Protective efficacy of Newcastle disease virus expressing soluble trimeric hemagglutinin against highly pathogenic H5N1 influenza in chickens and mice.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5(3)). A single intramuscular immunization with NDV-sH5(3) or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5(3) was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5(3) was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited. CONCLUSIONS/SIGNIFICANCE: Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles

Serum antibody responses in vaccinated chickens.

<p>HI antibody titers against A) NDV Herts/33 or B) AIV measured in individual serum samples collected on day 21 p.v. via ON/IT or IM route. Titres are expressed as the reciprocal of the highest serum dilution showing HI. The dashed line indicates the lower limit of detection. Titers below the detection limit were assigned a value of 2 or 4, respectively for calculation of the mean. Horizontal bars represent the geometric mean titers of the groups and vertical bars represent 95% confidence intervals. Serum titers from chickens that died after the challenge are indicated by filled circles, the open circles correspond to animals that survived the challenge. For reference, each panel includes the same PBS group.</p

Detection of H5N1 challenge virus in tracheal and cloacal swabs of vaccinated chickens.

a<p>Day (D) post infection on which tracheal and cloacal swabs were collected are indicated. Amount of RNA detected in swabs is expressed as log10 TCID50 ml-1 equivalents.− =  negative (C_T >39.0); x  =  not tested because chicken did not survive after challenge with HPAIV H5N1.</p

Vaccination of mice.

<p>Groups of 10 BALB/c mice were vaccinated with 10^6.5 TCID₅₀ of NDV-H5 or NDV-sH5³, either via the ON or IM route. One group of mice was mock-vaccinated (PBS) as challenge control. Three weeks after the vaccination, mice were infected with ∼10 LD₅₀ of HPAIV H5N1 and weighed daily and observed for clinical signs during 14 days. Graphed for each group are A and B) Kaplan-Meier survival curves indicating percentage of survival p.c., C and D) mean percentage of body weight changes relative to starting weights measured on the day of challenge (day 0) with error bars representing the standard deviation, and E and F) median clinical scores observed after challenge. For reference, each panel includes the same PBS group.</p

Vaccination of chickens.

<p>Groups of 10 SPF chickens were immunized with 10⁷ TCID₅₀ of NDV-H5 or NDV-sH5³, either via the ON/IT or IM route. Another group received an IM immunization with sH5³ adjuvanted in Stimune. As a challenge control, one group of chickens was mock-vaccinated with PBS. Three weeks after the vaccination, birds were challenged with ∼10⁵ TCID₅₀ of HPAIV H5N1. A and B) Kaplan-Meier survival curves indicating percentage of survival p.c. on each day for each group that was (mock-)vaccinated via the ON/IT (A) or IM (B) route. C and D) Clinical index calculated on basis of clinical signs (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044447#s2" target="_blank">materials and methods</a>) observed after challenge for each group that was (mock-)vaccinated ON/IT (C) or IM (D). An index of 3.0 means that all birds died within 24 hours. For reference, each panel includes the same PBS group.</p

Construction of recombinant NDV, expression and purification of H5 protein.

<p>A) Construction of pFL-HertsΔPBC expressing influenza H5N1 HA proteins. The sequence of the full-lenght cDNA of Herts/33, which codes for the multibasic cleavage site of the F protein was mutated into a monobasic cleavage site-encoding sequence (FΔPBC) resulting in pFL-HertsΔPBC. The HA gene of H5N1 A/Vietnam/1194/04, encoding either the full length or the soluble trimeric form, was cloned between the <i>Pme</i>I sites created in the intergenic region of the P and M genes of the pFL-HertsΔPBC vector following insertion of a linker containing NDV transcription termination and start signals. NP, nucleoprotein; P, phosphoprotein; M, membrane protein; F, fusion protein; HN, hemagglutinin-neuraminidase; L, large polymerase. B) Expression of HA protein by recombinant NDV in QM5 cells. Cells were infected with wt NDV (Herts/33), NDV-H5, NDV-sH5³ or were mock-infected and analyzed by immunocytochemistry using mAb specific for NDV F or influenza virus H5. Scale bars represent 400 μm. C) Western blot analysis with an anti-H5 mAb following SDS-PAGE of combined lysate of QM5 cells and the cell culture supernatant (20 μl/T75 flask). The MW of the marker proteins is indicated on the left. D) The sH5³ protein was purified from the supernatant of NDV-sH5³-infected QM5 cells and subjected to SDS-PAGE (∼ 1 μg/15 μl), after which proteins were stained with Blue stain reagent. A series of BSA dilutions were run in parallel. E) Blue native PAGE analysis of sH5³ purified from the cell culture supernatant of NDV-sH5³-infected QM5 cells (∼ 1.7 μg/20 μl). The position in the gel of the trimeric and monomeric form of the H5 protein is indicated. Prior to gel electrophoresis some samples were heated at 95°C for the indicated time periods.</p