A nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria

Abstract

We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)(2)D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2(b), H-2(d), and H-2(k) alleles. Immunized mice produced a CD4(+) T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

Figure 1. Assembly of P4c-Mal SAPN

(A) Monomeric building block of the P4c-Mal construct composed of the five-fold coiled-coil domain from COMP (green), the de-novo designed trimeric coiled-coil domain (blue) and the B cell epitope (red) in extended conformation. (B) Diagram of an assembled SAPN with icosahedral symmetry; the view is down the five-fold symmetry axis of the icosahedron. The B cell epitope is presented at the surface of the SAPN. The figure was prepared using the program DINO (http://www.dino3d.org). (C) Coomassie Blue stained SDS-PAGE gel analysis of P4c-Mal linear polypeptide. On left are relative molecular weight markers (kDa). (D). Transmission EM of P4c-Mal SAPN particle preparation. Bar is 200 nm.

Figure 2. A. Titers of anti-PbCSP peptide antibody

Balb/c mice were immunized at 0, 14 and 28 days with P4c in saline, P4c in Montanide ISA 720, R-PbCSP in saline, R-PbCSP in Montanide ISA 720, P4c-Mal in saline or P4c-Mal in Montanide ISA 720. Titers were determined on sera taken on days 0, 14, 28 or 42 as the dilution required to achieve and OD_(405nm) =1. Values are from a pool of sera from a representative cohort of 10 mice per group. Similar titers were obtained with C57BL/6 mice. (B). Survival of mice whose titers are depicted in Figure 2A 15 days following challenge with live sporozoites. Values are the combined results of two separate experiments (two cohorts of 10 mice) per group. Additional mice received irradiated sporozoites, saline or Montanide ISA 720 in saline as described. No mouse that developed parasitemia survived beyond day 15 and no mouse developed parasitemia after day 15.

Figure 3

Development of PbCSP peptide specific antibody in mice immunized with SAPN constructs containing different ratios of LP containing PbCSP peptide and a random peptide. Mice were immunized with SAPN containing 0, 10, 25, 50, 75 or 100% of LP on days 0, 14, 28. Titers were determined on sera obtained on days 14, 28 and 42 as the dilution required to achieve an OD_(405nm) =1.

Figure 4. P4c-Mal induces long term immunity that is boostable by challenge with live sporozoites

(A) Groups (n=5) mice received three doses of P4c-Mal or saline at 0, 2 and 4 wks. A group was challenged with live sporozoites either 14 days, or 1, 3 or 6 months later. (B) Groups (n=5) mice received three doses of P4c-Mal or saline at 0, 2 and 4 wks. All mice that received P4c-Mal were challenged with live sporozoites two weeks post 3^rd dose of vaccine. All mice survived challenge. A group (n=5) of vaccinated and challenged mice or mice receiving saline only were challenged a second time either 1, 3, 6, 9 or 15 months later with live sporozoites.

Figure 5. P4c-Mal induces the production of IL-2 in CD4⁺ T cells

Splenocytes from P4c-Mal immunized or naïve C57BL/6 mice were cultured with P4c-Mal nanoparticles, Con A or media with or without anti-CD4⁺ T cell blocking antibodies. After 18 h the numbers of cells secreting IL-2 were determined. Values are representative of one of four separate experiments.*: P>0.001.

Figure 6. P4c-Mal induces a mixed TH₁/TH₂ IgG subclass profile

C57BL/6 or Balb/C mice were immunized with P4c-Mal in saline or P4c-Mal in Montanide ISA 720. A group of C57BL/6 mice also received R-PbCSP in Montanide ISA 720. (A) Total IgG and (B) IgG subclass titers were determined on pooled sera obtained two weeks after the last immunization. Error bars depict the SD of the geometric mean values.

Figure 7. Avidity measurements of anti-PbCSP peptide total IgG and IgG subclasses

(A) C57BL/6 or (B) Balb/C mice were immunized with R-PbCSP, P4c-Mal or P4c-Mal/M. The amount of total IgG or IgG subclass antibody that bound to PbCSP peptide was determined after incubation with PBS (•) or NaSCN (▪). Antibody determined in sera obtained two weeks after the last immunization.

Figure 7. Avidity measurements of anti-PbCSP peptide total IgG and IgG subclasses

(A) C57BL/6 or (B) Balb/C mice were immunized with R-PbCSP, P4c-Mal or P4c-Mal/M. The amount of total IgG or IgG subclass antibody that bound to PbCSP peptide was determined after incubation with PBS (•) or NaSCN (▪). Antibody determined in sera obtained two weeks after the last immunization.