Immunogenicity of full-length P. vivax rPvs48/45 protein formulations in BALB/c mice

Abstract

Background: Pvs48/45 is a Plasmodium vivax gametocyte surface protein involved in the parasite fertilization process. Previous studies showed that Pvs48/45 proteins expressed in Escherichia coli (E. coli) and Chinese hamster ovary (CHO) cells were highly immunoreactive with sera from malaria-endemic areas and highly immunogenic in animal models. Here the immunogenicity in mice of three different vaccine formulations was compared.

Methods: Recombinant (r) Pvs48/45 proteins were expressed in E. coli and CHO, purified, formulated in Alhydrogel, GLA-SE and Montanide ISA-51 adjuvants and used to immunize BALB/c mice. Animals were immunized on days 0, 20 and 40, and serum samples were collected for serological analyses of specific antibody responses using ELISA and immunofluorescence (IFAT). Additionally, ex-vivo transmission-reducing activity (TRA) of sera on P. vivax gametocyte-infected human blood fed to Anopheles albimanus in direct membrane feeding assays (DMFA) was evaluated.

Results: Most immunized animals seroconverted after the first immunization, and some developed antibody peaks of 10⁶ with all adjuvants. However, the three adjuvant formulations induced different antibody responses and TRA efficacy. While GLA-SE formulations of both proteins induced similar antibody profiles, Montanide ISA-51 formulations resulted in higher and longer-lasting antibody titers with CHO-rPvs48/45 than with the E. coli formulation. Although the CHO protein formulated in Alhydrogel generated a high initial antibody peak, antibody responses to both proteins rapidly waned. Likewise, anti-Pvs48/45 antibodies displayed differential recognition of the parasite proteins in IFAT and ex vivo blockade of parasite transmission to mosquitoes. The CHO-rPvs48/45 formulated in Montanide ISA-51 induced the most effective ex vivo parasite blockage.

Conclusions: Three out of six vaccine formulations elicited antibodies with ex vivo TRA. The CHO-rPvs48/45 Montanide ISA-51 formulation induced the most stable antibody response, recognizing the native protein and the most robust ex vivo TRA. These results encourage further testing of the vaccine potential of this protein.

Keywords: Gametocytes; Immunogenicity; Malaria; Plasmodium vivax; Pvs48/45; Transmission Reducing Activity; Transmission blocking vaccine; Vaccines.

Figure 1.. Immunization and bleeding schedule.

Mice were immunized three times at 20 days intervals with 20 μg of rPvs48/45 formulated separately in Alhydrogel, GLA-SE or Montanide ISA-51 adjuvants. Blood samples were obtained at the indicated time points until day 180 for serology and functional assays. For days 0 and 40, the blood samples were collected before immunizations. EG: Experimental group; CG: Control group; Prot: recombinant protein

Figure 2.. Antibody Titers.

Kinetics of antibody titers in BALB/c mice upon immunization with the full-length P. vivax rPvs48/45 proteins produced E. coli (open symbols) or CHO (closed symbols) formulated with (A) Alhydrogel, (B) GLA-SE, or (C) Montanide ISA-51. Each plot has its respective control group per adjuvant (▲). Antibody titers correspond to the last dilution of the test sera in which OD405 values were above that of the cut-off. The cut-off value was defined as the mean plus 3SD of OD405 value of pooled naïve mouse sera tested at 1:300 dilution. The median +/− IQR of each group is shown. (D and E) The same data presented in 2A-2C are plotted to compare the kinetics of antibodies among adjuvant groups against either E. coli (D) or CHO-rPvs48/45 (E) proteins.

Figure 3.. IC50.

IC50 (mol/L) of sera samples from mice immunized with E. coli or CHO rPvs48/45 in each indicated adjuvant, collected at day 60 (A, B and C). Comparisons between CHO-rPvs48/45 and E. coli rPvs48/45 protein within each adjuvant are shown. Mann-Whitney analysis shows significant differences between E. coli and CHO proteins formulated in Alhydrogel (p=0.0079) and GLA-SE (p=0.0286). (D and E) comparison among adjuvants to each rPvs48/45 protein. Dunn’s multiple comparison tests showed significant differences for E. coli protein (D) (*p<0.05), but not for CHO protein (E). Bar corresponds to the median of each subgroup.

Figure 4.. IFAT.

Recognition of parasite protein by pools of sera of immunized mice with E. coli and CHO-rPvs48/45 formulated in Montanide ISA-51 at day 60 by IFAT. Mice serum was incubated with acetone-fixed smears containing P. vivax gametocyte enriched erythrocytic forms. Parasites seen under light (left) or epifluorescence (right) microscopy with a 40X objective lens are shown. Picture scale 238μm.