Comparison of mouse models of microbial experience reveals differences in microbial diversity and response to vaccination.

Specific pathogen-free (SPF) laboratory mice dominate preclinical studies for immunology and vaccinology. Unfortunately, SPF mice often fail to accurately model human responses to vaccination and other immunological perturbations. Several groups have taken different approaches to introduce additional microbial experience to SPF mice to better model human immune experience. How these different models compare is unknown. Here, we directly compare three models: housing SPF mice in a microbe-rich barn-like environment (feralizing), adding wild-caught mice to the barn-like environment (fer-cohoused), or cohousing SPF mice with pet store mice in a barrier facility (pet-cohoused); the two latter representing different murine sources of microbial transmission. Pet-cohousing mice resulted in the greatest microbial exposure. Feralizing alone did not result in the transmission of any pathogens tested, while fer-cohousing resulted in the transmission of several picornaviruses. Murine astrovirus 2, the most common pathogen from pet store mice, was absent from the other two model systems. Previously, we had shown that pet-cohousing reduced the antibody response to vaccination compared with SPF mice. This was not recapitulated in either the feralized or fer-cohoused mice. These data indicate that not all dirty mouse models are equivalent in either microbial experience or immune responses to vaccination. These disparities suggest that more cross model comparisons are needed but also represent opportunities to uncover microbe combination-specific phenotypes and develop more refined experimental models. Given the breadth of microbes encountered by humans across the globe, multiple model systems may be needed to accurately recapitulate heterogenous human immune responses.IMPORTANCEAnimal models are an essential tool for evaluating clinical interventions. Unfortunately, they can often fail to accurately predict outcomes when translated into humans. This failure is due in part to a lack of natural infections experienced by most laboratory animals. To improve the mouse model, we and others have exposed laboratory mice to microbes they would experience in the wild. Although these models have been growing in popularity, these different models have not been specifically compared. Here, we directly compare how three different models of microbial experience impact the immune response to influenza vaccination. We find that these models are not the same and that the degree of microbial exposure affects the magnitude of the response to vaccination. These results provide an opportunity for the field to continue comparing and contrasting these systems to determine which models best recapitulate different aspects of the human condition.

Rotavirus Infection in Swine: Genotypic Diversity, Immune Responses, and Role of Gut Microbiome in Rotavirus Immunity.

Rotaviruses (RVs) are endemic in swine populations, and all swine herds certainly have a history of RV infection and circulation. Rotavirus A (RVA) and C (RVC) are the most common among all RV species reported in swine. RVA was considered most prevalent and pathogenic in swine; however, RVC has been emerging as a significant cause of enteritis in newborn piglets. RV eradication from swine herds is not practically achievable, hence producers' mainly focus on minimizing the production impact of RV infections by reducing mortality and diarrhea. Since no intra-uterine passage of immunoglobulins occur in swine during gestation, newborn piglets are highly susceptible to RV infection at birth. Boosting lactogenic immunity in gilts by using vaccines and natural planned exposure (NPE) is currently the only way to prevent RV infections in piglets. RVs are highly diverse and multiple RV species have been reported from swine, which also contributes to the difficulties in preventing RV diarrhea in swine herds. Human RV-gut microbiome studies support a link between microbiome composition and oral RV immunogenicity. Such information is completely lacking for RVs in swine. It is not known how RV infection affects the functionality or structure of gut microbiome in swine. In this review, we provide a detailed overview of genotypic diversity of swine RVs, host-ranges, innate and adaptive immune responses to RVs, homotypic and heterotypic immunity to RVs, current methods used for RV management in swine herds, role of maternal immunity in piglet protection, and prospects of investigating swine gut microbiota in providing immunity against rotaviruses.

Respiratory Influenza Virus Infection Causes Dynamic Tuft Cell and Innate Lymphoid Cell Changes in the Small Intestine.

Influenza A viruses (IAV) can cause severe disease and death in humans. IAV infection and the accompanying immune response can result in systemic inflammation, leading to intestinal damage and disruption of the intestinal microbiome. Here, we demonstrate that a specific subset of epithelial cells, tuft cells, increase across the small intestine during active respiratory IAV infection. Upon viral clearance, tuft cell numbers return to baseline levels. Intestinal tuft cell increases were not protective against disease, as animals with either increased tuft cells or a lack of tuft cells did not have any change in disease morbidity after infection. Respiratory IAV infection also caused transient increases in type 1 and 2 innate lymphoid cells (ILC1 and ILC2, respectively) in the small intestine. ILC2 increases were significantly blunted in the absence of tuft cells, whereas ILC1s were unaffected. Unlike the intestines, ILCs in the lungs were not altered in the absence of tuft cells. This work establishes that respiratory IAV infection causes dynamic changes to tuft cells and ILCs in the small intestines and that tuft cells are necessary for the infection-induced increase in small intestine ILC2s. These intestinal changes in tuft cell and ILC populations may represent unexplored mechanisms preventing systemic infection and/or contributing to severe disease in humans with preexisting conditions. IMPORTANCE Influenza A virus (IAV) is a respiratory infection in humans that can lead to a wide range of symptoms and disease severity. Respiratory infection can cause systemic inflammation and damage in the intestines. Few studies have explored how inflammation alters the intestinal environment. We found that active infection caused an increase in the epithelial population called tuft cells as well as type 1 and 2 innate lymphoid cells (ILCs) in the small intestine. In the absence of tuft cells, this increase in type 2 ILCs was seriously blunted, whereas type 1 ILCs still increased. These findings indicate that tuft cells are necessary for infection-induced changes in small intestine type 2 ILCs and implicate tuft cells as regulators of the intestinal environment in response to systemic inflammation.

Natural rodent model of viral transmission reveals biological features of virus population dynamics.

Emerging viruses threaten global health, but few experimental models can characterize the virus and host factors necessary for within- and cross-species transmission. Here, we leverage a model whereby pet store mice or rats-which harbor natural rodent pathogens-are cohoused with laboratory mice. This "dirty" mouse model offers a platform for studying acute transmission of viruses between and within hosts via natural mechanisms. We identified numerous viruses and other microbial species that transmit to cohoused mice, including prospective new members of the Coronaviridae, Astroviridae, Picornaviridae, and Narnaviridae families, and uncovered pathogen interactions that promote or prevent virus transmission. We also evaluated transmission dynamics of murine astroviruses during transmission and spread within a new host. Finally, by cohousing our laboratory mice with the bedding of pet store rats, we identified cross-species transmission of a rat astrovirus. Overall, this model system allows for the analysis of transmission of natural rodent viruses and is a platform to further characterize barriers to zoonosis.

Mice with diverse microbial exposure histories as a model for preclinical vaccine testing.

Laboratory mice comprise an expeditious model for preclinical vaccine testing; however, vaccine immunogenicity in these models often inadequately translates to humans. Reconstituting physiologic microbial experience to specific pathogen-free (SPF) mice induces durable immunological changes that better recapitulate human immunity. We examined whether mice with diverse microbial experience better model human responses post vaccination. We co-housed laboratory mice with pet-store mice, which have varied microbial exposures, and then assessed immune responses to influenza vaccines. Human transcriptional responses to influenza vaccination are better recapitulated in co-housed mice. Although SPF and co-housed mice were comparably susceptible to acute influenza infection, vaccine-induced humoral responses were dampened in co-housed mice, resulting in poor control upon challenge. Additionally, protective heterosubtypic T cell immunity was compromised in co-housed mice. Because SPF mice exaggerated humoral and T cell protection upon influenza vaccination, reconstituting microbial experience in laboratory mice through co-housing may better inform preclinical vaccine testing.

Cutting Edge: Nucleocapsid Vaccine Elicits Spike-Independent SARS-CoV-2 Protective Immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing Abs target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with a human adenovirus type 5 vector expressing the SARS-CoV-2 nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and K18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral Ags in SARS-CoV-2 vaccines, even if they are not a target of neutralizing Abs, to broaden epitope coverage and immune effector mechanisms.

Nucleocapsid vaccine elicits spike-independent SARS-CoV-2 protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Neutralizing antibodies target the receptor binding domain of the spike (S) protein, a focus of successful vaccine efforts. Concerns have arisen that S-specific vaccine immunity may fail to neutralize emerging variants. We show that vaccination with HAd5 expressing the nucleocapsid (N) protein can establish protective immunity, defined by reduced weight loss and viral load, in both Syrian hamsters and k18-hACE2 mice. Challenge of vaccinated mice was associated with rapid N-specific T cell recall responses in the respiratory mucosa. This study supports the rationale for including additional viral antigens, even if they are not a target of neutralizing antibodies, to broaden epitope coverage and immune effector mechanisms.

Leveraging a Validated in silico Approach to Elucidate Genotype-Specific VP7 Epitopes and Antigenic Relationships of Porcine Rotavirus A.

Rotavirus A (RVA) remains one of the most widespread causes of diarrheal disease and mortality in piglets despite decades of research and efforts to boost lactogenic immunity for passive protection. Genetic changes at B cell epitopes (BCEs) may be driving failure of lactogenic immunity, which relies on production of IgA antibodies to passively neutralize RVA within the piglet gut, yet little research has mapped epitopes to swine-specific strains of RVA. Here we describe a bioinformatic approach to predict BCEs on the VP7 outer capsid protein using sequence data alone. We first validated the approach using a previously published dataset of VP7-specific cross-neutralization titers, and found that amino acid changes at predicted BCEs on the VP7 protein allowed for accurate recapitulation of antigenic relationships among the strains. Applying the approach to a dataset of swine RVA sequences identified 9 of the 11 known BCEs previously mapped to swine strains, indicating that epitope prediction can identify sites that are known to drive neutralization escape in vitro. Additional genotype-specific BCEs were also predicted that may be the cause of antigenic differences among strains of RVA on farms and should be targeted for further confirmatory work. The results of this work lay the groundwork for high throughput, immunologically-relevant analysis of swine RVA sequence data, and provide potential sites that can be targeted with vaccines to reduce piglet mortality and support farm health.

Evolution of rotavirus C in humans and several domestic animal species.

Rotavirus C (RVC) causes enteric disease in multiple species, including humans, swine, bovines, and canines. To date, the evolutionary relationships of RVC populations circulating in different host species are poorly understood, owing to the low availability of genetic sequence data. To address this gap, we sequenced 45 RVC complete genomes from swine samples collected in the United States and Mexico. A phylogenetic analysis of each genome segment indicates that RVC populations have been evolving independently in human, swine, canine, and bovine hosts for at least the last century, with inter-species transmission events occurring deep in the phylogenetic tree, and none in the last 100 years. Bovine and canine RVC populations clustered together nine of the 11 gene segments, indicating a shared common ancestor centuries ago. The evolutionary relationships of RVC in humans and swine were more complex, due to the extensive genetic diversity and multiple RVC clades identified in pigs, which were not structured geographically. Topological differences between trees inferred for different genome segments occurred frequently, including at nodes deep in the tree, indicating that RVC's evolutionary history includes multiple reassortment events that occurred a long time ago. Overall, we find that RVC is evolving within host-defined lineages, but the evolutionary history of RVC is more complex than previously recognized due to the high genetic diversity of RVC in swine, with a common ancestor dating back centuries. Pigs may act as a reservoir host for RVC, and a source of the lineages identified in other species, including humans, but additional sequencing is needed to understand the full diversity of this understudied pathogen across multiple host species.

Whole Genome Classification and Phylogenetic Analyses of Rotavirus B strains from the United States.

Rotaviruses (RVs) are a major etiological agent of acute viral gastroenteritis in humans and young animals, with rotavirus B (RVB) often detected in suckling and weaned pigs. Group A rotavirus classification is currently based on the two outer capsid proteins, VP7 and VP4, and the middle layer protein, VP6. Using RVB strains generated in this study and reference sequences from GenBank, pairwise identity frequency graphs and phylogenetic trees were constructed for the eleven gene segments of RVB to estimate the nucleotide identity cutoff values for different genotypes and determine the genotype diversity per gene segment. Phylogenetic analysis of VP7, VP4, VP6, VP1–VP3, and NSP1–NSP5 identified 26G, 5P, 13I, 5R, 5C, 5M, 8A, 10N, 6T, 4E, and 7H genotypes, respectively. The analysis supports the previously proposed cutoff values for the VP7, VP6, NSP1, and NSP3 gene segments (80%, 81%, 76% and 78%, respectively) and suggests new cutoff values for the VP4, VP1, VP2, VP3, NSP2, NSP4, and NSP5 (80%, 78%, 79%, 77% 83%, 76%, and 79%, respectively). Reassortment events were detected between the porcine RVB strains from our study. This research describes the genome constellations for the complete genome of Group B rotaviruses in different host species.

Genome Characterization of a Pathogenic Porcine Rotavirus B Strain Identified in Buryat Republic, Russia in 2015.

An outbreak of enteric disease of unknown etiology with 60% morbidity and 8% mortality in weaning piglets occurred in November 2015 on a farm in Buryat Republic, Russia. Metagenomic sequencing revealed the presence of rotavirus B in feces from diseased piglets while no other pathogens were identified. Clinical disease was reproduced in experimentally infected piglets, yielding the 11 RVB gene segments for strain Buryat15, with an RVB genotype constellation of G12-P[4]-I13-R4-C4-M4-A8-N10-T4-E4-H7. This genotype constellation has also been identified in the United States. While the Buryat15 VP7 protein lacked unique amino acid differences in the predicted neutralizing epitopes compared to the previously published swine RVB G12 strains, this report of RVB in Russian swine increases our epidemiological knowledge on the global prevalence and genetic diversity of RVB.

Longitudinal Surveillance of Porcine Rotavirus B Strains from the United States and Canada and In Silico Identification of Antigenically Important Sites.

Rotavirus B (RVB) is an important swine pathogen, but control and prevention strategies are limited without an available vaccine. To develop a subunit RVB vaccine with maximal effect, we characterized the amino acid sequence variability and predicted antigenicity of RVB viral protein 7 (VP7), a major neutralizing antibody target, from clinically infected pigs in the United States and Canada. We identified genotype-specific antigenic sites that may be antibody neutralization targets. While some antigenic sites had high amino acid functional group diversity, nine antigenic sites were completely conserved. Analysis of nucleotide substitution rates at amino acid sites (dN/dS) suggested that negative selection appeared to be playing a larger role in the evolution of the identified antigenic sites when compared to positive selection, and was identified in six of the nine conserved antigenic sites. These results identified important characteristics of RVB VP7 variability and evolution and suggest antigenic residues on RVB VP7 that are negatively selected and highly conserved may be good candidate regions to include in a subunit vaccine design due to their tendency to remain stable.