Examples

Immunogenicity of predicted universal T cell epitopes of malaria proteins.

 

Student

Ritesh Dhar, class of 2001

Primary Sponsor

Elizabeth H. Nardin, Ph.D.
Associate Professor
Dept. of Medical and Molecular Parasitology
New York University School of Medicine, New York, NY

Faculty Advisor

John Goldman, M.D.
Professor of Medicine
Chair, Dept. of Infectious Diseases
Penn State College of Medicine, Hershey, PA

Research Period

May 1998 - August 1998

Background

Introduction

Malaria is globally one of the most prevalent severe infectious diseases and remains a leading cause of human mortality and morbidity in over 90 countries, causing up to 500 million clinical cases and over 1 million deaths per year1,2. This resurgence is in part due to the emergence of multi-drug resistant Plasmodium strains and insecticide-resistant mosquito vectors. A reliable, highly protective malaria vaccine, in particular for P. falciparum, the parasite that causes the most severe and life-threatening complications, would provide the most cost-effective means of control2.

The successive developmental stages of the plasmodia involve a number of stage-specific antigens that provide potentially protective targets for immune intervention. Motile sporozoites, found in the Anopheles mosquito's salivary glands, are injected into the mammalian host and rapidly infect hepatocytes forming schizonts and constitute the asymptomatic, pre-erythrocytic stage. Subsequent maturation and multiplication results in rupture of the schizonts, releasing thousands of merozoites capable of erythrocyte entry, initiating the erythrocytic phase and clinical disease. Some intraerythrocytic merozoites which subsequently escape and lyse erythrocytes differentiate into gametes which can then be taken up by Anopheles and develop into sporozoites2.

These stages are the basis for three distinct approaches to vaccine development which aim to elicit immunity to the pre-erythrocytic and erythrocytic stages, as well as transmission blocking immunity2. The feasibility of developing an effective malaria vaccine was proven in the 1970s, and corroborated recently, in studies in human volunteers in which it was shown that exposure of previously uninfected subjects to irradiated malaria-infected mosquitoes protected individuals from P. falciparum and P. vivax. While demonstrating the presence of protective sporozoite-specific antigens, this approach is impossible and can not be used on a global scale3,4.

Currently, the major focus for pre-erythrocytic stage synthetic vaccine development has been the identification of relevant T and B cell epitopes on a major sporozoite cell surface protein, the circumsporozoite (CS) protein. A repeat region in the CS protein has been shown to contain an immunodominant B cell epitope, (NANP)3, that elicits polyclonal and monoclonal antibodies that neutralize sporozoite infectivity both in vivo and in vitro5. The first synthetic malarial vaccine, which used the immunodominant (NANP)3 epitope conjugated to a tetanus toxoid (TT) carrier protein, elicited low anti-sporozoite antibody titers, due in part to low peptide density, and limited protection in phase I and II6. T cell responses to the (NANP)3 peptide were restricted to murine H-2b cells and human T cell responses from vaccinees and naturally infected individuals living in endemic areas were poor7,8.

The development of Multiple Antigen Peptides (MAP) based on CS epitopes provided high density peptides without the requirement for a carrier macromolecule. Human T cell clones derived from irradiated P. falciparum sporozoite immunized volunteers were used to identify T cell epitopes for inclusion into the next generation vaccine9. A MAP using (NANP)3 and T1, a T cell epitope in the 5' repeat of P. falciparum (NF54 isolate) CS, successfully elicits high antibody titers in human volunteers, but only a limited number of class II molecules can function as restriction elements10,11. Phase I trials are in progress for this vaccine in order to validate the prediction of responders based on in vitro peptide/MHC interactions.

The second T cell epitope that was identified, T*, is contained in as 326-345 of the CS protein of the P. falciparum NF54 isolate12 In contrast to Tl, T* is recognized by both T helper and T cytotoxic cells, binds to multiple DQ and DR molecules and is immunogenic in all strains of mice11. This "universal" T cell epitope was incorporated into the existing MAP vaccine design to create a genetically unrestricted T1BT* vaccine that uses oxime bonds in order to provide a homogeneous synthetic product for unambiguous identification by mass spectrometry13. This vaccine is immunogenic in multiple strains of mice and Phase I trials are currently in progress13.

Eventually, a multicomponent MAP displaying T and B cell epitopes from various stages of plasmodia development and providing protection for individuals of all HLA allelic backgrounds is desired. Thus, a more efficient method to identify universal epitopes (i.e. those that bind to most, if not all, class II molecules) is needed since irradiated sporozoite immunized volunteers are a rare resource. In addition, a large number of potential antigens from various plasmodial stages are being identified by the efforts of the Malaria Genome Project. A new computer driven program developed by Hammer and Sinigaglia, TEPITOPE (T epitope), may have the capacity to predict and locate universal T cell epitopes of previously or newly identified antigens and therefore bypass the necessity of characterizing T cell clones from immunized volunteers14. These could then be tested for and used in a putative multicomponent, genetically unrestricted MAP polyoxime malaria vaccine.

Project: Are predicted universal T cell epitopes of the TRAP malarial antigen functional in vivo?

Rationale

The T* epitope identified from analysis of T cell clones from sporozoite immunized volunteers was also correctly identified from prediction by Hammer and Sinigaglia's TEPITOPE algorithm. With this encouraging result, potential universal T cell epitopes of the Thrombospondin Related Anonymous Protein (TRAP) were predicted using TEPITOPE and constructed. TRAP is another sporozoite-specific cell surface protein that is important for gliding motility and hepatocyte infection and has become a candidate for inclusion into a multicomponent vaccine15. Studies in mice have demonstrated that immunization with a combination of TRAP and CS protects the majority of mice, while significantly fewer mice are protected with either alone16. Of the three TEPITOPE predicted P. falciparum universal T cell epitopes, one was shown to bind to multiple HLA-DR molecules in peptide binding assays and an immunization of a MAP consisting of this epitope elicited anti-sporozoite antibody in two of three strains of mice tested (H-2a and H-2d, not H-2b). Since antibody was generated, the epitope must be a functional T helper epitope. Furthermore, immunization of MAP containing an analogous predicted universal T cell epitope from the murine plasmodium P. yoelii TRAP also elicited anti-sporozoite antibody in two of three strains of mice immunized (H-2b and H-2d, not H-2a). The antibody levels were, however, low and detectable by IFA only, not ELISA.

This study is the first to elucidate the ability of TEPITOPE predicted universal T cell epitopes to generate parasite-specific functional T cell responses. The analysis of these predicted TRAP T cell epitopes will help define the predictive capabilities of TEPITOPE and may uncover another universal protective epitope in a different sporozoite antigen for eventual incorporation into a multicomponent vaccine, and provide initial in vitro and in vivo data on the protective immune response to this sporozoite-specific antigen.

Experimental Design

  1. Immunize C57BL (H-2b), A/J (H-2a), and BALB/c (H-2d) mice with three peptides containing predicted TRAP universal T cell epitopes of P. falciparum and the corresponding sequences of P. yoelii. Linear TRAP peptide in Freund's complete adjuvant (FCA) will be used for immunization s.c. at the base of tail.
    1. Assay lymph node T cell proliferation by measuring incorporation of [3H]thymidine
    2. Assay lymphokine production by measuring supernatant content of IL-2 (using a IL-2 bioassay) and 1L-4 and  IFN-y (using ELISA)
  2. Assay protection in C57BL, A/J, and BALB/c mice immunized with P. yoelii TRAP universal epitope.
    1. Immunize mice with TRAP universal T cell epitope MAP emulsified in FCA
    2. Challenge with sporozoites i.v. or by bite
    3. Assay protection by measuring parasitemia by Giemsa blood smears or liver probes
  3. Test the binding of the remaining two predicted universal T cell P. falciparum TRAP epitopes to soluble HLA DR4 and DR13 molecules in vitro by using peptide competition ELISA11. The HLA DR4 and DR13 represent known and unknown restriction molecules, respectively, for the T1 and T* epitopes.

 

Student's Responsibilities

  1. Sound familiarization of relevant literature
  2. Successful completion of stated goals
  3. Develop proficiency in stated experimental assays and techniques
  4. Data analysis
  5. Understand relevance of conclusions in relation to ongoing projects
  6. Preparation of manuscript
  7. Presentation

 

Sponsor's Responsibilities

 The sponsor's responsibility will be to supervise the student in the performance of experiments, design of protocols and write-up of experiments; to ensure that reagents and supplies are available for completion of experiments in time period stated and to provide scientific and technical expertise.

Resources

Commercial preparations of peptides have been obtained. An animal facility is present in the Dept. of Medical and Molecular Parasitology, NYU School of Medicine; IACUC approval for protocols have been received. Proliferation, cytokine and serological assays are routinely performed in the lab. Rodent malaria life cycle in mosquitoes and mice maintained in core facility (insectary, etc.)

References

 

1.  WHO, 1997, World Malaria situation in 1994. Weekly Epidemiological Record, WHO.
2.  Nardin, E.H. and R.S. Nussenzweig, 1993. T cell responses to pre-erythrocytic stages of malaria: Role in protection and vaccine development against pre-erythrocytic stages. Annu. Rev. Immunology 11:687-727.
3.  Nussenzweig, R.S., J. Vanderberg, H. Most, C. Orten, 1967. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature 216:160.
4.  Herrington, D., J. Davis, E. Nardin, M. Beler, J. Cortese, H. Eddy, G. Losonsky, M. Hollingdale, M. Sztein, M. Levine, R. Nussenzweig, D. Clyde, R. Edelman, 1991. Successful immunization of humans with irradiated malaria sporozoites: Immoral and cellular responses of the protected individuals. Am. J. Trop. Med. Hyg. 45:535.
5.  Nussenzweig V., R.S. Nussenzweig, 1989. Rationale for the development of an engineered sporozoite malaria vaccine. Adv. Immunol. 45:283.
6.  Herrington, D.A., D. F. Clyde, G. Losonsky, M. Cortesia, J.R. Murphy, J. Davis, S. Baqar, A.M. Felix, E.P. Heimer, D. Gillessen, E. Nardin, R.S. Nussenzweig, V. Nussenzweig, M. Hollingdale, M. Levine, 1987. Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature 328:257.
7.  Good, M.F., J.A. Berzofsky, W.L. Maloy., et al., 1986. Genetic control of the immune response in mice to a Plasmodium falciparum sporozoite vaccine. Widespread nonresponsiveness to a single malaria T epitope in highly repetitive vaccine. J. Exp. Med. 164: 655-660.
8.  Del Guidice, G., J.A. Cooper, J. Merino, et al., 1986. The antibody response to mice to carrier free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab restricted: possible implications for malaria vaccines. J. Immunol. 137.2952-2955.
9. Nardin, E.H., D.A. Herrington, J. Davis, M. Levine, D. Stuber, B. Takacs, P. Caspers, P. Barr,, R. Altszuler, P. Clavijo, R.S. Nussenzweig, 1989. Conserved repetitive epitope recognized by CD4+ clones from a malaria immunized volunteer. Science 246:1603.
10.  Munesinghe, DY, P. Clavijo, J.M. Calvo-Calle, R.S. Nussenzweig, E.H. Nardin, 1991. Immunogenicity of multiple antigen peptides (MAP) containing T and B cell epitopes of the repeat region of the P. falciparum circumsporozoite protein. Eur. J. Irnmunol. 21:3015-3020.
11.  Calvo-Calle, J.M., J. Hammer, F. Sinigaglia, P. Clavijo, Z.R. Moya-Castro, E.H. Nardin, 1997. Binding of malaria T cell epitopes to DR and DQ molecules in vitro correlates with immunogenicity in vivo: Identification of a universal T cell epitope in the Plasmodium falciparum circumsporozoite protein. J. Immunol. 159:1362-1373.
12.  Moreno A., P. Clavijo, R. Edelman, J. Davis, M. Sztein, F. Sinigaglia, E. Nardin, 1993. CD4+ T cell clones obtained from Plasmodium falciparum sporozoite-immunized volunteers recognize polymorphic sequences of the circumsporozoite protein. J. Immunol. 151:489.
13.  Nardin, E.H., J.M. Calvo-Calle, G.A. Oliveira, P. Clavijo, R. Nussenzweig, R. Simon, W. Zeng, K. Rose, 1998. Plasmodium falciparum polyoximes: highly immunogenic synthetic vaccines constructed by chemoselective ligation of repeat B-cell epitopes and a universal T-cell epitope of CS protein. Vaccine 16: 590-600.
14.  Hammer, J., T. Sturniolo, F. Sinigaglia, 1997. HLA class II peptide binding specificity and autoimmunity. Adv. Immunol. 66:67-100.
15.  Sultan A., et al, 1997. TRAP is necessary for gliding motility and infectivity of Plasmodium sporozoites. Cell 90: I -12.
16.  Khusmith, S., et al, 1991. Protection against malaria by vaccination with Sporozoite Surface Protein 2 plus CS protein. Science 252: 715.

 

 

Signatures

  • Ritesh Dhar
  • Elizabeth H. Nardin, Ph.D.
  • John Goldman, M.D.

 

Web Statement

I do    x     or do not          give permission for my proposal to possibly be published on the College of Medicine website.