Plasmodium Preerythrocytic Vaccine Antigens Enhance Sterile Protection in Mice Induced by Circumsporozoite Protein

Abstract

Preerythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading preerythrocytic vaccine, RTS,S/AS01E (Mosquirix), entered implementation programs in 2019 and targets the major sporozoite surface antigen, circumsporozoite protein (CSP). However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination. Previously, we identified highly expressed liver-stage proteins that could potentially be used in combination with CSP; they are referred to as preerythrocytic vaccine antigens (PEVAs). Here, we developed heterologous prime-boost CSP vaccination models to confer partial sterilizing immunity against Plasmodium yoelii (protein prime-adenovirus 5 [Ad5] boost) and Plasmodium berghei (DNA prime-Ad5 boost) in mice. When combined as individual antigens with P. yoelii CSP (PyCSP), three of eight P. yoelii PEVAs significantly enhanced sterile protection against sporozoite challenge, compared to PyCSP alone. Similar results were obtained when three P. berghei PEVAs and P. berghei CSP were combined in a single vaccine regimen. In general, PyCSP antibody responses were similar after CSP alone versus CSP plus PEVA vaccinations. Both P. yoelii and P. berghei CSP plus PEVA combination vaccines induced robust CD8⁺ T cell responses, including signature gamma interferon (IFN-γ) increases. In the P. berghei model system, IFN-γ responses were significantly higher in hepatic versus splenic CD8⁺ T cells. The addition of novel antigens may enhance the degree and duration of sterile protective immunity conferred by a human vaccine such as RTS,S.

Keywords: circumsporozoite protein; malaria; preerythrocytic; sterile protection; vaccine.

FIG 1

(A) Purification of PEVAs. Coomassie-stained gel showing purified proteins expressed in either insect cells or E. coli with a His tag at the C terminus and purified using Ni-NTA resin. (B) Protein prime-Ad5 boost PyPEVA/PyCSP combination study design. Mice were immunized as described in Materials and Methods. On day 56, spleens were harvested posteuthanasia from 5 mice/group. The remaining 14 mice/group were challenged with 300 P. yoelii SPZ on day 63. Mice were monitored for parasitemia via blood smears for 14 days postchallenge. (C) Protection conferred by PEVAs combined with CSP. Following challenge with P. yoelii SPZ, mice were monitored by blood smears from day 4 to day 7 and on day 14 for the presence of parasites. Mice that stayed parasite free were considered protected. *, P < 0.004; ***, P < 0.0001. P values were based on the time to parasitemia, and P values of <0.004 were considered significant after Bonferroni correction for multiple comparisons. (D) Protection after rechallenge of protected animals. Protected animals (challenged animals that did not develop blood-stage parasitemia) were rechallenged, 16 weeks after the last vaccine dose, with 300 P. yoelii 17XNL SPZ to assess the duration of protection. Mice were monitored by blood smears from day 4 to day 7 and on day 14 postrechallenge for the presence of parasites. *, P < 0.006. P values were based on the time to parasitemia, and P values of <0.006 were considered significant after Bonferroni correction for multiple comparisons.

FIG 2

(A) DNA prime-Ad5 boost PbPEVA/PbCSP combination study design. (B) P. berghei LS antigens combined with CSP reduce parasite burden in B6 mice. B6 mice (5 mice per group) were immunized at 3-week intervals twice with DNA by GG (5 μg) and boosted with 4 × 10¹⁰ VP of Ad5 vector by IM injection. Control groups received DNA EV and Ad5 EV according to the same immunization regimen. Fourteen days after the last boost, mice were challenged with 10,000 P. berghei SPZ by IV injection. Animals were euthanized and livers were harvested 40 h after the challenge for LPB determination by real-time qPCR. Data represent the ratio of expression of P. berghei 18SrRNA to that of mouse β-actin for individual mice. **, P < 0.01. (C) Partial sterile protection in CD1 mice immunized with a combination of three PbPEVAs and CSP. CD1 outbred mice (14 animals per group) were immunized as for panel B. Fourteen days after the last boost, mice were challenged with 300 P. berghei SPZ by IV injection. Mice were monitored for parasitemia via blood smears for 14 days postchallenge. Mice that stayed parasite free were considered protected. Data represent percentages of surviving mice.

FIG 3

Antibody responses to PyPEVAs combined with PyCSP measured against CSP antigen (A) or individual PyPEVAs (B). Box and whisker plots show antibodies to CSP antigens or PEVAs measured by direct ELISA using sera from vaccinated mice collected 14 days after the last vaccine dose.

FIG 4

CD8⁺ T cell recall responses to PEVAs after immunization with PyPEVA/PyCSP combinations. Fourteen days after the final immunization, spleens were harvested posteuthanasia from 5 mice per group. Single-cell suspensions of splenocytes were used for in vitro stimulation with pools of antigen-specific peptides. Samples from PyPEVA- plus PyCSP-immunized animals were stimulated with peptides from the corresponding PyPEVA and PyCSP together; splenocytes from P. yoelii GFP- plus PyCSP-treated control animals and adjuvant-only-treated animals were stimulated with peptides only from CSP. To determine levels of CD8⁺ T cell activation, cells were stained for markers, including CD3ε, CD4, CD8α, CD11a, IL-2, and IFN-γ. Levels of expression of various T cell activation markers against each PyPEVA are shown as medians with interquartile ranges and were compared between each PyPEVA plus CSP group and the GFP plus CSP group using a nonparametric Mann-Whitney test. *, P < 0.05; **, P < 0.01.

FIG 5

Higher IFN-γ responses in IHMC than in spleens of mice immunized with three PbPEVAs or three PbPEVAs plus CSP. B6 mice (5 mice per group) were immunized using the same immunization schedule as for Fig. 2A. Fourteen days after the last boost immunization, IHMC and spleen cells were analyzed for IFN-γ production with ELISPOT and ICS assays (see Materials and Methods). Data represent IFN-γ responses in livers (A and B) and spleens (C and D) in ELISPOT (A and C) and ICS (B and D) assays.