CD8+ T-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells.

We have recently reported that CD8(+) T-cell memory maintenance after immunization with recombinant human adenovirus type 5 (rHuAd5) is dependent upon persistent transgene expression beyond the peak of the response. In this report, we have further investigated the location and nature of the cell populations responsible for this sustained response. The draining lymph nodes were found to be important for primary expansion but not for memory maintenance, suggesting that antigen presentation through a nonlymphoid source was required. Using bone marrow chimeric mice, we determined that antigen presentation by nonhematopoietic antigen-presenting cells (APCs) was sufficient for maintenance of CD8(+) T-cell numbers. However, antigen presentation by this mechanism alone yielded a memory population that displayed alterations in phenotype, cytokine production and protective capacity, indicating that antigen presentation through both hematopoietic and nonhematopoietic APCs ultimately defines the memory CD8(+) T-cell response produced by rHuAd5. These results shed new light on the immunobiology of rHuAd5 vectors and provide evidence for a mechanism of CD8(+) T-cell expansion and memory maintenance that relies upon both hematopoietic and nonhematopoietic APCs.

Persistence of transgene expression influences CD8+ T-cell expansion and maintenance following immunization with recombinant adenovirus.

Previous studies determined that the CD8(+) T-cell response elicited by recombinant adenovirus exhibited a protracted contraction phase that was associated with long-term presentation of antigen. To gain further insight into this process, a doxycycline-regulated adenovirus was constructed to enable controlled extinction of transgene expression in vivo. We investigated the impact of premature termination of transgene expression at various time points (day 3 to day 60) following immunization. When transgene expression was terminated before the maximum response had been attained, overall expansion was attenuated, yielding a small memory population. When transgene expression was terminated between day 13 and day 30, the memory population was not sustained, demonstrating that the early memory population was antigen dependent. Extinction of transgene expression at day 60 had no obvious impact on memory maintenance, indicating that maintenance of the memory population may ultimately become independent of transgene expression. Premature termination of antigen expression had significant but modest effects on the phenotype and cytokine profile of the memory population. These results offer new insights into the mechanisms of memory CD8(+) T-cell maintenance following immunization with a recombinant adenovirus.

The magnitude of the CD8+ T cell response produced by recombinant virus vectors is a function of both the antigen and the vector.

Virus-based recombinant vaccines have proven highly effective at generating protective CD8+ T cell responses. Multiple vector platforms are available, however, little is known about the relative influence of the different vectors on the transgene-specific CD8+ T cell population. To address this question, we compared several characteristics of the CD8+ T cell response elicited by recombinant adenovirus (rAd) and vaccinia virus (rVV). We found that following rAd immunization the transgene-specific CD8+ T cell response peaked around day 12 and was larger and more sustained than the response produced by rVV. In addition, the CD8+ T cell response generated by rAd was directed primarily against the transgene, whereas the CD8+ T cell response produced by rVV principally targeted the vector backbone. In addition, we also observed that transgene selection also impacted on the magnitude of the CD8+ T cell response elicited by both vectors. Despite differences in the magnitude of the anti-transgene CD8+ T cell response, both vectors elicited CD8+ T cell populations with similar cytokine production, functional avidity and cytolytic activity. In addition, plasmid priming prior to immunization with either rAd or rVV only impacted the magnitude of the transgene gene specific CD8+ T cell response. Our study demonstrates that both vector and transgene selection can influence the magnitude of the CD8+ T cell response, but they do not influence functionality.

Recent advances in the development of adenovirus- and poxvirus-vectored tuberculosis vaccines.

Tuberculosis vaccine research began with the search for a vaccine that might be better than, and thus could replace, the current Bacillus Calmette Guérin (BCG) vaccine. Over the last fifteen years or so, intense research effort has led to the identification of a number of novel tuberculosis (TB) vaccines which can be divided into 4 categories: genetically modified mycobacteria, protein, plasmid DNA and viral. However, it is increasingly believed that the current BCG vaccine will continue to be used as a childhood vaccine and that more effort should be directed to developing appropriate boosting vaccines. Mounting evidence suggests that recombinant genetic vaccines, particularly recombinant viral vaccines, are effective in boosting immune activation and protection by BCG vaccination. Since modified vaccinia virus Ankara (MVA)- and adenovirus-vectored TB vaccines have been most extensively studied, this review will focus on recent advances in the development and applications of these two viral TB vaccines.

T-cell immunity generated by recombinant adenovirus vaccines.

Recombinant adenovirus vaccines show great promise for generating protective immunity against infectious agents and tumors. Our studies have identified several interesting biological features of the adenovirus vector that influence the T-cell response. Notably, we have demonstrated that following immunization with adenovirus vaccines, the transgene antigen remains available to the system for a longer period than would be expected, resulting in a T-cell population with a sustained effector phenotype. The implications of these observations with regards to the utility of adenovirus vaccines are discussed.

On the role of CD4+ T cells in the CD8+ T-cell response elicited by recombinant adenovirus vaccines.

We have investigated the role of CD4(+) T cells in the development of the CD8(+) T-cell response after immunization with recombinant adenovirus (rAd). In the absence of CD4(+) T cells, the "unhelped" CD8(+) T-cell population exhibited a reduction in primary expansion and long-term survival that appeared to be due to inadequate priming of naïve T cells. There were few functional or phenotypic differences between the helped and unhelped CD8(+) T-cell populations with the exception of O-glycosylated CD43, a marker of effector cells, which was augmented on the unhelped CD8(+) T-cell population. In some cases, the unhelped CD8(+) T-cell population exhibited reduced ability to control virus infection; however, this appeared to be a function of the reduced frequency of antigen-specific CD8(+) T cells. Most notably, the unhelped CD8(+) T-cell population exhibited no defect in secondary expansion. These results provide insight into the role of CD4(+) T cells during the primary CD8(+) T-cell response generated by rAd vaccines and identify potential benefits and issues that must be considered when using adenovirus vaccines under conditions where CD4(+) T-cell function may be limiting, such as vaccination of human immunodeficiency virus patients.

Recombinant adenovirus vaccines can successfully elicit CD8+ T cell immunity under conditions of extreme leukopenia.

We have examined the efficacy of vaccination with recombinant adenovirus under conditions of extreme leukopenia in lethally irradiated mice reconstituted with autologous bone marrow. The expansion of antigen-specific CD8(+) T cells following immunization of lethally irradiated hosts paralleled the recovery of total CD8(+) T cells. Surprisingly, the numbers of antigen-specific CD8(+) T cells in lethally irradiated mice beyond 6 weeks postimmunization were comparable to the numbers found in nonirradiated controls. CD8(+) T cells elicited in the lethally irradiated hosts were functionally indistinguishable from those elicited in normal hosts. Antigen expression and presentation persisted for a longer period of time in the draining lymph nodes of irradiated mice compared to those of nonirradiated animals, suggesting that antigen presentation mechanisms were intact during the reconstitution period. Experiments employing allogeneic bone marrow demonstrated that radioresistant host antigen-presenting cells were responsible for antigen presentation during the process of immune reconstitution. These results demonstrate clear compatibility of adenovirus vaccines and cytotoxic therapy. Furthermore, these observations provide novel insights into the mechanisms of CD8(+) T cell activation following adenovirus immunization.

The CD8+ T cell population elicited by recombinant adenovirus displays a novel partially exhausted phenotype associated with prolonged antigen presentation that nonetheless provides long-term immunity.

We have previously reported that the CD8+ T cell response elicited by recombinant adenovirus vaccination displayed a delayed contraction in the spleen. In our current study, we demonstrate that this unusual kinetic is a general phenomenon observed in multiple tissues. Phenotypic analysis of transgene-specific CD8+ T cells present 30 days postimmunization with recombinant adenovirus revealed a population with evidence of partial exhaustion, suggesting that the cells had been chronically exposed to Ag. Although Ag expression could no longer be detected 3 wk after immunization, examination of Ag presentation within the draining lymph nodes demonstrated that APCs were loaded with Ag peptide for at least 40 days postimmunization, suggesting that Ag remains available to the system for a prolonged period, although the exact source of this Ag remains to be determined. At 60 days postimmunization, the CD8+ T cell population continued to exhibit a phenotype consistent with partially exhausted effector memory cells. Nonetheless, these CD8+ T cells conferred sterilizing immunity against virus challenge 7-12 wk postimmunization, suggesting that robust protective immunity can be provided by CD8+ T cells with an exhausted phenotype. These data demonstrate that prolonged exposure to Ag may not necessarily impair protective immunity and prompt a re-evaluation of the impact of persistent exposure to Ag on T cell function.

Inhalation tolerance is induced selectively in thoracic lymph nodes but executed pervasively at distant mucosal and nonmucosal tissues.

Under immunogenic conditions, both the site of initial Ag exposure and consequent T cell priming in specific draining lymph nodes (LNs) imprint the ensuing immune response with lasting tissue-selective tropism. With respect to immune tolerance, whether the site of tolerance induction leads to compartmentalized or, alternatively, pervasive tolerance has not been formally investigated. Using a murine model of inhalation tolerance, we investigated whether the induction of respiratory mucosal tolerance precludes the development of de novo Th2 sensitization upon subsequent exposure to the same Ag at distant mucosal (gut) and nonmucosal (cutaneous) sites. By tracking the proliferation of CFSE-labeled OVA-TCR transgenic CD4(+) T cells upon OVA inhalation in vivo, we defined the site of tolerance induction to be restricted to the thoracic LNs. Expectedly, inhalation tolerance prevented de novo Th2 sensitization upon subsequent exposure to the same Ag at the same site. Importantly, although gut- and skin-draining LNs were not used during tolerance induction, de novo Ag-specific proliferation and Th2 differentiation in these LNs, as well as memory/effector Th2 responses in the gut (allergic diarrhea) and skin (late-phase cutaneous responses) were inhibited upon immunogenic challenge to the same Ag. Interestingly, this pervasive tolerogenic phenotype was not associated with the presence of suppressive activity throughout the lymphatics; indeed, potent suppressive activity was detected solely in the spleen. These data indicate that while inhalation tolerance is selectively induced in local thoracic LNs, its tolerogenic activity resides systemically and leads to pervasive immune tolerance in distant mucosal and nonmucosal sites.

Detailed analysis of the CD8+ T-cell response following adenovirus vaccination.

We examined CD8(+) T-cell expansion and function following intramuscular immunization with a recombinant adenovirus. This study has identified a number of properties which may explain the strong immunogenicity of adenovirus vectors: (i) the ability to deliver large amounts of antigen into the lymphoid tissues, (ii) the ability to induce rapid expansion and migration of CD8(+) T cells throughout the lymphatics, and (iii) the ability to produce a sustained, high-level CD8(+) T-cell response.