Antigenic Characterization of Human Monoclonal Antibodies for Therapeutic Use against H7N9 Avian Influenza Virus.

Since 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1,500 human infections and the culling of millions of poultry. Despite large-scale poultry vaccination, H7N9 AIVs continue to circulate among poultry in China and pose a threat to human health. Previously, we isolated and generated four monoclonal antibodies (mAbs) derived from humans naturally infected with H7N9 AIV. Here, we investigated the hemagglutinin (HA) epitopes of H7N9 AIV targeted by these mAbs (L3A-44, K9B-122, L4A-14, and L4B-18) using immune escape studies. Our results revealed four key antigenic epitopes at HA amino acid positions 125, 133, 149, and 217. The mutant H7N9 viruses representing escape mutations containing an alanine-to-threonine substitution at residue 125 (A125T), a glycine-to-glutamic acid substitution at residue 133 (G133E), an asparagine-to-aspartic acid substitution at residue 149 (N149D), or a leucine-to-glutamine substitution at residue 217 (L217Q) showed reduced or completely abolished cross-reactivity with the mAbs, as measured by a hemagglutination inhibition (HI) assay. We further assessed the potential risk of these mutants to humans should they emerge following mAb treatment by measuring the impact of these HA mutations on virus fitness and evasion of host adaptive immunity. Here, we showed that the L4A-14 mAb had broad neutralizing capabilities, and its escape mutant N149D had reduced viral stability and human receptor binding and could be neutralized by both postinfection and antigen-induced sera. Therefore, the L4A-14 mAb could be a therapeutic candidate for H7N9 AIV infection in humans and warrants further investigation for therapeutic applications. IMPORTANCE Avian influenza virus (AIV) H7N9 continues to circulate and evolve in birds, posing a credible threat to humans. Antiviral drugs have proven useful for the treatment of severe influenza infections in humans; however, concerns have been raised as antiviral-resistant mutants have emerged. Monoclonal antibodies (mAbs) have been studied for both prophylactic and therapeutic applications in infectious disease control and have demonstrated great potential. For example, mAb treatment has significantly reduced the risk of people developing severe disease with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In addition to the protection efficiency, we should also consider the potential risk of the escape mutants generated by mAb treatment to public health by assessing their viral fitness and potential to compromise host adaptive immunity. Considering these parameters, we assessed four human mAbs derived from humans naturally infected with H7N9 AIV and showed that the mAb L4A-14 displayed potential as a therapeutic candidate.

Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential.

H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly in vitro and in ovo They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry.

Targeting Haemagglutinin Antigen of Avian Influenza Virus to Chicken Immune Cell Receptors Dec205 and CD11c Induces Differential Immune-Potentiating Responses.

Improving the immunogenicity and protective efficacy of vaccines is critical to reducing disease impacts. One strategy used to enhance the immunogenicity of vaccines is the selective delivery of protective antigens to the antigen presenting cells (APCs). In this study, we have developed a targeted antigen delivery vaccine (TADV) system by recombinantly fusing the ectodomain of hemagglutinin (HA) antigen of H9N2 influenza A virus to single chain fragment variable (scFv) antibodies specific for the receptors expressed on chicken APCs; Dec205 and CD11c. Vaccination of chickens with TADV containing recombinant H9HA Foldon-Dec205 scFv or H9HA Foldon-CD11c scFv proteins elicited faster (as early as day 6 post primary vaccination) and higher anti-H9HA IgM and IgY, haemagglutination inhibition, and virus neutralisation antibodies compared to the untargeted H9HA protein. Comparatively, CD11c scFv conjugated H9HA protein showed higher immunogenic potency compared to Dec205 scFv conjugated H9HA protein. The higher immune potentiating ability of CD11c scFv was also reflected in ex-vivo chicken splenocyte stimulation assay, whereby H9HA Foldon-CD11c scFv induced higher levels of cytokines (IFNγ, IL6, IL1ß, and IL4) compared to H9HA Foldon-Dec205 scFv. Overall, the results conclude that TADV could be a better alternative to the currently available inactivated virus vaccines.

Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza.

The immunogenicity and protective efficacy of vaccines can be enhanced by the selective delivery of antigens to the antigen-presenting cells (APCs). In this study, H9N2 avian influenza virus haemagglutinin (HA) antigen, was targeted by fusing it to single-chain fragment variable (scFv) antibodies specific to CD83 receptor expressed on chicken APCs. We observed an increased level of IFNγ, IL6, IL1ß, IL4, and CxCLi2 mRNA upon stimulation of chicken splenocytes ex vivo by CD83 scFv targeted H9HA. In addition, CD83 scFv targeted H9HA induced higher serum haemagglutinin inhibition activity and virus neutralising antibodies compared to untargeted H9HA, with induction of antibodies as early as day 6 post primary vaccination. Furthermore, chickens vaccinated with CD83 scFv targeted H9HA showed reduced H9N2 challenge virus shedding compared to untargeted H9HA. These results suggest that targeting antigens to CD83 receptors could improve the efficacy of poultry vaccines.

Presence of Antibodies to SARS-CoV-2 in Domestic Cats in Istanbul, Turkey, Before and After COVID-19 Pandemic.

Recent studies demonstrated that domestic cats can be naturally and experimentally infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This study was performed to investigate the presence of SARS-CoV-2-specific antibodies within the domestic cat population in Istanbul, Turkey, before the coronavirus disease 2019 (COVID-19) and during the COVID-19 pandemic. Overall, from 155 cat sera analyzed, 26.45% (41/155) tested positive in the spike protein-ELISA (S-ELISA), 28.38% (44/155) in the receptor-binding domain-ELISA (RBD-ELISA), and 21.9% (34/155) in both, the S- and RBD-ELISAs. Twenty-seven of those were also positive for the presence of antibodies to feline coronavirus (FCoV). Among the 34 SARS-CoV-2-positive sera, three of those were positive on serum neutralization assay. Six of the 30 cats before COVID-19 and 28 of the 125 cats during COVID-19 were found to be seropositive. About 20% of ELISA-positive cats exhibited mainly respiratory, gastrointestinal, and renal signs and skin lesions. Hematocrit, hemoglobin, white blood cells, lymphocyte, and platelet numbers were low in about 30% of ELISA-positive cats. The number of neutrophils and monocytes were above normal values in about 20% of ELISA-positive cats. The liver enzyme alanine aminotransferase levels were high in 23.5% ELISA-positive cats. In conclusion, this is the first report describing antibodies specific to SARS-CoV-2 antigens (S and RBD) in cats in Istanbul, Turkey, indicating the risk for domestic cats to contract SARS-CoV-2 from owners and/or household members with COVID-19. This study and others show that COVID-19-positive pet owners should limit their contact with companion animals and that pets with respiratory signs should be monitored for SARS-CoV-2 infections.

Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza.

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

Meeting Report: Global Alliance for Research on Avian Diseases 2018, International Conference, January 17 to 19, 2018, Hanoi, Vietnam.

Poultry production is one of the fastest growing sectors of the livestock industry, growing at a rate of around 5% per year (2015-16) to meet the global demands and food security, as shown by European Union Open Data Portal. One of the major challenges for the sustainable growth of this sector comes from the plethora of diseases, including viral diseases, which have devastating effects on productivity. With a significant growth in poultry production in Asia, South America, and Africa, most of the disease challenges are in these regions. Because of the global nature of these diseases, it is of vital importance to work collaboratively to generate effective mitigation opportunities via innovative strategies. In the spirit of this international collaboration, the second International Conference of the Global Alliance for Research on Avian Diseases (GARAD) was held from January 17 to January 19, 2018, in Hanoi, Vietnam. The conference, attended by over 150 delegates from academia, poultry breeding/farming, and the pharmaceutic industry, discussed the major challenges and research advances related to the control of poultry diseases. The topics reviewed included the continuous threat from avian influenza and its antigenic shifts/drifts, the risks of disease transmission within and from live bird markets, the challenges from antigenic diversity of other avian viruses, innovative approaches for poultry vaccine development, and the potential opportunities to introduce genetic resistance to infectious agents through novel gene editing techniques. In separate interactive sessions, delegates actively debated the challenges, priorities, and opportunities for academia in driving avian disease research, the importance of developing improved disease measures by industry, and the contribution by the farming sector in the low- and middle-income countries.

Reporte de caso- Reporte de la reunión: Alianza mundial para la investigación sobre enfermedades aviares 2018, Conferencia Internacional, 17 al 19 de enero de 2018, Hanói, Vietnam. La producción avícola es uno de los sectores de más rápido crecimiento de la industria pecuaria, que crece a una tasa de alrededor del 5% por año (2015-2016) para satisfacer las demandas globales y la seguridad alimentaria, como lo demuestra el Portal de Datos abiertos de la Unión Europea. Uno de los principales desafíos para el crecimiento sostenible de este sector proviene de la gran cantidad de enfermedades, incluyendo las enfermedades virales, que tienen efectos devastadores en la productividad. Con un crecimiento significativo en la producción en Asia, América del Sur y África, la mayoría de los desafíos relacionados con las enfermedades se encuentran en estas regiones. Debido a la naturaleza global de estas enfermedades, es de vital importancia trabajar en colaboración para generar oportunidades de mitigación efectivas a través de estrategias innovadoras. En el espíritu de esta colaboración internacional, la segunda Conferencia Internacional de la Alianza Global para la Investigación sobre las Enfermedades Aviares (con las siglas en inglés GARAD) se celebró del 17 al 19 de enero del 2018 en Hanói, Vietnam. Durante la conferencia, a la que asistieron más de 150 delegados de la academia, productores avícolas y la industria farmacéutica, se discutieron los principales desafíos y los avances de la investigación relacionado con el control de las enfermedades de la avicultura. Los temas se encuentran la amenaza continua de la influenza aviar y sus cambios antigénicos tanto drásticos como menores, los riesgos de transmisión de enfermedades dentro y desde los mercados de aves vivas, los desafíos de la diversidad antigénica de otros virus aviares, los enfoques innovadores para el desarrollo de vacunas para la avicultura y las oportunidades potenciales de introducir resistencia genética a los agentes infecciosos a través de nuevos técnicas de edición de genes. En sesiones interactivas separadas, los delegados debatieron activamente los desafíos, las prioridades y las oportunidades para la academia en la conducción de la investigación de enfermedades aviares, la importancia de desarrollar medidas mejoradas para el manejo de las enfermedades por parte de la industria y la contribución del sector agrícola en los países de ingresos bajos y medios. Abbreviations: AIV = avian influenza virus; GARAD = Global Alliance for Research on Avian Diseases; IBV = infectious bronchitis virus; IBDV = infectious bursal disease virus; NDV = Newcastle disease virus; ND = Newcastle disease; NLRC5 = NOD-like receptor caspase recruitment domain containing protein 5; UPM = University Putra Malaysia.

Current and Novel Approaches in Influenza Management.

Influenza is a disease that poses a significant health burden worldwide. Vaccination is the best way to prevent influenza virus infections. However, conventional vaccines are only effective for a short period of time due to the propensity of influenza viruses to undergo antigenic drift and antigenic shift. The efficacy of these vaccines is uncertain from year-to-year due to potential mismatch between the circulating viruses and vaccine strains, and mutations arising due to egg adaptation. Subsequently, the inability to store these vaccines long-term and vaccine shortages are challenges that need to be overcome. Conventional vaccines also have variable efficacies for certain populations, including the young, old, and immunocompromised. This warrants for diverse efficacious vaccine developmental approaches, involving both active and passive immunization. As opposed to active immunization platforms (requiring the use of whole or portions of pathogens as vaccines), the rapidly developing passive immunization involves administration of either pathogen-specific or broadly acting antibodies against a kind or class of pathogens as a treatment to corresponding acute infection. Several antibodies with broadly acting capacities have been discovered that may serve as means to suppress influenza viral infection and allow the process of natural immunity to engage opsonized pathogens whilst boosting immune system by antibody-dependent mechanisms that bridge the innate and adaptive arms. By that; passive immunotherapeutics approach assumes a robust tool that could aid control of influenza viruses. In this review, we comment on some improvements in influenza management and promising vaccine development platforms with an emphasis on the protective capacity of passive immunotherapeutics especially when coupled with the use of antivirals in the management of influenza infection.