Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV replication in M3+ Mauritian cynomolgus macaques initiated on ART two weeks post-infection.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Host Immunity to Mycobacterium tuberculosis Infection Is Similar in Simian Immunodeficiency Virus (SIV)-Infected, Antiretroviral Therapy-Treated and SIV-Naïve Juvenile Macaques.

Pre-existing HIV infection increases tuberculosis (TB) risk in children. Antiretroviral therapy (ART) reduces, but does not abolish, this risk in children with HIV. The immunologic mechanisms involved in TB progression in both HIV-naive and HIV-infected children have not been explored. Much of our current understanding is based on human studies in adults and adult animal models. In this study, we sought to model childhood HIV/Mycobacterium tuberculosis (Mtb) coinfection in the setting of ART and characterize T cells during TB progression. Macaques equivalent to 4 to 8 year-old children were intravenously infected with SIVmac239M, treated with ART 3 months later, and coinfected with Mtb 3 months after initiating ART. SIV-naive macaques were similarly infected with Mtb alone. TB pathology and total Mtb burden did not differ between SIV-infected, ART-treated and SIV-naive macaques, although lung Mtb burden was lower in SIV-infected, ART-treated macaques. No major differences in frequencies of CD4+ and CD8+ T cells and unconventional T cell subsets (Vγ9+ γδ T cells, MAIT cells, and NKT cells) in airways were observed between SIV-infected, ART-treated and SIV-naive macaques over the course of Mtb infection, with the exception of CCR5+ CD4+ and CD8+ T cells which were slightly lower. CD4+ and CD8+ T cell frequencies did not differ in the lung granulomas. Immune checkpoint marker levels were similar, although ki-67 levels in CD8+ T cells were elevated. Thus, ART treatment of juvenile macaques, 3 months after SIV infection, resulted in similar progression of Mtb and T cell responses compared to Mtb in SIV-naive macaques.

Control of Simian Immunodeficiency Virus Infection in Prophylactically Vaccinated, Antiretroviral Treatment-Naive Macaques Is Required for the Most Efficacious CD8 T Cell Response during Treatment with the Interleukin-15 Superagonist N-803.

The IL-15 superagonist N-803 has been shown to enhance the function of CD8 T cells and NK cells. We previously found that in a subset of vaccinated, ART-naive, SIV+ rhesus macaques, N-803 treatment led to a rapid but transient decline in plasma viremia that positively correlated with an increase in the frequency of CD8 T cells. Here, we tested the hypothesis that prophylactic vaccination was required for the N-803 mediated suppression of SIV plasma viremia. We either vaccinated rhesus macaques with a DNA prime/Ad5 boost regimen using vectors expressing SIVmac239 gag with or without a plasmid expressing IL-12 or left them unvaccinated. The animals were then intravenously infected with SIVmac239M. 6 months after infection, the animals were treated with N-803. We found no differences in the control of plasma viremia during N-803 treatment between vaccinated and unvaccinated macaques. Interestingly, when we divided the SIV+ animals based on their plasma viral load set-points prior to the N-803 treatment, N-803 increased the frequency of SIV-specific T cells expressing ki-67+ and granzyme B+ in animals with low plasma viremia (<104 copies/mL; SIV controllers) compared to animals with high plasma viremia (>104 copies/mL; SIV noncontrollers). In addition, Gag-specific CD8 T cells from the SIV+ controllers had a greater increase in CD8+CD107a+ T cells in ex vivo functional assays than did the SIV+ noncontrollers. Overall, our results indicate that N-803 is most effective in SIV+ animals with a preexisting immunological ability to control SIV replication. IMPORTANCE N-803 is a drug that boosts the immune cells involved in combating HIV/SIV infection. Here, we found that in SIV+ rhesus macaques that were not on antiretroviral therapy, N-803 increased the proliferation and potential capacity for killing of the SIV-specific immune cells to a greater degree in animals that spontaneously controlled SIV than in animals that did not control SIV. Understanding the mechanism of how N-803 might function differently in individuals that control HIV/SIV (for example, individuals on antiretroviral therapy or spontaneous controllers) compared to settings where HIV/SIV are not controlled, could impact the efficacy of N-803 utilization in the field of HIV cure.

Pre-existing Simian Immunodeficiency Virus Infection Increases Expression of T Cell Markers Associated with Activation during Early Mycobacterium tuberculosis Coinfection and Impairs TNF Responses in Granulomas.

Tuberculosis (TB) is the leading infectious cause of death among people living with HIV. People living with HIV are more susceptible to contracting Mycobacterium tuberculosis and often have worsened TB disease. Understanding the immunologic defects caused by HIV and the consequences it has on M. tuberculosis coinfection is critical in combating this global health epidemic. We previously showed in a model of SIV and M. tuberculosis coinfection in Mauritian cynomolgus macaques (MCM) that SIV/M. tuberculosis-coinfected MCM had rapidly progressive TB. We hypothesized that pre-existing SIV infection impairs early T cell responses to M. tuberculosis infection. We infected MCM with SIVmac239, followed by coinfection with M. tuberculosis Erdman 6 mo later. Although similar, TB progression was observed in both SIV+ and SIV-naive animals at 6 wk post-M. tuberculosis infection; longitudinal sampling of the blood (PBMC) and airways (bronchoalveolar lavage) revealed a significant reduction in circulating CD4+ T cells and an influx of CD8+ T cells in airways of SIV+ animals. At sites of M. tuberculosis infection (i.e., granulomas), SIV/M. tuberculosis-coinfected animals had a higher proportion of CD4+ and CD8+ T cells expressing PD-1 and TIGIT. In addition, there were fewer TNF-producing CD4+ T cells in granulomas of SIV/M. tuberculosis-coinfected animals. Taken together, we show that concurrent SIV infection alters T cell phenotypes in granulomas during the early stages of TB disease. As it is critical to establish control of M. tuberculosis replication soon postinfection, these phenotypic changes may distinguish the immune dysfunction that arises from pre-existing SIV infection, which promotes TB progression.

Zika Virus Infection of Pregnant Ifnar1-/- Mice Triggers Strain-Specific Differences in Fetal Outcomes.

Zika virus (ZIKV) is a flavivirus that causes a constellation of adverse fetal outcomes collectively termed congenital Zika syndrome (CZS). However, not all pregnancies exposed to ZIKV result in an infant with apparent defects. During the 2015 to 2016 American outbreak of ZIKV, CZS rates varied by geographic location. The underlying mechanisms responsible for this heterogeneity in outcomes have not been well defined. Therefore, we sought to characterize and compare the pathogenic potential of multiple Asian-/American-lineage ZIKV strains in an established Ifnar1-/- pregnant mouse model. Here, we show significant differences in the rate of fetal demise following maternal inoculation with ZIKV strains from Puerto Rico, Panama, Mexico, Brazil, and Cambodia. Rates of fetal demise broadly correlated with maternal viremia but were independent of fetus and placenta virus titer, indicating that additional underlying factors contribute to fetal outcome. Our results, in concert with those from other studies, suggest that subtle differences in ZIKV strains may have important phenotypic impacts. With ZIKV now endemic in the Americas, greater emphasis needs to be placed on elucidating and understanding the underlying mechanisms that contribute to fetal outcome. IMPORTANCE Zika virus (ZIKV) transmission has been reported in 87 countries and territories around the globe. ZIKV infection during pregnancy is associated with adverse fetal outcomes, including birth defects, microcephaly, neurological complications, and even spontaneous abortion. Rates of adverse fetal outcomes vary between regions, and not every pregnancy exposed to ZIKV results in birth defects. Not much is known about how or if the infecting ZIKV strain is linked to fetal outcomes. Our research provides evidence of phenotypic heterogeneity between Asian-/American-lineage ZIKV strains and provides insight into the underlying causes of adverse fetal outcomes. Understanding ZIKV strain-dependent pathogenic potential during pregnancy and elucidating underlying causes of diverse clinical sequelae observed during human infections is critical to understanding ZIKV on a global scale.

Substrate, temperature, and geographical patterns among nearly 2000 natural yeast isolates.

Yeasts have broad importance as industrially and clinically relevant microbes and as powerful models for fundamental research, but we are only beginning to understand the roles yeasts play in natural ecosystems. Yeast ecology is often more difficult to study compared to other, more abundant microbes, but growing collections of natural yeast isolates are beginning to shed light on fundamental ecological questions. Here, we used environmental sampling and isolation to assemble a dataset of 1962 isolates collected from throughout the contiguous United States of America (USA) and Alaska, which were then used to uncover geographic patterns, along with substrate and temperature associations among yeast taxa. We found some taxa, including the common yeasts Torulaspora delbrueckii and Saccharomyces paradoxus, to be repeatedly isolated from multiple sampled regions of the USA, and we classify these as broadly distributed cosmopolitan yeasts. A number of yeast taxon-substrate associations were identified, some of which were novel and some of which support previously reported associations. Further, we found a strong effect of isolation temperature on the phyla of yeasts recovered, as well as for many species. We speculate that substrate and isolation temperature associations reflect the ecological diversity of and niche partitioning by yeast taxa.

Validation of multiplex PCR sequencing assay of SIV.

BACKGROUND: The generation of accurate and reproducible viral sequence data is necessary to understand the diversity present in populations of RNA viruses isolated from clinical samples. While various sequencing methods are available, they often require high quality templates and high viral titer to ensure reliable data. METHODS: We modified a multiplex PCR and sequencing approach to characterize populations of simian immunodeficiency virus (SIV) isolated from nonhuman primates. We chose this approach with the aim of reducing the number of required input templates while maintaining fidelity and sensitivity. We conducted replicate sequencing experiments using different numbers of quantified viral RNA (vRNA) or viral cDNA as input material. We performed assays with clonal SIVmac239 to detect false positives, and we mixed SIVmac239 and a variant with 24 point mutations (SIVmac239-24X) to measure variant detection sensitivity. RESULTS: We found that utilizing a starting material of quantified viral cDNA templates had a lower rate of false positives and increased reproducibility when compared to that of quantified vRNA templates. This study identifies the importance of rigorously validating deep sequencing methods and including replicate samples when using a new method to characterize low frequency variants in a population with a small number of templates. CONCLUSIONS: Because the need to generate reproducible and accurate sequencing data from diverse viruses from low titer samples, we modified a multiplex PCR and sequencing approach to characterize SIV from populations from non-human primates. We found that increasing starting template numbers increased the reproducibility and decreased the number of false positives identified, and this was further seen when cDNA was used as a starting material. Ultimately, we highlight the importance of vigorously validating methods to prevent overinterpretation of low frequency variants in a sample.

Molecularly barcoded Zika virus libraries to probe in vivo evolutionary dynamics.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a "synthetic swarm" whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.

Acute-Phase CD4+ T Cell Responses Targeting Invariant Viral Regions Are Associated with Control of Live Attenuated Simian Immunodeficiency Virus.

We manipulated SIVmac239Δnef, a model of major histocompatibility complex (MHC)-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) that express the M3 MHC haplotype, which has been associated with poor control of pathogenic simian immunodeficiency virus (SIV). We created SIVΔnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8+ T cell responses targeting variable epitopes and that control is achievable in individuals lacking known "protective" MHC alleles. Full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays identified six newly targeted immunogenic regions following SIVΔnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVΔnef-8x had T cell responses that targeted at least one of the four invariant regions and had a lower set point viral load than two animals that did not have T cell responses that targeted any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8+ T cell responses that target variable regions during SIVmac239Δnef infection, individuals without protective MHC alleles developed predominantly CD4+ T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4+ T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection.IMPORTANCE Studies defining effective cellular immune responses to human immunodeficiency virus (HIV) and SIV have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8+ T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare "elite controllers," may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is of paramount importance to determine whether T cell responses can be redirected toward invariant viral regions in individuals without protective MHC alleles and if these responses improve control of virus replication.

Pathogenic budding yeasts isolated outside of clinical settings.

Budding yeasts are distributed across a wide range of habitats, including as human commensals. However, under some conditions, these commensals can cause superficial, invasive, and even lethal infections. Despite their importance to human health, little is known about the ecology of these opportunistic pathogens, aside from their associations with mammals and clinical environments. During a survey of approximately 1000 non-clinical samples across the United States of America, we isolated 54 strains of budding yeast species considered opportunistic pathogens, including Candida albicans and Candida (Nakaseomyces) glabrata. We found that, as a group, pathogenic yeasts were positively associated with fruits and soil environments, whereas the species Pichia kudriavzevii (syn. Candida krusei syn. Issatchenkia orientalis) had a significant association with plants. Of the four species that cause 95% of candidiasis, we found a positive association with soil. These results suggest that pathogenic yeast ecology is more complex and diverse than is currently appreciated and raises the possibility that these additional environments could be a point of contact for human infections.

Complex Ancestries of Lager-Brewing Hybrids Were Shaped by Standing Variation in the Wild Yeast Saccharomyces eubayanus.

Lager-style beers constitute the vast majority of the beer market, and yet, the genetic origin of the yeast strains that brew them has been shrouded in mystery and controversy. Unlike ale-style beers, which are generally brewed with Saccharomyces cerevisiae, lagers are brewed at colder temperatures with allopolyploid hybrids of Saccharomyces eubayanus x S. cerevisiae. Since the discovery of S. eubayanus in 2011, additional strains have been isolated from South America, North America, Australasia, and Asia, but only interspecies hybrids have been isolated in Europe. Here, using genome sequence data, we examine the relationships of these wild S. eubayanus strains to each other and to domesticated lager strains. Our results support the existence of a relatively low-diversity (π = 0.00197) lineage of S. eubayanus whose distribution stretches across the Holarctic ecozone and includes wild isolates from Tibet, new wild isolates from North America, and the S. eubayanus parents of lager yeasts. This Holarctic lineage is closely related to a population with higher diversity (π = 0.00275) that has been found primarily in South America but includes some widely distributed isolates. A second diverse South American population (π = 0.00354) and two early-diverging Asian subspecies are more distantly related. We further show that no single wild strain from the Holarctic lineage is the sole closest relative of lager yeasts. Instead, different parts of the genome portray different phylogenetic signals and ancestry, likely due to outcrossing and incomplete lineage sorting. Indeed, standing genetic variation within this wild Holarctic lineage of S. eubayanus is responsible for genetic variation still segregating among modern lager-brewing hybrids. We conclude that the relationships among wild strains of S. eubayanus and their domesticated hybrids reflect complex biogeographical and genetic processes.

Transiently boosting Vγ9+Vδ2+ γδ T cells early in Mtb coinfection of SIV-infected juvenile macaques does not improve Mtb host resistance.

Children living with HIV have a higher risk of developing tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Gamma delta (γδ) T cells in the context of HIV/Mtb coinfection have been understudied in children, despite in vitro evidence suggesting γδ T cells assist with Mtb control. We investigated whether boosting a specific subset of γδ T cells, phosphoantigen-reactive Vγ9+Vδ2+ cells, could improve TB outcome using a nonhuman primate model of pediatric HIV/Mtb coinfection. Juvenile Mauritian cynomolgus macaques (MCM), equivalent to 4-8-year-old children, were infected intravenously (i.v.) with SIV. After 6 months, MCM were coinfected with a low dose of Mtb and then randomized to receive zoledronate (ZOL), a drug that increases phosphoantigen levels, (n=5; i.v.) at 3- and 17- days after Mtb accompanied by recombinant human IL-2 (s.c.) for 5 days following each ZOL injection. A similarly coinfected MCM group (n=5) was injected with saline as a control. Vγ9+Vδ2+ γδ T cell frequencies spiked in the blood, but not airways, of ZOL+IL-2-treated MCM following the first dose, however, were refractory to the second dose. At necropsy eight weeks after Mtb, ZOL+IL-2 treatment did not reduce pathology or bacterial burden. γδ T cell subset frequencies in granulomas did not differ between treatment groups. These data show that transiently boosting peripheral γδ T cells with ZOL+IL-2 soon after Mtb coinfection of SIV-infected MCM did not improve Mtb host defense.

CD8+ cells and small viral reservoirs facilitate post-ART control of SIV in Mauritian cynomolgus macaques.

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). For six months after ART withdrawal, we observed undetectable or transient viremia in seven of eight MCMs. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the PTC was mediated, at least in part, by CD8α+ cells. We found that MCMs had smaller acute viral reservoirs than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. The mechanisms by which unusually small viral reservoirs and CD8α+ cell-mediated virus suppression enable PTC can be investigated using this MHC-haplomatched MCM model. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans.

Vaccination with intravenous BCG protects macaques with pre-existing SIV infection from tuberculosis.

Tuberculosis (TB) is the most common cause of death in people living with HIV. BCG delivered intradermally (ID) is the only licensed vaccine to prevent TB. However, it offers little protection from pulmonary TB in adults. Intravenous (IV) BCG, but not ID BCG, confers striking protection against Mycobacterium tuberculosis (Mtb) infection and disease in rhesus macaques. We investigated whether IV BCG could protect against TB in macaques with a pre-existing SIV infection. There was a robust influx of airway T cells following IV BCG in both SIV-infected and SIV-naïve animals, with elevated antibody titers in plasma and airways. Following Mtb challenge, all 7 SIV-naïve and 9 out of 12 SIV-infected vaccinated animals were completely protected, without any culturable bacilli in their tissues. PBMC responses post-challenge indicated early clearance of Mtb in vaccinated animals regardless of SIV infection. These data support that IV BCG is immunogenic and efficacious in SIV-infected animals.

Intravenous Bacille Calmette-Guérin vaccination protects simian immunodeficiency virus-infected macaques from tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the most common cause of death in people living with human immunodeficiency virus (HIV). Intra-dermal Bacille Calmette-Guérin (BCG) delivery is the only licensed vaccine against tuberculosis; however, it offers little protection from pulmonary tuberculosis in adults and is contraindicated in people living with HIV. Intravenous BCG confers protection against Mtb infection in rhesus macaques; we hypothesized that it might prevent tuberculosis in simian immunodeficiency virus (SIV)-infected macaques, a model for HIV infection. Here intravenous BCG-elicited robust airway T cell influx and elevated plasma and airway antibody titres in both SIV-infected and naive animals. Following Mtb challenge, all 7 vaccinated SIV-naive and 9 out of 12 vaccinated SIV-infected animals were protected, without any culturable bacteria detected from tissues. Peripheral blood mononuclear cell responses post-challenge indicated early clearance of Mtb in vaccinated animals, regardless of SIV infection. These data support that intravenous BCG is immunogenic and efficacious in SIV-infected animals.

Spontaneous Control of SIV Replication Does Not Prevent T Cell Dysregulation and Bacterial Dissemination in Animals Co-Infected with M. tuberculosis.

Individuals co-infected with HIV and Mycobacterium tuberculosis (Mtb) are more likely to develop severe tuberculosis (TB) disease than HIV-naive individuals. To understand how a chronic pre-existing Simian immunodeficiency virus (SIV) infection impairs the early immune response to Mtb, we used the Mauritian cynomolgus macaque (MCM) model of SIV/Mtb co-infection. We examined the relationship between peripheral viral control and Mtb burden, Mtb dissemination, and T cell function between SIV+ spontaneous controllers, SIV+ non-controllers, and SIV-naive MCM who were challenged with a barcoded Mtb Erdman strain 6 months post-SIV infection and necropsied 6 weeks post-Mtb infection. Mycobacterial burden was highest in the SIV+ non-controllers in all assessed tissues. In lung granulomas, the frequency of TNF-α-producing CD4+ T cells was reduced in all SIV+ MCM, but IFNγ-producing CD4+ T cells were only lower in the SIV+ non-controllers. Further, while all SIV+ MCM had more PD1+ and TIGIT+ T cells in the lung granulomas relative to SIV-naive MCM, SIV+ controllers exhibited the highest frequency of cells expressing these markers. To measure the effect of SIV infection on within-host bacterial dissemination, we sequenced the molecular barcodes of Mtb present in each tissue and characterized the Mtb population complexity. While Mtb population complexity was not associated with SIV infection group, lymph nodes had increased complexity when compared with lung granulomas across all groups. These results provide evidence that SIV+ animals, independent of viral control, exhibit a dysregulated T cell immune response and enhanced dissemination of Mtb, likely contributing to the poor TB disease course across all SIV/Mtb co-infected animals. IMPORTANCE HIV and TB remain significant global health issues, despite the availability of treatments. Individuals with HIV, including those who are virally suppressed, are at an increased risk to develop and succumb to severe TB disease when compared with HIV-naive individuals. Our study aims to understand the relationship between the extent of SIV replication, mycobacterial growth, and T cell function in the tissues of co-infected Mauritian cynomolgus macaques during the first 6 weeks of Mtb infection. Here we demonstrate that increased viral replication is associated with increased bacterial burden in the tissues and impaired T cell responses, and that the immunological damage attributed to virus infection is not fully eliminated when animals spontaneously control virus replication.

Monkeying around with MAIT Cells: Studying the Role of MAIT Cells in SIV and Mtb Co-Infection.

There were an estimated 10 million new cases of tuberculosis (TB) disease in 2019. While over 90% of individuals successfully control Mycobacterium tuberculosis (Mtb) infection, which causes TB disease, HIV co-infection often leads to active TB disease. Despite the co-endemic nature of HIV and TB, knowledge of the immune mechanisms contributing to the loss of control of Mtb replication during HIV infection is lacking. Mucosal-associated invariant T (MAIT) cells are innate-like T cells that target and destroy bacterially-infected cells and may contribute to the control of Mtb infection. Studies examining MAIT cells in human Mtb infection are commonly performed using peripheral blood samples. However, because Mtb infection occurs primarily in lung tissue and lung-associated lymph nodes, these studies may not be fully translatable to the tissues. Additionally, studies longitudinally examining MAIT cell dynamics during HIV/Mtb co-infection are rare, and lung and lymph node tissue samples from HIV+ patients are typically unavailable. Nonhuman primates (NHP) provide a model system to characterize MAIT cell activity during Mtb infection, both in Simian Immunodeficiency Virus (SIV)-infected and SIV-naïve animals. Using NHPs allows for a more comprehensive understanding of tissue-based MAIT cell dynamics during infection with both pathogens. NHP SIV and Mtb infection is similar to human HIV and Mtb infection, and MAIT cells are phenotypically similar in humans and NHPs. Here, we discuss current knowledge surrounding MAIT cells in SIV and Mtb infection, how SIV infection impairs MAIT cell function during Mtb co-infection, and knowledge gaps to address.

The mucosal barrier and anti-viral immune responses can eliminate portions of the viral population during transmission and early viral growth.

Little is known about how specific individual viral lineages replicating systemically during acute Human Immunodeficiency Virus or Simian Immunodeficiency Virus (HIV/SIV) infection persist into chronic infection. In this study, we use molecularly barcoded SIV (SIVmac239M) to track distinct viral lineages for 12 weeks after intravenous (IV) or intrarectal (IR) challenge in macaques. Two Mafa-A1*063+ cynomolgus macaques (Macaca fascicularis, CM) were challenged IV, and two Mamu-A1*001+ rhesus macaques (Macaca mulatta, RM) were challenged IR with 200,000 Infectious Units (IU) of SIVmac239M. We sequenced the molecular barcode of SIVmac239M from all animals over the 12 weeks of the study to characterize the diversity and persistence of virus lineages. During the first three weeks post-infection, we found ~70-560 times more unique viral lineages circulating in the animals challenged IV compared to those challenged IR, which is consistent with the hypothesis that the challenge route is the primary driver restricting the transmission of individual viral lineages. We also characterized the sequences of T cell epitopes targeted during acute SIV infection, and found that the emergence of escape variants in acutely targeted epitopes can occur on multiple virus templates simultaneously, but that elimination of some of these templates is likely a consequence of additional host factors. These data imply that virus lineages present during acute infection can still be eliminated from the systemic virus population even after initial selection.

A cautionary perspective regarding the isolation and serial propagation of SARS-CoV-2 in Vero cells.

An array of SARS-CoV-2 virus variants have been isolated, propagated and used in in vitro assays, in vivo animal studies and human clinical trials. Observations of working stocks of SARS-CoV-2 suggest that sequential propagation in Vero cells leads to critical changes in the region of the furin cleavage site, which significantly reduce the value of the working stock for critical research studies. Serially propagating SARS-CoV-2 in Vero E6 cells leads to rapid increases in genetic variants while propagation in other cell lines (e.g. Vero/hSLAM) appears to mitigate this risk thereby improving the overall genetic stability of working stocks. From these observations, investigators are urged to monitor genetic variants carefully when propagating SARS-CoV-2 in Vero cells.