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Immunoregulatory role of epitopes from infectious pathogens

Objective

The severe pathologies observed during infectious diseases such as Malaria and Tuberculosis are associated with, and in some instances caused by, an inappropriate immune response against the corresponding pathogen. The development of modern subunit vaccines against Plasmodia or Mycobacteria requires the knowledge of the regulatory roles of the different epitopes from the target antigen(s) during the human immune responses against these pathogens. New techniques for in vitro analysis of the interactions between the human immune cells must be devised to identify which subunits must be incorporated in synthetic immunogens, in order to elicit the immune mechanisms leading to resistance in a majority of individuals. The objectives were i: identification of T- and B-cell epitopes in defined antigens of P. falciparum and M. tuberculosis recognised by lymphocytes from immune individuals; ii: characterisation of the functions of the responding T cells in the immune response, such as their pattern of cytokines production; and iii: modelling of these functions for the production of an efficient immune response such as effector antibodies against the corresponding pathogen. For this purpose, artificial antigens have been produced, by either chemical synthesis or genetic engineering, and their capacity to induce this efficient immune response assessed in vivo in animal models and in vitro using lymphoid cells from immune individuals naturally exposed and/or resistant to the corresponding pathogen.
Aminoacid sequences of interest have been selected in three Plasmodium falciparum antigens, Pf332, Pf72 and PfMSA2, and the corresponding peptides have been synthesised. Mapping of T- and B-cell epitopes in those antigens has been completed on peripheral blood cells from individuals in the process of natural acquisition of immune resistance to malaria.
PfMSA2 genes from parasites found in these infected individuals have been analysed by PCR using specific primers. DNA sequences of alleles show an extensive polymorphism and diversity of the parasite population. Some aminoacid substitutions have been found in the region coding for one T-cell epitope.
Mapping of epitopes in Mt19 kDa, 38 kDa and 16 kDa Mycobacterial antigens has been performed, and changes in recognition of defined T and B-cell epitopes observed depending on the patients: active tuberculosis, or leprosy infection, or vaccination.
ELISPOT technologies have been set-up for testing the functions of the T cells responding to defined epitopes of P. falciparum and M. tuberculosis in correlation with protective immunity.
Artificial antigens have been synthesized from peptides corresponding to known B and T cell epitopes from Plasmodium falciparum antigens. The efficacy of such compounds has been first checked in animal models (mice and monkeys), and the effect of T-cell epitopes on the antibody responses to B-cell epitopes of Pf332 and Pf72 antigens, has confirmed the interest in using heterogenous multiple peptides `MAPs' for the study of regulatory T cell epitopes in modulating the humoral responses to B cell epitopes.
PfMSA2 genes from parasites found in hospitalized individuals have been analysed by PCR. DNA sequences of alleles show an extensive polymorphism and diversity. We found aminoacid substitutions in the region coding for one T-cell epitope, in patients suffering from severe, but not mild, malaria attack.
MAJOR SCIENTIFIC BREAKTHROUGHS:
There are general implications for vaccine design from the changes in the epitope specificity of T and B cells to the 38 kDa antigen and from the proportion of antibodies to linear and conformational epitopes when comparing subunit vaccination with live infection in tuberculosis.
The described results with peptides of the Mt19 kDa antigen indicate that cross-reactivity between structurally unrelated proteins may be of common occurence and may have biological importance particularly in relation to the pathogenesis of autoimmunity.
With the developed MAP methodology, potent T-cell epitopes can easily be included in diepitope MAPs to test in vitro on human cells their efficacy in B-cell help induction, and render efficient vaccine immunogens. Furthermore, by combining different B-cell epitopes derived from pathogen antigens into a diepitope MAP, efficient tools for serological screenings can be constructed.
Increase in the sensitivity of T cell diagnosis of tuberculous infection has been achieved on the basis of the additive effect between the most immunogenic peptides of the 16 kDa antigen. More than 90% of tested subjects responded to four peptides, each of which stimulated singly only about 50% of individuals.
Some amino acid substitutions were found in slightly polymorphic, semi-conserved T cell epitopes of PfMSA2 antigen. They could impair the proper response of memory T cells against new alleles. Changes in cytokine production can be expected during analysis of the T cell response upon restimulation with modified peptides.
We have shown that T cells expressing the gammadelta T cell receptor are stimulated by parasite extracts, and that they can play a prominent role during malaria as either an effector cell against the parasite blood-stages or an effector in the onset of severe malaria pathology by secreting lymphokines such as TNF (alpha and beta).

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Institut Pasteur
Address
25 Rue Du Docteur Roux
75724 Paris
France

Participants (1)

Stockholms Universitet
Sweden
Address

106 91 Stockholm