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.

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.

Infant rhesus macaques immunized against SARS-CoV-2 are protected against heterologous virus challenge 1 year later.

The U.S. Food and Drug Administration only gave emergency use authorization of the BNT162b2 and mRNA-1273 SARS-CoV-2 vaccines for infants 6 months and older in June 2022. Yet questions regarding the durability of vaccine efficacy, especially against emerging variants, in this age group remain. We demonstrated previously that a two-dose regimen of stabilized prefusion Washington SARS-CoV-2 S-2P spike (S) protein encoded by mRNA encapsulated in lipid nanoparticles (mRNA-LNP) or purified S-2P mixed with 3M-052, a synthetic Toll-like receptor (TLR) 7/8 agonist, in a squalene emulsion (Protein+3M-052-SE) was safe and immunogenic in infant rhesus macaques. Here, we demonstrate that broadly neutralizing and spike-binding antibodies against variants of concern (VOCs), as well as T cell responses, persisted for 12 months. At 1 year, corresponding to human toddler age, we challenged vaccinated rhesus macaques and age-matched nonvaccinated controls intranasally and intratracheally with a high dose of heterologous SARS-CoV-2 B.1.617.2 (Delta). Seven of eight control rhesus macaques exhibited severe interstitial pneumonia and high virus replication in the upper and lower respiratory tract. In contrast, vaccinated rhesus macaques had faster viral clearance with mild to no pneumonia. Neutralizing and binding antibody responses to the B.1.617.2 variant at the day of challenge correlated with lung pathology and reduced virus replication. Overall, the Protein+3M-052-SE vaccine provided superior protection to the mRNA-LNP vaccine, emphasizing opportunities for optimization of current vaccine platforms. The observed efficacy of both vaccines 1 year after vaccination supports the implementation of an early-life SARS-CoV-2 vaccine.

SARS-CoV-2 infects neurons and induces neuroinflammation in a non-human primate model of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of coronavirus disease 2019 (COVID-19), can induce a plethora of neurological complications in some patients. However, it is still under debate whether SARS-CoV-2 directly infects the brain or whether CNS sequelae result from systemic inflammatory responses triggered in the periphery. By using high-resolution microscopy, we investigated whether SARS-CoV-2 reaches the brain and how viral neurotropism can be modulated by aging in a non-human primate model of COVID-19. Seven days after infection, SARS-CoV-2 was detected in the olfactory cortex and interconnected regions and was accompanied by robust neuroinflammation and neuronal damage exacerbated in aged, diabetic animals. Our study provides an initial framework for identifying the molecular and cellular mechanisms underlying SARS-CoV-2 neurological complications, which will be essential to reducing both the short- and long-term burden of COVID-19.

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.

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.

Blood Pressure Reference Intervals for Ketamine-sedated Rhesus Macaques (Macaca mulatta).

Appropriate calculation and use of reference intervals have widespread clinical and research implications. Unfortunately, reference intervals for blood pressure in one of the most commonly used NHP species, rhesus macaques (Macaca mulatta), have never been calculated. Although anesthetic drugs and noninvasive methods of blood pressure measurement both have known effects on blood pressure values, their use provides the safest, fastest, and most widely used approach to clinical evaluation and blood pressure collection in this species. We analyzed noninvasive blood pressure measurements from 103 healthy, ketamine-sedated, adult (age, 8 to 16 y) rhesus macaques, representing both sexes, with various body condition scores by using 2 types of sphygmomanometers at 3 different anatomic locations. Reference intervals were calculated for each device, in each location, thus establishing normative data beneficial to clinical veterinarians assessing animal health and encouraging researchers to use noninvasive methods. Age, body condition score, sex, type of sphygmomanometer, and location of cuff placement were all found to influence blood pressure measurements significantly, providing important information necessary for the appropriate interpretation of noninvasive blood pressure values in rhesus macaques.

Metastatic hepatocellular carcinoma in a juvenile rhesus macaque (Macaca mulatta).

Neoplasia in juvenile (younger than 5 y) rhesus macaques has been estimated to represent only approximately 1.4% of all occurrences of spontaneous neoplasia. Here we report an unusual case of a 3.75-y-old primiparous female rhesus macaque that was euthanized due to poor prognosis associated with progressive anemia, marked hepatomegaly, and radiographic evidence of meta- static neoplasia. Postmortem examination revealed an invasive, hemorrhagic hepatic mass that effaced approximately 70% of the liver parenchyma and had evidence of metastatic spread to multiple abdominal organs, the lungs, and the pituitary gland. Neoplastic polygonal cells lined large necrohemorrhagic cavities and exhibited marked anisocytosis and anisokaryosis, with frequent multinucleate cells. There was no desmoplasia associated with the primary neoplasm or metastases. Immunohistochemical studies revealed the neoplastic cells to be diffusely reactive with pancytokeratin, cytokeratin 7, and cytokeratin 8/18 antibodies and rarely reactive with carcinoembryonic antigen antibodies. The cells did not react with vimentin, S100, CD31, or factor VIII antibodies. Tumor morphology and immunophenotype led to the diagnosis of anaplastic hepatocellular carcinoma. This report represents the first known case of metastatic liver neoplasia in a rhesus macaque. The young age of this animal and the aggressive nature of the neoplasm are highly unusual and reminiscent of adolescent onset hepatocellular carcinoma in humans.

Investigation of tularemia outbreak after natural infection of outdoor-housed rhesus macaques (Macaca mulatta) with Francisella tularensis.

In the summer and fall of 2010, a series of outdoor-housed rhesus macaques were diagnosed with tularemia. PCR analysis or positive culture confirmed 11 cases, and 9 additional animals with similar clinical signs responded to empiric antibiotic treatment. A serosurvey conducted in the 9 mo after the outbreak found 53% (43 of 81 macaques) seropositivity in the southern outdoor colony, which had an average population of 700 animals. A prospective survey of small mammal reservoirs and arthropod vectors was conducted during the late summer and fall of 2011. PCR analyses of tissues from all 135 mice, 18 ground squirrels, 1 rat, 3 raccoons, 2 cats, and 3 jackrabbits and their fleas were negative for DNA of Francisella tularensis. Conventional PCR evaluation of stored DNA from affected macaques identified the causative organism as F. tularensis subsp. holartica. DNA evaluated from historic cases of tularemia in nonhuman primates confirmed that the organism that infected the colony during the late 1980s likewise was F. tularensis subsp. holartica. The macaque tularemia epizootic of 2010 appears to have been an extreme example of the periodic resurgence of tularemia. No evidence of rodent disease was found in the immediate vicinity during the 2011 interepizootic period. The concurrent widespread seropositivity (53%) and low incidence of clinical disease (2.7%) in 2010 suggests that this strain of Francisella has low pathogenicity in macaques.