Patent Application
20170165352
The object of the invention is the influenza virus (IV) hemagglutinin (HA) protein as a vaccine antigen against influenza viruses, in particular the H5 hemagglutinin protein of a highly pathogenic avian influenza virus (HPAIV) H5N1 strain, a composition comprising the said hemagglutinin protein applicable in the development of vaccines against influenza virus, an antibody binding to the said protein and a method of obtaining the hemagglutinin protein.
Solving the spatial structure of various HA serotypes, the development of in silico drug design methods, biotechnology and protein engineering have opened new prospects for anti-influenza vaccines produced in a bacterial expression system. The current work on obtaining such a vaccine is focused on HA proteins based on the HA-1 subunit, the HA-2 subunit, the ectodomain and the polypeptides containing repeated sequences of a single epitope (single-epitope vaccines) or several epitopes (multi-epitope vaccines) of the influenza virus proteins, including HA.
The proteins comprising the HA-1 subunit and fragments thereof are intended to provide immunity against influenza viruses of a particular HA serotype. Longer HA fragments, despite having conserved HA-2 subunit neutralizing epitopes (Ekiert D C et al. 2009, Okuno Y et al. 1993, Sui J et al. 2009), are also considered to be the so-called serotype-specific vaccines. Presumably, the globular domain inhibits stalk region recognition by immune cells either by masking it or through immunodominance (Steel J et al. 2010). As a result, vaccines comprising e.g. the AIV strain of H5 serotype elicits protective immune response against H5 viral infections, while either providing no immunity against other virus serotypes or a very low one (van den Berg et al. 2008). Due to the high variability of HA dominant antigenic sites, induction of cross-immunity is problematic also in relation to vaccine HA antigenic variants of related influenza virus strains. In the case of AIV H5N1, weak cross-reactivity is observed between antibodies induced against clade 1 viruses and clade 2 viruses, despite their considerable similarity (Khurana S et al. 2011 b).
Studies on broadening of cross-immunity induced by subunit vaccines are focused on antigen properties and adjuvants. Comparative studies on the ability of various HA forms to induce protective immune response demonstrate that a significant role in this regard may be played by protein oligomerization (Khurana S et al. 2011b, Verma S et al. 2012). Attempts at generating universal vaccines are realized through designs of immunogens deprived of the globular domain, referred to as stalk domain-based HA proteins (Bommakanti G et al. 2010, 2012, Sagawa H et al. 1996, Steel J et al., 2010).
The possibility of enhancing the range of immune response by developing an efficacious immunogenic composition is indicated by studies of Khurana S et al. (2010a), who demonstrated, using MF59 in an HSN1-inactivated vaccine, an advantageous effect of an adjuvant on binding of induced antibodies to conformational epitopes of the HA-1 subunit, correlating with broadening of cross-neutralization between clades and the expected improved in vivo protection.
Overexpression in E. coli was used to produce the HPAIV H5N1 HA fragment comprising a signal sequence and a HA-1 subunit with (Chiu F F et al. 2009) and without basic amino acids of the cleavage site between the HA-1 and HA-2 subunits (Shen S et al. 2008).
Shorter HA-1 subunit fragments expressed in bacteria together with a signal sequence: 1-330 aa, 1-320 aa of the H1N1pdm09 and H5N1 viruses and the 1-320 aa H5 HA fragment of three strains belonging to the 1, 2.1 and 2.2 clades of the H5N1 virus were obtained by Khurana S et al. (2010b, 2011a, 2011b) and Verma S et al. (2012).
According to other designs, fragments of the HA-1 subunit: 57-264 aa, 57-272 aa, 50-280 aa of H1 HA from the H1N1pdm09 influenza virus (Xuan C et al. 2011) and the 91-261 aa H3 HA fragment of the H3N2 influenza virus (Jeon S H and Arnon R 2002), comprising mainly the protein globular domain (gH), were produced in a bacterial expression system. The 63-286 aa H1 HA fragment of the H1N1pdm09 virus was expressed in E. coli in the form of monomers—mHA63-286 (Aguilar-Yáñez J M et al. 2010, DuBois R M et al. 2011, Sánchez-Arreola P et al. 2013), as well as dimers—dHA63-286, in which the HA proteins are linked by a sequence of 10 amino acid residues (Sánchez-Arreola P et al. 2013).
Proteins of various lengths based on the H1 HA globular domain of the H1N1/PR8 influenza virus (101-276 aa, 53-324 aa, 62-284 aa) were also obtained, two of which (53-324 aa, 62-284 aa) were linked to the C-terminus of type 2 flagellin of the Salmonella typhimurium strain (STF2) through the SGSGSGS flexible linker (Song L et al., 2008). STF2 is a type 5 Toll-like receptor (TLR 5) ligand and according to the current state of knowledge, linking it to the HA protein should enhance the antigen ability to trigger immune response. Fusion proteins of STF with HA fragments of the H1N1/SI (Song L et al. 2008), H1N1pdm09 (Liu G et al. 2011) and H5N1 viruses (Song L et al. 2009) were obtained based on the same principle as the STF2:HA (62-284 aa) antigen of the H1N1/PR8 virus. Based on the H5 and H1pdm09 HA sequences the other formats of STF:HA protein than C-terminal ones were produced, differing in the number of HA molecules and their placement in fusion proteins (Liu G et al. 2011, 2012, Song L et al. 2009). The RO and R3 constructs were generated by replacing the flagellin D0 and D3 domains, respectively, with the HA protein, while the R3.2×HA construct was obtained by fusing one HA molecule to the flagellin C-terminus and the other in the site of the D3 domain. Certain formats of the STF2:HA fusion protein were used as immunogenic components of the VAX125 vaccine against seasonal influenza virus H1N/SI (Treanor J J et al. 2010, Taylor D N et al. 2011) and three variants of the VAX128 vaccine against pandemic influenza virus N1N1pdm09 (Taylor D N et al., 2012). Vaccine compositions based on solutions utilizing HA fusion protein linked to flagellin were disclosed in documents EP 2 476 432, WO2009128950, WO2014035989.
Eleven HA proteins based on the H1 HA globular domain (gH) of the H1N1/PR8 virus were designed and produced, forming four (group A), two (group B) or one (group C) disulfide bond(s) (Jegerlehner A et al. 2013). Group A and B proteins were fused in vitro to virus-like particles (VLPs) derived from the Qβ bacteriophage. By structural alignment to the prototype HA fragment (gH1_A_PR8) gH proteins of the following viruses were obtained: H1N1pdm09, H5N1, H1N1, H3N2 and type B influenza virus, and were subsequently linked to Qβ-VLPs (Jegerlehner A et al. 2013, Skibinski D A et al. 2013).
The HA-1 subunit and its fragments were expressed in E. coli in inclusion bodies as insoluble proteins, therefore obtaining an antigen intended for vaccination required solubilization of the proteins and HA refolding. With various procedures for obtaining protein, strong dependence of effectiveness and efficiency of the H5 HA HA-1 subunit refolding on the applied method was definitely shown (Chiu F F et al. 2009). In the case of the HA globular domain it was further shown that the effectiveness and efficiency of protein refolding is largely dependent on the fragment of the HA-1 subunit, which is selected to produce the vaccine antigen (Jegerlehner A et al. 2013, Song L et al. 2008, Xuan C et al. 2011), thus confirming the importance of secondary structures formed by peptides adjacent to the globular domain (Song L et al., 2008) and disulfide bonds (Jegerlehner A et al., 2013) for proper protein folding.
Purified and refolded the HA-1 subunit fragments (1-330 aa, 1-320 aa) of the H1N1pdm09 and H5N1 viruses, produced in a bacterial expression system, in contrast to the shorter H5 HA fragment (28-320 aa), were mostly present in the form of trimers and oligomers (Khurana S et al. 2010b, 2011a, 2011 b, Verma S et al. 2012). It has been shown that the HA-1 subunit oligomerization is mediated by the conserved amino acids in the protein signal sequence (Khurana S et al., 2011b) and that the 1-320 aa fragment of H5 and H1 HAs forms more stable oligomers than the 1-330 aa fragment (Khurana S et al., 2011a, 2011b). The HA protein oligomers, composed of trimers, agglutinated erythrocytes and bound to fetuin, which indicates the capacity of these proteins to bind to receptors containing sialic acid residues (Khurana S et al., 2010b, 2011b). As a result of purification and refolding of structurally corresponding HA-1 subunit fragments from a bacterial expression system, correctly folded fragments of hemagglutinins: H1pdm09, H5, H7 and H3, have been produced with high yields (Khurana S et al. 2011a, 2011b, Verma S et al. 2012). The obtained proteins comprised a large fraction of oligomers (≧70%) in the form of rosette structures composed of trimers, similar to those formed by native HA isolated from influenza viruses. In contrast, the refolded and purified HA-1 subunit fragments, smaller than the ones obtained by Khurana S et al. (2010b, 2011a, 2011b,) and Verma S et al. (2012), expressed in bacteria without the signal sequence, did not form oligomers and were present as monomers containing mainly the globular domain with correct conformation (Aguilar-Yáñez J M et al. 2010, DuBois R M et al. 2011, Jegerlehner A et al. 2013, Liu G et al. 2011, 2012, Skibinski D A et al. 2013, Song L et al. 2008, 2009, Xuan C et al. 2011) or formed dimers mediated by a peptide linker (Sánchez-Arreola P et al. 2013). The correctly folded proteins based on the HA globular domain exhibited erythrocyte agglutination activity, characteristic of oligomeric HA forms, after linking to Qβ-VLPs (Jegerlehner A et al. 2013).
Studies on the structure and/or antigenicity of the purified and refolded HA HA-1 subunit or its fragments have shown that the proteins produced in a bacterial expression system have well-preserved neutralizing conformational epitopes, essential for the stimulation of immune response (Aguilar-Yáñez J M et al. 2010, Chiu F F et al. 2009, DuBois R M et al. 2011, Hong et al. 2013, Khurana S et al. 2010b, 2011b, Sánchez-Arreola P et al. 2013, Song L et al. 2008, Verma S et al. 2012, Xuan C et al. 2011).
Comparative studies on various STF:HA fusion protein constructs have shown that antigenicity and activity of TLR5 depend on the site of the HA protein and flagellin linkage (Liu G et al. 2011, 2012, Song L et al. 2009).
In tests with influenza viruses homologous to the vaccine antigen it has been shown that sera of animals parenterally vaccinated with HA: 1-330 aa, 1-320 aa (Khurana S et al. 2010b, 2011a), 57-264 aa (Xuan C et al. 2011), 63-286 aa (Aguilar-Yáñez J M et al. 2010) of the H1N1pdm09 viruses and 1-320 aa of the H5N1 virus (Khurana S et al. 2011b, Verma S et al. 2012), as well as STF2:HA of the H1N1/SI virus (Song L et al. 2008) inhibit hemagglutination and/or neutralize the viruses in vitro. A varied capacity to induce HI antibodies in animals has been documented for the C-terminal, R0, R3, R3×2HA formats of STF2:HA proteins (Liu G et al. 2011, 2012, Song et al. 2009). Immunization studies in a mouse model have shown that the gH proteins conjugated to Qβ-VLPs elicit significantly higher titers of native HA-specific antibodies, HI antibodies and neutralizing antibodies in comparison to their non-conjugated counterparts, and that they induce a strong Th1 response (Jegerlehner A et al. 2013, Skibinski D A et al. 2013).
In the case of vaccinations with the HA protein (1-320 aa) of H5N1 viruses, the neutralizing activity of sera has also been shown against H5N1 viruses belonging to other clades than that, from which the vaccine antigen was derived (Khurana S et al. 2011a, 2011b). Comparative studies of the antisera induced by vaccination with the oligomeric (1-320 aa) and monomeric (28-320 aa) HA protein of the H5N1 virus have shown that the induction of high titer of neutralizing antibodies with a broad range of cross-reactivity is associated with oligomerization of the vaccine antigen (Khurana S et al. 2011b). The importance of HA protein oligomerization for immune response quality has been confirmed in immunization studies carried out with oligomeric and monomeric fractions of the 1-320 aa H5 HA protein (Verma S et al. 2012).
The ability to block membrane fusion, which correlates with the neutralizing activity of HA pseudotyped lentiviral particles, has been shown for sera of rabbits vaccinated with the HA protein (1-340 aa) of the H5N1 virus (Shen S et al. 2008). Immunization studies in mice with a HA-1 subunit fragment (91-261 aa) of the H3N2 virus have shown that the vaccine is capable of inducing IgG antibodies in serum on parenteral administration, but also IgG antibodies in sera and IgA in lungs on intranasal administration (Jeon S H and Arnon R. 2002). The induced antibodies recognized HA in intact particles of the H3N2, H1N1 and H2N2 viruses. Vaccinations of mice with the HA protein (91-261 aa) have shown that the protein induces both humoral and cellular response against influenza virus.
The final criteria in the assessment of vaccination efficacy with purified and refolded HA proteins based on the HA-1 subunit from a bacterial expression system are the results of studies on infection of vaccinated animals with influenza viruses under experimental conditions (challenge). The capacity of the vaccines to induce immunity against H1N1pdm09 virus has been assessed in the ferret (Aguilar-Yáñez J M et al. 2010, Khurana S et al. 2010b, Skibinski D A et al. 2013) and mouse models (Xuan C et al. 2011, Skibinski D A et al. 2013) using viruses homologous to the vaccine antigen to infect the animals. It has been shown that parenteral vaccination of animals with the HA proteins: 1-330 aa (Khurana S et al. 2010b), 57-264 aa (Xuan C et al. 2011), 63-286 (Aguilar-Yáñez J M et al. 2010) and the gH1pdm09-Qβ-VLPs (Skibinski D A et al. 2013) elicits protective immunity against influenza virus infection. Protective activity of the vaccinations was manifested in that challenged animals did not get sick from flu or the course of the disease was milder than in non-vaccinated animals. Efficacy of immunization with STF2:H1pdm09 fusion proteins was studied in a lethal challenge experiment using the H1N1pdm09 infectious virus adapted to mice (Liu G et al. 2011). All tested fusion protein formats at doses of 3 μg and 0.3 μg conferred protection of animals against infection. Using 0.03 μg-doses the relative efficacy of three vaccine formats was found to be: R3.2×H1>R3>C-terminal. In some challenge experiments it has been shown that vaccination induces a substantial reduction in infectious virus loads in the respiratory tract of animals (Liu G et al. 2011, Khurana S et al. 2010b, Skibinski D A et al. 2013).
Similar to the vaccines against H1N1pdm09, HA proteins based on the HA-1 subunit of HPAIV H5N1 produced in a bacterial expression system have been studied for their efficacy in challenge experiments. It has been shown that the H5 HA (1-320 aa) protein, when correctly folded and forming functional oligomers, in contrast to the monomeric fraction of the 1-320 aa protein and the monomeric 28-320 aa protein, elicits protective immune response in ferrets against HPAIV H5N1 when used in parenteral immunization (Khurana S et al. 2011b, Verma S et al. 2012). After challenge with a homologous virus the survival rate of animals vaccinated with a largely oligomeric antigen or an exclusively oligomeric one was 100%, while after challenge with a heterologous one it was 80% and 100%, respectively. In ferrets, which survived the experimental infection, reduced infectious virus loads were also found in the upper respiratory tract. Studies on the capacity of STF2:H5 proteins to induce protective immune response have shown that vaccination with the C-terminal, R3, R3.2×H5 protein formats effectively protects the animals against infection with homologous H5N1 viruses, in contrast to immunization with the RO format of fusion protein (Liu G et al. 2012, Song L et al. 2009). It has been further shown that the high survival rate of the vaccinated animals is accompanied by reduced virus loads in tissues and that the best vaccine antigen is the R3.2×HA format of STF2:H5 protein, effectively protecting animals against infection even when administered at sub-microgram doses.
Vaccines based on the HA-1 subunit of H3 and H1 HAs, produced in a bacterial expression system, have been tested in challenge experiments with infectious viruses: H3N2 and H1N1, respectively. In an experimental infection with the H3N2 virus it has been shown that intranasal administration of the HA (91-261 aa) protein induces 80% protection against infection in mice, measured by the number of animals, in which viruses applied in the challenge infection were detected in lungs (Jeon S H and Arnon R 2002). The prototype vaccine against influenza—STF2:H1_PR8 (62-284 aa) in the C-terminal configuration, administered parenterally, completely protected mice against lethal infection with the H1N1/PR8 influenza virus adapted to mice (Song L et al. 2008). Challenge experiments with vaccines containing type A and B gH proteins based on the H1 HA sequence of H1N1/PR8 virus have shown that these proteins fully protect mice against lethal infection with influenza viruses only after linking to QB-VLPs (Jegerlehner A et al. 2013). Additionally, it has been shown that vaccines based on QB-VLPs as carriers for H1N1/PR8 and H1N1pdm09 gH proteins provide a broad cross-immunity within the serotype and are affective even at sub-microgram doses.
By overexpressing in E. coli, a fragment of HA-2 subunit ectodomain (347-522 aa according to full-length HA numbering) of the H5N1 influenza virus was produced and purified by electroelution following denaturing polyacrylamide gel electrophoresis (SDS-PAGE), yielding at least partially denatured protein (Shen S et al. 2008). Sera of HA-2 protein vaccinated rabbits did not recognize HA surface-expressed on mammalian cells, did not block membrane fusion and did not neutralize pseudotyped lentiviral HA particles in vitro, whereas sera of animals immunized with the HA-1 (1-340 aa) protein, purified in the same manner as the HA-2 protein, bound to native HA and exhibited both activities.
The concept to apply proteins containing mainly the HA-2 subunit as vaccine antigens is realized by producing HA deprived of the globular domain, in which the HA-1 subunit fragments are retained, so that the produced protein maintains pre-fusion conformation and stalk region integrity (Steel J et al. 2010). Such HA proteins were produced in mammalian cells (Sagawa H et al. 1996, Steel J et al. 2010). Stalk domain-based immunogens, denoted as HA6 and H1HA0HA6, varying in linkage of the HA-2 subunit fragment with the HA-1 subunit fragments into one polypeptide chain, were produced in a bacterial expression system (Bommakanti G et al. 2010, 2012). Mutations stabilizing the HA-2 subunit pre-fusion conformation were introduced in both constructs. Stalk domain-based immunogens were produced using HA sequences of the H3N2, H1N1 and H1N1pdm09 viruses, while in certain proteins additional disulfide bond mutations or sequence-related mutations were introduced. The desired pre-fusion conformation of immunogens was confirmed in biophysical studies and antigenicity tests. In contrast to the HA0HA6-type protein forming random aggregates, H1HA6-type proteins were mostly present in the form of trimers. Conventional tests showed no neutralizing activity of the antisera obtained using stalk domain immunogens. In contrast, binding tests with rHA from heterologous viruses and competitive experiments with broadly neutralizing monoclonal antibodies showed the capacity of the proteins to induce cross-reacting antibodies. Vaccinations with H6- and/or H1HA0HA-type immunogens with sequences derived from HAs of various H1N1, H1N1pdm09 and H3N2 virus strains conferred full protection of mice against infection with homologous viruses, high survival rates (80-90%) of animals after challenge with heterologous viruses, whereas they did not provide heteroserotypic immunity.
The full-length H5 HA ectodomain of HPAIV H5N1 without a signal sequence was produced in a bacterial expression system, preserving basic amino acids at the cleavage site (Biesova Z et al. 2009). The obtained ˜60 kDa protein was recognized in Western blot by ferret antiserum antibodies against a homologous H5N1 virus. Mice immunization studies have demonstrated immunogenicity of the obtained protein and HI activity of the induced antibodies at a level meeting the FDA guidelines for vaccines against endemic and pandemic influenza. The H1 HA ectodomain fragment of the H1N1pdm09 virus was also expressed with the signal sequence in a bacterial expression system (Khurana S et al. 2010b). Purified and refolded protein (1-480 aa) comprising correctly folded monomers did not exhibit the oligomeric HA function (fetuin binding, erythrocyte agglutination). The correct conformation of the obtained HA ectodomain fragment, demonstrated using circular dichroism spectroscopy, as well as the presence of essential neutralizing conformational epitopes were confirmed by binding assays with antisera against the H1N1 virus and adsorption of neutralizing activity from these sera. Immunization studies in rabbits and ferrets with HA (1-480 aa) have shown that the protein is strongly immunogenic and the induced antibodies neutralize a homologous virus in vitro and inhibit its hemagglutinating activity. In a challenge experiment it was observed that vaccination with HA (1-480 aa) elicits protective immunity in ferrets against infection with a homologous influenza virus, manifested in that the animals exhibited substantially milder disease symptoms (temporary rise in temperature without weight loss) than non-vaccinated animals.
The H5 HA protein of HPAIV H5N1 with molecular weight of ˜63 kDa was produced in the E. coli LMG strain (Horthongkham N et al. 2007). Mice immunized with purified rH5 responded with the production of antibodies recognizing the antigen used for vaccination and neutralizing the heterologous H5N1 virus in vitro. The H5 HA protein of HPAIV H5N1 was produced in E. coli BL21(DE3), expressed as a soluble fusion protein with the msyB chaperone with molecular weight of ˜97 kDa (Xie Q M et al. 2009). The obtained msyB:HA protein was recognized in Western blot analysis by the H5N1-positive chicken sera. Monoclonal antibodies, obtained using the msyB:HA protein for mice immunization, detected AIV with high sensitivity, indicating the preservation of native HA epitopes in the recombinant protein. Vaccination of chickens with high msyB:HA doses induced HI antibody production in those animals, as well as conferred protection against HPAIV H5N1 infection under experimental conditions (survival rate—100%, moderate disease symptoms).
The main challenge in work on obtaining HA in a bacterial expression system is the method of refolding, which would provide an antigen resembling viral HA, despite the fact that HA may be efficiently produced in bacterial cells as a non-glycosylated protein, additionally deprived of regions involved in higher order structure formation of the protein during virus propagation. The HA native structure of the vaccine antigen needs to be preserved, as majority of neutralizing epitopes are conformational antigenic sites (Wiley D C et al. 1981). Published results of the studies on HA proteins produced in a eukaryotic expression system demonstrate that, apart from the correct conformation of HA monomer, the protein oligomerization status also has a significant impact on the level and quality of immune response. In contrast to monomers, rHA trimers and oligomers of H5N1 virus from mammalian and baculovirus expression systems were effective in inducing high levels of neutralizing antibodies in mice (Wei C J et al. 2008). It was also shown that the HA protein of H3N2 virus, produced in a baculovirus expression system with a trimer-stabilizing modification, has a significantly stronger capacity to induce virus specific antibodies and HI antibodies in mice when compared to the unmodified protein form, which correlates with increased immunity against infection with a homologous influenza virus under experimental conditions (Weldon W C et al. 2010). Probably the monomeric form of antigen is less immunogenic than the trimer/oligomer forms of the same protein (Wei C J et al. 2008), but the oligomerization status may also influence the repertoire of induced antibodies (Wei C J et al. 2008, Weldon W C et al. 2010, Wilson I A and Cox N J 1990). In order to obtain soluble HA forming stable oligomeric structures, the proteins constituting the antigen ectodomain, produced in mammalian or insect cells, are expressed together with intentionally added foreign trimerizing sequences (Bosch B J et al. 2009, Loeffen W L et al. 2011, Wei C J et al. 2008, Weldon W C et al. 2010).
A majority of the HA proteins described above, i.e. the HA-1 subunit and its fragments, the ectodomain fragment and the full-length ectodomain, were expressed in E. coli in inclusion bodies as insoluble proteins with an affinity label, typically 6×His, exceptionally with another label, e.g. GST (Shen S et al. 2008), or without an affinity label (Liu G et al. 2011, 2012, Song L et al. 2008, 2009, Xuan C et al. 2011). The STF2:HA proteins were expressed as soluble and/or insoluble proteins (Liu G et al. 2011, 2012, Song L et al. 2008, 2009). By the attachment of a signal sequence for periplasmic expression to the N-terminus, in addition to the affinity label (6×His), a HA-1 subunit fragment expressible in soluble form was also obtained (Aguilar-Yáñez J M et al. 2010). However, the yield of this protein was 2 orders of magnitude lower than that of its refolded counterpart expressed in inclusion bodies. The HA protein of 63 kDa (Horthongkham N et al. 2007) and the 97-kDa msyB:HA fusion protein (Xie Q M et al. 2009) were expressed in E. coli with a histidine tag as soluble protein. The HA proteins contained in inclusion bodies, after they had been solubilized in standard denaturing buffers, were refolded either through slow or rapid dilution or on a column with a metal affinity chromatography bed. To ensure effective HA protein refolding, either L-arginine or L-arginine and oxidized/reduced glutathione were used in some refolding procedures. Fusion proteins with a histidine tag were purified by metal affinity chromatography. Proteins expressed with no affinity label were purified by standard chromatography procedures (Liu G et al. 2011, 2012, Song L et al. 2008, 2009, Xuan C et al. 2011). The process utilizing E. coli to produce a vaccine comprising the H1 hemagglutinin globular domain of the H1N1pdm09 virus was demonstrated by Sánchez-Arreola P et al. in their recent publication (2013).
To summarize, studies conducted to date on obtaining the HA antigen in a bacterial expression system focused primarily on proteins expressed in inclusion bodies, in particular those containing the HA-1 subunit or its fragments and intended for production of a serotype-specific vaccine (Aguilar-Yáñez J M et al. 2010, Chiu F F et al. 2009, DuBois R M et al. 2011, Jegerlehner A et al. 2013, Jeon S H i Arnon R 2002, Khurana S et al. 2010b, 2011a, 2011 b, Liu G et al. 2011, 2012, Sánchez-Arreola P et al. 2013, Shen S et al. 2008, Skibinski D A et al. 2013, Song L et al. 2008, 2009, Verma S et al. 2012, Xuan C et al. 2011). In this way the HA proteins of serotypes: H1, H3, H5, H7 of pandemic, seasonal or prototype type A influenza viruses have been obtained. For most proteins based on the HA-1 subunit of HA conformational integrity has been documented by spectroscopy and/or reactivity assays with neutralizing antibodies. The capacity of the produced proteins to induce functional anti-HA antibodies has been shown in hemagglutination inhibition assays and in vitro influenza virus neutralization tests, while in challenge experiments they were shown to elicit protective immune response against influenza. This group of proteins is represented by the influenza vaccines tested in clinical trials and based on patented technologies: TLR (VaxInnate) and Qβ-VLPs (Cytos Biotechnology).
Another well-characterized bacterial HA protein is an HA ectodomain fragment expressed with a signal sequence (Khurana S et al. 2010b). The 1-480 aa protein was shown to possess the correct conformation, essential neutralizing conformational epitopes, the capacity to induce antibodies neutralizing a homologous virus in vitro and inhibiting its hemagglutinating activity, correlating with protection against influenza under experimental infection conditions. Properties of the full-length HA ectodomain synthesized without a signal sequence have not been fully documented (Biesova Z et al. 2009). The capacity of the protein to induce HI antibodies has been shown, although its potential to induce protective immune response by evaluating morbidity and survival rates of vaccinated animals after infection with influenza viruses has not been confirmed. Among the HA proteins expressed in bacteria in a soluble form (Horthongkham N et al. 2007, Xie Q M et al. 2009), the capacity to provide protective immune response in a challenge experiment has been shown only for the fusion protein with the msyB chaperone (Xie Q M et al. 2009).
Experiments with HA expression in bacterial cells have shown that the HA proteins of various lengths expressed in bacterial cells as soluble proteins or in the form of inclusion bodies may be valuable immunogens. The rHA-E. coli proteins may fold into correct conformation, despite not being glycosylated and even when they do not comprise regions involved in the formation of the protein native structure at viral infection, such as hydrophobic regions, i.e. the signal sequence and/or transmembrane domain. In the case of antigens with the dominant globular domain, the length of peptide fragments adjacent to the globular domain were shown to be crucial for its correct folding (Jegerlehner A et al. 2013, Song L et al. 2008, Xuan C et al. 2011). The rHA-E. coli proteins may also form functional oligomers, which is essential to induce protective immune response and has been demonstrated in studies on rHAs from both eukaryotic (Wei C J et al. 2008, Weldon W C et al. 2010) and prokaryotic expression systems (Khurana S et al. 2011b, Verma S et al. 2012).
Among the HA proteins produced in bacterial cells, also those with confirmed correct tertiary structure, the formation of functional oligomers has only been documented for specific HA-1 subunit fragments expressed with a signal sequence (Khurana S et al. 2010b, 2011a, 2011b, Verma S et al. 2012). Native HA oligomers induce high levels of neutralizing antibodies owing to the presence of trimer-dependent epitopes (Wilson I A, Cox N J 1990) and presumably also by limiting the accessibility of certain epitopes, e.g. those present on the interface between monomers (Wei C J et al. 2008, Weldon W C et al. 2010). Fragments of the HA-1 subunit of HA expressed without a signal sequence (Aguilar-Yáñez J M et al. 2010, DuBois R M et al. 2011, Jegerlehner A et al. 2013, Liu G et al. 2011, 2012, Skibinski D A et al. 2013, Song L et al. 2008, 2009, Xuan C et al. 2011), as well as the 1-480 aa fragment of ectodomain synthesized with a signal sequence (Khurana S et al. 2010b) were found in the form of monomers. The oligomerization capacity of other HA fragments produced in bacteria has not been studied. A change in the presentation of HA proteins based on the globular domain of HA was provided by the formation of HA dimers (dHA63-286) involving a peptide linker (Sánchez-Arreola P et al. 2013) or using Qβ-VLPs as an antigen carrier (Jegerlehner A et al. 2013, Skibinski D A et al. 2013). Results of dHA63-286 immunogenicity studies have not been published yet. The advantageous effect of conjugating gH proteins with Qβ-VLPs on the quality of immune response has been well documented.
Studies on production of HA proteins in bacterial inclusion bodies show that the basic requirement for obtaining an antigen with a correct conformation, capable of forming oligomeric structures, is to identify the protein fragment to be expressed, purified and refolded. The precise determination of the HA sequence for the production of vaccine antigen, particularly in the case of short protein fragments, often requires HA crystallographic screening and in silico analysis of its structure. Such analyses have been conducted in the course of projects, in which the globular domain was the dominant HA fragment in the vaccine (Aguilar-Yáñez J M et al. 2010, DuBois R M et al. 2011, Jegerlehner A et al. 2013, Song L et al. 2008, Xuan C et al. 2011). An essential requirement for the production of a valuable antigen is also to develop an effective refolding method, which has been clearly shown in comparative studies on antigen refolding using different procedures (Chiu F F et al. 2009). Most HA proteins overexpressed in bacterial cells were expressed with an affinity label and purified by metal affinity chromatography, which poses a problem of the final product quality associated with the presence of even trace amounts of metals.
Despite intensive research aiming at obtaining subunit vaccines against influenza, based on HA produced by genetic engineering, we still need to find an effective vaccine antigen as well as a technology facilitating efficient production of the vaccine antigen within a relatively short time, which would be an alternative for a lengthy procedure of conventional vaccine production.
The availability of such a technology is particularly significant in the case of influenza pandemic threat. The HPAIV H5N1 continues to be a current epidemiological problem with a pandemic potential, in view of the threat that the virus will acquire the capacity of direct transmission between humans. The emerging disease outbreaks among birds, often reaching the epizootic scale, cause high mortality among animals and require poultry culling, bringing heavy losses to the poultry industry. Furthermore, there is a continuous risk of reassortment of the circulating AI viruses with mammalian viruses, giving rise to new viral strains posing a threat to human and animal health.
Thus the aim of the invention is to provide a solution responding to the need for an inexpensive, safe vaccine against influenza, quickly and efficiently produced, particularly when facing a threat of pandemic or zoonosis, utilizing the potential of a bacterial expression system, verified in production of biopharmaceuticals, to produce a vaccine antigen.
The object of the invention is an isolated and purified influenza virus (IV) hemagglutinin (HA) polypeptide, consisting of a HA-1 subunit, forming the viral HA globular domain with a binding site to the host cells receptors and involved in the formation of the native HA stalk domain, and a HA-2 subunit fragment with an N-terminal fusion peptide, forming the native HA stalk domain, while the polypeptide is deprived of sequences present in the precursor (HA0) and/or mature viral HA form: the N-terminal signal peptide and the C-terminal protein region downstream of the bromelain cleavage site, in which the transmembrane and cytoplasmic domains are present.
Preferably, the influenza virus is HPAIV H5N1.
Preferably, the influenza virus is the HPAIV H5N1 A/swan/Poland/305-135V08/2006 strain (EpiFlu Access no. EPI156789).
Preferably, the polypeptide is comprised of HA 17-522 aa consisting of the HA-1 (17-340 aa) subunit and a portion of the HA-2 subunit (347-522 aa) with the N-terminal fusion peptide and a retained C-terminal bromelain cleavage site (521-522 aa).
Preferably, the polypeptide consists of an amino acid sequence, structurally corresponding to the HA 17-522 aa sequence of the H5N1 strain of avian influenza virus (AIV) A/swan/Poland/305-135V08/2006 (EpiFluDatabase Accession No. EPI156789).
Preferably, the polypeptide comprises a deletion of basic amino acids: lysine (K) and arginine (R) from the cleavage site between the protein HA-1 and HA-2 subunits (ΔRRRKKR, Δ341-346 aa).
Preferably, the polypeptide has the SEQ ID NO: 1 amino acid sequence or an amino acid sequence corresponding to SEQ ID NO: 1.
Preferably, the polypeptide has the SEQ ID NO: 2 amino acid sequence or an amino acid sequence corresponding to SEQ ID NO: 2.
Preferably, the polypeptide is produced in a prokaryotic expression system.
Preferably, the polypeptide is produced in E. coli.
Preferably, the polypeptide is expressed in the form of inclusion bodies.
A further object of the invention is a composition comprising a carrier and the said polypeptide in an effective amount to elicit immune response and/or treat an influenza virus infection.
Preferably, the carrier is an adjuvant stimulating humoral and/or cellular response, selected from the group comprising mineral salts, oil emulsions, mycobacterial products, saponins, synthetic products and cytokines.
Preferably, the adjuvant is aluminium hydroxide, chitosan salts, MF59, AS03, ISCOMATRIX or PROTASAN™ UP G 113 (NovaMatrix/FMC Corp.).
Preferably, the composition is administered subcutaneously, intradermally, intramuscularly or mucosally, including intranasally, via the gastrointestinal tract, and in the case of bird immunization also conjunctivally, naso-conjunctivally, in ovo.
A further object of the invention is the composition as defined above for prophylactic vaccination of humans or for immunization of birds against influenza virus, particularly of laying hens in commercial flocks and breeding flocks of laying hens and broilers.
Preferably, the influenza virus is the HPAIV H5N1 avian influenza strain.
Another object of the invention is an antibody specifically binding to the polypeptide, as defined above.
A further object of the invention is the method of obtaining a polypeptide inducing functional antibodies against influenza virus (IV) hemagglutinin (HA), comprising:
The influenza virus (IV) hemagglutinin (HA) protein according to the invention is a part of naturally occurring hemagglutinins of various serotypes and antigenic variants and constitutes a fragment of the protein ectodomain, containing sequences which in the native protein form epitopes for neutralizing antibodies, acting via two different mechanisms of infection inhibition, described for IV.
In one embodiment, the HA protein according to the invention is HPAIV H5N1 H5 HA. However, this does not impose limitations for the serotype or strain of the virus that the vaccine containing the HA protein according to the invention may target. The protein according to the invention may be isolated from all HA serotypes and antigenic variants, or combine HA fragments of different serotypes or antigenic variants, e.g. as a consensus sequence.
The presence of a HA-1 subunit and a HA-2 subunit fragment in the HA protein according to the invention results in the antigen containing sequences that in the native protein form epitopes for neutralizing antibodies that act via two mechanisms of infection inhibition, described for influenza viruses, i.e. blocking the binding of influenza viruses to host cells (HA-1), and inhibiting the fusion of the virus lipid envelope with the endosomal membrane of host cells (HA-2), and thus the penetration of pathogens into host cells. Retaining in the HA protein, according to the invention, of a long fragment of the HA-2 subunit, which is involved in formation of oligomeric structures during viral HA synthesis, creates advantageous conditions for optimal protein refolding.
The HA protein according to the invention has properties distinguishing it from other bacterial proteins—the HA sequence to be expressed was obtained by removing immunologically inert protein regions: the signal sequence, the transmembrane domain and the cytoplasmic domain, as well as the C-terminal protein ectodomain fragment downstream of the bromelain cleavage site, and in the case of highly pathogenic (HP) IV strains (HP HPAIV) also the basic amino acids from the cleavage site between the protein HA-1 and HA-2 subunits.
Removal of the HA hydrophobic regions, i.e. the signal peptide and the transmembrane domain, enables efficient HA expression in bacterial cells and is additionally justified by the fact that the signal peptide is not found in the mature HA protein and the removed fragments are not essential for antigenicity and immunogenicity of the protein. Since the protein does not contain a majority of native HA hydrophobic sequences, it is readily soluble in aqueous solutions and may be efficiently expressed in prokaryotic cells.
The HA protein of the HPAIV strains does not contain a sequence rich in basic amino acids: lysine (K) and arginine (R), susceptible to digestion by proteolytic enzymes of the trypsin and subtilisin family, which prevents cleavage and degradation to subunits during the production process.
The influenza virus HA protein, in a particular embodiment of the invention, is the HPAIV H5N1 H5 HA protein obtained by overexpression in E. coli in the form of inclusion bodies and constitutes an H5 HA ectodomain fragment (17-522 aa), soluble in aqueous solutions, deprived of a cleavage site susceptible to digestion by proteolytic enzymes (ΔRRRKKR). The H5 HA (17-522 aa) protein substantially corresponds to the HA particles released from influenza viruses using bromelain—BHA (bromelain-released hemagglutinin).
The HPAIV H5N1 H5 HA protein was produced based on the sequence of the Polish virus isolate: A/swan/Poland/305-135V08/2006(H5N1) (EpiFluDatabase Accession No. EPI156789). The Polish AIV isolate protein regions were identified by comparing its amino acid sequence with the HA sequence of the A/Hong Kong/156/97(H5N1) strain (GenBank Accession No. AAC32088). Individual HPAIV A/swan/Poland/305-135V08/2006(H5N1) HA regions are formed by the following amino acid sequences:
In the case of H5 HA of the Polish HPAIV isolate, the BHA molecule is formed by the 17-521 amino acids of the viral HA. Isolated BHA molecules constitute a non-infectious immunogen capable of inducing immune response, still utilized to produce reference antisera for single radial immunodiffusion (SRID) assay to determine vaccine potency against influenza (Khurana S et al. 2011a). Determination of the HA protein sequence according to the invention, for different type A influenza virus protein variants and serotypes for the vaccine antigen production, requires identification of the site where HA is cleaved from viral particles by bromelain, and protein crystallographic studies and their in silico structure analysis are not required.
Sequences structurally corresponding to the amino acid sequence of the selected HA polypeptide contain higher order homologous protein structures and as a result also the HA protein characteristic regions with specific properties and functions. Structurally corresponding sequences do not necessarily correspond to the linear amino acid sequence, thus amino acid numbering of the structurally corresponding protein fragments may differ.
For instance, the amino acid sequence structurally corresponding to the 17-522 aa HA sequence of the avian influenza virus (AIV) A/swan/Poland/305-135V08/2006 H5N1 strain consists of a HA-1 subunit, forming the viral HA globular domain with the binding site to the host cells receptors and is involved in the formation of the native HA stalk domain, and a HA-2 subunit fragment with the N-terminal fusion peptide, forming the native HA stalk domain, while the polypeptide is deprived of sequences present in the precursor (HA0) and/or mature viral HA form, i.e. the N-terminal signal peptide and the C-terminal protein region downstream of the bromelain cleavage site, in which the transmembrane and cytoplasmic domains are found.
To date the HA protein according to the invention has not been produced in a bacterial expression system, nor it has been studied and described as an effective antigen for vaccines against influenza, in particular against HPAIV H5N1.
In the preferred variants for the production of proteins according to the invention the pIGKesHA17522Δ, pDBHa17522Δ vectors are used, containing the HA17522Δ gene encoding the HA 17-522 aa protein of the HPAIV H5N1 A/swan/Poland/305-135V08/2006 strain (EpiFluDatabase Accession No. EPI156789) with deletion of lysine (K) and arginine (R) residues from the cleavage site (ΔRRRKKR, Δ341-346 aa), optimized for expression in E. coli and providing a high level of the antigen expression. Bacterial strains transformed with the aforementioned vectors are E. coli BL21(DE3) and E. coli Z 0526, respectively.
Purified and refolded HA (17-522 aa, ΔRRRKKR) protein from the HPAIV H5N1 strain, obtained by overexpression in bacteria E. coli in inclusion bodies—rH5-E. coli exhibits key properties of the vaccine HA, as it retains the neutralizing epitopes, recognized by antibodies specific towards the H5 serotype of HA and by hemagglutination inhibiting (HI) antibodies and is present, at least partly, in the form of oligomers.
In contrast to the 1-480 aa ectodomain found in the form of monomers, the rH5-E. coli forms functional oligomers in the absence of foreign trimerizing sequences, possibly mediated by the HA-2 subunit fragment. The HA protein oligomerization has an advantageous effect on immune response quality, while it also reduces the effective antigen dose and hence lowers the unit cost of the influenza vaccine.
The obtained rH5-E. coli protein induces in chickens the production of IgY antibodies, specific towards H5 HA, the antibodies active in the FLUAc H5 (IDVet) assay and inhibiting hemagglutination by the homologous HPAIV H5N1 and the heterologous LPAIV H5N2. The administration of HA as a vaccine antigen enables detection of infected birds within the population of vaccinated birds using differentiating serological tests, detecting antibodies against AIV proteins other than the antigen used for immunization of animals. Providing the potential for DIVA (differentiation of infected from vaccinated animals) is the basic requirement for vaccination against avian flu (Suarez DL 2005).
The vaccine against influenza contains the HA protein according to the invention, an adjuvant or adjuvants approved in human and animal immunization specific to the route of administration, as well as other components, e.g. conventional components of pharmaceutical formulations.
The vaccine contains hemagglutinin of a specific serotype and antigen variant (monovalent vaccine), or hemagglutinins of different serotypes or antigen variants (polyvalent vaccine), or HA with a sequence of different antigen variants of a specific serotype (serotype-specific vaccine with increased protectivity against viruses of various clades), or HA with a sequence of various serotypes, eg. a consensus (universal) vaccine.
The vaccine may comprise the above-mentioned HA proteins physically associated with peptides or polypeptides, including a label, a peptide with a protease cleavage site, a trimerizing sequence, immunomodulators or other antigens.
In a preferred embodiment of the invention, the vaccine against influenza contains the rH5-E. coli protein, aluminium hydroxide as an adjuvant, and elicits production of antibodies specific to H5 HA in subcutaneously vaccinated chickens, including functional antibodies active in the FLUAc H5 (IDVet) assay and in HI tests with homologous HPAIV H5N1 and heterologous LPAIV H5N2, as well as provides protection against infection with influenza viruses and reduces virus shedding in the experimental infection tests.
In a preferred embodiment of the invention, the vaccine contains the rH5-E. coli protein and PROTASAN™ UP G 113 (NovaMatrix/FMC Corp.) as an adjuvant, and exhibits the capacity to enhance production of antibodies specific to H5 HA when administered intranasally to chickens primed by subcutaneous administration of rH5-E. coli with aluminium hydroxide as an adjuvant, including functional antibodies active in the FLUAc H5 (IDVet) assay and in the HI test with heterologous LPAIV H5N2.
In the method of HA protein production according to the invention, the antigen is expressed in inclusion bodies of bacteria transformed using expression vectors, in which the protein coding sequence is optimized for a bacterial expression system. In the method of HA protein production according to the invention, a highly efficient and effective refolding method was applied without the use of amino acids: L-arginine, reduced and oxidized glutathione, frequently contained in buffers for bacterial rHA refolding, thus lowering the cost of vaccine HA production. In contrast to most bacterial rHAs, which are purified using metal affinity chromatography, standard chromatography methods are used in the method of HA production according to the invention. Accordingly, the presence of metal ions in the final product, produced by the method according to the invention, is not subject to assessment and quality evaluations may be performed using the procedures developed for biopharmaceuticals, commonly produced in a bacterial expression system.
The essence of the invention is also a method of eliciting protective immune response. The method of eliciting immune response comprises vaccinations using the HA protein obtained by the method according to the invention, by parenteral administration of the antigen, including subcutaneous, intradermal, intramuscular or mucosal, including intranasal, via the gastrointestinal tract, and in the case of bird immunization also conjunctivally, naso-conjunctivally, in ovo or any other method approved in bird vaccination; performed employing identical or different administration routes for the vaccine antigen in a given vaccination cycle; by administrating the antigen with adjuvants approved in human and animal immunization and specific to the route of administration; through mucosa in mass vaccinations. The immunization regimen for humans or a particular animal species using the protein obtained by the method of the invention is optimized towards increased efficacy, the range of the induced cross-immunity and reduction of the effective dosage. Optimization of the immunization regimen involves: the level and number of doses, the type of adjuvant used and the route of administration as well as the interval between the doses. The method of eliciting immune response induces production of antibodies against HA, including functional antibodies, such as serotype-specific neutralizing antibodies, antibodies inhibiting hemagglutination by influenza viruses, antibodies neutralizing influenza viruses in vitro, provides protection against influenza, reduces virus shedding and viral transmission to humans or animals susceptible to IV infection.
In the example solutions the method of eliciting immune response comprises subcutaneous immunization of broiler-type chickens with two 25 μg-doses of the rH5-E. coli protein with aluminium hydroxide as an adjuvant, at an interval of 4 weeks, as substantially more effective in inducing IgY antibodies specific to H5 HA, antibodies active in HI tests with the homologous HPAIV H5N1 and the heterologous LPAIV H5N2, while in the case of induction of antibodies active in the FLUAc H5 (IDVet) assay as the only effective one in comparison to immunization at a 2-week interval.
The subcutaneous immunization of broiler-type chickens repeated using 25 μg of the rH5-E. coli protein with aluminium hydroxide as an adjuvant, at an interval of 4 weeks, elicits production of IgY antibodies specific to H5 HA, antibodies active in the FLUAc H5 (IDVet) assay and in HI tests with the homologous HPAIV H5N1 and the heterologous LPAIV H5N2 (titer≧1:8) in 100%, 37.5%, 75% and 62.5% animals, respectively.
The subcutaneous immunization of laying-type chickens with the rH5-E. coli protein at doses of 5, 10, 15 and 25 μg with aluminium hydroxide as an adjuvant, repeated twice at an interval of 4 or 6 weeks, elicits production of IgY antibodies specific to H5 HA, antibodies active in the FLUAc H5 (IDVet) assay and in the HI test with the heterologous LPAIV H5N2 (protection titer≧1:16) in 100%, 74%±16.5 and 85%±14 animals, respectively.
Intranasal administration of a booster dose of the vaccine containing 20 μg of rH5-E. coli and PROTASAN™ UP G113 (NovaMatrix/FMC Corp.) as an adjuvant, 4 weeks after subcutaneous immunization using 25 μg of rH5-E. coli with aluminium hydroxide, enhances production of IgY antibodies against H5 HA, in 10 out of 15 vaccinated animals, and in some of them also of functional antibodies against H5 HA: active in the FLUAc H5 (IDVet) assay and in the HI test with the heterologous LPAIV H5N2.
Tab. 1 shows a quantitative analysis of the composition of one of the rH5-E. coli preparations. Results for hemagglutinin monomer and dimer are marked in bold.
Tab. 2 shows results of immunoreactivity assays of monoclonal (Mab) and polyclonal (Pab) antibodies against HA with rH5 of different H5 serotype AIV strains. HA proteins were obtained in the baculovirus (Oxford Expression Technologies) or mammalian (Immune Technology Corp.) expression systems. Hemagglutinin homology was determined for full-length protein (1-568 aa) and the HA-1 subunit (17-340 aa) in relation to HA from the A/swan/Poland/305-135V08/2006(H5N1) strain. Commercially available Mabs (Acris Antibodies, ABR/Thermo Scientific, USBiological) and Pabs (Immune Technology Corp.) were used in the assay. Analyses were conducted by ELISA on Ni-NTA plates (Qiagen).
Tab. 3 shows results of the hemagglutination assay (HA) conducted with chicken erythrocytes for an example rH5-E. coli preparation. The reduced rH5-E. coli protein was the negative control for the HA assay for hemagglutinin from a bacterial expression system. The HA protein (17-530 aa, ΔRRRKKR, 6×His), produced on the basis of the HA sequence from the A/swan/Poland/305-135V08/2006(H5N1) strain in a baculovius expression system (Oxford Expression Technologies)—rH5-BEVS (OET) and the A/Bar-headed Goose/Qinghai/12/05 (H5N1) strain in a mammalian expression system (Immune Technology Corp.)—mammalian rH5 (ITC), were reference antigens.
Tab. 4.1 shows results of the competitive ELISA-FLUAc H5 (IDVet) assay for detection of antibodies against H5 in bird sera, collected from chickens vaccinated subcutaneously with one 25 μg-dose of rH5-E. coli and aluminium hydroxide as an adjuvant. Serum samples that contained the highest levels of IgY antibodies against H5 in individual chickens, as shown in the H5-ELISA, were used for analysis and were prepared from blood collected 2 (*), 4 (**) or 5 (***) weeks after immunization. Studies were conducted in broiler-type chickens. Vaccinations and sample collection from the animals were performed according to group A schedule, shown in regimen A.
Tab. 4.2 shows results of the competitive ELISA-FLUAc H5 (IDVet) assay for detection of antibodies against H5 in bird sera, collected from chickens vaccinated twice subcutaneously at a 2-week interval with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant. Studies were conducted in broiler-type chickens. Vaccinations and sample collection from animals were performed according to group B schedule, shown in regimen A.
Tab. 4.3 shows results of the competitive ELISA-FLUAc H5 (IDVet) assay for detection of antibodies against H5 in bird sera, collected from chickens vaccinated twice subcutaneously at a 4-week interval with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant. Studies were conducted in broiler-type chickens. Vaccinations and sample collection from animals were performed according to group C schedule, shown in regimen A.
Tab. 5.1 shows results of HI tests obtained for chickens vaccinated subcutaneously with one 25 μg-dose of rH5-E. coli and aluminium hydroxide as an adjuvant. Tests were conducted at HIU=1:8 with homologous HPAIV H5N1 and heterologous LPAIV H5N2 as antigens, described in line-up A. Studies were conducted in broiler-type chickens. Vaccinations and sample collection from animals were performed according to group A schedule, shown in regimen A.
Tab. 5.2 shows results of HI tests obtained for chickens vaccinated twice subcutaneously at a 2-week interval with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant. Tests were conducted at HIU=1:8, using homologous HPAIV H5N1 and heterologous LPAIV H5N2 as antigens, described in line-up A. Studies were conducted in broiler-type chickens. Vaccinations and sample collection from animals were performed according to group B schedule, shown in regimen A.
Tab. 5.3 shows results of HI tests obtained for chickens vaccinated twice subcutaneously at a 4-week interval with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant. Tests were conducted at HIU=1:8 with homologous HPAIV H5N1 and heterologous LPAIV H5N2 as antigens, described in line-up A. Studies were conducted in broiler-type chickens. Vaccinations and sample collection from animals were performed according to group C schedule, shown in regimen A.
Tab. 6 shows results of immunization studies in broiler-type chickens, obtained using an indirect and competitive ELISAs for detection of antibodies against H5 in chicken sera (H5-ELISA; FLUAc H5, IDVet) and HI tests with AI viruses described in line-up A: homologous HPAIV H5N1 and heterologous LPAIV H5N2. Animals were administered subcutaneously one (group A) or two 25 μg-doses (groups: B, C) of rH5-E. coli with aluminium hydroxide as an adjuvant. Booster vaccination in group B was administered 2 weeks after and in group C—4 weeks after the priming vaccination. Vaccinations and sample collection from animals were performed according to schedules for groups A, B and C, shown in regimen A. The table includes results of analyses of available serum samples from all blood collections in groups A, B and C (*), from blood collections after rH5 booster dose administration to chickens in groups B and C (**), or from blood collected from group A animals, which contained the highest levels of IgY antibodies against H5 in individual chickens and were prepared 2, 4 or 5 weeks after immunization (***).
Tab. 7 shows results of the IDEXX AI MultiS-Screen (Idexx Laboratories) assay for detection of antibodies against AI viruses in bird sera from laying-type SPF chickens in a challenge experiment. Animals were immunized subcutaneously twice at a 4- or 4½-weeks interval using 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant. The infection was performed by intranasal/conjunctival (i.n/i.o.) administration of the virus at a dose of 106 EID50. Chicken vaccinations and infection were conducted according to regimens B and C. Serum samples collected 3 weeks after administering the booster dose of the antigen, i.e. directly before infection and 2 weeks after infection with clade 2.2 homologous (experiment 1) or clade 1 heterologous (experiment 2) HPAIV H5N1, were analyzed. Chickens that died 3, 2, 8, 7-8 dpi are indicated as a, b, c, d, respectively. Chickens exhibiting less or more severe disease symptoms are indicated *, ** respectively.
Tab. 8 shows results of real time RT-qPCR analyses of throat (T) and cloacal (C) swabs collected from laying-type SPF chickens vaccinated with rH5-E. coli and infected with HPAIV H5N1 and from contact chickens. Chickens were immunized subcutaneously twice at a 4- or 4½-weeks interval with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant. The infection was performed 3 weeks after the second immunization by intranasally/conjunctivally (i.n/i.o.) administering the clade 2.2 homologous or clade 1 heterologous HPAIV H5N1 viruses at a dose of 106 EID50. Chicken vaccinations and infection were conducted according to regimens B and C. Swabs were collected from animals that survived the infection 3, 7, 10 and 14 days post infection (dpi). Swabs from chickens that died at 3, 2, 8, 7-8 dpi, indicated as a, b, c, d, respectively, were collected post mortem (pm). Chickens exhibiting less or more severe disease symptoms are indicated *, ** respectively. The amount of viral RNA is expressed in PCR units EID50/ml and shown as log10 EID50/ml.
Tab. 9 shows results of the competitive ELISA-FLUAc H5 (IDVet) test for detection of antibodies against H5 in bird sera and the HI test at HIU=1:8 with heterologous LPAIV H5N2, described in line-up A, obtained in chicken immunization trials. Animals were vaccinated with rH5-E. coli subcutaneously twice at a 4-week (groups: 1A, 2A, 3A, 4A) or a 6-week interval (groups: 1B, 2B, 3B, 4B) with 25 μg (groups: 1A, 1B), 15 μg (groups: 2A, 2B), 10 μg (groups: 3A, 3B) or 5 μg (groups: 4A, 4B) of antigen per dose and aluminium hydroxide as an adjuvant. Results are shown as mean percentage of chickens seropositive in the FLUAc H5 assay and exhibiting a protective HI antibody titer (≧1:16) 1 and 2 weeks after the second administration of 25, 15, 10 or 5 μg of rH5-E. coli (n=4) and all administered antigen doses (n=16). The studies were conducted in laying-type chickens. Vaccinations and sample collection from experimental (groups: 1A-4A, 1B-4B) and control (group K) animals were conducted according to regimen D.
Tab. 10 shows results of the HI test, obtained from chickens vaccinated twice with rH5-E. coli at a 4-week interval by administering a priming dose (25 μg with aluminium hydroxide) subcutaneously and a booster dose (20 μg with PROTASAN™ UP G 113) intranasally. Analyses were conducted at HIU=1:8 with LPAIV H5N2 as a heterologous antigen, described in line-up A. Data from the analysis of sera are shown for chickens that, according to the indirect H5-ELISA responded to intranasal immunization (10/15). Studies were conducted in laying-type chickens. Vaccinations and sample collection from experimental (group no. 5) and control (group K) animals were performed according to regimen E.
The invention is presented in the following embodiments.
Production of the Hemagglutinin Fragment (17-522 as, ΔRRRKKR) from Highly Pathogenic Avian Influenza Virus (HPAIV) H5N1 in Escherichia coli BL21(DE3) Cells
A fragment of the HA 17-522 aa coding sequence was obtained with a deletion of the HA0 cleavage site between the HA-1 and HA-2 subunits (ΔRRRKKR). The sequence encodes the HA-1 subunit and a fragment of the HA-2 subunit and is deprived of the RRRKKR basic amino acids in the 341-346 aa region of the HA antigen.
The cDNA fragment containing the full-length reading frame for hemagglutinin was prepared by reverse transcription and subsequent amplification (RT-PCR) on the RNA template from the Polish influenza virus strain from 2006 (A/swan/Poland/305-135V08/2006; EpiFlu Database; access number EPI156789; http://platform.gisaid.org) (Gromadzka et al., 2008). Based on the obtained nucleotide sequence (
E. coli Strains Used During the Procedure:
NM522 supE thi Δ (lac-proAB) hsd5 F′[proAB+laclq lacZΔ M15]
BL21(DE3) hsdS gal (λcIts857 ind1 Sam7 nin5 lacUV5-T7 gene 1)
To clone the DNA fragments after PCR amplification a non-expression plasmid (sequence available in GenBank, accession no. X52324 S52394) was used. The plasmid contains: the ColE1 replication origin, T3 and T7 promoters (for phage RNA polymerases) flanking a polylinker and the ampicillin resistance gene.
A bacterial expression system, developed at the Institute of Biotechnology and Antibiotics in Warsaw was used to produce the recombinant protein. The system is the object of patent applications nos. P379905 (2007), PCT/PL2007/000037 and the American patent no. U.S. Pat. No. 8,628,954 B2. In this system proteins are produced in host cells, such as Escherichia coli, comprising the T7 phage RNA polymerase coding gene. A vector comprising the target gene operationally linked with a promoter recognized by the T7 phage RNA polymerase is introduced into the host cells and the host cells are cultured under controlled conditions promoting expression. The employed system enables protein expression that is stable over time thanks to the application of an expression cassette comprising a phage promoter operationally linked with the target polypeptide coding sequence and with the sequence encoding the selective marker that determines the survival of the host expressing the target polypeptide. The transcription terminator for promoters other than phage promoters is found in the cassette, between the target polypeptide coding sequence and the sequence coding for the selective marker determining host survival.
The application of the modified expression vector makes it possible to eliminate from the culture those bacterial cells, in which chromosome a mutation appeared preventing production of functional T7 phage polymerase and/or those in which, as a result of a mutation, the T7 phage promoter has lost its functionality. The expression stabilizing cassette is inserted into the vector pIGCmT7Kes (
Production of Vector pIGKesHA17522Δ with Inserted HPAIV H5N1 Hemagglutinin Fragment (17-522 Aa, ΔRRRKKR)
Scheme for Production of Vector pIGKesHA17522
Step I.
Construction of pGEM-T Easy Plasmid Comprising the Hemagglutinin (A/Swan/Poland/305-135V08/2006 (H5N1)) Coding Gene Sequence.
The first step was to acquire genetic material from avian influenza virus using an efficient method of viral genetic material purification. The starting material for those reactions were samples provided by veterinarians and ornithologists. The cDNA fragment was obtained by reverse transcription with subsequent amplification (RT-PCR) on the RNA template from the Polish influenza virus strain from 2006 (A/swan/Poland/305-135V08/2006). The fragment contained the full-length reading frame for hemagglutinin. The obtained fragment was cloned into the pGEM-T Easy plasmid (Promega) used in DNA sequence analysis.
Step II.
From the pGEM-T Easy plasmid comprising the hemagglutinin (A/swan/Poland/305-135V08/2006 (H5N1)) coding gene sequence, the hemagglutinin coding gene was amplified by PCR using the following primers: Ha17522
The PCR primers were designed on the basis of the DNA sequence encoding hemagglutinin. The primer sequence complementary to the amplified gene sequence is underlined.
Primer Ha17522 introduces sites recognized by the BamHI and NdeI restriction nucleases. Primer HaFr522 primer introduces sites recognized by the XhoI restriction nuclease. The product obtained in the PCR reaction was separated by electrophoresis and isolated from the polyacrylamide gel and then digested with the BamHI and XhoI restriction enzymes and deproteinated. The obtained fragment was ligated into pBluescript SK(−) digested with the same restriction enzymes. The ligation product was used to transform E. coli NM522 cells. After plasmid DNA isolation from the transformed E. coli cells, the presence of the cloned HA insert was confirmed by restriction analysis and the sequence accuracy was verified by sequencing.
Step III.
Codons of the HA insert sequence were optimized allowing for improved expression efficiency in E. coli. For this purpose selected codons in the virus-derived sequence were replaced so that it contained those most often found in E. coli. The replacement was performed in a site-directed mutagenesis reaction. The replaced codons are marked in bold in the sequence (
Step IV.
The cloned DNA fragment was digested out of the pBluescript vector with digestion enzymes NdeI and XhoI and deproteinated. The obtained DNA fragment was ligated with the vector pIGCmT7Kes, digested with the same restriction enzymes and deproteinated after digestion. The ligation product was used to transform E. coli NM522 cells. The presence of the cloned HA 17-522 aa insert in the obtained vector pIGKesHA17522 was confirmed by restriction analysis.
Scheme for Construction of Vector pIGKesHA17522Δ
The vector pIGKesHA17522 was used as the template in the amplification reaction. PCR was run using primers designed on the basis of the hemagglutinin coding sequence. The product was the HA 17-522 aa coding sequence without the fragment coding amino acids 341-346, i.e. RRRKKR. The deleted amino acid 341-346 region is the HA0 cleavage site between the HA-1 and HA-2 subunits. The delated amino acids are underlined in the nucleotide sequence (
The PCR product was separated by electrophoresis and isolated from agarose gel, digested with the NdeI and XhoI restriction enzymes and deproteinated. The obtained fragment was ligated into the vector pIGCmT7Kes digested with the same restriction enzymes. The ligation product was used to transform E. coli NM522 cells. After plasmid DNA was isolated from the transformed E. coli cells, the presence of the cloned HA17522Δ insert in the vector pIGKesHA17522Δ (
Construction of E. coli BL21(DE3) Strain Expressing the HPAIV H5N1 Hemagglutinin Fragment (17-522 Aa, ΔRRRKKR)
E. coli BL21(DE3) cells were transformed with the pIGKesHA17522Δ plasmid comprising the verified HA17522Δ coding sequence. Escherichia coli cells were tranformed using the method for competent E. cold cell transformation proposed by Chung and Miller [Chung C T, Miller R H. A rapid and convenient method for preparation and storage of competent bacterial cells. Nucleic Acids Res. 1988 Apr. 25; 16(8):3580]. Transformants were selected on LB medium supplemented with chloramphenicol (12 μg/ml).
The product was E. coli BL21(DE3) strain expressing the HA17522Δ gene with a deletion of the HA0 cleavage region between the HA-1 and HA-2 subunits (ΔRRRKKR). The strain expresses the rH5 recombinant protein comprising the HA-1 subunit and a fragment of the HA-2 subunit, and it is deprived of the region comprising the RRRKKR amino acids in the 341-346 aa site of the HA antigen. The amino acid sequence of the rH5 protein is shown on
Escherichia coli BL21(DE3) bacteria harboring the recombinant plasmid were cultured in LB medium with chloramphenicol (12 μg/ml) at 25° C. with shaking (150 rpm) until OD600˜0.6. The rH5 expression was induced by adding isopropyl-thio-β-D-galactoside (IPTG, up to the final concentration of 0.1 μg/ml). The bacteria were cultured for 4.5 h without changing culture conditions and then centrifuged.
Isolation of Inclusion Bodies Containing rH5 from E. coli
The cell pellet was suspended in lysis buffer (0.5 M NaCl; 0.05 M Tris HCl pH 7.5; 0.01 M EDTA pH 7.5; 0.005 M 2-Mercaptoethanol; 0.35 mg/ml lysozyme; 1% PMSF) and incubated for 30 minutes at 20° C. Triton X-100 was added up to concentration of 1%. The suspension was sonicated and centrifuged. The inclusion bodies pellet was suspended in PBS buffer containing 1% Triton X-100 and then in PBS containing 2M urea and centrifuged. The isolated inclusion bodies were washed twice with PBS buffer. Finally, the inclusion bodies were suspended in PBS buffer, divided into batches and frozen at −20° C. for further analysis.
Analysis of Inclusion Bodies Isolated from E. Col BL21(DE3)
The presence of the inclusion body fraction containing rH5 was confirmed by separating samples by electrophoresis in 15% polyacrylamide gel (SDS-PAGE). Proteins were visualized with Coomassie Brillant Blue G. Images for electrophoretic separation of isolated inclusion body samples are shown on
Production of the Hemagglutinin Fragment (17-522 Aa, ΔRRRKKR) from Highly Pathogenic Avian Influenza Virus (HPAIV) H5N1 in Escherichia coli Z0526
The product was the HA 17-522 aa coding sequence fragment with the HA0 cleavage region between the HA-1 and HA-2 subunits deleted (ΔRRRKKR). This sequence encodes the HA-1 subunit and a fragment of the HA-2 subunit and is deprived of basic amino acids RRRKKR in the 341-346 aa region of the HA antigen.
The cDNA fragment comprising the hemagglutinin full-length reading frame was prepared by reverse transcription and subsequent amplification (RT-PCR) on the RNA template from the Polish influenza virus strain from 2006 (A/swan/Poland/305-135V08/2006; EpiFlu Database; access number EPI156789; http://platform.gisaid.org) (Gromadzka et al., 2008). Based on the obtained nucleotide sequence (
E. coli Strains Used During the Procedure:
NM522 supE thi Δ (lac-proAB) hsd5 F′[proAB+laclq lacZΔ M15]
Z0526 F cyt R, strA
The pDB plasmid contains the constitutive deoP1P2 promoter. Expression of recombinant proteins produced using the pDB plasmid is regulated by the deoP1P2 promoter. The pDB plasmid also carries the tetracycline resistance gene facilitating cell selection after transformation with the plasmid.
Protein production in this system is based on host cells, such as Escherichia coli Z0526. This strain carries the gene coding for polymerase recognizing the deoP1P2 promoter. The recognized deoP1P2 promoter is found in the pDB expression plasmid. In Escherichia coli Z0526, the cytR and strA gene sequence was mutated within the chromosome. The vector comprising the target gene encoding the recombinant protein is introduced to the host cells by transformation. The transformed host cells are cultured under controlled conditions promoting expression. The used system allows for constitutive recombinant protein expression.
Production of the Vector pDBHA17522Δ with the Inserted HPAIV H5N1 Hemagglutinin Fragment (17-522 Aa, ΔRRRKKR)
Scheme for Construction of the Vector pDBHA17522Δ
In the amplification reaction the vector pIGKesHA17522Δ (
Construction of the E. coli Z0526 Strain Expressing the HPAIV H5N1 Hemagglutinin Fragment (17-522 Aa, ΔRRRKKR)
Cells of E. coli Z0526 were transformed with the pDBHA17522Δ plasmid comprising the verified HA17522Δ coding sequence. The method for competent E. colil cell transformation proposed by Chung and Miller [Chung C T, Miller R H. A rapid and convenient method for preparation and storage of competent bacterial cells. Nucleic Acids Res. 1988 Apr. 25; 16(8):3580] was applied to transform E. coli cells. Transformants were selected on LB medium with the addition of tetracycline (12.5 μg/ml).
The product was E. coli Z0526 strain expressing the HA17522Δ gene with deletion of the HA0 cleavage region between the HA-1 and HA-2 subunits (ΔRRRKKR). The strain expresses the rH5 recombinant protein comprising the HA-1 subunit and a fragment of the HA-2 subunit and is deprived of the region comprising the RRRKKR amino acids in the 341-346 aa site of the HA antigen. The amino acid sequence of the rH5 protein is shown on
Expression of the HPAIV H5N1 Hemagglutinin Fragment (17-522 Aa, ΔRRRKKR) in E. coli Z0526.
Bacteria E. coli Z0526 harboring the recombinant plasmid were cultured in mineral media containing tetracycline (12.5 μg/ml) at 37° C. with shaking (150 rpm) until reaching OD600˜1.2 and then centrifuged.
Isolation of Inclusion Bodies Containing rH5 from E. coli Z0526
The cell pellet was suspended in lysis buffer (0.5 M NaCl; 0.05 M Tris HCl pH 7.5; 0.01 M EDTA pH 7.5; 0.005 M 2-Mercaptoethanol; 0.35 mg/ml lysozyme; 1% PMSF) and incubated for 30 minutes at 20° C. Triton X-100 was added up to the concentration of 1%. The suspension was sonicated and centrifuged. The inclusion body pellet was suspended in PBS buffer containing 1% Triton X-100 and then in PBS containing 2M urea and centrifuged. The isolated inclusion bodies were washed twice with PBS buffer. Finally, the inclusion bodies were suspended in PBS buffer, divided into batches and frozen at −20° C. for further analysis.
Analysis of Inclusion Bodies Isolated from E. coli Z0526
The presence of the fraction of inclusion bodies containing rH5 was confirmed by separating the samples by electrophoresis in 15% polyacrylamide gel (SDS-PAGE). The proteins were visualized with Coomassie Brillant Blue G. The images for electrophoretic separation of the isolated inclusion bodies samples are shown on
Refolding and Purification of H5 Hemagglutinin (17-522 as, ΔRRRKKR) from a Bacterial Expression System (rH5-E. coli)
The isolated inclusion bodies were solubilized in DRCI buffer (50 mM TrisHCl, pH 8.0; 8 M urea; 10 mM beta-mercaptoethanol; 0.01% Triton X-100). The inclusion bodies were solubilized for 1-2 h at room temperature. After this time the solution was centrifuged and then filtered through 0.2 μm filters. At this stage protein concentration, as measured by the Bradford method (Bradford, 1976), was 5-6 mg/ml.
The protein solution, obtained by solubilization of inclusion bodies, was loaded onto a 10 ml column with the DEAE Sepharose Fast Flow bed (Amersham Pharmacia Biotech AB). The column with the bed was equilibrated with a calibration buffer (50 mM TrisHCl pH 8.0; 6 M urea; 0.1% Triton X-100). Unbound proteins were eluted with the calibration buffer. Bound proteins were eluted with an elution buffer (50 mM TrisHCl pH 8.0; 6 M urea; 800 mM NaCl). Fractions containing rH5-E. coli were collected. The concentration of eluted protein was determined by the Bradford method. An example chromatogram of protein separation at this stage is shown on
Proteins eluted from the DEAE Sepharose Fast Flow bed were refolded by diluting in BR buffer (40 mM TrisHCl pH 8.0; 100 mM NaCl). In this refolding method the protein was diluted to 0.07-0.09 mg/ml and then refolding was continued for 15-16 h at 4° C. with mixing. After refolding the solution was filtered through 0.4 μm filters and then protein concentration was determined by the Bradford method. At this stage protein loss was approx. 1-2%, making this developed refolding method particularly efficient.
The protein solution after refolding was loaded onto a 7 ml column with the Phenyl Sepharose 6 Fast Flow High Sub bed (Amersham Pharmacia Biotech AB). The column with the bed was equilibrated with a calibration buffer (40 mM TrisHCl pH 8.0; 100 mM NaCl). Unbound proteins were eluted with the calibration buffer, while bound proteins were eluted with deionized water. Fractions containing rH5-E. coli were collected. The concentration of eluted protein was determined by the Bradford method. An example chromatogram of the protein separation at this stage is shown on
The eluted protein solution was supplemented with 1 M TrisHCl pH 8.0 up to the final concentration of 40 mM and protease inhibitors were added (Sigma, cat. no. P8430) according to the manufacturer's instructions, next the protein solution was filtered through 0.2 μm filters and protein concentration was determined by the Bradford method. The rH5-E. coli preparation obtained as above was stored at 4° C.
All purification steps can be performed within the temperature range of 4-24° C.
Analysis of Purified and Refolded rH5-E. coli
Samples were collected after each step of production of the final rH5-E. coli preparation and their composition was analyzed by discontinuous SDS-PAGE electrophoresis under denaturing conditions (Laemmli, 1970). Protein polyacrylamide gel electrophoresis with SDS detergent was run in a discontinuous buffer system, in double polyacrylamide gel set-up (3.5% stacking gel, pH 6.8 and 15% resolving gel, pH 9.2). An example of one such analysis is shown on
The quantitative composition of the obtained rH5-E. coli preparation was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies) with High Sensitivity Protein 250 kit chips with increased sensitivity. The analysis was performed following the manufacturer's protocol. The method is capable of detecting proteins within the mass range of 10-250 kDa in the tested sample at concentrations starting from 1 pg/μl.
The results obtained by this method indicate a 75-85% rH5-E. coli content in the final protein product. An example result of qualitative analysis of the final rH5-E. coli preparations is given on
The exact mass of the rH5-E. coli hemagglutinin was determined using the MALDI TOF/TOF MDS SCIEX 4800 Plus mass spectrometer by Applied Biosystems, using the CHCA matrix, i.e. α-cyano-4-hydroxycinnamic acid (Fluka 70990-1G-F).
The rH5-E. coli molecular mass, determined using MS, is 56.7 kDa (
Antibodies for Analyses of rH5-E. coli Antigenicity
Antigenicity of purified and refolded HA (17-522 aa, ΔRRRKKR), produced by overexpression in E. coli (rH5-E. coli) was analyzed using commercially available monoclonal and polyclonal antibodies against AIV H5 HA. The monoclonal antibodies (Mabs) were supplied by the following companies: Acris Antibodies (5 clones), ABR/Thermo Scientific (1 clone) and USBiological (3 clones). Mabs were obtained with purified AIV H5N1 (8 clones) or recombinant AIV H5N1 HA from the A/Vietnam/1203/04 strain (1 clone) as immunogens. According to the manufacturers' specifications, the antibodies, referred to as Mab 1-2 and 6-9, react with the following influenza viruses: H5N1, H5N2 and H5N9, with H5 HA in the HI test and do not cross-react with hemagglutinins of serotypes other than H5. The specificity of the other Mab clones is described as capacity to recognize the H5 antigen in viral samples (Mab 3) or in the HI test (Mab 5). According to the manufacturer's data, the antibodies, referred to as Mab 4, are specific towards HA from A/Vietnam/1203/04 (H5N1) AIV, react with H5N1 influenza viruses of different strains (clades, subclades) and do not cross-react with hemagglutinins of other subtypes. Specifications for all the used Mab indicate that the antibodies recognize conformational epitopes. Immunoreactivity assays were also conducted with polyclonal antibodies (Pabs) against the HA-1 and HA-2 subunits of H5 HA. The antibodies, referred to as Pab 1 and Pab 2, were obtained using HA-1 (1-345 aa) from A/Bar-headed Goose/Qinghai/12/05(H5N1) and a HA-2 subunit fragment (347-523 aa) from A/VietNam/1203/2004(H5N1) (Immune Technology Corp.) as respective immunogens. It may be expected that the Pabs against HA will recognize both conformational and linear epitopes of the protein.
In order to determine the specificity of Mabs and Pabs to be used in analyses of rH5-E. coli properties, their reactivity was tested using commercially available recombinant hemagglutinins from different AIV strains. Based on the A/swan/Poland/305-135V08/2006(H5N1) HA sequence, a 17-530 aa fragment of the protein with deletion of the cleavage site (ΔRRRKKR) and a histidine tag—6×His (Oxford Expression Technologies) conjugated at C-terminus was produced in a baculovirus expression system. The hemagglutinins from 5 H5N1 and 1 H5N2 IV strains, used in the tests performed, were produced in a mammalian expression system without the signal sequence, the transmembrane or the cytoplasmic domains of the protein (Immune Technology Corp.). The HA fragments (17-530 aa, 18-530 aa or 19-506 aa), comprising the HA-1 subunit and a HA-2 subunit fragment, constituted the antigen. In 4 out of 5 hemagglutinins the cleavage site (ΔRRRKKR) was deleted and these proteins are, according to the specification, found primarily in the form of trimers/oligomers. Oligomerization of the produced antigens renders them similar to the HA in the viral envelope, where the protein is found in the form of trimers.
The set of hemagglutinins to be used in analyses of antibody reactivity against H5, included also recombinant HA fragments corresponding to the HA-1 subunit (Immune Technology Corp.), which in the native protein comprises essential protein neutralizing epitopes and is characterized by a greater sequence variation than the HA-2 subunit. The recombinant HA-1 from the selected H5N1 virus strains were expressed in mammalian cells with (1-345 aa) or without (17-346 aa) the signal sequence. The conformation of HA fragments corresponding to the HA-1 subunit was not specified. All recombinant hemagglutinins produced in a mammalian expression system comprised the histidine tag—6×His.
Analyses of Mab and Pab specificity against recombinant hemagglutinins from different H5 influenza virus strains were conducted by ELISA using Ni-NTA plates (Qiagen). HA proteins were applied to the plates, diluted in 1% BSA/PBS to 1 μg/ml and incubated overnight at 2-8° C. To control the level of nonspecific binding of the antibodies, some wells on the plate were filled with 1% BSA/PBS and incubated in parallel with the antigen coated wells. Anti-H5 Mab and Pab were added to the plates, diluted to 1 μg/ml in 2% BSA/PBS and incubated overnight at 2-8° C. Polyclonal antibodies against mouse IgG antibodies, specific towards the whole molecule, labeled with HRP (Sigma) or HRP-labeled monoclonal antibodies against rabbit IgG antibodies, specific towards the γ chain (Sigma) were used to develop plates. Secondary antibodies were diluted to 1:1000 with 1% BSA/PBST and incubated on plates at room temperature for 45 minutes. TMB (Sigma) was used as horseradish peroxidase substrate. The reaction was stopped after 30 minutes with 1.25 M H2SO4 solution. Sample absorption was read at λ=450 nm. The ELISA was not optimized for individual antigens and antibodies.
The results of reactivity analyses for the antibodies with rH5 from different H5 AIV strains are shown in Tab. 2 together with data concerning hemagglutinins used (EpiFluDatabase, GenBank Accession No., HA fragment, expression system for protein production, manufacturer). Hemagglutinin homology shown in Tab. 2 was established in relation to A/swan/Poland/305-135V08/2006(H5N1) HA, to the full-length protein (1-568 aa) and to the HA-1 subunit (17-340 aa). The hemagglutinins used in the assays were characterized by diverse homology with the HA from the Polish AIV isolate—from very high, as in the case of hemagglutinins from the H5N1 AIV: A/Bar-headed Goose/Qinghai/12/05 and A/chicken/India/NIV33487/2006, to the lowest, as demonstrated for the H5N2 AIV HA: A/American greenwinged teal/California/HKWF609/2007.
Monoclonal antibodies 1-5 bound to all rH5 used in the test (17-530 aa, ±ΔRRRKKR) and the rHA-1 (1-345 aa, 17-346 aa) from a mammalian expression system, while Mab 1-4 recognized all antigens with high affinity (A450>4). Monoclonal antibodies 6-8 demonstrated specificity towards some mammalian rH5, including HA with high homology with the Polish AIV isolate HA. Most mammalian rH5 immobilized on the Ni-NTA plate were detected by polyclonal antibodies (Pab 1, 2). Seven out of eight tested Mabs recognized rH5 (17-530 aa, ΔRRRKKR) from a baculovirus expression system with high affinity. Reactivity of Mab 4, Pab 1 and Pab 2 with the baculoviral rH5 was much lower than with proteins of similar lengths from a mammalian expression system, which probably resulted from the differences in binding of these antigens on the Ni-NTA plates.
Immunoreactivity assays for monoclonal (Mab) and polyclonal (Pab) antibodies with rH5 and rHA-1 (Tab. 2) produced in a eukaryotic expression system, combined with data from the specifications of used Mabs, Pabs and rH5 HAs, indicate that:
Analyses of rH5-E. coli properties with a panel of Mabs and Pabs against H5 HA with the above described specificity were conducted in the presence of two reference antigens. One of those was the H5 HA fragment (17-530 aa, ΔRRRKKR, 6×His), produced in a baculovirus expression system (Oxford Expression Technologies) based on the HA sequence from the same AIV strain as rH5-E. coli (A/swan/Poland/305-135V08/2006(H5N1)). The other reference antigen was the HA fragment (17-530 aa, ΔRRRKKR, 6×His) from A/Bar-headed Goose/Qinghai/12/05(H5N1), produced in a mammalian expression system (Immune Technology Corp.). The Qinghai HA sequence was the most similar to that of the Polish AIV isolate among the commercially available HA produced in a mammalian expression system. Protein homology measured by the number of identical amino acids is 99%, while the conservative amino acid replacement is found in the signal sequence, whereas the other 3 replacements are semiconservative and are in the HA-1 and HA-2 subunits of the protein. According to the specification, the protein, purified to at least 95%, is found primarily in the form of trimers/oligomers. In contrast to rH5-E. coli, reference hemagglutinins produced in an eukaryotic expression system are glycosylated proteins, similarly to native AIV HA.
Antigenicity of rH5-E. coli was analyzed in the presence of reference antigens by ELISA method. MediSorp (NUNC) plates were coated with rH5-E. coli, mammalian rH5 and baculoviral rH5 at 5 μg/ml PBS at 2-8° C. overnight. Nonspecific binding sites on the plates were blocked using 10% FBS/PBS. Anti-H5 Mabs and Pabs diluted to 1 μg/ml (Mab) or 10 μg/ml (Pab) in 2% BSA/PBS were applied to the plates. The plates were incubated overnight at 2-8° C. To detect antigen-bound Mabs the HRP-labeled polyclonal antibodies against murine IgG antibodies, specific towards the γ chain (Sigma), were used diluted to 1:1000 in 2% BSA/PBS. To detect antigen-bound Pabs the HRP labeled monoclonal antibodies against rabbit IgG antibodies, specific towards the γ chain (Sigma) were used, diluted 1:1000 in 1% BSA/PBS. Secondary antibodies were incubated on the plates for 1 hour at 37° C. TMB (Sigma) was used as HRP substrate. The reaction was stopped after 30 minutes with a 0.5 M H2SO4 solution. Absorption of the samples was read at λ=450 nm.
Analyses of rH5-E. coli have shown that the HA from a bacterial expression system is recognized by all used antibodies, similarly to reference hemagglutinins (
Hemagglutinin with the correct conformation has the oligomerization capacity, as it is found in the native form as trimer. Such protein complexes have erythrocyte agglutinating properties, which is utilized in the hemagglutination assay (HA). Erythrocytes that are agglutinated do not settle at the bottom, but form a uniform suspension. Non-agglutinated erythrocytes precipitate rapidly, forming a distinct red dot at the bottom of the test tube. This test is a simple method of evaluating HA antigen quality.
In the test, a fresh preparation of erythrocytes collected from blood of SPF chickens was used, obtained from a sterile culture at the Department of Poultry Diseases, the National Veterinary Research Institute (NVRI) in Pulawy. The hemagglutination test was performed using a 0.5% chicken erythrocyte suspension in PBS on 96-well V-bottomed plates by Cellstar. Each cell was supplemented with 50 μl PBS, followed by a suitable amount of antigen and made up to 100 μl with PBS. Subsequently, 50 μl of 0.5% chicken erythrocyte suspension in PBS was added to each well and mixed gently by pipetting. The plates were incubated at 20° C. for 45 min. and then the result was read. The AI-H5N2 virus (x-OvO) was used as positive control. Negative controls were: 50 mM TrisHCl pH 8.0; 50 mM TrisHCl pH 8.0 with 30 mM β-mercaptoethanol; PBS and the rH5-E. coli protein reduced with 30 mM β-mercaptoethanol.
To compare rH5-E. coli with hemagglutinins from other expression systems, rH5 HA (17-530 aa, ΔRRRKKR, 6×His): from the same AIV strain as rH5-E. coli (A/swan/Poland/305-135V08/2006(H5N1), produced in a baculovirus expression system by Oxford Expression Technologies (OET) and from AIV A/Bar-headed Goose/Qinghai/12/05(H5N1), produced in a mammalian expression system by Immune Technology Corp. (ITC) were also tested in the hemagglutination assay. Hemagglutinins tested in the HA assay were also used as reference antigens in the antigenicity analyses described above for rH5-E. coli. According to the specification, rH5 from a mammalian expression system (ITC), with purity of at least 95%, was found primarily as trimer/oligomer.
The HA assay conducted with chicken erythrocytes showed that rH5-E. coli exhibits the hemagglutinating capacity, similarly to baculoviral rH5 and mammalian rH5 (Tab. 3). For complete agglutination of chicken erythrocytes, a lower concentration of the rH5-E. coli protein than of the HA from a baculovirus expression system is required. The obtained results indicate that the produced rH5-E. coli protein is at least partially found in the form of oligomers, which is an advantageous characteristic for a vaccine HA.
Preparation of Vaccine Containing rH5-E. coli, Chicken Immunization and Collection of Samples for Serological Analyses
To evaluate the applicability of purified and refolded HA (17-522 aa, ΔRRRKKR) as a vaccine antigen, produced by overexpression in E. coli (rH5-E. coli), a vaccine composition was developed using rH5-E. coli and immunization trials were performed on chickens as one of the target groups for vaccinations against avian influenza.
To prepare a vaccine against avian influenza using rH5-E. coli, aluminium hydroxide was used as an adjuvant. Protein content in rH5-E. coli preparations for vaccine production was determined by the Bradford method. The HA content in rH5-E. coli preparations was estimated on the basis of its quantitative composition, analyzed using increased sensitivity chips of the High Sensitivity Protein 250 kit and the Agilent 2100 Bioanalyzer by Agilent Technologies. The result of the analysis for one of the protein preparations is shown in Tab. 1. To prepare the vaccine, an rH5-E. coli preparation was used in the amount providing the desired rH5 concentration per dose. Aluminium hydroxide—1.3% Alhydrogel (Brenntag) constituted ¼ of the vaccine volume. The rH5-E. coli preparation was added to aluminium hydroxide gel in PBS, shaken on a vortex mixer at 2500 rpm for 5 minutes and then incubated at room temperature for 20 min. Following incubation, the vaccine was again shaken on a vortex mixer for 5 minutes at 2500 rpm. The vaccine was prepared directly before vaccination.
Immunization trials using rH5-E. coli were conducted under commercial rearing conditions on broiler-type Ross 308 chickens. Experiments were performed in parallel on 4 animal groups designated A, B, C and K. Chickens in groups A, B and C, with 8 animals in each, were vaccinated, while 15 non-immunized chicken constituted the control (K). The first immunization was performed at day 7 of life. Group A chickens were given one antigen dose, whereas groups B and C were administered two doses. Group B chickens were booster vaccinated at day 21 of life, i.e. 2 weeks after the administration of priming vaccination, while group C at day 35 of life, i.e. 4 weeks after primary vaccination. In chicken immunizations identical antigen doses were used for all groups, administered in the same manner. One vaccine dose contained 25 μg rH5-E. coli and aluminium hydroxide as an adjuvant. Vaccine was administered subcutaneously in three areas on the back of the neck in volume of 200 μl.
To monitor chickens' response to immunization, blood was collected from the vaccinated animals at days 21, 35, 42 and 48 of life. Thus samples for serological analyses were prepared from the following stages of the experiment in individual groups: in group A—2, 4, 5 and 6 weeks after immunization, in group B—2 weeks after priming immunization and 2, 3 and 4 weeks after booster immunization, while in group C—2 and 4 weeks after priming immunization and 1 and 2 weeks after booster immunization. Samples collected in parallel from non-vaccinated chickens were the control material. Blood, collected from the wing vein was left to coagulate at room temperature for 1 h and 30 min., afterwards it was stored at 2-8° C. to allow for clotting. Serum, obtained by clot centrifugation (5000×g, 10 min., 4° C.) was aliquoted and stored at −70° C. until analysis. Chicken immunization and blood collection schedules for vaccinated and control animals were performed according to regimen A, presented below.
Sera prepared during the experiments were analyzed using indirect and competitive ELISA to detect antibodies against H5 HA of AIV. Serum samples were also analyzed for the erythrocyte agglutination inhibition activity by homologous HPAIV H5N1 and heterologous LPAIV H5N2 (HI). The assays were performed on serum samples from all blood collections or from blood collected from selected experiment stages. Some samples were not analyzed in all tests due to a lack or insufficient amounts of serum, which is indicated in figures and in tables as n.a.
Immune Response Analysis with ELISA
Serological analyses of the samples collected during the immunization experiments described in Example 6 were carried out by ELISA to detect antibodies against H5 HA of AIV: an indirect one (H5-ELISA) and the competitive one (FLUAc H5, IDVet).
Indirect ELISA (H5-ELISA) was designed using recombinant HA H5 (17-530 aa, ΔRRRKKR, 6×His) homologous to the vaccine antigen and it was produced in a baculovirus expression system (Oxford Expression Technologies). Anti-HA antibody levels were treated as the main indicator of chicken humoral response to the vaccination and of rH5-E coli immunogenicity.
To carry out the H5-ELISA test, MediSorp plates (NUNC) were coated with rH5 (17-530 aa, ΔRRRKKR, 6×His) of (A/swan/Poland/305-135V08/2006(H5N1)) from a baculovirus expression system (Oxford Expression Technologies) at 3 μg/ml of PBS or were filled with PBS (non-specific binding control). The plates were incubated overnight at 2-8° C. and then blocked with “Protein-Free T20 (PBS) Blocking Buffer” (Pierce/Thermo Scientific) for 1 h at room temperature. Chicken sera were diluted 1:200 using 2% BSA/PBSS (phosphate buffer containing 0.3 M NaCl+KCl). In order to determine the endpoint titer, sera from individual blood collections from the analyzed chicken group were pooled and then serially diluted in 2% BSA/PBSS. The plates with serum samples and control samples—BLK (2% BSA/PBSS) were incubated overnight at 2-8° C. Assay development involved 1-hour incubation at 37° C. with HRP labeled antibodies against IgY chicken antibodies, specific towards the Fc fragment (Pierce/Thermo Scientific), diluted at 1:13000. Plates were washed with PBS buffer with Tween 20 at 0.05% (PBST) or 0.1% (PBSTT). The PBSTT buffer was used to wash only the plates with serum samples. After coating, 4 washing cycles were used, 2 after blocking and 5 between the other stages of the procedure. Color reaction for horseradish peroxidase was developed using TMB (Sigma). Plates were incubated with substrate for 30 minutes and then 0.5 M H2SO4 solution was added to stop the HRP reaction. Sample absorption was read at λ=450 nm.
The A450 values obtained for the BLK samples were subtracted from the A450 values measured for serum samples. Assay results for sera on plates not coated with rH5 constituted the control for the non-specific serum binding levels (background). Serum samples were considered H5-positive if the A450 value for the samples was higher than the mean A450 value for the control increased by the doubled standard deviation value (cut off). The endpoint titer was defined as the highest dilution of an immunized chicken serum giving an absorption value 4-times higher than the mean absorption value for the control.
Analyzing the chicken humoral response to subcutaneous administration of one 25 μg-dose of rH5-E. coli in the presence of aluminium hydroxide, conducted using the H5-ELISA on samples collected 2, 4, 5 and 6, and 2 or 2 and 4 weeks after immunization in chicken groups A, B and C showed the presence of IgY antibodies against H5 in sera diluted 200-fold from 23 out of 24 (96%) immunized animals (
In 6 out of 8 (75%) group A animals vaccinated once with rH5-E. coli the anti-H5 antibody levels were the highest 2 weeks after immunization (
Serological analyses of chickens after subcutaneous priming immunization with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, conducted using the H5-ELISA on samples collected directly before administering the booster dose of the antigen, i.e. 2 or 4 weeks after priming vaccination of group B and C chickens, demonstrated detectable IgY antibodies against H5 in sera diluted 1:200 amounting to 100% and 50%, respectively (
Assays of the endpoint titer in sera analyzed 2, 3 and 4 weeks after administering the II antigen dose to group B chickens and 1 and 2 weeks after administering the 2nd antigen dose to group C chicken, demonstrated that rH5-E. coli induces a high titer of IgY antibodies against H5 at the second subcutaneous administration of the antigen in the presence of aluminium hydroxide (
To detect antibodies against H5 HA, a commercially available ELISA “IDScreen® Influenza H5 Competition ELISA kit” (FLUAc H5, IDVet) was also used. The assay is used in the diagnostics of H5 serotype AIV infections in birds. Results of this assay were considered as an indicator for the induction of antibodies recognizing a presumably serotype-specific, neutralizing conformational AIV epitope. The FLUAc H5 test, beside the HI test, provided a functional evaluation of antisera produced as a result of vaccination with rH5-E. coli and potency of HA vaccine (17-522 aa, ΔRRRKKR), produced in a bacterial expression system.
The FLUAc H5 test was conducted according to the manufacturer's (IDVet) instructions, applying the conditions that enhance test sensitivity (serum samples diluted 1:5, incubated on plates overnight at 2-8° C.). Each analysis of serum samples from rH5-E. coli immunized chickens met the test's validation requirements. The competition level was determined by calculating the ratio of mean absorption values of the analyzed samples at λ=450 (A450) to the mean A450 value for the negative control (NC). The competition values for serum samples were expressed in percent. According to the FLUAc H5 manufacturer's data, competition values of 240%, between 35 and 40% and 535% indicate that the samples are seronegative, doubtful and seropositive, respectively.
Analysis of humoral response in group A chickens to subcutaneous administration of 25 μg of rH5-E. coli in the presence of aluminium hydroxide, conducted using the FLUAc H5 test on samples having the highest anti-H5 IgY antibody levels in individual chickens (
The serological analysis conducted using the FLUAc H5 test on samples collected 2, 3 and 4 weeks after booster immunization in group B chickens vaccinated subcutaneously twice at a 2-week interval with 25 μg-doses of rH5-E. coli in the presence of aluminium hydroxide, demonstrated varied competition levels for the analyzed sera (Tab 4.2). The calculated competition levels for serum samples from group B chickens were 47-97% (Tab. 4.2) and were usually lower than those recorded in the group of chickens immunized once (Tab. 4.1). None of the analyzed sera from the group vaccinated twice at a 2-week interval was positive or doubtful, according to the FLUAc H5 test classification criteria.
The competition percentage values for individual group B animals increased in successive assays (Tab. 4.2). As a result, the range of serum competition values obtained 2 weeks after booster immunization (47-69%) shifted towards higher values with time from the second immunization, amounting to 59-86% after 3 weeks and 72-97% 4 weeks from the 2nd antigen dose administration. Results of the analyses, obtained with the FLUAc H5 test (Tab. 4.2) indicate a decrease in the levels of antibodies recognizing a presumably serotype-specific epitope in group B chicken sera within 4 weeks after the second immunization, which may be associated with a decrease in total levels of anti-H5 antibodies in the sera, shown by H5-ELISA (
The analyses performed on samples collected 1 and 2 weeks after booster immunization from group C chickens, vaccinated twice subcutaneously with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant at an interval of 4 weeks, demonstrated competitive activity against the FLUAc H5 test antibodies for a large proportion of analyzed sera (Tab. 4.3). The competition range for group C chicken sera was 10-95% (Tab. 4.3) and included markedly lower values than those obtained in group B chickens, vaccinated twice at a 2-week interval (Tab. 4.2). Among the 14 analyzed group C sera, 7 samples showed competition % below 40% (Tab. 4.3), indicating that the samples are positive or doubtful, according to the test classification. These samples were collected from 5 out of 8 examined chickens, among which 3 should be considered seropositive according to the FLUAc H5 test criteria (competition≦35%). The seropositivity of the other 2 chickens was doubtful, since samples of their collected sera showed competition at 37 and 38%.
In 3 of the analyzed group C chickens, serum competition % decreased by 6-13% between the 1st and the 2nd week after booster dose administration and in 3 others it increased during that time by 4-9% (Tab. 4.3). Slight fluctuations in antibody levels within 2 weeks after booster immunization were also observed in the H5-ELISA detecting total anti-H5 antibody levels (
The analysis of humoral response in broiler-type Ross 308 chickens to subcutaneous administration of one or two doses of vaccine containing 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, performed by ELISAs (H5-ELISA, FLUAc H5) demonstrated that:
Analysis of Immune Response with Hemagglutination Inhibition (HI) Tests
Serum samples collected during the immunization experiments described in example 6 were analyzed for the presence of antibodies inhibiting hemagglutination (HI) by the H5 serotype AN. Titers of HI antibodies in chicken sera were determined by hemagglutination inhibition tests with homologous HPAIV H5N1 and heterologous LPAIV H5N2 as antigens and the hemagglutination inhibition unit (HIU) of 1:8. The HI tests, commonly used in influenza virus infection diagnostics, provided functional evaluation of antibodies produced as a result of vaccination with rH5-E. coli and their potential capacity to protect against homologous and heterologous AIV infection. The principle of the HI test and its performance are described below.
Hemagglutinin—a protein found on the surface of influenza viruses—has the capacity to agglutinate erythrocytes. This characteristic is the basis for identification of influenza virus isolates. The HI test utilizes the fact that specific binding of antibodies to antigenic sites on the HA molecule alters the capacity of this protein to bind erythrocyte surface receptors, thus preventing agglutination. The tests were conducted according to the current recommendations (Minta, 2008; Avian Influenza in: OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2012).
HI Tests with AI Viruses
Two HI tests were performed using two H5 AI viruses and antisera described in line-up A.
The tests were conducted with fresh erythrocyte preparation collected from blood of SPF chickens, obtained from a sterile culture at DPD NVRI. For each HI test a hemagglutination unit (HAU) was determined. Each conducted HI test included 3 controls: the blood cell control (internal control for the assay), i.e. samples that did not contain the virus, the positive control and the negative control containing components specified in line-up A. The hemagglutination inhibition effect, evaluated visually in comparison to blood cell control samples, is considered to be a positive result. In both cases the cells do not hemagglutinate, settling freely to the bottom of the well on a 96-well plate with V-bottomed wells.
For the purpose of evaluating chicken sera obtained as a result of vaccinations, a serum dilution protocol was employed, starting at 1:8. Each analyzed and control serum had its own blood cell control in order to exclude the potential presence of non-specific inhibitors in the sample. Using a series of twofold dilutions a range of 1:8 to 1:512 was investigated. Samples of 25 μl from successive dilutions of the analyzed sera were incubated with 4 HA units (25 μl) of inactivated AIV. Viral strains used in each HI test are described in line-up A. After 25 minutes of preliminary incubation at room temperature, 25 μl of 1% chicken erythrocyte suspension was added. The result was observed after 30 min incubation. The inversion of the highest dilution inhibiting chicken erythrocyte agglutination defined the HI titer for the analyzed sera. Samples were considered positive if they reached minimal tite value of 1:8. Titer of ≧1:16 was considered a result indicating a protective activity of induced antibodies, according to the currently binding requirements for influenza vaccines.
Analyses of humoral response in chickens to subcutaneous administration of one (group A) or two 25 μg-doses of rH5-E. coli with aluminium hydroxide, at an interval of 2 (group B) or 4 weeks (group C), based on HI tests on samples collected at 2, 4, 5 and 6 and 2 or 2 and 4 weeks after immunization in the chicken groups A, B and C, demonstrated a strong dependence of the induction of HI antibodies on the schedule of immunization with rH5-E. coli (Tab. 5.1, 5.2, 5.3). In chicken group A, vaccinated once with rH5-E. coli, only 1 positive serum sample was found, showing the lowest assayed titer (1:8) and this was only in the presence of the homologous H5N1 virus (Tab. 5.1). Among the samples collected from group B chickens, vaccinated twice with rH5-E. coli at a 2-week interval, 9 samples seropositive for the H5N1 virus were detected at an HI titer of 1:8 or 1:16, and 2 samples seropositive for the H5N2 virus with an HI titer of 1:8 (Tab. 5.2). A minimal protective HI titer, i.e. 1:16, was shown only with the homologous AIV and with sera collected after the administration of the 2nd antigen dose, from 2 group B chickens.
In group C chickens, vaccinated twice with rH5-E. coli at an interval of 4 weeks, 32 samples were analyzed in the HI test using homologous and heterologous AIV and positivity was demonstrated for 15 and 8 serum samples, respectively (Tab. 5.3). Sera collected from all chickens in group C were positive in the test with the H5N1 and/or H5N2 virus, while HI titers of sera of 5 chickens exceeded 1:8. A minimal protective serum HI antibody level (titer of 1:16) was shown for 2 immunized chickens, a desired level (≧1:64) for 2 other chickens, whereas for the other animals the maximum HI titer was 1:32.
Analyses of humoral response in broiler-type Ross 308 chickens to subcutaneous administration of vaccine comprising 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, conducted using the HI tests with homologous HPAIV H5N1 and heterologous LPAIV H5N2, demonstrated that:
Effectiveness of Chicken Immunization with rH5-E. coli at Different Immunization Schedules
In order to evaluate rH5-E. coli vaccine potency the results of analyses of sera prepared during immunization trials of broiler-type Ross 308 chickens, described in example 6, are summarized in the present example. Assays were performed using indirect and competitive ELISA to detect anti-H5 antibodies in chicken serum (H5-ELISA; FLUAc H5, IDVet). The HI tests were conducted using the following AI viruses as antigens: homologous HP H5N1 and heterologous LP H5N2 and the hemagglutination inhibition unit (HIU) of 1:8. The method of serological assays (ELISA, HI) and the results of these tests for chickens immunized with rH5-E. coli were described in detail in examples 7 and 8, with regard to the kinetics of humoral immune response and the individual nature of the response.
The analysis of immune response to vaccination in chickens, shown in the present example, includes results obtained from all blood collections in groups A, B and C (antibody detection, H5-ELISA), from blood collections after the administration of the 2nd rH5 dose to group B and C chickens (endpoint titer, H5-ELISA, FLUAc H5) or blood collected 2, 4 or 5 weeks after rH5 immunization of group B animals (FLUAc H5). The positivity of individual animals in the tests was evaluated here independently of the experimental stage at which it was assayed.
Efficacy of immunization of broiler-type chickens using rH5-E. coli is evaluated based on the statistics of animal response to the vaccination, the titer of IgY antibodies against H5, the titer of active antibodies in the FLUAc H5 test and the titer of HI antibodies. The titer of induced antibodies inhibiting erythrocyte agglutination by AIV should preferably be at least 1:16 (minimal protection level), it is optimal to reach 1:64 or higher. Assessment of the potential applicability of the tested vaccine to immunize birds against avian influenza, apart from the efficacy of vaccinations applying it, should also take into account the unit production cost of the vaccine and animal immunization costs. The number and size of antigen doses required to elicit immune response in animals and the type of adjuvant in the vaccine composition are significant components of these costs.
The results of analysis of broiler-type Ross 308 breed chicken humoral response to subcutaneous administration of one or two doses of vaccine comprising 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, conducted using ELISAs (H5-ELISA, FLUAc H5) and HI tests (HI-H5N1, HI-H5N2) are summarized in Tab. 6. The data shown indicate that:
Good results for immunization of broiler-type chickens with rH5-E. coli were obtained by administering only two doses of the antigen in the presence of aluminium hydroxide as an adjuvant. Aluminium hydroxide adjuvant has been commonly used for more than 80 years, primarily to enhance humoral immune response. Its activity enhancing cellular response is debatable. Efficacy of an inexpensive adjuvant, approved for animal and human immunization, such as aluminium hydroxide, in a vaccine composition with rH5-E. coli would result in the estimated unit cost of the vaccine not being excessively burdened by the price of its additional components. The efficacy of double vaccinations with rH5-E. coli in the presence of aluminium hydroxide has a positive effect on the costs of animal immunization using the tested vaccine.
Serological analyses of chickens vaccinated twice at a 4-week interval with 25 μg-doses of rH5-E. coli with aluminium hydroxide showed that all animals were responsive to the vaccination, as indicated by the high anti-H5 IgY antibody levels in animal sera (Tab. 6). The activity of the induced antibodies in tests evaluating their immunoprotective properties (FLUAc H5, HI-H5N1, HI-H5N2) varied within the vaccinated animal group (Tab. 6). Positivity in the group, reaching 37.5% in the FLUAc H5 test, 75% in the HI-H5N1 test and 62.5% in the HI-H5N2 test, demonstrates the vaccine potency in terms of the induction of desired functional antibodies and simultaneously indicates the necessity to optimize vaccinations such to increase the number of seropositive animals after vaccination in the aforementioned tests. Demonstration that two immunizations of broiler-type chickens at a 4-week interval are much more effective, if not exclusively effective, in inducing active antibodies in the FLUAc H5 test and in HI tests in comparison to two vaccinations at a 2-week interval (Tab. 6) shows the direction for optimization of animal vaccinations against avian influenza. Specification of the optimal time interval between the priming and booster doses seems to be crucial to increase efficacy of vaccinations of broiler-type chickens with the rH5-E. coli vaccine.
Immunizations of broiler-type chicken and vaccination response tests were performed during the period between day 7 of life, when the immunological system of birds is already mature and blood levels of maternal antibodies decrease, and the end of chicken production cycle (day 45 of life). This made it possible to examine immunogenicity of the rH5 (17-522 aa, ΔRRRKKR) fragment produced by overexpression in E. coli (rH5-E. coli) and to test the efficacy of various immunization regimens. Short life-cycle of broiler-type chickens in commercial rearing systems considerably limits the potential for optimization of vaccinations; moreover, there is no economic justification for the use of the tested vaccine for mass vaccinations in this animal group. Studies on the optimization of rH5-E. coli dosage and vaccination regimen will target chicken groups with long life-cycles, such as laying hens in commercial flocks and breeding flocks of hens and broilers.
The final criteria in assessment of efficacy of rH5-E. coli vaccination are provided by the results of experimental infection of chickens with AI viruses. The challenge experiment was performed in laying-type SPF chickens. Chickens vaccinated with rH5-E. coli were infected with HPAIV H5N1. The method of the experiment and its results are described in example 10.
Experimental Infection of Chickens Vaccinated with rH5-E. coli with Highly Pathogenic (HP) Avian Influenza Viruses (AIV) H5N1 (Challenge)
In order to evaluate the capacity of rH5-E. coli vaccine to provide protection against AIV infection, an experimental infection of vaccinated chickens was performed using the following HPIV H5N1 viruses: homologous from clade 2.2 (experiment 1) and heterologous from clade 1 (experiment 2). The challenge experiments were conducted in laying-type SPF White Leghorn chickens, at the National Veterinary Research Institute (NVRI) in Pulawy, in PCL3 containment.
Immunization with rH5-E. coli, Infection with HPAIV H5N1 and Sample Collection for Analyses
A group of 3-week old (experiment 1) or 3½-week old (experiment 2) SPF chickens (10 animals) were vaccinated subcutaneously by administering 25 μg of rH5-E. coli in the volume of 200 μl with aluminium hydroxide as an adjuvant. The vaccine composition was prepared as described in example 6.
The booster dose—25 μg of rH5-E. coli with aluminium hydroxide, was administered 4 (experiment 1) or 4 (experiment 2) weeks later. Two and four weeks after the administration of the 1st dose and two and three weeks after administration of the 2nd antigen dose, blood was collected for serological analyses.
Three weeks after the second immunization the chickens were infected with homologous HPAIV-A/turkey/Poland/35/07(H5N1) from clade 2.2 (experiment 1) or heterologous HPAIV-A/crested eagle/Belgium/01/2004(H5N1) from clade 1 (experiment 2). The viral infection titer was 109 EID50/ml. Viruses were administered intranasally and conjunctivally (i.n/i.o.) at the dose of 106 EID50 in 0.1 ml (1:100 dilution). To analyze the potential of AI virus transmission from chickens infected after vaccination to fully susceptible birds, approx. 24 hours after HPAIV infection, 2 non-vaccinated SPF chickens (contact animals) were introduced to each experimental group. During 14 days after infection clinical observations of the animals were conducted. Three, seven and ten days after infection, throat and cloacal swabs for real time RT-qPCR analyses were collected from vaccinated and contact chickens. Fourteen days after infection, blood samples for serological analyses were collected from all challenge surviving birds, as well as throat and cloacal swabs for real time RT-qPCR analyses.
The positive control for the challenge experiments comprised SPF chickens, 5 in each group, that at the same age as the experimental group chickens were infected with A/turkey/Poland/35/07(H5N1) from clade 2.2 (experiment 1) or with A/crested eagle/Belgium/01/2004(H5N1) from clade 1 (experiment 2), applying identical doses and administration method as for vaccinated animals, i.e. i.n/i.o., dose of 106 EID50 in 0.1 ml (1:100 dilution). Throat and cloacal swabs as well as body organs (lungs, spleen, kidneys, brain) were collected for real time RT-qPCR from the chickens that died during the observation period.
The experiments were conducted according to the requirements for vaccine testing in challenge experiments, e.g. regarding the dose (106 EID50) and the interval between vaccination and experimental infection (3 weeks), as described in “The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2012” OIE.
The course of immunization, infection and sample collection for analyses in experiments 1 and 2 were applied according to regimens B and C, respectively, as shown below.
Serum samples collected during the experiment were analyzed using the hemagglutination inhibition (HI) tests and commercial ELISA tests: FLUAc H5 (IDVet) and IDEXX AI MultiS-Screen (Idexx Laboratories). The results of the HI tests with H5 viruses and the FLUAc H5 test to detect antibodies against AIV H5 made it possible to evaluate humoral response of chickens to rH5-E. coli vaccination and serum functional antibody levels in vaccinated animals directly before the HPAIV H5N1 infection. The IDEXX AI MultiS-Screen (Idexx Laboratories) is designed to detect anti-AIV antibodies in bird sera. The application of this test on the samples collected during the challenge experiments was to monitor exposure of chickens to AI viruses. Serological analyses of samples collected after HPAIV infection from vaccinated and contact chickens with the IDEXX AI MultiS-Screen test were complementary in relation to the analyses of viral genetic material levels in swabs that were performed using real time RT-qPCR.
The HI activity of chicken sera collected during the challenge experiments was tested using inactivated HPAIV H5N1: homologous and heterologous. Viruses and antisera used in the HI tests are described in line-up B.
The HI test was conducted with SPF chicken erythrocytes using the hemagglutination inhibition unit (HIU) of 1:8. The tests were performed as described in example 8. Serum HI titers≧1:16 were considered as indicative of the protective activity of induced antibodies, according to the currently binding requirements for influenza vaccines.
The FLUAc H5 test was performed according to the manufacturer's (IDVet) instructions, however, conditions enhancing test sensitivity were not applied. According to the basic protocol, serum samples were diluted 1:5 and then incubated on plates for 1 hour at 37° C. The method to calculate competition levels and interpretation of the results, according to the manufacturer's data, are described in example 7. The IDEXX AI MultiS-Screen test was conducted according to the manufacturer's (Idexx Laboratories) instructions. Interpretation of results in this test depends on the value of absorption reading for the analyzed sample (S) in relation to the value read for the negative control (N). Calculated values S/N≧0.5 and <0.5 obtained for the analyzed samples indicate that the samples are negative and positive, respectively, with respect to anti-AIV antibodies.
Determination of AIV Levels after Infection
The aim of real time RT-qPCR analyses was to detect viral genetic material in throat and cloacal swabs and in the case of dead animals also in their organs (lungs, spleen, kidneys, brain). Results of these analyses made it possible to determine the degree of AIV propagation and multiplication in the infected birds and the probability of virus shedding to the environment and its transmission to other birds.
Samples collected for analyses with real time RT-qPCR were stored in a universal transport medium (COPAN Diagnostics Inc.). Total RNA was extracted from 0.1 ml medium using the RNeasy Mini Kit (Qiagen). Real time mRT-PCR was performed as described by Spackman E et al. (2002). The following oligonucleotides: M-25 (5′-AGATGAGTCTTCTAACCGAGGTCG-3′) and M-124 (5′-TGCAAAAACATCTICAAGTCTCT-3′) were used as primers, while the probe was M-64 (5′-FAMTCAGGCCCCCTC AAAGCCGA-TAMRA-3′). Qualitative RNA standards with known virus titers, extracted from 10-fold dilutions of viruses used in experimental infection of chickens were used to convert the Ct value of RT-qPCR to values equivalent to EID50 (eqEID50) per ml of swab fluid or per gram of tissue. The amounts of viral RNA in the tested samples were extrapolated from the standard curve.
The results of serological analyses of laying-type SPF chickens after subcutaneous administration of two rH5-E. coli doses, 25 μg each, with aluminium hydroxide as an adjuvant, conducted on samples collected 3 weeks after administration of the 2nd dose of antigen using the HI tests are shown in
Chickens denoted 2-2 . . . 2-10 responded more strongly to rH5-E. coli vaccination with production of HI antibodies active against homologous HPAIV H5N1 than chickens denoted 1-1 . . . 1-10, as indicated by the number of seropositive chickens in the group (100% vs. 50%) and the HI antibody titers (mean 65.8 vs 26.4). In the FLUAc H5 test, seropositivity of chickens 2-2 . . . 2-10 vaccinated before infection with heterologous HPAIV H5N1, was 56% and it was lower than that for chickens 1-1 . . . 1-10 from the group vaccinated in the challenge experiment with homologous HPAIB H5N1, in which 100% animals were seropositive (
The results of experimental infection of vaccinated and control laying-type SPF chickens with the following HPAI H5N1 viruses: a clade 2.2 homologous one (experiment 1) and a clade 1 heterologous one (experiment 2), expressed as % survival rate of infected and contact animals during 14 days after infection are shown in
In the experiment with heterologous HPAIV H5N1 from clade 1, 3 out of 10 vaccinated chickens died—one animal on the second and two others on the third day after infection (
The results of the FLUAc H5 test and the HI test with homologous HPAIV H5N1 recorded for chickens directly before infection with homologous HPAIV H5N1 (
The results of the FLUAc H5 test and the HI test with heterologous HPAIV H5N1 obtained for chickens directly before infection with heterologous HPAIV H5N1 (
The results of serological analyses of laying-type SPF chickens, conducted using the IDEXX AI MultiS-Screen test (Idexx Laboratories) on samples collected 3 weeks after administration of the 2nd dose of rH5-E. coli and 2 weeks after experimental infection with homologous (experiment 1) and heterologous (experiment 2) HPAIV H5N1 and in parallel from contact chickens are shown in Tab. 7. Directly before infection with HPAI viruses, all studied chickens from the rH5-E. coli vaccinated and contact groups were seronegative. Two weeks after infection with homologous HPAIV H5N1 all vaccinated chickens and 1 out of 2 in-contact animals, denoted 1-KK-2, produced antibodies against AIV. Among the 7 chickens that survived infection with heterologous HPAIV H5N1, 4 were seropositive, while 3 others remained seronegative in the IDEXX AI MultiS-Screen test. Among the 3 chickens, 2-3, 2-5 and 2-6, showing symptoms of influenza after infection with heterologous HPAIV H5N1 animals 2-5 and 2-6, in contrast to animal 2-3, produced antibodies detectable by the test.
a3 dpi,
b2 dpi,
c8 dpi,
d7-8 dpi
The obtained results confirmed that chickens selected for challenge experiments did not have contact with AIV before the experimental infection and immunity against the infection was induced exclusively by rH5-E. coli vaccination. Among the 17 vaccinated chickens that survived infection with HPAI viruses, a majority, i.e. 82%, of animals produced antibodies against AIV, detected with the IDEXX AI MultiS-Screen test. Assays of anti-AIV antibody levels in contact chickens that were introduced to the group infected with homologous HPAIV H5N1 after vaccination, demonstrate that only one of the two fully susceptible animals had contact with AIV originating from the infected animals.
Amount of HPAIV RNA in Swabs after Infection
The results of viral RNA level analysis in throat (T) and cloacal (C) swabs collected from chickens vaccinated with rH5-E. coli and from contact chickens after infection with the HPAI H5N1 viruses—3, 7, 10 and 14 days post infection (dpi) or post mortem, are shown in Tab.8. In the group of vaccinated chickens infected with the homologous HPAIV H5N1, genetic material of the virus was detected in 8 out of 10 chickens 3 dpi and only in throat swabs, wherein levels thereof reached values in the range of 2.4-3.8 log10 EID50/ml. In the next days after infection, i.e. 7 and 10 dpi, viral RNA was not detected in swabs from this animal group. Seven days post infection of the vaccinated animals, viral RNA was found in throat swab from 1 out of 2 contact chickens (1-KK-2) but was no longer detected on 10 dpi.
a3 dpi.
b2 dpi.
c8 dpi.
d7-8 dpi - swabs collected post mortem (pm)
In the group of chickens vaccinated with rH5-E. coli and then infected using the heterologous HPAIV H5N1, genetic material of the virus was not detected in throat and cloacal swabs of 5 out of 10 vaccinated animals in none of the assays performed on samples collected in the 2-week observation period. Analysis of samples collected post mortem from 3 chickens—denoted 2-8, 2-9 and 2-10 that died 2 or 3 dpi, demonstrated the presence of substantial amounts of viral RNA in the range of 6.7-8.3 log10 EID50/ml in throat or throat and cloacal swabs. Among the chickens that survived infection with the heterologous HPAIV, viral genetic material was detected in 2 animals. For the 2-6 denoted animal viral genetic material was detected at the level of 4.9 and 4.6 log10 EID50/ml in throat swabs collected 3 and 7 dpi and for the 2-7 denoted animal in throat, in throat and cloacal and in cloacal swabs, on 3, 7 and 10 dpi respectively at the level in the range of 3.8-7.1 log10 EID50/ml. The results of virus level analysis in swabs collected from the 2-7 animal additionally confirm the hypothesis of the crucial role of antibodies active in the FLUAc H5 test in the immunity against infection, described in the ‘Anti-H5 antibodies and immunity against infection’ chapter. Two weeks after infection, viral RNA was not detected in swabs collected from all animals that survived the challenge (7/10). Among the 3 chickens: 2-3, 2-5 and 2-6 showing symptoms of influenza after infection with the heterologous HPAIV H5N1, viral presence in swabs was found only for the animal with more severe disease symptoms, denoted 2-6. In the 2 other chickens: 2-3 and 2-5 no viral RNA was found in samples collected from throat and cloaca, which may indicate a systemic nature of the infection. In the contact chickens no viral genetic material was detected on 3 dpi, however on 7 dpi viral RNA was found in the range of 5.0-5.4 log10 EID/ml in both throat and cloacal swabs, suggesting replication of AI viruses deriving from vaccinated chickens with detectable levels of viral particles. Analysis of samples collected post mortem from contact chickens on 8 dpi demonstrated an even greater amounts of viral RNA in swabs, wherein levels thereof reached values in the range of 6.8-7.7 log10 EID50/ml.
The results of the first challenge experiment unambiguously demonstrated that rH5-E. coli vaccination induces immune response in chickens, preventing multiplication of homologous HPAIV H5N1. Furthermore, the vaccination limited shedding of HPAI viruses to a few days after infection at most, maximally to 7 dpi, and eliminated effective HPAIV transmission to fully susceptible contact chickens. The results of the second challenge experiment demonstrated that rH5-E. coli vaccination induces immune response in 7 out of 10 chickens, preventing (5 chickens) or limiting to a large extent (2 chickens) multiplication of heterologous HPAIV H5N1. Moreover, vaccination decreased by 70% the number of chickens excreting the heterologous AIV during a period longer than 10-14 dpi and in that sense caused a decrease in shedding of HPAI viruses as well as delayed effective HPAIV transmission to fully susceptible contact animals, but did not prevent it.
The results of challenge experiments, wherein laying-type SPF chickens (White Leghorn) were vaccinated twice at an interval of 4 or 4½ weeks, using 25 μg-doses or rH5-E. coli (17-522 aa, ΔRRRKKR) with aluminium hydroxide and next infected with HPAIV H5N1: a homologous one from clade 2.2 or a heterologous one from clade 1, demonstrated that:
Optimization of Vaccination Regimen with rH5-E. coli
In order to determine the optimal rH5-E. coli dose and the interval between the doses that is advantageous for immunization effectiveness, immunization studies were performed on laying-type chickens, using antigen doses in range of 5-25 μg and 4- and 6-week interval between the I and the II immunization.
The vaccine containing rH5-E. coli and aluminium hydroxide as an adjuvant was prepared directly before use, as described in example 6. Experiments were performed simultaneously in 8 groups of laying-type Rossa 1 breed chickens under production rearing conditions. Chickens from groups 1A-4A, 1B-4B, 10 animals each, were vaccinated, whereas 15 non-immunized chickens formed control group K. First immunization was done in the 3rd week of life of the animals. Chickens from groups 1A-4A were immunized for the second time in the 7th week of life, i.e. 4 weeks after administering the I dose, while groups 1B-4B in the 9th week of life, i.e. 6 weeks after administering the I dose. rH5-E. coli was used for animal immunization at doses of 25, 15, 10 and 5 μg, administered subcutaneously in three areas on the back of the neck in volume of 200 μl with aluminium hydroxide as an adjuvant.
In order to monitor chicken response to immunization, blood was collected from vaccinated animals directly before and 1 and 2 weeks after administering the II antigen dose. Samples collected in parallel from non-vaccinated chickens were the control material. Serum samples were prepared and stored as described in example 6. Schedule of chicken immunizations and blood collection from vaccinated and control animals was according to regimen D, shown below.
Immune Response Analysis with Serological Assays
Sera prepared during the experiment were analyzed using ELISAs: indirect—H5-ELISA and competitive—FLUAc H5 (IDVet) for detection of antibodies against AIV H5 HA and the hemagglutination inhibition (HI) test. The test detecting antibodies recognizing the presumably serotype-specific neutralizing conformational epitope of AIV (FLUAc H5) and the HI test, detecting antibodies recognizing HA antigenic sites in viral particles which are essential for binding to cellular receptors, enabled a functional analysis of the antisera produced as a result of vaccination.
The H5-ELISA was done according to the description in example 7. The FLUAc H5 test was conducted according to the manufacturer's (IDVet) instructions, applying the conditions increasing the test sensitivity (serum samples diluted 1:1, incubated on plates for 1 hour at temperature of 37° C.). The method of calculating competition levels and of interpretation of the results, according to the manufacturer's data, is described in example 7. The HI test was conducted with SPF chicken erythrocytes, using the heterologous LPAIV H5N2 as an antigen and hemagglutination inhibition unit (HIU) of 1:8. The principle of the HI test and the method of conducting it are shown in example 8. Assays were done on serum samples from all blood collections. Samples were considered positive if they reached the titer of ≧1:8. It was considered that, according to the requirements for influenza vaccines currently in force, the HI titer indicating a protective effect of the induced antibodies should be ≧1:16.
Serological studies of laying-type chickens after subcutaneous priming immunization using 25 μg (groups: 1A, 1B), 15 μg (groups: 2A, 2B), 10 μg (groups: 3A, 3B) or 5 μg (groups: 4A, 4B) of rH5-E. coli and aluminium hydroxide as an adjuvant, performed with the H5-ELISA on samples collected 4 weeks (groups: 1A-4A) or 6 weeks (groups: 1B-4B) after vaccination, demonstrated detectable IgY antibodies against H5 in the sera, diluted 1:200, at the level in range of 60%-100% and 0%-33%, respectively (FIG. 8.1). One and two weeks after administering the antigen booster dose, high anti-H5 antibody levels in all vaccinated chickens were found indicating 100% seropositivity. Endpoint titer for serum anti-H5 IgY antibodies in all animal groups after two immunizations was very high—1 week and 2 weeks after administering the II antigen dose it reached values in range of: 320 000-850 000 and 200 000-430 000, respectively (
Changes in anti-H5 IgY antibody levels after second immunization were accompanied by changes in levels of functional antibodies, detected with the FLUAc H5 (IDVet) and HI test with the heterologous LPAIV H5N2. Directly before administering the II antigen dose, no positive samples were found in the FLUAc H5 test (
In Tab. 9 the results of chicken positivity assays during 2 weeks after vaccinations are summarized, the results being obtained using the FLUAc H5 and the HI-H5N2 tests. Positivity in the FLUAc H5 test in groups vaccinated with rH5-E. coli at doses of 10, 15 and 25 μg reached values in range of 68-90%, in the group vaccinated with 5 μg of antigen it was 52.5%±10, whereas the percentage of chickens vaccinated twice using 5-25 μg of rH5-E. coli showing protective HI antibody titer was in range of 81-87%.
87% ± 12.5
87% ± 12.5
The results of analyses of sera collected 1 and 2 weeks after administering two doses of rH5-E. coli, 5, 10, 15 or 25 μg each, shown in
Analyses of laying-type Rossa 1 breed chicken humoral response to subcutaneous administration, at an interval of 4 or 6 weeks, of two vaccine doses comprising 5-25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, conducted on 8 chicken groups of 10 animals each, using ELISAs (H5-ELISA; FLUAc H5, IDVet) and the HI test with the heterologous LPAIV H5N2, demonstrated that:
Immunization studies of laying-type Rossa 1 breed chickens, wherein the animals were administered twice at a 4 week interval with 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, described in example 11, demonstrated a much higher vaccination effectiveness than the immunization studies conducted using an identical antigen dose, adjuvant and interval between the doses in broiler-type Ross 308 breed chickens, described in examples: 6-9. Immunization regimens differed only in that the immunization of laying-type chickens was started in the third week of life, whereas that of broiler-type chickens out of necessity in the first week. However, the difference in humoral response to the vaccination for laying-type and broiler-type chickens, primarily concerning the presence and levels of functional anti-H5 antibodies (
Enhancing Immune Response by Intranasal Administration of rH5-E. coli
In order to investigate the potential of rH5-E. coli to induce immune response in birds by mucosa, a vaccine composition was developed comprising rH5-E. coli and a mucoadhesive adjuvant (chitosan glutamate) for mucosal immunization and an immunization study was conducted on laying-type chickens, wherein the parenterally primed animals were given an intranasal booster dose.
Protein and hemagglutinin content in the rH5-E. coli preparations for production of vaccines, were determined according to the description in example 6. The vaccine for parenteral immunization, comprising rH5-E. coli and aluminium hydroxide as an adjuvant, was prepared directly before use as described in example 6. For the preparation of the intranasal vaccine with rH5-E. coli, PROTASAN™ UP G 113 (NovaMatrix/FMC Corp.) was used, being a highly purified chitosan glutamate—a bioadhesive polymer with <20 mPa·s viscosity, molecular mass of <200 kDa and deacetylation level of 75-90%. PROTASAN was dissolved in deionized water by intense shaking on a vortex mixer, and next a 10× concentrated PBS solution, pH=7.4 was added in volume ensuring buffer dilution and it was shaken on a vortex mixer for 1 minute at 2500 rpm, thus obtaining a 1% (w/v) adjuvant solution in PBS, pH˜6. The rH5-E. coli preparation was added to the prepared PROTASAN solution in 1:1 ratio (v/v), followed by shaking the mixture on a vortex mixer at 2500 rpm for 5 minutes. The vaccine composition with pH˜6.5, comprising the antigen and 0.5% (w/v) PROTASAN™ UP G 113, was prepared directly before performing the vaccinations.
Immunization studies were done in a group of 15 laying-type Rossa 1 breed chickens (group no. 5) under production rearing conditions. The control group (K) was composed of 15 non-immunized chickens. The first vaccination was performed in the 3rd week of animal life by subcutaneous administration of 25 μg of rH5-E. coli with aluminium hydroxide in three areas on the back of the neck in volume of 200 μl. In the 7th week of life, i.e. 4 weeks after administering the I dose, intranasal vaccination of the animals was performed by administering 25 μg of rH5-E. coli with PROTASAN in volume of 272 μl, interchangeably to the left and right nostril. In order to monitor chicken response to immunization, blood was collected from vaccinated animals directly before administering the booster dose and within one month after administering the II antigen dose at one-week intervals. Samples collected in parallel from non-vaccinated chickens were the control material. Serum samples were prepared and stored as described in example 6. The schedule for immunizations and blood collection from vaccinated and control animals was according to regimen E, shown below.
Sera prepared during the experiment were analyzed using tests detecting antibodies against H5 AIV: the indirect and the competitive ELISAs (H5-ELISA; FLUAc H5 IDVet) and the hemagglutination inhibition (HI) test. The tests were conducted in order to investigate chicken humoral response to vaccination by administering a subcutaneous priming dose and an intranasal booster dose. The H5-ELISA test allowed to determine antigen-specific IgY antibodies, whereas the FLUAc H5 and HI tests allowed to evaluate functional properties of induced antibodies.
The H5-ELISA was performed as described in example 7, but the endpoint titer was determined for selected samples collected from individual animals. The method of calculating the cut-off value for H5-positive serum samples and the definition of the endpoint titer were shown in example 7. The FLUAc H5 test was done according to the manufacturer's (IDVet) instruction, applying the conditions increasing the test sensitivity (serum samples diluted 1:1, incubated on plates for 1 hour at temperature of 37° C.). The method of calculating competition levels and of interpretation of the results, according to the manufacturer's data, is described in example 7. The HI test was conducted with SPF chicken erythrocytes, using the heterologous LPAIV HSN2 as an antigen and hemagglutination inhibition unit (HIU) of 1:8. The principle of the HI test and the method of conducting it are shown in example 8. Assays were done on serum samples from all blood collections. Samples were considered positive if they reached the titer of at least 1:8.
Serological studies of chickens after subcutaneous priming immunization using 25 μg of rH5-E. coli and aluminium hydroxide as an adjuvant, conducted using the H5-ELISA on samples collected directly before administering the II antigen dose, i.e. 4 weeks after the first chicken vaccination, demonstrated the presence of anti-H5 IgY antibodies in 200-fold diluted sera from 13 out of 15 (87%) immunized animals. After intranasal administration of the vaccine comprising 20 μg of rH5-E. coli and PROTASAN™ UP G 113 as an adjuvant, in 10 out of 15 vaccinated animals an increase of serum antibody levels was observed, typical for secondary immune response (
Analyses of sera obtained from chickens 4 weeks after subcutaneous administration of priming dose of antigen using the FLUAc H5 test demonstrated that none of the studied samples was seropositive according to the classification criteria of the test and that competition values are within the range of 49-93% (
All of the conducted serological tests demonstrated a considerable variation in animal response to intranasal immunization, which was most probably caused by the observed partial swallowing of the vaccine by chickens when it was administered in too large volumes (272 lal).
Analyses of humoral response of laying-type Rossa 1 breed chickens to intranasal administration of the booster dose, comprising 20 μg of rH5-E. coli and PROTASAN™ UP G 113 (NovaMatrix/FMC Corp.) as an adjuvant, conducted using serological tests (H5-ELISA, FLUAc H5, HI-H5N2) suggest that:
| Number | Date | Country | Kind |
|---|---|---|---|
| P.408649 | Jun 2014 | PL | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/PL2015/050025 | 6/24/2015 | WO | 00 |
