Dam-Infant Rhesus Macaque Pairs to Dissect Age-Dependent Responses to SARS-CoV-2 Infection.

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for other respiratory viral diseases. To investigate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analyses of the respiratory tract and evaluated systemic cytokine and Ab responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum at day 1 postinfection whereas CXCL10 was induced in all animals. Both groups mounted neutralizing Ab responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal airway cells isolated at day 14 postinfection revealed significant upregulation of multiple IFN-stimulated genes in infants compared with adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared with infants. Our study in rhesus macaque monkey dam-infant pairs suggests age-dependent differential airway responses to SARS-CoV-2 infection and describes a model that can be used to investigate SARS-CoV-2 pathogenesis between infants and adults.

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity had no antiviral effects but moderately reduced lung inflammation.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable but low levels of antiviral antibodies after infusion. In comparison to the control animals, CCP-treated animals had similar levels of viral RNA in upper and lower respiratory tract secretions, similar detection of viral RNA in lung tissues by in situ hybridization, but lower amounts of infectious virus in the lungs. CCP-treated animals had a moderate, but statistically significant reduction in interstitial pneumonia, as measured by comprehensive lung histology. Thus overall, therapeutic benefits of CCP were marginal and inferior to results obtained earlier with monoclonal antibodies in this animal model. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses.

Common and Not-So-Common Pathologic Findings of the Gastrointestinal Tract of Rhesus and Cynomolgus Macaques.

Rhesus and cynomolgus macaques are the most frequently used nonhuman primate (NHP) species for biomedical research and toxicology studies of novel therapeutics. In recent years, there has been a shortage of laboratory macaques due to a variety of competing factors. This was most recently exacerbated by the surge in NHP research required to address the severe acute respiratory syndrome (SARS)-coronavirus 2 pandemic. Continued support of these important studies has required the use of more varied cohorts of macaques, including animals with different origins, increased exposure to naturally occurring pathogens, and a wider age range. Diarrhea and diseases of the gastrointestinal tract are the most frequently occurring spontaneous findings in macaques of all origins and ages. The purpose of this review is to alert pathologists and scientists involved in NHP research to these findings and their impact on animal health and study endpoints, which may otherwise confound the interpretation of data generated using macaques.

Rhesus Macaque CODEX Multiplexed Immunohistochemistry Panel for Studying Immune Responses During Ebola Infection.

Non-human primate (NHP) animal models are an integral part of the drug research and development process. For some biothreat pathogens, animal model challenge studies may offer the only possibility to evaluate medical countermeasure efficacy. A thorough understanding of host immune responses in such NHP models is therefore vital. However, applying antibody-based immune characterization techniques to NHP models requires extensive reagent development for species compatibility. In the case of studies involving high consequence pathogens, further optimization for use of inactivated samples may be required. Here, we describe the first optimized CO-Detection by indEXing (CODEX) multiplexed tissue imaging antibody panel for deep profiling of spatially resolved single-cell immune responses in rhesus macaques. This 21-marker panel is composed of a set of 18 antibodies that stratify major immune cell types along with a set three Ebola virus (EBOV)-specific antibodies. We validated these two sets of markers using immunohistochemistry and CODEX in fully inactivated Formalin-Fixed Paraffin-Embedded (FFPE) tissues from mock and EBOV challenged macaques respectively and provide an efficient framework for orthogonal validation of multiple antibody clones using CODEX multiplexed tissue imaging. We also provide the antibody clones and oligonucleotide tag sequences as a valuable resource for other researchers to recreate this reagent set for future studies of tissue immune responses to EBOV infection and other diseases.

SARS-CoV-2 Infection of Rhesus Macaques Treated Early with Human COVID-19 Convalescent Plasma.

Human clinical studies investigating use of convalescent plasma (CP) for treatment of coronavirus disease 2019 (COVID-19) have produced conflicting results. Outcomes in these studies may vary at least partly due to different timing of CP administration relative to symptom onset. The mechanisms of action of CP include neutralizing antibodies but may extend beyond virus neutralization to include normalization of blood clotting and dampening of inflammation. Unresolved questions include the minimum therapeutic titer in the CP units or CP recipient as well as the optimal timing of administration. Here, we show that treatment of macaques with CP within 24 h of infection does not reduce viral shedding in nasal or lung secretions compared to controls and does not detectably improve any clinical endpoint. We also demonstrate that CP administration does not impact viral sequence diversity in vivo, although the selection of a viral sequence variant in both macaques receiving normal human plasma was suggestive of immune pressure. Our results suggest that CP, administered to medium titers, has limited efficacy, even when given very early after infection. Our findings also contribute information important for the continued development of the nonhuman primate model of COVID-19. These results should inform interpretation of clinical studies of CP in addition to providing insights useful for developing other passive immunotherapies and vaccine strategies. IMPORTANCE Antiviral treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain very limited. One treatment that was explored beginning early in the pandemic (and that is likely to be tested early in future pandemics) is plasma collected from people who have recovered from coronavirus disease 2019 (COVID-19), known as convalescent plasma (CP). We tested if CP reduces viral shedding or disease in a nonhuman primate model. Our results demonstrate that administration of CP 1 day after SARS-CoV-2 infection had no significant impact on viral loads, clinical disease, or sequence diversity, although treatment with normal human plasma resulted in selection of a specific viral variant. Our results demonstrate that passive immunization with CP, even during early infection, provided no significant benefit in a nonhuman primate model of SARS-CoV-2 infection.

Monoclonal antibodies protect aged rhesus macaques from SARS-CoV-2-induced immune activation and neuroinflammation.

Anti-viral monoclonal antibody (mAb) treatments may provide immediate but short-term immunity from coronavirus disease 2019 (COVID-19) in high-risk populations, such as people with diabetes and the elderly; however, data on their efficacy in these populations are limited. We demonstrate that prophylactic mAb treatment blocks viral replication in both the upper and lower respiratory tracts in aged, type 2 diabetic rhesus macaques. mAb infusion dramatically curtails severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated stimulation of interferon-induced chemokines and T cell activation, significantly reducing development of interstitial pneumonia. Furthermore, mAb infusion significantly dampens the greater than 3-fold increase in SARS-CoV-2-induced effector CD4 T cell influx into the cerebrospinal fluid. Our data show that neutralizing mAbs administered preventatively to high-risk populations may mitigate the adverse inflammatory consequences of SARS-CoV-2 exposure.

Early post-infection treatment of SARS-CoV-2 infected macaques with human convalescent plasma with high neutralizing activity reduces lung inflammation.

Early in the SARS-CoV-2 pandemic, there was a high level of optimism based on observational studies and small controlled trials that treating hospitalized patients with convalescent plasma from COVID-19 survivors (CCP) would be an important immunotherapy. However, as more data from controlled trials became available, the results became disappointing, with at best moderate evidence of efficacy when CCP with high titers of neutralizing antibodies was used early in infection. To better understand the potential therapeutic efficacy of CCP, and to further validate SARS-CoV-2 infection of macaques as a reliable animal model for testing such strategies, we inoculated 12 adult rhesus macaques with SARS-CoV-2 by intratracheal and intranasal routes. One day later, 8 animals were infused with pooled human CCP with a high titer of neutralizing antibodies (RVPN NT 50 value of 3,003), while 4 control animals received normal human plasma. Animals were monitored for 7 days. Animals treated with CCP had detectable levels of antiviral antibodies after infusion. In comparison to the control animals, they had similar levels of virus replication in the upper and lower respiratory tract, but had significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. By highlighting strengths and weaknesses, data of this study can help to further optimize nonhuman primate models to provide proof-of-concept of intervention strategies, and guide the future use of convalescent plasma against SARS-CoV-2 and potentially other newly emerging respiratory viruses. AUTHOR SUMMARY: The results of treating SARS-CoV-2 infected hospitalized patients with COVID-19 convalescent plasma (CCP), collected from survivors of natural infection, have been disappointing. The available data from various studies indicate at best moderate clinical benefits only when CCP with high titer of neutralizing antibodies was infused early in infection. The macaque model of SARS-CoV-2 infection can be useful to gain further insights in the value of CCP therapy. In this study, animals were infected with SARS-CoV-2 and the next day, were infused with pooled human convalescent plasma, selected to have a very high titer of neutralizing antibodies. While administration of CCP did not result in a detectable reduction in virus replication in the respiratory tract, it significantly reduced lung inflammation. These data, combined with the results of monoclonal antibody studies, emphasize the need to use products with high titers of neutralizing antibodies, and guide the future development of CCP-based therapies.

Early treatment with a combination of two potent neutralizing antibodies improves clinical outcomes and reduces virus replication and lung inflammation in SARS-CoV-2 infected macaques.

There is an urgent need for effective therapeutic interventions against SARS-CoV-2, including new variants that continue to arise. Neutralizing monoclonal antibodies have shown promise in clinical studies. We investigated the therapeutic efficacy of a combination of two potent monoclonal antibodies, C135-LS and C144-LS that carry half-life extension mutations, in the rhesus macaque model of COVID-19. Twelve young adult macaques (three groups of four animals) were inoculated intranasally and intra-tracheally with a high dose of SARS-CoV-2 and 24 hours later, treated intravenously with a high (40 mg/kg) or low (12 mg/kg) dose of the C135-LS and C144-LS antibody combination, or a control monoclonal antibody. Animals were monitored for 7 days. Compared to the control animals, animals treated with either dose of the anti-SARS-CoV-2 antibodies showed similarly improved clinical scores, lower levels of virus replication in upper and lower respiratory tract, and significantly reduced interstitial pneumonia, as measured by comprehensive lung histology. In conclusion, this study provides proof-of-concept in support of further clinical development of these monoclonal antibodies against COVID-19 during early infection.

Respiratory Tract Explant Infection Dynamics of Influenza A Virus in California Sea Lions, Northern Elephant Seals, and Rhesus Macaques.

To understand susceptibility of wild California sea lions and Northern elephant seals to influenza A virus (IAV), we developed an ex vivo respiratory explant model and used it to compare infection kinetics for multiple IAV subtypes. We first established the approach using explants from colonized rhesus macaques, a model for human IAV. Trachea, bronchi, and lungs from 11 California sea lions, 2 Northern elephant seals, and 10 rhesus macaques were inoculated within 24 h postmortem with 6 strains representing 4 IAV subtypes. Explants from the 3 species showed similar IAV infection kinetics, with peak viral titers 48 to 72 h post-inoculation that increased by 2 to 4 log10 PFU/explant relative to the inoculum. Immunohistochemistry localized IAV infection to apical epithelial cells. These results demonstrate that respiratory tissue explants from wild marine mammals support IAV infection. In the absence of the ability to perform experimental infections of marine mammals, this ex vivo culture of respiratory tissues mirrors the in vivo environment and serves as a tool to study IAV susceptibility, host range, and tissue tropism. IMPORTANCE Although influenza A virus can infect marine mammals, a dearth of marine mammal cell lines and ethical and logistical challenges prohibiting experimental infections of living marine mammals mean that little is known about IAV infection kinetics in these species. We circumvented these limitations by adapting a respiratory tract explant model first to establish the approach with rhesus macaques and then for use with explants from wild marine mammals euthanized for nonrespiratory medical conditions. We observed that multiple strains representing 4 IAV subtypes infected trachea, bronchi, and lungs of macaques and marine mammals with variable peak titers and kinetics. This ex vivo model can define infection dynamics for IAV in marine mammals. Further, use of explants from animals euthanized for other reasons reduces use of animals in research.

SARS-CoV-2 induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques.

CD4 T follicular helper (Tfh) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates Tfh cells and stimulates the germinal center (GC) response is an important question as we investigate vaccine induced immunity against COVID-19. Here, we report that SARS-CoV-2 infection in rhesus macaques, either infused with convalescent plasma, normal plasma, or receiving no infusion, resulted in transient accumulation of pro-inflammatory monocytes and proliferating Tfh cells with a Th1 profile in peripheral blood. CD4 helper cell responses skewed predominantly toward a Th1 response in blood, lung, and lymph nodes. SARS-CoV-2 Infection induced GC Tfh cells specific for the SARS-CoV-2 spike and nucleocapsid proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Collectively, the data show induction of GC responses in a rhesus model of mild COVID-19.

SARS-CoV-2 infection induces robust germinal center CD4 T follicular helper cell responses in rhesus macaques.

CD4 T follicular helper (T fh ) cells are important for the generation of durable and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T fh cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that SARS-CoV-2 infection resulted in transient accumulation of pro-inflammatory monocytes and proliferating T fh cells with a T h 1 profile in peripheral blood. CD4 helper cell responses were skewed predominantly toward a T h 1 response in blood, lung, and lymph nodes. We observed the generation of germinal center T fh cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies. Our data suggest that a vaccine promoting T h 1-type T fh responses that target the S protein may lead to protective immunity.

MYBPC3 Haplotype Linked to Hypertrophic Cardiomyopathy in Rhesus Macaques (Macaca mulatta).

In humans, abnormal thickening of the left ventricle of the heart clinically defines hypertrophic cardiomyopathy (HCM), a common inherited cardiovascular disorder that can precede a sudden cardiac death event. The wide range of clinical presentations in HCM obscures genetic variants that may influence an individual's susceptibility to sudden cardiac death. Although exon sequencing of major sarcomere genes can be used to detect high-impact causal mutations, this strategy is successful in only half of patient cases. The incidence of left ventricular hypertrophy (LVH) in a managed research colony of rhesus macaques provides an excellent comparative model in which to explore the genomic etiology of severe HCM and sudden cardiac death. Because no rhesus HCM-associated mutations have been reported, we used a next-generation genotyping assay that targets 7 sarcomeric rhesus genes within 63 genomic sites that are orthologous to human genomic regions known to harbor HCM disease variants. Amplicon sequencing was performed on 52 macaques with confirmed LVH and 42 unrelated, unaffected animals representing both the Indian and Chinese rhesus macaque subspecies. Bias-reduced logistic regression uncovered a risk haplotype in the rhesus MYBPC3 gene, which is frequently disrupted in both human and feline HCM; this haplotype implicates an intronic variant strongly associated with disease in either homozygous or carrier form. Our results highlight that leveraging evolutionary genomic data provides a unique, practical strategy for minimizing population bias in complex disease studies.

SARS-CoV-2 infection induces germinal center responses with robust stimulation of CD4 T follicular helper cells in rhesus macaques.

CD4 T follicular helper (T fh ) cells are important for the generation of long-lasting and specific humoral protection against viral infections. The degree to which SARS-CoV-2 infection generates T fh cells and stimulates the germinal center response is an important question as we investigate vaccine options for the current pandemic. Here we report that, following infection with SARS-CoV-2, adult rhesus macaques exhibited transient accumulation of activated, proliferating T fh cells in their peripheral blood on a transitory basis. The CD4 helper cell responses were skewed predominantly toward a T h 1 response in blood, lung, and lymph nodes, reflective of the interferon-rich cytokine environment following infection. We also observed the generation of germinal center T fh cells specific for the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins, and a corresponding early appearance of antiviral serum IgG antibodies but delayed or absent IgA antibodies. Our data suggest that a vaccine promoting Th1-type Tfh responses that target the S protein may lead to protective immunity.

Clinical and Pathologic Features of Spontaneous Klebsiella pneumoniae Infection in 9 Rhesus Macaques (Macaca mulatta).

Klebsiella pneumoniae is a gram-negative bacterium found in the environment and as a commensal in humans and animals. In humans, K. pneumoniae is one of the most serious nosocomial infections encountered currently and is characterized by liver abscesses, pneumonia, and bacteremia resulting in meningoencephalitis and endophthalmitis. K. pneumoniae in veterinary medicine is rarely reported in NHP, and recent literature describing this disease is sparse. In our colony of predominantly outdoor-housed rhesus macaques (Macaca mulatta), K. pneumoniae is cultured infrequently from healthy animals during routine screening and is even rarer in sick animals. This report summarizes the clinical and postmortem findings associated with this pathogen in 9 rhesus macaques and compares these results with the disease outcomes reported for humans. In these cases, K. pneumoniae infection was confirmed through culture or PCR testing or both. In our experience, when this bacterium does cause clinical signs, the disease is rapidly progressive and severe. At necropsy of NHP, the findings are strikingly similar to opportunistic Klebsiella-associated syndromes described in humans and include liver abscesses, meningoencephalitis, and endophthalmitis. In addition, many of the affected macaques had similar risk factors to humans that succumb to disease, thus perhaps indicating that rhesus macaques could be a viable model for investigating these syndromes.

Pulmonary amniotic fluid embolism in a rhesus macaque (Macaca mulatta).

A pregnant female rhesus macaque died spontaneously during stage two labor. Gross and histopathologic findings included severe pulmonary edema, with low numbers of blood vessels containing pale basophilic mucinous material (Alcian Blue positive and PTAH negative), consistent with intravascular amniotic fluid-derived mucin resulting in pulmonary amniotic fluid embolism.

Mixed Malignant Pancreatic Tumour in a Rhesus Macaque (Macaca mullata).

Pancreatic islet cell tumours are rare in non-human primates. The majority of reported cases are benign islet cell adenomas in rhesus macaques (Macaca mullata). Here we describe a pancreatic tumour composed of both exocrine and endocrine cells known as a mixed acinar-neuroendocrine carcinoma in a captive rhesus macaque. A diagnosis of a mixed tumour requires intermingling of neoplastic exocrine and neuroendocrine cells and must be differentiated from ductal adenocarcinomas in which only the ductal component is neoplastic with interspersed normal neuroendocrine cells. Immunohistochemistry, including antibodies against cytokeratin 7 and chromogranin A, was used to demonstrate that both exocrine and endocrine neoplastic cells exhibited cellular atypia, invasion into the adjacent parenchyma and intraparenchymal metastasis consistent with a mixed malignant tumour. Expression of multiple hormones such as gastrin, insulin, pancreatic polypeptide and somatostatin was also observed throughout the neoplastic cell population, while the endocrine component of the neoplasm was predominantly positive for glucagon.

Primary Myxoid Liposarcoma of the Greater Omentum in a Rhesus Macaque (Macaca mulatta).

We here report a spontaneous case of primary myxoid liposarcoma of the greater omentum with subsequent transperitoneal recurrence. The primary mass was incidentally found during an exploratory laparotomy for a presumed diagnosis of trichobezoar or phytobezoar and was removed surgically. Histopathologic examination of the mass revealed the presence of a myxoid liposarcoma. Eleven months later, recurrence with severe transperitoneal sarcomatosis of the myxoid liposarcoma was noted and confirmed by necropsy and histopathology. A review of the literature revealed that liposarcoma in NHP conforms to the behavior and prognosis of this neoplasm in humans.

Intraamniotic Zika virus inoculation of pregnant rhesus macaques produces fetal neurologic disease.

Zika virus (ZIKV) infection of pregnant women can cause fetal microcephaly and other neurologic defects. We describe the development of a non-human primate model to better understand fetal pathogenesis. To reliably induce fetal infection at defined times, four pregnant rhesus macaques are inoculated intravenously and intraamniotically with ZIKV at gestational day (GD) 41, 50, 64, or 90, corresponding to first and second trimester of gestation. The GD41-inoculated animal, experiencing fetal death 7 days later, has high virus levels in fetal and placental tissues, implicating ZIKV as cause of death. The other three fetuses are carried to near term and euthanized; while none display gross microcephaly, all show ZIKV RNA in many tissues, especially in the brain, which exhibits calcifications and reduced neural precursor cells. Given that this model consistently recapitulates neurologic defects of human congenital Zika syndrome, it is highly relevant to unravel determinants of fetal neuropathogenesis and to explore interventions.

Evaluation of a Zinc Gluconate Neutralized with Arginine Product as a Nonsurgical Method for Sterilization of Rhesus Macaques (Macaca mulatta).

Because rhesus macaques (Macaca mulatta) are prolific breeders, overpopulation can be problematic in both research and feral populations. Currently, the most effective contraceptive methods are hormonal control in female macaques and vasectomies in males. These methods each come with innate challenges, the foremost being the alteration of necessary hormonal patterns. In this study, we assessed the use of zinc gluconate neutralized with arginine as a novel, nonsurgical alternative to male contraception in 12 rhesus macaques. This FDA-approved product for dogs is given as a one-time, intratesticular injection to cause permanent infertility yet theoretically spare the testosterone-producing Leydig cells of the testis. CBC counts, serum biochemistry analyses, testosterone levels, and testicular widths were evaluated at the time of injection and at 1 wk, 1 mo, 2 mo, or 3 mo afterward. Daily postinjection observations revealed transient scrotal enlargement in 8 of the 12 macaques but no indications of pain. In addition, full necropsies including testicular histopathology were assessed at study endpoints. Although some portion of every testis had evidence of seminiferous tubule loss, normal spermatogenesis was present in 22 of the 24 testes. In conclusion, chemical castration with the tested zinc gluconate neutralized with arginine product is not an effective method for sterilization of male rhesus macaques.

Pneumatosis intestinalis in an SIV-infected rhesus macaque (Macaca mulatta).

Pneumatosis intestinalis (PI) has been described as an incidental finding in domestic animals and humans where it is associated with human immunodeficiency virus infection among other comorbidities. This report describes emphysematous changes consistent with PI in a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta).