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

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

Influenza viruses used in this work.

<p>Influenza viruses used in this work.</p

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

<div><p>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 <i>Escherichia coli</i>, 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-<i>E</i>. <i>coli</i>, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses <i>in vitro</i>. 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-<i>E</i>. <i>coli</i> 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-<i>E</i>. <i>coli</i> 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.</p></div

Viral RNA detection after challenging the chickens.

<p>The chickens were vaccinated and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172008#pone.0172008.g001" target="_blank">Fig 1</a>. The amount of viral RNA in the (A-B) swabs and (C) organs of chickens after infection with (A, C) homologous (Exp 1) and (B, C) heterologous (Exp 2) H5N1 HPAIVs was determined by quantitative real-time mRT-PCR. Analyses were conducted as described in the Materials and Methods. Viral RNA titers were expressed as log₁₀ eqEID₅₀ (50% egg infectious dose) <i>per</i> milliliter of swabs or gram of tissue. The results for the vaccinated, contact and control chickens in Exp 1 and Exp 2 are presented. For animals that survived the (A) homologous or (B) heterologous challenge, data on viral RNA in oropharyngeal and cloacal swabs collected at (A) 3, 7 and 10 (Exp 1) or (B) 3, 7, 10 and 14 (Exp 2) days post-infection (dpi) are provided. The data are completed with results from analyses of (A-B) swab samples and (C) brains, lungs, kidneys and spleens collected postmortem (pm) from chickens that died upon infection at the indicated number of days post-infection. Filled symbols, differentiated between (A-B) oropharyngeal and cloacal swabs or (C) organs, represent the results for individual or pooled samples collected from chickens as indicated by the numbers on the horizontal axes. A value of zero indicates that viral RNA was not detected. Bars represent the Geometric Mean Titers (GMT) of viral RNA in (A, B) oropharyngeal or cloacal swabs and (C) organs collected from individual chicken groups at the subsequent number of days post-infection and/or postmortem. Only virus-positive samples were considered in the GMT calculations.</p

Survival rates in the challenge experiments.

<p>A group of 3-week-old specific pathogen-free (SPF) chickens denoted 1–1 to 1–10 (Exp 1) and 3½-week-old ones denoted 2–1 to 2–10 (Exp 2) were vaccinated subcutaneously twice at a 4-week interval with 25 μg of rH5-<i>E</i>. <i>coli</i> and aluminum hydroxide adjuvant (Alhydrogel). Three weeks after the boosts, the chickens were inoculated intranasally/intraocularly (in/io) with 10⁶ 50% egg infectious doses (EID₅₀) of (A) clade 2.2 homologous (Exp 1) or (B) clade 1 heterologous (Exp 2) H5N1 HPAIVs, as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172008#pone.0172008.t001" target="_blank">Table 1</a>. Approximately 24 hours after inoculation, non-vaccinated contact SPF chickens denoted 1-CC-1, 1-CC-2 (Exp 1) and 2-CC-1, 2-CC-2 (Exp 2) were introduced to the tested groups. Untreated, fully susceptible SPF chickens denoted 1-C-1 to 1-C-5 (Exp 1) and 2-C-1 to 2-C-5 (Exp 2) that were infected in/io with challenge viruses at the same age as the vaccinated animals served as positive controls. The data are presented as the survival percentage in the respective groups on each day during the 2-week observation period.</p