Abstract Panel Presentation Purpose and appropriate sample types This 28-color panel was developed to identify classical and non-classical T lymphocytes in decidual leukocytes and peripheral blood mononuclear cells (PBMC) of pregnant rhesus macaques ( Table 1 ). By profiling these T lymphocytes, we can investigate how maternal immunity balances tolerance to fetal antigens with protection against vertically transmitted pathogens. The selected markers define memory populations and characterize tissue residency, activation, proliferation, cytotoxicity, trafficking, and exhaustion status. This panel also delineates B lymphocytes and NK cells to confirm expected frequencies. The utility of this panel is aimed at evaluating cellular immune correlates of protection against congenital infections at the maternal-fetal interface and PBMC in rhesus macaques.

Background: Human immunodeficiency virus (HIV) and Plasmodium spp., which causes malaria, are co-endemic. Previously, we showed that during antiretroviral therapy (ART)-treated simian immunodeficiency virus (SIV)/Plasmodium fragile co-infection, peripheral markers of neutrophil extracellular trap (NET) formation positively correlated with peripheral markers of disease and gastrointestinal (GI) dysfunction. However, the impact of co-infection directly in the GI mucosa is unclear. We hypothesized that ART-treated SIV/P. fragile co-infection would result in peripheral and GI immune disruption associated with exacerbated clinical manifestations of SIV and P. fragile. Methods: Adult male rhesus macaques (RMs; n=6) were inoculated with SIVmac239, initiated ART at week 8 post-SIV infection (p.i.), were inoculated with P. fragile at week 12 p.i., and were followed until week 20 p.i. Plasma viral loads, peripheral parasitemia, and peripheral and GI immune cell frequencies and function were assessed longitudinally. Results: We observed significant CCR5+ CD4+ T cell decline in the periphery, colon, and duodenum following SIV infection. Neutrophil frequencies were unchanged throughout ART-treated SIV/P. fragile co-infection. Notably, duodenum NET-forming granulocyte frequencies were significantly positively associated with peripheral SIV burden following P. fragile co-infection but were unassociated with peripheral parasitemia and CD4+ T cell frequencies. Finally, although P. fragile was present in the duodenum, GI parasite burden was not associated with NET-forming granulocyte frequencies, peripheral viral loads, or CD4+ T cell frequencies. Conclusions: P. fragile co-infection during ART-treated SIV could cause mucosal disruptions that contribute to peripheral SIV replication despite ART. These data may have implications for HIV and malaria disease progression and treatment strategies.

Traditional immunohistochemistry techniques are limited in the number of fluorescent detection channels, markers, and staining resolution, which restricts the ability to fully evaluate bulk tissue samples and biopsies. Recent technical advancements, such as co-detection by indexing and spatial proteomics, have enabled in situ visualization of tissues by combining multiplex assays, repetitive staining, and quantitative image analysis. These technologies can identify cellular co-expression, cellular spatial relationships, tissue heterogeneity, and detect low-abundance molecules, which are critical for basic immunology, disease evaluation, and therapeutic evaluation studies. However, these methods require specially conjugated antibodies and protocols to analyze highly multiplexed tissue imaging (HMTI) readouts. Here, we utilize imaging cytometry as a viable alternative that also enables individual cellular cytometry analyses in two-dimensional formats. This technique has been used to investigate human and mouse tissues but is underexplored in the translationally relevant rhesus macaque (RM) model. Here, we demonstrate the use of this platform to image RM placenta, jejunum, kidney, and liver stored in OCT, using commercially available fluorophore-conjugated antibodies to identify structural markers (cytokeratin and vimentin), pan-immune cells (CD45), T cells (CD3 and CD8), natural killer cells (NKG2A/C), monocytes (CD16) and macrophages (CD68 and CD163), without any customization. We compared these preclinical samples to human samples to emphasize the potential for cross-species translational analyses using this platform. The flexibility to perform multiple rounds of photobleaching and fluorophore-based staining, combined with the ability to compensate for autofluorescence, makes this technology extremely valuable for deciphering tissue architecture and the spatial distribution of immune cells. Furthermore, we leveraged the platform's ability to export data in HMTI format and flow cytometry standard format, compatible with other quantitative downstream analysis pipelines, to simultaneously visualize the spatial distribution of various cell populations.

ABSTRACT:Compared with total body irradiation (TBI) or chemotherapy, antibody-drug conjugates (ADCs) offer a more targeted and potentially less toxic method for transplantation conditioning. CD45-ADC targets a pan-leukocyte antigen expressed on hematopoietic stem and progenitor cells (HSPCs) and immune cells, offering a promising strategy for gene therapy or allogeneic transplantation. We evaluated reconstitution, clonal dynamics, immune tolerance, and toxicity after conditioning with a DGN549-C-conjugated CD45-ADC followed by autologous transplantation in rhesus macaques. Two doses (0.2 and 0.3 mg/kg) were tested, with HSPC infusion 10 days after conditioning. All animals received barcoded, copepod green fluorescent protein (CopGFP)-expressing lentivirally transduced HSPCs. Both doses resulted in profound depletion of HSPCs and expected cytopenias, with incomplete lymphocyte depletion. With 0.2 mg/kg CD45-ADC conditioning, 2 animals showed robust multilineage engraftment with gene-modified cells and high clonal diversity, comparable with TBI. However, CopGFP+ cell levels and clonal diversity declined at 4 to 8 months, accompanied by development of anti-CopGFP antibodies, suggesting immune rejection. Incomplete T-cell depletion may have contributed. Notably, this rejection was slower and less complete than that after busulfan conditioning, suggesting partial immune tolerance. Dexamethasone treatment in 1 animal reversed rejection and stabilized CopGFP+ levels for >2 years. At 0.3 mg/kg CD45-ADC, 2 animals developed severe respiratory distress 4 to 6 days after transplantation, requiring humane euthanasia, accompanied by elevated inflammatory cytokines. This severe syndrome was not seen in 9 additional animals conditioned with CD45-ADC. These findings highlight the importance of preclinical evaluation of experimental therapeutics. Combining lower-dose CD45-ADC with immune suppression may enable durable engraftment in settings of alloantigen or neoantigen expression.

Modern antiretroviral therapy (ART) regimens have revolutionized the management of human immunodeficiency virus (HIV) and transformed it from a life-threatening disease to a manageable chronic condition. Despite the durable viral suppression associated with ART adherence, people with HIV (PWH) continue to experience chronic immune activation and inflammation, which has been linked with increased risk of developing non-acquired immunodeficiency syndrome (AIDS) comorbidities, including cardiovascular disease, liver disease, or neurocognitive disorders. Importantly, the mechanisms underlying establishment and maintenance of immune activation in ART-treated PWH remain incompletely defined. Here, we used a nonhuman primate model to evaluate associations between markers of systemic immune activation and peripheral neutrophils in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs), both before and after ART. As expected, peripheral frequencies of activated CD4+ and CD8+ T cells were elevated during acute SIV infection and returned to baseline levels following ART initiation. Neutrophil dynamics were impacted during acute SIV infection, including decreased peripheral neutrophil frequencies, increased neutrophil degranulation, and the potential for increased neutrophil extracellular trap (NET) formation. Treatment with ART mitigated these inflammatory neutrophil effector functions. Finally, frequencies of HLA-DR+ CD4+ and CD8+ T cells were significantly positively correlated with frequencies of inflammatory CD62Ldim neutrophils and plasma levels of myeloperoxidase, a component of neutrophil granules. Taken together, these data indicate that neutrophil activity and systemic T cell activation are correlated during acute SIV and early ART. Our work provides insight into associations between neutrophil dynamics and immune activation during HIV/SIV in the context of ART.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform maternal vaccine design remain elusive due to the lack of a translational animal model. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection in pregnant, immunocompetent, RhCMV-naïve rhesus macaques. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) dams following early second trimester gestation RhCMV inoculation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). RhCMV DNA detection in the amniotic fluid was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. Maternal RhCMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection, as evidenced by ubiquitous maternal-fetal interface infection, and reduced placental efficiency and small-for-gestation age fetuses compared to control pregnancies. This model provides new insights into the complexity of CMV vertical transmission and can be used to evaluate immune and viral determinants of protection against cCMV.

Cytomegalovirus (CMV) is a prevalent β-herpesvirus that persists asymptomatically in immunocompetent hosts. In people with HIV-1 (PWH), CMV is associated with HIV-1 persistence and particular inflammatory-related comorbidities. The true causative role of CMV in HIV-associated pathologies, however, remains unclear given that nearly all PWH are coinfected with CMV. In this study, we examined acute phase immune and virological dynamics in cohorts of SIV-infected rhesus macaques (RMs) that were naturally seropositive or -negative for rhesus CMV (RhCMV). We observed prior to SIV, RhCMV expanded a polyclonal population of target CCR5+CD4+ T cells in gut and lymph nodes that expressed the chemotactic receptor CXCR3 and were largely not specific for RhCMV. Upon SIV infection, RhCMV+ RMs exhibited higher peak viremia and elevated levels of SIV DNA in the upper and lower intestine. Greater seeding of SIV DNA was associated with a maintenance of CCR5-expressing CD4+ T cells that were enriched within the RhCMV+ gut along a CXCR3/CXCL9 chemotactic axis. Overall, the data suggest that RhCMV can promote SIV susceptibility within a diverse, polyclonal pool of CD4+ T cells that are not entirely RhCMV specific.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of neonatal neurological impairment worldwide, but the viral factors enabling vertical spread across the placenta remain undetermined. The pentameric complex (PC), composed of the subunits gH/gL/UL128/UL130/UL131A, has been demonstrated to be important for entry into nonfibroblast cells in vitro. These findings link the PC to broad cell tropism and virus dissemination in vivo, denoting all subunits as potential targets for intervention strategies and vaccine development. To determine the relevance of the PC for congenital transmission in a translational nonhuman primate model, we engineered a rhesus CMV (RhCMV) mutant lacking the orthologs of UL128 and UL130, which demonstrated diminished infection of epithelial cells in vitro. However, intravenous inoculation of either CD4+ T cell-depleted or immunocompetent RhCMV-seronegative pregnant rhesus macaques (RMs) in the early second trimester with the PC-deficient mutant resulted in maternal RhCMV peak plasma viremia similar to inoculations with PC-intact RhCMV, although virus shedding in saliva and urine was limited. Infections with the PC-intact virus induced IgG responses that neutralized RhCMV entry into epithelial cells in tissue culture. These responses were reduced, but not absent, from animals infected with the PC-deficient virus, which also induced IgG responses against gH. Moreover, congenital CMV transmission was confirmed in multiple animals infected with PC-deficient virus by detecting viral DNA in the amniotic fluid, indicating that transplacental transmission in RMs is not contingent on the PC.

Neutrophils, traditionally recognized for their role in innate immunity, have emerged as a key cell population at the maternal-fetal interface, during both uncomplicated and pathological pregnancies. Neutrophil effector functions, including phagocytosis, neutrophil extracellular trap formation, and degranulation, can play protective roles, such as preventing infection and facilitating tissue remodeling during pregnancy. However, these effector functions may also contribute to excessive inflammation, tissue damage, and adverse pregnancy outcomes in the context of sterile inflammation or maternal infection, underscoring the dual nature of neutrophils at the maternal-fetal interface. In this review, we examine the paradoxical nature of neutrophils at the maternal-fetal interface. Further, the protective and deleterious roles of neutrophils during pregnancy are evaluated in the context of bacterial, viral, and parasitic infections. Insights from this review are anticipated to inform basic and clinical research aimed at identifying neutrophils or neutrophil components as biomarkers and therapeutic targets in obstetric conditions and infectious diseases during pregnancy.

Throughout evolution, the placenta has diversified in structure and composition while maintaining its essential role in supporting fetal development. Trophoblasts, cells responsible for nutrient exchange and immune modulation, are a conserved feature of all placentas. Although primate placentas share morphological similarities, species-specific differences in gene expression remain poorly characterized, largely due to the lack of tractable in vitro models. To address this gap, we developed rhesus macaque placental organoids representing trophoblast and maternal-derived decidual cell types and compared them with human placental organoids. Using integrated single-cell and single-nucleus RNA sequencing, we identified shared and species-specific transcriptional programs across corresponding trophoblast lineages. We further reconstructed lineage trajectories to multinucleated syncytiotrophoblast and extravillous trophoblast populations, revealing conserved differentiation pathways alongside divergent gene expression signatures. This work establishes in vitro models of the nonhuman primate placenta and defines molecular distinctions between human and rhesus trophoblasts, offering insights into evolutionary adaptations underlying placental development.

Human cytomegalovirus (HCMV) encodes four viral Fc-gamma receptors (vFcγRs) that counteract antibody-mediated activation in vitro, but their role in infection and pathogenesis is unknown. To examine their in vivo function in an animal model evolutionarily closely related to humans, we identified and characterized Rh05, Rh152/151 and Rh173 as the complete set of vFcγRs encoded by rhesus CMV (RhCMV). Each one of these proteins displays functional similarities to their prospective HCMV orthologs with respect to antagonizing host FcγR activation in vitro. When RhCMV-naïve male rhesus macaques were infected with vFcγR-deleted RhCMV, peak plasma DNAemia levels and anti-RhCMV antibody responses were comparable to wildtype infections of both male and female animals. However, the duration of plasma DNAemia was significantly shortened in immunocompetent, but not in CD4 + T cell-depleted animals. Since vFcγRs were not required for superinfection of rhesus macaques, we conclude that these proteins can prolong lytic replication during primary infection by evading virus-specific adaptive immune responses, particularly antibodies. The role of viral Fc-gamma receptors in rhesus cytomegalovirus (RhCMV) infection is unclear. Here, the authors characterized RhCMV vFcγRs and report that their deletion did not affect virus replication, tropism or superinfection in rhesus macaques but increased susceptibility of the virus to antibody control.

ABSTRACT The success of chimeric antigen receptor (CAR)-T cell (Tc) immunotherapy in refractory B-cell acute lymphoblastic leukemia (B-ALL) suggests adaptation of this strategy toward HIV. Because cytomegalovirus (CMV) vaccine vectors generated Tc responses that controlled viral replication, these studies aim to genetically modify CMV-specific Tc with HIV-CAR2 vectors and link HIV immunotherapy to persistent CMV antigen stimulation. To mimic a clinical scenario, rhesus macaques were challenged with the CCR5-tropic simian/human immunodeficiency virus (SHIV-D) prior to antiretroviral therapy (ART). Autologous CMV-specific Tc were transduced with the control CEA-CAR2 or CD4-CAR2/maC46 vectors and reinfused. After stopping ART, the plasma viral load (PVL) in the control rebounded and was sustained above 1.7 × 10 4 copies/mL; PVL in CD4-CAR2-treated animals was delayed up to 6 weeks and 10-fold lower. The CD4 CAR-Tc frequency peaked at day 7 and was detected in lymphoid tissues at 6 weeks. Both CEA-CAR2 and CD4-CAR2 persisted in PBMCs for about 2 years, which indicates that the CMV-specific CAR Tc were maintained based on their CMV specificity. However, long-term PVL was stable in all animals. Thus, CMV-specific CAR-Tc were active initially, persisted long term, but failed to control viral replication. IMPORTANCE Because of latent viral reservoirs and a dysfunctional immune response, HIV replication rebounds when antiretroviral therapy is interrupted. Therefore, cytomegalovirus (CMV)-specific Tc were genetically modified with anti-HIV CD4-CAR2 vectors to link the targeting of the HIV envelope to the persistent CMV immune response. In this clinical scenario with simian/human immunodeficiency virus (SHIV) challenge and antiretroviral therapy (ART) suppression, early activity of the CAR Tc delayed rebound in the rhesus macaque/SHIV challenge model. However, even with long-term persistence of CAR Tc in the blood, control of viral replication was not achieved. These data suggest that CAR Tc will require additional interventions to cure HIV infection.

Congenital cytomegalovirus (cCMV) is the leading infectious cause of birth defects worldwide, yet immune determinants of protection to inform maternal vaccine design remain elusive due to the lack of a translational animal model. Here, we characterized the outcome of primary rhesus CMV (RhCMV) infection in pregnant, immunocompetent, CMV-naïve rhesus macaques. RhCMV DNA was detected in amniotic fluid and/or fetal tissues in six of 12 (50% placental transmission) dams following early second trimester gestation RhCMV inoculation. Widespread tissue dissemination dominated by one of two inoculated RhCMV strains was present in one fetus (8.3% cCMV disease). Placental transmission was associated with elevated fetal and maternal plasma TNF-alpha and reduced maternal brain-derived neurotrophic factor and IL-10 levels. CMV exposure during pregnancy had a broad impact on the placenta and fetus even in the absence of congenital infection, as evidenced by ubiquitous maternal-fetal interface infection, and reduced placental efficiency and small-for-gestation age fetuses compared to control pregnancies. This model recapitulates key aspects of human cCMV and provides new insights into the complexity of CMV vertical transmission.

Human immunodeficiency virus (HIV) and malaria, caused by infection with Plasmodium spp., are endemic in similar geographical locations. As a result, there is high potential for HIV/Plasmodium co-infection, which increases the pathology of both diseases. However, the immunological mechanisms underlying the exacerbated disease pathology observed in co-infected individuals are poorly understood. Here, we used the rhesus macaque (RM) model to characterize the immunopathogenic impact of Plasmodium fragile co-infection during antiretroviral therapy (ART)-treated simian immunodeficiency virus (SIV)-infection. We observed that P. fragile co-infection resulted in parasitemia and anemia, as well as persistently detectable viral loads (VL) and decreased absolute CD4+ T-cell counts despite daily ART treatment. Notably, P. fragile co-infection was associated with increased levels of inflammatory cytokines linked with neutrophil function, including monocyte chemoattractant protein 1 (MCP-1). P. fragile co-infection was associated with increased levels of neutrophil elastase, a plasma marker of neutrophil extracellular trap (NET) formation, but significant decreases in markers of neutrophil degranulation, potentially indicating a shift in neutrophil functionality during co-infection. Finally, we characterized levels of plasma markers of gastrointestinal (GI) barrier permeability and microbial translocation and observed significant correlations between indicators of GI dysfunction, clinical markers of SIV and Plasmodium infection, and neutrophil frequency and function. Taken together, these data indicate that neutrophil-driven inflammation and GI dysfunction may underlie heightened SIV/P. fragile co-infection pathogenesis.

Human immunodeficiency virus (HIV) and malaria, caused by infection with Plasmodium spp., are endemic in similar geographical locations. As a result, there is high potential for HIV/Plasmodium co-infection, which increases the pathology of both diseases. However, the immunological mechanisms underlying the exacerbated disease pathology observed in co-infected individuals are poorly understood. Moreover, there is limited data available on the impact of Plasmodium co-infection on antiretroviral (ART)-treated HIV infection. Here, we used the rhesus macaque (RM) model to conduct a pilot study to establish a model of Plasmodium fragile co-infection during ART-treated simian immunodeficiency virus (SIV) infection, and to begin to characterize the immunopathogenic effect of co-infection in the context of ART. We observed that P. fragile co-infection resulted in parasitemia and anemia, as well as persistently detectable viral loads (VLs) and decreased absolute CD4+ T-cell counts despite daily ART treatment. Notably, P. fragile co-infection was associated with increased levels of inflammatory cytokines, including monocyte chemoattractant protein 1 (MCP-1). P. fragile co-infection was also associated with increased levels of neutrophil elastase, a plasma marker of neutrophil extracellular trap (NET) formation, but significant decreases in markers of neutrophil degranulation, potentially indicating a shift in the neutrophil functionality during co-infection. Finally, we characterized the levels of plasma markers of gastrointestinal (GI) barrier permeability and microbial translocation and observed significant correlations between indicators of GI dysfunction, clinical markers of SIV and Plasmodium infection, and neutrophil frequency and function. Taken together, these pilot data verify the utility of using the RM model to examine ART-treated SIV/P. fragile co-infection, and indicate that neutrophil-driven inflammation and GI dysfunction may underlie heightened SIV/P. fragile co-infection pathogenesis.

The human immunodeficiency virus (HIV) continues to pose a significant global health challenge, with millions of people affected and new cases emerging each year. While various treatment and prevention methods exist, including antiretroviral therapy and non-vaccine approaches, developing an effective vaccine remains the most crucial and cost-effective solution to combating the HIV epidemic. Despite significant advancements in HIV research, the HIV vaccine field has faced numerous challenges, and only one clinical trial has demonstrated a modest level of efficacy. This review delves into the history of HIV vaccines and the current efforts in HIV prevention, emphasizing pre-clinical vaccine development using the non-human primate model (NHP) of HIV infection. NHP models offer valuable insights into potential preventive strategies for combating HIV, and they play a vital role in informing and guiding the development of novel vaccine candidates before they can proceed to human clinical trials.

Cytomegalovirus (CMV) is a master manipulator of host metabolic pathways. The impact of CMV metabolic rewiring during congenital CMV on immune function is unknown. CMV infection can directly alter glycolytic and oxidative phosphorylation pathways in infected cells. Recent data suggests CMV may alter metabolism in uninfected neighboring cells. In this mini review, we discuss how CMV infection may impact immune function through metabolic pathways. We discuss how immune cells differ between maternal and decidual compartments and how altered immunometabolism may contribute to congenital infections.

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 (B GC ) cells that last for at least 6 months. A 186-fold increase in B GC 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 B GC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding B GC 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 cells 1 , 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 B GC 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.