Infant diarrheal disease in rhesus macaques impedes microbiome maturation and is linked to uncultured Campylobacter species.

Diarrheal diseases remain one of the leading causes of death for children under 5 globally, disproportionately impacting those living in low- and middle-income countries (LMIC). Campylobacter spp., a zoonotic pathogen, is one of the leading causes of food-borne infection in humans. Yet to be cultured Campylobacter spp. contribute to the total burden in diarrheal disease in children living in LMIC thus hampering interventions. We performed microbiome profiling and metagenomic genome assembly on samples collected from over 100 infant rhesus macaques longitudinally and during cases of clinical diarrhea within the first year of life. Acute diarrhea was associated with long-lasting taxonomic and functional shifts of the infant gut microbiome indicative of microbiome immaturity. We constructed 36 Campylobacter metagenomic assembled genomes (MAGs), many of which fell within 4 yet to be cultured species. Finally, we compared the uncultured Campylobacter MAGs assembled from infant macaques with publicly available human metagenomes to show that these uncultured species are also found in human fecal samples from LMIC. These data highlight the importance of unculturable Campylobacter spp. as an important target for reducing disease burden in LMIC children.

Immunological and microbial shifts in the aging rhesus macaque lung during nontuberculous mycobacterial infection.

Nontuberculous mycobacteria (NTM) are environmentally ubiquitous organisms that predominately cause NTM pulmonary disease (NTMPD) in individuals over the age of 65. The incidence of NTMPD has increased in the U.S., exceeding that of Mycobacterium tuberculosis. However, the mechanisms leading to higher susceptibility and severity of NTMPD with aging are poorly defined in part due to the lack of animal models that accurately recapitulate human disease. Here, we compared bacterial load, microbial communities, and host responses longitudinally between three young (two female and one male) and two aged (two female) rhesus macaques inoculated with Mycobacterium avium subsp. hominissuis (MAH) in the right caudal lobe. Unilateral infection resulted in a low bacterial load in both young and aged animals confined to the infected side. Although a robust inflammatory response was only observed in the inoculated lung, immune cell infiltration and antigen-specific T cells were detected in both lungs. Computed tomography, gross pathology, and histopathology revealed increased disease severity and persistence of bacterial DNA in aged animals. Additional analyses showed the translocation of gut and oral-pharyngeal bacterial DNA into the lower respiratory microbiome. Finally, single-cell RNA sequencing revealed a heightened inflammatory response to MAH infection by alveolar macrophages in aged animals. These data are consistent with the model that increased disease severity in the aged is mediated by a dysregulated macrophage response that may be sustained through persistent antigen presence. IMPORTANCE: Nontuberculous mycobacteria (NTM) are emerging as pathogens of high consequence, as cases of NTM pulmonary disease (NTMPD) have exceeded those of Mycobacterium tuberculosis. NTMPD can be debilitating, particularly in patients over 65 years of age, as it causes chronic cough and fatigue requiring prolonged treatments with antibiotics. The underlying mechanisms of this increased disease severity with age are poorly understood, hampering the development of therapeutics and vaccines. Here, we use a rhesus macaque model to investigate the impact of age on host-NTM interactions. This work shows that aging is associated with increased disease severity and bacterial persistence in aged rhesus macaques, thus providing a preclinical model to develop and test novel therapeutics and interventions.

Campylobacter vaccination reduces diarrheal disease and infant growth stunting among rhesus macaques.

Campylobacter-associated enteric disease is estimated to be responsible for more than 160 million cases of gastroenteritis each year and is linked to growth stunting of infants living under conditions of poor sanitation and hygiene. Here, we examine naturally occurring Campylobacter-associated diarrhea among rhesus macaques as a model to determine if vaccination could reduce severe diarrheal disease and infant growth stunting. Compared to unvaccinated controls, there are no Campylobacter diarrhea-associated deaths observed among vaccinated infant macaques and all-cause diarrhea-associated infant mortality is decreased by 76% (P = 0.03). By 9 months of age, there is a 1.3 cm increase in dorsal length that equaled a significant 1.28 LAZ (Length-for-Age Z score) improvement in linear growth among vaccinated infants compared to their unvaccinated counterparts (P = 0.001). In this work, we show that Campylobacter vaccination not only reduces diarrheal disease but also potentially serves as an effective intervention that improves infant growth trajectories.

Taxonomic and Functional Shifts in the Perinatal Gut Microbiome of Rhesus Macaques.

Pregnancy and the postpartum period result in some of the most dramatic metabolic, hormonal, and physiological changes that can be experienced by an otherwise healthy adult. The timing and magnitude of these changes is key for both maternal and fetal health. One of the factors believed to critically modulate these physiological changes is the maternal gut microbiome. However, the dynamic changes in this community during the perinatal period remain understudied. Clinical studies can be complicated by confounding variables like diet and other drivers of heterogeneity in the human microbiome. Therefore, in this study, we conducted a longitudinal analysis of the fecal microbiome obtained during the pregnancy and postpartum periods in 26 captive rhesus macaques using 16S rRNA gene amplicon sequencing and shotgun metagenomics. Shifts at both the taxonomic and functional potential level were detected when comparing pregnancy to postpartum samples. Taxonomically, Alloprevotella, Actinobacillus, and Anaerovibrio were enriched in the gut microbiome during pregnancy, while Treponema, Lachnospiraceae, and Methanosphaera were more abundant postpartum. Functionally, the gut microbiome during pregnancy was associated with increased abundance in pathways involving the production of the short-chain fatty acid (SCFA) butyrate, while pathways associated with starch degradation and folate transformation were more abundant during the postpartum period. These data demonstrate dramatic changes in the maternal gut microbiome even in the absence of dietary changes and suggest that rhesus macaques could provide a valuable model to determine how changes in the microbiome correlate to other physiological changes in pregnancy. IMPORTANCE Pregnancy and the postpartum period are characterized by a myriad of metabolic and physiological adaptations needed to support fetal growth and maternal health. The maternal gut microbiome is believed to play a key role during this period but remains underexplored. Here, we report significant shifts in the taxonomic landscape and functional potential of the gut microbiome in 26 pregnant rhesus macaques during the transition from pregnancy to the postpartum period, despite shared dietary and environmental exposures. Increased abundance of pathways involved in the production of the short-chain fatty acid butyrate could play a critical role in modulating the maternal immune system and regulating fetal tolerance. On the other hand, increased abundance of pathways associated with starch degradation and folate transformation during the postpartum period could be important for meeting the metabolic demands of breastfeeding and neonatal growth.

Functional, transcriptional, and microbial shifts associated with healthy pulmonary aging in rhesus macaques.

Older individuals are at increased risk of developing severe respiratory infections. However, our understanding of the impact of aging on the respiratory tract remains limited as samples from healthy humans are challenging to obtain and results can be confounded by variables such as smoking and diet. Here, we carry out a comprehensive cross-sectional study (n = 34 adult, n = 49 aged) to define the consequences of aging on the lung using the rhesus macaque model. Pulmonary function testing establishes similar age and sex differences as humans. Additionally, we report increased abundance of alveolar and infiltrating macrophages and a concomitant decrease in T cells were in aged animals. scRNAseq reveals shifts from GRZMB to IFN expressing CD8+ T cells in the lungs. These data provide insight into age-related changes in the lungs' functional, microbial, and immunological landscape that explain increased prevalence and severity of respiratory diseases in the elderly.

Reduced infant rhesus macaque growth rates due to environmental enteric dysfunction and association with histopathology in the large intestine.

Environmental enteric dysfunction is associated with malnutrition as well as infant growth stunting and has been classically defined by villous blunting, decreased crypt-to-villus ratio, and inflammation in the small intestine. Here, we characterized environmental enteric dysfunction among infant rhesus macaques that are naturally exposed to enteric pathogens commonly linked to human growth stunting. Remarkably, despite villous atrophy and histological abnormalities observed in the small intestine, poor growth trajectories and low serum tryptophan levels were correlated with increased histopathology in the large intestine. This work provides insight into the mechanisms underlying this disease and indicates that the large intestine may be an important target for therapeutic intervention.

Acute SARS-CoV-2 infection is associated with an expansion of bacteria pathogens in the nose including Pseudomonas Aeruginosa.

Much of the research conducted on SARS-CoV-2 and COVID-19 has focused on the systemic host response, especially that generated by severely ill patients. Very few studies have investigated the impact of acute SARS-CoV-2 within the nasopharynx, the site of initial infection and viral replication. In this study we profiled changes in the nasal microbial communities as well as in host transcriptional profile during acute SARS-CoV-2 infection using 16S amplicon sequencing and RNA sequencing. These analyses were coupled to viral genome sequencing. Our microbiome analysis revealed that the nasal microbiome of COVID patients was unique and was marked by an expansion of bacterial pathogens. Some of these microbes (i.e. Acinetobacter ) were shared with COVID negative health care providers from the same medical center but absent in COVID negative outpatients seeking care at the same institutions suggesting acquisition of nosocomial respiratory pathogens. Specifically, we report a distinct increase in the prevalence and abundance of the pathogen Pseudomonas aeruginosa in COVID patients that correlated with viral RNA load. These data suggest that the inflammatory environment caused by SARS-CoV-2 infection and potentially exposure to the hospital environment leads to an expansion of bacterial pathogens in the nasal cavity that could contribute to increased incidence of secondary bacterial infections. Additionally, we observed a robust host transcriptional response in the nasal epithelia of COVID patients, indicative of an antiviral innate immune repones and neuronal damage. Finally, analysis of viral genomes did not reveal an association between viral loads and viral sequences.

Acute SARS-CoV-2 infection is associated with an increased abundance of bacterial pathogens, including Pseudomonas aeruginosa in the nose.

Research conducted on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and coronavirus disease 2019 (COVID-19) generally focuses on the systemic host response, especially that generated by severely ill patients, with few studies investigating the impact of acute SARS-CoV-2 at the site of infection. We show that the nasal microbiome of SARS-CoV-2-positive patients (CoV+, n = 68) at the time of diagnosis is unique when compared to CoV- healthcare workers (n = 45) and CoV- outpatients (n = 21). This shift is marked by an increased abundance of bacterial pathogens, including Pseudomonas aeruginosa, which is also positively associated with viral RNA load. Additionally, we observe a robust host transcriptional response in the nasal epithelia of CoV+ patients, indicative of an antiviral innate immune response and neuronal damage. These data suggest that the inflammatory response caused by SARS-CoV-2 infection is associated with an increased abundance of bacterial pathogens in the nasal cavity that could contribute to increased incidence of secondary bacterial infections.

Growth faltering regardless of chronic diarrhea is associated with mucosal immune dysfunction and microbial dysbiosis in the gut lumen.

Despite the impact of childhood diarrhea on morbidity and mortality, our understanding of its sequelae has been significantly hampered by the lack of studies that examine samples across the entire intestinal tract. Infant rhesus macaques are naturally susceptible to human enteric pathogens and recapitulate the hallmarks of diarrheal disease such as intestinal inflammation and growth faltering. Here, we examined intestinal biopsies, lamina propria leukocytes, luminal contents, and fecal samples from healthy infants and those experiencing growth faltering with distant acute or chronic active diarrhea. We show that growth faltering in the presence or absence of active diarrhea is associated with a heightened systemic and mucosal pro-inflammatory state centered in the colon. Moreover, polyclonal stimulation of colonic lamina propria leukocytes resulted in a dampened cytokine response, indicative of immune exhaustion. We also detected a functional and taxonomic shift in the luminal microbiome across multiple gut sites including the migration of Streptococcus and Prevotella species between the small and large intestine, suggesting a decompartmentalization of gut microbial communities. Our studies provide valuable insight into the outcomes of diarrheal diseases and growth faltering not attainable in humans and lays the groundwork to test interventions in a controlled and reproducible setting.

Longitudinal Profiling of the Macaque Vaginal Microbiome Reveals Similarities to Diverse Human Vaginal Communities.

The vaginal microbiota plays an important role in women's reproductive and urogenital health. It is now well accepted that a "healthy" vaginal microbiome is dominated by Lactobacillus species. Disturbances in this microbial community can lead to several adverse outcomes, including pelvic inflammatory disease and bacterial vaginosis (BV), as well as increased susceptibility to sexually transmitted infections, miscarriage, and preterm births. However, vaginal communities, especially those of women in the developing world, can be comprised of a diverse set of microorganisms in the absence of overt clinical symptoms. The implications of these diverse vaginal microbiomes for women's health remain poorly understood. Rhesus macaques are an excellent translational animal model to address these questions due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from 16 reproductive-age female rhesus macaques. At both the taxonomic and functional levels, the rhesus macaque vaginal microbiome was most similar to that of women who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Specifically, rhesus macaque vaginal microbiomes harbored a diverse set of anaerobic Gram-negative bacteria, including Sneathia, Prevotella, Porphyromonas, and Mobiluncus Interestingly, some animals were transiently colonized by Lactobacillus and some with Gardnerella Our in-depth and comprehensive analysis highlights the importance of the model to understand the health implications of a diverse vaginal microbiome and test interventions for manipulating this community.IMPORTANCE It is widely accepted that the "healthy" vaginal microbiome of women in the developed world is dominated by Lactobacillus species. However, in the developing world, many asymptomatic women harbor diverse vaginal microbial communities that are typically associated with bacterial vaginosis. Many questions remain about the drivers and health implications of a diverse vaginal microbial community. Rhesus macaques provide an excellent translational model to address these questions due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from a large cohort of reproductive-age rhesus macaques. At the taxonomic, genomic, and functional levels, the rhesus macaque vaginal microbiome was most similar to that of humans, who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Our in-depth and comprehensive analysis highlights the utility of macaques as a model to study diverse vaginal community state types and test interventions for manipulating the vaginal microbiome.

Differential dynamics of peripheral immune responses to acute SARS-CoV-2 infection in older adults.

In this study, peripheral blood mononuclear cells from young and old patients with COVID-19 were examined phenotypically, transcriptionally and functionally to reveal age-, time- and severity-specific adaptations. Gene signatures within memory B cells and plasmablasts correlated with reduced frequency of antigen-specific B cells and neutralizing antibodies in older patients with severe COVID-19. Moreover, these patients exhibited exacerbated T cell lymphopenia, which correlated with lower plasma interleukin-2, and diminished antigen-specific T cell responses. Single-cell RNA sequencing revealed augmented signatures of activation, exhaustion, cytotoxicity and type I interferon signaling in memory T and natural killer cells with age. Although cytokine storm was evident in both age groups, older individuals exhibited elevated levels of myeloid cell recruiting factors. Furthermore, we observed redistribution of monocyte and dendritic cell subsets and emergence of a suppressive phenotype with severe disease, which was reversed only in young patients over time. This analysis provides new insights into the impact of aging on COVID-19.

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.