Immunogenicity and protection of a recombinant human adenovirus serotype 35-based malaria vaccine against Plasmodium yoelii in mice

Abstract

Given the promise of recombinant adenovirus type 5 (rAd5) as a malaria vaccine carrier in preclinical models, we evaluated the potency of rAd35 coding for Plasmodium yoelii circumsporozoite protein (rAd35PyCS). We chose rAd35 since a survey with serum samples from African subjects demonstrated that human Ad35 has a much lower seroprevalence of 20% and a much lower geometric mean neutralizing antibody titer (GMT) of 48 compared to Ad5 (seroprevalence, 85%; GMT, 1,261) in countries with a high malaria incidence. We also demonstrated that immunization with rAd35PyCS induced a dose-dependent and potent, CS-specific CD8(+) cellular and humoral immune response and conferred significant inhibition (92 to 94%) of liver infection upon high-dose sporozoite challenge. Furthermore, we showed that in mice carrying neutralizing antibody activity against Ad5, mimicking a human situation, CS-specific T- and B-cell responses were significantly dampened after rAd5PyCS vaccination, resulting in loss of inhibition of liver infection upon sporozoite challenge. In contrast, rAd35 vaccine was as potent in naive mice as in Ad5-preimmunized mice. Finally, we showed that heterologous rAd35-rAd5 prime-boost regimens were more potent than rAd35-rAd35 because of induction of anti-Ad35 antibodies after rAd35 priming. The latter data provide a further rationale for developing rAd prime-boost regimens but indicate that priming and boosting Ad vectors must be immunologically distinct and also should be distinct from Ad5. Collectively, the data presented warrant further development of rAd35-based vaccines against human malaria.

Figures

FIG. 1.

Prevalence of Ad vector neutralizing antibodies in Africa. Sera collected from healthy adults from Africa were analyzed for neutralizing activity against Ad35 or Ad5. A total of 153 serum samples from 20 countries where malaria is endemic and 53 serum samples from 9 other African countries were available for screening (A). Corresponding GMTs are depicted in panel B.

FIG. 2.

Dose dependence of Ad35PyCS- or rAd5PyCS-induced immune responses and protection upon challenge. Groups of naive BALB/c mice (four per group) were immunized i.m. with 106 to 1010 vp of rAd35PyCS (left panel) or rAd5PyCS (right panel). After 2 weeks, CS-specific cellular and humoral immune responses were determined by ELISPOT (A, C) (data from two independent experiments) and ELISA (B, D) (data from four independent experiments). Bars represent median values.

FIG. 3.

Comparison between rAd35PyCS- and rAd5PyCS-induced immune responses and protection. BALB/c mice were immunized with 109 vp of rAd5PyCS or rAd35PyCS. Splenocytes were isolated 2 weeks later, and the number of gamma interferon-secreting, CS-specific CD8+ T cells was determined in an ELISPOT assay (A). CS-specific humoral responses were assessed in an ELISA (B). The parasite burdens in the livers of immunized and naive mice, as determined by real-time PCR, are depicted in panel C. The protection against a sporozoite challenge (i.e., inhibition of liver infection) that was achieved by immunization is shown in panel D. Data from three independent experiments are shown. Bars represent average values. SFU, spot-forming units.

FIG. 4.

rAd35PyCS immunogenicity and protection in the presence of anti-Ad5 immunity. BALB/c mice were preimmunized with 1010 vp of rAd5empty 8 weeks before immunization. Groups of naive mice or mice with anti-Ad5 immunity (three to five per group) were immunized with 109 vp of rAd35PyCS or rAd5PyCS. CS-specific responses were analyzed 2 weeks after the immunization by ELISPOT (A) and ELISA (B). Parasite burdens in the livers of sporozoite-challenged mice are depicted in panel C, while protection (inhibition of liver infection) is shown in panel D. Bars represent geometric values. SFU, spot-forming units.

FIG. 5.

Immunogenicity and protection in prime-boost immunization regimens. Groups of naive BALB/c mice (five to six per group) were primed with 109 vp of rAd35PyCS or rAd5PyCS at week 0 and boosted with 109 vp of homologous or heterologous vector at week 8. Mean percentages of Kd/CS tetramer-positive, CD8+ T cells at various time points after boost immunization are shown (A). Bars represent standard deviations of the means. After 8 weeks, CS-specific cellular and humoral immune responses were determined by ELISPOT (B) and ELISA (C), respectively. Parasite burdens in the livers of sporozoite-challenged mice are shown in panel D. Percent inhibition of liver infection was calculated on the basis of rRNA data. In homologous prime-boost regimens, 62% inhibition was observed, compared to 92% and 94%, respectively, in heterologous prime-boost regimens. Bars represent geometric means. SFU, spot-forming units.