12 June 2026
Since 2020, the global dissemination of Gs/GD-lineage H5Nx viruses belonging to clade 2.3.4.4b has caused unprecedented losses in poultry production worldwide. With more than 200 million domestic birds either dying or being culled across multiple continents between 2020 and 2025, biosecurity measures alone have repeatedly proven insufficient to withstand the persistent infection pressure originating from migratory wild bird populations.
Although emergency vaccination strategies have gained increasing international acceptance, regulatory authorities have historically expressed concerns regarding the phenomenon of subclinical viral replication. Conventional vaccines may protect birds from clinical disease and mortality while failing to completely prevent viral shedding, thereby enabling undetected virus circulation within vaccinated populations. Such "silent circulation" can compromise traditional passive surveillance systems. To determine whether modern vaccine platforms can overcome this limitation throughout the entire production cycle, a Dutch research consortium conducted a comprehensive field evaluation.
The investigators established an experimental model involving commercial Novogen Brown Light layer hens maintained under authentic commercial production conditions on two farms in the Netherlands. On the day of hatch, chicks received a subcutaneous primary vaccination with VAXXITEK® HVT+IBD+H5, a cell-associated recombinant turkey herpesvirus vector expressing an H5 hemagglutinin sequence modified to resemble low-pathogenicity motifs to ensure safety.
To evaluate the durability of protection, the study included two vaccination regimens:
Regimen 1: A single day-of-hatch vaccination with VAXXITEK® HVT+IBD+H5.
Regimen 2: The primary day-of-hatch vaccination followed by a subcutaneous booster dose of the inactivated Volvac® B.E.S.T. AI+ND vaccine at 12 weeks of age.
At 8, 24, 54, and 84 weeks of age, subgroups of hens were transferred from commercial facilities to Biosafety Level 3 (BSL-3) containment units at Wageningen Bioveterinary Research (WBVR). Birds were challenged intra-choanally with a virulent field isolate of HPAI H5N1 (A/chicken/Netherlands/2021; GISAID: EPI_ISL_6101848).
The study generated important epidemiological insights into age-dependent transmission dynamics. In unvaccinated control groups, transmission efficiency increased substantially with age. The basic reproduction number (R₀) was estimated at 1.6 in 8-week-old pullets but increased to 6.4 in adult laying hens, indicating that a single infected adult layer could infect more than six susceptible flock-mates under experimental conditions.
Vaccination substantially altered these parameters:
- Vaccinated birds developed robust and long-lasting humoral immunity. Although birds receiving only the primary vaccination exhibited fluctuations in antibody levels at 30 and 60 weeks of age, hens receiving the heterologous booster maintained consistently high hemagglutination inhibition (HI) antibody titers (≥7 log₂) through 85 weeks of age.
- Following HPAI challenge, all unvaccinated control birds succumbed to infection, resulting in 100% mortality. In contrast, hens receiving the prime-boost regimen achieved complete clinical protection with 0% mortality. Even vaccinated birds exhibiting comparatively lower pre-challenge antibody responses remained protected from severe systemic disease, limiting mortality across vaccinated groups to a maximum of 7.1%.
- Vaccination markedly reduced viral shedding. Unvaccinated birds exhibited high levels of viral excretion from the upper respiratory tract, whereas hens with elevated antibody titers induced by VAXXITEK® showed substantial suppression of respiratory shedding. Furthermore, the booster regimen nearly eliminated gastrointestinal shedding: only 4 of 14 infected boosted hens shed detectable virus in feces, compared with universal and substantial fecal shedding among unvaccinated controls.
From a macro-epidemiological perspective, modeling the experimental data at the national level demonstrated that the dual-vaccination strategy fundamentally altered outbreak dynamics. Following introduction of a field virus into a boosted flock, major outbreaks became uncommon. Approximately 90% of simulated introductions resulted in negligible transmission, with fewer than 0.1% of birds becoming infected. When larger outbreaks occurred, the effective reproduction number (Rₑ) decreased from 6.4 to a median of 1.5–1.7. At the national scale, this translated into a median epidemic size of only one to two affected farms, compared with a median of 14 infected premises in unvaccinated scenarios.
The investigators also identified important implications for disease surveillance. Because vaccinated birds generally remained clinically normal and did not exhibit elevated mortality, passive surveillance alone proved inadequate. In simulation models, routine passive monitoring detected only 0.0–19.2% of infections within vaccinated populations.
To address this limitation, the authors recommended implementation of active surveillance through routine PCR testing of mortality samples collected during daily flock inspections ("bucket sampling"). Modeling demonstrated that PCR testing of naturally deceased birds at intervals of two to seven days achieved outbreak detection probabilities ranging from 93.7% to 99.9%. Such surveillance enables rapid identification and containment of index cases before onward transmission to neighboring farms can occur.
The study further demonstrated compatibility of this surveillance approach with DIVA (Differentiating Infected from Vaccinated Animals) principles. Across 8,630 field serum samples tested using commercial NP-ELISA assays, diagnostic specificity reached 99.8%. Because the vector vaccine expresses only the H5 hemagglutinin antigen, vaccinated birds remained negative for antibodies against the influenza nucleoprotein (NP). Consequently, detection of NP-specific antibodies provides a reliable indicator of field-virus exposure, supporting disease monitoring and maintaining trade transparency.
The authors concluded that although vaccination alone may not completely eliminate subclinical transmission under all field conditions, the combination of a robust prime-boost vaccination strategy and structured active surveillance provides an effective, scientifically validated approach for long-term control of highly pathogenic avian influenza.
This study provided the scientific foundation for the Netherlands' decision to implement HPAI vaccination in commercial egg-laying hens.
Bouwman et al. (2026). 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. Wageningen Bioveterinary Research, Lelystad; Royal GD, Deventer; Wageningen University & Research, Wageningen; Utrecht University, Utrecht. Report number 2529108
