Designing multivalent immunogens for alphavirus vaccine optimization

Abstract

There is a pressing need for vaccines against mosquito-borne alphaviruses such as Venezualen and eastern equine encephalitis viruses (VEEV, EEEV). We demonstrate an approach to vaccine development based on physicochemical properties (PCP) of amino acids to design a PCP-consensus sequence of the epitope-rich B domain of the VEEV major antigenic E2 protein. The consensus "spike" domain was incorporated into a live-attenuated VEEV vaccine candidate (ZPC/IRESv1). Mice inoculated with either ZPC/IRESv1 or the same virus containing the consensus E2 protein fragment (VEEVconE2) were protected against lethal challenge with VEEV strains ZPC-738 and 3908, and Mucambo virus (MUCV, related to VEEV), and had comparable neutralizing antibody titers against each virus. Both vaccines induced partial protection against Madariaga virus (MADV), a close relative of EEEV, lowering mortality from 60% to 20%. Thus PCP-consensus sequences can be integrated into a replicating virus that could, with further optimization, provide a broad-spectrum vaccine against encephalitic alphaviruses.

Keywords: Alphaviruses; Computational and structural vaccine design; Eastern equine encephalitis virus; Murine model; Physicochemical property (PCP) consensus; Venezuelan equine encephalitis virus.

Figure 1:. Model of the altered region of the VEEV E2 protein.

Residues that have been altered from the wild-type ZPC-738 E2 protein are highlighted in red. Residue alterations were based on the PCP-consensus of 8 different VEEV strains (see table 1).

Figure 2:. Sera from vaccinated mice neutralized MUCV.

PRNT₈₀ titers at 3- and 6-weeks post-vaccination (A, B). Sera were first diluted 10-fold and then serially 2-fold; therefore the limit of detection (LOD) was set at 10. (** - p < 0.01, Kruskal-Wallis Test)

Figure 3:. Survival curve for mice inoculated with ZPC/IRESv1 or VEEVconE2.

Female CD-1 mice were challenged subcutaneously, 6 weeks after initial vaccination with 10³ pfu of either ZPC-738 (A), MUCV (B), or intranasally with 6×10⁴ pfu 3908 (C). For both the ZPC-738 and MUCV infections, 3 mice received PBS, 4 were vaccinated with ZPC/IRESv1, and 5 received the VEEVconE2 vaccine. In the 3908 study, two groups of 4 female mice received either ZPC/IRESv1 or VEEVconE2 while one group of 3 male mice received the mock vaccine. (*** - p <0.001, ** - p < 0.01, * - p < 0.05, Log-rank test)

Figure 4:. Percent weight change over time post-challenge.

Mice were weighed daily after challenge with ZPC-738 (A) or MUCV (B). Weight data were not collected after challenge with 3908. Data points represent average weights of all surviving mice in each group on the day post-challenge. Error bars indicate standard deviation. Asterisks denote where the consensus vaccine differed significantly from ZPC/IRESV1. (* - p < 0.05, ** - p < 0.01, **** - p < 0.001, Two-way ANOVA)

Figure 5:. Vaccinated mice had no detectable viremia two days post-challenge.

Serum was collected two days after challenge and tested for presence of ZPC-738 (A) and MUCV (B). Limit of detection, 100 pfu, (LOD) was determined by the lowest number of plaques detected by the assay. (* - p < 0.05, **** - p < 0.0001, One-way ANOVA)

Figure 6:. ZPC/IRESv1 and VEEVconE2 immunization provided partial protection against MADV.

Three groups of 5 female CD-1 mice received either the ZPC/IRESv1 vaccine, the VEEVconE2 vaccine, or PBS. These mice were challenged subcutaneously with 10³ pfu of MADV at 6 weeks post-vaccination. Survival (A), weight (B), viremia (C), and neutralizing antibody titers at 3- and 6-weeks post-vaccination (D,E) were measured using the same methods as those for the mice challenged with wild-type strains of viruses from the VEE complex. Statistical analysis was performed with the same tests used in figures 2–5.