Establishment Of H5 Haemagglutinin Monoclonal Antibodies Panel, Identification Of Broad Reactive Neutralizing Monoclonal Antibodies And Their Cross-Protection Against H5N1 Infection

Long-term endemicity of highly pathogenic avian influenza H5N1 viruses inbirds and sporadic human transmissions by H5N 1 viruses since 2003 have raised theconcern of a potential pandemic. It has been explored actively in the development ofthe H5N1 pre-pandemic vaccine and treatment. Hemagglutinin (HA) of H5N1 virus isthe key protein producing neutralizing antibody and becomes a hot target.Given the high variation, the key work is to select an ideal virus candidate forvaccine development. Based on phylogeneitc analysis of HA sequence, H5N 1 viruseshave been divided into at least 10 genetical clades. The antibody was traditionallyused to analysis the real antigenecity of virus, however, co-circulation of multipleantigenic variants of H5N1 virus and low immunogenecity of H5N1 viurs hamper thetimely production of standard H5N 1-specific ferret antisera. Thus, it is useful toestablish a panel of H5 HA specific MAb for antigenic analysis and vaccine selectionof H5N1 virus. The result as bellows: A panel of 476 H5 HA specific MAb has beenestablished and characterized by HI assay to 40 H5N1 viruses representing differentgenetic lineages from 1997 to 2007. A panel of 60 broadly cross-reactive MAbs havebeen found due to their almost cross-reactive with all the 40 H5N1 virus. Accordingto their HI reactive spectrums, 15 representive MAbs were further selected as a panelto characterize the antigenecity of H5N1 virus. Four antigenic groups, A, B C and D,were identified among 40 H5N1 viruses based on their cross-reactive spectra with thispanel of MAbs in neutralisation assay. Group A consists mostly of clade 2.1 viruses,which are circulating mainly in Indonesia; Group B consists of the most geneticallydiversified variants, including viruses from clade 0, 1,4, 5, 7 and 9; Group C containsprimarily clade 2.2 Qinghai-like viruses; and Group D is composed almostexclusively of clade 2.3.4 Fujian-like viruses. In summary, this study established auseful panel of H5 HA MAbs and will facilitate for the recognition of emergingantigenic variants of the H5N 1 virus and help in the selection or evaluation of vaccinestrains.Human H5N 1 infections cause rapid dissemination of virus to multiple organsand are associated with severe disease and high mortality. Difficulties of currentanti-viral drugs, such as ion channel blockers and neuraminidase inhibitors, intreatment and high mortality rates may be attributable to failures in rapidneutralization and control dissemination of H5N1 virus in humans after symptoms emerge. Passive immunotherapy has been used increasingly in treatment of infectiousdiseases. However, to achieve neutralization, therapeutic antibodies rely on theirspecific binding to epitopes in the HA protein of influenza viruses. Continuousevolution and co-circulation of multiple genetic H5N1 lineages in broad regions have,however, generated antigenically diverse variants that pose significant challenges fordeveloping broadly cross protective therapeutic MAbs. Thus, the ideal therapeuticalantibody is the broadly cross-reactive H5 specific MAbs. One of these MAbs, 13D4,has been demonstrated to protect mice against lethal challenge by 4 H5N 1 strainsrepresenting the current major genetic populations, clades 1,2.1,2.2, and 2.3, even ata stage of infection when H5N1 virus has disseminated beyond the pulmonary system.Complete neutralization of virus in lung tissue of infected animals was observed 24 hafter treatment with 13D4. Compared the protection efficacy between differentisotypes of cross-reactive MAb, it was found that both IgG2a and IgG2b are betterthan IgG 1,IgM, IgG3. This suggested that Fc fragment of MAb may be related toprotection. Further study also showed that efficacy of F(ab')₂ is less than wholeantibody but better than oseltamivir (Tamiflu) in protect H5N1 infected mice. It wasalso observed that MAb 13D4 provide a good heterologous protection efficacy forH5N 1 infected cat. Mapping of this MAb with escape mutants showed it to bind to 2conserved, possibly critical, sites of H5N1 hemagglutinin, 152 and 182. It was alsofound that their escape mutants were partially attenuated in mice. Such evidencesupports the idea that these sites may be critical to virus function. HI assay showedthese escape mutants still retain hemagglutination inhibition, albeit at a lower levelthan their parental viruses. 13D4 MAb was still able to protect mice against a lethalchallenge of N182K virus, although with lower efficiency. In summary, H5broad-reactive MAbs such as 13D4 may lie in the fact that they target conserved,critical, and, therefore, preserved HA antigenic sites in H5N1 viruses and may havetherapeutic value in controlling infection due to current and future H5N1 variants.