Immunization with Transgenic Rodent Malaria Parasites Expressing Pfs25 Induces Potent Transmission-Blocking Activity

Abstract

An anti-malarial transmission blocking vaccine (TBV) would be an important tool for disease control or elimination, though current candidates have failed to induce high efficacy in clinical studies. The ookinete surface protein P25 is a primary target for TBV development, but heterologous expression of P25 with appropriate conformation is problematic and a pre-requisite for achieving functional titers. A potential alternative to recombinant/sub-unit vaccine is immunization with a non-pathogenic, whole-parasite vaccine. This study examines the ability of a purified transgenic rodent-malaria parasite (PbPfs25DR3), expressing Plasmodium falciparum P25 in native conformation on the P. berghei ookinete surface, to act as a TBV. Vaccination with purified PbPfs25DR3 ookinetes produces a potent anti-Pfs25 response and high transmission-blocking efficacy in the laboratory, findings that are then translated to experimentation on natural field isolates of P. falciparum from infected individuals in Burkina Faso. Efficacy is demonstrated in the lab and the field (up to 93.3%/97.1% reductions in transmission intensity respectively), with both a homologous strategy with one and two boosts, and as part of a prime-boost regime, providing support for the future development of a whole-parasite TBV.

Figure 1

Anti-PbPfs25DR3 immunization regimes. Groups of 5 mice received each vaccine regime. For each individual experimental regime, the corresponding (negative) control regime is to its immediate right. In each regime, for DFA 5 mice were challenged with P. berghei PbPfs25DR3 to assess for transmission blockade. In regimes 1–6 mice were immunized to attempt to induce a Pfs25 response (‘experimental regimes’). In regimes 7–12 mice were immunized with carrier protein or empty vector controls (‘control regimes’). Regimes 1,2,7,8 use Matrix M as adjuvant, 3,4,9,10 use alhydrogel, 5,6,11,12 are prime/boost regimes, with ChAd63 prime and ookinete boost. All immunizations were performed i.m.

Figure 2

Induction of antibody following immunization with PbPfs25DR3 ookinetes. The ability of each regime to generate Pfs25-specific antibody responses after administration was tested by ELISA against recombinant Pfs25 protein^, and IFA against P. falciparum (NF54) ookinete/retort stages within the mosquito midgut 26 hours post-feed. (A) End -point anti-Pfs25 titers in serum. Bars show mean titers from 5 mice. Pre-immune/non-immunized serum did not recognize recombinant Pfs25. Error bars represent SEM. Bars show mean titers from 5 mice. Error bars represent SEM. (B) IFA against P. falciparum (NF54) ookinete/retort stages. Ability of generated serum to recognize native Pfs25 on the surface of sexual stages of P. falciparum post-fertilization was assessed by immunofluorescence on fixed, non-permeabilized parasites probed with anti-serum from each regime. To control for non Pfs25-specific signal, IFA was performed using serum from control regimes (7–12). Each panel shows an overlay of anti-Pfs25 signal (turquoise) and DNA labelled with DAPI (blue). Scalebar  =  5 µm. (C and D) The ability of induced titers to reduce both oocyst intensity (C) and infection prevalence (D) by DFA, and relationship with anti-Pfs25 titers. Small open circles denote estimates generated from mosquitoes fed on individual mice (Fig. 3) compared to the average from mosquitoes feeding on unimmunized mice) whilst the large filled dots show the average for the regimen as estimated using a generalized-linear mixed effect model. Vertical lines show 95% confidence interval on overall estimates. Point colours indicate the regimen tested, be it regime 1 (blue), 2 (green), 3 (purple), 4 (orange), 5 (red) or 6 (brown).

Figure 3

Assessment of in vivo transmission blockade following immunization with PbPfs25DR3 ookinetes by DFA. To assess transmission-blocking activity following immunization, 5 mice per regime (experimental and control) were infected/challenged with P. berghei PbPfs25DR3 and three days later, mosquitoes were exposed to individual mice to perform DFA. DFAs were performed in three individual tranches to account for varying experimental timings within multiple regimes (A) regimes 1,2,7,8; (B) regimes 3,4,9,10; (C) regimes 5,6,11,12). Transmission blockade was assessed as mean reduction in oocyst intensity/ prevalence with respect to cohorts of non-immunized challenged mice within each DFA. Regimes 5 and 6 contain four mice due to death before challenge. Individual data points represent the number of oocysts found in individual mosquitoes 12 days post feed. Horizontal bars indicate mean intensity, whilst error bars indicate S.E.M. For each regime, mean reductions in intensity and prevalence for each group are reported in Table 1.

Figure 4

Transmission blocking efficacy of serum derived from immunization with PbPfs25DR3 against field isolates of P. falciparum. To assess the ability of serum generated from regimes 3 (PbPfs25DR3/Alhydrogel 2 boosts), 4 (PbPfs25DR3/Matrix M 2 boosts) and 6 (ChAd63-Pfs25-prime/PbPfs25DR3 Matrix M-boost) to block transmission of malarial field isolates, P. falciparum gametocytes were collected from naturally infected volunteers recruited in malaria endemic localities, and DMFA subsequently performed. A range of naturally occurring gametocyte densities were assessed (A,B,C) were examined at dilutions of 1:5, 1:10 and 1:100, and control (pre-immune) serum at 1:5. Individual data points represent the number of oocysts found in individual mosquitoes 12 days post feed. Horizontal bars indicate mean intensity of infection, whilst error bars indicate S.E.M. within individual samples. For each regime, the mean reductions in intensity and prevalence at each dilution are reported in Table 2.