Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Immune restoration by TIGIT blockade is insufficient to control chronic SIV infection.

TIGIT is a negative immune checkpoint receptor associated with T cell exhaustion in cancer and HIV. TIGIT upregulation in virus-specific CD8+ T cells and NK cells during HIV/SIV infection results in dysfunctional effector capabilities. In vitro studies targeting TIGIT on CD8+ T cells suggest TIGIT blockade as a viable strategy to restore SIV-specific T cell responses. Here, we extend these studies in vivo using TIGIT blockage in nonhuman primates in an effort to reverse T cell and NK cell exhaustion in the setting of SIV infection. We demonstrate that in vivo administration of a humanized anti-TIGIT monoclonal antibody (mAb) is well tolerated in both cynomolgus macaques and rhesus macaques. Despite sustained plasma concentrations of anti-TIGIT mAb, we observed no consistent improvement in NK or T cell cytolytic capacity. TIGIT blockade minimally enhanced T cell proliferation and virus-specific T cell responses in both magnitude and breadth though plasma viral loads in treated animals remained stable indicating that anti-TIGIT mAb treatment alone was insufficient to increase anti-SIV CD8+ T cell function. The enhancement of virus-specific T cell proliferative responses observed in vitro with single or dual blockade of TIGIT and/or PD-1 highlights TIGIT as a potential target to reverse T cell dysfunction. Our studies, however, reveal that targeting the TIGIT pathway alone may be insufficient in the setting of viremia and that combining immune checkpoint blockade with other immunotherapeutics may be a future path forward for improved viral control or elimination of HIV.IMPORTANCEUpregulation of the immune checkpoint receptor TIGIT is associated with HIV-mediated T cell dysfunction and correlates with HIV disease progression. Compelling evidence exists for targeting immune checkpoint receptor pathways that would potentially enhance immunity and refocus effector cell efforts toward viral clearance. In this report, we investigate TIGIT blockade as an immunotherapeutic approach to reverse immune exhaustion during chronic SIV/SHIV infection in a nonhuman primate model of HIV infection. We show that interfering with the TIGIT signaling axis alone is insufficient to improve viral control despite modest improvement in T cell immunity. Our data substantiate the use of targeting multiple immune checkpoint receptors to promote synergy and ultimately eliminate HIV-infected cells.

Compartmentalized SARS-CoV-2 replication in upper versus lower respiratory tract after intranasal inoculation or aerosol exposure.

Non-human primate models are essential for the development of vaccines and antivirals against infectious diseases. Rhesus macaques are a widely utilized infection model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We compared cellular tropism and virus replication in rhesus macaques inoculated with SARS-CoV-2 via the intranasal route, or via exposure to aerosols. Intranasal inoculation results in replication in the upper respiratory tract and limited lower respiratory tract involvement, whereas exposure to aerosols results in infection throughout the respiratory tract. In comparison to multi-route inoculation, the intranasal and aerosol inoculation routes result in reduced SARS-CoV-2 replication in the respiratory tract.

A 3-month-old neonatal rhesus macaque HFMD model caused by coxsackievirus B1 infection and viral tissue tropism.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

Excess prenatal loss and respiratory illnesses of infant macaques living outdoors and exposed to wildfire smoke.

Global climate change has transformed predictions of fire seasons in the near future, and record-breaking wildfire events have had catastrophic consequences in recent years. In September 2020, multiple wildfires subjected Oregon to hazardous air quality for several days. In this retrospective cohort study, we aimed to examine prenatal loss, morbidity, and mortality of rhesus (Macaca mulatta) and Japanese macaques (Macaca fuscata) exposed to poor air quality from the nearby wildfires. Detailed medical records from 2014 to 2020 of 580 macaques housed outdoors at a research facility in Beaverton, Oregon were used to evaluate the association between these health outcomes and wildfire smoke exposure. Logistic regression models estimated excess prenatal loss, hospitalization rates, respiratory problems, and mortality during and following the wildfire event, and Kruskal-Wallis statistics were used to determine if infant growth was affected by wildfire smoke exposure. Risk of pregnancy loss (relative risk = 4.1; p < 0.001) and odds of diagnosis with a respiratory problem (odds ratio = 4.47; p = 0.003) were higher in exposed infant macaques compared to nonexposed infants. Infant growth was not affected by poor air quality exposure. Our findings suggest wildfire smoke exposure poses a risk to the health of infants and pregnant individuals and should be monitored more closely in the future.

Hepatitis E virus infection upregulates ING5 expression in vitro and in vivo.

Hepatitis E virus (HEV) is the major pathogen of viral hepatitis. Immunocompromised individuals infected by HEV are prone to chronic hepatitis and increase the risk of hepato-cellular carcinoma (HCC). Inhibitor of growth family member 5 (ING5) is a tumor suppressor that is expressed at low levels in cancer tumors or cells. However, the underlying relationship between ING5 and HEV infection is unclear. In the present study, acute and chronic HEV animal models are used to explore the interaction between ING5 and HEV. Notably, the expression of ING5 is significantly increased in both the livers of acute HEV-infected BALB/c mice and chronic HEV-infected rhesus macaques. In addition, the relationship between HEV infection and ING5 expression is further identified in human hepatoma (HepG-2) cells. In conclusion, HEV infection strongly upregulates ING5 expression both in vivo and in vitro, which has significant implications for further understanding the pathogenic mechanism of HEV infection.

Sex- and species-associated differences in complement-mediated immunity in humans and rhesus macaques.

The complement system can be viewed as a "moderator" of innate immunity, "instructor" of humoral immunity, and "regulator" of adaptive immunity. While sex is known to affect humoral and cellular immune systems, its impact on complement in humans and rhesus macaques, a commonly used non-human primate model system, has not been well studied. To address this knowledge gap, we analyzed serum samples from 90 humans and 72 rhesus macaques for the abundance and activity of the complement system components. While sequences of cascade proteins were highly conserved, dramatically different levels were observed between species. Whereas the low levels detected in rhesus samples raised questions about the suitability of the test for use with macaque samples, differences in levels of complement proteins were observed in male and female humans. Levels of total and antibody-dependent deposition of C1q and C3b on a glycosylated antigen differed between humans and rhesus, suggesting differential recognition of glycans and balance between classical and alternative activation pathways. Functional differences in complement-mediated lysis of antibody-sensitized cells were observed in multiple assays and showed that human females frequently exhibited higher lytic activity than human males or rhesus macaques, which typically did not exhibit such sex-associated differences. Other differences between species and sexes were observed in more narrow contexts-for only certain antibodies, antigens, or assays. Collectively, these results expand knowledge of sex-associated differences in the complement system in humans, identifying differences absent from rhesus macaques.IMPORTANCEThe complement system is a critical part of host defense to many bacterial, fungal, and viral infections. In parallel, rich epidemiological, clinical, and biomedical research evidence demonstrates that sex is an important biological variable in immunity, and many sex-specific differences in immune system are intimately tied with disease outcomes. This study focuses on the intersection of these two factors to define the impact of sex on complement pathway components and activities. This work expands our knowledge of sex-associated differences in the complement system in humans and also identifies the differences that appear to be absent in rhesus macaques, a popular non-human primate model. Whereas differences between species suggest potential limitations in the ability of macaque model to recapitulate human biology, knowledge of sex-based differences in humans has the potential to inform clinical research and practice.

Antimicrobial resistance spectrum and virulence characterization of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis isolated from asymptomatic and diarrheal rhesus monkeys.

This study aims to deepen our understanding of the drug resistance and virulence characterization among gut bacteria in asymptomatic and diarrheal captive rhesus macaques (RMs). A total of 31 samples, including 8 asymptomatic RMs, 10 diarrheal RMs, and 1 dead RM, were collected from a breeding base in Sichuan, China, for bacterial isolation. As a result, Escherichia coli (n = 23), Klebsiella (n = 22), Proteus mirabilis (n = 10), Enterococcus (n = 10), Salmonella (n = 2), and Staphylococcus (n = 2) were isolated. All isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing, among which some E. coli, K. pneumoniae, and P. mirabilis were subjected to the Galleria mellonella and mice infection testing. The antimicrobial resistance rates of levofloxacin, enrofloxacin, and cefotaxime in diarrhea-associated isolates were higher than those of asymptomatic isolates. Consistent with the antimicrobial resistance phenotype, diarrheal isolates had a higher prevalence rate to qnrS1, blaTEM-1B and blaCTX-M-27 than asymptomatic isolates. Furthermore, compared with asymptomatic isolates, diarrheal isolates demonstrated a higher pathogenic potential against larvae and mice. Additionally, sequence types (STs) 14179-14181 in E. coli and ST 625 and ST 630-631 in Klebsiella aerogenes were firstly characterized. Our evidence underscores the considerable challenge posed by high rates of bacterial drug resistance in the effective treatment of diarrheal RMs.

Effects of healthy aging on tongue-jaw kinematics during feeding behavior in rhesus macaques.

Several age-related oral health problems have been associated with neurodegenerative diseases such as Alzheimer's Disease (AD), yet how oromotor dysfunction in healthy aging differ from those found in pathological aging is still unknown. This is partly because changes in the cortical and biomechanical ("neuromechanical") control of oromotor behavior in healthy aging are poorly understood. To this end, we investigated the natural feeding behavior of young and aged rhesus macaques (Macaca mulatta) to understand the age-related differences in tongue and jaw kinematics. We tracked tongue and jaw movements in 3D using high-resolution biplanar videoradiography and X-ray Reconstruction of Moving Morphology (XROMM). Older subjects exhibited a reduced stereotypy in tongue movements during chews and a greater lag in tongue movements relative to jaw movements compared to younger subjects. Overall, our findings reveal age-related changes in tongue and jaw kinematics, which may indicate impaired tongue-jaw coordination. Our results have important implications for the discovery of potential neuromechanical biomarkers for early diagnosis of AD.

A SMART method for isolating monoclonal antibodies from individual rhesus macaque memory B cells.

Characterizing antigen-specific B cells is a critical component of vaccine and infectious disease studies in rhesus macaques (RMs). However, it is challenging to capture immunoglobulin variable (IgV) genes from individual RM B cells using 5' multiplex (MTPX) primers in nested PCR reactions. In particular, the diversity within RM IgV gene leader sequences necessitates large 5' MTPX primer sets to amplify IgV genes, decreasing PCR efficiency. To address this problem, we developed a switching mechanism at the 5' ends of the RNA transcript (SMART)-based method for amplifying IgV genes from single RM B cells to capture Ig heavy and light chain pairs. We demonstrate this technique by isolating simian immunodeficiency virus (SIV) envelope-specific antibodies from single-sorted RM memory B cells. This approach has several advantages over existing methods for cloning antibodies from RMs. First, optimized PCR conditions and SMART 5' and 3' rapid amplification of cDNA ends (RACE) reactions generate full-length cDNAs from individual B cells. Second, it appends synthetic primer binding sites to the 5' and 3' ends of cDNA during synthesis, allowing for PCR amplification of low-abundance antibody templates. Third, the nested PCR primer mixes are simplified by employing universal 5' primers, eliminating the need for complex 5' MTPX primer sets. We anticipate this method will enhance the isolation of antibodies from individual RM B cells, supporting the genetic and functional characterization of antigen-specific B cells.

Comparative transcriptome analysis between rhesus macaques ( Macaca mulatta) and crab-eating macaques ( M. fascicularis).

Understanding gene expression variations between species is pivotal for deciphering the evolutionary diversity in phenotypes. Rhesus macaques ( Macaca mulatta, MMU) and crab-eating macaques ( M. fascicularis, MFA) serve as crucial nonhuman primate biomedical models with different phenotypes. To date, however, large-scale comparative transcriptome research between these two species has not yet been fully explored. Here, we conducted systematic comparisons utilizing newly sequenced RNA-seq data from 84 samples (41 MFA samples and 43 MMU samples) encompassing 14 common tissues. Our findings revealed a small fraction of genes (3.7%) with differential expression between the two species, as well as 36.5% of genes with tissue-specific expression in both macaques. Comparison of gene expression between macaques and humans indicated that 22.6% of orthologous genes displayed differential expression in at least two tissues. Moreover, 19.41% of genes that overlapped with macaque-specific structural variants showed differential expression between humans and macaques. Of these, the FAM220A gene exhibited elevated expression in humans compared to macaques due to lineage-specific duplication. In summary, this study presents a large-scale transcriptomic comparison between MMU and MFA and between macaques and humans. The discovery of gene expression variations not only enhances the biomedical utility of macaque models but also contributes to the wider field of primate genomics.

Lethal dog attacks on adult rhesus macaques (Macaca mulatta) in an anthropogenic landscape.

For nonhuman primates living in anthropogenic areas, predation by larger predators is relatively rare. However, smaller predators, such as free-ranging as well as domesticated dogs, can shape the socioecology of urban nonhuman primates, either directly by attacking and killing them or indirectly by modifying their activity patterns. Here, we describe three (two probably fatal) cases of dog attacks on adult rhesus macaques inhabiting an anthropogenic landscape in Northern India and the circumstances surrounding these incidents. We discuss the importance of considering human presence and intervention in dog-nonhuman primate relationships while studying nonhuman primate populations across anthropogenic gradients, and its potential influences on group social dynamics and transmission of zoonotic agents.

Metagenomic comparison of intestinal microbiota between normal and liver fibrotic rhesus macaques (Macaca mulatta).

Liver fibrosis is an important pathological process in chronic liver disease and cirrhosis. Recent studies have found a close association between intestinal microbiota and the development of liver fibrosis. To determine whether there are differences in the intestinal microbiota between rhesus macaques with liver fibrosis (MG) and normal rhesus macaques (MN), fecal samples were collected from 8 male MG and 12 male MN. The biological composition of the intestinal microbiota was then detected using 16S rRNA gene sequencing. The results revealed statistically significant differences in ASVs and Chao1 in the alpha-diversity and the beta-diversity of intestinal microbiota between MG and MN. Both groups shared Prevotella and Lactobacillus as common dominant microbiota. However, beneficial bacteria such as Lactobacillus were significantly less abundant in MG (P = 0.02). Predictive functional analysis using PICRUSt2 gene prediction revealed that MG exhibited a higher relative abundance of functions related to substance transport and metabolic pathways. This study may provide insight into further exploration of the mechanisms by which intestinal microbiota affect liver fibrosis and its potential future use in treating liver fibrosis.

Development of a hydrogen peroxide-inactivated vaccine that protects against viscerotropic yellow fever in a non-human primate model.

Yellow fever virus (YFV) is endemic in >40 countries and causes viscerotropic disease with up to 20%-60% mortality. Successful live-attenuated yellow fever (YF) vaccines were developed in the mid-1930s, but their use is restricted or formally contraindicated in vulnerable populations including infants, the elderly, and people with compromised immune systems. In these studies, we describe the development of a next-generation hydrogen peroxide-inactivated YF vaccine and determine immune correlates of protection based on log neutralizing index (LNI) and neutralizing titer-50% (NT50) studies. In addition, we compare neutralizing antibody responses and protective efficacy of hydrogen peroxide-inactivated YF vaccine candidates to live-attenuated YFV-17D (YF-VAX) in a rhesus macaque model of viscerotropic YF. Our results indicate that an optimized, inactivated YF vaccine elicits protective antibody responses that prevent viral dissemination and lethal infection in rhesus macaques and may be a suitable alternative for vaccinating vulnerable populations who are not eligible to receive replicating live-attenuated YF vaccines.

Taking identity-by-descent analysis into the wild: Estimating realized relatedness in free-ranging macaques.

Biological relatedness is a key consideration in studies of behavior, population structure, and trait evolution. Except for parent-offspring dyads, pedigrees capture relatedness imperfectly. The number and length of DNA segments that are identical-by-descent (IBD) yield the most precise estimates of relatedness. Here, we leverage novel methods for estimating locus-specific IBD from low coverage whole genome resequencing data to demonstrate the feasibility and value of resolving fine-scaled gradients of relatedness in free-living animals. Using primarily 4-6× coverage data from a rhesus macaque (Macaca mulatta) population with available long-term pedigree data, we show that we can call the number and length of IBD segments across the genome with high accuracy even at 0.5× coverage. The resulting estimates demonstrate substantial variation in genetic relatedness within kin classes, leading to overlapping distributions between kin classes. They identify cryptic genetic relatives that are not represented in the pedigree and reveal elevated recombination rates in females relative to males, which allows us to discriminate maternal and paternal kin using genotype data alone. Our findings represent a breakthrough in the ability to understand the predictors and consequences of genetic relatedness in natural populations, contributing to our understanding of a fundamental component of population structure in the wild.

Multivariate analysis of FcR-mediated NK cell functions identifies unique clustering among humans and rhesus macaques.

Rhesus macaques (RMs) are a common pre-clinical model used to test HIV vaccine efficacy and passive immunization strategies. Yet, it remains unclear to what extent the Fc-Fc receptor (FcR) interactions impacting antiviral activities of antibodies in RMs recapitulate those in humans. Here, we evaluated the FcR-related functionality of natural killer cells (NKs) from peripheral blood of uninfected humans and RMs to identify intra- and inter-species variation. NKs were screened for FcγRIIIa (human) and FcγRIII (RM) genotypes (FcγRIII(a)), receptor signaling, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by a cocktail of monoclonal IgG1 antibodies with human or RM Fc. FcγRIII(a) genetic polymorphisms alone did not explain differences in NK effector functionality in either species cohort. Using the same parameters, hierarchical clustering separated each species into two clusters. Importantly, in principal components analyses, ADCC magnitude, NK contribution to ADCC, FcγRIII(a) cell-surface expression, and frequency of phosphorylated CD3ζ NK cells all contributed similarly to the first principal component within each species, demonstrating the importance of measuring multiple facets of NK cell function. Although ADCC potency was similar between species, we detected significant differences in frequencies of NK cells and pCD3ζ+ cells, level of cell-surface FcγRIII(a) expression, and NK-mediated ADCC (P<0.001), indicating that a combination of Fc-FcR parameters contribute to overall inter-species functional differences. These data strongly support the importance of multi-parameter analyses of Fc-FcR NK-mediated functions when evaluating efficacy of passive and active immunizations in pre- and clinical trials and identifying correlates of protection. The results also suggest that pre-screening animals for multiple FcR-mediated NK function would ensure even distribution of animals among treatment groups in future preclinical trials.

Defining genetic diversity of rhesus macaque Fcγ receptors with long-read RNA sequencing.

Fcγ receptors (FcγRs) are membrane-bound glycoproteins that bind to the fragment crystallizable (Fc) constant regions of IgG antibodies. Interactions between IgG immune complexes and FcγRs can initiate signal transduction that mediates important components of the immune response including activation of immune cells for clearance of opsonized pathogens or infected host cells. In humans, many studies have identified associations between FcγR gene polymorphisms and risk of infection, or progression of disease, suggesting a gene-level impact on FcγR-dependent immune responses. Rhesus macaques are an important translational model for most human health interventions, yet little is known about the breadth of rhesus macaque FcγR genetic diversity. This lack of knowledge prevents evaluation of the impact of FcγR polymorphisms on outcomes of preclinical studies performed in rhesus macaques. In this study we used long-read RNA sequencing to define the genetic diversity of FcγRs in 206 Indian-origin Rhesus macaques, Macaca mulatta. We describe the frequency of single nucleotide polymorphisms, insertions, deletions, frame-shift mutations, and isoforms. We also index the identified diversity using predicted and known rhesus macaque FcγR and Fc-FcγR structures. Future studies that define the functional significance of this genetic diversity will facilitate a better understanding of the correlation between human and macaque FcγR biology that is needed for effective translation of studies with antibody-mediated outcomes performed in rhesus macaques.

Histopathologic findings of pulmonary acariasis in a rhesus monkeys breeding unit / Aspectos histopatológicos da acaríase pulmonar em uma criação de macacos rhesus

Histological lesions in the lungs of rhesus monkeys (Macaca mulatta) related with Pneumonyssus simicola were evidenced. The most prominent pathologic alterations included numerous thin-walled cysts 1-5 mm diameter scattered throughout the lungs, bronchiolitis, peribronchiolitis where the mites were found associated with pigmented and non-pigmented materials. Our study included data from 347 rhesus monkeys submitted to necropsies during 20 years. Four adult debilitated animals were found with pulmonary acariasis which showed a very low incidence of parasite (1.2 percent) in the colony. Most of the published literature described as common and widespread pulmonary acariasis in Old World monkeys. The present study confirms the ubiquity of P. simicola in captive born and raised rhesus monkeys that would compromise experimental studies involving the respiratory system.

Foram evidenciadas lesões histológicas nos pulmões de macacos rhesus (Macaca mulatta) relacionadas ao Pneumonyssus simicola. As principais alterações incluíram numerosos cistos variando de 1-5 mm de diâmetro, com paredes finas e amplamente distribuídos nos pulmões; bronquiolite e peribronquiolite, onde os ácaros foram encontrados associados com materiais particulados pigmentados ou não. Nosso estudo incluiu dados de 347 macacos rhesus submetidos a necropsias no decorrer de 20 anos. A acaríase pulmonar foi diagnosticada em quatro animais adultos debilitados, o que representou uma incidência muito baixa do parasita (1,2 por cento) na colônia. A literatura descreve uma alta incidência de acaríase pulmonar em macacos do Velho Mundo. O presente estudo confirma a ubiqüidade do P. simicola em macacos rhesus nascidos e manejados em cativeiro, que compromete sua utilização em estudos experimentais envolvendo o sistema respiratório.