A vaccine prepared from a non-pathogenic H5N1 avian influenza virus strain confers protective immunity against highly pathogenic avian influenza virus infection in cynomolgus macaques
Introduction
H5N1 highly pathogenic avian influenza (HPAI) viruses have become a threat not only for poultry but also for humans, because infection is associated with a high mortality rate, although they are not yet transmissible among humans [1], [2]. If these viruses acquire the ability of human-to-human transmission, an influenza pandemic will occur worldwide, since most humans do not possess immunity against H5N1 influenza viruses. Humans infected with H5N1 HPAI viruses have developed severe symptoms with a tissue tropism that differs from that noted in those infected with seasonal influenza viruses; the H5N1 HPAI viruses replicated not only in the upper respiratory tract but also in the lungs and intestine [3], [4]. Therefore, the development of a vaccine is an urgent issue, and the results of several trials have been reported [5], [6], [7]. However, the protective efficacy of the vaccine against H5N1 influenza viruses has not been examined in humans that have been challenged with H5N1 HPAI viruses, although it has been shown in mice, chickens, and ferrets [8], [9], [10], [11], [12], [13], [14], [15]. Therefore, we assessed the immunogenic potency of vaccines against H5N1 HPAI virus challenge using cynomolgus macaques as a primate model.
It is thought that, in the airway mucosa, avian influenza viruses bind to sialic acids linked to galactose (Gal) by α2, 3-linkage, but that during pandemics, human influenza viruses bind to sialic acids by α2, 6-linkage [16], [17], [18]. In humans, sialic acids with an α2, 6-linkage are present on the mucosa located in the upper respiratory tract, and sialic acids with an α2, 3-linkage are present on lung mucosa [19], [20]. Therefore, in humans, a high titer of an HPAI virus would be required for the virus to reach the lungs and then to replicate. Previously, a 2.5 × 104 mean tissue culture infectious dose (TCID50) of HPAI viruses was inoculated onto the conjunctivas, tonsils, and tracheas of macaques; however, this dose did not induce severe symptoms, as it does in human patients [21], [22]. It is possible that, in macaques, the inoculum titer of HPAI viruses was not sufficient to reach the alveoli and induce severe symptoms. Thus, in the present study, we inoculated the macaques intratracheally or intranasally using high titers of HPAI viruses.
We have been establishing a library of non-pathogenic influenza A virus strains of different subtypes without using reverse genetics technology [23]. Influenza A viruses of 56 combinations of 15 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes out of 135 theoretical combinations have been isolated from fecal samples of migratory ducks. In addition, 79 other combinations have been generated by genetic reassortment in chicken embryos. Thus, non-pathogenic avian influenza viruses with 135 different combinations of HA and NA subtypes have been stocked as vaccine strain candidates. We have been analyzing their pathogenicity, antigenicity, genetic information, and yields in chicken embryos and have entered all of these data in the database.
Highly pathogenic strains are not suitable for use as vaccine strains due to the risk of infection during preparation, a low virus recovery yield, and the need for the facilities to have a high biosafety level. In order to test the immunogenic potential of non-pathogenic influenza A viruses, we selected an H5N1 vaccine strain from the virus library. Next, inactivated whole virus particle vaccines were prepared, since it has been shown that formalin-inactivated whole virus particles (whole particle vaccines) induce both cytotoxic T lymphocyte (CTL) and antibody responses that protect mice from HPAI virus infection [15].
In the present study, it was found that an H5N1 HPAI virus isolated from a human patient replicated in the upper respiratory tract but did not induce severe clinical symptoms in cynomolgus macaques. This suggests that the avian influenza viruses replicate in the upper respiratory tract of macaques regardless of the expression of sialic acids with α2, 3-linkage. It was also found that inactivated whole particle vaccines of a non-pathogenic H5N1 avian influenza virus obtained from our influenza virus library conferred protective immunity against HPAI virus infection in cynomolgus macaques.
Section snippets
Viruses
Influenza virus A/R (duck/Mongolia/54/01-duck/Mongolia/47/01) (H5N1) (R (Mong-Mong), National Center for Biotechnology Information taxonomy database ID: 376899) is a genetic reassortant generated by co-infection with A/duck/Mongolia/54/01 (H5N2) and A/duck/Mongolia/47/01 (H7N1) in chicken embryos [24]. NA and nonstructural protein (NS) 1 genes of R (Mong-Mong) were derived from the H7N1 virus, and the other genes were derived from the H5N2 virus. HPAI virus A/Vietnam/1194/04 (H5N1) (VN1194) was
Infection of cynomolgus macaques with a highly pathogenic avian influenza virus isolated from a human patient
The pathogenicity of the HPAI virus strain, A/Vietnam/1194/2004 (H5N1) (VN1194), which was isolated from a patient living in Vietnam, was assessed in cynomolgus macaques [3]. A high dose of VN1194 (5 × 107.33 TCID50) was inoculated into the trachea or nasal cavities, since close contact with poultry has been suggested as inducing transmission to human patients [3], and a low dose of an HPAI virus strain, A/HongKong/156/97 (H5N1), was found to not cause severe symptoms, unlike in human patients
Discussion
The protective efficacy of a vaccine against H5N1 HPAI virus was assessed using cynomolgus macaques as a primate model. In this study, two major findings were obtained. Firstly, in infected macaques that were not vaccinated, the viruses were recovered from nasal swabs for up to 5 days post-inoculation and from tracheal swabs for up to 7 days post-inoculation, although the macaques did not show the severe symptoms seen in human patients. In addition, the presence of viral antigen in the alveoli
Acknowledgements
This study was supported by the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases, MEXT Japan. We thank Drs. Keiji Terao, Hirofumi Akari, and Akihiko Uda for their technical consultations, Drs. Takahiro Nakagawa and Norio Okahara for assisting animal care, and Dr. Akira Yokoe for the histological examinations.
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These authors contributed equally to this work.