HIV-1 vaccine development : evaluating mechanisms of CD8+ T cell avidity

CD8+ T cells play pivotal roles in anti-viral immunity. In particular, CD8+ T cells that make high avidity interactions with virus-infected cells are extremely efficient in controlling virus infections including human immunodeficiency virus (HIV)-1. Therefore, induction of high avidity anti-viral CD8+ T cells following vaccination is expected to be beneficial for conferring protection against HIV-1. Our laboratory has shown that mucosal HIV-1 recombinant pox viral prime-boost vaccination can induce HIV-specific CD8+ T cells with reduced interleukin (IL)-4 and IL-13 cytokine expression and higher avidity compared to systemic vaccine delivery. To understand how these cytokines regulated CD8+ T cell avidity, the PhD studies initially evaluated the expression of receptors for these cytokines on immune cells using flow cytometry following a range of viral infections (e.g. pox viruses and influenza virus) in mice. Results indicated that unlike other IL-4/IL-13 receptor subunits, IL-4 receptor alpha (IL-4Ra) was significantly down-regulated on anti-viral CD8+ T cells in a T cell receptor (TCR) and cognate antigen dependent manner. These studies also showed that up-regulation of IL-4Ra on naive CD8+ T cells most likely resulting from signal transducer and activator of transcription 6 (STAT6) signaling correlated with poor avidity anti-viral CD8+ T cell cytokine responses. Poor avidity anti-viral CD8+ T cells that developed in this instance as well as following systemic HIV-1 recombinant pox viral prime-boost vaccination expressed lower CD8 co-receptor densities. Interestingly, mucosal and IL-13 inhibitor HIV-1 recombinant pox viral prime-boost vaccination strategies prevented significant down-regulation of CD8 densities and enhanced avidity of anti-viral CD8+ T cells. Collectively, data suggest that enhancing responsiveness of naive CD8+ T cells to IL-4 and IL-13 (i.e. up-regulation of IL-4Ra) primes poor avidity anti-viral CD8+ T cells with reduced CD8 densities during virus infection and recombinant HIV-1 pox viral prime-boost vaccination. Given the poor capacity of current assays to evaluate CD8+ T cell avidity in vivo following HIV-1 vaccination, the current PhD studies also evaluated the use of a fluorescent target array (FTA) for this purpose. The FTA assay involves measuring in vivo T cell responses against peptide-pulsed splenocytes (targets) that have unique fluorescent signatures (e.g. over 200 signatures) after injection into vaccinated mice. This assay was utilized to effectively screen for 24 HIV-1 recombinant pox viral vaccination regimes for the capacity to induce high avidity and epitope variant cross-reactive CD8+ T cells as well as T helper responses in vivo. Overall, the FTA was found to be an extremely versatile assay for screening vaccines that could induce high quality T cell responses in vivo using pre-clinical models. Currently, the lack of knowledge regarding mechanisms that affect CD8+ T cell avidity and methods that evaluate CD8+ T cell avidity in vivo is a major barrier for developing efficacious HIV-1 vaccines. Therefore, the findings from the PhD research studies regarding how IL-4 and IL-13 regulate avidity and the use of a FTA to measure avidity in vivo could be exploited to foster future development of more efficacious HIV-1 vaccines

Unraveling the Convoluted Biological Roles of Type I Interferons in Infection and Immunity: A Way Forward for Therapeutics and Vaccine Design

It has been well-established that type I interferons (IFN-Is) have pleiotropic effects and play an early central role in the control of many acute viral infections. However, their pleiotropic effects are not always beneficial to the host and in fact several reports suggest that the induction of IFN-Is exacerbate disease outcomes against some bacterial and chronic viral infections. In this brief review, we probe into this mystery and try to develop answers based on past and recent studies evaluating the roles of IFN-Is in infection and immunity as this is vital for developing effective IFN-Is based therapeutics and vaccines. We also discuss the biological roles of an emerging IFN-I, namely IFN-ε, and discuss its potential use as a mucosal therapeutic and/or vaccine adjuvant. Overall, we anticipate the discussions generated in this review will provide new insights for better exploiting the biological functions of IFN-Is in developing efficacious therapeutics and vaccines in the future.This work was supported by Australian National Health and Medical Research Council project grant award 525431 (Charani Ranasinghe) and ACH2 EOI grants (Charani Ranasinghe)

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.This work was supported by the Australian National Health and Medical Research Council project grant award 525431 (CR) and development grant award APP1000703 (CR), the Australian Centre for Hepatitis and HIV Virology EOI grant 2010/12 (CR&RJ), and Bill and Melinda Gates Foundation GCE Phase I grant OPP1015149 (CR)

MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα(-/-) (I-Aα(-/-)), MHC-IIAβ(-/-) (I-β(-/-)) and the double knockout (I-Aαxβ(-/-)). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα(-/-), but not in I-β(-/-) or I-Aαxβ(-/-), mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα(-/-) mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα(-/-) bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα(-/-) mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα(-/-) compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα(-/-) mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.This work was supported by institutional research support to Prof Mullbacher laboratory at the John Curtin School of Medical Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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.This work was supported by the Australian National Health and Medical Research Council project grant award 525431 (CR) and development grant award APP1000703 (CR) and the Australian Centre for Hepatitis and HIV Virology EOI grant 2010 (CR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Emerging targets for developing T cell-mediated vaccines for human immunodeficiency virus (HIV)-1

Human immunodeficiency virus (HIV)-1 has infected >75 million individuals globally, and, according to the UN, is responsible for ~2.1 million new infections and 1.1 million deaths each year. Currently, there are ~37 million individuals with HIV infection and the epidemic has already resulted in 35 million deaths. Despite the advances of anti-retroviral therapy (ART), a cost-effective vaccine remains the best long-term solution to end the HIV-1 epidemic especially given that the vast majority of infected individuals live in poor socio-economic regions of the world such as Sub-Saharan Africa which limits their accessibility to ART. The modest efficacy of the RV144 Thai trial provides hope that a vaccine for HIV-1 is possible, but as markers for sterilizing immunity are unknown, the design of an effective vaccine is empirical, although broadly cross-reactive neutralizing antibodies (bNAb) that can neutralize various quasispecies of HIV-1 are considered crucial. Since HIV-1 transmission often occurs at the genito-rectal mucosa and is cell-associated, there is a need to develop vaccines that can elicit CD8+ T cell immunity with the capacity to kill virus infected cells at the genito-rectal mucosa and the gut. Here we discuss the recent progress made in developing T cell-mediated vaccines for HIV-1 and emphasize the need to elicit mucosal tissue-resident memory CD8+ T (CD8+ Trm) cells. CD8+ Trm cells will likely form a robust front-line defense against HIV-1 and eliminate transmitter/founder virus-infected cells which are responsible for propagating HIV-1 infections following transmission in vast majority of cases.From the National Health and Medical Research Council (NHMRC): grants APP1026293 (EG), APP525431 (CR), APP543139 (EG), and APP543143 (EG). From the Australian Centre for HIV and Hepatitis Virology Research, CR received an EOI gran

MHC class II-alpha chain knockout mice support increased viral replication that is independent of their lack of MHC class II cell surface expression and associated immune function deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.Mohammed Alsharifi, Aulikki Koskinen, Danushka K. Wijesundara, Jayaram Bettadapura, Arno Müllbache

Unravelling the convoluted biological roles of Type I interferons (IFN-Is) in infection and immunity: a way forward for therapeutics and vaccine design

It has been well-established that type I interferons (IFN-Is) have pleiotropic effects and play an early central role in the control of many acute viral infections. However, their pleiotropic effects are not always beneficial to the host and in fact several reports suggest that the induction of IFN-Is exacerbate disease outcomes against bacterial and chronic viral infections. In this brief review, we probe into this mystery and try to develop answers based on past and recent studies evaluating the roles of IFN-Is in infection and immunity as this is vital for developing effective IFN-Is based therapeutics and vaccines. We also discuss the biological roles of an emerging IFN-I, namely IFN-ε, and discuss its potential use as a mucosal therapeutic and/or vaccine adjuvant. Overall, we anticipate the discussions generated in this review will provide new insights for better exploiting the biological functions of IFN-Is in developing efficacious therapeutics and vaccines in the future

Prime boost regimens for enhancing immunity: magnitude, quality of mucosal and systemic gene vaccines

The use of therapeutic or prophylactic vaccines against numerous infectious agents has been well documented. The hallmark of most vaccine strategies is to mimic infection and induce immunological memory that has the potential to confer protection. The use of live attenuated viruses, killed viruses and recombinant viral proteins for vaccination are classical vaccine approaches that have successfully induced protective antibody and cell mediated immune responses (CMI) against viruses such as small pox, influenza, polio, mumps, measles, and rubella. Despite the successes of classical vaccine approaches, these have been ineffective against chronic and highly pathogenic diseases such as human immunodeficiency virus-1 (HIV-1), tuberculosis (TB) or malaria. Hence, this paved the way for the emergence of range of novel vaccine approaches. In particular, the use of recombinant vector technologies such as the use of recombinant DNA (rDNA) or recombinant live virus vectors encoding single or multiple epitopes used in prime-boost vaccination strategies have exhibited immense potential for generating excellent immunity against intracellular pathogens such as M. tuberculosis, HIV-1, simian immunodeficiency virus (SIV), Plasmodium, Leishmania, S. mansoni, hepatitis C virus, herpes simplex virus and hepatitis B virus. In this chapter, we discuss the use of gene based heterologous prime-boost immunisation strategies for improved vaccination against intracellular pathogens, particularly with respect to HIV-1, given that prime-boost vaccine strategies have been extensively tested and studied against this virus in animal models as well as in humans

Fluorescent target array killing assay: A multiplex cytotoxic T-cell assay to measure detailed T-cell antigen specificity and avidity in vivo

Here we describe a multiplex, fluorescence-based, in vivo cytotoxic T-cell assay using the three vital dyes carboxyfluorescein diacetate succinimidyl ester, cell trace violet, and cell proliferation dye efluor 670. When used to label cells in combination, these dyes can discriminate >200 different target cell populations in the one animal due to each target population having a unique fluorescence signature based on fluorescence intensity and the different emission wavelengths of the dyes. This allows the simultaneous measurement of the in vivo killing of target cells pulsed with numerous peptides at different concentrations and the inclusion of many replicates. This fluorescent target array killing assay can be used to measure the fine antigen specificity and avidity of polyclonal cytotoxic T-cell responses in vivo, immunological parameters that were previously impossible to monitor