Human Monoclonal Antibody 81.39a Effectively Neutralizes Emerging Influenza A Viruses of Group 1 and 2 Hemagglutinins

ABSTRACT The pandemic threat posed by emerging zoonotic influenza A viruses necessitates development of antiviral agents effective against various antigenic subtypes. Human monoclonal antibody (hMAb) targeting the hemagglutinin (HA) stalk offers a promising approach to control influenza virus infections. Here, we investigated the ability of the hMAb 81.39a to inhibit in vitro replication of human and zoonotic viruses, representing 16 HA subtypes. The majority of viruses were effectively neutralized by 81.39a at a 50% effective concentration (EC 50 ) of <0.01 to 4.9 μg/ml. Among group 2 HA viruses tested, a single A(H7N9) virus was not neutralized at 50 μg/ml; it contained HA 2 -Asp19Gly, an amino acid position previously associated with resistance to neutralization by the group 2 HA-neutralizing MAb CR8020. Notably, among group 1 HA viruses, H11-H13 and H16 subtypes were not neutralized at 50 μg/ml; they shared the substitution HA 2 -Asp19Asn/Ala. Conversely, H9 viruses harboring HA 2 -Asp19Ala were fully susceptible to neutralization. Therefore, amino acid variance at HA 2 -Asp19 has subtype-specific adverse effects on in vitro neutralization. Mice given a single injection (15 or 45 mg/kg of body weight) at 24 or 48 h after infection with recently emerged A(H5N2), A(H5N8), A(H6N1), or A(H7N9) viruses were protected from mortality and showed drastically reduced lung viral titers. Furthermore, 81.39a protected mice infected with A(H7N9) harboring HA 2 -Asp19Gly, although the antiviral effect was lessened. A(H1N1)pdm09-infected ferrets receiving a single dose (25 mg/kg) had reduced viral titers and showed less lung tissue injury, despite 24- to 72-h-delayed treatment. Taken together, this study provides experimental evidence for the therapeutic potential of 81.39a against diverse influenza A viruses. IMPORTANCE Zoonotic influenza viruses, such as A(H5N1) and A(H7N9) subtypes, have caused severe disease and deaths in humans, raising public health concerns. Development of novel anti-influenza therapeutics with a broad spectrum of activity against various subtypes is necessary to mitigate disease severity. Here, we demonstrate that the hemagglutinin (HA) stalk-targeting human monoclonal antibody 81.39a effectively neutralized the majority of influenza A viruses tested, representing 16 HA subtypes. Furthermore, delayed treatment with 81.39a significantly suppressed virus replication in the lungs, prevented dramatic body weight loss, and increased survival rates of mice infected with A(H5Nx), A(H6N1), or A(H7N9) viruses. When tested in ferrets, delayed 81.39a treatment reduced viral titers, particularly in the lower respiratory tract, and substantially alleviated disease symptoms associated with severe A(H1N1)pdm09 influenza. Collectively, our data demonstrated the effectiveness of 81.39a against both seasonal and emerging influenza A viruses.

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Heterosubtypic Protection Conferred by the Human Monoclonal Antibody PN-SIA28 against Influenza A Virus Lethal Infections in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00118-15 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2647-2653 ◽

Cited By ~ 1

Author(s):

Miguel Retamal ◽

Yacine Abed ◽

Chantal Rhéaume ◽

Francesca Cappelletti ◽

Nicola Clementi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Body Weight ◽

Influenza Virus ◽

Influenza A ◽

Human Monoclonal Antibody ◽

Influenza A Viruses ◽

Virus Challenge ◽

Influenza A H1n1

ABSTRACTPN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011,http://dx.doi.org/10.1371/journal.pone.0028001). Previousin vitrostudies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypesin vitrobut also, more importantly, protect from a lethal influenza virus challengein vivo.

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In VitroAssessment of the Immunological Significance of a Human Monoclonal Antibody Directed to the Influenza A Virus Nucleoprotein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00339-13 ◽

2013 ◽

Vol 20 (8) ◽

pp. 1333-1337 ◽

Cited By ~ 24

Author(s):

Rogier Bodewes ◽

Martina M. Geelhoed-Mieras ◽

Jens Wrammert ◽

Rafi Ahmed ◽

Patrick C. Wilson ◽

...

Keyword(s):

Monoclonal Antibody ◽

Influenza A Virus ◽

Influenza A ◽

Viral Antigen ◽

Human Monoclonal Antibody ◽

Cell Cytotoxicity ◽

Influenza A Viruses ◽

Dependent Cell ◽

Infected Cells

ABSTRACTInfluenza A viruses cause annual epidemics and occasionally pandemics. Antibodies directed to the conserved viral nucleoprotein (NP) may play a role in immunity against various influenza A virus subtypes. Here, we assessed the immunological significance of a human monoclonal antibody directed to NPin vitro. This antibody bound to virus-infected cells but did not display virus-neutralizing activity, complement-dependent cell cytotoxicity, or opsonization of viral antigen for improved antigen presentation to CD8+T cells by dendritic cells.

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Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires

Immunity ◽

10.1016/j.immuni.2017.12.009 ◽

2018 ◽

Vol 48 (1) ◽

pp. 174-184.e9 ◽

Cited By ~ 63

Author(s):

Kevin R. McCarthy ◽

Akiko Watanabe ◽

Masayuki Kuraoka ◽

Khoi T. Do ◽

Charles E. McGee ◽

...

Keyword(s):

B Cells ◽

Influenza A ◽

Memory B Cells ◽

Influenza A Viruses ◽

Adult Human ◽

Group 2 ◽

Group 1

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A broadly neutralizing human monoclonal antibody is effective against H7N9

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502374112 ◽

2015 ◽

Vol 112 (35) ◽

pp. 10890-10895 ◽

Cited By ~ 52

Author(s):

Kannan Tharakaraman ◽

Vidya Subramanian ◽

Karthik Viswanathan ◽

Susan Sloan ◽

Hui-Ling Yen ◽

...

Keyword(s):

Single Dose ◽

Influenza A ◽

Human Monoclonal Antibody ◽

Human Antibody ◽

Significant Survival ◽

Significant Public Health ◽

Group 2 ◽

Conserved Epitope

Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (−12 h) combined with oseltamivir at 50 mg/kg (−12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.

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Hemagglutinin stalk-based monoclonal antibody elicits broadly reactivity against group 1 influenza A virus

Virology Journal ◽

10.1186/s12985-020-01458-z ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Jingjin Huang ◽

Nan Huang ◽

Menglu Fan ◽

Lingcai Zhao ◽

Yan Luo ◽

...

Keyword(s):

Monoclonal Antibody ◽

Broad Spectrum ◽

Influenza A ◽

Lethal Dose ◽

Influenza Viruses ◽

Antigenic Drift ◽

Linear Epitope ◽

Group 1

Abstract Background Influenza virus remains a continuous and severe threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. Methods A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments. Results The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What’s more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy. Conclusions Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

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Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses

Cell ◽

10.1016/j.cell.2016.06.043 ◽

2016 ◽

Vol 166 (3) ◽

pp. 609-623 ◽

Cited By ~ 158

Author(s):

M. Gordon Joyce ◽

Adam K. Wheatley ◽

Paul V. Thomas ◽

Gwo-Yu Chuang ◽

Cinque Soto ◽

...

Keyword(s):

Influenza A ◽

Influenza A Viruses ◽

Group 2 ◽

Group 1

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Hemagglutinin Stalk-based Monoclonal Antibody Elicits Broadly Reactivity Against Group 1 Influenza A Virus

10.21203/rs.3.rs-97761/v1 ◽

2020 ◽

Author(s):

Jingjin Huang ◽

Nan Huang ◽

Menglu Fan ◽

Lingcai Zhao ◽

Yan Luo ◽

...

Keyword(s):

Monoclonal Antibody ◽

Broad Spectrum ◽

Influenza A ◽

Lethal Dose ◽

Influenza Viruses ◽

Antigenic Drift ◽

Linear Epitope ◽

Group 1

Abstract Background: Influenza virus remains a continuous and serious threat to public health worldwide, and its prevention and treatment have always been a major international issue. Because of its ability to evade immune surveillance through rapid antigenic drift and antigenic shift, broad-spectrum vaccines seem increasingly important. Methods: A mAb named 3C12 from an immortalized hybrid cell was generated via immunizing mice with HA2 protein from A/chicken/Anhui/BRI99/2016 (AH/BRI99/16, H9N2) generated by prokaryotic expression. Then, its broad-spectrum activity was analyzed by WB and IFA. Next, the minimal linear epitope was identified via analyzing the reaction of a series of HA truncations with 3C12. Finally, the protective effects of 3C12 were evaluated in vitro and in vivo infection experiments.Results: The mAb could react with the viruses of subtypes H1, H2, H5, H8, H9, H12, H13, H16, and HA protein of H18 in group 1, but failed to react with viruses in group 2. The minimal linear epitope targeted by the mAb was 433NAELLVL439 in full length of HA and localized in the C-helix region of HA2 (residue 95-101, HA2 numbering). What’s more, the mAb 3C12 inhibited H1, H2, H5, H8, H9, H12, H13 and H16 virus-replication in vitro and also has shown effectiveness in preventing and treating disease in mice challenged with lethal dose of AH/BRI99/16 (H9N2) virus in vivo. These results suggested that the broadly reactive anti-HA stem mAb 3C12 exhibited prophylactic and therapeutic efficacy.Conclusions: Here, we have demonstrated that the linear epitope identified in this study could be a novel target for developing broad-spectrum influenza diagnostics or vaccine design, and the HA2-based monoclonal antibody is indeed a promising strategy for broad-spectrum protection against seasonal and pandemic influenza viruses.

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A novel linear epitope crossing Group 1 and Group 2 influenza A viruses located in the helix A of HA2 derived from H7N9

Veterinary Microbiology ◽

10.1016/j.vetmic.2018.11.002 ◽

2019 ◽

Vol 228 ◽

pp. 39-44 ◽

Cited By ~ 6

Author(s):

Zhanping Li ◽

Zhimin Wan ◽

Tuofan Li ◽

Quan Xie ◽

Haiwei Sun ◽

...

Keyword(s):

Influenza A ◽

Linear Epitope ◽

Influenza A Viruses ◽

Group 2 ◽

Group 1

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A Broad and Potent H1-Specific Human Monoclonal Antibody Produced in Plants Prevents Influenza Virus Infection and Transmission in Guinea Pigs

Viruses ◽

10.3390/v12020167 ◽

2020 ◽

Vol 12 (2) ◽

pp. 167 ◽

Cited By ~ 2

Author(s):

Jun-Gyu Park ◽

Chengjin Ye ◽

Michael S. Piepenbrink ◽

Aitor Nogales ◽

Haifeng Wang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Influenza Virus ◽

Guinea Pigs ◽

Influenza A ◽

Human Monoclonal Antibody ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Antigenic Drift

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift. Previously, we described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV) in vitro, and prophylactic and therapeutic activities in vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrated how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian-produced KPF1-HEK hMAb. KPF1-Antx hMAb showed broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, which was comparable to that observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrated, for the first time, a plant-produced influenza hMAb with in vitro and in vivo activity against influenza virus. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

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