Rescue of a H3N2 Influenza Virus Containing a Deficient Neuraminidase Protein by a Hemagglutinin with a Low Receptor-Binding Affinity

Influenza viruses possess at their surface two glycoproteins, the hemagglutinin and the neuraminidase, of which the antagonistic functions have to be well balanced for the virus to grow efficiently. Ferraris et al. isolated in 2003–2004 viruses lacking both a NA gene and protein (H3NA- viruses) (Ferraris O., 2006, Vaccine, 24(44–46):6656-9). In this study we showed that the hemagglutinins of two of the H3NA- viruses have reduced affinity for SAα2.6Gal receptors, between 49 and 128 times lower than that of the A/Moscow/10/99 (H3N2) virus and no detectable affinity for SAα2.3Gal receptors. We also showed that the low hemagglutinin affinity of the H3NA- viruses compensates for the lack of NA activity and allows the restoration of the growth of an A/Moscow/10/99 virus deficient in neuraminidase. These observations increase our understanding of H3NA- viruses in relation to the balance between the functional activities of the neuraminidase and hemagglutinin

Cross-Protective Potential of a Novel Monoclonal Antibody Directed against Antigenic Site B of the Hemagglutinin of Influenza A Viruses

The hemagglutinin (HA) of influenza A viruses has been classified into sixteen distinct subtypes (H1–H16) to date. The HA subtypes of influenza A viruses are principally defined as serotypes determined by neutralization or hemagglutination inhibition tests using polyclonal antisera to the respective HA subtypes, which have little cross-reactivity to the other HA subtypes. Thus, it is generally believed that the neutralizing antibodies are not broadly cross-reactive among HA subtypes. In this study, we generated a novel monoclonal antibody (MAb) specific to HA, designated MAb S139/1, which showed heterosubtypic cross-reactive neutralization and hemagglutination inhibition of influenza A viruses. This MAb was found to have broad reactivity to many other viruses (H1, H2, H3, H5, H9, and H13 subtypes) in enzyme-linked immunosorbent assays. We further found that MAb S139/1 showed neutralization and hemagglutination-inhibition activities against particular strains of H1, H2, H3, and H13 subtypes of influenza A viruses. Mutant viruses that escaped neutralization by MAb S139/1 were selected from the A/Aichi/2/68 (H3N2), A/Adachi/2/57 (H2N2), and A/WSN/33 (H1N1) strains, and sequence analysis of the HA genes of these escape mutants revealed amino acid substitutions at positions 156, 158, and 193 (H3 numbering). A molecular modeling study showed that these amino acids were located on the globular head of the HA and formed a novel conformational epitope adjacent to the receptor-binding domain of HA. Furthermore, passive immunization of mice with MAb S139/1 provided heterosubtypic protection. These results demonstrate that MAb S139/1 binds to a common antigenic site shared among a variety of HA subtypes and neutralizes viral infectivity in vitro and in vivo by affecting viral attachment to cells. The present study supports the notion that cross-reactive antibodies play some roles in heterosubtypic immunity against influenza A virus infection, and underscores the potential therapeutic utility of cross-reactive antibodies against influenza

The Epitope and Neutralization Mechanism of AVFluIgG01, a Broad-Reactive Human Monoclonal Antibody against H5N1 Influenza Virus

The continued spread of highly pathogenic avian influenza (HPAI) H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb) showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA). By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III) that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus

Antigenic Fingerprinting of H5N1 Avian Influenza Using Convalescent Sera and Monoclonal Antibodies Reveals Potential Vaccine and Diagnostic Targets

Using whole-genome-fragment phage display libraries, Hana Golding and colleagues identify the viral epitopes recognized by serum antibodies in humans who have recovered from infection with H5N1 avian influenza

Generation, Characterization and Epitope Mapping of Two Neutralizing and Protective Human Recombinant Antibodies against Influenza A H5N1 Viruses

The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development.We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs), AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model.Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines

Selective Pressure to Increase Charge in Immunodominant Epitopes of the H3 Hemagglutinin Influenza Protein

The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein–protein interactions, including antibody–antigen binding and ligand–receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R2 > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies

Mimotope ELISA for Detection of Broad Spectrum Antibody against Avian H5N1 Influenza Virus

Science and Technology Foundation of Fujian Province [2009YZ0002]; National Natural Science Foundation of China [30901077]; National High Technology Research and Development Program [2010AA022801]Background: We have raised a panel of broad spectrum neutralizing monoclonal antibodies against the highly pathogenic H5N1 avian influenza virus, which neutralize the infectivity of, and afford protection against infection by, most of the major genetic groups of the virus evolved since 1997. Peptide mimics reactive with one of these broad spectrum H5N1 neutralizing antibodies, 8H5, were identified from random phage display libraries. Method: The amino acid residues of the most reactive 12mer peptide, p125 (DTPLTTAALRLV), were randomly substituted to improve its mimicry of the natural 8H5 epitope. Result: 133 reactive peptides with unique amino acid sequences were identified from 5 sub-libraries of p125. Four residues (2,4,5.9) of the parental peptide were preserved among all the derived peptides and probably essential for 8H5 binding. These are interspersed among four other residues (1,3,8,10), which exhibit restricted substitution and probably could contribute to binding, and another four (6,7,11,12) which could be randomly substituted and probably are not essential for binding. One peptide, V-1b, derived by substituting 5 of the latter residues is the most reactive and has a binding constant of 3.16x10(-9) M, which is 38 fold higher than the affinity of the parental p125. Immunoassay produced with this peptide is specifically reactive with 8H5 but not also the other related broad spectrum H5N1 avian influenza virus neutralizing antibodies. Serum samples from 29 chickens infected with H5N1 avian influenza virus gave a positive result by this assay and those from 12 uninfected animals gave a negative test result. Conclusion: The immunoassay produced with the 12 mer peptide, V1-b, is specific for the natural 8H5 epitope and can be used for detection of antibody against the broad spectrum neutralization site of H5N1 avian influenza virus

Combination Therapy Using Chimeric Monoclonal Antibodies Protects Mice from Lethal H5N1 Infection and Prevents Formation of Escape Mutants

10.1371/journal.pone.0005672PLoS ONE4

Recombinant Trimeric HA Protein Immunogenicity of H5N1 Avian Influenza Viruses and Their Combined Use with Inactivated or Adenovirus Vaccines

[[abstract]]Background:The highly pathogenic avian influenza (HPAI) H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.Methodology/Principal Findings:We used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA) proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers.Conclusion/Significance:Our findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development