Humoral responses against coimmunized protein antigen but not against alphavirus-encoded antigens require alpha/beta interferon signaling

Abstract

Viruses typically elicit potent adaptive immune responses, and live-virus-based vaccines are among the most efficient human vaccines known. The mechanisms by which viruses stimulate adaptive immune responses are not fully understood, but activation of innate immune signaling pathways in the early phase of the infection may be of importance. In addition to stimulating immune responses to viral antigens expressed in infected cells, viruses can also provide adjuvant signals to coimmunized protein antigens. Using recombinant Semliki Forest virus (rSFV)-based vaccines, we show that rSFV potently enhanced antibody responses against coimmunized protein antigens in the absence of other exogenously added adjuvants. Elicitation of antibody responses against both virus-encoded antigens and coimmunized protein antigens was independent of the signaling via Toll-like receptors (TLRs) previously implicated in antiviral responses. In contrast, the adjuvant effect of rSFV on coimmunized protein was completely abolished in mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor (IFN-AR1), demonstrating that IFN-alpha/beta signaling was critical for mediating this effect. Antibody responses directed against virus-encoded antigens were intact in IFN-AR1(-/-) mice, suggesting that other signals are sufficient to drive immune responses against virally encoded antigens. These data provide a basis for the adjuvant effect of rSFV and show that different signals are required to stimulate antibody responses to virally encoded antigens and to antigens administered as purified protein vaccines, together with viral particles.

FIG. 1.

rSFV confers an adjuvant effect on antibody responses against coimmunized protein. (A) Cartoon illustrating the experimental system. The adjuvant effect of rSFV was investigated by mixing recombinant protein antigens with single-round infectious rSFV particles, and antibody responses against the protein antigens were measured. (B) Rabbits (two per group) were immunized with a mixture of purified HIV-1 envelope glycoprotein (Env) and 5 × 10⁷ IU rSFV, or twice with 25 μg Env alone. The rabbits were prebled before immunization (Naïve). Sera from immunized rabbits were drawn 10 days after the final immunization, and antibodies against Env were analyzed by fivefold serial dilutions starting at a 50-fold dilution. Data from individual rabbits are shown. (C) SV129 mice were immunized s.c. a single time with 10⁷ IU rSFV-NP mixed with 15 μg β-Gal protein (n = 7) or with 15 μg β-Gal protein alone (n = 3). Sera from immunized mice were drawn 12 days after the immunization, and β-Gal-specific IgG was analyzed by threefold serial dilutions starting at a 50-fold dilution. Mean titers from each group are shown. The error bars represent standard deviations. (D) IgG2a:IgG1 ratios in sera from the immunized mice were calculated by dividing the IgG2a with the IgG1 OD values for each individual mouse. Mean values are shown. The protein antigen is underlined.

FIG. 2.

Signaling via MyD88 or TLR3 is not required for the adjuvant effect of rSFV. (A) MyD88^−/− (n = 6) and TLR3^−/− (n = 7) mice and their respective wt control strains (n = 7 each) were immunized s.c. a single time with 10⁷ IU rSFV-NP particles mixed with 15 μg β-Gal protein or with 15 μg β-gal protein alone (n = 3). Sera were drawn 12 days after the immunization, and IgG responses against the coimmunized protein (β-Gal; left) and the virus-encoded antigen (NP; right) were measured using threefold serial dilutions of the sera starting at a 50-fold dilution. Mean OD values from each group are shown. The error bars represent standard deviations. (B) IFN-γ ELISPOT analysis was performed on splenocytes from the immunized MyD88^−/− (top) and TLR3^−/− (bottom) mice and their respective wt controls. Mean ELISPOT values from quadruplicate wells, stimulated with NP peptide, are shown. Cytokine-producing cells are shown as spot-forming units (SFU)/10⁶ cells. Mean values for the groups are indicated by the bars. The protein antigen is underlined, and the mouse strain is italic.

FIG. 3.

Viral particles provide an adjuvant effect to protein antigen administered via a different route. To determine if rSFV particles stimulated an adjuvant effect that could act at distant sites, B6 wt mice (n = 5) were immunized s.c. with 10 μg β-Gal protein mixed with 10⁷ IU rSFV-NP particles or with 10 μg β-Gal protein administered s.c. and 10⁷ IU rSFV-NP particles administered i.v. Control mice were immunized s.c. with 10 μg β-Gal protein alone. Sera were drawn 12 days after immunizations, and β-Gal-specific IgG levels were determined using fivefold serial dilutions starting at a 50-fold dilution. The differences in OD values between the animals that received β-Gal alone s.c. and those that in addition received rSFV-NP i.v. were statistically significant (P < 0.05) for four consecutive serial dilutions. The error bars represent standard deviations.

FIG. 4.

IFN-α/β receptor signaling is required for the adjuvant effect of rSFV on coimmunized protein. SV129 wt mice (n = 8) and IFN-AR1^−/− mice (n = 9) were immunized with 10⁶ IU of rSFV-NP mixed with 10 μg β-Gal protein each or with 10 μg β-Gal protein alone (n = 3 for each strain). Sera drawn 12 days after the immunization were analyzed for total (A) β-Gal-specific IgG and (B) NP-specific IgG. All samples were analyzed using threefold serial dilutions starting at a 50-fold dilution. The protein antigen is underlined, and the mouse strain is italic. The error bars represent standard deviations.

FIG. 5.

IFN-α/β levels in sera of rSFV-infected wt and TLR3^−/− mice and adjuvant effects of viruses that induce different levels of IFN-α/β. (A) In vivo IFN-α/β levels were measured to determine if TLR3^−/− mice retained the capacity to produce IFN-α/β in response to rSFV infection. IFN-α/β levels in sera from individual wt and TLR3^−/− mice at 6 h after injection with 10⁷ IU rSFV particles were measured using a bioassay. Data from individual mice are shown, and the bars indicate mean values. (B) The dose dependence of the adjuvant effect of IFN-α/β was determined by coimmunizing β-Gal protein with 10⁷ IU rSFV that had been subjected to different doses of UV treatment. Mildly UV-inactivated rSFV (UV′ rSFV-NP) has previously been shown to induce lower levels IFN-α/β than rSFV-NP, while harshly UV-inactivated rSFV-NP (UV" rSFV-NP) does not induce any detectable IFN-α/β. Sera were drawn 12 days after the immunizations, and β-Gal-specific IgG levels were determined using fivefold serial dilutions starting at a 50-fold dilution. The differences in OD values between UV′ rSFV-NP and UV" rSFV-NP were statistically significant (P < 0.01) for six consecutive serial dilutions. The error bars represent standard deviations.

FIG. 6.

Antibody responses against virus-encoded antigens are not defective in the absence of IFN-α/β signaling. (A) SV129 wt mice (n = 5) and IFN-AR1^−/− mice (n = 5) were immunized once with 10⁶ IU of rSFV-LacZ. After 14 days, total anti-β-Gal specific IgG levels were determined using fivefold serial dilutions starting at a 50-fold dilution. The error bars represent standard deviations. (B) IgG2a:IgG1 ratio between SV129 and IFN-AR1^−/− mice from the sample shown in panel A. (C) SV129 (n = 4) and IFN-AR1^−/− (n = 4) mice were immunized twice with 10⁶ IU of rSFV-LacZ, 4 weeks apart. Total anti-β-Gal-specific IgG levels were measured in sera 14 days after the first immunization (Prime) and 14 days (Boost) and 20 weeks (long term [LT]) after the second immunization. Endpoint titers for each individual mouse are shown. Geometric means are indicated. The protein antigen is underlined, and the mouse strain is italic.

FIG. 7.

Role of CD4⁺ T cells and activation of DCs by rSFV and rSFV-infected cells. (A) CD4⁺-T-cell requirement for antibody responses elicited against the coadministered protein antigen (β-Gal) and against the virus-encoded antigen (NP). Wt and CD4^−/− mice were immunized with a mixture of 10⁶ IU rSFV-NP particles and 10 μg β-Gal protein. Total anti-β-Gal or NP-specific IgG responses were determined using threefold serial dilutions of the respective sera starting at 50-fold dilutions. The error bars represent standard deviations. (B) Apoptosis induction in rSFV-infected and uninfected MEFs was analyzed by flow cytometry using forward scatter (FCS) and side scatter (SCS) analyses (left) and Annexin V-FITC and PI staining (right; ungated cells) at 16 h postinfection. The apoptotic population was Annexin V positive and PI dim. (C) CD40 expression on bone marrow-derived mDCs from SV129 and IFN-AR1^−/− mice. Bone marrow-derived DCs (10⁶ per stimulation) were incubated as indicated with 50 μg, 5 μg, or 0.5 μg of pI-C; with rSFV particles at a multiplicity of infection of 20; or with 5 × 10⁵ rSFV-infected MEFs. CD40 expression was analyzed by flow cytometry after 20 h of incubation. CD40 expression on CD11c and CD11b double-positive cells is shown. Unstimulated cells are shown as filled histograms and stimulated cells as overlaid empty histograms. The protein antigen is underlined, and the mouse strain is italic.