Codelivery of the chemokine CCL3 by an adenovirus-based vaccine improves protection from retrovirus infection

Abstract

Processing and presentation of vaccine antigens by professional antigen-presenting cells (APCs) is of great importance for the efficient induction of protective immunity. We analyzed whether the efficacy of an adenovirus-based retroviral vaccine can be enhanced by coadministration of adenovirus-encoded chemokines that attract and stimulate APCs. In the Friend retrovirus (FV) mouse model we coexpressed CCL3, CCL20, CCL21, or CXCL14 from adenoviral vectors, together with FV Gag and Env antigens, and then analyzed immune responses and protection from pathogenic FV infection. Although most tested chemokines did not improve protection against FV challenge, mice that received adenoviral vectors encoding CCL3 together with FV antigens showed significantly better control over viral loads and FV-induced disease than mice immunized with the viral antigens only. Improved protection correlated with enhanced virus-specific CD4+ T cell responses and higher neutralizing antibody titers. To apply these results to an HIV vaccine, mice were immunized with adenoviral vectors encoding the HIV antigens Env and Gag-Pol and coadministered vectors encoding CCL3. Again, this combination vaccine induced higher virus-specific antibody titers and CD4+ T cell responses than did the HIV antigens alone. These results indicate that coexpression of the chemokine CCL3 by adenovirus-based vectors may be a promising tool to improve antiretroviral vaccination strategies.

Fig 1

Vaccine-induced control over FV-induced disease. CB6F1 mice were immunized twice with adenoviral vectors encoding FV Env and Gag in combination with adenoviral vectors encoding the murine chemokines CCL3, CCL20, CCL21, or CXCL14. For prime immunization, Ad5-based vectors were used, while Ad5F35-based vectors were used for boost immunization. To ensure that all mice received equal amounts of viral particles, mice immunized with Env/Gag alone were coadministered an adenoviral vector encoding luciferase as an irrelevant transgene. (A) Three weeks after the boost immunization, the mice were challenged with 250 SFFU of FV and the development of splenomegaly as a surrogate marker for disease progression was monitored. (B) On day 21 p.c., the spleens were removed and weighed. The graphs show mean values and standard errors of the means for eight mice per group. Statistically significant differences (P < 0.05, Kruskal-Wallis ANOVA on ranks with a Student-Newman-Keuls multiple-comparison test) compared to the unvaccinated control group or to mice immunized with Env/Gag alone are indicated by “*” and “#”, respectively; significant differences of the other chemokine groups to Env/Gag/CCL3 are indicated by “^”. The results were obtained in two independent experiments.

Fig 2

Reduction of virus load in the plasma and spleen in vaccinated mice. CB6F1 mice were prime and boost immunized with Ad5- and Ad5F35-based vectors, respectively, encoding FV Env and Gag and coadministered vectors encoding the chemokine CCL3, CCL20, CCL21, or CXCL14 and challenged with FV 3 weeks after the boost immunization. The viral loads in plasma (A) and spleen (B) samples were analyzed at days 10 and 21 p.c., respectively. The graphs show the mean values and standard errors of the means for six mice per group. Statistically significant differences (P < 0.05, one-way ANOVA [A] and Kruskal-Wallis ANOVA on ranks [B] with Student-Newman-Keuls multiple-comparison test) compared to the unvaccinated control group or to mice immunized with Env/Gag alone are indicated by “*” and “#”, respectively; significant differences of the other chemokine groups to Env/Gag/CCL3 are indicated by “^”. The results were obtained in two independent experiments.

Fig 3

Induction of FV-specific CD4⁺ T cell responses by MHC-II tetramer staining. For the analysis of CD4⁺ T cell responses, CB6F1 mice were immunized twice with Ad5 and Ad5F35 vectors, respectively, encoding FV Env and Gag and coadministered vectors encoding CCL3, CCL20, CCL21, or CXCL14. To enhance immune responses, mice were challenged with FV and T cell responses were analyzed 3 days later by MHC-II tetramer staining of spleen cells. (A) The graph shows mean values with standard errors of the means of four mice per group. Statistically significant differences (P < 0.05, Kruskal-Wallis ANOVA on ranks with Student-Newman-Keuls multiple-comparison test) compared to the unvaccinated control group or to mice immunized with Env/Gag alone are indicated by “*” and “#”, respectively; significant differences of the other chemokine groups to Env/Gag/CCL3 are indicated by “^”. The results are representative of two independent experiments. (B) Dot plots from representative mice showing the percentage of MHC II tetramer⁺ cells after gating on the lymphocyte population in a forward- versus sideward-scatter plot and on CD4⁺ CD11b⁻ cells.

Fig 4

Effect of depletion of CD4⁺ or CD8⁺ T cells during vaccination on vaccine-mediated protection from FV infection. To determine the influence of CD4⁺ and CD8⁺ T cells on vaccine-mediated protection from FV infection, mice were depleted of the respective T cell subsets around the time of vaccination, and challenged with FV as before. FV infection was monitored by palpation of the spleens (A) and by analysis of FV loads in plasma on day 10 p.c. (B) and in spleen cells on day 21 p.c. (C). The graphs show mean values with standard errors of the means of four mice per group. Statistically significant differences (P < 0.05, Kruskal-Wallis ANOVA on ranks with Student-Newman-Keuls multiple-comparison test [A], one-way ANOVA with Student-Newman-Keuls multiple-comparison test [B and C]) compared to the unvaccinated control group, to mice immunized with Env/Gag alone or to the Env/Gag/CCL3 group are indicated by “*”, “#”, and “^”, respectively, significant differences between CD4- and CD8-depleted mice are indicated by “°”.

Fig 5

Antibody response to adenovirus-based vaccination. CB6F1 mice were prime and boost immunized with Ad5- and Ad5F35-based vectors, respectively, encoding FV Env and Gag and coadministered vectors encoding the chemokines CCL3, CCL20, CCL21, or CXCL14. The binding antibody titers of vaccinated mice were analyzed 2 weeks after boost immunization (A), and neutralizing antibody titers were analyzed 10 days after FV challenge infection (B). The dots represent values of individual mice, the lines indicate median values. Statistically significant differences (P < 0.05, Kruskal-Wallis ANOVA on ranks with Student-Newman-Keuls multiple-comparison test) compared to the unvaccinated control group or to mice immunized with Env/Gag alone are indicated by “*” and “#”, respectively, significant differences of the other chemokine groups to Env/Gag/CCL3 are indicated by “^”. The data were obtained in two independent experiments.

Fig 6

Humoral immune response to adenovirus-based immunization against HIV proteins. BALB/c mice were immunized twice with adenoviral vectors encoding HIV Env and Gag-Pol, sera were collected 3 weeks after the boost immunization, and the titers of Env-binding IgG1 and IgG2a antibodies were analyzed. Each dot represents an individual mouse, the bar marks the median value. No significant differences were detected in a Mann-Whitney rank sum test (P > 0.05). n.s., not significant.

Fig 7

Cellular immune responses to adenovirus-based vaccination against HIV proteins. Cellular immune responses were analyzed after a single immunization of BALB/c mice with adenoviral vectors encoding HIV Env and Gag-Pol proteins with or without codelivery of CCL3. Two weeks after immunization the spleen cells were removed, and the expression of IFN-γ, TNF-α, and IL-2 after restimulation with Gag-derived peptides in vitro was analyzed by flow cytometry. The graphs show the percentages of IFN-γ-, TNF-α-, and IL-2-producing CD4⁺ (A to C) and CD8⁺ T cells (D to F). The graphs show the mean values and standard errors of the means of eight mice per group. The data were acquired in two independent experiments. Statistically significant differences (P < 0.05, Kruskal-Wallis ANOVA on ranks with Student-Newman-Keuls multiple-comparison test) compared to the unvaccinated control group or to mice immunized with HIV Env and Gag-Pol alone are indicated by “*” and “#”, respectively.