Coadministration of DNA encoding interleukin-6 and hemagglutinin confers protection from influenza virus challenge in mice

Abstract

This study was conducted to investigate whether Accell gene gun coadministration of DNA encoding human interleukin-6 (IL-6) would enhance protective immune responses in mice to an equine influenza A virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone exhibited accelerated clearance of homologous challenge virus but were not protected from infection. In contrast, mice that received both HA and IL-6 DNA had no detectable virus in their lungs after challenge. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.

FIG. 1

The mean titers, and standard errors of the means, of virus in the lungs of mice following vaccination with control pWRG7077 DNA (▪), pWRGHA plus pWRG7077 DNA (▥), pWRGHA plus pWRGIL-6 DNA (░⃞), or pWRGIL-6 plus pWRG7077 DNA (▨). Mice were challenged with 6.65 × 10⁴ EID₅₀s of Eq/KY virus by intranasal instillation under light methoxyflurane (Pittman Moore) sedation, 2 weeks after the second vaccinations. (A) Results from experiment 1. (B) Results from experiment 2. Virus was detected by inoculation (in triplicate) of serial dilutions of each of the mouse lungs into the allantoic cavities of 10-day-old embryonated chicken eggs (23). Mice were euthanatized for collection of lung samples either 3 or 5 days after challenge (p.i.). *, the reduction in titer achieved by administration of HA DNA compared to controls is statistically significant (P = 0.001) (one-way ANOVA and pairwise contrasts); **, the reduction in titer achieved by coadministration of HA plus IL-6 DNA compared to administration of HA DNA alone is highly statistically significant (P < 0.0001) (one-way ANOVA and pairwise contrasts).

FIG. 2

(A and B) Virus-specific serum IgG (A) and IgA (B) titers, as measured by ELISA, in mice vaccinated with control pWRG7077 DNA, pWRGHA plus pWRG7077 DNA (▥), or pWRGHA plus pWRGIL-6 DNA (▪). The ELISAs were performed as previously described (23). ELISA titers are defined as the reciprocal of the highest dilution of serum for which the optical density was a least twice as great as the optical density of the negative control sample on that plate. (C) VN-Ab titers in mice vaccinated with control pWRG7077 DNA, pWRGHA plus pWRG7077 DNA (▥), or pWRGHA plus pWRGIL-6 DNA (▪). VN Abs were measured by inoculation (in triplicate) of MDCK cells with serial dilutions of serum incubated with 50% tissue culture infective doses (50 doses) of Eq/KY virus (23). The VN-Ab titers are defined as the reciprocal of the highest dilution of serum that completely inhibited the Eq/KY virus-induced cytopathic effect. In each panel, the times of sampling are shown. pre, immediately prior to the first vaccination; 3wk, immediately prior to the second vaccination; 5wk, immediately prior to challenge; and 3dp.i. or 5dp.i., 3 or 5 days after challenge infection.

FIG. 3

Virus-specific IgG titers, as measured by ELISA, in nasal wash specimens from mice vaccinated with control pWRG7077 DNA, pWRGHA plus pWRG7077 DNA (▥), pWRGHA plus pWRGIL-6 DNA (▪), or pWRGIL-6 plus pWRG7077 DNA. Titers are defined as the reciprocal of the highest dilution of sample for which the optical density was a least twice the optical density of the negative control samples on that plate. The times of sampling are shown. 5wk, immediately prior to challenge; 3dp.i., 3 days after challenge infection.