African Green Monkeys Research Articles

Replacement substance P reduces cardiac fibrosis in monkeys with type 2 diabetes

BackgroundType 2 diabetes mellitus (T2DM)-associated cardiac fibrosis contributes to heart failure. We previously showed that diabetic mice with cardiomyopathy, including cardiac fibrosis, exhibit low levels of the neuropeptide substance P; exogenous replacement of substance P reversed cardiac fibrosis, independent of body weight, blood glucose and blood pressure. We sought to elucidate the effectiveness and safety of replacement substance P to ameliorate or reverse cardiac fibrosis in type 2 diabetic monkeys. MethodsFour female T2DM African Green monkeys receive substance P (0.5 mg/Kg/day S.Q. injection) for 8 weeks. We obtained cardiac magnetic resonance imaging and blood samples to assess left ventricular function and fibrosis by T1 map-derived extracellular volume as well as circulating procollagen type I C-terminal propeptide. Hematological parameters for toxicities were also assessed in these monkeys and compared with three female T2DM monkeys receiving saline S.Q. as a safety comparison group. ResultsDiabetic monkeys receiving replacement substance P exhibited a ∼20% decrease in extracellular volume (p = 0.01), concomitant with ∼25% decrease procollagen type I C-terminal propeptide levels (p = 0.008). Left ventricular ejection fraction was unchanged with substance P (p = 0.42); however, circumferential strain was improved (p < 0.01). Complete blood counts, glycosylated hemoglobin A1c, lipids, liver and pancreatic enzymes, and inflammation markers were unchanged (p > 0.05). ConclusionsReplacement substance P reversed cardiac fibrosis in a large preclinical model of type 2 diabetes, independent of glycemic control. No hematological or organ-related toxicity was associated with replacement substance P. These results strongly support a potential application for replacement substance P as safe therapy for diabetic cardiac fibrosis.

Recombinant vesicular stomatitis virus-vectored vaccine induces long-lasting immunity against Nipah virus disease.

The emergence of the novel henipavirus, Langya virus, received global attention after the virus sickened over three dozen people in China. There is heightened concern that henipaviruses, as respiratory pathogens, could spark another pandemic, most notably the deadly Nipah virus (NiV). NiV causes near-annual outbreaks in Bangladesh and India and induces a highly fatal respiratory disease and encephalitis in humans. No licensed countermeasures against this pathogen exist. An ideal NiV vaccine would confer both fast-acting and long-lived protection. Recently, we reported the generation of a recombinant vesicular stomatitis virus-based (rVSV-based) vaccine expressing the NiV glycoprotein (rVSV-ΔG-NiVBG) that protected 100% of nonhuman primates from NiV-associated lethality within a week. Here, to evaluate the durability of rVSV-ΔG-NiVBG, we vaccinated African green monkeys (AGMs) one year before challenge with an uniformly lethal dose of NiV. The rVSV-ΔG-NiVBG vaccine induced stable and robust humoral responses, whereas cellular responses were modest. All immunized AGMs (whether receiving a single dose or prime-boosted) survived with no detectable clinical signs or NiV replication. Transcriptomic analyses indicated that adaptive immune signatures correlated with vaccine-mediated protection. While vaccines for certain respiratory infections (e.g., COVID-19) have yet to provide durable protection, our results suggest that rVSV-ΔG-NiVBG elicits long-lasting immunity.

Walk on the wild side: SIV infection in African non-human primate hosts-from the field to the laboratory.

HIV emerged following cross-species transmissions of simian immunodeficiency viruses (SIVs) that naturally infect non-human primates (NHPs) from Africa. While HIV replication and CD4+ T-cell depletion lead to increased gut permeability, microbial translocation, chronic immune activation, and systemic inflammation, the natural hosts of SIVs generally avoid these deleterious consequences when infected with their species-specific SIVs and do not progress to AIDS despite persistent lifelong high viremia due to long-term coevolution with their SIV pathogens. The benign course of natural SIV infection in the natural hosts is in stark contrast to the experimental SIV infection of Asian macaques, which progresses to simian AIDS. The mechanisms of non-pathogenic SIV infections are studied mainly in African green monkeys, sooty mangabeys, and mandrills, while progressing SIV infection is experimentally modeled in macaques: rhesus macaques, pigtailed macaques, and cynomolgus macaques. Here, we focus on the distinctive features of SIV infection in natural hosts, particularly (1): the superior healing properties of the intestinal mucosa, which enable them to maintain the integrity of the gut barrier and prevent microbial translocation, thus avoiding excessive/pathologic immune activation and inflammation usually perpetrated by the leaking of the microbial products into the circulation; (2) the gut microbiome, the disruption of which is an important factor in some inflammatory diseases, yet not completely understood in the course of lentiviral infection; (3) cell population shifts resulting in target cell restriction (downregulation of CD4 or CCR5 surface molecules that bind to SIV), control of viral replication in the lymph nodes (expansion of natural killer cells), and anti-inflammatory effects in the gut (NKG2a/c+ CD8+ T cells); and (4) the genes and biological pathways that can shape genetic adaptations to viral pathogens and are associated with the non-pathogenic outcome of the natural SIV infection. Deciphering the protective mechanisms against SIV disease progression to immunodeficiency, which have been established through long-term coevolution between the natural hosts and their species-specific SIVs, may prompt the development of novel therapeutic interventions, such as drugs that can control gut inflammation, enhance gut healing capacities, or modulate the gut microbiome. These developments can go beyond HIV infection and open up large avenues for correcting gut damage, which is common in many diseases.

ChAdOx1 NiV vaccination protects against lethal Nipah Bangladesh virus infection in African green monkeys

Nipah virus (NiV) is a highly pathogenic and re-emerging virus, which causes sporadic but severe infections in humans. Currently, no vaccines against NiV have been approved. We previously showed that ChAdOx1 NiV provides full protection against a lethal challenge with NiV Bangladesh (NiV-B) in hamsters. Here, we investigated the efficacy of ChAdOx1 NiV in the lethal African green monkey (AGM) NiV challenge model. AGMs were vaccinated either 4 weeks before challenge (prime vaccination), or 8 and 4 weeks before challenge with ChAdOx1 NiV (prime-boost vaccination). A robust humoral and cellular response was detected starting 14 days post-initial vaccination. Upon challenge, control animals displayed a variety of signs and had to be euthanized between 5 and 7 days post inoculation. In contrast, vaccinated animals showed no signs of disease, and we were unable to detect infectious virus in tissues and all but one swab. No to limited antibodies against fusion protein or nucleoprotein antigen could be detected 42 days post challenge, suggesting that vaccination induced a very robust protective immune response preventing extensive virus replication.

1960. Mucosal rAd5 Immunization against SARS-CoV-2 Spike Elicits Cross-Reactive Nasal and Serum Neutralizing Antibodies and Protects Against Beta Variant Challenge in African Green Monkeys

Abstract Background Mucosal vaccination may offer increased protection against SARS-CoV-2 compared to parental immunization. Here, we describe immunogenicity and efficacy following viral challenge in non-human primates after intranasal delivery of three unique non-replicating adenoviral vector vaccine (rAd5) candidates. Methods African green monkeys (AMG) were prime boost immunized 29 days apart with vaccine candidates either expressing the parental spike protein alone (Wuhan-S), spike plus nucleocapsid (Wuhan-S-N), or the spike protein from the beta variant (beta-S). Serum and nasal swabs were collected every 14 days and humoral responses to full length spike (S) and receptor binding domain (RBD) were assessed. All AMGs were challenged with SARS-CoV-2 B.1.351 (beta variant) on day 56. Viral loads measured every two days by TCID50 in nasal washes and bronchial lavage fluid post challenge. Results Mucosal immunization with Wuhan-S induced significant increases in serum IgG and IgA responses against the homologous parental lineage, as well as beta, delta, and omicron variants. In nasal samples, Wuhan-S immunization elicited over 500-fold increases in in cross-reactive IgA against multiple variants of concern including delta and omicron. While the beta-S rAd5 vaccine candidate induced enhanced serum IgG responses to homologous S and RBD proteins, this approach resulted in less cross-reactive antibodies to other variants compared to Wuhan-S rAd5 vaccine. Despite the differences in the ability to elicit cross-reactive antibody responses, all vaccinated AMGs challenged with SARS-CoV-2 B.1.351 (beta variant), had a significant reduction in viral titers by TCID50 in the nasal passages and reduced viral load in bronchial lavage fluid compared to unvaccinated controls. Conclusion These results demonstrate mucosal administration of rAd5 clinical candidate vaccine, Wuhan-S, is immunogenic and offers cross-protective humoral responses in both serum and nasal compartments against a mismatched SARS-CoV-2 challenge virus. Disclosures Becca A. Flitter, PhD, MPH, Vaxart Inc: Stocks/Bonds Sarah N. Tedjakusuma, n/a, Vaxart Inc: Stocks/Bonds Colin A. Lester, n/a, Vaxart Inc: Stocks/Bonds Elena D. Neuhaus, n/a, Vaxart Inc: Stocks/Bonds Susan Johnson, PhD, Vaxart Inc: Stocks/Bonds Emery G. Dora, n/a, Vaxart Inc: Stocks/Bonds Nadine Peinovich, MPH, Vaxart Inc: Stocks/Bonds Clara B. Jegede, n/a, Vaxart Inc: Stocks/Bonds Sean N. Tucker, PhD, Vaxart Inc: Stocks/Bonds.

Second Generation Adeno-Associated Viral Vector Using a Novel Factor VIII Variant with Improved Secretion Achieves Higher Factor VIII Expression in Non-Human Primates

Hemophilia A (HA) is an X-linked bleeding disorder caused by a deficiency in functional factor VIII (FVIII). Adeno-associated virus (AAV)-mediated gene therapy for HA is actively in clinical development, primarily utilizing codon-optimized (CO) wild type (WT) B-domain deleted human FVIII (hFVIII) transgenes. Limitations of these approaches highlight the need to develop novel second generation AAV approaches that improve transgene expression while reducing the vector dose with the fundamental goal of achieving durable levels of FVIII in the normal range while minimizing the potential risk of anti-AAV immune responses and genotoxicity. One of the main challenges of HA gene therapy development is that hFVIII is not expressed efficiently in vitro or in vivo compared to other proteins of similar size. Notably, the FVIII proteins of other species, such as canine and porcine, have superior expression. Previously, we reported that canine FVIII is expressed at higher levels than hFVIII which prompted us to conduct a comparative study between these two proteins (Sabatino 2009). Those studies led us to engineer and characterize a series of FVIII variants around the furin cleavage recognition site (1645-48)(Nguyen 2017) and a secondary cleavage site at 1657-58 in the acidic region 3 (a3). After investigating 15 variants, we identified our best performing variant, hFVIII-Δ3-SP/DE, carrying a deletion of three residues at the furin site (1645-47) (Δ3) (Nguyen 2017) and two amino acid substitutions S1657P and D1658E at the a3 cleavage site. With these minimal modifications, hFVIII-Δ3-SP/DE is secreted significantly more efficiently than WT hFVIII with and without codon-optimization and in conjunction with other improvements in the expression cassette. In the setting of AAV gene therapy, we used AAV8 to deliver WT hFVIII-CO or variant hFVIII-Δ3-SP/DE-CO driven by the modified transthyretin (TTRm) promoter and compared levels of FVIII expression in vivo. Immuno-deficient HA/CD4 KO mice administered AAV8-TTRm-hFVIII-Δ3-SP/DE-CO expressed FVIII levels that were 2-3-fold higher than animals delivered WT FVIII-CO. To investigate this variant in a large animal model, we previously injected AAV8-hFVIII-CO and AAV8-hFVIII-Δ3-SP/DE-CO (2x1012 vg/kg) in an HA dog model tolerized to hFVIII. We observed that the levels of hFVIII expression using the variant were 2 to 4-fold higher than WT hFVIII. Given the success of our variant in HA mice and dogs, we investigated hFVIII-Δ3-SP/DE in non-human primates (NHPs). Animals were negative for neutralizing antibodies to AAV8 prior to vector administration. We delivered AAV8-TTRm-hFVIII-CO or AAV8-TTRm-hFVIII-Δ3-SP/DE-CO to male African green monkeys via intravenous infusion at three vector doses (2x1012, 5x1012, 1x1013 vg/kg)(n=3 per dose per AAV construct). hFVIII antigen levels were measured by hFVIII specific ELISA and are expressed as a percentage of normal levels. Two weeks after AAV administration, NHPs treated with low dose (2x1012 vg/kg) of AAV8-hFVIII-CO expressed a median of 7.4% (range 5.2-8.8%) and AAV8-hFVIII-Δ3-SP/DE-CO expressed 19.2% (range 11.4-72.0%). The mid-dose cohort (5x1012 vg/kg) expressed 17.7% (range 11.8-128.2%) after AAV8-hFVIII-CO and 38.2% (range 12.1-97.0%) after AAV8-hFVIII-Δ3-SP/DE-CO delivery. At 1x1013 vg/kg the AAV8-hFVIII-CO treated NHPs expressed 17.9% (range 12.6-54.3%) while the AAV8-hFVIII-Δ3-SP/DE-CO expressed 114.2% (range 20.2-195.6%). As noted in clinical studies, there is some variability in hFVIII expression in our cohorts. Nevertheless, our data suggests that the delivery of the WT hFVIII transgene did not show a dose response. In contrast, there is a dose-dependent increase in hFVIII-Δ3-SP/DE expression, implying improved secretion correlating with higher levels of FVIII expression. As anticipated, anti-hFVIII antibodies developed to the non-species specific transgene in 16 of 18 animals after 3 weeks post vector administration, leading to a decline in hFVIII expression in most animals. In summary, consistent with our observations in HA mice and dogs, AAV delivery of the Δ3-SP/DE variant in NHPs results in 2 to 5-fold higher dose-dependent levels of FVIII expression than the WT hFVIII. This novel hFVIII variant provides the opportunity to overcome the challenge of inefficient FVIII secretion to improve the efficacy of AAV gene therapy for hemophilia A.

The epitranscriptome of Vero cells infected with SARS-CoV-2 assessed by direct RNA sequencing reveals m6A pattern changes and DRACH motif biases in viral and cellular RNAs.

The epitranscriptomics of the SARS-CoV-2 infected cell reveals its response to viral replication. Among various types of RNA nucleotide modifications, the m6A is the most common and is involved in several crucial processes of RNA intracellular location, maturation, half-life and translatability. This epitranscriptome contains a mixture of viral RNAs and cellular transcripts. In a previous study we presented the analysis of the SARS-CoV-2 RNA m6A methylation based on direct RNA sequencing and characterized DRACH motif mutations in different viral lineages. Here we present the analysis of the m6A transcript methylation of Vero cells (derived from African Green Monkeys) and Calu-3 cells (human) upon infection by SARS-CoV-2 using direct RNA sequencing data. Analysis of these data by nonparametric statistics and two computational methods (m6anet and EpiNano) show that m6A levels are higher in RNAs of infected cells. Functional enrichment analysis reveals increased m6A methylation of transcripts involved in translation, peptide and amine metabolism. This analysis allowed the identification of differentially methylated transcripts and m6A unique sites in the infected cell transcripts. Results here presented indicate that the cell response to viral infection not only changes the levels of mRNAs, as previously shown, but also its epitranscriptional pattern. Also, transcriptome-wide analysis shows strong nucleotide biases in DRACH motifs of cellular transcripts, both in Vero and Calu-3 cells, which use the signature GGACU whereas in viral RNAs the signature is GAACU. We hypothesize that the differences of DRACH motif biases, might force the convergent evolution of the viral genome resulting in better adaptation to target sequence preferences of writer, reader and eraser enzymes. To our knowledge, this is the first report on m6A epitranscriptome of the SARS-CoV-2 infected Vero cells by direct RNA sequencing, which is the sensu stricto RNA-seq.

Mucosal administration of a live attenuated recombinant COVID-19 vaccine protects nonhuman primates from SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 global pandemic. SARS-CoV-2 is an enveloped RNA virus that relies on its trimeric surface glycoprotein spike for entry into host cells. Here we describe the COVID-19 vaccine candidate MV-014-212, a live, attenuated, recombinant human respiratory syncytial virus expressing a chimeric SARS-CoV-2 spike as the only viral envelope protein. MV-014-212 was attenuated and immunogenic in African green monkeys (AGMs). One mucosal administration of MV-014-212 in AGMs protected against SARS-CoV-2 challenge, reducing by more than 200-fold the peak shedding of SARS-CoV-2 in the nose. MV-014-212 elicited mucosal immunoglobulin A in the nose and neutralizing antibodies in serum that exhibited cross-neutralization against virus variants of concern Alpha, Beta, and Delta. Intranasally delivered, live attenuated vaccines such as MV-014-212 entail low-cost manufacturing suitable for global deployment. MV-014-212 is currently in Phase 1 clinical trials as an intranasal COVID-19 vaccine.

Epigenetic Reprogramming Leads to Downregulation of CD4 and Functional Changes in African Green Monkey Memory CD4+ T Cells.

African green monkeys (AGMs), Chlorocebus pygerythrus, are a natural host for a lentivirus related to HIV, SIV. SIV-infected AGMs rarely progress to AIDS despite robust viral replication. Though multiple mechanisms are involved, a primary component is the animals' ability to downregulate CD4 expression on mature CD4+ Th cells, rendering these cells resistant to infection by SIV. These CD8αα+ T cells retain functional characteristics of CD4+ Th cells while simultaneously acquiring abilities of cytotoxic CD8αβ+ T cells. To determine mechanisms underlying functional differences between T cell subsets in AGMs, chromatin accessibility in purified populations was determined by assay for transposase-accessible chromatin sequencing. Differences in chromatin accessibility alone were sufficient to cluster cells by subtype, and accessibility at the CD4 locus reflected changes in CD4 expression. DNA methylation at the CD4 locus also correlated with inaccessible chromatin. By associating accessible regions with nearby genes, gene expression was found to correlate with accessibility changes. T cell and immune system activation pathways were identified when comparing regions that changed accessibility from CD4+ T cells to CD8αα+ T cells. Different transcription factor binding sites are revealed as chromatin accessibility changes, and these differences may elicit downstream changes in differentiation. This comprehensive description of the epigenetic landscape of AGM T cells identified genes and pathways that could have translational value in therapeutic approaches recapitulating the protective effects CD4 downregulation.

NK cell spatial dynamics and IgA responses in gut-associated lymphoid tissues during SIV infections

HIV infection induces tissue damage including lymph node (LN) fibrosis and intestinal epithelial barrier disruption leading to bacterial translocation and systemic inflammation. Natural hosts of SIV, such as African Green Monkeys (AGM), do not display tissue damage despite high viral load in blood and intestinal mucosa. AGM mount a NK cell-mediated control of SIVagm replication in peripheral LN. We analyzed if NK cells also control SIVagm in mesenteric (mes) LN and if this has an impact on gut humoral responses and the production of IgA known for their anti-inflammatory role in the gut. We show that CXCR5 + NK cell frequencies increase in mesLN upon SIVagm infection and that NK cells migrate into and control viral replication in B cell follicles (BCF) of mesLN. The proportion of IgA+ memory B cells were increased in mesLN during SIVagm infection in contrast to SIVmac infection. Total IgA levels in gut remained normal during SIVagm infection, while strongly decreased in intestine of chronically SIVmac-infected macaques. Our data suggest an indirect impact of NK cell-mediated viral control in mesLN during SIVagm infection on preserved BCF function and IgA production in intestinal tissues.

Exposure modality influences viral kinetics but not respiratory outcome of COVID-19 in multiple nonhuman primate species.

The novel coronavirus SARS-CoV-2 emerged in late 2019, rapidly reached pandemic status, and has maintained global ubiquity through the emergence of variants of concern. Efforts to develop animal models have mostly fallen short of recapitulating severe disease, diminishing their utility for research focusing on severe disease pathogenesis and life-saving medical countermeasures. We tested whether route of experimental infection substantially changes COVID-19 disease characteristics in two species of nonhuman primates (Macaca mulatta; rhesus macaques; RM, Chlorocebus atheiops; African green monkeys; AGM). Species-specific cohorts were experimentally infected with SARS-CoV-2 by either direct mucosal (intratracheal + intranasal) instillation or small particle aerosol in route-discrete subcohorts. Both species demonstrated analogous viral loads in all compartments by either exposure route although the magnitude and duration of viral loading was marginally greater in AGMs than RMs. Clinical onset was nearly immediate (+1dpi) in the mucosal exposure cohort whereas clinical signs and cytokine responses in aerosol exposure animals began +7dpi. Pathologies conserved in both species and both exposure modalities include pulmonary myeloid cell influx, development of pleuritis, and extended lack of regenerative capacity in the pulmonary compartment. Demonstration of conserved pulmonary pathology regardless of species and exposure route expands our understanding of how SARS-CoV-2 infection may lead to ARDS and/or functional lung damage and demonstrates the near clinical response of the nonhuman primate model for anti-fibrotic therapeutic evaluation studies.

Abstract 482: Assessing myocardial stiffness changes in a non-human primate anthracycline-induced cardiotoxicity model utilizing a biomechanical elasticity imaging methodology

Abstract Scope: Anthracyclines are a routine regimen for adjuvant therapy of breast cancer, but pose dose-dependent cardiotoxicity risks. With breast cancer diagnoses trending younger, there is a critical need for accurate non-invasive methods for assessing early cardiac degradation for mitigating cardiotoxicity due to cancer therapies. Methods: We developed a biomechanical model-based elasticity imaging method (BEIM) for assessing direct, functional mechanical stiffness of the left ventricle (LV) to indicate early cardiac degradation1. In this study, we examined the use of BEIM in a cohort of 5 African Green monkeys (AGM) receiving human equivalent doses of doxorubicin (cumulative dose: 240mg/m2) over 10 weeks. Cardiac magnetic resonance (CMR) imaging was acquired before and 15 weeks after final doxorubicin dose. Mechanical elasticity was estimated throughout the mid-plane of the LV utilizing BEIM. After necropsy, histopathology with picrosirius red of the matching LV mid-plane was conducted at 15 weeks after the final doxorubicin dose to calculate collagen volume fraction (CVF) indicative of anthracycline-associated cardiac fibrosis. Archival, age-and gender-matched LVs from healthy, chemotherapy naïve AGM were used as histopathological controls. Results: Two-dimensional circumferential, radial, and shear elasticity maps of the mid-plane of the LV indicate substantial changes in mechanical elasticity between baseline and 15 weeks post-doxorubicin CMR acquisitions. Average global change between baseline and post-doxorubicin image acquisitions in circumferential, radial, and shear modulus were 4.8, 5.7, and 5.2-fold increases respectively, with an average 1.9-fold change of CVF. Global circumferential, radial, and shear modulus values at baseline and 15 weeks post-doxorubicin were compared to CVF values for each subject and indicate a very strong correlation between circumferential, radial, and shear modulus with CVF, represented by Pearson’s correlation coefficients of 0.91, 0.92, and 0.89, respectively. Conclusions: We developed BEIM to assess changes in LV mechanical elasticity utilizing routine CMR imaging data with a goal of non-invasively identifying cardiac stiffening associated with cardiotoxicity. To validate our methodology, we utilized CMR data from an AGM model of anthracycline-induced cardiotoxicity to estimate changes in LV elasticity and compare to CVF values from histopathology. Our results show an increase in both CVFs and mechanical elasticity between baseline and 15 weeks following the last doxorubicin dose. Elasticity measures exhibit a very strong correlation with histopathological assessment of cardiac fibrosis. Results demonstrate the ability of our BEIM to identify changes in elasticity related to increased collagen deposition from anthracycline-induced cardiotoxicity. Citation Format: Caroline Miller, Jennifer H. Jordan, Alexandra Thomas, Giselle Meléndez, Jared A. Weis. Assessing myocardial stiffness changes in a non-human primate anthracycline-induced cardiotoxicity model utilizing a biomechanical elasticity imaging methodology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 482.

Circulating S-Glutathionylated cMyBP-C as a Biomarker for Cardiac Diastolic Dysfunction.

BackgroundcMyBP‐C (Cardiac myosin binding protein‐C) regulates cardiac contraction and relaxation. Previously, we demonstrated that elevated myocardial S‐glutathionylation of cMyBP‐C correlates with diastolic dysfunction (DD) in animal models. In this study, we tested whether circulating S‐glutathionylated cMyBP‐C would be a biomarker for DD.Methods and ResultsHumans, African Green monkeys, and mice had DD determined by echocardiography. Blood samples were acquired and analyzed for S‐glutathionylated cMyBP‐C by immunoprecipitation. Circulating S‐glutathionylated cMyBP‐C in human participants with DD (n=24) was elevated (1.46±0.13‐fold, P=0.014) when compared with the non‐DD controls (n=13). Similarly, circulating S‐glutathionylated cMyBP‐C was upregulated by 2.13±0.47‐fold (P=0.047) in DD monkeys (n=6), and by 1.49 (1.22–2.06)‐fold (P=0.031) in DD mice (n=5) compared with the respective non‐DD controls. Circulating S‐glutathionylated cMyBP‐C was positively correlated with DD in humans.ConclusionsCirculating S‐glutathionylated cMyBP‐C was elevated in humans, monkeys, and mice with DD. S‐glutathionylated cMyBP‐C may represent a novel biomarker for the presence of DD.

Intravenous delivery of GS-441524 is efficacious in the African green monkey model of SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.

A Non‐Human Primate Model of Carfentanil Overdose: Preventing Renarcotization with Superior Countermeasures

We established a physiological and overt intoxication model using African green monkeys (Chlorocebus aethiops sabeus) and a high dose of injected carfentanil. Intoxication is rapid and rescue is typically required within 10 minutes. Naloxone, administered at a 10 mg human‐equivalent dose (HED; 0.355 mg/kg), is immediately effective but is limited by its short duration of action. Specifically, animals typically return to a state of severe respiratory depression within about 2.5 hours, necessitating further medical intervention. Therefore, we studied two novel medical countermeasures: nalmefene and CS‐1131. Nalmefene is a mu‐opioid antagonist with high affinity and a much longer duration of action, but little is known about its efficacy against carfentanil. Therefore, we covered a range of doses from 3‐30 mg estimated HED (based upon allometric scaling) through both the intramuscular and intranasal routes. CS‐1131 is a novel opioid scavenger (created by Clear Scientific, LLC) capable of binding carfentanil stoichiometrically, inactivating the synthetic opioid, and then eliminating the scavenger‐opioid complex through renal excretion. Results show that both countermeasures were superior to naloxone alone and exhibited dose‐dependent efficacy. This research provides strong proof‐of‐concept and sets the stage for additional studies of longer‐lasting opioid antagonists administered through various routes, and for the further development, characterization, and refinement of novel opioid scavenger molecules. This non‐human primate model can be utilized to study potent synthetic opioids, new analogues, and other novel countermeasures in a way that yields predictive results to support human clinical trials.

Sex Differences in Insulin Sensitivity and Glomerular Function in African Green Monkeys

In the United States, it is estimated that approximately 10.5% of the population has diabetes of which 90% are classified as insulin resistant expressing Type 2 Diabetes Mellitus (T2DM). T2DM is one of the leading causes of end stage renal disease (ESRD) accounting for approximately 40% of new ESRD cases. Previous studies have reported a more prevalent decrease in renal function in glucose intolerant males when compared to pre‐menopausal females. Males also have been reported to have earlier onset and higher prevalence of T2DM compared to pre‐menopausal females. Renal injury can be present before the onset of T2DM however, sex differences in renal injury have yet to be assessed in humans. A subpopulation of African Green Monkeys (AGM; Chlorocebus aethiops sabaeus) in the Caribbean exhibit spontaneous glucose intolerance and co‐morbidities such as insulin insensitivity, and hypertension. The AGM also shares similar genomic expression and metabolic phenotypes to humans making them optimal for translatable diabetes research. We hypothesize that female AGM are protected against renal injury and early onset T2DM. Systolic blood pressure (SBP) was measured by forearm plethysmography. Glucose tolerance was assessed via conscious and anesthetized oral glucose tolerance tests (OGTT). All AGM were fasted 12‐18 hours prior to study with water provided ad libitum. Blood glucose was measured at 0, 15, 30, 60, 90, and 120 min after an oral glucose load. Conscious AGM OGTT were delivered as 0.75g glucose/kg body weight. Sedated AGM (ketamine, 15mg/kg, i.m.), received 5g/dl glucose and 15 ml/kg in water by oral gavage. Venous blood samples were collected at timepoints 0, 30, 60, and 120 minutes post glucose administration. Urine samples were collected for 3 days post OGTT. Kidney histology was performed by staining 10µm sections with Periodic‐Acid Schiff stain (PAS). A minimum of 10 glomeruli were identified at 400x magnification per animal then analyzed with ImageJ. There were no differences between lightly sedated or conscious OGTT. All AGM in this study were normotensive and there were no sex differences in blood pressure between male and female AGM (F=11; 113.9±3.73 mmHg, M=6; 117.6 ± 6.3mmHg). Female AGM OGTT area under the curve (AUC) over 120 minutes post glucose load were lower compared to male AGM (F; 11,961.5± 710.6 min x mg/dl, M; 14,076.3±710.6 min x mg/dl, p&lt;0.05). There were no significant differences in plasma insulin at all time points between male and female AGM (F; 2,013± 1,007 min x mIU/L, M; 5,940± 1,424.6 min x mIU/L) despite the lower glucose AUC in females. Renal glomerular function was assessed by measuring plasma creatinine, urinary albumin, and urinary albumin to creatinine ratio (UACR). Plasma creatinine was not different between male and females (F; 1.35±0.2 mg/dl, M; 1.77±0.25 mg/dl). Microalbuminuria was present in both male and female AGMs, in the absence of sex differences (F; 98.43±23.1 ng/ml, M; 81.15±29.2 ng/ml). UACRs were not different between male and female AGM. Glomerular injury was assessed by determining the intensity of glomerular PAS stain. PAS stain intensity was not different in male and female AGM (F=3; 178.3 ± 8.6, M=3; 181.6± 8.6) regardless of OGTT sensitivity. Together these data suggest that female AGM are more insulin sensitive than male AGM and may be protected against early onset T2DM. Despite differences in OGTT in male and female AGM, microalbuminuria suggested onset of renal dysfunction. We conclude that the AGM provides a strong nonhuman primate model of T2DM for the long‐term study of sex differences in the mechanisms of ESRD.

A Comprehensive Atlas of Immunological Differences Between Humans, Mice, and Non-Human Primates.

Animal models are an integral part of the drug development and evaluation process. However, they are unsurprisingly imperfect reflections of humans, and the extent and nature of many immunological differences are unknown. With the rise of targeted and biological therapeutics, it is increasingly important that we understand the molecular differences in the immunological behavior of humans and model organisms. However, very few antibodies are raised against non-human primate antigens, and databases of cross-reactivity between species are incomplete. Thus, we screened 332 antibodies in five immune cell populations in blood from humans and four non-human primate species generating a comprehensive cross-reactivity catalog that includes cell type-specificity. We used this catalog to create large mass cytometry universal cross-species phenotyping and signaling panels for humans, along with three of the model organisms most similar to humans: rhesus and cynomolgus macaques and African green monkeys; and one of the mammalian models most widely used in drug development: C57BL/6 mice. As a proof-of-principle, we measured immune cell signaling responses across all five species to an array of 15 stimuli using mass cytometry. We found numerous instances of different cellular phenotypes and immune signaling events occurring within and between species, and detailed three examples (double-positive T cell frequency and signaling; granulocyte response to Bacillus anthracis antigen; and B cell subsets). We also explore the correlation of herpes simian B virus serostatus on the immune profile. Antibody panels and the full dataset generated are available online as a resource to enable future studies comparing immune responses across species during the evaluation of therapeutics.

Intrathecal amyloid-beta oligomer administration increases tau phosphorylation in the medial temporal lobe in the African green monkey: A nonhuman primate model of Alzheimer's disease.

An obstacle to developing new treatment strategies for Alzheimer's disease (AD) has been the inadequate translation of findings in current AD transgenic rodent models to the prediction of clinical outcomes. By contrast, nonhuman primates (NHPs) share a close neurobiology with humans in virtually all aspects relevant to developing a translational AD model. The present investigation used African green monkeys (AGMs) to refine an inducible NHP model of AD based on the administration of amyloid-beta oligomers (AβOs), a key upstream initiator of AD pathology. AβOs or vehicle were repeatedly delivered over 4 weeks to age-matched young adult AGMs by intracerebroventricular (ICV) or intrathecal (IT) injections. Induction of AD-like pathology was assessed in subregions of the medial temporal lobe (MTL) by quantitative immunohistochemistry (IHC) using the AT8 antibody to detect hyperphosphorylated tau. Hippocampal volume was measured by magnetic resonance imaging (MRI) scans prior to, and after, intrathecal injections. IT administration of AβOs in young adult AGMs revealed an elevation of tau phosphorylation in the MTL cortical memory circuit compared with controls. The largest increases were detected in the entorhinal cortex that persisted for at least 12 weeks after dosing. MRI scans showed a reduction in hippocampal volume following AβO injections. Repeated IT delivery of AβOs in young adult AGMs led to an accelerated AD-like neuropathology in MTL, similar to human AD, supporting the value of this translational model to de-risk the clinical trial of diagnostic and therapeutic strategies.

Detailed analysis of the pathologic hallmarks of Nipah virus (Malaysia) disease in the African green monkey infected by the intratracheal route.

Disease associated with Nipah virus infection causes a devastating and often fatal spectrum of syndromes predominated by both respiratory and neurologic conditions. Additionally, neurologic sequelae may manifest months to years later after virus exposure or apparent recovery. In the two decades since this disease emerged, much work has been completed in an attempt to understand the pathogenesis and facilitate development of medical countermeasures. Here we provide detailed organ system-specific pathologic findings following exposure of four African green monkeys to 2.41×105 pfu of the Malaysian strain of Nipah virus. Our results further substantiate the African green monkey as a model of human Nipah virus disease, by demonstrating both the respiratory and neurologic components of disease. Additionally, we demonstrate that a chronic phase of disease exists in this model, that may provide an important opportunity to study the enigmatic late onset and relapse encephalitis as it is described in human disease.

Chronic Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta) with Cisterna Magna Ports: Update on Refinements.

More than 20 y ago, we developed an animal model for chronic and continuous collection of cerebrospinal fluid (CSF) from conscious rhesus macaques. Since our previous publication in 2003, we have successfully implanted 168 rhesus macaques using this approach. Our experience enables us to provide up-to-date information regarding the model, including refine- ments to our implant design, reductions in maintenance, and new procedures for dealing with contamination. The results of our experiences have reduced the number of surgeries required and helped to increase the longevity of the implant, with some functioning for more than 18 y. Building on our success in rhesus macaques, we attempted to develop similar animal models in the African green monkeys and dogs but have been unable to develop reliable chronic models for CSF collection in these species.