A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

Abstract

The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

Prime-Boost Vaccination With a Novel Hemagglutinin Protein Produced in Bacteria Induces Neutralizing Antibody Responses Against H5-Subtype Influenza Viruses in Commercial Chickens

“…Previously, we confirmed the protective efficacy of rH5- E. coli by means of experimental infections performed in specific pathogen-free (SPF) layer chickens (41). Prime-boost immunizations with rH5- E. coli (25 μg per dose) and aluminum hydroxide (alum) adjuvant at 4-week intervals protected 100% and 70% of chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively.…”

“…The pIGCmT7Kes expression plasmid (44) containing cDNA encoding a fragment (aa 17–522, Δ341–346) of the H5 HA (pIGKesHA17522Δ) was obtained as described previously (41). The source sequence originated from the H5N1 A/swan/Poland 305-135V08-2006 strain of HPAIV (EpiFlu Database Accession No.…”

“…The target protein was expressed, refolded, and purified according to the quality-focused procedure for laboratory scale production, as described previously (41). Briefly, transformed BL21 (DE3) E. coli cells were cultured in LB medium with chloramphenicol and IPTG induction.…”

“…Our previous (41) work and the work presented here respond to the demand for an inexpensive, safe vaccine against HPAIVs that can be used in preventive or emergency vaccinations in compliance with the DIVA strategy. A novel, ectodomain-based HA protein, with the sequence of the H5N1 HP viral strain, was expressed in Escherichia coli , refolded, and chromatographically purified from IBs.…”

Recent outbreaks of highly pathogenic avian influenza viruses in South Korea

“…The use of current conventional egg-based H5 AI vaccines are limited by subtype-specific vaccine efficacy and long period of vaccine production (approximately 6 months) [ 38 ]. Therefore, previous studies have suggested the use of recombinant protein vaccines based on bacterial [ 60 ], mammalian [ 61 ], and recombinant baculovirus/insect cell expression system [ 62 ] for controlling HPAIVs. Among the viral proteins of H5 AI viruses, the HA and matrix protein 2 ectodomains (M2e) have been developed as candidate antigens for recombinant protein vaccines [ 60 62 63 ].…”