inhibitory depending
on its concentration.
Studies by the Dintzis' indicate the existence of a sharp threshold in the
immunogenic response elicited by various polymer preparations. In particular, all
polymers with <12-16 appropriately spaced hapten groups per molecule were non-
immunogenic at MW<100Kd, whereas those polymers with this number or more
were fully immunogenic, at MW>100Kd. These results indicated that the
immunological response at its most elementary level is quantized. In other words, a
minimum specific number of antigen receptors, approximately 12-16 for the work
reported, must be connected together as a spatially continuous cluster before an
immunogenic signal is delivered to the responding B cell44-47.
Though the physical and chemical requirements for inherent immunogenicty
were defined by the Dintzis', the application of the Dintzis technology has been
limited to homogeneous receptor conjugates wherein multiple, identical epitopes have
been arrayed on a polymer backbone. Activation was limited to B cells and the
concomitant production of IgM antibodies. We sought to extend this concept and
develop an inherently immunogenic vaccine which would be a T-dependent antigen
leading to the production of immunological memory.
19


1.3 Scope
This thesis focuses on a means for changing the quality of the immune
response in as much as the presence of the T cell epitope contained within an
inherently immunogenic conjugate can cause the antibody response to the B cell
epitope to switch from an IgM antibody response to an IgG, IgA or E response and
additionally elicit the induction of immunological memory and affinity maturation.
Because the conjugates are able to cause a switch in antibody class and induce the
production of memory cells, the constructs containing co-arrays of B and T epitopes
have great potential as vaccines.
The studies discussed here use a controlled number of B cell epitopes and T
cell epitopes both of which are conjugated to and simultaneously contained on inert
carriers of high molecular weight (> lOOKd), termed co-arrays. By controlled it is
meant that the number and spacing of B epitopes must be sufficient to deliver an
immunogenic signal to the responding B cell with this number being greater than 20.
In co-arraying B cell and T cell epitopes, an inherently immunogenic conjugate is
created which does not require the addition of adjuvant in order to elicit an antibody
response. For applications to the vaccine field, these epitopes were derived from
Plasmodium falciparum, Plasmodium berghei and the principle neutralizing (PND)
20


determinant of human immunodeficiency virus (HIV). Another hapten model,
fluorescein (FI), was also included in these studies.
21


2.
Materials and Methods
2.1 Chemistry
2.1.1 Preparation of Peptides
Peptides were synthesized by solid-phase peptide synthesis using an
automated peptide synthesizer (Milligen Bioresearch, Novato CA or Applied
Biosystems Inc., Foster City, CA)48 The amino acids used in these syntheses were
ordered from various standard suppliers. A-tert-Butyloxycarbonyl protection was
employed for all peptide syntheses, and finished peptides were cleaved from the
synthesis resin using standard hydrogen fluoride (HF) cleavage procedures49. Free
peptides were extracted from the resin, purified by preparative reversed-phase high
performance liquid chromatography (HPLC), and lyophilized. Amino acid analysis
of purified peptides was carried out via the WATERS PICO-TAG chemistry
following 22-24 hour vapor-phase hydrolysis with constantly boiling in 6M HC150.
Amino acid analyses were within +1-5% of the values predicted by the respective
22


peptide sequence.
2.2 Epitopes used in these Studies
2.2.1 Descriptive Information
The hapten, fluorescein (FI), has been extensively studied as a B cell epitope
using the Immunon Model by the Dintzis44-47, ourselves and colleagues at Cortech51.
Each of the peptides derived from either the circumsporozoite of
Plasmodifalciparum52, the causative agent of human malaria, or Plasmodium
berghei53,54, the causative agent of murine malaria, have also been extensively studied
by others for their B cell52'54 as well as T cell activity38,55'59. The T cell epitope
chosen to afford help for the antibody response to these epitopes was the 323-339
sequence of chicken ovalbumin (OA323.339). This epitope has also been shown to be a
strong T helper epitope by Shimonkevitz60. It has been studied, using the ASIM
technology, by ourselves51 and colleagues at Cortech, Inc.61. The Principal
Neutralizing Determinant (PND) epitope derived from the envelope glycoprotein,
gpl20, of human immunodeficiency virus (HIV) was also used in these studies. It
has been described as a B epitope62 and as a T helper epitope57 for cytotoxic T cell
(CTL) responses in mice.
23


2.2.2 Modifications of the Native Sequence:
In every case, the amino termini of the peptides were acetylated (Ac) and
peptides were synthesized as C-terminal amides using methods described by Stewart
and Young48. Most peptides were assumed to be derived from the internal sequence
of a protein; thus, modifying the peptide via these two methods made it appear to be
an internal peptide sequence rather than one derived from the actual -TV or -C
terminus of the protein48. Both modifications ultimately served to stabilize the
peptides64. Additionally, single cysteine residues were incorporated into the native
sequence for conjugation purposes.
2.2.3 The Epitopes
The P. falciparum epitope used in these studies had the sequence Ac-Cys-
(Asn-Ala-Asn-Pro)3-CONH2 and was conjugated to the various backbones via the TV-
terminal cysteine residue. It is referred to herein by its single letter amino acid code
(NANP)3. The P. berghei epitope had the sequence Ac-(Asp-Pro-Pro-Pro-Pro-Arg-
Pro-Arg)2Asp-Gly-Cys-CONH2 and was conjugated to the various backbones via the
C-terminal cysteine. It is referred to herein by its single letter amino acid code
(DPPPNPN)2DGC. The cOA(323.339) epitope had the sequence Ac-Cys-(ACA)-Glu-
24


Ala-323Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg339-
CONH2. It is referred to herein as OA(323_339). Amino acids not native to the OA(323_339)
peptide were included for the following reasons. In order to counteract a potential for
overall cationicity in the peptide, a charge-balancing glutamic acid residue was
included near the TV-terminus. Further, epsilon amino caproic acid (ACA) and
alanine were included as spacer moieties between the biologically relevant portion of
the peptide and the portion included purely for conjugation, i.e., the cysteine residue.
The peptide was conjugated to the various backbones via the iV-terminal cysteine.
Finally, the PND epitope used in these studies had the sequence Ac-Cys-Asn-Asn-
Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Thr-Ile-
Gly-Lys-Ile-Gly-CONH2 and was conjugated to the various backbones via the N-
terminal cysteine. It is referred to herein as the PND epitope. Blocked monomeric
versions of these peptides were prepared from the free thiol (-SH)-containing
monomer via carboxyamidomethylation65. Conjugates used throughout this work are
shown in Table 1.
25


Table 1
CONJUGATES
Note: The B epitope refers to the P. berghei sequence whereas the T epitope
refers to the cOA(323.339). The first number under B or T epitope substitution density
refers to the experiment perfromed at the Fort Collins facility while the second
number refers to the substitution density of conjugates used in experiments done at
Cortech. Copies of the B or T cell epitopes refer to the number of molecules of the
epitope per molecule of dex500.
Plasmodium berehei related compounds
Cortech # Carrier____________________ # B epitopes # T epitopes
CI-0676 none, iodoacetylated peptide
CI-0562 Bovine Serum Albumin(BSA) 27
0-0561/ CI-0561B deX500 351/342
0-0560/ CI-0560B dex5oo 323/375 126/172
0-0564 gelatin 16
26


Plasmodium falciparum related compounds
Cortech#________ Backbone_________________ Copies B epitope Copies T epitope
C-I0662 none, iodoacetylated peptide
CI-0496 BSA 32.9
CI-0499 711
CI-0500 dex,nn 273 202
CI-0502 gelatin 35
Plasmodium falciparum related compounds used for the varied T epitope substitution
density experiments.
Cortech#__________Backbone Copies B epitope Copies T epitope
CI-0576 dex500 281
CI-0577 ^ex5nn 285 8
CI-0578 dexsnn 286 34
CI-0579 dex,nn 270 45
CI-0580 ^X500 210 206
27


T epitope cOA. r? related compounds
C-0I454 none, iodoacetylated peptide

CI-0115 gelatin 32
Note: The B epitope now refers to the cOA in its capacity as a B epitope
FI related compounds (stimulation experiments')
Cortech#_______Backbone_______ Copies B epitope Copies T epitope
CI-0239 dexsn(1 40
C-I0241 dex,nn 40 150
CI-0387 BSA 34
Fl3 gelatin 3
CI-0422 gelatin 0.2
FI related compounds (suppression experiments')
Cortech#______ Backbone ___________Copies B epitope _________Copies T epitope
C-I0390 cOVA 5
CI-0323F dex7(1 54
CI-0460D dex70 . 21 41
28


2.3 Preparation and Maleimidation of High Molecular Weight Dexamine and
Subsequent Peptide Conjugation
Most of the dexamine used in these studies was kindly supplied by Drs. Howard and
Renee Dintzis, Johns Hopkins University, Baltimore, MD. Generally, suppliers of
dextran provide the material as crude, size fractionated preparation containing a
statistically normal distribution of molecular weights surrounding a mean molecular
weight. The Dintzis' purchased the dextran from Pharmacia while Cortech purchased
it from Sigma. An average molecular weight of 500Kd was desired for the
stimulation studies undertaken in the context of the current work. To obtain a dextran
of higher average molecular weight and, thus, with less contamination from low
molecular weight dexamines than would be present in unfractionated preparations,
high molecular weight dexamine fractions were further size fractionated via
chromatography on Superose 6 or 12 resin (Pharmacia).
The dexamine prepared at Cortech from size fractionated dextran was NOT carried
through any additional type of fine sizing procedures prior to its use. At the
dexamine stage of conjugate production, the product was purified via an ultrafiltrative
process that would just served to remove extremely low molecular weight compounds
from the dexamine, i.e. not dexamines of low MW, however. Dexamine was acylated
with a five-fold molar excess, relative to total amine content, of gamma malamide n-
29


butyric acid jV-hydroxysuccinimide ester in HEPES buffer, 0.2 M, pH=8. The
resulting gamma malamide n-butyryl GMB-dexamine was purified by repetitive
ultrafiltration using Phosphate-Buffered Saline (PBS) as the buffer medium. This
yielded the form of dextran capable of undergoing conjugation with the relevant
cysteine-containing peptides (Figure 3), and is referred to herein as dex500.
Subsequent covalent peptide conjugation was accomplished by addition of the desired
cysteine-containing peptide(s) to the maleimide-containing (GMB)-dexamine in PBS
via a conjugate addition process66 Conjugation reactions were terminated by the
addition of excess mercaptoethanol. Peptide-containing dextran conjugates were
purified by repetitive ultrafiltration, again using PBS. Lyophilization afforded the
conjugates as fluffy white powders. Assessment of peptide substitution density was
made by PICO-TAG amino acid analysis50 (see below). Peptide-gelatin (J.T Baker)
or peptide-carrier (Bovine serum albumin [BSA, Initial Fractionation by Heat Shock,
98-99% fatty acid free #A7030] or chicken ovalbumin [cOA, Grade V #A5503]
Sigma Chemical Company, ST. Louis, MO.) conjugates were prepared similarly.
30


Legend for Figure 3
Preparation and Analysis of Conjugates
SYNTHESIS: Peptide-dextran conjugates were produced from size fractionated
dextran. Dextran was derivatized with choloroacetic acid, ethylene diamine and
finally gamma-maleimido w-butyric acid jV-hydroxysuccinimide ester (GMBS) to
yield gamma-maleimido-nbutyryl dexamine (GMB-dexamine), the form of dextran
capable of undergoing conjugation with the relevant cysteine containing peptides.
For fluorescein (FI) conjugates, the linkage was of the thiourea type in which the
dexamine was modified directly with FI. The resulting compound was the acetylated
with GMBS to incoporate the maleimide groups. The conugates containing only FI
were then quenched with mercaptoethoanl whereas the conjugaes containing FI and
peptide were quenched following the conjugation of the peptide.
ANALYSIS: Acid hydrolysis of the conjugate (6M, Hcl, 110C) produced
ethylenediamine, gamma-aminobutyric acid (GABA, an indicator of dextran
recovery), S-2(2R,2S-succinyl)-L-cysteine (an indicator of overall covalent
attachment between peptide and dextran) and the peptide component amino acids.
Quantitation of the amount of covalently-linked peptide was possible via amino acid
analysis of the liberated residues. Dextan recovery was assessed via gamma-
aminobutyric acid quantitation. Peptide substituion density was thus established. For
the FI conjugates, comparisions were dranw between the theoretical recovery of
GABA and the GABA actually recovered. The difference between the two was an
indication of the number of dextran amino groups occupied by the FI, and thus the
copy number of FI per dextran molecule.
31


32


2.4 Fluoresceination and Maleimidation of High Molecular Weight Dexamine
and Subsequent Peptide Conjugation
Dex500 was acylated with 0.3 equivalents, relative to total amine content, of
fluorescein isothiocyanate (FI, isomer 1 Aldrich, Milwaukee, WI) in carbonate buffer,
0.2M, pH 9. The resulting fluorescein-containing dex500( Fl-dex500) was purified by
repetitive ultrafiltration using PBS as the buffer medium, then acylated with a five-
fold molar excess (relative to total amine content) of gamma malamide n-butyric acid
vV-hydroxysuccinimide ester, and purified by size exclusion chromatography on
Sephadex G-25 resin using PBS as the eluent. Subsequent conjugation (Figure 3)
was accomplished by addition of either the desired cysteine-containing peptide, i.e.,
the cOA(323_339) peptide, and then mercaptoethanol OR by addition of mercaptoethanol
alone. Fluorescein-peptide containing dextran conjugates were purified by dialysis
using PBS as the buffer medium. Lyophilization afforded the conjugates as fluffy
orange powders. Assessment of fluorescein and/or peptide substitution densities
were made by PICO-TAG amino acid analysis. Fluorescein-gelatin or fluorescein-
carrier conjugates were prepared similarly.
33


2.5 Amino Acid Analysis and Substitution Density Determinations
Acid hydrolysis of the conjugate (6M, HC1,110C) produced the following: 1)
ethylenediamine, 2) gamma aminobutyric acid (GABA), an indicator of dextran
recovery, 3) S-2(2R,2S-succinyl)-L-cysteine, an indicator of covalent attachment
between peptide and dextran, 4) the peptide component amino acids. Quantification
of the amount of covalently-linked peptide was possible via amino acid analysis
(PICO-TAG) of the liberated residues (Figure 3). Dextran recovery was assessed via
gamma-amino butyric acid (GABA) quantification. Peptide substitution density was
thus established. For the fluorescein conjugates, comparisons were drawn between
the theoretical recovery of GABA and the GABA actually recovered. The difference
between the two was an indication of the number of dexamino groups occupied by
fluorescein and thus the copy number of fluorescein molecules per dextran molecule.
Figure 1 shows the composition of all peptides and conjugates used in these studies.
34


2.6 Animals
Balb/c (H-2d) or C57/B16 (H-2b) or A/J (H^) female mice, 6-8 weeks of age,
(Jackson Laboratories, Bar Harbor, Maine) were used for these experiments. Since
the literature is replete with examples of how latent and unsuspected viral infections
may complicate research, as summarized by Hamm et al67, it was critical that animals
purchased were anti-viral antibody free. Jackson Laboratories routinely screen
animals for Sendai virus (SEN), Pneumonia virus (PVM), Mouse Hepatitis virus
(MHV),Minute virus of mice (MVM),Mouse polio virus (GD-7),Reovirus type 3
(REO-3), and Mycoplasma pulmonis (MPUL), monthly, and for Lymphocytic
choriomeningitis virus (LCMV), Mouse adenovirus F1/K87 (MAD), Ectromelia virus
(ECTRO), Mouse pneumonitis virus (K), Poloma virus (POLY), quarterly and for
Epizootic diarrhea of infant mice virus (EDIM),Mouse cytomegalovirus (MCMV),
Mouse thymic virus (MTLV),Hantaan virus (HANT), Encephalitozoon cuniculi
(ECUN), Cilia-Associated Respiratory Bacillus (CARB) and Bacillus piliformis
(BPIL), annually68 Additionally, animals were tested, in house, for Sendai virus,
Mouse Hepatitis Virus and Mycoplasma by the Murine Immuno-COMB (Charles
Rivers Laboratories, Wilmington, MA.). All animal work was approved by the
Cortech Animal Care and Use Committee which strictly adheres to United States
Department of Agriculture (USD A) Guidelines.
35


2.7 Preparation of Adjuvant
Adjuvanted immunizations (to specifically direct the production of antibody) were
performed using aluminum hydroxide18 Al(OH)3, as the source of adjuvant. This was
either a purchased source of Al(OH)3, REHYDRAGEL (Reheis, Inc., Berkeley
Heights, N. J.) or a preparation of Al(OH)3 made in-house by the following method69.
To prepare the adjuvant, approximately 100 ml of 1M Aluminum Potassium Sulfate
Dodecahydrate, A1K(S04)2.12H20 was added dropwise to 600 ml of lM'Tris-Base.
The pH of the solution was monitored during the addition until a pH of 8.5-8.7 was
achieved indicating the endpoint of the reaction. The precipitate was washed five
times in a 0.0075M NaCl, 0.005 M Tris-Cl buffer by centrifugation at 4C, 1250-
2500 x g. The solution was lyophilized, weighed and the Al(OH)3 adjusted to a 30
mg/ml concentration in the NaCl/Tris-Cl buffer described above. The final product
was stored at room temperature until use.
2.8 Immunizations for Antibody Production
All conjugates were injected intraperitoneally, i.p., as a 0.5 ml volume per mouse
with or without 1 mg of Al(OH)3 using PBS as the vehicle. To absorb the antigen
onto Al(OH)370, the appropriate weight of Al(OH)3 was dispensed and brought to
36


three times its volume with PBS. In a separate test tube, the appropriate quantity of
antigen was brought to the same volume as the Al(OH)3. The Al(OH)3 was placed on
ice and antigen was added, slowly and dropwise (2 ml/minute) to the slurry, while
sonicating with a probe sonicator at 20 kHz (Ultrasonic Homogenizer, 4710 Series,
Cole-Parmer Instrument CO., Chicago IL.). Importantly, after sonication, the
suspension was washed with PBS by centrifugation, three times at 1250-2500 x g for
10 minutes at 4C to remove antigen that did not absorb to the adjuvant.
Subsequently, the adjuvanted antigen preparation was resuspended in PBS to the
appropriate volume for immunizations. (Note: lyophilized antigens or conjugates
were, generally, rehydrated in PBS at a concentration of 1 mg/ml. Some aliquots of
antigens were stored at -20C until use. However, samples were never subjected to
multiple freeze-thaw cycles). Serum samples, obtained at regular intervals, were
collected from anaesthetized mice via retro-orbital plexus bleeds and were stored at -
70C until use.
37


2.9 Measurement of Antibody Titers
2.9.1 Solid-Phase ELISA
Antigen-specific antibody titers were measured by solid phase ELISA (Enzyme Linked
Immuno-Sorbent Assay)71,72. Immulon II microtiter plates (Dynatech Laboratories,
Chantilly, VA) were coated overnight at 4C with 0.1 /ig/well (as fxg of carrier molecule,
i.e. gelatin, dextran) of the appropriate readout antigen in PBS. Generally, the readout
antigen consisted of the epitope conjugated to gelatin. The following day, unbound
antigen was removed from the wells by manually flicking off the antigen solution.
Non-specific sites on the wells were blocked with 200 fA of PBS buffer, containing 0.1%
gelatin (A: From Porcine Skin, approx. 300 Bloom, Sigma) Chemical Co., St. Louis,
MO.) and 0.01% thimerosal, for one hour at ambient temperature. Plates were then
washed three times with a PBS buffer containing 0.01% thimerosal and 5 ml/L Tween-
20 (V/V) using a DYNATECH automated plate washer.
Test sera were thawed at ambient temperature and diluted into a PBS buffer containing
0.5% bovine gamma globulins (Cohn Fraction Type II,III, Sigma) and 0.1% gelatin
with 5 ml/L (V/V) Tween-20, referred to herein as Serum Diluent. Subsequently, 100 /A
of diluted sera were added to the microtiter plates and incubated for two hours at
ambient temperature.
38


The plates were again washed three times, as described above, after which 100 /A of
enzyme conjugated antibody was added This antibody was either Goat anti-Murine
IgG or IgM (heavy chain specific) horseradish peroxidase labeled (Kirkegaard and Perry
Laboratories, Gaithersburg, MD) at a concentration of 0.5 Atg/ml in the Serum Diluent
Buffer. For those ELISA's investigating the IgG subclass specific antibodies the enzyme
conjugated antibody was purchased from Fisher Laboratories and used at a concentration
of 1 //g/ml. The enzyme conjugated antibody was allowed to incubate at ambient
temperature for 1 1/2 hours at which time the plates were washed as described above.
Two additional manual washes were done with PBS buffer alone. Finally, 100 /A of the
substrate consisting of McClivain's Buffer (0.084 M Na2HP04, 0.04788M citric acid),
0.1% 2,2'-Azino-6w- (3-Ethylbenz-Thiazoline-6-Sulfonic Acid, ABTS, Sigma), and 1.65
x lO^M H202 was added to the microtiter plates. Plates were incubated at ambient
temperature for 30 minutes after which the amount of ABTS product was measured with
a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA.) at 405nm. Data
are expressed as Optical Density (O.D.). Backgrounds, defined as the O.D. produced by
binding of peroxidase-labeled antibody only, were subtracted from individual samples.
39


2.9.2 Competition ELISA
For the competition ELISA, in which sera were tested for their ability to recognize
antigen in solution phase72 titers of the sera were first determined by solid phase
ELISA as described above. The dilution of sera corresponding to an O.D. within the
linear range of the assay were then used in the competition assay. The competition
ELISA was performed as described for the solid phase ELISA with the following
exception.
Sera were added to 12 x 75 mm borosillicate glass test tubes with various
concentrations of competing antigen in solution (Serum Diluent) and incubated for
one hour at ambient temperature with intermittent vortexing before being added to the
microtiter wells. The ability of the antigen in solution to compete for or to inhibit the
binding of sera to solid phase antigen was expressed as follows:
Percent Inhibition of Binding=
(O.D. achieved in the presence of competing antigen/ O.D. achieved with no antigen) X 100.
40


2.10 In Vitro Peptide Stability Studies
Peptides from the current stimulation studies as well as from other antigen-specific
immunomuodulation (ASIM) studies were analyzed in an in vitro peptide stability
assay. Individual peptides in PBS at ImM were placed in separate 1.5 ml
polypropylene tubes and stored on ice until use. Trichloroacetic acid (TCA) or
trifluoroacetic acid (TFA), 10% in water, served as a protein precipitation solution.
Acetonitrile, water, TFA and TCA were all purchased as HPLC-grade reagents
(Waters/Millipore, Milford, PA).
Whole blood was collected from Balb/c female mice into tubes containing sodium
heparin. Blood was allowed to clot for 1 hour on ice at which time tubes were spun
in an IEC Centra08 refrigerated centrifuge (750 x g, 10 minutes, 4C). Plasma
(supernatant) was removed by aspiration, aliquoted and stored at -20C. Plasma was
thawed once at the time of use but was not subjected to multiple freeze-thaw cycles.
Although general enzyme activity appears to be retained upon freezing as indicated
by activity and degradation of historic, control peptides (personal communication,
Sherman Ross, Cortech, Inc.) plasma was not subjected to multiple freeze-thaw
cycles since loss of enzyme activity over time was anticipated. For the assay, plasma
was thawed to ambient temperature and stored on ice until use.
41


Ten microliters of each peptide sample (ImM) plus 90 fA of plasma were placed into
1.5 ml polypropylene tubes and incubated for various lengths of time. At each time
point, an aliquot (100 iA) of protein precipitation73 solution was added. After
vigorous mixing tubes were centrifuged in an Ependorf 5415C centrifuge (17,500 x
g, 10-15 minutes). 150 [A aliquots were analyzed with reverse-phase HPLC (Waters)
using a 3.6 72% CH3CN gradient in 0.1% TFA on a Vydac C]g column (4.6 x
150mm, 5u, 300A). Peak integration and processing was performed using Baseline
Model 810 application software (Millipore). Sample stability (t1/2) was determined by
analyzing data using ENZFITTER Regression application software (Elsevier, UK).
2.11 Pyrogenicity Testing
For one batch of the P. berghei compounds, sufficient compound was available for
pyrogenicity testing. Test samples were sent as lyophilized material to The
Biological Test Center, Irvine CA. for testing in the USP Rabbit Pyrogen testing.
This is a validated procedure for the detection of endotoxin. Rabbits were injected
subcutaneously (s.c.) with doses of the conjugates using pyrogen-free conditions.
Rectal temperatures were recorded hourly If temperature increases of less than
3.3C were obtained for the cumulative rise in temperature for three rabbits, a sample
was deemed to be non-pyrogenic. Data were expressed as pass or fail74 of this test.
42


2.12 In Vitro Infectivity Testing of P. falciparum
Pools of sera which contained anti-(NANP)3 antibodies as well as sera containing
antibodies specific for an unrelated epitope were sent to Dr. Michael Hollingdale
(Biomedical Research Institute, Bethesda, MD) who kindly performed the P.
falciparum in vitro infectivity assay75,76 In this assay, a hepatocyte cell line was first
incubated with the sera and then challenged with live sporozoites. The sera were
evaluated for their ability to prevent infection of the hepatocytes by the live
sporozoites.
2.13 In vitro T cell Proliferation Assays (Lymph Node Proliferation Assay)
The lymph node proliferation assay was performed as previously described by
Corradin et al77 Antigens were emulsified 1:1 in Complete Freund's Adjuvant (CFA,
Difco Laboratories, Detroit MI). 100 //g of emulsified antigen was injected
subcutaneously at two sites at the base of the tail in a total emulsion volume of 100
/A. Ten days later, inguinal and periaortic lymph node (LN) were used as a source of
cells for the proliferation assay. They were removed aseptically from animals
sacrificed by cervical dislocation and placed in Hank's Balanced Salt Solution
(HBSS, Bio Whittaker, Walkersville, MD.). A single cell suspension was achieved
43


by gently homogenizing the cells manually with a standard glass homogenizer. Cells
were washed twice in Click's Medium (Irvine Scientific, Santa Ana, CA.), containing
0.5% Normal Mouse Serum (NMS, Pel-Freeze Laboratories, Rogers, AR.), 500
units/ml penicillin-streptomycin (BioWhittaker), 2mM 1-glutamine (BioWhittaker)
and 5 x 105M2-mercaptoethanol. Cells were resuspended to 4 x 106 cells/ml in the
same buffer. Two hundred A of cells and 20 A of three-fold dilutions of antigen,
pre-filtered through a 0.2 // filter, were added to flat-bottomed microtiter plates
(Coming Laboratories, Coming, N.Y.). Cells were incubated at 31C, 5%C02, in a
humidified incubator for four days. Wells were pulsed with one uCi per well of
thymidine, [Methyl-3H] (3HTdr), ICN Biochemicals, Irvine, CA.) and cells were
incubated for a further 16-18 hours DNA was harvested onto glass fiber (A) filter
mats (Wallac Inc., Gaithersburg, MD.) with a Tomtec cell harvester. Filters were
allowed to dry and then were placed into scintillation bags with 4-5 ml of Beta Plate
scintillation fluid (Wallac, Inc.). Radiometric measurements were made with a
Microbeta Counter (Wallac, Inc.). All determinations were made in triplicate. Data
are expressed as a stimulation index (S.I.)
S.I.= cpm test sample/ cpm cells alone
44


3. Presentation and Discussion of Results
3.1 Hapten B Epitope (FI), Peptide T Epitope Co-arrays
As discussed in the introduction, the Dintzis' originally defined the Immunon in terms
of the size and valence of conjugates which lead to the production of an IgM antibody
response44. Whereas they focused on the response for hapten model systems and on
the primary immune response for T-independent antigens, this thesis focuses on
producing a T-dependent, memory response.
The first study to determine whether the presence of a T cell epitope in a co-array
with a B cell epitope could switch the antibody response to IgG, with concomitant
development of memory, used the small organic hapten, fluorescein, as the B cell
epitope. The primary response to this and other hapten epitopes was first documented
by Dintzis et al.45"47 whereas the memory response was first studied by Coeshott78.
The T cell epitope chosen was a tryptic product of chicken ovalbumin, cOA(323.
339), which is well documented in the literature as a potent T cell epitope in mice60
regardless of the strain of mouse in which the TH function was examined (personal
communication Wegman and Coeshott). The scaffold chosen for attachment of the
45


epitopes was dextran for all studies herein. Constructs consisted of either the hapten
conjugated alone to dextran or the hapten and the synthetic peptide co-conjugated to
the same dextran molecule.
Groups of Balb/c female mice, n=8, were immunized i.p. with either the FI
conjugated to dex500 at a substitution density of 40 copies per molecule of dextran
(Fl40-dex500) or with a construct containing both FI and cOA(323-339) also conjugated to
dex500, referred to herein as the co-array. For the co-array, the FI was present at a
substitution density of 40 copies per dextran molecule whereas the cOA(323.339) was
present at a substitution density of 150 copies per dextran molecule ([Fl40 +
peptide150]-dex500). Six weeks later half of each group was boosted with Fl-dex500
whereas the other half received the co-array. Importantly, no adjuvant was used for
immunizations with the conjugates though an adjuvanted Fl-BSA was included as a
control. Sera were obtained at various time points after immunization. Levels of IgM
and IgG antibodies to FI or cOA{323_339) were assayed.
46


3.1.1 Antibody Response to the B cell (FI) Epitope
In mice receiving the primary immunization with the Fl-dex500, IgM antibody
responses peaked after 7 days and persisted at moderate levels for six weeks. The
dose did not appear to affect the response (Figure 4). In the situation where a second
dose of Fl-dex500 was administered to these mice, the IgM persisted but did not
exceed the pre-boost levels (Figure 4). Low levels of IgG antibodies to FI were
detected after the primary immunization with the Fl-dexsoo conjugates. As shown in
Figure 5, titers peaked 21 days after immunization but did not reach the levels
attained when animals were immunized with the co-array (Figure 6). In contrast to
the response to Fl-dex500, a second immunization with the co-array increased the anti-
FI IgG response (Figure 5). However, this response did not have the profile of a
memory response but instead was comparable to the response obtained after a
primary immunization with the co-array (Figure 6) (The IgG response to the Fl-dex500
will be discussed further at the end of this section). Therefore, it appeared that this
antigen, Fl-dex500, did not cause the induction of memory B cells which would have
given rise to a secondary response consisting of IgG antibodies.
In mice receiving the primary immunization with [Fl+peptide]-dex500, IgM responses
also peaked at 7 days and persisted at moderate levels for six weeks. The dose again
47


did not appear to affect the response (Figure 7). In contrast to animals which received
the Fl-dex500 animals which received the co-array developed a significant and
considerable IgG response to the FI epitope after the primary immunization (Figure
6). This response peaked twenty-one days after immunization followed by a waning
of titers over time, a profile typical of a primary response. Here, a dose response was
evident; the response to the 10 //g dose was larger than that to the 100 ^g dose
(Figure 6). It has been suggested that large doses of conjugate could be inhibitory
rather than stimulatory due to insufficient clustering of receptors for Immunon
formation46. In the extreme situation for a large dose of conjugate, one epitope on
each dextran molecule could interact with an individual receptor thereby preventing
formation of the Immunon. With a lower dose, multiple epitopes arrayed on the same
backbone would interact with the receptors on the B cell resulting in the formation of
more clusters, or Immunons, capable of causing an antibody response.
In the situation where mice received the primary immunization with the co-array and
the boost with Fl-dex500, IgG titers detected in the primary response continued to
wane over time and a secondary IgG response did not occur (Figure 6). IgM titers
also did not increase after the boost (Figure 7). In contrast, when the mice were both
immunized and boosted with the co-array containing the T cell epitope ([Fl+peptide]-
dex500), animals which received 100 /xg of the co-array for the primary immunization
and 10 /j,g for the second immunization showed a large and rapid increase in titers to
48


the FI epitope indicative of a secondary memory response (Figure 6). Though the
titers for animals that received 10 yUg of the co-array for both immunizations also
increased after the boost, they did not dramatically exceed the pre-boost levels.
Thus the addition of the T cell epitope to the hapten array caused the switch in
isotype from IgM to IgG and the secondary response had some of the hallmarks of a
memory response in the absence of adjuvant. It is assumed that the IgG antibodies in
the secondary response would be of higher affinity than those produced in the
primary response since affinity maturation would have taken place as a consequence
of memory development. To date, this has not been measured, however.
3.1.2 Antibody Response to the T cell cOA Epitope
The antibody response to the T cell epitope was also monitored. For the primary
response, IgM titers to this epitope were low when the animals were immunized with
the co-array (Figure 8) whereas no antibody response was detectable when animals
were immunized with Fl-dex500 as expected (data not shown). The only IgG response
detectable after the primary immunization with the co-array was in animals
immunized with the 100 pg dose (Figure 9). After the secondary immunization with
the co-array, the IgG response to the T cell epitope was boosted although the
response exhibited by animals immunized with the 10 yug dose was approximately
49


two-fold higher than the response for animals immunized with the 100 //g dose.
Animals that received their primary immunization with Fl-dex500 but were boosted
with the co-array also had an IgG response to the T cell epitope (Figure9). It was
interesting that immunization with the cOA(323.339) epitope on the co-array resulted in
only low levels of IgM production to itself. The ability of this epitope to direct the
immune response immediately to IgG antibody production has been noted
previously78. Since the cOA epitope was available as its tryptic cleavage product, it
presumably required no further processing before presentation. Therefore, the result
was the immediate production of IgG antibodies to the epitope.
Thus, in addition to causing the switch in isotype from IgM to IgG for the hapten
epitope, the T cell epitope was able to produce an IgG response to itself with the
secondary response having some of the hallmarks of a memory response in the
absence of adjuvant. Again, it is assumed that the IgG antibodies in the secondary
response would be of higher affinity than those produced in the primary response
since affinity maturation would have taken place as a consequence of memory
development. To date, this has not been measured, however.
50


Legend for Figures 4-10
Group# Primary Immunization Secondary Immunization
A 1 Fl-dex500,10 fj.g Fl-dex500,10 /xg
2 Fl-dex,nn,10 Atg [FI + peptide ]dex,nn,10 /xg
V 3 Fl-dexsoo,100 /xg Fl-dex500,10 /xg
T 4 Fl-dex O 5 [FI + peptide ]dexsnn,10 /xg Fl-dexsnn,10 /^g
6 fFl + peptide ]dexsnft,10 /xg [FI + peptide ]dex5nn,10 /xg
7 [FI + peptide]dex5nn,100 fxg Fl-dexsnn,10 /xg
8 [FI + peptide]dex500,100 fxg [FI + peptide] dex500,10 /xg
Balb/c mice were immunized on day 0 and boosted 62 days after the
primary immunization with the conjugates as described above. Antibody titers to
the FI or the cOA epitopes were measured by ELISA. Sera were diluted 1:300 for
measurement of IgM and 1:900 for measurement of IgG Doses for injection were
based on the weight of the dextran molecule. When the dose was expressed as
mmoles of the B or T cell epitope mice received 8 x 107 mmoles of the FI epitope
for the 100 /xg dose and 8 x 108 mmoles for the 10 fu.g dose. For the T cell epitope,
animals received 2.8 x 10'6 mmoles for the 100 /xg dose and 2.8 x 1 O'7 mmoles for
the 10 dose. See Table 1 for the description of the conjugates and their
substitution densities.
51


OPTICAL DENSITY
FIGURE 4
IgM Antibody Response to FI: Primary immunization Fl-dexggg
0 10 20 30 40 50 60 70 80 90 100
TIME (DAYS)
Primary immunization Fl-dex^gg 10ug (triangles) or 100ug (inverted triangles).
Boost 10 ug FI-dex50Q (open figures) or 10 ug [FI + peptide]-dex5g0 (closed figures).
52


OPTICAL DENSITY
FIGURE 5
IgG Antibody Response to FI: Primary Immunization Fl-dexggg
Primary immunization FI-dex50Q 10 ug (triangles) or 100 ug (inverted triangles).
Boost 10 ug Fl-dex500 (open figures) or 10 ug [FI + peptide]-dex500 (closed figures).
53


OPTICAL DENSITY
FIGURE 6
IgG Antibody Response to FI: Primary Immunization [FI + peptide]-dex500
Primary immunization [FI + peptide]-dex50Q 10 ug (circles) or 100ug (squares).
Boost 10 ug FI-dexgQQ (open figures) or 10 ug [FI + peptidej-dexggg (closed figures).
54


OPTICAL DENSITY
FIGURE 7
IgM Antibody Response to FI: Primary Immunization [FI + peptide]-dex50g
TIME (DAYS)
Primary immunization [FI + peptide]-dex500 10ug (circles) or 100 ug (squares).
Boost 10 ug FI-dex5QQ (open figures) or 10 ug [FI + peptide]-dex500 (closed figures).
55


OPTICAL DENSITY
FIGURE 8
IgM Antibody Response to the cOA (323-339) epitope:
Primary lmmunization:[FI + peptideJ-dexgQQ
TIME (DAYS)
Primary immunization [FI+ peptide]-dex500 10 ug (circles) or 100 ug (squares).
Boost 10 ug FI-dex5Q0 (open figures) or 10 ug [FI + peptide]-dex500 (closed figures).
56


OPTICAL DENSITY
FIGURE 9
IgG Antibody Response to the £04^323.339) epitope
0 10 20 30 40 50 60 70 80 90 100110
TIME( DAYS)
Primary immunization Fl-dex500 10 ug (triangles) or 100 ug (inverted triangles).
Primary immunization [FI + peptide]-dex500 10 ug (circles) or 100 ug (squares).
Boost 10 ug FI-dex5Q0 (open figures) or 10 ug [FI + peptide]-dex500(closed figures)
57


FIGURE 10
IgG Antibody Response to FI
High Substitution Density Readout Antigen
TIME( DAYS)
Primary immunization Fl-dex500 10 ug (triangles) or 100 ug (inverted triangles)
Primary immunization! FI+ peptide]-dex500 10 ug (circles) or 100 ug (squares)
Boost 10 ug Fl-dex50g( open figures) or 10 ug [FI+ peptide]-dex5g0(closed figures)
58


3.1.3 IgG Antibody Response to Conjugates Lacking the T cell Epitope
As mentioned above, some low levels of IgG antibodies to FI could be detected in
animals receiving Fl-dex500 (Figure 5). This would seem to contradict the hypothesis
that the T cell epitope in the co-array with the B cell epitope was required to achieve
an IgG response. However, these antibodies were only detectable when the assays
described above were setup in a particular fashion, i.e. using a densely substituted Fl-
gelatin as a readout antigen in ELISA (Figure 5).
One possibility for this result is that using high substitution density readout
antigen presumably favors the detection of a mixed population of both high and low
affinity antibodies whereas low substitution density antigen favors detection of the
higher affinity antibodies in the population72,79. A primary antibody response consists
of a mixed population of predominantly low affinity antibodies. This antibody
population undergoes affinity maturation upon additional challenge with antigen13
When these sera were tested using a low substitution density Fl-gelatin conjugate as a
readout antigen, only those animals immunized with the co-arrays had IgG titers to
the FI. This result suggests the IgG antibodies obtained with the Fl-dex500 conjugate
were of low affinity (Figure 10).
59


Another explanation for the appearance of IgG antibodies in animals
immunized with Fl-dex500 was a possible preference for a particular IgG subclass
distribution in these animals. When these sera were tested on high substitution
density readout antigen, the subclass distribution of the IgG in the primary response
was found to be IgGl or IgG3 only with no contribution from IgG2aor IgG2b (Figure
11). IgG3 is known to be produced in a T cell independent fashion perhaps induced
as a result of cytokine production by non-T cells80. The explanation for the presence
of IgGl was not readily apparent. However, it was also possible that environmental
stimulation of the mice had occurred by some unknown T dependent antigen which
raised antibodies cross-reactive with FI. When these sera were tested for the IgG
isotype distribution of anti-FI antibodies using low substitution density readout
antigen to overcome the potential problem of low affinity non-specific antibody
binding, a different result was obtained. Under these conditions IgG was
predominantly detectable only in those animals receiving the co-array and this IgG
was of the IgGl isotype (Figure 11). These data do not conflict with the hypothesis
that a T cell epitope is necessary to achieve an IgG memory response.
60


Legend for Figure 11
ISOTYPES
Sera from animals 14 days after primary immunization with either Fl-dex500 or
the [FI + peptide]-dex500 were obtained and tested by ELISA for the presence of IgG
subclass specific antibodies at 1:500 dilution. The readout antigen was either a highly
substituted Fl-gelatin (3-8 copies of FI per gelatin molecule) or a low substition density
Fl-gelatin (0.02 copies of FI per gelatin molecule). See Table 1 for a description of the
conjugates and their substitution densities.
61


FIGURE 11
ISOTYPES
Os
tO
4.00
3.75
3.50
3.25
3.00
b 2.75
^ 2.50
g 2.25
2.00
g 1.75
F 1.50
O 1-25
1.00
0.75
0.50
0.25
0.00
0 1 ca XI CO
u> 0 CM CM 0
J? 0 0 O)
"(3 o H ) O)
Isotype-Specific, HRP-labelled Antibody
] FI-dexgQQ 0.02 copies FI per gelatin
Co-array, 0.02 copies FI per gelatin
liiiil FI-dex5Qg 3 copies FI per gelatin
llllllllllll Co-array 3 copies FI per gelatin


3.1.4 Summary of Section 3.1
As hypothesized, the addition of the cOA T cell epitope resulted in IgG
antibody production to both the FI hapten epitope and to itself, in the absence of
adjuvant.
63


3.2 Peptide B Epitope,(NANP)3, Peptide T Epitope (cOA323_339) Co-array:
Part I
The co-array concept was shown to be efficacious with a hapten B cell epitope
system. To test the co-arrays in a system that would have more relevance to the
vaccine field, co-arrays were prepared using a B cell epitope from a known pathogen,
the (NANP)3 repeat sequence from the circumsporozoite protein (CSP) of the human
malarial parasite, Plasmodium falciparum29,81. Anti-sera containing antibodies
specific for this epitope have been shown to confer protection against both in vitro
and in vivo challenge with viable parasite29,81.
The literature also reports that the circumsporozoite protein can be recognized
as a T cell epitope in H-2b mice38,55"59. To test the ability of the (NANP)3 peptide to
provide T cell helper function for itself, C57B1/6, H-2b mice and Balb/c, H-2d mice
were used in these studies. This strategy permitted testing of an alternative T cell
epitope in the co-array as well as the ability of two peptide T cell epitopes in a co-
array to induce antibody production to either themselves or to each other.
The peptide comprising the B cell epitope had the following amino acid
sequence: C(NANP)3 and thus was conjugated via its TV-terminal cysteine residue to
the dex500 backbone. The T epitope chosen was once again the OA(323.339) peptide.
The (NANP)3 epitope was conjugated alone or with the cOA T cell epitope on dex500.
64


The substitution densities on the co-array were 203 copies of (NANP)3 and 273
copies of cOA peptide per dex500 whereas the (NANP)3 epitope when arrayed alone
was present in 711 copies per dex500 molecule.
Groups of Balb/c or C57/B16 female mice (n=8) were immunized i.p. with 10
Hg of the co-array or with either of two doses: 3.3 peg or 10 /ug, of the (NANP)3
epitope arrayed alone on dex500. Additionally, 10 /ig of a control immunogen
(NANP)3-BSA and the co-array were administered with 1 mg Al(OH)3 as adjuvant.
Doses administered were based on the weight of the dextran (or control, BSA)
backbone. Animals received a second injection of the same construct 35 days after
the primary injection. Sera were obtained a various times and were assayed at 1:100
dilution for the presence of IgG anti-(NANP)3 or anti-cOA(323.339) antibodies by
ELISA using the epitope conjugated to gelatin as the readout antigen.
3.2.1 (NANP)3 Antibody Response in H-2d mice
In the Balb/c mice when the adjuvanted (NANP)3-BSA was used as the
immunogen, the IgG response to (NANP)3 was poor for the primary response and was
65


varied (O.D =0.315 to 2.589) though reasonable after the boost (Figure 12). In
contrast, when the adjuvanted co-array was used as the immunogen, a primary
response was detected prior to the boost and achieved levels higher than the control
(NANP)3-BSA immunogen (Figure 12). Furthermore, the standard error of the mean
for the adjuvanted co-array indicated an overall more uniform antibody response
when compared to that for the adjuvanted (NANP)3-BSA. In the absence of
adjuvant, no IgG titers were detected to the (NANP)3 sequence whether the co-array
or the (NANP)3 sequence alone, on dex500, was the immunogen.
3.2.2 (NANP)3 Antibody Response in H-2b mice
The results for the H-2bmice were similar to the results obtained for the H-2d
mice. The IgG anti-(NANP)3 response to adjuvanted (NANP)3-BSA was poor after
either the primary or secondary immunizations (Figure 13). After the boost only two
of eight animals responded to the (NANP)3 epitope. As with the Balb/c mice, when
the adjuvanted co-array was the immunogen, a primary antibody response was
detectable and achieved levels higher than the control (NANP)3-BSA immunogen
post-boost (Figure 13). Again, the standard error of the mean for the adjuvanted co-
array indicated a more uniform antibody response than the adjuvanted (NANP)3-
BSA. Furthermore, when the adjuvanted co-array was the immunogen, only one
66


animal did not develop an IgG titer to the (NANP)3 after the boost. In the absence of
adjuvant, no IgG titers were detected to the (NANP)3 sequence whether the co-array
or the (NANP)3 arrayed alone, on dex500, was the immunogen.
3.2.3 cOA(323.339) Antibody Response
As shown in Figures 14 and 15, for both strains of mice IgG antibodies to the
cOA epitope were detectable after a primary immunization with the co-array in the
absence of adjuvant as well as in the presence of adjuvant, and these levels were
increased after the boost. However, the response to the cOA epitope using the
adjuvanted co-array was greater than that for the non-adjuvanted co-array for either
the primary or secondary response. Furthermore, both strains of mice had low (most
likely anomalous) titers to the T cell epitope after the boost with (NANP)3-dex500.
This is mentioned here because anomalous titers were detected in various systems
used during these studies. Many reasons for anomalous titers have been proposed and
these will be discussed in the P. berghei data section 3.5.7.
67


3.2.4 (NANP)3 as a T helper epitope for IgG Antibody Production
In either strain of mice receiving (NANP)3-dex500, no IgG antibodies to the
(NANP)3 sequence were detectable (Figures 14 and 15). In contrast to the cOA T cell
epitope which was able to provide T helper function for both FI and for the peptide
(NANP)3, the (NANP)3 epitope was unable to provide T helper function for itself
under these immunization conditions and dose schedules.
68


Legend for Figures 12-15
Group#__________Immunogen______________________Adjuvant
1 PBS
2 [(NANP)3lr,-BSA, 10 nz 1 mg, Al(OH)3
3 f(NANP)317, i dex^n, 10 Aig
4 ff(NANP)3l3+ peptide77l-dexsnn,10 Kg
5 ff(NANP)3l7 + peptide7n7l-dex,nn,10 Kg 1 mg ,Al(OH)7
6 [(NANP)3]71i-dexs00,3.3 Kg
Balb/c or C57B1/6 mice (n=8) were immunized on day 0 and boosted 35 days after
the primary immunization with the conjugates as indicated above.
Animals that received 10 /ug of the co-array were immunized with 5.46 x 10"4 mmoles
of the B cell epitope and 4.04 x 10-4 mmoles of the T cell epitope. Animals that
received 10 /^g of the B cell epitope alone were immunized with 1.42 x 10'3 mmoles
whereas animals that received 3.3 /ug received 4.7 xlO^mmoles of the epitope.
Antibody titers to the circumsporozoite repeat sequence, (NANP)3,or the cOA(323.339)
T epitope were measured by solid phase ELISA with either epitope conjugated to
gelatin. Sera were diluted 1:100 for measurement of IgG levels. Substitution
densities are shown as subscripts.
69


OPTICAL DENSITY
FIGURE 12
gj
IgG Antibody Response to the (NANP)3 epitope : H-2
TIME(DAYS)
70


OPTICAL DENSITY
FIGURE 13
IgG Antibody Response to the (NANP)3 epitope,
H-2^5 mice
TIME(DAYS)
71


OPTICAL DENSITY
FIGURE 14
IgG ab response to the cOA(323-339) epitope, H-2d mice
TIME(DAYS)
72


OPTICAL DENSITY
FIGURE 15
IgG Antibody Response to the cOA^323.33g^ epitope,
mice
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 5 10 15 20 25 30 35 40 45 50 55 60
TIME(DAYS)
73


3.2.5 (NANP)3 as a T Cell Epitope via a Lymph Node Proliferation Assay
While the cOA epitope provides T helper function to stimulate an IgG antibody
response to the (NANP)3 epitope in H-2b and H-2d mice, the (NANP)3 epitope was
unable to provide T helper function for itself when arrayed on dex500. The conjugate
was then tested as a T epitope in a lymph node proliferation assay In agreement with
the literature, there was no proliferative response to the (NANP)3-dex500 in the
Balb/c mice while a maximal stimulation index of 10 was observed in C57B1/6 mice
(Figure 16). This result was similar to that obtained by Good et al41,55"57. Therefore,
although a T cell proliferative response was obtained with CFA as the adjuvant, no T
helper function was detectable when Al(OH)3 was used as the adjuvant and antibody
production was the readout for activity.
74


Legend for Figure 16
Lymph Node Proliferation
C57B1/6 (H-2b) or Balb/c (H-2d) mice were immunized with 100 jxg (1.42 x 10'
2mmoles of B epitope) of (NANP)3-dex500 in CFA. Draining lymph nodes were
harvested 10 days later and 4 x 105 cells were challenged in vitro with varying
concentrations of immunizing antigen or unrelated antigen (digoxin264-dex50()). Five
days later wells were pulsed with 1.0 uCi per well of3H-Tdr and harvested 18 hours
later for determination of 3H-Tdr uptake by liquid scintillation. Data are expressed as
a stimulation index
CPM test wells with antigen / CPM cells without antigen
75


FIGURE 16
Lymph Node Proliferation Assay
[(NANPy^-dex^ H-2d
[(NANP)3]71,-dex500, H-2b
digoxin-dex500, H-2d
Q digoxin-dex500, H-2b
76


3.2.6 Summary of Antibody Response and Lymph Node Proliferation
Data (Sections 3.2-3.2.5)
For both strains of mice, the adjuvanted co-array produced a better and more
consistent IgG response to the (NANP)3 sequence than the adjuvanted (NANP)3-BSA.
The reasons for these differences are potentially numerous. Possibly, there are more
T epitopes available with the co-array as the immunogen than when (NANP)3-BSA
was the immunogen. These epitopes could thus afford more TH activity for the
antibody response. Furthermore, the cOA epitope may be a more potent T cell
epitope than any of the T epitopes available within the protein BSA. In addition, the
cOA epitope, as a tryptic cleavage product of cOA, may be presented to the immune
system in a form more readily available for activation of TH cells. This could allow
for a more rapid and directed immune response. Also, in its processed state, the cOA
epitope could provide TH activity in a more uniform fashion than BSA. Specifically,
since the epitopes in the co-array are all cOA(323.339), they would be expected to
stimulate a specific subset of the TH cells. The assumption is that this group of cells
would be activated at a similar time and in a similar fashion. In contrast, BSA must
be processed into its TH peptide components and then presented to the immune
system. Within this protein, multiple TH epitopes could potentially exist with varying
degrees of stimulatory capacity. The assumption here is this group of cells would be
77


activated at different times and a fashion unique to each TH epitope.
In contrast to the co-array, the (NANP)3-dex500 conjugated did not produce an
IgG response to the (NANP)3 sequence. This lends support to the idea that the
strength of the T cell epitope is critical for its ability to provide T helper function.
The (NANP)3 epitope did not provide T helper function for antibody production in
the H-2b mice though it was reported to be a T cell epitope in the literature and shown
to be a T helper epitope herein by the lymph node proliferation assay. Only the cOA
epitope, known as a powerful T cell epitope, was able to provide T helper activity for
itself or for a co-arrayed B cell epitope. Though the (NANP)3 epitope acted as a T cell
epitope in a lymph node proliferation assay with CFA as adjuvant, it did not afford T
helper activity with alum as adjuvant. It is possible that the peptide takes on different
conformation when administered in different adjuvants, i.e. alum vs CFA/water vs oil
I8. It is also possible that there are different cell types that encounter antigen and are
thus activated in response to the antigen in the peritoneum, for the i.p.-alum injection
and near or in lymph nodes for the tail base injection-CFA injection. Further, the
trafficking of the antigen may be different for these different type of injections
resulting again in differential activation of cells. Unfortunately, in this experiment,
mice were not immunized with (NANP)3-dex500 on alum so that comparisons of the
immunogenicity of this conjugate in the two forms of adjuvant are not possible at this
time. It is possible, though unlikely, that the cOA(323.339) epitope alone may not have
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been able to drive an IgG response to the (NANP)3 epitope. Possibly, alum was
required to exert a non-specific stimulatory effect which ultimately drove antibody
production to the (NANP)3 epitope in conjunction with the cOA(323-339) epitope.
Potentially, the presence of alum with the (NANP)3-dex500 conjugate may have
provided a similar non-specific stimulus which would have resulted in an IgG
response to the (NANP)3 epitope.
Two additional points to mention are the following: 1) overall, the IgG response
to the (NANP)3 epitope was higher in the Balb/c than the C57B1/6 mice. A
reasonable explanation for these differences lies in the genetic differences between
these strains of mice and thus their differences in repertoire both in the B cell
compartment and the T cell compartment, however, determining the exact differences
are possible from these data; 2) After a single injection without adjuvant mice
receiving the co-array made IgG antibodies to the T cell epitope and the levels were
increased after a second injection of the co-array in either strain of mice. The IgG
response to the T cell epitope was both more pronounced and less variable when
adjuvant was used than when adjuvant was not used in the system. This would be
predicted if adjuvant serves as a depot for the recruitment of immunocompetent cells
and/or allows the slow release of antigen to the cells of the immune system.
Therefore, as with FI, the cO (323.339) TH cell epitope was required to elicit an IgG
antibody response to the arrayed B cell epitope. In contrast to the FI co-arrays,
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adjuvant was necessary to establish an IgG response to the B cell epitope in this
system. Again, adjuvant was not necessary to establish an IgG response to the cOA T
cell epitope.
3.2.7 In Vitro Protective Ability of anti-(NANP)3 Antibodies
To assess whether antibodies raised to the (NANP)3 sequence with the co-
arrays could recognize the native pathogen, sera containing anti-(NANP)3 antibodies
were tested in an in vitro infectivity assay for their ability to protect hepatocytes from
infection by P. falciparum. A pool of sera was obtained from the mice involved in
the previously described immunogenicity experiment; these mice had been boosted
two weeks previously with the co-array in the presence of adjuvant. A pool of
control sera was included from mice that had been immunized with an unrelated
protein also in adjuvant. These pooled sera, at a 1:100 dilution, were subsequently
tested in the in vitro infectivity assay. Control sera did not inhibit infection of the
hepatocytes by P. falciparum sporozoites (100% infectivity) whereas the pool of sera
from mice immunized with the co-array reduced infectivity to 11%. Thus these
antibodies protected against pathogenic challenge in an in vitro system.
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3.2.8 Anti-dextran Antibody Response
Ideally, antibodies should not be generated to the scaffold. However, it was
anticipated that if antibodies could be generated to the B cell epitope and/or the T
epitope in co-arrays, it was also possible that an antibody response to the
polysaccharide scaffold82 might be induced. The presence of antibodies to the
scaffold would most likely be problematic if this scaffold was to be employed for
more than one vaccination. Upon subsequent exposure to the immunogen, these
antibodies would be expected to cause delayed-type hypersensitivity, anaphylaxis
and/or serum sickness83'88 or they might may bind to the scaffold and remove it from
circulation or misdirect the immune response to the B and/or T epitopes. Up to this
point, no antibody response to the dextran scaffold had been detected regardless of
the size of the scaffold or the presence of T epitopes (data not shown). However,
since the vaccine technology would require injection of multiple, large doses of
conjugate to be most efficacious and since most vaccines, regardless of their efficacy,
would utilize the immune response-enhancing qualities of adjuvant, it was necessary
to test these systems for the presence of anti-dextran antibodies. Sera from the three
week post boost bleed were tested for IgG antibodies to dextran at a 1:100 dilution.
The readout antigen was the 70Kd dexamine plus linker used for many Cortech
studies. Only those animals immunized with the co-array in the presence of adjuvant
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had an anti-dextran response. Whereas seven of seven Balb/c mice had O.D.'s greater
than 1.8, only three of the seven C57B1/6 animals had O.D.'s and these levels varied
from 0.7 to 1.2.
It was possible that these apparent anti-dex antibodies were non-specific for the
dextran. Since the linker used for the immunizations was present in the readout
antigen, it was possible the antibodies could have been anti-linker or anti-dextran or a
combination or both. Thus the assay was repeated with the readout antigens being
either dextran only or linker coupled to an unrelated antigen. Sera were again tested
at both a 1:100 and a 1:500 dilution for the presence of IgG antibodies to dextran.
Though some linker titers were detectable, the majority of the antibody response was
directed to the dextran as the antigen (Table 2). These results were of concern and
will be discussed further in section 3.5.6 and section 4.
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Table 2
Anti-Dextran IgG Titers: Optical Densities @ 450 nm
Animal anti-Dextran IgG anti-Dextran IgG Anti-linker Anti-linker
# 1:100 1:500 IgG 1:100 IgG 1:500
33 1.184 0.55 0.059 0.01
34 0.93 0.283 0.177 0.023
35 0.348 0.101 0.362 0.096
36 1.022 0.585 0.035 0
37 1.057 0.477 0.018 0
38 0.976 0.517 0.008 0
39 0.927 0.319 0.308 0.059
Animals were immunized with the 10 /^g of the (NANP)3-cOA co-array in the presence of
adjuvant. Sera from the day 21 post-boost bleed was tested by ELISA for the presence of IgG
antibodies to the scaffold dex500 @ a 1:100 and a 1:500 dilution.
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3.3 Peptide B Cell Epitope, (NANP)3, Peptide T Cell Epitope Co-arrays:
Part II
The initial experiment with the (NANP)3 and cOA (323.339) co-array seemed to indicate
that the presence of the T cell epitope was required for the antibody response to the
(NANP)3 sequence. However, this response occurred only in the presence of adjuvant.
To eliminate the possibility that the presence of adjuvant with the (NANP)3 -peptide-
dex500 conjugate was responsible for the IgG response to the (NANP)3 sequence and
was not due solely to the T cell epitope, a group of animals immunized with (NANP)3-
dex500 was included and the study repeated in part. A non-adjuvanted response to the
peptide co-array was still anticipated, based on the working hypothesis and the data
obtained with the FI co-array. Therefore, a ten-fold higher dose of conjugate was used
in the attempt to achieve a non-adjuvanted response. Conjugates used for this
experiment were the same as those used in the previous experiment, section 3.2 .
Groups of Balb/c female mice (n=4) were immunized i.p. with 10 /^g or 100 /ug of
either the co-array or the B cell epitope alone, on dex500 with or without Al(OH)3 as
adjuvant. Animals received a second injection of the same construct 21 days after the
primary injection. Sera were assayed for the presence of IgG and IgM antibodies to the
(NANP)3 sequence or the cOA(323.339) epitope.
84