Vaccination of poultry with HVT-based H5 vaccine against highly pathogenic avian influenza (HPAI) H5N1 virus (clade 2.3.4.4b): VAXXITEK HVT+IBD+H5 and VAXXITEK HVT+IBD+H5 + Volvac® B.E.S.T. AI+ND vaccine

This longitudinal field study on two commercial layer farms in the Netherlands provides comprehensive evidence on the safety, effectiveness, and population-level impact of vaccination against highly pathogenic avian influenza (HPAI) H5 throughout the full production cycle of laying hens. All chicks received standard Dutch hatchery vaccinations, while test groups additionally received a subcutaneous avian influenza (AI) vaccination at day of hatch. Throughout the rearing period, chicks received the standard Dutch vaccination program as prescribed by the veterinary advisors of the rearing company, and in one test group, a heterologous to the prime booster vaccination was provided (at approx. 12 weeks of age). Chickens were monitored under field conditions from hatch through end of lay, with detailed assessment of immunity, health, mortality, production, virus transmission, poultry product safety, and surveillance performance. Vaccine safety and flock health. (Chapter 2) AI vaccination was well tolerated: no local vaccine reactions were observed, and monthly clinical inspections revealed no major abnormalities. Vaccine related mortality in vaccinated flocks remained low over the rearing period (~1%) and production period (4.4-7.6%), and well within expected ranges for healthy brown layer flocks during the production period. The non AI-vaccinated control group on farm A showed higher cumulative mortality (12.4%), attributable to non AI causes (e.g. E. coli septicaemia, feather pecking, red mite infestation, and a single fright-related event). There was no indication that AI vaccination negatively affected flock health, welfare, or overall performance. Immune responses and DIVA performance. Vaccinated chickens developed robust and durable humoral immunity. Mean homologous H5 HI titers ranged from approximately log₂ 4.5 at 5 weeks of age to log₂ 7.9–9.1 (VAXXITEK HVT+IBD+H5 and VAXXITEK HVT+IBD+H5 + Volvac® B.E.S.T. AI+ND) at 85 weeks, with similar trends on both farms. Antibody levels fluctuated over time, with temporary dips around 30–35 weeks and 60–65 weeks in chickens that received only a VAXXITEK HVT+IBD+H5 vaccination, whereas prime-heterologous booster-vaccinated groups maintained consistently high titers throughout the production cycle. DIVA surveillance using NP-ELISA (IDEXX) was highly specific: 8,630 serum samples were tested, of which 99.8% were NP-ELISA negative. The small fraction of positives (0.2%, n=17) fell within the expected falsepositive range and showed no evidence of field infection. Vaccinated and non AI-vaccinated groups had comparable NP-ELISA negativity rates (99.95 and 99.91% vs 99.81% respectively). Absence of field infection. Extensive PCR surveillance, as well as the serological results mentioned above, supported AI freedom of both vaccinated and non AI-vaccinated groups during the field study. All 196 pooled tracheal and cloacal swabs collected from mortality cases tested negative by M-gene RRT-PCR. This indicates that there was no evidence of silent circulation of avian influenza field infections during the study period. The effect of vaccination on the cellular immune response. The whole blood staining assay developed in this study can be used to monitor vaccine-induced changes in Tcell activation in blood. In field situations, including this field study, where multiple vaccines are administered, the assay cannot attribute responses to a single vaccine. However, under controlled experimental conditions the use of this assay showed that vaccinated chickens mounted a rapid and strong T cell response within 3-7 days post challenge which is reflected by increased numbers of activated CD4+ and CD8+ T cells as well as the presence of IFNγ positive cells in the blood. This response is consistent with a vaccine-induced memory cell response originating from vaccination at day of hatch. Transmission, mortality, production effects and poultry product safety. (Chapters 3 and 5) Experimental transmission studies (containing two duplo groups A and B) demonstrated strong life stage - dependent transmission (pullets versus layers)in the non AI-vaccinated control group: the estimated basic reproduction number (R₀) was 1.6 (95% CI 0.6–3.1) in 8-week-old pullets and 6.4 (2.5–12.0) in adult layers. This means that one infected adult layer could infect on average more than 6 other birds in a fully susceptible flock. This means that one infected adult layer could infect on average more than 6 other birds in a fully susceptible flock. This difference between pullets and adult layers was due to a significant difference in infection rate (β). No significant differences in transmission rates were observed among layers of different ages. Vaccination reduced transmission, virus shedding, and mortality, particularly in chickens with homologous HI titers ≥ log₂ 7. Vaccination reduced mortality to 0.0% (0.0-10.3%) in chickens vaccinated with VAXXITEK HVT+IBD+H5 + Volvac® B.E.S.T. AI+ND, compared to non AI-vaccinated controls (mortality: 100% (94-100%). Among vaccinated chickens, no significant difference in mortality was observed despite having high or low HI-titers. Egg production in group B remained stable during experimental challenge at 84 weeks of age, with no detectable production losses in vaccinated groups before and after challenge. In contrast, group A showed a drop in egg production, declining from 0.8 pre-challenge to 0.5 post-challenge. Under field conditions, vaccinated flocks reached a normal peak of lay despite subclinical avian metapneumovirus (AMPV) infection, whereas reduced egg production was observed only in non AI-vaccinated chickens of farm A (Chapter 2.2.7). In experimentally infected chickens, viral RNA (of the challenge virus) was detected in choanal and/or cloacal swabs in 10 of 12 chickens vaccinated with VAXXITEK HVT+IBD+H5, with feather positivity in 6 of 12 chickens. Infectious virus was isolated (3 days post challenge) only from a chicken lacking HI antibodies (pre-challenge), indicating systemic infection in the absence of detectable humoral immunity. Egg contents were mostly negative for viral RNA detection. Occasional positivity detected on eggshells or in a single egg white, along with limited eggshell contamination, was consistent with external contamination rather than internal infection. This indicates that the risk to egg safety and poultry products is minimal. Population-level impact and surveillance implications. (Chapter 6) Epidemiological modeling, integrating field and experimental data, demonstrated that vaccination substantially reduced both outbreak probability and outbreak size. The predicted effective reproduction number (Rₑ) fell below 1 during approximately 25-69% of the production period (4 and 11/16 monthly samplings done for Flocks A and B respectively) only in the VAXXITEK HVT+IBD+H5 + Volvac® B.E.S.T. AI+ND groups. This was also dependent on the antibody cut-off used, and was reduced from 6.4 to a median of ~1.5-1.7 during the remaining weeks (Chapter 3). This R value above 1 means transmission is still possible, but the substantial reduction indicates that spread progresses more slowly and affects fewer chickens before detection. For the flocks without booster vaccination, the level of induced antibody responses were insufficient to drop the predicted Re below 1 for the duration of the production period. As a result, vaccination reduced the probability of outbreaks to 10-28% vs 96% without vaccination and reduced the median epidemic size to 1–2 farms instead of a median of 14 farms if vaccination is not applied. However, because vaccination lowers mortality, passive surveillance alone detected only 0.0-19.2% of outbreaks in vaccinated flocks. This reduced visibility of disease highlights the need for structured active surveillance, including regular testing of dead chickens, to ensure timely detection and rapid response. Overall conclusion. This longitudinal field study demonstrates that AI vaccination in layer chickens is safe, compatible with commercial production vaccination schedules, provides robust clinical protection, safeguards poultry products, and reduces within flock transmission, although it does not consistently reach the R<1 threshold required to effectively halt transmission within a flock. Although an R>1 indicates a theoretical potential for sustained transmission in a AI-vaccinated flock, effective surveillance enables early detection and rapid intervention. The significant reduction in R in vaccinated flocks in combination with enhanced surveillance is, however, sufficient to prevent most of the onward transmission, within the flock, poultry flocks, and humans. As a result, onward transmission can be halted despite an underlying R greater than 1. In conclusion, while vaccination alone may not fully stop within-flock transmission under all circumstances, the combined strategy of additional vaccination and surveillance on top of existing biosecurity measures, is a robust, evidence-based, strategy for effective long term control of avian influenza.