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Yellow fever virus (YFV) is a reemerging global health threat, driven by several factors, including increased spread of the mosquito vector and rapid urbanization. Although a prophylactic vaccine exists, vaccine hesitancy, supply deficits, and distribution difficulties leave specific populations at risk of severe YFV disease, as evidenced by recent outbreaks in South America. To establish a treatment for patients with severe YFV infection, we tested 37 YFV-specific monoclonal antibodies isolated from vaccinated humans and identified two capable of potently neutralizing multiple pathogenic primary YFV isolates. Using both hamster and nonhuman primate models of lethal YFV infection, we demonstrate that a single administration of either of these two potently neutralizing antibodies during acute infection fully controlled viremia and prevented severe disease and death in treated animals. Given the potential severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing YFV cases during outbreaks.

In the absence of a prophylactic vaccine, the use of antiretroviral therapy (ART) as pre-exposure prophylaxis (PrEP) to prevent HIV acquisition by uninfected individuals is a promising approach to slowing the epidemic, but its efficacy is hampered by incomplete patient adherence and ART-resistant variants. Here, we report that competitive inhibition of HIV Env-CCR5 binding via the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of CCR5-tropic SHIVSF162P3. Injection of Leronlimab weekly at 10 mg/kg provides significant but partial protection, while biweekly 50 mg/kg provides complete protection from SHIV acquisition. Tissue biopsies from protected macaques post challenge show complete CCR5 receptor occupancy and an absence of viral nucleic acids. After Leronlimab washout, protected macaques remain aviremic, and adoptive transfer of hematologic cells into naïve macaques does not transmit viral infection. These data identify CCR5 blockade with Leronlimab as a promising approach to HIV prophylaxis and support initiation of clinical trials., CCR5 is a co-receptor for many transmitted HIV strains. Here, the authors show that biweekly injection of the CCR5-specific antibody Leronlimab protects rhesus macaques against infection following repeated intrarectal challenges of a CCR5-tropic SHIV.

The strain 68-1 rhesus cytomegalovirus (RhCMV)–based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8 + T cells that recognize epitopes presented by major histocompatibility complex (MHC)–II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II– and/or MHC-Ia–restricted CD8 + T cells do not protect against SIV, it remains unclear whether MHC-E–restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II–restricted component. Using host microRNA (miR)–mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope–targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142–mediated tropism restriction eliminated MHC-E epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-II epitope–targeted response. Inhibition with the endothelial cell–selective miR-126 eliminated MHC-II epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-E epitope–targeted response. Dual miR-142 + miR-126–mediated tropism restriction reverted CD8 + T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8 + T cell responses were generally similar, only the vectors programmed to elicit MHC-E–restricted CD8 + T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

Few countermeasures to treat Yellow Fever virus (YFV) infection are under development, because vaccines have helped to limit new infections. Unfortunately, vaccine hesitancy, supply deficits, and a paucity of therapeutic options have left individuals at risk. Here, we tested potent YFV-specific neutralizing monoclonal antibodies in rodents and non-human primates. We administered antibodies during acute pathogenic YFV infection and demonstrate that we can prevent severe disease and death. Given the severity of YFV-induced disease, our results show that these antibodies could be effective in saving lives and fill a much-needed void in managing Yellow Fever cases during outbreaks around the world.One Sentence SummaryTherapeutic monoclonal antibodies prevent death from YFV infection.

Nonbronchoscopic bronchoalveolar lavage (NB-BAL) is a minimally invasive diagnostic and research tool used to sample the cells of lower airways and alveoli without using a bronchoscope. Our study compared NB-BAL and bronchoscopic bronchoalveolar lavage (B-BAL) in terms of costs, cell yields, and the number of post-procedural complications in macaques. We also analyzed procedure times, BAL fluid volume yields, and vital signs in a subset of animals that underwent NB-BAL. Compared with the B-BAL technique, NB-BAL was less expensive to perform, with fewer complications, fewer animals requiring temporary or permanent cessation of BALs, and higher cell yields per mL of recovered saline. The average procedure time for NB-BAL was 6.8 ± 1.6 min, and the average NB-BAL lavage volume yield was 76 ± 9%. We found no significant differences in respiration rate before, during, or after NB-BAL but did find significant differences in heart rate and oxygen saturation (SpO2). This study demonstrates that NB-BAL is a simple, cost-effective, and safe alternative to B-BAL that results in higher cell yields per mL, improved animal welfare, and fewer missed time points, and thus constitutes a refinement over the B-BAL in macaques.

Macaques are physiologically relevant animal models of human immunology and infectious disease that have provided key insights and advanced clinical treatment in transplantation, vaccinology, and HIV/AIDS. However, the small size of macaques is a stumbling block for studies requiring large numbers of cells, such as hematopoietic stem cells (HSCs) for transplantation, antigen-specific lymphocytes for in-depth immunological analysis, and latently-infected CD4+ T-cells for HIV cure studies. Here, we provide a detailed protocol for collection of large numbers of HSCs and T-cells from cynomolgus macaques as small as 3 kilograms using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 x 10(9) total nucleated cells from mobilized animals and 1.2 x 10(9) total nucleated cells from non-mobilized animals per procedure. This report provides sufficient detail to adapt this apheresis technique at other institutions, which will facilitate more efficient and detailed analysis of HSCs and their progeny blood cells.

The diverse bacterial communities that colonize the gastrointestinal tract play an essential role in maintaining immune homeostasis through the production of critical metabolites such as short-chain fatty acids (SCFAs) and this can be disrupted by antibiotic use. However, few studies have addressed the effects of specific antibiotics longitudinally on the microbiome and immunity. We evaluated the effects of four specific antibiotics: enrofloxacin, cephalexin, paromomycin, and clindamycin, in healthy female rhesus macaques. All antibiotics disrupted the microbiome, including reduced abundances of fermentative bacteria and increased abundances of potentially pathogenic bacteria, including Enterobacteriaceae in the stool, and decreased Helicobacteraceae in the colon. This was associated with decreased SCFAs, indicating altered bacterial metabolism. Importantly, antibiotic use also substantially altered local immune responses, including increased neutrophils and Th17 cells in the colon. Furthermore, we observed increased soluble CD14 in plasma, indicating microbial translocation. These data provide a longitudinal evaluation of antibiotic-induced changes to the composition and function of colonic bacterial communities associated with specific alterations in mucosal and systemic immunity.

The CCR5-specific antibody Leronlimab is being investigated as a novel immunotherapy that can suppress HIV replication with minimal side effects. Here we studied the virological and immunological consequences of Leronlimab in chronically CCR5-tropic HIV-1 infected humans (n = 5) on suppressive antiretroviral therapy (ART) and in ART-naïve acutely CCR5-tropic SHIV infected rhesus macaques (n = 4). All five human participants transitioned from daily combination ART to self-administered weekly subcutaneous (SC) injections of 350 mg or 700 mg Leronlimab and to date all participants have sustained virologic suppression for over seven years. In all participants, Leronlimab fully occupied CCR5 receptors on peripheral blood CD4+ T cells and monocytes. In ART-naïve rhesus macaques acutely infected with CCR5-tropic SHIV, weekly SC injections of 50 mg/kg Leronlimab fully suppressed plasma viremia in half of the macaques. CCR5 receptor occupancy by Leronlimab occurred concomitant with rebound of CD4+ CCR5+ T-cells in peripheral blood, and full CCR5 receptor occupancy was found in multiple anatomical compartments. Our results demonstrate that weekly, self-administered Leronlimab was safe, well-tolerated, and efficacious for long-term virologic suppression and should be included in the arsenal of safe, easily administered, longer-acting antiretroviral treatments for people living with HIV-1. Trial Registration: ClinicalTrials.gov Identifiers: NCT02175680 and NCT02355184.

CCR5 plays a central role in infectious disease, host defense, and cancer progression, thereby making it an ideal target for therapeutic development. Notably, CCR5 is the major HIV entry co-receptor, where its surface density correlates with HIV plasma viremia. The level of CCR5 receptor occupancy (RO) achieved by a CCR5-targeting therapeutic is therefore a critical predictor of its efficacy. However, current methods to measure CCR5 RO lack sensitivity, resulting in high background and overcalculation. Here, we report on two independent, flow cytometric methods of calculating CCR5 RO using the anti-CCR5 antibody, Leronlimab. We show that both methods led to comparable CCR5 RO values, with low background on untreated CCR5+CD4+ T cells and sensitive measurements of occupancy on both blood and tissue-resident CD4+ T cells that correlated longitudinally with plasma concentrations in Leronlimab-treated macaques. Using these assays, we found that Leronlimab stabilized cell surface CCR5, leading to an increase in the levels of circulating and tissue-resident CCR5+CD4+ T cells in vivo in Leronlimab-treated macaques. Weekly Leronlimab treatment in a chronically SIV-infected macaque led to increased CCR5+CD4+ T cells levels and fully suppressed plasma viremia, both concomitant with full CCR5 RO on peripheral blood CD4+ T cells, demonstrating that CCR5+CD4+ T cells were protected from viral replication by Leronlimab binding. Finally, we extended these results to Leronlimab-treated humans and found that weekly 700 mg Leronlimab led to complete CCR5 RO on peripheral blood CD4+ T cells and a statistically significant increase in CCR5+CD4+ T cells in peripheral blood. Collectively, these results establish two RO calculation methods for longitudinal monitoring of anti-CCR5 therapeutic antibody blockade efficacy in both macaques and humans, demonstrate that CCR5+CD4+ T cell levels temporarily increase with Leronlimab treatment, and facilitate future detailed investigations into the immunological impacts of CCR5 inhibition in multiple pathophysiological processes.

Preventing human immunodeficiency virus (HIV) infection in newborns by vertical transmission remains an important unmet medical need in resource-poor areas where antiretroviral therapy (ART) is not available and mothers and infants cannot be treated prepartum or during the breastfeeding period. In the present study, the protective efficacy of the potent HIV-neutralizing antibodies PGT121 and VRC07-523, both produced in plants, were assessed in a multiple-SHIV (simian-human immunodeficiency virus)-challenge breastfeeding macaque model. Newborn macaques received either six weekly subcutaneous injections with PGT121 alone or as a cocktail of PGT121-LS plus VRC07-523-LS injected three times every 2 weeks. Viral challenge with SHIV(SF162P3) was twice weekly over 5.5 weeks using 11 exposures. Despite the transient presence of plasma viral RNA either immediately after the first challenge or as single-point blips, the antibodies prevented a productive infection in all babies with no sustained plasma viremia, compared to viral loads ranging from 10(3) to 5 × 10(8) virions/ml in four untreated controls. No virus was detected in peripheral blood mononuclear cells (PBMCs), and only 3 of 159 tissue samples were weakly positive in the treated babies. Newborn macaques proved to be immunocompetent, producing transient anti-Env antibodies and anti-drug antibody (ADA), which were maintained in the circulation after passive broadly neutralizing antibody clearance. ADA responses were directed to the IgG1 Fc CH2-CH3 domains, which has not been observed to date in adult monkeys passively treated with PGT121 or VRC01. In addition, high levels of VRC07-523 anti-idiotypic antibodies in the circulation of one newborn was concomitant with the rapid elimination of VRC07. Plant-expressed antibodies show promise as passive immunoprophylaxis in a breastfeeding model in newborns. IMPORTANCE Plant-produced human neutralizing antibody prophylaxis is highly effective in preventing infection in newborn monkeys during repeated oral exposure, modeling virus in breastmilk, and offers advantages in cost of production and safety. These findings raise the possibility that anti-Env antibodies may contribute to the control of viral replication in this newborn model and that the observed immune responsiveness may be driven by the long-lived presence of immune complexes.

Depletion of gut T helper 17 (Th17) cells during HIV infection leads to decreased mucosal integrity and increased disease progression. Conversely, T regulatory (Treg) cells may inhibit antiviral responses or immune activation. In HIV elite controllers, a balanced Th17/Treg ratio is maintained in the blood, suggesting a role for these responses in controlling inflammation and viral replication. HIV-infected individuals exhibit a range in responsiveness to combination antiretroviral therapy (cART). Given the link between the Th17/Treg ratio and HIV disease, we reasoned these responses may play a role in cART responsiveness. In this study, we investigated the relationship between the mucosal Th17/Treg ratio to acute simian immunodeficiency virus (SIV) viremia and the response to cART. Nineteen rhesus macaques were infected with highly pathogenic SIVΔB670 virus and cART was initiated 6 weeks postinfection. Mucosal CD4 T cell subsets were assessed by intracellular cytokine staining in the colon and mesenteric lymph nodes. Higher baseline Th17/Treg ratios corresponded with increased acute SIV viremia. Th17/Treg ratios decreased during acute SIV infection and were not restored during cART, and this corresponded to increased gut immune activation (Ki67(+)), markers of microbial translocation (sCD14), and T cell exhaustion (TIGIT(+)). Animals that maintained a more balanced mucosal Th17/Treg ratio at the time of cART initiation exhibited a better virological response to cART and maintained higher peripheral CD4 counts. These results suggest mucosal Th17 and Treg homeostasis influences acute viremia and the response to cART, a result that suggests therapeutic interventions that improve the Th17/Treg ratio before or during cART may improve treatment of HIV.

Strain 68-1 rhesus cytomegalovirus expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) primes MHC-E-restricted CD8+ T cells that control SIV replication in 50%-60% of the vaccinated rhesus macaques. Whether this unconventional SIV-specific immunity and protection is unique to rhesus macaques or RhCMV or is intrinsic to CMV remains unknown. Here, using cynomolgus CMV vectors expressing SIV antigens (CyCMV/SIV) and Mauritian cynomolgus macaques, we demonstrate that the induction of MHC-E-restricted CD8+ T cells requires matching CMV to its host species. RhCMV does not elicit MHC-E-restricted CD8+ T cells in cynomolgus macaques. However, cynomolgus macaques vaccinated with species-matched 68-1-like CyCMV/SIV mounted MHC-E-restricted CD8+ T cells, and half of the vaccinees stringently controlled SIV post-challenge. Protected animals manifested a vaccine-induced IL-15 transcriptomic signature that is associated with efficacy in rhesus macaques. These findings demonstrate that the ability of species-matched CMV vectors to elicit MHC-E-restricted CD8+ T cells that are required for anti-SIV efficacy is conserved in nonhuman primates, and these data support the development of HCMV/HIV for a prophylactic HIV vaccine.

Selection of a pre-clinical non-human primate (NHP) model is essential when evaluating therapeutic vaccine and treatment strategies for HIV. SIV and SHIV-infected NHPs exhibit a range of viral burdens, pathologies, and responses to combinatorial antiretroviral therapy (cART) regimens and the choice of the NHP model for AIDS could influence outcomes in studies investigating interventions. Previously, in rhesus macaques (RMs) we showed that maintenance of mucosal Th17/Treg homeostasis during SIV infection correlated with a better virological response to cART. Here, in RMs we compared viral kinetics and dysregulation of gut homeostasis, defined by T cell subset disruption, during highly pathogenic SIVΔB670 compared to SHIV-1157ipd3N4 infection. SHIV infection resulted in lower acute viremia and less disruption to gut CD4 T-cell homeostasis. Additionally, 24/24 SHIV-infected versus 10/19 SIV-infected animals had sustained viral suppression &lt, 100 copies/mL of plasma after 5 months of cART. Significantly, the more profound viral suppression during cART in a subset of SIV and all SHIV-infected RMs corresponded with less gut immune dysregulation during acute SIV/SHIV infection, defined by maintenance of the Th17/Treg ratio. These results highlight significant differences in viral control during cART and gut dysregulation in NHP AIDS models and suggest that selection of a model may impact the evaluation of candidate therapeutic interventions for HIV treatment and cure strategies.

To define the contribution of CD8(+) T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8(+) T cell depletion using a rhesusized anti-CD8β monoclonal antibody on barcoded SIVmac239 dynamics on stable ART and after ART cessation in rhesus macaques (RMs). Among the RMs with full CD8(+) T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA(+) cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RMs during ART. Upon ART cessation, both CD8(+) T cell–depleted and control RMs rebounded in fewer than 12 days, with no difference in the time to viral rebound or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8(+) T cell–depleted RMs showed a stable, approximately 2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent antiviral CD8(+) T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.

An effective vaccine to prevent HIV transmission has not yet been achieved. Modulation of the microbiome via probiotic therapy has been suggested to result in enhanced mucosal immunity. Here, we evaluated whether probiotic therapy could improve the immunogenicity and protective efficacy of SIV/HIV vaccination. Rhesus macaques were co-immunized with an SIV/HIV DNA vaccine via particle-mediated epidermal delivery and an HIV protein vaccine administered intramuscularly with Adjuplex™ adjuvant, while receiving daily oral Visbiome® probiotics. Probiotic therapy alone led to reduced frequencies of colonic CCR5+ and CCR6+ CD4+ T cells. Probiotics with SIV/HIV vaccination led to similar reductions in colonic CCR5+ CD4+ T cell frequencies. SIV/HIV-specific T cell and antibody responses were readily detected in the periphery of vaccinated animals but were not enhanced with probiotic treatment. Combination probiotics and vaccination did not impact rectal SIV/HIV target populations or reduce the rate of heterologous SHIV acquisition during the intrarectal challenge. Finally, post-infection viral kinetics were similar between all groups. Thus, although probiotics were well-tolerated when administered with SIV/HIV vaccination, vaccine-specific responses were not significantly enhanced. Additional work will be necessary to develop more effective strategies of microbiome modulation in order to enhance mucosal vaccine immunogenicity and improve protective immune responses.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope., Graphical abstract, Corey and colleagues evaluated CAR T cells targeting the envelope of simian immunodeficiency virus. They show that antibodies blocking the interaction between the CAR and its epitope might be responsible for the lack of in vivo proliferation of anti-SIV CAR T cells and killing of SIV-infected cells after infusion in rhesus macaques.

Non-alcoholic fatty liver disease (NAFLD) contributes to morbidity and mortality in HIV-infected individuals. To evaluate liver immune changes during SIV infection of macaques, we obtained liver biopsies laparoscopically at 2, 6, and 16 weeks post-infection as well as at necropsy (32 weeks). SIV infection was associated with liver dysfunction that included elevated liver enzymes during acute infection, higher liver weight at necropsy, and histologic changes within the liver lobule (functional unit of the liver) including sinusoidal dilatation near central veins and portal expansion. The type-1 interferon (IFN-1) response (MX1 expression) increased over time post-SIV infection, initially being observed in macrophages (week 2) with a switch to a mixed hepatocyte/monocyte/macrophage response by 16–32 weeks. Over the course of SIV infection, macrophage frequencies also increased – both infiltrating (CD68+CD163+CD206-) and resident (CD68+CD163+CD206+) tissue macrophages – and these macrophages were found more near central veins over time. In contrast, CD68+CD163-CD206− trafficking myeloid cells/monocytes were detected predominantly in the periportal zone of the lobule and associated with elevated MX1 expression in that zone. Our findings identify pathologic changes within the livers of SIV infected macaques as early as 2 weeks post-infection, IFN-1 responses that are initially observed in liver macrophages, and an accumulation of macrophages associated with signs of inflammation by week 32. These studies offer quantitative and spatial insights to inform macrophage-targeted therapeutic approaches that may improve liver function during HIV infection. Supported by grants from NIH (R01AI134630)

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens demonstrate a vaccine efficacy where 50–60% of vaccinated rhesus macaques are protected from SIV challenge. Intriguingly, RhCMV/SIV vectors elicit CD8+ T cells recognizing epitopes presented by MHC-II and MHC-E instead of MHC-Ia. We are studying how these unconventional T cell responses are elicited and contribute to the efficacy against SIV challenge. Here we utilize host microRNA (miRNA)-mediated vector tropism restriction to show that MHC-II- and MHC-E-restricted responses are primed by directly infected, non-overlapping cell types in rhesus macaques. Targeting essential RhCMV genes with myeloid cell-selective miR-142-3p eliminated MHC-E-restricted CD8+ T cell priming, yielding an exclusively MHC-II-restricted response, whereas endothelial cell-selective miR-126-3p targeting eliminated MHC-II-restricted CD8+ T cell priming, yielding an exclusively MHC-E-restricted response. Incorporation of both restriction elements reverts CD8+ T cell responses back to conventional MHC-Ia restriction. Using these otherwise isogenic vectors we show that although they demonstrate similar overall immunogenicity, only the vectors programmed to elicit MHC-E-restricted CD8+ T cell responses provided protection against SIV challenge. The MHC-E-only RhCMV/SIV vaccine efficacy did not exceed that of the parental 68-1 RhCMV/SIV vectors (that elicits both MHC-II and MHC-E responses) indicating that while the MHC-II-restricted CD8+ T cell responses are neutral to overall vaccine efficacy, an additional component of 68-1 RhCMV/SIV-induced immunity contributes to overall vaccine efficacy. This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) grants UM1 AI124377 and U19 AI128741 to LJP; the Oregon National Primate Research Center Core grant from the National Institutes of Health, Office of the Director (P51 OD011092); contracts from the National Cancer Institute (# HHSN261200800001E) to JDL; and the Bill and Melinda Gates Foundation grant OPP1107409.

Background: Liver disease remains a significant contributor to morbidity and mortality in HIV-infected individuals, even during successful treatment with combination antiretroviral therapy (cART). In non-human primates, SIV infection is associated with gut microbiome dysbiosis as well as bacterial translocation into the colonic lamina propria and liver via the portal vein. Here the liver microbiome was evaluated in rhesus macaques to discern the influence of SIV infection alone (SIV+) and during cART administration (SIV+cART) on liver bacterial dysbiosis and neutrophil infiltration.Results: Dysbiosis in liver bacterial composition was observed, encompassing changes in a number of genera, during SIV infection in the absence and presence of cART. The most striking finding was an increase in the level of Mycobacterium, which while barely detectable in the uninfected macaques, was the most abundant genus observed in the livers of a majority SIV+ and SIV+cART macaques. Multi-gene sequencing analyses identified a species of environmental mycobacteria similar to the opportunistic pathogen M. smegmatis. The effect of M. smegmatis on host gene expression in primary hepatocytes was evaluated in vitro utilizing PILAM, a glycolipid cell wall component found in atypical Mycobacteria. PILAM induced an upregulation of inflammatory responses, including an increase in the chemokines associated with neutrophil chemotaxis (CXCL1, CXCL5, and CXCL6). Assessment of the macaque livers by microscopy determined that neutrophil levels were reduced in SIV+cART macaques, suggesting that the SIV infection and/or cART treatment influence the liver-associated neutrophil response. Conclusions: A number of liver bacteria genera were altered following SIV infection even in the context of cART, possibly as a consequence of reduced neutrophil recruitment. Mycobacteria became a major component of the SIV infected macaque liver microbiome, raising the possibility that bacteria of this genus might contribute to liver disease in HIV infected patients.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68–1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68–1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68–1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68–1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68–1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques., Author summary Human cytomegalovirus (HCMV) infections are generally asymptomatic in healthy immunocompetent individuals, but HCMV can cause serious disease after congenital infection and in individuals with immunocompromised immune systems. Since HCMV is highly species specific and cannot productively infect immunocompetent laboratory animals, experimental infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a closely related animal model for HCMV. By employing the unique ability of CMV to elicit robust and lasting cellular immunity, this model has also been instrumental in developing novel CMV-based vaccines against chronic and recurring infections with pathogens such as the human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb). However, most of this work was conducted with derivatives of the 68–1 strain of RhCMV which has acquired multiple genomic alterations in tissue culture. To model pathogenesis and immunology of clinical HCMV isolates we generated a full-length (FL) RhCMV clone representative of low passage isolates. Infection of RhCMV-naïve RM with FL-RhCMV demonstrated viremia and tissue dissemination that was comparable to that of non-clonal low passage isolates. We further demonstrate that FL-RhCMV is strongly attenuated upon deletion of gene regions absent in 68–1 thus demonstrating the usefulness of FL-RhCMV to study RhCMV pathogenesis.

Development of a successful HIV vaccine is dependent upon a determination of the optimum antigen and adjuvant as well as choosing an optimal site for vaccine delivery. The site of delivery is particularly relevant as HIV transmission generally requires that the virus crosses a mucosal membrane to infect a new host. Here we undertake a pilot study comparing three vaccine delivery routes, two to the oral cavity (intraepithelial (iEp) and needle-free (NF-Injex)) as well as intramuscular (IM) delivery. These vaccinations utilized a recombinant HIV-1 Env trimer 10042.05 from an elite neutralizer, subject VC10042, that has previously induced high titers of cross-clade reactive V1V2 antibodies. The 10042.05.SOSIP fused trimer was administered with adjuvants R848 (Resiquimod), MPLA and Alhydrogel to characterize the innate cellular and anti-HIV Envelope (Env) antibody responses following the administration of the vaccine to the oral mucosa. Oral delivery of the 10042.05.SOSIP induced high titers of anti-V1V2 antibodies, which together with previous studies, indicates an immunogenic bias toward the V1V2 regions in 10042-derived Envs. Both types of oral vaccine delivery resulted in immunologic and serologic responses that were comparable to the IM delivery route. Furthermore, induction of anti-V1-V2 specific antibodies was best following iEp delivery of the oral vaccine identifying this as the optimal method to orally deliver this vaccine formulation.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential “shock and kill” therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir., Author summary IL-15 regulates NK and memory T cell homeostasis and is therefore being explored for clinical immunotherapy of chronic diseases like cancer and HIV. To explore the applicability of the clinical grade IL-15 superagonist N-803 to HIV cure strategies we tested the impact of N-803 on host immunity and latent virus in SHIV-infected rhesus macaques. Our results suggest that N-803 beneficially modulates effector NK and CD8+ T cells by expanding the numbers of these cells and redistributing them to lymph node B cell follicles, a site known to harbor persistent latent virus during ART. However, our results further suggest that N-803 does not perturb the viral reservoir present in memory CD4+ T cells and that in order to fully unlock the immunotherapeutic potential of N-803 it must be paired with latency reversal agents.

The immunological and virological events that contribute to the establishment of Zika virus (ZIKV) infection in humans are unclear. Here, we show that robust cellular innate immune responses arising early in the blood and tissues in response to ZIKV infection are significantly stronger in males and correlate with increased viral persistence. In particular, early peripheral blood recruitment of plasmacytoid dendritic cells and higher production of monocyte chemoattractant protein (MCP-1) correspond with greater viral persistence and tissue dissemination. We also identify non-classical monocytes as primary in vivo targets of ZIKV infection in the blood and peripheral lymph node. These results demonstrate the potential differences in ZIKV pathogenesis between males and females and a key role for early cellular innate immune responses in the blood in viral dissemination and ZIKV pathogenesis., The immune response to Zika virus is required to curtail the infection and avoid immunopathology, but may be involved in the associated pathophysiology. Here the authors show that viral persistence and tissue tropism is shaped by an early innate immune response in a pigtail macaque model of infection.

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human Cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients, and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.

Simian-human immunodeficiency viruses (SHIVs) have been utilized to test vaccine efficacy and characterize mechanisms of viral transmission and pathogenesis. However, the majority of SHIVs currently available have significant limitations in that they were developed using sequences from chronically HIV-infected individuals or uncommon HIV subtypes or were optimized for the macaque model by serially passaging the engineered virus in vitro or in vivo. Recently, a newly developed SHIV, SHIV.C.CH505.375H.dCT (SHIV.CH505), which incorporates vpu-env (gp140) sequences from a transmitted/founder HIV-1 subtype C strain, was shown to retain attributes of primary HIV-1 strains. However, a comprehensive analysis of the immunopathology that results from infection with this virus, especially in critical tissue compartments like the intestinal mucosa, has not been completed. In this study, we evaluated the viral dynamics and immunopathology of SHIV.CH505 in rhesus macaques. In line with previous findings, we found that SHIV.CH505 is capable of infecting and replicating efficiently in rhesus macaques, resulting in peripheral viral kinetics similar to that seen in pathogenic SIV and HIV infection. Furthermore, we observed significant and persistent depletions of CCR5(+) and CCR6(+) CD4(+) T cells in mucosal tissues, decreases in CD4(+) T cells producing Th17 cell-associated cytokines, CD8(+) T cell dysfunction, and alterations of B cell and innate immune cell function, indicating that SHIV.CH505 elicits intestinal immunopathology typical of SIV/HIV infection. These findings suggest that SHIV.CH505 recapitulates the early viral replication dynamics and immunopathogenesis of HIV-1 infection of humans and thus can serve as a new model for HIV-1 pathogenesis, treatment, and prevention research. IMPORTANCE The development of chimeric SHIVs has been instrumental in advancing our understanding of HIV-host interactions and allowing for in vivo testing of novel treatments. However, many of the currently available SHIVs have distinct drawbacks and are unable to fully reflect the features characteristic of primary SIV and HIV strains. Here, we utilize rhesus macaques to define the immunopathogenesis of the recently developed SHIV.CH505, which was designed without many of the limitations of previous SHIVs. We observed that infection with SHIV.CH505 leads to peripheral viral kinetics and mucosal immunopathogenesis comparable with those caused by pathogenic SIV and HIV. Overall, these data provide evidence of the value of SHIV.CH505 as an effective model of SIV/HIV infection and an important tool that can be used in future studies, including preclinical testing of new therapies or prevention strategies.

Viral dynamics and host responses to vaginal infection of SIV in nonhuman primates may identify viral vulnerabilities for HIV., Understanding HIV transmission is critical to guide the development of prophylactic interventions to prevent infection. We used a nonhuman primate (NHP) model with a synthetic swarm of sequence-tagged variants of SIVmac239 (“SIVmac239X”) and scheduled necropsy during primary infection (days 3 to 14 after challenge) to study viral dynamics and host responses to the establishment and dissemination of infection following vaginal challenge. We demonstrate that local replication was initiated at multiple sites within the female genital tract (FGT), with each site having multiple viral variants. Local replication and spread in the FGT preceded lymphatic dissemination. Innate viral restriction factors were observed but appeared to follow viral replication and were ineffective at blocking initial viral establishment and dissemination. However, major delays were observed in time to dissemination in animals and among different viral variants within the same animal. It will be important to assess how phenotypic differences affect early viral dynamics.