ABSTRACT
The effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. In this pilot study, we exposed two pigtail macaques (PTMs) chronically infected with SIVmac239, exhibiting from very low to no CD4 T cells across all compartments, to SARS-CoV-2. We monitored the disease progression, viral replication, and evolution, and compared these outcomes with SIV-naïve PTMs infected with SARS-CoV-2. No overt signs of COVID-19 disease were observed in either animal, and the SARS-CoV-2 viral kinetics and evolution in the SIVmac239 PTMs were indistinguishable from those in the SIV-naïve PTMs in all sampled mucosal sites. However, the single-cell RNA sequencing of bronchoalveolar lavage cells revealed an infiltration of functionally inert monocytes after SARS-CoV-2 infection. Critically, neither of the SIV-infected PTMs mounted detectable anti-SARS-CoV-2 T-cell responses nor anti-SARS-CoV-2 binding or neutralizing antibodies. Thus, HIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants but may remove the ability of infected individuals to mount adaptive immune responses against SARS-CoV-2.
Subject(s)
COVID-19 , Coinfection , Disease Models, Animal , SARS-CoV-2 , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Simian Immunodeficiency Virus/immunology , COVID-19/immunology , COVID-19/virology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , SARS-CoV-2/immunology , Coinfection/immunology , Coinfection/virology , Virus Replication , Macaca nemestrina , Pilot Projects , Antibodies, Viral/immunology , Antibodies, Viral/blood , Viral Load , CD4-Positive T-Lymphocytes/immunology , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/bloodABSTRACT
An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.
Subject(s)
CD4-Positive T-Lymphocytes , Cytomegalovirus , Influenza A Virus, H1N1 Subtype , Influenza Vaccines , Macaca fascicularis , Animals , Influenza Vaccines/immunology , Influenza Vaccines/administration & dosage , CD4-Positive T-Lymphocytes/immunology , Influenza A Virus, H1N1 Subtype/immunology , Cytomegalovirus/immunology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/prevention & control , Influenza A Virus, H5N1 Subtype/immunology , Lung/immunology , Lung/virology , Lung/pathology , Genetic Vectors/genetics , Genetic Vectors/immunology , Male , Female , Memory T Cells/immunology , Immunologic Memory/immunology , VaccinationABSTRACT
Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.
Subject(s)
HIV Infections , Immunity, Humoral , Animals , Germinal Center , Adjuvants, Immunologic/pharmacology , Antigens , Primates , Antibodies, Neutralizing , HIV Antibodies , env Gene Products, Human Immunodeficiency VirusABSTRACT
Persistent and uncontrolled SARS-CoV-2 replication in immunocompromised individuals has been observed and may be a contributing source of novel viral variants that continue to drive the pandemic. Importantly, the effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. Here we conducted a pilot study wherein two pigtail macaques (PTM) chronically infected with SIVmac239 were exposed to SARS-CoV-2 and monitored for six weeks for clinical disease, viral replication, and viral evolution, and compared to our previously published cohort of SIV-naïve PTM infected with SARS-CoV-2. At the time of SARS-CoV-2 infection, one PTM had minimal to no detectable CD4+ T cells in gut, blood, or bronchoalveolar lavage (BAL), while the other PTM harbored a small population of CD4+ T cells in all compartments. Clinical signs were not observed in either PTM; however, the more immunocompromised PTM exhibited a progressive increase in pulmonary infiltrating monocytes throughout SARS-CoV-2 infection. Single-cell RNA sequencing (scRNAseq) of the infiltrating monocytes revealed a less activated/inert phenotype. Neither SIV-infected PTM mounted detectable anti-SARS-CoV-2 T cell responses in blood or BAL, nor anti-SARS-CoV-2 neutralizing antibodies. Interestingly, despite the diminished cellular and humoral immune responses, SARS-CoV-2 viral kinetics and evolution were indistinguishable from SIV-naïve PTM in all sampled mucosal sites (nasal, oral, and rectal), with clearance of virus by 3-4 weeks post infection. SIV-induced immunodeficiency significantly impacted immune responses to SARS-CoV-2 but did not alter disease progression, viral kinetics or evolution in the PTM model. SIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants.
ABSTRACT
Previous studies have indicated that the loss of CD161-expressing CD4+ Th17 cells is linked to the progression of chronic HIV. These cells are significantly depleted in peripheral blood and gut mucosa of HIV-infected individuals, contributing to inflammation and disruption of the gut barrier. However, the impact of HIV infection on CD161-expressing CD8+ T cells remain unclear. Here, we examined the functions of peripheral blood and mucosal CD161+CD8+ T cells in the macaque model of HIV infection. In contrast to the significant loss of CD161+CD4+ T cells, CD161+CD8+ T cell frequencies were maintained in blood and gut during chronic SIV infection. Furthermore, gut CD161+CD8+ T cells displayed greater IL-17 production and maintained Th1-type and cytolytic functions, contrary to impaired IL-17 and granzyme-B production in CD161+CD4+ T cells of SIV-infected macaques. These results suggest that augmented Th17-type effector functions of CD161+CD8+ T cells during SIV infection is a likely mechanism to compensate for the sustained loss of gut mucosal Th17 cells. Targeting the cytokine and cytolytic effector functions of CD161+CD8+ T cells in the preclinical setting of chronic SIV infection with antiretroviral therapy has implications in the restoration of gut barrier disruption in persons with HIV infection.
Subject(s)
HIV Infections , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Humans , CD8-Positive T-Lymphocytes , Macaca mulatta , Interleukin-17/therapeutic use , Intestinal Mucosa , Simian Acquired Immunodeficiency Syndrome/drug therapyABSTRACT
BACKGROUND: Fine-needle aspiration (FNA) has been reported since 1912 beginning with the use of trocars and other specialized instruments that were impractical. Since then, FNA has proven to be a successful alternative technique to excisional biopsy for some assays despite a few limitations. METHODS: In this study, we compared four different techniques for FNA in rhesus macaques by evaluating total live cells recovered and cell viability using a standard 6 mL syringe and 1.5-inch 22-gauge needle. RESULTS: Technique B which was the only technique in which the needle was removed from the syringe after collection of the sample to allow forced air through the needle to expel the contents into media followed by flushing of the syringe and needle resulted in the highest total cell count and second highest cell viability in recovered cells. CONCLUSION: Based on our results, Technique B appears to be the superior method.
Subject(s)
Biopsy, Fine-Needle , Animals , Biopsy, Fine-Needle/veterinary , Biopsy, Fine-Needle/methods , Macaca mulattaABSTRACT
HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.
Subject(s)
Chickenpox , SAIDS Vaccines , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Female , Simian Immunodeficiency Virus/genetics , Macaca mulatta , Vaccines, Synthetic/genetics , SAIDS Vaccines/genetics , Antibodies, Neutralizing , Cytokines , Antibodies, ViralABSTRACT
Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.
Subject(s)
Antibody Affinity , B-Lymphocytes , Cell Movement , Clone Cells , Germinal Center , HIV Antibodies , Immunization , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibody Affinity/genetics , Antibody Affinity/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Clone Cells/cytology , Clone Cells/immunology , Epitopes, B-Lymphocyte/immunology , Gene Expression Profiling , Germinal Center/cytology , Germinal Center/immunology , HIV Antibodies/genetics , HIV Antibodies/immunology , HIV Infections/immunology , HIV-1/immunology , Humans , Immunization, Secondary , Macaca mulatta/immunology , Macaca mulatta/virology , Memory B Cells/cytology , Memory B Cells/immunology , Single-Cell Analysis , Somatic Hypermutation, Immunoglobulin/genetics , Somatic Hypermutation, Immunoglobulin/immunology , Time Factors , env Gene Products, Human Immunodeficiency Virus/administration & dosage , env Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734-736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.
Subject(s)
Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Endocytosis , Gene Products, env/genetics , Macaca mulatta/metabolism , Macaca nemestrina , Simian Acquired Immunodeficiency Syndrome/genetics , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/metabolismABSTRACT
The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over five million people worldwide as of December 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics.
Subject(s)
COVID-19 , Disease Models, Animal , Macaca nemestrina , Monkey Diseases/virology , Animals , COVID-19/immunology , COVID-19/pathology , COVID-19/physiopathology , COVID-19/virology , Humans , Immunity, Humoral , Lung/immunology , Lung/virology , Male , Monkey Diseases/immunology , Monkey Diseases/pathology , Monkey Diseases/physiopathology , T-Lymphocytes/immunologyABSTRACT
While T cell immunity is an important component of the immune response to Zika virus (ZIKV) infection generally, the efficacy of these responses during pregnancy remains unknown. Here, we tested the capacity of CD8 lymphocytes to protect from secondary challenge in four macaques, two of which were depleted of CD8+ cells prior to rechallenge with a heterologous ZIKV isolate. The initial challenge during pregnancy produced transcriptional signatures suggesting complex patterns of immune modulation as well as neutralizing antibodies that persisted until rechallenge, which all animals efficiently controlled, demonstrating that the primary infection conferred adequate protection. The secondary challenge promoted activation of innate and adaptive immune cells, possibly suggesting a brief period of infection prior to clearance. These data confirm that ZIKV infection during pregnancy induces sufficient immunity to protect from a secondary challenge and suggest that this protection is not dependent on CD8 T cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Coinfection/immunology , Coinfection/prevention & control , Zika Virus Infection/immunology , Zika Virus/genetics , Zika Virus/immunology , Animals , Antibodies, Neutralizing/blood , Chlorocebus aethiops , Female , Gene Expression Profiling , Kinetics , Macaca , Pregnancy , Vero Cells , Zika Virus Infection/virologyABSTRACT
We compare immunogenicity and protective efficacy of an HIV vaccine comprised of env and gag DNA and Env (Envelope) proteins by co-administration of the vaccine components in the same muscles or by separate administration of DNA + protein in contralateral sites in female rhesus macaques. The 6-valent vaccine includes gp145 Env DNAs, representing six sequentially isolated Envs from the HIV-infected individual CH505, and matching GLA-SE-adjuvanted gp120 Env proteins. Interestingly, only macaques in the co-administration vaccine group are protected against SHIV CH505 acquisition after repeated low-dose intravaginal challenge and show 67% risk reduction per exposure. Macaques in the co-administration group develop higher Env-specific humoral and cellular immune responses. Non-neutralizing Env antibodies, ADCC, and antibodies binding to FcγRIIIa are associated with decreased transmission risk. These data suggest that simultaneous recognition, processing, and presentation of DNA + Env protein in the same draining lymph nodes play a critical role in the development of protective immunity.
Subject(s)
DNA/genetics , Immunization/methods , Macaca/genetics , Proteins/genetics , Simian Immunodeficiency Virus/immunology , Animals , HumansABSTRACT
Recent data in a nonhuman primate model showed that infants postnatally infected with Zika virus (ZIKV) were acutely susceptible to high viremia and neurological damage, suggesting the window of vulnerability extends beyond gestation. In this pilot study, we addressed the susceptibility of two infant rhesus macaques born healthy to dams infected with Zika virus during pregnancy. Passively acquired neutralizing antibody titers dropped below detection limits between 2 and 3 months of age, while binding antibodies remained detectable until viral infection at 5 months. Acute serum viremia was comparatively lower than adults infected with the same Brazilian isolate of ZIKV (n = 11 pregnant females, 4 males, and 4 non-pregnant females). Virus was never detected in cerebrospinal fluid nor in neural tissues at necropsy two weeks after infection. However, viral RNA was detected in lymph nodes, confirming some tissue dissemination. Though protection was not absolute and our study lacks an important comparison with postnatally infected infants born to naïve dams, our data suggest infants born healthy to infected mothers may harbor a modest but important level of protection from postnatally acquired ZIKV for several months after birth, an encouraging result given the potentially severe infection outcomes of this population.
Subject(s)
Infectious Disease Transmission, Vertical , Macaca mulatta , Pregnancy Complications, Infectious/veterinary , Zika Virus Infection/transmission , Animals , Animals, Newborn/immunology , Animals, Newborn/virology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Female , Male , Pilot Projects , Pregnancy , Pregnancy Complications, Infectious/virology , Zika Virus , Zika Virus Infection/immunology , Zika Virus Infection/virologyABSTRACT
The composition of gastrointestinal tract viromes has been associated with multiple diseases. Our understanding of virus communities in the GI tract is still very limited due to challenges in sampling from different GI sites. Here we defined the GI viromes of 15 rhesus macaques with chronic diarrhea. Luminal content samples from terminal ileum, proximal and distal colon were collected at necropsy while samples from the rectum were collected antemortem using a fecal loop. The composition of and ecological parameters associated with the terminal ileum virome were distinct from the colon and rectum samples; these differences were driven by bacteriophages rather than eukaryotic viruses. The six contigs that were most discriminative of the viromes were distantly related to bacteriophages from three different families. Our analysis provides support for using fecal loop sampling of the rectum as a proxy of the colonic virome in humans.
Subject(s)
Bacteriophages/physiology , Biodiversity , Diarrhea/veterinary , Lower Gastrointestinal Tract/virology , Macaca mulatta , Primate Diseases/virology , Animals , Bacteriophages/classification , Bacteriophages/genetics , Chronic Disease , Colon/pathology , Colon/virology , Contig Mapping , Diarrhea/virology , Feces/virology , Ileum/pathology , Ileum/virology , Lower Gastrointestinal Tract/pathology , Metagenome , Rectum/virologyABSTRACT
Breast milk HIV-1 transmission is currently the predominant contributor to pediatric HIV infections. Yet, only ~10% of breastfeeding infants born to untreated HIV-infected mothers become infected. This study assessed the protective capacity of natural HIV envelope-specific antibodies isolated from the milk of HIV-infected women in an infant rhesus monkey (RM), tier 2 SHIV oral challenge model. To mimic placental and milk maternal antibody transfer, infant RMs were i.v. infused and orally treated at the time of challenge with a single weakly neutralizing milk monoclonal antibody (mAb), a tri-mAb cocktail with weakly neutralizing and ADCC functionalities, or an anti-influenza control mAb. Of these groups, the fewest tri-mAb-treated infants had SHIV detectable in plasma or tissues (2/6, 5/6, and 7/8 animals infected in tri-mAb, single-mAb, and control-mAb groups, respectively). Tri-mAb-treated infants demonstrated significantly fewer plasma transmitted/founder variants and reduced peripheral CD4+ T cell proviral loads at 8 weeks post-challenge compared to control mAb-treated infants. Abortive infection was observed as detectable CD4+ T cell provirus in non-viremic control mAb- and single mAb-, but not in tri-mAb-treated animals. These results suggest that polyfunctional milk antibodies contribute to the natural inefficiency of HIV-1 transmission through breastfeeding and infant vaccinations eliciting non-neutralizing antibody responses could reduce postnatal HIV transmission.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1/physiology , Macaca mulatta/immunology , Milk, Human/virology , Animals , Animals, Newborn , Antibodies, Monoclonal/blood , Disease Models, Animal , Disease Transmission, Infectious , Female , HIV Antibodies/blood , HIV Infections/transmission , HIV-1/pathogenicity , Humans , Immunization, Passive , Viral LoadABSTRACT
Zika virus (ZIKV) infection of pregnant women is associated with pathologic complications of fetal development. Here, we infect pregnant rhesus macaques (Macaca mulatta) with a minimally passaged ZIKV isolate from Rio de Janeiro, where a high rate of fetal development complications was observed. The infection of pregnant macaques with this virus results in maternal viremia, virus crossing into the amniotic fluid (AF), and in utero fetal deaths. We also treated three additional ZIKV-infected pregnant macaques with a cocktail of ZIKV-neutralizing human monoclonal antibodies (nmAbs) at peak viremia. While the nmAbs can be effective in clearing the virus from the maternal sera of treated monkeys, it is not sufficient to clear ZIKV from AF. Our report suggests that ZIKV from Brazil causes fetal demise in non-human primates (NHPs) without additional mutations or confounding co-factors. Treatment with a neutralizing anti-ZIKV nmAb cocktail is insufficient to fully stop vertical transmission.
Subject(s)
Antibodies, Viral/administration & dosage , Pregnancy Complications/drug therapy , Zika Virus Infection/drug therapy , Zika Virus/physiology , Animals , Antibodies, Neutralizing/administration & dosage , Female , Fetal Death , Humans , Macaca mulatta , Pregnancy , Pregnancy Complications/mortality , Pregnancy Complications/virology , Zika Virus/drug effects , Zika Virus/genetics , Zika Virus Infection/mortality , Zika Virus Infection/virologyABSTRACT
UNLABELLED: Chronic immune activation/inflammation driven by factors like microbial translocation is a key determinant of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) disease progression. Although extensive research on inflammation has focused on studying protein regulators, increasing evidence suggests a critical role for microRNAs (miRNAs) in regulating several aspects of the immune/inflammatory response and immune cell proliferation, differentiation, and activation. To understand their immunoregulatory role, we profiled miRNA expression sequentially in intestinal lamina propria leukocytes (LPLs) of eight macaques before and at 21, 90, and 180 days postinfection (dpi). At 21 dpi, â¼20 and 9 miRNAs were up- and downregulated, respectively. However, at 90 dpi (n = 60) and 180 dpi (n = 44), ≥75% of miRNAs showed decreased expression. Notably, the T-cell activation-associated miR-15b, miR-142-3p, miR-142-5p, and miR-150 expression was significantly downregulated at 90 and 180 dpi. Out of â¼10 downregulated miRNAs predicted to regulate CD69, we confirmed miR-92a to directly target CD69. Interestingly, the SIV-induced miR-190b expression was elevated at all time points. Additionally, elevated lipopolysaccharide (LPS)-responsive miR-146b-5p expression at 180 dpi was confirmed in primary intestinal macrophages following LPS treatment in vitro Further, reporter and overexpression assays validated IRAK1 (interleukin-1 receptor 1 kinase) as a direct miR-150 target. Furthermore, IRAK1 protein levels were markedly elevated in intestinal LPLs and epithelium. Finally, blockade of CD8(+) T-cell activation/proliferation with delta-9 tetrahydrocannabinol (Δ(9)-THC) significantly prevented miR-150 downregulation and IRAK1 upregulation. Our findings suggest that miR-150 downregulation during T-cell activation disrupts the translational control of IRAK1, facilitating persistent gastrointestinal (GI) inflammation. Finally, the ability of Δ(9)-THC to block the miR-150-IRAK1 regulatory cascade highlights the potential of cannabinoids to inhibit persistent inflammation/immune activation in HIV/SIV infection. IMPORTANCE: Persistent GI tract disease/inflammation is a cardinal feature of HIV/SIV infection. Increasing evidence points to a critical role for miRNAs in controlling several aspects of the immune/inflammatory response. Here, we show significant dysregulation of miRNA expression exclusively in the intestinal lamina propria cellular compartment through the course of SIV infection. Specifically, the study identified miRNA signatures associated with key pathogenic events, such as viral replication, T-cell activation, and microbial translocation. The T-cell-enriched miR-150 showed significant downregulation throughout SIV infection and was confirmed to target IRAK1, a critical signal-transducing component of the IL-1 receptor and TLR signaling pathways. Reduced miR-150 expression was associated with markedly elevated IRAK1 expression in the intestines of chronically SIV-infected macaques. Finally, Δ(9)-THC-mediated blockade of CD8(+) T-cell activation in vitro significantly inhibited miR-150 downregulation and IRAK1 upregulation, suggesting its potential for targeted immune modulation in HIV infection.
Subject(s)
Gene Expression Regulation , Lymphocyte Activation , MicroRNAs/genetics , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunology , Animals , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , CD4-Positive T-Lymphocytes/immunology , Down-Regulation , Gene Expression Profiling , HIV Infections , Humans , Inflammation/immunology , Interleukin-1 Receptor-Associated Kinases/genetics , Intestinal Mucosa/immunology , Intestinal Mucosa/virology , Lectins, C-Type/genetics , Macaca mulatta , MicroRNAs/biosynthesis , Signal Transduction , Up-Regulation , Viral Load , Virus ReplicationABSTRACT
UNLABELLED: Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques (Macaca mulatta). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4(+) T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques (Macaca nemestrina), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4(+) T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of <15 to 50 RNA copies/ml. CD4(+) T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4(+) T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8(+) cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE: The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.
Subject(s)
Amino Acid Motifs , CD4-Positive T-Lymphocytes/immunology , Immunity, Mucosal , Macaca nemestrina/virology , Sequence Deletion , Simian Acquired Immunodeficiency Syndrome/immunology , Viral Envelope Proteins/immunology , Animals , CD4-Positive T-Lymphocytes/virology , Disease Progression , Female , Gene Expression , Intestines/immunology , Intestines/virology , Macaca mulatta/virology , Male , Molecular Sequence Data , Mucous Membrane/immunology , Mucous Membrane/virology , Protein Sorting Signals , Protein Structure, Tertiary , Protein Transport , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/immunology , Species Specificity , Viral Envelope Proteins/deficiency , Viral Envelope Proteins/genetics , Viral Load/genetics , Viral Load/immunology , Viremia/immunology , Viremia/pathology , Virus Replication/genetics , Virus Replication/immunologyABSTRACT
Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles.