Infant antibody and B-cell responses following confirmed pediatric GII.17 norovirus infections functionally distinguish GII.17 genetic clusters.

Genogroup II (GII) noroviruses are a major cause of diarrheal disease burden in children in both high- and low-income countries. GII.17 noroviruses are composed of distinct genetic clusters (I, II, IIIa, and IIIb) and have shown potential for replacing historically more prevalent GII.4 strains, but the serological basis for GII.17 antigenic diversity has not been studied in children. Utilizing samples from a birth cohort, we investigated antibody and B-cell responses to GII.17 cluster variants in confirmed GII.17 infections in young children as well as demonstrated that the distinct genetic clusters co-circulate. Polyclonal serum antibodies bound multiple clusters but showed cluster-specific blockade activity in a surrogate virus neutralization assay. Antibodies secreted by immortalized memory B cells (MBCs) from an infant GII.17 case were highly specific to GII.17 and exhibited blockade activity against this genotype. We isolated an MBC-derived GII.17-specific Immunoglobulin A (IgA) monoclonal antibody called NVA.1 that potently and selectively blocked GII.17 cluster IIIb and recognized an epitope targeted in serum from cluster IIIb-infected children. These data indicate that multiple antigenically distinct GII.17 variants co-circulate in young children, suggesting retention of cluster diversity alongside potential for immune escape given the existence of antibody-defined cluster-specific epitopes elicited during infection.

Emergent variant modeling of the serological repertoire to norovirus in young children.

Human norovirus is the leading cause of acute gastroenteritis. Young children and the elderly bear the greatest burden of disease, representing more than 200,000 deaths annually. Infection prevalence peaks at younger than 2 years and is driven by novel GII.4 variants that emerge and spread globally. Using a surrogate neutralization assay, we characterize the evolution of the serological neutralizing antibody (nAb) landscape in young children as they transition between sequential GII.4 pandemic variants. Following upsurge of the replacement variant, antigenic cartography illustrates remodeling of the nAb landscape to the new variant accompanied by improved nAb titer. However, nAb relative avidity remains focused on the preceding variant. These data support immune imprinting as a mechanism of immune evasion and GII.4 virus persistence across a population. Understanding the complexities of immunity to rapidly evolving and co-circulating viral variants, like those of norovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), and dengue viruses, will fundamentally inform vaccine design for emerging pathogens.

Immune Imprinting Drives Human Norovirus Potential for Global Spread.

Understanding the complex interactions between virus and host that drive new strain evolution is key to predicting the emergence potential of variants and informing vaccine development. Under our hypothesis, future dominant human norovirus GII.4 variants with critical antigenic properties that allow them to spread are currently circulating undetected, having diverged years earlier. Through large-scale sequencing of GII.4 surveillance samples, we identified two variants with extensive divergence within domains that mediate neutralizing antibody binding. Subsequent serological characterization of these strains using temporally resolved adult and child sera suggests that neither candidate could spread globally in adults with multiple GII.4 exposures, yet young children with minimal GII.4 exposure appear susceptible. Antigenic cartography of surveillance and outbreak sera indicates that continued population exposure to GII.4 Sydney 2012 and antigenically related variants over a 6-year period resulted in a broadening of immunity to heterogeneous GII.4 variants, including those identified here. We show that the strongest antibody responses in adults exposed to GII.4 Sydney 2012 are directed to previously circulating GII.4 viruses. Our data suggest that the broadening of antibody responses compromises establishment of strong GII.4 Sydney 2012 immunity, thereby allowing the continued persistence of GII.4 Sydney 2012 and modulating the cycle of norovirus GII.4 variant replacement. Our results indicate a cycle of norovirus GII.4 variant replacement dependent upon population immunity. Young children are susceptible to divergent variants; therefore, emergence of these strains worldwide is driven proximally by changes in adult serological immunity and distally by viral evolution that confers fitness in the context of immunity. IMPORTANCE In our model, preepidemic human norovirus variants harbor genetic diversification that translates into novel antigenic features without compromising viral fitness. Through surveillance, we identified two viruses fitting this profile, forming long branches on a phylogenetic tree. Neither evades current adult immunity, yet young children are likely susceptible. By comparing serological responses, we demonstrate that population immunity varies by age/exposure, impacting predicted susceptibility to variants. Repeat exposure to antigenically similar variants broadens antibody responses, providing immunological coverage of diverse variants but compromising response to the infecting variant, allowing continued circulation. These data indicate norovirus GII.4 variant replacement is driven distally by virus evolution and proximally by immunity in adults.

Norovirus Infection in Young Nicaraguan Children Induces Durable and Genotype-Specific Antibody Immunity.

There are significant challenges to the development of a pediatric norovirus vaccine, mainly due to the antigenic diversity among strains infecting young children. Characterizing human norovirus serotypes and understanding norovirus immunity in naïve children would provide key information for designing rational vaccine platforms. In this study, 26 Nicaraguan children experiencing their first norovirus acute gastroenteritis (AGE) episode during the first 18 months of life were investigated. We used a surrogate neutralization assay that measured antibodies blocking the binding of 13 different norovirus virus-like particles (VLPs) to histo-blood group antigens (HBGAs) in pre- and post-infection sera. To assess for asymptomatic norovirus infections, stools from asymptomatic children were collected monthly, screened for norovirus by RT-qPCR and genotyped by sequencing. Seroconversion of an HBGA-blocking antibody matched the infecting genotype in 25 (96%) of the 26 children. A subset of 13 (50%) and 4 (15%) of the 26 children experienced monotypic GII and GI seroconversion, respectively, strongly suggesting a type-specific response in naïve children, and 9 (35%) showed multitypic seroconversion. The most frequent pairing in multitypic seroconversion (8/12) were GII.4 Sydney and GII.12 noroviruses, both co-circulating at the time. Blocking antibody titers to these two genotypes did not correlate with each other, suggesting multiple exposure rather than cross-reactivity between genotypes. In addition, GII titers remained consistent for at least 19 months post-infection, demonstrating durable immunity. In conclusion, the first natural norovirus gastroenteritis episodes in these young children were dominated by a limited number of genotypes and induced responses of antibodies blocking binding of norovirus VLPs in a genotype-specific manner, suggesting that an effective pediatric norovirus vaccine likely needs to be multivalent and include globally dominant genotypes. The duration of protection from natural infections provides optimism for pediatric norovirus vaccines administered early in life.

Preexisting Heterotypic Ligand-blocking Antibody Does Not Protect Against Genogroup II Norovirus Episodes in Young Children.

A birth cohort design was used to understand whether heterotypic ligand-blocking norovirus antibodies provide cross-protection within the GII genogroup. We found that almost one-half of children who experienced a norovirus GII episode had preexisting antibodies heterotypic to the infecting genotype; therefore, these antibodies did not provide cross-protection.

Antigenic Site Immunodominance Redirection Following Repeat Variant Exposure.

Human norovirus is a leading cause of acute gastroenteritis, driven by antigenic variants within the GII.4 genotype. Antibody responses to GII.4 vaccination in adults are shaped by immune memory. How children without extensive immune memory will respond to GII.4 vaccination has not been reported. Here, we characterized the GII.4 neutralizing antibody (nAb) landscape following natural infection using a surrogate assay and antigenic site chimera virus-like particles. We demonstrate that the nAb landscape changes with age and virus exposure. Among sites A, C, and G, nAbs from first infections are focused on sites A and C. As immunity develops with age/exposure, site A is supplemented with antibodies that bridge site A to sites C and G. Cross-site nAbs continue to develop into adulthood, accompanied by an increase in nAb to site G. Continued exposure to GII.4 2012 Sydney correlated with a shift to co-dominance of sites A and G. Furthermore, site G nAbs correlated with the broadening of nAb titer across antigenically divergent variants. These data describe fundamental steps in the development of immunity to GII.4 over a lifetime, and illustrate how the antigenicity of one pandemic variant could influence the pandemic potential of another variant through the redirection of immunodominant epitopes.

Norovirus-Specific CD8+ T Cell Responses in Human Blood and Tissues.

BACKGROUND & AIMS: Noroviruses (NoVs) are the leading cause of acute gastroenteritis worldwide and are associated with significant morbidity and mortality. Moreover, an asymptomatic carrier state can persist following acute infection, promoting NoV spread and evolution. Thus, defining immune correlates of NoV protection and persistence is needed to guide the development of future vaccines and limit viral spread. Whereas antibody responses following NoV infection or vaccination have been studied extensively, cellular immunity has received less attention. Data from the mouse NoV model suggest that T cells are critical for preventing persistence and achieving viral clearance, but little is known about NoV-specific T-cell immunity in humans, particularly at mucosal sites. METHODS: We screened peripheral blood mononuclear cells from 3 volunteers with an overlapping NoV peptide library. We then used HLA-peptide tetramers to track virus-specific CD8+ T cells in peripheral, lymphoid, and intestinal tissues. Tetramer+ cells were further characterized using markers for cellular trafficking, exhaustion, cytotoxicity, and proliferation. RESULTS: We defined 7 HLA-restricted immunodominant class I epitopes that were highly conserved across pandemic strains from genogroup II.4. NoV-specific CD8+ T cells with central, effector, or tissue-resident memory phenotypes were present at all sites and were especially abundant in the intestinal lamina propria. The properties and differentiation states of tetramer+ cells varied across donors and epitopes. CONCLUSIONS: Our findings are an important step toward defining the breadth, distribution, and properties of human NoV T-cell immunity. Moreover, the molecular tools we have developed can be used to evaluate future vaccines and engineer novel cellular therapeutics.

Disulfide stabilization of human norovirus GI.1 virus-like particles focuses immune response toward blockade epitopes.

Human noroviruses are non-enveloped, single-strand RNA viruses that cause pandemic outbreaks of acute gastroenteritis. A bivalent vaccine containing GI.1 and GII.4 virus-like particles (VLPs) has been shown to be safe and highly immunogenic, but its efficacy and durability have been limited. Here, we show that norovirus GI.1 VLPs are unstable and contain a substantial fraction of dissociated VLP components. Broadly reactive, non-neutralizing antibodies isolated from vaccinated donors bound to the dissociated components, but not to the intact VLPs. Engineering of interprotomer disulfide bonds within the shell domain prevented disassembly of the VLPs, while preserving antibody accessibility to blockade epitopes. Without adjuvant, mice immunized with stabilized GI.1 VLPs developed faster blockade antibody titers compared to immunization with wild-type GI.1 VLPs. In addition, immunization with stabilized particles focused immune responses toward surface-exposed epitopes and away from occluded epitopes. Overall, disulfide-stabilized norovirus GI.1 VLPs elicited improved responses over the non-disulfide-stabilized version, suggesting their promise as candidate vaccines.

Preadaptation of pandemic GII.4 noroviruses in unsampled virus reservoirs years before emergence.

The control of re-occurring pandemic pathogens requires understanding the origins of new pandemic variants and the factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have hypothesized that new GII.4 pandemic viruses arise when previously circulating pandemic or pre-pandemic variants undergo substitutions in antigenic regions that enable evasion of host population immunity, as described by conventional models of antigenic drift. In contrast, we show here that the acquisition of new genetic and antigenic characteristics cannot be the proximal driver of new pandemics. Pandemic GII.4 viruses diversify and spread over wide geographical areas over several years prior to simultaneous pandemic emergence of multiple lineages, indicating that the necessary sequence changes must have occurred before diversification, years prior to pandemic emergence. We confirm this result through serological assays of reconstructed ancestral virus capsids, demonstrating that by 2003, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics.

Bile Facilitates Human Norovirus Interactions with Diverse Histoblood Group Antigens, Compensating for Capsid Microvariation Observed in 2016-2017 GII.2 Strains.

Human norovirus (HuNoV) is the leading cause of global infectious acute gastroenteritis, causing ~20% of reported diarrheal episodes. Typically, GII.4 strains cause 50-70% of yearly outbreaks, and pandemic waves of disease approximately every 2-7 years due to rapid evolution. Importantly, GII.4 dominance is occasionally challenged by the sudden emergence of other GII strains, most recently by GII.2 strains which peaked in 2016-2017, dramatically increasing from 1% to 20% of total HuNoV outbreaks. To determine if viral capsid evolution may account for the sudden rise in GII.2 outbreaks, Virus Like Particles (VLPs) of two 2016-2017 GII.2 strains were compared by antigenic and histo blood group antigen (HBGA) binding profiles to the prototypic 1976 GII.2 Snow Mountain Virus (SMV) strain. Despite >50 years of GII.2 strain persistence in human populations, limited sequence diversity and antigenic differences were identified between strains. However, capsid microvariation did affect HBGA binding patterns, with contemporary strains demonstrating decreased avidity for type A saliva. Furthermore, bile salts increased GII.2 VLP avidity for HBGAs, but did not alter antigenicity. These data indicate that large changes in antigenicity or receptor binding are unlikely to explain GII.2 emergence, in contrast to the pandemic GII.4 strains, and indicate that host factors such as waning or remodeling of serum or mucosal immunity likely contributed to the surge in GII.2 prevalence.

Virus-Host Interactions Between Nonsecretors and Human Norovirus.

BACKGROUND & AIMS: Human norovirus infection is the leading cause of acute gastroenteritis. Genetic polymorphisms, mediated by the FUT2 gene (secretor enzyme), define strain susceptibility. Secretors express a diverse set of fucosylated histoblood group antigen carbohydrates (HBGA) on mucosal cells; nonsecretors (FUT2-/-) express a limited array of HBGAs. Thus, nonsecretors have less diverse norovirus strain infections, including resistance to the epidemiologically dominant GII.4 strains. Because future human norovirus vaccines will comprise GII.4 antigen and because secretor phenotype impacts GII.4 infection and immunity, nonsecretors may mimic young children immunologically in response to GII.4 vaccination, providing a needed model to study cross-protection in the context of limited pre-exposure. METHODS: By using specimens collected from the first characterized nonsecretor cohort naturally infected with GII.2 human norovirus, we evaluated the breadth of serologic immunity by surrogate neutralization assays, and cellular activation and cytokine production by flow cytometry. RESULTS: GII.2 infection resulted in broad antibody and cellular immunity activation that persisted for at least 30 days for T cells, monocytes, and dendritic cells, and for 180 days for blocking antibody. Multiple cellular lineages expressing interferon-γ and tumor necrosis factor-α dominated the response. Both T-cell and B-cell responses were cross-reactive with other GII strains, but not GI strains. To promote entry mechanisms, inclusion of bile acids was essential for GII.2 binding to nonsecretor HBGAs. CONCLUSIONS: These data support development of within-genogroup, cross-reactive antibody and T-cell immunity, key outcomes that may provide the foundation for eliciting broad immune responses after GII.4 vaccination in individuals with limited GII.4 immunity, including young children.

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.

Human Norovirus Epitope D Plasticity Allows Escape from Antibody Immunity without Loss of Capacity for Binding Cellular Ligands.

Emergent strains of human norovirus seed pandemic waves of disease. These new strains have altered ligand binding and antigenicity characteristics. Study of viral variants isolated from immunosuppressed patients with long-term norovirus infection indicates that initial virus in vivo evolution occurs at the same antigenic sites as in pandemic strains. Here, cellular ligand binding and antigenicity of two cocirculating strains isolated from a patient with long-term norovirus infection were characterized. The isolated GII.4 viruses differed from previous strains and from each other at known blockade antibody epitopes. One strain had a unique sequence in epitope D, including loss of an insertion at residue 394, corresponding to a decreased relative affinity for carbohydrate ligands. Replacement of 394 with alanine or restoration of the contemporary strain epitope D consensus sequence STT improved ligand binding relative affinity. However, monoclonal antibody blockade of binding potency was only gained for the consensus sequence, not by the alanine insertion. In-depth study of unique changes in epitope D indicated that ligand binding, but not antibody blockade of ligand binding, is maintained despite sequence diversity, allowing escape from blockade antibodies without loss of capacity for binding cellular ligands.IMPORTANCE Human norovirus causes ∼20% of all acute gastroenteritis and ∼200,000 deaths per year, primarily in young children. Most epidemic and all pandemic waves of disease over the past 30 years have been caused by type GII.4 human norovirus strains. The capsid sequence of GII.4 strains is changing over time, resulting in viruses with altered ligand and antibody binding characteristics. The carbohydrate binding pocket of these strains does not vary over time. Here, utilizing unique viral sequences, we study how residues in GII.4 epitope D balance the dual roles of variable antibody binding site and cellular ligand binding stabilization domain, demonstrating that amino acid changes in epitope D can result in loss of antibody binding without ablating ligand binding. This flexibility in epitope D likely contributes to GII.4 strain persistence by both allowing escape from antibody-mediated herd immunity and maintenance of cellular ligand binding and infectivity.

Conformational Occlusion of Blockade Antibody Epitopes, a Novel Mechanism of GII.4 Human Norovirus Immune Evasion.

Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo-evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle "breathing core." Together, these data suggest that limiting antibody access to blockade antibody epitopes may be a frequent mechanism of immune evasion for GII.4 human noroviruses. Mapping blockade antibody epitopes, the interaction between adjacent epitopes on the particle, and the breathing core that mediates antibody access to epitopes provides greater mechanistic understanding of epitope camouflage strategies utilized by human viral pathogens to evade immunity.

Antigenic Characterization of a Novel Recombinant GII.P16-GII.4 Sydney Norovirus Strain With Minor Sequence Variation Leading to Antibody Escape.

Background: Human noroviruses are the leading cause of acute gastroenteritis. Strains of the GII.4 genotype cause pandemic waves associated with viral evolution and subsequent antigenic drift and ligand-binding modulation. In November 2015, a novel GII.4 Sydney recombinant variant (GII.P16-GII.4 Sydney) emerged and replaced GII.Pe-GII.4 Sydney as the predominant cause of acute gastroenteritis in the 2016-2017 season in the United States. Methods: Virus-like particles of GII.4 2012 and GII.4 2015 were compared for ligand binding and antibody reactivity, using a surrogate neutralization assay. Results: Residue changes in the capsid between GII.4 2012 and GII.4 2015 decreased the potency of human polyclonal sera and monoclonal antibodies. A change in epitope A resulted in the complete loss of reactivity of a class of blockade antibodies and reduced levels of a second antibody class. Epitope D changes modulated monoclonal antibody potency and ligand-binding patterns. Conclusions: Substitutions in blockade antibody epitopes between GII.4 2012 and GII.4 2015 influenced antigenicity and ligand-binding properties. Although the impact of polymerases on fitness remains uncertain, antigenic variation resulting in decreased potency of antibodies to epitope A, coupled with altered ligand binding, likely contributed significantly to the spread of GII.4 2015 and its replacement of GII.4 2012 as the predominant norovirus outbreak strain.

Emergence of Novel Human Norovirus GII.17 Strains Correlates With Changes in Blockade Antibody Epitopes.

Background: Human norovirus is a significant public health burden, with >30 genotypes causing endemic levels of disease and strains from the GII.4 genotype causing serial pandemics as the virus evolves new ligand binding and antigenicity features. During 2014-2015, genotype GII.17 cluster IIIb strains emerged as the leading cause of norovirus infection in select global locations. Comparison of capsid sequences indicates that GII.17 is evolving at previously defined GII.4 antibody epitopes. Methods: Antigenicity of virus-like particles (VLPs) representative of clusters I, II, and IIIb GII.17 strains were compared by a surrogate neutralization assay based on antibody blockade of ligand binding. Results: Sera from mice immunized with a single GII.17 VLP identified antigenic shifts between each cluster of GII.17 strains. Ligand binding of GII.17 cluster IIIb VLP was blocked only by antisera from mice immunized with cluster IIIb VLPs. Exchange of residues 393-396 from GII.17.2015 into GII.17.1978 ablated ligand binding and altered antigenicity, defining an important varying epitope in GII.17. Conclusions: The capsid sequence changes in GII.17 strains result in loss of blockade antibody binding, indicating that viral evolution, specifically at residues 393-396, may have contributed to the emergence of cluster IIIb strains and the persistence of GII.17 in human populations.