| Influenza is a contagious,acute respiratory disease caused by influenza viruses.Since the pandemic of“1918 Spanish Influenza"(H1N1)in the early 20th century,flu epidemics have occurred almost every year.Currently,only H1,H3 subtype viruses and type B virus are circulating in humans while sporadic human infections with novel influenza viruses such as avian H5N1?H5N6?H6N2?H9N2?H7N9 and H10N8 were identified.It poses a great threat to human health so we need to find effective flu prevention and control measures.Hemagglutinin(HA)is an important glycoprotein on the surface of influenza virus which can induce protective humoral immunity.The Abs induced by influenza virus infection or vaccine were conventionally thought to be mainly directed against the variable HA head region and could neutralize viruses by blocking the binding process.However,the protections performed by this type of the Abs were limited to the specific viral strain and faild to counteract newly-emerging influenza virus strains.Recently,the heterosubtypic Abs targeting HA have been isolated and could cross-protect against a broad range of influenza viruses either within or across subtype even groups.These Abs mainly target the conserved epitopes on HA head or HA stem.To isolate the cross-reactive antibodies,we recovered the antibodies gene reservoir by culturing B cells in peripheral blood from human H5N1 cases and screening by HA cross-binding and neutralization test using H5N1 pseudotype particles,and then cloning and expressing the varible genes of heavy and light chain.After the Ab gene pairing screening by transient transfection of human kidney 293T cells,the candidate Abs were further generated and characterized.The results were summarized as the following:1.H5-reactive B cells could be detected in the B cell culture of four specimens collecting at different time points from three H5N1 patients and even some of them reacted with both H5 and H7.2.In specimen 2#,we discovered 5.78%of H5-reactive B cells in the sample of day 12 after disease onset.Additionally,1.07%of the H5/H7-cross reactive B cells were also presented.Seven wells(0.33%)displayed 80-90%of inhibition on Clade2.3.4 H5 pseudotype viral particles(pps)(A/Guangxi/1/2008(H5N1),GX-H5N1)infectivity.It suggested that the cross-reactive anti-HA B lymphocytes might pre-exist in host and could be reactivated into peripheral circulation by H5N1 infection.3.In specimen 3#and 4#,collecting on 21 and 18 days after disease onset respectively,1.3%and 3.17%of H5-reactive B cells were detected while 0.63%of H5-reactive B cells with a major of H5/H7-reavtive B cells(80%)were found in specimen 6#collected at 357 days after disease onset.It suggested that the number of H5-reactive B cells in peripheral circulation reduced along with the increased duration of recovery.4.Forty-four functional mAbs were obtained and 41 among them exhibited cross-reactivity with both H5 and H7.Two neutralizing Abs,encoding by VH1-69 and VH1-2,respectively,were obtained.5.The VH1-69 mAbs Ab2-12D5 and Ab2-12G8 shared the common HA stem-targeted IFY motif with VH1-69-encoded Abs.Ab2-12D5 could bind with Group 1 HAs and neutralize 2009pdm H1N1,H5N1 and H5N6 influenza viruses.Ab2-12G8 has broader binding activity with Group 1 and 2 influenza subtypes but cannot neutralize any influenza virus.Both of them demonstrated Ab-dependent cellular cytotoxicity(ADCC)activity.6.VH1-2 mAb from specimen 3#,Ab3-32G7.1,has a broader and strong binding with Group 1 HA and also could neutralize 2009pdm H1N1,H5N1 and H5N6 influenza viruses.7.The Group 1 and 2 HA-reactive mAbs derived from VH3-30 displayed genetic diversity and different affinity to H5 or H7.Their bindings to the Helix A of HA stem might be mediated by the hydrophobic domain at Site 111 of CDR3,MIVGAVL as well as the D99 of LCDR3.Conclusions:1.Broadly HA cross-reactive Abs could be detected in H5N1 cases and performed neutralizing or ADCC activity.2.The cross-reactive memory B cell preexisted in host could be rapidly activated by H5N1 virus infection. |
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