We investigated sequential episodes of acute norovirus gastroenteritis in a young child within an 11-month period. The infections were caused by 2 distinct genotypes (GII.4 and GII.6). Failure to ...achieve cross-protective immunity was linked to absence of an enduring and cross-reactive mucosal immune response, a critical consideration for vaccine design.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
GII.4 noroviruses are a major cause of acute gastroenteritis. Their dominance has been partially explained by the continuous emergence of antigenically distinct variants. To gain insights into the ...mechanisms of viral emergence and population dynamics of GII.4 noroviruses, we performed large-scale genomics, structural, and mutational analyses of the viral capsid protein (VP1). GII.4 noroviruses exhibited a periodic replacement of predominant variants with accumulation of amino acid substitutions. Genomic analyses revealed (i) a large proportion (87%) of conserved residues; (ii) variable residues that map on the previously determined antigenic sites; and (iii) variable residues that map outside the antigenic sites. Residues in the third pattern category formed motifs on the surface of VP1, which suggested extensions of previously predicted and new uncharacterized antigenic sites. The role of two motifs (C and G) in the antigenic makeup of the GII.4 capsid protein was confirmed with monoclonal antibodies and carbohydrate blocking assays. Amino acid profiles from antigenic sites (A, C, D, E, and G) correlated with the circulation patterns of GII.4 variants, with three of them (A, C, and G) containing residues (352, 357, 368, and 378) linked with the diversifying selective pressure on the emergence of new GII.4 variants. Notably, the emergence of each variant was followed by stochastic diversification with minimal changes that did not progress toward the next variant. This report provides a methodological framework for antigenic characterization of viruses and expands our understanding of the dynamics of GII.4 noroviruses and could facilitate the design of cross-reactive vaccines.
Noroviruses are an important cause of viral gastroenteritis around the world. An obstacle delaying the development of norovirus vaccines is inadequate understanding of the role of norovirus diversity in immunity. Using a population genomics approach, we identified new residues on the viral capsid protein (VP1) from GII.4 noroviruses, the predominant genotype, that appear to be involved in the emergence and antigenic topology of GII.4 variants. Careful monitoring of the substitutions in those residues involved in the diversification and emergence of new viruses could help in the early detection of future novel variants with pandemic potential. Therefore, this novel information on the antigenic diversification could facilitate GII.4 norovirus vaccine design.
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.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Noroviruses are major pathogens associated with acute gastroenteritis. They are diverse viruses, with at least six genogroups (GI-GVI) and multiple genotypes defined by differences in the major ...capsid protein, VP1. This diversity has challenged the development of broadly cross-reactive vaccines as well as efficient detection methods. Here, we report the characterization of a broadly cross-reactive monoclonal antibody (MAb) raised against the capsid protein of a GII.3 norovirus strain. The MAb reacted with VLPs and denatured VP1 protein from GI, GII, GIV and GV noroviruses, and mapped to a linear epitope located in the inner shell domain. An alignment of all available VP1 sequences showed that the putative epitope (residues 52-56) is highly conserved across the genus Norovirus. This broadly cross-reactive MAb thus constitutes a valuable reagent for the diagnosis and study of these diverse viruses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that ...rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.
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•Rotaviruses and noroviruses are shed in stool as viral clusters inside vesicles•Vesicles containing virus clusters remain intact during fecal-oral transmission•Vesicles achieve a high MOI and induce severe disease•Vesicle-cloaked viral clusters are more virulent units than free viral particles
Freely disseminating standalone viral particles are considered the optimal agents for spreading infection. Santiana et al. discover that enteric viruses are also shed in feces as viral clusters cloaked in vesicles. These virus-containing vesicles are a more potent infectious form that enhances the MOI and disease severity.
Noroviruses are global agents of acute gastroenteritis, but the development of control strategies has been hampered by the absence of a robust animal model. Studies in chimpanzees have played a key ...role in the characterization of several fastidious hepatitis viruses, and we investigated the feasibility of such studies for the noroviruses. Seronegative chimpanzees inoculated i.v. with the human norovirus strain Norwalk virus (NV) did not show clinical signs of gastroenteritis, but the onset and duration of virus shedding in stool and serum antibody responses were similar to that observed in humans. NV RNA was detected in intestinal and liver biopsies concurrent with the detection of viral shedding in stool, and NV antigen expression was observed in cells of the small intestinal lamina propria. Two infected chimpanzees rechallenged 4, 10, or 24 mo later with NV were resistant to reinfection, and the presence of NV-specific serum antibodies correlated with protection. We evaluated the immunogenicity and efficacy of virus-like particles (VLPs) derived from NV (genogroup I, GI) and MD145 (genogroup II, GII) noroviruses as vaccines. Chimpanzees vaccinated intramuscularly with GI VLPs were protected from NV infection when challenged 2 and 18 mo after vaccination, whereas chimpanzees that received GII VLPs vaccine or a placebo were not. This study established the chimpanzee as a viable animal model for the study of norovirus replication and immunity, and shows that NV VLP vaccines could induce protective homologous immunity even after extended periods of time.
Human noroviruses are the major viral cause of acute gastroenteritis around the world. Although norovirus symptoms are in most cases mild and self-limited, severe and prolonged symptoms can occur in ...the elderly and in immunocompromised individuals. Thus, there is a great need for the development of specific therapeutics that can help mitigate infection. In this study, we sought to characterize a panel of human monoclonal antibodies (mAbs; NORO-123, -115, -273A, -263, -315B, and -250B) that showed carbohydrate blocking activity against the current pandemic variant, GII.4 Sydney 2012. All antibodies tested showed potent neutralization against GII.4 Sydney virus in human intestinal enteroid culture. While all mAbs recognized only GII.4 viruses, they exhibited differential binding patterns against a panel of virus-like particles (VLPs) representing major and minor GII.4 variants spanning twenty-five years. Using mutant VLPs, we mapped five of the mAbs to variable antigenic sites A (NORO-123, -263, -315B, and -250B) or C (NORO-115) on the major capsid protein. Those mapping to the antigenic site A showed blocking activity against multiple variants dating back to 1987, with one mAb (NORO-123) showing reactivity to all variants tested. NORO-115, which maps to antigenic site C, showed reactivity against multiple variants due to the low susceptibility for mutations presented by naturally-occurring variants at the proposed binding site. Notably, we show that cross-blocking and neutralizing antibodies can be elicited against variable antigenic sites. These data provide new insights into norovirus immunity and suggest potential for the development of cross-protective vaccines and therapeutics.
Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant ...monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human sapovirus is a causative agent of acute gastroenteritis in all age groups. The use of full-length viral genomes has proven beneficial to investigate evolutionary dynamics and transmission ...chains. In this study, we developed a full-length genome sequencing platform for human sapovirus and sequenced the oldest available strains (collected in the 1970s) to analyse diversification of sapoviruses. Sequence analyses from five major genotypes (GI.1, GI.2, GII.1, GII.3, and GIV.1) showed limited intra-genotypic diversification for over 20-40 years. The accumulation of amino acid mutations in VP1 was detected for GI.2 and GIV.1 viruses, while having a similar rate of nucleotide evolution to the other genotypes. Differences in the phylogenetic clustering were detected between RdRp and VP1 sequences of our archival strains as well as other reported putative recombinants. However, the lack of the parental strains and differences in diversification among genomic regions suggest that discrepancies in the phylogenetic clustering of sapoviruses could be explained, not only by recombination, but also by disparate nucleotide substitution patterns between RdRp and VP1 sequences. Together, this study shows that, contrary to noroviruses, sapoviruses present limited diversification by means of intra-genotype variation and recombination.
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.
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.
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