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Sapoviruses, like noroviruses, are single-stranded positive-sense RNA viruses classified in the family Caliciviridae and are recognized as a causative pathogen of diarrhea in infants and the elderly. Like human norovirus, human sapovirus (HuSaV) has long been difficult to replicate in vitro. Recently, it has been reported that HuSaV can be replicated in vitro by using intestinal epithelial cells (IECs) derived from human tissues and cell lines derived from testicular and duodenal cancers. In this study, we report that multiple genotypes of HuSaV can sufficiently infect and replicate in human-induced pluripotent stem cell-derived IECs. We also show that this HuSaV replication system can be used to investigate the conditions for inactivation of HuSaV by heat and alcohol, and the effects of virus neutralization of antisera obtained by immunization with vaccine antigens, under conditions closer to the living environment. The results of this study confirm that HuSaV can also infect and replicate in human normal IECs regardless of their origin and are expected to contribute to future virological studies.

Passive administration of neutralizing antibodies (Abs) is an attractive strategy for the control of gastrointestinal infections. However, an unanswered practical concern is the need to assure the stability of sufficient amounts of orally administered neutralizing Abs against intestinal pathogens (e.g., norovirus) in the harsh environment of the gastrointestinal tract. To this end, we expressed a single-domain Ab (VHH, nanobody) against norovirus on the cell surface of Lactobacillus, a natural and beneficial commensal component of the gut microbiome. First, we used intestinal epithelial cells generated from human induced pluripotent stem cells to confirm that VHH 1E4 showed neutralizing activity against GII.17 norovirus. We then expressed VHH 1E4 as a cell-wall-anchored form in Lactobacillus paracasei BL23. Flow cytometry confirmed the expression of VHH 1E4 on the surface of lactobacilli, and L. paracasei that expressed VHH 1E4 inhibited the replication of GII.17 norovirus in vitro. We then orally administered VHH 1E4-expressing L. paracasei BL23 to germ-free BALB/c mice and confirmed the presence of lactobacilli with neutralizing activity in the intestine for at least 10 days after administration. Thus, cell-wall-anchored VHH-displaying lactobacilli are attractive oral nanobody deliver vectors for passive immunization against norovirus infection.

We investigated the molecular epidemiology of human norovirus (HuNoV) in all age groups using samples from April 2019 to March 2023, before and after the COVID-19 countermeasures were implemented. GII.2[P16] and GII.4[P31], the prevalent strains in Japan before COVID-19 countermeasures, remained prevalent during the COVID-19 pandemic, except from April to November 2020; in 2021, the prevalence of GII.2[P16] increased among children. Furthermore, there was an increase in the prevalence of GII.4[P16] after December 2022. Phylogenetic analysis of GII.P31 RdRp showed that some strains detected in 2022 belonged to a different cluster of other strains obtained during the present study period, suggesting that HuNoV strains will evolve differently even if they have the same type of RdRp. An analysis of the amino acid sequence of VP1 showed that some antigenic sites of GII.4[P16] were different from those of GII.4[P31]. The present study showed high infectivity of HuNoV despite the COVID-19 countermeasures and revealed changes in the prevalent genotypes and mutations of each genotype. In the future, we will investigate whether GII.4[P16] becomes more prevalent, providing new insights by comparing the new data with those analyzed in the present study. [ABSTRACT FROM AUTHOR]

Passive administration of neutralizing antibodies (Abs) is an attractive strategy for the control of gastrointestinal infections. However, an unanswered practical concern is the need to assure the stability of sufficient amounts of orally administered neutralizing Abs against intestinal pathogens (e.g., norovirus) in the harsh environment of the gastrointestinal tract. To this end, we expressed a single-domain Ab (VHH, nanobody) against norovirus on the cell surface of Lactobacillus, a natural and beneficial commensal component of the gut microbiome. First, we used intestinal epithelial cells generated from human induced pluripotent stem cells to confirm that VHH 1E4 showed neutralizing activity against GII.17 norovirus. We then expressed VHH 1E4 as a cell-wall–anchored form in Lactobacillus paracasei BL23. Flow cytometry confirmed the expression of VHH 1E4 on the surface of lactobacilli, and L. paracasei that expressed VHH 1E4 inhibited the replication of GII.17 norovirus in vitro. We then orally administered VHH 1E4-expressing L. paracasei BL23 to germ-free BALB/c mice and confirmed the presence of lactobacilli with neutralizing activity in the intestine for at least 10 days after administration. Thus, cell-wall-anchored VHH-displaying lactobacilli are attractive oral nanobody deliver vectors for passive immunization against norovirus infection. [ABSTRACT FROM AUTHOR]

Background: Human norovirus (HuNoV) is a major cause of enteric infectious gastroenteritis and is classified into several genotypes based on its capsid protein amino acid sequence and nucleotide sequence of the polymerase gene. Among these, GII.4 is the major genotype worldwide. Epidemiological studies have highlighted the prevalence of GII.2. Although recent advances using human tissue- and induced pluripotent stem cell (iPSC)-derived intestinal epithelial cells (IECs) have enabled in vitro replication of multiple HuNoV genotypes, GII.2 HuNoV could replicate only in tissue-derived IECs and not in iPSC-derived IECs., Methods: We investigated the factors influencing GII.2 HuNoV replication in IECs, focusing on histo-blood group antigens. We also assessed the immunogenicity of GII.2 virus-like particles (VLPs) and their ability to induce neutralizing antibodies. Antibody cross-reactivity was tested to determine whether GII.2 VLPs could neutralize other HuNoV genotypes, including GII.4, GII.3, GII.6, and GII.17., Results: Our findings indicated that GII.2 HuNoV replication in vitro requires the presence of both H and B antigens. Moreover, GII.2 VLPs generated neutralizing antibodies effective against both GII.2 and GII.4 but not against GII.3, GII.6, or GII.17. Comparatively, GII.2 and GII.17 VLPs induced broader neutralizing responses than GII.4 VLPs., Conclusions: The findings of this study suggests that GII.2 and GII.17 VLPs may be advantageous as HuNoV vaccine candidates because they elicit neutralizing antibodies against the predominant GII.4 genotype, which could be particularly beneficial for infants without prior HuNoV exposure. These insights will contribute to the development of effective HuNoV vaccines., Competing Interests: Potential conflicts of interest. Y. Y. and H. K. are the cofounders and directors of HanaVax, which provided research funding to S. S. Y. Y., H. K., and S. S. have filed a patent application related to the content of this article. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)