Pseudotyping of vesicular stomatitis virus with the envelope glycoproteins of highly pathogenic avian influenza viruses

Pseudotype viruses are useful for studying the envelope proteins of harmful viruses. This work describes the pseudotyping of vesicular stomatitis virus (VSV) with the envelope glycoproteins of highly pathogenic avian influenza viruses. VSV lacking the homotypic glycoprotein (G) gene (VSVΔG) was used to express haemagglutinin (HA), neuraminidase (NA) or the combination of both. Propagation-competent pseudotype viruses were only obtained when HA and NA were expressed from the same vector genome. Pseudotype viruses containing HA from different H5 clades were neutralized specifically by immune sera directed against the corresponding clade. Fast and sensitive reading of test results was achieved by vector-mediated expression of GFP. Pseudotype viruses expressing a mutant VSV matrix protein showed restricted spread in IFN-competent cells. This pseudotype system will facilitate the detection of neutralizing antibodies against virulent influenza viruses, circumventing the need for high-level biosafety containment

Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry

Biological and protective properties of immune sera directed to the influenza virus neuraminidase

The envelope of influenza A viruses contains two large antigens, hemagglutinin (HA) and neuraminidase (NA). Conventional influenza virus vaccines induce neutralizing antibodies that are predominantly directed to the HA globular head, a domain that is subject to extensive antigenic drift. Antibodies directed to NA are induced at much lower levels, probably as a consequence of the immunodominance of the HA antigen. Although antibodies to NA may affect virus release by inhibiting the sialidase function of the glycoprotein, the antigen has been largely neglected in past vaccine design. In this study, we characterized the protective properties of monospecific immune sera that were generated by vaccination with recombinant RNA replicon particles encoding NA. These immune sera inhibited hemagglutination in an NA subtype-specific and HA subtype-independent manner and interfered with infection of MDCK cells. In addition, they inhibited the sialidase activities of various influenza viruses of the same and even different NA subtypes. With this, the anti-NA immune sera inhibited the spread of H5N1 highly pathogenic avian influenza virus and HA/NA-pseudotyped viruses in MDCK cells in a concentration-dependent manner. When chickens were immunized with NA recombinant replicon particles and subsequently infected with low-pathogenic avian influenza virus, inflammatory serum markers were significantly reduced and virus shedding was limited or eliminated. These findings suggest that NA antibodies can inhibit virus dissemination by interfering with both virus attachment and egress. Our results underline the potential of high-quality NA antibodies for controlling influenza virus replication and place emphasis on NA as a vaccine antigen. IMPORTANCE The neuraminidase of influenza A viruses is a sialidase that acts as a receptor-destroying enzyme facilitating the release of progeny virus from infected cells. Here, we demonstrate that monospecific anti-NA immune sera inhibited not only sialidase activity, but also influenza virus hemagglutination and infection of MDCK cells, suggesting that NA antibodies can interfere with virus attachment. Inhibition of both processes, virus release and virus binding, may explain why NA antibodies efficiently blocked virus dissemination in vitro and in vivo. Anti-NA immune sera showed broader reactivity than anti-HA sera in hemagglutination inhibition tests and demonstrated cross-subtype activity in sialidase inhibition tests. These remarkable features of NA antibodies highlight the importance of the NA antigen for the development of next-generation influenza virus vaccines

Virus neutralizing activity of serum antibodies from vaccinated SPF chickens.

a<p>SPF chickens were immunized (i.m.) once or twice with the indicated doses of either VSV*ΔG or VSV*ΔG(HA_H5-HP).</p>b<p>Blood was collected from vaccinated chickens at 9 weeks of age.</p><p>Serum was prepared and inactivated for 30 minutes at 56°C. Virus neutralisation assays were performed with HPAIV A/whooper swan/Mongolia/3/2005 (H5N1).</p

Fusion inhibitory activity of chicken immune sera.

a<p>Vero cells were infected with the indicated recombinant VRPs using an MOI of 5 ffu/cell.</p><p>Five hours p.i., HA_H5/H1 and HA_H1 were proteolytically activated with trypsin. Six hours p.i., the cells were incubated with serial dilutions of the indicated chicken sera or Mab C179, shortly exposed to pH 5.4 and incubated for 2 hours in normal medium prior to fixation. The serum dilution causing 50% inhibition of syncytia formation was calculated. Mean values and standard deviations of three independent experiments are shown.</p

Expression of functional hemagglutinin (HA) with recombinant VRPs.

<p>(a) Genome maps of recombinant VSV: the parental VSV genome contains five transcription units encoding for the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the large RNA polymerase (L). VSV*ΔG lacks the glycoprotein G gene but contains the eGFP gene instead (the asterisk denotes for eGFP). VSV*ΔG(HA) expresses the influenza virus HA from the fourth gene position while eGFP is expressed from an additional transcription unit downstream of HA. (b) Immunofluorescence analysis of Vero cells 8 hours p.i. with either VSV*ΔG, VSV*ΔG(HA_H5-HP) or VSV*ΔG(HA_H5-LP). At 5 hours p.i. with VSV*ΔG(HA_H5-LP), the cells were treated for 60 minutes with trypsin (+T) or were left untreated (−T). Thereafter, the cells were exposed for 5 minutes to either pH 5.4 or pH 7.4, incubated for 60 minutes at 37°C with normal medium, fixed with formalin, and finally processed for immunofluorescence using a swine antiserum to HA_H5 (red fluorescence). Nuclei were stained with DAPI (blue fluorescence). Expression of eGFP is indicated by green fluorescence. Scale bar represents 20 µm.</p

Inhibition of HA-mediated syncytia formation by chicken immune sera.

<p>(a) Diagrams of chimeric hemagglutinins. The chimeric H1/H5 hemagglutinin was constructed by swapping the globular head domain located between C42 and C274 of A/chicken/Yamaguchi/8/2004 (H5N1) HA_H5 (grey) with that of the corresponding region from A/duck/Italy/1447/2005 (H1N1) HA_H1 (green). The chimeric H5/H1 hemagglutinin was constructed accordingly by replacing the HA_H1 globular head domain with the corresponding HA_H5 domain. The proteolytic cleavage site (arrow), fusion peptide (FP) and transmembrane (TM) domain are indicated. (b) Flow cytometric analysis of BHK-21 cells expressing parental and chimeric HAs. Cells were infected with either VSV*ΔG (blue graphs) or VRPs expressing the indicated HAs (red graphs). At 6 hours p.i., cells were stained with chicken immune sera specific for either HA_H5 or HA_H1 and goat anti-chicken IgY Alexa-546 conjugates. (c) Inhibition of syncytia formation. Vero cells were infected with VRPs expressing the indicated HAs using an MOI of 5 ffu/cell. At 5 hours p.i., cells were treated for 60 minutes with acetylated trypsin to proteolytically activate HA_H1 and HA_H5/H1. At 6 hours p.i., the cells were incubated for 30 minutes at 37°C with the indicated chicken immune sera (diluted 1∶20 in MEM), exposed for 5 minutes at 37°C to pH 5.4, further incubated in medium for 2 hours, and fixed with paraformaldehyde. The nuclei were stained with DAPI (blue fluorescence). Expression of eGFP is indicated by green fluorescence. Scale bar represents 20 µm.</p

Western-blot analysis of purified LPAIV particles with different chicken immune sera.

<p>A/duck/Hokkaido/Vac-01/2004 (H5N1) was grown on MDCK cells in the presence of trypsin and pelleted from the cell culture supernatant by ultracentrifugation. The virus particles were separated by SDS-PAGE under non-reducing (− βME) or reducing (+ βME) conditions and blotted onto a nitrocellulose membrane. HA antigen was detected with the chicken immune sera indicated on top of the blot. The positions of marker proteins of known molecular masses are indicated on the left, the positions of the HA subunits on the right hand side.</p

Survival rate and seroconversion of vaccinated and sentinel SPF chickens following challenge infection with A/whooper swan/Mongolia/3/2005 (H5N1).

a<p>SPF chickens (n = 6) were immunized twice with either VSV*ΔG or VSV*ΔG(HA_H5-LP) and subsequently challenged with A/whooper swan/Mongolia/3/2005 (H5N1).</p><p>At 1 day post challenge, sentinel birds (n = 6) were housed along with the infected chickens. The animals were serologically tested directly before infection and 12 days post infection for the presence of NP and HA_H5 antibodies, respectively. The number of surviving and seropositive animals are shown. NA, not applicable.</p