Screening And Study Of Broad - Spectrum Reactive Antibody Targeting Group2 Influenza Virus Hemagglutinin Stem

Antigenic diversity in seasonal influenza virus as well as in the newly-emerging influenza virus is being concerned. Furthermore, type A virus has a broad host range and is highly variant. As to type A, there are two groups of HA (Hemagglutinin) subtypes, including Group1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17 and H18) and Group2 (H3, H4, H7, H10, H14 and H15).HA contains a globular head and a relatively conserved stalk domain (Cys52-Cys277 (H3 numbering)).The antibodies(Abs) induced by influenza virus infection or tranditional flu vaccine were conventionally thought to be mainly directed against the variable HA head region. However, a low level of HA stem-reactive Abs could indeed be detected in the sera from H1N1-or H3N2-infected humans and mice. Importantly, HA stem-reactive broad-spectrum neutralzing monoclonal Abs(MAbs) with neutralizing activity to either within or across subtype even group viruses were isolated. Most of them target the Groupl HA influenza viruses, and the Abs against Group2 HA viruses is rare. Until now, we could list the MAbs reacting with Group2 HA viruses (such as CR8020, CR8043 and VIS410) or with H3 (such as 12D1, C05 and F045-092) or Group 1 and 2 (such as FI6, CR9114 and CT149). The effective 50% HA-binding concentrations of the MAbs range from 1 to 40μg/ml and some of them show no reactivity with H7 or H10 subtype virus.Besides the neutralizing effects mediated by Fab domain, the MAbs could function with Fc in vivo via antibody-dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) to lyze virus-infected cells. For example, CR9114, displayed its protective effects in mice model while showed no neutralizing effect in vitro.Here, we investigated the Group2 HA-stem reactive Abs among the cohhort vaccinated by a trivalent seasonal flu vaccine in fall 2009.Our study includes:1. The Recombinant HAs, a chimer H7/3 HA (cH7/3) bearing the head of A/Netherlands/219/03(H7N7) and the stalk of A/Aichi/2/1968(H3N2) and ZJH7 HA of A/Zhejiang/1/2013(H7N9), were expressed by baculovirus expression system. The HAs were purificated and their functions were tested.2. The paired sera on Day 0 and 21 after vaccination from 49 healthy adult volunteers were screened for the Group2 HA-stem reactive Abs. Initially, the binding to cH7/3 were tested. Then the HA-binding was re-identificated by using ZJH7. The affinity index of the ZJH7-binding sera and their targeting epitope domains were assayed.3. The in vitro culture of human memory B cells were performed. Cells from positive wells of ZJH7-binding were harvested and total RNA was isolated. Human VH and Vk or VX, genes were amplified by RT-PCR and cloned into IgGl and k or X expressing vectors. The candidate MAbs were obtained and tested for their binding to different HAs as well as their hemagglutination inhibition (HI) and ADCC activity.Results:1. Recombinant protein cH7/3 and ZJH7HAs secreted from the insect cells and then-size is approximately 70kD. The HAs in the trimer forms show hemagglutination activity and bind to specific Abs.2. The sera on Day 21 after vaccination from 6 donors in the cohhort displayed enhanced binding to cH7/3HA. Only Sample 152 was also positive to ZJH7-binding, its EC50 value of the sera on Day 21 is 2.1-fold higher than that on Day 0. The affinity index of sera increased after imminization and were around 1. The epitope domains recognizeby the sera Abs targeted HA stalk domain and showed somewhat similar as those of FI6-370 or F10.3. Nine wells of peripheral memory B cells from Donor 152 were selectd for further Ab gene-coloning from 39 wells of B cell supernatants positive to ZJHA-binding. The MAb, H3-2/k3-1, which were encoded by VH1-69*O1 and VK3-20*01, has a 19-amino acids(AA) HCDR3 and 12 AA substitutions in VH. H3-2/k3-1 could bind to HI, H3, H5, H7, H9 and H10 antigen. Its lowest concentrations for postive-binding varied from 3.0to7.0μg/ml. No HI activity was observed in the MAb and have ADCC activity was being tested.Conclusions:The Abs targeting Group2 HA stalk domain could be detected in the sera from seasonal flu vaccinees and the positive rate is 1 out of 49 (2%). The obtained MAb showed widely cross-reactive with Group1 and 2 HA (H1,3,5,7,9 and 10) and its affinities are relatively lower(the lowest concentrations for binding are 3.0-7.0μg/ml).