New candidate vaccines against blood-stage Plasmodium falciparum malaria: prime-boost immunization regimens incorporating human and simian adenoviral vectors and poxviral vectors expressing an optimized antigen based on merozoite surface protein 1

Abstract

Although merozoite surface protein 1 (MSP-1) is a leading candidate vaccine antigen for blood-stage malaria, its efficacy in clinical trials has been limited in part by antigenic polymorphism and potentially by the inability of protein-in-adjuvant vaccines to induce strong cellular immunity. Here we report the design of novel vectored Plasmodium falciparum vaccines capable of overcoming such limitations. We optimized an antigenic insert comprising the four conserved blocks of MSP-1 fused to tandemly arranged sequences that represent both allelic forms of the dimorphic 42-kDa C-terminal region. Inserts were expressed by adenoviral and poxviral vectors and employed in heterologous prime-boost regimens. Simian adenoviral vectors were used in an effort to circumvent preexisting immunity to human adenoviruses. In preclinical studies these vaccines induced potent cellular immune responses and high-titer antibodies directed against MSP-1. The antibodies induced were found to have growth-inhibitory activity against dimorphic allelic families of P. falciparum. These vectored vaccines should allow assessment in humans of the safety and efficacy of inducing strong cellular as well as cross-strain humoral immunity to P. falciparum MSP-1.

FIG. 1.

A schematic representation of the design of the composite MSP-1 antigen inserts. The design of PfM115 and PfM128 was based on the structure of merozoite surface protein 1 and included the four more-conserved blocks (blocks 1, 3, 5, and 12) and FVO and 3D7 allelic variants of blocks 16 and 17 as shown.

FIG. 2.

T cell responses following immunization with viral vectors expressing PfM115. MSP-1-specific cytokine production from splenocytes of mice previously immunized with AdHu5 or C7 expressing PfM115, followed by a boost immunization at 8 weeks with C6, C7, or MVA (M) expressing PfM115 (prime_boost), was assessed at 2 weeks after the final immunization. Multiparameter flow cytometry was used to determine the total frequencies of IFN-γ-, TNF-α-, and IL-2-producing T cells. CD4⁺ (A) and CD8⁺ (B to D) T cell responses to vaccination are shown. The murine strains used are shown in the figure. In panel D The fraction of the total response comprising cells expressing one (+), two (++) or, three (+++) cytokines in the experiment for panel B is shown. Adenoviral vectors and poxviral vectors were given at doses of 5 × 10¹⁰ VP or 5 × 10⁷ PFU, respectively. Data are means ± standard errors of the means (SEM) (n = 6).

FIG. 3.

T cell responses following immunization with viral vectors expressing PfM128. MSP-1-specific cytokine production from splenocytes of C57BL/6 mice previously immunized with AdHu5 or AdCh63 expressing PfM128, followed by a boost immunization at 8 weeks with MVA (M) expressing PfM128 (prime_boost), was assessed at 2 weeks after the final immunization. Multiparameter flow cytometry was used to determine the total frequencies of IFN-γ-, TNF-α-, and IL-2-producing T cells. CD4⁺ (A and C) and CD8⁺ (B and D) T cell responses to vaccination are shown. Panels C and D show the fraction of the total response comprising cells expressing one (+), two (++), or three (+++) cytokines. Adenoviral vectors and poxviral vectors were given at doses of 10¹⁰ VP or 10⁷ PFU, respectively. Data are means ± SEM (n = 6).

FIG. 4.

Vaccine-induced antibody responses to MSP-1₁₉. BALB/c mice were immunized with adenoviral vectors (AdHu5, C6, C7, and C9), and total IgG titers to GST-MSP-1₁₉ (ETSR) were measured by ELISA (unless stated otherwise). (A) Comparison of antibody responses at week 8 following a single immunization with 5 × 10¹⁰ adenoviral particles (VP) expressing PfM115. (B) Comparison of antibody responses at week 2 following a single immunization with 10¹⁰ VP expressing PfM128. (C) Total IgG responses (geometric mean titer [GMT]) over time following immunization as for panel A, followed at week 8 by a boost with 5 × 10⁷ PFU MVA PfM115 (M) or 5 × 10¹⁰ VP AdHu5 or C6 PfM115 as shown (prime_boost). (D) Isotype ELISAs were performed at week 10 on samples from panel C. Total IgG titers to GST-MSP-1₁₉ ETSR (A to D) and GST-MSP-1₁₉ QKNG (C) were measured. Geometric mean titers (± 95% confidence intervals [CIs]) are shown. ***, Different from week 8 GMT (P < 0.001).

FIG. 5.

Antibody responses to MSP-1 allelic variants. BALB/c mice (A and C) and New Zealand White rabbits (B and D) were immunized with vaccines incorporating the 3D7 allelic variant in PfM115 (A and B) or both allelic variants in PfM128 (C and D) of MSP-1₁₉. Mice were immunized as described for Fig. 4 and 6. Rabbits were immunized as described for Fig. 7. Animals received a range of prime_boost immunization regimens expressing PfM115 or AdHu5 PfM128 and a boost immunization of MVA PfM128 (C and D). Sera were taken at week 10. Total IgG ELISAs were performed for GST-MSP-1₁₉ ETSR (3D7 allelic variant) and GST-MSP-1₁₉ QKNG (FVO allelic variant). Antibody titers from individual animals and linear regression lines are shown, along with Pearson rank correlations (r²) and P values.

FIG. 6.

Vaccine-induced antibody responses to MSP-1₁₉ following AdHu5_M PfM115 or AdHu5_M PfM128. BALB/c mice were immunized with 10¹⁰ adenoviral particles of AdHu5 expressing either PfM115 or PfM128 as indicated and were boosted at week 8 by immunization with 10⁷ PFU of MVA expressing the same antigen. Sera were taken at 2 weeks following the final immunization, and total IgG titers to GST-MSP-1₁₉ (ETSR or QKNG, as shown) were measured by ELISA. *, different from PfM115 QKNG (P < 0.05).

FIG. 7.

Functional antibodies against whole P. falciparum. Rabbits (n = 3/group) were immunized with AdHu5 or C7 expressing PfM115 and boosted 8 weeks later with MVA PfM115 (M), AdHu5, or C6. Alternatively, rabbits were immunized with AdHu5 or AdCh63 expressing PfM128 and boosted with MVA expressing PfM128. Doses used were 5 × 10¹⁰ VP (adenoviruses) or 10⁸ PFU (MVA). Both vaccines were administered i.d. or i.m. (§). Groups were as follows: A§, AdHu5_M PfM115 i.m.; B, AdHu5_M PfM115 i.d.; C§, AdHu5_M PfM128; D§, AdCh63_M PfM128; E, C7_M PfM115 i.d.; F, C7_C6 PfM115 i.d.; G, C7_AdHu5 PfM115 i.d. Purified polyclonal IgG for GIA was obtained from sera collected at 2 weeks after the final immunization. (A) IFAs were performed with sera from immunized rabbits. Areas in green are stained with anti-rabbit IgG secondary antibody, while areas in blue are stained with DAPI. No significant green staining was seen with naïve sera. A representative slide from a rabbit from group A is shown (1:1,000). (B) GIA responses were correlated with total IgG ELISA titer to 3D7 MSP-1₄₂. (C) GIA against 3D7 strains in vitro at 10 mg/ml IgG was determined. (D) GIA against 3D7 (circles) and FVO (squares) at 2.5 mg/ml IgG was determined. Individual and median values are shown.