Incorporation of porcine adenovirus 4 fiber protein enhances infectivity of adenovirus vector on dendritic cells: implications for immune-mediated cancer therapy.

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.

A porcine adenovirus with low human seroprevalence is a promising alternative vaccine vector to human adenovirus 5 in an H5N1 virus disease model.

Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5.

Evaluation of cross-reactive cell-mediated immune responses among human, bovine and porcine adenoviruses.

The absence of preexisting immunity against porcine adenovirus (Ad) serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) in humans makes them attractive alternatives to human Ad serotype 5 (HAd5) vectors. To determine whether there is significant cross-reactivity among HAd5, BAd3 and PAd3 at the level of cell-mediated immune responses, BALB/c mice were inoculated intraperitoneally with wild-type (WT) or replication-defective (RD) HAd5, BAd3 or PAd3. After 35 days of the first inoculation, cross-reactive CD8+ cytotoxic T cells, as well as CD4+ Th1- and Th2-helper T cells, in the spleen were analyzed by enzyme-linked-immunospot, flow cytometry and cytotoxic T lymphocyte assays. Virus-neutralization assays were used to evaluate humoral cross-reactivity. CD8+ or CD4+ T cells primed with WT or RD HAd5, PAd3 or BAd3 showed significant (P<0.005) reactivity with homologous Ad antigens, whereas only minimal cross-reactivity was observed on stimulation with heterologous Ad antigens. Ad-neutralizing antibodies were found to be homologous Ad specific. Overall, these results suggest that there is no significant immunological cross-reactivity among HAd5, BAd3 and PAd3, thereby supporting the rationale for the use of BAd3 and PAd3 as alternative HAd vectors to circumvent anti-HAd immunity in humans.

Porcine adenovirus type 3 E1B large protein downregulates the induction of IL-8.

Replication-defective (E1-E3 deleted) adenovirus vector based gene delivery results in the induction of cytokines including IL-8, which may contribute to the development of inflammatory immune responses. Like other adenoviruses, E1 + E3 deleted porcine adenovirus (PAdV) 3 induces the production of IL-8 in infected cells. In contrast, no IL-8 production could be detected in cells infected with wild-type or mutant PAdV-3s containing deletion in E1A + E3 (PAV211) or E1Bsmall + E3 (PAV212). Expression of PAdV-3 E1Blarge inhibited the NF-kappaB dependent transcription of luciferase from IL-8 promoter. Imunofluorescence and electrophoretic mobility shift assays suggested that constitutive expression of PAdV-3 E1Blarge inhibited the nuclear translocation of NF-kappaB and its subsequent binding to DNA. These results suggest that E1Blarge interacts with NF-kappaB to prevent transcription and down regulate proinflammatory cytokine IL-8 production.

Porcine adenovirus serotype 3 internalization is independent of CAR and alphavbeta3 or alphavbeta5 integrin.

Nonhuman adenoviruses including porcine adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus-adenovirus receptor (CAR) serves as a primary receptor and alphavbeta3 or alphavbeta5 integrin as a secondary receptor for several human adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, alphavbeta3 or alphavbeta5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-alphavbeta3 or anti-alphavbeta5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition in transduction by HAd-GFP was observed in antibody-treated cells as compared to untreated cells, whereas transduction by PAd-GFP remained to similar levels irrespective of the treatment. To study the adenoviral fiber knob-mediated virus interference, MCF-10A cells were treated with or without the recombinant HAd5 or PAd3 knob followed by infection with HAd-GFP or PAd-GFP. Significant (P <0.05) inhibition was observed only in transduction of the homologous vector. These results suggested that PAd3 internalization was CAR- as well as alphavbeta3 or alphavbeta5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and alphavbeta3 or alphavbeta5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.

Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture.

Preexisting immunity against human adenoviruses (HAd) limits the efficiency of transduction of HAd vectors in humans. In addition, development of a vector-specific immune response after the first inoculation with a HAd vector further lowers vector uptake following readministration. We investigated the usefulness of porcine adenovirus serotype 3 (PAd3)-based vectors as a supplement to HAd vectors. Here we demonstrate that preexisting HAd-specific neutralizing antibodies in humans do not cross-neutralize PAd3. In order to generate E1A-deleted PAd3 vectors, an E1-complementing cell line of porcine origin was produced. E1A-deleted PAd3 vector expressing green fluorescent protein; GFP (PAd-GFP) and E1-deleted HAd5 vector expressing GFP (HAd-GFP) transduced human cell lines with comparable efficiencies. Both of these vectors efficiently transduced murine MT1A2 breast cancer cell line, while PAd-GFP transduced murine NIH 3T3 fibroblast cell line significantly better (P < 0.05) than HAd-GFP. These results suggest that PAd3 vectors would be promising supplement to HAd vectors as a delivery vehicle for recombinant vaccines and gene therapy applications.

Analysis of early region 4 of porcine adenovirus type 3.

The early region 4 (E4) of porcine adenovirus (PAdV)-3, located at the right-hand end of the genome is transcribed in a leftward direction and has the potential to encode seven (p1-p7) open reading frames (ORFs). To determine the role of each protein in viral replication, we constructed full-length PAdV-3 genomic clones containing deletions of individual E4 ORF or combined deletions of the neighboring ORFs. Transfection of swine testicular (ST) cells with individual E4 mutant plasmid DNAs generated PAdV-3 E4 mutant viruses except with plasmids containing a deletion of ORF p3, ORF p2+ p3 or ORF p3+ p4. Each of the mutants was further analyzed for growth kinetics, and early/late protein synthesis. Mutant viruses carrying deletions in ORF p1, ORF p2 or ORF p4 showed growth characteristics similar to that of wild-type PAdV-3. Early/late protein synthesis was also indistinguishable from that of wild-type PAdV-3. However, mutant viruses carrying deletions in ORF p5, ORF p6 or ORF p7 showed a modest effect in their ability to grow in porcine cells and express early proteins. These results suggest that the E4 ORF p3 (showing low homology with non-essential human adenovirus (HAdV)-9-E4 ORF1 encoded proteins) is essential for the replication of PAdV-3 in vitro. In contrast, the E4 ORF p7 (showing homology to essential HAdV-2 34 kDa protein) is not essential for replication of PAdV-3 in vitro. Moreover, successful deletion of 1.957 kb fragment in E4 region increased the available capacity of replication-competent PAdV-3 (E3 + E4 deleted) to approximately 4.3 kb and that of replication-defective PAdV-3 (E1 + E3 + E4 deleted) to approximately 7 kb. This is extremely useful for the construction of PAdV-3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.