miRNA-mediated silencing in hepatocytes can increase adaptive immune responses to adenovirus vector-delivered transgenic antigens.

Adenovirus vectors based on human serotype 5 can induce potent CD8 T cell responses to vector-encoded transgenic antigens. However, the individual contribution of different cell types expressing antigen upon adenovirus vector injection to the generation of antigen-directed adaptive immune responses is poorly understood so far. We investigated the role of hepatocytes, skeletal muscle, and hematopoietic cells for the induction of cellular and humoral immune responses by miRNA-mediated tissue-specific silencing of antigen expression. Using hepatitis B small surface antigen (HBsAg) as the vector-encoded transgene we show that adenovirus vector dissemination from an intramuscular (i.m.) injection site into the liver followed by HBsAg expression in hepatocytes can limit early priming of CD8 T cells and the generation of anti-HBsAg antibody responses. However, hepatocyte-specific miRNA122a-mediated silencing of HBsAg expression overcame these limitations. Early clonal expansion of K(b)/S(190-197)-specific CD8 T cells was significantly enhanced and improved polyfunctionality of CD8 T cells was found. Furthermore, miRNA122a-mediated antigen silencing induced significantly higher anti-HBsAg antibody titers allowing an up to 100-fold vector dose reduction. These results indicate that miRNA-mediated regulation of antigen expression in the context of adenovirus vectors can significantly improve transgene product-directed immune responses. This finding could be of interest for future adenovirus vaccine vector development.

ΔE1 and high-capacity adenoviral vectors expressing full-length codon-optimized merozoite surface protein 1 for vaccination against Plasmodium falciparum.

BACKGROUND: The merozoite surface protein (MSP)-1 of Plasmodium falciparum, the causative agent of malaria tropica, is considered to be a promising vaccine candidate. Although its stable cloning and expression has been difficult in the past, adenoviral vectors expressing the complex protein are described in the present study. METHODS: Codon-optimized msp-1 was used to construct a set of first generation (ΔE1Ad) and high-capacity adenovirus (HC-Ad) vectors, and cellular and humoral immune responses induced by the vectors were characterized in detail in mice. RESULTS: Generation of stable ΔE1Ad and HC-Ad vectors expressing full-length MSP-1 and their production to high vector titers was found to be feasible. Epitope identification and analysis of frequencies of specific CD8 T-cells revealed that MSP-1 expressing HC-Ad vectors induced higher frequencies of interferon-γ + CD8 T-cells than ΔE1 vectors. Irrespective of the vector format, higher titers of MSP-1 specific antibodies were generated by Ad vectors expressing MSP-1 from a chicken ß-actin (CAG) promoter comprising the cytomegalovirus early enhancer element and the chicken ß-actin promoter. CONCLUSIONS: The findings of the present study suggest that Ad vectors expressing full-length codon-optimized MSP-1 are promising candidate vaccines against P. falciparum infections. Use of the HC-Ad vector type for delivery, as well as the CAG promoter to control MSP-1 expression, may further increase the efficacy of this vaccine candidate.

High-capacity adenoviral vectors circumvent the limitations of ΔE1 and ΔE1/ΔE3 adenovirus vectors to induce multispecific transgene product-directed CD8 T-cell responses.

BACKGROUND: The ability to induce cytotoxic T lymphocyte (CTL) responses that are multispecific is considered to comprise an essential feature for an efficacious genetic vaccine against many pathogens including HIV and hepatitis C virus. ΔE1Ad vectors are promising vectored vaccines but have been shown to induce antigen-specific CTLs with only limited multispecificity. In the present study, we investigated the applicability of gene-deleted high-capacity adenovirus (HC-Ad) vectors and focused on the induction of multispecific CTL responses. METHODS: We generated Δ E1 and HC-Ad vectors expressing hepatitis B virus small surface antigen (HBsAg). We comparatively analyzed the CTL profiles against various transgene product- and vector-derived epitopes in several mouse strains and HBsAg- and vector-directed antibody responses. RESULTS: HC-Ad vectors efficiently induced multispecific HBsAg-directed CTLs. By contrast, ΔE1Ad vectors mainly primed CTLs against one immunodominant epitope of HBsAg. This absence of multispecific CTL responses correlated with the induction of CTLs against viral epitopes generated by de novo expression of Ad genes from the ΔE1Ad vector. However, Ad-specific CTLs induced in trans did not impair HC-AdS-induced multispecific CTL responses against HBsAg. Finally, HC-Ad vectors also induced higher HBsAg antibody titers compared to ΔE1Ad vectors. CONCLUSIONS: De novo expression of viral genes from ΔE1Ad vector genomes restricts the multispecificity of transgene product-specific CTLs by immunodominance effects. HC-Ad vectors devoid of Ad genes are favorable for the induction of both multispecific CD8 T-cell responses and high antibody responses. Our results suggest the deletion of Ad genes as an important means for developing potent Ad-based vectored vaccines.

Covalent decoration of adenovirus vector capsids with the carbohydrate epitope αGal does not improve vector immunogenicity, but allows to study the in vivo fate of adenovirus immunocomplexes.

Adenovirus-based vectors are promising tools for genetic vaccination. However, several obstacles have to be overcome prior to a routine clinical application of adenovirus-based vectors as efficacious vectored vaccines. The linear trisaccharide epitope αGal (alpha-Gal) with the carbohydrate sequence galactose-α-1,3-galactosyl-ß-1,4-N-acetylglucosamine has been described as a potent adjuvant for recombinant or attenuated vaccines. Humans and α-1,3-galactosyltransferase knockout mice do not express this epitope. Upon exposure of α-1,3-galactosyltransferase-deficient organisms to αGal in the environment, large amounts of circulating anti-Gal antibodies are produced consistently. Immunocomplexes formed between recombinant αGal-decorated vaccines and anti-Gal antibodies exhibit superior immunogenicity. We studied the effects of the trisaccharide epitope on CD8 T cell responses that are directed specifically to vector-encoded transgenic antigens. For that, covalently αGal-decorated adenovirus vectors were delivered to anti-Gal α-1,3-galactosyltransferase knockout mice. We generated replication-defective, E1-deleted adenovirus type 5 vectors that were decorated with αGal at the hexon hypervariable regions 1 or 5, at fiber knob, or at penton base. Surprisingly, none of the adenovirus immunocomplexes being formed from αGal-decorated adenovirus vectors and anti-Gal immunoglobulins improved the frequencies of CD8 T cell responses against the transgenic antigen ovalbumin. Humoral immunity directed to the adenovirus vector was neither increased. However, our data indicated that decoration of Ad vectors with the αGal epitope is a powerful tool to analyze the fate of adenovirus immunocomplexes in vivo.

Adenovirus vectors and subviral particles for protein and peptide delivery.

Adenovirus vectors belong to the most frequently used vector type in gene therapy approaches. In addition, adenovirus vector particles and adenovirus subviral particles offer a great potential for the direct delivery of proteins into cells. In this review we discuss this potential and the technology of adenovirus as a protein delivery platform for applications ranging from vaccination to gene therapy.