Histo-blood Group Antigen Blocking Research Articles

A Bivalent Human Norovirus Vaccine Induces Homotypic and Heterotypic Neutralizing Antibodies.

A GII.2 outbreak in an efficacy study of a bivalent virus-like particle norovirus vaccine, TAK-214, in healthy US adults provided an opportunity to examine GII.4 homotypic vs GII.2 heterotypic responses to vaccination and infection. Three serologic assays-virus-like particle binding, histoblood group antigen blocking, and neutralizing-were performed for each genotype. Results were highly correlated within a genotype but not between genotypes. Although the vaccine provided protection from GII.2-associated disease, little GII.2-specific neutralization occurred after vaccination. Choice of antibody assay can affect assessments of human norovirus vaccine immunogenicity.

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.

Immunogenicity of a bivalent virus-like particle norovirus vaccine in children from 1 to 8 years of age: A phase 2 randomized, double-blind study

BackgroundYoung children can suffer severe consequences of norovirus gastroenteritis. We performed a dose-finding study of a bivalent virus-like particle (VLP) vaccine candidate (TAK-214) in healthy 1–8-year-old children. MethodsIn this phase 2 study two age cohorts (1–3 and 4–8 years of age inclusive, N = 120 per cohort) of children enrolled from Finland, Panama and Colombia were initially randomized 1:1:1:1 to four groups which were further split into two equal subgroups, to receive one or two intramuscular doses of four TAK-214 formulations containing 15/15, 15/50, 50/50 or 50/150 μg of GI.1/GII.4c genotype VLPs and 0.5 mg Al(OH)3 at 28 days interval. ELISA Pan-Ig and histoblood group antigen-blocking (HBGA) antibodies against each VLP were measured on days 1, 29, 57 and 210. Parents/guardians recorded solicited local and systemic adverse events (AE) and any unsolicited or serious AEs (SAE). ResultsAll formulations were well-tolerated across both age cohorts and dosage groups with no vaccine-related SAEs reported. Solicited AEs were mostly mild-to-moderate, resolved quickly, and did not increase after the second dose. Pan-Ig and HBGA responses induced after one dose were only slightly increased by the second dose. Across dose groups at Day 29 after one dose GI.1 Pan Ig seroresponse rates (SRR) were 82–97% and 81–96% and GII.4c SRR were 79–97% and 80–91% in 1–3 and 4–8 year-olds, respectively. Respective rates were to 92–93% and 73–92% for GI.1, and 77–100% and 62–83% for GII.4c at Day 57 following two doses. HBGA responses had similar profiles. Both Pan Ig and HBGA geometric mean titers persisted above baseline up to Day 210. ConclusionsAll dosages of TAK-214 displayed acceptable reactogenicity in 1–8-year-old children and induced robust, durable immune responses after one dose which are further increased after two doses.

Broadly cross-reactive human antibodies that inhibit genogroup I and II noroviruses

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features.

The endemic GII.4 norovirus-like-particle induced-antibody lacks of cross-reactivity against the epidemic GII.17 strain.

Norovirus‐like particle (VLP) vaccine is promising against human norovirus infection. Unfortunately, genetic diversity of norovirus hindered the development of this vaccine. In this study, the immunogenicity of norovirus VLPs induced by the endemic GII.4 and the epidemic GII.17 genotypes, and the cross‐reactivity between them as well as GI.1 and GII.3 VLPs were evaluated in mice by using serum IgG and histo‐blood group antigen (HBGA) blocking antibodies as index. Results showed well immunogenicity of both GII.4 and GII.17 VLPs in mice. Serum IgG GMT (Geometric Mean Titer) were 3.63 (GII.4) and 3.88 (GII.17) respectively, and sustained to the 15th week. The HBGA blocking antibodies were 130 (GII.4) and 360 (GII.17) respectively at the end of the 4th week. Additionally, there was a dramatically statistical difference found in the cross‐reactivity within genogroup (GII.3, GII.4 and GII.17) (p < .001), and also showed similar difference between genogroups (GI.1 vs. GII.3, GII.4 and GII.17) (p < .001). Summarized the pPICZa pichi pichia expression system showed a potential to be the alternative for expression of norovirus VLPs in secretion form, and the little cross‐reactivity found between the endemic strain and the epidemic strain provides an evident for the consideration of selecting candidates of norovirus vaccine strains.

Serum antibodies against norovirus GI.1 and GII.4 in populations in central and eastern China

Objective To detect norovirus (NoV) GⅠ.1- and GⅡ.4-specific IgG, IgA and histo-blood group antigen (HBGA)-blocking antibodies in healthy populations of all age groups in China for better understanding the epidemiological features of norovirus in China from a serological point of view and providing basic data for vaccine development and clinical trial design. Methods Indirect ELISA and HBGA-blocking assay were used to detect NoV-specific IgG, IgA and HBGA-blocking antibodies in serum samples collected from healthy natural populations (n=839, aged from six months to 88 years old) in Guangzhou, Fuyang and Yantai. The results were statistically analyzed. Results The total positive rates of NoV GⅠ.1- and GⅡ.4-specific IgG antibodies were 91.9% and 93.0%. The positive rates of GⅠ.1- and GⅡ.4-specific IgA antibodies were 48.6% and 75.6%, and the titers of HBGA-blocking antibodies to GⅠ.1 and GⅡ.4 norovirus were 5.04 (95%CI: 4.63-5.49) and 18.15 (95%CI: 16.11-20.44). The positive rates of IgG and IgA antibodies generally showed an increasing trend with age. The positive rates of GⅠ.1- and GⅡ.4-specific IgG antibodies ranged from 79.2% to 100.0% and 76.7% to 100.0% in different age groups. They were 81.7% and 85.0% in the age group of 0.5-<1 year, 79.2% and 76.7% in the age group of 1-<2 years, and 98.1% and 96.3% in the age group of 12-<18 years, and maintained at 96% and 98% in the older age groups. The positive rates of GⅠ.1-specific IgA antibody ranged from 11.7% to 93.8% in different age groups and rapidly increased with age. It was 11.7% in the age group of 0.5-<1 year, and reached 93.3% in people aged 45-<60 years and 93.8% in people aged ≥60 years. The positive rates of GⅡ.4-specific IgA antibody ranged from 50.8% to 88.8% in different age groups with 50.8% in people aged 0.5-<1 year, and 86.7%-90.7% in people aged 12-<18 years and older. The titer of GⅠ.1 HBGA-blocking antibody generally increased with age. The antibody titer in populations aged 0.5-<12 years old was lower than that in those aged 18 years and above (GMT: 2.98-4.07 vs 8.21-11.62, P<0.001), and the titer in people of 12-<18 years old was lower than that in those of 45 years old and above (GMT: 5.21 vs 11.03-11.62, P<0.05). No obvious change with age was observed in the titer of GⅡ.4 HBGA-blocking antibody excepting the significant difference between populations of 2-<5 and 22-<45 years old (GMT: 26.73 vs 11.87, P<0.01). Conclusions This study revealed the characteristics of serum NoV GⅠ.1- and GⅡ.4-specific IgG, IgA and HBGA blocking antibodies in populations of different age groups in central and eastern China through analyzing their positive rates and titers and provided preliminary seroepidemiological data for the development of NoV vaccines in China. Key words: Norovirus; Serum antibody; Receptor-blocking assay

Comparison of Microneutralization and Histo-Blood Group Antigen-Blocking Assays for Functional Norovirus Antibody Detection.

The development of an in vitro cultivation system for human noroviruses allows the measurement of neutralizing antibody levels. Serum neutralizing antibody levels were determined using a GII.4/Sydney/2012-like virus in human intestinal enteroids in samples collected before and 4 weeks after administration of an investigational norovirus vaccine and were compared with those measured in histo-blood group antigen (HBGA)-blocking assays. Neutralizing antibody seroresponses were observed in 71% of 24 vaccinated adults, and antibody levels were highly correlated (r = 0.82, P < .001) with those measured by HBGA blocking. HBGA-blocking antibodies are a surrogate for neutralization in human noroviruses. NCT02475278.

Evaluation of immune effects of virus-like particles of recombinant GII. 17 norovirus

Objective To evaluate the immune effects of virus-like particles (VLPs) assembled from the capsid protein VP1 of a recombinant norovirus (NoV) GⅡ.17 genotype. Methods The recombinant NoV GⅡ.17 VP1 VLPs were purified, and then tested by SDS-PAGE and Western blot to analyze the purity. The size, morphology and diameter distribution of the recombinant VLPs were detected by transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyzer. The recombinant VP1 VLPs adsorbed by aluminium adjuvant were used to immunize BALB/c mice. Serum samples were collected after immunization. Specific antibody level and neutralizing antibody activity were evaluated with enzyme linked immunosorbent assay (ELISA) and histo-blood group antigen (HBGA)-VLP blocking test. Cross-reactivity of serum samples with GⅠ.1 and GⅡ.4 VP1 VLPs were detected. Moreover, cross-protection against GⅠ.1 and GⅡ.4 VP1 VLPs was analyzed. Results The purity of the recombinant NoV GⅡ.17 VP1 VLP was greater than 90% and specific bands were detected by Western blot. TEM images and DLS experiments showed that VLPs were 30-50 nm in size with good morphology and uniformity, indicating that the recombinant VLPs were similar to the wildtype virus. High titers of specific antibodies were detected in serum samples of the immunized mice. A certain degree of cross-reactions between serum samples and VP1 VLPs of NoV GⅠ.1 and GⅡ.4 were observed, but no cross-protection was detected. Conclusion The recombinant GⅡ. 17 VP1 VLPs in combination with aluminum adjuvant can induce higher titers of HBGA blocking antibodies in mice, suggesting that it could be used as a candidate target antigen for norovirus vaccine. Key words: Norovirus (NoV); GⅡ.17 genotype; Virus like particles (VLPs); Histo-blood group antigen (HBGA)

Development of a Surrogate Neutralization Assay for Norovirus Vaccine Evaluation at the Cellular Level.

Noroviruses (NoVs) are the main pathogens responsible for sporadic and epidemic nonbacterial gastroenteritis, causing an estimated 219,000 deaths annually worldwide. There is no commercially available vaccine for NoVs, due partly to the difficulty in establishing NoV cell culture models. The histo-blood group antigen (HBGA) blocking assay is used extensively to assess the protective potential of candidate vaccine-elicited antibodies, but there is still no widely used cellular evaluation model. In this study, we have established a cell line-based NoV vaccine evaluation model through the construction of human α1,2-fucosyltransferase 2-overexpressing 293T (293T-FUT2) cell lines. The 293T-FUT2 cells stably expressed H type 2 and Lewis y antigens. Virus-like particles (VLPs) of the NoV prototype strain genogroup I.1 (GI.1) and the predominant strains GII.4 and GII.17 could attach to the cell line efficiently in a dose-dependent manner. Importantly, antisera against these NoV VLPs could inhibit the attachment of the VLPs, where the inhibitory effects measured by the attachment inhibition assay correlated significantly with the antibody levels determined by the HBGA blocking assay. Collectively, our attachment inhibition assay could serve as a surrogate neutralization assay for the evaluation of NoV vaccines at the cellular level.

Bivalent Norovirus VLP Vaccine Candidate in Older Adults: Impact of MPL and a Second Dose in a Randomized, Controlled, Double-Blind Clinical Trial

BackgroundAcute norovirus (NoV) gastroenteritis may cause significant morbidity in healthy adults and can prove fatal in older subjects. We investigated the safety and immunogenicity in older adults of one or two doses of an intramuscular bivalent virus-like particle (VLP) vaccine candidate (genotypes GI.1 and multivalent consensus GII.4c) formulated with alum and with and without MPL (3-O-deacyl-4′-monophosphoryl lipid A) adjuvant.MethodsIn a phase II, double-blind, controlled trial, 294 healthy adults ≥ 60 years of age randomized to 4 equal groups received one or two immunizations 28 days apart. One dose groups received placebo (saline) on Day 1. Vaccine formulations contained 500μg Al(OH)3adjuvant with 15μg GI.1 and 50μg GII.4c VLP antigens, with or without 15μg MPL adjuvant. A fifth group of 26 healthy 18–49 year-olds received one dose of MPL-free vaccine. Humoral immunity was assessed as ELISA pan-Ig and histo-blood group antigen blocking (HBGA) antibody titers at Days 1, 8, 29 and 57. Cell-mediated immunity (CMI) and avidity indices (AI) were also measured. Safety was assessed as solicited local and systemic adverse events (AE) for 7 days, and unsolicited AEs until Day 28 after each vaccination.ResultsMarked increases in pan-Ig and HBGA to both genotypes occurred by Day 8 after first vaccination. Geometric mean titers were similar in magnitude in all groups and persisted at similar levels through Day 56. No increases were observed with a second vaccine dose on Day 29 or with the formulations containing MPL. Responses were similar in magnitude when assessed by age groups (60–74, 75–84 and ≥ 85 years of age) and when compared with those to a single vaccine dose in 18–49 year-olds. No clinically relevant differences in CMI responses or changes in antibody avidity were observed between formulations. Both formulations were generally well tolerated, the most frequent reaction being mild pain at the injection site. No vaccine-related SAEs were reported.ConclusionOlder adults aged over 60 years displayed immune responses to NoV VLP vaccines that were similar to those in younger adults with no apparent signs of immunosenescence. These data support the further development of the MPL-free vaccine candidate in older adults.Disclosures J. Sherwood, Takeda Pharmaceuticals International AG: Employee, Salary; J. Cramer, Takeda Pharmaceuticals International AG: Employee, Salary; N. Bouveret Le Cam, Takeda Vaccines Inc.: Employee, Salary; S. Lin, Takeda Vaccines Inc.: Employee, Salary; F. Baehner, Takeda Pharmaceuticals International AG: Employee, Salary; P. Mendelman, Takeda Vaccines Inc.: Employee, Salary; A. Borkowski, Takeda Pharmaceuticals International AG: Employee, Salary

Characterization of Antigenic Relatedness between GII.4 and GII.17 Noroviruses by Use of Serum Samples from Norovirus-Infected Patients.

A novel GII.17 norovirus variant caused major gastroenteritis epidemics in China in 2014 to 2016. To explore the host immune factors in selection of the emergence of this new variant, we characterized its antigenic relatedness with the GII.4 noroviruses that have dominated in China for decades. Through an enzyme-linked immunosorbent assay (ELISA) and a histo-blood group antigen (HBGA) blocking assay using sera from GII.4 and the GII.17 variant-infected patients, respectively, we observed limited cross-immune reactivity by the ELISA but little reactivity by the HBGA blocking assay between GII.4 norovirus and the new GII.17 variant. Our data suggest that, among other possible factors, GII.4-specific herd immunity had little role in the emergence of the new GII.17 variant. Thus, GII.17 may be an important active antigenic type or immunotype that needs to be considered for future vaccine strategies against human noroviruses.

Evaluation of immune effects of recombinant norovirus GI.1 and GII.4 virus-like particles

Objective To evaluate the immune effects of virus-like particles (VLPs) of VP1 proteins derived from norovirus GⅠ.1 and GⅡ.4 genotypes expressed in Hansenula polymorpha expression system. Methods SDS-PAGE and Western blot assay were performed to detect the purity of GⅠ.1 and GⅡ.4 VP1 proteins after purification. Morphologies of the recombinant VLPs were observed under transmission electron microscopy (TEM). Sizes and distributions of the VLPs were analyzed by dynamic light scattering analyzer. BT50 (50% of blocking titer) was detected by HBGA (histo-blood group antigen) blocking assay in BALB/c mice immunized with different regimens. Results SDS-PAGE analysis of the purified recombinant GⅠ.1 and GⅡ.4 VP1 proteins showed that their purity were greater than 90%. Western blot assay confirmed the specific bands of VLPs. TEM images showed that the sizes of purified GⅠ.1 and GⅡ.4 VP1 VLPs were at a mean diameter of 30-50 nm with clear border and high homogeneity, which was similar to that of wild virus. BT50 significantly increased in the groups, in which Al(OH)3 was used as adjuvant. Conclusion Animal studies have shown that administration of GⅠ.1 and GⅡ.4 VP1 VLPs in the presence of Al(OH)3 induces detectable HBGA-blocking antibody, indicating that GⅠ.1 and GⅡ.4 VP1 VLPs are promising candidates for norovirus vaccine. Key words: Norovirus (NoV); Virus-like particles (VLPs); Histo-blood group antigen (HBGA)

Structural basis for norovirus neutralization by an HBGA blocking human IgA antibody

Human noroviruses (HuNoVs) cause sporadic and epidemic gastroenteritis worldwide. They are classified into two major genogroups (GI and GII), with each genogroup further divided into multiple genotypes. Susceptibility to these viruses is influenced by genetically determined histo-blood group antigen (HBGA) expression. HBGAs function as cell attachment factors by binding to a surface-exposed region in the protruding (P) domain of the capsid protein. Sequence variations in this region that result in differential HBGA binding patterns and antigenicity are suggested to form a basis for strain diversification. Recent studies show that serum antibodies that block HBGA binding correlate with protection against illness. Although genogroup-dependent variation in HBGA binding specificity is structurally well characterized, an understanding of how antibodies block HBGA binding and how genotypic variations affect such blockade is lacking. Our crystallographic studies of the GI.1 P domain in complex with the Fab fragment of a human IgA monoclonal antibody (IgA 5I2) with HBGA blocking activity show that the antibody recognizes a conformational epitope formed by two surface-exposed loop clusters in the P domain. The antibody engulfs the HBGA binding site but does not affect its structural integrity. An unusual feature of the antigen recognition by IgA 5I2 is the predominant involvement of the CDR light chain 1 in contrast to the commonly observed CDR heavy chain 3, providing a unique perspective into antibody diversity in antigen recognition. Identification of the antigenic site in the P domain shows how genotypic variations might allow escape from antibody neutralization and exemplifies the interplay between antigenicity and HBGA specificity in HuNoV evolution.