Vaccine-induced FV-neutralizing antibody responses.

<p>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 (<i>P</i> < 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.</p

Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

<div><p>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 T_H2 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.</p> </div

FV-induced splenomegaly in mice immunized with adenoviral vectors.

<p>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 (<i>P</i> < 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.</p

Viral loads of vaccinated mice after FV challenge infection.

<p>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 (<i>P</i> < 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.</p

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

<p>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 (<i>P</i> < 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.</p

Functional analysis of splenic and hepatic WHV-specific T-cells elicited by vaccinations in WHV transgenic mice.

<p>(A) Degranulation capacity of IFNγ⁺ CD8⁺ splenic T-cells expanded <i>in vitro</i> for 7 days with peptide c13-21. (B) Frequencies of IFNγ- TNFα- and IL-2-producing CD8⁺ T-cells detected <i>ex vivo</i> in splenocytes stimulated for 6 h with the epitope c13-21. (C) Summary of WHcAg-specific CD8⁺ T-cells detected <i>ex vivo</i> in the populations of splenic and hepatic lymphocytes. (D) Representative dot-plots of WHcAg-specific CD8⁺ T-cells detected <i>ex vivo</i> in the spleen and the liver of one DNA prime – AdV boost immunized WHV transgenic mouse. (E–F) Frequencies of IFNγ- TNFα- and IL-2-positive CD8⁺ and CD4+ T cells detected ex vivo in splenic and hepatic lymphocytes stimulated 6 h with CD8⁺ T-cell epitope c13-21 or CD4⁺ T-cell epitope c131-145, respectively. The bars represent the mean value obtained for each group of mice including SEM. Asterisks mark the statistically significant difference (<b>*</b><0.05;<b>**</b><0.005; <b>***</b><0.0005; <i>ns</i> – not significant).</p

Determination of WHV infection markers in immunized and only entecavir-treated chronic WHV carrier woodchucks.

<p>Six chronically WHV-infected woodchucks were treated with ETV for 23 weeks. Four of them (61792, 61793, 61786 and 61789) received subsequently 9 intramuscular immunizations with DNA plasmids, expressing WHcAg and WHsAg (blue arrows), Ad5WHc (red arrows), and Ad35WHc (green arrows) (A). Two animals (number 61791 and 61795) were treated only with ETV and served as controls (B). Summarized comparison of WHV load (C) and WHsAg levels (D) detected in woodchucks receiving immunizations and only ETV-treated controls at representative time points of the therapy. The black arrow represents beginning of the immunization regimen at week 8. The viral DNA was extracted from woodchuck sera and the viral loads were quantified per ml of serum, using real-time PCR analysis. Serum WHsAg concentration was determined by electroimmunodiffusion using rabbit anti-WHs serum. WHsAg-specific antibodies were (anti-WHs) detected in woodchuck sera using protein G coupled to peroxidase. The GOT levels in woodchuck sera were quantified using the standard diagnostic methods. The GOT value above 50 IU/l was considered elevated (GE – genome equivalents; n.d. – not done; † - dead).</p

Quantification of the WHV loads before and after the immunization trials in WHV transgenic mice.

<p>The viral load was evaluated by quantitative real-time PCR with the detection limit of 10³ genome equivalents (GE) per milliliter serum. For analysis DNA samples obtained from the serum of mice were used. The single pair of dots connected with the line represents values obtained from one mouse before (time point of cardiotoxin pretreatment) and after the immunization trials (week 2 after the last immunization). The correlations of the WHV loads for 4 immunization groups of mice are presented: (A) control group primed with ‘empty’ pCG and boosted with Ad5 expressing GFP, (B) group immunized only with DNA vaccine – pCGWHc, (C) group primed with pCGWHc and boosted with Ad5WHc, and (D) group primed with pCGWHc and boosted twice with Ad5WHc and Ad35WHc. The statistical analysis was performed using the Wilcoxon signed rank test (<b>*</b><0.05; <b>***</b><0.0005; <i>ns</i> – not significant).</p

Cellular immune response induced by DNA prime – AdV boost immunization in WHV transgenic mice.

<p>Mice were primed two times by immunization with the pCGWHc plasmid. Four weeks later boosting immunization with Ad5WHc, Ad35WHc, or pCGWHc was performed. The group of mice immunized twice with pCGWHc in combination with Ad5WHc was boosted 4 weeks later for a second time with Ad35WHc. Mice immunized with ‘empty’ pCG and boosted with Ad5 expressing GFP served as controls. (A–B) Representative and summarised frequencies of WHcAg-specific CD8⁺ T-cells detected <i>ex vivo</i> in the population of splenic lymphocytes. Antigen-specific cells were detected using DimerX H2-Db fusion protein loaded with H2-Db-restricted CD8⁺ epitope c13-21. (C,E) Representative and summarised WHcAg-specific IFNγ⁺ CD8⁺ T-cell responses detected in the splenocytes expanded <i>in vitro</i> for 7 days with CD8⁺ T-cell epitope c13-21. (D,F) Representative and summarised WHcAg-specific IFNγ⁺ CD4⁺ T-cell responses in the splenocytes expanded <i>in vitro</i> for 7 days with CD4⁺ T-cell epitope c131-145. The bars represent the mean value obtained for each group of mice including SEM. Asterisks mark the statistically significant difference (<b>*</b><0.05;<b>**</b><0.005; <b>***</b><0.0005; <i>ns</i> – not significant).</p

Cytotoxic T-cell responses detected in chronically WHV-infected woodchucks treated with the combination therapy.

<p>Summary of WHcAg-specific (A) and WHsAg-specific (B) degranulation responses evaluated by CD107a assay at representative time points of the experiment. PBMCs were expanded <i>in vitro</i> for 3 days with WHcAg-derived epitope c96-110 or WHsAg-derived epitope s220-234. The background value was calculated as a mean of all values detected for negative controls in all woodchucks at all time points. Presented values show the percentage of CD107a⁺ CD3⁺ CD4⁻ T-cells in the CD3⁺ CD4⁻ T-cell population. The black arrow represents beginning of the immunization regimen at week 8. (C–D) The kinetics of CD107a⁺ degranulation responses in WHV chronic carriers at all monitored time points of therapy. Chronically WHV-infected woodchucks were treated with ETV for 23 weeks. Four of them received subsequently 9 intramuscular immunizations with DNA plasmids, expressing WHcAg and WHsAg (blue arrows), Ad5WHc (red arrows), and Ad35WHc (green arrows). Two animals (number 61791 and 61795) were treated only with ETV and served as controls. The positive CTL responses are marked with “+” sign.</p