After incubation, equal volume of 1% horse erythrocytes was added and incubated at RT for 1 h

After incubation, equal volume of 1% horse erythrocytes was added and incubated at RT for 1 h. pandemic viruses. Keywords: Virus like particles, subunit vaccine, influenza virus, H5N1, protection, memory immune responses Introduction Wild aquatic birds are a continuous source of influenza viruses and provide a reservoir for each of the 16 known subtypes of hemagglutinin (HA) and Resiquimod 9 subtypes of neuraminidase (NA) (Fouchier et al., 2005; Rohm et al., 1996; Webster et al., 1992). The emergence or re-emergence of highly pathogenic avian influenza (HPAI) viruses in domestic poultry and the increasing numbers of direct transmission of avian viruses to humans underscore the persistent threat to public health. Six fatal cases of human infection with HPAI H5N1 virus were first reported during an outbreak in 1997 in Hong Kong (Claas et al., 1998; Subbarao et al., 1998). Since then, repeated outbreaks of HPAI H5N1 virus have emerged and spread to countries across Asia, Europe, the Middle East, Africa, and the Pacific. Human cases have increased with a high fatality rate of 60%, and some H5N1 isolates shown resistance to the antiviral drugs amantadine and rimantadine (Cheung et al., 2006; de Jong et al., 2005; Le et al., 2005). Currently, H5N1, H7N2, H7N3, H7N7 and H9N2 avian origin Resiquimod influenza viruses have been shown to cause human infections on multiple occasions (Fouchier et al., 2004; Peiris et al., 1999; Wong and Yuen, 2006). Most recently, the 2009 2009 outbreak of a new H1N1 virus illustrates how fast a new pandemic virus can spread in the human population once it acquires the ability to transmit among humans (Nava et al., 2009; Solovyov et al., 2009). Vaccination is considered to be the most effective preventive measure against potential epidemic and pandemic influenza viruses. Among the licensed vaccine formulations, split and subunit vaccines are most frequently used for immunization against the yearly influenza epidemics (Nicholson et al., 1979; Wright et al., 1977). Resiquimod In preclinical studies, two doses of inactivated split vaccine against H5 subtype provided protection against lethal challenges with homologous or heterologous H5N1 viruses in mice or ferrets (Govorkova et al., 2006; Lipatov et al., 2006; Nicolson et al., 2005; Subbarao et al., 2003; Webby et al., 2004). Phase I clinical trials indicate that inactivated split vaccines are not optimally immunogenic (Nicholson et al., 2001) and require multiple doses (Stephenson et al., 2003) or the inclusion of an adjuvant to induce a protective immune response (Atmar et al., 2006; Bresson et al., 2006; Hehme et al., 2002; Lin et al., 2006). A baculovirus-expressed recombinant H5 protein or subvirion H5 influenza vaccine was poorly immunogenic and two doses of 90 g HA were required to develop a desired antibody response to the vaccine (Treanor et al., 2006; Treanor et al., 2001). Although live, attenuated TNFRSF11A virus vaccines are licensed in an intranasal spray form, there are some challenges for developing an effective and safe live attenuated vaccine against avian influenza viruses (Subbarao and Joseph, 2007). There is a risk of gene reassortment of the live vaccine virus with human influenza viruses. The currently licensed influenza vaccines are produced in embryonated hen eggs, and the manufacturing process usually takes approximately 6 months when a new virus seed is required. If the pandemic virus causes widespread morbidity and mortality in poultry, the supply of embryonated eggs needed for producing seasonal and pandemic vaccines at the same time might be significantly compromised. Therefore, developing an alternative vaccine production system not reliant on egg substrates is highly desirable. Most pandemic vaccine candidates such as inactivated split or live attenuated virus contain a modified HA with a deletion of the multibasic amino-acid motif that is involved in broadening the tropism and in enhancing the virulence for HPAI viruses (Steinhauer, 1999). The effect of the multibasic amino-acid motif on inducing protective immunity by pandemic vaccines has not been well studied. Also, we do not understand the basis for the low immunogenicity of split or subunit H5 vaccines in na?ve individuals. In addition, protective immune correlates, doses of vaccines, and frequencies of vaccination required for inducing protection against pandemic viruses are not well understood. Therefore, we investigated some of these important issues in this study using the novel vaccine platform of non-infectious influenza VLPs. Our laboratory and others have developed H5 influenza VLP vaccines produced Resiquimod in an egg-free cell culture system using recombinant technology (Bright et al., 2008; Haynes et al., 2009; Kang et al., 2009). Influenza VLP vaccines do not require handling of live influenza virus during the development, manufacturing, or administration. In this study, we have used a mouse model to.