The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

Avian influenza A(H9N2) viruses are an increasing threat to global poultry production and, through zoonotic infection, to human health where they are considered viruses with pandemic potential. Vaccination of poultry is a key element of disease control in endemic countries, but vaccine effectiveness is persistently challenged by the emergence of antigenic variants. Here we employed a combination of techniques to investigate the genetic basis of H9N2 antigenic variability and evaluate the role of different molecular mechanisms of immune escape. We systematically tested the influence of published H9N2 monoclonal antibody escape mutants on chicken antisera binding, determining that many have no significant effect. Substitutions introducing additional glycosylation sites were a notable exception, though these are relatively rare among circulating viruses. To identify substitutions responsible for antigenic variation in circulating viruses, we performed an integrated meta-analysis of all published H9 haemagglutinin sequences and antigenic data. We validated this statistical analysis experimentally and allocated several new residues to H9N2 antigenic sites, providing molecular markers that will help explain vaccine breakdown in the field and inform vaccine selection decisions. We find evidence for the importance of alternative mechanisms of immune escape, beyond simple modulation of epitope structure, with substitutions increasing glycosylation or receptor-binding avidity, exhibiting the largest impacts on chicken antisera binding. Of these, meta-analysis indicates avidity regulation to be more relevant to the evolution of circulating viruses, suggesting that a specific focus on avidity regulation is required to fully understand the molecular basis of immune escape by influenza, and potentially other viruses

Analysis of immune responses induced by avian pathogenic Escherichia coli infection in turkeys and their association with resistance to homologous re-challenge.

International audienceAvian pathogenic Escherichia coli (APEC) cause severe respiratory and systemic disease in poultry yet the nature and consequences of host immune responses to infection are poorly understood. Here, we describe a turkey sub-acute respiratory challenge model and cytokine, cell-mediated and humoral responses associated with protection against homologous re-challenge. Intra-airsac inoculation of turkeys with 105 colony-forming units of APEC O78:H9 strain χ 7122nalR induced transient and mild clinical signs of colibacillosis followed by clearance of the bacteria from the lungs and visceral organs. Upon re-challenge with 107χ 7122nalR, primed birds were solidly protected against clinical signs and exhibited negligible bacterial loads in visceral organs, whereas age-matched control birds exhibited high lesion scores and bacterial loads in the organs. Levels of mRNA for signature cytokines suggested induction of a Th1 response in the lung, whereas a distinct anti-inflammatory cytokine profile was detected in the liver. Proliferative responses of splenocytes to either Concanavalin A or soluble χ 7122nalR antigens were negligible prior to clearance of bacteria, but APEC-specific responses were significantly elevated at later time intervals and at re-challenge relative to control birds. Primary infection also induced significantly elevated χ 7122nalR-specific serum IgY and bile IgA responses which were bactericidal against χ 7122nalR and an isogenic Δrfb mutant. Bactericidal activity was observed in the presence of immune, but not heat-inactivated immune serum, indicating that the antibodies can fix complement and are not directed solely at the lipopolysaccharide O-antigen. Such data inform the rational design of strategies to control a recalcitrant endemic disease of poultry

The early immune response to infection of chickens with Infectious Bronchitis Virus (IBV) in susceptible and resistant birds

Background: Infectious Bronchitis is a highly contagious respiratory disease which causes tracheal lesions and also affects the reproductive tract and is responsible for large economic losses to the poultry industry every year. This is due to both mortality (either directly provoked by IBV itself or due to subsequent bacterial infection) and lost egg production. The virus is difficult to control by vaccination, so new methods to curb the impact of the disease need to be sought. Here, we seek to identify genes conferring resistance to this coronavirus, which could help in selective breeding programs to rear chickens which do not succumb to the effects of this disease. Methods: Whole genome gene expression microarrays were used to analyse the gene expression differences, which occur upon infection of birds with Infectious Bronchitis Virus (IBV). Tracheal tissue was examined from control and infected birds at 2, 3 and 4 days post-infection in birds known to be either susceptible or resistant to the virus. The host innate immune response was evaluated over these 3 days and differences between the susceptible and resistant lines examined. Results: Genes and biological pathways involved in the early host response to IBV infection were determined andgene expression differences between susceptible and resistant birds were identified. Potential candidate genes for resistance to IBV are highlighted. Conclusions: The early host response to IBV is analysed and potential candidate genes for disease resistance are identified. These putative resistance genes can be used as targets for future genetic and functional studies to prove a causative link with resistance to IBV

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

Analysis of the early immune response to infection by infectious bursal disease virus in chickens differing in their resistance to the disease

Chicken whole-genome gene expression arrays were used to analyze the host response to infection by infectious bursal disease virus (IBDV). Spleen and bursal tissue were examined from control and infected birds at 2, 3, and 4 days postinfection from two lines that differ in their resistance to IBDV infection. The host response was evaluated over this period, and differences between susceptible and resistant chicken lines were examined. Antiviral genes, including IFNA, IFNG, MX1, IFITM1, IFITM3, and IFITM5, were upregulated in response to infection. Evaluation of this gene expression data allowed us to predict several genes as candidates for involvement in resistance to IBDV. © 2015, American Society for Microbiology

Characterization of the haemagglutinin properties of the H5N1 avian influenza virus that caused human infections in Cambodia

High pathogenicity avian influenza (HPAI) H5N1 is a subtype of the influenza A virus primarily found in birds. The subtype emerged in China in 1996 and has spread globally, causing significant morbidity and mortality in birds and humans. In Cambodia, a lethal case was reported in February 2023 involving an 11-year-old girl, marking the first human HPAI H5N1 infection in the country since 2014. This research examined the zoonotic potential of the human H5N1 isolate, A/Cambodia/NPH230032/2023 (KHM/23), by assessing its receptor binding, fusion pH, HA thermal stability, and antigenicity. Results showed that KHM/23 exhibits similar receptor binding and antigenicity as the early clade 2.3.2.1c HPAI H5N1 strain, and it does not bind to human-like receptors. Despite showing limited zoonotic risk, the increased thermal stability and reduced pH of fusion in KHM/23 indicate a potential threat to poultry, emphasizing the need for vigilant monitoring

Mapping the interaction sites of human and avian influenza A viruses and complement factor H

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects “self” cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated

Influence de l'hôte sur l'évolution d'une infection à salmonella (rôle des composants de l'immunité innée dans le contrôle du portage intestinal de Salmonella Enteritidis chez la volaille)

Asymptomatic persistence of Salmonella Enteritidis in the digestive tract of fowls is a major concern of food safety. The mechanisms involved in this carriage are still unknown. The objective of this thesis was to identify intestinal innate immunity factors which could be deficient in high carrier birds. Therefore we analysed the differential expression of genes of inflammatory and anti-infectious mediators between lines of birds which vary in the level of Salmonella colonisation. We have particularly shown that the resistance to bacterial colonization was related to the age of the animals, and that the high expression of inflammatory cytokine and defensin genes was related to a better control of Salmonella colonisation in adult but not in young birds. These results suggest that the efficiency of these factors in fighting against Salmonella carriage depends on the maturation of the immune system.La persistance asymptomatique de Salmonella Enteritidis au niveau du tube digestif des volailles est une préoccupation majeure pour la sécurité alimentaire. Les mécanismes de ce portage sont encore mal connus. L'objectif de la thèse était de mettre en évidence les facteurs de l'immunité innée intestinale qui pouvaient être déficients chez les oiseaux fortement porteurs. Pour cela nous avons analysé l'expression différentielle des gènes de médiateurs inflammatoires et anti-infectieux en comparant des lignées de volaille plus ou moins porteuses de salmonelles. Nous avons pu montrer en particulier que la résistance au portage variait selon l'âge des animaux et que l'expression élevée des gènes de cytokines inflammatoires et des défensines était liée à un meilleur contrôle de la colonisation chez l'adulte mais pas chez le jeune. Ces résultats suggèrent que l'efficacité de ces facteurs dans la lutte des oiseaux contre le portage dépend de la maturation du système immunitaire.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Enhancing Protective Efficacy of Poultry Vaccines through Targeted Delivery of Antigens to Antigen-Presenting Cells

Avian viral diseases including avian influenza, Marek’s disease and Newcastle disease are detrimental to economies around the world that depend on the poultry trade. A significant zoonotic threat is also posed by avian influenza viruses. Vaccination is an important and widely used method for controlling these poultry diseases. However, the current vaccines do not provide full protection or sterile immunity. Hence, there is a need to develop improved vaccines. The major aim of developing improved vaccines is to induce strong and specific humoral and cellular immunity in vaccinated animals. One strategy used to enhance the immunogenicity of vaccines is the selective delivery of protective antigens to antigen-presenting cells (APCs) including dendritic cells, macrophages and B cells. APCs have a central role in the initiation and maintenance of immune responses through their ability to capture, process and present antigens to T and B cells. Vaccine technology that selectively targets APCs has been achieved by coupling antigens to monoclonal antibodies or ligands that are targeted by APCs. The aim of this review is to discuss existing strategies of selective delivery of antigens to APCs for effective vaccine development in poultry