Influenza-pseudotyped Gag virus-like particles (VLPs) were produced via the expression of influenza hemagglutinin (HA), neuraminidase (NA) and the murine leukemia virus Gag product in the baculovirus-insect cell expression system. Hemagglutination specific activities of sucrose gradient-purified VLPs were similar to those of egg-grown influenza viruses but particle morphologies were gamma retrovirus-like in the form of consistent 100nm spheres. Immunization of mice and ferrets demonstrated robust immunogenicity and protection from challenge with no measurable morbidity. Ferret data were striking in that immunization with H5N1 VLPs representing either A/Vietnam/1203/04 or A/Indonesia/5/05 resulted in solid protection against highly pathogenic A/Vietnam/1203/04 challenge with no detectable virus in the upper respiratory tract post-challenge in either group. H1N1 VLP immunization of ferrets resulted in partial protection against H5N1 challenge with markedly accelerated virus clearance from the upper respiratory tract relative to controls. The immunogenicity of influenza-pseudotyped VLPs was not dependent on the adjuvant properties of replication competent contaminating baculovirus. These data demonstrate robust vaccine protection of Gag-based, influenza-pseudotyped VLPs carrying a variety of influenza antigens and suggest applicability toward a number of additional respiratory viruses.
"This study demonstrates that modification of the H5-HA MBCS may be beneficial to the development of new candidate vaccines from mammalian cells to combat H5N1 HPAIV. Influenza pseudotypes can be used to elicit antibodies in immunized mice, and can be employed in the study of HA and in the detection of HA antibodies, which is important for vaccine evaluation and sera surveillance (Garcia J M, et al., 2010; Haynes J R, et al., 2009; Ho J W, et al., 2008; Kong W P, et al., 2006; Lin A H, et al., 2002; Nefkens I, et al., 2007; Tsai C G, et al., 2009; Wang S Y, et al., 2009; Wang W, et al., 2010; Wang W, et al., 2008; Wu Q, et al., 2009; Zhang S, et al., 2008). Here, we generated influenza pseudotypes packaging mammalian expressed HA (wtH5 or mtH5) and N1 proteins around the core of MLV using a strategy similar to that previously described by Szecsi et al (Szecsi J, et al., 2006). "
[Show abstract][Hide abstract] ABSTRACT: Traditionally, the multibasic cleavage site (MBCS) of surface protein H5-hemagglutinin (HA) is converted to a monobasic one so as to weaken the virulence of recombinant H5N1 influenza viruses and to produce inactivated and live attenuated vaccines. Whether such modification benefits new candidate vaccines has not been adequately investigated. We previously used retroviral vectors to generate wtH5N1 pseudotypes containing the wild-type HA (wtH5) from A/swine/Anhui/ca/2004 (H5N1) virus. Here, we generated mtH5N1 pseudotypes, which contained a mutant-type HA (mtH5) with a modified monobasic cleavage site. Groups of mice were subcutaneously injected with the two types of influenza pseudotypes. Compared to the group immunized with wtH5N1 pseudotypes, the inoculation of mtH5N1 pseudotypes induced significantly higher levels of HA specific IgG and IFN-γ in immunized mice, and enhanced protection against the challenge of mouse-adapted avian influenza virus A/Chicken/Henan/12/2004 (H5N1). This study suggests modification of the H5-hemagglutinin MBCS in retroviral pseudotypes enhances protection efficacy in mice and this information may be helpful for development of vaccines from mammalian cells to fight against H5N1 influenza viruses.
"produced chimeric VLPs (RVF chimVLPs) in HEK-293 cells constitutively expressing a Moloney murine leukaemia virus (MoMLV) gag protein (293-gag). The presence of gag in VLPs has been shown to increase yield and stability (Gheysen et al., 1989; Haffar et al., 1990; Rovinski et al., 1992; Hammonds et al., 2003; Haynes et al., 2009). RVFV chimVLPs were produced by transient transfection of 293-gag cells with expression plasmids encoding the RVFV glycoproteins and nucleo- protein. "
[Show abstract][Hide abstract] ABSTRACT: Rift Valley fever virus (RVFV) is an arbovirus that causes significant morbidity and mortality in both humans and livestock. With increased world travel and the threat of bioterrorism, there is a real risk of RVFV spreading to naïve geographical areas (Trans. R. Soc. Trop. Med. Hyg., 73, 1979, 618; MMWR Morb. Mortal. Wkly Rep., 49, 2000, 905). The introduction of RVFV would cause critical public health, agricultural and economic damage. Despite the clear need for an efficacious vaccine, there are no United States (US) Food and Drug Administration or US Department of Agriculture approved vaccines against RVFV. To address this need, a virus-like particle (VLP)-based vaccine candidate was developed. First, a non-replicating chimeric RVF VLP vaccine candidate was generated that protected mice and rats against a lethal RVFV challenge. This was followed by the development and optimization of conditions for production of RVF VLPs in insect and mammalian cells. Immunological studies demonstrated that VLP-based vaccine candidates elicit both humoral and cellular immune responses. Subsequent challenge studies using a lethal wild-type RVFV strain under high-containment conditions showed that RVF VLP vaccine candidates can completely protect mice and rats.
Zoonoses and Public Health 09/2012; 59((Suppl 1)):1-9. DOI:10.1111/j.1863-2378.2012.01478.x · 2.37 Impact Factor
"Influenza VLPs represent a promising candidate vaccine because they are nonreplicating and safe, contain viral structural proteins, and show robust immunogenicity (Kang et al. 2009a). By expressing 3 structural proteins, HA, NA, and M1 (Bright et al. 2008; Bright et al. 2007; Crevar and Ross 2008; Kang et al. 2009b; Mahmood et al. 2008), or HA, NA, and the Gag protein of MLV (Haynes et al. 2009), a variety of H5N1 influenza VLPs are produced from baculovirus–insect cell systems.. These VLPs are effective for inducing broad, Fig. 3. Characterization of influenza virus-like particles (VLPs). "
[Show abstract][Hide abstract] ABSTRACT: Vaccination is an effective way to protect from influenza virus infection. Among the new candidates of influenza vaccines, influenza virus-like particles (VLPs) seem to be promising. Here, we generated 2 types of H5N1 influenza VLPs by co-expressing influenza virus Env (envelope protein) and murine leukemia virus (MLV) Gag-Pol. VLPs generated by co-transfection of pHCMV-wtH5 or pHCMV-mtH5 with pSV-Mo-MLVgagpol and pHCMV-N1 were named as wtH5N1 VLPs or mtH5N1 VLPs. The plasmid of pHCMV-wtH5 encoded the wild-type hemagglutinin (HA) (wtH5) from A/swine/Anhui/ca/2004 (H5N1) with a multibasic cleavage site, while pHCMV-mtH5 encoded the modified mutant-type (mtH5) with a monobasic cleavage site. Influenza virus HA VLPs were characterized and equal amounts of them were used to immunize mice subcutaneously, intraperitoneally, or intramuscularly. The levels of HA-specific IgG1, IFN-γ, and neutralization antibodies were significantly induced in mice immunized with wtH5N1 VLPs or mtH5N1 VLPs via all 3 routes, while HA-specific IgG2a was barely detectable. IL-4 secretion was detected in mice subcutaneously immunized with wtH5N1 VLPs or mtH5N1 VLPs, or intramuscularly immunized with mtH5N1 VLPs. Our results indicated that both H5N1 influenza VLPs could induce specific humoral and cellular immune responses in immunized mice. In conclusion, our study provides helpful information for designing new candidate vaccines against H5N1 influenza viruses.
Canadian Journal of Microbiology 03/2012; 58(4):391-401. DOI:10.1139/w2012-006 · 1.22 Impact Factor
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