Blake Frey

Vaccine-induced myeloid cell population dampens protective immunity to SIV

Vaccines are largely evaluated for their ability to promote adaptive immunity, with little focus on the induction of negative immune regulators. Adjuvants facilitate and enhance vaccine-induced immune responses and have been explored for mediating protection against HIV. Using a regimen of peptide priming followed by a modified vaccinia Ankara (MVA) boost in a nonhuman primate model, we found that an SIV vaccine incorporating molecular adjuvants mediated partial protection against rectal SIVmac251 challenges. Animals treated with vaccine and multiple adjuvants exhibited a reduced viral load (VL) compared with those treated with vaccine only. Surprisingly, animals treated with adjuvant alone had reduced VLs that were comparable to or better than those of the vaccine-treated group. VL reduction was greatest in animals with the MHC class I allele Mamu-A*01 that were treated with adjuvant only and was largely dependent on CD8+ T cells. Early VLs correlated with Ki67+CCR5+CD4+ T cell frequency, while set-point VL was associated with expansion of a myeloid cell population that was phenotypically similar to myeloid-derived suppressor cells (MDSCs) and that suppressed T cell responses in vitro. MDSC expansion occurred in animals receiving vaccine and was not observed in the adjuvant-only group. Collectively, these results indicate that vaccine-induced MDSCs inhibit protective cellular immunity and suggest that preventing MDSC induction may be critical for effective AIDS vaccination.

Comparative analysis of SIV-specific cellular immune responses induced by different vaccine platforms in rhesus macaques

To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.

Low Antigen Dose in Adjuvant-Based Vaccination Selectively Induces CD4 T Cells with Enhanced Functional Avidity and Protective Efficacy

T cells with high functional avidity can sense and respond to low levels of cognate Ag, a characteristic that is associated with more potent responses against tumors and many infections, including HIV. Although an important determinant of T cell efficacy, it has proven difficult to selectively induce T cells of high functional avidity through vaccination. Attempts to induce high-avidity T cells by low-dose in vivo vaccination failed because this strategy simply gave no response. Instead, selective induction of high-avidity T cells has required in vitro culturing of specific T cells with low Ag concentrations. In this study, we combined low vaccine Ag doses with a novel potent cationic liposomal adjuvant, cationic adjuvant formulation 09, consisting of dimethyldioctadecylammonium liposomes incorporating two immunomodulators (monomycolyl glycerol analog and polyinosinic-polycytidylic acid) that efficiently induces CD4 Th cells, as well as cross-primes CD8 CTL responses. We show that vaccination with low Ag dose selectively primes CD4 T cells of higher functional avidity, whereas CD8 T cell functional avidity was unrelated to vaccine dose in mice. Importantly, CD4 T cells of higher functional avidity induced by low-dose vaccinations showed higher cytokine release per cell and lower inhibitory receptor expression (PD-1, CTLA-4, and the apoptosis-inducing Fas death receptor) compared with their lower-avidity CD4 counterparts. Notably, increased functional CD4 T cell avidity improved antiviral efficacy of CD8 T cells. These data suggest that potent adjuvants, such as cationic adjuvant formulation 09, render low-dose vaccination a feasible and promising approach for generating high-avidity T cells through vaccination.

Humoral immunity induced by mucosal and/or systemic SIV-specific vaccine platforms suggests novel combinatorial approaches for enhancing responses.

Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in the bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest that combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.

Paradoxical myeloid-derived suppressor cell reduction in the bone marrow of SIV chronically infected macaques

Myeloid derived suppressor cells (MDSCs), which suppress anti-tumor or anti-viral immune responses, are expanded in the peripheral blood and tissues of patients/animals with cancer or viral infectious diseases. We here show that in chronic SIV infection of Indian rhesus macaques, the frequency of MDSCs in the bone marrow (BM) was paradoxically and unexpectedly decreased, but increased in peripheral blood. Reduction of BM MDSCs was found in both CD14+MDSC and Lin-CD15+MDSC subsets. The reduction of MDSCs correlated with high plasma viral loads and low CD4+ T cell counts, suggesting that depletion of BM MDSCs was associated with SIV/AIDS disease progression. Of note, in SHIVSF162P4-infected macaques, which naturally control viral replication within a few months of infection, the frequency of MDSCs in the bone marrow was unchanged. To investigate the mechanisms by which BM MDSCs were reduced during chronic SIV infection, we tested several hypotheses: depletion due to viral infection, alterations in MDSC trafficking, and/or poor MDSC replenishment. We found that the possible mobilization of MDSCs from BM to peripheral tissues and the slow self-replenishment of MDSCs in the BM, along with the viral infection-induced depletion, all contribute to the observed BM MDSC reduction. We first demonstrate MDSC SIV infection in vivo. Correlation between BM CD14+MDSC reduction and CD8+ T cell activation in tissues is consistent with decreased immune suppression by MDSCs. Thus, depletion of BM MDSCs may contribute to the pathologic immune activation during chronic SIV infection and by extension HIV infection.

Lack of the programmed death‐1 receptor renders host susceptible to enteric microbial infection through impairing the production of the mucosal natural killer cell effector molecules

The programmed death‐1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death‐1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death‐1 expression is increased on conventional natural killer cells but not on CD4+, CD8+ or natural killer T cells, or CD11b+ or CD11c+ macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death‐1 or treated with anti–programmed death‐1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild‐type but not programmed death‐1–deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death‐1–deficient mice had impaired expression of those mediators. Consistent with programmed death‐1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin‐deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death‐1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.

Early SIV Dissemination After Intrarectal SIVmac251 Challenge Was Associated With Proliferating Virus-Susceptible Cells in the Colorectum.

Objective:Few studies have examined the eclipse time of simian immunodeficiency virus/HIV infection through the anal route. We aimed to measure the eclipse time after SIVmac251 intrarectal inoculation, and to investigate the factor(s) associated with early dissemination. Design:Forty macaques were intrarectally challenged with SIVmac251 3 times at 2-week intervals. Methods:Plasma viral RNA was monitored at 4, 7, 11, 14, 21, and 28 days after infection. Rectal/vaginal tissues were obtained and tissue viral loads (VLs) were measured at day 14 postinfection. Results:Of 40 macaques 26 (65%) had first detectable viral RNAs in the plasma at day 7 after the challenge that led to productive infection. Strikingly, 6 animals (15%) had detectable viral RNA in the plasma as early as at day 4. The Ki67+ viral target CD4+ T cells in the colorectal tissues were significantly higher in the early or middle-transmitter groups than those in the late-transmitter group. The rectal VL did not correlate with plasma VL at 14-day postinoculation, but did positively correlate with plasma VLs at days 21 and 28 postinfection. Conclusions:The median eclipse time after intrarectal challenge was 7 days, with a few early transmitters at 4 days. More rapid viral dissemination was associated with a high frequency of colorectal Ki67+CCR5+CD4+T cells, which fuel the local viral replication. Furthermore, local viral replication in the colorectal tissue during the early stage might affect the plasma VL in a delayed manner. Therefore, to reduce/limit these target cells at the portal of viral entry is essential.

Interleukin-15 Constrains Mucosal T Helper 17 Cell Generation: Influence of Mononuclear Phagocytes.

Interleukin (IL)-15 has multiple roles in innate and adaptive immunity, especially regarding CD8+ T cells and natural killer cells. However, the role of IL-15 in regulating differentiation of T helper cell subsets and mononuclear phagocytes (MPs) in different tissues in vivo is unknown. Here we report that IL-15 indirectly regulates Th17 but not other Th subsets in the intestinal lamina propria (LP), apparently through effects on MPs. Th17 cells in the LP were more prevalent in IL-15 KO mice than their wild-type counterparts, and less prevalent in IL-15 transgenic mice than their wild-type littermates, even co-caged. MPs from the LP of these mice were sufficient to mimic the in vivo finding in vitro by skewing of cocultured wild type OVA-specific CD4+ T cells. However, production of IL-15 or lack thereof by these MPs was not sufficient to explain the skewing, as addition or blockade of IL-15 in the cultures had no effect. Rather, a skewing of the relative proportion of CD11b+, CD103+ and double positive LP MP subsets in transgenic and KO could explain the differences in Th17 cells. Thus, IL-15 may influence MP subsets in the gut in a novel way that alters the frequency of LP Th17 cells.

A panel of correlates predicts vaccine-induced protection of rats against respiratory challenge with virulent Francisella tularensis

There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat-killed LVS, which were poorly protective. These genes included IFN-γ, IL-21, NOS2, LTA, T-bet, IL-12rβ2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5–7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens.

Differential T cell homing to colon vs. small intestine is imprinted by local CD11c+ APCs that determine homing receptors

Mechanisms that imprint T cell homing to the small intestine have been well studied; however, those for homing to the colon are poorly understood. Recently, we found that these are distinct subcompartments of the gut mucosal immune system, which implies differential homing. Here, we show that colonic CD11c+ APCs imprint CD8+ T cell preferential homing to the colon, in contrast to those from the small intestine that imprint CD8+ T cell homing to the small intestine, and that the differences are related to the variable ability of APCs to induce α4β7‐integrin and CCR9 expression on T cells. Colon APCs also expressed lower levels of retinoic acid–producing enzymes that are known to control the mucosal homing of T cells. These findings are the first to our knowledge to directly demonstrate that colon APCs imprint T cells to selectively home to the large bowel, which is critical for the design of successful T cell–based therapies and vaccines, such as colon cancer immunotherapy and HIV vaccines.