Improved vaccine protection against retrovirus infection after co-administration of adenoviral vectors encoding viral antigens and type I interferon subtypes

Abstract

Background: Type I interferons (IFNs) exhibit direct antiviral effects, but also distinct immunomodulatory properties. In this study, we analyzed type I IFN subtypes for their effect on prophylactic adenovirus-based anti-retroviral vaccination of mice against Friend retrovirus (FV) or HIV.

Results: Mice were vaccinated with adenoviral vectors encoding FV Env and Gag proteins alone or in combination with vectors encoding IFNα1, IFNα2, IFNα4, IFNα5, IFNα6, IFNα9 or IFNβ. Only the co-administration of adenoviral vectors encoding IFNα2, IFNα4, IFNα6 and IFNα9 resulted in strongly improved immune protection of vaccinated mice from subsequent FV challenge infection with high control over FV-induced splenomegaly and reduced viral loads. The level of protection correlated with augmented virus-specific CD4(+) T cell responses and enhanced antibody titers. Similar results were obtained when mice were vaccinated against HIV with adenoviral vectors encoding HIV Env and Gag-Pol in combination with various type I IFN encoding vectors. Here mainly CD4(+) T cell responses were enhanced by IFNα subtypes.

Conclusions: Our results indicate that certain IFNα subtypes have the potential to improve the protective effect of adenovirus-based vaccines against retroviruses. This correlated with augmented virus-specific CD4(+) T cell and antibody responses. Thus, co-expression of select type I IFNs may be a valuable tool for the development of anti-retroviral vaccines.

Figure 1

FV-induced splenomegaly in adenoviral vector immunized mice. CB6F1 mice were immunized with Ad5 and Ad5F35 based vectors encoding F-MuLV Env and Gag with or without co-administration of a specific vectored type I IFN subtype, as indicated. Ad5-based vectors were used for the prime immunization and Ad5F35 vectors for the boost immunization. Mice of the group "env+gag" received an equal amount of luciferase encoding adenoviral vectors instead of IFN encoding vectors to ensure that the total amount of particles used for immunization was the same in all groups. Three weeks after the boost immunization mice were challenged with FV. Disease progression was monitored by palpation of the spleen twice a week. The categorized spleens of six mice per group on day 14 p.c. (A) and day 17 p.c. (B) are shown (means + standard error of the means). On day 21 p.c. spleens were removed and weighed (C). Statistically significant differences (P < 0.05) compared to unvaccinated control mice (*) or mice vaccinated with Env- plus Gag-encoding vectors (#) are indicated. Data are representative of two independent experiments. (D) CB6F1 mice were immunized twice with Ad5 and Ad5F35 based vectors encoding the indicated type I interferons alone and infected with FV three weeks after the second application of IFN vectors. The disease progression was monitored by twice-weekly palpations of the spleen, the graph shows the categorized spleen sizes (mean + standard error of the means) at the indicated time points after FV infection.

Figure 2

Viral loads after FV challenge infection of vaccinated mice. CB6F1 mice were prime- and boost-immunized with Ad5 and Ad5F35 based vectors, respectively, encoding F-MuLV Env and Gag with or without co-administration of a specific vectored type I IFN subtype, as indicated. Three weeks after the boost immunization mice were challenged with FV. Viral loads in the plasma of FV-infected mice were analyzed on day 10 p.c. (A) shows viremia levels as FFU/ml, median values are indicated by lines. On day 21 p.c. the viral loads in spleen were analyzed (B), the graph shows the viral load as IC/spleen, the horizontal lines mark median values. Statistically significant differences (P < 0.05) compared to unvaccinated control mice (*) or mice vaccinated with Env- plus Gag-encoding vectors (#) are indicated. Each dot represents an individual mouse. Data are representative of two independent experiments with similar results.

Figure 3

Vaccine-induced F-MuLV Env-specific CD4⁺ T cell responses. CB6F1 mice were prime- and boost-immunized with Ad5 and Ad5F35 based vectors, respectively, encoding F-MuLV Env and Gag with or without co-administration of a specific vectored type I IFN subtype, as indicated. Three weeks after the boost immunization mice were challenged with FV and the F-MuLV Env-specific CD4⁺ T cell response was analyzed 3 days p.c. by staining with MHC II tetramers presenting an F-MuLV Env gp70-derived epitope. (A) The graph shows the percentage of MHC II tetramer⁺ CD4⁺ T cells, the line designates the mean value. Statistically significant differences (P < 0.05) compared to unvaccinated control mice (*) or mice vaccinated with Env- plus Gag-encoding vectors (#) are indicated. Each dot represents an individual mouse. Data are representative of two independent experiments with similar results. (B) Representative dot plots from an unvaccinated mouse and mice vaccinated with env+gag or env+gag+IFNα4 are shown.

Figure 4

Depletion of CD4⁺ and CD8⁺ T cells during vaccination. CB6F1 mice were vaccinated with adenoviral vectors encoding F-MuLV Env and Gag with or without co-administration of vectored IFNα4 as described before. On day -3, -1, +1, +3, and +5 of vaccination, mice were injected i.p. with antibodies against CD4 or CD8 to deplete the respective T cell subset. After FV challenge infection, spleens were palpated twice a week to monitor disease progression (A). Viral loads in plasma were determined on day 10 p.c. (B), viral loads in spleen were analyzed on day 21 p.c. (C). For the analysis of T cell induction, mice were immunized once with the indicated vectors and the expression of CD43, CD44, CD62L and CCR7 on CD4+ T cells in the draining lymph nodes was analyzed 10 days after immunization (D). The graphs show data of four mice per group. Statistically significant differences (P < 0.05) compared to unvaccinated control mice (*) or mice vaccinated with Env- plus Gag-encoding vectors (#) are indicated.

Figure 5

Vaccine-induced FV-specific antibody response. CB6F1 mice were prime- and boost-immunized with Ad5 and Ad5F35 based vectors, respectively, encoding F-MuLV Env and Gag with or without co-administration of a specific vectored type I IFN subtype, as indicated. Binding antibodies were analyzed 18 days after boost immunization (A), whereas neutralizing antibodies were analyzed 10 days after FV challenge infection (B). The graphs show the reciprocal titers and horizontal lines mark the mean values. Statistically significant differences (P < 0.05) compared to unvaccinated control mice (*) or mice vaccinated with Env- plus Gag-encoding vectors (#) are indicated. Each dot represents an individual mouse. Data are representative of two independent experiments with similar results.

Figure 6

HIV Env- and Gag-specific T cell responses after adenovirus-based vaccination. Two weeks after a single immunization with HIV Env and Gag encoding Ad5-based vectors in combination with Ad5 vectors encoding specific type I IFN subtypes, spleens were removed and after in vitro restimulation of spleen cells with HIV Gag-derived peptides the expression of IL-2, TNF-α and IFN-γ by CD4⁺ (A-C) and CD8⁺ T cells (D-F) was analyzed. The graphs show mean percentages with standard error of the means for six mice per group. Statistically significant differences (P < 0.05) compared to unvaccinated control mice (*) or mice vaccinated with Env- plus Gag-encoding vectors (#) are indicated. Data were acquired in two independent experiments.