EVALUATION OF T-CELL AND B-CELL EPITOPES AND DESIGN OF MULTIVALENT VACCINES AGAINST HTLV-1 DISEASES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Roshni Sundaram, M.S.
* * * * *
The Ohio State University 2003
Dissertation Committee: Approved by
Professor Pravin T.P. Kaumaya, Adviser
Professor Christopher M. Walker Adviser
Professor Neil R. Baker Department of Microbiology
Professor Marshall V. Williams
ABSTRACT
Human T-cell lymphotropic virus type I (HTLV-1) is a C type retrovirus that is the
causative agent of an aggressive T-cell malignancy, adult T-cell leukemia/lymphoma (ATLL).
The virus is also implicated in a number of inflammatory disorders, the most prominent
among them being HTLV-1 associated myelopathy or tropical spastic paraparesis
(HAM/TSP). HTLV-1, like many viruses that cause chronic infection, has adapted to persist
in the face of an active immune response in infected individuals. The viral transactivator Tax
is the primary target of the cellular immune response and humoral responses are mainly
directed against the envelope protein. Vaccination against HTLV-1 is a feasible option as
there is very little genetic and antigenic variability. Vaccination regimes against chronic
viruses must be aimed at augmenting the immune response to a level that is sufficient to
clear the virus. This requires that the vaccine delivers a potent stimulus to the immune
system that closely resembles natural infection to activate both the humoral arm and the
cellular arm. It is also clear that multicomponent vaccines may be more beneficial in terms of
increasing the breadth of the immune response as well as being applicable in an outbred
population. As a step in this direction, this dissertation work first describes the identification and evaluation of human T-cell and B-cell epitope based vaccines that are capable of inducing robust immune responses. A novel strategy was designed for delivery of multiple
Tax protein derived CTL epitopes into the same antigen presenting cell for the simultaneous
ii priming of anigen specific T-cells. This design allowed antigen processing by cellular proteasomes to efficiently liberate the individual minimal epitopes endogenously for presentation via MHC Class I. This type of construct was effective in vivo in HLA-A*0201 transgenic mice in inducing cellular immune responses against all individual epitopes. A statistically significant reduction in viral replication was observed in mice that were immunized with a multiepitope Tax CTL construct and challenged with recombinant Tax vaccinia virus, by the induction of antiviral IFN-γ and cytolysis of infected target cells. This reduction was determined to be the result of immune responses specifically targeting the Tax protein and was dependent on the presence of CD8+ T-cells.
Studies were also undertaken to engineer peptides that have a high propensity to fold into native protein like structure for the induction of antibodies that have a high affinity for the complex transient secondary structure adopted by the native protein. A single matrix multicomponent template strategy was applied to design a peptide from the central region of the TM (residues 347-374) that forms a parallel trimeric hairpin structure. Mutational studies have implicated this region to be critical for the fusion process after receptor binding. The template design served to constrain the individual peptide strands at one end and bring them in close proximity to promote hydrophobic interactions to initiate a coiled coil formation.
Key mutations in the native peptide sequence were also made to increase hydrophobic interactions. We observed that the mutations combined with the template design resulted in a peptide that had a high helical content in aqueous medium and antibodies raised against the peptide recognized native viral protein with higher affinity compared with the wild type template peptide antibodies. These results suggested the presence of native sequence like conformation. Furthermore, a chimeric peptide incorporating a “promiscuous” T-helper
iii epitope derived from the region of chain reversal in the TM subunit (residues 392-415 of gp21) implicated in the fusion process was tested for its ability to induce neutralizing antibodies. This peptide induced high titered antibodies that were capable of inhibiting
HTLV-1 infected cell induced syncytia formation. These data, taken together may have implications in the development of multivalent vaccines against HTLV-1.
iv
Dedicated to my father
v
ACKNOWLEDGMENTS
I express my deepest gratitude to my mentor Dr. Pravin Kaumaya for his patience
and support. His enthusiasm and encouragement in pursuing a multitude of novel ideas was
the key to the success of this project. I would like to express my sincere thanks to Dr.
Christopher Walker for his assistance in overcoming many technical difficulties and for all
the useful suggestions and insightful discussions to improve on my results. I extend my
appreciation to my committee members Dr. Neil Baker and Dr. Marshall Williams for their
time and effort. I also thank Dr. Francois Lemonnier for providing us with the transgenic
animal model and cell lines, Dr. Steven Jacobson for reagents necessary to carry out this
project and Dr. Don Young for all the statistical analysis. I thank Dr. Valerie Bergdall, Carrie
Kraly and the vivarium staff for training me in animal handling and assistance in the
maintenance of large breeding colonies. Further thanks are also due to Dr. Sharad Rawale
for synthesis of peptides and circular dichroism experiments.
Finally, I am indebted to my parents and my family for their unconditional love,
support and encouragement. I am also eternally grateful to my husband who helped and
stood by me through all the challenging times over the years.
vi
VITA
1992-1995……………………………………………….B.S. Microbiology University of Bombay, India
1995-1997……………………………………………….M.S. Microbiology University of Bombay, India
1997-2003………………………………………………Graduate Teaching and Research Associate, The Ohio State University `
PUBLICATIONS
Sundaram, R., Sun, Y., Walker, C.M., Lemonnier, F.A., Jacobson, S., and Kaumaya, P.T.P (2003) A Novel Multivalent Human CTL Peptide Construct Elicits Robust Cellular Immune Responses in HLA-A*0201 Transgenic Mice: Implications for HTLV-1 Vaccine Design. Vaccine 21(21-22), 2767-2781
Sundaram, R., Dakappagari, N., and Kaumaya P.T.P. (2002). Synthetic Peptides as Cancer Vaccines. Peptide Science. Biopolymers 66(3), 200-16.
Sundaram, R., and Kaumaya P.T.P. (2001). Multivalent Vaccine Studies for HTLV-1 Associated Diseases. In Peptides: The Wave of the Future. (Lebl, M., and Houghten, R.A., Eds) Kluwer Academic Publishers, The Netherlands 1006-1007.
Sundaram, R., Walker, C.M., and Kaumaya P.T.P. (2001). Evaluation of HTLV-1 Cytotoxic T-Cell Epitopes in HLA-A*0201 Transgenic Mice. In Peptides: The Wave of the Future. (Lebl, M., and Houghten, R.A., Eds) Kluwer Academic Publishers, The Netherlands 1008- 1009.
FIELDS OF STUDY
Major Field: Microbiology vii
TABLE OF CONTENTS Page
Abstract ...... ii Dedication...... v Acknowledgments...... vi Vita ...... vii List of Tables...... xi List of Figures ...... xii Chapters: 1. Introduction History and Epidemiology...... 1 Genetic Organization of HTLV-1 ...... 2 The Tax protein and its role in transformation...... 5 Disease Association and Pathogenesis...... 6 Adult T-cell Leukemia/Lymphoma (ATLL) ...... 8 HTLV-1 Associated Myelopathy ...... 8 Other diseases linked to HTLV-1 infection...... 9 Immune Responses...... 10 Humoral response...... 10 The helper T-cell response ...... 11 The cytotoxic T-cell response...... 11 Current Therapy ...... 11 Vaccine Studies...... 13 Mapping humoral determinants ...... 15 Mapping T-cell determinants...... 18
viii Animal models and protection studies...... 21 Peptide Vaccine Approach...... 25 2. Design and Characterization of a Multiepitope CTL Peptide Construct in HLA-A*0201 Transgenic β2M Db Double Knockout Mice Rationale ...... 28 Summary...... 31 Materials and methods...... 31 Results ...... 38 Discussion...... 44 Supplement ...... 51 3. Protective Efficacy of Multiepitope CTL Peptide Construct against Challenge with Recombinant Vaccine Virus Expressing Tax Protein Rationale...... 71 Summary...... 73 Materials and Methods...... 74 Results...... 79 Discussion...... 88 4. Design and Immunological Characterization of Peptides that Mimic the Coiled Coil Region of HTLV-1 Transmembrane Subunit Rationale...... 110 Summary...... 113 Materials and Methods...... 114 Results...... 118 Discussion...... 123 5. Evaluation of Other B-cell Epitopes from the HTLV-1 Envelope Protein for Induction of Neutralizing Antibodies Rationale...... 140 Summary...... 141 Materials and Methods...... 142 Results...... 144 Discussion...... 148 ix
Ongoing and Future Studies...... 157 Concluding Remarks...... 161 Appendices: A. Purification and Mass Spectrometry Profiles of HTLV-1 Tax CTL Epitopes, Envelope Chimeric B-cell Epitopes and Template Peptides ...... 162 B. Mass Spectrometry profiles of Proteasome Digestion of Multiepitope CTL Constructs...... 180 Bibliography...... 185
x
LIST OF TABLES
Table Page
2.1. List of predicted CTL epitopes from the Tax protein of HTLV-1...... 55
2.2. List of predicted CTL epitopes from the Envelope protein of HTLV-1 ...... 56
3.1. Variants of multiepitope constructs from Tax protein...... 92
3.2. Immunoproteasomal cleavage analysis of multiepitope constructs ...... 93
4.1. Chimeric template peptides from gp21 envelope protein...... 128
4.2. Concentration dependence analysis by circular dichroism ...... 129
xi
LIST OF FIGURES
Figure Page
2.1. Frequency of IFN-γ secreting cells in HAM/TSP patients PBMCs...... 57
2.2. Design of multiepitope CTL peptide construct...... 58
2.3. Proteasomal cleavage analysis of multiepitope construct...... 59
2.4. Proteasomal cleavage products ...... 61
2.5. Immunogenicity of multiepitope peptide construct...... 63
2.6. Cumulative cytotoxic responses in multiepitope construct immunized
mice...... 64
2.7. Frequency of IFN-γ secreting CD8+ T-cell after multiepitope construct
immunization ...... 65
2.8. Immunogenicity of individual epitopes ...... 66
2.9. Mixture immunization of HHD mice...... 67
2.10. Frequency of envelope specific IFN-γ secreting cells in HAM/TSP
patient PBMCs ...... 68
2.11. Immunization of HHD mice with Env 175-218 for CTL induction ...... 69
2.12. Chimeric peptide approach for delivery of CTL epitopes...... 70
3.1. Immunogenicity of predicted CTL epitopes ...... 94
3.2. In vivo proteasomal processing of 236 multiepitope construct...... 95
3.3. Cytolytic responses against variants of multiepitope constructs...... 96
xii
3.4. Cytokine release by multiepitope variants...... 98
3.5. Affinity of CTLs induced by single epitope and multiepitope
immunization ...... 99
3.6. Comparison of IFN-γ release from single epitope and multiepitope
construct immunization ...... 100
3.7. Tax (p40) exression analysis in EL4/HHD of HeLa/HHD cells...... 101
3.8. Cytolysis of p40-VV infected target cells ...... 102
3.9. Activation of IFN-γ secretion by p40-VV infected target cells ...... 104
3.10. Vaccinia virus titers in ovaries of 236 immunized mice ...... 105
3.11. Specificity of protection by 236 immunization...... 106
3.12. Analysis of in vivo depletion of CD8+ T-cells by flow cytometry ...... 107
3.13. Protection is dependent on CD8+ T-cells...... 108
3.14. Comparison of protection elicited by single epitope, mixture
and multiepitope immunization ...... 109
4.1. Template design for synthesis of coiled coil region of gp21 envelope
subunit...... 130
4.2. CD spectra of B-cell epitope constructs from gp21 coiled coil region ...... 131
4.3. Guanidinium hydrochloride denaturation curve for CCR2T...... 132
4.4. Immunogenicity of template peptide constructs ...... 133
4.5. Antibody isotyping of CCR2T and WCCR2T antisera ...... 134
xiii 4.6. Analysis of relative binding of CCR2T and WCCR2T antisera to gp21
protein by live cell immunofluorescence staining of HTLV-1 infected
cells ...... 135
4.7. Cross reactivity of peptide antibodies to native gp21 protein...... 136
4.8. Competitive inhibition ELISA curves for CCR2T and WCCR2T
antisera using template peptide coated plates ...... 137
4.9. Competitve inhibition ELISA curves for CCR2T and WCCR2T antisera
using gp21 coated plates...... 139
5.1. Molecular modeling of chimeric B-cell epitopes from HTLV-1 envelope
protein...... 151
5.2. CD spectra of chimeric B-cell epitopes...... 152
5.3. Immunogenicity of chimeric B-cell epitopes...... 153
5.4. Live cell immunfluorescence staining of HTLV-1 infected cells...... 154
5.5. Cross reactivity of TT3-gp21-(392-415) antibodies to gp21 protein ...... 155
5.6. TT3-gp21-(392-415) antibodies inhibit HTLV-1 infected cell induced
syncytia ...... 156
6.1. Immunogenicity of MVF-gp46-(175-218) in squirrel monkeys...... 159
6.2. Lymphoproliferative assay of PBMCs from MVF-gp46-(175-218) + 236
multiepitope immunized squirrel monkeys ...... 160
A.1. RP-HPLC traces and ESI profiles of purified Env239-247 and Env339-347...... 163
A.2. RP-HPLC traces and ESI profiles of purified Env346-354 and Env395-403...... 164
A.3. RP-HPLC traces and ESI profiles of purified Env402-410 and Env175-183...... 165
A.4. RP-HPLC traces and ESI profiles of purified Env182-190 and Env210-218...... 166
xiv A.5. RP-HPLC traces and ESI profiles of purified Tax155-163, Tax11-19
and Tax178-186...... 167
A.6. RP-HPLC traces and ESI profiles of purified Tax233-241, Tax307-315
and Tax306-315...... 168
A.7. RP-HPLC traces and ESI profiles of purified multiepitope construct...... 169
A.8. RP-HPLC traces and ESI profiles of purified multiepitope
construct 236...... 170
A.9. RP-HPLC traces and ESI profiles of purified multiepitope
construct 362...... 171
A.10. RP-HPLC traces and ESI profiles of purified multiepitope
construct 632...... 172
A.11. RP-HPLC traces and ESI profiles of purified multiepitope
construct 326...... 173
A.12. RP-HPLC traces and ESI profiles of purified T-helper epitope TT3 ...... 174
A.13. RP-HPLC traces and ESI profiles of purified Env175-218 ...... 175
A.14. RP-HPLC traces and ESI profiles of purified CCR2T and CCR2E ...... 176
A.15. RP-HPLC traces and ESI profiles of purified WCCR2T and WCCR2E. 177
A.16. RP-HPLC traces and ESI profiles of purified TT3-gp21-(392-415) ...... 178
A.17. RP-HPLC traces and ESI profiles of purified TT3-gp46-(136-160) ...... 179
B.1. Proteasome digestion profiles for 236 construct ...... 181
B.2. Proteasome digestion profiles for 362 construct ...... 182
B.3. Proteasome digestion profiles for 632 construct ...... 183
B.4. Proteasome digestion profiles for 326 construct ...... 184
xv
CHAPTER 1
INTRODUCTION
History and Epidemiology
The suggested involvement of a transmissible agent in Adult T-cell
Leukemia/Lymphoma (ATLL) in certain endemic regions of Japan led to the discovery of the first human retrovirus to be associated with human disease (Uchiyama et al., 1977). The
Human T-cell Lymphotropic Virus Type I (HTLV-1) was first isolated from a T- lymphoblastoid cell line (HUT 102) that was established from a patient with cutaneous T-cell lymphoma (Poiesz et al., 1980). Around the same time, a cell line derived from a patient with leukemia (MT-2) was also found to harbor a retrovirus and produce antigens that were reactive against sera from ATLL patients (Hinuma et al., 1981). The viruses from the two cell lines were found to be identical and thus HTLV-1 was confirmed to be the etiological agent of Adult T-cell Leukemia/Lymphoma (Yoshida et al., 1982). Later, in 1985, patients with Tropical Spastic Paraparesis in Martinique and patients with a myelopathy in Southern
Japan, were found to have seroreactivity against HTLV-1. Comparative studies also showed that these two diseases are identical and were proposed to be called HTLV-1 Associated
Myelopathy or Tropical Spastic Paraparesis (HAM/TSP) (Gessain et al., 1985; Osame et al.,
1986b). HTLV-1 infection has also been implicated in a variety of other inflammatory disorders such as polymyositis, chronic arthropathy, uveitis and infective dermatitis (Nera et al., 1989) although no direct association has been established.
1 Recent estimates indicate that there are approximately 10-20 million people
worldwide that are infected with HTLV-1. HTLV-1 is widespread in the tropics and
subtropics. The endemic regions include southern Japan, central and South Africa, the
Caribbean and eastern parts of South America and Brazil (Bangham, 2000a; Johnson et al.,
2001). The virus is also found in southern Africa, southern India, Northern Iran and among
the aboriginal populations of Australia. In the United States, the virus is found mostly
among certain immigrant groups, the African American population and in intravenous drug
users. Very few cases of ATLL and HAM/TSP have been reported in European countries
(Franchini, 1995). The distribution of HTLV-1 within endemic regions is observed to be uneven and there seems to be a predilection for coastal areas. The major modes of transmission of HTLV-1 are through breast feeding from mother to child and sexual transmission, mainly between males and females. Male to female transmission is four times more prevalent than female to male. Transmission via contaminated blood products is also another major mode in endemic regions especially in Japan and blood screening is now routine in many countries including Japan, the USA and Brazil. Unlike other retroviruses such as HIV, there is very little evidence to support the transmission of HTLV-1 through cell-free body fluids. HTLV-1 transmission occurs almost exclusively via cell-to-cell contact within the host as well as in vitro. Infection by cell-free HTLV-1 has been found to be less efficient.
Genetic Organization of HTLV-1
The HTLV-1 virus is an enveloped single-stranded diploid RNA retrovirus. The genome is approximately 9 kb and encodes for the structural proteins envelope and gag, enzymes reverse transcriptase, protease and integrase (Seiki et al., 1983). The viral promoter
2 and other regulatory elements are contained within the long terminal repeats (LTR) located
at the 5’ and the 3’ ends of the genome and is divided into the U3, R and the U5 regions.
Proviral transcription, mRNA termination and polyadenylation signals are controlled by
elements located within the U3 region. Additionally, the U3 region also contains three
repetitive 21 bp-enhancer elements referred to as TRE (Tax responsive elements) (Beimling
and Moelling, 1992; Paca-Uccaralertkun et al., 1994; Zhao and Giam, 1992). Similar to other
retroviruses, a full length mRNA encodes the gag protein (p55) and the pol protein that is
synthesized by ribosomal frameshifting. The gag protein is then further cleaved by the viral
protease (encoded by a reading frame consisting of the 3’ end of the gag and the 5’ end of
the pol protein coding regions that results from a ribosomal frameshifting) to generate the
matrix (MA, p19), capsid (CA, p24) and the nucleocapsid (NC, p15) proteins. In addition to
these structural proteins, the HTLV-1 genome complexity is highly increased by several
alternative splicing events within the pX region that results in many accessory and regulatory
protein. These genes are encoded in four open reading frames (ORF) in the pX region
(Berneman et al., 1992; Cereseto et al., 1997; Ciminale et al., 1996; Ciminale et al., 1992;
Koralnik et al., 1992; Nagashima et al., 1986; Orita et al., 1993). Two of these open reading
frames encode for the Rex and the Tax protein (ORF III and ORF IV respectively). Rex is a
27-kDa phosphoprotein that localizes in the nucleolus and functions to increase the
cytoplasmic accumulation of unspliced and singly spliced RNA. The Tax protein is a 40kDa protein comprised of 353 amino acids that localizes in the nucleus and serves to increase the
transcription from the viral long terminal repeats and also several host genes that are
involved in cellular proliferation. The four accessory proteins are the p12I, p27I, p13II and
p30II that are encoded by pX ORF I and II. Some recent studies have highlighted the
3 importance of these accessory proteins in the successful establishment of infection. The p12I protein is a small protein of 99 amino acids that is localized within the cell membranes. It has been shown to associate with immature forms of the IL-2R β and γ chains, thus retaining them within the Golgi apparatus and reducing their number on the cell surface.
Hence, it is believed to play a role in downmodulation of the immune response. The protein is also known to have atleast four proline rich SH3 binding motifs (PXXP) indicating a possible role in intracellular signaling pathways. Further, recent studies have demonstrated that p12I mutant infectious clones of HTLV-1 have a reduced capacity to infect quiescent T-
lymphocytes. However, activated target cells such as those stimulated with
phytohaemagglutinin (PHA) and IL-12 can be efficiently infected by the mutant clones. This
suggests a role of p12I in the activation of primary naïve T-lymphocytes during the early
stages of infection (Albrecht et al., 2000).
The p30II protein shares homology with the POU family of transcription factors.
The p13II and p30II proteins localize to the nucleus and nucleolus. P13II has also been
demonstrated in the mitochondrial membranes (Ciminale et al., 1999; Koralnik et al., 1993).
Their cellular location suggests a role in the regulation of HTLV-1 replication or virus-cell
interactions. In this regard, there have been reports that a mutation in p30II results in lower
proviral loads in vivo (Lairmore et al., 2000).
At an early stage of viral gene expression, the Tax and Rex proteins predominate.
Tax transactivates the transcription of HTLV-1 and the Rex protein serves to upregulate the
expression of unspliced and single-spliced viral genome RNA and viral envelope and
gag/pol proteins. The Rex protein also suppresses further expression of tax/rex mRNA
4 resulting in transient expression of HTLV-1. This may be one of the ways in which HTLV-1
may be able to evade immune surveillance by the host.
The Tax protein and its role in cellular transformation
HTLV-1 mediated transformation may include two distinct stages. The first stage is
the polyclonal expansion of infected T-lymphocytes that is IL-2 dependent. Hence these
cells are considered immortal. These immortal T-cells, over a period of decades, gradually
undergo changes and display chromosomal abnormalities. Finally, the cells are transformed
when they become IL-2 independent (Iwanaga et al., 1999; Ressler et al., 1996). The Tax
protein is implicated in the conversion to the IL-2 independent stage which is considered a hallmark of T-cell transformation and leukemogenesis during ATLL development.
The Tax protein exerts its transactivation of viral and cellular genes by various
mechanisms for which the three 21-bp repeats in the U3 region of the LTR are crucial.
However, it does not directly bind to enhancer elements. It interacts with various
transcription factors which can bind to different sites within the enhancer elements. Tax can
bind to CREB (cyclic AMP responsive elements binding proteins)/ATF (activating
transcription factor) family which form homo or heterodimers and bind to either CRE (c-
AMP responsive element) or to the 21-bp repeat regions and change the affinity of these
proteins for various DNA-binding sites. Other heterologous promoters that are
transactivated by Tax are the cellular IL-2Rα chain, IL-2, tumor growth factor-β (TGF-β),
granulocyte/macrophage colony stimulating factor (GMCSF), tumor necrosis factor-
β (TNF-β), the OX40 ligand and c-myc (Ballard et al., 1988; Duyao et al., 1992; Kim et al.,
1990; Leung and Nabel, 1988; Miura et al., 1991; Miyatake et al., 1988). It also binds to cell
cycle regulators such as cyclin D3, mitotic checkpoint regulator MAD1, the cyclin dependent
5 kinases (Cdk) Cdk4 and Cdk6, Cdk inhibitors (p16INK4A inhibitor of Cdk 4) and the tumor
suppressor p53 (Jin et al., 1998; Low et al., 1997; Mulloy et al., 1998; Neuveut et al., 1998;
Pise-Masison et al., 1998a; Pise-Masison et al., 1998b). Hence the Tax protein alters the
proliferation of cells thorough various mechanisms. One of the main mechanisms is through the activation of the NF-κB family of transcription factors. The Tax protein binds to the
NF-κB inhibitory proteins (I-κBs) which results in the release and nuclear translocation of
NF-κB to activate various cellular genes encoding growth factors, proinflammatory
cytokines and apoptotic regulators (McKinsey et al., 1996; Sun et al., 1994).
In summary, the enhanced expression of cellular genes involved in T-cell proliferation and cell growth, cell cycle or cell death appears to be the mechanism of Tax induced transformation of T-cells that are infected by HTLV-1. Further evidence of the oncogenic potential of the Tax comes from the observation that Tax transgenic mice display different phenotypes including Sjogren’s syndrome, brain and lymphoid tumors and large granular lymphocyte leukemia (Benvenisty et al., 1992; Green et al., 1989; Grossman et al.,
1995).
Disease Association and Pathogenesis
As the name suggests the HTLV-1 virus primarily infects T-cells and cause polyclonal or monoclonal T-cell proliferation. It can also infect a variety of other cell types including some from non-human species such as rabbits as well as mice, as recently demonstrated by Kushida et al (Fang et al., 1998; Kushida et al., 1997). Although the receptor to which the envelope protein binds has not been identified it is linked to chromosome 17q and may be a member of the VCAM family (Hildreth et al., 1997). The
6 receptor is widely distributed because peripheral blood cells from many species such as
monkey, murine and rabbit are susceptible to HTLV-1 mediated infection.
HTLV-1 induces T-cell activation and proliferation. It has been observed that activated or dividing T-cells have a greater susceptibility to infection by HTLV-1 as compared to quiescent non-dividing T-cells (Merl et al., 1984), suggesting that T-cell activation may be necessary for the virus to establish active infection after entry into the cell.
The viral envelope is the first element to contact the target cell and plays a major role in the infection process. It is thought to be responsible for receptor recognition and subsequent receptor mediated fusion of virus with the target cell membrane, leading to the delivery of the capsid into the host cell (Rosenberg et al., 1997). Additionally, the fusion between infected cells expressing viral envelope and other receptor bearing uninfected cells leads to the formation of multinucleated giant cells termed syncytia (Kiyokawa et al., 1984).
The envelope is made up of a host derived lipid bilayer that is studded with virally encoded glycoproteins. The HTLV-1 glycoprotein like many other retroviral glycoproteins is synthesized initially as a precursor molecule (gp61) within the host cell and subsequently transported into the Golgi apparatus where it is cleaved into a surface glycoproteins gp46
(SU) of 312 amino acids and a transmembrane glycoprotein gp21(TM ) of 176 amino acids which is partly extracellular and partly traverses the lipid bilayer (Hunter and Swanstrom,
1990). The two subunits are non-covalently associated with each other. The entire complex is anchored via the TM to the virion membrane or the infected cell surface (Delamarre et al.,
1996).
7 Adult T-cell Leukemia/Lymphoma (ATLL)
ATLL is an aggressive T-cell malignancy, that develops after a long latency period of decades (Tajima, 1990) and is classified into four clinical subtypes: acute, chronic, smouldering and lymphoma depending on the number of abnormal T-cells in the periphery, the serum lactic acid dehydrogenase (LDH) levels, tumor lesions in the various organs and the clinical course. Acute ATLL is characterized by acute presentation of symptoms including general malaise, cough, fever abdominal fullness, drowsiness, lymph node enlargement, hepatosplenomegaly and jaundice. High levels of serum LDH and interleukin-2 receptor (IL-2Rα) chain are also observed. Characteristic leukemic cells with large deeply convoluted nuclei become apparent described as “flower cells”. The typical phenotype of
ATLL cells is CD3+, CD4+, CD8- and CD25+. (Hattori et al., 1981; Kamihira et al., 1992).
Chronic ATLL symptoms are milder with a longer clinical course. In smoldering ATLL, patients display fewer leukemic cells in the periphery but skin lesions such as papules, nodules and erythema are common. Lymphoma on the other hand, does not manifest leukemic cells; however there is significant enlargement of the lymph nodes. Suppression of the cellular arm of the immune system is a characteristic feature of ATLL. The major complications in ATLL are hypercalcemia and opportunistic infections by bacteria, fungi, viruses and protozoa. The prognosis for ATLL is very poor since the even the combination of chemotherapy with anti-cancer drugs produces partial or transient remission and death usually results within 4-6 months.
HTLV-1 Associated Myelopathy or Tropical Spastic Paraparesis (HAM/TSP)
HAM/TSP is a chronic progressive disease of the central nervous system. The typical onset of the disease is usually around the age of 40 or more, however the onset could
8 also be as short as 18 weeks following infection with HTLV-1 contaminated blood (Gout et al., 1990; Osame et al., 1986a). The incubation period is shorter than ATLL. Women are more frequently affected than men. The disease is not as fatal as ATLL and the mean duration of disease is about 10 years at the time death. HAM/TSP is characterized by spasticity and muscle weakness that is symmetrical and progressive, hyperreflexia, Babinsky signs of the lower extremities and urinary bladder and bowel disturbances (Hollsberg and
Hafler, 1993; McFarlin and Blattner, 1991; Nakagawa et al., 1995; Osame et al., 1990). Motor disfunctions are also more common than sensory symptoms. Although clinically many of the symptoms resemble that of multiple sclerosis (MS), the relapsing-remitting course that is characteristic of MS is almost never observed in HAM/TSP and most patients demonstrate progressive ascending myelopathy (Levin and Jacobson, 1997b). Cerebrospinal fluid (CSF) analysis shows a mild lymphocytic pleocytosis, mild protein elevation, elevated IgG synthesis and IgG index and oligoclonal bands (Ceroni et al., 1988; Jacobson et al., 1990a). The major causes of death are complications by infections and cancers.
Other diseases linked to HTLV-1 infection
There are several other inflammatory disorders that are linked to HTLV-1 such as
HTLV-1 uveitis (HAU), HTLV-1 associated Arthropathy (HAAP) and Cutaneous T-cell
Lymphoma. HAU and HAAP are distinct clinical diseases. HAU is characterized by visual dysfunctions with acute or subacute onset. Patients with HAAP demonstrate symptoms that are similar to rheumatoid arthritis. Diseases such as polymyositis, respiratory diseases and lymphadenitis and dermatitis are also linked to HTLV-1 but these diseases are yet to be established as distinct clinical entities (Uchiyama, 1997).
9 The factors that predispose an infected individual to develop HAM/TSP or ATL are not completely clear. Genetic influences have been implicated (Sonoda et al., 1996). The
presence of HLA-A*0201 gene is associated with both a reduction in the proviral load in
asymptomatic carriers and protection against HAM/TSP. The MHC Class II HLA-
DRB1*0101 was also found to be associated with high incidence of HAM/TSP but only in
the absence of the protective effects of HLA-A*0201. These results are supportive of
antiviral vaccines against HTLV-1 that induce a strong cell mediated response (Jeffery et al.,
2000).
Immune Responses
Humoral responses
The natural immune response an infected individual mounts against HTLV-1
infection is believed to be protective in terms of active virus infection and the production of
infectious virus particles and virus-infected cells. However there are other studies which
seem to implicate the immune response to be a contributing factor to the development of
disease especially in the case of HAM/TSP and other inflammatory diseases linked with
HTLV-1 infection. The first specific antibodies to appear after infection are directed against
the gag protein. These antibodies persist for about two months after which anti-envelope
antibodies appear. Finally, approximately 50% of the infected populations also produce
antibodies against the Tax protein although Tax is predominantly the target of the cellular
arm of the immune system (Franchini, 1995; Manns et al., 1991). The titer of antibodies
correlates well with the proviral load. In general, the serum titers of anti-HTLV-1 antibodies
are much higher in HAM/TSP patients than in ATLL patients or asymptomatic carriers.
Further, neutralizing antibodies have been demonstrated in HTLV-1 infected patients
10 (Kuroki et al., 1992b; Lyerly et al., 1987; Matsushita et al., 1986; Tanaka et al., 1991). Several
immunodominant regions have been mapped within the envelope protein for the purpose of vaccine development and diagnostic reagents, some of which have been described in the sections that follow.
The helper T-cell response
Th1 responses are more predominant than the Th2 responses among the circulating
CD4+ cells in HTLV-1 infected individuals (Horiuchi et al., 2000). An HLA-DRB1*0101 restricted epitope has also been mapped to the envelope protein (Kitze et al., 1998; Yamano et al., 1997). There is as yet not much information on the CD4+ antigen specific T-cell response against HTLV-1.
The cytotoxic T-cell response
HTLV-1 infected individuals mount a strong CD8+ T-cell response. These cells are highly abundant and competent when isolated fresh from blood which implies that they have encountered antigen recently in vivo (Bangham, 2000b). The cellular immune response is highly unusual because the vast majority of the cytotoxic response is targeted against the Tax protein, particularly against one specific epitope spanning sequence 11-19 of the Tax protein.
Current Therapy
There has not been much success in the treatment of the aggressive forms of ATLL.
Chemotherapy in the chronic and smouldering type of ATLL appears to do more harm than benefit the individual as it causes an increase in the immune deficiency already present
(Nakada et al., 1987; Shimoyama, 1991). Significant improvement was achieved in ATLL treatment with the combined administration of zidovudine (AZT) and interferon (IFN)-
α. This combination had minimal side effects. These results may be further improved by
11 higher doses and the inclusion of additional anti-retroviral agents. A combination of arsenic
trioxide and IFN-α which induced the degradation of Tax followed by cell cycle arrest and
apoptosis of HTLV-1 infected cells may also be promising (Bazarbachi et al., 1999;
Bazarbachi and Hermine, 2001). The use of biological mediators such as retinoid acid is now being investigated. Retinoids are analogues of vitamin A and have varied effects on different
cell types. Retinoid acid increases the apoptosis of ATLL cells in vitro and reduces drug
resistance to stimulate the immune system and the anti-tumor activity against ATLL cells
(Fujimura et al., 1998; Goodman, 1984; Miyatake et al., 1998; Miyatake and Maeda, 1997; Y
et al., 1995).
Most treatments for HAM/TSP on the other hand, have been aimed to reduce the
inflammation in the affected tissues. Zidovudine has been used for the treatment of
HAM/TSP. (Taylor, 1998). The anti-retroviral agent lamuvidine has also shown a 10-fold
decrease in the proviral load (Taylor et al., 1999). Treatment with a variety of antibiotics such
as erythromycin and fosfomycin which cause immunomodulation as well as anti-
inflammatory agents like salazosulphapyridine reported 50% clinical benefit in patients
(Taylor, 1998). Recently, passive therapy of HAM/TSP was demonstrated with the use of
humanized anti-IL2Rα monoclonal antibody (anti-Tac) (Waldmann et al., 1993). Among
other genes the Tax protein activates the IL2Rα expression in HTLV-1 infected as well as
uninfected cells, resulting in the expansion of IL2Rα bearing cells in the peripheral blood
following proliferation induced by an IL2 and IL2Rα autocrine loop. Administration of anti-
tac for a short duration caused a 52% reduction in the proviral loads in these patients, with
no effect on the HTLV-1 specific cytotoxic T-cells. This reduction was due to the apoptotic
death of IL2Rα bearing cells (Lehky et al., 1998). In addition to IL-2, IL-15 expression has
12 also been found to be upregulated in these patients and as both cytokines have a similar
function in the activation and proliferation of T-cells, this may contribute to the
inflammatory responses characteristic of the disease state in a similar manner as IL-2 by
engaging the IL-2β receptor. Thus it has been shown that administration of a combination of anti-IL-2 and anti-IL-15 has a more pronounced effect in arresting T-cell proliferation than by administering anti-IL-2 alone. Antibodies against the receptor are more effective than antibodies to the cytokine (Azimi et al., 1999). However, as with all non-specific therapies large doses are required and there is always the potential risk of non-specifically deleting T-cells which are protective in nature or the toxicity of various agents may spread to other healthy tissues. Thus the ideal approach would be an active specific immunotherapy that selectively kills virus infected cells and the cytotoxic cells which result in tissue damage and mediate disease.
Vaccine Studies
The HTLV-1 genome is fairly stable which makes vaccine development a feasible proposition. There is a high degree of genetic and antigenic relatedness among the different
HTLV-1 isolates around the world. The sequence variation among the individual genes such as the envelope ranges from 5-7% in certain isolates from Melanesia to 2% in other isolates from Europe and South America (Hart et al., 1995). Besides, HTLV-1 isolates from
HAM/TSP as well as ATLL have been found to be genotypically identical or with minor variations (de The and Kazanji, 1996). Thus, in comparison to other retroviruses such as
HIV, antigenic variation may not be a stumbling block. More importantly, immune responses against viral proteins have been observed in infected individuals and studies to map the highly immunogenic regions may be useful in the design of vaccination strategies.
13 Another encouraging fact is that there have been several successful vaccines developed
against animal oncoretroviral agents that have been used to prevent certain malignancies in
animals (Earl et al., 1986; Lewis et al., 1981; Marx et al., 1986; Miyazawa et al., 1992;
Osterhaus et al., 1989; Plata et al., 1987; Schaller et al., 1977; Tartaglia et al., 1993).
An effective vaccine should ideally harness the enormous potential of the immune
system and activate all arms (humoral or antibody and cell-mediated) of the immune system.
Prevention of disease is obviously the first choice when one considers the potential of a
vaccine. However, in the case of HTLV-1 where disease may occur decades after initial
infection, therapeutic vaccination strategies aimed at ameliorating disease in already infected
individuals would be the best choice. In the last decade or so, many investigators have
focused their efforts in delineating the immunological determinants that have the capacity to
elicit neutralizing antibodies or a protective cell mediated immune response. For the purpose
of therapeutic interventions, it is important that those regions in the HTLV-1 encoded
proteins be identified that are able to elicit functional antibodies which will be orchestrative
in the elimination of the virus in infected individuals. In this respect, there are several
mechanisms by which antibodies can mediate virus neutralization. Antibodies can directly
prevent virus spread in vivo by preventing syncytia formation. Syncytia formation is very
important for HTLV-1 as transmission occurs via cell to cell fusion and infection by free
virus has been found to be very inefficient. Thus those antibodies that bind to regions of the
envelope proteins that are involved in fusion with the host cell membrane would be effective
in preventing spread of the disease. Also free virus may be opsonized by specific antibodies
and thus are rendered more susceptible to phagocytosis. Antibodies also can indirectly kill virus infected cells by recruiting the innate arm of the immune system in the form of
14 antibody dependent cellular cytotoxicity (ADCC) mediated by natural killer cells or
complement mediated cytotoxicity where the Complement system comes into play (Burton,
2002). Both these mechanisms serve to eliminate HTLV-1 infected cells in vivo and thus prevent further spread of the virus. Although NK cells need not be sensitized and can be activated before acquired immunity develops, its activity has been enhanced in the presence of anti-HIV antibodies and thus one can assume that the same may occur in the case of
HTLV-1 infection.
Mapping humoral determinants
Several linear determinants that elicit protective antibody responses have been mapped using sera from infected individuals and synthetic peptides derived from different regions of the envelope glycoprotein. Matsushita et al were among the first to make a human monoclonal antibody against gp46 surface glycoprotein of HTLV-1 which was found to have neutralizing properties in tissue culture. The epitope for this antibody was subsequently narrowed down to amino acids 186-195 of gp46 (Matsushita et al., 1986; Ralston et al.,
1989). In addition, a human monoclonal antibody to this region exhibited antibody- dependent cellular cytotoxicity against an HTLV-1 producing cell line (Kuroki et al., 1992a).
In studies with human monoclonal antibodies, Baba et al further identified three more epitopes within the same region namely, amino acids 187-193, 191-196, and 193-199. They also made a conjugated peptide vaccine using a branched polylysine oligomer and obtained good proliferative responses in rat, rabbit and human PBMCs thus illustrating the presence of T-helper epitopes within this region (Baba et al., 1995). Based on these studies the central region between amino acids 187-209 was defined as the principle neutralizing region of gp46
15 as antibodies to this region abrogated the infectivity of HTLV-1 as demonstrated by the inhibition of HTLV-1 mediated syncytium formation and cellular transformation.
Besides this immunodominant region, other regions in gp46 as well as gp21 have
been described by various studies. One of them lies between gp46 residues 91-105 (Horal et
al., 1991). Parker et al have shown that amino acid sequence 90-98 especially the asparagines
at position 93 and 95 are critical for the peptides to recognize neutralizing antibodies. These
sequences were also type specific in that they were unable to adsorb HTLV-II sera raised in
a similar manner. Studies conducted by Inoue et al further confirm this region to have
neutralizing properties in terms of ability to interfere with syncytia formation (Inoue et al.,
1992).
Another region described by Baba et al and Inoue et al (Baba et al., 1993; Inoue et al.,
1992) as neutralizing lies between amino acids 287-317. Although this region showed
promise in earlier studies, recently, Grange et al failed to elicit neutralizing antibody
responses using this and other peptides in conjunction with carrier proteins (Grange et al.,
1998). This may be due to the fact that carrier protein associated peptides do not mimic the
native structure of the envelope protein due to interference of a large carrier molecule in the
folding of a relatively small peptide and therefore, other forms of immunization may have to
be resorted to in order to achieve higher immunogenicity with these peptides.
A third region of interest is the central region of gp46 spanning amino acids 239-261,
as determined by the high reactivity of this region with HTLV-1 positive sera. There is
regional homology between human IL-2 receptor and gp46 residues 246-253 and this may be responsible for triggering cellular proliferation independently of HTLV-1 infection via the
IL-2β receptor. This particular consensus sequence has also been found in the C-terminal
16 end of v-erb protein that encodes a truncated version of the epidermal growth factor
receptor (EGFR) which plays a functional role in signal transduction leading to cell
proliferation (Lal, 1991; Londos-Galgliardi et al., 1997). The region has characteristic N-
linked glycosylation sites. Many investigators have tested a panel of peptides from this region
and have found that they do mimic the native protein as they show reactivity to gp46
recombinant protein and also show high reactivity against sera from infected individuals.
However the antibodies did not prevent syncytia formation (Lal et al., 1991). This may be
attributed to the fact that these antibodies were raised to a non-glycosylated synthetic
peptide from that region and glycosylation may have been important for the production of
neutralizing antibodies. However, Kaumaya et al in studies to examine the role of
glycosylation in immune recognition and antibody responses showed that there was no
significant difference in the titers of antibodies obtained with the glycosylated and non-
glycosylated versions of a similar peptide derived from this region (Kaumaya et al., 1995).
Thus it could be possible that the correct B-cell epitope has not been mapped although the
region is important for infectivity and further work needs to be done with this region to
identify the correct B-cell epitope.
The transmembrane protein of the HTLV-1 envelope, gp21 has only received attention in recent years although seroreactivity to epitopes from this region has been
demonstrated earlier (Horal et al., 1991). Sagara et al have also studied this region and have shown that antibodies to central residues 400-429 abrogate syncytium formation to an extent of 97% (Sagara et al., 1996). Further, mutations within these residues observed in different isolates do not affect the ability of this region to aid syncytium formation. It has been shown that antibodies against epitopes within this sequence inhibit syncytium formation at a post
17 binding step in the fusion process (Jinno et al., 1999). T-helper epitopes have been mapped to this region in HAM/TSP patients and it has been suggested that the gp21 region is somehow involved in the immunopathology of HAM/TSP as the responses to gp21 in these patients is relatively high as compared to the asymptomatic individuals (Yamano et al., 1997).
Recently the crystal structure of the gp21 protein has been solved as a maltose binding protein chimera. The structure reveals an N-terminal hydrophobic fusion peptide, a coiled- coil forming sequence, a disulphide bonded loop followed by a C-terminal segment that has
α-helical character similar to other retroviral transmembrane domains that have been studied
(Kobe et al., 1999). The structure data enables better understanding of the sequences that are
involved in the multi-step fusion process and the conformational changes that are brought
about in the TM region of the envelope to facilitate fusion. This in turn would help us to better design conformation dependent epitopes that would elicit antibodies that target these transitional structures and prevent further infection by the virus.
Mapping T-cell determinants
In the case of viruses that persist within the host, humoral responses may not be sufficient to clear infection and residual infected cells could pose as reservoirs for virus reactivation and disease development. Cytotoxic T-cells have been shown to eliminate virus infected cells and are activated by peptides derived from various viral antigens that are presented in the context of MHC Class I by antigen presenting cells as well as the cells which are infected with the virus. This recognition is specific for both the peptide as well as the
MHC haplotype. Factors which influence the immunodominance of a peptide epitope are dependent on the efficiency of the generation of the epitope within the cell, transport of the peptide into the endoplasmic reticulum (ER) for loading onto nascent MHC Class I
18 molecules which in turn is dependent on its affinity for Transporter Associated with Antigen
Processing (TAP) and the affinity of the peptide for the particular MHC Class I molecule
itself.
Approximately 5% of the individuals infected with HTLV-1 develop disease; some of them develop fatal Adult T-cell Leukemia while the others develop a slowly progressive
demyelinating disorder of the central nervous system. The cellular immune response against
HTLV-1 is very unconventional. A vast majority of infected individuals suffering from
HAM/TSP exhibit chronically activated virus specific CTLs that are directed predominantly
against the Tax protein, specifically to the Tax residues 11-19 (LLFGYPVYV). These specific CTLs which are of the CD8+ phenotype are seen both in the peripheral blood as well as in the cerebrospinal fluid (CNS) in HAM/TSP patients and are thought to play a role in the pathogenesis of the disease (Jacobson et al., 1990b). This is a phenomenon unique to
HAM/TSP and other HTLV-1-associated inflammatory disorders. Other studies however report conflicting results with asymptomatic HTLV-1 infected individuals that suggest that although the activated antigen specific CTL may contribute to the pathogenesis of
HAM/TSP they may not be the actual cause of the disorder (Kannagi et al., 1993). The specific sequence is also highly conserved and alanine scanning mutagenesis has shown that mutations in any of the aromatic residues or in the internal valine causes a reduction in the lytic responses, although it is not clear if this is due to weakened binding to the Class I molecule or loss of recognition by the TCR (Koenig et al., 1993). The frequency of CD8+ activated CTLs in HAM/TSP have been found to be as high as 1 in 86 and 1 in 60 in some of the patients diagnosed, while the frequency of precursor CTL specific for certain other viruses such as mumps and measles are 1 in a million or less lymphocytes (Levin and
19 Jacobson, 1997a). However, the frequency of activated CD8+ CTLs was not as high in patients with ATL or in seropositive asymptomatic individuals (Jacobson et al., 1990a). The apparent absence or low antigen-specific cytotoxic responses in ATL patients and the observation that HTLV-1 specific CTL are able to recognize and lyse leukemic cells in vitro leads us to believe that low cytotoxic cell activity in these patients may be related to the uncontrolled proliferation of ATL cells (Ohashi et al., 1999). The high frequency of cytotoxic cells is often accompanied by a high proviral load in the PBLs of HAM/TSP patients (Kubota et al., 2000). One of the hypothetical models that have been put forth to explain this peculiar immune response seen in HAM/TSP patients is that the T-lymphocytes directly recognize viral antigens or cross-reactive self peptides on the surface of certain target cells in the CNS leading to tissue damage and autoimmune disorder. The disparity seen in patients suffering from ATL is not fully understood but it could be possible that there are suppressor mechanisms that are at play or that the T-helper functions are not optimum
(Kannagi et al., 1994).
The Tax protein expression is chronic and is seen in the early stages of virus infection. It is thought to influence virus replication as it is expressed early. Besides the tax region has been described as a viral oncogene and has the capacity to control cellular transcription as well as prevent DNA repair promoting mutations in the genome. It also has been shown to induce apoptosis in Tax expressing cells. Tax mRNA is regularly detected in infected cells in vivo.
Activated CTLs to other viral products such as envelope and polymerase are also seen but to a lesser extent. Other CTL epitopes that have been described lie in the middle region of the
Tax protein as well as one from the Pol protein, from amino acid 863-877 (Parker et al.,
1992). Cytotoxic cells of the CD4+ HLA Class II restricted type were also identified in
20 HAM/TSP patients. These CTLs were found to be specific for epitopes within the gp46
envelope protein between amino acids 196-209. This sequence lies within the principle
immunodominant region of the HTLV-1 envelope protein as described earlier and overlaps
with the strong B-cell epitope but is distinct from the region that reacts with antibodies from
patients (Jacobson et al., 1991). Several studies have also identified CTL epitopes that are restricted to other HLA haplotypes such as HLA-B14 and HLA-A3 (Elovaara et al., 1993).
Animal models and protection studies
Over the past several years many models of HTLV-1 infection have been developed to assess the protective efficacy of various vaccines formulations. The nature of HTLV-1 infection such as the highly cell associated properties and the fact that disease is only observed in a small proportion of the infected population, make it challenging to develop pertinent animal models. There are studies which demonstrate the protective efficacy of entire envelope protein, both gp46 and gp21 in monkeys that were challenged with the
HTLV-1 producing cell line MT-2. The protected monkeys had low titered neutralizing
antibodies but there were no studies to demonstrate the mechanism of protection. Besides, a
large number of boosters were required to achieve protection (Nakamura et al., 1987).
Different types of vectors, including adenovirus vectors or recombinant vaccinia virus and
poxvirus vectors expressing HTLV-1 envelope proteins have been used in rats and/or
rabbits that resulted in partial protection as judged by the reduction in proviral loads. A
study by Ibuki et al in cynomogus monkeys demonstrated protective envelope specific
humoral and cell mediated activity after vaccination with a recombinant vaccinia virus expressing the gp46 envelope protein (Ibuki et al., 1997). The rabbit model for infection has
been extensively used to study infection, lymphoproliferation associated with HTLV-1
21 disease and for vaccine studies (Akagi et al., 1985; Cockerell et al., 1990; Collins et al., 1996;
Franchini et al., 1995; Lairmore et al., 1992; Miyoshi et al., 1985; Sawasdikosol et al., 1993;
Seto et al., 1987; Simpson et al., 1996; Zhao et al., 1995). Challenge studies have also been conducted in rabbits immunized with vaccinia virus constructs expressing HTLV-1 envelope glycoproteins. High titered neutralizing antibodies were demonstrated but the cellular arm of
the immune system was not investigated (Shida et al., 1987). In contrast, in a more recent
study, Hakoda et al failed to demonstrate protection in most of the vaccinated rabbits using
recombinant vaccinia virus carrying the HTLV-1 envelope gene (Hakoda et al., 1995).
Attempts have also been made to establish a chronic infection in rats to provide a small
animal model for vaccine studies. However, infection in rats is highly suppressed and
correlates with transient or low to negligible cellular or humoral responses. In this regard,
one study reported the induction of ATL like lymphoproliferative disease in adult nude rats
within two weeks of subcutaneous inoculation of HTLV-1-immortalized rat cell lines. In
addition, cytotoxic T-cells obtained from syngeneic immunocompetent HTLV-1-immunized
rats were able to lyse the tumors in the nude rats. Further, it has also been shown that
blockade of CD80 and CD86 costimulatory signals in rats leads to the formation of tumors
which regressed when treatment with monoclonal antibodies to CD80/CD86 was
withdrawn (Hanabuchi et al., 2000; Ohashi et al., 1999). In another report, Hanabuchi et al
fine mapped a Tax CTL epitope (sequence 180-188) recognized by rats and showed that
adoptive transfer of T-cells from rats immunized with the peptide into athymic rats
prevented the development of lymphomas when challenged with an HTLV-1 infected rat
cell line (Hanabuchi et al., 2001). These studies taken together underscore the importance of
cytotoxic responses in tumor surveillence. For the purpose of studying human immune
22 responses to vaccine candidates non-human primates may be the best choice in terms of similarity and relatedness to the human system. Several non-human primates have been found to be susceptible to HTLV-1 infection. Some of these include the marmosets
(Callithrix jacchus), cynomolgus macaques (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina). Recently Kazanji et al have developed a squirrel monkey model (Kazanji, 2000).
While these monkeys are susceptible to infection, the main advantage over other primate models is that squirrel monkeys are New World monkeys and they do not harbor the STLV virus or other related retrovirus, which may cause complications especially in vaccine studies.
The peripheral blood mononuclear cells (PBMCs), spleen and lymph nodes are the major reservoirs for HTLV-1 in these monkeys during the early stages of disease (Kazanji et al.,
2000). Further, env-DNA prime, vaccinia-virus derived NYVAC (gag + env) boost regimen could protect the monkeys against subsequent challenge with HTLV-1 infected cells
(Kazanji et al., 2001).
Essentially, there have been two approaches to vaccine development, those that elicit strong protective humoral responses and the other, which focuses on cellular responses because CTLs play a very significant role in the protection against exogenous pathogens such as viruses and other intracellular pathogens and is required for the clearance of virus and/or pathogen infected cells. In one study, candidate CTL epitopes from various HTLV-1 proteins have been identified based on their ability to strongly bind the HLA-A2 molecule
(Pique et al., 1996b). This molecule has been widely studied in the context of CTL vaccine development because it is the most frequently occurring haplotype in the human population and thus vaccines incorporating such MHC restricted constructs would be applicable in an
23 outbred population. Interest has been shown in the gag protein for cytotoxic responses against HTLV-1 but extensive studies are still awaited.
The studies of cytotoxic responses to HTLV-1 or for that matter any virus associated disorders, is limited by the problem of MHC restriction and the need of appropriate animals models with compatible MHC expression is an absolute requirement to evaluate the cellular responses to candidate epitopes in vivo prior to challenge studies or clinical trials. In this regard studies with the HLA-A2/Kb transgenic mice have been carried out for the purpose of evaluating candidate predicted epitopes of influenza virus where it has been possible to generate epitope specific CTL responses in mice that have the transgenic chimeric HLA-
A2/Kb molecule (Vitiello et al., 1991). More recently, Pascolo et al have developed another mouse model that expresses a chimeric human HLA-A2/Db molecule but additionally these mice are β2m and H-2Db knockouts. Thus they are devoid of classical murine MHC Class I molecules. T-cells in these mice are educated only in context of the chimeric molecule and this would prevent any bias toward the murine Class I molecule (Pascolo et al., 1997). This is an interesting model to study the induction of HLA-A2 restricted cytotoxic responses in an in vivo situation without the potential interference from bonafide murine Class I molecules.
Schonbach et al tested the immunogenic potential of putative HLA-B*3501 restricted CTL epitopes derived from both Tax, Envelope as well as Gag and Pol proteins by immunization of HLA-B*3501 transgenic mice with peptide constructs in conjunction with
water soluble, amphiphilic P3C-Ser-(Lys)4 as adjuvant. These mice expressed the cognate human HLA-B*3501 molecule, the rationale being to assess the CTL responses in the context of weak allogenic CD8+-HLA-B*3501 interactions and its effects on the immunogenicity of the binding peptides. Four HLA-B*3501 restricted CTL epitopes were
24 identified which induced cytotoxic responses against the epitope as well as the naturally processed antigen (Schonbach et al., 1996). This study is the first to use a transgenic mouse model to demonstrate immunogenic potential of peptides against HTLV-1.
Peptide vaccine approach
Over the past several years our laboratory has focused on the development and testing of molecularly defined epitope based peptide vaccine approaches that will specifically activate B-cells, helper T-cells and cytotoxic T-cells. Such epitope based vaccines have the advantage of being specific in their target as opposed to whole protein based vaccinations where multiple responses are generated, some of which may be suppressive in nature.
Further, peptide vaccines are easy to produce with high reproducibility, offer high safety with low toxicity than other forms of vaccinations such as recombinant vaccines especially those that require the use of live viral vectors, which may have infectious or oncogenic potential. The focus has been in the design of peptides that have a defined secondary structure which closely mimics that observed in the native protein. This is crucial especially in the design of peptides to elicit antibodies that have neutralizing properties. Our laboratory has conducted extensive studies on the testing of chimeric B-cell epitope peptide vaccines against HTLV-1 based on the principle immunodominant region (Frangione-Beebe et al.,
2000; Lairmore et al., 1995a; Lairmore et al., 1995b). These vaccines incorporated a B-cell epitope and a promiscuous T-helper epitope synthesized in a colinear fashion and separated by a turn sequence such that the two peptides are able to fold independently of one another.
One main obstacle in the design of subunit peptide vaccines is MHC restriction. T- cells only recognize peptide in the context of the cognate MHC allele. B-cells require “help” from T-helper cells to activate them so they can differentiate into antibody producing plasma
25 cells. Cytotoxic T-cells (CTL) are also activated by cytokines produced by specific T-helper
cells. A three-cell model has been proposed to explain the activation of antigen specific
CTLs. In this model the antigen presenting cell (APC) engages both the antigen specific CTL
and the T-helper cell by displayed peptides in the context of MHC Class I and MHC Class II respectively. The T-helper cell then produces cytokines and expresses costimulatory
molecules that lead to the highly activated state of the APC which can then directly prime
the CTL (Stuhler and Schlossman, 1997). In the classical method of immunizing peptides
conjugated with carrier proteins, the carrier proteins provided the necessary T-helper
epitopes in an outbred population. However, this method of immunization is beset with
problems such as poor reproducibility during carrier protein conjugation, hypersensitivity
and most importantly epitope suppression that could arise due to the presence of
suppressive epitopes in the carrier protein or undesired immune response directed towards
the carrier protein itself. There have been several reports describing certain T-helper
epitopes that have the capacity to bind several different MHC molecules across species and
hence have been described as “promiscuous”. These epitopes have been derived from
different infectious organisms such as measles virus, tetanus toxoid, Hepatitis B and malaria
(Fern and Good, 1992; Francis et al., 1987; Ho et al., 1990; Panina-Bordignon et al., 1989a;
Partidos and Steward, 1990). To bypass MHC restriction our lab proposed the use of these
promiscuous T-helper epitopes as a component of subunit vaccines to achieve broad
coverage of the spectrum of MHC alleles present in the human population (Kaumaya et al.,
1993; Kaumaya, 1992; Kaumaya, 1993b). Additionally the use of alternative adjuvants and
delivery systems for peptide based vaccines have also been tested and optimized for
induction of high immune responses (Frangione-Beebe et al., 2001; Kaumaya, 1997).
26 Given the above advancements, the long term goal was to develop a universal multiepitope vaccine that activates the humoral arm and the cellular arm of the immune system for effective clearance of infection. In pursuit of this broad goal, these studies describe the evaluation of B-cell epitopes derived from the gp46 and gp21 regions of the envelope protein and the engineering of peptides to fold into complex secondary structures found in the native protein. Likewise, peptides constructs designed to elicit cytotoxic T-cell responses to multiple determinants simultaneously was also attempted and its protective efficacy was demonstrated in a relevant preclinical model of infection. These results indicate that immunotherapy of HTLV-1 associated diseases is feasible and further advancements and studies to enhance protective immunity should be pursued.
27 CHAPTER 2
DESIGN AND CHARACTERIZATION OF A MULTIEPITOPE CTL PEPTIDE
CONSTRUCT IN HLA-A*0201 TRANSGENIC β2M, Db DOUBLE KNOCKOUT
MICE
2.1 RATIONALE
The humoral response against HTLV-1 is mainly directed against the envelope
glycoproteins gp46 and gp21 which play a critical role in protection. The cell mediated responses are largely directed against the HTLV-1 regulatory protein Tax which is known to
interact with various cellular transcription factors promoting genetic mutations besides
driving viral transcription (Johnson et al., 2001). Several studies have demonstrated the
presence of highly activated cytotoxic T-lymphocytes in HAM/TSP patients as outlined in
the previous chapter.
It is clear that to develop a more potent long lived antiviral response, a vaccine that
would stimulate multiple components of the immune system is absolutely essential to prime
the immune system in a manner similar to that seen during a natural immune response
against an infectious agent. This requires identifying immunogens that can elicit broadly
neutralizing antibody (B-cell) responses as well as a broadly reactive T-cell response (CD4+
helper T-cell and CD8+ cytotoxic T-cell) that is long lasting. This chapter describes the
characterization of CTL epitopes that are critical in eliciting cell-mediated immunity which is
a prominent effector mechanism for controlling and eliminating viral infection/replication.
28 The Tax protein is an ideal candidate for the design of HTLV-1 T-cell vaccines because the presence of activated CTLs to a regulatory protein that is expressed early in the
infection process and which is essential for the replication of the virus would be useful to
inhibit virus replication at an early stage (Parker et al., 1994a). Furthermore, studies have
demonstrated a negative correlation between the presence of the Tax-specific CTLs and the
percentage of infected cells in peripheral blood of HTLV-1 positive individuals (Hanon et
al., 2000). Cytotoxic T-cell epitopes have also been identified and isolated from patients and
these CTLs were efficient in lysing Tax specific targets in vitro (Koenig et al., 1993; Parker et
al., 1994a; Pique et al., 1996a). These studies are encouraging and suggest that the immune
system may be manipulated by vaccinations so that natural response to virus infection could
be enhanced sufficiently to effectively arrest the further replication of the virus. However,
none of these human CTL epitopes have been tested in an active immunization setting to
determine their immunogenicity in animal models. An in vivo assessment of these epitopes in appropriate transgenic animal models is crucial in the development of immunotherapeutic strategies against HTLV-1.
Given the advances in the elucidation of MHC peptide complexes and the activation of cytotoxic T-cells, several strategies are being used to amplify the CTL response. These include the use of minigene, string of beads protocols, and more recently, provision of genes encoding interleukins (IL-12, GM-CSF) and costimulatory molecules (Ahlers et al., 2001;
Kalus et al., 1999; Kim et al., 1998; Oldstone et al., 1993; Whitton et al., 1993). Polyvalent synthetic peptides may offer greater safety, especially in immunocompromised individuals as opposed to recombinant vaccines. There have been some studies to elicit CTL responses of multiple specificities using long peptides derived from the antigenic protein, that encompass
29 one or more CTL epitopes to increase the stability and hence the probability of the relevant
epitope being presented for CTL activation (Correale et al., 1998; Eberl et al., 1999). Multiple factors determine the antigenicity of determinants that include antigen fragmentation, binding to the MHC molecules, and resultant stability of peptides. Both the preferential proteasomal degradation pathway and the TAP transport system have been shown to restrict the repertoire of peptides that are available for binding MHC Class I molecules (Driscoll et al., 1993; Gaczynska et al., 1993; Ossendorp et al., 1996; Savory et al., 1993). The delivery of peptides into antigen presenting cells for endogenous presentation in the context of MHC
Class I may potentially restrict the applicability of peptides to induce long lived CTL responses. It is also becoming increasingly clear that single epitopes may not be sufficient in clearing viral infections and that multiple epitopes are highly desirable that may result in an enhanced biological effect against the invading virus. This is especially true in a diverse population where differential responses may be observed against single epitopes. Hence the inclusion of multiple epitopes would broaden population coverage and increase the efficacy of the vaccine. In addition, multiple epitopes would be more effective in the case of viral escape mutants, which poses another major concern (Furukawa et al., 2001).
We hypothesized that a specifically engineered peptide comprising several optimum length CTL epitopes separated by an intervening sequences comprised of proteolytic cleavage sites for intracellular processing could be utilized to efficiently elicit cytotoxic responses in vivo. The goal was to examine the intracellular delivery, processing and presentation of multiple epitopes by the same antigen presenting cell for activation of CTLs of multiple specificities and to test the immunogenicity of various CTL epitopes mapped from the Tax protein of HTLV-1.
30 2.2 SUMMARY
To address our hypothesis, three HLA-A*0201 epitopes from the Tax protein of
HTLV-1 were selected and synthesized as one multiepitope construct separated by double
arginine residues (Arg-Arg) in tandem to allow for intracellular processing by cellular proteasomes. Peptide digestion analysis by mass spectrometry demonstrated the liberation of the individual CTL epitopes by 20s proteasome. The peptide construct was highly
immunogenic in HLA-A*0201 transgenic, Db, β2m double knockout mice eliciting robust
CTL responses in vivo against each intended epitope in the multiepitope construct, as
assessed by cytolytic and ELISPOT assays, indicating that the peptide was being efficiently
internalized and processed by antigen presenting cells to prime the specific CTLs. These data
are supportive of a multiepitope peptide T-cell vaccine strategy to simultaneously prime
multispecific CTL responses against HTLV-1.
2.3 MATERIALS AND METHODS
Peptide Synthesis and Purification
All peptides were synthesized either on a Milligen/Biosearch 9600 peptide synthesizer
(Bedford, MA) or on a multiple peptide synthesizer (Model 396; Advanced Chemtech,
Louisville, KY) using a 4-methylbenzhydrylamine resin as the solid support (substitution
0.54mmol/g) as described earlier (Kobs-Conrad et al., 1993). The Fmoc/t-butyl synthetic
method was used, using 4-(hydroxymethyl) phenoxyacetic acid as the linker. After the final
deprotection step, protecting groups and peptide resin bond were cleaved with 90%
trifluoroacetic acid, 5% anisole, 3% thioanisole, and 2% ethanedithiol. The crude peptides
were purified by semi-preparative reverse-phase high performance liquid chromatography
(RP-HPLC) using a Vydac C4 or C18 column (10mm by 25cm) and were >95% pure before
31 immunization. The identity of the peptides was confirmed by matrix assisted laser
desorption ionization-time of flight mass spectrometry (Kratos IV MALDI-TOF) at the The
Campus Chemical Instrument Center (CCIC, Columbus, OH).
In Vitro Proteasomal Digestion of Peptide Substrates
Twenty micrograms of the synthetic 31mer multiepitope peptide substrate was incubated
with 1µg of 20s constitutive proteasome purified from human erythrocytes or 20s
immunoproteasomes purified from LCL 721 cells, containing the IFN-γ inducible subunits
LMP2, LMP7 and MECL-1 (Toes et al., 2001) (Immatics Biotechnologies Inc, Germany) at
37 0C for indicated time points in a total volume of 300µl of assay buffer (1mM HEPES pH
7.8, 2mM MgAc2 and 1mM dithiothreitol) as previously described (Kessler et al., 2001;
Theobald et al., 1998). The reaction was stopped using 30µl of TFA (Pierce) and stored at –
20 0C until analysis.
Peptide Digestion Analysis by Electrospray Mass Spectrometry
Capillary liquid chromatography-electrospray mass spectrometry (CapLC-MS) was
performed on a hybrid quadrupole time-of-flight mass spectrometer Q-TOF2 (Micromass
Inc., Manchester, UK) for the standard proteasome analysis. The CapLC system was from
Waters Co. (Milford, MA). Pumps A and B were used for gradient elution and pump C was used for loading samples onto a trapping/desalting column (0.3 mm x 5 mm, C18PM, LC-
Packings, CA). The solvent A was water containing 0.02% trifluoroacetic acid (TFA) and
the solvent B was acetonitrile containing 0.02% TFA. Five microliters of each sample was
first loaded onto the trapping column at a flow rate of 30 µl/min for 3 minutes, and then back-flushed onto a 0.32 mm x 15 cm Symmetry C18 column (5 um, 300 A, Waters) with a
10-port switching valve. Peptides were eluted into the Q-TOF system using a gradient of 3-
32 80% B over 20 minutes, with a flow rate of 5µl/min. The total run time was 35 minutes. A standard electrospray ionization interface was used for the CapLC-MS experiments, with the
electrospray capillary voltage at 3 kV and cone at 35 V. The source and desolvation
temperatures were maintained at 80 0C and 150 0C, respectively, and the desolvation and
cone gas flow rates were kept at 300 L/h and 50 L/h. Mass spectra were recorded from
mass 300-5,000 daltons every 2 seconds with a resolution of 10,000 (FWHM). For the 20s
immunoproteasome digestion analysis, capillary liquid chromatography-nanospray tandem
mass spectrometry was performed on a separate Q-TOF2 system equipped with an
orthogonal nanospray source from New Objective, Inc. (Woburn, MA) operated in positive
ion mode. The LC system was a Waters Alliance 2690 Separation Module (Waters, Milford,
MA). The solvent A was water containing 50mM acetic acid and the solvent B was
acetonitrile. 10µl of each sample was first injected on to the trapping column, and then
washed with 50 mM acetic acid. The injector port was switched to inject and the peptides
were eluted off of the trap onto the column. A 10 cm 50 mM ID BioBasic C18 column
packed directly in the nanospray tip was used for chromatographic separations. Peptides
were eluted directly off the column into the Q-TOF system using a gradient of 3-80%B over
35 minutes, with a flow rate of 280µl/min with a pre-column split of about 500nl/min. The
total run time was 70 minutes. The nanospray capillary voltage was set at 2.8 kV and cone at
55 V. The source temperature was maintained at 100 0C. Mass spectra were recorded using
MassLynx 3.5 automatic switching functions. Mass spectra were acquired from mass 300-
2,000 daltons every 1 second with a resolution of 8,000 (FWHM). When the desired peak
(using include tables) was detected at a minimum of 8 ion counts, the mass spectrometer
automatically switched to acquire CID MS/MS spectrum of the individual peptide. Collision
33 energy was set dependent on charge state recognition properties. Sequence information
from the MS/MS data was processed using the MassLynx 3.5 Biolynx software. The
intensity of the peaks in the mass spectra was used to estimate the relative amounts of peptides generated after digestion and expressed as a percentage of the total amount of peptide digested at the given time point.
Mice and Immunizations
The transgenic HHD mice have a chimeric HLA-A*0201/Db transgene consisting of the α1
(H) and α2 (H) domains of the HLA-A*0201 linked to the α3 transmembrane and
cytoplasmic domain of H-2Db(D). Also, the α1 region is covalently linked to human β2
microglobulin. The transgene was introduced into mice that are H-2Db and β2 microglobulin
knockouts. Hence the only Class I molecules that these mice express is the chimeric HLA-
A*0201/Db (Pascolo et al., 1997). The transgenic mice were provided by Dr. Francois
Lemonnier (Pasteur Institute, France). The mice were rendered pathogen free by caesarian
rederivation in collaboration with Dr. Maurizio Zanetti (UCSD, San Diego). Pathogen free
mice were bred and housed at the Ohio State University in an AAALAC accredited facility.
All animal procedures were approved by the institutional laboratory animal care and use
committee.
For all peptide immunizations, each mouse received 100µg of peptide mixed with 140µg
TT3, a promiscuous T-helper epitope from tetanus toxoid (residues 947-967) and 100µg of
adjuvant N-acetyl-glucosamine-3-acetyl-L-alanyl-D-isoglutamine (nor-MDP) (Moulton et al.,
2002) (Peninsula Laboratories, Belmont, CA) and emulsified 50:50 in 4:1 squalene:arlacel A
(Sigma, St Louis). For the equimolar mixture immunizations, each mouse received a mixture
of 33µg of each peptide + 140µg of TT3 in one emulsion. The emulsions were injected 34 subcutaneously at the base of the tail. All mice were boosted once, 3 weeks after primary immunization and spleens were harvested 10 days later.
Cell Lines
All cell culture media, FCS and supplements were purchased from Life Technologies, Inc.
(Grand Island, NY). The murine EL-4S3-Rob (HHD) (EL4/HHD) cell line was obtained from Dr. Francois Lemonnier and has been described elsewhere (Pascolo et al., 1997). These are EL-4 cells that are β2-microgloblin and H-2Db knockouts transfected with the HLA-
A*0201/Db (HHD) chimeric gene. This cell line was maintained in RPMI 1640 medium supplemented with 10% FCS, p/s and 0.5mg/ml G418. Frozen human PBMCs from an
HLA-A*0201 positive HAM/TSP patient was provided by Dr. Steve Jacobson. The PBMCs were cryopreserved until they were used for the ELISPOT assay.
Purification of CD8+ T-cells
Single cell suspensions from spleens of immunized mice were RBC depleted (0.83% NH4Cl) and washed once in PBS containing 0.5%BSA and 2mM EDTA (MACS buffer). The CD8+
T-cells from splenocytes were positively selected using MACS microbeads and positive selection column type LS+ according to manufacturer’s instructions (Miltenyi Biotech,
Auburn, CA). Briefly, 50-75x106 cells were incubated with CD8a (Ly-2) microbeads for 15 minutes at 6o C. Cells were then washed in MACS buffer and applied onto the LS+ column placed in a magnetic field. Negative cells were passed through the column by rinsing with buffer. The positive cells were then flushed out of the column after removing the column from the magnetic field.
35 Measurement of Cytolytic Activity
10 days after the first boost, spleens were harvested from immunized mice and pooled.
Single cell suspensions were prepared by teasing out and crushing the spleens using a cell strainer. 4x106 splenocytes were stimulated with 1x106 splenocytes from the immunized pool
that were pulsed separately with 100µg of relevant nonamer peptide at 37 0C for 1 hour and
washed twice before use. In the case of multiepitope peptide construct immunized mice or
mice immunized with an equimolar mixture of the three epitopes, splenocytes were pulsed
µ separately with individual epitopes (Tax 11-19, Tax 178-186 or Tax 233-241) or a mixture of 100 g of
each epitope. Cells were cultured in RPMI 1640 with 10% FCS, 1% pen/strep and 25mM
Hepes buffer supplemented with 5% Rat-T-Stim without ConA (BD Labware, Bedford,
MA). 1 ml of culture medium was replaced every 48 hours. On day 7 the cells were harvested for use as effectors in a standard 4-hour 51Cr release assay using EL4/HHD cells as targets.
µ 51 6 Target cells were radiolabelled with 100 Ci Na2 CrO4 (Perkin Elmer, Boston, MA)/10 cells for 1 hour. The target cells were also sensitized with 100µg relevant peptide or a mixture of the three peptides at the time of radiolabelling. The cells were washed 3 times
(RPMI 1640 + 10% FCS and 1% pen/strep) before use. 5000 target cells were plated onto triplicate wells of 96-well round bottom tissue culture plates and used at different effector to target ratios (E:T) as indicated. After 4 hours the supernatants were collected and counted on a γ counter (Beckman model 5500). Spontaneous and maximum (100%) release was determined from wells containing either medium alone or 5% SDS. Specific Lysis was calculated in triplicate as (Experimental release – Spontaneous release)/(100% release- spontaneous)X100.
36 Enzyme Linked Immunospot (ELISPOT) Assay for IFN-γ Detection
The ELISPOT assay was carried out as previously described (Allen et al., 2000). Briefly 96-
well flat-bottom plates (U-Cytech, Utrecht, The Netherlands) were coated with 5µg/ml of
anti-IFN-γ mAb (U-Cytech) overnight at 40C. The plates were washed 5-10 times with PBST
(PBS containing 0.05% Tween 20) and blocked with PBS containing 2% BSA for 1 hour at
370C. The BSA was discarded and purified CD8+ T-cells from freshly harvested and pooled splenocytes of immunized mice were added along with 2.5 X 105 naïve syngeneic splenocytes
irradiated at 5000 rads. Cells were cultured for 18 hours in RPMI 1640 containing 5% FCS,
1% pen/strep and 25mM Hepes buffer in the presence or absence of 10µg/ml final
concentration of relevant peptide. The cells were then removed by flicking the plate and
200µl of ice-cold deionized water was added and incubated in melting ice for 10 minutes to
lyse any remaining cells. The plates were then washed 5-10 times with PBST before adding
1µg/well of anti-IFN-γ detector antibody (U-Cytech). The plates were incubated overnight
at 40C and then washed again 5-10 times with PBST. 50µl of gold-labeled anti-biotin IgG
was added (U-Cytech) was added for 1 hour at 370C and washed 10 times with PBST.
30µl/well of the activator mix (U-Cytech) was then added and the plate was developed for
5-7 minutes. The reaction was stopped by washing with tap water and air dried before counting the spots on an Immunospot Image Analyzer (Zeiss, Oberkochen, Germany). The
# of spot forming cells was calculated after subtraction of background spots from wells containing cells and medium only and no peptide.
For the ELISPOT using human PBMCs, multiscreen filtration plates (MAIPS4510,
Millipore, Bedford, MA) were coated with 15µg/ml anti-IFN-γ mAb (1-D1K, Mabtech,
Sweden) overnight at 40C. The plates were then washed 5-6 times with RPMI-1640 medium 37 and blocked with RPMI 1640 supplemented with L-glu, pen/strep, and 10% HI human AB
serum for 1 hour. 1X105 of freshly thawed cryopreserved PBMCs in the above medium was
added in a total volume of 200µl in the presence or absence of 10µg/ml of relevant peptide
0 and incubated for 24 hours at 37 C and 5%CO2. The cells were then washed off with PBS-
0.05% Tween 20 (PBST) and the plate was incubated with 100µl of 1µg/ml biotinylated
anti-IFN-γ mAb 7-B6-1 (Mabtech, Sweden) for 3 hours at room temperature. The plates were then washed 5 times with PBST and 100µl of a 1:1000 diluted Streptavidin alkaline
phosphatase (Mabtech, Sweden) was added and incubated for a further 2 hours at room
temperature. Next, the plates were washed again in PBST and 200µl of substrate
BCIP/NBT (Kirkegaard and Perry Labs, Gaithersburg, MD) was added and incubated in the dark for 5-10 minutes until the appearance of dark blue spots. The reaction was then stopped by washing with cold tap water and spots were allowed to air dry before counting on an image analyzer.
2.4 RESULTS
Selection and screening of HLA-A*0201 Epitopes from the Tax Protein of HTLV-1
Potential HLA-A*0201 binders from the Tax protein of HTLV-1 were predicted using the computer program developed by Parker et al (Parker et al., 1994b) at the Bioinformatics and
Molecular Analysis Section website (http://bimas.cit.nih.gov/molbio/index.html). The results were compared to HLA-A*0201 restricted epitopes from the Tax protein of HTLV-1 that were previously reported based on in vitro peptide binding assays (Pique et al., 1996a).
The relatively higher scoring epitopes in our prediction analysis namely Tax 11-19 and Tax 178-
186 and Tax 307-315 correlated well with the binding data and were classified as strong binders.
Tax 155-163 was shown to promote HLA-A*0201 assembly at a lower concentration than Tax
38 233-241 implying strong binding, although it scored lower in the prediction analysis. We selected strong binders from the Tax protein (Tax 11-19, Tax 307-315, Tax 155-163 and Tax 178-186) and one moderate binder Tax 233-241. The rationale for the selecting high affinity binders was that the
affinity of a potential epitope is directly related to its capacity to induce CTL responses
(Kubo et al., 1994; Sette et al., 1994). All five selected peptides were synthesized and purified
by reverse phase high performance liquid chromatography. The identity of the peptides was
confirmed by MALDI-TOF mass spectrometry. The predicted epitopes along with their
amino acid sequences and binding classifications are indicated in Table 2.1.
In order to determine if these HLA-*A*0201 binders are presented as epitopes during the
course of natural infection, we determined whether the peptide nonamers could stimulate
peripheral blood mononuclear cells from a HLA-A*0201 positive HAM/TSP patient to
produce IFN-γ in an ELISPOT assay. The rationale for this study was that the cytotoxic T-
lymphocytes in the peripheral blood mononuclear cells in HTLV-1 infected patients would
have already been primed against naturally presented epitopes and hence could be used as an
indication of the immunogenicity of the selected epitopes. Out of the five epitopes that were
γ tested, four epitopes namely Tax 11-19, Tax 178-186, Tax 233-241 and Tax 155-163 stimulated IFN-
production in the PBMCs (Fig.2.1). The Tax 233-241 which was described as a moderate HLA-
5 A*0201 binder gave a high number of spots (44.5 per 10 PBMCs) while the Tax 307-315 which
is a strong HLA-A*0201 binder, failed to elicit IFN-γ production in the PBMCs. The
peripheral blood mononuclear cells specific to the Tax 11-19 epitope were the highest in
5 frequency, with spots in excess of 200/10 PBMCs. This was followed by Tax 178-186 with 60
5 5 spots/10 PBMCs and then Tax 233-241 with 44.5 spots/10 PBMCs.
39 Design of the Multiepitope Peptide Construct
Based on the epitope screening studies, we designed a multiepitope peptide construct using
the three epitopes that were most efficient in stimulating an IFN-γ response in patient
PBMCs. The three epitopes namely Tax 11-19, Tax 178-186 and Tax 233-241 were synthesized colinearly with a dibasic Arg-Arg sequence in tandem as depicted in Fig.2.2. The arginine residues were included as spacers between the CTL epitopes in order to provide for proteolytic cleavage sites to allow for in vivo antigen processing and the generation of intended CTL epitopes. Cytosolic proteasomes are non-lysosomal high molecular weight proteases which play a major role in the generation of MHC Class I ligands by preferentially cleaving peptide substrates after hydrophobic and basic amino acids (Niedermann et al.,
1996). We also reasoned that many high-affinity CTL epitopes are very hydrophobic in nature making them difficult to dissolve in aqueous buffers necessary for immunization and biological testing. Hence polar side chains such as those of arginine would greatly facilitate the solubility of these peptides. Furthermore, in contrast to nonamer peptides, a longer peptide would be less likely to degrade rapidly and allow for prolonged antigen presentation.
Individual Intended Epitopes are efficiently liberated in vitro by 20s Constitutive and
Immunoproteasomes
To determine if the engineered peptide could be effectively cleaved to liberate the individual
CTL epitopes from the precursor peptide constructed using arginine spacers, the
multiepitope peptide was digested in vitro using 20s proteasome. We utilized both the
constitutive and the immunoproteasomes in the in vitro digestion studies, since, although
both types are present in immature dendritic cells, only the immunoproteasomes are
exclusively expressed on mature dendritic cells (Van den Eynde and Morel, 2001).
40 Furthermore, during immunization, as antigen could be taken up by different cell types, we
wanted to delineate any differences in the cleavage patterns. The polyepitope was digested
for 12, 24 and 48 hours. The precursor peptide was completely digested after 48 hours
generating all the three intended nonameric epitopes by both proteasomes (Fig.2.3). The 12
and 24 hour digestion showed the presence of precursor peptide (Mol. Wt. 3605) indicating
incomplete digestion. Fig.2.3A and Fig.2.3B represents the total ion chromatogram traces of
the immunoproteasome and standard proteasome digestion reactions respectively. The
relevant ions for each of the intended epitopes [Mol Wt. 1069.5 (Tax 11-19), 961.3 (Tax 178-186)
and 983.2 (Tax 233-241)] were extracted from the total ion traces and their relative abundance
was calculated as described in section 2.3 and shown in Fig.2.3C and Fig.2.3D. The Tax 11-19 was found to be the most abundant (58%) followed by the Tax 233-241 (7.5%) and then the
Tax 178-186 (0.8%) after cleavage with the 20s immunoproteasome. In the case of the
constitutive proteasome, the relative abundance obtained was lower, i.e. 1.1% for Tax 11-19,
0.3% for Tax 178-186 and 0.4% for Tax 233-241. The sequence of the ion peaks corresponding to
Tax 11-19 and Tax 233-241 peptides was further confirmed by MS/MS analysis. The relative
abundance of Tax 178-186 was insufficient to determine the sequence by MS/MS. In the case
of the constitutive proteasomes, we found that the three intended epitopes were liberated
although with lower efficiency as compared to the immunoproteasome. In both the
proteasome digestions, the Tax 11-19 epitope (m/z 1070.5) was the most abundant and the
Tax 178-186 epitope (m/z 962.3) was the least abundant. No spontaneous hydrolysis was
observed when the multiepitope peptide was incubated in the buffer without proteasome for
48 hours.
41 The different fragments that were generated using both the standard and
immunoproteasome are shown schematically in Fig.2.4. The cleavage with the standard
proteasome resulted in efficient liberation of the C-terminus of the intended epitopes that
was also accompanied by a further trimming from the N-terminus. There was no significant
N-terminus trimming with the immunoproteasomes. These results are consistent with the
observation that immunoproteasomes are more precise in the generation of antigenic
epitopes from infectious organisms.
Multiepitope peptide construct is effective in eliciting CTL responses against
individual epitopes in HHD mice
To test if our multiepitope construct was immunogenic in vivo and could elicit HLA- A*0201
restricted cellular immune responses against the individual epitopes, HHD mice were
coimmunized with the multiepitope construct mixed with a promiscuous T-helper epitope
derived from tetanus toxoid (designated TT3, aa 947-967). CD4+ T-cell help has been
shown to be essential in the priming of naïve CTLs and for the maintanenace of effective
CTL responses (Kalams and Walker, 1998; Sauzet et al., 1996). Robust cellular immune
responses were induced against the individual HLA-A*0201 restricted epitopes as detected
in a standard 51Cr release assay using single epitope pulsed, HHD transfected EL-4 cells as
targets (EL4/HHD) (Fig.2.5). However, splenocytes from naïve mice stimulated in vitro with
the relevant peptides did not lyse the same target cells, indicating that the priming of CTLs
was due to immunization with the multiepitope construct. Tax 11-19 epitope and the Tax 178-186 epitope elicited lysis of 32% and 34% respectively (Fig.2.5A and Fig.2.5B). However, the
response against the Tax 233-241 was very weak (approximately 6% lysis, Fig.2.5C). These
results were consistent between independent experiments and clearly indicate that the
42 multiepitope peptide construct was being internalized and processed by antigen presenting
cells and that two of the three individual epitopes were highly immunogenic and were being presented in the context of MHC Class I to prime the peptide specific CTL population.
Furthermore, the splenocytes were also stimulated using equimolar concentrations of all the three individual epitopes and tested for their lytic activity against target cells that were pulsed with equimolar concentrations of the 3 epitopes. The mixture yielded higher lytic responses as compared to that obtained against the individual epitopes alone (Fig.2.6). Thus higher responses may be obtained by the use of multiple epitopes rather than single epitope immunizations.
Frequency of Peptide Specific CD8+ T-cells in HHD Mice Immunized with the
Multiepitope Peptide Construct
The frequency of peptide specific CTLs elicited by the multiepitope construct against individual epitopes was evaluated using the single cell ELISPOT assay for IFN-γ production.
The CD8+ T-cells purified from freshly harvested spleen cells from immunized HHD mice
were used in an 18-24 hour ELISPOT assay. The frequency of Tax 11-19 specific spot forming
units was the highest with over a 100 spots/0.1 million CD8+ T-cells followed by Tax 178-186 with about 50 spots per 0.1 million and then Tax 233-241 with 40 spots/0.1 million CD8+ T-
cells (Fig.2.7). This was as expected since the Tax 11-19 was the strongest MHC Class I binder and also the most efficient in stimulating patient PBMCs. The Tax 233-241 epitope which gave
very low cytolytic responses was very efficient in stimulating IFN-γ production.
Immunogenicity of Individual Epitopes
Since the multiepitope construct was efficient in inducing CTL responses against the
individual epitopes in HHD mice, we next asked the question, is the immunogenicity of the
43 individual epitopes being affected in any way when they are part of a longer peptide
construct as opposed to when they are individual optimum length epitopes? To address this
question, we immunized groups of HHD mice separately with each of the 3 individual
epitopes, Tax 11-19, Tax 178-186 and Tax 233-241 in conjunction with the promiscuous T-helper
epitope. Mice were also immunized with an equimolar mixture of the three epitopes and
TT3 T-helper epitope in one emulsion. Purified CD8 + T-cells from pooled splenocytes
from groups of 6 immunized mice each were tested separately for their ability to lyse HHD
51 transfected EL-4 cells in a Cr release assay. The results indicated that both Tax 11-19 and Tax
178-186 were highly immunogenic in mice. However, Tax 233-241 was again a very weak
immunogen (Fig.2.8 and Fig.2.9). The same trend was observed whether the three epitopes
were used separate, as an equimolar mixture or as one multiepitope construct. The important observation however, was that the responses were raised to multiple epitopes simultaneously with no immunosuppression.
In summary, we observed a similar pattern whether the three epitopes chosen were used as
part of a multiepitope construct or as single epitopes. The Tax 11-19 and Tax 178-186 epitopes were strong immunogens and elicited a strong CTL response in HHD mice and also a high
γ level of IFN- secretion consistent with the patient ELISPOT data. However, the Tax 233-241,
although efficient in inducing IFN-γ production in primed human PBMCs and HHD mice,
was not very effective as an immunogen to stimulate naïve CD8+ T-cells to lyse antigen
expressing target cells.
2.5 DISCUSSION
In the development of vaccine strategies designed to elicit long lived CTL responses,
it is evident that activation of multi-specific CTLs against a given antigen or multiple
44 antigens would be more beneficial than the use of single CTL epitopes. This concept stems
from the observation that during natural viral infection there is a massive expansion of
antigen specific T-cell responses (Callan et al., 1998). Hence in a vaccination regime this enormous potential of the immune system should be harnessed to prime a large and diverse population of antigen specific T-cells to increase the potency of the immune response and also increase the population coverage as different epitopes may predominate in different individuals. This would be especially useful in the case of viral escape mutants that may arise due to selective pressures by single immunodominant epitopes (Gould and Bangham, 1998).
Although the Tax 11-19 epitope is the primary immunodominant target of the immune system
in HTLV-1 infected individuals, mutations in the tax gene that result in an altered
immunodominant epitope and prematurely truncated forms of Tax that are non-functional
have been reported (Furukawa et al., 2001). The Tax protein is required for the
transactivation of viral genes during the initial stages of infection. It may be possible that mutations in Tax occur after infection has been established when the Tax protein is not needed anymore for proliferation and the infected cells undergo transformation. The altered
Tax 11-19 peptide in the context of HLA-A*0201 is no longer recognized by the specific
CTLs. Therefore at some stage in ATL development, HTLV-1 infected cells escape
detection by the CTLs and hence increase their chances of survival and malignant
transformation. Under such circumstances, the presence of CTLs specific to other epitopes
would be beneficial in the control of infection. Studies by Hanabuchi et al have outlined the
importance of virus specific CTL responses in the immunosurveillance against HTLV-1
transformed tumor cells in their rat model of ATL (Hanabuchi et al., 2000). They also
demonstrated protection induced by Tax encoding DNA immunization in adoptive transfer
45 experiments (Ohashi et al., 2000). More recently, fine mapping studies of the immune
response against the Tax protein have shown that Tax 180-188 is the immunodominant epitope to be recognized in their rat model. Immunization with this peptide elicited CTL responses that were protective in adoptive transfer experiments in athymic rats (Hanabuchi et al.,
2001). These studies taken together underscore the potential of peptide based vaccines to elicit CTL responses against HTLV-1. The studies described here is the first demonstration of successful generation of CTL responses against human epitopes from the Tax protein in a relevant preclinical mouse model for CTL evaluation. The results constitute an important preliminary observation in the development of peptide vaccination strategies against HTLV-
1. Peptides are safe and easy to administer and peptide-based T-cell vaccines have shown promise in clinical trials against various types of spontaneous cancers e.g., melanoma and virus associated cancers e.g., HPV positive cervical cancer (Bartlett et al., 1998; Muderspach et al., 2000; Slingluff et al., 2001; Wang et al., 1999). These studies made use of single or multiple epitopes either unmodified or modified to increase their immunogenicity or strengthen binding to MHC Class I. Further innovative strategies to deliver multiple epitopes may have greater potential in the development of immunotherapeutic strategies. In this study, the possibility of delivering multiple epitopes in a single peptide construct into the same antigen presenting cell to activate different antigen specific CTLs was evaluated in order to mimic a natural virus infection, when multiple viral epitopes would be presented by virus infected cells or antigen presenting cells during cross priming of viral antigens. This can be better achieved if all of the epitopes are part of one single construct rather than a mixture of individual epitopes. Further, delivery of multiple epitopes as single constructs, offer several distinct advantages over simple admixing of the individual epitopes. Non-covalent
46 oligomerization and other deleterious interpeptide interactions may preclude the induction
of responses against certain epitopes in the mixture. In addition, peptides have different
solubility requirements which may further complicate the vaccine formulation, (Elliott et al.,
1999) although this was not a limiting factor with the present set of epitopes we evaluated
since both Tax 178-186 and Tax 233-241 were water soluble. However, in general, these problems may be overcome with the use of multiepitope polypeptides.
This study demonstrated the feasibility of using a single multiepitope peptide construct to elicit cytotoxic T-cell responses to multiple Tax derived epitopes simultaneously in HLA-A*0201 transgenic mice. One of the criteria used to select epitopes for inclusion into the multiepitope construct was based on their ability to bind the HLA-A*0201 molecule. High affinity peptides would better stabilize the MHC-peptide complex and hence result in stronger MHC-peptide TCR complexes to better prime naïve antigen specific CTLs.
Additionally, this data was correlated with the induction of IFN-γ in PBMCs from a HLA-
A*0201 positive HAM/TSP patient, which gave an indication of the functional relevance of these strong binding peptides. Another criteria in assessing T-cell responses in mice for use in human clinical trials, is to select an appropriate animal model. The HLA-A*0201/Db transgenic mice which express a chimeric HLA-A*0201/Db molecule as their MHC Class I
molecule (Pascolo et al., 1997) was used. Since they are devoid of the murine MHC Class I
molecules, the T-cell repertoire is educated exclusively in the context of the chimeric HLA-
A*0201 molecule with no bias contributed by endogenous MHC Class I molecules. These
mice have been shown to be a versatile preclinical animal model to evaluate HLA-A*0201
restricted responses (Firat et al., 1999; Ureta-Vidal et al., 1999).
47 The peptide construct was designed to incorporate three minimum length CTL
epitopes with intervening dibasic Arg-Arg residues. Immunization studies with DNA
encoding various C-terminal flanking residues for CTL epitopes have shown that the
presence of basic and hydrophobic amino acids facilitates intracellular processing and subsequent presentation in the context of MHC Class I (Bergmann et al., 1996; Livingston et al., 2001). We hypothesized that a peptide construct comprising appropriate proteasomal cleavage sites between the epitopes would favor intracellular processing to generate the individual epitopes that may then be presented in the context of HLA-A*0201 on antigen presenting cells to prime multispecific CTLs. The incorporation of double arginine residues also took into consideration the possibility of generating junctional epitopes as a result of multiple epitopes being brought together in one single construct. In this particular design, no junctional epitopes were found by the predictive algorithms. To confirm the integrity of the
processed epitopes we performed proteasomal digestion studies which are known to reliably
yield MHC Class I ligands from viral or other protein derived polypeptides (Kessler et al.,
2001). Mass spectrometry analysis of 20s proteasome digested samples showed that indeed
the individual epitopes were being released from the precursor peptide as determined by the respective molecular weights. Careful analysis of the fragments generated, revealed that the
arginine residues were efficient in preventing the formation of junctional sequences and the
correct carboxy terminus of the epitopes were generated. The Tax 11-19 epitope was
abundantly generated while the levels of Tax 178-186 and Tax 233-241 were very low. The absence
of chaperone proteins such as TAP in such digestion studies may have resulted in the
continued trimming of the peptides generated. It is possible that the Tax 11-19 is very resistant
to clipping while the other two epitopes are more susceptible which could account for their
48 low abundance. Nevertheless, the cytolytic responses and the activation of release of IFN-γ by these epitopes suggest that the peptide-MHC complexes are presented at sufficient densities to elicit an immune response. Finally, the digestion analysis with the immunoproteasomes was more specific in generating the intended epitopes as compared to the standard proteasomes which resulted in extensive N-terminal clipping of the epitopes.
This type of biochemical analysis of polyepitopes constructs may be a useful tool to prescreen multiepitope constructs composed of several minimal epitopes to get a prior indication of whether the correct epitopes are being released.
With a longterm goal of developing a HTLV-1 vaccine, we determined the potential of eliciting CTL responses by our novel construct and also compared the responses obtained by immunization with the individual epitopes versus immunization with the multiepitope peptide construct. Additionally, since helper T-cell responses are necessary for the induction of CTL responses, especially memory T-cells (Keene and Forman, 1982; Sauzet et al., 1996), we co-immunized the mice by admixing the CTL epitopes with a promiscuous T-helper epitope derived from tetanus toxin (amino acids 947-967). Promiscuous T-cell epitopes interact with a wide range of MHC Class II haplotypes and have been shown to be effective in inducing strong helper cell responses to prime naïve CTLs when used as a mixture in combination with the CTL epitopes (BenMohamed et al., 2000). Further, the efficacy of helper T-cell epitopes administered mixed with CTL epitopes in the same water-in-oil emulsion is comparable with covalent linkage of helper T-cell epitopes with the CTL epitope
(Berzofsky et al., 1999). Hence we included the promiscuous helper T-cell epitope in the same emulsion. Cellular immune responses were evaluated using two functional assays, a standard 51Cr release assay for cytotoxicity and the ELISPOT assay for the frequency of
49 IFN-γ secreting CD8+ T-cells. These studies showed that multiepitope peptide was
processed and presented in vivo in the transgenic mice. The Tax 11-19 and the Tax 178-186 epitopes generated strong CTL responses. Tax 233-241 epitope proved to be a very weak
immunogen either by itself or when administered as part of the multiepitope construct.
However, it was efficient in stimulating IFN-γ secretion in both the human PBMCs as well
as the HLA-A*0201/Db transgenic mice. No epitope suppressive effects were observed as a
result of one or more of the epitopes being immunodominant. It is not exactly clear why the
γ Tax 233-241 epitope stimulated IFN- secretion effectively but failed to elicit lytic responses in
the transgenic mice. It is possible that this epitope was classified as a moderate binder to
MHC Class I and did not stay bound to MHC Class I in the 51Cr release cytolytic assay.
However, the secretion of IFN-γ is also an important antiviral function of cytotoxic T-cells
(Karupiah et al., 1990; Kohonen-Corish et al., 1990) in addition to their lytic function.
Antiviral cytokines such as IFN-γ and TNF-α produced by CTLs at the site of infection
serve to purge viruses out of the infected cells by noncytolytic mechanisms. These cytokines
may be effective by modulating immune functions such as increasing antigen presentation by
APCs and influencing homing functions of effector populations (Guidotti and Chisari, 2001;
Tishon et al., 1995). Recently, another study by Alexander et al., was reported, in which the
authors showed that a decaepitope polypeptide incorporating 9 minimal CTL epitopes and a
universal T-helper epitope (PADRE) was able to simultaneously induce both CTL and T-
helper cell responses to all intended epitopes (Alexander et al., 2002). The decaepitope
sequence contains no spacers, and therefore is distinct from the construct described in the
present study. These results taken together, are supportive of a multiepitope design implying
that the strategy may have universal application in the design of future peptide vaccines to
50 deliver multiple immunodominant epitopes from different antigens or from different MHC
Class I alleles to increase population coverage.
The efficacy of such multiepitope vaccine constructs in challenge experiments in the HLA-
A*0201 transgenic mice using recombinant vaccinia virus expressing the Tax protein is described in the following chapter.
2.6 SUPPLEMENT
Prediction and evaluation of CTL epitopes from the envelope protein of HTLV-1
In addition to evaluating epitopes derived from the Tax protein we also predicted potential
HLA-A*0201 epitopes from the envelope protein. Although the envelope protein is predominantly recognized by the humoral arm of the immune system, some reports describe the presence of envelope CTL epitopes in HTLV-1 infected individuals. Jacobson et al reported the presence of a CTL epitope within the immunodominant region 175-209 of the envelope protein (Jacobson et al., 1991). Pique et al tested the binding affinity of HLA-
A*0201 restricted envelope derived epitopes. Potential HLA-A*0201 binders from the envelope protein were predicted using the computer program developed by Parker et al
(Parker et al., 1994b) at the Bioinformatics and Molecular Analysis Section website
(http://bimas.cit.nih.gov/molbio/index.html). Prediction analysis gave 8 “hits”. These epitopes were further compared to the binding data available for some peptides (Pique et al.,
1996a). The peptides along with the molecular weight, sequence and binding data are shown in Table 2.2. We evaluated these peptides for their ability to activate the secretion of IFN-γ in PBMCs of a HAM/TSP patient. The number of positive spots obtained with the envelope peptides were in general lower than that obtained with the Tax epitopes (Fig.2.10).
The most optimal responses were obtained with the Env 175-183 and Env 182-190 which showed
51 a frequency of 55 spots/105 cells and 33 spots/105 cells respectively. Env 395-403 also
5 showed a frequency of 39 spots/10 cells. Env 175-183 and Env 182-190 and Env 210-218 lie within the principle immunodominant region identified for humoral responses. A chimeric B-cell epitope derived from this region was extensively studied by our group (Frangione-Beebe et
al., 2000). Hence we used the same peptide Env 175-218 to test if CTL responses could be
generated to the epitopes predicted within this region. This region also harbors an endogenous T-helper epitope (residues 196-209) (Kurata et al., 1989). To examine the ability of the endogenous T-helper epitope to induce CTL responses, we immunized mice with Env
175-218 mixed with or without the promiscuous T-helper epitope. In both groups no cytolytic
responses were observed to Env 175-218 or Env 182-190. Another epitope within this region Env
210-218 was highly immunogenic. Surprisingly, we observed higher responses in the group
immunized with Env 175-218 alone than the group immunized with a mixture of the
promiscuous T-helper epitope and Env 175-218 (Fig.2.11). This suggested that the identified
HTLV-1 specific T-helper epitope may be effective in inducing responses against HTLV-1
antigens. This observation may be important in the development of vaccines for HTLV-1 where an endogenous T-helper epitope may be incorporated in the vaccine. It may be
possible that due to improper processing Env 175-218 and Env 182-190 were not efficiently
generated and hence were poorly immunogenic. It may be useful to evaluate these epitopes
in a multiepitope construct with arginine spacers as described to test whether the
immunogenicity of these epitopes may be optimized by enhancing the processing efficiency.
52 Chimeric peptide approach incorporating the Tat basic domain for the intracellular delivery of CTL epitopes
to elicit CTL responses
Protein transduction domains (PTDs) which can mediate the receptor-independent transport of protein complexes into different cell types have been identified and characterized from a variety of cellular and viral proteins. The first PTD identified by virtue of its high cationic
content was the HIV Tat protein (Frankel and Pabo, 1988). Other domains include the
Drosophila antennapedia homeodomain (Ant PTD) (Derossi et al., 1998; Derossi et al., 1994)
and the VP-22 from herpes simplex virus type 1 (HSV-1) (Elliott and O'Hare, 1997). Recent
studies have demonstrated the efficient uptake of any protein or peptide that is conjugated
or fused to the Tat protein. The conjugated proteins or peptides transported into target cells
were also able to be processed and presented in the context of MHC Class I (Moy et al.,
1996) on target cells for CTL mediated cytolysis. The protein transducing property maps to
the 11 amino acid basic domain from the HIV Tat protein (Fawell et al., 1994). More
recently, this region has also been previously utilized for the delivery of OVA protein into
dendritic cells which were then used to generate CTL responses against ovalbumin epitopes
(Kim et al., 1997). In our studies to develop strategies for the intracellular delivery of CTL
epitopes for presentation via MHC Class I, we were interested in testing whether the basic
Tat domain could mediate the intracellular delivery of peptides into APCs in vivo by peptide
immunization that would result in endogenous processing and prolonged presentation of
peptide-MHC complexes and subsequent induction of CTL responses. We engineered a
chimeric construct that incorporates a CTL epitope and the basic domain of Tat as one
colinear sequence. This sequence mixed with the T-helper epitope from tetanus toxoid was
used to immunize HHD mice intraperitoneally or subcutaneously. Our attempts to induce
53 CTL responses against Tax 11-19 were not successful and no cytolytic responses were
observed in HHD mice (Fig. 2.12). These results were consistent with those of Kim et al.
They were able to demonstrate the in vivo priming of CTLs using dendritic cells treated with
Tat (aa 49-57)-OVA protein conjugate, however there was no priming when Tat (49-57)-
OVA conjugate was used as a protein immunogen (Kim et al., 1997). There are other studies that report the successful generation of protective cell mediated responses using cell penetrating peptides (Wang and Wang, 2002). All of these studies made use of in vitro manipulated mature dendritic cells to take up and present various epitopes conjugated to cell penetrating peptides. However immunization with peptide may result in the uptake of peptide by different cells and hence the processing and presentation efficiencies may vary from mature dendritic cells which are the most potent antigen presenting cells. This may account for the differential results observed. Another speculation is the fact that most protein transduction domains are all highly cationic with a high content of arginine residues.
Basic residues such as arginine and lysine are also recognized as cleavage sites for intracellular proteases and this may result in the rapid degradation of the peptide with less efficient presentation in the context of MHC Class I. Furthermore, Tat mediated delivery is not only restricted to the cytoplasm and nuclear localization has also been reported (Mi et al.,
2000). Nuclear translocation coupled with rapid degradation would result in inefficient priming in vivo. In summary, it may be possible to use these strategies more efficiently for the delivery of proteins first into dendritic cells which can then be adoptively transferred in vivo for induction of cytolytic responses.
54
Epitope Sequence Molecular Weights Ranking based on
(Daltons) binding data a
Tax 11-19 LLFGYPVYV 1069.5 Strong
Tax 178-186 QLGAFLTNV 961.3 Strong
Tax 307-315 LLFEEYTNI 1139.6 Strong
Tax 233-241 GLLPFHSTL 983.2 Moderate
Tax 155-163 YLYQLSPPI 1092.3 Strong
Table 2.1. List of predicted CTL epitopes from the Tax protein of HTLV-1. aRanking data are presented for information only and have been described elsewhere (Pique et al., 1996a).
55
Epitope Sequence Molecular weight Ranking based on (Daltons) binding dataa Env 175-183 FLNTEPSQL 1048.16 Strong
Env 182-190 QLPPTAPPL 933.11 Strong
Env 210-218 LLTLVQLTL 1013.31 Moderate
Strong Env VLYGPNVSV 947.10 239-247 ND Env SLASGKSLL 875.03 339-347 ND Env LLHEVDKDI 1081.24 346-354 Strong Env ALQEQCRFL 1107.30 395-403 Strong Env FLNITNSHV 1044.18 402-410
Table 2.2. List of predicted CTL epitopes from the envelope protein of HTLV-1. aRanking data are presented for information only and have been described elsewhere (Pique et al.,
1996a). ND indicates not determined.
56
250
200 PBMCs 5
150
100
50 Spot forming cells (SFC) / 10 (SFC) cells forming Spot γ # IFN- 0
63 19 86 41 15 5-1 11- 8-1 3-2 7-3 15 ax 17 23 30 Tax T Tax Tax Tax µ Predicted Epitopes (10 g/ml)
Fig.2.1. Frequency of IFN-γ secreting cells in HTLV-1 infected HAM/TSP patient peripheral blood mononuclear cells after culture with predicted CTL epitopes from Tax protein. 105 cells were added to anti-IFN-γ coated ELISPOT 96-well plates and stimulated with 10µg/ml of each peptide in duplicate wells for 24 hrs as described in section 2.3. The
IFN-γ secreting cells were counted using an ELISPOT image analyzer. Each point represents the mean of duplicate wells after subtraction of background with SEM. This experiment was repeated twice with similar results.
57
CTL Epitope1- RR -CTL Epitope 2- RR - CTL Epitope 3
dibasic Arg-Arg residues for intracellular proteolytic processing
Fig.2.2. Design of the multiepitope CTL peptide construct. Three epitopes most efficient in inducing the secretion of IFN-γ from patient’s PBMCc were selected and synthesized as one contiguous sequence with intervening double arginine residues in tandem (shown as boxed
residues). CTL epitope 1 represents Tax 11-19, CTL epitope 2 represents Tax 178-186 and CTL epitope 3 represents Tax 233-241. The predicted sites for cleavage are also indicated with arrows.
58
Fig.2.3. Proteasomal cleavage analysis of the 31-mer multiepitope peptide construct. Panel
(A,C,E) show digestion analysis of the multiepitope peptide by 20s immunoproteasome and panel (B,D,F) show the digestion analysis by 20s standard proteasome. The multiepitope peptide was digested and analyzed by mass spectrometry. A and B, total ion chromatogram
(TIC) traces of the bulk peptide products derived after 48 hour cleavage of 31-mer peptide.
59 C and D, the ions corresponding to the intended epitopes namely Tax 11-19, Tax 178-186 and Tax
233-241 extracted from the respective TICs. The relative abundance of each of the epitopes calculated as described in section 2.3 are also indicated. E and F, mass spectra for each of the
extracted ions. Tax 11-19 (m/z 1070.5), Tax 178-186 (m/z 962.3) and Tax 233-241 (m/z 984.1). mass/charge (m/z) represented on the x-axis and relative intensities on the y-axis.
60 N--LLFGYPVYVRRQLGAFLTNVRRGLLPFHSTL-- C
Fig.2.4. Cleavage products identified by their mass/charge (m/z) obtained after digestion of multiepitope peptide with 20s proteasomes. Peptide fragments common to both 20s immunoproteasomes and 20s standard proteasomes are indicated by solid lines. Fragments generated by 20s immunoproteasome alone is indicated by dotted lines and fragments generated by standard proteasomes alone are indicated by dashed lines.
61
Fig.2.5. Immunization with multiepitope peptide construct elicits CTL responses against
individual intended epitopes. CD8+ T-cells from in vitro stimulated splenocytes of multiepitope peptide immunized mice selected using magnetic beads are shown on the left of each panel depicted as clear histograms. The depleted population is shown as gray histograms. The CD8+ selected population was tested for cytotoxicity at the indicated E/T in a 4 hour 51Cr release assay against EL-4/HHD syngenic target cells pulsed with the either
Tax 11-19 (upper panel) or Tax 178-186 (middle panel) or Tax 233-241 (lower panel) peptides (solid
circles). Background lysis with peptide unpulsed target cells (clear cirlces) are also shown. As an internal control, lysis with naïve splenocytes from unimmunized mice, stimulated in vitro separately with each of the three relevant peptides and tested against the same peptide pulsed targets are indicated (solid triangles). The results shown are representative of three independent experiments.
62 A 35
EL4- HHD + Tax 11-19 30 EL4-HHD unpulsed Naive effectors
25
20 Events
15 % Specific Lysis Specific % 10 075 100 101 102 103 104 PE LOG 5
0 50 : 1 25 : 1 12.5 : 1 E : T Ratio
40 B
EL4-HHD + Tax 178-186 35 EL4-HHD unpulsed Naive effectors 30
25
20 Events 15 % Specific% Lysis
10
5
085 0 1 2 3 4 10 10 10 10 10 0 PE LOG 50 : 1 25 : 1 12.5 : 1 E : T Ratio
10 C
EL4-HHD + Tax 233-241 EL4-HHD unpulsed Naive effectors
5 Events % Specific % Lysis
090 0 1 2 3 4 10 10 10 10 10 0 PE LOG 50 : 1 25 : 1 12.5 : 1 E : T Ratio
63 Events 070 10 0 101 102 10 3 10 4 PE LOG
50 EL4/HHD + Tax 11-19, Tax178-186 & Tax 233-241 40 EL4/HHD unpulsed
30
20 % Specific Lysis % Specific
10
0 50 : 1 25 : 1 12.5 : 1 E : T Ratio
Fig.2.6. Cumulative cytotoxic responses of CD8+ T-cells from spleens of multiepitope construct immunized mice. The top panel shows the selection of CD8+ T-cells (clear histogram) from splenocytes after stimulation with equimolar concentrations of all three
intended epitopes Tax 11-19, Tax 178-186 and Tax 233-241 for 7 days. Gray histogram shows CD8
staining of depleted fraction. CD8+ cells selected were tested for cytotoxicity (bottom panel) against EL4/HHD target cells pulsed with all three epitopes simultaneously at the indicated
E/T ratios (solid circle). Background lysis with peptide unpulsed target cells is also shown (clear
circles).
64 Tax 233-241Tax 178-186 Tax 11-19 Tax
0 153045607590105120
# IFN-γ Spots / 0.1 million CD8+ T-cells
Fig.2.7. Frequency of epitope-specific IFN-γ secreting CD8+ T-cells in mouse splenocytes after immunization with the multiepitope peptide. CD8+ T-cells isolated from freshly harvested splenocytes of peptide immunized HLA-A*0201 mice were cultured in the presence of 10µg/ml relevant peptide as indicated for 24 hours. IFN-γ releasing cells were evaluated in an IFN-γ ELISPOT assay. Background levels were measured in wells containing medium only. The number of peptide specific spots was obtained by subtracting the background from the number of spots appearing after peptide stimulation. Bars represent means and SEM from duplicate wells for 105 CD8+ T- cells. Similar results were obtained in
two independent experiments.
65
70 EL4/HHD + Tax 11-19 60 EL4/HHD unpulsed
50
40
30 % Specific Lysis % Specific 20
10
0 100 : 1 50 : 1 25 : 1
E : T Ratio 40
EL4/HHD + Tax 178-186 EL4/HHD unpulsed 30
20 % Specific Lysis Specific %
10
0 100 : 1 50 : 1 25 : 1
E : T Ratio 10
EL4/HHD + Tax 233-241 EL4/HHD unpulsed
5 % Specific Lysis Specific %
0 100 : 1 50 : 1 25 : 1
E : T Ratio
Fig.2.8. Immunogenicity of individual epitopes when administered as single minimum length
epitopes. CD8+ T-cells from groups of mice each immunized with Tax 11-19, Tax 178-186 or Tax
233-241 separately were tested for cytotoxic activity against EL4/HHD cells pulsed with the relevant peptide, Tax 11-19, Tax 178-186 or Tax 233-241 (shown from top to bottom).
66
40 80 EL4/HHD + Tax 11-19 EL4/HHD + Tax 178-186 EL4/HHD unpulsed 35 70 EL4/HHD unpulsed
30 60
25 50
20 40
15 30 % Specific Lysis% % Specific% Lysis
10 20
5 10
0 0 50 : 1 25 : 1 12.5 : 1 50 : 1 25 : 1 12.5 : 1
E : T Ratio E : T Ratio
100 20 El4/HHD + Tax 11-19, EL4/HHD + Tax 233-241 90 EL4/HHD unpulsed Tax 178-186 & Tax 233-241 EL4/HHD unpulsed 15 80
70
10 60
50
5 40 % Specific Lysis% Specific % Specific Lysis % 30
0 20
10
0 50 : 1 25 : 1 12.5 : 1 50 : 1 25 : 1 12.5 : 1
E : T Ratio E : T Ratio
Fig.2.9. Mixture immunization with Tax 11-19, Tax 178-186 and Tax 233-241 (33µgs each).
HHD mice were immunized with a mixture of the three Tax derived epitopes as described in section 2.3. Lysis of target cells using splenocytes stimulated for 7 days with individual epitopes are shown.
67 Env182-190
Env175-183
Env402-410
Env395-403
Env346-354 peptides (10ug/ml) Env339-347
Env239-247
-10 0 10 20 30 40 50 60 70 80 90 100
# SFC/105 PBMCs
Fig.2.10. Frequency of IFN-γ secreting cells in HTLV-1 infected HAM/TSP patients’ peripheral blood mononuclear cells after culture with predicted CTL epitopes from envelope protein. 105 cells were added to anti-IFN-γ coated ELISPOT 96-well plates and stimulated
with 10µg/ml of each peptide in duplicate wells for 24 hours as described in section 2.3.
γ Env 210-218 was not evaluated by ELISPOT. The IFN- secreting cells were counted using an
ELISPOT image analyzer. Each point represents the mean of duplicate wells after
subtraction of background with SEM.
68
50
EL4/HHD + Env 210-218 EL4/HHD unpulsed 40
30
20 % Specific Lysis %
10
0 100 : 1 50 : 1 25 : 1 E : T Ratio
20
El4/HHD + Env 210-218 El4/HHD unpulsed
15
10
% Specific Lysis
5
0 50 : 1 25 : 1 12.5 : 1
E : T Ratio
Fig.2.11. Cytolytic responses against Env 210-218 CTL epitope in HHD mice immunized with
Env 175-218. Mice were immunized either without T-helper epitope from tetanus toxoid (TT3)
(upper panel) or mixed with 140µg TT3 (lower panel). No responses were obtained with
Env 175-183 and Env 182-190.
69
H2N-YGRKKRRQRRR-LLFGYPVYV-COOH
Tat sequence
20
EL4/HHD + Tax 11-19 (i.p.) EL4/HHD unpulsed (i.p.) EL4/HHD unpulsed (s.c.) EL4/HHD unpulsed (s.c.)
10 % Specific Lysis %
0 100 : 1 50 : 1 25 : 1 E : T Ratio
Fig.2.12. Chimeric peptide approach for the delivery of CTL epitopes. Tax 11-19 was synthesized as a chimeric construct with the basic domain of HIV Tat protein at the N- terminus as depicted. Splenocytes from HHD immunized mice were stimulated in vitro with
the Tax 11-19 peptide for 6 days and tested for cytolysis against EL4/HHD target cells pulsed
with Tax 11-19.
70
CHAPTER 3
PROTECTIVE EFFICACY OF MULTIEPITOPE CTL PEPTIDE CONSTRUCT
AGAINST CHALLENGE WITH RECOMBINANT VACCINIA VIRUS EXPRESSING
TAX PROTEIN
3.1 RATIONALE
In Chapter 2, a multiepitope CTL peptide from the Tax protein was designed,
synthesized and characterized both immunologically and biochemically. Cellular proteases
are strongly influenced by residues that flank the C-terminal putative cleavage site which may
in turn determine whether an epitope is generated or not (Mo et al., 2000). The identification
of biologically relevant epitopes to be incorporated into multiepitope constructs is the first
of the obstacles to be overcome. The second important criterium is the relative location of
the individual epitope in a multiepitope construct which may influence the processing
efficiency of the immunogenic sequence. Therefore the positioning of CTL epitopes is an
important consideration when designing multiepitope vaccines because each construct will
be seen by the proteasome to be a different substrate. In Chapter 2, the orientation of the
individual Tax epitopes was set arbitrarily from the N-terminus as Tax11-19, Tax 178-186 and Tax
233-241 based on the occurrence of the epitope in the native protein sequence. This orientation
gave rise to cleavage products encompassing all three individual epitopes with Tax 11-19 epitope being the most abundant product, whereas the other two epitopes were generated to a smaller extent quantitatively, although immune responses were induced against all three
71 epitopes. We wished to further examine whether the positioning of the epitopes within the
construct would give rise to a different pattern or rate of cleavage by the proteasomes and
whether this would influence the relative immunogenicity of individual epitopes. In addition
to the epitopes described previously, we evaluated two other predicted Tax epitopes that
gave positive responses when tested with peripheral blood cells (PBMCs) from a HAM/TSP
patient as single immunogens. One of the epitopes, Tax 306-315 elicited high cytotoxic
responses in HHD transgenic mice when immunized with a T-helper epitope from tetanus
toxoid. We designed several new multiepitope immunogens incorporating the Tax11-19, Tax
178-186 and Tax306-315 epitopes. Four constructs were designed in which the three epitopes were placed in different orientations relative to one another, with intervening double arginine
residues. These were Tax 11-19-RR-Tax 178-186-RR-Tax 306-315 (construct 236); Tax 178-186-RR-Tax
306-315-RR-Tax 11-19 (construct 362); Tax 178-186-RR-Tax 11-19-RR-Tax 306-315 (construct 326) and
Tax 306-315-RR-Tax 178-186-RR-Tax 11-19 (construct 632) each designated from the N-terminus to
C-terminus.
Another crucial step in the development of vaccine candidates that can be translated
to humans requires the demonstration of protection in an appropriate animal model of
disease or cancer. There are several animal models, both small animal and non-human
primate models of HTLV-1 infection. These models have been useful in the study of the
pathogenesis and molecular mechanisms of infection as well as vaccine studies using
recombinant and protein based candidates. Some of these studies included the
demonstration of immune responses, either humoral or cell mediated or both being involved
in protection against infection (described in previous chapter). However, these immune
responses were all species restricted and to date there is no animal model that can be used to
72 evaluate human determinants of protection due in part to the problem of MHC restriction and more importantly, that the evaluation is achieved by indirect means. Hence, it is not possible to evaluate MHC restricted cell mediated responses in the context of human infection and immunity. Furthermore, HTLV-1 does not replicate well in murine cells. There are reports describing the utilization of recombinant vaccinia viruses expressing the desired target antigen from different viruses that can infect mice. The vaccinia virus preferentially replicates in the ovaries of the mice and hence the ovaries may be harvested and titered for the presence of virus. For example, Hepatitis C virus (HCV)-recombinant vaccinia virus has been used to demonstrate the synergism between IL-12, TNF-α and GMCSF to elicit higher protection in HLA-A*0201 transgenic mice (Ahlers et al., 2001). HCV-recombinant vaccinia virus has also been used to demonstrate the efficacy of various genetic vaccine candidates
(Arribillaga et al., 2002; Pancholi et al., 2003). Recombinant vaccinia viruses expressing the
HIV envelope protein has also been used to test the efficacy of DNA immunization (Kiszka et al., 2002). In the absence of an appropriate animal model, this surrogate model of using recombinant HTLV-1 vaccinia virus and HHD mice is an attractive alternative for preclinical protection studies using human vaccine candidates.
3.2 SUMMARY
Four triple epitope constructs were designed and tested by immunoproteasomal cleavage analysis for the liberation of the individual epitopes. Different time points of cleavage tested showed that the four constructs differed in the rates at which the three epitopes were being generated. One of the constructs, 236 was most efficiently digested by the immunoproteasome and the three individual epitopes were liberated at the earliest time point tested. Furthermore, these triple epitope constructs were also used to immunize HHD
73 transgenic mice to evaluate their in vivo immunogenicity. Although all four constructs were
immunogenic in vivo, it was observed that the responses were differentially generated to all
four constructs which was somewhat correlative with the proteasome cleavage data. Thus
epitope orientation seemed to have altered the rate of generation of the epitopes which in turn may have influenced the immune responses. The 236 construct which elicited the highest responses against all three individual epitopes was selected for evaluating the protective efficacy of multiepitope Tax peptide constructs using HHD mice and recombinant vaccinia virus (WR strain) expressing the Tax protein of HTLV-1. Significant reduction in viral replication in the ovaries of immunized mice was observed as compared to naïve control mice. This protection was dependent on the presence of CD8+ T-cells. In addition, there was no protection against challenge with a control recombinant vaccinia virus expressing the influenza protein indicating successful antigen specific immunization.
3.3 MATERIALS AND METHODS
Peptide synthesis
The four triple epitope constructs were synthesized on Milligen/Biosearch 9600 peptide synthesizer (PE Biosystems, Foster City, CA). The peptides were synthesized using a 4- methylbenzhydrylamine resin as the solid support (substitution 0.54mmol/g). Peptides were cleaved and purified to 95% purity as described in section 2.3. The identities of all purified peptides were confirmed by MALDI-TOF mass spectrometry.
In vitro proteasomal cleavage analysis
Twenty micrograms of each of the four peptides was incubated separately with 2µg of the immunoproteasome for 12, 24 or 48 hours. The digestion was stopped and the digested samples were stored and processed as described in section 2.3.
74 Mass spectrometry
The digested peptide samples were analyzed by nano-LC-MS as described in section 2.3 for the immunoproteasome using 2µg of the proteasome.
Animals and immunizations
HHD mice as described in Chapter 2 were used for these studies. To determine the
immunogenicity of the various multiepitope constructs, HHD mice were immunized once with 100µg of peptide mixed with 140µg TT3, a promiscuous T-helper epitope from tetanus
toxoid (residues 947-967) and 100µg of adjuvant N-acetyl-glucosamine-3-acetyl-L-alanyl-D- isoglutamine (nor-MDP) (Peninsula Laboratories, Belmont, CA) emulsified 50:50 in 4:1 squalene:arlacel A (Sigma, St Louis). The emulsions were injected subcutaneously at the base of the tail. Eleven days post immunization, the mice were sacrificed and the spleens removed. For single epitope immunizations, a booster was administered three weeks after primary immunization and the splenocytes harvested 10 days after the final boost. The splenocytes were then stimulated in vitro for 5-6 days as described in section 2.3 using splenocytes pulsed with 1µM of the various individual epitopes. For the challenge experiments HHD mice were immunized twice, three weeks apart prior to challenge.
Cell culture
EL4/HHD cells and HeLa-HHD cells were provided by Dr. Francois Lemonnier, Pasteur
Institute, France and have been described earlier (Pascolo et al., 1997). All cells were maintained in RPMI, 10% FCS, 1% p/s. HHD transfected cells were maintained in selection medium (RPMI, 10% FCS, 1% p/s and 0.5% G418). Hu143 TK- and BSC-1 cells were provided by Dr. Christopher Walker. Hu143TK- cells are osteosarcoma cells and were
75 maintained in DMEM, 10% FCS, 1%p/s. BSC-1 (monkey kidney cell line) also was maintained in DMEM, 10% FCS and 1%p/s.
51Cr release assay
Cytolytic properties of the splenocytes from immunized mice was examined as described in section 2.3. For the multiepitope pulsed target cells, the target cells were pulsed overnight in the presence or absence of 50µM proteasome inhibitor LLNL (Sigma), with 1µM of the relevant multiepitope construct. The excess peptide was washed off before the cells were used as targets in the cytolytic assay. For the vaccinia infected targets, target cells were infected with the relevant vaccinia construct at an MOI of 50:1 overnight for 18 hours prior to the 51Cr release assay.
IFN-γ release assay
Splenocytes from immunized mice were stimulated in vitro for 6 days with splenocytes pulsed with 1µM relevant peptide as described previously. After 6 days the splenocytes were harvested and further incubated with various target cells as indicated for 24 hours in RPMI
10% FCS, 1% pen/strep in a total volume of 200µl. After 24 hours the cultures were centrifuged and the supernatant was collected and stored at -200C. The amount of IFN-γ released into the supernatant was estimated using a sandwich ELISA as per the manufacturer’s protocols (Pharmingen, CA).
Western Blotting
3-5 X 106 EL4/HHD cells or HeLa-HHD cells were infected with either p40-VV or HA-VV at an MOI of 50:1. After overnight incubation the cells were washed once with PBS and resuspended in 100µl HBSS per sample and lysed in 1ml of ice cold Nonidet P-40 lysis buffer (150mM NaCl; 50 mM Tris, pH 8; 10mM EDTA, 10mM sodium pyrophosphate,
76 10mM sodium fluoride; 1% NP-40, 0.1% SDS) containing 10µg/ml each of aprotinin and
leupeptin. Lysis was achieved by gentle rotation at 4oC for 20 minutes. After centrifugation
(14,000g, 10 minutes) to remove cell debris, lysates were boiled in SDS sample buffer for 3
minutes. Proteins were resolved by 10% SDS-PAGE, transferred to nitrocellulose and then
probed with 1:5 dilution of a Tax protein specific hybridoma supernatant. The hybridoma
was obtained through the AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH (168A51-A) from Dr. Beatrice Langton. Protein transfer was monitored with pre-stained molecular weight standards (Bio-Rad). Immunoreactive bands are detected using horse-radish peroxidase conjugated goat anti-mouse immunoglobulin
(1:10,000 dilution) by enhanced chemiluminescence (Pierce).
Flow cytometry
Peripheral blood was collected by retro-orbital bleeding and peripheral blood cells were
prepared by RBC depletion of whole blood. Cells were stained for 1 hour with phycoerythrin
(PE) conjugated rat anti-mouse CD8a antibody (Ly-2) (Pharmingen, CA) for 1 hour in FACS
buffer (PBS, 2% FCS, 0.02% Na azide). Cells were then washed with PBS and fixed using
1% paraformaldehyde in PBS and analyzed by flow cytometry.
Propagation and purification of recombinant vaccinia virus
Recombinant vaccinia virus expressing the Tax protein of HTLV-1 and recombinant
vaccinia virus expressing the hemagglutinin protein of influenza virus has been previously
described (Siomi et al., 1988). The vaccinia virus constructs were a kind gift from Dr. Steven
Jacobson. The vaccinia constructs were propagated in Hu143 TK- cells as per the described
protocols. Briefly, Hu143 TK- cells were grown to confluence in 175cm2 tissue culture
flasks. The cells were infected with the vaccinia virus constructs in DMEM, 5% FCS, 1%
77 0 pen/strep and incubated at 37 C and 5% CO2 for 24-48 hours or until marked cytopathic effects were observed (rounding up of cells and loss of adherence to the flask). The cells
were scraped off the flask and spun down and resuspended in 2ml of medium and frozen at
-700C until purification.
Purification of vaccinia virus constructs
The vaccinia constructs were purified by zonal sucrose gradient centrifugation. Briefly, the
infected cell pellets were freeze-thawed twice. The cells were then homogenized in 10ml of
1mM Tris buffer pH 9.0 with 20 stokes of the pestle in a 15ml tissue grinder and centrifuged
at 2500 rpm for 5 minutes. The supernatant was collected and saved on ice. The
homogenization process was repeated three times and the supernatants were pooled and
sonicated for 5 minutes twice with 20 second intervals. The supernatant was then layered (5-
6ml) on 5ml of 36% sucrose in 10mM Tris pH 9.0 in polyallomar tubes (Beckman) 14 X
89mm and centrifuged at 18K for 80 minutes at 40C in an SW41 (Beckman) rotor. The virus
pellet at the bottom was resuspended in 1ml of 1mM Tris pH 9.0. Fresh 1mM Tris buffer pH 9.0 was used to rinse the tubes once. The suspension was then homogenized once again as before and 200µl aliquots were transferred to freezing vials and stored at -700C.
Plaque assay for viral titers
BSC-1 cells were used as indicator cells. Briefly, serial 10-fold dilutions of the virus
preparation were made in DMEM, 2% FCS, 1% pen/strep. 200µl of the various dilutions
were plated on confluent BSC-1 indicator cells in 6-well tissue culture plates. After 48 hours
of incubation, the medium was aspirated out and the cells were stained with 0.1% crystal
violet in 20% ethanol. The plates were washed once with PBS, dried and the plaques were
counted to estimate the viral titer as plaque forming units (pfu).
78 Challenge studies
Ten days after the last immunization, HHD mice were challenged intraperitoneally (i.p) with
5X106 pfu of recombinant vaccinia virus expressing the Tax protein of HTLV-1 (p40-VV) or
the control recombinant vaccinia virus expressing the hemagglutinin protein from influenza
virus. In some experiments, the mice were depleted of CD8+ T-cells by i.p. injection of
100µg of anti-CD8 antibody (YTS 169.4.2.1) (Cobbold et al., 1984) on day -4, -3, -1, 0 and
+1, day 0 being the day of challenge with vaccinia virus. 5 days post challenge, the mice were
sacrificed and the ovaries removed, freeze-thawed twice, homogenized and sonicated and assayed for vaccinia virus titers by plating serial 10-fold dilutions on BSC-1 indicator cells as previously described.
Statistical Analysis
Statistical analyses compared the effect of various peptide vaccines on vaccinia titer
[pfu/ovary]. Since titer data were not normally distributed, we used non-parametric Kruskal-
Wallis and Mann-Whitney U tests to compare groups with Bonferroni adjustment. Based on the number of tests performed a two-sided level of significance of 0.005 was defined as statistically significant.
3.4 RESULTS
Immunogenicity testing of additional Tax derived epitopes
In Chapter 2, we predicted five HLA-A*0201 restricted epitopes in the Tax protein.
These epitopes were evaluated in an ELISPOT assay for the induction of IFN-γ by peripheral blood cells (PBMCs) from a HAM/TSP patient. Three out of the five epitopes
namely Tax11-19, Tax 178-186 and Tax 233-241 gave a high number of spots indicating that they are
recognized during natural infection. Another epitope Tax 155-163 gave a lower number of spots
79 although it was classified as a strong binder (Pique et al., 1996a) and Tax 307-315 being a strong
binder did not give any spots. We also evaluated the immunogenicity of the two additional
epitopes, Tax 155-163 and a decamer Tax 306-315 in HHD transgenic mice. Groups of 2-3 HHD
mice each were immunized with the epitope separately and boosted three weeks apart. After
the final boost the splenocytes from each group were pooled and stimulated with the
relevant peptide for one week. Only the Tax 306-315 epitope proved to be a strong epitope in its ability to induce cytolytic responses as shown in Fig.3.1. In comparison, the Tax 155-163 epitope was not highly immunogenic and gave lower cytolytic responses when tested against peptide pulsed target cells.
Design and synthesis of the multiepitope CTL peptide constructs
In Chapter 2, out of the three epitopes in the multepitope peptide construct Tax11-19 and Tax 178-186 elicited strong cytolytic responses in the HHD mice. The Tax 233-241 was not
immunogenic in its ability to induce cytotoxicity administered either singly or as part of a
multiepitope construct. We evaluated additional epitopes and we found that the decamer
Tax 306-315 was highly immunogenic in HHD mice. Hence this epitope was incorporated in a new multiepitope with three CTL epitopes Tax 11-19, Tax 178-186 and Tax 306-315. In order to
determine if the orientation of the individual epitopes in the multiepitope construct has any
effect on the rate of liberation of the individual epitopes as well as immunogenicity, four
different variants of the multiepitope peptide construct were synthesized. The orientation of
the individual epitopes and their designations are depicted in Table 3.1. All peptides were
purified to greater than 95% by reverse phase HPLC and their identity confirmed by
MALDI-TOF mass spectrometry.
80 Immunoproteasomal liberation of individual peptides from multiepitope constructs
Each of the four multiepitope CTL peptide constructs was digested at three different
time points using purified immunoproteasome. In the earlier time point of 12 hours
digestion, the 236 peptide orientation was successfully digested and all the three individual epitopes could be detected by mass spectrometry (Table 3.2). Furthermore, there was complete substrate turnover because the undigested peptide could not be detected at the earliest 12 hour time point. The relative abundance of each epitope calculated as described in
section 3.3 increased marginally at the 24 hour time point. By 48 hours the relative
abundance of all three epitopes in the digest had decreased. For peptide 362, the Tax 11-19 epitope was observed in decreasing abundance in 12, 24 and 48 hour time points. Tax 178-186 epitope was observed only at 24 hours and Tax 306-315 was observed at both 24 and 48 hours,
although the signal was very weak. Likewise, for 326, Tax 11-19 and Tax 178-186 were observed in
marginally increasing abundance at all time points but the Tax 306-315 could only be detected at
24 and 48 hours, again at extremely low levels. The 632 construct had the slowest rate of
digestion and initially none of the epitopes could be detected at 12 hours, with very low to
negligible substrate turnover. At 24 and 48 hours however, all three epitopes Tax 11-19, Tax
178-186 and Tax 306-315 could be detected with complete substrate turnover comparable with the
236 construct. Overall, in all four constructs the relative abundance of Tax 11-19 was the highest and the Tax 306-315 was the lowest. The 236 construct had the highest rate of
digestion.
We further tested whether the presentation of epitopes in the context of MHC Class
I correlated with the trend of liberation of the individual epitopes in proteasomal digestion
experiments. EL4/HHD cells were incubated overnight with 1µM of the 236 multiepitope
81 construct. The cells were washed free of any peptide and used as target cells in a cytolytic assay using effectors from mice immunized with the 236 construct and stimulated in vitro with the each individual epitope for 6 days. It was observed that the level of lysis obtained
with the Tax 11-19 effectors was comparable to the that observed with epitope pulsed targets
(Fig.3.2). However, the lysis obtained with Tax 178-186 effectors was significantly lower than
the respective peptide pulsed targets. This indicated that the Tax 11-19 epitope was liberated
and presented at higher levels as compared to the Tax 178-186 epitope which was the case with the proteasomal experiments in which the relative abundance of the Tax 11-19 was the highest.
In the case of the Tax 306-315 epitope, very low lysis was observed against epitope pulsed
targets and there was no significant difference between epitope pulsed and 236 treated
targets. Furthermore, when the target cells were pulsed overnight with 236 construct in the
presence of the proteasomal inhibitor LLNL (Castilleja et al., 2001), a reduction in the level
of lysis by the Tax 11-19 effectors was observed, while that of Tax 178-186 and Tax 306-315 was not affected confirming that the epitope was being processed by the cellular proteasomes.
Immunogenicity studies of multiepitope variants in HHD transgenic mice
The four variants of the multiepitope peptide construct were tested individually for their ability to induce a cell mediated response in HHD transgenic mice. Groups of 4-5 mice
per construct were immunized once with each of the four constructs. The cytolytic
responses against relevant peptide pulsed EL4/HHD target cells are shown in Fig.3.3. It was
observed that although the Tax 306-315 epitope was highly immunogenic in HHD mice when
administered as a single epitope, the cytolytic responses observed when administered as part
of a multiepitope construct was very weak. This was the case in all the four multiepitope
variants. The Tax 11-19 epitope was immunogenic as part of all four constructs with the
82 highest lysis seen in 236 and 632 constructs. The Tax 178-186 epitope also induced cytolytic
responses as a multiepitope construct and the highest responses were again obtained in the
236 construct followed by 632 and 326 constructs. In summary, the 236 construct gave the
most optimum responses against all individual epitopes as compared to the other
multiepitope variants.
The multiepitope constructs were also evaluated for their ability to induce the release
of IFN-γ by stimulation of splenocytes. Freshly harvested splenocytes from HHD mice
immunized with the various multiepitope constructs were stimulated for 6 days with the
relevant epitope and then tested against epitope pulsed EL4/HHD target cells for their
ability to be activated to release IFN-γ. Splenocytes from the 236 construct immunized mice
were the most efficient in releasing IFN-γ followed by the 632 construct (Fig.3.4). In
contrast, the 362 and 326 constructs elicited very low levels of IFN-γ against all individual epitopes. Surprisingly with the 236 construct the levels of IFN-γ induced by splenocytes
stimulated with the Tax 306-315 epitope was also very high (8000pg/ml). This was in contrast
to the other multiepitope constructs that did not elicit a high response against this epitope.
Thus, overall, the position of the different epitopes in the 236 construct was the most
optimum in inducing immune responses in HHD mice. The data was somewhat correlative
with the peptide digestion studies because cytolytic responses and IFN-γ secretion was best
observed with the 236 peptide followed by the 632 peptide; the 632 peptide was digested at a
slowest rate but the relative abundance of the epitopes generated were comparable with the
236 construct.
83 Affinity of CTLs induced by multiepitope immunization for peptide pulsed target
cells
We tested the affinity of CTLs induced by multiepitope 236 immunization and those
induced by immunization with the individual epitopes separately. In vitro stimulated CTLs from both groups were tested against EL4/HHD pulsed with various concentrations of the individual epitopes. We did not observe significant differences between the multiepitope immunization and the single epitope immunization since CTLs from both groups were able
to lyse target cells pulsed with very low concentrations of peptide (Fig. 3.5). The Tax 11-19 specific CTL showed the highest affinity and high lytic responses were observed with very
µ low doses of 0.0001 M epitope pulsed target cells. Tax 178-186 CTLs were also of high affinity.
The Tax 306-315 was not very immunogenic as a multiepitope construct and a titration was
observed with only the single epitope immunized mice, however, the affinity of these CTLs
was much lower compared to the other two epitopes. Furthermore, IFN-γ release was also
compared between single epitope immunized mice and multiepitope 236 immunized mice
(Fig.3.6). While there was no difference in the IFN-γ levels induced by splenocytes
immunized with Tax 11-19 and those from 236 immunized mice, the levels were approximately
double for the Tax 178-186 and Tax 306-315 in the case of 236 immunization in comparison to the
respective single epitope immunization.
Splenocytes from multiepitope peptide immunized mice recognize vaccinia virus
infected target cells
Although high cytolytic responses or IFN-γ secretion were observed against target
cells pulsed with the individual epitope, it is imperative to test the cross-reactivity of antigen
specific CTLs induced by peptide vaccination with target cells that express the native protein
84 antigen. CTLs stimulated in vitro with the relevant epitopes were tested against target cells
that were infected overnight with recombinant Tax vaccinia virus (p40-VV). EL4/HHD
cells and HeLa-HHD cells were first tested for Tax protein expression after overnight
infection with p40-VV by Western blotting using whole cell lysates. Both cell lines expressed
the Tax protein after infection as indicated by the 40kDa band in Fig.3.7 (upper and lower
panels). HTLV- infected MT-2 cells were used as a positive control. Bands migrating at
approximately 80kDa and 120kDa are dimmers and trimers of the Tax protein. We then
tested three individual constructs (236, 362 and 632) against EL4/HHD target cells infected
with p40-VV. Cytolytic responses were obtained with splenocyte effector CTLs from 236
and 362 immunized mice expanded separately with each individual epitope (Fig.3.8A). These
responses however, were very weak. Splenocytes from 632 immunized mice failed to lyse
p40-VV infected targets. This trend was consistent with the low levels of lysis obtained for
epitope pulsed targets in the case of the 362 construct. Cytotoxicity of splenocytes from 236
immunized mice was further tested using an alternative HeLa-HHD cell line (HeLa cells
transfected with the HHD construct). We observed higher p40-VV specific cytotoxic
responses against the HeLa-HHD vaccinia infected cells as compared with EL4/HHD
infected cells (3.8B).
We also examined the activation of release of IFN-γ in the same splenocytes by p40-
VV infected target cells. Comparable responses were observed between in vitro Tax 11-19 stimulated splenocytes from the 236 construct and the 362 construct immunization (Fig.3.9).
However, in the case of Tax 178-186 in vitro stimulated effectors from 236 construct
immunization elicited more than double the amount of IFN-γ as compared with the 362
construct (670pg/ml v/s 148pg/ml). The 632 construct did not elicit any IFN-γ release in
85 response to vaccinia infected target cells. In summary, lytic responses and cytokine release was best observed with the 236 construct immunized mice.
Multiepitope peptide immunization protects against challenge with recombinant
Tax vaccinia virus
The four variants of the multiepitope vaccines were differentially immunogenic in the HHD mice as determined by functional cytolytic and cytokine release assays. Based on these results we examined the protective efficacy of the multiepitope T-cell vaccine candidate against viral infection. Since HTLV-1 does not infect mice, we used a surrogate model of recombinant vaccinia virus expressing the Tax protein to challenge previously immunized HHD mice. The 236 multiepitope construct was utilized for all the challenge studies since the highest lytic responses and cytokine release were observed with this construct. HHD mice were immunized with the 236 construct mixed with a T-helper epitope (TT3) and boosted three weeks later. Ten days after the last boost the mice were challenged i.p. with 5X106pfu each of recombinant vaccinia virus expressing the Tax protein
(p40-VV). Immunized mice showed a statistically significant reduction in vaccinia virus
replication in the ovaries that corresponded to a 3-5 log decrease in the viral titers compared with naïve mice or mice that were immunized with TT3 by itself in emulsion (mock immunization) that were challenged with the same vaccinia virus (Fig.3.10). Furthermore, the reduction in viral titers was also specific to Tax expressing vaccinia virus because groups of
236 immunized mice did not show any reduction in viral replication when challenged with an irrelevant recombinant vaccinia virus expressing the influenza hemagglutinin protein (HA-
VV) (Fig. 3.11) (Average HA-VV titers 1013pfu/ovary in immunized and naïve mice).
86 Protection is dependent on the presence of CD8+ T-cells
To demonstrate the role of CD8+ T-cells in mediating the reduction in viral titers in the 236 peptide construct vaccinated mice, we immunized HHD mice with the 236 peptide construct. Prior to challenge with p40-VV the mice were injected with anti-CD8 antibodies to deplete the CD8+ T-cell population. The mice were bled prior to challenge and analyzed for the efficacy of depletion (Fig.3.12.). The viral titers observed in the CD8+ T-cell depleted mice were comparable with unimmunized control mice viral titers as shown in
Fig.3.13.
Comparison of protection mediated by multiepitope and single epitope immunization
We hypothesized that a multiepitope vaccine comprised of several CD8+ T-cell epitopes would mediate synergistic or additive anti-viral effects in challenge models. To examine our hypothesis we immunized groups of HHD mice with each of the three individual epitopes or the 236 peptide construct. After boosting the mice once we challenged them i.p. with 5X106pfu p40-VV. As indicated by the p values in Fig.3.13, the decrease in
viral replication in mice that were immunized with the Tax 178-186 or Tax 306-316 single epitopes was less significant than the mice immunized with the multiepitope 236 peptide construct.
Another important observation was that the Tax 11-19 epitope mediated protection was not statistically significant. We also tested the protective efficacy of immunization with an equimolar mixture of the three epitopes in one emulsion. The reduction in the viral load observed with immunization with the mixture was statistically significant only when compared with the titers in naïve mice but not when compared with the titers obtained in
87 mock (TT3) immunized mice. The same was true for all three single epitope immunized
mice.
3.5 DISCUSSION
Recombinant vaccines such as vaccinia virus vectors expressing HTLV-1 proteins or
whole protein vaccines have shown protection in various HTLV-1 animal models. However,
to date there is no effective vaccine against HTLV-1 infection. Whole protein vaccinations
are largely undefined and that may also lead to epitope suppression. Multipepitope vaccines
have shown promise in multiple animal models of viral infections (Hanke et al., 1999; Hanke
et al., 1998; Thomson et al., 1995; Thomson et al., 1998; Velders et al., 2001). Epitope based vaccines have the advantage of being defined and amenable to changes that may enhance the protective efficacy of vaccine formulations because epitopes which elicit weak or deleterious
immune responses may be selectively avoided. However, the formulation of multiepitope
vaccines may be complicated and careful design of the immunogen is required to optimize
processing and consequently the immunogenicity of each epitope selected. It is believed that
the phenomenon of immunodominance may pose a hindrance where only some epitopes
within a protein will be immunogenic. While the reasons behind this are still a topic of
intense investigation, factors such as the affinity of the particular epitope for its cognate
MHC or “holes” in the T-cell repertoire or the fact that these epitopes are not generated at
sufficiently high levels by the natural cellular antigen processing machinery for priming of
naïve CTLs (Chen et al., 2001; Pamer and Cresswell, 1998; Yewdell and Bennink, 2001) may
influence the outcome of such immunizations. Hence, after selection of epitopes that bind
with sufficiently high affinity for MHC Class I, it is necessary to design multiepitope
constructs such that the processing of each epitope is maximized and sufficient levels of
88 each epitope are generated within the antigen presenting cell to elicit a strong immune response.
The multicatalytic proteasomes that are responsible for the generation of the correct
C-terminal end of the antigenic epitope are largely influenced by the flanking residues of the putative epitope (York et al., 1999). Different arrangements of the same set of epitopes result in different peptide substrates that differ with respect to the flanking residues for each epitope. Therefore, we hypothesized that there would be a difference in the processing rates which may subsequently influence the immunogenicity of the individual epitopes. In support of our hypothesis, we observed that indeed there were differences in the processing rates.
Although all four constructs were immunogenic, they varied in the degree of immunogenicity. This effect was pronounced especially in the activation of IFN-γ release by splenocytes from immunized mice. Only the 236 orientation construct successfully activated the release of IFN-γ against target cells pulsed with each of the three epitopes and more importantly against p40-VV infected vaccinia virus. It is not clear why the 632 construct was effective in inducing IFN-γ against target cells pulsed with peptide but not against p40-VV infected targets. We speculate that since the 236 peptide was cleaved at a much higher rate than the other constructs, relatively higher number of peptide-MHC complexes may have been expressed on the cell surface which may be responsible for higher activation levels of antigen specific T-cells. Additionally, these results show that there are different thresholds or requirements for lytic activity and the secretion of anti-viral cytokines. It is therefore important to utilize multiple read-out assays to completely characterize cellular responses against epitopes (Alexander et al., 1998).
89 Another important observation was that there were pronounced differences in the
secretion of IFN-γ against individual epitopes when mice were immunized with the multiepitope construct as opposed to single epitope immunization, especially in the case of
γ the less immunodominant Tax 178-186 and Tax 306-315 epitopes. IFN- secretion is an important
antiviral mechanism which may enhance the antiviral potential while avoiding the destructive
effects of lytic activity of induced CTLs against infected target cells. Epitopes that only
induce IFN-γ secretion but not lysis of specific target cells have been previously reported
(Alexander et al., 1998; Arribillaga et al., 2002; Wedemeyer et al., 2001). Such epitopes may
be particularly useful for infections such as HTLV-1 where the primary cells infected are the
CD4+ T-cells that are instrumental during initial priming of immune responses and for
maintenance of memory (Altfeld and Rosenberg, 2000; Borrow et al., 1998; Ossendorp et al.,
1998).
The challenge model that we utilized in this study allowed us to efficiently evaluate
the efficacy of human epitopes that have been previously identified in subjects infected with
HTLV-1. The Tax 11-19 epitope has been widely studied with respect to its binding affinity
and its possible role in the pathogenesis of HAM/TSP (Jacobson, 1996; Jacobson et al.,
1997; Jacobson et al., 1992). However, there are other conflicting reports that demonstrate
that this epitope is protective and that HLA-A*0201 positive HTLV-1 infected individuals
are less likely to develop disease (Bangham, 2000b; Jeffery et al., 1999). Likewise, immune
responses against the Tax 178-186 epitope was detected in peripheral blood of HTLV-1 infected individuals several years ago and the epitope was also classified as a strong MHC
Class I binder. Furthermore, protective studies in athymic rats using recombinant Tax
protein also demonstrate the applicability of Tax based epitopes for therapeutic purposes.
90 HTLV-1 like many other persistent viruses has evolved mechanisms to survive in the face of
an active immune response. The challenge studies described in this chapter can serve as a
proof of principle that the epitopes identified during natural infection are also immunogenic
in an active immunization setting. Hence, these epitopes may be used in a vaccination or
therapeutic setting to amplify the existent cell mediated response which may aid in the
clearance of the virus. Such multiepitope vaccines combined with adjuvants or cytokines
such as IL-12 or costimulatory molecules may provide greater clinical benefit against HTLV-
1 infections.
It is not clear why the Tax 11-19 was not as effective in protecting mice against
vaccinia challenge. However, there are numerous reports where immunodominant epitopes
are shown to be less effective in protecting against infections as compared to subdominant
epitopes. This was the case with our results as well where the Tax 178-186 and the Tax 306-315 epitopes were more effective. The statistical analysis showed that the multiepitope immunizations and the mixture immunizations were more effective in protecting the animals than single epitope immunizations. This could be due to an additive or synergistic effect between the different activated CTL. Furthermore, we believe that the 236 immunization more closely mimics natural infection and hence qualitatively, the immune responses may have been superior to the mixture immunization.
91
Name Orientation Sequence
236 Tax 11-19-RR-Tax 178-186-RR-Tax 306-315 LLFGYPVYVRRQLGAFLTNVRRHLLFEEYTNI
326 Tax 178-186-RR-Tax 11-19-RR-Tax 306-315 QLGAFLTNVRRLLFGYPVYVRRHLLFEEYTNI
632 Tax 306-315-RR-Tax 178-186-RR-Tax 11-19 HLLFEEYTNIRRQLGAFLTNVRRLLFGYPVYV
362 Tax 178-186-RR-Tax 306-315-RR-Tax 11-19 QLGAFLTNVRRHLLFEEYTNIRRLLFGYPVYV
Table 3.1. Variants of multiepitope constructs synthesized to determine the effect of the relative positioning of individual epitopes on the rate of processing of the construct by immunoproteasomes. Epitope sequences are shown in bold.
92 HOURS PEPTIDE CONSTRUCT 236 362 632 326
12 Tax 11-19 + + - + 4.68% 0.53% 0.18%
Tax 178-186 + - + 0.47% - 0.5%
Tax 306-315 + - - - 0.01% Substrate - ++ +++ ++
24 Tax 11-19 + + + + 6.53% 0.35% 6.84% 0.27%
Tax 178-186 + + + + 1.24% 1.15% 1.64% 1.51%
Tax 306-315 + + + + 0.02% 0.03% 0.02% 0.03% Substrate - + + +
48 Tax 11-19 + + + + 0.56% 0.15% 0.77% 0.24%
Tax 178-186 + - + + 0.07% 0.01% 0.09%
Tax 306-315 + + + + 0.02% 0.02% 0.02% 0.02% Substrate - + - -
Table 3.2. Immunoproteasomal cleavage analysis of variants of multiepitope constructs. The
four multiepitope constructs were digested as described in section 3.3. Digested samples
were analyzed by capillary liquid chromatography-nanospray tandem mass spectrometry.
Presence or absence of the individual epitope or the undigested peptide substrate in each
sample at every time point is indicated as +/- [+++>++>+]. The relative abundance of
each individual epitope detected in the sample is expressed as a percentage of the total
summed mass-peak intensities of the digested peptide substrate at the indicated incubation times.
93 Tax 306-315 Tax 155-163 50 50
El4/HHD + Tax 306-315 EL4/HHD + Tax 155-163 El4/HHD unpulsed El4/HHD unpulsed 40 40
30 30
20 20 % Specific % Specific Lysis % Specific Lysis Specific %
10 10
0 0 100 : 1 10 : 1 1 : 1 100 : 1 10 : 1 1 : 1
E : T Ratio E : T Ratio
Fig.3.1. Immunogenicity of predicted Tax epitopes. HHD mice were immunized with the indicated epitopes mixed with a T-helper epitope (TT3). After one boost, the splenocytes were tested for cytotoxicity against relevant epitope pulsed target cells at the indicated E:T ratios in a standard 51Cr release assay.
94 60 El4/HHD + 9mer epitope El4/HHD + 236 construct EL4/HHD + 236 construct 50 + LLNL inhibitor
40
30
% Specific Lysis % Specific 20
10
0 9 6 5 -1 11 8-18 6-31
Tax ax 17 ax 30 T T
Effectors
Fig.3.2. In vivo proteasomal processing of 236 multiepitope construct. Splenocytes from 236 immunized mice were stimulated in vitro with individual epitopes for 6 days and tested for cytolytic activity against EL4/HHD target cells that were pulsed overnight with 1µM of the
236 multiepitope construct in the presence or absence of 50µM proteasome inhibitor LLNL as detailed in section 3.3.
95 236 Construct
60 60 12
50 50 10 EL4/HHD + Tax 11-19 EL4/HHD + Tax 178-186 EL4/HHD + Tax 306-315 EL4/HHD unpulsed EL4/HHD unpulsed EL4/HHD unpulsed
40 40 8
30 30 6
20 20 4
10 10 2
0 0 0 100 : 1 20 : 1 4 : 1 100 : 1 20 : 1 4 : 1 100 : 1 20 : 1 4 : 1
362 Construct
25 14 25
12 20 EL4/HHD + Tax 11-19 EL4/HHD + Tax 178-186 20 El4/HHD + Tax 306-315 EL4/HHD unpulsed EL4/HHD unpulsed EL4/HHD unpulsed 10
15 15 8
6 10 10
4
5 5 2
0 0 0 100 : 1 20 : 1 4 : 1 100 : 1 20 : 1 4 : 1 100 : 1 20 : 1 4 : 1 632 Construct 60 60 6
50 EL4/HHD + Tax 11-19 50 EL4/HHD + Tax 178-186 5 EL4/HHD + Tax 306-315 EL4/HHD unpulsed EL4/HHD unpulsed EL4/HHD unpulsed
% Specific Lysis 40 40 4
30 30 3
20 20 2
10 10 1
0 0 0 100 :1 20 : 1 4 : 1 100 :1 20 : 1 4 : 1 100 :1 20 : 1 4 : 1 326 Construct 25 60 40
50 EL4/HHD + Tax 306-315 20 EL4/HHD + Tax 11-19 EL4/HHD + Tax 178-186 EL4/HHD unpulsed EL4/HHD unpulsed 30 El4/HHD unpulsed
40 15
30 20
10 20
10 5 10
0 0 0 100 : 1 20 : 1 4 : 1 100 : 1 20 : 1 4 : 1 100 : 1 20 : 1 4 : 1
E : T Ratio
Fig.3.3. Cytolytic responses of four variants of multiepitope constructs. Splenocytes from groups of HHD mice immunized once with each multiepitope construct were stimulated in vitro for 6 days with the relevant individual epitope as described in section 3.3. Cytolysis was
96 tested in a standard 51Cr release assay against EL4/HHD cells pulsed 10µM of the indicated epitope. Data represents mean responses with std. error from two independent experiments.
97 12000 236 construct 362 construct 632 construct 10000 326 construct
8000 pg/ml γ 6000 IFN-
4000
2000
0 Tax 11-19 Tax 178-186 Tax 306-315
Peptide stimulation (10µg/ml)
Fig.3.4. Cytokine release by multiepitope variants. Splenocytes from groups of HHD mice immunized once with each multiepitope construct were stimulated in vitro for 6 days with the each individual epitope as described in section 3.3. Activation of IFN-γ release was tested against relevant epitope pulsed EL4/HHD target cells in a 24 hour assay.
98 100 100 236 immunization 236 immunization Tax 11-19 immunization Tax 178-186 immunization
80 80
60 60
40 40 % specific lysis specific % % specific lysis specific %
20 20
0 0 10uM 1uM 0.01uM 0.0001uM unpulsed 10uM 1uM 0.01uM 0.0001uM unpulsed
peptide concentration peptide concentration
100
236 immunization Tax 306-315 immunization 80
60
40 % specific lysis specific %
20
0 10uM 1uM 0.01uM 0.0001uM unpulsed
peptide concentration
Fig.3.5. Affinity of CTLs induced by single epitope and multiepitope immunization.
Splenocytes from HHD mice immunized with either the single epitopes separately or 236 multiepitope construct were tested for cytotoxicity against EL4/HHD targets pulsed with decreasing amounts of relevant epitopes as indicated at a 100:1 E:T ratio. Data are representative of multiple independent experiments.
99 236 immunization single epitope immunization g/ml µ Peptide stimulation 10 stimulation Peptide Tax 306-315 Tax 178-186 Tax 11-19
0 2000 4000 6000 8000 10000
IFN-γ (pg/ml)
Fig.3.6. Comparison of IFN-γ release by splenocytes from single epitope and multiepitope
construct immunized mice. Splenocytes from each group were stimulated in vitro for 6 days
with the relevant epitope and tested against EL4/HHD target cells pulsed with 10µM indicated epitope in a 24 hour assay. IFN-γ released into the supernatant was quantitated using a sandwich ELISA as described in section 3.3.
100
l) l) rs o o tr tr V -V rke n on 0V A a co c 4 H d m (- P - te . (+ - D c t D D H e W -2 H H H nf . T H /H i 4/ 4 4/ k M M L L L E E E oc (m 207kDa
121kDa
81kDa
51kDa
33kDa
28kDa
uninfectedHA-VV infected P40-VV infected 207kDa 121kDa 81kDa
51kDa
33kDa 28kDa
21kDa
Fig. 3.7.Tax (p40) protein expression analysis in EL4/HHD cells (upper panel) or HeLa-HHD cells (lower panel) infected overnight with p40-VV by Western Blotting. Whole cell lysates of
HA-VV control virus infected cells or uninfected control cells were prepared as described in section 3.2. HTLV-1 infected MT-2 cells were used as positive control. Membrane was probed with Tax specific monoclonal antibody at a 1:5 dilution.
101
A 10
Tax 11-19 effectors Tax 178-186 effectors 8 Tax 306-315 effectors
6
4 % Specific Lysis % Specific
2
0 236 632 362 Immunization
B 25
p40 infected targets HA infected targets 20
15
10 % Specific Lysis %
5
0 Tax 11-19 Tax 178-186 Tax 306-315 Effectors
Fig.3.8. Cytolysis of p40-VV infected target cells. (A) Splenocytes from various multiepitope immunized HHD mice expanded in vitro with individual epitopes for 6 days as described in section 3.2 were tested for cytolysis against p40-VV EL4/HHD infected target cells at 100:1
102 E:T ratio. Background lysis from HA-VV control vaccinia infected EL4/HHD target cells were subtracted for each group. Bars represent mean values from two independent experiments with standard error. (B) Splenocytes from 236 immunized HHD mice expanded in vitro with individual epitopes were tested for cytotoxicity against HeLa-HHD cells infected with p40-VV or control HA-VV at 100:1 E:T ratio.
103 800
Tax 11-19 Tax 178-186 Tax 306-315 600
pg/ml 400 γ IFN-
200
0 236 362 632 Immunization
Fig.3.9. Activation of IFN-γ secretion by effector splenocytes from mice immunized
separately with each multiepitope variant. Splenocytes were tested against EL4/HHD target cells infected with recombinant Tax vaccinia virus (p40-VV) in a 24 hour assay. Level of
IFN-γ secreted was estimated using a sandwich ELISA. Background secretion observed against EL4/HHD target cells infected with control recombinant vaccinia virus expressing influenza hemagglutinin (HA-VV) was subtracted from above values.
104 p < 0.001 1015
1014 p < 0.001 ) 1013 ovary / 12 u 10 f p ( 11 er 10 tit 1010 rus i
a v 9 i 10 n i 108 vacc 10 107 og L 106
105 236 immunized mock immunized naive control Treatment
Fig.3.10. Vaccinia virus titers in the ovaries of HHD mice challenged with p40-VV. 10 days
after last immunization with 236 multiepitope peptide construct, mice were challenged i.p with 5X106 pfu of p40-VV. 5 days post challenge, ovaries were extracted and vaccinia titers
were measured by plaque assay on BSC-1 cells. As a control HHD mice were also
immunized with the T-helper epitope alone in emulsion (mock immunization). Viral load is
expressed as the log of the virus titer per ovary of each mouse. Statistical analysis to evaluate the therapeutic effect of 236 peptide immunization was performed as described in section
3.2.
105 1014 236 immunized 1013 naive
1012 p < 0.001 1011 * 1010
109
108
Vaccinia virusVaccinia titers (pfu/ovary) 107
106
105 p40 challenged HA challenged Treatment
Fig.3.11. Specificity of protection by 236 immunization. Average vaccinia virus titers in ovaries of 236 immunized mice or naïve mice challenged with either p40-VV or control irrelevant vaccinia virus expressing the influenza hemagglutinin protein (HA-VV). Asterisk indicates a statistically significant reduction in p40-VV viral loads (p < 0.001).
106
Naïve 236 immunized
1 2 3 4
56 7 8
Events
PE log
Fig.3.12. Flow cytometric analysis of in vivo depletion of CD8+ T-cells in HHD mice.
Peripheral blood cells from naïve, 236 immunized (upper panels) or 236 immunized mice
that were in vivo CD8+ T-cell depleted (lower panels) were stained with 1µg PE-labeled rat
anti-mouse CD8 antibodies prior to challenge with p40-VV. Peripheral blood staining of each individual CD8 depleted mouse is shown.
107 15 p < 0.001
14 p = 0.005 p = 0.025
13
12
11
10
9
Vaccinia titer (pfu/ovary) 8 10
7 Log
6
5 236 immunized CD8+ depleted naive control Treatment
Fig.3.13. Protection is dependent of the presence of CD8+ T-cells. Vaccinia virus titers in
the ovaries of HHD mice challenged with p40-VV. 10 days after last immunization with 236
multiepitope peptide construct mice were CD8+ T-cell depleted by i.p injection of 100µg
anti-CD8 antibodies on Day -4, -3, -1 and +1, day 0 being the day of challenge with 5X106 pfu of p40-VV. 5 days post challenge, ovaries were extracted and vaccinia titers were measured by plaque assay on BSC-1 cells. Viral load is expressed as the log of the virus titer per ovary of each mouse. p values of 0.005 or lower were considered significant.
108 1015 = = 0.003 = 0.001 = p p 1014
13 = 0.001
10 * p
1012 < < 0.001 1011 p
1010
109
108 Vaccinia virus titer (pfu/ovary) titer virus Vaccinia 10 107 Log Log 106
105
ed ed d iz iz ntrol nize un un o u ax 11-19 178-186 306-315 m e c T x x v Ta Ta im nai ck imm 236 imm o ixture m m Treatment
Fig.3.14. Comparison of vaccinia virus titers in the ovaries of HHD mice challenged with
p40-VV. 10 days after last immunization with 236 multiepitope peptide construct or single
epitopes as indicated or with an equivalent mixture of the three epitopes, mice were
challenged i.p with 5X106 pfu of p40-VV. 5 days post challenge, ovaries were extracted and
vaccinia titers were measured by plaque assay on BSC-1 cells. Viral load is expressed as the log of the virus titer per ovary of each mouse. p values of 0.005 or lower were considered significant. Asterisk indicates statistically significant (p < 0.001) difference in viral loads as
compared to mock immunized mice observed only with 236 immunization.
109
CHAPTER 4
DESIGN AND IMMUNOLOGICAL CHARACTERIZATION OF PEPTIDES THAT
MIMIC THE COILED COIL REGION OF HTLV-1 TRANSMEMBRANE SUBUNIT
4.1 RATIONALE
The HTLV-1 envelope proteins expressed on the surface of virus infected cells and on viral particles are the first to be recognized by the host in the course of a natural immune response (Nagy et al., 1983; Palker et al., 1989). Several studies have focused on linear
immunodominant regions of the surface glycoprotein (SU) to elicit neutralizing antibody
responses against HTLV-1 for the purpose of vaccine development and diagnostic screening
(Baba et al., 1995; Baba et al., 1993; Desgranges et al., 1994; Grange et al., 1998; Inoue et al.,
1992; Kuroki et al., 1992b; Palker et al., 1992; Tanaka et al., 1994; Tanaka et al., 1991).
However, there have been far less focus on the gp21 transmembrane (TM) subunit. Recent
data has shown that the transmembrane gp21 domain plays a critical role in the postbinding
steps during infection that is required for the viral core to be delivered into the target cell
cytoplasm (Rosenberg et al., 1997). Peptides derived from the gp21 region encompassing
amino acids 361-430 showed specific reactivity to sera from HTLV-1 infected individuals
(Hadlock et al., 1995). Likewise, later studies with overlapping synthetic peptides revealed
the importance of amino acids 400-429 in inhibiting the formation of syncytia between
infected cells (Sagara et al., 1996).
110 There is little structural homology between the SU subunits of various retroviral
envelope proteins however, the TM subunits of retroviruses display remarkable homology
with conserved spatial conformations (Pancino et al., 1994). In general, they can be divided
into an N-terminal hydrophobic fusion peptide, an adjacent leucine-zipper like motif that is
capable of self-assembly into a coiled coil, a disulphide bonded region followed by a C-
terminal region that contains α-helical segments. The transmembrane and cytoplasmic tail
regions are located at the C-terminus. The recently solved crystal structure of the central segment of the HTLV-1 transmembrane subunit as a chimera with maltose binding protein shows that the HTLV-1 TM does not deviate from the conserved spatial organization and general organization (Kobe et al., 1999).
The gp46 subunit is believed to be responsible for the recognition of the cellular receptor. Following receptor recognition, conformational changes in the transmembrane region exposes the fusion peptide that brings the viral and cell membranes in close proximity for fusion to occur. Fusion is required for the introduction of the viral core into the cytoplasm of target cells. Although there is limited information regarding the precise mechanism of envelope mediated fusion and the role of the TM glycoproteins, their critical role in the fusion process is suggested by their similarity in sequence and structure.
Mutational studies have defined certain regions such as the coiled coil segment and the disulphide bonded region of chain reversal C-terminal to the coiled coil segment as being critically involved during the later stages of the fusion process after receptor binding (Maerz et al., 2000; Rosenberg et al., 1997).
Based on the above observations, we hypothesized that a rationally designed vaccine consisting of B-cell epitopes derived from these important regions of the TM subunit should
111 elicit antibodies that may interfere with the fusion process by binding to the relevant region
on the envelope protein. We focused on a B-cell epitope derived from the central coiled
region for our studies that is critical for fusion. Over the last several years, our laboratory has
focused on the development of a chimeric strategy to elicit antibody responses. These
constructs consist of a “promiscuous” T-helper cell epitope linked via a 4-residue turn
sequence, (GPSL) to a B-cell epitope. These chimeric peptides allow independent folding of
the B-cell and T-cell epitope and are very effective in overcoming MHC restriction in
various strains of mice and in eliciting of high titered antibody responses (Frangione-Beebe
et al., 2000; Kaumaya, 1992; Kaumaya, 1994a; Lairmore et al., 1995a). The incorporation of a
promiscuous T-helper epitope was effective in generating the required “help” for the induction of antigen specific antibodies without the problems associated with carrier proteins. Furthermore, in order to construct epitopes in various defined orientations and conformations, a novel single-matrix multicomponent template strategy was also developed in the past (Kaumaya, 1994a; Kaumaya, 1994b; Kaumaya, 1993a). In this approach, a core β- sheet template consisting of alternating Gly/Leu or Lys is synthesized such that the Σ side
chains of the lysine residues allow for the growth of individual epitopes in varying
permutations. Model peptides incorporating different combinations of B-cell epitopes and
T-cell epitopes from LDH-C4 antigen and the HTLV-1 envelope SU subunit synthesized in
a template format resulted in enhanced immunogenicity in several inbred and outbred strains
of mice and rabbits eliciting antibodies reactive with the native protein (Kaumaya et al.,
1992; Kaumaya, 1996; Kobs-Conrad et al., 1993; Lairmore et al., 1995a). This chapter
describes the structural characterization and immunogenicity studies of a peptide derived
from the central region of the TM subunit that required a new template design and a
112 different synthetic approach to synthesize a peptide that would assemble into a triple helical coiled coil conformation mimicking the gp21 solved crystal structure. The rationale for this design was to constrict the three strands at one end and also to bring them in close proximity to allow for hydrophobic interactions to form a trimeric coiled coil.
4.2 SUMMARY
The region corresponding to the coiled coil (residues 347-374) was synthesized as three separate strands, off of three lysines of a Gly-Lys template. The same peptide was also synthesized with five leucine substitutions at the a and d positions of the heptad repeat to maximize hydrophobic interactions between these residues that would potentiate the triple helical coiled coil formation. Circular dichroism (CD) measurements of the mutated coiled coil peptide revealed a stable α-helical structure that was concentration independent. The wild type peptide on the other hand was less helical under similar conditions. Both peptides were highly immunogenic in outbred mice. Isotyping of the antibodies showed that the two peptides elicited different isotype subclasses of antibodies. Antibodies against both peptides were able to cross react with the native envelope protein as determined by ELISA with the recombinant gp21 protein and flow cytometric analysis using HTLV-1 infected cells.
Competition experiments using various peptides or the gp21 chimeric protein as inhibitor revealed that antibodies against the leucine substituted template peptide are highly specific to the conformation of the immunogen. Furthermore, these antibodies bound with higher affinity to the native whole viral protein indicating that the peptide was folding in the native conformation.
113 4.3 MATERIALS AND METHODS
Peptide synthesis and purification
Template peptides were synthesized by solid phase peptide synthesis following 9- fluorenylmethoxycarbonyl-t-butyl (Fmoc) chemistry with benzotriazole-1-yl-oxy-tris-
(dimethylamino)-phosphoniumhexafluorophosphate/N-hydroxybenzotriazole
(BOP/HOBt) as a coupling reagent on a fully automated peptide synthesizer (Model 9600
Peptide Synthesizer MilliGen/Biosearch) as described previously (Kobs-Conrad et al., 1993) with modifications. The template peptide (GKGKGKG), with Lys side chain protected
(ivDde) was assembled on Rink-Amide-CLEAR Resin (substitution 0.41 mmol amino groups/g, (Peptides International Louisville, KY) using Fmoc-Lys(ivDde) (BACHEM
California). The N-terminus of the template peptide was acetylated using Acetylimidazole
(Aldrich, Milwaukee, WI) in DMF. The Lys side chain deprotection (ivDde) was achieved using 2% hydrazine hydrate in DMF (3 min. and 10 min, positive Kaiser Test). The gp21
(residues 347-374) peptides were assembled on the template using peptide synthesis protocol described above and acetylated at N-terminus. The peptides were cleaved from support using Reagent R. The crude peptides were purified by semipreparative reversed phase HPLC using a C-4 column (Vydac, Hesperia, CA). Analytical HPLC was performed using a Vydac
Column using a linear gradient of 90% acetonitrile in water containing 0.1% trifluoroacetic acid. The identity of peptides was confirmed by electrospray ionization time of flight mass spectroscopy.
Circular dichrosim measurements
Circular dichroism measurements were performed on an AVIV model 62A DS CD instrument (Lakewood, NJ). All spectral measurements were obtained at 250C under
114 continuous nitrogen purging of the sample chamber, using a quartz cuvette of 0.1cm path
length. Spectral measurements of the peptides were obtained over a range of concentrations
(25-100µM) in water or in 50% trifluoroethanol (TFE). Water or 50% TFE blanks were
subtracted from the CD spectra. Molar ellipticity values were calculated using the formula
θ = (θ 100 Μ / ( λ), θ [ ]M, λ x x r) n x c x where is the recorded ellipticity (deg), Mr is the molecular weight of the peptide, n=number of amino acid residues in peptide, c=peptide concentration (mg/ml) and l=path length of cuvette (Kaumaya, 1990). Helicity of the peptides were calculated according to Chen’s (Chen et al., 1974) equation with reference to
α θ the mean residue ellipticity of polylysine for 100% -helix ( )222nm = -33,000.
Guanidinium hydrochloride denaturation
The structural stability of the CCR2T and WCCR2T template peptides were determined by
chemical denaturation experiments using guanidinium hydrochloride (GnHCL) as described
previously (Causton and Sherman, 2002). Peptide concentration was maintained at 50µM
and the concentration of GnHCL was increased from 0-13M in water. The ellipticity of the
peptide was then measured at 222nm similar to that described above. Ellipticity was then
θ normalized to the fraction folded by using the following equation: f folded = ( observed-
θ θ θ unfolded)/( folded- unfolded) (Litowski and Hodges, 2001).
Animal immunizations
Six to eight-week old female ICR outbred mice were purchased from Jackson Laboratories
(Bar Harbor, ME). Groups of 8-9 mice were immunized separately with 100µg of either
CCR2T or WCCR2T. The peptides were dissolved in water with 100µg of a muramyl
dipeptide adjuvant, nor MDP (N–acetyl-glucosamine-3 yl–acetyl L–alanyl–D–isoglutamine),
and emulsified (50:50) in Squalene/Arlacel A oil (4:1) as described elsewhere (Jones et al., 115 1988). Booster immunizations were administered at 3, 6 and 9 weeks for CCR2T and at 3
weeks and 6 weeks for WCCR2T. Mice were bled retro-orbitally every week for antibody
titer determination and sera collected was heat inactivated by heating to 560C for 30 minutes.
ELISA
Antibody titers were determined using ELISA as previously described (Kaumaya et al.,
1992). Briefly, 96-well plates were coated overnight with 100µl of a 2µg/ml solution of the
peptide or gp21 protein in PBS at 40C. The plates were then blocked for 1 hour with 200µl
PBS-1% BSA and plates rinsed with PBS-Tween 0.05% and 1% horse serum (wash buffer).
Mouse antiserum was diluted in wash buffer and added to the antigen coated plates in
duplicate wells, serially diluted 1:2 in wash buffer, and incubated 2 hours at room
temperature. After washing the plates, 100µl of 1:500 goat anti-mouse IgG conjugated to
horseradish peroxidase (Pierce) was added to each well and further incubated for 1 hour.
µ After washing, the bound antibody was detected using 50 l of 0.15% H2O2 in 24mM citric
acid, 5mM sodium phosphate buffer, pH 5.2, with 0.5mg/ml 2,2’-aminobis(3-
ethylbenzthiazoline-6-sulfonic acid) as the chromophore. Color development was allowed to
proceed for 10 minutes and the reaction stopped with the addition of 25µl of 1% SDS.
Absorbance was determined at 415nm using a Benchmark Microplate Reader (Bio-Rad,
Hercules, CA). Titers were defined as the highest dilution of sera with an absorbance greater than 0.2 after subtracting the background. All data represent the average of duplicate samples.
Competitive ELISA
Plates were coated with peptide/antigen and blocked as described for the direct ELISA.
Primary antibody was added as 50µl of a constant dilution (50 % maximal binding) with 50
116 µl of a serially diluted competitive inhibitor, with concentrations ranging from 0 to 60µM.
Positive inhibitors were peptide/antigen for which the antibodies were specific. Inhibitor and antiserum were incubated for 2 h. HRP linked goat anti-mouse IgG addition and color development were performed as described for the direct ELISA protocol.
Whole Virus ELI SA
Reactivity of peptide antisera to native HTLV-1 whole viral lysate was determined using whole virus ELISA. A Vironostika HTLV-I/II Microelisa assay (Organon Teknika, Durham,
NC) was performed according to manufacturer’s instructions, modified for mouse serum.
Antibody dilutions used were 1/100 for immune and preimmune negative control serum. A
1/2000 dilution of HRP linked goat anti-mouse IgG (0.8mg/ml) secondary antibody was
used. Plates were read at 450nm and the data represented as absorbance units.
Antibody isotyping
The mouse antisera were isotyped using a Mouse Typer Sub-Isotyping kit (Bio-Rad,
Hercules, CA). The assay was performed as per manufacturer’s instructions; except that a
1/250 dilution of the primary serum and HRP linked goat anti-rabbit IgG were used.
Flow cytometry
Binding of peptide antibodies to HTLV-1 infected cells was tested by flow cytometry as adopted from Hudziak et al (Hudziak et al., 1989). ACH cells were grown in RPMI 1640 with
10% FCS and 1% pen/strep. For the ACH cells, 5% of human IL-2 was also added to the
culture media. 0.5 X 106 cells were incubated with a 1/5 dilution of the peptide antibodies.
The same dilution of pre-immune serum was used as negative controls. HTLV-1 infected
rabbit serum was used as a positive control. The cells were stained with primary antibodies
for 2 hours at 40C in 100µl of PBS-2% FCS. The cells were then washed twice with PBS and
117 stained by incubation with FITC labeled F(ab)2 fragment goat anti-mouse IgG secondary
antibody (1:50 dilution) for 45 minutes at 40C in 100µl of PBS-2%FCS, then fixed in PBS-
2% paraformadehyde and analyzed by coulter ELITE flow cytometer (Coulter, Hialeah, FL).
A total of 10,000 gated events were collected and final processing performed. Debris, cell clusters and dead cells were gated out by light scatter assessment before single parameter histograms were drawn
4.4 RESULTS
Engineering of template peptides to mimic coiled coil conformation
It has been previously shown that the minimum peptide length for stable formation of a coiled coil was 29 residues (Lau et al., 1984) that corresponds to approximately 4 heptads with 7 residues/heptad. Coiled coils are usually made up of heptad (abcdefg) repeats which are stabilized by hydrophobic interactions between the a and d positions in the different strands (Hodges, 1996; Hodges et al., 1981). The HTLV-1 coiled coil segment consists of amino acids 340-392. We selected the region between amino acids 347-374 which spans the central region of the coiled coil and has four potential heptad repeats. In order to increase the proximity of the three peptide strands such that they interact with one another
to form a coiled coil, the peptides were synthesized simultaneously through the Σ NH2 side
chains of 3 lysyl residues separated by intervening glycine residues as depicted in Fig.4.1. To
limit charge repulsions at the N-terminus between each strand the three strands were
acetylated. A promiscuous T-helper epitope was also synthesized colinear with the template
sequence at the N-terminus. First, we synthesized the wild type sequence 347-374 designated
WCCR2T. Additionally, another template construct designated CCR2T, was designed that incorporated five substitutions at the a and d positions (V349L, I353L, I360L, N363L and
118 I370L). The rationale for these substitutions was to increase the hydrophobic interactions that should further stabilize the coiled coil conformation. We also synthesized the two peptides as single strands that corresponded to only the B-cell epitopes that were acetylated and amidated to stabilize the helix dipole. The peptides were purified by reverse phase
HPLC and the identities of all peptides were confirmed by MALDI-TOF or electrospray ionization mass spectrometry. All peptides synthesized along with their molecular weights and sequences are shown in Table 4.1.
Conformational characterization of template peptides in aqueous solution by circular dichrosim measurements
We used circular dichroism measurements to evaluate the structural characteristics of the two template peptides. Spectra typical of α-helices, was obtained showing a double minima at 222nm and 208nm and a miximum at 193nm (Chen et al., 1972) as shown in
Fig.4.2. Concentration dependencies of each peptide over a wide range of concentrations, ranging from 25µM-100µM were investigated. The helicities of the different peptides observed in aqueous solution and in 1:1 water: TFE are shown in Table 4.2. High helical content (approximately 55%) that was stable over a wide range of concentrations was observed in water with the CCR2T peptide. When the measurements were taken in 50%
TFE there was a marginal increase in the helicity of the peptide. This implies that the peptide was attaining its maximal helical potential in water and that TFE did not have any major effect in inducing a helical conformation. Furthermore the concentration independent helical content implied that there was no oligomerization or aggregation of the peptide at higher concentrations. The WCCR2T peptide, on the other hand, showed a much lower helicity in water as compared to the CCR2T (approximately 13%) which increased significantly to 55%
119 when TFE was added. TFE stabilizes α-helical structure (Jasanoff and Fersht, 1994); hence
the wild type template peptide did not fold completely in aqueous solution. The individual
B-cell epitopes synthesized as free peptides were also tested for their helical potential.
CCR2E and WCCR2E showed similar levels of helicity of (approximately 12% and 10%
respectively) in water. However, upon addition of TFE the helicity increased to about 49%
for WCCR2E and 55% for CCR2E. Here again there was no increase in helicity as the
concentration was increased suggesting that there was no oligomerization.
Additionally, the stability of the CCR2T and WCCR2T peptide in water was tested
by chemical denaturation experiments using increasing concentrations of guanidinium
hydrochloride (GnHCL). As observed in Fig.4.3, the CCR2T peptide was highly resistant to
chemical denaturation and significant unfolding was observed only at higher concentrations
of >6M GnHCL. In contrast, WCCR2T revealed a steady unfolding of the secondary
structure with increasing concentrations of GnHCL. This implied that the CCR2T template
peptide had adopted a more stable α-helical secondary which was probably due to the
increased hydrophobic interactions with the leucine substitutions.
Immunogenicity studies of template peptides
The two template peptides were synthesized to include a promiscuous T-helper
epitope TT from tetanus toxoid (aa 580-599) (Kaumaya, 1994b). This epitope was chosen
since it was devoid of lysine residues that may interfere with the synthesis of the three
strands of the gp21 B-cell epitope. WCCR2T and CCR2T were emulsified as described in
section 4.3 and used to immunize groups of female outbred mice. Both template peptides
were highly immunogenic and high titered antibodies were obtained (Fig.4.4). Both CCR2T
and WCCR2T elicited high antibody responses after the first booster immunization and
120 subsequent booster injections did not increase the titers. The WCCR2T peptide was slightly
more immunogenic and higher titers in the range of 32000-64000 (reciprocal of highest sera dilution with absorbance units > 0.2) were obtained two weeks after the first booster.
Overall, WCCR2T titers were higher than CCR2T titers although both peptides elicited high
titered antibodies in outbred mice. Furthermore, the antibody isotypes induced by
immunization with the template constructs were also determined. The isotypes of the
antibodies that were reactive with the template peptides and those reactive with the native
protein on viral particles were tested separately. Both CCR2T and WCCR2T peptides elicited
antibodies of the IgG subtype, predominantly IgG2b in the case of CCR2T (Fig.4.5) specific
to the template peptide and equivalent amounts of IgG1, IgG2a and IgG2b in the case of
WCCR2T. Likewise, antibodies that were cross reactive with the native protein on HTLV-1
viral particles were also predominantly of the IgG2b type for CCR2T antisera but
predominantly IgG2a type for WCCR2T antisera and to a lesser extent IgG2b. Hence the
two template peptides induced a slightly different antibody profile that may be related to the
difference in sequence and/or conformation.
Cross reactivity of peptide antibodies to native protein
For peptide antibodies to be effective in neutralizing virus infection, it is imperative
that the antibodies cross react with the native protein from which the epitope was derived.
To test the cross reactivity of template peptide antibodies to the native gp21 protein we
utilized three separate assays; 1) flow cytometry using HTLV-1 infected cells, 2) direct
ELISA using recombinant maltose binding protein (MBP)-gp21 chimera (Center et al., 1998)
and 3) whole virus ELISA using plates coated with HTLV-1 viral lysate.
121 ACH cells are primary human peripheral blood cells that were immortalized using an infectious molecular clone of HTLV-1 (Collins et al., 1996). These cells express the envelope protein of HTLV-1 and were used to test the binding of CCR2T and WCCR2T mouse antisera to ACH cells. As depicted in Fig.4.6 we observed that both the antibodies bound the surface of ACH cells indicating that both antibodies were capable of recognizing the native protein. Preimmune serum from outbred mice was used as a negative control. These results were further confirmed using direct ELISA and recombinant MBP-gp21 protein chimera.
The antibodies against CCR2T and WCCR2T were able to specifically bind the gp21 protein although the absorbance observed was lower that that seen with the template peptide immunogen (Fig.4.7, upper panel).
Virus neutralization has been defined as the “loss of infectivity” that occurs as a result of anti-viral antibodies binding to free virus particles (Burton, 2002; Parren and
Burton, 2001). The ability of peptide antibodies to bind native protein on viral particles was tested in a whole virus ELISA system using microtiter plates coated with an HTLV-1 viral lysate and various dilutions of CCR2T and WCCR2T antisera. As indicated in Fig.4.7 (lower panel), both CCR2T and WCCR2T antisera were able to recognize whole virus. A significant difference in the levels of binding of CCR2T antisera and WCCR2T antisera was observed.
The CCR2T antisera showed much higher binding as compared with the binding observed for the WCCR2T antisera. The preimmune mouse serum did not show any binding under similar conditions. These results indicated that the CCR2T antisera had higher levels of native protein cross reactive antibodies than WCCR2T.
122 Specificity of peptide antibodies
The relative affinities or specificity of the two template peptide antisera for the native
gp21 protein, the corresponding template peptide immunogen and the B-cell epitope for
each template by itself was assayed using competitive inhibition ELISA. Immune CCR2T
antisera (1/16000 dilution) could be effectively inhibited for binding to immobilized
immunogen CCR2T by the CCR2T inhibitor in solution. In the same analysis 16-fold higher
concentrations of the B-cell epitope CCR2E was required to completely abrogate binding of
CCR2T antisera to the immobilized immunogen (Fig.4.8). These results indicated that the
CCR2T antiserum was highly specific to the immunizing template peptide or to the specific conformation. In contrast, on WCCR2T template peptide coated plates, both WCCR2T template peptide and WCCR2E B-cell epitope peptide in solution were effective in inhibiting binding of WCCR2T antisera (1/16000 dilution); the WCCR2T template peptide being only slightly more effective than WCCR2E.
We also tested the relative affinity of the fraction of gp21 protein cross reactive antibodies in CCR2T antisera and WCCR2T antisera. Both antisera (1/20 dilution) were tested for their binding to gp21 protein immobilized on the plate in the presence of increasing concentrations of gp21 protein inhibitor in solution. As seen in Fig.4.9, CCR2T antisera and WCCR2T antisera binding to immobilized protein could be inhibited by gp21
protein in solution with similar affinity.
4.5 DISCUSSION
Peptides that mimic the native structure in the antigenic protein are more likely to
elicit antibodies that have high affinity for the native protein which may have neutralizing
potential. Likewise, many of the neutralizing antibodies that have been identified during
123 natural infection have been specific for conformational epitopes i.e those that arise due to
specific folding of the protein or those antibodies that recognize specific conformations
adopted by immunogenic contiguous segments of the protein. Hence, in recent years there
has been a great thrust in the design of peptides that are able to adopt a defined
conformation in solution (Barthe et al., 2000; Cooper et al., 1997; Starovasnik et al., 1997).
The central region of the HTLV-1 TM subunit has been implicated to be very important in
the fusion process between the viral or infected cell membrane and the receptor expressing
target cells. This region forms a leucine-zipper like motif that undergoes conformational
changes to adopt a parallel trimeric coiled coil structure that brings the fusion peptide in close proximity with the target cell membrane leading to fusion. Indeed the crystal structure of the gp21 subunit in the fusion activated state shows the central region to be a coiled coil.
Furthermore, most of the point mutations within this region result in loss of infectivity
(Maerz et al., 2000). From a vaccine standpoint, we hypothesized that antibodies specific to these transitional structures would be able to interfere with the fusion process and hence arrest viral infection. The goal of the present study therefore was to design peptides that mimic the native coiled coil conformation of the gp21 central region and to generate antibodies that bind with high affinity to the native protein. The HTLV-1 coiled coil region is naturally stabilized by various internal interactions. This includes among other interactions, an N-terminal hydrophobic cap consisting of the Met338 side chain that makes extensive contact with the first helical residue Leu340 and a hydrophobic core structure formed by
Leu384, Leu385, Phe386 and Ala395 at the C-terminus. The N-terminal cap is implicated to stabilize the fusion activated coiled coil state since the HA2 capping structure is superimposable with the HTLV-1 (Maerz et al., 2000). α-helical segments as isolated
124 peptides are generally devoid of any structure and assume random-like conformations in aqueous medium unless constrained by disulphide bridges and appropriate cyclizations
(Hodges, 1996). To compensate for the lack of additional interactions with adjacent residues when designing the coiled coil peptide segment as an isolated species, a template strategy was applied that has been successfully used in the past by our laboratory to synthesize peptides that have defined structure. This strategy involves a single-matrix multicomponent synthesis that constrains the individual peptide strands at one end on a β-sheet template and serves to bring each strand in close proximity to promote hydrophobic interactions to fold into a coiled coil. Similar strategies such as the use of rigid organic macromolecules have been applied for the synthesis of model coiled coils from other proteins referred to as template assembled synthetic proteins (TASPs) (Causton and Sherman, 2002; Futaki, 1998; Suich et al., 2000). Additionally, based on studies of proteins that form helical bundles, leucine was found to be the most frequent occupant at postions a and d of the heptad repeat (Islam and
Weaver, 1990; Parry, 1982; Zhou et al., 1992). Hence, we synthesized a similar template peptide where all the residues at the a and d positions were mutated to leucine (CCR2T) as the latter residue displays side chain orientation that is conducive to ideal packing. The interdigitation of the hydrophobic core allows the display of hydrophilic residues that are important for antibody recognition. Circular dichroism experiments revealed that the peptide with leucine substitutions had a high helical content in aqueous solution and the addition of helix-inducing TFE solvent did not increase the helicity significantly. Furthermore, the
CCR2T peptide was highly resistant to chemical denaturation and the peptide remained almost completely folded in 6M GnHCL. This was in sharp constrast to the wild type peptide WCCR2T synthesized on a similar template whose structure showed steady
125 disruption with lower concentrations of GnHCL. Likewise the individual B-cell epitope
CCR2E also displayed very low helical content in water. These results taken together suggested that the leucine substitutions combined with the template design for synthesis was essential for the formation of a defined α-helical structure. A further confirmation of a defined helical structure was obtained in whole virus ELISA assays in which the CCR2T antisera showed higher binding to the HTLV-1 viral lysate than WCCR2T wild type antisera.
These results indicated that the CCR2T template peptide was able to better mimic the native protein conformation and the antibodies induced had higher affinity for the native protein.
Competitive inhibition experiments further confirmed the specificity of CCR2T antisera to a defined secondary structure. In addition, peptide competition analysis showed the CCR2T template peptide was a better inhibitor for CCR2T antisera than the corresponding B-cell epitope which lacked a defined secondary structure in aqueous solution. In constrast,
WCCR2T antisera could be efficiently inhibited by both WCCR2T template peptide and the corresponding B-cell epitope. Similar analysis using recombinant gp21 protein however, showed CCR2T and WCCR2T antisera to have similar affinity. Furthermore, in direct
ELISA very low levels of reactivity were observed with both antisera. One possible explanation is that the gp21 protein used in the ELISA and competition studies was a chimera of maltose binding protein (MBP) and gp21 (amino acids 338-425), with the gp21 segmant forming a very small part of the entire chimera. This could lead to steric hindrances in binding analysis when the protein is immobilized onto ELISA plates. Hence, the binding and titers we observed may not be the accuarate representation of actual affinity for the native protein. This could also explain the results obtained in the competition experiments
126 using the gp21 protein as inhibitor. However, in the whole virus ELISA experiments we tested the binding of CCR2T and WCCR2T against a disrupted virus which is more relevant.
Although both constructs were immunogenic in mice the isotypes of antibodies induced was different indicating a differential activation of the immune response. CCR2T predominantly induced an IgG2b response when tested against the immunogen as well as whole virus while WCCR2T predominantly induced an IgG2a response in the case of whole virus. IgG2a and IgG2b antibodies are known to effectively fix complement in mice (Ey et al., 1980; Neuberger and Rajewsky, 1981); hence these antibodies may be important in the control of viral infections. IgG2a is also effective in interacting with macrophage and NK cell Fcγ receptors that are involved in indirect functions of antibodies such as antibody dependent cell mediated cytotoxicity. However, high affinity binding to the cognate protein is a prerequisite for these functions.
In conclusion we utilized a novel strategy to synthesize peptides that would structurally resemble the fusion activated coiled coil conformation of the gp21 subunit to elicit antibodies that would bind with high affinity to the native protein. While the antisera was able to recognize native protein, further modifications aimed at stabilizing the structure such as the introduction of disulphide bridges to covalently link the different strands on the template can be attempted that may serve to increase helix stabilizing interactions. Indeed, a recent report by Lu et al (Lu and Hodges, 2002) describes the use of a template strategy with a disulphide bridge to elicit antibodies that are specific to a α-helical structure. These kinds of studies are not only important from a vaccine perspective to elicit neutralizing antibodies against transient structures of the viral fusion process but also form important tools to probe
the structure function relationship in the envelope protein of HTLV-1
127 Designation Peptide Sequence M.Wt. (da) Wild type * * * WCCR2T template Ac-NSVDDALINSTIYSYFPSV-GKGKGKG-Am 12,605.8 peptide *LHEVDKDILSQLTQAIVKNHKNLLKIAQY Leucine * * * substituted Ac-NSVDDALINSTIYSYFPSV-GKGKGKG-Am CCR2T 12,652.8 template *LHELDKDLLSQLTQALVKLHKNLLKLAQY peptide Wild type WCCR2E B-cell Ac-LHEVDKDILSQLTQAIVKNHKNLLKIAQY-Am 3299 epitope Leucine substituted CCR2E Ac-LHELDKDLLSQLTQALVKLHKNLLKLAQY-Am 3312 B-cell epitope
Table 4.1. Chimeric template peptides synthesized from central region of gp21 envelope subunit. Leucine substitutions introduced into the wild type sequence are bolded. Lysine residues used for extension of the gp21 B-cell epitope (aa 347-374) are indicated by an asterisk. CCR2E and WCCR2E are the B-cell epitopes synthesized separately as free peptides. All peptides were acetylated (Ac) at the N-terminus and amidated at the C-terminus
(Am).
128 Peptide WCCR2T CCR2T WCCR2E CCR2E (µM) % helicity % helicity % helicity % helicity 50% 50% 50% 50% water water water water TFE TFE TFE TFE 25 15.8 63 ND ND 10.2 51.9 12.1 61.3 50 13.7 61.9 45.5 54.1 11.6 48.5 12.2 52.8 75 12.2 52.1 43.9 62.3 10 46.8 12.6 53.4 100 12.3 50.6 43.8 52.2 9.1 49.9 13.6 54.1
Table 4.2. Concentration dependence analysis by circular dichroism measurements. The % helicity of the various peptides were determined at 222nm as a function of peptide concentration. Helicity of peptides were calculated according to Chen’s equation (Chen et al.,
α θ 1974) with reference to the mean residue ellipticity of polylysine for 100% -helix ( ) 222 = -
33,000. ND= not determined.
129
CH3CO CH3CO CH3CO
gp21 Coiled Coil B-cell Epitope
Promiscous T-helper Epitope (TT) K K K
G G G G CONH2 N-terminus C-terminus
Fig.4.1. Template design for synthesis of the coiled coil region of gp21 envelope subunit.
Amino acids 347-374 correponding to the coiled coil region was synthesized on a template of three Gly-Lys repeats (shown as boxed residues). A promiscuous T-helper epitope (TT) derived from tetanus toxoid was synthesized colinear with the template. The peptide was acetylated and amidated for greater stability.
130 CCR2T 15000 WCCR2T 5000
10000 0 5000 /dmol) 2 /dmol) 2 0 -5000
-5000 -10000
-10000 -15000 -15000 water Mean Residue Ellipticity (deg cm water 50% TFE Mean Residue Ellipticity (deg cm -20000 -20000 50% TFE
-25000 -25000 200 220 240 260 280 300 200 220 240 260 280 300
Wavelength (nm) Wavelength (nm)
50000 40000 CCR2E WCCR2E 40000 30000
/dmol) 30000 2 /dmol) 2 20000
20000 10000 10000
0 0
-10000 water -10000 Mean Residue Ellipticity (degcm water MeanResidue Ellipticity (deg cm 50% TFE 50% TFE
-20000 -20000
200 220 240 260 280 300 200 220 240 260 280 300
Wavelength (nm) Wavelength (nm)
Fig.4.2. CD spectra of the different B-cell epitope constructs synthesized from the gp21
coiled coil region. Spectroscopy measurements were made either in water or water:TFE (1:1) at 50µM peptide concentration. Changes in the spectra indicate the presence of secondary
structure in the peptides. Spectra characteristic of α-helices with minima at 208nm and
222nm and a maximum at 193nm was observed in all peptides in TFE indicating the
formation of α-helices. High helical content in water that was comparable with TFE was
observed with the CCR2T template construct only.
131 1.2 50µM CCR2T 50µM WCCR2T 1.0
0.8
0.6 fraction folded fraction 0.4
0.2
0.0 0 1 2 3 4 5 6 7 8 9 10 11 12 [GnHCL] (M)
Fig.4.3. Guanidinium hydrochloride denaturation curve of CCR2T and WCCR2T at 250C in water. The fraction of folded peptide was calculated at described in Section 4.2.
132
CCR2T
100000
10000
Antibody Titers Antibody 1000
100
3 4 5 6 7 8 9 10 11 12
Weeks post immunization
WCCR2T
100000
10000
Antibody Titers Antibody 1000
100
3 4 5 6 7 8 9 10
Weeks post immunization
Fig.4.4. Immune responses to CCR2T and WCCR2T in ICR outbred mice (represented by individual bars). Antibody titers against the corresponding immunogen in the sera of each individual mouse were determined by direct ELISA. Arrows represent booster immunizations.
133
Immunogen coated plates IgG1 IgG2a 60 IgG2b
50
40
30
20 Serum concentration (%) concentration Serum
10
0 CCR2T WCCR2T
Peptide immunogen
HTLV-1 lysate coated plates
60 IgG1 IgG2a IgG2b 50
40
30
20 Serum concentration (%) concentration Serum
10
0 CCR2T WCCR2T
Peptide immunogen
Fig.4.5. Antibody isotypes induced by CCR2T and WCCR2T template peptides. The concentrations of each antibody isotype present in sera collected from mice immunized with the template peptides are indicated. The serum concentrations of IgG3, IgA and IgGM were less than 10%.
134
CCR2T antisera WCCR2T antisera
55
Events Events
0
055 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 FITC LOG FITC LOG
Fig.4.6. Relative binding of CCR2T and WCCR2T antisera to gp21 protein on the surface of
HTLV-1 infectious molecular clone immortalized ACH primary cells was determined by indirect immunofluorescence staining. Gray histograms represent staining with preimmune serum, solid line histograms represent sera from an HTLV-1 infected individual used as a positive control. CCR2T and WCCR2T antisera (dashed line) were tested at 1:5 dilution.
135
1.0
CCR2T antisera WCCR2T antisera 0.8 Preimmune serum
415nm 415nm
0.6 λ
0.4
Absorbance
0.2
0.0 10 20 40 80 160 320 640 1280 Reciprocal sera dilution
4.0 CCR2T antisera WCCR2T antisera 3.5 preimmune serum
3.0
2.5 450nm
λ 2.0 1.5
Absorbance 1.0
0.5
0.0 100 200 400 800 1600 3200 6400 Reciprocal sera dilution
Fig.4.7. Cross reactivity of peptide antibodies to native gp21 protein. Reactivity of CCR2T and WCCR2T mouse antisera was tested against recombinant MBP-gp21 protein chimera
(upper panel) in a direct ELISA or against inactivated HTLV-1 viral lysate in a whole virus
ELISA (lower panel). Preimmune mouse serum did not show any reactivity against gp21 protein or whole viral lysate.
136
0.4 1.2 immunogen CCR2T 1.0 B-cell epitope CCR2E 0.8 0.6 0.3 0.4 Absorbance l 415nm 0.2 415nm 0.0 λ 4000 8000 16000 32000 64000 128000 256000 512000 sera dilution 0.2
Absorbance Absorbance
0.1
0.0 60 30 15 7.5 3.75 1.875 0.9375 0 inhibitor concentration (µM)
0.8 1.8 1.6 immunogen WCCR2T
1.4 WCCR2T immunogen 0.7 1.2 415nm
λ 1.0 0.6 0.8 0.6 Absorbance 0.5 0.4 415nm 0.2 λ
0.0 0.4 4000 8000 16000 32000 64000 128000 256000 512000 sera dilution 0.3
Absorbance 0.2
0.1
0.0 60 30 15 7.5 3.75 1.875 0.9375 0
inhibitor concentration (µM) Fig.4.8. Competitive inhibition ELISA curves for CCR2T and WCCR2T antisera using corresponding immunogen coated plates. Using CCR2T coated plates and 1/16000 dilution
CCR2T antisera (upper panel), or WCCR2T coated plates and 1/16000 dilution WCCR2T
(lower panel) antisera, the inhibitory capacity of increasing concentrations of various peptide 137 inhibitors as indicated was tested. The binding of both antisera was most efficiently inhibited by its corresponding immunogen. The insets in both panels show the serum dilution curve for each antibody on the same microtiter plates.
138
0.7 0.8
0.7 CCR2T antisera CCR2T antisera against gp21 protein 0.6
WCCR2T antisera 0.5
0.6 415nm
λ
0.4
0.3 Absorbance 0.5 0.2 0.1
0.0 10 20 40 80 160 320 640 1280
415nm reciprocal sera dilution
λ 0.4
1.0
WCCR2T antisera against gp21 protein
0.8 0.3
0.6 415nm
λ
Absorbance Absorbance 0.4 0.2 Absorbance 0.2
0.0 0.1 10 20 40 80 160 320 640 1280 reciprocal sera dilution
0.0 40 20 10 5 2.5 1.25 0.625 0 inhibitor concentration µM
Fig.4.9. Competitive inhibition ELISA curves for CCR2T and WCCR2T antisera using recombinant gp21 protein coated plates. The inhibitory capacity of increasing concentrations of gp21 protein inhibitor on the binding of CCR2T and WCCR2T antisera (1/20 dilution) to immobilized gp21 protein on the microtiter plate was tested. The insets show the serum dilution curve against the recombinant gp21 protein for each antibody on the same microtiter plates.
139 CHAPTER 5
EVALUATION OF OTHER B-CELL EPITOPES FROM THE HTLV-1 ENVELOPE
PROTEIN TO ELICIT NEUTRALIZING ANTIBODIES
5.1 RATIONALE
In chapter 4, a peptide that mimics the α-helical structure of the coiled coil segment of the native gp21 envelope subunit was described. Another segment of interest in the gp21
TM subunit for the development of vaccines to elicit neutralizing antibodies is the region of chain reversal that lies adjacent to the coiled coil region towards the C-terminus. In studies by Hadlock et al. using recombinant proteins encoding select regions of gp21, epitope 361-
404 showed 100% specific seroreactivity with sera from patients infected with HTLV-1 and
HTLV-II (Hadlock et al., 1995). Addtionally, 397-430 was recognized by 61% of sera tested
(Hadlock et al., 1997). Previously serum reactivity to this region has been shown to range between 7-59% (Horal et al., 1991). The region 383-403 has been described as neutralizing by Inoue et al in studies using region- specific human antibodies derived from seropositive blood donors (Inoue et al., 1992). Studies done by Sagara et al. describe the region 400-429 to be a neutralizing region and have further shown that peptides between amino acids 397-
406 is involved in inhibition of syncytia formation (97% ) (Sagara et al., 1996). The amino terminal part of gp21 has a 29 residue hydrophobic stretch with characteristics of a fusion peptide. The anchorage domain corresponds to the hydrophobic sequence 446-465 (C- terminus). Peptide 400-429 which appears to affect syncytia formation lies between the
140 anchorage domain and the fusion domain. Syncytia formation induced by virus bearing cells
consists of many stages that include binding to the target cell receptor and folding of the
envelope protein to expose fusion domain and finally membrane fusion. Thus antibodies to
epitopes that lie in the region 400-429 may directly inhibit binding or act on a subsequent
step of the fusion process involved in syncytia formation. Investigation in this direction by a
plaque assay using a pseudotype of VSV bearing the envelope of HTLV-1 has recently
shown that gp21 (400-429) plays a major role in the post binding stages of HTLV-1
infection and inhibits HTLV-1 infection to an extent of about 60% (Jinno et al., 1999). Thus
we predicted this region to be useful as a therapeutic agent against HTLV-1 and investigated the possibility of eliciting neutralizing antibodies against this region.
In addition to the gp21 epitope an epitope from the SU subunit corresponding to residues 136-160 was also selected for evaluation. This region shows 30% seroreactivity,
(Desgranges et al., 1994) and has also been described as a neutralizing epitope (Inoue et al.,
1992) using human antibodies from HTLV-1 positive patients. Residues 141-146 have been identified as a good T-helper cell epitope in studies using gp46 native protein immunization as determined by lymph node proliferation assays. (Kurata et al., 1989).
5.2 SUMMARY
Two potential B-cell epitopes, one from the gp46 region (amino acids 136-160) and one from the gp21 region (amino acids 392-415) of the HTVL-1 envelope glycoprotein were predicted by computer aided algorithms. We designed these peptides as chimeric constructs with a promiscous T-helper epitope derived from tetanus toxoid (TT3, aa 967-987) and tested their immunogenicity and efficacy in eliciting antibodies capable of neutralizing viral
infection. One out of the 2 epitopes tested, namely the epitope derived from the gp21
141 subunit elicited antibodies that were effective in preventing syncytia formation. The
implications of these studies in the context of vaccine design are discussed.
5.3 MATERIALS AND METHODS
Peptide synthesis
The selected chimeric B-cell epitopes were synthesized as detailed in section 2.3
Molecular Modeling
Energy minimization calculations for all three possible cysteine paired epitopes for gp21
(392-415) were performed using the Hyperchem molecular modeling software (release 5.0,
Hypercube, Canada). Calculations were performed with default settings using the AMBER
forcefield. A cut-off of 500 cycles was used with the Polak-Ribere algorithm.
Circular dichroism spectroscopy measurements
CD measurements to evaluate the structural attributes of the chimeric peptides constructs
were performed as described in section 4.3 using 100µM concentrations of the chimeric B-
cell epitopes in 10mM PO4 buffer or 50% TFE in 10mM PO4 buffer.
Immunization of Rabbits and Mice
Female New Zealand outbred white rabbits were purchased from Mohican Valley Rabbitry
(Loudenville, OH). A pair of rabbits was immunized s.c in the thigh muscle with a total of
1mg of the chimeric peptide dissolved in PBS and 100µg of a muramyl dipeptide adjuvant, norMDP (N-acetyl-glucosamine-3-yl-acetyl-L-alanyl-D-isoglutamine), and emulsified (50:50) in squalene/Arlacel A vehicle (4:1). Booster injections were given 3-5 weeks apart with
500µg of peptide.
142 Immunogenicity testing
Sera were collected weekly for rabbits and complement inactivated at 560C for 30 minutes.
High titered sera was purified on protein A/G agarose column (Pierce, Rockford, IL) and eluted antibodies were concentrated and exchanged in PBS using 100kDa cutoff centrifuge filter units (Millipore, Bedford MA). Antibody concentration was determined by Coomassie plus protein assay reagent kit (Pierce).
Antibody titers were determined using ELISA as described in section 4.3 using 100µl of
1:500 goat anti-rabbit IgG conjugated to horseradish peroxidase (Pierce)
Flow cytometry
Binding of peptide antibodies to HTLV-1 infected cells was tested by flow cytometry as
described in section 4.3 using ACH and MT-2 cells. MT-2 cells were cultured in RPMI 1640
with 10% FCS and 1% pen/strep. 10µg of purified rabbit antisera or pre-immune control serum and FITC labeled goat anti-rabbit IgG secondary antibody (1:50 dilution) was used for staining.
Syncytia Inhibition assay
Rous sarcoma transformed rat epithelial XC cells were maintained in DMEM (10% FCS, 1% p/s) and used as target cells in the syncytia inhibition assay. Briefly, 0.25 X 106 ACH
(HTLV-1 infected) cells were added to 96 well plates and purified peptide antibodies added as indicated. The cells with antibodies were incubated for 2 hours at 370C. XC cells (0.25 X
6 0 10 ) were then added to the plates and incubated at 37 C, 5% CO2 for 36 hours. The plates
were then washed gently with sterile PBS and the cells fixed with ice-cold methanol for 10
minutes. After fixing, the cells were stained with giemsa stain diluted 1:20 for 45 minutes at
room temperature. Syncytia (cells with 4 or more nuclei) were counted using an inverted
143 microscope, counting 4 fields per well. The counts from duplicate wells were averaged and
the % inhibition was calculated as (average # syncytia in the absence of antibody-average # syncytia in presence of antibody)/(average # syncytia in the absence of antibody) X 100.
5.4. RESULTS
Selection, design and synthesis of the chimeric B-cell epitopes
Computer aided analysis of protein sequences using various correlates of protein antigenicity has been reviewed previously by Kaumaya et al (Kaumaya, 1994b). These 10 algorithms locate regions in the protein that are solvent exposed and therefore more likely to be available for antibody binding. The region corresponding to amino acids 389-423 was selected as a potential B-cell epitope as it ranked high in several of the algorithms considered such as hydrophilicity, antigenicity, hydropathy, hydrophobicity and acrophilicity.
Furthermore, Sagara et al have shown that peptides encompassing regions between 397-404 was effective in inhibiting the formation of syncytia to an extent of 97%. Based on the above observations, we chose the region 392-415 for our study. Further, molecular modeling of the region 392-415 showed that this region has a stable secondary structure with some residues involved in the formation of a α-helix and the others a β-sheet. Thus the epitope may mimic a native conformation. To avoid the formation unwanted of interchain or intrachain disulphide bonds due to the presence of 3 cysteine residues that may interfere with the native structure, a Cys401Ala mutation was introduced. Molecular modeling showed that the
Cys401Ala mutation results in minimum changes in the energy of the structure
(27.93kcal.mol for the Cys401Ala mutation v/s 27.9kcal.mol for the wild type peptide with no mutations) as compared with mutating any of the other two cysteines. The recent crystal structure data obtained for the gp21 protein showed that our molecular modeling results of
144 the peptide correlated very well with the crystal structure analysis which indeed confirms the
presence of a disulphide bridge between Cys393 and Cys400 while Cys401 remains reduced
(Kobe et al., 1999). Epitope 136-165 from the gp46 region ranked high in 4 of the 10
algorithms studied. Furthermore, this region was shown to have 31% syncytia inhibition
activity using sera from healthy seropositive HTLV-1 carriers as described by Inoue et al.
The potential B-cell epitope was synthesized collinearly with a promiscuous T-helper epitope
derived from tetanus toxoid (aa 947-967) at the N-terminus, with a 4-residue linker GPSL
separating the T-cell epitope and the B-cell epitope {TT3-(392-415), and TT3-(136-163)}.
The linker provides the flexibility such that the B-cell epitope is allowed to fold
independently of the T-cell epitope (Kaumaya, 1996; Partidos et al., 1999). The T-helper
epitope has been shown to bind multiple MHC haplotypes (Ho et al., 1990; Panina-
Bordignon et al., 1989b) and could be effective in an outbred population to elicit antibody responses. The crude peptides were purified by reverse phase HPLC and were found to be >
95% pure. The identity of the peptide was confirmed by mass spectrometry.
Secondary Structural analysis of chimeric B-cell epitopes
Circular dichroism was used to analyze the secondary structure of the chimeric B-cell
epitope constructs. A wavelength spectrum showing a double minimum at 208nm and
222nm with a maximum value at 193nm is typical of α-helical secondary structures. The
wavelength spectra of TT3-(392-415) displayed a double minimum at 208nm and 222nm as
characterized for peptides having α-helical structures (Fig.5.2). CD measurements of the chimeric construct TT3-(392-415) showed a mean residue ellipticity of -5062 deg cm2/dmol at 222nm, corresponding to 16.2% helicity in aqueous phosphate buffer. Increased helicity was observed in 50% trifluoroethanol (TFE) which is thought to mimic the membrane
145 environment in vivo (Jasanoff and Fersht, 1994) and also has helix stabilizing effects with a
mean residue ellipticity of -13164.8 deg cm2/dmol at 222nm, corresponding to 42% helicity.
Chow-Fasman secondary structure prediction (Chou and Fasman, 1974) for the chimeric
constructs however predicted a 27% helical content which is lower than that observed with
the CD measurements in TFE. Analysis of the crystal structure revealed the presence of a
short α-helix at the base of the coiled coil (Thr387) just after the region of chain reversal and
yet another short α-helix between residues 408-415. This would predict a helical content of
approximately 41% for the chimeric peptide encompassing residues 392-415. These data
suggested that the peptide does possess a stable secondary structure in TFE which could be
mimicking the structure of the native HTLV-1 gp21 protein region and hence had the
potential to elicit antibodies that can react with the native gp21 protein. TT3-(136-165)
displayed a mean residue ellipticity of -6754.43 deg cm2/dmol at 222nm, which corresponds
to 22% helicity. This value was 100% of the value predicted according to Chow Fasman
calculations. In the absence of a crystal structure for gp46, one must rely on the predictive
and empirical methods to get an indication of the folding pattern of peptides. CD
measurements of the TT3-turn peptide alone in similar buffer conditions revealed the
presence of mainly β-sheet and hence it was concluded that the observed helicity of the
chimeric constructs were due to the folding of the B-cell epitopes themselves.
Immunogenicity of the chimeric peptide constructs
To test the immunogenic potential of the chimeric peptides, two New Zealand white
rabbits were immunized with 1mg each of TT3-(392-415) of TT3-(136-160) peptide in
water-in-oil emulsion. TT3-(392-415) peptide was highly immunogenic in both rabbits
(Fig.5.3). Antibody titers against the immunogen were 32000 (reciprocal sera dilution with
146 absorbance units > 0.2) as detected by ELISA just three weeks after the first immunization
and increased to > 250000 when the first booster was administered. The titers remained at
maximal levels 5 weeks after the first booster and did not increase any further with another
subsequent third booster but were maintained almost the same for three weeks post the
third and final booster. Epitope TT3-gp46-(136-160) also elicited high titers against the
immunogen with titers reaching over 200,000 in one rabbit.
Binding of peptide antibodies to the native HTLV-1 envelope protein
We used two different approaches to test the binding of peptide antibodies to the
native HTLV-1 envelope protein. We first tested if the peptide antibodies against TT3-gp46-
(392-415) would bind to the gp21 protein in a standard ELISA. The gp21 protein is a
chimera with MBP consisting of residues 338-425 within which lies the sequence of TT3-
gp21(392-415). The peptide antibodies were able to cross-react with the gp21 chimeric protein after the third immunization (Fig.5.4). To confirm the reactivity of the peptide antibodies to the native envelope protein, we also tested for antibody binding to the envelope protein on the surface of two HTLV-1 infected cells lines MT-2 and ACH
(Fig.5.5). The ACH cells are primary peripheral blood mononuclear cells that have been transformed using an infectious molecular clone of HTLV-1 (Collins et al., 1996).
Antibodies to TT3-gp21(392-415) bind both the MT-2 and the ACH cell lines, thus confirming their cross-reactivity to the native envelope protein. The extent of binding however was higher with the ACH cells (with a mean log fluorescence of 8.9) as compared to the MT-2 cells (mean log fluorescence 6.9) which could be attributed to the slight sequence variability (Ser411Pro) of the envelope protein in the MT-2 cells. However, TT3-
147 gp46(136-160) antisera was not able to bind the cells under the same conditions suggesting that the peptide antibodies had very low affinity for the native protein.
Peptide antibodies inhibit the formation of virus induced syncytia
The effect of affinity purified peptide antibodies on envelope function and hence virus infectivity was examined by a syncytia inhibition assay between HTLV-1 infected cell line ACH and a rous sarcoma immortalized rat cell line. We used antibodies against TT3- gp21(392-415) since these antibodies were able to bind the surface of the HTLV-1 infected cells. The antibodies against TT3-gp21(392-415) inhibited syncytia formation to an extent of about 30% as shown in Fig.5.6. Purified sera from an HTLV-1 infected patient used as a positive control gave about 45% inhibition. TT3-gp46-(136-160) antisera was not tested for syncytia inhibition since these antibodies did not bind the surface of HTLV-1 infected cells.
5.5. DISCUSSION
We engineered two different peptide constructs that contain B-cell epitopes and a promiscuous T-helper cell epitope that showed variable immunogenicity in rabbits. Without the structural restraints in the native protein, free peptides in solution often display random conformations most of which may be non-native (Dyson and Wright, 1995). However, the peptides synthesized in a chimeric fashion along with a promiscuous epitope showed a stable secondary structure as characterized by circular dichroism measurements that seemed to correlate well with the recently solved crystal structure in the case of TT3-gp21(392-415) or the predicted helicity TT3-gp46(136-160). At the time of inception of these experiments the crystal structure of the gp21 central region was not available; hence we relied on computer aided algorithms for protein antigenicity, molecular modeling and the reactivities of HTLV-1 positive human sera to design the chimeric peptides that have the propensity to induce
148 antibodies that would recognize the native protein with high affinity. One of the peptide antibodies TT3-gp21(392-415) was able to recognize and bind native HTLV-1 envelope gp21 suggesting that the peptide was mimicking the native HTLV-1 structure. The crystal structure shows the region to be packed against the coiled coil segment (338-387) and it is speculated that this region serves to stabilize the transient fusion activated hairpin structure.
Indeed, mutational studies have shown that the C-terminal segment of the gp21 protein namely Glu398, Asn407, Ser408 and Leu413 play an important role in the fusion process.
Mutations in these residues, cause severe inhibition of the fusion process but have no effect on the maturation of the gp61 precursor. Upon receptor engagement by the SU subunit, the region of chain reversal including the disulphide bonded domain is involved in inducing the fusion-activated hairpin structure (Jinno et al., 1999; Maerz et al., 2000). Hence this region may be critical for the infection process. More recent studies have focused on the development of inhibitory peptides derived from the region of chain reversal that competitively interferes with the infection process. One such peptide is P-197 (residues 197-
216 of the SU subunit) that is speculated to mediate its inhibitor effects by binding to a cellular coreceptor during the initial stages of infection (Brighty and Jassal, 2001) and the other P-400 (residues 400-429 of the TM subunit) was shown to mediate its inhibitory effects by competitively binding to the coiled coil segment of the TM subunit in a manner similar to that observed in the crystal structure (Pinon et al., 2003). More importantly, the peptide P-400 was modified to include three substitutions that resulted in higher levels of binding to the TM coiled coil segment. Two of those substitutions namely Leu403 and
Ser411 are also present in our chimeric construct TT3-gp21-(392-415) as this sequence was modeled based on the envelope sequence from the infectious molecular clone ACH (Kimata
149 et al., 1994). Consistent with the above observations, we were also able to demonstrate the functional significance of these antibodies in their ability to inhibit the formation of virus- mediated cell fusion. It is possible that the antibodies induced against this region have a similar mechanism of interference in the fusion process. Although peptide mimotopes have great clinical potential, the stability of these peptides when injected in vivo may pose a limitation. In this regard we believe that active immunization to induce antibodies and memory responses may provide superior long lasting effects.
The epitope from the SU subunit TT3-(136-160) elicited high titers of antibodies that were unable to recognize the native protein by flow cytometric analysis. This peptide harbors a cysteine residue. Furthermore there are several cysteine residues around this region in the SU subunit. Hence it is possible that there exist several disulphide bonds within this region that results in complex folding that is poorly mimicked by the chimeric peptide construct we synthesized. A solution structure of this region would contribute to better understanding of the folding patterns within this region that can be used to design immunogenic sequences from the SU ubunit that would inhibit infection at the early binding stages.
In summary, we showed that a chimeric B-cell epitope designed from the C-terminal portion of the TM subunit was able to inhibit viral infection in tissue culture. Additional modeling of the peptide sequence to increase its stability in vivo may provide a longer stimulus to induce antibodies with higher affinity for the native sequence.
150
TT3-gp21-(392-415) L C K A L Q E Q C A F L N I T N S H V S I L Q E
Q Q D V N F T Q E V S H L N I N L H F S K C G F S
TT3-gp46-(136-160) Q Q D V N F T Q E V S H L N I N L H F S K C G F S
Fig.5.1. Molecular modeling of chimeric B-cell epitopes from the HTLV-1 TM subunit (top panel) and SU subunit (bottom panel). Sequences of the B-cell epitope are also shown. The
Cys401Ala mutation introduced in the gp21-(392-415) is shown in bold.
151 15000 TT3-gp21(392-415) 10000
5000 /dmol) 2
0
-5000
-10000
Mean Residue Ellipticity (deg cm (deg Ellipticity Residue Mean -15000 50% TFE in 10mM PO 4 buffer -20000 water 190 200 210 220 23 0 240 25 0 260 2 70
W avelength (nm )
TT3-gp46(139-160) 15000
10000 /dmol) 2 5000 (deg cm (deg θ 0
-5000
-10000
Mean Residue Ellipticity Ellipticity Residue Mean 50% TFE in 10mM PO buffer -15000 4 water
-20000 190 200 210 220 230 240 250 260 270
Wavelength (nm) Fig.5.2. CD spectra of chimeric B-cell epitopes. Spectroscopy measurements were made
either in 10mM phosphate buffer or 10mM phosphate buffer : TFE (1:1) at 100µM peptide concentration. Changes in the spectra indicate the presence of secondary structure in the peptides. Spectra characteristic of α-helices with minima at 208nm and 222nm and a maximum at 193nm was observed in all peptides in TFE indicating the formation of α- helices.
152 TT3-gp21-(392-415)
Rabbit 1 Rabbit 2
100000
10000
Antibody Titers 1000
100
0 3 4 5 6 7 8 9 10 11
Weeks post immunization
TT3-gp46-(136-160)
Rabbit 1 Rabbit 2 100000
10000
Antibody Titers Antibody 1000
100
0 3 4 5 6 7 8 9 10 11
Weeks post immunization
Fig.5.3. Immune responses to TT3-gp21-(392-415) and TT3-gp46-(136-160) in pairs of New
Zealand white outbred rabbits. Antibody titers against the corresponding immunogen in the sera of each individual rabbit were determined by direct ELISA. Arrows represent booster immunizations.
153
ACH MT-2
TT3-gp21-(392-415)
Events
Events
064 10 0 101 10 2 103 10 4 064 FITC Log 100 101 102 103 10 4 FITC Log TT3-gp46-(136-160)
Events Events
064 064 10 0 101 10 2 103 10 4 100 101 102 103 10 4 FITC Log FITC Log
Fig.5.4. Live cell immunofluorescence staining of HTLV-1 infected ACH or MT-2 cells.
Antibodies were purified on protein A/G columns as described in section 4.3 and used at
10µg/ml for cell staining (solid line histogram). Purified rabbit pre-immune serum was used at the same concentration as negative control (gray histograms).
154 2.5
TT3-gp21-(392-415) antisera preimmune serum 2.0
410nm 1.5 λ
1.0 Absorbance Absorbance
0.5
0.0 1/2000 1/4000 1/8000 1/16000 1/32000 1/64000 Sera Dilution
Fig.5.5. Cross reactivity of TT3-gp21-(392-415) peptide antibodies to gp21 recombinant protein was tested by direct ELISA on microtiter plates coated with gp21 recombinant protein. Rabbit preimmune serum did not show any binding.
155 Antibodies
20µg µ presera gp21 392-415 control + 40 g 80µg
0204060 % syncytia inhibition
Fig.5.6. Syncytia inhibition assay of peptide antibodies. Various indicated concentrations of affinity purified peptide antibodies against TT3-gp21-(392-415) were combined with HTLV-
1 infected ACH cells and XC target cells and the number of syncytia formed was enumerated after 24 hours incubation. The percent inhibition was calculated as described in section 5.3. Each point represents the mean of two separate assays each done in duplicates
156 ONGOING AND FUTURE STUDIES
The confirmation for any vaccine strategy is the demonstration of protection in a
relevant model of the particular disease which can be extrapolated into humans. HTLV-1 is a
complex virus that persists in the face of an active antiviral response. In recent years, a non- human primate model of HTLV-1 infection has been developed by Kazanji et al (Kazanji,
2000). The high proviral load in these monkeys together with high antibody titers are very
similar to that observed in humans. Although other non human-primates such as marmosets, cynomolgus macaques and pig-tailed macaques were found to be susceptible to infection by
HTLV-1 the main advantage with the use of the New World squirrel monkey is that these monkeys do not harbor STLV-1 which genetically and immunologically resembles HTLV-1.
This makes the squirrel monkey model more useful for the evaluation of immune responses to vaccine candidates.
Based on published data on a chimeric vaccine candidate derived from the principle immunodominant region of the SU subunit (Frangione-Beebe et al., 2000) and the work described in this dissertation, a combination vaccine consisting of a chimeric B-cell epitope
(MVF-gp46-175-218) and a multiepitope Tax CTL peptide construct is currently being tested for protective efficacy against challenge with HTLV-1 transformed and producing squirrel monkey cell line EVO/1540 (Kazanji et al., 2001). These experiments are being conducted at the primate facility in French Guiana in collaboration with Dr. Mirdad Kazanji. Four monkeys were immunized subcutaneously with the chimeric B-cell epitope four weeks apart.
After two more weeks the animals were immunized with the multiepitope 236 peptide 157 construct. Three weeks after the last immunization, monkeys were boosted once more with
both the chimeric B-cell epitope and the multepitope 236 construct at different sites.
Antibody responses against the chimeric B-cell epitope was tested by direct ELISA using
sera collected weekly by bleeding the animals. Furthermore, PBMCs from vaccinated
monkeys were also tested for Tax CTL epitope specific activation using lymphoproliferative
assays (LPA). The chimeric B-cell epitope induced high titered antibody responses of 600 in
all the monkeys by the 9th week as shown in Fig.6.1. The titers increased to 1280 after the
booster immunization by the 11th week. There were no titers against the T-helper epitope
MVF. The LPA assays for T-cell activation showed responses against the Tax 178-186 epitope
in one of the monkeys (Fig.6.2). Tax 11-19 and Tax 306-315 did not induce any proliferative
responses in the LPA tests.
The immune responses observed thus far are promising. Challenge studies are
currently underway to evaluate the protective efficacy of the combination vaccine. Results
from these preliminary experiments will provide important information regarding the design
of the immunogens and more importantly the effectiveness of administering both B-cell and
T-cell immunogens that may aid in the better design of a universal vaccine.
158 AO47C AO51C
1400 700 MVF-gp46 (175-218) immunogen MVF-gp46 (175-218) immunogen gp46 (175-218) B-cell epitope 1200 600 gp46 (175-218) B-cell epitope
1000 500
800 400
600 300 Antibody Titers Antibody Antibody Titers Antibody
400 200
200 100
0 0 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10
Weeks post immunization Weeks post immunization
AO56C 1400 AO64C 1400 MVF-gp46(175-218) immunogen MVF-gp46 (175-218) immunogen 1200 gp46 (175-218) B-cell epitope gp46 (175-218) B-cell epitope 1200
1000 1000
800 800
600 600 Antibody Titers Antibody
400 Antibody Titers 400
200 200
0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11 12
Weeks post immunization Weeks post immunization
Fig.6.1. Immune responses against MVF-gp46 (175-218) in 4 (represented by individual panels) squirrel monkeys were determined by titering the sera against the chimeric immunogen or against the B-cell epitope as indicated by an indirect ELISA. Arrows show booster administration. No antibodies were detected against the promiscuous T-helper epitope MVF (data not shown).
159
Fig.6.2. Lymphoproliferative assay for the detection of Tax specific CTL in 236 immunized squirrel monkeys. Ficoll purified monkey PBMCs were stimulated in the presence or absence of 10µg/ml of each of the individual Tax peptides of the 236 construct. IL-2 stimulation was used as a positive control and 3H incorporation was measuresd in a scintillation counter.
The top panel shows PBMCs from two monkeys vaccinated with the 236 construct. The bottom panel shows control monkeys immunized with an irrelevant multiepitope construct.
This experiment was repeated twice with similar results. 160 CONCLUDING REMARKS
The long term goal of this project is the development of a universal multivalent vaccine
against HTLV-1 diseases that is applicable to the general population and which efficiently
activates all arms of the immune system. Towards this broad goal this dissertation work was
aimed at first defining immunogenic determinants and subsequently the development of
strategies that would present these determinants in their most native form to the immune
system for priming, in a manner similar to natural viral infection. The results reported
contribute important findings regarding the immunogenic potential and protective efficacy
of human T-cell epitopes in a relevant preclinical model for HTLV-1 infection that has not been previously documented. The benefit of these vaccines may be further enhanced by combining the immunomodulatory effects of cytokines such as IL12 or co-stimulatory
molecules which are known to have profound effects on the strength and quality of the
immune response elicited by vaccination. The ongoing studies in a non-human primate
model of HTLV-1 infection may provide further insights into the understanding of the
immune responses triggered by these vaccinations that may lead to more potent vaccines to provide greater clinical benefit.
161 APPENDIX A
PURIFICATION AND MASS SPECTROMETRY PROFILES OF HTLV-1 TAX CTL
EPITOPES, ENVELOPE CHIMERIC B-CELL EPITOPES AND TEMPLATE
PEPTIDES
SUMMARY
Presented here are the HPLC traces and mass spectrometry profiles of purified peptides used in all the described studies.
162
Fig.A.1. RP-HPLC traces (left) and ESI profiles (right) of purified Envelope peptides. Env
239-247 (expected molecular weight = 947.1) and Env 339-347 (expected molecular weight = 875) {bottom panel}.
163
Fig.A.2. RP-HPLC traces (left) and ESI profiles (right) of purified Envelope peptides. Env
346-354 (expected molecular weight = 1081.2) {top panel} and Env 395-403 (expected molecular weight = 1107.3) {bottom panel}.
164
Fig.A.3. RP-HPLC traces (left) and ESI profiles (right) of purified Envelope peptides. Env
402-410 (expected molecular weight = 1044.18) {top panel} and Env 175-183 (expected molecular weight = 1048.16) {bottom panel}.
165
Fig.A.4. RP-HPLC traces (left) and ESI profiles (right) of purified Envelope peptides. Env
182-190 (expected molecular weight = 933.1) {top panel} and Env 210-218 (expected molecular weight = 1013.31) {bottom panel}.
166
Fig.A.5. RP-HPLC traces (left) and ESI profiles (right) of purified Tax peptides. Tax 155-163 (expected molecular weight = 1092.3) {top panel} and Tax 11-19 (expected molecular weight = 1069.5) {middle panel} and Tax 178-186 (expected molecular weight = 961.3) {bottom panel}.
167
Fig.A.6. RP-HPLC traces (left) and ESI profiles (right) of purified Tax peptides. Tax 233-241 (expected molecular weight = 983.2) {top panel} and Tax 307-315 (expected molecular weight = 1139.6) {middle panel} and Tax 306-315 (expected molecular weight = 1278.9) {bottom panel}.
168
Fig.A.7. RP-HPLC traces (top) and ESI profiles (bottom) of purified multiepitope construct
Tax 11-19-RR-Tax 178-186-RR-Tax 233-241Tax 233-241 (expected molecular weight = 3605).
169
Fig.A.8. RP-HPLC traces (top) and ESI profiles (bottom) of purified multiepitope construct 236 (expected molecular weight = 3899).
170
Fig.A.9. RP-HPLC traces (top) and ESI profiles (bottom) of purified multiepitope construct 362 (expected molecular weight = 3899).
171
Fig.A.10. RP-HPLC traces (top) and ESI profiles (bottom) of purified multiepitope construct 632 (expected molecular weight = 3899).
172
Fig.A.11. RP-HPLC traces (top) and ESI profiles (bottom) of purified multiepitope construct. 326 (expected molecular weight = 3899).
173
Fig.A.12. RP-HPLC traces (top) and ESI profiles (bottom) of purified T-helper epitope TT3 (expected molecular weight = 2478).
174
Fig.A.13. RP-HPLC traces (top) and ESI profiles (bottom) of purified Env 175-218 (expected molecular weight = 4910.9).
175
A
B
Fig.A.14. RP-HPLC traces (top) and ESI profiles (bottom) of purified (A) CCR2T (expected molecular weight = 12,652.8) and (B) CCR2E (expected molecular weight = 3312).
176
A
B
Fig.A.15. RP-HPLC traces (top) and ESI profiles (bottom) of purified (A) WCCR2T (expected molecular weight = 12,605.8) and (B) WCCR2E (expected molecular weight = 3299).
177
Fig.A.16. RP-HPLC traces (top) and ESI profiles (bottom) of purified TT3-gp21-(392-415) (expected molecular weight =5541).
178
Fig.A.17. RP-HPLC traces (top) and ESI profiles (bottom) of purified TT3-gp46-(136-160) (expected molecular weight = 5704).
179 APPENDIX B
PROTEASOMAL DIGESTION ANALYSIS OF MULTIEPITOPE CTL PEPTIDE
VARIANTS
SUMMARY
Presented here are the averaged mass peaks of the digested multiepitope constructs at the different time points tested; 12 hours, 24 hours and 48 hours. Samples were analyzed by capillary liquid chromatography-electrospray ionization mass spectrometry (CapLC-MS) as described in section 2.3.
180
Fig.B.1. Proteasomal digestion profiles of 236 construct at 12 hours (top panel), 24 hours
(middle panel) and 48 hours (bottom panel). Expected mass peaks were 1070.7 (Tax 11-19), 962.5 (Tax 178-186) and 1278 (Tax 306-315). The Tax 306-315 peaks were too weak to be seen on the scan presented.
181
Fig.B.2. Proteasomal digestion profiles of 362 construct at 12 hours (top panel), 24 hours
(middle panel) and 48 hours (bottom panel). Expected mass peaks were 1070.7 (Tax 11-19), 962.5 (Tax 178-186) and 1278 (Tax 306-315). The Tax 306-315 peaks were too weak to be seen on the scan presented. Undigested substrate peaks are seen as +3 (m/z 1300) or +4 charge (m/z 975).
182
Fig.B.3. Proteasomal digestion profiles of 632 construct at 12 hours (top panel), 24 hours
(middle panel) and 48 hours (bottom panel). Expected mass peaks were 1070.7 (Tax 11-19), 962.5 (Tax 178-186) and 1278 (Tax 306-315). The Tax 306-315 peaks were too weak to be seen on the scan presented. Undigested substrate peaks are seen as +3 (m/z 1300) or +4 charge (m/z 975).
183
Fig.B.4. Proteasomal digestion profiles of 326 construct at 12 hours (top panel), 24 hours
(middle panel) and 48 hours (bottom panel). Expected mass peaks were 1070.7 (Tax 11-19), 962.5 (Tax 178-186) and 1278 (Tax 306-315). The Tax 306-315 peaks were too weak to be seen on the scan presented. Undigested substrate peaks are seen as +3 (m/z 1300) or +4 charge (m/z 975).
184
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