Interleukin-encoding adenoviral vectors as genetic adjuvant for vaccination against retroviral infection

Abstract

Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4(+) T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4(+) T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4(+) T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity.

Figure 1. FV-induced splenomegaly in mice immunized with adenoviral vectors.

CB6F1 mice were immunized twice with Ad.pIXgp70 in combination with adenoviral vectors encoding specific interleukins. Mice were immunized first with Ad5 vectors and after 4 weeks, mice were boost-immunized with Ad5F35 vectors. Mice of the group Ad.pIXgp70 received in addition a GFP-encoding or empty adenoviral vector so that the total amount of adenoviral particles in all vaccinated groups was the same. Three weeks after the second immunization, mice were challenged with a high dose of FV and spleens were removed and weighed 3 weeks after challenge infection. Statistically significant differences (P < 0.05; Kruskal-Wallis one-way analysis of variance on ranks with Dunns multiple comparison procedure) compared to unvaccinated control mice (#) are indicated. Each dot shows an individual mouse, the horizontal lines indicate mean values. Data are results of two independent experiments with similar outcome.

Figure 2. Viral loads of vaccinated mice after FV challenge infection.

CB6F1 mice were prime- and boost-immunized with Ad.pIXgp70 in combination with interleukin-encoding adenoviral vectors. Mice were challenged with FV 3 weeks after boost immunization. Plasma viremia (A) in FV infected mice was analyzed on day 10 p.i. and is shown as focus forming units (FFU) / ml plasma, median values are indicated by lines. On day 21 p.i. viral loads in spleen (B) were analyzed and are shown as infectious centers (IC) / spleen, the horizontal lines mark median values. Statistically significant differences (P < 0.05; Kruskal-Wallis one-way analysis of variance on ranks with Dunns multiple comparison procedure) compared to unvaccinated mice (#) or mice immunized with Ad.pIXgp70 alone (*) are indicated. For statistical analysis, mice with viral loads below the detection limits were assigned the values 20 for viral load in blood, or 2 for viral load in spleen. Values were subjected to statistical analysis without logarithmic transformation. Each dot represents an individual animal. The dashed lines indicate the detection limits of the assays. Data are results of two independent experiments with similar outcome.

Figure 3. Vaccine-induced FV-neutralizing antibody responses.

CB6F1 mice were prime- and boost-immunized with Ad5 and Ad5F35 based vectors of Ad.pIXgp70 combined with interleukin-encoding vectors as indicated. Total neutralizing antibody titers (A) and neutralizing IgG antibody titers (B) were analyzed 10 days after FV challenge infection. Statistically significant differences (P < 0.05; Kruskal-Wallis one-way analysis of variance on ranks with Dunns multiple comparison procedure) compared to unvaccinated mice (#) or mice immunized with Ad.pIXgp70 alone (*) are indicated. For statistical analysis, mice with viral loads below the detection limits were assigned the value 2. Values were subjected to statistical analysis without logarithmic transformation. Each dot represents an individual mouse, horizontal lines indicate median values. The dashed lines indicate the detection limit. Data are results of two independent experiments with similar outcome.

Figure 4. Vaccine-induced F-MuLV Env-specific CD4⁺ T cell responses.

CB6F1 mice were immunized twice with Ad.pIXgp70 in combination with adenoviral vectors encoding different interleukins. Three weeks after the second immunization, mice were challenged with 500 SFFU FV. Virus-specific CD4⁺ T cell responses were analyzed 3 days p.i. by tetramer staining of spleen cells. Statistically significant differences (P < 0.05; Kruskal-Wallis one-way analysis of variance on ranks with Dunns multiple comparison procedure) compared to unvaccinated mice (#) are indicated. Each dot represents an individual animal, horizontal lines indicate mean values. Data are results of two independent experiments with similar outcome.