Long-acting lenacapavir protects macaques against intravenous challenge with simian-tropic HIV.

BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.

Bacterial Genotype, Carrier Risk Factors, and an Antimicrobial Stewardship Approach Relevant to Methicillin-resistant Staphylococcus aureus Prevalence in a Population of Macaques Housed in a Research Facility.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a significant problem for human and animal health and can negatively affect the health status of macaques and other nonhuman primates (NHP) in research colonies. However, few publications provide guidance on the prevalence, genotype, or risk factors for macaques with MRSA and even fewer on how to effectively respond to MRSA once identified in a population. After having a clinical case of MRSA in a rhesus macaque, we sought to determine the MRSA carrier prevalence, risk factors, and genotypes of MRSA in a population of research NHPs. Over a 6-wk period in 2015, we collected nasal swabs from 298 NHPs. MRSA was isolated from 28% (n = 83). We then reviewed each macaque's medical record for a variety of variables including animal housing room, sex, age, number of antibiotic courses, number of surgical interventions, and SIV status. Analysis of these data suggests that MRSA carriage is associated with the room location, age of the animal, SIV status, and the number of antibiotic courses. We used multilocus sequence typing and spa typing on a subset of MRSA and MSSA isolates to determine whether the MRSA present in NHPs was comparable with common human strains. Two MRSA sequence types were predominant: ST188 and a novel MRSA genotype, neither of which is a common human isolate in the United States. We subsequently implemented antimicrobial stewardship practices (significantly reducing antimicrobial use) and then resampled the colony in 2018 and found that MRSA carriage had fallen to 9% (26/285). These data suggest that, as in humans, macaques may have a high carrier status of MRSA despite low clinically apparent disease. Implementing strategic antimicrobial stewardship practices resulted in a marked reduction in MRSA carriage in the NHP colony, highlighting the importance of limiting antimicrobial use when possible.

Preferential Small Intestine Homing and Persistence of CD8 T Cells in Rhesus Macaques Achieved by Molecularly Engineered Expression of CCR9 and Reduced Ex Vivo Manipulation.

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.

Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice.

RAS signaling is central to many cellular processes and SOS proteins promote RAS activation. To investigate the role of SOS proteins in T cell biology, we crossed Sos1f/fSos2-/- mice to CD4-Cre transgenic mice. We previously reported an effect of these mutations on T cell signaling and T cell migration. Unexpectedly, we observed nodules on the joints of greater than 90% of these mutant mice at 5 months of age, especially on the carpal joints. As the mice aged further, some also displayed joint stiffness, hind limb paralysis, and lameness. Histological analysis indicated that the abnormal growth in joints originated from dysplastic chondrocytes. Second harmonic generation imaging of the carpal nodules revealed that nodules were encased by rich collagen fibrous networks. Nodules formed in mice also deficient in RAG2, indicating that conventional T cells, which undergo rearrangement of the T cell antigen receptor, are not required for this phenotype. CD4-Cre expression in a subset of cells, either immune lineage cells (e.g., non-conventional T cells) or non-immune lineage cells (e.g., chondrocytes) likely mediates the dramatic phenotype observed in this study. Disruptions of genes in the RAS signaling pathway are especially likely to cause this phenotype. These results also serve as a cautionary tale to those intending to use CD4-Cre transgenic mice to specifically delete genes in conventional T cells.

CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering.

Follicular helper CD4 T cells, TFH, residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8hCXCR5) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8hCXCR5 T cells were present throughout the follicles with some observed near infected TFH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication.IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, TFH, present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the TFH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on TFH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.

Geminin Is Essential for Pluripotent Cell Viability During Teratoma Formation, but Not for Differentiated Cell Viability During Teratoma Expansion.

Pluripotent embryonic stem cells (ESCs) are unusual in that geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes or to prevent DNA rereplication-dependent apoptosis. To distinguish between these two incompatible hypotheses, immune-compromised mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen-dependent Cre recombinase gene. Mice were then injected with tamoxifen at various times during which the ESCs proliferated and differentiated into a teratoma. For comparison, the same ESCs were cultured in vitro in the presence of monohydroxytamoxifen. The results revealed that geminin is a haplosufficient gene that is essential for ESC viability before they differentiate into a teratoma, but once a teratoma is established, the differentiated cells can continue to proliferate in the absence of Gmnn alleles, geminin protein, and pluripotent stem cells. Thus, differentiated cells did not require geminin for efficient proliferation within the context of a solid tissue, although they did when teratoma cells were cultured in vitro. These results provide proof-of-principle that preventing geminin function could prevent malignancy in tumors derived from pluripotent cells by selectively eliminating the progenitor cells with little harm to normal cells.

Acute Liver Damage Associated with Innate Immune Activation in a Small Nonhuman Primate Model of Hepacivirus Infection.

UNLABELLED: Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection. IMPORTANCE: Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study.

Polymer multilayer tattooing for enhanced DNA vaccination.

DNA vaccines have many potential benefits but have failed to generate robust immune responses in humans. Recently, methods such as in vivo electroporation have demonstrated improved performance, but an optimal strategy for safe, reproducible, and pain-free DNA vaccination remains elusive. Here we report an approach for rapid implantation of vaccine-loaded polymer films carrying DNA, immune-stimulatory RNA, and biodegradable polycations into the immune-cell-rich epidermis, using microneedles coated with releasable polyelectrolyte multilayers. Films transferred into the skin following brief microneedle application promoted local transfection and controlled the persistence of DNA and adjuvants in the skin from days to weeks, with kinetics determined by the film composition. These 'multilayer tattoo' DNA vaccines induced immune responses against a model HIV antigen comparable to electroporation in mice, enhanced memory T-cell generation, and elicited 140-fold higher gene expression in non-human primate skin than intradermal DNA injection, indicating the potential of this strategy for enhancing DNA vaccination.

Psychogenic alopecia in rhesus macaques presenting as focally extensive alopecia of the distal limb.

Focally extensive alopecia affecting the distal limbs is a common clinical finding in rhesus macaque (Macaca mulatta) colonies and is both a regulatory and colony-health concern. We performed diagnostic examinations including physical exams, bloodwork, skin scrapes, surface cytology, and surface bacterial-fungal cultures on 17 rhesus macaques with this presentation of alopecia. Skin biopsies from alopecic skin obtained from each macaque were compared with those of normal skin from the same animal. Immunohistochemistry and metachromatic staining for inflammatory cells were performed to compare alopecic and normal skin. In addition, we compared these biopsies with those previously obtained from macaques with generalized alopecia and dermal inflammatory infiltrates consistent with cutaneous hypersensitivity disorders and with those from animals with normal haircoats. Bacterial and fungal cultures, skin scrapes, surface cytology, and bloodwork were unremarkable. Affected skin showed only mild histologic alteration, with rare evidence of trichomalacia and follicular loss. Numbers of mast cells and CD3+ lymphocytes did not differ between alopecic and normally haired skin from the same animal. The number of mast cells in alopecic skin from animals in the current cohort was significantly lower than that in skin of animals previously diagnosed with a cutaneous hypersensitivity disorder. Numbers of both mast cells and CD3+ lymphocytes in alopecic skin from the current cohort were similar to those from biopsies of animals with normal haircoats. Together, the clinical findings and pathology are consistent with a psychogenic origin for this pattern of alopecia in rhesus macaques.

Hypergammaglobulinemia in an SIV-infected rhesus macaque with a B-cell neoplasm with plasma cell differentiation.

An SIV-infected rhesus macaque presented with anemia, hypercalcemia, and hyperglobulinemia. Neoplastic round cells with plasma cell morphology infiltrated multiple organs and stained immunohistochemically positive for CD45, MUM1/IRF4, CD138, VS38C, and Kappa light chain and variably positive for CD20 and CD79a, consistent with a B-cell neoplasm with plasma cell differentiation.

Differential contribution of dietary fat and monosaccharide to metabolic syndrome in the common marmoset (Callithrix jacchus).

There is a critical need for animal models to study aspects type 2 diabetes (T2D) pathogenesis and prevention. While the rhesus macaque is such an established model, the common marmoset has added benefits including reduced zoonotic risks, shorter life span, and a predisposition to birth twins demonstrating chimerism. The marmoset as a model organism for the study of metabolic syndrome has not been fully evaluated. Marmosets fed high-fat or glucose-enriched diets were followed longitudinally to observe effects on morphometric and metabolic measures. Effects on pancreatic histomorphometry and vascular pathology were examined terminally. The glucose-enriched diet group developed an obese phenotype and a prolonged hyperglycemic state evidenced by a rapid and persistent increase in mean glycosylated hemoglobin (HgbA1c) observed as early as week 16. In contrast, marmosets fed a high-fat diet did not maintain an obese phenotype and demonstrated a delayed increase in HgbA1) that did not reach statistical significance until week 40. Consumption of either diet resulted in profound pancreatic islet hyperplasia suggesting a compensation for increased insulin requirements. Although the high-fat diet group developed atherosclerosis of increased severity, the presence of lesions correlated with glucose intolerance only in the glucose-enriched diet group. The altered timing of glucose dysregulation, differential contribution to obesity, and variation in vascular pathology suggests mechanisms of effect specific to dietary nutrient content. Feeding nutritionally modified diets to common marmosets recapitulates aspects of metabolic disease and represents a model that may prove instrumental to elucidating the contribution of nutrient excess to disease development.

Treatment of giardiasis in common marmosets (Callithrix jacchus) with tinidazole.

Giardia intestinalis is a common protozoan parasite that can infect many laboratory animal primates, although its role as a contributor to the induction of gastrointestinal disease remains unclear. This study sought to investigate the prevalence of Giardia in a colony of common marmosets by using a Giardia antigen-capture assay and to address the possible eradication of this infection by using tinidazole, an antiprotozoal similar to metronidazole but requiring fewer doses. Among 31 colony marmosets, 13 (42%) were positive for Giardia. Two doses of oral tinidazole eliminated the infection in all animals. Repeat testing of the 13 Giardia-positive monkeys 1 y later showed that 11 remained negative and that treated animals had a significant increase in weight at 1 y. Giardia antigen is common in common marmoset feces, and treatment using oral tinidazole is possible and highly effective.