Chemically attenuated Plasmodium sporozoites induce specific immune responses, sterile immunity and cross-protection against heterologous challenge

Abstract

Vaccination with Plasmodium sporozoites attenuated by irradiation or genetic manipulation induces a protective immune response in rodent malaria models. Recently, vaccination with chemically attenuated P. berghei sporozoites (CAS) has also been shown to elicit sterile immunity in mice. Here we show that vaccination with CAS of P. yoelii also protects against homologous infection and that a P. berghei CAS vaccine cross protects against heterologous challenge with P. yoelii sporozoites. Vaccination with P. yoelii or P. berghei CAS induced parasite-specific antibodies and IFN-gamma-producing CD8(+) T cells at levels not significantly different from radiation-attenuated sporozoites. Our findings provide an initial characterization of the immune response generated by CAS vaccination and suggest that this attenuation process could be used in the production of an effective cross-protective liver stage vaccine for malaria.

Figure 1. Treatment of P. yoelii 17 XNL sporozoites in vitro does not affect sporozoite membrane integrity

Sporozoites were incubated with vehicle (gray bars) or 2mM centanamycin (black bars) for 30, 60, or 90 min before addition of propidium iodide. Control sporozoites were either tested immediately following dissection (open bar) or heat killed (striped bar) for 15 min at 65°C before counting. For each sample, 200 sporozoites were counted in two separate experiments, and the average percentage of staining with propidium iodide is shown.

Figure 2. Antigen-specific IFN-γ producing CD8⁺ T cell responses in mice following a prime-boost vaccination with P. yoelii 17 XNL or P. berghei ANKA CAS or RAS

The CD8⁺ T-cell response to a H-2K^d-specific CD8⁺ peptide (SYVPSAEQI) from the P. yoelii CSP (A) or P. berghei CSP (SYIPSAEKI ) (B) was measured by an IFN-γ ELISPOT assay of BALB/c mouse splenocytes. Grey bars represent the number of spot-forming cells per 10⁶ splenocytes incubated with antigen-presenting cells loaded with CSP peptide. White bars represent the background control splenocytes incubated with unprimed antigen-presenting cells. Splenocytes were harvested from five (A) or three (B) mice and assayed individually. The experiment was completed in triplicate for each animal and the mean of all animals is shown for both groups. *Indicates significant difference: p=0.0031, ANOVA, n=5 (A) or p=0.0088, ANOVA, n=3 (B). There is no significant difference between the number of spot-forming cells in RAS and CAS in (A) or (B).

Figure 3. Specific antibody titers in mice to P. yoelii and P. berghei sporozoites following a prime-boost vaccination regimen with CAS or RAS

Sera from animals immunized with P. yoelii CAS or RAS was obtained and titers were determined by immunofluorescence using whole sporozoites from P. yoelii (A) or sera from animals immunized with P. berghei was used to determine titers and P. berghei sporozoites (B). Each circle represents data from one mouse. Black circles represent data where sporozoites were florescent, while open circles represent the lowest dilution of serum that did not produce florescent sporozoites. The experiment was completed in triplicate at each concentration for sera from each animal.