Toward DNA-Based T-Cell Mediated Vaccines to Target HIV-1 and Hepatitis C Virus: Approaches to Elicit Localized Immunity for Protection.

Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are major contributors to the global disease burden with many experts recognizing the requirement of an effective vaccine to bring a durable end to these viral epidemics. The most promising vaccine candidates that have advanced into pre-clinical models and the clinic to eliminate or provide protection against these chronic viruses are viral vectors [e.g., recombinant cytomegalovirus, Adenovirus, and modified vaccinia Ankara (MVA)]. This raises the question, is there a need to develop DNA vaccines against HIV-1 and HCV? Since the initial study from Wolff and colleagues which showed that DNA represents a vector that can be used to express transgenes durably in vivo, DNA has been regularly evaluated as a vaccine vector albeit with limited success in large animal models and humans. However, several recent studies in Phase I-IIb trials showed that vaccination of patients with recombinant DNA represents a feasible therapeutic intervention to even cure cervical cancer, highlighting the potential of using DNA for human vaccinations. In this review, we will discuss the limitations and the strategies of using DNA as a vector to develop prophylactic T cell-mediated vaccines against HIV-1 and HCV. In particular, we focus on potential strategies exploiting DNA vectors to elicit protective localized CD8+ T cell immunity in the liver for HCV and in the cervicovaginal mucosa for HIV-1 as localized immunity will be an important, if not critical component, of an efficacious vaccine against these viral infections.

Identification of biomarkers to measure HIV-specific mucosal and systemic CD8(+) T-cell immunity using single cell Fluidigm 48.48 Dynamic arrays.

Thirty genes composed of cytokines, chemokines, granzymes, perforin and integrins were evaluated in gut and splenic K(d)Gag197-205-specific single CD8(+) T cells using Fluidigm 48.48 Dynamic arrays, with the aim of identifying biomarkers to predict effective mucosal and systemic vaccine efficacy. The mRNA expression profiles were analyzed in three ways: (i) the "number" of K(d)Gag197-205-specific CD8(+) T cells expressing the biomarker, (ii) "level" of mRNA expression using principal component analysis (PCA) and (iii) poly-functionality in relation to RANTES expression. In total, 21 genes were found to be differentially expressed between the vaccine groups and the immune compartments tested. Overall, the PCA indicated that IL-13Rα2 or IL-4R antagonist adjuvanted vaccines that previously induced high-avidity mucosal/systemic CD8(+) T cells with better protective efficacy, the "level" of mRNA expression, specifically RANTES, MIP-1ß, and integrin α4 in gut K(d)Gag197-205-specific single CD8(+) T cells, were significantly elevated compared to unadjuvanted vaccine. Furthermore, significantly elevated granzymes/perforin levels were detected in IL-13(-/-) mice given the unadjuvanted vaccine, indicating that the degree of IL-13 inhibition (total, transient or no inhibition) can considerably alter the level of T-cell activity/poly-functionality. When splenic- and gut-K(d)Gag197-205-specific CD8(+) T cells were compared, PC1 vs. PC2 scores revealed that not only RANTES, MIP-1ß, and integrin α4 mRNA, but also perforin, granzymes A/B, and integrins ß1 and ß2 mRNA were elevated in spleen. Collectively, data suggest that RANTES, MIP-1ß, perforin, and integrins α4, ß1 and ß7 mRNA in single HIV-specific CD8(+) T cells could be used as a measure of effective mucosal and systemic vaccine efficacy.

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 receptors: Roles in immunity and powerful vaccine adjuvants.

The roles of interleukin (IL)-4 and IL-13 during both innate and adaptive Th2 mediated immunity have received considerable scrutiny, however, mechanisms by which these cytokines influence the cellular interactions involved in negatively modulating the development of effective Th1 immunity are poorly characterized. In this article we discuss the recent advances in IL-4/IL-13 biology, mainly (i) role of these cytokines in allergic inflammation, atopic dermatitis, cancer, transplant rejection, bacterial/viral infections, and specifically the therapeutic potential of IL-13Rα2, (ii) insights into how "alarmin" stimulation activate IL-4/IL-13 at the lung mucosae, (iii) how these two cytokines modulate antigen-specific CD8(+) T cell quality/avidity in a vaccine route dependent manner and (iv) finally discuss the potential of using transient inhibition of IL-4 and/or IL-13 at the vaccination site as a platform vaccine technology to induce strong sustained high quality CD8(+) T cell immunity for protection against many chronic mucosal pathogens such as HIV-1.

New advances in mucosal vaccination.

The ICI 2013 Mucosal Vaccine Workshop presentations covered a wide range of topics, these mainly fell into three categories: (i) Understanding the interactions of host and microbes, specifically commensal pathogens and improving the antigen uptake via the (microfold cells) M cells to induce effective IgA antibody immunity at the gut mucosa; (ii) effective plant-based vaccines and (iii) development of prophylactic and therapeutic mucosal-based vaccine strategies for virus infections such as human immunodeficiency virus (HIV), influenza and human papillomavirus (HPV) associated head and neck cancers. How to improve the efficacy of oral vaccines, novel intranasal mucosal adjuvants and a unique intra-cheek delivery method were also discussed. Presenters emphasized the differences associated with systemic and mucosal vaccination, specifically, how mucosal vaccines unlike systemic delivery can induce effective immunity at the first line of defence. Collectively, the workshop provided insights into recent developments in the mucosal vaccine research field, highlighting the complexities associated with designing safe and effective mucosal vaccines.

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.

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.

Fluorescent target array T helper assay: a multiplex flow cytometry assay to measure antigen-specific CD4+ T cell-mediated B cell help in vivo.

CD4(+) T cells play a central role in regulating the immune response. Their effector function is commonly assessed by their capacity to secrete cytokines detected by ELISPOT and intracellular cytokine staining. However, one aspect of their effector function that is often overlooked is their ability to help activation of cognate B cells directly, a process that is initiated through the engagement of their T cell-receptor (TCR) with cognate peptide presented on major histocompatibility complex class II (MHC-II) molecules by B cells. Here we report a method to monitor CD4(+) T cell-mediated B cell help in vivo using a multiplex high throughput assay. This assay utilizes a fluorescent target array (FTA), which is composed of lymphocytes labeled with numerous (>200) unique fluorescence signatures that can be delineated in a single recipient animal based on combination labeling with the three vital dyes carboxyfluorescein diacetate succinimidyl ester (CFSE), CellTrace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD). By pulsing different B cell populations in a FTA with titrated amounts of cognate MHC-II binding peptides, CD4(+) T cell help could be assessed by measuring induction of the B cell activation markers CD69 and CD44 by antibody labeling and flow cytometry. We call this the "FTA T helper assay", and have found it to be a robust and sensitive assay to measure CD4(+) T cell helper activity across a multitude of peptide-pulsed B "target" cells in real time in vivo. Furthermore, the technique can be used simultaneously with the FTA killing assay that measures cytotoxic T cell function, to provide a comprehensive tool for measuring both CD4(+) and CD8(+) T cell activity during an immune response in vivo.

Immunisation route-dependent expression of IL-4/IL-13 can modulate HIV-specific CD8(+) CTL avidity.

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-gamma, IL-4 and/or IL-13 by K(d)Gag(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(d)Gag(197-205)-specific CTL was also regulated by IL-4/IL-13.

Differential effects of the type I interferons alpha4, beta, and epsilon on antiviral activity and vaccine efficacy.

The type I IFNs exert a range of activities that include antiviral, antiproliferative, and immunomodulatory effects. To study this further, we have constructed recombinant vaccinia viruses expressing HIV or hemagglutinin (HA) Ags along with murine type I IFNs, IFN-alpha(4) (HA-VV-IFN-alpha(4)), IFN-beta (HA-VV-IFN-beta), or IFN-epsilon (HIV-VV-IFN-epsilon), a recently discovered member of this family. Our aims were to characterize IFN-epsilon functionality as a type I IFN and also to study the biological properties of these factors toward the development of safer and more effective vector-based vaccines. HIV-VV-IFN-epsilon and HA-VV-IFN-beta grew to lower titers than did their parental controls in murine cell lines. In vivo, however, HIV-VV-IFN-epsilon growth was not attenuated, while IFN-beta demonstrated potent local antiviral activity with no replication of HA-VV-IFN-beta detected. Flow cytofluorometric analysis of B lymphocytes incubated with virally encoded IFN-epsilon showed up-regulation of activation markers CD69 and CD86, while RT-PCR of IFN-epsilon-treated cells revealed that gene expression levels of antiviral proteins were elevated, indicating the induction of an antiviral state. The use of these constructs in a poxvirus prime-boost immunization regime led to robust humoral and cellular immune responses against the encoded Ags, despite the lack of replication in the case of HA-VV-IFN-beta. Thus, coexpression of these factors may be beneficial in the design of safer vector-based vaccines. Our data also indicate that while IFN-epsilon exhibits certain biological traits similar to other type I IFNs, it may also have a specific role in mucosal immune regulation that is quite distinct.

Mucosal HIV-1 pox virus prime-boost immunization induces high-avidity CD8+ T cells with regime-dependent cytokine/granzyme B profiles.

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 K(d)Gag(197-205) (amino acid sequence AMQMLKETI; H-2K(d) binding) compared with i.m. immunization regime. Single-cell RT-PCR of K(d)Gag(197-205)-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.

4-1BBL coexpression enhances HIV-specific CD8 T cell memory in a poxvirus prime-boost vaccine.

We have constructed a recombinant fowlpox virus expressing HIV antigens and the costimulatory molecule 4-1BBL. When included in the boost, but not the prime of a poxvirus prime-boost strategy, 4-1BBL significantly enhanced the anti-HIV T cell response generated to this vaccination in BALB/c mice, as detected by ex vivo IFNgamma ELISPOT responses, intracellular cytokine staining to HIV Gag antigens, and enumeration of Gag-reactive CD8 T cells. 4-1BBL however, is not capable of modulating the CD4 T cell response, nor the antibody response to this vaccination strategy. Enhancement of the T cell response by 4-1BBL continues into the memory phase, as detected 2 months post vaccination. This data is the first to show modulation of the immune response to a viral vaccine by coexpression of 4-1BBL and supports this strategy as an exciting approach for enhancement of T cell memory in prime-boost vaccines.

Development of a synthetic consensus sequence scrambled antigen HIV-1 vaccine designed for global use.

Induction of high levels of broadly reactive cytotoxic T lymphocytes (CTL) remains a promising approach for an effective HIV-1 vaccine. We have developed a novel genetic-based vaccine strategy that encodes consensus overlapping peptide sets from all HIV-1 proteins scrambled together. This synthetic scrambled antigen vaccine (SAVINE) strategy has significant advantages, e.g. capacity to encode more antigens safely and is very flexible compared to traditional isolate-based strategies. The SAVINE vaccine strategy is clearly immunogenic, being able to restimulate a range of human HIV-1 specific responses in vitro and induce HIV-1 specific immunity in vivo in mice. Interestingly, different in vivo delivery strategies affected the resulting immunity and immunodominance pattern in mice. This platform strategy could be used for other infections and cancers where T cell responses are important for protection.

Mucosally-administered human-simian immunodeficiency virus DNA and fowlpoxvirus-based recombinant vaccines reduce acute phase viral replication in macaques following vaginal challenge with CCR5-tropic SHIVSF162P3.

Further advances are required in understanding protection from AIDS by T cell immunity across mucosal sites of virus transmission. We analysed a set of multigenic HIV and SHIV DNA and Fowlpoxvirus (FPV) prime and boost vaccines for immunogenicity and protective efficacy in outbred pigtail macaques when delivered via mucosal surfaces (intranasally or intrarectally). Intranasally delivered DNA, even when adjuvanted and given as a fine droplet spray, was neither immunogenic nor protective in macaques. Some protection from acute infection with a pathogenic vaginal SHIVSF162P3 challenge was, however, observed with a regimen involving intramuscular DNA vaccine priming followed by either intranasally or intrarectally delivered rFPV boosting. Interestingly, animals boosted with rFPV vaccine via either of these mucosal routes had poor circulating T cell responses prior to challenge with SHIV compared to those boosted via the intramuscular route. Nevertheless, the mucosally-vaccinated animals generated equivalent anamnestic mucosal and systemic SHIV-specific CD4 and CD8 T cell responses following SHIV administration, with significant reduction in acute plasma viremia against this vaginal challenge. Our data suggest strategies for effective priming of partial immunity to mucosal HIV-1 exposure utilizing systemic prime and mucosal boost vaccination strategies.