Characterization Of Norovirus Sydney Strain P Particle Bind To HBGA Receptor And Preparation Of GⅡ.4 Broad-spectrum Monoclonal Antibodies

Background and ObjectiveNoroviruses represent the most important cause of acute nonbacterial gastroenteritis. Noroviruses are highly contagious, transmitted through oral/fecal route and contaminated water and/or food. The infection is typically characterized by vomiting and/or diarrhea for 24-48 hours. This disease is often self-limited. Norovirus is the second most common viral cause of gastroenteritis after rotavirus in children below 5 years old in many countries. Recently, with the effective rotavirus vaccines being used in some developed countries, norovirus are rapidly replacing rotavirus as the most common cause of viral gastroenteritis in these countries. Not only can norovirus infect children below 5 years of age, but also can cause gastroenteritis among adults. A large of data support that the incidence of norovirus among the adults is increasing. Norovirus can cause chronic infection, severe disease, even death in elderly and immunocompromised individuals. It can infect population of all ages in developing country and developed country. Norovirus has been the significant public health issue all over the world.Noroviruses are nonenveloped, single-stranded, positive sense RNA viruses. The norovirus genome encodes three open reading frames （ORFs）. ORF2 encodes the major capsid protein VP1. VP1 has two major domains, a shell（S） domain and protrusion domain （P）. There is a short hinge region to link these two domains. The P domain consists of P1 and P2 subdomains located on the surfaces of the capsid protein. The majority of the genetic variants are identified in the P2 domain. The analysis of mutagenesis and X-ray crystallography show that P2 domain binds to histo-blood group antigen （HBGA）. Because of human norovirus cannot grow in cell culture, and lack of an appropriate small animal model, the study of pathogenic mechanisms is limited. Technique for gene engineering is the priority manner to research norovirus. VP1 protein can be expressed in insect cells by a recombinant baculovirus, assemble into virus-like particle. Its structure and antigenicity are similar to native virus without RNA. VLPs have been served as a surrogate for studying norovirus. The P particle has the same property and is easy to product, thus more and more researches regard P particle as the tool of NoV-HBGA binding reaction to predict the susceptible population.Based on genetic differences in the capsid gene, noroviruses are genetically highly diverse and divided into six genogroup （G Ⅰ、GⅡ、GⅢ、GⅣ、GⅤ、G Ⅵ）. Noroviruses from GⅠ、GⅡ are responsible for disease in humans. They are further subdivided into several different genotypes, at least 9 genotypes of G I,22 genotypes of GⅡ. In humans, GⅡ.4 viruses cause the majority of norovirus-related gastroenteritis outbreaks worldwide; New variants of the GⅡ.4 strain emerge every 2-4 years. The most recent emergent strain, GⅡ.4 Sydney, was first described in Australia in 2012 and replaced GⅡ.4 New Orleans as the dominant norovirus strain in the woridwide. It rapidly spreaded all over the world and caused vary degrees of acut gastroenteritis outbreaks in the United States, Britain, the Netherlands, Japan, Australia, France, New Zealand and other countries. It was also reportd in Beijing, Shanghai, Guangzhou, Jiangsu, Huzhou, Jingzhou, Hong Kong, Taiwan and other places in China.This study, we will express the GⅡ.4 NoV Sydney strain P particle by prokaryotic expression system, E.coli BL21, then the correlation between Sydney strain and HBGA receptor will detect by saliva-based binding assays to analysis the HBGA binding pattern of new variant. To clear the host range of Sydney strains infection and provide evidence for people at high risk prevention strategies. In this study, we will detect the serum antibody level of anti-NoV Sydney strain in health examination population in Guangzhou shanwei area. To provide scientific information for NoV prevention and control. The preparation of broad-spectrum anti-GⅡ.4 NoV monoclonal antibodies will be used immunization of combined multiple GⅡ.4 NoV variants P particle. To provide the experimental basis of the NoV P Particle subunit vaccine reserch.Method1、The extraction of P gene segmentStool samples were collected from the scene of the NoV outbreak, SH 201205. The P gene segment was amplified by PCR. Sequences were aligned and compared by phylogenetic tree.2、The construction and identification of pGEX-4T-1-P recombinant expression vectorThe P gene segment was attached to the pGEX-4T-1 expression vector. The recombinant expression plasmid was transformed into TOP 10 competent cells, and confirmed by colony PCR and restriction enzyme analysis.3、Expression, purification, and identification of P particleRecombinant plasmid of pGEX-4T-1-P was expressed in E.coli BL21 competent cells. After sonication of the bacteria, recombinant protein was purified by GST-beads. Then removed GST fusion protein tags by thrombin. We analysed the activity and antigenicity of P particle by 10%SDS-PAGE and Westernblot.4、The bingding experiment of P particle and HBGA receptorThe binding patterns of P particle with saliva-based HBGA receptor, which phenotype was known, were identified by indirect ELISA, and readed OD value in the 450 nm wavelength. OD450≥0.12 was judged to be positive.5、The serum antibody of GⅡ.4 NoV Sydney in healthy population in ShanweiThe serum antibody of GⅡ.4 NoV Sydney strain was tested by indirect ELISA with the envelope antigen of GⅡ.4 NoV Sydney P particle. OD450≥0.15 was judged to be positive.6、The preparation of monoclonal antibodies against GⅡ.4 NoVBALB/c mice were immunized with the multiple GⅡ.4 NoV P particles, according to the conventional technology to fuse the hybridoma cell. The positive hybridoma cell was screened by indirect ELISA, and gained the stable cell lines by 3 times subcloning. The subclass、specificity、neutralization of the monoclonal antibody were identified by MAbs subclass kit、ELISA and blocking assays.Results1、The obtainment of P gene segmentThe 1% agarose gel electrophoresis analysis showed the size of the PCR product was about 1000bp. The results of sequencing and phylogenetic tree analysis showed that the P gene sequence of SH 2012₀5 was identical to the GⅡ.4 NoV Sydney/2012（accession number:KC175323） with 99.9% homoligy.2、The identification of pGEX-4T-1-P recombinant expression vectorThe result of colony PCR shows that the size of the PCR product was about 1000bp and the restriction enzyme analysis showed the size of the segments were about 4.9kb and 1000bp, which were consistent with our expectation. The results demonstrated that we had constructed the recombinant expression vector pGEX-4T-1-P of GⅡ.4 NoV Sydney strain successfully.3、The identification of GⅡ.4 NoV Sydney P particle10%SDS-PAGE analysis showed the relative molecular weight of P particle was about 36kD, which were consistent with our expectation. And the antigen-specificity of GII.4 NoV Sydney strain P particle was demonstrated by Westernblot.4、The binding patterns of GⅡ.4 NoV Sydney P particle and saliva-based HBGA receptorThe results showed that GII.4 NoV Sydney strain P particle had strong binding activity to A-type, B-type, AB-type, and O-type secretor HBGA rececptor （OD450 ≥0.12）but not to nonsecretors （OD450≤0.09）, and the affinity was the highest in B-type secretor HBGA recptor.5、Detection of GⅡ.4 NoV Sydney strain serum antibody in healthy population in Shanwei areaThe positive of serum antibody against GⅡ.4 NoV Sydney strain was 88.14% （171/194）,86.11%（62/72） in male and 81.15%（99/122） in female. Gender distribution was statistically significant difference among different phenotypes of HBGA receptors （x2=14.041, P=0.003<0.05）Compared the serum antibody level among different phenotypes of HBGA receptor in male, the positive rate of serum antibody against GⅡ.4 NoV Sydney strain in A-type、B-type、O-type secretor HBGA receptor and nonsecretor were 95.00%（19/20）、95.83%（23/24）、78.95%（15/19） and 55.56%（5/9）. （The value of Fisher exact test is 9.289, P=0.013）; Compared the serum antibody level among different phenotypes of HBGA receptor in female, the positive rate of serum antibody against GII.4 NoV Sydney strain in A-type、B-type、O-type secretor HBGA receptor and nonsecretor were 83.33%（20/24）、100.00%（19/19）、81.97%（50/61） and 55.56% （10/18）. （The value of Fisher exact test is 11.709, P=0.007）.6、Identification of monoclonal antibodies against GⅡ.4 NoVHybridoma cells showed string shape distribution which were round and bright under the microscope. The fusion rete of the hybridoma was 33.07%（127/384）, and the positive rate of hybridoma was 28.35%（36/127）. Thirteen hybridoma cell lines were obtained. The subtypes of the MAbs were immunoglobulin G1（IgG1）. All of the MAbs coulud specifically bind to different GⅡ.4 NoV variants and three MAbs had blocking ability.Conclusion1、The P particle of GⅡ.4 NoV Sydney strain with the high antigen specificity could only bind to all kind of secretor HBGA receptors, including A-type, B-type, AB-type, O-type, not nonsecretor HBGA receptors.2、The positive rate of serum antibody against GⅡ.4 NoV Sydney strain was 88.1% （171/194）, and the positive rate of serum antibody with all kinds of secretor HBGA receptors was higher than nonsecretor. It was prompted that GⅡ.4 NoV Sydney strain spreaded widely among surveyed populations. It showed that the individuals of secretor HBGA receptor were susceptible to GⅡ.4 NoV Sydney strain.3、The GⅡ.4 NoV broad-spectrum monoclonal antibodies were prepared successfully and had the blocking ability.