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

Abstract

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.

Figure 1. Expression of functional hemagglutinin (HA) with recombinant VRPs.

(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.

Figure 2. VSV replicon vaccines protect chickens from HPAIV challenge in a dose-dependent manner.

Chickens (group size n = 5) were immunized (i.m.) once or twice with VSVΔG(HA_H5-HP) using the indicated doses and subsequently challenged by intranasal inoculation with 10⁶ TCID₅₀ of A/whooper swan/Mongolia/3/2005 (H5N1). The animals were surveyed daily for survival (a) and clinical symptoms (b). The animals were scored as follows: (0) healthy, (1) ill (animals show one of the following symptoms: apathy, ruffled feather, anorexia, diarrhea, cyanosis of the exposed skin, comb and wattles, edemas of the face and/or head, neurological symptoms), (2) severely ill (severe or more than one of the symptoms mentioned above), and (3) dead. A daily clinical index representing the mean value of all chickens per group was calculated. For analysis of virus shedding oropharyngeal (c) and cloacal (d) swabs were taken from each animal at daily intervals for a period of 8 days post infection. RNA was extracted from the swabs and analyzed for the presence of viral RNA segment 7 by quantitative RT-PCR.

Figure 3. Western-blot analysis of purified LPAIV particles with different chicken immune sera.

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.

Figure 4. Inhibition of HA-mediated syncytia formation by chicken immune sera.

(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.