Immunogenicity and protection efficacy of monovalent and polyvalent poxvirus vaccines that include the D8 antigen

Abstract

Recent studies have established the feasibility of subunit-based experimental vaccines to protect animals from lethal poxvirus infection. Individual outer membrane proteins from intracellular and extracellular virions of vaccinia virus, when delivered in the form of either DNA vaccines or recombinant protein vaccines produced from baculovirus-infected insect cells, were able to protect mice from the vaccinia virus challenge and rhesus macaques from the monkeypox virus challenge. The polyvalent formulations with various combinations of the four poxvirus antigens (A27, L1, B5 and A33) achieved better protection than the monovalent formulation using only one of these antigens. However, it is not clear whether any of the remaining outer membrane poxvirus proteins can further improve the efficacy of the current polyvalent formulations. In this study, we conducted detailed analysis on the immunogenicity of D8, a previously reported protective antigen from intracellular mature virions. Our results indicated that D8 induced strong protective antibody responses and was effective in improving the efficacy of previously reported polyvalent poxvirus vaccine formulations. Therefore, D8 is an excellent candidate antigen to be included in the final polyvalent subunit-based poxvirus vaccines.

Fig. 1

(A) Schematic designs of D8L gene inserts. Transmembrane (TM) domain and the number of amino acid residues are marked. (B) Western blot analysis of transiently expressed D8 proteins from the supernatant (sup) and lysate (lys) of 293T cells transfected with two D8L DNA vaccine plasmids (tPA-D8L and wtD8L). The empty DNA vector (pSW3891) was included as the negative control. (C) Deglycosylation of D8 proteins with PNGase. D8 proteins, either produced in lysate (lys) or supernatant (sup) of 293T cells transfected with the tPA-D8L DNA plasmid, or from lysate of Vero cells infected with the vaccinia virus WR strain (VACV), were treated (+) or not treated (−) with PNGaseF before subjecting to SDS-PAGE and Western blot analysis. Vero cells without vaccinia infection (Vero) and 293T cells transfected with the empty DNA vector (vector) were also included as the negative controls. (D) Detection of D8 (lane 2) expressed in the supernatant of tPA-D8 DNA plasmid transfected 293T cells with mouse sera immunized with vaccinia virus WR strain. Supernatant 293T cells transfected with the empty DNA vector plasmid (lane 1) were included as the negative control. A D8-specific rabbit serum R274 (1:300 dilution) was used as the detecting antibody for Western blot analyses (B–C).

Fig. 2

Titers of antibody responses in immune sera of mice received either the tPA-D8L or wtD8L DNA vaccines. IgG antibody responses as measured by ELISA against either recombinant D8 antigen produced from 293T cells (A) or lysate of Vero cells infected with vaccinia virus WR strain (VACV) (B). Control groups of mice received either the empty DNA vector (Vector) or the vaccinia virus WR strain (WR). Each group included 5 mice and data shown are geometric means of the end titration titers for each group. (C) Titers of neutralizing antibodies for the same groups of mouse sera in an IMV-based, plaque reduction assay. Data shown are geometric means of neutralizing antibody titers which are the highest sera dilution that could inhibit 50% of virus infection in this assay.

Fig. 3

Protection of BALB/c mice against challenge of vaccinia virus WR strain by the intraperitoneal route. Mice were challenged with 5 × 10⁷ pfu of vaccinia virus WR strain 2 weeks after the final DNA immunization. The positive control group (WR) received immunization with 10⁵ pfu of vaccinia virus WR strain 2 weeks prior to challenge. The negative control group (vector) received the empty DNA vector. (A) Protection of mice immunized with individual D8L DNA vaccines. Body weight loss as the percentage of pre-challenge weight was measured. Each curve showed the group average weight loss (10 mice per group). (B) Protection of mice immunized with a bi-valent pox DNA vaccine formulation (A27L and B5R), with or without the third component (tPA-D8L DNA vaccine). Each curve showed the group average weight loss (10 mice per group). (C) Protection of mice immunized with a 4-valent formulation (A27L, B5R, L1R and A33R) with or without the addition of the fifth component (tPA-D8L DNA vaccine). Each curve showed the group average weight loss (10 mice per group).

Fig. 4

Protection of BALB/c mice against lethal challenge of vaccinia virus WR strain (5 × 10⁶ pfu) by the intranasal route at 2-week after the last DNA immunization. Mice were immunized with a 4-valent pox DNA vaccine formulation (A27L, B5R, L1R and A33R) with or without the addition of the fifth component (tPA-D8L DNA vaccine). The negative control group (vector) received the empty DNA vector. (A) Body weight loss as the percentage of pre-challenge weight was measured. Each curve showed the group average weight loss of surviving mice (15 per group initially). (B) Survival curves of the same mice groups showing the percentage of mice alive at each day post-challenge.

Fig. 5

Pre-challenge IgG antibody responses induced by individual D8L or polyvalent pox DNA vaccine formulations against either the individual pox antigens including A27 (A), B5 (B), D8 (C), L1 (D), A33 (E) or vero cell lysate infected with vaccinia virus (VACV) (F). Data are shown as the geometric means of end titration titers as determined by ELISA for each group (5 mice per group). Titers of neutralizing antibody responses against IMV were also shown (G) as the geometric means of highest serum dilutions that inhibit 50% of virus infection in the plaque reduction assay.