Previous studies have demonstrated the ability of T cells to discriminate sequence variation in vaccine candidate proteins including circumsporozoite protein and merozoite surface protein-1. This has been interpreted to necessitate multi-allelic vaccines to overcome geographic diversity. The extensive study herein of T cell peptide responses spanning conserved, allelic and polymorphic blocks of amino acid sequence derived from P. falciparum MSPl may locate region/s to be manipulated for future subunit vaccine/s circumventing and/or improving on nature.
Human immunization with a three antigen cocktail induced dose-dependent T and B cell responses to the P. falciparum MSPl-derived antigen, 190LCS.T3 with no antigenic competition evident. Specific T cell responses were induced with 4µg yet antibody was detected only after the booster dose. Significantly enhanced antigen-specific T cell and antibody responses (p<0.05) were recorded for up to 12 months. Significantly increased responses were recorded to conserved peptide K22, and to peptides D37 and D38 from the spectrin-binding sequence of 190L, the MSPl component of the vaccine. In addition to the universal peptide, CS.T3, peptides K22 and K29 induced responses in individuals expressing a range of MHC class II haplotypes.
An 8-day challenge infection induced weak T cell responses in placebo recipients (n=5) to antigen and peptides. Immunized, parasite-challenged individuals showed decreased peptide responses, some of which recovered while others continued to decline. The capacity to prime an anamnestic response was noted for one donor not given the booster dose. For this individual long-lived T cell responses and anti-190LCS.T3 were observed following challenge infection. Immunization enhanced In vitro 190LCS.T3- induced IFN-γ and TNF-β (Th1-type) and IL-13 (Th2-type) cytokine secretion, but day 8 parasite density was comparable in the two treatment groups. Challenge infection diminished secretion of IFN-γ and TNF-β at week 14 and diminished production of IL-13 at week 14 and week 18. Secretion of IL-5 and IL-10 (Th2-type) was indistinguishable between the treatment groups.
MSPl-derived peptides inducing proliferation predominantly from malaria-infected PBMNC donors were identified. Sequence-variant peptides D7 and D9 (block 2), D25 (block 3), D37, D38 and D41 (block 4) showed statistically significant differences (p<0.05) between naive and malaria exposed donor groups. Differences were recorded for peptides D36, D39, D40, M121 and M186 but were not significant (p<0.1).
CD4+ T cells were found responsible for In vitro peptide-specif ic proliferation by subset depletion. MSPl-derived peptide-specific T cell lines from malaria exposed and 190LCS.T3-immunized but not naive and placebo-treated donors responded to native antigen in iRBC preparations. This effect was most evident when the peptide sequence was conserved. T cell epitope prediction models (PHD-sec, AMPHI) confirmed In vitro proliferation data for 52% (n=50) of the MSPl peptides.
Immunization of human volunteers with 190LCS.T3 induced strong T cell proliferation to the recombinant protein and modified the peptideresponse pattern. However, immunization of MHC class Il-defined strains of mice with 190LCS.T3 or MSPI19 only induced weak proliferation to the respective protein (BIO.BR mice only) and to a very limited range of spanning peptides.
Immunization of mice with pools of peptides defined from human responses to 190LCS.T3 (pool L) or MSPI19 (pool G and pool S), failed to induce proliferation to the parent protein but CD4+ T cell proliferation to several peptides from each pool in at least one mouse strain (n=6 MHC class Il-defined strains; BALE and BIO backgrounds) were recorded. Immunization of mice with pool L primed BIO and BIO.BR mice for an anamnestic antibody response when injected with soluble 190LCS.T3. Similarly pool G appeared to prime against MSPI19 in BIO and BIO.BR mice. However, it was later noted that several of the MSPI19 peptides contained B cell epitopes. The anti-(E-TSR)-MSPli9 response was partially reactive with each of the other 3 variants.
Remaining MSPl peptides inducing In vitro proliferation in human studies were segregated in peptide pools by regional location for murine immunization. Conserved peptides Dl (block 1), D18 (conserved block 3), D116, D119, D123, D125 and D132 (variant/allelic blocks 12 and 13) were active in most of the 5 mouse strains tested suggesting a capacity for "promiscuous" T cell stimulating activity not modified substantially in the two genetic series tested (BALB and C57B1/10). Infection-specific, variant peptides D9 and DlO rarely induced proliferation in mice. Competition between peptides was negligible and proliferation was dependent on the presence of CD4+ T cells.
These observations offer encouragement for the development of a future subunit vaccine that could stimulate cell mediated immune responses in a range of populations without the complication of geographic parasite population antigenic variation. Peptides representing T cell epitopes from the 190L region of MSPl primed an antibody response in mice undetected after protein immunization alone. This infers that peptides stimulated T cells to provide immunological "help" for the induction of an antigen-specific (anti-190LCS.T3) antibody response.