Interferon-γ, a valuable surrogate marker of Plasmodium falciparum pre-erythrocytic stages protective immunity.

Immunity against the pre-erythrocytic stages of malaria is the most promising, as it is strong and fully sterilizing. Yet, the underlying immune effectors against the human Plasmodium falciparum pre-erythrocytic stages remain surprisingly poorly known and have been little explored, which in turn prevents any rational vaccine progress. Evidence that has been gathered in vitro and in vivo, in higher primates and in humans, is reviewed here, emphasizing the significant role of IFN-γ, either as a critical immune mediator or at least as a valuable surrogate marker of protection. One may hope that these results will trigger investigations in volunteers immunized either by optimally irradiated or over-irradiated sporozoites, to quickly delineate better surrogates of protection, which are essential for the development of a successful malaria vaccine.

Evidence for multiple B- and T-cell epitopes in Plasmodium falciparum liver-stage antigen 3.

Liver-stage antigen 3 (LSA-3) is a new vaccine candidate that can induce protection against Plasmodium falciparum sporozoite challenge. Using a series of long synthetic peptides (LSP) encompassing most of the 210-kDa LSA-3 protein, a study of the antigenicity of this protein was carried out in 203 inhabitants from the villages of Dielmo (n = 143) and Ndiop (n = 60) in Senegal (the level of malaria transmission differs in these two villages). Lymphocyte responses to each individual LSA-3 peptide were recorded, some at high prevalences (up to 43%). Antibodies were also detected to each of the 20 peptides, many at high prevalence (up to 84% of responders), and were directed to both nonrepeat and repeat regions. Immune responses to LSA-3 were detectable even in individuals of less than 5 years of age and increased with age and hence exposure to malaria, although they were not directly related to the level of malaria transmission. Thus, several valuable T- and B-cell epitopes were characterized all along the LSA-3 protein, supporting the antigenicity of this P. falciparum vaccine candidate. Finally, antibodies specific for peptide LSP10 located in a nonrepeat region of LSA-3 were found significantly associated with a lower risk of malaria attack over 1 year of daily clinical follow-up in children between the ages of 7 and 15 years, but not in older individuals.

Protection against Plasmodium falciparum challenge induced in Aotus monkeys by liver-stage antigen-3-derived long synthetic peptides.

The vaccine potential of Plasmodium falciparum liver stage antigen-3 (LSA3) was investigated in Aotus monkeys using two long synthetic peptides corresponding respectively to an N-terminal non-repeat peptide (NRP) and repeat 2 (R2) region of the LSA3, adjuvanted by ASO2. Both 100-222 (NRP) and 501-596 repeat peptides induced effector B- and T-cell responses in terms of antigen-driven antibodies and/or specific IFN-gamma secretion. Animals challenged with P. falciparum sporozoites were protected following immunization with either the NRP region alone or the NRP combined with the R2 repeat region, as compared with controls receiving the adjuvant alone. These results indicate that the NRP may be sufficient to induce full, sterile protection and confirm the vaccine potential of LSA3 previously demonstrated in chimpanzees and in Aotus.

Genetic immunisation by liver stage antigen 3 protects chimpanzees against malaria despite low immune responses.

BACKGROUND: The true interest of genetic immunisation might have been hastily underestimated based on overall immunogenicity data in humans and lack of parallelism with other, more classical immunisation methods. PRINCIPAL FINDINGS: Using malaria Liver Stage Antigen-3 (LSA-3), we report that genetic immunization induces in chimpanzees, the closest relative of humans, immune responses which are as scarce as those reported using other DNA vaccines in humans, but which nonetheless confer strong, sterile and reproducible protection. The pattern was consistent in 3/4 immunized apes against two high dose sporozoite challenges performed as late as 98 and 238 days post-immunization and by a heterologous strain. CONCLUSIONS: These results should, in our opinion, lead to a revisiting of the value of this unusual means of immunisation, using as a model a disease, malaria, in which virulent challenges of volunteers are ethically acceptable.

A role for the Plasmodium falciparum RESA protein in resistance against heat shock demonstrated using gene disruption.

During erythrocyte invasion, the Plasmodium falciparum Ring-infected erythrocyte surface antigen (RESA) establishes specific interactions with spectrin. Based on analysis of strains with a large chromosome 1 deletion, RESA has been assigned several functions, none of which is firmly established. Analysis of parasites with a disrupted resa1 gene and isogenic parental or resa3-disrupted controls confirmed the critical role of RESA in the surface reactivity of immune adult sera on glutaraldehyde-fixed ring stages. Absence of RESA did not influence merozoite invasion or erythrocyte membrane rigidity, was associated with a modest increase of cytoadhesion to CD36 under conditions of flow, but resulted in marked susceptibility to heat shock. resa1-KO-infected erythrocytes were prone to heat-induced vesiculation like uninfected erythrocytes, whereas parental or resa3-KO infected erythrocytes remained undamaged. Furthermore, a 6 h exposure of ring stages at 41 degrees C resulted in 33% culture inhibition of resa1-KO parasites while marginally impacting parental and resa3-KO parasite growth. This points to a role for RESA in protecting the infected erythrocyte cytoskeleton during febrile episodes. Infection patterns of resa1-KO and parental parasites in Saimiri sciureus indicated that RESA does not, at least on its own, modulate virulence in the squirrel monkey, as had been previously suggested.

Immunogenicity and protective efficacy of Plasmodium falciparum liver-stage Ag-3 in Aotus lemurinus griseimembra monkeys.

Three recombinant proteins spanning the Plasmodium falciparum liver-stage Ag-3 (LSA-3) were used to immunize Aotus monkeys. The proteins were delivered subcutaneously without adjuvant, adsorbed onto polystyrene 0.5 microm particles at a concentration of 2 microg per immunization. Control animals received glutathione-S-transferase formulated similarly. Animals were challenged as late as 5 months after the last immunization, by intravenous inoculation of 100,000 P. falciparum sporozoites of a strain heterologous to the one from which the immunogens were derived. Sterile protection was achieved in three of the five immunized monkeys but in none of four controls. Antibodies were at low titer, but reacted with the native parasite protein and were boosted by parasite challenge. Ag-specific IFN-gamma secretion was detectable in all LSA-3-immunized animals in response to the LSA-3-derived Ag. The protection was apparently associated with high levels of IFN-gamma production in response to in vitro recall Ag. These results lend support to the vaccine potential of LSA-3 indicated by previous results obtained in chimpanzees, as well as the value of yet another Ag-delivery system. They also support the value of the Aotus model for the pre-clinical development of pre-erythrocytic-stage vaccines.

Analysis of intra-hepatic peptide-specific cell recruitment in mice immunised with Plasmodium falciparum antigens.

The liver stage of Plasmodium spp. now appears as a relevant target of immune effectors triggered by the so-called "anti-sporozoite" vaccine. Since the monitoring of immune responses at the systemic level may not faithfully reflect the local protective mechanisms, the aim of the present work was to set up a model to study the local intra-hepatic cellular responses and to compare these with the peripheral immune responses. This was achieved by intra-portal delivery of epitopic peptides, i.e. peptides containing B and T cell epitopes, which were coated onto the surface of polystyrene microbeads. The peptide-coated beads presumably mimic the hepatic schizont, and when distinct peptides are administered separately, this method of delivery allows us to decipher the immune responses resulting in mice immunised with recombinant proteins spanning several such epitopes. Using the P. falciparum liver stage antigen-3 (LSA3) molecule, which can induce protection against a sporozoite challenge, our results show that 25-microm microbeads could easily access the liver parenchyma by intra-portal injection and were distributed evenly in the liver. Also, LSA3-derived synthetic peptides coated onto microbeads initiated specific cell recruitment within 6 h. Depending on the LSA3 peptide used, the infiltrates induced differed in size, with the strongest cell recruitment obtained using nonrepeat II peptide (NR2)-coated microbeads with a mean leukocyte number of 79 per granuloma. Immunohistological studies of liver sections revealed that, irrespective of the delivered peptide, cells infiltrating the liver towards microbeads were mainly CD3(+) T lymphocytes, both CD4(+) (70 to 80%) and CD8(+) (20 to 30%) subtypes, macrophages and dendritic cells. Cells infiltrating the granuloma had features of activated cells, with evidence of VLA-4 cell-surface expression, and production of IFN-gamma and IL-4. Analysis of the peripheral B and T-cell responses in the same animals revealed that, whereas the local responses were directed mainly towards NR2 and repeat peptides (RE), the peripheral T-cell response to these peptides was weak and infrequent, although antibody production was high.