Shubhanshi Trivedi

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.

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.

Human mucosal-associated invariant T (MAIT) cells possess capacity for B cell help

Mucosal‐associated invariant T (MAIT) cells are an innate‐like T cell subset, restricted by the nonclassic MHC class I‐related protein MR1 and enriched at mucosal sites. Human studies have shown an association between MAIT cells and pathogen‐specific antibody responses. In this study, we investigate the effect of human MAIT cells on B cells ex vivo. We found that supernatants from microbe‐ or cytokine‐stimulated MAIT cells, when added to purified autologous B cells, increase frequencies of plasmablasts and promote IgA, IgG, and IgM production. We found effects to be mostly MR1‐dependent and that the increases in plasmablasts are likely a result of increased differentiation from memory B cells. Furthermore, microbe‐activated MAIT cell supernatant contains multiple cytokines known to stimulate B cells, including IL‐6, ‐10, and ‐21. This study thus provides the first direct evidence of a newly identified role of MAIT cells in providing help to B cells.

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.

The Influence of Immunization Route, Tissue Microenvironment, and Cytokine Cell Milieu on HIV-Specific CD8 + T Cells Measured Using Fluidigm Dynamic Arrays

Thirty different genes including cytokines, chemokines, granzymes, perforin and specifically integrins were evaluated in Peyers patch-KdGag197–205-specific CD8+ T cells (pools of 100 cells) using Fluidigm 48.48 Dynamic arrays following three different prime-boost immunization strategies. Data revealed that the route of prime or the booster immunization differentially influenced the integrin expression profile on gut KdGag197–205-specific CD8+ T cells. Specifically, elevated numbers of integrin αE and αD expressing gut KdGag197–205-specific CD8+ T cells were detected following mucosal but not systemic priming. Also, αE/β7 and αD/β2 heterodimerization were more noticeable in an intranasal (i.n.)/i.n. vaccination setting compared to i.n./intramuscular (i.m) or i.m./i.m. vaccinations. Moreover, in all vaccine groups tested α4 appeared to heterodimerize more closely with β7 then β1. Also MIP-1β, RANTES, CCR5, perforin and integrin α4 bio-markers were significantly elevated in i.n./i.m. and i.m./i.m. immunization groups compared to purely mucosal i.n./i.n. delivery. Furthermore, when wild type (WT) BALB/c and IL-13 knockout (KO) mice were immunized using i.n./i.m. strategy, MIP-1α, MIP-1β, RANTES, integrins α4, β1 and β7 mRNA expression levels were found to be significantly different, in mucosal verses systemic KdGag197–205-specific CD8+ T cells. Interestingly, the numbers of gut KdGag197–205-specific CD8+ T cells expressing gut-homing markers α4β7 and CCR9 protein were also significantly elevated in IL-13 KO compared to WT control. Collectively, our findings further corroborate that the route of vaccine delivery, tissue microenvironment and IL-13 depleted cytokine milieu can significantly alter the antigen-specific CD8+ T cell gene expression profiles and in turn modulate their functional avidities as well as homing capabilities.

Recombinant fowlpox virus vector-based vaccines: Expression kinetics, dissemination and safety profile following intranasal delivery.

We have previously established that mucosal uptake of recombinant fowlpox virus (rFPV) vaccines is far superior to other vector-based vaccines. Specifically, intranasal priming with rFPV vaccines can recruit unique antigen-presenting cells, which induce excellent mucosal and systemic HIV-specific CD8+ T-cell immunity. In this study, we have for the first time investigated the in vivo dissemination, safety and expression kinetics of rFPV post intranasal delivery using recombinant viruses expressing green fluorescent protein or mCherry. Both confocal microscopy of tissue sections using green fluorescent protein and in vivo Imaging System (IVIS) spectrum live animal and whole organ imaging studies using mCherry revealed that (i) the peak antigen expression occurs 12 to 24 h post vaccination and no active viral gene expression is detected 96 h post vaccination. (ii) The virus only infects the initial vaccination site (lung and nasal cavity) and does not disseminate to distal sites such as the spleen or gut. (iii) More importantly, rFPV does not cross the olfactory receptor neuron pathway. Collectively, our findings indicate that rFPV vector-based vaccines have all the hallmarks of a safe and effective mucosal delivery vector, suitable for clinical evaluation.