Evaluation of Commercial Vaccines for Efficacy and Transmission Control Against the Emergent H5N8 (Clade 2.3.4.4b) Avian Influenza Virus in Kazakhstan

Evaluation of Commercial Vaccines for Efficacy and Transmission Control Against the Emergent H5N8 (Clade 2.3.4.4b) Avian Influenza Virus in Kazakhstan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of Commercial Vaccines for Efficacy and Transmission Control Against the Emergent H5N8 (Clade 2.3.4.4b) Avian Influenza Virus in Kazakhstan Kairat Tabynov, Aidana Kuanyshbek, Kuantay Zharmambet, Leila Yelchibayeva, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6142334/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Highly pathogenic avian influenza H5N8 (clade 2.3.4.4b) has caused devastating poultry outbreaks globally, including in Kazakhstan, underscoring the need for vaccines that protect birds and curb virus transmission. We evaluated the efficacy of three commercial H5 vaccines and an experimental homologous H5N8 vaccine in chickens. Methods: Chickens received a single dose of each vaccine, and antibody titers were measured over 4 weeks. At 30 days post-vaccination, birds were challenged intranasally with a virulent H5N8 strain and monitored for 10 days for survival and clinical signs. Virus titers in tracheal and cloacal swabs (days 1, 3, 5 post-challenge) measured shedding, and unvaccinated sentinel chickens were co-housed to assess transmission. Results: The homologous H5N8 vaccine and a closely related commercial vaccine elicited rapid, high antibody responses and conferred 100% survival. In contrast, two antigenically mismatched vaccines induced slower, lower immunity, resulting in 40-60% mortality and high virus shedding after challenge. Only the homologous vaccine sharply reduced viral shedding and significantly decreased transmission to contacts (protecting 2 of 3 sentinel birds), whereas the other vaccines failed to prevent transmission. Conclusion: An antigenically matched H5N8 vaccine with a potent adjuvant provided near-sterilizing immunity, preventing disease and significantly limiting viral shedding and transmission. These findings highlight the importance of using strain-matched vaccines in HPAI control strategies to avoid silent viral spread in vaccinated flocks. Vaccine Development Virology Immunology Avian Influenza Commercial Vaccine H5N8 Clade 2.3.4.4b Efficacy Adjuvant Transmission Chicken Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights • Antigenically matched H5N8 vaccine provided robust immunity and 100% survival post-challenge. • Mismatched H5 vaccines resulted in delayed immunity, high viral shedding, and 40–60% mortality. • Only the homologous vaccine significantly reduced virus transmission to contact birds. • Findings emphasize vaccine updates to prevent silent spread of HPAI in vaccinated flocks. • Adjuvant formulation influenced immunogenicity, underscoring its role in optimizing vaccine efficacy. 1. Introduction Highly Pathogenic Avian Influenza (HPAI) virus subtype H5N8, belonging to clade 2.3.4.4b, first emerged in China in 2016 [ 1 ] and experienced a widespread outbreak from 2020, affecting Europe, Asia, Africa, and the Americas, where it became the dominant viral variant [ 2 , 3 ]. This subtype has caused considerable economic losses in the global poultry industry. Additionally, sporadic cases of infection have been reported in various mammalian species, including foxes, bobcats, coyotes, raccoons, skunks, seals, otters, lynxes, ferrets, badgers, sea lions, dogs, cats, and cows [ 4 , 5 ], as well as in humans [ 6 ]. These occurrences highlight their potential as a serious public health threat and a possible candidate for a future pandemic [ 7 ]. Since the fall of 2020, Kazakhstan has faced numerous large-scale outbreaks of HPAI among domestic poultry, identified as the H5N8 subtype of the clade 2.3.4.4b virus [ 8 , 9 ]. The initial outbreaks were reported in densely populated areas near the Kazakhstan-Russia border. The disease had spread to 11 regions across Kazakhstan. In response, the Veterinary Service of Kazakhstan implemented measures to contain the outbreak, including enforcing strict quarantines in affected areas, imposing export bans on poultry and poultry products, and organizing rapid vaccination campaigns for poultry [ 9 ]. Our research group from the outbreak in Almaty Province isolated from chicken ( G. gallus domesticus ) and characterized a genetically variant A/chicken/Kazakhstan/23/2020 strain of avian influenza virus (AIV) subtype H5N8 belongs to the clade 2.3.4.4b and also confirmed its typical pathotype based on the PLREKRRRRKRGLF cleavage site in the hemagglutinin (HA) gene. The success of measures against HPAI is largely dependent on vaccination, which in recent years has been widely implemented in more than 30 countries affected by this infection [ 10 ]. In Kazakhstan’s poultry industry, several types of injectable vaccines are currently used for bird immunization against HPAI. These vaccines are registered in the state register of veterinary drugs and feed additives under the Ministry of Agriculture of the Republic of Kazakhstan and within the customs territory of the Eurasian Economic Union (including Russia, Belarus, Kyrgyzstan, and Armenia). However, the protective efficacy of these vaccines against the circulating HPAI virus in Kazakhstan, specifically the H5N8 subtype of clade 2.3.4.4b, has not yet been studied. Despite the widespread immunization of poultry at farms across Kazakhstan, outbreaks of mortality and reduced productivity due to HPAI continue to be reported at some facilities [ 11 ]. The effectiveness of vaccination is influenced by several factors, including the type of vaccine used, the immunization protocol, the bird species, and, most critically, the antigenic similarity between the vaccine strain and the circulating field strain of the virus [ 10 ]. The antigenic mismatch between the vaccine strain and the circulating field strain is a key factor not only in the partial protection of vaccinated birds against HPAI but also in the potential emergence of vaccine-induced mutant strains capable of asymptomatic transmission among birds [ 12 , 13 ]. While mortality data and quantification of viral shedding from directly infected birds post-vaccination are commonly used to evaluate vaccine efficacy, these measures do not necessarily account for the potential of virus transmission [ 14 ]. Currently, data on the efficacy of commercial inactivated influenza vaccines, the most widely used type, against viral transmission of the HPAI clade 2.3.4.4b virus are either unavailable or scarce in the literature. Considering this, the present study aimed to evaluate the immunogenicity and protective efficacy, including protection against viral transmission, of three commercial inactivated vaccines produced by global, regional, and local manufacturers. A vaccine formulated with a strain homologous to the field virus was included as a reference product for comparison. 2. Materials and methods 2.1 Facility and Biosafety Statement The experiments were conducted following the biosafety protocols detailed in our previous publication [ 11 ]. Briefly, all work with the infectious HPAI virus was carried out in BSL-3 and ABSL-3 facilities at the M. Aikimbayev National Scientific Center for Especially Dangerous Infections (NSCEDI), Ministry of Health of the Republic of Kazakhstan. These ISO 35001:2019-accredited facilities adhered to strict biorisk management protocols, including the use of powered air-purifying respirators (PAPRs), comprehensive decontamination procedures, and regular staff training. At the conclusion of the experiments, all biohazardous materials were autoclaved and incinerated to ensure safety. 2.2 Ethics statement All animal experiments were conducted in strict accordance with the ARRIVE guidelines, following the U.K. Animals (Scientific Procedures) Act, 1986, and related regulations, as well as the EU Directive 2010/63/EU. The animals' sex was recorded, and any potential impact or correlation of sex with the study's outcomes was thoroughly analyzed and documented. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of NSCEDI under Protocol No. 17, dated November 1, 2022. The birds were housed in specialized cages (3 chickens/m²) within a facility maintained at 22–23°C with 35–45% humidity and an air exchange rate of at least 16 times per hour. They were kept on deep bedding with drinkers, which were regularly monitored and replaced, and had unrestricted access to feed. Each group of birds was housed separately in designated rooms within the ABSL-3 laboratory of the Central Reference laboratory (CRL). Daily veterinary supervision was provided to ensure the birds' health, and conditions were carefully managed to meet their physiological and behavioral needs. Any factors causing stress or discomfort were promptly addressed. After HPAI challenge, all surviving chickens were humanely euthanized with sodium pentobarbital (5 g/mL). Humane endpoint criteria for post-infection included a body weight loss of 35% or more or the inability to maintain an upright position. 2.3 Viruses, commercial vaccines, cell culture, and birds The influenza virus strain A/chicken/Kazakhstan/23/2020 (H5N8; clade 2.3.4.4b; GISAID accession number: EPI_ISL_13632084; GenBank accession numbers: ON943051, ON943052, ON943053, ON943054, ON943055, ON943056, ON943057, and ON943058) was isolated in 10-day-old SAN embryonated chicken eggs (ECEs) from the tracheal swab collected from affected domestic chicken during the HPAI outbreak in Almaty Province in 2020 [ 15 ]. The 50% egg infective dose (EID 50 ) of the virus was measured and aliquots of allantoic fluid were stored at − 80°C until use. The three most commonly used commercially available vaccines, registered in the state register of veterinary drugs and feed additives under the Ministry of Agriculture of the Republic of Kazakhstan and within the customs territory of the Eurasian Economic Union (EAEU), were procured from poultry vaccine distributors in Kazakhstan (Table 1 ). The commercial bivalent vaccine, Volvac® B.E.S.T. AI + ND (Volvac; Baculovirus Expression System Technology), targets avian influenza subtype H5 and Newcastle disease. This vaccine is based on a recombinant baculovirus produced in insect cells, with its hemagglutinin (HA) derived from the A/duck/China/E319-2/03 (H5N1, clade 2.3.2) strain. Additionally, two commercial and one experimental inactivated oil-adjuvanted vaccines were included in the study. One commercial vaccine, produced by RIBSP, is formulated using the recombinant strain IDCDC-RG43A, developed through reverse genetics (H5N8, clade 2.3.4.4c), while Flu Protect H5 is based on a HPAI H5N1 strain (clade 2.2). The experimental homologous vaccine, developed by KazNARU, was prepared using the HPAI H5N8 virus A/chicken/Kazakhstan/23/2020 (clade 2.3.4.4b). Table 1 List of H5 commercial and experimental vaccines used for the immunization of poultry against HPAI in Kazakhstan and Eurasian Economic Union countries Vaccine trade name Virus strain used Lineage Manufacturer, Country HA nucleotide sequence % similarity to Kazakh H5N8 Flu Protect H5 inactivated emulsified avian influenza vaccine A/duck/Novosibirsk/02/05 (H5N1) Clade 2.2 Stavropol Biofactory, Russia 90 Volvac® B.E.S.T. AI + ND inactivated vaccine against avian influenza and Newcastle disease A/duck/China/E319-2/2003 (H5N1) + ND (LaSota) Clade 2.3.2 Boehringer Ingelheim, Mexico 92 RIBSP inactivated emulsified avian influenza vaccine A/gyrfalcon/Washington/41088-6/2014(H5N8)-PR8-IDCDC-RG43A Clade 2.3.4.4c RIBSP, Kazakhstan 93 X-vac1 experimental homologous inactivated emulsified avian influenza vaccine A/chicken/Kazakhstan/23/2020 (H5N8) Clade 2.3.4.4b KazNARU, Kazakhstan 100 Madin-Darby Canine Kidney (MDCK) cells (ATCC® CCL-34™, NBL-2) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, UK) and antibiotics (penicillin at 100 units/mL and streptomycin at 100 µg/mL; Gibco, UK). The cells were maintained at 37°C in a 5% CO 2 incubator. To determine viral titers from tracheal and cloacal swab samples collected post-challenge, MDCK cells seeded in 96-well tissue culture plates were utilized. Viral titers were quantified using the Reed and Muench method [ 16 ] and expressed as log 10 TCID 50 /0.2 mL. Three-week-old specific antibody negative (SAN) White Leghorn chickens ( G. gallus domesticus ), sourced from a commercial poultry farm in Almaty, Kazakhstan, were used for this experiment. The birds had not received any vaccinations at the farm and underwent serological testing upon arrival at the ABSL-3 facility. Testing was conducted using the hemagglutination inhibition (HI) assay with 0.5% chicken red blood cells (RBCs). All chickens were confirmed seronegative for H5 avian influenza virus (AIV). 2.4 Preparation of experimental vaccine formulation The HPAI H5N8 virus A/chicken/Kazakhstan/23/2020 (clade 2.3.4.4b) was inoculated at a dose of 10³ EID₅₀ in 0.1 mL into the allantoic cavity of 10-day-old SAN embryonated chicken eggs (ECEs) and incubated at 36°C in a BSL-3 facility. At 36 h post-infection, the allantoic fluids were harvested and clarified by centrifugation at 1,800× g for 30 min at 4°C using a centrifuge with aerosol-tight caps to ensure the safe handling of hazardous samples. The clarified allantoic fluid was then titrated using the EID₅₀ and hemagglutination assays. To prepare the X-vac1 experimental homologous inactivated vaccine, the A/chicken/Kazakhstan/23/2020 virus was treated with 0.1% formaldehyde (Sigma, Germany) for 30 h at 37°C. Formaldehyde neutralization was performed by adding sodium bisulfite (NaHSO₃) to a final concentration of 0.4%. Complete virus inactivation was confirmed by inoculating treated samples into the allantoic cavity of 10-day-old ECEs, followed by HA testing using 1% chicken RBCs. The inactivated experimental vaccine strain, referred to as "Antigen" was emulsified with Montanide ISA 78 VG (Seppic, France) for subcutaneous (SC) administration. To prepare the experimental vaccine formulation, the aqueous antigen phase (256 HA units) and Montanide ISA 78 VG adjuvant were mixed at a 70:30 weight ratio. Mixing was performed using an IKA Ultra Turrax® Tube Drive Basic high-shear stirrer (Ref. 3646000; IKA, Germany) equipped with a DT-20 rotor-stator insert tube (Ref. 3703100; IKA, Germany) with a working volume of 2–15 mL. The adjuvant was first added to the DT-20 rotor-stator tube, and the aqueous antigen phase was carefully introduced without stirring to maintain the emulsion temperature below 20°C. The pre-emulsification step was performed by docking the tube and mixing at 1,100 rpm (speed level "3") for 2 min. During the emulsification step, the speed was increased to 4,000 rpm (speed level "9") and mixing continued for 10 min. The final emulsified formulation was dispensed into sterile 10 mL vials, sealed, and stored at 4°C until further testing. 2.5 Study design Fifty SAN White Leghorn chickens were used to assess the immunogenicity, efficacy, and transmissibility of the commercial HPAI vaccines in comparison with experimental vaccine homologous to the H5N8 field virus, administered via parenteral route in a single immunization regimen in chickens according to the study design (Table 2 ). Table 2 Study design of immunogenicity, efficacy, and transmissibility of the commercial HPAI vaccines in comparison with experimental vaccine homologous to the H5N8 field virus Flu Protect H5 Subtype (Clade): H5N1 (2.2) Adjuvant: Oil-based Volvac® B.E.S.T. AI + ND Subtype (Clade): recH5 (2.3.2) Adjuvant: Oil-based RIBSP Subtype (Clade): RG-H5N8 (2.3.4.4c) Adjuvant: Oil-based X-vac 1 Subtype (Clade): H5N8 (2.3.4.4b) Adjuvant: Oil-based Negative control : PBS Adjuvant: None Volume of vaccine or negative control administered 0.5 mL 0.5 mL 0.5 mL 0.5 mL 0.5 mL Single immunization schedule SC SC SC SC SC Number of chickens per group (SAN White Leghorn) N = 7 N = 7 N = 7 N = 7 N = 7 A 28-day clinical follow-up with weekly weight monitoring N = 7 N = 7 N = 7 N = 7 N = 7 Blood test for anti-hemagglutinin antibodies 0, 7, 14, 21 and 28 days after vaccination (immunogenicity) N = 7 N = 7 N = 7 N = 7 N = 7 Intranasal challenge of chickens with virulent strain A/chicken/Kazakhstan/23/2020 (H5N8; clade 2.3.4.4b) at 30 days after vaccination and 10 days of clinical observation with daily body temperature monitoring N = 5 N = 5 N = 5 N = 5 N = 5 Introduction of contact chickens 12 h after the challenge (transmissibility) and 10 days of clinical observation with daily body temperature monitoring N = 3 N = 3 N = 3 N = 3 N = 3 Collection of tracheal and cloacal swabs on days 1, 3, and 5 after challenge N = 5 N = 5 N = 5 N = 5 N = 5 Collection of tracheal and cloacal swabs on days 2 and 4 after the introduction of contact chickens N = 3 N = 3 N = 3 N = 3 N = 3 recH5 – recombinant baculovirus recH5 encoding HA of the A/dk/China/E319-2/2003 (H5N1) strain of HPAI virus belonging to clade 2.3.2 (Genbank accession AY518362.1); RG-H5N8 – reverse genetics, candidate vaccine strain IDCDC-RG43A (H5N8; clade 2.3.4.4b); ISA-78 VG – water-in-oil emulsion adjuvant Montanide ISA 78 VG, developed by SEPPIC; PBS – phosphate-buffered saline; SC – subcutaneous; SAN – specific antibody negative. The chickens at the age of 3 weeks were divided into 5 experimental groups by randomization with 7 White Leghorns in each group on single subcutaneous immunization regimen (Groups 1–4). In Group 5 (negative control), PBS was administered instead of the vaccine subcutaneously. 2.5.1 Sample collection Blood samples were collected from the wing vein for antibody analysis at 0-, 7-, 14-, 21-, and 28-days post vaccination in the vaccinated and control groups. After blood clotting the samples were centrifuged at 5000×g for 10 min at 4°C to collect serum, which was stored in aliquots at − 80°C until tested. The tracheal and cloacal swab samples were collected at 1-, 3-, and 5-days post-challenge and 2-, 4-days post-contact (virus transmission study) in all groups (Table 2 ). The swab samples were resuspended in 1 mL of DMEM (Gibco, UK) supplemented with 2000 mg/mL streptomycin, and 2000 IU/mL penicillin. The suspensions were centrifuged at 3000×g for 10 min, and 0.2 mL of the supernatants from the tracheal or cloacal swabs were used to inoculate the MDCK cells. 2.5.2 Immunogenicity The immunogenicity of the commercial vaccines and experimental formulation in chickens was determined by hemagglutination inhibition (HI) assay to measure anti-hemagglutinin antibody levels. The HI assay was performed using the following standard protocol [ 17 ]. Briefly, cholera filtrate was used as a receptor-destroying enzyme (RDE) according to the WHO protocol [ 18 ] to remove innate inhibitors from the serum that could interfere with the assay. The serum was then heated to 56°C for 30 min to remove nonspecific hemagglutination inhibition factors and to inactivate the cholera filtrate. The RDE-treated serum samples (25 µL) were diluted twofold with PBS (25 µL) in 96-well V-bottom plates and incubated with 4 HA units (HAU) of the experimental vaccine strain A/chicken/Kazakhstan/23/2020 (H5N8) for 30 min at room temperature (RT). Then, 50 µL of a 1% suspension of RBCs was added to each well and incubated at RT for 30 min for the readout. The HI titer was expressed as the reciprocal (log 2 titers) of the highest serum dilution that completely inhibited hemagglutination. According to the WOAH Terrestrial Manual [ 19 ], serum HI titers ≥ 1:32 (≥ 5 log₂) in 80% of vaccinated chickens are considered the seroprotective threshold for preventing mortality, whereas HI titers ≥ 1:128 (≥ 7 log₂) in 80% of vaccinated birds indicate seroprotection against virus replication and shedding [ 20 ]. The limit of detection was at dilution 1:8 (3 log 2 ). 2.5.3 Efficacy To test the efficacy of the commercial vaccines and experimental formulation, 28 days after immunization, 5 chickens from each group were transferred to the ABSL-3 facility and challenged at day 30 with a dose of 10 6 EID 50 of the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI in a volume of 500 µL by the intranasal (IN) route. Virus back-titration was performed in ECEs immediately following inoculation, confirming that birds received 10 6 EID 50 . The chickens were monitored daily for 10 days for clinical signs through visual inspection. The efficacy (protection) of the vaccine was calculated according to the following equation [ 21 ]: Protection (%) = \(\:\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{s}\text{u}\text{r}\text{v}\text{i}\text{v}\text{a}\text{l}\text{s}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{u}\text{m}\text{b}\text{e}\text{r}\:\text{o}\text{f}\:\text{c}\text{h}\text{a}\text{l}\text{l}\text{e}\text{n}\text{g}\text{e}\text{d}\:\text{b}\text{i}\text{r}\text{d}\text{s}\:}\) × 100 Tracheal and cloacal swabs were collected from each bird on days 1, 3, and 5 after challenge to measure the level of viral shedding across the different groups by calculating the tissue culture infectious dose 50% (TCID 50 ) per 1 mL of swab sample in 96-well plates of MDCK cells. After incubation at 37°C for 120 h plates were observed daily for the presence of cytopathic effect (CPE) by means of an inverted optical microscope, then the cell supernatants were harvested and transferred to V-bottom 96-wells plates. The presence of the virus was detected using a hemagglutination assay [ 22 ]. The endpoint titers were calculated according to the Reed and Muench method [ 23 ] based on six replicates for titration. Virus titers are expressed as log 10 TCID 50 /mL. 2.5.4 Virus transmission To evaluate virus transmission, three sentinel chickens were cohoused into a new, clean cage 12 hours post-challenge for each experimental group. These birds were housed under the same conditions and monitored for 10 days for clinical signs through visual inspection. To assess viral shedding, tracheal and cloacal swabs were collected from each sentinel chicken on days 2 and 4 post-cohabitation. Viral titers were quantified by calculating the TCID₅₀ per 1 mL of swab sample using 96-well plates of MDCK cells. 2.6 Statistical Analysis GraphPad Prism 10.0.0 (GraphPad Software, San Diego, CA, USA) was used for preparing graphs and statistical analysis of the experimental data. Differences in antibody titers, viral load in swabs between bird groups were assessed using Tukey's multiple-comparisons test or Tukey's honestly significant difference (HSD) test. To determine the p-values for survival rate comparisons among the groups, Fisher’s exact test was used. The detection limit of the infectivity titer was 0.7 log 10 TCID 50 /mL. For all comparisons, P < 0.05 was considered a significant difference. 3. Results 3.1 Humoral immune response and seroprotection dynamics induced by commercial and experimental vaccines in chickens The immunogenicity of the commercial vaccines and experimental formulation in chickens was evaluated by measuring anti-hemagglutinin (HA) antibody levels at 7, -14, 21, and 28 days post-single immunization (Fig. 1 ). By day 7, early HI antibody responses were detected in the RIBSP and X-vac 1 groups, with 40% and 80% seroprotection against mortality, respectively. On the same day, only the X-vac 1 group exhibited a 40% seroprotection rate against virus shedding. By day 14, both the RIBSP and X-vac 1 groups achieved 100% seroprotection against virus shedding, which was maintained at 21- and 28-days post-immunization. In contrast, the Flu Protect H5 and Volvac groups demonstrated only 20% seroprotection at this time point. By day 21, the Flu Protect H5 group exhibited an increase in seroprotection against mortality to 60%, while seroprotection against virus shedding reached 20%, similar to that observed in the Volvac group. By day 28, seroprotection against virus shedding reached 80% in the Flu Protect H5 group and 20% in the Volvac group. Additionally, the Volvac group demonstrated 20% seroprotection against mortality. These findings indicate that the X-vac 1 and RIBSP vaccines induced the most robust and rapid immune responses, while Flu Protect H5 and Volvac exhibited delayed and suboptimal immunogenicity. The X-vac 1 and RIBSP groups exhibited significantly higher HI antibody levels (P < 0.0001) on days 14, 21, and 28 post-vaccination compared to the Control (PBS), Flu Protect H5, and Volvac groups. Notably, in the X-vac 1 group, HI antibody levels were slightly higher (P = 0.0328) than those in the RIBSP group only at 7 days post-vaccination. In the Volvac group, a modest increase in HI antibody titers (P = 0.0145) was observed compared to the Control (PBS) group only at 28 days post-vaccination. Overall, the X-vac 1 and RIBSP commercial vaccines demonstrated the strongest and earliest immune responses, while Flu Protect H5 and Volvac showed delayed and lower immunogenicity. Given these findings, further studies on vaccine efficacy and virus transmission, as outlined in the following sections, are necessary to assess their protective potential. 3.2 Protective efficacy and viral shedding dynamics in chickens vaccinated with commercial and experimental H5Nx vaccines To assess the efficacy of vaccines, chickens at 30 days post single-immunization were transferred to our ABSL-3 facility and challenged with 10⁶ EID 50 of the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI via the IN route. Birds were monitored for 10 days to assess clinical signs through visual inspection and mortality rates. Vaccine efficacy was evaluated based on survival rates (Fig. 2 ). All chickens vaccinated with the RIBSP commercial vaccine and X-vac 1 experimental vaccine formulation survived until 10 days post-infection without showing clinical signs of avian influenza virus (AIV) infection. In contrast, chickens vaccinated with the remaining commercial vaccines succumbed to viral infection, with mortality rates ranging from 40% (Volvac) to 60% (Flu Protect H5), although clinical signs of AIV infection were milder compared to unvaccinated chickens (Control, PBS). Nevertheless, these groups exhibited significantly higher survival rates compared to the Control group (Flu Protect H5: P = 0.0152; Volvac: P = 0.0048). Moreover, both RIBSP and X-vac 1 groups demonstrated significantly improved survival rates compared to Flu Protect H5 (P = 0.0021, P = 0.0013) and Volvac (P = 0.0009, P = 0.0006), respectively. To investigate the capability of different commercial vaccines and experimental vaccine formulation to control viral shedding after challenge infection with the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI, virus shedding in tracheal (Fig. 3 A) and cloacal (Fig. 3 B) swabs was measured at days 1, 3, and 5 post-infection. In the unvaccinated chickens (Control, PBS), the virus was detected in both tracheal and cloacal swabs on days 1 and 3 post-challenge. The mean viral titers in tracheal swabs were 4.2 log₁₀ EID₅₀/0.2 mL on day 1 (detected in 4/5 chickens) and 5.0 log₁₀ EID₅₀/0.2 mL on day 3 (detected in 5/5 chickens). The mean viral titers in cloacal swabs were 3.9 log₁₀ EID₅₀/0.2 mL on day 1 (detected in 3/5 chickens) and 4.2 log₁₀ EID₅₀/0.2 mL on day 3 (detected in 4/5 chickens). No virus was detected in tracheal or cloacal swabs collected from chickens vaccinated with the X-vac 1 experimental vaccine on days 3 and 5 post-infection. However, on day 1 post-challenge, the virus was recovered from a single tracheal and a single cloacal swab, each with a titer of 2.2 log₁₀ EID₅₀/0.2 mL. By day 4 post-challenge, all control group chickens succumbed to infection, preventing further viral titer analysis on day 5. On day 3 post-infection, viral titers in tracheal swabs from the experimental vaccine group were significantly lower compared to the Control group (P = 0.0383). The RIBSP vaccine provided 100% protection against mortality, but virus shedding was detected in tracheal swabs on days 1, 3, and 5 post-infection (mean titers: 5.2, 4.5, and 2.2 log₁₀ EID₅₀/0.2 mL, respectively) and in cloacal swabs (mean titers: 3.2, 5.2, and 2.2 log₁₀ EID₅₀/0.2 mL, respectively). Tracheal shedding on days 1, 3, and 5 post-challenge was detected in chickens vaccinated with Flu Protect H5 (40% protection) and Volvac (60% protection). Mean viral titers for Flu Protect H5 were 3.2 (3/5), 3.9 (4/5), and 7.2 (2/4) log₁₀ EID₅₀/0.2 mL, while for Volvac, titers were 7.7 (2/5), 2.2 (2/5), and 3.7 (4/5) log₁₀ EID₅₀/0.2 mL, respectively. Cloacal shedding followed a similar pattern, with mean titers of 2.2 (2/5), 7.2 (1/5), and 4.2 (3/5) log₁₀ EID₅₀/0.2 mL for Flu Protect H5 and 2.2 (2/5), 8.2 (1/5), and 3.7 (4/5) log₁₀ EID₅₀/0.2 mL for Volvac. The results demonstrate that the RIBSP commercial vaccine and the X-vac 1 experimental formulation provided 100% protection against mortality following HPAI (H5N8) challenge, with X-vac 1 showing superior control of viral shedding. In contrast, other commercial vaccines exhibited partial protection, reducing clinical signs but failing to prevent virus shedding, with mortality rates reaching 40–60%. Notably, X-vac 1 significantly reduced viral shedding, suggesting its potential as a more effective intervention. To fully assess the impact of these vaccines on disease transmission, further studies on virus transmission dynamics are necessary, as detailed in the following section. 3.3 Transmission potential of vaccinated birds to sentinel chickens To evaluate the effect of vaccination on virus transmission, three sentinel chickens were introduced into a separate, clean cage 12 hours post-challenge for each experimental group. Only birds immunized with the X-vac 1 experimental vaccine exhibited partial prevention of virus transmission, as evidenced by a 66.6% survival rate among sentinel chickens (Fig. 4 ), with one bird succumbing on day 4 post-cohabitation. Viral shedding was detected in only one of three sentinel chickens in the X-vac 1 group, with virus titers of 2.2 and 4.2 log₁₀ TCID₅₀/0.2 mL in tracheal swabs on days 2 and 4, respectively (Fig. 5 A). Cloacal shedding was observed only on day 2, with a titer of 3.2 log₁₀ TCID₅₀/0.2 mL (Fig. 5 B). Additionally, on day 4 post-cohabitation, X-vac 1-immunized sentinels exhibited significantly lower viral titers in tracheal swabs compared to unvaccinated contact chickens (P = 0.0232). These findings suggest that while X-vac 1 reduced viral shedding and transmission, however, it did not fully prevent it. By contrast, sentinels housed with unvaccinated, infected chickens (Control group) demonstrated clear evidence of virus transmission, as indicated by a 0% survival rate (mean death time (MDT) = 4.0 days). High viral titers were detected in their tracheal swabs (6.20 ± 0.82 and 8.53 ± 0.94 log₁₀ TCID₅₀/0.2 mL) and cloacal swabs (4.53 ± 3.29 and 3.86 ± 1.69 log₁₀ TCID₅₀/0.2 mL) on days 2 and 4, respectively. Control sentinels also exhibited clinical signs of AIV infection (data not shown). Similarly, sentinels in all vaccinated groups (Flu Protect H5, Volvac, and RIBSP) demonstrated virus transmission, with viral titers in tracheal swabs ranging from 1.20 ± 0.70 to 6.03 ± 3.79 log₁₀ TCID₅₀/0.2 mL on day 2 and 1.87 ± 1.65 to 8.53 ± 0.94 log₁₀ TCID₅₀/0.2 mL on day 4. Cloacal shedding was also detected, with titers ranging from 1.53 ± 1.18 to 5.20 ± 2.00 log₁₀ TCID₅₀/0.2 mL on day 2 and 1.20 ± 0.70 to 3.20 ± 0.00 log₁₀ TCID₅₀/0.2 mL on day 4. The MDT of sentinels housed with Volvac-vaccinated birds (MDT = 7.0 days) was significantly longer than that of sentinels in the Flu Protect H5 (MDT = 3.3 days, P = 0.0340) and RIBSP (MDT = 2.7 days, P = 0.0277) groups. However, no significant difference in MDT was observed between the Control group and any of the vaccinated groups. The RIBSP group had the shortest MDT, but this difference was not statistically significant compared to the Control and Flu Protect H5 groups. These findings suggest that Volvac vaccination delayed mortality, while other vaccines had less impact on survival. X-vac 1 showed promising results, reducing viral shedding and improving survival, though none fully prevented transmission, warranting further investigation. 4. Discussion HPAI H5Nx viruses of clade 2.3.4.4b (exemplified by H5N8) have spread explosively across the globe in recent years, causing devastating poultry outbreaks in Europe, Asia, Africa, and beyond​ [ 1 – 3 , 6 , 24 ]. Kazakhstan experienced its first large-scale incursions of H5N8 (clade 2.3.4.4b) in 2020, with the virus spreading to multiple regions despite control measures​ [ 8 , 9 ]. These events underscore an urgent need for effective vaccination strategies against this continually evolving lineage. Vaccination is increasingly viewed as a critical tool for HPAI control, provided that vaccines are antigenically well-matched to circulating strains​ [ 10 , 12 ]. An effective H5 vaccine must not only prevent disease in immunized birds but also curtail virus shedding and onward transmission​ [ 10 , 25 ]. Achieving such sterilizing immunity is essential to halt virus circulation, given that HPAI outbreaks can otherwise persist via silent transmission in vaccinated flocks​ [ 26 ]. In this context, our study evaluated the efficacy of several H5 vaccines against a clade 2.3.4.4b H5N8 challenge in chickens, yielding important insights into antigenic match, adjuvant effects, and transmission dynamics. Our findings demonstrate a clear link between antigenic matching and vaccine performance. The experimental H5N8 (clade 2.3.4.4b) vaccine formulated from a homologous strain (X-vac 1) induced a rapid and robust immune response, achieving full (100%) protection against mortality upon challenge. In parallel, a commercial vaccine (RIBSP) based on a closely related H5 seed strain provided complete protection from death. In contrast, two other commercial vaccines (Flu Protect H5 and Volvac), whose H5 antigens were antigenically more distant from the challenge virus, conferred only partial protection. Birds vaccinated with these mismatched vaccines showed significant mortality (up to 40–60% in our trials) and developed moderate clinical signs post-challenge, whereas all chickens in the X-vac 1 and RIBSP groups survived without signs. These outcome disparities correlated with immunogenicity: by two weeks post-immunization, the X-vac 1 and RIBSP groups had elicited high hemagglutination-inhibition antibody titers, meeting WOAH’s efficacy benchmark of ≥ 80% protective seroconversion [ 19 , 20 ]. Notably, HI titers in the X-vac 1 group were slightly higher than those in the RIBSP group, reflecting the closer antigenic match. In comparison, Flu Protect H5 and Volvac elicited significantly lower and slower antibody responses, with many birds not reaching protective titer thresholds until four weeks post-vaccination​. Overall, the antigenically matched vaccines (X-vac 1 and to a lesser extent RIBSP) stimulated the strongest and earliest immunity, whereas the more divergent vaccines lagged in both magnitude and timing of response. These results underscore that even within the same clade, subtle genetic differences can translate into major differences in protective immunity, reinforcing antigenic matching as a critical factor for H5 vaccine efficacy​ [ 27 ]. Apart from antigenic relatedness, our data suggest that vaccine formulation, particularly adjuvant type, also influenced efficacy. All vaccines in this study were oil-emulsified inactivated formulations, but the potency of the immune response varied, likely reflecting differences in adjuvant composition or dose. The X-vac 1 vaccine was formulated with a high-quality water-in-oil adjuvant (Montanide ISA-78 VG), which is known to enhance immunogenicity [ 11 , 28 , 29 ]. This may have contributed to the rapid antibody rise and strong protection observed in the X-vac 1 group. Consistent with this, recent studies have shown that the choice of adjuvant can significantly alter H5 vaccine outcomes​ [ 29 – 31 ]. For example, a single immunization with an H5 virus-like particle vaccine protected chickens against diverse H5 strains only when combined with an appropriate adjuvant and dosage​ [ 22 , 32 ]. In our study, even though the RIBSP and X-vac 1 vaccines both used oil-based adjuvants, X-vac 1 achieved slightly better control of virus replication, hinting that Montanide (or a higher antigen dose) provided an edge. Conversely, the Volvac vaccine, which induced the weakest antibody response, might have utilized a less potent formulation or lower antigen content. These findings highlight that adjuvants are not interchangeable, the specific adjuvant (and its formulation) can profoundly impact the quality of the immune response, and thus the degree of protection, afforded by an H5 vaccine. Future vaccine development and evaluation should therefore weigh adjuvant selection as heavily as antigen selection to maximize efficacy. A paramount goal of HPAI vaccination is to reduce viral shedding from infected hosts, thereby limiting transmission [ 26 , 33 , 34 ]. In our challenge experiments, only the homologous X-vac 1 vaccine achieved substantial suppression of viral shedding. X-vac 1-immunized chickens had either no detectable virus or markedly lower titers in tracheal and cloacal swabs after challenge, indicating that this vaccine nearly achieved sterilizing immunity in most individuals. In stark contrast, chickens vaccinated with the less-matched commercial vaccines shed high levels of virus from both the respiratory tract and gastrointestinal tract, comparable to or only marginally lower than unvaccinated controls in the first days post-challenge. Even the RIBSP vaccine, while fully protective against death, did not completely prevent virus replication, as low to moderate titers of challenge virus were recovered from RIBSP-vaccinated birds. Thus, a gap emerged between clinical protection (prevention of illness/death) and virological protection (prevention of infection/shedding) for the mismatched vaccines. This finding is in line with field observations in Egypt where most commercial H5 vaccines protected chickens from mortality but failed to stop virus shedding when challenged with an emergent H5N8 strain​ [ 12 ]. The danger of such an outcome is that vaccinated flocks may appear healthy yet continue to propagate the virus. Indeed, if a vaccine mitigates disease signs but does not block transmission, HPAI can spread silently and even generate new escape variants under immune pressure​ [ 12 ]. Our results empirically reinforce this concern: only the vaccine with optimal antigenic match significantly curtailed shedding, whereas suboptimal vaccines would likely allow onward transmission of H5N8 in a flock or farm setting. To directly address the issue of transmission, we incorporated a sentinel bird experiment. Unvaccinated contact chickens housed with infected birds universally became infected and succumbed, affirming the high transmissibility of the challenge strain. Sentinels exposed to vaccinated, infected birds fared only marginally better in most groups: in the Flu Protect H5, Volvac, and RIBSP groups, all contact birds eventually contracted fatal infections, indicating that those vaccines did not halt bird-to-bird spread. Only in the X-vac 1 group transmission partially impeded, two out of three sentinel chickens were completely protected (surviving and remaining virus-negative), and only one became infected, with a delayed infection timeline. This 66.6% reduction in transmission in the X-vac 1 group is a noteworthy outcome, given that no current inactivated H5 vaccine is expected to entirely prevent viral shedding after a single dose. The X-vac 1 formula’s ability to diminish shedding and confer some herd immunity suggests that improved vaccines can contribute to transmission blockade, not just protection of individual birds. Nevertheless, even X-vac 1 did not achieve 100% transmission prevention, as one contact bird did succumb, meaning that while greatly reduced, infectious virus was still eventually emitted by some vaccinated-challenged hosts. This underscores that further enhancements (e.g. booster doses or more immunogenic platforms) would be needed to reach full transmission stoppage. Importantly, our transmission findings align with the principle that vaccines should be evaluated on their capacity to reduce shedding and secondary spread, not solely on preventing disease in vaccinated individuals​ [ 35 ]. Incorporating such transmission assessments into vaccine evaluation (as we have done) provides a more rigorous test of a vaccine’s true epidemiological impact. The outcomes of this study carry significant implications for HPAI control policies, both in Kazakhstan and globally. For Kazakhstan, where emergency vaccination has been deployed to contain H5N8 outbreaks​ [ 9 ], our data suggest that vaccine choice is crucial: using an antigenically mismatched vaccine may fail to stop the virus from spreading in poultry populations, potentially undermining control efforts. It will be important for veterinary authorities to update and match vaccine seed strains to the currently circulating viruses. Given that clade 2.3.4.4b viruses continue to evolve and new variants (like the recent H5N1 2.3.4.4b strain) have emerged [ 15 ], a dynamic vaccination strategy is warranted, one that can rapidly incorporate new antigenic variants into vaccine formulations, analogous to how seasonal influenza vaccines are updated. On a global scale, there is growing interest in adopting poultry vaccination in regions experiencing relentless H5N1/H5N8 outbreaks, as evidenced by recent recommendations from the European Food Safety Authority on HPAI vaccination strategies​ [ 10 ]. Our findings provide a timely evidence-based message for such initiatives: vaccination can be highly effective at protecting flocks and reducing virus dissemination, but only if the vaccine antigen closely matches the outbreak strain and induces sufficiently potent immunity. Using vaccines that only prevent mortality without stopping infection would risk creating reservoirs of infection in ostensibly “protected” flocks, a scenario that has played out in past HPAI epizootics [ 35 ]. ​Therefore, any vaccination program should be accompanied by robust surveillance (to detect breakthrough infections and viral evolution) and, ideally, the use of DIVA (Differentiating Infected from Vaccinated Animals) strategies to monitor field virus circulation in vaccinated populations. In light of the partial success of the best-performing vaccine in this study, future research should aim to optimize vaccine formulations for complete transmission blocking. One approach could be the use of prime-boost regimens or multiple doses to elevate mucosal and systemic immunity to levels that eliminate virus more rapidly. Another promising avenue is exploring alternative vaccine platforms: for instance, vectored vaccines have shown the ability to protect against heterologous H5Nx challenges and even prevent onward transmission​ [ 36 ]. A recombinant turkey herpesvirus-vectored H5 vaccine, for example, was reported to halt transmission of a clade 2.3.4.4b H5N8 challenge in chickens​ [ 37 ], highlighting that next-generation vaccines might achieve what conventional inactivated vaccines cannot. Additionally, novel adjuvants or delivery routes (e.g. mucosal immunization) could further enhance local immunity in the respiratory tract, which is key to stopping virus shedding at the source. Our group’s ongoing work in evaluating adjuvant combinations and mucosal vaccine delivery (Tabynov et al., 2025; In press) [ 11 ] is geared toward this goal. Ultimately, an ideal HPAI vaccine for poultry would induce fast, robust, and broad immunity that protects the bird and its flock-mates by sharply curtailing viral shedding. The limitations of this study include several factors that may affect the generalizability and robustness of the findings. One primary limitation is the challenge model itself, as only a single H5N8 strain was used for post-vaccination challenge, which may not fully represent the broad diversity of H5 avian influenza strains. This approach may limit the generalizability of findings to other clades. Furthermore, antigen-specific cytokine production, such as IFN-gamma, was not assessed in this study. The absence of an IFN-gamma ELISPOT assay limits the understanding of the functional T-cell responses elicited by the vaccines. Future studies will incorporate this assay to provide a more comprehensive evaluation of T-cell activation and its role in protective immunity. Additionally, the study was conducted with SAN chickens, which may exhibit different immune responses than poultry populations with prior exposures to low pathogenic avian influenza viruses. This factor should be considered when interpreting the real-world applicability of the vaccine efficacy results. The study also employed relatively small group sizes, potentially impacting the statistical power and robustness of the findings. Another limitation is the lack of assessment for the durability of protection, as the study focused on short-term outcomes post-vaccination. This leaves open questions about the long-term immunity provided by the vaccines. Finally, apart from HI activity, the study did not measure functional antibodies, such as neutralizing antibodies (MN). These assays are crucial for assessing the quality of immune responses and understanding the protective mechanisms of the vaccines. Future studies will incorporate MN assays to comprehensively evaluate the functional antibody responses elicited by the vaccine formulations. Together, these limitations highlight the need for future studies with larger sample sizes, repeat trials, and extended observation periods to assess the durability of protection, which would provide a more comprehensive understanding of vaccine efficacy. In conclusion , this study highlights that an inactivated H5N8 vaccine with a close antigenic match and a potent adjuvant can provide excellent protection to chickens and significantly reduce virus shedding and transmission. These findings have immediate relevance for enhancing avian influenza control programs in Kazakhstan and other affected regions. By prioritizing antigenic matching in vaccine selection and striving for transmission-blocking immunity, veterinary authorities can improve vaccine effectiveness and reduce the threat of HPAI in poultry. Such optimized vaccine strategies, combined with biosecurity and surveillance, will be critical to curtail the spread of clade 2.3.4.4b H5Nx viruses and diminish the risk of emergent variants that could impact both animal and public health. The mechanistic insights gained from our experimental trials, linking antigenic match and adjuvant-driven immune potency to real-world outcomes in infected birds, provide a strong foundation for designing better vaccines and policies to combat HPAI now and in the future. Declarations CRediT authorship contribution statement Conceptualization: KaissarT. Data curation: KairatT. Formal analysis: KaissarT, KairatT. Funding acquisition: KaissarT. Investigation: KairatT, TK, LY, MB, LY, KZ, AK, ZZ. Methodology: KaissarT, KairatT. Project administration: KaissarT. Resources: KaissarT, ZZ. Software: KaissarT, KairatT. Supervision: KaissarT. Validation: KairatT. Visualization: KaissarT, KairatT. Writing—original draft: KairatT. Writing—review and editing: KaissarT, KairatT, TK, LY, MB, LY, KZ, AK, ZZ. All authors contributed to the article and approved the submitted version. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KaissarT and KairatT are affiliated with T&TvaX. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was funded by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP19675939). Partial funding was also provided by the startup company T&TvaX LLC (Kazakhstan). The funders had no role in the study design, data collection, analysis, or interpretation, nor in the writing of the manuscript or the decision to submit it for publication. 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Vaccine. 2021 Feb 12;39(7):1072-1079. doi: 10.1016/j.vaccine.2021.01.018. Nassif S, Zaki F, Mourad A, Fouad E, Saad A, Setta A, Felföldi B, Mató T, Kiss I, Palya V. Herpesvirus of turkey-vectored avian influenza vaccine offers cross-protection against antigenically drifted H5Nx highly pathogenic avian influenza virus strains. Avian Pathol. 2020 Dec;49(6):547-556. doi: 10.1080/03079457.2020.1790502. Additional Declarations The authors declare potential competing interests as follows: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KaissarT and KairatT are affiliated with T&TvaX. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6142334","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":423094126,"identity":"a6221716-7058-4f28-83b9-902ad1da34af","order_by":0,"name":"Kairat Tabynov","email":"","orcid":"https://orcid.org/0000-0001-9411-7952","institution":"International Center for Vaccinology, Kazakh National Agrarian Research University, Almaty, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Kairat","middleName":"","lastName":"Tabynov","suffix":""},{"id":423094319,"identity":"1aaa1428-c6d8-452c-9119-41df6311ab2b","order_by":1,"name":"Aidana Kuanyshbek","email":"","orcid":"https://orcid.org/0009-0005-0967-8269","institution":"National Collection of Deposited Strains, Almaty Branch of National Reference Veterinary Center, Almaty, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Aidana","middleName":"","lastName":"Kuanyshbek","suffix":""},{"id":423094697,"identity":"95378dc0-b4ec-44cb-aa9b-7498b5ff2473","order_by":2,"name":"Kuantay Zharmambet","email":"","orcid":"https://orcid.org/0009-0004-7226-8249","institution":"International Center for Vaccinology, Kazakh National Agrarian Research University, Almaty, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Kuantay","middleName":"","lastName":"Zharmambet","suffix":""},{"id":423094698,"identity":"d525c446-b020-4f26-b21d-36f92622053d","order_by":3,"name":"Leila Yelchibayeva","email":"","orcid":"https://orcid.org/0000-0001-6895-7720","institution":"International Center for Vaccinology, Kazakh National Agrarian Research University, Almaty, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Leila","middleName":"","lastName":"Yelchibayeva","suffix":""},{"id":423094699,"identity":"4e747a46-4645-44c3-ad02-a9edc569c9a5","order_by":4,"name":"Talgat Karibayev","email":"","orcid":"https://orcid.org/0000-0003-4463-127X","institution":"Infectious Disease Diagnostic Laboratory, National Reference Veterinary Center, Astana, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Talgat","middleName":"","lastName":"Karibayev","suffix":""},{"id":423097846,"identity":"8848c09f-c675-4bab-af42-348b20cdec57","order_by":5,"name":"Maxat Berdikulov","email":"","orcid":"https://orcid.org/0000-0003-1304-0354","institution":"Infectious Disease Diagnostic Laboratory, National Reference Veterinary Center, Astana, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Maxat","middleName":"","lastName":"Berdikulov","suffix":""},{"id":423097847,"identity":"e74ec3e9-5b74-480f-8363-85e28087234f","order_by":6,"name":"Zauresh Zhumadilova","email":"","orcid":"https://orcid.org/0009-0002-7489-2737","institution":"Central Reference Laboratory, M. Aikimbayev National Scientific Center for Especially Dangerous Infections, Almaty, Kazakhstan","correspondingAuthor":false,"prefix":"","firstName":"Zauresh","middleName":"","lastName":"Zhumadilova","suffix":""},{"id":423097848,"identity":"f7fbae6c-d127-4543-868f-2c5a7af035d1","order_by":7,"name":"Kaissar Tabynov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYBACCQYGNjCDH0QkFBCjhQ2qRbIBpMWAFC0GB8AkEVok5zc/e8y7wyZx8/nViR8eGDDI84sdwK9Fmo3N3Jj3TFrithtvN0sAHWY4c3YCfi1ybAxm0rxth43NbpzdANKSYHCboBb2b2AtxjPObv5BlBZpNh6wLXIG/L3biLNFsi2n3HBuW5qcxA3ebRYJBhKE/SJx+Pi2B2/bbHj4+89uvvmjwkaeX5qAFhBg4gFrBquUIKwcBBh/gEj+A8SpHgWjYBSMgpEHABNmPvDkd6xNAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5823-1280","institution":"International Center for Vaccinology, Kazakh National Agrarian Research University, Almaty, Kazakhstan","correspondingAuthor":true,"prefix":"","firstName":"Kaissar","middleName":"","lastName":"Tabynov","suffix":""}],"badges":[],"createdAt":"2025-03-03 03:40:48","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6142334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6142334/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77687036,"identity":"d0b928de-2457-45e0-b552-c13434c19438","added_by":"auto","created_at":"2025-03-04 09:09:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":860484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of anti-hemagglutinin antibodies in chickens vaccinated with commercial vaccines and experimental formulation by HI assay. \u003c/strong\u003eSerum HI titers ≥1:32 (≥5 log₂) in vaccinated chickens are considered the seroprotective threshold for preventing mortality, whereas HI titers ≥1:128 (≥7 log₂) in vaccinated birds indicate seroprotection against virus shedding. The limit of detection (LoD) was at dilution 3 log\u003csub\u003e2\u003c/sub\u003e (horizontal black dotted line). Data are mean ± SD titer of 7 chickens in each group. Statistical difference between groups were assessed using the Tukey's multiple comparisons test. For all comparisons, P\u0026lt;0.05 was considered a significant difference. *P=0.0328 and 0.0145; ****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6142334/v1/e8424293e05e0cec3c91625c.png"},{"id":77689306,"identity":"c1b133cd-3117-42a0-b090-aa9260f18e54","added_by":"auto","created_at":"2025-03-04 09:25:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan-Meyer\u003c/strong\u003e \u003cstrong\u003esurvival curve of challenged chickens after immunization with commercial vaccines and experimental formulation.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6142334/v1/b05640c3273eb967182304aa.png"},{"id":77687032,"identity":"863d20b9-a03e-418a-8f66-85616716c278","added_by":"auto","created_at":"2025-03-04 09:09:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":270421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral shedding from collected tracheal and cloacal swabs of the immunized chickens at 1-, 3- and 5-days post-challenge. \u003c/strong\u003e(A) tracheal swabs and (B) cloacal swabs.\u003cstrong\u003e \u003c/strong\u003eThe immunized chickens with commercial vaccines and experimental formulation were subjected to challenge with 10⁶ EID\u003csub\u003e50\u003c/sub\u003e of the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI via the intranasal route. The limit of detection (LoD) was at titer 0.7 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e (horizontal dotted line). Data are mean ± SD titer of 5 chickens in each group. Statistical differences between groups were assessed using Tukey’s multiple-comparisons test.\u003cstrong\u003e \u003c/strong\u003eFor all comparisons, P\u0026lt;0.05 was considered a significant difference. *P= 0.0383.\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6142334/v1/ed09c5d5d33e2b7ac72eef6d.png"},{"id":77687030,"identity":"3b438064-8837-4328-b03f-fd019a1c2244","added_by":"auto","created_at":"2025-03-04 09:09:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":248103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan-Meyer\u003c/strong\u003e \u003cstrong\u003esurvival curve of sentinel chickens after challenged birds after immunization with commercial vaccines and experimental formulation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6142334/v1/f480e9120b6bf6fbd71831ad.png"},{"id":77688012,"identity":"0d051d6c-6f11-4597-b5c4-62e4ba4e447d","added_by":"auto","created_at":"2025-03-04 09:17:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":552297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViral shedding from collected tracheal and cloacal swabs of the contact sentinel chickens at 2 and 4-days post-cohabitation. \u003c/strong\u003e(A) tracheal swabs sentinels and (B) cloacal swabs sentinels.\u003cstrong\u003e \u003c/strong\u003ethree sentinel chickens were cohoused in a separate, clean cage 12 h after challenge for each experimental group. The limit of detection (LoD) was at titer 0.7 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e (horizontal dotted line). Data are mean ± SD titer of 5 chickens in each group. For all comparisons, P\u0026lt;0.05 was considered a significant difference. *P=0.0232.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6142334/v1/7a4ce6fdc0209c97da39135b.png"},{"id":77689911,"identity":"79a2c244-1595-4b63-b103-1325fd6827fc","added_by":"auto","created_at":"2025-03-04 09:33:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3495831,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6142334/v1/4a53895f-ac24-4d1e-9d60-f898d4282d85.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KaissarT and KairatT are affiliated with T\u0026TvaX. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluation of Commercial Vaccines for Efficacy and Transmission Control Against the Emergent H5N8 (Clade 2.3.4.4b) Avian Influenza Virus in Kazakhstan\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; Antigenically matched H5N8 vaccine provided robust immunity and 100% survival post-challenge.\u003c/p\u003e\u003cp\u003e\u0026bull; Mismatched H5 vaccines resulted in delayed immunity, high viral shedding, and 40\u0026ndash;60% mortality.\u003c/p\u003e\u003cp\u003e\u0026bull; Only the homologous vaccine significantly reduced virus transmission to contact birds.\u003c/p\u003e\u003cp\u003e\u0026bull; Findings emphasize vaccine updates to prevent silent spread of HPAI in vaccinated flocks.\u003c/p\u003e\u003cp\u003e\u0026bull; Adjuvant formulation influenced immunogenicity, underscoring its role in optimizing vaccine efficacy.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eHighly Pathogenic Avian Influenza (HPAI) virus subtype H5N8, belonging to clade 2.3.4.4b, first emerged in China in 2016 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and experienced a widespread outbreak from 2020, affecting Europe, Asia, Africa, and the Americas, where it became the dominant viral variant [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This subtype has caused considerable economic losses in the global poultry industry. Additionally, sporadic cases of infection have been reported in various mammalian species, including foxes, bobcats, coyotes, raccoons, skunks, seals, otters, lynxes, ferrets, badgers, sea lions, dogs, cats, and cows [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], as well as in humans [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These occurrences highlight their potential as a serious public health threat and a possible candidate for a future pandemic [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the fall of 2020, Kazakhstan has faced numerous large-scale outbreaks of HPAI among domestic poultry, identified as the H5N8 subtype of the clade 2.3.4.4b virus [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The initial outbreaks were reported in densely populated areas near the Kazakhstan-Russia border. The disease had spread to 11 regions across Kazakhstan. In response, the Veterinary Service of Kazakhstan implemented measures to contain the outbreak, including enforcing strict quarantines in affected areas, imposing export bans on poultry and poultry products, and organizing rapid vaccination campaigns for poultry [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Our research group from the outbreak in Almaty Province isolated from chicken (\u003cem\u003eG. gallus domesticus\u003c/em\u003e) and characterized a genetically variant A/chicken/Kazakhstan/23/2020 strain of avian influenza virus (AIV) subtype H5N8 belongs to the clade 2.3.4.4b and also confirmed its typical pathotype based on the PLREKRRRRKRGLF cleavage site in the hemagglutinin (HA) gene.\u003c/p\u003e \u003cp\u003eThe success of measures against HPAI is largely dependent on vaccination, which in recent years has been widely implemented in more than 30 countries affected by this infection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In Kazakhstan\u0026rsquo;s poultry industry, several types of injectable vaccines are currently used for bird immunization against HPAI. These vaccines are registered in the state register of veterinary drugs and feed additives under the Ministry of Agriculture of the Republic of Kazakhstan and within the customs territory of the Eurasian Economic Union (including Russia, Belarus, Kyrgyzstan, and Armenia). However, the protective efficacy of these vaccines against the circulating HPAI virus in Kazakhstan, specifically the H5N8 subtype of clade 2.3.4.4b, has not yet been studied. Despite the widespread immunization of poultry at farms across Kazakhstan, outbreaks of mortality and reduced productivity due to HPAI continue to be reported at some facilities [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The effectiveness of vaccination is influenced by several factors, including the type of vaccine used, the immunization protocol, the bird species, and, most critically, the antigenic similarity between the vaccine strain and the circulating field strain of the virus [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The antigenic mismatch between the vaccine strain and the circulating field strain is a key factor not only in the partial protection of vaccinated birds against HPAI but also in the potential emergence of vaccine-induced mutant strains capable of asymptomatic transmission among birds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While mortality data and quantification of viral shedding from directly infected birds post-vaccination are commonly used to evaluate vaccine efficacy, these measures do not necessarily account for the potential of virus transmission [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, data on the efficacy of commercial inactivated influenza vaccines, the most widely used type, against viral transmission of the HPAI clade 2.3.4.4b virus are either unavailable or scarce in the literature. Considering this, the present study aimed to evaluate the immunogenicity and protective efficacy, including protection against viral transmission, of three commercial inactivated vaccines produced by global, regional, and local manufacturers. A vaccine formulated with a strain homologous to the field virus was included as a reference product for comparison.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Facility and Biosafety Statement\u003c/h2\u003e \u003cp\u003eThe experiments were conducted following the biosafety protocols detailed in our previous publication [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, all work with the infectious HPAI virus was carried out in BSL-3 and ABSL-3 facilities at the M. Aikimbayev National Scientific Center for Especially Dangerous Infections (NSCEDI), Ministry of Health of the Republic of Kazakhstan. These ISO 35001:2019-accredited facilities adhered to strict biorisk management protocols, including the use of powered air-purifying respirators (PAPRs), comprehensive decontamination procedures, and regular staff training. At the conclusion of the experiments, all biohazardous materials were autoclaved and incinerated to ensure safety.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Ethics statement\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted in strict accordance with the ARRIVE guidelines, following the U.K. Animals (Scientific Procedures) Act, 1986, and related regulations, as well as the EU Directive 2010/63/EU. The animals' sex was recorded, and any potential impact or correlation of sex with the study's outcomes was thoroughly analyzed and documented. The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of NSCEDI under Protocol No. 17, dated November 1, 2022. The birds were housed in specialized cages (3 chickens/m\u0026sup2;) within a facility maintained at 22\u0026ndash;23\u0026deg;C with 35\u0026ndash;45% humidity and an air exchange rate of at least 16 times per hour. They were kept on deep bedding with drinkers, which were regularly monitored and replaced, and had unrestricted access to feed. Each group of birds was housed separately in designated rooms within the ABSL-3 laboratory of the Central Reference laboratory (CRL). Daily veterinary supervision was provided to ensure the birds' health, and conditions were carefully managed to meet their physiological and behavioral needs. Any factors causing stress or discomfort were promptly addressed. After HPAI challenge, all surviving chickens were humanely euthanized with sodium pentobarbital (5 g/mL). Humane endpoint criteria for post-infection included a body weight loss of 35% or more or the inability to maintain an upright position.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Viruses, commercial vaccines, cell culture, and birds\u003c/h2\u003e \u003cp\u003eThe influenza virus strain A/chicken/Kazakhstan/23/2020 (H5N8; clade 2.3.4.4b; GISAID accession number: EPI_ISL_13632084; GenBank accession numbers: ON943051, ON943052, ON943053, ON943054, ON943055, ON943056, ON943057, and ON943058) was isolated in 10-day-old SAN embryonated chicken eggs (ECEs) from the tracheal swab collected from affected domestic chicken during the HPAI outbreak in Almaty Province in 2020 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The 50% egg infective dose (EID\u003csub\u003e50\u003c/sub\u003e) of the virus was measured and aliquots of allantoic fluid were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eThe three most commonly used commercially available vaccines, registered in the state register of veterinary drugs and feed additives under the Ministry of Agriculture of the Republic of Kazakhstan and within the customs territory of the Eurasian Economic Union (EAEU), were procured from poultry vaccine distributors in Kazakhstan (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The commercial bivalent vaccine, Volvac\u0026reg; B.E.S.T. AI\u0026thinsp;+\u0026thinsp;ND (Volvac; Baculovirus Expression System Technology), targets avian influenza subtype H5 and Newcastle disease. This vaccine is based on a recombinant baculovirus produced in insect cells, with its hemagglutinin (HA) derived from the A/duck/China/E319-2/03 (H5N1, clade 2.3.2) strain. Additionally, two commercial and one experimental inactivated oil-adjuvanted vaccines were included in the study. One commercial vaccine, produced by RIBSP, is formulated using the recombinant strain IDCDC-RG43A, developed through reverse genetics (H5N8, clade 2.3.4.4c), while Flu Protect H5 is based on a HPAI H5N1 strain (clade 2.2). The experimental homologous vaccine, developed by KazNARU, was prepared using the HPAI H5N8 virus A/chicken/Kazakhstan/23/2020 (clade 2.3.4.4b).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of H5 commercial and experimental vaccines used for the immunization of poultry against HPAI in Kazakhstan and Eurasian Economic Union countries\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVaccine trade name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVirus strain used\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLineage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eManufacturer, Country\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHA nucleotide sequence % similarity to Kazakh H5N8\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlu Protect H5 inactivated emulsified avian influenza vaccine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA/duck/Novosibirsk/02/05 (H5N1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClade\u003c/p\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStavropol Biofactory, Russia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolvac\u0026reg; B.E.S.T. AI\u0026thinsp;+\u0026thinsp;ND\u003c/p\u003e \u003cp\u003einactivated vaccine against avian influenza and Newcastle disease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA/duck/China/E319-2/2003 (H5N1)\u0026thinsp;+\u0026thinsp;ND (LaSota)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClade\u003c/p\u003e \u003cp\u003e2.3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBoehringer Ingelheim, Mexico\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRIBSP inactivated emulsified avian influenza vaccine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA/gyrfalcon/Washington/41088-6/2014(H5N8)-PR8-IDCDC-RG43A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClade\u003c/p\u003e \u003cp\u003e2.3.4.4c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRIBSP, Kazakhstan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eX-vac1 experimental homologous inactivated emulsified avian influenza vaccine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA/chicken/Kazakhstan/23/2020 (H5N8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eClade\u003c/p\u003e \u003cp\u003e2.3.4.4b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKazNARU, Kazakhstan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMadin-Darby Canine Kidney (MDCK) cells (ATCC\u0026reg; CCL-34\u0026trade;, NBL-2) were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Gibco, UK) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, UK) and antibiotics (penicillin at 100 units/mL and streptomycin at 100 \u0026micro;g/mL; Gibco, UK). The cells were maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. To determine viral titers from tracheal and cloacal swab samples collected post-challenge, MDCK cells seeded in 96-well tissue culture plates were utilized. Viral titers were quantified using the Reed and Muench method [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and expressed as log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/0.2 mL.\u003c/p\u003e \u003cp\u003eThree-week-old specific antibody negative (SAN) White Leghorn chickens (\u003cem\u003eG. gallus domesticus\u003c/em\u003e), sourced from a commercial poultry farm in Almaty, Kazakhstan, were used for this experiment. The birds had not received any vaccinations at the farm and underwent serological testing upon arrival at the ABSL-3 facility. Testing was conducted using the hemagglutination inhibition (HI) assay with 0.5% chicken red blood cells (RBCs). All chickens were confirmed seronegative for H5 avian influenza virus (AIV).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Preparation of experimental vaccine formulation\u003c/h2\u003e \u003cp\u003eThe HPAI H5N8 virus A/chicken/Kazakhstan/23/2020 (clade 2.3.4.4b) was inoculated at a dose of 10\u0026sup3; EID₅₀ in 0.1 mL into the allantoic cavity of 10-day-old SAN embryonated chicken eggs (ECEs) and incubated at 36\u0026deg;C in a BSL-3 facility. At 36 h post-infection, the allantoic fluids were harvested and clarified by centrifugation at 1,800\u0026times; g for 30 min at 4\u0026deg;C using a centrifuge with aerosol-tight caps to ensure the safe handling of hazardous samples. The clarified allantoic fluid was then titrated using the EID₅₀ and hemagglutination assays. To prepare the X-vac1 experimental homologous inactivated vaccine, the A/chicken/Kazakhstan/23/2020 virus was treated with 0.1% formaldehyde (Sigma, Germany) for 30 h at 37\u0026deg;C. Formaldehyde neutralization was performed by adding sodium bisulfite (NaHSO₃) to a final concentration of 0.4%. Complete virus inactivation was confirmed by inoculating treated samples into the allantoic cavity of 10-day-old ECEs, followed by HA testing using 1% chicken RBCs. The inactivated experimental vaccine strain, referred to as \"Antigen\" was emulsified with Montanide ISA 78 VG (Seppic, France) for subcutaneous (SC) administration. To prepare the experimental vaccine formulation, the aqueous antigen phase (256 HA units) and Montanide ISA 78 VG adjuvant were mixed at a 70:30 weight ratio. Mixing was performed using an IKA Ultra Turrax\u0026reg; Tube Drive Basic high-shear stirrer (Ref. 3646000; IKA, Germany) equipped with a DT-20 rotor-stator insert tube (Ref. 3703100; IKA, Germany) with a working volume of 2\u0026ndash;15 mL. The adjuvant was first added to the DT-20 rotor-stator tube, and the aqueous antigen phase was carefully introduced without stirring to maintain the emulsion temperature below 20\u0026deg;C. The pre-emulsification step was performed by docking the tube and mixing at 1,100 rpm (speed level \"3\") for 2 min. During the emulsification step, the speed was increased to 4,000 rpm (speed level \"9\") and mixing continued for 10 min. The final emulsified formulation was dispensed into sterile 10 mL vials, sealed, and stored at 4\u0026deg;C until further testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Study design\u003c/h2\u003e \u003cp\u003eFifty SAN White Leghorn chickens were used to assess the immunogenicity, efficacy, and transmissibility of the commercial HPAI vaccines in comparison with experimental vaccine homologous to the H5N8 field virus, administered via parenteral route in a single immunization regimen in chickens according to the study design (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy design of immunogenicity, efficacy, and transmissibility of the commercial HPAI vaccines in comparison with experimental vaccine homologous to the H5N8 field virus\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFlu Protect H5\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSubtype (Clade):\u003c/p\u003e \u003cp\u003e\u003cem\u003eH5N1 (2.2)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAdjuvant:\u003c/p\u003e \u003cp\u003e\u003cem\u003eOil-based\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eVolvac\u0026reg; B.E.S.T. AI\u0026thinsp;+\u0026thinsp;ND\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSubtype (Clade):\u003c/p\u003e \u003cp\u003e\u003cem\u003erecH5 (2.3.2)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAdjuvant:\u003c/p\u003e \u003cp\u003e\u003cem\u003eOil-based\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eRIBSP\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSubtype (Clade):\u003c/p\u003e \u003cp\u003e\u003cem\u003eRG-H5N8 (2.3.4.4c)\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAdjuvant:\u003c/p\u003e \u003cp\u003e\u003cem\u003eOil-based\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eX-vac 1\u003c/em\u003e\u003c/p\u003e \u003cp\u003eSubtype (Clade):\u003c/p\u003e \u003cp\u003eH5N8 (2.3.4.4b)\u003c/p\u003e \u003cp\u003eAdjuvant:\u003c/p\u003e \u003cp\u003eOil-based\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eNegative control\u003c/em\u003e:\u003c/p\u003e \u003cp\u003ePBS\u003c/p\u003e \u003cp\u003eAdjuvant:\u003c/p\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eVolume of vaccine or negative control administered\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.5 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5 mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eSingle immunization schedule\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNumber of chickens per group (SAN White Leghorn)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eA 28-day clinical follow-up with weekly weight monitoring\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eBlood test for anti-hemagglutinin antibodies 0, 7, 14, 21 and 28 days after vaccination (immunogenicity)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eIntranasal challenge of chickens with virulent strain A/chicken/Kazakhstan/23/2020 (H5N8; clade 2.3.4.4b) at 30 days after vaccination and 10 days of clinical observation with daily body temperature monitoring\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eIntroduction of contact chickens 12 h after the challenge (transmissibility) and 10 days of clinical observation with daily body temperature monitoring\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eCollection of tracheal and cloacal swabs on days 1, 3, and 5 after challenge\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eCollection of tracheal and cloacal swabs on days 2 and 4 after the introduction of contact chickens\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003erecH5 \u0026ndash; recombinant baculovirus recH5 encoding HA of the A/dk/China/E319-2/2003 (H5N1) strain of HPAI virus belonging to clade 2.3.2 (Genbank accession AY518362.1); RG-H5N8 \u0026ndash; reverse genetics, candidate vaccine strain IDCDC-RG43A (H5N8; clade 2.3.4.4b); ISA-78 VG \u0026ndash; water-in-oil emulsion adjuvant Montanide ISA 78 VG, developed by SEPPIC; PBS \u0026ndash; phosphate-buffered saline; SC \u0026ndash; subcutaneous; SAN \u0026ndash; specific antibody negative.\u003c/p\u003e \u003cp\u003eThe chickens at the age of 3 weeks were divided into 5 experimental groups by randomization with 7 White Leghorns in each group on single subcutaneous immunization regimen (Groups 1\u0026ndash;4). In Group 5 (negative control), PBS was administered instead of the vaccine subcutaneously.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Sample collection\u003c/h2\u003e \u003cp\u003eBlood samples were collected from the wing vein for antibody analysis at 0-, 7-, 14-, 21-, and 28-days post vaccination in the vaccinated and control groups. After blood clotting the samples were centrifuged at 5000\u0026times;g for 10 min at 4\u0026deg;C to collect serum, which was stored in aliquots at \u0026minus;\u0026thinsp;80\u0026deg;C until tested. The tracheal and cloacal swab samples were collected at 1-, 3-, and 5-days post-challenge and 2-, 4-days post-contact (virus transmission study) in all groups (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The swab samples were resuspended in 1 mL of DMEM (Gibco, UK) supplemented with 2000 mg/mL streptomycin, and 2000 IU/mL penicillin. The suspensions were centrifuged at 3000\u0026times;g for 10 min, and 0.2 mL of the supernatants from the tracheal or cloacal swabs were used to inoculate the MDCK cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Immunogenicity\u003c/h2\u003e \u003cp\u003eThe immunogenicity of the commercial vaccines and experimental formulation in chickens was determined by hemagglutination inhibition (HI) assay to measure anti-hemagglutinin antibody levels. The HI assay was performed using the following standard protocol [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, cholera filtrate was used as a receptor-destroying enzyme (RDE) according to the WHO protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] to remove innate inhibitors from the serum that could interfere with the assay. The serum was then heated to 56\u0026deg;C for 30 min to remove nonspecific hemagglutination inhibition factors and to inactivate the cholera filtrate. The RDE-treated serum samples (25 \u0026micro;L) were diluted twofold with PBS (25 \u0026micro;L) in 96-well V-bottom plates and incubated with 4 HA units (HAU) of the experimental vaccine strain A/chicken/Kazakhstan/23/2020 (H5N8) for 30 min at room temperature (RT). Then, 50 \u0026micro;L of a 1% suspension of RBCs was added to each well and incubated at RT for 30 min for the readout. The HI titer was expressed as the reciprocal (log\u003csub\u003e2\u003c/sub\u003e titers) of the highest serum dilution that completely inhibited hemagglutination. According to the WOAH Terrestrial Manual [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], serum HI titers\u0026thinsp;\u0026ge;\u0026thinsp;1:32 (\u0026ge;\u0026thinsp;5 log₂) in 80% of vaccinated chickens are considered the seroprotective threshold for preventing mortality, whereas HI titers\u0026thinsp;\u0026ge;\u0026thinsp;1:128 (\u0026ge;\u0026thinsp;7 log₂) in 80% of vaccinated birds indicate seroprotection against virus replication and shedding [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The limit of detection was at dilution 1:8 (3 log\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3 Efficacy\u003c/h2\u003e \u003cp\u003eTo test the efficacy of the commercial vaccines and experimental formulation, 28 days after immunization, 5 chickens from each group were transferred to the ABSL-3 facility and challenged at day 30 with a dose of 10\u003csup\u003e6\u003c/sup\u003e EID\u003csub\u003e50\u003c/sub\u003e of the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI in a volume of 500 \u0026micro;L by the intranasal (IN) route. Virus back-titration was performed in ECEs immediately following inoculation, confirming that birds received 10\u003csup\u003e6\u003c/sup\u003e EID\u003csub\u003e50\u003c/sub\u003e. The chickens were monitored daily for 10 days for clinical signs through visual inspection. The efficacy (protection) of the vaccine was calculated according to the following equation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003eProtection (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{N}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{s}\\text{u}\\text{r}\\text{v}\\text{i}\\text{v}\\text{a}\\text{l}\\text{s}}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}\\:\\text{o}\\text{f}\\:\\text{c}\\text{h}\\text{a}\\text{l}\\text{l}\\text{e}\\text{n}\\text{g}\\text{e}\\text{d}\\:\\text{b}\\text{i}\\text{r}\\text{d}\\text{s}\\:}\\)\u003c/span\u003e\u003c/span\u003e \u003cem\u003e\u0026times;\u003c/em\u003e 100\u003c/p\u003e \u003cp\u003eTracheal and cloacal swabs were collected from each bird on days 1, 3, and 5 after challenge to measure the level of viral shedding across the different groups by calculating the tissue culture infectious dose 50% (TCID\u003csub\u003e50\u003c/sub\u003e) per 1 mL of swab sample in 96-well plates of MDCK cells. After incubation at 37\u0026deg;C for 120 h plates were observed daily for the presence of cytopathic effect (CPE) by means of an inverted optical microscope, then the cell supernatants were harvested and transferred to V-bottom 96-wells plates. The presence of the virus was detected using a hemagglutination assay [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The endpoint titers were calculated according to the Reed and Muench method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] based on six replicates for titration. Virus titers are expressed as log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4 Virus transmission\u003c/h2\u003e \u003cp\u003eTo evaluate virus transmission, three sentinel chickens were cohoused into a new, clean cage 12 hours post-challenge for each experimental group. These birds were housed under the same conditions and monitored for 10 days for clinical signs through visual inspection. To assess viral shedding, tracheal and cloacal swabs were collected from each sentinel chicken on days 2 and 4 post-cohabitation. Viral titers were quantified by calculating the TCID₅₀ per 1 mL of swab sample using 96-well plates of MDCK cells.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 10.0.0 (GraphPad Software, San Diego, CA, USA) was used for preparing graphs and statistical analysis of the experimental data. Differences in antibody titers, viral load in swabs between bird groups were assessed using Tukey's multiple-comparisons test or Tukey's honestly significant difference (HSD) test. To determine the p-values for survival rate comparisons among the groups, Fisher\u0026rsquo;s exact test was used. The detection limit of the infectivity titer was 0.7 log\u003csub\u003e10\u003c/sub\u003e TCID\u003csub\u003e50\u003c/sub\u003e/mL. For all comparisons, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered a significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Humoral immune response and seroprotection dynamics induced by commercial and experimental vaccines in chickens\u003c/h2\u003e \u003cp\u003eThe immunogenicity of the commercial vaccines and experimental formulation in chickens was evaluated by measuring anti-hemagglutinin (HA) antibody levels at 7, -14, 21, and 28 days post-single immunization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By day 7, early HI antibody responses were detected in the RIBSP and X-vac 1 groups, with 40% and 80% seroprotection against mortality, respectively. On the same day, only the X-vac 1 group exhibited a 40% seroprotection rate against virus shedding. By day 14, both the RIBSP and X-vac 1 groups achieved 100% seroprotection against virus shedding, which was maintained at 21- and 28-days post-immunization. In contrast, the Flu Protect H5 and Volvac groups demonstrated only 20% seroprotection at this time point. By day 21, the Flu Protect H5 group exhibited an increase in seroprotection against mortality to 60%, while seroprotection against virus shedding reached 20%, similar to that observed in the Volvac group. By day 28, seroprotection against virus shedding reached 80% in the Flu Protect H5 group and 20% in the Volvac group. Additionally, the Volvac group demonstrated 20% seroprotection against mortality.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings indicate that the X-vac 1 and RIBSP vaccines induced the most robust and rapid immune responses, while Flu Protect H5 and Volvac exhibited delayed and suboptimal immunogenicity.\u003c/p\u003e \u003cp\u003eThe X-vac 1 and RIBSP groups exhibited significantly higher HI antibody levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) on days 14, 21, and 28 post-vaccination compared to the Control (PBS), Flu Protect H5, and Volvac groups. Notably, in the X-vac 1 group, HI antibody levels were slightly higher (P\u0026thinsp;=\u0026thinsp;0.0328) than those in the RIBSP group only at 7 days post-vaccination. In the Volvac group, a modest increase in HI antibody titers (P\u0026thinsp;=\u0026thinsp;0.0145) was observed compared to the Control (PBS) group only at 28 days post-vaccination. Overall, the X-vac 1 and RIBSP commercial vaccines demonstrated the strongest and earliest immune responses, while Flu Protect H5 and Volvac showed delayed and lower immunogenicity. Given these findings, further studies on vaccine efficacy and virus transmission, as outlined in the following sections, are necessary to assess their protective potential.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Protective efficacy and viral shedding dynamics in chickens vaccinated with commercial and experimental H5Nx vaccines\u003c/h2\u003e \u003cp\u003eTo assess the efficacy of vaccines, chickens at 30 days post single-immunization were transferred to our ABSL-3 facility and challenged with 10⁶ EID\u003csub\u003e50\u003c/sub\u003e of the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI via the IN route. Birds were monitored for 10 days to assess clinical signs through visual inspection and mortality rates. Vaccine efficacy was evaluated based on survival rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll chickens vaccinated with the RIBSP commercial vaccine and X-vac 1 experimental vaccine formulation survived until 10 days post-infection without showing clinical signs of avian influenza virus (AIV) infection. In contrast, chickens vaccinated with the remaining commercial vaccines succumbed to viral infection, with mortality rates ranging from 40% (Volvac) to 60% (Flu Protect H5), although clinical signs of AIV infection were milder compared to unvaccinated chickens (Control, PBS). Nevertheless, these groups exhibited significantly higher survival rates compared to the Control group (Flu Protect H5: P\u0026thinsp;=\u0026thinsp;0.0152; Volvac: P\u0026thinsp;=\u0026thinsp;0.0048). Moreover, both RIBSP and X-vac 1 groups demonstrated significantly improved survival rates compared to Flu Protect H5 (P\u0026thinsp;=\u0026thinsp;0.0021, P\u0026thinsp;=\u0026thinsp;0.0013) and Volvac (P\u0026thinsp;=\u0026thinsp;0.0009, P\u0026thinsp;=\u0026thinsp;0.0006), respectively.\u003c/p\u003e \u003cp\u003eTo investigate the capability of different commercial vaccines and experimental vaccine formulation to control viral shedding after challenge infection with the A/chicken/Kazakhstan/23/2020 (H5N8) strain of HPAI, virus shedding in tracheal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and cloacal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) swabs was measured at days 1, 3, and 5 post-infection. In the unvaccinated chickens (Control, PBS), the virus was detected in both tracheal and cloacal swabs on days 1 and 3 post-challenge. The mean viral titers in tracheal swabs were 4.2 log₁₀ EID₅₀/0.2 mL on day 1 (detected in 4/5 chickens) and 5.0 log₁₀ EID₅₀/0.2 mL on day 3 (detected in 5/5 chickens). The mean viral titers in cloacal swabs were 3.9 log₁₀ EID₅₀/0.2 mL on day 1 (detected in 3/5 chickens) and 4.2 log₁₀ EID₅₀/0.2 mL on day 3 (detected in 4/5 chickens). No virus was detected in tracheal or cloacal swabs collected from chickens vaccinated with the X-vac 1 experimental vaccine on days 3 and 5 post-infection. However, on day 1 post-challenge, the virus was recovered from a single tracheal and a single cloacal swab, each with a titer of 2.2 log₁₀ EID₅₀/0.2 mL. By day 4 post-challenge, all control group chickens succumbed to infection, preventing further viral titer analysis on day 5. On day 3 post-infection, viral titers in tracheal swabs from the experimental vaccine group were significantly lower compared to the Control group (P\u0026thinsp;=\u0026thinsp;0.0383). The RIBSP vaccine provided 100% protection against mortality, but virus shedding was detected in tracheal swabs on days 1, 3, and 5 post-infection (mean titers: 5.2, 4.5, and 2.2 log₁₀ EID₅₀/0.2 mL, respectively) and in cloacal swabs (mean titers: 3.2, 5.2, and 2.2 log₁₀ EID₅₀/0.2 mL, respectively). Tracheal shedding on days 1, 3, and 5 post-challenge was detected in chickens vaccinated with Flu Protect H5 (40% protection) and Volvac (60% protection). Mean viral titers for Flu Protect H5 were 3.2 (3/5), 3.9 (4/5), and 7.2 (2/4) log₁₀ EID₅₀/0.2 mL, while for Volvac, titers were 7.7 (2/5), 2.2 (2/5), and 3.7 (4/5) log₁₀ EID₅₀/0.2 mL, respectively. Cloacal shedding followed a similar pattern, with mean titers of 2.2 (2/5), 7.2 (1/5), and 4.2 (3/5) log₁₀ EID₅₀/0.2 mL for Flu Protect H5 and 2.2 (2/5), 8.2 (1/5), and 3.7 (4/5) log₁₀ EID₅₀/0.2 mL for Volvac.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results demonstrate that the RIBSP commercial vaccine and the X-vac 1 experimental formulation provided 100% protection against mortality following HPAI (H5N8) challenge, with X-vac 1 showing superior control of viral shedding. In contrast, other commercial vaccines exhibited partial protection, reducing clinical signs but failing to prevent virus shedding, with mortality rates reaching 40\u0026ndash;60%. Notably, X-vac 1 significantly reduced viral shedding, suggesting its potential as a more effective intervention. To fully assess the impact of these vaccines on disease transmission, further studies on virus transmission dynamics are necessary, as detailed in the following section.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Transmission potential of vaccinated birds to sentinel chickens\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of vaccination on virus transmission, three sentinel chickens were introduced into a separate, clean cage 12 hours post-challenge for each experimental group. Only birds immunized with the X-vac 1 experimental vaccine exhibited partial prevention of virus transmission, as evidenced by a 66.6% survival rate among sentinel chickens (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), with one bird succumbing on day 4 post-cohabitation. Viral shedding was detected in only one of three sentinel chickens in the X-vac 1 group, with virus titers of 2.2 and 4.2 log₁₀ TCID₅₀/0.2 mL in tracheal swabs on days 2 and 4, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Cloacal shedding was observed only on day 2, with a titer of 3.2 log₁₀ TCID₅₀/0.2 mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Additionally, on day 4 post-cohabitation, X-vac 1-immunized sentinels exhibited significantly lower viral titers in tracheal swabs compared to unvaccinated contact chickens (P\u0026thinsp;=\u0026thinsp;0.0232). These findings suggest that while X-vac 1 reduced viral shedding and transmission, however, it did not fully prevent it.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy contrast, sentinels housed with unvaccinated, infected chickens (Control group) demonstrated clear evidence of virus transmission, as indicated by a 0% survival rate (mean death time (MDT)\u0026thinsp;=\u0026thinsp;4.0 days). High viral titers were detected in their tracheal swabs (6.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 and 8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 log₁₀ TCID₅₀/0.2 mL) and cloacal swabs (4.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.29 and 3.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69 log₁₀ TCID₅₀/0.2 mL) on days 2 and 4, respectively. Control sentinels also exhibited clinical signs of AIV infection (data not shown). Similarly, sentinels in all vaccinated groups (Flu Protect H5, Volvac, and RIBSP) demonstrated virus transmission, with viral titers in tracheal swabs ranging from 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70 to 6.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.79 log₁₀ TCID₅₀/0.2 mL on day 2 and 1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65 to 8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 log₁₀ TCID₅₀/0.2 mL on day 4. Cloacal shedding was also detected, with titers ranging from 1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 to 5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 log₁₀ TCID₅₀/0.2 mL on day 2 and 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70 to 3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 log₁₀ TCID₅₀/0.2 mL on day 4. The MDT of sentinels housed with Volvac-vaccinated birds (MDT\u0026thinsp;=\u0026thinsp;7.0 days) was significantly longer than that of sentinels in the Flu Protect H5 (MDT\u0026thinsp;=\u0026thinsp;3.3 days, P\u0026thinsp;=\u0026thinsp;0.0340) and RIBSP (MDT\u0026thinsp;=\u0026thinsp;2.7 days, P\u0026thinsp;=\u0026thinsp;0.0277) groups. However, no significant difference in MDT was observed between the Control group and any of the vaccinated groups. The RIBSP group had the shortest MDT, but this difference was not statistically significant compared to the Control and Flu Protect H5 groups.\u003c/p\u003e \u003cp\u003eThese findings suggest that Volvac vaccination delayed mortality, while other vaccines had less impact on survival. X-vac 1 showed promising results, reducing viral shedding and improving survival, though none fully prevented transmission, warranting further investigation.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHPAI H5Nx viruses of clade 2.3.4.4b (exemplified by H5N8) have spread explosively across the globe in recent years, causing devastating poultry outbreaks in Europe, Asia, Africa, and beyond​ [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Kazakhstan experienced its first large-scale incursions of H5N8 (clade 2.3.4.4b) in 2020, with the virus spreading to multiple regions despite control measures​ [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. These events underscore an urgent need for effective vaccination strategies against this continually evolving lineage. Vaccination is increasingly viewed as a critical tool for HPAI control, provided that vaccines are antigenically well-matched to circulating strains​ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. An effective H5 vaccine must not only prevent disease in immunized birds but also curtail virus shedding and onward transmission​ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Achieving such sterilizing immunity is essential to halt virus circulation, given that HPAI outbreaks can otherwise persist via silent transmission in vaccinated flocks​ [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this context, our study evaluated the efficacy of several H5 vaccines against a clade 2.3.4.4b H5N8 challenge in chickens, yielding important insights into antigenic match, adjuvant effects, and transmission dynamics.\u003c/p\u003e \u003cp\u003eOur findings demonstrate a clear link between antigenic matching and vaccine performance. The experimental H5N8 (clade 2.3.4.4b) vaccine formulated from a homologous strain (X-vac 1) induced a rapid and robust immune response, achieving full (100%) protection against mortality upon challenge. In parallel, a commercial vaccine (RIBSP) based on a closely related H5 seed strain provided complete protection from death. In contrast, two other commercial vaccines (Flu Protect H5 and Volvac), whose H5 antigens were antigenically more distant from the challenge virus, conferred only partial protection. Birds vaccinated with these mismatched vaccines showed significant mortality (up to 40\u0026ndash;60% in our trials) and developed moderate clinical signs post-challenge, whereas all chickens in the X-vac 1 and RIBSP groups survived without signs. These outcome disparities correlated with immunogenicity: by two weeks post-immunization, the X-vac 1 and RIBSP groups had elicited high hemagglutination-inhibition antibody titers, meeting WOAH\u0026rsquo;s efficacy benchmark of \u0026ge;\u0026thinsp;80% protective seroconversion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, HI titers in the X-vac 1 group were slightly higher than those in the RIBSP group, reflecting the closer antigenic match. In comparison, Flu Protect H5 and Volvac elicited significantly lower and slower antibody responses, with many birds not reaching protective titer thresholds until four weeks post-vaccination​. Overall, the antigenically matched vaccines (X-vac 1 and to a lesser extent RIBSP) stimulated the strongest and earliest immunity, whereas the more divergent vaccines lagged in both magnitude and timing of response. These results underscore that even within the same clade, subtle genetic differences can translate into major differences in protective immunity, reinforcing antigenic matching as a critical factor for H5 vaccine efficacy​ [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApart from antigenic relatedness, our data suggest that vaccine formulation, particularly adjuvant type, also influenced efficacy. All vaccines in this study were oil-emulsified inactivated formulations, but the potency of the immune response varied, likely reflecting differences in adjuvant composition or dose. The X-vac 1 vaccine was formulated with a high-quality water-in-oil adjuvant (Montanide ISA-78 VG), which is known to enhance immunogenicity [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This may have contributed to the rapid antibody rise and strong protection observed in the X-vac 1 group. Consistent with this, recent studies have shown that the choice of adjuvant can significantly alter H5 vaccine outcomes​ [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For example, a single immunization with an H5 virus-like particle vaccine protected chickens against diverse H5 strains only when combined with an appropriate adjuvant and dosage​ [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In our study, even though the RIBSP and X-vac 1 vaccines both used oil-based adjuvants, X-vac 1 achieved slightly better control of virus replication, hinting that Montanide (or a higher antigen dose) provided an edge. Conversely, the Volvac vaccine, which induced the weakest antibody response, might have utilized a less potent formulation or lower antigen content. These findings highlight that adjuvants are not interchangeable, the specific adjuvant (and its formulation) can profoundly impact the quality of the immune response, and thus the degree of protection, afforded by an H5 vaccine. Future vaccine development and evaluation should therefore weigh adjuvant selection as heavily as antigen selection to maximize efficacy.\u003c/p\u003e \u003cp\u003eA paramount goal of HPAI vaccination is to reduce viral shedding from infected hosts, thereby limiting transmission [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In our challenge experiments, only the homologous X-vac 1 vaccine achieved substantial suppression of viral shedding. X-vac 1-immunized chickens had either no detectable virus or markedly lower titers in tracheal and cloacal swabs after challenge, indicating that this vaccine nearly achieved sterilizing immunity in most individuals. In stark contrast, chickens vaccinated with the less-matched commercial vaccines shed high levels of virus from both the respiratory tract and gastrointestinal tract, comparable to or only marginally lower than unvaccinated controls in the first days post-challenge. Even the RIBSP vaccine, while fully protective against death, did not completely prevent virus replication, as low to moderate titers of challenge virus were recovered from RIBSP-vaccinated birds. Thus, a gap emerged between clinical protection (prevention of illness/death) and virological protection (prevention of infection/shedding) for the mismatched vaccines. This finding is in line with field observations in Egypt where most commercial H5 vaccines protected chickens from mortality but failed to stop virus shedding when challenged with an emergent H5N8 strain​ [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The danger of such an outcome is that vaccinated flocks may appear healthy yet continue to propagate the virus. Indeed, if a vaccine mitigates disease signs but does not block transmission, HPAI can spread silently and even generate new escape variants under immune pressure​ [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our results empirically reinforce this concern: only the vaccine with optimal antigenic match significantly curtailed shedding, whereas suboptimal vaccines would likely allow onward transmission of H5N8 in a flock or farm setting.\u003c/p\u003e \u003cp\u003eTo directly address the issue of transmission, we incorporated a sentinel bird experiment. Unvaccinated contact chickens housed with infected birds universally became infected and succumbed, affirming the high transmissibility of the challenge strain. Sentinels exposed to vaccinated, infected birds fared only marginally better in most groups: in the Flu Protect H5, Volvac, and RIBSP groups, all contact birds eventually contracted fatal infections, indicating that those vaccines did not halt bird-to-bird spread. Only in the X-vac 1 group transmission partially impeded, two out of three sentinel chickens were completely protected (surviving and remaining virus-negative), and only one became infected, with a delayed infection timeline. This 66.6% reduction in transmission in the X-vac 1 group is a noteworthy outcome, given that no current inactivated H5 vaccine is expected to entirely prevent viral shedding after a single dose. The X-vac 1 formula\u0026rsquo;s ability to diminish shedding and confer some herd immunity suggests that improved vaccines can contribute to transmission blockade, not just protection of individual birds. Nevertheless, even X-vac 1 did not achieve 100% transmission prevention, as one contact bird did succumb, meaning that while greatly reduced, infectious virus was still eventually emitted by some vaccinated-challenged hosts. This underscores that further enhancements (e.g. booster doses or more immunogenic platforms) would be needed to reach full transmission stoppage. Importantly, our transmission findings align with the principle that vaccines should be evaluated on their capacity to reduce shedding and secondary spread, not solely on preventing disease in vaccinated individuals​ [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Incorporating such transmission assessments into vaccine evaluation (as we have done) provides a more rigorous test of a vaccine\u0026rsquo;s true epidemiological impact.\u003c/p\u003e \u003cp\u003eThe outcomes of this study carry significant implications for HPAI control policies, both in Kazakhstan and globally. For Kazakhstan, where emergency vaccination has been deployed to contain H5N8 outbreaks​ [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], our data suggest that vaccine choice is crucial: using an antigenically mismatched vaccine may fail to stop the virus from spreading in poultry populations, potentially undermining control efforts. It will be important for veterinary authorities to update and match vaccine seed strains to the currently circulating viruses. Given that clade 2.3.4.4b viruses continue to evolve and new variants (like the recent H5N1 2.3.4.4b strain) have emerged [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], a dynamic vaccination strategy is warranted, one that can rapidly incorporate new antigenic variants into vaccine formulations, analogous to how seasonal influenza vaccines are updated. On a global scale, there is growing interest in adopting poultry vaccination in regions experiencing relentless H5N1/H5N8 outbreaks, as evidenced by recent recommendations from the European Food Safety Authority on HPAI vaccination strategies​ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our findings provide a timely evidence-based message for such initiatives: vaccination can be highly effective at protecting flocks and reducing virus dissemination, but only if the vaccine antigen closely matches the outbreak strain and induces sufficiently potent immunity. Using vaccines that only prevent mortality without stopping infection would risk creating reservoirs of infection in ostensibly \u0026ldquo;protected\u0026rdquo; flocks, a scenario that has played out in past HPAI epizootics [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. ​Therefore, any vaccination program should be accompanied by robust surveillance (to detect breakthrough infections and viral evolution) and, ideally, the use of DIVA (Differentiating Infected from Vaccinated Animals) strategies to monitor field virus circulation in vaccinated populations.\u003c/p\u003e \u003cp\u003eIn light of the partial success of the best-performing vaccine in this study, future research should aim to optimize vaccine formulations for complete transmission blocking. One approach could be the use of prime-boost regimens or multiple doses to elevate mucosal and systemic immunity to levels that eliminate virus more rapidly. Another promising avenue is exploring alternative vaccine platforms: for instance, vectored vaccines have shown the ability to protect against heterologous H5Nx challenges and even prevent onward transmission​ [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. A recombinant turkey herpesvirus-vectored H5 vaccine, for example, was reported to halt transmission of a clade 2.3.4.4b H5N8 challenge in chickens​ [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], highlighting that next-generation vaccines might achieve what conventional inactivated vaccines cannot. Additionally, novel adjuvants or delivery routes (e.g. mucosal immunization) could further enhance local immunity in the respiratory tract, which is key to stopping virus shedding at the source. Our group\u0026rsquo;s ongoing work in evaluating adjuvant combinations and mucosal vaccine delivery (Tabynov et al., 2025; In press) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] is geared toward this goal. Ultimately, an ideal HPAI vaccine for poultry would induce fast, robust, and broad immunity that protects the bird and its flock-mates by sharply curtailing viral shedding.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe limitations\u003c/b\u003e of this study include several factors that may affect the generalizability and robustness of the findings. One primary limitation is the challenge model itself, as only a single H5N8 strain was used for post-vaccination challenge, which may not fully represent the broad diversity of H5 avian influenza strains. This approach may limit the generalizability of findings to other clades. Furthermore, antigen-specific cytokine production, such as IFN-gamma, was not assessed in this study. The absence of an IFN-gamma ELISPOT assay limits the understanding of the functional T-cell responses elicited by the vaccines. Future studies will incorporate this assay to provide a more comprehensive evaluation of T-cell activation and its role in protective immunity. Additionally, the study was conducted with SAN chickens, which may exhibit different immune responses than poultry populations with prior exposures to low pathogenic avian influenza viruses. This factor should be considered when interpreting the real-world applicability of the vaccine efficacy results. The study also employed relatively small group sizes, potentially impacting the statistical power and robustness of the findings. Another limitation is the lack of assessment for the durability of protection, as the study focused on short-term outcomes post-vaccination. This leaves open questions about the long-term immunity provided by the vaccines. Finally, apart from HI activity, the study did not measure functional antibodies, such as neutralizing antibodies (MN). These assays are crucial for assessing the quality of immune responses and understanding the protective mechanisms of the vaccines. Future studies will incorporate MN assays to comprehensively evaluate the functional antibody responses elicited by the vaccine formulations. Together, these limitations highlight the need for future studies with larger sample sizes, repeat trials, and extended observation periods to assess the durability of protection, which would provide a more comprehensive understanding of vaccine efficacy.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn conclusion\u003c/b\u003e, this study highlights that an inactivated H5N8 vaccine with a close antigenic match and a potent adjuvant can provide excellent protection to chickens and significantly reduce virus shedding and transmission. These findings have immediate relevance for enhancing avian influenza control programs in Kazakhstan and other affected regions. By prioritizing antigenic matching in vaccine selection and striving for transmission-blocking immunity, veterinary authorities can improve vaccine effectiveness and reduce the threat of HPAI in poultry. Such optimized vaccine strategies, combined with biosecurity and surveillance, will be critical to curtail the spread of clade 2.3.4.4b H5Nx viruses and diminish the risk of emergent variants that could impact both animal and public health. The mechanistic insights gained from our experimental trials, linking antigenic match and adjuvant-driven immune potency to real-world outcomes in infected birds, provide a strong foundation for designing better vaccines and policies to combat HPAI now and in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: KaissarT. Data curation: KairatT. Formal analysis: KaissarT, KairatT. Funding acquisition: KaissarT. Investigation: KairatT, TK, LY, MB, LY, KZ, AK, ZZ. Methodology: KaissarT, KairatT. Project administration: KaissarT. Resources: KaissarT, ZZ. Software: KaissarT, KairatT. Supervision: KaissarT. Validation: KairatT. Visualization: KaissarT, KairatT. Writing—original draft: KairatT. Writing—review and editing: KaissarT, KairatT, TK, LY, MB, LY, KZ, AK, ZZ. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following financial interests/personal relationships which may be considered as potential competing interests: KaissarT and KairatT are affiliated with T\u0026amp;TvaX. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. AP19675939). Partial funding was also provided by the startup company T\u0026amp;TvaX LLC (Kazakhstan). The funders had no role in the study design, data collection, analysis, or interpretation, nor in the writing of the manuscript or the decision to submit it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to Zhambyrbayeva L. and Mukhambetova A. for their dedicated care and maintenance of the laboratory animals. Special thanks are extended to Dr. Turebekov N. for providing valuable guidance on biosafety and biosecurity during experiments involving highly pathogenic avian influenza.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChang N, Zhang C, Mei X, Du F, Li J, Zhang L, Du H, Yun F, Aji D, Shi W, Bi Y, Ma Z. Novel reassortment 2.3.4.4b H5N8 highly pathogenic avian influenza viruses circulating in Xinjiang, China. 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Efficacy of a Recombinant Turkey Herpesvirus AI (H5) Vaccine in Preventing Transmission of Heterologous Highly Pathogenic H5N8 Clade 2.3.4.4b Challenge Virus in Commercial Broilers and Layer Pullets. J Immunol Res. 2018 Nov 21;2018:3143189. doi: 10.1155/2018/3143189.\u003c/li\u003e\n\u003cli\u003eTabynov K, Strochkov V, Sandybayev N, Karibayev T, Berdikulov M, Yelchibayeva L, Zharmambet K, Kuanyshbek A, Zhumadilova Z, Tabynov K. Detection and genomic characterization of an avian influenza virus A/mute swan/Mangystau/1-S24R-2/2024 (H5N1; clade 2.3.4.4b) strain isolated from the lung of a dead swan in Kazakhstan. Microbiol Resour Announc. 2024 Aug 13;13(8):e0026024. doi: 10.1128/mra.00260-24.\u003c/li\u003e\n\u003cli\u003eReed LJ, Muench H. A simple method for estimating fifty percent endpoints. Am J Hyg. 1938;27:493\u0026ndash;497.\u003c/li\u003e\n\u003cli\u003eKaufmann L, Syedbasha M, Vogt D, Hollenstein Y, Hartmann J, Linnik JE, Egli A. An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers. J Vis Exp. 2017 Dec 1;(130):55833. doi: 10.3791/55833.\u003c/li\u003e\n\u003cli\u003eWebster R, Cox N, St\u0026ouml;hr K. WHO Animal Influenza Manual. WHO/CDS/CSR/NCS. 2002;2002.5:1\u0026ndash;99.\u003c/li\u003e\n\u003cli\u003eWorld Organisation for Animal Health. WOAH terrestrial manual 2021, Chapter 3.3.4. Avian influenza (including infection with high pathogenicity avian influenza viruses. https://www.woah.org/fileadmin/Home/eng/Health_standards/tahm/3.03.04_AI.pdf \u003c/li\u003e\n\u003cli\u003eSwayne DE, Sims L. Avian influenza. In: Metwally S, El Idrissi M, Viljoen G, editors. Veterinary Vaccines: Principles and Applications. Chichester (UK): Wiley; 2020. p. 229\u0026ndash;251.\u003c/li\u003e\n\u003cli\u003eMahmoud SH, Khalil AA, Abo Shama NM, El Sayed MF, Soliman RA, Hagag NM, Yehia N, Naguib MM, Arafa AS, Ali MA, El-Safty MM, Mostafa A. 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Avian Pathol. 2023 Jun;52(3):176-184. doi: 10.1080/03079457.2023.2181145.\u003c/li\u003e\n\u003cli\u003evan der Goot JA, Koch G, de Jong MC, van Boven M. Quantification of the effect of vaccination on transmission of avian influenza (H7N7) in chickens. Proc Natl Acad Sci U S A. 2005 Dec 13;102(50):18141-6. doi: 10.1073/pnas.0505098102.\u003c/li\u003e\n\u003cli\u003ePoetri ON, Van Boven M, Claassen I, Koch G, Wibawan IW, Stegeman A, Van den Broek J, Bouma A. Silent spread of highly pathogenic Avian Influenza H5N1 virus amongst vaccinated commercial layers. Res Vet Sci. 2014 Dec;97(3):637-41. doi: 10.1016/j.rvsc.2014.09.013.\u003c/li\u003e\n\u003cli\u003eZhang Y, Cui P, Shi J, Zeng X, Jiang Y, Chen Y, Zhang J, Wang C, Wang Y, Tian G, Chen H, Kong H, Deng G. A broad-spectrum vaccine candidate against H5 viruses bearing different sub-clade 2.3.4.4 HA genes. 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Estimation of transmission parameters of H5N1 avian influenza virus in chickens. PLoS Pathog. 2009 Jan;5(1):e1000281. doi: 10.1371/journal.ppat.1000281.\u003c/li\u003e\n\u003cli\u003eSwayne DE, Spackman E, Pantin-Jackwood M. Success factors for avian influenza vaccine use in poultry and potential impact at the wild bird-agricultural interface. Ecohealth. 2014;11(1):94-108. doi: 10.1007/s10393-013-0861-3.\u003c/li\u003e\n\u003cli\u003eReemers S, Verstegen I, Basten S, Hubers W, van de Zande S. A broad spectrum HVT-H5 avian influenza vector vaccine which induces a rapid onset of immunity. Vaccine. 2021 Feb 12;39(7):1072-1079. doi: 10.1016/j.vaccine.2021.01.018.\u003c/li\u003e\n\u003cli\u003eNassif S, Zaki F, Mourad A, Fouad E, Saad A, Setta A, Felf\u0026ouml;ldi B, Mat\u0026oacute; T, Kiss I, Palya V. Herpesvirus of turkey-vectored avian influenza vaccine offers cross-protection against antigenically drifted H5Nx highly pathogenic avian influenza virus strains. Avian Pathol. 2020 Dec;49(6):547-556. doi: 10.1080/03079457.2020.1790502.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Kazakh National Agrarian Research University","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Avian Influenza, Commercial Vaccine, H5N8, Clade 2.3.4.4b, Efficacy, Adjuvant, Transmission, Chicken","lastPublishedDoi":"10.21203/rs.3.rs-6142334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6142334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Highly pathogenic avian influenza H5N8 (clade 2.3.4.4b) has caused devastating poultry outbreaks globally, including in Kazakhstan, underscoring the need for vaccines that protect birds and curb virus transmission. We evaluated the efficacy of three commercial H5 vaccines and an experimental homologous H5N8 vaccine in chickens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Chickens received a single dose of each vaccine, and antibody titers were measured over 4 weeks. At 30 days post-vaccination, birds were challenged intranasally with a virulent H5N8 strain and monitored for 10 days for survival and clinical signs. Virus titers in tracheal and cloacal swabs (days 1, 3, 5 post-challenge) measured shedding, and unvaccinated sentinel chickens were co-housed to assess transmission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The homologous H5N8 vaccine and a closely related commercial vaccine elicited rapid, high antibody responses and conferred 100% survival. In contrast, two antigenically mismatched vaccines induced slower, lower immunity, resulting in 40-60% mortality and high virus shedding after challenge. Only the homologous vaccine sharply reduced viral shedding and significantly decreased transmission to contacts (protecting 2 of 3 sentinel birds), whereas the other vaccines failed to prevent transmission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e An antigenically matched H5N8 vaccine with a potent adjuvant provided near-sterilizing immunity, preventing disease and significantly limiting viral shedding and transmission. These findings highlight the importance of using strain-matched vaccines in HPAI control strategies to avoid silent viral spread in vaccinated flocks.\u003c/p\u003e","manuscriptTitle":"Evaluation of Commercial Vaccines for Efficacy and Transmission Control Against the Emergent H5N8 (Clade 2.3.4.4b) Avian Influenza Virus in Kazakhstan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-04 09:08:59","doi":"10.21203/rs.3.rs-6142334/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68c4b22d-f867-45ac-94d5-532128c15c98","owner":[],"postedDate":"March 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45090056,"name":"Vaccine Development"},{"id":45090057,"name":"Virology"},{"id":45090058,"name":"Immunology"}],"tags":[],"updatedAt":"2025-03-04T09:08:59+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-04 09:08:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6142334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6142334","identity":"rs-6142334","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}