Different immunogens and prime-boost vaccination strategies affect the efficacy of recombinant candidate vaccines against pathogenic orthopoxviruses

Abstract

Although not as lethal as variola virus (VARV), the cause of smallpox, monkeypox virus (MPXV) represents a threat to public health, with important infection rates and mortality in several African countries and signs of spreading worldwide. MPXV may establish new reservoirs in non-endemic countries and can be considered a possible biological weapon. Human-to-human MPXV transmission is increasing with a growing susceptibility, coincident with the declining herd immunity against smallpox. The emerging threat of MPXV highlights the urgent need for protection from new zoonotic infections, as mankind is completely unprepared for encounters with new viruses. Preventive vaccination remains the most effective control against orthopoxviruses (OPXVs) such as MPXV and prime-boost vaccination strategies can significantly influence vaccine efficacy and enhance immune responses. Our study aimed at characterizing potential vaccine candidates against OPXV infections in a murine model using DNA, viral and protein recombinant vaccines using different prime-boost regimens. The experiments employed Vaccinia virus (VACV) A33, B5, L1, and A27 envelope proteins as immunogens for both priming and boosting. Priming was carried out using a mixture of four plasmids (4pVAXmix), and boosts employed fowlpox (FWPV) recombinants (4FPmix) and/or the purified recombinant proteins (4protmix), all of them expressing the same antigens. One or two doses of the same immunogens were tested and identical protocols were also compared for intranasal (i.n.) or intramuscular (i.m.) viral administration, before challenge with the highly pathogenic VACV VV_IHD-J strain. Our results show that a single dose of any combined immunogen elicited a very low antibody response. Protein mixtures administered twice boosted the humoral response of DNA immunizations by electroporation (e. p.), but did not protect from viral challenge. The antibody neutralizing titer was inversely correlated with animals' weight loss, which was initially similar in all of the groups after the challenge, but was then reversed in mice that had been primed twice with the DNA recombinants and boosted twice with the FWPV recombinants.

Keywords: Prime-boost immunization regimens; Enhancement of the immune response; Orthopoxvirus vaccines; Recombinant vaccines against MPXV.

Fig. 1

Immunization protocols. Eight different regimens (G1-G8) were followed using 7 mice per group. All of the immunogens expressed the VACV L1R, A27L, A33R, and B5R gene products and were administered twice (G1-G5) or once (G6-G8). DNA recombinants (4pVAXmix, in blue) were always used for priming and injected s.c. on the back (12 µg/mouse, 3 µg/recombinant) or i.m./e.p on the leg (48 µg/mouse, 12 µg /recombinant). Viral recombinants (4FPmix, in red) were used as boosts and administered i.n. (4 × 10⁶/mouse, 1 × 10⁶ PFU/recombinant) except for G4 where i.m. administration was performed. Protein recombinants (4protmix, in green) were used for the boost and administered s.c. (40 µg/mouse, 10 µg of each recombinant protein). pVAXgp and FPgp recombinants, containing HIV-1 gag/pro genes, were used as irrelevant immunogens. The VV_IHD−J challenge virus was administered i.n. at 1 × 10⁷ PFU/mouse (i.e. 5-fold the LD₅₀). Blood samples were obtained from the mice before the first immunization (T0), before the boosts (T1, T2) and just before the VV_IHD−J challenge (T3)

Fig. 2

Analysis of specific humoral responses by ELISA. The sera of the mice from the eight groups were examined at the different times post immunization, using the individual proteins as plate-bound antigens. (A) when using the A33 protein, the antibody response, with a 1:100 serum dilution, was significantly greater for G3 and G5 (G3 and G5 vs. G1; p < 0.001); (B) for the A27 protein, the response, with 1:100 and 1:2000 serum dilutions, was significantly higher for G3, G4, G5 (G3, G4, G5 vs. G1; p < 0.001); (C) for the B5 protein, the response, with 1:100 and 1:500 serum dilutions, was significantly higher only for G5 (G5 vs. G1; p < 0.001); (D) for the L1 protein, the response, with 1:2000 and 1:10000 serum dilutions, was significantly higher for all of the groups (p < 0.001) except for G8. Statistical differences are shown (one-way ANOVA parametric tests, Bonferroni analysis of variance): ***, p < 0.001

Fig. 3

Inhibition of viral infectivity by the different immunization protocols and effects on weight loss induced by the different immunization regimens after VV_IHD−Jchallenge. Viral plaque reduction neutralization tests (PRNT) were performed on Vero cells using sera before the first immunization (T0) and just before the challenge (T2 or T3). Plaque reduction was quantified, and expressed as percentages of inhibition of infectivity. (A) Using pooled sera from each group of mice, G2, G3, G4, G5, G6 showed significant inhibition of infectivity when compared with G1 (G2, G3, G4, G5 vs. G1, p < 0.001). Neutralization activity was significantly higher in G3 and G4 compared to G2 and G6 (G3 and G4 vs. G2 and G6; p < 0.001, internal comparisons). (B) All of the mice challenged with VV_IHD−J were monitored daily for the post-challenge (p.c.) percentage weight loss. Data are means of percentage weight loss of each group. Mice rapidly lost weight by day 3 p.c., as 15–22% with no relevant differences among most of the groups, but G3 and G4 mice started to regain weight after day 6 p.c. All of the G3 and G4 mice were protected and survived. G1 mice and mice of the remaining groups had to be euthanized from day 4 to 6 p.c. when their weight diminished by 25–30%, which represented the humane endpoint. Statistical differences are shown (one-way ANOVA parametric tests, Bonferroni analysis of variance): *, p < 0.05; **, p < 0.01; ***, p < 0.001; internal comparisons are indicated in red