Review the following articles and citations

Biomed Res Int. 2015;2015:901817. doi: 10.1155/2015/901817. Epub 2015 Feb 12.

Protection against multiple subtypes of influenza viruses by virus-like particle vaccines based on a hemagglutinin conserved epitope.

Chen S1, Zheng D1, Li C2, Zhang W3, Xu W1, Liu X1, Fang F4, Chen Z5.

Author information

1Shanghai Institute of Biological Products, Shanghai 200052, China.

2National Institutes for Food and Drug Control and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China.

3Xinhua Hospital Affiliated to Shanghai Jiaotong University of Medicine, Shanghai 200092, China.

4College of Life Sciences, Hunan Normal University, Hunan, Changsha 410081, China.

5Shanghai Institute of Biological Products, Shanghai 200052, China ; College of Life Sciences, Hunan Normal University, Hunan, Changsha 410081, China.

Abstract

We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA) trimmer, the long alpha helix (LAH), as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR) of hepatitis B virus core protein (HBc), and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP). Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB(*)) adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8) (H1N1)). In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB(*) adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections.

Vet Microbiol. 2014 Dec 5;174(3-4):333-41. doi: 10.1016/j.vetmic.2014.10.008. Epub 2014 Oct 25.

A monoclonal antibody recognizes a highly conserved neutralizing epitope on hemagglutinin of H6N1 avian influenza virus.

He JL1, Hsieh MS1, Juang RH2, Wang CH3.

Author information

1Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan.

2Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan. Electronic address: [email protected].

3School of Veterinary Medicine, National Taiwan University, No. 1, Sec 4, Roosevelt Road, Taipei 10617, Taiwan. Electronic address: [email protected].

Abstract

Neutralizing antibodies on the globular head of the hemagglutinin (HA) of avian influenza virus (AIV) are crucial for controlling this disease. However, most neutralizing antibodies lack cross reaction. This report describes the identification of a hemagglutinin epitope on the globular head near the receptor binding site of the H6N1 AIV. A monoclonal antibody named EB2 was prepared against the H6N1 AIV HA. Flow cytometry of AIV-infected chicken embryo fibroblast, DF-1 cells and specific-pathogen-free embryonated eggs were used to verify the neutralizing activity of this mAb. To narrow down the binding region, partially overlapping HA fragments and synthetic peptides were used to map the epitope by immune-blotting. The linear motif RYVRMGTESMN, located on the surface on the globular head of the HA protein, was identified as the epitope bound by EB2 mAb. Alignment of the EB2-defined epitope with other H6 AIVs showed that this epitope was conserved and specific to H6. We propose that this motif is a linear B-cell epitope of the HA protein and is near the receptor binding site. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the AIV HA protein.

J Virol. 2014 Nov;88(21):12326-38. doi: 10.1128/JVI.01542-14. Epub 2014 Aug 13.

The N terminus of the influenza B virus nucleoprotein is essential for virus viability, nuclear localization, and optimal transcription and replication of the viral genome.

Sherry L1, Smith M1, Davidson S1, Jackson D2.

Author information

1Biomolecular Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, United Kingdom.

2Biomolecular Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, United Kingdom [email protected].

Abstract

The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However, NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50-amino-acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension; however, this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. The truncation of more than three amino acids prevented virus recovery, suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in the nuclear localization of BNP, with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first 10 residues of BNP demonstrated few differences in nuclear localization; however, the viruses exhibited significant reductions in viral mRNA transcription and genome replication, resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall, our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP but also for regulating viral mRNA transcription and genome replication.

IMPORTANCE:

The multifunctional NP of influenza viruses has roles throughout the viral replication cycle; therefore, it is essential for virus viability. Despite high levels of homology between the NP of influenza A and B viruses, the NP of influenza B virus contains an evolutionarily conserved 50-amino-acid N-terminal extension that is absent from the NP of influenza A viruses. In this study, we show that this N-terminal extension is essential for virus viability, and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore, we demonstrate for the first time that the N terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension, these viruses may have evolved separate mechanisms to regulate these processes.

Virology. 2014 Apr;454-455:40-7. doi: 10.1016/j.virol.2014.01.023. Epub 2014 Feb 22.

Large-scale analysis of influenza A virus nucleoprotein sequence conservation reveals potential drug-target sites.

Kukol A1, Hughes DJ2.

Author information

1School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK. Electronic address: [email protected].

2School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK.

Abstract

The nucleoprotein (NP) of the influenza A virus encapsidates the viral RNA and participates in the infectious life cycle of the virus. The aims of this study were to find the degree of conservation of NP among all virus subtypes and hosts and to identify conserved binding sites, which may be utilised as potential drug target sites. The analysis of conservation based on 4430 amino acid sequences identified high conservation in known functional regions as well as novel highly conserved sites. Highly variable clusters identified on the surface of NP may be associated with adaptation to different hosts and avoidance of the host immune defence. Ligand binding potential overlapping with high conservation was found in the tail-loop binding site and near the putative RNA binding region. The results provide the basis for developing antivirals that may be universally effective and have a reduced potential to induce resistance through mutations.

8.J Virol. 2014 Jul;88(13):7130-44. doi: 10.1128/JVI.00420-14. Epub 2014 Apr 9.

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

Iba Y1, Fujii Y2, Ohshima N1, Sumida T2, Kubota-Koketsu R3, Ikeda M2, Wakiyama M2, Shirouzu M2, Okada J4, Okuno Y5, Kurosawa Y6, Yokoyama S2.

Author information

1Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan.

2RIKEN Systems and Structural Biology Center, Suehiro, Tsurumi, Yokohama, Japan.

3Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

4Sales and Marketing Division, Medical and Biological Laboratories Co., Ltd., Nagoya, Aichi, Japan.

5Kanonji Institute, The Research Foundation for Microbial Diseases, Osaka University, Kanonji, Kagawa, Japan.

6Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan [email protected].

Abstract

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses.

IMPORTANCE:

Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

J Virol. 2014 Jun;88(12):7083-92. doi: 10.1128/JVI.00178-14. Epub 2014 Apr 9.

Alternative recognition of the conserved stem epitope in influenza A virus hemagglutinin by a VH3-30-encoded heterosubtypic antibody.

Wyrzucki A1, Dreyfus C2, Kohler I1, Steck M1, Wilson IA3, Hangartner L4.

Author information

1Institute of Medical Virology, University of Zurich, Zürich, Switzerland.

2Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

3Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [email protected] [email protected].

4Institute of Medical Virology, University of Zurich, Zürich, Switzerland [email protected] [email protected].

Abstract

A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes.

IMPORTANCE:

Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic protein HB36.3. Thus, besides providing novel insights into the molecular interactions between heterosubtypic antibodies and influenza virus hemagglutinin, MAb 3.1 demonstrates that de novo in silico-designed and affinity-matured synthetic proteins can foretell naturally selected antibody binding. This knowledge will aid development of a pan-influenza virus vaccine.

J Virol. 2014 Jun;88(11):5977-86. doi: 10.1128/JVI.00508-14. Epub 2014 Mar 12.

Conserved features of the PB2 627 domain impact influenza virus polymerase function and replication.

Kirui J1, Bucci MD, Poole DS, Mehle A.

Author information

1Cellular and Molecular Biology Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.

Abstract

Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation.

IMPORTANCE:

Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

Hi,

Do you need primers only for nucleoprotein and PB2 protein? You will can design a primers for matrix protein. There are much conservative regions in this gene.

PB2 and the nucleoprotein gene are the most conserved regions of Influenza A viral genome. Apart from this certain internal regions of conservation are also observed in the PA gene. 

Review the following articles and citations

Biomed Res Int. 2015;2015:901817. doi: 10.1155/2015/901817. Epub 2015 Feb 12.

Protection against multiple subtypes of influenza viruses by virus-like particle vaccines based on a hemagglutinin conserved epitope.

Chen S1, Zheng D1, Li C2, Zhang W3, Xu W1, Liu X1, Fang F4, Chen Z5.

Author information

1Shanghai Institute of Biological Products, Shanghai 200052, China.

2National Institutes for Food and Drug Control and WHO Collaborating Center for Standardization and Evaluation of Biologicals, Beijing, China.

3Xinhua Hospital Affiliated to Shanghai Jiaotong University of Medicine, Shanghai 200092, China.

4College of Life Sciences, Hunan Normal University, Hunan, Changsha 410081, China.

5Shanghai Institute of Biological Products, Shanghai 200052, China ; College of Life Sciences, Hunan Normal University, Hunan, Changsha 410081, China.

Abstract

We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA) trimmer, the long alpha helix (LAH), as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR) of hepatitis B virus core protein (HBc), and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP). Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB(*)) adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8) (H1N1)). In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB(*) adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections.

Vet Microbiol. 2014 Dec 5;174(3-4):333-41. doi: 10.1016/j.vetmic.2014.10.008. Epub 2014 Oct 25.

A monoclonal antibody recognizes a highly conserved neutralizing epitope on hemagglutinin of H6N1 avian influenza virus.

He JL1, Hsieh MS1, Juang RH2, Wang CH3.

Author information

1Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan.

2Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, Taipei, Taiwan. Electronic address: [email protected].

3School of Veterinary Medicine, National Taiwan University, No. 1, Sec 4, Roosevelt Road, Taipei 10617, Taiwan. Electronic address: [email protected].

Abstract

Neutralizing antibodies on the globular head of the hemagglutinin (HA) of avian influenza virus (AIV) are crucial for controlling this disease. However, most neutralizing antibodies lack cross reaction. This report describes the identification of a hemagglutinin epitope on the globular head near the receptor binding site of the H6N1 AIV. A monoclonal antibody named EB2 was prepared against the H6N1 AIV HA. Flow cytometry of AIV-infected chicken embryo fibroblast, DF-1 cells and specific-pathogen-free embryonated eggs were used to verify the neutralizing activity of this mAb. To narrow down the binding region, partially overlapping HA fragments and synthetic peptides were used to map the epitope by immune-blotting. The linear motif RYVRMGTESMN, located on the surface on the globular head of the HA protein, was identified as the epitope bound by EB2 mAb. Alignment of the EB2-defined epitope with other H6 AIVs showed that this epitope was conserved and specific to H6. We propose that this motif is a linear B-cell epitope of the HA protein and is near the receptor binding site. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the AIV HA protein.

J Virol. 2014 Nov;88(21):12326-38. doi: 10.1128/JVI.01542-14. Epub 2014 Aug 13.

The N terminus of the influenza B virus nucleoprotein is essential for virus viability, nuclear localization, and optimal transcription and replication of the viral genome.

Sherry L1, Smith M1, Davidson S1, Jackson D2.

Author information

1Biomolecular Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, United Kingdom.

2Biomolecular Sciences Research Complex, University of St. Andrews, North Haugh, St. Andrews, United Kingdom [email protected].

Abstract

The nucleoprotein (NP) of influenza viruses is a multifunctional protein with essential roles throughout viral replication. Despite influenza A and B viruses belonging to separate genera of the Orthomyxoviridae family, their NP proteins share a relatively high level of sequence conservation. However, NP of influenza B viruses (BNP) contains an evolutionarily conserved N-terminal 50-amino-acid extension that is absent from NP of influenza A viruses. There is conflicting evidence as to the functions of the BNP N-terminal extension; however, this has never been assessed in the context of viral infection. We have used reverse genetics to assess the significance of this region on the functions of BNP and virus viability. The truncation of more than three amino acids prevented virus recovery, suggesting that the N-terminal extension is essential for virus viability. Mutational analysis indicated that multiple regions of the protein are involved in the nuclear localization of BNP, with the entire N-terminal extension required for this to function efficiently. Viruses containing mutations in the first 10 residues of BNP demonstrated few differences in nuclear localization; however, the viruses exhibited significant reductions in viral mRNA transcription and genome replication, resulting in significantly attenuated phenotypes. Mutations introduced to ablate a previously reported nuclear localization signal also resulted in a significant decrease in mRNA production during early stages of viral replication. Overall, our results demonstrate that the N-terminal extension of BNP is essential to virus viability not only for directing nuclear localization of BNP but also for regulating viral mRNA transcription and genome replication.

IMPORTANCE:

The multifunctional NP of influenza viruses has roles throughout the viral replication cycle; therefore, it is essential for virus viability. Despite high levels of homology between the NP of influenza A and B viruses, the NP of influenza B virus contains an evolutionarily conserved 50-amino-acid N-terminal extension that is absent from the NP of influenza A viruses. In this study, we show that this N-terminal extension is essential for virus viability, and we confirm and expand upon recent findings that this region of BNP is required for nuclear localization of the protein. Furthermore, we demonstrate for the first time that the N terminus of BNP is involved in regulating viral mRNA transcription and replication of the viral genome. As the NP of influenza A virus lacks this N-terminal extension, these viruses may have evolved separate mechanisms to regulate these processes.

Virology. 2014 Apr;454-455:40-7. doi: 10.1016/j.virol.2014.01.023. Epub 2014 Feb 22.

Large-scale analysis of influenza A virus nucleoprotein sequence conservation reveals potential drug-target sites.

Kukol A1, Hughes DJ2.

Author information

1School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK. Electronic address: [email protected].

2School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK.

Abstract

The nucleoprotein (NP) of the influenza A virus encapsidates the viral RNA and participates in the infectious life cycle of the virus. The aims of this study were to find the degree of conservation of NP among all virus subtypes and hosts and to identify conserved binding sites, which may be utilised as potential drug target sites. The analysis of conservation based on 4430 amino acid sequences identified high conservation in known functional regions as well as novel highly conserved sites. Highly variable clusters identified on the surface of NP may be associated with adaptation to different hosts and avoidance of the host immune defence. Ligand binding potential overlapping with high conservation was found in the tail-loop binding site and near the putative RNA binding region. The results provide the basis for developing antivirals that may be universally effective and have a reduced potential to induce resistance through mutations.

8.J Virol. 2014 Jul;88(13):7130-44. doi: 10.1128/JVI.00420-14. Epub 2014 Apr 9.

Conserved neutralizing epitope at globular head of hemagglutinin in H3N2 influenza viruses.

Iba Y1, Fujii Y2, Ohshima N1, Sumida T2, Kubota-Koketsu R3, Ikeda M2, Wakiyama M2, Shirouzu M2, Okada J4, Okuno Y5, Kurosawa Y6, Yokoyama S2.

Author information

1Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan.

2RIKEN Systems and Structural Biology Center, Suehiro, Tsurumi, Yokohama, Japan.

3Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

4Sales and Marketing Division, Medical and Biological Laboratories Co., Ltd., Nagoya, Aichi, Japan.

5Kanonji Institute, The Research Foundation for Microbial Diseases, Osaka University, Kanonji, Kagawa, Japan.

6Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan [email protected].

Abstract

Neutralizing antibodies that target the hemagglutinin of influenza virus either inhibit binding of hemagglutinin to cellular receptors or prevent the low-pH-induced conformational change in hemagglutinin required for membrane fusion. In general, the former type of antibody binds to the globular head formed by HA1 and has narrow strain specificity, while the latter type binds to the stem mainly formed by HA2 and has broad strain specificity. In the present study, we analyzed the epitope and function of a broadly neutralizing human antibody against H3N2 viruses, F005-126. The crystal structure of F005-126 Fab in complex with hemagglutinin revealed that the antibody binds to the globular head, spans a cleft formed by two hemagglutinin monomers in a hemagglutinin trimer, and cross-links them. It recognizes two peptide portions (sites L and R) and a glycan linked to asparagine at residue 285 using three complementarity-determining regions and framework 3 in the heavy chain. Binding of the antibody to sites L (residues 171 to 173, 239, and 240) and R (residues 91, 92, 270 to 273, 284, and 285) is mediated mainly by van der Waals contacts with the main chains of the peptides in these sites and secondarily by hydrogen bonds with a few side chains of conserved sequences in HA1. Furthermore, the glycan recognized by F005-126 is conserved among H3N2 viruses. F005-126 has the ability to prevent low-pH-induced conformational changes in hemagglutinin. The newly identified conserved epitope, including the glycan, should be immunogenic in humans and may induce production of broadly neutralizing antibodies against H3 viruses.

IMPORTANCE:

Antibodies play an important role in protection against influenza virus, and hemagglutinin is the major target for virus neutralizing antibodies. It has long been believed that all effective neutralizing antibodies bind to the surrounding regions of the sialic acid-binding pocket and inhibit the binding of hemagglutinin to the cellular receptor. Since mutations are readily introduced into such epitopes, this type of antibody shows narrow strain specificity. Recently, however, broadly neutralizing antibodies have been isolated. Most of these bind either to conserved sites in the stem region or to the sialic acid-binding pocket itself. In the present study, we identified a new neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against H3N2. This epitope may be useful for design of vaccines.

J Virol. 2014 Jun;88(12):7083-92. doi: 10.1128/JVI.00178-14. Epub 2014 Apr 9.

Alternative recognition of the conserved stem epitope in influenza A virus hemagglutinin by a VH3-30-encoded heterosubtypic antibody.

Wyrzucki A1, Dreyfus C2, Kohler I1, Steck M1, Wilson IA3, Hangartner L4.

Author information

1Institute of Medical Virology, University of Zurich, Zürich, Switzerland.

2Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

3Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [email protected] [email protected].

4Institute of Medical Virology, University of Zurich, Zürich, Switzerland [email protected] [email protected].

Abstract

A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes.

IMPORTANCE:

Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic protein HB36.3. Thus, besides providing novel insights into the molecular interactions between heterosubtypic antibodies and influenza virus hemagglutinin, MAb 3.1 demonstrates that de novo in silico-designed and affinity-matured synthetic proteins can foretell naturally selected antibody binding. This knowledge will aid development of a pan-influenza virus vaccine.

J Virol. 2014 Jun;88(11):5977-86. doi: 10.1128/JVI.00508-14. Epub 2014 Mar 12.

Conserved features of the PB2 627 domain impact influenza virus polymerase function and replication.

Kirui J1, Bucci MD, Poole DS, Mehle A.

Author information

1Cellular and Molecular Biology Graduate Program, University of Wisconsin-Madison, Madison, Wisconsin, USA.

Abstract

Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved "P[F/P]AAAPP" sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation.

IMPORTANCE:

Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

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