Murine Rotavirus Research Articles

Chimeric Viruses Enable Study of Antibody Responses to Human Rotaviruses in Mice.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human-rotavirus-specific immunity to the outer capsid, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

Reverse Genetics of Murine Rotavirus: A Comparative Analysis of the Wild-Type and Cell-Culture-Adapted Murine Rotavirus VP4 in Replication and Virulence in Neonatal Mice.

Small-animal models and reverse genetics systems are powerful tools for investigating the molecular mechanisms underlying viral replication, virulence, and interaction with the host immune response in vivo. Rotavirus (RV) causes acute gastroenteritis in many young animals and infants worldwide. Murine RV replicates efficiently in the intestines of inoculated suckling pups, causing diarrhea, and spreads efficiently to uninoculated littermates. Because RVs derived from human and other non-mouse animal species do not replicate efficiently in mice, murine RVs are uniquely useful in probing the viral and host determinants of efficient replication and pathogenesis in a species-matched mouse model. Previously, we established an optimized reverse genetics protocol for RV and successfully generated a murine-like RV rD6/2-2g strain that replicates well in both cultured cell lines and in the intestines of inoculated pups. However, rD6/2-2g possesses three out of eleven gene segments derived from simian RV strains, and these three heterologous segments may attenuate viral pathogenicity in vivo. Here, we rescued the first recombinant RV with all 11 gene segments of murine RV origin. Using this virus as a genetic background, we generated a panel of recombinant murine RVs with either N-terminal VP8* or C-terminal VP5* regions chimerized between a cell-culture-adapted murine ETD strain and a non-tissue-culture-adapted murine EW strain and compared the diarrhea rate and fecal RV shedding in pups. The recombinant viruses with VP5* domains derived from the murine EW strain showed slightly more fecal shedding than those with VP5* domains from the ETD strain. The newly characterized full-genome murine RV will be a useful tool for dissecting virus-host interactions and for studying the mechanism of pathogenesis in neonatal mice.

Purinergic Signaling Drives Multiple Aspects of Rotavirus Pathophysiology

Background: Rotavirus causes life-threatening diarrhea in children, resulting in ~200,000 deaths each year. Rotavirus infects a limited number of cells at the tips of the villi in the small intestine; however, rotavirus is known to dysregulate cells distant from the site of infection through paracrine signaling. We recently identified that rotavirus infected cells release the purinergic signaling molecule ADP, which binds to the P2Y1 receptor on nearby uninfected cells; causing a signaling cascade that affects distant uninfected cells. In vitro P2Y1 was found to contribute to chloride and serotonin secretion; suggesting P2Y1 may activate pathways responsible for diarrhea. Hypothesis: We hypothesize that P2Y1 orchestrates cell-cell communication involved in diarrhea and infection severity. Methods: To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection, we used the mouse-like rotavirus (rD6/2) to investigate the effects of purinergic signaling in the context of homologous murine rotavirus infection in vivo. C57Black6 mouse pups were orally gavaged rD6/2 rotavirus at day 4-6 of age and assessed over the course of 5 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4mg/kg MRS2500, a P2Y1-selective, competitive antagonist. Stool was collected and scored for diarrhea daily prior to each treatment. Pups were euthanized and small intestine tissue was collected at 3- and 5-days post infection for immunostaining, qRT-PCR and luminal contents. Intestinal organoids derived from Balb/C pup jejunum were inoculated with rD6/2 rotavirus, followed by supplementation with vehicle or MRS2500 to assess fluid secretion by organoid swelling over time. Results: Rotavirus-infected pups exhibited significant diarrhea incidence and high diarrhea scores on days 2, 3, and 4 post infection. Treatment of rotavirus-infected mouse pups with MRS2500 resulted in decreased severity and incidence of diarrhea compared to vehicle treated controls. This was consistent with in vitro, rD6/2-infected organoids exhibiting a significant increase in organoid swelling compared to uninfected organoids, which was significantly prevented by inhibition of P2Y1 signaling. Viral stool shedding from MRS2500 treated pups was significantly lower than saline-treated pups starting at day 4 post infection, which suggests P2Y1 signaling may amplify rotavirus replication in vivo. Finally, transmission experiments in litters with infected (but not treated) and pretreated MRS2500 uninfected pups show a decreased incidence in diarrhea suggesting that P2Y1 inhibition has a potential protective role in reducing the effects of transmission. Conclusion: Collectively these findings establish a conserved role of purinergic signaling in the pathophysiology of rotavirus infection. Additionally, our data indicates P2Y1 as a new candidate for host-targeted therapeutics that could have both antiviral and antidiarrheal effects against rotavirus pathophysiology. R01 DK115507 (Hyser) R01 AI158683 (Hyser)APS Postdoctoral Fellowship (Engevik) F32 DK130288 (Engevik). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses.

Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.

Interactions Between Infectious Foodborne Viruses and Bacterial Biofilms Formed on Different Food Contact Surfaces

Bacterial biofilms contribute to contamination, spoilage, persistence, and hygiene failure in the food industry, but relatively little is known about the behavior of foodborne viruses evolving in the complex communities that make up biofilm. The aim of this study was to evaluate the association between enteric viruses and biofilms on food contact surfaces. Formed biofilms of mono- and multispecies cultures were prepared on glass, stainless steel, and polystyrene coupons and 105 pfu/ml of murine norovirus, rotavirus, and hepatitis A virus were added and incubated for 15 min, 90 min, and 24 h. The data obtained clearly demonstrate that the presence of biofilms generally influences the adhesion of enteric viruses to different surfaces. Many significant increases in attachment rates were observed, particularly with rotavirus whose rate of viral infectious particles increased 7000 times in the presence of Pseudomonas fluorescens on polystyrene after 24 h of incubation and with hepatitis A virus, which seems to have an affinity for the biofilms formed by lactic acid bacteria. Murine norovirus seems to be the least influenced by the presence of biofilms with few significant increases. However, the different factors surrounding this association are unknown and seem to vary according to the viruses, the environmental conditions, and the composition of the biofilm.

The Role of the VP4 Attachment Protein in Rotavirus Host Range Restriction in an In Vivo Suckling Mouse Model.

The basis for rotavirus (RV) host range restriction (HRR) is not fully understood but is likely multigenic. RV genes encoding VP3, VP4, NSP1, NSP2, NSP3, and NSP4 have been associated with HRR in various studies. With the exception of NSP1, little is known about the relative contribution of the other RV genes to HRR. VP4 has been linked to HRR because it functions as the RV cell attachment protein, but its actual role in HRR has not been fully assessed. We generated a collection of recombinant RVs (rRVs) in an isogenic murine-like RV genetic background, harboring either heterologous or homologous VP4 genes from simian, bovine, porcine, human, and murine RV strains, and characterized these rRVs in vitro and in vivo. We found that a murine-like rRV encoding a simian VP4 was shed, spread to uninoculated littermates, and induced diarrhea comparably to rRV harboring a murine VP4. However, rRVs carrying VP4s from both bovine and porcine RVs had reduced diarrhea, but no change in fecal shedding was observed. Both diarrhea and shedding were reduced when VP4 originated from a human RV strain. rRVs harboring VP4s from human or bovine RVs did not transmit to uninoculated littermates. We also generated two rRVs harboring reciprocal chimeric murine or bovine VP4. Both chimeras replicated and caused disease as efficiently as the parental strain with a fully murine VP4. These data suggest that the genetic origin of VP4 partially modulates HRR in the suckling mouse and that both the VP8* and VP5* domains independently contribute to pathogenesis and transmission. IMPORTANCE Human group A rotaviruses (RVs) remain the most important cause of severe acute gastroenteritis among infants and young children worldwide despite the introduction of several safe and effective live attenuated vaccines. The lack of knowledge regarding fundamental aspects of RV biology, such as the genetic basis of host range restriction (HRR), has made it difficult to predictively and efficiently design improved, next-generation live attenuated rotavirus vaccines. Here, we engineered a collection of VP4 monoreassortant RVs to systematically explore the role of VP4 in replication, pathogenicity, and spread, as measures of HRR, in a suckling mouse model. The genetic and mechanistic bases of HRR have substantial clinical relevance given that this restriction forms the basis of attenuation for several replication-competent human RV vaccines. In addition, a better understanding of RV pathogenesis and the determinants of RV spread is likely to enhance our ability to improve antiviral drug and therapy development.

Vesicle-Cloaked Rotavirus Clusters are Environmentally Persistent and Resistant to Free Chlorine Disinfection.

Recent discovery of vesicle-cloaked virus clusters (i.e., viral vesicles) has greatly challenged the central paradigm of viral transmission and infection as a single virion. To understand the environmental transmission of viral vesicles, we used an in vivo model to investigate their environmental persistence and engineering control by disinfection. Murine rotavirus vesicles maintained both their integrity and infectivity after incubation in filtered freshwater and wastewater for at least 7 days, with 24.5-27.5% of the vesicles still intact at 16 weeks after exposure to both waters. Free chlorine disinfection at a dosage of 13.3 mg min L-1 did not decompose murine rotavirus vesicles, and it was much less effective in inactivating rotaviruses inside vesicles than free rotaviruses based on the quantification of rotavirus shedding in mouse stool and rotavirus replication in small intestines. Rotavirus vesicles may be more environmentally transmissible than free rotaviruses regardless of disinfection. Vesicle-mediated en bloc transmission could be responsible for vesicles' resistance to disinfection due to an increased multiplicity of infection and/or genetic recombination or reassortment to promote infection. Our work highlights the environmental, biological, and public health significance of viral vesicles, and the findings call for urgent action in advancing disinfection for pathogen control.

Rotavirus infection elicits host responses and amplifies viral replication via P2Y1 purinergic signaling

Rotavirus causes life‐threatening diarrhea in children, resulting in ~130,000 deaths each year. Rotavirus infects a limited number of cells at the tips of the villi in the small intestine. Yet, rotavirus dysregulates cells far away from the site of infection. We recently identified, using simian (SA11) and rhesus rotavirus (RRV), that infected cells release the purinergic signaling molecule ADP, which binds to the P2Y1 receptor on nearby uninfected cells. Furthermore, using the in vivo mouse model, mild rotavirus diarrhea in mouse pups was alleviated by daily treatment with a P2Y1 inhibitor. To elucidate the role of purinergic signaling via P2Y1 receptors during rotavirus infection, we used the mouse‐like rotavirus (D6/2) to investigate the effects of purinergic signaling in the context of severe rotavirus infection in vivo. C57Black6 mouse pups were orally gavaged D6/2 rotavirus at day 4‐6 of age and assessed over the course of 5 days. Beginning at day 1 post infection, infected pups were treated daily by oral gavage with saline or 4mg/kg MRS2500, a selective P2Y1 antagonist. Stool was collected and scored for diarrhea daily prior to each treatment. Pups were euthanized and small intestine tissue was collected at 3 and 5 days post infection for immunostaining, qRT‐PCR and luminal contents. Similar to mild rotavirus infection, treatment of D6/2‐infected mouse pups with MRS2500 results in decreased severity and incidence of diarrhea. MRS2500 treated pups also exhibit decreased luminal serotonin and chloride content compared to control infected pups. Together, these results confirm that P2Y1 signaling is also involved in the pathogenesis of a homologous murine rotavirus strain. Viral stool shedding, assessed by qRT‐PCR for rotavirus gene 11 levels, revealed that MRS2500 treated pups had significantly lower viral shedding starting at day 4 post infection compared to saline treated pups, which suggests P2Y1 signaling may amplify rotavirus replication. Collectively these findings point to the conserved role of purinergic signaling in the pathophysiology of rotavirus infection, and indicate P2Y1 is a new candidate for host‐targeted therapeutics that could have both antiviral and antidiarrheal effects against rotavirus pathophysiology.

Homeostatic interferon-lambda response to bacterial microbiota stimulates preemptive antiviral defense within discrete pockets of intestinal epithelium.

Interferon-lambda (IFN-λ) protects intestinal epithelial cells (IECs) from enteric viruses by inducing expression of antiviral IFN-stimulated genes (ISGs). Here, we find that bacterial microbiota stimulate a homeostatic ISG signature in the intestine of specific pathogen-free mice. This homeostatic ISG expression is restricted to IECs, depends on IEC-intrinsic expression of IFN-λ receptor (Ifnlr1), and is associated with IFN-λ production by leukocytes. Strikingly, imaging of these homeostatic ISGs reveals localization to pockets of the epithelium and concentration in mature IECs. Correspondingly, a minority of mature IECs express these ISGs in public single-cell RNA sequencing datasets from mice and humans. Furthermore, we assessed the ability of orally administered bacterial components to restore localized ISGs in mice lacking bacterial microbiota. Lastly, we find that IECs lacking Ifnlr1 are hyper-susceptible to initiation of murine rotavirus infection. These observations indicate that bacterial microbiota stimulate ISGs in localized regions of the intestinal epithelium at homeostasis, thereby preemptively activating antiviral defenses in vulnerable IECs to improve host defense against enteric viruses.

Perspectives for the optimization and utility of the rotavirus reverse genetics system

Following Kobayashi and colleagues’ seminal paper in 20171, in the past four years the rotavirus (RV) field has witnessed a burst in research and publications based on the use of a fully plasmid-based RV reverse genetics systems and subsequent modifications2,3. However, in most cases, the rotaviral strain under interrogation has been the prototypic simian RV SA11-L2 strain (G3P[2]). Of note, a variety of other weakly-to-modestly replication-competent animal and human RV strains, bioluminescent and fluorescent reporter viruses, and clinical isolates of human RVs have proved hard or impossible to rescue using the original reverse genetics system2,4, highlighting a critical need to further enhance the recovery efficiency and expand the rescue tool kit. A number of further modifications of the initial reverse genetics system have enabled the rescue of other RV strains such as the human RV KU and CDC-9 strains, and a murine RV D6/2-like strain4,5. Here, we discuss future possible modifications of existing RV reverse genetics systems to further increase efficiency based on past experience with the improvement of influenza A virus recovery. The development of RV to accommodate the insertion and expression of heterologous sequences has substantial potential in the design of next-generation RV vaccine candidates and enteric viral vectors.

Minor alterations in the intestinal microbiota composition upon Rotavirus infection do not affect susceptibility to DSS colitis

Viral triggers at the intestinal mucosa can have multiple global effects on intestinal integrity, causing elevated intestinal barrier strength and relative protection from subsequent inflammatory bowel disease (IBD) induction in various models. As viruses can interfere with the intestinal immune system both directly and indirectly through commensal bacteria, cause-effect relationships are difficult to define. Due to the complexity of putatively causative factors, our understanding of such virus-mediated protection is currently very limited. We here set out to better understand the impact that adult enteric infection with rotavirus (RV) might have on the composition of the intestinal microbiome and on the severity of IBD. We found that RV infection neither induced significant long-lasting microbiota community changes in the small or large intestine nor affected the severity of subsequent dextran sulfate sodium-induced colitis. Hence, adult murine RV infection does not exert lasting effects on intestinal homeostasis.

The enteric microbiome stimulates localized interferon responses and provides preemptive antiviral protection of intestinal epithelial cells through IFNLR1 signaling

Abstract Specialized immune responses have evolved to eliminate pathogens in the gastrointestinal tract. In particular, intestinal epithelial cells (IECs) are uniquely responsive to interferon lambda (IFN-λ) and depend on IFN-λ for antiviral defense to a greater extent than other types of IFN. IFN-λ signaling protects IECs by inducing expression of antiviral IFN-stimulated genes (ISGs), but it is unclear if the intestinal microbiome interacts with the IFN-λ pathway. We find that commensal microbes stimulate a steady-state ISG signal in the gastrointestinal tract that is depleted with broad-spectrum antibiotics, as assessed by whole-tissue RNA sequencing. Steady-state ISG expression is also significantly reduced in mice lacking IFN-λ receptor (IFNLR) specifically in IECs. These data suggest that enteric bacteria stimulate expression of IFN-λ and, subsequently, epithelial ISGs at homeostasis. Strikingly, imaging data reveals that these steady-state ISGs are present in distinct pockets throughout the small intestinal epithelium and are concentrated in IECs at the tips of individual villi. Furthermore, we find that IFNLR protects IECs during initiation of murine rotavirus infection at early time-points post-infection, suggesting that steady-state ISGs are protective against viral infection. Lastly, we find that steady-state ISGs are expressed in a subpopulation of human IECs when we re-analyze publicly available single-cell RNA sequencing datasets. These novel observations indicate that commensal microbes stimulate ISGs in localized regions of the intestinal epithelium at homeostasis and may preemptively activate antiviral defenses in vulnerable IECs to improve host fitness against enteric viruses.

Genipin inhibits rotavirus-induced diarrhea by suppressing viral replication and regulating inflammatory responses

Rotavirus is the leading cause of acute gastroenteritis among young children worldwide. However, agents specifically designed to treat rotavirus infection have not been developed yet. In this study, the anti-rotavirus and anti-inflammatory effects of genipin, a chemical compound found in the fruit of Gardenia jasminoides, were evaluated. Genipin had an antiviral effect against the human rotavirus Wa and SA-11 strains in vitro, and it inhibited two distinct stages of the viral replication cycle: attachment and penetration (early stage) in pre-treatment and assembly and release (late stage) in post-treatment. Additionally, genipin downregulated nitric oxide synthase and pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW264.7 cells and rotavirus-infected Caco-2 cells. Oral administration of genipin before and after viral infection with the murine rotavirus epidemic diarrhea of infant mice strain led to a reduced duration of diarrhea and faecal viral shedding and to decreased destruction of the enteric epithelium. Genipin could have potential as a natural compound with preventive and therapeutic effects against infection and colitis caused by rotavirus.

Intracellular neutralisation of rotavirus by VP6-specific IgG.

Rotavirus is a major cause of gastroenteritis in children, with infection typically inducing high levels of protective antibodies. Antibodies targeting the middle capsid protein VP6 are particularly abundant, and as VP6 is only exposed inside cells, neutralisation must be post-entry. However, while a system of poly immune globulin receptor (pIgR) transcytosis has been proposed for anti-VP6 IgAs, the mechanism by which VP6-specific IgG mediates protection remains less clear. We have developed an intracellular neutralisation assay to examine how antibodies neutralise rotavirus inside cells, enabling comparison between IgG and IgA isotypes. Unexpectedly we found that neutralisation by VP6-specific IgG was much more efficient than by VP6-specific IgA. This observation was highly dependent on the activity of the cytosolic antibody receptor TRIM21 and was confirmed using an in vivo model of murine rotavirus infection. Furthermore, mice deficient in only IgG and not other antibody isotypes had a serious deficit in intracellular antibody-mediated protection. The finding that VP6-specific IgG protect mice against rotavirus infection has important implications for rotavirus vaccination. Current assays determine protection in humans predominantly by measuring rotavirus-specific IgA titres. Measurements of VP6-specific IgG may add to existing mechanistic correlates of protection.

Elucidating the Role of Purinergic and Calcium Signaling During Rotavirus Infection

Calcium (Ca2+) is a ubiquitous messenger that influences numerous cellular processes, and therefore Ca2+ signaling is tightly regulated by cells. Ca2+ signaling dysregulation results in severe and potentially life‐threatening diseases, which is exemplified by rotavirus (RV) infection. RV is an enteric virus that causes life‐threatening diarrhea in children, resulting in ~198,000 deaths each year. While pathophysiological consequences of RV infection are widely studied, host Ca2+ signaling pathways and the mechanisms by which RV exploits them to cause diarrhea remain incompletely characterized. We have previously found that RV infection increases Ca2+ signaling both within infected enterocytes and in surrounding uninfected cells through paracrine signaling. This manifests as intercellular Ca2+ waves that originate from the infected cell and propagates to surrounding uninfected cells mainly through ADP activation of the P2Y1 receptor, which is expressed in many cell types in the intestine. We hypothesize that RV‐induced paracrine purinergic signaling functionally dysregulates neighboring uninfected enterocytes and modulates innate immune responses from both epithelial and intestinal immune cells. We generated cell lines and human intestinal enteroids (HIEs) stably expressing cytosolic genetically‐encoded Ca2+ indicators to characterize Ca2+ signaling throughout RV infection by time‐lapse imaging. We found that P2Y1‐mediated signaling was critical for activation of secretory epithelial cells, including induction of serotonin secretion of enterochromaffin cells and mucus secretion from goblet cells in human intestinal enteroids (HIEs) and mucin‐producing intestinal cell lines. Further, signaling from RV‐infected MA104 monkey kidney cells chemoattracted mouse bone‐marrow derived macrophages, which was blocked by pharmacological inhibitors of the P2Y1 receptor. Consistent with our in vitro findings, we observed that murine RV infection promoted secretion of serotonin, mucin and accumulation of macrophages. These effects of minimized in the presence of P2Y1 inhibitors and in P2Y1 knock out mice. Furthermore, we found that the RV‐induced increase in NFKb and IL‐1α are significantly attenuated by P2Y1 receptor blockers. Collectively these findings indicate that intercellular Ca2+ waves caused by paracrine purinergic signaling have a broad influence on intestinal pathophysiology during RV infection. Understanding the cellular consequences of P2Y1 activation during RV will generate new mechanistic insights into the homeostatic function of purinergic signaling in the GI tract.Support or Funding InformationR01 DK115507 (Hyser)T32 DK007664 (Engevik)

Immune response and protective efficacy of the S particle presented rotavirus VP8* vaccine in mice

Rotaviruses cause severe diarrhea in infants and young children, leading to significant morbidity and mortality. Despite implementation of current rotavirus vaccines, severe diarrhea caused by rotaviruses still claims ∼200,000 lives of children with great economic loss worldwide each year. Thus, new prevention strategies with high efficacy are highly demanded. Recently, we have developed a polyvalent protein nanoparticle derived from norovirus VP1, the S particle, and applied it to display rotavirus neutralizing antigen VP8* as a vaccine candidate (S-VP8*) against rotavirus, which showed promise as a vaccine based on mouse immunization and in vitro neutralization studies. Here we further evaluated this S-VP8* nanoparticle vaccine in a mouse rotavirus challenge model. S-VP8* vaccines containing the murine rotavirus (EDIM strain) VP8* antigens (S-mVP8*) were constructed and immunized mice, resulting in high titers of anti-EDIM VP8* IgG. The S-mVP8* nanoparticle vaccine protected immunized mice against challenge of the homologous murine EDIM rotavirus at a high efficacy of 97% based on virus shedding reduction in stools compared with unimmunized controls. Our study further supports the polyvalent S-VP8* nanoparticles as a promising vaccine candidate against rotavirus and warrants further development.

The distinct impact of maternal antibodies on the immunogenicity of live and recombinant rotavirus vaccines

A high titre of maternal antibodies is one of the possible factors associated with decreased rotavirus vaccine efficacy in low-income countries where rotavirus-associated morbidity and mortality are high. Although some studies show a negative correlation between maternal antibody levels and seroconversion after vaccination, withholding breastfeeding does not improve rotavirus vaccine efficacy. Different types of recombined vaccines were developed as an alternative to produce higher protection in developing areas. In previous studies, we found that recombinantly expressed, truncated VP4* can stimulate high titres of neutralizing antibodies and can confer protection against rotavirus infections and rotavirus-induced diarrhoea. In this study, the impact of maternal antibodies on live and recombinant rotavirus vaccines (VP4*) was evaluated in a mouse model. Dams were infected orally with murine rotavirus 7 days after delivery to mimic a natural rotavirus infection in infants and to evaluate the separate effects of trans-placentally acquired and milk-acquired maternal antibodies, pups were half exchanged. After immunization with live rotavirus, both the neutralizing antibody and IgA antibody responses were inhibited by maternal antibodies, especially by milk antibodies; however, the neutralizing antibody responses after immunization with recombinant VP4* were enhanced. In addition, the in vitro incubation of VP4* with immune sera of rotavirus could also enhance the immune responses could also enhance the immune responses. Our finding provides a basis for the development of non-replicating vaccines to address the problem of live attenuated vaccines in low- and middle-income countries.

Viral complementation of immunodeficiency confers protection against enteric pathogens via interferon-λ.

Commensal microbes profoundly impact host immunity to enteric viral infections1. We have shown that the bacterial microbiota and host antiviral cytokine interferon-lambda (IFN-λ) determine the persistence of murine norovirus in the gut2,3. However, the effects of the virome in modulating enteric infections remain unexplored. Here we report that murine astrovirus can complement primary immunodeficiency to protect against murine norovirus and rotavirus infections. Protection against infection was horizontally transferable between immunocompromised mouse strains by cohousing and fecal transplantation. Furthermore, protection against enteric pathogens corresponded with the presence of a specific strain of murine astrovirus in the gut, and this complementation of immunodeficiency required IFN-λ signaling in gut epithelial cells. Our study demonstrates that elements of the virome can protect against enteric pathogens in an immunodeficient host.

The Nontoxic Cholera B Subunit Is a Potent Adjuvant for Intradermal DC-Targeted Vaccination.

CD4+ T cells are major players in the immune response against several diseases; including AIDS, leishmaniasis, tuberculosis, influenza and cancer. Their activation has been successfully achieved by administering antigen coupled with antibodies, against DC-specific receptors in combination with adjuvants. Unfortunately, most of the adjuvants used so far in experimental models are unsuitable for human use. Therefore, human DC-targeted vaccination awaits the description of potent, yet nontoxic adjuvants. The nontoxic cholera B subunit (CTB) can be safely used in humans and it has the potential to activate CD4+ T cell responses. However, it remains unclear whether CTB can promote DC activation and can act as an adjuvant for DC-targeted antigens. Here, we evaluated the CTB's capacity to activate DCs and CD4+ T cell responses, and to generate long-lasting protective immunity. Intradermal (i.d.) administration of CTB promoted late and prolonged activation and accumulation of skin and lymphoid-resident DCs. When CTB was co-administered with anti-DEC205-OVA, it promoted CD4+ T cell expansion, differentiation, and infiltration to peripheral nonlymphoid tissues, i.e., the skin, lungs and intestine. Indeed, CTB promoted a polyfunctional CD4+ T cell response, including the priming of Th1 and Th17 cells, as well as resident memory T (RM) cell differentiation in peripheral nonlymphoid tissues. It is worth noting that CTB together with a DC-targeted antigen promoted local and systemic protection against experimental melanoma and murine rotavirus. We conclude that CTB administered i.d. can be used as an adjuvant to DC-targeted antigens for the induction of broad CD4+ T cell responses as well as for promoting long-lasting protective immunity.

Highly sensitive ELISA for the serological detection of murine rotavirus EDIM based on its major immunogen VP6

Precise health monitoring of laboratory animals is a critical factor for surveillance and accuracy of animal experiments. Rotavirus epizootic diarrhea of infant mice (EDIM) leads to infections in mice that can influence animal studies, e.g., by altering the intestinal physiology. Thus, the aim of this study was establishing a highly sensitive and specific ELISA for the serological detection of EDIM infections in rodents. First, virus proteins were separated by SDS-PAGE and immunogenic proteins were visualized by immunoblotting and identified after in-gel digestion by tandem mass spectrometry. Subsequently, the major immunogen VP6 (virus protein 6) was expressed in Escherichia coli in high yields, purified by affinity chromatography, and used to establish an indirect ELISA. The diagnostic sensitivity and specificity were both above 99 % and the selectivity better than 98.7 % for animals infected by other pathogens listed by the Federation of Laboratory Animal Science Associations. Importantly, the Strep-rVP6-His-ELISA was more sensitive than a commercial virus-based ELISA and is a time- and cost-efficient complement to EDIM-specific immune-fluorescence assays. In conclusion, the assay can improve health monitoring by reducing the risk of missed EDIM infections in animal housing facilities, thereby improving animal welfare, reliability of animal studies, and protection of precious mice breeds.