Long synthetic peptides encompassing the Plasmodium falciparum LSA3 are the target of human B and T cells and are potent inducers of B helper, T helper and cytolytic T cell responses in mice.

We synthesized 17 long synthetic peptides (LSP) spanning the whole 200-kDa Plasmodium falciparum liver stage antigen-3 (LSA3), an antigen that induces protection in chimpanzee, and analyzed their immunogenicity in BALB/c mice and their antigenicity in individuals living in a hyper-endemic malaria area. Our findings show that both specific antibodies and T cell proliferation against most LSA3-LSP develop in malaria-exposed adults. All individuals studied had detectable antibodies against a minimum of 6 and a maximum of 15 polypeptides. It is noteworthy that antibody prevalence and titers were as high against non-repeat as repeat regions. Although the extent of T cell reactivity was lower than that observed for B cells, most of the sequences contained at least one T helper epitope, indicating that the majority of LSA3-LSP contain both B and T cell epitopes within the same sequence. Injection of LSA3-LSP with SBSA2 adjuvant in mice, showed strong immunogenicity for most of them, eliciting both T cell responses and specific antibody production. While all the peptides were immunogenic for B cells, different patterns of T cell responses were induced. These peptides were thus classified in three sets according to the levels of the T cell proliferative and of the IFN-gamma-specific responses. Importantly, antibodies and T cells against some of the LSP were able to recognize LSA3 native protein on P. falciparum sporozoites. Additionally, some LSP (44-119, 1026-1095, 1601-1712) also contained epitopes recognized by H-2(d) class I-restricted T cells. These results led to the identification of numerous domains that are highly antigenic and immunogenic within the LSA3 protein, and underline the value of the LSP approach for vaccine development.

DNA immunization by Plasmodium falciparum liver-stage antigen 3 induces protection against Plasmodium yoelii sporozoite challenge.

DNA-based immunization of mice by Plasmodium falciparum liver-stage antigen 3 (PfLSA3), a novel highly conserved P. falciparum preerythrocytic antigen, was evaluated. Animals developed a dominant Th1 immune response (high gamma interferon T-cell responses and predominance of immunoglobulin G2a) to each of three recombinant proteins spanning the molecule. We have exploited the immunological cross-reactivity of PfLSA3 with its putative homologue on sporozoites of the rodent parasite Plasmodium yoelii, and we show for the first time that responses induced by PfLSA3 in mice significantly protect against a heterologous challenge by P. yoelii sporozoites. These results support a significant effect of DNA-induced immune responses on preerythrocytic stages.

Immunogenicity of four Plasmodium falciparum preerythrocytic antigens in Aotus lemurinus monkeys.

Aotus lemurinus monkeys were immunized with pools of either lipid-tailed peptides injected in PBS or peptides in Montanide ISA-51, all derived from four Plasmodium falciparum pre-erythrocytic antigens, namely, LSA1, LSA3, SALSA, and STARP. These formulations were well tolerated. Their immunogenicity was demonstrated by the induction of both B- and T-cell responses to most of the peptides studied (of the 12, 10 induced antibody production, 9 induced T-cell proliferative responses, and all 12 induced gamma interferon secretion). Immune responses proved to be long lasting, since some were still detectable 210 days after immunization. Of particular importance is the fact that B- and T-cell responses elicited in this way by synthetic peptides were specific for native parasite proteins on P. falciparum sporozoites and liver stage parasites.

Antigenicity and immunogenicity of multiple antigen peptides (MAP) containing P. vivax CS epitopes in Aotus monkeys.

Using linear synthetic peptides corresponding to the Plasmodium vivax circumsporozoite (CS) protein of the common type, we have identified several T and B-cell epitopes recognized by human individuals. Three T-cell epitopes studied (p6) from the amino, (p11) from the central and (p25) from the carboxyl regions, were widely recognized by lymphocytes of immune donors. A series of six peptides, in addition to p11, representing the central repeat domain of the CS(p11-p17) protein were used in ELISA assays to map the B-cell epitopes of this region. P11 was the peptide most frequently recognized by sera containing antibodies to the homologous CS protein as determined by IFAT. The sequences corresponding to peptides p6, p11 and P25 as well as that representing a universal T-cell epitope derived from the tetanus toxin were used to assemble eight different Multiple Antigen Peptides (MAP). The immunogenicity of these MAP was analysed in Aotus monkeys. Groups of two animals were immunized with each MAP and both antibody response, T-lymphocyte proliferation and in vitro gamma-IFN production were evaluated. Two MAPs containing the same B-cell epitope and either a promiscuous CS-protein derived T-cell epitope (p25) or the tetanus toxin epitope (p-tt30) proved to be the most immunogenic and induced high levels of anti-peptide antibodies that recognized the native protein. Except for animals immunized with MAP VII, there was no correlation between antibody levels, lymphocyte proliferation or gamma-IFN production in vitro. The broad recognition of these epitopes by individuals which had been exposed to malaria, the capacity of these MAPs to induce antibodies, recognize the cognate protein, and in vitro gamma-IFN production encourages further analyses of the potential of these proteins as malaria vaccine candidates for human use.

Leishmania major infection in mice primes for specific major histocompatibility complex class I-restricted CD8+ cytotoxic T cell responses.

This report shows that lymphoid tissues of mice which have resolved a primary infection with Leisihmania major contain parasite-specific major histocompatibility complex (MHC) class I-restricted cytolytic CD8+ T cell precusors that can be expanded after specific restimulation in vitro with syngeneic antigen-presenting cells pulsed with a cyanogen bromide digest of L. major. In H-2b mice, two distinct populations of CD8+ T cells were identified which both lysed target cells pulsed with L. major-derived peptides but were restricted by a different H-2b class I gene product. Interestingly, these two populations appear to recognize different parasite-derived peptides. It is noteworthy that one Kb-restricted CD8+ T cell line was able to specifically lyse syngeneic macrophages infected with viable L. major, indicating that some L. major-derived peptides may reach the MHC class I pathway of presentation from the phagolysosomal compartment where the parasites are confined in infected macrophages. The importance of these parasite-specific MHC class I restricted cytolytic CD8+ T cells for the elimination of L. major by the infected host remains to be determined.

Serum factors inhibitory for in vitro development of Plasmodium falciparum blood-stage parasites.

Sera from 29 individuals residing in a malaria-endemic region of Colombia were evaluated by an inhibition assay for their capacity to retard the growth of Plasmodium falciparum in vitro. The inhibitory activity was found to be independent of antibody activity. Furthermore, the degree of inhibition of parasite development was variable, depending on the parasite isolate used for the assay and the season of malaria transmission. We selected sera with high inhibitory activity and carried out partial analytical characterization by anion exchange fast protein liquid chromatography (FPLC) to identify the chemical nature of the inhibitory factor(s). The results suggested that the in vitro inhibitory activity might result from the additive effect of different molecules. It appears that these molecules could be non-specifically induced by stimulation of the immune system, they seem to play a role in the immunity to malaria.

Malaria crisis activity in sera from individuals of different ethnic groups of Colombia.

Sera of negroes of African origin and of indians, living in a malaria endemic village on the Pacific Coast of Colombia, were analyzed to see if they could block intraerythrocytic Plasmodium falciparum growth in vitro. A group of mestizos from a malaria-free city in Colombia was used as a negative control. Blood of each individual was studied for the presence of circulating parasites by thick and thin smears and their sera for antimalarial antibodies by IFAT and IRMA techniques. The inhibition of the intraerythrocytic growth induced by these sera was assessed by [3H]Hypoxanthine incorporation. All groups showed inhibitory activity independent of their exposure to malaria. Negro sera had the highest inhibitory activity even following the removal of antibody, and also the highest antimalarial antibody titers. The group of indians had reduced inhibitory activity and lower antibody titers compared to the negro sera. In the group of mestizos, who reported no malaria exposure, 14% had antibodies to asexual blood forms of P. falciparum and 60% induced significant inhibition.

Antibody-independent inhibition of Plasmodium falciparum in vitro cultures.

The sera of 100 Colombian individuals of African origin living in a malaria-endemic area of the Pacific coast were studied with regard to their capacity to inhibit Plasmodium falciparum cultures in vitro. Antimalarial antibody levels determined by indirect immunofluorescence were higher in the group of infected individuals than in the noninfected individuals, and inhibitory activity assessed by the inhibition of parasite incorporation of 3H-hypoxanthine in vitro was present in the sera of both the infected and noninfected patients. We believe that the noninfected patients were probably immune. The sera of some of the infected patients had high inhibitory capacities for the P. falciparum FCB-1 isolate. When the inhibitory effects of some of the sera were tested by using four parasite isolates from different regions of the world, striking differences among them were found.

Immunization of Aotus monkeys with Plasmodium falciparum blood-stage recombinant proteins.

The current spread of multidrug-resistant malaria demands rapid vaccine development against the major pathogen Plasmodium falciparum. The high quantities of protein required for a worldwide vaccination campaign select recombinant DNA technology as a practical approach for large-scale antigen production. We describe the vaccination of Aotus monkeys with two recombinant blood-stage antigens (recombinant p41 and 190N) that were considered as vaccine candidates because parasite-derived antigen preparations could protect susceptible monkeys from an otherwise lethal malaria infection. In contrast to the natural antigen, recombinant p41 protein (P. falciparum aldolase) could not protect monkeys, although all animals seroconverted. 190N antigen, a recombinant protein containing conserved sequences of the major merozoite surface antigen p190, protected two of five monkeys from critical levels of infection with the highly virulent FVO isolate of P. falciparum. However, the B- and T-cell responses to 190N antigen were similar in protected and unprotected animals so that other unknown factors may contribute to protection. Higher purity or lack of protective epitopes or different structure of protective epitopes in the recombinant proteins might explain the better performance of parasite-derived antigens in vaccination trials. The partial protection obtained with 190N antigen suggests that this molecule could contribute to a vaccine mixture against P. falciparum.