PLoS One. 2017 Feb 17;12(2):e0172008. doi: 10.1371/journal.pone.0172008.

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

Sączyńska V1, Romanik A1, Florys K1, Cecuda-Adamczewska V1, Kęsik-Brodacka M1, Śmietanka K2, Olszewska M2, Domańska-Blicharz K2, Minta Z2, Szewczyk B3, Płucienniczak G1, Płucienniczak A1.
1 Institute of Biotechnology and Antibiotics, Warsaw, Poland.
2 Department of Poultry Diseases, National Veterinary Research Institute, Puławy, Poland.
3 Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland.



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.
PMID: 28212428



A novel HA antigen was produced in bacteria (rH5-E. coli)

The H5N1 HPAIVs remain a serious epidemiological problem. Our work responds to the demand for an inexpensive, safe vaccine against influenza that can be quickly and efficiently manufactured in case of a pandemic threat. The antigen produced in bacteria was designed as the ectodomain-based, soluble HA protein of H5N1 HPAIV with a polybasic cleavage site deletion [1]. Consistently, the designed protein contained the full-length HA1 subunit encompassing the viral HA globular domain with the host cell receptor-binding site. It also contained a fragment of the stalk domain-forming HA2 subunit with a fusion peptide and bromelain cleavage site at N- and C-termini, respectively. The target protein was deprived of the signal peptide, characteristic only of the precursor form of the antigen, as well as the transmembrane and cytoplasmic domains of mature viral HA. The structures of the source H5 HA protein and the target antigen, referred to as rH5-E. coli, are presented schematically in Fig 1.

The viral HA fragment, corresponding to rH5-E. coli, contains immunodominant and subtype-, clade- and strain-specific neutralizing epitopes of the HA1 subunit as well as hetero-subtype-neutralizing epitopes of the HA2 subunit. Antibodies against HA1 subunit are known to act mainly by blocking the binding of the viral HA to cellular receptors, and against HA2 subunit by interfering with the subsequent HA-mediated membrane fusion.

The prototype vaccine H5 HA (aa 17-522, ΔRRRKKR) was efficiently expressed in E. coli due to the removal of the native HA hydrophobic regions and to codon optimization [1]. Because the target antigen was expressed in the form of inclusion bodies, the main challenge of our bacterial HA production was to develop a refolding method that would allow to obtain a similar antigen to viral HA. The lack of a signal sequence and an anchoring peptide participating in HA folding during biosynthesis and the truncation of the HA2 subunit involved in native HA trimer formation were potential difficulties. As produced in a prokaryotic expression system, the protein did not undergo glycosylation known to play an important role in the proper folding and oligomerization of viral HA and trimer stabilization.

To evaluate the proof-of-concept for our bacterial H5 HA, a quality-focused procedure was used in the laboratory level production of the target antigen [1]. According to this procedure, proteins solubilized from inclusion bodies were subjected sequentially to purification on a DEAE-Sepharose column, refolding by dilution and purification on a Phenyl-Sepharose column.



Fig 1. Schematic representation of the source (A) and the target (B) H5 HA proteins. (A) A full-length HA from the H5N1 HPAIV (A/swan/Poland 305- 135V08-2006). (B) Bacterially expressed H5 HA (rH5-E. coli). (Data adapted from Sączyńska V et al. (2017). PLoS ONE 12(2): e0172008. doi:10.1371/journal.pone.0172008)


rH5-E. coli displays native HA characteristics

The value of the rH5-E. coli was verified by studies on its antigenicity, ability to bind to sialic acid-containing receptors and oligomerization status [1]. Determination of rH5-E-coli antigenicity showed that the protein is recognized by H5-subtype specific, inhibiting hemagglutination and/or virus neutralizing antibodies (Fig 2A). This means that the protein contains well-preserved conformational epitopes of native HA, which are essential for the induction of the protective immune responses. Hemagglutination test showed that rH5-E. coli can bind and agglutinate erythrocytes, in contrast to its 2-mercaptoethanol-reduced form (Fig 2B). This indicates that the receptor binding and oligomeric HA activities of viral antigen were preserved. Beneficial effect of protein oligomerization on its protective efficacy, cross-protection and/or dose sparing is widely recognized.

Our bacterial HA has several unique features and presents some advantages over both the longer and the shorter bacterial HAs (for review, see [2]). This topic is discussed extensively in the original paper [1].



Fig 2. Properties of rH5-E. coli. (A) Antigenicity. The studies were performed by ELISA using commercially available monoclonal and polyclonal antibodies (mAbs, pAbs, respectively) against H5 HA. Most of the mAbs, denoted mAb 1 to mAb 9, were specified as displaying characteristics of H5 subtype-specific Abs (6/9) and recognizing H5 HAs in the hemagglutination inhibition (HI) and/or virus neutralization (VN) tests (8/9). pAb 1 was directed against HA1 and pAb 2 against HA2 subunit of H5 HA. (B) Hemagglutination activity. rH5-E. coli was tested in parallel with its 2-mercaptoethanol-reduced form using chicken erythrocytes. (Data adapted from Sączyńska V et al. (2017). PLoS ONE 12(2): e0172008. doi:10.1371/journal.pone.0172008)


rH5-E. coli protects chickens against H5N1 HPAIVs

The immunogenicity and efficacy of rH5-E. coli were verified by the high-dose challenge exposure of the specific pathogen-free, layer-type chickens to H5N1 HPAIVs [1]. Consistent with the biochemical characteristics (Fig 2A-B), the administration of two rH5-E. coli doses in the presence of aluminum hydroxide adjuvant resulted in 100% and 70% survival rates in the homologous (Exp 1) and heterologous (Exp 2) challenge experiments, respectively (Fig 3A-B). Because rH5-E. coli only partially exists as a functional oligomer [1], a solution for providing higher inter-clade protection with our bacterial HA may be augmented oligomerization and/or improved vaccine composition.

The vaccinated, contact and control chickens in Exp 1 and Exp 2 were evaluated for the presence of homologous and heterologous H5N1 HPAIVs following challenges with the respective viruses [1]. Viral RNA determinations in oropharyngeal and cloacal swabs allowed to establish that vaccination with rH5-E. coli greatly prevented viral replication in 41% of survivors and contributed to the complete clearance of replicating viruses from respiratory and digestive tracts in 59% of survivors. Thus, a total of 85% chickens surviving challenges with homologous and heterologous H5N1 HPAIVs did not or only transiently shed the viruses.

As a consequence of complete chicken protection against homologous H5N1 HPAIV challenge, which was accompanied by the lack of or greatly reduced and transient viral replication, effective virus transmission to fully susceptible contact birds did not occur, resulting in animal survival (Fig 3A). The strong multiplication of heterologous H5N1 HPAIV in 30% of vaccinated chickens that died upon infection led to virus transmission and death of the contact birds (Fig 3B). In the control group animals that quickly succumbed to infection (Fig 3A-B), large amounts of the viruses were determined postmortem.

Our studies clearly indicate the potential of the novel bacterial H5 HA to control H5N1 HPAIV infection and transmission rates in the chicken flocks as well as to reduce viral shedding. In addition, these studies prove once again that non-glycosylated HA protein expressed in the form of inclusion bodies can be a valuable vaccine antigen when appropriate folding and purification methods are applied to the rationally designed protein.



Fig 3. Survival rates in the challenge experiments. A group of 3-week-old specific pathogen-free, layer-type 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-E. coli and aluminum hydroxide adjuvant. Three weeks after the boosts, the chickens were inoculated intranasally/intraocularly with 106 50% egg infectious doses (EID50) of (A) clade 2.2 homologous (Exp 1) or (B) clade 1 heterologous (Exp 2) H5N1 HPAIVs. Approximately 24 hours after inoculation, non-vaccinated contact 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 chickens denoted 1-C-1 to 1-C-5 (Exp 1) and 2-C-1 to 2-C-5 (Exp 2) that were infected with challenge viruses 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. (Data adapted from Sączyńska V et al. (2017). PLoS ONE 12(2): e0172008. doi:10.1371/journal.pone.0172008)


Protection was conferred by H5 subtype-specific neutralizing antibodies

To evaluate the immune responses of chickens to vaccinations with adjuvanted rH5-E. coli and challenges with H5N1 HPAIVs, the anti-H5 HA antibodies in sera were measured using hemagglutination inhibition (HI) and ID Screen Influenza H5 Antibody Competition (FluAC H5, IDVet) assays [1]. The HI test detects antibodies that block virus binding to cellular receptors. FluAC H5 is a diagnostic kit designed to specifically detect antibodies directed against the H5 antigen of influenza A viruses in bird sera.

The first finding from the analysis of the anti-H5 antibody profiles in sera prior to animal infections was that prime-boost vaccination elicits in the chickens antibodies that were active in the HI assay with homologous H5N1 HPAIV and/or in the FluAC H5 test. Chickens that were subjected to studies in the homologous (Exp 1) and heterologous (Exp 2) challenge experiments responded differently to vaccination (50% vs. 100% HI-positivity and 100% vs. 60% FluAC H5-positivity).

The second finding relates the vaccine-induced antibodies against H5 HA in individual chickens to their survival from viral infection. Five chickens of Exp 1 (1-1 to 1-4, 1-9), classified as positive in the HI and FluAC H5 tests after vaccination, as well as the remaining birds in the group (1-5 to 1-8, 1-10), being HI negative but FluAC H5 positive, survived infection with the homologous H5N1 HPAIV (Figs 4A, C and 3A, respectively). Six chickens of Exp 2 (2-1 to 2-6), both HI and FluAC H5 positive after vaccination, and one animal (2-7), HI positive and FluAC H5 negative, survived challenge with heterologous H5N1 HPAIV (Figs 4B, D and 3B, respectively). In contrast, the other three birds (2-8 to 2-10), determined to be HI positive but FluAC H5 negative, died upon infection (Figs 4B, D and 3B, respectively).

The conclusion from obtained results is that the protection of chickens against challenging viruses was primarily provided by antibodies detected by the FluAC H5 test (IDVet) but not, if at all, by HI antibodies. Accordingly, our work showed novel functional antibodies with inter-clade reactivity, which were reasonably assumed to be H5 subtype-specific, non-HI neutralizing antibodies. Possible mechanism of virus neutralization by these antibodies is discussed in the original paper [1]. It remains to be established, similarly as the target epitopes. Identification of novel anti-HA functional antibodies has important implications for the pre-challenge efficacy evaluation of vaccines against H5N1 HPAIVs and their designs.

In influenza vaccine studies, the level of HI antibody titer is widely considered as the correlate of vaccine-induced protection. Nevertheless, protection against challenge in the absence of detectable HI antibodies has been reported, and our work adds new examples. Moreover, we met the demand for the novel and specific correlates of vaccine-induced immunity and accompanying tests in relation to anti-H5N1 HPAIV vaccines.



Fig 4. Chicken immune responses in the course of the challenge experiments. Chickens were vaccinated and challenged as described in Fig 3. Antibodies to influenza A virus antigens in chicken sera from (A, C) homologous (Exp 1) and (B, D) heterologous (Exp 2) challenge experiments were measured by (A-B) hemagglutination inhibition (HI) and (C-D) FluAC H5 (IDVet) tests. The HI activities of sera towards homologous H5N1 virus (A-B) are displayed as HI antibody titers. The levels of anti-H5 HA antibodies in sera (C-D) are presented as a competition percentage. Annotations of the horizontal axes refer to chicken numbering (the same as in Fig 3). Each symbol represents the result for one chicken at the indicated time point. The 1.0 values, marked with symbols on charts A and B, indicate that the HI antibody titers were, if any, lower than the detection limit (1:8). Signs at the horizontal axes denote that determinations were not performed due to (x) serum loss or (+) chicken death. The bars represent the (A-B) geometric or (C-D) arithmetic means of the results from the respective tests, calculated for each vaccine group at individual time points. Geometric mean titers of HI antibodies were obtained considering only HI-positive sera (HI titers ≥1:16). The dashed lines indicate cut-off values in the respective tests. (Data adapted from Sączyńska V et al. (2017). PLoS ONE 12(2): e0172008. doi:10.1371/journal.pone.0172008)



1.      Sączyńska V, Romanik A, Florys K, Cecuda-Adamczewska V, Kęsik-Brodacka M, Śmietanka K, Olszewska M, Domańska-Blicharz K, Minta Z, Szewczyk B, Płucienniczak G, Płucienniczak A. A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies. PLoS One. 2017;12: e0172008. doi: 10.1371/journal.pone.0172008.

2.      Sączyńska V. Influenza virus hemagglutinin as a vaccine antigen produced in bacteria. Acta Biochim Pol. 2014;61: 561-572.