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1.
Immunity ; 56(7): 1649-1663.e5, 2023 07 11.
Article in English | MEDLINE | ID: mdl-37236188

ABSTRACT

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.


Subject(s)
HIV Infections , Hematopoietic Stem Cell Transplantation , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Animals , Macaca fascicularis , Viral Load
3.
PLoS Pathog ; 17(5): e1009565, 2021 05.
Article in English | MEDLINE | ID: mdl-33970966

ABSTRACT

Here, we assessed the efficacy of a short-course multimodal therapy (enrofloxacin, azithromycin, fenbendazole, and paromomycin) to eliminate common macaque endemic pathogens (EPs) and evaluated its impact on gastrointestinal (GI) microbiota, mucosal integrity, and local and systemic inflammation in sixteen clinically healthy macaques. Treatment combined with expanded practices resulted in successful maintenance of rhesus macaques (RM) free of common EPs, with no evidence of overt microbiota diversity loss or dysbiosis and instead resulted in a more defined luminal microbiota across study subjects. Creation of a GI pathogen free (GPF) status resulted in improved colonic mucosal barrier function (histologically, reduced colonic MPO+, and reduced pan-bacterial 16s rRNA in the MLN), reduced local and systemic innate and adaptive inflammation with reduction of colonic Mx1 and pSTAT1, decreased intermediate (CD14+CD16+) and non-classical monocytes (CD14-CD16+), reduced populations of peripheral dendritic cells, Ki-67+ and CD38+ CD4+ T cells, Ki-67+IgG+, and Ki-67+IgD+ B cells indicating lower levels of background inflammation in the distal descending colon, draining mesenteric lymph nodes, and systemically in peripheral blood, spleen, and axillary lymph nodes. A more controlled rate of viral acquisition resulted when untreated and treated macaques were challenged by low dose intrarectal SIVmac239x, with an ~100 fold increase in dose required to infect 50% (AID50) of the animals receiving treatment compared to untreated controls. Reduction in and increased consistency of number of transmitted founder variants resulting from challenge seen in the proof of concept study directly correlated with post-treatment GPF animal's improved barrier function and reduction of key target cell populations (Ki-67+ CD4+T cells) at the site of viral acquisition in the follow up study. These data demonstrate that a therapeutic and operational strategy can successfully eliminate varying background levels of EPs and their associated aberrant immunomodulatory effects within a captive macaque cohort, leading to a more consistent, better defined and reproducible research model.


Subject(s)
Inflammation/therapy , Microbiota/drug effects , Simian Acquired Immunodeficiency Syndrome/therapy , Simian Immunodeficiency Virus/immunology , Adaptive Immunity , Animals , B-Lymphocytes , CD4-Positive T-Lymphocytes , Cell Proliferation , Combined Modality Therapy , Gastrointestinal Tract/immunology , Gastrointestinal Tract/microbiology , Humans , Immunity, Innate , Intestinal Mucosa , Lymph Nodes , Macaca mulatta , Male , Monocytes , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology
4.
J Clin Apher ; 36(1): 67-77, 2021 Feb.
Article in English | MEDLINE | ID: mdl-32941672

ABSTRACT

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 kg using the Terumo Spectra Optia apheresis system, yielding an average of 5.0 × 109 total nucleated cells from mobilized animals and 1.2 × 109 total nucleated cells from nonmobilized 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.


Subject(s)
Blood Component Removal/methods , Hematopoietic Stem Cells/cytology , T-Lymphocytes/cytology , Animals , Benzylamines/pharmacology , Creatinine/blood , Cyclams/pharmacology , Female , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization/methods , Macaca fascicularis , Male
5.
Xenotransplantation ; 27(4): e12578, 2020 07.
Article in English | MEDLINE | ID: mdl-31930750

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Hematopoietic Stem Cell Transplantation , Opportunistic Infections , Virus Diseases , Allografts , Animals , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Macaca fascicularis , Opportunistic Infections/virology
7.
PLoS Pathog ; 13(3): e1006219, 2017 03.
Article in English | MEDLINE | ID: mdl-28278237

ABSTRACT

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.


Subject(s)
Zika Virus Infection/pathology , Zika Virus Infection/virology , Animals , Cell Separation , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , In Situ Hybridization , Macaca mulatta , Male , Neutralization Tests , Polymerase Chain Reaction , Viremia/virology , Zika Virus
8.
Nature ; 473(7348): 523-7, 2011 May 26.
Article in English | MEDLINE | ID: mdl-21562493

ABSTRACT

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (T(EM)) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIV(MAC239) infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4(+) memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8(+) T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8(+) or CD4(+) lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated T(EM) responses might significantly contribute to an efficacious HIV/AIDS vaccine.


Subject(s)
Immunologic Memory/immunology , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Immunodeficiency Virus/immunology , Simian Immunodeficiency Virus/pathogenicity , T-Lymphocytes/immunology , AIDS Vaccines/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytomegalovirus/genetics , DNA, Viral/analysis , Genetic Vectors/genetics , Immunity, Mucosal/immunology , Macaca mulatta/blood , Macaca mulatta/immunology , Macaca mulatta/virology , Male , RNA, Viral/analysis , SAIDS Vaccines/genetics , Simian Acquired Immunodeficiency Syndrome/blood , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/growth & development , Simian Immunodeficiency Virus/isolation & purification , Time Factors , Vaccines, DNA/genetics , Vaccines, DNA/immunology , Viral Load , Virus Replication
9.
Front Immunol ; 14: 1244637, 2023.
Article in English | MEDLINE | ID: mdl-37675101

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) is a S. aureus strain with resistance to beta-lactam antibiotics, making it a global human and veterinary health concern. Specifically, immunosuppressed patients have a remarkably higher risk of clinical MRSA infections with significantly increased rates of prolonged clinical recovery, morbidity, and mortality. The current treatment of choice for MRSA is vancomycin. Importantly, we report the first known vancomycin-resistant S. aureus (VRSA) carriers in a cohort of Mauritian cynomolgus macaques (CM) imported to the Oregon National Primate Research Center (ONPRC), with a MRSA carrier rate of 76.9% (10/13 animals). All MRSA isolates also demonstrated resistance to vancomycin with prevalence of vancomycin-intermediate Staphylococcus aureus (VISA) at 30% (3/10 MRSA-positive CMs) and VRSA at 70% (7/10 MRSA-positive CMs). Additionally, we identified VRSA in a rhesus macaque (RM) housed within the same room as the VRSA-positive CMs and identified a MRSA/VISA carrier rate of 18.8% in RMs (3/16 positive for both MRSA and VISA) in unexposed recently assigned animals directly from the ONPRC RM breeding colony. Considering that the MRSA and VRSA/VISA-positive CMs future study aims included significant immunosuppression, MRSA/VRSA/VISA decolonization treatment and expanded "MRSA-free" practices were employed to maintain this status. We report the first controlled study using in-depth analyses with appropriate diagnostic serial testing to definitively show an MRSA decolonization therapy (90% success rate) and expanded barrier practice techniques to successfully prevent recolonization (100%) of a cohort of CMs MRSA-free (up to 529 days with a total of 4,806 MRSA-free NHP days).


Subject(s)
Methicillin-Resistant Staphylococcus aureus , Animals , Humans , Macaca fascicularis , Vancomycin Resistance , Macaca mulatta , Staphylococcus aureus , Vancomycin/pharmacology , Vancomycin/therapeutic use
10.
Comp Med ; 70(6): 520-525, 2020 12 01.
Article in English | MEDLINE | ID: mdl-33153516

ABSTRACT

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.


Subject(s)
Lung , Macaca , Animals , Bronchoalveolar Lavage , Bronchoalveolar Lavage Fluid
11.
Nat Commun ; 9(1): 263, 2018 01 17.
Article in English | MEDLINE | ID: mdl-29343712

ABSTRACT

Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.


Subject(s)
Placenta/metabolism , Placental Circulation , Pregnancy Complications, Infectious/immunology , Zika Virus Infection/immunology , Adaptive Immunity , Animals , Brain/embryology , Brain/pathology , Cytokines/blood , Disease Models, Animal , Female , Fetal Development , Fetus/pathology , Immunity, Innate , Macaca mulatta , Magnetic Resonance Imaging , Oxygen/metabolism , Permeability , Placenta/immunology , Placenta/pathology , Placenta/virology , Pregnancy , Pregnancy Complications, Infectious/metabolism , Pregnancy Complications, Infectious/pathology , Pregnancy Complications, Infectious/physiopathology , Viral Load , Zika Virus Infection/metabolism , Zika Virus Infection/pathology , Zika Virus Infection/physiopathology
12.
Nat Commun ; 8(1): 2146, 2017 12 15.
Article in English | MEDLINE | ID: mdl-29247188

ABSTRACT

Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.


Subject(s)
Hepatitis B virus/physiology , Hepatocytes/metabolism , Hepatocytes/virology , Organic Anion Transporters, Sodium-Dependent/metabolism , Symporters/metabolism , Animals , Cells, Cultured , DNA, Viral/metabolism , Hepatitis B/genetics , Hepatitis B/metabolism , Hepatitis B/virology , Hepatitis B Core Antigens/metabolism , Hepatitis B virus/genetics , Hepatitis B virus/metabolism , Hepatocytes/cytology , Host-Pathogen Interactions , Humans , Macaca mulatta , Organic Anion Transporters, Sodium-Dependent/genetics , RNA, Viral/metabolism , Symporters/genetics
13.
Nat Commun ; 8(1): 1418, 2017 11 10.
Article in English | MEDLINE | ID: mdl-29127275

ABSTRACT

Allogeneic hematopoietic stem cell transplantation (HSCT) is a critically important therapy for hematological malignancies, inborn errors of metabolism, and immunodeficiency disorders, yet complications such as graft-vs.-host disease (GvHD) limit survival. Development of anti-GvHD therapies that do not adversely affect susceptibility to infection or graft-vs.-tumor immunity are hampered by the lack of a physiologically relevant, preclinical model of allogeneic HSCT. Here we show a spectrum of diverse clinical HSCT outcomes including primary and secondary graft failure, lethal GvHD, and stable, disease-free full donor engraftment using reduced intensity conditioning and mobilized peripheral blood HSCT in unrelated, fully MHC-matched Mauritian-origin cynomolgus macaques. Anti-GvHD prophylaxis of tacrolimus, post-transplant cyclophosphamide, and CD28 blockade induces multi-lineage, full donor chimerism and recipient-specific tolerance while maintaining pathogen-specific immunity. These results establish a new preclinical allogeneic HSCT model for evaluation of GvHD prophylaxis and next-generation HSCT-mediated therapies for solid organ tolerance, cure of non-malignant hematological disease, and HIV reservoir clearance.


Subject(s)
Hematopoietic Stem Cell Transplantation/methods , Macaca fascicularis/immunology , Major Histocompatibility Complex , Animals , Female , Graft vs Host Disease/prevention & control , Histocompatibility Testing , Humans , Macaca fascicularis/genetics , Male , Models, Animal , Species Specificity , Transplantation Chimera/genetics , Transplantation Chimera/immunology , Transplantation Tolerance/genetics , Transplantation Tolerance/immunology , Transplantation, Homologous , Treatment Outcome
14.
Nat Med ; 21(2): 132-9, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25599132

ABSTRACT

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.


Subject(s)
B-Lymphocytes/immunology , Lymph Nodes/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/physiology , T-Lymphocytes, Helper-Inducer/immunology , Virus Replication , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Lymphocyte Depletion , Macaca mulatta , Viral Load
15.
Nat Med ; 18(11): 1673-81, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22961108

ABSTRACT

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , SAIDS Vaccines , Simian Acquired Immunodeficiency Syndrome , Simian Immunodeficiency Virus , Vaccines, Attenuated , Animals , CD8-Positive T-Lymphocytes/cytology , Humans , Immunity, Innate , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Macaca mulatta/immunology , Macaca mulatta/virology , Male , SAIDS Vaccines/administration & dosage , SAIDS Vaccines/immunology , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/immunology , Simian Immunodeficiency Virus/pathogenicity , Tissue Distribution , Vaccines, Attenuated/immunology , Virus Replication/genetics
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