Immunization with pre-erythrocytic antigen CelTOS from Plasmodium falciparum elicits cross-species protection against heterologous challenge with Plasmodium berghei

Abstract

Background: The Plasmodium protein Cell-traversal protein for ookinetes and sporozoites (CelTOS) plays an important role in cell traversal of host cells in both, mosquito and vertebrates, and is required for successful malaria infections. CelTOS is highly conserved among the Plasmodium species, suggesting an important functional role across all species. Therefore, targeting the immune response to this highly conserved protein and thus potentially interfering with its biological function may result in protection against infection even by heterologous species of Plasmodium.

Methodology/principal findings: To test this hypothesis, we developed a recombinant codon-harmonized P. falciparum CelTOS protein that can be produced to high yields in the E. coli expression system. Inbred Balb/c and outbred CD-1 mice were immunized with various doses of the recombinant protein adjuvanted with Montanide ISA 720 and characterized using in vitro and in vivo analyses.

Conclusions/significance: Immunization with PfCelTOS resulted in potent humoral and cellular immune responses and most importantly induced sterile protection against a heterologous challenge with P. berghei sporozoites in a proportion of both inbred and outbred mice. The biological activity of CelTOS-specific antibodies against the malaria parasite is likely linked to the impairment of sporozoite motility and hepatocyte infectivity. The results underscore the potential of this antigen as a pre-erythrocytic vaccine candidate and demonstrate for the first time a malaria vaccine that is cross-protective between species.

Figure 1. Immunization with recombinant PfCelTOS induces sterile protection against a heterologous sporozoite challenge with P. berghei.

Inbred Balb/c mice and outbred CD-1 mice were immunized three times with the indicated doses and challenged two weeks after the last immunizations by subcutaneous injection of either 4,000 (for Balb/c) or 15,000 (for CD-1 mice) sporozoites. Vaccine efficacy was calculated using the formula: Efficacy  =  [1-[(number of infected animals (I)_vaccine/total number of animals (n)_vaccine)÷(number of infected animals _control (I)/total number of animals (n) _control)]]*100. The numbers above bars represent the number of protected animals/total number of animals in the group. Statistical significance was determined with Fisher Exact test (two-tailed) and is indicated by asterisks (* p = 0.01, ** p = 0.001).

Figure 2. PfCelTOS induces a strong humoral immune response in inbred and outbred mice.

Sere were collected three weeks after the first and second immunization (post-1, post-2) and two days prior to challenge (post-3). Antibody kinetics for Balb/c mice (Panel A) and CD-1 mice (Panel B) were determined using a quantitative ELISA. Data are expressed as the mean µg/ml, error bars represent the SEM of n = 15 mice/group (i.e., 10 challenged mice and 5 reagent mice/group).

Figure 3. PfCelTOS specific antibody concentration does not predict protective status of mice.

Data of pre-challenge sera from all tested dose groups were stratified by protective status of the animals. Box plot indicates the median and the boundaries indicate the 25^th and the 75^th percentile.

Figure 4. PfCelTOS-specific antibodies raised by protein immunization react with P. falciparum and P. berghei sporozoites.

Reactivity of polyclonal anti-PfCelTOS antibodies against P. falciparum (Panel A) and P. berghei sporozoites (Panel B) and reactivity of PfCelTOS-specific mAb 2C7 against P. falciparum (Panel C) and P. berghei-specific mAb 15E2 (Panel D) against P. berghei sporozoites. The antisera used for the staining were from the post-3 sera ( =  prechallenge) and were tested at 1∶200 dilutions. Images were taken at 1,000x magnification.

Figure 5. CelTOS-specific antibodies inhibit invasion of hepatocytes by P. falciparum sporozoites.

Sera from mice immunized either with CelTOS (and protected against challenge) or adjuvant alone were tested in triplicate for their ability to inhibit hepatocyte infection in vitro. HepG2A16 cells were incubated with sporozoites in the presence or absence of antisera at either 1∶100 (black bars) or 1∶400 (white bars). Percent inhibition was calculated based on the number of infected hepatocytes in the control cultures.

Figure 6. PfCelTOS specific IFN-γ and IL-4 responses show significant cross-reactivity to the heterologous protein.

Splenocytes from Balb/c mice (Panel A, B) and CD-1 mice (Panel C, D) immunized three times with recombinant protein in Montanide ISA 720 were tested in IFN-γ (Panel A,C) and IL-4 (Panel B,D) specific ELISpot assays. Data are expressed as the mean number plus standard error of the mean (SEM) of cytokine producing cells (spot forming cells, SFC)/10⁶ splenocytes of individual mice (n = 5/group). Immunization doses for Balb/c  = 10 µg (high dose), 1 µg (low dose), immunization dose for CD-1  = 25 µg (high dose), 10 µg (low dose). Antigens used for ex vivo stimulation: no antigen (white bar), 10 µg/ml PfCelTOS (black bar), 10 µg/ml PbCelTOS (hatched bar). For the statistical analysis, vaccinated groups were compared with the Montanide ISA 720 control group stimulated with the same antigen (* indicates p<0.05, two-sided T-test).