Human Monoclonal Antibody Derived from Transchromosomic Cattle Neutralizes Multiple H1 Clades of Influenza A Virus by Recognizing a Novel Conformational Epitope in the Hemagglutinin Head Domain

ABSTRACT Influenza remains a global health risk and challenge. Currently, neuraminidase (NA) inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug-resistant variants. Neutralizing antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. However, the major hurdle for the extensive application of antibody therapies lies in the difficulty of generating nonimmunogenic antibodies in large quantities rapidly. Here, we report that one human monoclonal antibody (MAb), 53C10, isolated from transchromosomic (Tc) cattle exhibits potent neutralization and hemagglutination inhibition titers against different clades of H1N1 subtype influenza A viruses. In vitro selection of antibody escape mutants revealed that 53C10 recognizes a novel noncontinuous epitope in the hemagglutinin (HA) head domain involving three amino acid residues, glycine (G), serine (S), and glutamic acid (E) at positions 172, 207, and 212, respectively. The results of our experiments supported a critical role for substitution of arginine at position 207 (S207R) in mediating resistance to 53C10, while substitutions at either G172E or E212A did not alter antibody recognition and neutralization. The E212A mutation may provide structural stability for the epitope, while the substitution G172E probably compensates for loss of fitness introduced by S207R. Our results offer novel insights into the mechanism of action of MAb 53C10 and indicate its potential role in therapeutic treatment of H1 influenza virus infection in humans. IMPORTANCE Respiratory diseases caused by influenza viruses still pose a serious concern to global health, and neutralizing antibodies constitute a promising area of antiviral therapeutics. However, the potential application of antibodies is often hampered by the challenge in generating nonimmunogenic antibodies in large scale. In the present study, transchromosomic (Tc) cattle were used for the generation of nonimmunogenic monoclonal antibodies (MAbs), and characterization of such MAbs revealed one monoclonal antibody, 53C10, exhibiting a potent neutralization activity against H1N1 influenza viruses. Further characterization of the neutralization escape mutant generated using this MAb showed that three amino acid substitutions in the HA head domain contributed to the resistance. These findings emphasize the importance of Tc cattle in the production of nonimmunogenic MAbs and highlight the potential of MAb 53C10 in the therapeutic application against H1 influenza virus infection in humans.

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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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A broad and potent H1-specific human monoclonal antibody produced in plants prevents influenza virus infection and transmission in guinea pigs

10.1101/2020.01.13.902841 ◽

2020 ◽

Author(s):

Jun-Gyu Park ◽

Chengjin Ye ◽

Michael S. Piepenbrink ◽

Aitor Nogales ◽

Haifeng Wang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Guinea Pigs ◽

Mammalian Cells ◽

Influenza A ◽

Human Monoclonal Antibody ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Antigenic Drift

AbstractAlthough 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, respectively. Previously, we have 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 demonstrate how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian produced KPF1-HEK hMAb. KPF1-Antx hMAb shows broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1) in vitro, that are comparable to those 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 demonstrates, for the first time, that plant-produced influenza hMAbs have similar in vitro and in vivo biological properties to those produced in mammalian cells. 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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A Host-Restricted Self-Attenuated Influenza Virus Provides Broad Pan-Influenza A Protection in a Mouse Model

Frontiers in Immunology ◽

10.3389/fimmu.2021.779223 ◽

2021 ◽

Vol 12 ◽

Author(s):

Minjin Kim ◽

Yucheol Cheong ◽

Jinhee Lee ◽

Jongkwan Lim ◽

Sanguine Byun ◽

...

Keyword(s):

Influenza Virus ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Influenza A ◽

Protective Efficacy ◽

Influenza Viruses ◽

Cross Protection ◽

Infection Model ◽

Wild Type ◽

Group 1

Influenza virus infections can cause a broad range of symptoms, form mild respiratory problems to severe and fatal complications. While influenza virus poses a global health threat, the frequent antigenic change often significantly compromises the protective efficacy of seasonal vaccines, further increasing the vulnerability to viral infection. Therefore, it is in great need to employ strategies for the development of universal influenza vaccines (UIVs) which can elicit broad protection against diverse influenza viruses. Using a mouse infection model, we examined the breadth of protection of the caspase-triggered live attenuated influenza vaccine (ctLAIV), which was self-attenuated by the host caspase-dependent cleavage of internal viral proteins. A single vaccination in mice induced a broad reactive antibody response against four different influenza viruses, H1 and rH5 (HA group 1) and H3 and rH7 subtypes (HA group 2). Notably, despite the lack of detectable neutralizing antibodies, the vaccination provided heterosubtypic protection against the lethal challenge with the viruses. Sterile protection was confirmed by the complete absence of viral titers in the lungs and nasal turbinates after the challenge. Antibody-dependent cellular cytotoxicity (ADCC) activities of non-neutralizing antibodies contributed to cross-protection. The cross-protection remained robust even after in vivo depletion of T cells or NK cells, reflecting the strength and breadth of the antibody-dependent effector function. The robust mucosal secretion of sIgA reflects an additional level of cross-protection. Our data show that the host-restricted designer vaccine serves an option for developing a UIV, providing pan-influenza A protection against both group 1 and 2 influenza viruses. The present results of potency and breadth of protection from wild type and reassortant viruses addressed in the mouse model by single immunization merits further confirmation and validation, preferably in clinically relevant ferret models with wild type challenges.

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Methods for Molecular Surveillance of Influenza Used in Macedonia

PRILOZI ◽

10.2478/prilozi-2014-0003 ◽

2014 ◽

Vol 35 (2) ◽

pp. 25-30

Author(s):

Golubinka Bosevska ◽

Elizabeta Janceska ◽

Gordana Kuzmanovska ◽

Vladimir Mikik ◽

Nikola Panovski

Keyword(s):

Influenza Virus ◽

Predictive Value ◽

Influenza A ◽

Rna Extraction ◽

Influenza Virus Infection ◽

Laboratory Diagnosis ◽

Influenza Viruses ◽

Rt Pcr ◽

Two Samples ◽

Nose And Throat

AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.

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Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00793-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4962-4973 ◽

Cited By ~ 32

Author(s):

Yasushi Itoh ◽

Shintaro Shichinohe ◽

Misako Nakayama ◽

Manabu Igarashi ◽

Akihiro Ishii ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Cynomolgus Macaque ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

H7n9 Virus ◽

Macaque Model ◽

H7n9 Influenza Virus ◽

Number Of Patients ◽

H7n9 Influenza

ABSTRACTThe number of patients infected with H7N9 influenza virus has been increasing since 2013. We examined the efficacy of neuraminidase (NA) inhibitors and the efficacy of a vaccine against an H7N9 influenza virus, A/Anhui/1/2013 (H7N9), isolated from a patient in a cynomolgus macaque model. NA inhibitors (oseltamivir and peramivir) barely reduced the total virus amount because of the emergence of resistant variants with R289K or I219T in NA [residues 289 and 219 in N9 of A/Anhui/1/2013 (H7N9) correspond to 292 and 222 in N2, respectively] in three of the six treated macaques, whereas subcutaneous immunization of an inactivated vaccine derived from A/duck/Mongolia/119/2008 (H7N9) prevented propagation of A/Anhui/1/2013 (H7N9) in all vaccinated macaques. The percentage of macaques in which variant H7N9 viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitorsin vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.

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Potency of an Inactivated Influenza Vaccine against a Challenge with A/Swine/Missouri/A01727926/2015 (H4N6) in Mice for Pandemic Preparedness

Vaccines ◽

10.3390/vaccines8040768 ◽

2020 ◽

Vol 8 (4) ◽

pp. 768

Author(s):

Hirotaka Hayashi ◽

Norikazu Isoda ◽

Enkhbold Bazarragchaa ◽

Naoki Nomura ◽

Keita Matsuno ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Neutralizing Antibodies ◽

Influenza Pandemic ◽

Influenza Virus Infection ◽

Cross Reactivity ◽

Influenza Viruses ◽

Pandemic Preparedness ◽

Antigenic Relation ◽

High Immunogenicity

H4 influenza viruses have been isolated from birds across the world. In recent years, an H4 influenza virus infection has been confirmed in pigs. Pigs play an important role in the transmission of influenza viruses to human hosts. Therefore, it is important to develop a new vaccine in the case of an H4 influenza virus infection in humans, considering that this virus has a different antigenicity from seasonal human influenza viruses. In this study, after selecting vaccine candidate strains based on their antigenic relation to one of the pig isolates, A/swine/Missouri/A01727926/2015 (H4N6) (MO/15), an inactivated whole-particle vaccine was prepared from A/swan/Hokkaido/481102/2017 (H4N6). This vaccine showed high immunogenicity in mice, and the antibody induced by the vaccine showed high cross-reactivity to the MO/15 virus. This vaccine induced sufficient neutralizing antibodies and mitigated the effects of an MO/15 infection in a mouse model. This study is the first to suggest that an inactivated whole-particle vaccine prepared from an influenza virus isolated from wild birds is an effective countermeasure in case of a future influenza pandemic caused by the H4 influenza virus.

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Phase III Randomized, Double-Blind Study Comparing Single-Dose Intravenous Peramivir with Oral Oseltamivir in Patients with Seasonal Influenza Virus Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00360-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5267-5276 ◽

Cited By ~ 73

Author(s):

Shigeru Kohno ◽

Muh-Yong Yen ◽

Hee-Jin Cheong ◽

Nobuo Hirotsu ◽

Tadashi Ishida ◽

...

Keyword(s):

Influenza Virus ◽

Virus Infection ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Phase Iii ◽

Controlled Study ◽

Double Blind Study ◽

Double Blind

ABSTRACTAntiviral medications with activity against influenza viruses are important in controlling influenza. We compared intravenous peramivir, a potent neuraminidase inhibitor, with oseltamivir in patients with seasonal influenza virus infection. In a multinational, multicenter, double-blind, double-dummy randomized controlled study, patients aged ≥20 years with influenza A or B virus infection were randomly assigned to receive either a single intravenous infusion of peramivir (300 or 600 mg) or oral administration of oseltamivir (75 mg twice a day [b.i.d.] for 5 days). To demonstrate the noninferiority of peramivir in reducing the time to alleviation of influenza symptoms with hazard model analysis and a noninferiority margin of 0.170, we planned to recruit 1,050 patients in South Korea, Japan, and Taiwan. A total of 1,091 patients (364 receiving 300 mg and 362 receiving 600 mg of peramivir; 365 receiving oseltamivir) were included in the intent-to-treat infected population. The median durations of influenza symptoms were 78.0, 81.0, and 81.8 h in the groups treated with 300 mg of peramivir, 600 mg of peramivir, and oseltamivir, respectively. The hazard ratios of the 300- and 600-mg-peramivir groups compared to the oseltamivir group were 0.946 (97.5% confidence interval [CI], 0.793, 1.129) and 0.970 (97.5% CI, 0.814, 1.157), respectively. Both peramivir groups were noninferior to the oseltamivir group (97.5% CI, <1.170). The overall incidence of adverse drug reactions was significantly lower in the 300-mg-peramivir group, but the incidence of severe reactions in either peramivir group was not different from that in the oseltamivir group. Thus, a single intravenous dose of peramivir may be an alternative to a 5-day oral dose of oseltamivir for patients with seasonal influenza virus infection.

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Bacterial Sinusitis and Otitis Media following Influenza Virus Infection in Ferrets

Infection and Immunity ◽

10.1128/iai.74.5.2562-2567.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2562-2567 ◽

Cited By ~ 59

Author(s):

Ville T. Peltola ◽

Kelli L. Boyd ◽

Julie L. McAuley ◽

Jerold E. Rehg ◽

Jonathan A. McCullers

Keyword(s):

Influenza Virus ◽

Otitis Media ◽

Influenza A ◽

Bacterial Colonization ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Influenza B Virus ◽

Influenza B ◽

Complication Rates ◽

Pneumococcal Infections

ABSTRACT Streptococcus pneumoniae is the leading cause of otitis media, sinusitis, and pneumonia. Many of these infections result from antecedent influenza virus infections. In this study we sought to determine whether the frequency and character of secondary pneumococcal infections differed depending on the strain of influenza virus that preceded bacterial challenge. In young ferrets infected with influenza virus and then challenged with pneumococcus, influenza viruses of any subtype increased bacterial colonization of the nasopharynx. Nine out of 10 ferrets infected with H3N2 subtype influenza A viruses developed either sinusitis or otitis media, while only 1 out of 11 ferrets infected with either an H1N1 influenza A virus or an influenza B virus did so. These data may partially explain why bacterial complication rates are higher during seasons when H3N2 viruses predominate. This animal model will be useful for further study of the mechanisms that underlie viral-bacterial synergism.

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Reemerging H5N1 Influenza Viruses in Hong Kong in 2002 Are Highly Pathogenic to Ducks

Journal of Virology ◽

10.1128/jvi.78.9.4892-4901.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4892-4901 ◽

Cited By ~ 271

Author(s):

Katharine M. Sturm-Ramirez ◽

Trevor Ellis ◽

Barry Bousfield ◽

Lucy Bissett ◽

Kitman Dyrting ◽

...

Keyword(s):

Influenza Virus ◽

Hong Kong ◽

Influenza A ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Neurological Dysfunction ◽

Aquatic Birds ◽

Antigenic Analysis ◽

Highly Pathogenic ◽

H5n1 Influenza

ABSTRACT Waterfowl are the natural reservoir of all influenza A viruses, which are usually nonpathogenic in wild aquatic birds. However, in late 2002, outbreaks of highly pathogenic H5N1 influenza virus caused deaths among wild migratory birds and resident waterfowl, including ducks, in two Hong Kong parks. In February 2003, an avian H5N1 virus closely related to one of these viruses was isolated from two humans with acute respiratory distress, one of whom died. Antigenic analysis of the new avian isolates showed a reactivity pattern different from that of H5N1 viruses isolated in 1997 and 2001. This finding suggests that significant antigenic variation has recently occurred among H5N1 viruses. We inoculated mallards with antigenically different H5N1 influenza viruses isolated between 1997 and 2003. The new 2002 avian isolates caused systemic infection in the ducks, with high virus titers and pathology in multiple organs, particularly the brain. Ducks developed acute disease, including severe neurological dysfunction and death. Virus was also isolated at high titers from the birds' drinking water and from contact birds, demonstrating efficient transmission. In contrast, H5N1 isolates from 1997 and 2001 were not consistently transmitted efficiently among ducks and did not cause significant disease. Despite a high level of genomic homology, the human isolate showed striking biological differences from its avian homologue in a duck model. This is the first reported case of lethal influenza virus infection in wild aquatic birds since 1961.

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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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