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.

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.

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.

Cross-reactive neutralizing human monoclonal antibodies mapping to variable antigenic sites on the norovirus major capsid protein.

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.

Global and regional circulation trends of norovirus genotypes and recombinants, 1995-2019: A comprehensive review of sequences from public databases.

Human noroviruses are the leading global cause of viral gastroenteritis. Attempts at developing effective vaccines and treatments against norovirus disease have been stymied by the extreme genetic diversity and rapid geographic distribution of these viruses. The emergence and replacement of predominantly circulating norovirus genotypes has primarily been attributed to mutations on the VP1 capsid protein leading to genetic drift, and more recently to recombination events between the ORF1/ORF2 junction. However, large-scale research into the historical and geographic distribution of recombinant norovirus strains has been limited in the literature. We performed a comprehensive historical analysis on 30,810 human norovirus sequences submitted to public databases between the years 1995 and 2019. During this time, 37 capsid genotypes and 56 polymerase types were detected across 90 different countries, and 97 unique recombinant genomes were also identified. GII.4, both capsid and polymerase, was the predominately circulating type worldwide for the majority of this time span, save for a brief swell of GII.17 and GII.2 capsid genotypes and a near-total eclipse by GII.P16, GII.P21 and GII.P31 beginning in 2013. Interestingly, an analysis of 4067 recombinants found that 50.2% (N = 2039) of all recorded sequences belonged to three recently emerged recombinant strains: GII.2[P16], GII.4[P31], and GII.4[P16]. This analysis should provide an important historical foundation for future studies that evaluate the emergence and distribution of noroviruses, as well as the design of cross-protective vaccines.

Dynamic immunodominance hierarchy of neutralizing antibody responses to evolving GII.4 noroviruses.

A paradigm of RNA viruses is their ability to mutate and escape from herd immunity. Because antibody responses are a major effector for viral immunity, antigenic sites are usually under strong diversifying pressure. Here, we use norovirus as a model to study mechanisms of antigenic diversification of non-enveloped, fast-evolving RNA viruses. We comprehensively characterize all variable antigenic sites involved in virus neutralization and find that single neutralizing monoclonal antibodies (mAbs) map to multiple antigenic sites of GII.4 norovirus. Interactions of multiple epitopes on the viral capsid surface provide a broad mAb-binding repertoire with a remarkable difference in the mAb-binding profiles and immunodominance hierarchy for two distantly related GII.4 variants. Time-ordered mutant viruses confirm a progressive change of antibody immunodominance along with point mutations during the process of norovirus evolution. Thus, in addition to point mutations, switches in immunodominance that redirect immune responses could facilitate immune escape in RNA viruses.

Antigenic cartography reveals complexities of genetic determinants that lead to antigenic differences among pandemic GII.4 noroviruses.

Noroviruses are the predominant cause of acute gastroenteritis, with a single genotype (GII.4) responsible for the majority of infections. This prevalence is characterized by the periodic emergence of new variants that present substitutions at antigenic sites of the major structural protein (VP1), facilitating escape from herd immunity. Notably, the contribution of intravariant mutations to changes in antigenic properties is unknown. We performed a comprehensive antigenic analysis on a virus-like particle panel representing major chronological GII.4 variants to investigate diversification at the inter- and intravariant level. Immunoassays, neutralization data, and cartography analyses showed antigenic similarities between phylogenetically related variants, with major switches to antigenic properties observed over the evolution of GII.4 variants. Genetic analysis indicated that multiple coevolving amino acid changes-primarily at antigenic sites-are associated with the antigenic diversification of GII.4 variants. These data highlight complexities of the genetic determinants and provide a framework for the antigenic characterization of emerging GII.4 noroviruses.

Functional and structural characterization of the chikungunya virus translational recoding signals.

Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations. One such emerging virus of public health concern is chikungunya virus (CHIKV), a member of the Togaviridae family, genus Alphavirus CHIKV pathogenesis is predominately characterized by acute febrile symptoms and severe arthralgia, which can persist in the host long after viral clearance. CHIKV has also been implicated in cases of acute encephalomyelitis, and its vertical transmission has been reported. Currently, no FDA-approved treatments exist for this virus. Recoding elements help expand the coding capacity in many viruses and therefore represent potential therapeutic targets in antiviral treatments. Here, we report the molecular and structural characterization of two CHIKV translational recoding signals: a termination codon read-through (TCR) element located between the nonstructural protein 3 and 4 genes and a programmed -1 ribosomal frameshift (-1 PRF) signal located toward the 3' end of the CHIKV 6K gene. Using Dual-Luciferase and immunoblot assays in HEK293T and U87MG mammalian cell lines, we validated and genetically characterized efficient TCR and -1 PRF. Analyses of RNA chemical modification data with selective 2'-hydroxyl acylation and primer extension (SHAPE) assays revealed that CHIKV -1 PRF is stimulated by a tightly structured, triple-stem hairpin element, consistent with previous observations in alphaviruses, and that the TCR signal is composed of a single large multibulged hairpin element. These findings illuminate the roles of RNA structure in translational recoding and provide critical information relevant for design of live-attenuated vaccines against CHIKV and related viruses.

Ablation of Programmed -1 Ribosomal Frameshifting in Venezuelan Equine Encephalitis Virus Results in Attenuated Neuropathogenicity.

The alphaviruses Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are arthropod-borne positive-strand RNA viruses that are capable of causing acute and fatal encephalitis in many mammals, including humans. VEEV was weaponized during the Cold War and is recognized as a select agent. Currently, there are no FDA-approved vaccines or therapeutics for these viruses. The spread of VEEV and other members of this family due to climate change-mediated vector range expansion underscores the need for research aimed at developing medical countermeasures. These viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to synthesize the viral trans-frame (TF) protein, which has previously been shown to be important for neuropathogenesis in the related Sindbis virus. Here, the alphavirus -1 PRF signals were characterized, revealing novel -1 PRF stimulatory structures. -1 PRF attenuation mildly affected the kinetics of VEEV accumulation in cultured cells but strongly inhibited its pathogenesis in an aerosol infection mouse model. Importantly, the decreased viral titers in the brains of mice infected with the mutant virus suggest that the alphavirus TF protein is important for passage through the blood-brain barrier and/or for neuroinvasiveness. These findings suggest a novel approach to the development of safe and effective live attenuated vaccines directed against VEEV and perhaps other closely related -1 PRF-utilizing viruses. IMPORTANCE: Venezuelan equine encephalitis virus (VEEV) is a select agent that has been weaponized. This arthropod-borne positive-strand RNA virus causes acute and fatal encephalitis in many mammals, including humans. There is no vaccine or other approved therapeutic. VEEV and related alphaviruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to synthesize the viral trans-frame (TF) protein, which is important for neuropathogenesis. -1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that the TF protein is critical for neurological disease. These findings suggest a new approach to the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.