inch) before infection and transferred to Model 934C1 isolators (900 sq. 1 day of age. A single dose of pc4-LAIV was able to induce stronger innate and mucosal IgA responses and protect young immunologically C527 immature chickens better than a single dose of TCF10 IIV. Most importantly, when 1-day-old chickens were intranasally primed with pc4-LAIV and subcutaneously boosted with IIV three weeks later, they showed a rapid, robust, and highly cross-reactive serum antibody response and a high level of mucosal IgA antibody response. This vaccination regimen warrants further optimization to increase its range of protection. Introduction Avian influenza (AI) is a major zoonotic viral disease that causes significant adverse impacts on poultry production, the global trade, and public health [1C4]. Despite decades of research and control efforts, the incidences and severity of AI outbreaks have not been alleviated but rather increased [5, 6]. The strategies currently being employed to control AI are focused on prevention of virus introduction by maintaining strict biosecurity procedures [7C10]. However, the current biosecurity systems have repeatedly failed to protect poultry farms from introduction of novel strains that continue to cause major outbreaks [11, 12]. To overcome this challenge, some countries have incorporated vaccination with inactivated influenza vaccine (IIV) in their control programs [10, 13]. The United States has been using stamping out as the primary control strategy, but the occurrence of recent highly pathogenic avian influenza (HPAI) outbreaks [13, 14] further proves the limitations of stamping out in combination with biosecurity control. For this reason, vaccination is being C527 more seriously considered to be added to the C527 countrys AI control arsenal. The IIV is currently in use in countries like Bangladesh, China, Egypt, Indonesia and Viet Nam where HPAI viruses are endemic [7]. Unfortunately, although IIV can provide good protection from homologous field strains [13], it is weak against heterologous strains that arise from random genetic mutations and not protective against heterosubtypic strains [9, 15, 16]. In addition to having a narrow range of protection, there are serious concerns that a long-term use of IIV without eradication of heterologous (mismatched) strains may result in the selection of antigenically divergent strains [17]. Live vaccines have numerous advantages over IIV that can be exploited further to develop new broadly protective vaccines and vaccination regimens. Since a live agent can mimic the natural infection, it can elicit a broad range of immune responses including humoral, cell-mediated and mucosal immunity [18C20]. Importantly, live vaccine can be directly administered on the mucosal surface by spray or through drinking water, which not only elicits local mucosal immunity but also significantly reduces the cost of mass administration [19, 21]. Currently, the only live influenza vaccines available for use in the poultry industry are C527 live viral-vectored vaccines [10, 16]. Live viral-vectored vaccines can induce a broader range of protection compared to IIV, but they have several shortcomings including reduced efficacy due to preexisting immunity to the viral vector and the difficulty of expressing two or more C527 influenza virus proteins in the same vector [16, 22, 23]. Live-attenuated influenza vaccine (LAIV) is an excellent alternative to the vectored vaccine or IIV since it contains all proteins that are naturally found in influenza virus particles [19, 24]. In humans, LAIV has been used for more than a decade and it has been reported to protect young individuals better than IIV [25C27]. Importantly, recent studies showed that LAIV can pre-sensitize the population and, subsequently, synergistically boost the efficacy of IIV [28, 29]. It should be noted that the use of LAIV in poultry requires strict safety standards due to concerns about the possibility that wild type strains may circulate in domestic poultry without apparent clinical symptoms, undergo genetic reassortment with the vaccine virus, and produce novel virulent strains [30, 31]. An ideal poultry LAIV should not be able to revert to wild type virus or undergo reassortment with field strains. One of the promising candidates for poultry LAIV is pc4-LAIV, an influenza virus mutant that expresses a C-terminally truncated nonstructural protein.