Aerosol delivery of SARS-CoV-2 human monoclonal antibodies in macaques limits viral replication and lung pathology.

Passively administered monoclonal antibodies (mAbs) given before or after viral infection can prevent or blunt disease. Here, we examine the efficacy of aerosol mAb delivery to prevent infection and disease in rhesus macaques inoculated with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant via intranasal and intratracheal routes. SARS-CoV-2 human mAbs or a human mAb directed to respiratory syncytial virus (RSV) are nebulized and delivered using positive airflow via facemask to sedated macaques pre- and post-infection. Nebulized human mAbs are detectable in nasal, oropharyngeal, and bronchoalveolar lavage (BAL) samples. SARS-CoV-2 mAb treatment significantly reduces levels of SARS-CoV-2 viral RNA and infectious virus in the upper and lower respiratory tracts relative to controls. Reductions in lung and BAL virus levels correspond to reduced BAL inflammatory cytokines and lung pathology. Aerosolized antibody therapy for SARS-CoV-2 could be effective for reducing viral burden and limiting disease severity.

Rhesus macaque fetal and placental growth demographics: A resource for laboratory animal researchers.

Rhesus macaques (Macaca mulatta) are amongst the most common nonhuman primate species used in biomedical research. These animals provide a precious resource for translational studies and opportunities to maximize rhesus data use are encouraged. Here we compile data produced from 10 years of investigator-driven pregnancy studies conducted at the Oregon National Primate Research Center (ONPRC). All pregnancies were generated within the consistent and reproducible protocols of the ONPRC time-mated breeding program. The data included are from control animals who did not experience in utero perturbations or experimental manipulations. A total of 86 pregnant rhesus macaques were delivered by cesarean section over a range of gestational days (G) 50 to G159 (where term is G165 ± 10 days in the rhesus macaque), with subsequent immediate tissue harvesting following standardized protocols. Fetal and placental growth measures, and all major organ weights are reported. All data are presented relative to gestational age for the entire cohort and in addition, data are stratified by fetal sex. The outcome is a large reference resource for use by laboratory animal researchers in future comparative fetal development studies.

Nonhuman primate genetic models for the study of rare diseases.

Pre-clinical research and development relies heavily upon translationally valid models of disease. A major difficulty in understanding the biology of, and developing treatments for, rare disease is the lack of animal models. It is important that these models not only recapitulate the presentation of the disease in humans, but also that they share functionally equivalent underlying genetic causes. Nonhuman primates share physiological, anatomical, and behavioral similarities with humans resulting from close evolutionary relationships and high genetic homology. As the post-genomic era develops and next generation sequencing allows for the resequencing and screening of large populations of research animals, naturally occurring genetic variation in nonhuman primates with clinically relevant phenotypes is regularly emerging. Here we review nonhuman primate models of multiple rare genetic diseases with a focus on the similarities and differences in manifestation and etiologies across species. We discuss how these models are being developed and how they can offer new tools and opportunities for researchers interested in exploring novel therapeutics for these and other genetic diseases. Modeling human genetic diseases in translationally relevant nonhuman primates presents new prospects for development of therapeutics and a better understanding of rare diseases. The post-genomic era offers the opportunity for the discovery and further development of more models like those discussed here.

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.

A novel non-human primate model of Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is a severe hypomyelinating disorder of the central nervous system (CNS) linked to mutations in the proteolipid protein-1 (PLP1) gene. Although there are multiple animal models of PMD, few of them fully mimic the human disease. Here, we report three spontaneous cases of male neonatal rhesus macaques with the clinical symptoms of hypomyelinating disease, including intention tremors, progressively worsening motor dysfunction, and nystagmus. These animals demonstrated a paucity of CNS myelination accompanied by reactive astrogliosis, and a lack of PLP1 expression throughout white matter. Genetic analysis revealed that these animals were related to one another and that their parents carried a rare, hemizygous missense variant in exon 5 of the PLP1 gene. These animals therefore represent the first reported non-human primate model of PMD, providing a novel and valuable opportunity for preclinical studies that aim to promote myelination in pediatric hypomyelinating diseases.

Molecular and Phylogenetic Characterization of Novel Papillomaviruses Isolated from Oral and Anogenital Neoplasms of Japanese Macaques (Macaca fuscata).

Papillomaviruses (PVs) are a diverse group of host species-specific DNA viruses, etiologically linked with various benign and malignant neoplasms of cutaneous and mucosal epithelia. Here, we describe the detection and characterization of the first two PVs naturally infecting Japanese macaques (Macaca fuscata), including the determination of their etiological association(s) with the development of original neoplasms. The molecular and phylogenetic analyses were performed on complete genome sequences of Macaca fuscata PV types 1 (MfuPV1) and 2 (MfuPV2), which were completely sequenced in samples of a malignant oral tumor and benign anogenital neoplasm of Japanese macaques, respectively. Subsequently, two type-specific quantitative real-time PCRs were developed to estimate viral loads of MfuPV1 and MfuPV2 and to evaluate their etiological roles. The in silico molecular analyses revealed that both viral genomes encode characteristic PV proteins with conserved functional domains and have a non-coding genomic region with regulatory sequences to regulate and complete the viral life cycle. However, additional experimental evidence is needed to finally confirm the presence and biological functionality of the molecular features of both novel PVs. While MfuPV1, together with PVs identified in other macaques, is classified into the Alphapapillomavirus (Alpha-PV) species 12, MfuPV2 is most likely a representative of the novel viral species within the Alpha-PV genus. Their relatively high viral loads suggest that both PVs are etiologically linked with the development of the original neoplasms.

Colon volvulus in rhesus macaques (Macaca mulatta) at the Oregon National Primate Research Center-Retrospective analysis.

Colonic volvulus is an uncommon, often life-threatening condition, in non-human primates. Twenty-six cases of colonic volvulus in rhesus macaques (Macaca mulatta) were identified in necropsy records spanning 38 years at the Oregon National Primate Research Center (ONPRC). This report represents the largest collection of colonic volvuli in rhesus macaques.

Two cases of cryptococcus gattii infection in the rhesus macaque (Macaca Mulatta).

Cryptococcus gattii was diagnosed in two female indoor-housed rhesus macaques. Gross and histopathologic findings included an isolated pulmonary cryptococcoma in a non-SIV-infected macaque and disseminated disease centered on the lungs of an SIV-infected macaque. Fungal yeast were positive with special stains, and the diagnoses were confirmed with a lateral flow assay and PCR.

Vaccine-mediated protection against Campylobacter-associated enteric disease.

Campylobacter coli and Campylobacter jejuni are responsible for 400 million to 500 million cases of enteric disease each year and represent the most common cause of bacterial gastroenteritis worldwide. Despite its global importance, Campylobacter vaccine development has been hampered by the lack of animal models that recapitulate human disease pathogenesis. Here, we describe a naturally occurring Campylobacter-associated diarrhea model in outdoor-housed rhesus macaques. Using this model, we developed novel next-generation H2O2-based Campylobacter vaccines that induced strong antibacterial antibodies to multiple Campylobacter proteins including flagellin and provided up to 83% protection against severe C. coli-associated diarrhea. Whole-genome sequencing of circulating Campylobacter strains revealed little to no homology within lipooligosaccharide or capsular polysaccharide loci with the Campylobacter vaccine strains used in these studies, indicating that vaccine-mediated immunity was not restricted to a single homologous serotype. Together, these results demonstrate an important advance in vaccine development and a new approach to reducing Campylobacter-associated enteric disease.

Nurse-Led Initiative to Improve Addiction Services: An Interdisciplinary Approach.

Local efforts to provide treatment for individuals with substance use disorder have had some success in addressing the continued nationwide drug epidemic. A nurse-led withdrawal and stabilization program was developed that includes an interdisciplinary team of physicians, advanced practice providers, nurses, social workers, and substance abuse counselors. Utilizing a shared vision of optimal patient care, we describe how principles of high reliability were incorporated to build a successful program within a community hospital setting from the design of the unit, development of treatment protocols, staffing, and ongoing program refinement that is based on patient and program staff feedback to final thoughts for future program changes.

Spontaneous KRT5 Gene Mutation in Rhesus Macaques (Macaca mulatta): A Novel Nonhuman Primate Model of Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS) is an inherited skin disorder characterized by increased skin and mucous membrane fragility. Most cases are caused by mutations in keratin 5 (KRT5) and keratin 14 (KRT14). Mutations of these genes result in cytoskeletal disruption of the basal keratinocytes. Gross and histopathologic findings of 2 clinically affected homozygous rhesus macaques with an insertion variant mutation in KRT5 are described and compared with 6 deceased phenotypically normal animals that were heterozygous for the KRT5 insertion variant. Animals that were homozygous for the KRT5 insertion variant were stillborn and had widespread loss of the epidermis. Microscopic examination confirmed severe ulceration and basal cell vacuolation with basilar vesicle formation in the remaining intact epidermis. Immunohistochemistry for cytokeratin 5 demonstrated lack of epidermal immunoreactivity in homozygotes. DNA sequencing identified a 34-base pair insertion variant in exon 5 of the KRT5 gene. To our knowledge, this is the first report of epidermolysis bullosa in rhesus macaques.

A neonatal nonhuman primate model of gestational Zika virus infection with evidence of microencephaly, seizures and cardiomyopathy.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

Viral opportunistic infections in Mauritian cynomolgus macaques undergoing allogeneic stem cell transplantation mirror human transplant infectious disease complications.

Allogeneic hematopoietic stem cell transplantation (HSCT) and xenotransplantation are accompanied by viral reactivations and virus-associated complications resulting from immune deficiency. Here, in a Mauritian cynomolgus macaque model of fully MHC-matched allogeneic HSCT, we report reactivations of cynomolgus polyomavirus, lymphocryptovirus, and cytomegalovirus, macaque viruses analogous to HSCT-associated human counterparts BK virus, Epstein-Barr virus, and human cytomegalovirus. Viral replication in recipient macaques resulted in characteristic disease manifestations observed in HSCT patients, such as polyomavirus-associated hemorrhagic cystitis and tubulointerstitial nephritis or lymphocryptovirus-associated post-transplant lymphoproliferative disorder. However, in most cases, the reconstituted immune system, alone or in combination with short-term pharmacological intervention, exerted control over viral replication, suggesting engraftment of functional donor-derived immunity. Indeed, the donor-derived reconstituted immune systems of two long-term engrafted HSCT recipient macaques responded to live attenuated yellow fever 17D vaccine (YFV 17D) indistinguishably from untransplanted controls, mounting 17D-targeted neutralizing antibody responses and clearing YFV 17D within 14 days. Together, these data demonstrate that this macaque model of allogeneic HSCT recapitulates clinical situations of opportunistic viral infections in transplant patients and provides a pre-clinical model to test novel prophylactic and therapeutic modalities.

Single-dose bNAb cocktail or abbreviated ART post-exposure regimens achieve tight SHIV control without adaptive immunity.

Vertical transmission accounts for most human immunodeficiency virus (HIV) infection in children, and treatments for newborns are needed to abrogate infection or limit disease progression. We showed previously that short-term broadly neutralizing antibody (bNAb) therapy given 24 h after oral exposure cleared simian-human immunodeficiency virus (SHIV) in a macaque model of perinatal infection. Here, we report that all infants given either a single dose of bNAbs at 30 h, or a 21-day triple-drug ART regimen at 48 h, are aviremic with almost no virus in tissues. In contrast, bNAb treatment beginning at 48 h leads to tight control without adaptive immune responses in half of animals. We conclude that both bNAbs and ART mediate effective post-exposure prophylaxis in infant macaques within 30-48 h of oral SHIV exposure. Our findings suggest that optimizing the treatment regimen may extend the window of opportunity for preventing perinatal HIV infection when treatment is delayed.

Research Relevant Conditions and Pathology in Nonhuman Primates.

Biomedical research involving animal models continues to provide important insights into disease pathogenesis and treatment of diseases that impact human health. In particular, nonhuman primates (NHPs) have been used extensively in translational research due to their phylogenetic proximity to humans and similarities to disease pathogenesis and treatment responses as assessed in clinical trials. Microscopic changes in tissues remain a significant endpoint in studies involving these models. Spontaneous, expected (ie, incidental or background) histopathologic changes are commonly encountered and influenced by species, genetic variations, age, and geographical origin of animals, including exposure to infectious or parasitic agents. Often, the background findings confound study-related changes, because numbers of NHPs used in research are limited by animal welfare and other considerations. Moreover, background findings in NHPs can be exacerbated by experimental conditions such as treatment with xenobiotics (eg, infectious morphological changes related to immunosuppressive therapy). This review and summary of research-relevant conditions and pathology in rhesus and cynomolgus macaques, baboons, African green monkeys, common marmosets, tamarins, and squirrel and owl monkeys aims to improve the interpretation and validity of NHP studies.

Quality of investigations into unexpected deaths of infants and young children in England after implementation of national child death review procedures in 2008: a retrospective assessment.

OBJECTIVES: In 2008, new statutory national procedures for responding to unexpected child deaths were introduced throughout England. There has, to date, been no national audit of these procedures. STUDY DESIGN: Families bereaved by the unexpected death of a child under 4 years of age since 2008 were invited to participate. Factors contributing to the death and investigations after the death were explored. Telephone interviews were conducted, and coroners' documents were obtained. The nature and quality of investigations was compared with the required procedures; information on each case was reviewed by a multiagency panel; and the death was categorised using the Avon clinicopathological classification. RESULTS: Data were obtained from 91 bereaved families (64 infant deaths and 27 children aged 1-3 years); 85 remained unexplained after postmortem examination. Documentation of multiagency assessments was poorly recorded. Most (88%) families received a home visit from the police, but few (37%) received joint visits by police and healthcare professionals. Postmortem examinations closely followed national guidance; 94% involved paediatric pathologists; 61% of families had a final meeting with a paediatrician to explain the investigation outcome. There was no improvement in frequency of home visits by health professionals or final meetings with paediatricians between 2008-2013 and 2014-2017 and no improvement in parental satisfaction with the process. CONCLUSIONS: Statutory procedures need to be followed more closely. The implementation of a national child mortality database from 2019 will allow continuing audit of the quality of investigations after unexpected child deaths. An important area amenable to improvement is increased involvement by paediatricians.

Role of IL-15 Signaling in the Pathogenesis of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15-neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7-9 RM per group). In both cohorts, anti-IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti-IL-15- and control-treated groups in both cohorts. However, RM treated with anti-IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

Bardet-Biedl Syndrome in rhesus macaques: A nonhuman primate model of retinitis pigmentosa.

The development of therapies for retinal disorders is hampered by a lack of appropriate animal models. Higher nonhuman primates are the only animals with retinal structure similar to humans, including the presence of a macula and fovea. However, few nonhuman primate models of genetic retinal disease are known. We identified a lineage of rhesus macaques with a frameshift mutation in exon 3 of the BBS7 gene c.160delG (p.Ala54fs) that is predicted to produce a non-functional protein. In humans, mutations in this and other BBS genes cause Bardet-Biedl syndrome, a ciliopathy and a syndromic form of retinitis pigmentosa generally occurring in conjunction with kidney dysfunction, polydactyly, obesity, and/or hypogonadism. Three full- or half-sibling monkeys homozygous for the BBS7 c.160delG variant, at ages 3.5, 4 and 6 years old, displayed a combination of severe photoreceptor degeneration and progressive kidney disease. In vivo retinal imaging revealed features of severe macular degeneration, including absence of photoreceptor layers, degeneration of the retinal pigment epithelium, and retinal vasculature atrophy. Electroretinography in the 3.5-year-old case demonstrated loss of scotopic and photopic a-waves and markedly reduced and delayed b-waves. Histological assessments in the 4- and 6-year-old cases confirmed profound loss of photoreceptors and inner retinal neurons across the posterior retina, with dramatic thinning and disorganization of all cell layers, abundant microglia, absent or displaced RPE cells, and significant gliosis in the subretinal space. Retinal structure, including presence of photoreceptors, was preserved only in the far periphery. Ultrasound imaging of the kidneys revealed deranged architecture, and renal histopathology identified distorted contours with depressed, fibrotic foci and firmly adhered renal capsules; renal failure occurred in the 6-year-old case. Magnetic resonance imaging obtained in one case revealed abnormally low total brain volume and unilateral ventricular enlargement. The one male had abnormally small testes at 4 years of age, but polydactyly and obesity were not observed. Thus, monkeys homozygous for the BBS7 c.160delG variant closely mirrored several key features of the human BBS syndrome. This finding represents the first identification of a naturally-occurring nonhuman primate model of BBS, and more broadly the first such model of retinitis pigmentosa and a ciliopathy with an associated genetic mutation. This important new preclinical model will provide the basis for better understanding of disease progression and for the testing of new therapeutic options, including gene and cell-based therapies, not only for BBS but also for multiple forms of photoreceptor degeneration.

Cerebral cysts of ependymal or ventricular origin in a juvenile rhesus macaque (Macaca mulatta) with neurologic signs.

Naturally occurring neurologic disease in non-human primates may be attributable to a wide-range of causes, including infectious agents, congenital or acquired malformations, degenerative diseases, and, rarely, neoplasia. We report a case of ataxia and paresis in a juvenile rhesus macaque with ependymal-lined cerebral cysts.

Risk of Zika microcephaly correlates with features of maternal antibodies.

Zika virus (ZIKV) infection during pregnancy causes congenital abnormalities, including microcephaly. However, rates vary widely, and the contributing risk factors remain unclear. We examined the serum antibody response to ZIKV and other flaviviruses in Brazilian women giving birth during the 2015-2016 outbreak. Infected pregnancies with intermediate or higher ZIKV antibody enhancement titers were at increased risk to give birth to microcephalic infants compared with those with lower titers (P < 0.0001). Similarly, analysis of ZIKV-infected pregnant macaques revealed that fetal brain damage was more frequent in mothers with higher enhancement titers. Thus, features of the maternal antibodies are associated with and may contribute to the genesis of ZIKV-associated microcephaly.