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

Abstract

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.

FIG. 1.

(A) P. falciparum (NF54) CS protein showing the B-cell epitope (NANP)n within the major repeat region (bars), the T1 epitope within the 5′ minor repeat region (stippled), and the universal T∗ epitope in the C terminus (black). (B) The synthetic dsDNA fragment encoding the malaria epitope inserted in each plasmid is shown. Amino acid insertions contributed by the restriction sites are underlined, and the stop codons are indicated by asterisks. (C) Hybrid HBc protein showing the CS T1 and B repeat epitopes (hatched box) inserted between HBc amino acids 78 and 79, a region located at the tip of the core particle surface spikes (3, 8), and the T∗ epitope fused to amino acid Val₁₄₉ at the C terminus of the truncated HBc protein (solid box).

FIG. 2.

(A) Physiochemical characterization of truncated HBc149 protein (lane 1) and purified ICC-1132 (lane 2) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Coomassie blue-stained gels and Western blots, using anti-CS or anti-HBc antibodies, demonstrate migration at the expected molecular mass of the reduced ICC-1132, 22,000 Da, and of the C-terminally truncated HBc149, 17,000 Da (M, molecular mass markers). In the Western blots, a monoclonal antirepeat antibody (MAB 2A10) stained ICC-1132 (lane 2) but not HBc149, which lacks the CS repeats (lane 1). (B) Electron microscopy of purified ICC-1132 particles. Images were generated using 1% ammonium acetate as a negative stain. Magnification, ×40,000; scale bar = 250 nm.

FIG. 3.

Antibody titers in BALB/c mouse sera obtained 3 weeks after each dose of ICC-1132-Freund's adjuvant. (A) Kinetics and fine specificity of IgG responses measured by IFA using P. falciparum sporozoites and by ELISA using HBc immunogen or malaria peptides as antigen. (B) IgG subtypes of antirepeat antibody response following each immunization. O.D., optical density.

FIG. 4.

Antibody titers of class II^−/− knockout mice (open symbols) or wild-type mice (closed symbols) as measured by ELISA using P. falciparum CS repeats (A) or HBc protein (B) as antigen. Samples were obtained following immunization (arrows) with ICC-1132 (circles) or HBc149 (squares) in Freund's adjuvant.

FIG. 5.

(A) Lymph node (LN) cells of BALB/c mice immunized with ICC-1132 in Freund's adjuvant or adjuvant-only controls were assayed for proliferation (solid bars) and IL-2 responses (hatched bars). Results are shown as the SI obtained following stimulation with HBc or ICC-1132 core particles (10 μg/ml) or malaria CS peptides T1, B, and T∗ (20 μg/ml). (B) IFN-γ in supernatants of immune lymph node cells (dark bars) stimulated with 10-fold dilutions of antigen, starting at 10 μg/ml for core particles and 20 μg/ml for malaria CS peptides. Results for only the highest antigen concentration are shown for control nonimmune lymph nodes (open bars).

FIG. 6.

Cytokine production by spleen cells of mice primed with a single injection of ICC-1132-Montanide ISA 720 or followed by a booster injection of ICC-1132 either without adjuvant (in saline), adsorbed to alum, or emulsified in Montanide ISA 720. The spleen cells obtained from immunized mice or naïve mice were stimulated with malaria CS peptides or superantigen staphylococcal enterotoxin B (SEB), and levels of cytokine (in picograms per milliliter) were measured in culture supernatants.

FIG. 7.

Cynomolgous monkeys were immunized with ICC-1132 formulated with adjuvants acceptable for human use: Adju-Phos, Alhydrogel, oil-in-water emulsion, or Montanide ISA 720. Two monkeys were immunized per group. (A) ELISA and IFA titers for the first monkey of each group are shown as black and grey bars, respectively, and for the second monkey as hatched and diagonal bars. (B) Kinetics of ELISA and IFA responses in the two cynomolgous monkeys immunized with ICC-1132 in Montanide ISA 720.