Live Attenuated Influenza Vaccines against Highly Pathogenic H5N1avian Influenza: Development and Preclinical Characterization

Abstract

In this paper we describe the development and the outcomes of the preclinical studies of temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine (LAIV) based on highly pathogenic avian influenza viruses A/H5N1 with pandemic potential. The LAIV candidates were developed by methods of classical reassortment between H2N2 Master Donor Virus for Russian LAIV and H5N1 viruses generated by reverse genetics for inactivated vaccine. These reverse genetically generated viruses were chosen as a source of H5 hemagglutinin and contained a modified protease cleavage site believed to be associated with high virulence. The progeny of reassortment had 7:1 gene composition and were characterized by antigen specificity of the HA of the pandemic virus, a high growth rate in chicken embryos and their parameters of temperature sensitivity and cold adaptation confirmed preserved attenuation of the Master Donor Virus. In addition, one H5N1 LAIV 6:2 reassortant was generated by reverse genetics. When tested in appropriate animal models, all candidates for H5N2 LAIV vaccine were found to be safe, immunogenic and effective in protecting from severe disease, mortality and pathology caused by the homologues lethal challenge virus and they almost completely eliminated challenge virus replication in vaccinated animals. They did not appear to differ in tested properties from the reverse genetically generated H5N1 LAIV 6:2 reassortant which contained wild-type N1-neuraminidase in addition to H5-hemagglutinin. The most promising for further clinical trials has been considered a LAIV candidate to virus A/turkey/Turkey/1/2005 (H5N1, clade 2.2).

Live Attenuated Reassortant Vaccines Based on A/Leningrad/134/17/57 Master Donor Virus Against H5 Avian Influenza

“…Then, another prepandemic LAIV candidate, A/17/Vietnam/04/65107 (H5N2) (Len–VN), based on H5N1 virus of clade 1, A/Vietnam/1203/2004 was developed [17]. To date, these strains are immunogenically obsolete.…”

“…To date, these strains are immunogenically obsolete. For this reason, a potentially pandemic A/17/turkey/Turkey/05/133 (H5N2) (Len–tT) vaccine candidate related to more modern antigenic clade 2.2 was generated [17]. …”

“…In the next experiments, to reduce a risk of manipulations with highly pathogenic for humans avian influenza viruses, PR8–based reassortants for parenterally administered, IIV of subtype H5N1, VN/PR/CDC–RG and NIBRG–23 were used as a source of external glycoproteins [17]. The HA of these H5N1/PR8 viruses is engineered by Reverse Genetics (RG) to remove four basic amino acid codons from the cleavage site of HA as described in [20], resulting in a virus that is considered to be attenuated for natural hosts and safe for humans.…”

“…Shtyrya et al [25] suggested that combination of avian HA and human NA may result in decreasing the replicative potential of reassortant virus because of disturbance of a functional balance between “alien” HA and NA. However, Larionova et al [17] demonstrated that combination of avian H5HA and human N2NA resulted in significant increasing the infectivity of 7:1 reassortants in compare with H5N1 parental viruses which contained both HA and NA of avian origin. In addition, phenotypical pattern of H5N2 LAIV candidates did not differ from such of the Len–MDV – transferring seven genes from Len–MDV into the H5N1/PR8 genome led to a dramatic decrease in infectivity of resulting H5N2 reassortants at the temperature of 38–39 degrees C ( ts phenotype) and significantly increased their ability to grow at the temperature of 26 o C ( ca phenotype).…”

“…Guinea pigs and mice are the most commonly used species. These two species were used for determination of acute and sub–acute toxicity of Len–tT LAIV candidate [17]. Administration of Len–tT LAIV did not cause death, did not change the external appearance and behavior of animals, did not affect their consumption of food or water, and had no significant effect on body weight.…”