Molecular Epidemiological Characteristics Of Neisserial Heparin Binding Antigen (NHBA)

Cerebrospinal meningitis is a global serious respiratory infectious disease caused by Neisseria meningitidis(Nm)infection,and it is also a legally reported serogroup B infectious disease in China.According to the structure and antigenicity of capsular polysaccharides on the bacterial surface,it can be divided into 12 serogroups,among which serogroup A,serogroup B,serogroup C,serogroup W,serogroup Y and serogroup X were the main pathogenic serogroups.Other strains that do not belong to the above capsular polysaccharides or missing capsular polysaccharides are classified as non-groupable(NG).In recent years,the epidemic dominant serogroup of meningococcal disease has changed in China.Before 2000,Nm serogroup A has been in the dominant serogroup in China.After 2000,other serogroups such as serogroup B,serogroup C,serogroup W,serogroup Y and serogroup X began to appear or increase,especially the number of meningococcal cases in serogroup B increased significantly in recent years.At present,the prevalent serogroups in China include serogroup B and serogroup C of ST-4821 clonal complex(CC4821)and serogroup W of CC11.Among them,serogroup C has been included in the National expanded immunization program,but serogroup B and serogroup W have not been included.For the prevention and control of serogroup B Nm strains,serogroup B meningococcal vaccines have been used internationally,including Bexsero(?)4CMenB and Trumenba(?),but there was no serogroup B meningococcal vaccine in China.Neisseria heparin binding protein(NHBA)also known as genome-derived Neisseria antigen 2132(GNA2132)which was one of the main components of four-component serogroup B meningococcal vaccine.The nhba gene which encodes NHBA protein,was ubiquitous in Nm,and existed in Neisseria gonorrhoeae,Neisseria polysaccharides,Neisseria lactamica,Neisseria flavescens and other Neisseria strains.There probably was cross-protection between different variants of NHBA.Therefore,NHBA is a potential universal vaccine ingredient to prevent N.gonorrhoeae infection.The ST sequence type and nhba gene sequence of 621 Nm strains isolated from different serogroups in China from 1956 to 2019 were obtained by means of multi-locus sequence typing(MLST),polymerase chain reaction(PCR)and gene sequencing.And the genotypic characteristics,nhba gene distribution and amino acid sequence characteristics were analyzed.Based on the PubMLST public database,the nhba gene and strain information of all Neisseria strains were downloaded,to determine the NHBA protein type.The amino acid sequences of NHBA were compared and analyzed.The cluster tree was constructed by MEGA 7.0 software.The main prevalent NHBA variants(688 and 20)in China were amplified by PCR(removing the 63 bp signal peptide sequence at the N-terminal of the NHBA amino acid sequence)and inserted into the pET-28a(+)plasmid to construct the recombinant plasmid.The recombinant plasmid was induced and expressed in the prokaryotic expression system Escherichia coli,further the protein was purified and detected.The results showed that in addition to Nm strains,there were seven other Neisseria species with complete nhba gene sequences(N.gonorrhoeae,N.lactamica,N.polysaccharides,N.bergeri,N.flavescens,N.subflava,and Neisseria unassigned species).The dominant variants of NHBA in different Neisseria species were various.There were eight variants of NHBA in different Neisseria species.The peptide 9 and peptide 262 existed in Nm,N.gonorrhoeae and N.lactamica.There were four identical NHBA variants between N.lactamica and Neisseria polysaccharea,which were peptide 38,peptide 92,peptide 291 and peptide 545.The peptide 470 existed in N.polysaccharides and Neisseria unassigned species.The peptide 547 existed in N.flavescens and N.subflava.All Chinese Nm strains contained nhba gene,and their amino acid variants shared 65.70%-100.00%identity with the NHBA variant of Bexsero(?)4CMenB(peptide 2).The main NHBA variant of serogroup A CC1 strains(n=38)was peptide 347(n=26).The main type of serogroup A CC5 was peptide 126(n=38).There was polymorphism of NHBA in serogroup B strains,and the main variants were peptide 688(n=31)and peptide 20(n=27),and a few strains had gene frameshift mutation(n=2).The peptide 503 was the dominant peptide of NHBA(n=30)in serogroup C strains.The main variants of serogroup W were peptide 29(n=28)and peptide 697(n=18).The primary variants of serogroup X were peptide 1068(n=9)and peptide 1416(n=5).The nhba gene of serogroup Y mainly encoded peptide 9(n=11)and peptide 1060(n=6).The molecular clonal expression of nhba gene showed that both peptide 20 and peptide 688 were expressed.The molecular weight was about 70 kDa by SDS-PAGE and Western blot analysis.The peptide 20 had a little degradation,and peptide 688 had a large amount of protein degradation(and producing four fragments).In summary,this study analyzed the NHBA molecular epidemiological characteristics of different Neisseria species,and further carried out molecular cloning and protein expression of the dominant variants of NHBA for Chinese Nm strains.These results systematically showed the epidemiological distribution characteristics of NHBA and the basic biological characteristics related to expression.This work can provide basic data and experience for the follow-up researches,such as screening the Nm serogroup B protein vaccine candidates of China,investigating the variation of vaccine proteins,and performing related studies on other Neisseria species.