Immunity to enteric viruses.

Pathogenic enteric viruses are a major cause of morbidity and mortality, particularly among children in developing countries. The host response to enteric viruses occurs primarily within the mucosa, where the intestinal immune system must balance protection against pathogens with tissue protection and tolerance to harmless commensal bacteria and food. Here, we summarize current knowledge in natural immunity to enteric viruses, highlighting specialized features of the intestinal immune system. We further discuss how knowledge of intestinal anti-viral mechanisms can be translated into vaccine development with particular focus on immunization in the oral route. Research reveals that the intestine is a complex interface between enteric viruses and the host where environmental factors influence susceptibility and immunity to infection, while viral infections can have lasting implications for host health. A deeper mechanistic understanding of enteric anti-viral immunity with this broader context can ultimately lead to better vaccines for existing and emerging viruses.

Sera Antibody Repertoire Analyses Reveal Mechanisms of Broad and Pandemic Strain Neutralizing Responses after Human Norovirus Vaccination.

Rapidly evolving RNA viruses, such as the GII.4 strain of human norovirus (HuNoV), and their vaccines elicit complex serological responses associated with previous exposure. Specific correlates of protection, moreover, remain poorly understood. Here, we report the GII.4-serological antibody repertoire-pre- and post-vaccination-and select several antibody clonotypes for epitope and structural analysis. The humoral response was dominated by GII.4-specific antibodies that blocked ancestral strains or by antibodies that bound to divergent genotypes and did not block viral-entry-ligand interactions. However, one antibody, A1431, showed broad blockade toward tested GII.4 strains and neutralized the pandemic GII.P16-GII.4 Sydney strain. Structural mapping revealed conserved epitopes, which were occluded on the virion or partially exposed, allowing for broad blockade with neutralizing activity. Overall, our results provide high-resolution molecular information on humoral immune responses after HuNoV vaccination and demonstrate that infection-derived and vaccine-elicited antibodies can exhibit broad blockade and neutralization against this prevalent human pathogen.

Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Mucosal and systemic neutralizing antibodies to norovirus induced in infant mice orally inoculated with recombinant rotaviruses.

Rotaviruses (RVs) preferentially replicate in the small intestine and frequently cause severe diarrheal disease, and the following enteric infection generally induces variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid only-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a newly available opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued replication-competent recombinant RRVs harboring bicistronic gene segment 7 that encodes the native RV nonstructural protein 3 (NSP3) protein and a human norovirus (HuNoV) VP1 protein or P domain from the predominant genotype GII.4. The rescued viruses expressed HuNoV VP1 or P protein in infected cells in vitro and elicited systemic and local antibody responses to HuNoV and RRV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RRV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens.

Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

Production of infectious reporter murine norovirus by VP2 trans-complementation.

Human norovirus (HuNoV) causes gastroenteritis, a disease with no effective therapy or vaccine, and does not grow well in culture. Murine norovirus (MNV) easily replicates in cell cultures and small animals and has often been used as a model to elucidate the structural and functional characteristics of HuNoV. An MNV plasmid-based reverse genetics system was developed to produce the modified recombinant virus. In this study, we attempted to construct the recombinant virus by integrating a foreign gene into MNV ORF3, which encodes the minor structural protein VP2. Deletion of VP2 expression abolished infectious particles from MNV cDNA clones, and supplying exogenous VP2 to the cells rescued the infectivity of cDNA clones without VP2 expression. In addition, the coding sequence of C-terminal ORF3 was essential for cDNA clones compensated with VP2 to produce infectious particles. Furthermore, the recombinant virus with exogenous reporter genes in place of the dispensable region of ORF3 was propagated when VP2 was constitutively supplied. Our findings indicate that foreign genes can be transduced into the norovirus ORF3 region when VP2 is supplied and that successive propagation of modified recombinant norovirus could lead to the development of norovirus-based vaccines or therapeutics.IMPORTANCEIn this study, we revealed that some of the coding regions of ORF3 could be replaced by a foreign gene and infectious virus could be produced when VP2 was supplied. Propagation of this virus depended on VP2 being supplied in trans, indicating that this virus could infect only once. Our findings help to elucidate the functions of VP2 in the virus lifecycle and to develop other caliciviral vectors for recombinant attenuated live enteric virus vaccines or therapeutics tools.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Analysis of Archival Sera from Norovirus-Infected Individuals Demonstrates that Cross-Blocking of Emerging Viruses is Genotype-Specific.

BACKGROUND: Rapidly evolving RNA viruses, such as human norovirus, generate extraordinary sequence diversity, posing a significant challenge to vaccine design. This diversity coupled with short-lasting natural immunity leads to re-infection throughout one's lifetime. How re-exposure shapes humoral immunity to future norovirus strains remains incompletely understood. METHODS: We profiled the antibody responses following two community gastroenteritis outbreaks with GII.2 and GII.6 noroviruses in 1971. Using diverse VLPs, ELISA, and carbohydrate-blocking assays (surrogate for neutralization), we examined the antibody response at acute and convalescent timepoints following GII.6 infection. RESULTS: Convalescent sera displayed strong homologous blocking, demonstrating a 5-fold increase in GII.6 carbohydrate-blockade over acute samples, and broad blocking of diverse archival and modern GII.6 noroviruses. Convalescent sera displayed limited carbohydrate-blocking of heterotypic VLPs, despite high ELISA binding titers. Select individuals developed broad cross-genotype blockade, but this response was established before the second outbreak. Finally, we applied a novel competitive carbohydrate-blocking assay to demonstrate the epitope-specificity and discrete compartments of the neutralizing response. CONCLUSIONS: Our data show that infection generates narrow, focused immunity directed towards the infecting genotype. We did detect broad cross-blocking in specific individuals, but these responses could be attributed to diverse, genotype-specific antibodies pre-dating GII.6 infection.

GI.1 Norovirus Neutralizing Antibody Levels Are Correlated with GI.1 Histo-blood Group Antigen-Blocking Antibody Levels.

BACKGROUND: The in vitro cultivation of human noroviruses allows a comparison of antibody levels measured in neutralization and histoblood group antigen (HBGA)-blocking assays. METHODS: Serum samples collected during the evaluation of an investigational norovirus vaccine (HIL-214 [formerly TAK-214]) were assayed for neutralizing antibody levels against the vaccine's prototype Norwalk virus/GI.1 (P1) virus strain. Results were compared to those previously determined using HBGA-blocking assays. RESULTS: Neutralizing antibody seroresponses were observed in 83% of 24 vaccinated adults, and antibody levels were highly correlated (r=0.81, P<0.001) with those measured by HBGA-blocking. CONCLUSIONS: GI.1-specific HBGA-blocking antibodies are a surrogate for neutralization of GI.1 norovirus.

N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Emergence of Novel Norovirus GII.4 Variant.

We detected a novel GII.4 variant with an amino acid insertion at the start of epitope A in viral protein 1 of noroviruses from the United States, Gabon, South Africa, and the United Kingdom collected during 2017-2022. Early identification of GII.4 variants is crucial for assessing pandemic potential and informing vaccine development.

Acute Gastroenteritis Associated with Norovirus GII.8[P8], Thailand, 2023.

Acute gastroenteritis associated with human norovirus infection was reported in Phuket, Thailand, in June 2023. We amplified GII.8[P8] from the outbreak stool specimens. Retrospective sample analysis identified infrequent GII.8[P8] in the country beginning in 2018. In all, the 10 whole-genome GII.8[P8] sequences from Thailand we examined had no evidence of genotypic recombination.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

Favipiravir induces HuNoV viral mutagenesis and infectivity loss with clinical improvement in immunocompromised patients.

Chronic human norovirus (HuNoV) infections in immunocompromised patients result in severe disease, yet approved antivirals are lacking. RNA-dependent RNA polymerase (RdRp) inhibitors inducing viral mutagenesis display broad-spectrum in vitro antiviral activity, but clinical efficacy in HuNoV infections is anecdotal and the potential emergence of drug-resistant variants is concerning. Upon favipiravir (and nitazoxanide) treatment of four immunocompromised patients with life-threatening HuNoV infections, viral whole-genome sequencing showed accumulation of favipiravir-induced mutations which coincided with clinical improvement although treatment failed to clear HuNoV. Infection of zebrafish larvae demonstrated drug-associated loss of viral infectivity and favipiravir treatment showed efficacy despite occurrence of RdRp variants potentially causing favipiravir resistance. This indicates that within-host resistance evolution did not reverse loss of viral fitness caused by genome-wide accumulation of sequence changes. This off-label approach supports the use of mutagenic antivirals for treating prolonged RNA viral infections and further informs the debate surrounding their impact on virus evolution.

Direct Blockade of the Norovirus Histo-Blood Group Antigen Binding Pocket by Nanobodies.

Noroviruses are the leading cause of outbreaks of acute gastroenteritis. These viruses usually interact with histo-blood group antigens (HBGAs), which are considered essential cofactors for norovirus infection. This study structurally characterizes nanobodies developed against the clinically important GII.4 and GII.17 noroviruses with a focus on the identification of novel nanobodies that efficiently block the HBGA binding site. Using X-ray crystallography, we have characterized nine different nanobodies that bound to the top, side, or bottom of the P domain. The eight nanobodies that bound to the top or side of the P domain were mainly genotype specific, while one nanobody that bound to the bottom cross-reacted against several genotypes and showed HBGA blocking potential. The four nanobodies that bound to the top of the P domain also inhibited HBGA binding, and structural analysis revealed that these nanobodies interacted with several GII.4 and GII.17 P domain residues that commonly engaged HBGAs. Moreover, these nanobody complementarity-determining regions (CDRs) extended completely into the cofactor pockets and would likely impede HBGA engagement. The atomic level information for these nanobodies and their corresponding binding sites provide a valuable template for the discovery of additional "designer" nanobodies. These next-generation nanobodies would be designed to target other important genotypes and variants, while maintaining cofactor interference. Finally, our results clearly demonstrate for the first time that nanobodies directly targeting the HBGA binding site can function as potent norovirus inhibitors. IMPORTANCE Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics. We successfully developed and characterized four norovirus nanobodies that bound at the HBGA pockets. Compared with previously developed norovirus nanobodies that inhibited HBGA through disrupted particle stability, these four novel nanobodies directly inhibited HBGA engagement and interacted with HBGA binding residues. Importantly, these new nanobodies specifically target two genotypes that have caused the majority of outbreaks worldwide and consequently would have an enormous benefit if they could be further developed as norovirus therapeutics. To date, we have structurally characterized 16 different GII nanobody complexes, a number of which block HBGA binding. These structural data could be used to design multivalent nanobody constructs with improved inhibition properties.

Minimal Antigenic Evolution after a Decade of Norovirus GII.4 Sydney_2012 Circulation in Humans.

Norovirus is a major human pathogen that can cause severe gastroenteritis in vulnerable populations. The extensive viral diversity presented by human noroviruses constitutes a major roadblock for the development of effective vaccines. In addition to the large number of genotypes, antigenically distinct variants of GII.4 noroviruses have chronologically emerged over the last 3 decades. The last variant to emerge, Sydney_2012, has been circulating at high incidence worldwide for over a decade. We analyzed 1449 capsid sequences from GII.4 Sydney_2012 viruses to determine genetic changes indicative of antigenic diversification. Phylogenetic analyses show that Sydney_2012 viruses scattered within the tree topology with no single cluster dominating during a given year or geographical location. Fourteen residues presented high variability, 7 of which mapped to 4 antigenic sites. Notably, ~52% of viruses presented mutations at 2 or more antigenic sites. Mutational patterns showed that residues 297 and 372, which map to antigenic site A, changed over time. Virus-like particles (VLPs) developed from wild-type Sydney_2012 viruses and engineered to display all mutations detected at antigenic sites were tested against polyclonal sera and monoclonal antibodies raised against Sydney_2012 and Farmington_Hills_2002 VLPs. Minimal changes in reactivity were detected with polyclonal sera and only 4 MAbs lost binding, with all mapping to antigenic site A. Notably, reversion of residues from Sydney_2012 reconstituted epitopes from ancestral GII.4 variants. Overall, this study demonstrates that, despite circulating for over a decade, Sydney_2012 viruses present minimal antigenic diversification and provides novel insights on the diversification of GII.4 noroviruses that could inform vaccine design. IMPORTANCE GII.4 noroviruses are the major cause of acute gastroenteritis in all age groups. This predominance has been attributed to the continued emergence of phylogenetically discrete variants that escape immune responses to previous infections. The last GII.4 variant to emerge, Sydney_2012, has been circulating at high incidence for over a decade, raising the question of whether this variant is undergoing antigenic diversification without presenting a major distinction at the phylogenetic level. Sequence analyses that include >1400 capsid sequences from GII.4 Sydney_2012 showed changes in 4 out of the 6 major antigenic sites. Notably, while changes were detected in one of the most immunodominant sites over time, these resulted in minimal changes in the antigenic profile of these viruses. This study provides new insights on the mechanism governing the antigenic diversification of GII.4 norovirus that could help in the development of cross-protective vaccines to human noroviruses.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Chimeric virus-like particles of human norovirus constructed by structure-guided epitope grafting elicit cross-reactive immunity against both GI.1 and GII.4 genotypes.

IMPORTANCE: Human norovirus (HuNoV) is highly infectious and can result in severe illnesses in the elderly and children. So far, there is no effective antiviral drug to treat HuNoV infection, and thus, the development of HuNoV vaccines is urgent. However, NoV evolves rapidly, and currently, at least 10 genogroups with numerous genotypes have been found. The genetic diversity of NoV and the lack of cross-protection between different genotypes pose challenges to the development of broadly protective vaccines. In this study, guided by structural alignment between GI.1 and GII.4 HuNoV VP1 proteins, several chimeric-type virus-like particles (VLPs) were designed through surface-exposed loop grafting. Mouse immunization studies show that two of the designed chimeric VLPs induced cross-immunity against both GI.1 and GII.4 HuNoVs. To our knowledge, this is the first designed chimeric VLPs that can induce cross-immune activities across different genogroups of HuNoV, which provides valuable strategies for the development of cross-reactive HuNoV vaccines.