Immunogenicity and protective efficacy of cold-adapted X-31 live attenuated pre-pandemic H5N1 influenza vaccines
Introduction
Since the first report of human infections in 1997, the highly pathogenic avian influenza (HPAI) H5N1 virus has continued to spread among avian species and occasionally transmitted to humans, resulting in high case-fatality rates. Furthermore, the sudden emergence and global circulation of the 2009 pandemic H1N1 virus (pdmH1N1) by direct human-to-human transmission caused a concern for potential genetic exchange between HPAI H5N1 and the pdmH1N1, which would give rise to reassortants with both high virulence and transmissibility among humans [1], [2].
Such persistent and increasing threats posed by the HPAI emphasize the need of effective vaccines. Several inactivated H5N1 vaccines elicited protective humoral immune responses in animals or humans in multiple doses with adjutants [3], [4], [5], [6]. Baculovirus-expressed recombinant protein vaccine and subvirion vaccine were also tested for their immunogenicity but were poorly immunogenic, requiring at least two doses and the use of adjuvant [7], [8]. Live attenuated influenza vaccine (LAIV) is becoming a more attractive alternative because of several distinctive advantages, including stimulation of both systemic and mucosal immunity, ease of intranasal administration, induction of cell-mediated immunity, and cross-reactivity against antigenically distinct strains [9], [10], [11]. To date, three different cold-adapted influenza A (H2N2) viruses have been developed as the donor strains of LAIVs; A/Ann Arbor/6/60 ca (H2N2), A/Leningrad/134/17/57 ca (H2N2), and A/Leningrad/134/47/57 ca (H2N2) [12], [13], [14]. These cold-adapted strains not only have served as general platforms for generating LAIVs against seasonal influenza viruses, but also for pre-pandemic vaccines against avian H5N1 or H5N2 virus, with robust immunogenicity and protective efficacy in various clinical and preclinical trials [15], [16], [17], [18], [19].
Previously, we generated a cold-adapted influenza A virus, X-31 ca, which demonstrated excellent profiles of productivity, safety, immunogenicity, and protective efficacy in the mouse model [20]. The vaccine also provided extremely early protection against heterologous and heterosubtypic challenges, mediated by an antibody-independent innate immune response [21]. We also established a reverse genetics platform to generate this X-31 ca virus from cloned cDNAs derived from influenza viral RNAs [22], making it possible to generate 6:2 reassortant vaccine candidates through a simple DNA transfection protocol [23]. Using the reverse genetic system with X-31 ca we recently generated live attenuated influenza vaccine against the 2009 pandemic H1N1 virus and evaluated its immunogenicity and protective efficacy against homologous and heterologous infections in animal models [24], [25]. Here we extended the vaccine study to H5N1 virus and evaluated immunogenicity and protective efficacy using a mouse model. We generated two different live attenuated H5N1 pandemic vaccines candidates, each carrying HA and NA from A/Indonesia/05/2005 (H5N1) or A/chicken/Korea/ES/2003 (H5N1). These two H5N1 vaccine candidates were evaluated for their attenuated phenotypes, immunogenicity against both homologous and heterologous H5N1 strains, and protection against lethal heterosubtypic challenges with H5N2 virus.
Section snippets
Generation of H5N1 ca vaccines
The HA and NA of two reassortant H5N1 ca vaccines, R-IN2005 and R-CK2003, were derived from A/Indonesia/05/2005 (clade 2.1) and A/Chicken/Korea/ES/2003 (clade 2.5), respectively. Another reassortant H5N1 virus, R-VN2004, consisting of HA and NA derived from A/Vietnam/1203/2004 (clade 1) was also generated with the same backbone for the analysis of cross-reactivity. Each HA and NA gene was synthesized and cloned into pHW2000 vector via reverse genetics as previously described [23]. To ensure the
Generation and growth properties of H5N1 ca vaccines
Using a reverse genetics system, we generated two 6:2 reassortant viruses, R-IN2005 and R-CK2003, containing the HA and NA genes from the A/Indonesia/05/2005 (clade 2.1) or A/Chicken/Korea/ES/2003 (clade 2.5), in the genetic background of the cold-adapted X-31 (X-31 ca) [20], [22]. To determine whether these two viruses maintain the cold-adapted (ca) and temperature-sensitive (ts) phenotypes, their growth properties at various temperatures in cell cultures and embryonated chicken eggs were
Discussion
The X-31 ca-based H5N1 vaccines exhibited ca and ts phenotypes in eggs demonstrating both robust growth at lower temperatures and restrictive growth at higher temperatures. Based on the previous results that the X-31 ca backbone manifested such phenotypes in its growth ability in eggs [20], it is reasonable to conclude that a limited growth of the H5N1 ca vaccines at higher temperature is afforded by the six internal genes from the backbone strain. However, both vaccines exhibited slightly
Role of the funding source
This work was supported by the R&D Programs of Korean governments: MKE [grant number: 10031969], MEST [grant number: 2010-0001932], MHW [grant number: A085105], and Korea CDC [grant number: 2009-E00522-00]. This work was also supported from Yonsei University Research Fund 2012.
Conflicts of interest
The authors declare no conflicts of financial interests.
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2021, Nanomedicine: Nanotechnology, Biology, and MedicineCitation Excerpt :All serum samples were aliquoted and stored at −80 °C until further use. Three weeks after administering secondary boosting, all mice were challenged under anesthesia via intranasal administration of 5 MLD50, 5 × 106 PFU of A/Indonesia/5/2005 (H5N1) reassortant virus, or 2 MLD50 of A/aquatic bird/Korea/w81/2005 (H5N2) virus with a mixture of PBS, alfaxalone, and xylazine (volume ratio 2:2:1; 100 μL/mouse).36 The survival rate and weight change of the challenged mice were measured every day for 8 days.
Cell-cultured, live attenuated, X-31ca-based H5N1 pre-pandemic influenza vaccine
2017, VirologyCitation Excerpt :Here we explored the potential of the cold-adapted influenza vaccine (CAIV) strain, X-31ca, as a safe and protective cell-cultured live vaccine donor strain. The X-31ca strain has been used as a master donor for the production of reassortant vaccines including the seasonal trivalent influenza vaccine (Jang et al., 2014) and the H5N1 pre-pandemic vaccine (Jang et al., 2013b), as well as the 2009 H1N1 pandemic vaccine (Jang et al., 2013a). Recent studies also showed that the genetic mutations accumulated during cold-adaptation contribute to the attenuation phenotypes of the X-31 ca, supporting its general favorability as a CAIV against many other strains as well (Jang et al., 2016; Lee et al., 2016).
Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin
2016, VirologyCitation Excerpt :It should be noted, however, that the X-31ca MDV showed desired levels of the ts, ca, att phenotypes (Lee et al., 2006b), and the X-31ca-based reassortant vaccines against seasonal H1N1 or H3N2 influenza (Jang et al., 2012b), and the 2009 pandemic influenza (Jang et al., 2013b) closely resembled the X-31 MDV. An exception was the X-31ca-bsed highly pathogenic avian influenza (HPAI) H5N1 vaccine that showed high level of replication ability at 39 °C and exhibited increased virulence than the X-31ca MDV and other X-31ca-based vaccines (Jang et al., 2013d). In addition, the temperature profiles of growth properties of caPhil2-HAN81K in both MDCK cells and embryonated eggs suggest that the N81K mutation is an additional ts marker in the LAIV as well.
Genetic analysis of attenuation markers of cold-adapted X-31 influenza live vaccine donor strain
2016, VaccineCitation Excerpt :The X-31 ca virus exhibited the ca, ts, and att phenotypes and also genetic stability in MDCK cells and mice [17]. This donor strain served as a general platform for CAIVs against the seasonal influenza A viruses, the highly pathogenic H5N1 influenza virus, and the 2009 pandemic A/H1N1 virus [18–21]. The X-31 ca-derived CAIVs against these strains consistently demonstrated desired levels of safety, immunogenicity, and protective efficacy, offering X-31 as a suitable substrate in the development of effective and safe CAIVs.
Protective efficacy in mice of monovalent and trivalent live attenuated influenza vaccines in the background of cold-adapted A/X-31 and B/Lee/40 donor strains
2014, VaccineCitation Excerpt :Here we demonstrated that reassortant trivalent vaccines in the background of A/X-31 ca and B/Lee/40 ca donor strains were immunogenic and protective against wild type challenges in the mouse model. In line with our previous studies on monovalent vaccines from two independent donor strains [14–17], the present results also demonstrated acceptable levels of safety, immunogenicity, and protective efficacy both as monovalent and trivalent formulations. A single dose of each monovalent vaccine or the trivalent vaccine elicited protective levels of neutralizing antibodies and efficiently restricted the challenge viral replication to the respiratory tracts, providing complete protection against lethal challenges.
The Quest for a Truly Universal Influenza Vaccine
2019, Frontiers in Cellular and Infection Microbiology
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These authors contributed equally to this article.
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Present address: CJ Pharmaceutics, South Korea.
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Present address: Department of Biotechnology, The Catholic University, South Korea.