A recombinant flagellin-poxvirus fusion protein vaccine elicits complement-dependent protection against respiratory challenge with vaccinia virus in mice

Abstract

Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of protein antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore we evaluated the humoral response to these antigens in mice when flagellin was used as an adjuvant. Flagellin-L1R and flagellin-B5R fusion proteins were more potent than flagellin, L1R, and B5R as separate proteins. At least three immunizations with flagellin-L1R and flagellin-B5R fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Immune mice exhibited only limited signs of disease following challenge. Additionally, virus neutralization titers correlated with protection. Depletion of complement using cobra venom factor resulted in a marked decrease in the survival of immunized mice after challenge with vaccinia virus. Our results are consistent with the conclusion that flagellin-L1R and flagellin-B5R fusion proteins are effective in eliciting protective immunity against vaccinia virus that is dependent, in large part, on complement.

FIG. 1.

Recombinant proteins used in this study. Chimeric proteins were produced containing the ectodomains of L1R and B5R linked to flagellin. Diagrams illustrate antigen placement in the fusion proteins. L1R is present at the N-terminus of full length flagellin. B5R was substituted for the hypervariable domain of flagellin. TLR5-stimulating activity is calculated as units per milligram. A unit is the inverse of the concentration yielding a half maximal response. This value is then standardized to units per milligram.

FIG. 2.

Vaccine efficacy can be enhanced using flagellin-poxvirus antigen fusion proteins. (A and B) Groups of 7 BALB/c mice were immunized two times IM with 5 μg of the flagellin-antigen fusion proteins LF and FB or equimolar doses of the separate proteins. Asterisks indicate significant differences in titer (p < 0.05).

FIG. 3.

IgG, IgG₁, and IgG_2a responses following immunization with LF and FB. Mice were immunized IM with 5 μg of LF and 5 μg of FB. (A and B) Anti-B5R and L1R total IgG. (C and D) IgG₁. (E and F) IgG_2a titers after each immunization were determined by ELISA. Each symbol represents the results from an individual mouse, and the dashed lines at 10³ are the limit of detection for the assay. Numbers below the dashed lines indicate the number of mice whose titers were below the limit of detection (non-responders). In A–D all groups are significantly different unless noted by n.s. In E and F significant differences are indicated by asterisks.

FIG. 4.

Three or more immunizations with flagellin/poxvirus fusion proteins confers protection against vaccinia virus challenge. Groups of 10 BALB/c mice who received two to four IM immunizations of LF and FB at the indicated doses were challenged with 20 MTD₅₀ vaccinia virus. (A) Survival, (B) weight loss, and (C) disease index were measured following infection.

FIG. 4.

Three or more immunizations with flagellin/poxvirus fusion proteins confers protection against vaccinia virus challenge. Groups of 10 BALB/c mice who received two to four IM immunizations of LF and FB at the indicated doses were challenged with 20 MTD₅₀ vaccinia virus. (A) Survival, (B) weight loss, and (C) disease index were measured following infection.

FIG. 5.

In-vitro neutralization of vaccinia virus by plasma from mice immunized with LF + BF. Pooled plasma from mice receiving two to four immunizations with 5 μg of LF and FB was incubated at three dilutions with eGFP-expressing vaccinia virus in vitro at 37°C for 1 h. The plasma-treated virus was added to HeLa cells and incubated for 6 h at 37°C. Infected cells were detected by flow cytometry and the percentage of neutralization was calculated by standardizing to mock-infected cells or cells infected with virus that was not treated with plasma. Neutralizing titer is defined as the dilution at which 50% of virus is neutralized, and is calculated by non-linear regression. The dashed line shows 50% neutralization.

FIG. 6.

Complement depletion reduces the protective effect of flagellin/poxvirus fusion protein immunization. BALB/c mice were immunized three times IM with LF and FB. Six mice were treated with cobra venom factor (CVF) 1 d before challenge and 3 d after challenge with 20 MTD₅₀ vaccinia virus. (A) Survival, (B) weight loss, and (C) disease index were measured following infection.

FIG. 6.

Complement depletion reduces the protective effect of flagellin/poxvirus fusion protein immunization. BALB/c mice were immunized three times IM with LF and FB. Six mice were treated with cobra venom factor (CVF) 1 d before challenge and 3 d after challenge with 20 MTD₅₀ vaccinia virus. (A) Survival, (B) weight loss, and (C) disease index were measured following infection.

FIG. 7.

Time course for the restoration of complement activity in mice treated with CVF. Mice were treated with PBS (A) or CVF (B) as described in Fig. 6. At the indicated time points following treatment, the mice were bled and samples were assayed for the ability to promote red blood cell lysis.