The quality of virus-specific CD8+ CTL immune responses generated by mucosal and systemic poxvirus prime-boost vaccines were evaluated in terms of T cell avidity and single-cell analysis of effector gene expression. Intranasal (I.N.) immunization regimes generated higher avidity CTL responses specific for HIV KdGag197–205 (amino acid sequence AMQMLKETI; H-2Kd binding) compared with i.m. immunization regime. Single-cell RT-PCR of KdGag197–205-specific mucosal and systemic CTL revealed that the cytokine and granzyme B expression profiles were dependent on both the route and time after immunization. The I.N./i.m.-immunized group elicited elevated number of CTL-expressing granzyme B mRNA from the genitomucosal sites compared with the i.m./i.m. regime. Interestingly, CTL generated after both I.N. or i.m. immunization demonstrated expression of Th2 cytokine IL-4 mRNA that was constitutively expressed over time, although lower numbers were observed after I.N./I.N. immunization. Results suggest that after immunization, Ag-specific CTL expression of IL-4 may be an inherent property of the highly evolved poxvirus vectors. Current observations indicate that the quality of CTL immunity generated after immunization can be influenced by the inherent property of vaccine vectors and route of vaccine delivery. A greater understanding of these factors will be crucial for the development of effective vaccines in the future.

Intranasal infection with vaccinia virus co-expressing interferon epsilon (VV-HIV-IFN-ε) was used to evaluate the role of IFN-ε in mucosal immunity. VV-HIV- IFN-ε infection induced a rapid VV clearance in lung that correlated with (i) an elevated lung VV-specific CD8⁺CD107a⁺IFN-γ⁺ population expressing activation markers CD69/CD103, (ii) enhanced lymphocyte recruitment to lung alveoli with reduced inflammation, and (iii) an heightened functional/cytotoxic CD8⁺CD4⁺ T-cell subset (CD3^hiCCR7^hiCD62L^lo) in lung lymph nodes. These responses were different to that observed with intranasal VV-HA-IFN-α₄ or VV-HA-IFN-β infections. When IFN-ε was used in an intranasal/intramuscular heterologous HIV prime-boost immunization, elevated HIV-specific effector, but not memory CD8⁺T cells responses, were observed in spleen, genito-rectal nodes, and Peyer's patch. Homing marker α4β7 and CCR9 analysis indicated that unlike other type I IFNs, IFN-ε could promote migration of antigen-specific CD8⁺T cells to the gut. Our results indicate that IFN-ε has a unique role in the mucosae and most likely can be used to control local lung and/or gut infections (i.e., microbicide) such as tuberculosis, HIV-1, or sexually transmitted diseases.

In this article, we discuss the advantages and progress made with heterologous prime-boost vaccination strategies. Although the consecutive use of DNA and recombinant viral vectors induce greatly enhanced and sustained levels of both cell-mediated and humoral immunity in preclinical models, the results have not yet been translated to clinical use. Despite this, there is still a high level of optimism that these strategies offer the best hope for the development of vaccines against diseases for which there are no effective vaccines currently available. In this article, we discuss how prime-boost immunization can elicit improved mucosal immunity, how 'mucosal' regimes also elicit 'high-quality' (high-avidity) T-cell responses to vaccine antigens, and the use of cytokines/chemokines as genetic adjuvants.

All HIV‐1 ‘systemic vaccine trials’ in humans have yielded poor outcomes. Thus, it is important to understand whether the route of delivery influences the quality of protective CTL immunity. Using heterologous poxvirus immunisation we have shown that systemically (i.m./i.m.) immunised CD8⁺ T cells generated higher levels of IL‐4/IL‐13 compared to mucosal delivery and expression also correlated with i.m./i.m. immunised mice eliciting CTL of lower avidity. Studies using IL‐4^−/− and IL‐13^−/− KO mice have shown that the capacity to express IFN‐γ, IL‐4 and/or IL‐13 by K^dGag_197–205‐specific CTL differed between these groups and was inversely correlated with CTL avidity (IL‐13^−/−>IL‐4^−/−>BALB/c), although no significant differences in the magnitude of CTL responses were observed between IL‐13^−/− and wild type mice. When IL‐13 was reconstituted in IL‐13^−/− splenocytes in vitro, their ability to bind tetramers also decreased significantly. Our data reveal that total absence of IL‐13 can greatly enhance CTL avidity. In contrast, extracellular IL‐4 appears to be important in maintaining long‐term Th1/Th2 balance in CTL, even though expression of IL‐4 by CTL markedly reduced avidity. STAT6^−/− mice also showed memory CTL of higher avidity. Furthermore, CCL5 expression in K^dGag_197–205‐specific CTL was also regulated by IL‐4/IL‐13.