A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in preclinical analyses in rodent and primate hosts.

Despite extensive public health efforts, there are presently 200 to 400 million malaria infections and 1 to 2 million deaths each year due to the Plasmodium parasite. A prime target for malaria vaccine development is the circumsporozoite (CS) protein, which is expressed on the extracellular sporozoite and the intracellular hepatic stages of the parasite. Previous studies in rodent malaria models have shown that CS repeat B-cell epitopes expressed in a recombinant hepatitis B virus core (HBc) protein can elicit protective immunity. To design a vaccine for human use, a series of recombinant HBc proteins containing epitopes of Plasmodium falciparum CS protein were assayed for immunogenicity in mice [A. Birkett, B. Thornton, D. Milich, G. A. Oliveira, A. Siddique, R. Nussenzweig, J. M. Calvo-Calle, and E. H. Nardin, abstract from the 50th Annual Meeting of the American Society of Tropical Medicine and Hygiene 2001, Am. J. Trop. Med. Hyg. 65(Suppl. 3):258, 2001; D. R. Milich, J. Hughes, J. Jones, M. Sallberg, and T. R. Phillips, Vaccine 20:771-788, 2001]. The present paper summarizes preclinical analyses of the optimal P. falciparum HBc vaccine candidate, termed ICC-1132, which contains T- and B-cell epitopes from the repeat region and a universal T-cell epitope from the C terminus of the CS protein. The vaccine was highly immunogenic in mice and in Macaca fascicularis (cynomolgus) monkeys. When formulated in adjuvants suitable for human use, the vaccine elicited antisporozoite antibody titers that were logs higher than those obtained in previous studies. Human malaria-specific CD4(+)-T-cell clones and T cells of ICC-1132-immunized mice specifically recognized malaria T-cell epitopes contained in the vaccine. In addition to inducing strong malaria-specific immune responses in naïve hosts, ICC-1132 elicited potent anamnestic antibody responses in mice primed with P. falciparum sporozoites, suggesting potential efficacy in enhancing the sporozoite-primed immune responses of individuals living in areas where malaria is endemic.

A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types.

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.

Synthetic malaria peptide vaccine elicits high levels of antibodies in vaccinees of defined HLA genotypes.

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.

Preclinical evaluation of a synthetic Plasmodium falciparum MAP malaria vaccine in Aotus monkeys and mice.

Multiple antigen peptides (MAPs) containing epitopes of the major surface protein of the malaria sporozoite, the circumsporozoite (CS) protein, have been shown in previous studies to elicit antibody-mediated protection against sporozoite challenge in experimental murine and simian hosts. For the preparation for a phase I trial of a P. falciparum (T1B)4 MAP, which contains T and B cell epitopes from the CS repeat region, pre-clinical immunogenicity and adjuvant formulation studies were carried out in mice and Aotus monkeys. The (T1B)4 MAP was found to be immunogenic in three different species of owl monkeys, Aotus nancymae, A. vociferans and A. nigriceps. Optimal antibody responses were obtained in A. nancymae immunized s.c. with (T1B)4 MAP emulsified in Freund's, in which peak titers of over 10(6) were obtained in individual monkeys. MAP immunized A. vociferans also developed high levels of anti-sporozoite antibodies, although the kinetics and the magnitude of the response differed from A. nancymae. (T1B)4 MAP adsorbed to alum (aluminum hydroxide), a formulation that is acceptable for human use, was less immunogenic in naive A. nancymae, as well as A. nigriceps. The injection of MAPs/alum, however, significantly enhanced antibody responses in sporozoite-primed monkeys, suggesting that the administration of the MAP vaccine may be an effective means to increase the low levels of antibody present in individuals living in malaria endemic areas. The addition of a co-adjuvant QS-21, a purified saponin, significantly increased the immunogenicity of the alum-adsorbed MAP in both mice and monkeys, providing a vaccine formulation suitable for phase I trials in human volunteers.

Plasmodium falciparum polyoximes: highly immunogenic synthetic vaccines constructed by chemoselective ligation of repeat B-cell epitopes and a universal T-cell epitope of CS protein.

Effective immunoprophylaxis directed against the pre-erythrocytic stages of the malaria parasite requires a vaccine that can elicit humoral and cell mediated immunity in individuals of diverse genetic background. In order for a synthetic peptide malaria vaccine to meet these requirements, problems associated with genetic restriction, peptide chemistry, adjuvant formulation and physiochemical characterization of the final synthetic vaccine product must first be overcome. To address these issues, five polyoxime vaccine candidates have been constructed by ligating purified peptide epitopes of the P. falciparum CS protein to a branched template via oxime bonds. All five constructs, including two based on templates containing the synthetic adjuvant tripalmitoyl-S-glyceryl cysteine (Pam3Cys), were of sufficient purity for characterization by mass spectrometry. The immunogenicity of the malaria polyoximes in different murine strains was compared to that of multiple antigen peptide (MAP) constructs synthesized by standard step-wise synthesis. A tri-epitope polyoxime-Pam3Cys construct, based on the repeats and a universal T-cell epitope that contains both helper and CTL epitopes of the CS protein, was shown to be a precisely-defined synthetic malaria vaccine candidate that was highly immunogenic in murine strains of diverse H-2 haplotypes.

Binding of malaria T cell epitopes to DR and DQ molecules in vitro correlates with immunogenicity in vivo: identification of a universal T cell epitope in the Plasmodium falciparum circumsporozoite protein.

The efficacy of a malaria peptide vaccine would be enhanced by the inclusion of a parasite-derived universal T cell epitope to ensure that all vaccinees develop parasite-specific cellular and humoral immunity. Two circumsporozoite (CS) protein T cell epitopes, previously identified by CD4+ T cell clones derived from Plasmodium falciparum sporozoite-immunized volunteers, were studied to determine their HLA class II binding potential. One epitope, located in amino acid (aa) 326-345 of the P. falciparum (NF54 strain) CS protein, was "universal" in that it could bind to multiple DR and DQ molecules in vitro. In contrast, the second epitope, T1, which is located in the CS repeat region, was recognized by T cells in the context of DQ6 (DQB1*0603) and did not bind with high affinity to any of the class II molecules tested in the peptide binding assays. The in vitro patterns of peptide/HLA interactions correlated with immunogenicity in vivo. A multiple antigen peptide (MAP) containing the aa 326-345 epitope elicited responses in eight inbred strains (H-2(a,b,d,k,p,q,r,s)), while the T1 MAP was recognized by only a single haplotype, H-2b. The combination of the universal aa 326-345 T cell epitope and the T1 repeat in a di-epitope MAP overcame the genetic restriction to the P. falciparum CS repeat region and elicited antisporozoite Ab responses in all of the MAP-immunized mice. Synthetic peptide malaria vaccines containing the aa 326-345 universal T cell epitope would be expected to elicit parasite-specific immune responses in both sporozoite-primed and naive individuals of diverse genetic backgrounds.

In vitro development of infectious liver stages of P. yoelii and P. berghei malaria in human cell lines.

The preerythrocytic stages of the malaria parasite are the focus of intense efforts to identify new immunological and pharmacological methods for the control of the malaria parasite. The study of the malaria hepatic stages requires an in vitro system to facilitate the analysis of parasite/host cell interactions and the characterization of exoerythrocytic form (EEF) antigens. At the present time, only the rodent malaria, Plasmodium berghei, and the human malaria, Plasmodium vivax, develop into mature infectious EEF within established cell lines in vitro. We therefore used the rodent malaria, Plasmodium yoelii, which lacks a cell line for in vitro cultivation of EEF, to screen a series of human cell lines as potential host cells for the intracellular stage of the malaria parasite. Two human hepatomas, huH-1 and huH-2, were identified that supported the development of P. yoelii EEF which were antigenically and morphologically mature. Infectious merozoites that were shown to be capable of inducing P. yoelii blood infections in vivo developed within these in vitro EEF. In addition, mature, infectious EEF of P. berghei, but not P. yoelii, were found to develop within human HeLa cells. Preliminary results suggest that the cell lines that support the intracellular growth of P. yoelii might also serve as host cells for EEF of Plasmodium falciparum. The identification and characterization of the unique intracellular requirements for differentiation and development of P. berghei and P. yoelii EEF may provide new approaches to the prophylaxis of the human malaria species.

Immunogenicity of multiple antigen peptides containing B and non-repeat T cell epitopes of the circumsporozoite protein of Plasmodium falciparum.

We have characterized the immune response of mice to multiple Ag peptide systems (MAP) containing the immunodominant B cell epitope (NANP)3 and one of three distinct Th epitopes, Th2R, Th3R, and CS.T3, of the C terminal region of the circumsporozoite protein of Plasmodium falciparum, a human malaria parasite. Mice of three different MHC haplotypes (H-2k, H-2d, and H-2a) were immunized with the various MAP constructs. Mice of all three strains produced antibodies, but their anti-sporozoite titers were considerably lower than their anti-peptide titers as detected by ELISA. These antibodies reacted at high titers not only with the repeat polymer (NANP)50, but also with MAP that contained only the respective Th sequence. The antibody binding site within each of the Th sequences was mapped, using truncated peptides, in an inhibition assay. A primary antibody response, induced by a single i.v. inoculation of sporozoites, was greatly enhanced by the injection of MAP.