Vaccines against influenza A viruses in poultry and swine: Status and future developments.

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Review

Vaccines against influenza A viruses in poultry and swine: Status and future developments J. Rahn, D. Hoffmann, T.C. Harder, M. Beer ∗ Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald-Insel Riems, Germany

a r t i c l e

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Article history: Received 17 December 2014 Received in revised form 1 March 2015 Accepted 18 March 2015 Available online xxx Keywords: Influenza virus vaccines Swine Chicken Novel developments

a b s t r a c t Influenza A viruses are important pathogens with a very broad host spectrum including domestic poultry and swine. For preventing clinical disease and controlling the spread, vaccination is one of the most efficient tools. Classical influenza vaccines for domestic poultry and swine are conventional inactivated preparations. However, a very broad range of novel vaccine types ranging from (i) nucleic acid-based vaccines, (ii) replicon particles, (iii) subunits and virus-like particles, (iv) vectored vaccines, or (v) liveattenuated vaccines has been described, and some of them are now also used in the field. The different novel approaches for vaccines against avian and swine influenza virus infections are reviewed, and additional features like universal vaccines, novel application approaches and the “differentiating infected from vaccinated animals” (DIVA)-strategy are summarized. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Avian influenza (AI) in poultry and swine influenza (SI) in pigs are economically important diseases, which are caused by influenza A viruses (IAV) [1–3]. Certain phenotypes of IAV subtypes H5 and H7, termed highly pathogenic (HP) AI viruses, cause devastating mortality in gallinaceous poultry, as well as affect water fowl in a more strain dependent manner [4]. Furthermore, AI viruses (AIV) of low pathogenicity (LP) may cause economically tangible losses in gallinaceous poultry due to a drop in egg production in layer flocks and reduced daily weight gains in fattening poultry. In addition, substantial mortality in galliforme poultry may be noticed also in association with LP AIV infection in concert with bacterial or viral co-infections [5]. Influenza virus infections of swine are characterized by an acute febrile respiratory disease of usually short duration that often is complicated by opportunistic bacterial infections and then may lead to reduction in daily weight gains of fattening pigs; also, fertility disorders such as fever-induced abortion in sows have been associated with SI virus infections (reviewed by [6]). Influenza virus infected poultry and swine are also of pivotal importance for public health as they may play a role as sources of parental influenza virus from which, by reassortment and adaptation processes, yield virus strains with zoonotic and even pandemic potential that by spill-over transmission, may spread to human populations [7–9].

∗ Corresponding author. Tel.: +49 38351 7 1200; fax: +49 38351 7 1275. E-mail address: martin.beer@fli.bund.de (M. Beer).

Education of stakeholders, strict biosecurity regimes combined with rapid diagnosis and surveillance programs and rigorous eradication measures (“stamping out”) comprise the recommended primary line of defense against AI in especially poultry industrial holdings [10–13]. Such regimes, however, require comprehensive investments in stable facilities and farm management, veterinary administration and laboratory capacities, and are out of scope for production units in developing countries, smallholders and backyard rearing communities [14–16]. Preventive vaccination against AI in poultry is generally considered as a secondary line of defense and its pro’s and con’s should be weighted with respect to the specific epidemiologic situation including, in particular, (i) extent and impact of AI related disease, (ii) incursions into areas with a high density of poultry populations, (iii) establishment of endemic infections, and (iv) increased risks of spill-over infections of zoonotic IAV to humans [17–21]. In case of outbreaks of notifiable AI, i.e., caused by subtype H5 or H7 viruses, further caution is recommended and the use of vaccination might require explicit legal permissions by the competent authorities [22]. Protection against clinical disease caused by SI and its economical impact are of primary interest in case of vaccination against SI; SI viruses, some of them with zoonotic potential, are endemic to the worlds swine population and there are no mandatory eradication programs (at least in Europe). Vaccination against AI in poultry and SI in pigs follows two primary intentions: (i) Successful vaccination reduces clinical signs of disease upon infection and, hence, prevents economic losses, (ii) vaccinated animals excrete significantly less virus following infection with field virus whereby reducing risks of further spread of

http://dx.doi.org/10.1016/j.vaccine.2015.03.052 0264-410X/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Rahn J, et al. Vaccines against influenza A viruses in poultry and swine: Status and future developments. Vaccine (2015), http://dx.doi.org/10.1016/j.vaccine.2015.03.052

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field virus. A reduction of the amount of circulating virus is of particular interest if outbreaks are caused by zoonotic influenza viruses that may cause spill-over infection in humans, e.g., in the course of the on-going hemiglobal epizootic of Asian origin H5N1 HPAI virus or the emerging H7N9 virus in China [18].

[13]. Reaching every individual animal by needle and syringe for prime-and-boost vaccination rounds becomes practically near impossible [13]. The available licensed vaccines and approved vaccine strategies provide no universal solution to these problems and intensive research, as reviewed here, is required to provide new solutions to vaccination against IAV infections in pigs and poultry.

1.1. Classical vaccines: inactivated influenza virus Historically, influenza vaccination is based on culture-grown IAVs. Usually, embryonated chicken eggs from specific pathogen free (SPF) flocks are used for vaccine virus propagation, mainly because of their unsurpassed yield of virus compared to cell culture systems. For use in pigs or poultry, culture-derived virions in crude allantoic fluids are chemically inactivated and then formulated into mineral oil emulsion vaccines whereby avoiding, in contrast to vaccine formulations for human use, sophisticated and costly purification steps for the enrichment of the immunodominant envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) [22]. Depending on the host species, primary and booster vaccinations by the subcutaneous or intramuscular routes are required to induce protective levels of systemic hemagglutinationinhibiting (HI) antibodies, and at least annual re-vaccinations are required to ensure that such antibody levels are maintained [23]. Success in prevention largely depends on a maximized antigenic match between the virus strain used for vaccine preparation and the virus(es) circulating in the field [13]. Epidemiological data supported by model calculations indicated that achieving and maintaining a broad and resilient herd immunity is another prerequisite for successful influenza vaccination in pigs and poultry [24]. Vaccine-induced immunity that imprecisely fits the antigenic make-up of the circulating viruses and patchy herd immunity are major drivers of antigenic drift and silent virus spread [25]. Viral antigenic drift by continuing accidental point mutations in the hemagglutinin gene and consecutive selection of mutants that escape vaccine-induced immunity will lead to the emergence of virus escape mutants that gradually replace older circulating virus strains (see e.g., [26,27] and references therein). In such case, spread of circulating field viruses under the cover of a vaccination campaign, i.e., “silent spread”, ensues: clinically the animals may still be protected to an extent that incursions of virulent virus into these flocks would easily be missed by syndrome surveillance [28]. This situation prompts an update of the vaccine to re-attain efficacy by using the escape mutant strain for production [29]. Escape mutants have repeatedly emerged at different geographic localizations (China, Indonesia, Vietnam, Egypt) in the course of the HPAI H5N1 epizootic [26,27] and there is evidence for antigenic drift also in SIV [2,30,31]. By the use of reverse genetics the antigenic makeup of vaccine virus strains is adjustable to circulating viruses within a relative short time period: recombinant viruses with the HA and NA antigenic determinants specifically replaced have been successfully applied against HPAI H5N1 [13]. Furthermore the use of inactivated influenza vaccines and serology techniques based on detection of antibodies directed against NS1 protein permits the establishment of the “differentiating infected from vaccinated animals” concept (DIVA) (see Section 2.8) and are therefore an option for eradication programs [32]. The ideal vaccine, in an infectological sense, should induce sterilizing immunity that blocks infection with circulating field viruses completely after a single immunization. Protection should be generated against changing field strains, i.e., a broader cross protection, at best even across different subtypes, is needed. Maternally derived immunity should be circumvented to enable protection as early as possible in life. High numbers of animals in industrial settings, sequestration of backyard poultry in difficult-to-reach locations and rapid turnover rates of swine and poultry populations are mounting further practical problems of vaccine delivery

2. Novel approaches in influenza virus vaccination in swine and chickens 2.1. Nucleic acid based vaccines Nucleic acid-based vaccines combine the advantages of (i) a molecularly defined antigen, (ii) the induction of both humoral and cellular immune response associated with MHC class I and class II molecules, (iii) a fast and cheap development without the need for embryonated eggs and even without handling the potentially hazardous pathogen when synthetic nucleic acids are used, as well as (iv) the reduced need of an adjuvant, as the nuclide acid in it itself represents a potent target for the immune system. 2.1.1. Messenger RNA-based vaccines The proof-of-principle of mRNA vaccines has first been described in the mouse model 20 years ago with an mRNA of the influenza virus nucleoprotein (NP) [33], and due to its simple and safe production and its tailored immunogenicity, this strategy has been further improved in the recent years. These vaccines typically comprise of a simple vector carrying the information for the antigen of interest, as well as 5 and 3 untranslated regions (UTR), the CAP structure, and the poly-A-tail for an efficient translation and the stability of the vector within the eukaryotic cell [34]. Optimizing these cis-acting structures is one goal of the recent improvements of the mRNA vectors [35–39]. The mRNA vaccines are produced by an enzymatic in vitro transcription from the tailored DNA template downstream of a suitable promoter, followed by purification steps. Thus, only sequence information of the nucleic acid of the antigen of interest is needed [34]. This process can be easily adapted to new antigens in emergency scenarios, allowing cost-effective manufacturing processes in classical clean-room settings. Recent improvements have been made regarding the enzymatic reactions [40–42], purification [43], delivering [37,44] and complexing with stabilizing proteins such as protamine [45]. In contrast to DNA, exogenous RNA only has to cross the cell membrane to facilitate protein expression in the cytoplasm and several cell types tend to take up mRNAs by receptor-mediated endocytosis [46–49]. The physical delivery, improved uptake, increased protein expression and induction of the immune system are also under development [45,50–53]. In the host, the RNA is recognized by pattern recognition receptors (PRRs) like toll-like receptors (TLRs), retinoic acid inducible gene I (RIG-I), or protein kinase R (PKR) [54–56], leading to the activation of CD8+ T cells (by delivering the antigen to the MHC-I processing pathway) and B cells directly or via the induction of interferon expression [49,57,58]. Dendritic cells (DCs) represent the best target as mRNA-mediated antigen expression in these cells or in secondary lymphoid tissue induces cellular immunity [36,59], but it was suggested that also other cell types, that take up the mRNA after intramuscular or intradermal application, are able to transfer it to DCs and other immune cells and induce the immunity via cross-priming [48,60–62]. In addition, antigen expression activates the signaling pathway for B cells [63]. As most of the studies are done in the mouse model, further studies are needed to adapt these principles to pigs and/or poultry species. Recently, Petsch et al. showed that vaccination with the mRNAs of HA, NA, and NP of H1N1, H3N2, and H5N1 viruses, which have previously been optimized regarding GC content and the UTR sequence composition,




induced immunological correlates of protection in pigs as well as in mice and ferrets, and protected them against clinical signs and reduced viral shedding in challenge experiments [37]. 2.1.2. DNA-based vaccines DNA-based vaccines are based on bacterial plasmids that are constructed to express an encoded protein, e.g. an antigen, and are used for the transfection of cells, resulting in the induction of the cellular and humoral immune response [64]. The first description of a DNA vaccine was made by Wolff et al. in the mouse model [60], and since then improvements of this technique have been made, especially regarding the stability, expression, delivery, and immunogenicity [65–71]. Numerous studies showed the induction of a strong cytotoxic T lymphocyte (CTL) response with DNA vaccines expressing HA, NP, or M1 in various animal models [72–76]. A strong antibody response mediated by DNA vaccination of pigs followed by viral challenge [77] was observed in the study of Larsen et al., in which pigs were immunized with a priming dose of a HA DNA vaccine followed by a boost immunization with a conventional, inactivated whole-virus vaccine, resulting in an enhanced immune response and protection from challenge infection [78]. A further promising approach was made by Gorres et al., who designed a DNA vaccine comprising of trivalent or monovalent HA-genes in a backbone with the cytomegalovirus enhancer/promoter and the human T-cell leukemia virus type 1 R region [79]. The trivalent vaccine induced both humoral and IFN-␥ responses, protected pigs against viral shedding and lung disease after H1N1 challenge, and reduced viral shedding after H3N2 challenge, regardless of the application route (i.m. or needle-free s.c.). Macklin et al. used DNA-coated gold particles to deliver a HA-based DNA vaccine to the epidermis or the tongue of pigs with a gene gun and thereby induced an immune response that confers protection against homologous challenge [80]. In recent studies, Lim and colleagues examined the immunogenicity of different plasmid DNAs encoding the HA, NA and NP genes from an AIV combined with chicken IL-15 and IL-18 [81,82]. Prime vaccinated and boostered chickens (both i.m.) developed high antibody titers against N1 and an increase in CD8+ T cells or CD4+ T cells for the IL-15 or IL-18 adjuvanted groups, respectively, indicating that IL-15 enhances the immunogenicity of the DNA vaccine in chickens and, in accordance with other studies, showing that the NP alone is less immunogenic, but nevertheless induces cellmediated immune response leading to the survival of challenged chicken [83,84]. Jiang et al. reported DNA vaccines based on the codon-optimized HA-genes of an H5N1 or an H7N1 virus that after i.m. injection induced high antibody titers and a solid protection of chickens against lethal H5N1 or H7N1 challenge and of quails against lethal H5N1 challenge, respectively [85–87]. A significant advantage of DNA vaccines is their ability to encode multiple genes of interest [88], but in contrast to mRNA vaccines, are poorly transported into target cells [77,89,90]. Therefore, different modes of application have been studied (see Section 2.7). Nevertheless, big concerns for the use of DNA vaccines are the danger of genomic integration into the host cell and the presence of selective markers, such as antibiotic resistance genes [91]. 2.2. RNA replicon particle-based vaccines RNA replicon vaccines are ((+)ss, (−)ss or ds) RNA viruses from which at least one essential structural protein has been deleted and that possess an engineered virus genome to express foreign antigens [92,93]. They undergo autonomous replication and transcription of the genome, thereby stimulating both humoral and cellular immune response, but are unable to produce infectious progeny. Replicon particles are mainly based on alphaviruses, such as the Venezuelan equine encephalitis virus (VEEV), that are

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produced in mammalian cell lines after transfection of a set of in vitro transcribed RNAs (replicon RNA with viral non-structural proteins and heterologous antigen plus helper RNA that encode the viral structural proteins) [94]. Others have used the vesicular stomatitis virus (VSV) that lacks the envelope glycoprotein G (VSVG) [95,96]. An alphavirus replicon RNA for the expression of the HA from swine influenza viruses or the pH1N1 has been developed containing the alphavirus ORF1 (with deleted non-structural proteins rendering the replicon replication-defective) and the heterologous genes in ORF2 [93,97,98]. The corresponding genes responsible for replication of the viral RNA have to be provided in trans together with the replicon (during electroporation). The HA protein was expressed in cells and after combination with an adjuvant induced high HA-specific antibody titers after vaccination of pigs, resulting in reduced lung lesions and viral shedding after homologous challenge. The tropism of the constructs for DCs promotes a fast immune response. Also an attenuated variant of the VEEV was used as a replicon vaccine expressing the HA-gene of a human H3N2 virus or the pH1N1 strain and induced high HI antibody titers in vaccinated pigs, and also protected them from homologous challenge infection [99,100]. The use of an alphavirus replicon system for the expression of the HA and NP genes of a pH1N1 and a H3N2 virus, respectively, eliminated viral shedding and reduced pulmonary damage in vaccinated and pH1N1-challenged pigs [101]. Avian species are naturally not infected by VEEV and VSV making these viruses perfect vectors for replicon-based vaccines [92]. In a study by Schultz-Cherry et al., immunization with a VEEV expressing the HA of an H5N1 virus protected chickens against a lethal challenge with the homologous virus to differing extends, depending on the age of the bird [102], whereas Sylte et al. used a similar approach for the expression of the NA of an H5N2 or H3N2 virus, that could only mediate a partial protection in vaccinated and challenged chickens [103]. Kalharo et al. used the VSVG approach to express the HA or NP of an HPAIV H7N1 strain, that was able to protect vaccinated chickens against a challenge with a heterologous H7N1 strain [104]. More recently, Halbherr et al. used a VSVG replicon expressing the HA of an H5N1 virus [105]. Chickens receiving one vaccination were completely protected from challenge and chickens boostered with the same vaccine were even protected from viral shedding. After establishment of the vaccine vector, the development of a new and multivalent vaccine is possible within a short time period. Replicon-based vaccines are safe, as they are not spread to the environment and cannot integrate into the host genome [92,100]. Furthermore, the host immunity of birds to the alphavirus is described to be reduced, allowing several replicon-based vaccinations of the same animal [88]. 2.3. Synthetic peptides, subunit vaccines, and virus-like particles 2.3.1. Synthetic peptides The potential of synthetic peptides as a vaccine candidate has recently been shown by Ma et al., who designed a tetra-branched multiple antigenic peptide based vaccine constructed by fusing four copies of M2e to one copy of a foreign T-helper cell epitope, which was able to induce a strong immune response and protected mice from otherwise lethal challenge [106]. Several studies reported synthetic peptide vaccines that, when administered to mice induced antibodies directed against the HA-stalk, which have a broad neutralizing activity due to the conserved sequence among different influenza virus subtypes [107–109]. Vergara-Alert et al. developed a broadly protective influenza vaccine based on a theoretical prediction of highly conserved peptide sequences from either H5 or H1 viruses [110]. After (i.m.) immunization with the synthetic peptides pigs developed humoral responses and the


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induced antibodies were able to recognize heterologous influenza viruses in vitro (pH1N1, SwH1N1, SwH3N2, H5N1 HPAIV). However, more research has to be done to evaluate the potential of these vaccines to elicit broader immune responses in pigs and poultry species than conventional vaccines.

against a lethal challenge with the heterologous strain [131]. Thus, these approaches may also be considered as vaccine candidates for swine and poultry [88].

2.3.2. Subunit vaccines A subunit vaccine is an immunizing agent that contains viral proteins, but no viral nucleic acid and that therefore induces a focused immune response [111]. It comprises of at least one recombinant Influenza virus protein, which in most cases is the HA, as it induces hemagglutination inhibiting antibodies [112,113]. Liu et al. used a baculovirus system for the expression of the HA of an H5N1 HPAIV and were able to induce a strong antibody response and fully protected chicken (and mice) from lethal challenge [114]. Crawford et al. designed baculoviruses, that express the HA of an H5 or an H7 virus [115]. These purified proteins completely protected (s.c.) vaccinated SPF-chickens against challenge with the homologous virus and abolished or reduced viral shedding. Wu et al. used a baculovirus pseudotyped with VSV glycoprotein as a vector to express the hemagglutinin (HA) protein of an H5N1 HPAIV that offered complete protection of chickens from homologous challenge [116]. Sylte et al. found that the recombinant baculovirus-expressed N2 induced high levels of NA inhibiting activity and protected 88% of the vaccinated chicken from challenge with a H5N2 virus with the homologous NA [103]. Kalthoff et al. developed a viral vector for the expression of a fulllength HA in plants, which was able to reduce viral shedding and to protect chickens from otherwise lethal challenge with a heterologous H5N1 virus [117]. In other studies, an ‘immuno-stimulating complex’ (ISCOM) obtained from n-octyl-beta-d-glucopyranosidetreated H5N2 viruses was used to induce high antibody titers and a cellular immune response in turkeys, and were able to protect them from both homologous and heterologous challenge infection as shown by reduced viral titers in the lung and trachea [118,119].

Vector vaccines represent modified live-attenuated viruses obtained with reverse genetics technology, that contain genomic material that can be expressed in the cell after entry via infection by the vector used, leading to endogenous antigen processing and MHC class I restricted presentation [88]. Thus, the vector has to have a wide host range and the host species should not have preexisting antibodies against the vector. Several vectors such as paramyxoviruses, herpesviruses, adenoviruses, or poxviruses have been studied for the use in poultry and swine. The Human adenovirus 5 with a deleted transcription region, rendering the vector replication-defective, was used to express the HA and NP genes of an H3N2 virus, and was shown to protect (i.m.) vaccinated pigs from viral shedding and the development of lung lesions after challenge with the homologous virus [136,137]. This vaccine candidate can only be used as a prime vaccination, as pigs develop antibodies against the adenovirus vector, and these antibodies could reduce the boost reaction [138]. The pseudorabies virus (PrV) represents a further potential vector, as several non-essential genes of the virus can be replaced by other genes, leading to a reduced virulence of the vector virus [139]. Such a virus was used to express the HA of an H3N2 strain and tested successfully in the mouse model [140]. Klingbeil et al. inserted the codon-optimized HA of a pH1N1 virus into the PrV genome and immunized pigs (i.n.) with the respective vector vaccine [141]. The pigs developed HA-specific antibodies, showed only minimal respiratory symptoms and less nasal shedding after challenge infection with a related pH1N1 virus. As the host develops antibodies against the vector, multiple vaccinations of the same animal are also not possible and may in addition hamper PrV surveillance [142]. For AI, other vaccine types have been licensed as well. In general, these were built on viral vectors, which expressed at least the HA as the major target of a protective humoral immune response. In particular, fowl pox virus recombinant vaccines expressing the hemagglutinin gene of AIV subtypes H5 or H7, and, more recently, further vector vaccines based on Newcastle Disease virus, herpesvirus of turkey or duck enteritis virus have been licensed for use in the field. However, it has been estimated that these vaccines account for less than 5% of vaccine doses used for immunization of poultry in the field in Central America (rFPV), Southeast Asia and Egypt (rNDV) [13,143]. In a recent study, Said et al. employed an equine herpesvirus (EHV-1) as a vector for the expression of the HA-gene of a pH1N1 virus and demonstrated that the vaccine induces influenza virusspecific antibody responses in pigs and was able to protect at least partially against challenge infection as evidenced by decreased nasal virus shedding and faster virus clearance [144]. Furthermore, a Modified Vaccinia Ankara (MVA) virus was used to express the HA and NP genes of a classical H1N1 Swine influenza strain and vaccination of pigs (i.n. or i.m.) resulted in shorter duration and lower titers of viral shedding after homologous challenge as well as in the prevention of the lung lesion development [145]. As an addition poxvirus vector, the highly attenuated vaccinia virus NYVAC strain [146] was used for the expression of a H5N1 HPAIV HA to induce potent neutralizing antibody responses in swine, which were protected against viral replication after challenge infection with a low-pathogenic H5N2 strain [147]. In several studies, recombinant Newcastle Disease viruses (rNDV) were tested as a vaccine vector for HPAIV-challenge infection [148–156]. Swayne et al. described an NDV with an insertion of the HA-gene from an H7N2 strain that partially protected chicken

2.3.3. Virus-like particles The expression and self-assembling of viral structural proteins into virus-like particles (VLPs) represents another promising type of vaccine [120]. They resemble infectious virus particles in structure and morphology, induces a sufficient immune response but do not contain any viral genetic material and are therefore not infectious. Several systems for the production of influenza VLPs have been described, including baculovirus [121–125], transient plasmid expression [126–128], stable cell-line transformation [129] and expression in plants [130], but mostly mice or ferrets were used for vaccine studies. Chang et al. generated a baculovirus-based VLP which incorporated the H7 HA alone and showed that it was immunogenic in chickens [131], however, its protective efficacy has not been tested. Using also a baculovirus expression system, Lee et al. developed VLPs with HA and M1 from a H9N2 virus [132]. Chickens immunized with the adjuvanted vaccine (i.m.) elicited high levels of antibody responses and lessened viral shedding. In similar approaches, other groups generated VLPs comprising HA, NA and/or M1 from a H5N1 HPAIV and showed, that immunized chickens were protected against a lethal homologous challenge, showing no clinical signs of infection [133,134]. Pyo et al. generated VLPs consisting of HA, NA and M1 from pandemic H1N1/2009 virus that elicited robust levels of humoral and mucosal immune responses in immunized pigs which after challenge infection displayed reduced lung lesions and inhibition of virus replication in the lung [135]. A plant-based transient expression of the HA proteins of an H5N1 and an H1N1 virus revealed that HA was predominantly assembled into high-molecular-weight structures, triggered a strong immune response against the homologous virus in immunized mice and conferred complete protection

2.4. Vectored vaccines




from homologous HPAIV challenge [153]. Veits et al. inserted the HA-gene from an H5N2 HPAIV and demonstrated that immunization of chickens with that recombinant vector induced NDV- and AIV H5-specific antibodies and protected chickens against clinical disease and shedding of AIV after challenge infection with a lethal dose of velogenic NDV or highly pathogenic AIV, respectively [157]. Nayak et al. generated recombinant rNDVs expressing the HA, NA, or M2 protein of an H5N1 HPAIV, and showed that the immunization of chickens with HA alone or in combination with NA induced a complete protection against HPAIV infection [155]. In the study of Lardois et al., it could be shown that an NDV expressing the HA of an H5N1 HPAIV induced an H5 serologic response and afforded a high degree of clinical protection against challenge infection with a related H5N1 virus, independent of the inoculation route (oculonasal versus drinking water), but dose-dependent [156]. In order to overcome pre-existing NDV antibodies within the host, Steglich et al., constructed a NDV vector which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of the avian paramyxovirus type 8 instead of the corresponding NDV proteins, and inserted a gene expressing the HA of an H5 HPAIV between the F and HN genes [158]. This chimeric virus induced full protection against lethal HPAIV-infection in chickens without as well as with maternally derived NDV-specific antibodies. Today, rNDVs are approved as bivalent vaccines in poultry against NDV and AIV, and are widely used in China [159,160]. The Gallid Herpesvirus (infectious laryngotracheitis virus (ILTV)) was used as a vector for the insertion of the HA-gene of an H5 or H7 virus at the non-essential UL50 gene locus [157,161]. Immunized chickens produced specific antibodies against ILTV and AIV HA, and were protected against challenge infections with either virulent ILTV, or two different highly pathogenic AIV strains, but developed minimal clinical signs. Therefore, Pavlova et al. designed an attenuated ILTV variant for the insertion of the H5 (or N1) gene, which proved to offer protection of chickens, when vaccinated with the H5-expressing vector or the combination of the H5 and N1 expressing ILTVs, without causing clinical signs [162]. A genetically engineered Marek’s Disease vaccine of serotype 3 (turkey Herpesvirus, HVT) expressing the H5-protein is commercialized in Egypt and a recent study in layer chicken breed flocks attested a considerable protection after a one shot immunization strategy in day-old-chicks [163]. Toro et al. have shown that a non-replicating human adenovirus vector encoding the HA of an H5N9 avian influenza virus induced a protective immunity against avian influenza virus in chickens by a single-dose in ovo vaccination [164]. In another study, Gao et al. demonstrated that an adenovirus-based vaccine expressing different portions of the HA of a H5N1 virus completely protected chickens after a single subcutaneous immunization from homologous challenge [165]. Wu et al. designed a pseudotyped baculovirus, carrying the G protein of VSV and the HA-gene of a H5N1 HPAIV [116]. Immunized chickens (and mice) elicited strong serum antibody responses and protected from H5N1 challenge. Finally, also the fowlpox virus provides a suitable candidate for the development of a species-specific recombinant viral vector as it has a natural host range limited to avian species. Already in the late 80s, Taylor et al. reported a recombinant fowlpox virus expressing the HA from a HPAIV, that provided protection of immunized chickens and turkeys against a lethal challenge with either the homologous or a heterologous influenza virus strain [166]. Since then, several recombinant fowlpox viruses expressing the HA of H5 viruses have been described [167–173] and successfully used in an HPAIV outbreak [174]. Currently, at least ten poxvirus-vectored vaccines have been licensed for veterinary use [175], including an MVA virus expressing the HA which is licensed as a vectored influenza vaccine in poultry and equines [147].

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2.5. Live attenuated influenza vaccines In 2003 a live attenuated influenza vaccine (LAIV) was approved for humans by the US Food and Drug Administration (FDA). While since 2012 a quadrivalent LAIV is licensed by the FDA for immunization of humans, such vaccines did yet not enter the market of swine/poultry production. Several proof-of-principle-studies demonstrated extraordinary protection induced by LAIV in swine and poultry ([176–187]. Attenuation of the vaccine virus may be achieved by truncation of the Non-structural protein (NS1), which in turn attenuates the virus´ı ability to antagonize the effects of the host’s type I interferon antiviral response [176,181,182,184,188]) by genetic mutations within the HA leading to an altered protease dependency and impaired replication [177–180] or by engineering mutations into the polymerase genes, impairing polymerase activity and restricting virus replication at elevated temperatures [185]. Masic et al. generated an eight-segment SIV harboring two different SIV HA (H1 and H3) by fusing the H3 HA ectodomain to the cytoplasmic tail, transmembrane domain and stalk region of the NA [189]. The resulting LAIV was attenuated in pigs, and even after intranasal application conferred reduction of fever and other clinical signs as well as the development of gross lesions in the lungs after challenge infection with both H1 and H3 SIV [186]. The use of LAIV in poultry remains prohibited to date and their use is not recommended by relevant international organizations such as OIE and FAO since adaptation to gallinaceous birds and/or reassortment events may potentially lead to the generation of HPAI viruses. However, Röhrs and colleagues demonstrated exceptional protective propensity of a live neuraminidase deleted H5-virus, which induced full protection in chickens seven days after a single shot immunization [187]. The most efficient and beneficial applications for LAIV may be toward emerging vaccination campaigns rather than for routine mass vaccination. In addition Hai and colleagues attempted to generate a reassortment-incompetent virus by manipulating an influenza B virus to express different HA proteins originating from influenza A virus strains. Although the authors did not perform a vaccine/challenge experiment, they demonstrated seroconversion of chicken after intravenous inoculation [190]. 2.6. “Universal” vaccines The optimal universal vaccine should cover all relevant subtypes of influenza A viruses, however, it is more realistic to develop a vaccine that is more broadly cross-protective than the currently licensed vaccines, and that induces a longer lasting, sterile immunity. Most neutralizing antibodies are directed against the globular head of the HA and are, therefore, in most cases very strain specific. Additionally, both HA and NA glycoproteins are highly variable and are drifting under immune pressure [191]. Regions of the HA that carry out functions essential for infection and replication present sites of vulnerability for recognition by more cross-reactive and broadly neutralizing antibodies [192]. Thus, the receptor binding site on the HA1-subunit and the fusion machinery of the HA2subunit are prime targets for antibody intervention. Several groups described (human or murine) antibodies directed against the HA-stem that neutralize several group 1 viruses (H2, H5, H6, H9) and/or some group 2 viruses (H3, H7, H10), respectively, and proofed their therapeutic potential in mice [193–197]. Other groups identified antibodies, that neutralize both group 1 and group 2 viruses, and that are directed against a conserved epitope near the receptor binding site [198–200]. However, recent publications from swine experiments verified the contribution of non-neutralizing, but cross-reactive antibodies against the HA2 domain to induction of vaccine-associated enhanced




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respiratory disease after infection with a heterologous virus [201,202]. While this phenomenon is also a problem of whole virus inactivated vaccines (primarily used today within the swine industry) it impacts the idea of a universal vaccine negatively. Antibodies raised against regions with high amino acid conservation within the NA-protein were shown to be cross-reactive by Western Blot to representative NAs from nine subtypes [203], yet the in vivo efficacy has not been investigated. Unfortunately, literature lacks studies performed in more relevant host species than mice. In contrast to the HA, the internal viral proteins including NP, PA, M1 and M2 are highly conserved across the subtypes and induce cellular immune response, mainly CTL response against processed peptides [204], thus, represent promising targets for a universal vaccine. Boyd et al. developed recombinant MVA and Adenoviruses expressing a fusion construct of NP and M1 and, as a proof of concept, prime-boost vaccinated chickens showed an increased T-cell response, and cloacal shedding was at least reduced after challenge infection with a low pathogenic H7N7 virus [205]. As the extracellular domain of the M2-protein (M2e) is conserved among the influenza subtypes [206], this domain is thought to mediate heterosubtypic immunity, although the peptide itself is relatively low immunogenic [207]. Several studies analyzed different approaches using the M2e-epitope in mice, revealing its potential [125,207–210]. Kim et al. produced VLPs expressing tandem repeats of M2e peptides containing two human, two avian and one swine origin M2e peptide sequence fused with HA transmembrane and cytoplasmic domains [211,212]. Sera from immunized mice reacted with a range of Influenza A viruses, including H1N1, H3N2, and H5N1 viruses. Heinen et al. developed a DNA construct by the fusion of M2e to the Hepatitis B core protein or the influenza NP and vaccinated pigs with the resulting DNA vaccines [213]. Unfortunately, although vaccinated pigs developed antibodies against M2e, and even virus-specific lymphoproliferation in case of the M2e-NP vaccine, clinical signs and mortality were exacerbated after challenge infection, indicating that further research is needed in terms of using M2e as immunogenic protein. 2.7. Novel application approaches Needle-mediated injections (i.m., s.c., i.d.) are the most common application techniques, as upon pathogen contact these tissues rapidly deploy innate defenses to contain the infection and engender protective adaptive immunity [214,215]. Needlefree/non-invasive application is associated with less pain, an easier application, improved vaccine acceptance, reduced costs, reduced safety risks, and no need for trained personnel [216]. Intranasal administration of IAV vaccines has been attempted as an alternative method to protect pigs and induce local immune responses [217]. Furthermore, for DNA-based vaccines the delivery via “gene gun” to the skin or the tongue has been shown to enhance the transfection efficiency and thereby the induction of antibodies after immunization [77,78,218,219]. Chen et al. have shown that mice immunized via electroporation (i.m. administration followed by electrical stimulation) with a DNA-plasmid containing a consensus sequence of the HA of an H5N1 virus – optimized for an improved protein expression – elicited neutralizing antibodies against viruses from different H5N1 clades, and were completely or significantly protected against challenge viruses [220]. Also linear DNA-cassettes have been successfully tested in mice [221]. Finally, by the use of a conventional whole virus inactivated influenza vaccine (without adjuvant) applied as a dry powder aerosol vaccine at the syrinx of chickens, Peeters and co-workers obtained significantly reduced replication of the challenge virus [222]. The authors also tested vaccination by passive inhalation as

this method might be suitable for mass application, however by this technique only partial protection could be achieved [222]. 2.8. DIVA vaccines The acronym DIVA stands for ‘differentiating infected from vaccinated animals’. In order to fulfill the criteria of the DIVA-principle, a marker vaccine and its corresponding companion serological assay have to be available. While international organizations like OIE recommend DIVA vaccines for pigs against pseudorabies and classical swine fever [223], swine influenza vaccines are applied without any DIVA strategy until now. However, vaccines from any of the former presented types are described to fit in principal to the DIVA concept: A) For inactivated whole virus vaccine: several DIVA-vaccines based on a heterologous NA-concept were developed for avian influenza (reviewed in [32]). The ultimate heterologous NArecombinant virus was generated by Peeters et al. containing the (modified low-pathogenic) HA-gene of a contemporary H5N1 HPAIV strain in combination with the NA gene of a human type B influenza virus (which only occur in humans and not in birds), that, as inactivated vaccine, protected chickens against clinical disease and completely prevented virus shedding after H5N1 HPAIV challenge [224]. As inactivated virus preparations do not induce antibodies against non-structural proteins such vaccines and the sensitive detection of NS1 antibodies do meet the criteria of the DIVA principle. The advantage versus the heterologous NA-vaccine is that the NS1 DIVA is working irrespective of the antigenic subtype of vaccine strain and circulating virus strain. Although the general suitability was demonstrated for several avian species, vaccinated and infected animals maybe missed due to reduced seroconversion rates (reviewed in [32]). B) For nucleic acid-based vaccines, RNA replicon particle-based vaccines, synthetic peptides, subunit vaccines, virus-like particles and vector vaccines induce an antibody response only against the influenza proteins that are specifically encoded by the relevant vaccine, which is in most cases the HA alone or in combination with the NA protein. Therefore, sera from vaccinated animals consist of antibodies against the HA (and the NA), but not against the internal proteins like the nucleoprotein NP. NPELISAs can therefore be used as a possible accompanying marker assay. C) There are live attenuated influenza vaccines that meet the criteria of the DIVA concept. The NA-deleted H5-vaccine virus from Röhrs and co-workers [187], and the chimeric influenza B and influenza A construct from Hai and colleagues [190] both induce an antibody response, which is distinct from wildtype infection induced antibodies and NA-specific ELISA systems can be used as marker assays. Finally, these universal vaccine approaches that work with the expression of the M2-protein are likewise appropriate DIVA vaccines. However, while serological reactions against the M2 protein were demonstrated in general, more work is needed to show their suitability [32]. 3. Conclusions In conclusion, a whole range of novel influenza vaccine approaches is available for both poultry and swine. In order to overcome the obstacles associated with the conventional vaccines, several different inactivated and attenuated vaccine types are in the focus of influenza vaccine research. An overview of the most promising examples is given in Table 1.




7

Table 1 Overview of vaccine types and their features. Vaccine type

Species

Main application routes

DIVA

Universal

Selected literature references

Inactivated homologous whole virus vaccines Inactivated heterologous whole virus vaccines Live attenuated vaccines mRNA vaccines DNA vaccines Replicon vaccines Synthetic peptides Subunit vaccines Virus-like particles Vectored vaccines

Swine, chicken Chicken Chicken, swine Swine Swine, chicken Swine, chicken Swine Chicken, swine Chicken, swine Swine, chicken

i.m.a i.m. i.m., i.n.b i.m., i.d.c i.m., i.d., s.c.d , n.f.e i.m, i.n., s.c. i.m., s.c. i.m., s.c. i.m., s.c. i.m., s.c., n.f., i.n., o.n.f

− + − + + + + + + +

− − − − − − + − + +

[172] [227] [180,187,190] [34] [64,228] [92,93,98] [106,110] [114,116] [125,131] [229]

a b c d e f

Intramuscularly. Intranasally. Intradermally. Subcutaneously. Needle free. Oronasally.

Modified live vaccines seem to be the most efficient vaccines with a very early onset of immunity and the potential of mucosal immunization, and cold-adapted MLV (achieved by inducing combined mutations in PB1 and PB2 genes) are registered for humans and horses [225,226]; but there are still safety issues including the risk of reassortment and reversion to virulence [179]. In addition, not all approaches allow a DIVA-strategy, and licensing of genetically modified organisms could be an additional hurdle. In contrast, the very safe subunit preparations enable different DIVA concepts, but still need two applications, have to be injected, and reports are mainly available from the immunization of chickens and turkeys. Therefore, vector-based vaccines seem to be an alternative combining the subunit idea with a replicating viral vector. However, vector-specific immunity is a major problem for the continuous use of the same vector and is difficult to overcome. Beside the classic approaches, promising results were also obtained from studies with mRNA-based vaccines, inducing a good protection level in several mammalian species including pigs, and such vaccines should therefore also be tested in poultry. Furthermore, these vaccines are considered to be safe, since, in contrast to DNA-based vaccines, integration into the host genome is not possible. Nevertheless, those vaccine types have to be injected and a needle-free application should be considered in future studies. One of the major future goals in influenza vaccine research is the development of a “universal” vaccine, providing protection against a broad range of influenza subtypes. Therefore, further research targeting the relevant livestock species is required. Finally, vaccine application by injection of individual animals is nowadays still obligatory for nearly all of the registered and most of the novel vaccine types. However, mass application is only feasible e.g. by mucosal immunization especially in industrial settings. Hence, research has to focus on mucosal/needle-free immunization of poultry and pigs also. These obstacles pose significant challenges of vaccine development for avian/swine influenza. The ongoing influenza A endemic/epidemic or even pandemic situations clearly indicate that further efforts are needed to control Influenza A virus infections in livestock animals and, to the authors´ı opinion, vaccines are a very promising tool. In summary, promising novel approaches have been reported in recent years, and there continues to be progress in the development of novel influenza virus vaccines for livestock animals. However, the “ideal influenza vaccine” for poultry and pigs is still missing. A safe and efficacious vaccine with a very broad reactivity against several subtypes, with DIVA-features and which allows mass application e.g. by oral immunization, would be the perfect tool in the future, and further efforts are necessary to reach this goal.

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