Interleukin-12 (IL-12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. Clinical applications for this lymphokine include resolution of infectious disease, cancer immunotherapy, and boosting cellular immunity in AIDS patients. When using IL-12 and other cytokines therapeutically, an approach designed to obtain localized cytokine expression would be beneficial, because this could reduce the problem of systemic toxicity. As a means of developing a suitable delivery vehicle for IL-12, we have produced double-recombinant adenovirus vectors containing the p35 subunit cDNA of murine IL-12 in early region 1 of adenovirus type 5 and the cDNA for p40 in early region 3 (AdmIL-12). Cell lines infected with AdmIL-12 produced up to 42,000 units of IL-12/10(6) cells per 24 hr. Biological activity of the virally expressed product was demonstrated in vitro through its ability to induce proliferation of phytohemagglutinin (PHA)-stimulated lymphoblasts and to stimulate natural killer (NK) activity in naive splenocytes. Mice injected intraperitoneally with these vectors displayed serum IL-12 levels that increased proportionately with the amount of virus administered. IL-12 production in vivo caused a dose-dependent increase in splenic and lung NK cell activity. This work represents the first demonstration of a double-recombinant adenovirus vector expressing a functional heterodimeric protein. The results of these studies support the use of AdmIL-12 as an efficient delivery vehicle for IL-12, and direct studies of its ability to modulate cellular immunity in vivo are currently underway.
A recombinant adenovirus (Ad) expressing glycoprotein B (gB) of herpes simplex virus (HSV) type 1 (AdgB8) was evaluated as a mucosal vaccine candidate. When administered intranasally (inl) to C57B1/6 mice, AdgB8 induced levels of serum anti-HSV gB IgG antibodies similar to those of mice immunized intraperitoneally (ip), which neutralized both HSV-1 and -2. Mice immunized inl with AdgB8 produced secretory IgA specific for HSV gB, but mice immunized ip did not. Splenic anti-HSV cytotoxic T lymphocytes (CTL) were observed after inl and ip immunization; however, there was a time-dependent decrease in the anti-HSV CTL activity from spleens of inl immunized mice. Anti-HSV CTL were also present in the mediastinal lymph nodes after inl but not ip AdgB8 immunization. Furthermore, mice immunized inl with AdgB8 were protected against heterologous inl challenge with HSV-2, and this protection lasted longer than in ip-immunized mice. These results indicate that mucosal immunization with a recombinant adenovirus can induce mucosal and systemic immune responses and provide long-term protection from mucosally or sexually transmitted viruses.
SUMMARYAn infectious recombinant human adenovirus type 5 (Ad5) vector, AdG12, which carries the glycoprotein gene of vesicular stomatitis virus (VSV) and expresses that gene in cultured HeLa cells was used to examine the host range of insert expression by human Ad vectors. The VSV glycoprotein was expressed in bovine, canine and murine cells when infected with AdG 12 in culture. These cell lines are respectively permissive, non-permissive and semi-permissive for human Ad5 replication. Administration of the AdG 12 vector to calves, piglets or dogs by either the subcutaneous or oral route resulted in the production of high titres of neutralizing antibodies to VSV. Mice injected intraperitoneally with the vector produced neutralizing antibodies and were protected against subsequent intravenous challenge with normally lethal doses of VSV. This work demonstrates the utility of human adenoviral vectors for antigen expression in a number of non-human cell lines and for the induction of an immune response to the delivered antigen in a number of species.
Pulmonary tuberculosis (TB) remains to be a major global health problem despite many decades of parenteral use of Bacillus Calmette-Guérin (BCG) vaccine. Developing safe and effective respiratory mucosal TB vaccines represents a unique challenge. Over the past decade or so, the human serotype 5 adenovirus (AdHu5)-based TB vaccine has emerged as one of the most promising candidates based on a plethora of preclinical and early clinical studies. However, anti-AdHu5 immunity widely present in the lung of humans poses a serious gap and limitation to its real-world applications. In this study we have developed a novel chimpanzee adenovirus 68 (AdCh68)-vectored TB vaccine amenable to the respiratory route of vaccination. We have evaluated AdCh68-based TB vaccine for its safety, T-cell immunogenicity, and protective efficacy in relevant animal models of human pulmonary TB with or without parenteral BCG priming. We have also compared AdCh68-based TB vaccine with its AdHu5 counterpart in both naive animals and those with preexisting anti-AdHu5 immunity in the lung. We provide compelling evidence that AdCh68-based TB vaccine is not only safe when delivered to the respiratory tract but, importantly, is also superior to its AdHu5 counterpart in induction of T-cell responses and immune protection, and limiting lung immunopathology in the presence of preexisting anti-AdHu5 immunity in the lung. Our findings thus suggest AdCh68-based TB vaccine to be an ideal candidate for respiratory mucosal immunization, endorsing its further clinical development in humans.
Cohort studies of female commercial sex workers (CSWs) in Kenya were among the first to identify highly HIV-1-exposed seronegative (HESN) individuals. As natural resistance is usually mediated by innate immune mechanisms, we focused on determining whether expression and function of innate signaling pathways were altered locally in the genital mucosa of HESN CSWs. Our results demonstrated that selected pattern-recognition receptors (PRRs) were significantly reduced in expression in cervical mononuclear cells (CMCs) from HESN compared with the new HIV-negative (HIV-N) and HIV-positive (HIV-P) groups. Although baseline levels of secreted cytokines were reduced in CMCs of HESN, they were highly stimulated following exposure to ssRNA40 in vitro. Importantly, cervical epithelial cells from HESN also expressed reduced levels of PRRs, but Toll-like receptor 3 (TLR3) and TLR7 as well as nuclear factor-κB and activator protein 1 were highly expressed and activated. Lastly, inflammatory cytokines interleukin (IL)-1β, IL-8, and RANTES (regulated and normal T cell expressed and secreted) were detected at lower levels in cervicovaginal lavage of HESN compared with the HIV-N and HIV-P groups. Overall, our study reveals a local microenvironment of HIV resistance in the genital mucosa consisting of a finely controlled balance of basal immune quiescence with a focused and potent innate anti-viral response critical to resistance to sexual transmission of HIV-1.
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