Ronald J. Jackson

Role of novel type I interferon epsilon in viral infection and mucosal immunity

Intranasal infection with vaccinia virus co-expressing interferon epsilon (VV-HIV-IFN-ɛ) was used to evaluate the role of IFN-ɛ in mucosal immunity. VV-HIV- IFN-ɛ 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 (CD3hiCCR7hiCD62Llo) 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-ɛ 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 Peyers patch. Homing marker α4β7 and CCR9 analysis indicated that unlike other type I IFNs, IFN-ɛ could promote migration of antigen-specific CD8+T cells to the gut. Our results indicate that IFN-ɛ 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.

Human immunodeficiency virus-1 vaccine design: where do we go now?

Numerous human immunodeficiency virus (HIV)‐1 vaccines have been developed over the last three decades, but to date an effective HIV‐1 vaccine that can be used for prophylactic or therapeutic purposes in humans has not been identified. The failures and limited successes of HIV‐1 vaccines have highlighted the gaps in our knowledge with regard to fundamental immunity against HIV‐1 and have provided insights for vaccine strategies that may be implemented for designing more effective HIV‐1 vaccines in the future. Recent studies have shown that robust mucosal immunity, high avidity and polyfunctional T cells, and broadly neutralizing antibodies are important factors governing the induction of protective immunity against HIV‐1. Furthermore, optimization of vaccine delivery methods for DNA or live viral vector‐based vaccines, elucidating the immune responses of individuals who remain resistant to HIV‐1 infections and also understanding the core immune responses mediating protection against simian immunodeficiency viruses (SIV) and HIV‐1 in animal models following vaccination, are key aspects to be regarded for designing more effective HIV‐1 vaccines in the future.

Different HIV pox viral vector-based vaccines and adjuvants can induce unique antigen presenting cells that modulate CD8 T cell avidity.

The lung-derived dendritic cell (LDC) recruitment following intranasal (i.n.) vaccination of different poxviral vector-based vaccines/adjuvants were evaluated to decipher how these factors influenced CD8(+) T cell avidity. Compared to the standard i.n. recombinant fowlpox virus (FPV)-HIV vaccination, the FPV-HIV IL-13Rα2 or IL-4Rα antagonist adjuvanted vaccines that induced higher avidity CD8(+) T cells, also recruited significantly elevated MHCII(+) CD11c(+) CD11b(+) CD103(-) CD64(-) MAR-1(-) conventional DC (cDCs) to the lung mucosae (hierarchy: IL-4R antagonist>IL-13Rα2>unadjuvanted). In contrast, elevated CD11b(-) CD103(+) LDCs were detected in animals that received recombinant HIV vaccinia virus (rVV) or Modified Vaccinia Ankara virus (MVA) vector-based vaccines. Adoptive transfer studies indicated that CD11b(-) CD103(+) LDCs significantly dampened HIV-specific CD8(+) T cell avidity compared to CD11b(+) CD103(-) LDCs. Collectively; our observations revealed that rFPV vector prime and transient inhibition of IL-4/IL-13 at the vaccination site favoured the recruitment of unique LDCs, associated with the induction of high quality immunity.

Reduced Interleukin-4 Receptor α Expression on CD8+ T Cells Correlates with Higher Quality Anti-Viral Immunity

With the hope of understanding how interleukin (IL)-4 and IL-13 modulated quality of anti-viral CD8+ T cells, we evaluated the expression of receptors for these cytokines following a range of viral infections (e.g. pox viruses and influenza virus). Results clearly indicated that unlike other IL-4/IL-13 receptor subunits, IL-4 receptor α (IL-4Rα) was significantly down-regulated on anti-viral CD8+ T cells in a cognate antigen dependent manner. The infection of gene knockout mice and wild-type (WT) mice with vaccinia virus (VV) or VV expressing IL-4 confirmed that IL-4, IL-13 and signal transducer and activator of transcription 6 (STAT6) were required to increase IL-4Rα expression on CD8+ T cells, but not interferon (IFN)-γ. STAT6 dependent elevation of IL-4Rα expression on CD8+ T cells was a feature of poor quality anti-viral CD8+ T cell immunity as measured by the production of IFN-γ and tumor necrosis factor α (TNF-α) in response to VV antigen stimulation in vitro. We propose that down-regulation of IL-4Rα, but not the other IL-4/IL-13 receptor subunits, is a mechanism by which CD8+ T cells reduce responsiveness to IL-4 and IL-13. This can improve the quality of anti-viral CD8+ T cell immunity. Our findings have important implications in understanding anti-viral CD8+ T cell immunity and designing effective vaccines against chronic viral infections.

The mousepox experience

Much of the debate about science policy in recent years has focused on ‘the dual‐use dilemma’, which arises when well‐intentioned scientific research has the potential to be misused by state and non‐state actors for nefarious purposes. In the context of the life sciences, for example, the same discoveries that lead to advancements in medicine could also be used to facilitate the development of biological weapons. Although all life science techniques and discoveries might be inherently dual‐use (Atlas, 2009), current debates are concerned primarily with cases where the consequences of malevolent use would be especially severe (Selgelid, 2009). The dual‐use dilemma is not new. When physicists observed atomic fission and the nuclear chain reaction early in the twentieth century, they realized that these discoveries might have beneficial applications in medicine and energy production; but they also realized that they could lead to the production of new, horribly efficient weapons. The manufacture and use of the first atomic bombs—and the nuclear arms race that followed—demonstrated that their fears were justified. According to the American biologist Matthew Meselson, this is not specific to nuclear physics: “Every major technology—metallurgy, explosives, internal combustion, aviation, electronics, nuclear energy—has been intensively exploited, not only for peaceful purposes but also for hostile ones” (Meselson, 2000). Similarly, recent advances in biology and genetics in particular raise the possibility of a new generation of biological weapons. > …recent advances in biology and genetics in particular raise the possibility of a new generation of biological weapons One of the most cited examples of dual‐use research is that of Australian researchers who inadvertently developed a lethal mouse virus. In this now‐famous study, the researchers used standard genetic engineering techniques to insert the gene for interleukin‐4 (IL‐4) into the mousepox virus. They hoped that the altered virus would induce infertility in mice—which are …

Novel HIV IL-4R antagonist vaccine strategy can induce both high avidity CD8 T and B cell immunity with greater protective efficacy

We have established that the efficacy of a heterologous poxvirus vectored HIV vaccine, fowlpox virus (FPV)-HIV gag/pol prime followed by attenuated vaccinia virus (VV)-HIV gag/pol booster immunisation, is strongly influenced by the cytokine milieu at the priming vaccination site, with endogenous IL-13 detrimental to the quality of the HIV specific CD8+ T cell response induced. We have now developed a novel HIV vaccine that co-expresses a C-terminal deletion mutant of the mouse IL-4, deleted for the essential tyrosine (Y119) required for signalling. In our vaccine system, the mutant IL-4C118 can bind to IL-4 type I and II receptors with high affinity, and transiently prevent the signalling of both IL-4 and IL-13 at the vaccination site. When this IL-4C118 adjuvanted vaccine was used in an intranasal rFPV/intramuscular rVV prime-boost immunisation strategy, greatly enhanced mucosal/systemic HIV specific CD8+ T cells with higher functional avidity, expressing IFN-γ, TNF-α and IL-2 and greater protective efficacy were detected. Surprisingly, the IL-4C118 adjuvanted vaccines also induced robust long-lived HIV gag-specific serum antibody responses, specifically IgG1 and IgG2a. The p55-gag IgG2a responses induced were of a higher magnitude relative to the IL-13Rα2 adjuvant vaccine. More interestingly, our recently tested IL-13Rα2 adjuvanted vaccine which only inhibited IL-13 activity, even though induced excellent high avidity HIV-specific CD8+ T cells, had a detrimental impact on the induction of gag-specific IgG2a antibody immunity. Our observations suggest that (i) IL-4 cell-signalling in the absence of IL-13 retarded gag-specific antibody isotype class switching, or (ii) IL-13Rα2 signalling was involved in inducing good gag-specific B cell immunity. Thus, we believe our novel IL-4R antagonist adjuvant strategy offers great promise not only for HIV-1 vaccines, but also against a range of chronic infections where sustained high quality mucosal and systemic T and B cell immunity are required for protection.

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection.

Use of an In Vivo FTA Assay to Assess the Magnitude, Functional Avidity and Epitope Variant Cross-Reactivity of T Cell Responses Following HIV-1 Recombinant Poxvirus Vaccination

Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

IL-17A Expression in HIV-Specific CD8 T Cells Is Regulated by IL-4/IL-13 Following HIV-1 Prime-Boost Immunization

Although Th1 and Th2 cytokines can inhibit interleukin (IL)-17-secreting T cells, how these cells are regulated under different infectious conditions is still debated. Our previous studies have shown that vaccination of IL-4 and IL-13 gene knockout (KO) mice can induce high-avidity HIV K(d)Gag197-205-specific CD8 T cells with better protective efficacy. In this study, when IL-13, IL-4, STAT6 KO, and wild-type BALB/c mice were prime-boost immunized with an HIV poxviral modality, elevated numbers of IL-17A(+) splenic K(d)Gag197-205-specific CD8 T cells were observed in all the KO mice compared with the wt BALB/c control. Similarly, when wt BALB/c mice were immunized with IL-13Rα2-adjuvanted HIV vaccines (that transiently inhibited IL-13 activity and induced high-avidity CD8 T cells with enhanced protective efficacy), elevated IL-17A(+) K(d)Gag197-205-specific CD8 T cells were detected both in the lung and the spleen. However, at the transcriptional level, elevated TGF-β, IL-6, ROR-γt, and IL-17A mRNA copy numbers were mainly detected in IL-4 KO, but not the IL-13 KO mice. These data suggested that TGF-β, IL-6, ROR-γt, but not IL-23a, played a role in IL-17A regulation in K(d)Gag197-205-specific CD8 T cells. Collectively, our findings suggest that IL-4 and IL-13 differentially regulate the expression of IL-17A in K(d)Gag197-205-specific CD8 T cells at the transcriptional and translational level, respectively, implicating IL-17A as an indirect modulator of CD8 T cell avidity and protective immunity.

Interleukin-3 and granulocyte-macrophage colony-stimulating factor expression, a biomarker of memory CD8+ T cell immunity and vaccine efficacy

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) exhibit overlapping activities and are produced by activated T cells. In this study, the role of IL-3 and GM-CSF in CD8+ T cells were assessed following HIV-1 prime-boost immunization. Data indicate that the expression of IL-3/GM-CSF by HIV-specific effector CD8+ T cells is vaccine delivery route and time dependent, where purely systemic, intramuscular i.m./i.m. vaccination induce elevated levels of IL-3 in HIV-specific CD8+ T cells compared to purely mucosal, intranasal i.n./i.n. immunization. Interestingly, the GM-CSF expression was optimal only following i.n./i.m. delivery. Data also revealed that peak IL-3 mRNA and protein expression in CD8+ T cells were detected 16-20 h of KdGag197-205 peptide stimulation, whereas the expression kinetics of GM-CSF was similar to IFN-γ. Next the IL-3 and GM-CSF expression in HIV-specific CD8+ T cells were assessed at acute, effector and memory stages of immunity using i.n./i.m. delivery of FPV-HIV/VVHIV control vaccine compared to a novel IL-13Rα2 adjuvanted HIV-vaccine (FPV-HIV IL-13Rα2/VV-HIV IL-13Rα) that has shown to induce excellent high avidity CD8+ T cells with greater protective immunity. The IL-13Rα2 adjuvanted vaccine induced greatly elevated HIV-specific memory CD8+IL-3+ and also CD8+IL-3+IFN-γ+ T cells compared to the control vaccine, where the expression in memory phase was greater than effector T cells. Both the control and IL- 13Rα2 adjuvanted vaccines, elicited elevated but similar numbers of antigen-specific GM-CSF+ memory CD8+ T cells. Data suggest that induction of both IL-3 and GM-CSF play a role in maintenance of antigen-specific memory CD8+ T cells which is linked to better protective immunity. These results also demonstrate that route of delivery, time post vaccination, expression kinetics/ length of antigen exposure, should not be neglected when evaluating the vaccine efficacy. Also IL-3/GM-CSF expression by memory CD8+ T cells could be a biomarker of protective immunity.