NI activity against Vac-3 and NRT1 was detected in plasma of the vaccinated macaques, and the NI activity in two macaques (NR4 and NR5) was increased after challenge infection. reported since 1997 (http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/). Although H5N1 HPAIVs did not appear to transmit L-methionine very easily among humans (http://www.who.int/influenza/human_animal_interface/Influenza_Summary_IRA_HA_interface_04Jun13.pdf), the public health risks associated with H5N1 HPAIVs remain unchanged since most humans do not possess immunity against H5N1 disease and H5N1 HPAIVs have been detected in poultry and swine[1],[2], of which the L-methionine second option is thought to be an source of recent pandemic disease[3][5]. Therefore, Col4a5 development of vaccines against H5N1 HPAIVs has been required. Mutation rates in hemagglutinin (HA) genes of avian and swine influenza viruses were lower than those in HA genes of human being seasonal influenza viruses[1],[6]. However, H5N1 HPAIVs have genetically been divided into several clades relating to HA sequences, and further development of the disease offers led to the appearance of fresh clades and subclades as of 2012[7],[8]. Therefore, it is thought that vaccine strains should be renewed relating to circulating strains, and the development of a vaccine that is effective against a broad spectrum of different clades is definitely required[9]. We have founded a vaccine library comprising 144 different subtypes of non- or low pathogenic influenza viruses with mixtures of 16 hemagglutinins (HA) and 9 neuraminidases (NA)[10]. We previously selected vaccine candidate strains from your library to examine their effectiveness against H5N1, H7N7, and H1N1 disease infections in cynomolgus macaques[11][13]. To upgrade vaccine candidates, we developed a second strain of H5N1 subtype low pathogenic reassortant influenza disease, A/duck/Hokkaido/Vac-3/2007 (Vac-3)[14]. The Vac-3 disease propagated more vigorously in embryonated eggs than did Vac-1, which was the 1st nonpathogenic H5N1 disease in the disease library[11]. Consequently, if Vac-3 induced protecting immunity against H5N1 HPAIVs, it would be a suitable vaccine candidate for vaccine production to reduce the number of embryonated eggs required and to create vaccines more rapidly at pandemics[15]. In the present study, immunogenicity of the Vac-3 vaccine and its protective effectiveness against two H5N1 HPAIVs in different clades in macaques were analyzed. Whole disease particles of Vac-3 inactivated by formalin were subcutaneously inoculated into macaques. Neutralization activity of plasma against the vaccine strain was detected in all macaques. In challenge infections, period of disease detection in vaccinated macaques infected with the two different clades of H5N1 HPAIVs was shorter than that of disease detection L-methionine in unvaccinated macaques. Furthermore, propagation of a pandemic (H1N1) 2009 disease in macaques vaccinated with Vac-3 was prevented. The safety of vaccinated macaques from H5N1 HPAIV and pandemic (H1N1) 2009 disease infection was due to antibody reactions against HA and NA and to T lymphocyte reactions against viral antigens. Therefore, the whole particle vaccine of Vac-3 induced immune reactions against multiple clades and subtypes. == Results == == Pathogenicity of Two H5N1 Highly Pathogenic Avian Influenza Disease Strains in Cynomolgus Macaques == Firstly, we examined the pathogenicity of highly pathogenic avian influenza viruses, A/Vietnam/UT3040/2004 (H5N1) (clade 1, VN3040) and A/whooper swan/Hokkaido/1/2008 (H5N1) (clade 2.3.2.1, HOK1), in cynomolgus macaques. After inoculation of the disease into nose cavities, oral cavities, and tracheas, all macaques infected with either disease showed higher body temps over 40C than those before illness (Number 1). The average of clinical scores diagnosed relating toTable S4in macaques inoculated with HOK1 was higher than that in macaques inoculated with VN3040 although.