Biomaterials that modulate innate and adaptive immune responses are receiving increasing interest as adjuvants for eliciting protective immunity against a variety of diseases. Previous results have indicated that self-assembling β-sheet peptides, when fused with short peptide epitopes, can act as effective adjuvants and elicit robust and long-lived antibody responses. Here we investigated the mechanism of immunogenicity and the quality of antibody responses raised by a peptide epitope from P. falciparum circumsporozoite (CS) protein, (NANP)3,conjugated to the self-assembling peptide domain Q11. The mechanism of adjuvant action was investigated in knockout mice with impaired MyD88, NALP3, TLR-2, or TLR-5 function, and the quality of antibodies raised against (NANP)3-Q11 was assessed using a transgenic sporozoite neutralizing (TSN) assay for malaria infection. (NANP)3-Q11 self-assembled into nanofibers, and antibody responses lasted up to 40 weeks in C57BL/6 mice. The antibody responses were T cell- and MyD88-dependent. Sera from mice primed with either irradiated sporozoites or a synthetic peptide, (T1BT*)4-P3C, and boosted with (NANP)3-Q11 showed significant increases in antibody titers and significant inhibition of sporozoite infection in TSN assays. In addition, two different epitopes could be self-assembled together without compromising the strength or duration of the antibody responses raised against either of them, making these materials promising platforms for self-adjuvanting multi-antigenic immunotherapies.

A 12 amino-acid synthetic peptide (NANP)3 comprising the immunodominant epitope of Plasmodium falciparum circumsporozoite protein was conjugated to tetanus toxoid (TT), adjuvanted with aluminium hydroxide, and administered intramuscularly in three doses at monthly intervals to 35 healthy males as a malaria vaccine. No significant adverse reactions were noted, with mild soreness at the injection site the only common symptom. Seroconversions against NANP occurred in 53% and 71% of recipients of 100 or 160 micrograms, respectively, measured by enzyme-linked immunosorbent assay (ELISA). Most ELISA-positive sera reacted with sporozoites by indirect immunofluorescence (IFA). Three vaccinees with the highest ELISA and IFA titres and four unimmunized controls were challenged with P. falciparum sporozoites introduced via the bites of infective Anopheles mosquitoes. Blood stage parasites were detected in all controls by 10 days (mean 8.5 days, range 7-10). In contrast, the two vaccinees who became infected did not manifest parasitaemia until day 11 and the third vacinee showed neither parasites nor symptoms during the 29 day observation period. This first synthetic peptide parenteral vaccine against a communicable disease tested in man is safe and stimulates biologically active antibodies. These observations encourage the development of improved vaccine formulations which, by enhancing immunogenicity, may lead to practical vaccines to assist in the control of falciparum malaria.

We have used panels of monoclonal antibodies to circumsporozoite (CS) proteins of Plasmodium falciparium, P. vivax, and P. knowlesi to determine the number of topographically independent epitopes of these antigens. The results of competition binding assays indicated that single regions of the CS molecules were recognized by the homologous monoclonal antibodies. Competition binding assays were also used to study the specificity of antibodies contained in the sera of humans and monkeys that had developed sterile immunity after immunization with irradiated, intact sporozoites. We found that single monoclonal antibodies inhibited 70-95% of the specific binding of the polyclonal antibodies to crude extracts of sporozoites. It appears, therefore, that CS proteins are among the most immunogenic constituents of sporozoites, and that a single region of these molecules contains most of the immunogenic activity. An additional finding was that the immunodominant region of CS molecules is multivalent with regard to the expression of a single epitope. This was demonstrated by the ability of monomers of CS proteins to bind simultaneously two or more molecules of the same monoclonal antibody.

This open-labeled phase I study provides the first demonstration of the immunogenicity of a precisely defined synthetic polyoxime malaria vaccine in volunteers of diverse HLA types. The polyoxime, designated (T1BT*)4-P3C, was constructed by chemoselective ligation, via oxime bonds, of a tetrabranched core with a peptide module containing B cell epitopes and a universal T cell epitope of the Plasmodium falciparum circumsporozoite protein. The triepitope polyoxime malaria vaccine was immunogenic in the absence of any exogenous adjuvant, using instead a core modified with the lipopeptide P3C as an endogenous adjuvant. This totally synthetic vaccine formulation can be characterized by mass spectroscopy, thus enabling the reproducible production of precisely defined vaccines for human use. The majority of the polyoxime-immunized volunteers (7/10) developed high levels of anti-repeat Abs that reacted with the native circumsporozoite on P. falciparum sporozoites. In addition, these seven volunteers all developed T cells specific for the universal epitope, termed T*, which was originally defined using CD4+ T cells from protected volunteers immunized with irradiated P. falciparum sporozoites. The excellent correlation of T*-specific cellular responses with high anti-repeat Ab titers suggests that the T* epitope functioned as a universal Th cell epitope, as predicted by previous peptide/HLA binding assays and by immunogenicity studies in mice of diverse H-2 haplotypes. The current phase I trial suggests that polyoximes may prove useful for the development of highly immunogenic, multicomponent synthetic vaccines for malaria, as well as for other pathogens.

A multiple antigen peptide (MAP) malaria vaccine containing minimal Plasmodium falciparum circumsporozoite protein repeat epitopes was assessed for safety and immunogenicity in volunteers of known class II genotypes. The MAP/alum/QS-21 vaccine formulation elicited high levels of parasite-specific antibodies in 10 of 12 volunteers expressing DQB1*0603, DRB1*0401, or DRB1*1101 class II molecules. In contrast, volunteers of other HLA genotypes were low responders or nonresponders. A second study of 7 volunteers confirmed the correlation of class II genotype and high responder phenotype. This is the first demonstration in humans that a peptide vaccine containing minimal T and B cell epitopes composed of only 5 amino acids (N, A, V, D, and P) can elicit antibody titers comparable to multiple exposures to irradiated P. falciparum-infected mosquitoes. Moreover, the high-responder phenotypes were predicted by analysis of peptide/HLA interactions in vitro, thus facilitating the rational design of epitope-based peptide vaccines for malaria, as well as for other pathogens.