A DNA vaccine-encoded nucleoprotein of influenza virus fails to induce cellular immune responses in a diabetic mouse model

Abstract

Influenza virus infections cause yearly epidemics and are a major cause of lower respiratory tract illnesses in humans worldwide. Influenza virus has long been recognized to be associated with higher morbidity and mortality in diabetic patients. Vaccination is an effective tool to prevent influenza virus infection in this group of patients. Vaccines employing recombinant-DNA technologies are an alternative to inactivated virus and live attenuated virus vaccines. Internal highly conserved viral nucleoprotein (NP) can be delivered as a DNA vaccine to provide heterosubtypic immunity, offering resistance against various influenza virus strains. In this study, we investigated the efficacy of an NP DNA vaccine for induction of cell-mediated immune responses and protection against influenza virus infection in a mouse model of diabetes. Healthy and diabetic BALB/c mice were immunized on days 0, 14, and 28 by injection of NP DNA vaccine. Two weeks after the last immunization, the cellular immune response was evaluated by gamma interferon (IFN-gamma), lymphocyte proliferation, and cytotoxicity assays. The mice were challenged with influenza virus, and the viral titers in the lungs were measured on day 4. Diabetic mice showed significantly smaller amounts of IFN-gamma production, lymphocyte proliferation, and cytotoxicity responses than nondiabetic mice. Furthermore, higher titers of the influenza virus were detected after challenge in the lungs of the diabetic mice. The present data suggest that the NP DNA vaccine with the protocol of immunization described here is not able to induce efficient cellular immune responses against influenza virus infection in diabetic mice.

FIG. 1.

pcDNA3-NP expression in BHK-21 cells. NP expression in BHK-21 cells was detected by immunofluorescence. BHK-21 cells were transfected with 1 μg of pcDNA3-NP or pcDNA3 vector using Lipofectamine 2000. At 2 days posttransfection, the cells were fixed with 4% formaldehyde-PBS. Next, the cells were treated with Triton X-100 and then anti-NP monoclonal Ab, followed by incubation with anti-mouse IgG-rhodamine conjugate. (A) BHK-21 cells were transfected with pcDNA3-NP plasmid. (B) BHK-21 cells were transfected with pcDNA3 plasmid as a control.

FIG. 2.

Cytotoxicity responses induced by pNP immunization in diabetic mice. Healthy and diabetic mice were immunized three times intradermally with pcDNA3-NP (pNP and pNP-Dia groups, respectively) or PBS (PBS and PBS-Dia groups, respectively) on days 0, 14, and 28. Two weeks after final immunization, splenocytes from immunized and mock-immunized mice were prepared as described in Materials and Methods. LDH release assays were performed in triplicate with splenocytes as effector cells and A/New Caledonia\20\99 H1N1 p815 cells as target cells. All experiments were performed more than three times, and each group consisted of five mice. The cytotoxicity activity of the pNP group was significantly higher than that of the pNP-Dia group (***, P < 0.001). There was no significant difference between the cytotoxicity responses of the pNP-Dia and PBS-Dia groups. E/T, effector-to-target-cell ratio.

FIG. 3.

Lymphocyte proliferation responses induced by pNP immunization in diabetic mice. Healthy and diabetic mice were immunized three times intradermally with pcDNA3-NP (pNP and pNP-Dia groups, respectively) or PBS (PBS and PBS-Dia groups, respectively) on days 0, 14, and 28. Two weeks after final immunization, spleens of individual mice (five per group) were removed, and lymphocyte proliferation was evaluated using the MTT method. Values are the means ± standard errors of the means for five experiments. The lymphocyte proliferation of the pNP group was significantly higher than that of the pNP-Dia group (***, P < 0.001). There was no significant difference between the lymphocyte proliferation of the pNP-Dia and PBS-Dia groups.

FIG. 4.

IFN-γ production induced by pNP immunization in diabetic mice. Healthy and diabetic mice were immunized three times intradermally with pcDNA3-NP (pNP and pNP-Dia groups, respectively) or PBS (PBS and PBS-Dia groups, respectively) on days 0, 14, and 28. Two weeks after final immunization, spleens of individual mice (five per group) were removed, and IFN-γ production was measured with an ELISA kit. IFN-γ production of the pNP group was significantly higher than that of the pNP-Dia group (***, P < 0.001). There was no significant difference between the IFN-γ production of the pNP-Dia and PBS-Dia groups.

FIG. 5.

Replication of influenza A viruses in the lungs of diabetic and healthy immunized mice. Healthy and diabetic mice were immunized three times intradermally with pcDNA3-NP (pNP and pNP-Dia groups, respectively) or PBS (PBS and PBS-Dia groups, respectively) on days 0, 14, and 28. Two weeks after final immunization, virus titers in the lungs of diabetic and healthy mice (five per group) were measured 4 days after intranasal inoculation with influenza virus. Values are the means ± standard errors of the means for three experiments. The horizontal line shows the minimum inoculation of decimal dilutions (10⁻²) of lung in MDCK cells. ***, lung virus titers for pNP group were significantly lower than those for the pNP-Dia group (P < 0.001), PBS group (P = 0.006), and PBS-Dia group (P < 0.001). **, lung virus titers of PBS group were significantly lower than those of the PBS-Dia group (P = 0.009). There was no significant difference between the lung virus titers of the pNP-Dia and PBS-Dia groups.