An improved reverse genetics system for influenza A virus generation and its implications for vaccine production

Neumann, Gabriele; Fujii, Ken; Kino, Yoichiro; Kawaoka, Yoshihiro

doi:10.1073/pnas.0505587102

Cited by 155 publications

(118 citation statements)

References 24 publications

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“…Reverse genetics systems, developed in the 1990s, still remain a challenging and promising technology, enabling the creation of influenza viruses with modified replicative, pathogenic, antigenic, and immunogenic properties (20,42). We and others have recently shown that it is possible to generate replication-deficient and thus safe, but still highly immunogenic, influenza A viruses by genetic engineering of the NS gene segment (15,18,32,48,51,61).…”

Section: Discussionmentioning

confidence: 99%

Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts

Ferko

Kittel

Romanova

et al. 2006

J Virol

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Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8؉ T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8 ؉ T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8 ؉ T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.

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Section: Discussionmentioning

confidence: 99%

Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts

Ferko

Kittel

Romanova

et al. 2006

J Virol

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“…The HA mutants A/WSN/33 HA-MAY and A/WSN/33 HA-MAC were created. The pTM and pC plasmids required for reverse genetics were transfected into 293T cells according to a published protocol (45). After 48 h of transfection, cytopathic effects were monitored, and hemagglutinin assays were performed to confirm the presence of viruses.…”

Section: Methodsmentioning

confidence: 99%

“…HA (A/Aichi/2/68) and NA (A/Singapore/1/57) DNA sequences cloned into eukaryotic expression vector pCAGGS (58) (45). A QuikChange II XL site-directed mutagenesis kit (Agilent) was used to create site-directed mutations in three acylation sites of influenza virus hemagglutinin (Cys 555, Cys 562, and Cys 565) located in the protein C terminus.…”

Section: Methodsmentioning

confidence: 99%

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

Chlanda

Mekhedov

Waters

et al. 2017

J Virol

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The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, posttranslationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. Deacylation interrupted HA-M1 interactions since deacylated mutant HA failed to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild-type HA, deacylated HA is correlated with released particles with flat envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strainindependent failure of fusion pore enlargement among H2 (A/Japan/305/57), H3 (A/ Aichi/2/68), and H3 (A/Udorn/72) viruses. In contradistinction, acylation made no difference in the low-pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded, as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HA's interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins which help lower the energetic barrier to pore expansion. IMPORTANCE Influenza A virus is an airborne pathogen causing seasonal epidemics and occasional pandemics. Hemagglutinin (HA), a glycoprotein abundant on the virion surface, is important in both influenza A virus assembly and entry. HA is modified by acylation whose removal abrogates viral replication. Here, we used cryoelectron tomography to obtain three-dimensional images to elucidate a role for HA acylation in VLP assembly. Our data indicate that HA acylation contributes to the capability of HA to bend membranes and to HA's interaction with the M1 scaffold protein during virus assembly. Furthermore, our data on VLP and, by hypothesis, virus suggests that HA acylation, while not critical to fusion pore formation, contributes to pore expansion in a target-dependent fashion.KEYWORDS cryo-electron microscopy, electron microscopy, membrane fusion, palmitoylation, protein acylation

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“…Another type of influenza vaccine is the cold-adapted live influenza virus vaccine (CAIV) which has been shown to be more immunogenic than TIV in inducing protective immunity and may be associated with a longer-lasting and more cross-protective immune response than is elicited by TIV [5]. A new technology termed 'reverse genetics' has been developed to generate high growth reassortants [6][7][8][9][10][11] and combines viral genes from the high growth yield laboratory strain of influenza A virus A/PR/8/34 (H1N1) with genes encoding protective antigens of the target viral strains [12]. However, this technology does not change the subsequent manufacturing process needed to produce large stocks of vaccine viruses to make the final TIV or CAIV formulations.…”

Section: Introductionmentioning

confidence: 99%

Heterologous HA DNA vaccine prime—inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses

Wang¹,

Parker²,

Taaffe³

et al. 2008

Vaccine

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The trivalent inactivated vaccine (TIV) is used to prevent seasonal influenza virus infection in humans, however, the immunogenicity of this vaccine may be influenced by the priming effect of previous influenza vaccinations or exposure to antigenically-related influenza viruses. The current study examines the immunogenicity of a clinically licensed TIV in rabbits naïve to influenza antigens. Animals were immunized with either the licensed TIV, a bivalent (H1 and H3) HA DNA vaccine or the combination of both. Temporal and peak level serum anti-influenza virus IgG responses were determined by ELISA. Functional antibody responses were measured by hemagglutination inhibition and microneutralization against either A/NewCaledonia//20/99 (H1N1) or A/Panama/2007/99 (H3N2) influenza viruses. Our results demonstrate that the immunogenicity of the TIV is low in seronegative animals. More significantly, the heterologous DNA prime-TIV boost regimen was more immunogenic than the homologous prime-boost using either TIV or DNA vaccines alone. This finding justifies further investigation of HA DNA vaccines as a priming immunogen for the next generation of vaccines against seasonal or pandemic influenza virus infections.

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An improved reverse genetics system for influenza A virus generation and its implications for vaccine production

Cited by 155 publications

References 24 publications

Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts

Live Attenuated Influenza Virus Expressing Human Interleukin-2 Reveals Increased Immunogenic Potential in Young and Aged Hosts

Palmitoylation Contributes to Membrane Curvature in Influenza A Virus Assembly and Hemagglutinin-Mediated Membrane Fusion

Heterologous HA DNA vaccine prime—inactivated influenza vaccine boost is more effective than using DNA or inactivated vaccine alone in eliciting antibody responses against H1 or H3 serotype influenza viruses

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