Histo-blood Group Antigen Binding Sites Research Articles

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.

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.

Long Term Norovirus Infection in a Patient with Severe Common Variable Immunodeficiency.

Norovirus is the most common cause of acute non-bacterial gastroenteritis. Immunocompromised patients can become chronically infected, with or without symptoms. In Europe, common variable immunodeficiency (CVID) is one of the most common inborn errors of immunity. A potentially severe complication is CVID-associated enteropathy, a disorder with similar histopathology to celiac disease. Studies suggest that chronic norovirus infection may be a contributor to CVID enteropathy, and that the antiviral drug ribavirin can be effective against norovirus. Here, a patient with CVID-like disease with combined B- and T-cell deficiency, had chronic norovirus infection and enteropathy. The patient was routinely administered subcutaneous and intravenous immunoglobulin replacement therapy (SCIg and IVIg). The patient was also administered ribavirin for ~7.5 months to clear the infection. Stool samples (collected 2013–2016) and archived paraffin embedded duodenal biopsies were screened for norovirus by qPCR, confirming a chronic infection. Norovirus genotyping was done in 25 stool samples. For evolutionary analysis, the capsid (VP1) and polymerase (RdRp) genes were sequenced in 10 and 12 stool samples, respectively, collected before, during, and after ribavirin treatment. Secretor phenotyping was done in saliva, and serum was analyzed for histo-blood group antigen (HBGA) blocking titers. The chronic norovirus strain formed a unique variant subcluster, with GII.4 Den Haag [P4] variant, circulating around 2009, as the most recent common ancestor. This corresponded to the documented debut of symptoms. The patient was a secretor and had HBGA blocking titers associated with protection in immunocompetent individuals. Several unique amino acid substitutions were detected in immunodominant epitopes of VP1. However, HBGA binding sites were conserved. Ribavirin failed in treating the infection and no clear association between ribavirin-levels and quantity of norovirus shedding was observed. In conclusion, long term infection with norovirus in a patient with severe CVID led to the evolution of a unique norovirus strain with amino acid substitutions in immunodominant epitopes, but conservation within HBGA binding pockets. Regularly administered SCIg, IVIg, and ~7.5-month ribavirin treatment failed to clear the infection.

Identification of Human Norovirus GII.3 Blockade Antibody Epitopes.

Human noroviruses are a common pathogen causing acute gastroenteritis worldwide. Among all norovirus genotypes, GII.3 is particularly prevalent in the pediatric population. Here we report the identification of two distinct blockade antibody epitopes on the GII.3 capsid. We generated a panel of monoclonal antibodies (mAbs) from mice immunized with virus-like particle (VLP) of a GII.3 cluster 3 strain. Two of these mAbs, namely 8C7 and 8D1, specifically bound the parental GII.3 VLP but not VLPs of GII.4, GII.17, or GI.1. In addition, 8C7 and 8D1 efficiently blocked GII.3 VLP binding with its ligand, histo-blood group antigens (HBGA). These data demonstrate that 8C7 and 8D1 are GII.3-specific blockade antibodies. By using a series of chimeric VLPs, we mapped the epitopes of 8C7 and 8D1 to residues 385–400 and 401–420 of the VP1 capsid protein, respectively. These two blockade antibody epitopes are highly conserved among GII.3 cluster 3 strains. Structural modeling shows that the 8C7 epitope partially overlaps with the HBGA binding site (HBS) while the 8D1 epitope is spatially adjacent to HBS. These findings may enhance our understanding of the immunology and evolution of GII.3 noroviruses.

Full-genome sequences of GII.13[P21] recombinant norovirus strains from an outbreak in Changsha, China.

On 31 March 2019, 68 school students suffered from vomiting, diarrhea, and abdominal pain after participating in a group activity at a commercial park. In this outbreak, multiple norovirus genotypes were observed, including GII.2[P16], GII.17[P17], and GII.13[P21]. Further, we determined the full-genome sequences of two strains of GII.13[P21] recombinant noroviruses, which were 7434 nt long. Phylogenetic analysis based on open reading frames (ORFs) 1 and 2 revealed that these recombinants were related to stains of different genotypes from different countries. The full genome nucleotide sequences of the two isolates were 97.0% and 98.0% identical to those of strains from London and Thailand, respectively. Simplot analysis revealed the presence of a break point at nt 5059 in the ORF1 region. The histo-blood group antigen binding sites were conserved in both recombinant viruses. Our findings not only provide valuable genetic information about a recombinant norovirus but also contribute to our general understanding of the evolution, genetic diversity, and distribution of noroviruses.

A post-translational modification of human Norovirus capsid protein attenuates glycan binding

Attachment of human noroviruses to histo blood group antigens (HBGAs) is essential for infection, but how this binding event promotes the infection of host cells is unknown. Here, we employ protein NMR experiments supported by mass spectrometry and crystallography to study HBGA binding to the P-domain of a prevalent virus strain (GII.4). We report a highly selective transformation of asparagine 373, located in an antigenic loop adjoining the HBGA binding site, into an iso-aspartate residue. This spontaneous post-translational modification (PTM) proceeds with an estimated half-life of a few days at physiological temperatures, independent of the presence of HBGAs but dramatically affecting HBGA recognition. Sequence conservation and the surface-exposed position of this PTM suggest an important role in infection and immune recognition for many norovirus strains.

Human norovirus inhibition by a human milk oligosaccharide

Human noroviruses are the leading cause of outbreaks of acute gastroenteritis. Norovirus interactions with histo-blood group antigens (HBGAs) are known to be important for an infection. In this study, we identified the HBGA binding pocket for an emerging GII genotype 17 (GII.17) variant using X-ray crystallography. The GII.17 variant bound the HBGA with an equivalent set of residues as the leading pandemic GII.4 variants. These structural data highlights the conserved nature of HBGA binding site between prevalent GII noroviruses. Noroviruses also interact with human milk oligosaccharides (HMOs), which mimic HBGAs and may function as receptor decoys. We previously showed that HMOs inhibited the binding of rarely detected GII.10 norovirus to HBGAs. We now found that an HMO, 2′-fucosyllactose (2′FL), additionally blocked both the GI.1 and GII.17 noroviruses from binding to HBGAs. Together, these findings provide evidence that 2′FL might function as a broadly reactive antiviral against multiple norovirus genogroups.

Molecular Evolution of the Capsid Gene in Norovirus Genogroup I.

We studied the molecular evolution of the capsid gene in all genotypes (genotypes 1–9) of human norovirus (NoV) genogroup I. The evolutionary time scale and rate were estimated by the Bayesian Markov chain Monte Carlo (MCMC) method. We also performed selective pressure analysis and B-cell linear epitope prediction in the deduced NoV GI capsid protein. Furthermore, we analysed the effective population size of the virus using Bayesian skyline plot (BSP) analysis. A phylogenetic tree by MCMC showed that NoV GI diverged from the common ancestor of NoV GII, GIII, and GIV approximately 2,800 years ago with rapid evolution (about 10−3 substitutions/site/year). Some positive selection sites and over 400 negative selection sites were estimated in the deduced capsid protein. Many epitopes were estimated in the deduced virus capsid proteins. An epitope of GI.1 may be associated with histo-blood group antigen binding sites (Ser377, Pro378, and Ser380). Moreover, BSP suggested that the adaptation of NoV GI strains to humans was affected by natural selection. The results suggested that NoV GI strains evolved rapidly and date back to many years ago. Additionally, the virus may have undergone locally affected natural selection in the host resulting in its adaptation to humans.

Structural analysis of a rabbit hemorrhagic disease virus binding to histo-blood group antigens.

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family (Lagovirus genus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) recently has emerged, and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae, Norovirus genus), RHDV binds histo-blood group antigens (HBGAs), and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains also could bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains. We identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system also could work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.

Characterization of Emerging GII.g/GII.12 Noroviruses from a Gastroenteritis Outbreak in the United States in 2010

Norovirus is a major cause of acute gastroenteritis in humans. A norovirus outbreak occurred in Ohio in January 2010. Stool and saliva samples were obtained from six infected individuals. The full-length genomes of two representative strains (HS206 and HS210) were characterized. They belonged to GII.12 in the capsid but GII.g in the RNA polymerase region. Interestingly, an immunocompetent 2-year-old male shed virus for up to 30 days, as detected by real-time reverse transcription (RT)-PCR. Histo-blood group antigen (HBGA) typing of saliva showed that the norovirus strains infected various types of secretor-positive individuals (types A, B, and O). The viruslike particles of strain HS206 did not bind substantially to A/B/O antigens by synthetic HBGA binding, hemagglutination, or saliva binding assays. These results suggest that infection by this strain may not be A/B/O antigen dependent or that in vitro binding patterns do not always accurately reflect in vivo HBGA usage. This is different from the HBGA binding pattern of the previously reported GII.12/Aichi76 strain. Structural analysis of the predicted capsid of these GII.12 strains revealed two amino acid mismatches located near the HBGA binding sites. Four gnotobiotic pigs were inoculated orally with HS206 (6 × 10(10) genomic equivalents [GE]/pig). Virus shedding began at postinoculation days (PID) 1 to 3 and continued up to PID 16 (1 × 10(5) to 2 × 10(7) GE/ml). Gastroenteritis cases caused by GII.12 noroviruses have been recently reported worldwide. We observed that this emerging GII.12 norovirus infected humans regardless of A/B/O blood type. The infection of pigs by strain HS206 suggests that interspecies transmission of this strain is possible under experimental conditions.

Targeting Norovirus Infection—Multivalent Entry Inhibitor Design Based on NMR Experiments

Noroviruses attach to their host cells through histo blood group antigens (HBGAs), and compounds that interfere with this interaction are likely to be of therapeutic or diagnostic interest. It is shown that NMR binding studies can simultaneously identify and differentiate the site for binding HBGA ligands and complementary ligands from a large compound library, thereby facilitating the design of potent heterobifunctional ligands. Saturation transfer difference (STD) NMR experiments, spin-lock filtered NMR experiments, and interligand NOE (ILOE) experiments in the presence of virus-like particles (VLPs), identified compounds that bind to the HBGA binding site of human norovirus. Based on these data two multivalent prototype entry-inhibitors against norovirus infection were synthesized. A surface plasmon resonance based inhibition assay showed avidity gains of 1000 and one million fold over a millimolar univalent ligand. This suggests that further rational design of multivalent inhibitors based on our strategy will identify potent entry-inhibitors against norovirus infections.