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Recombinant fowlpox virus vaccines against Australian virulent Marek's disease virus. Gene sequence analysis and comparison of vaccine efficacy in specific pathogen free and production chickens
Abstract
We have cloned and sequenced the glycoprotein genes gB, gC and gD of the Australian virulent Marek's disease virus (MDV) isolate Woodlands No. 1. The glycoprotein gB and gC sequences were identical to the homologs of other virulent MDV type 1 strains, and the glycoprotein gD sequence contained only one unique amino acid substitution. Recombinant fowlpox viruses (rFPVs) expressing the MDV glycoprotein genes were constructed and their efficacy as vaccines was evaluated in specific pathogen free (SPF) and production chickens. Vaccination with the FPV-gB recombinant protected SPF chickens from Marek's disease mortality and tumour formation following challenge with virulent MDV Woodlands No. 1. The degree of protection from Marek's disease was dependent on the vaccine dose and route of inoculation. The rFPVs expressing gC or gD did not provide protection from Marek's disease. A rFPV expressing both gB and gC did not provide enhanced protection in comparison with the rFPV-gB alone. The rFPV-gB vaccine failed to protect commercial chickens from MD mortality and provided little protection from tumour formation in comparison with the commercial herpesvirus of turkey (HVT) vaccine. The failure to provide protection against MD may be related to the impact of maternally derived immunity to MDV and FPV and possibly the genotype of the chickens.
Supplementary resources (3)
Nucleotide Sequence
June 1996
Nucleotide Sequence
June 1996
Nucleotide Sequence
November 1995
... In these studies, DNA sequences encoding influenza A haemagglutinin were inserted with the aid of "shuttle vectors" into the non-essential thymidine kinase gene of the mild vaccine strain of FPV (FPV-M3) (Boyle and Coupar 1986;Boyle, Coupar et al. 1987;Boyle and Coupar 1988). Chickens vaccinated with this rFPV developed peak and Coupar 1988;Moss 1991;Boyle 1992;Boyle and Heine 1993;Heine and Boyle 1993;Boyle and Heine 1994;Heine, Hyatt et al. 1994;Boyle, Pye et al. 1997;Heine, Foord et al. 1997;Hertig, Coupar et al. 1997;Boyle, Anderson et al. 2004). under the transcriptional control of early FPV promoters (Fenner 1979;Boyle and Coupar 1986;Boyle, Coupar et al. 1987;Boyle, Anderson et al. 2004;Coupar, Purcell et al. 2005). ...
... These responses are presumably due to the intracellular expression of viral antigens possibly in local APC and presentation to T cells in the context of MHC I and/or MHC II proteins. rFPV have been used for vaccine antigen delivery in a number of studies Heine, Hyatt et al. 1994;Heine, Foord et al. 1997;Hertig, Coupar et al. 1997;Kent, Zhao et al. 1998;Dale, Zhao et al. 2000;Kent, Zhao et al. 2000;Boyle, Anderson et al. 2004;Dale, De Rose et al. 2004). Importantly, different rFPV vaccines, expressing vaccine antigens from diverse infectious agents such as HIV and malaria, have now been produced and approved for use in humans (Moorthy, Imoukhuede et al. 2004;Hutchings, Gilbert et al. 2005;Coupar, Purcell et al. 2006;Dale, Thomson et al. 2006). ...
... This finding suggested that inclusion of novel vaccines designed to induce both enhanced humoral and cellular immune responses with ETV could increase the proportion of ducks that are able to resolve their DHBV infection. It had long been known that recombinant pox viruses were effective delivery vehicles for vaccine antigens (Roizman and Jenkins 1985;Boyle and Coupar 1986;Boyle and Coupar 1988;Moss 1991;Lancet 1992;Boyle and Heine 1993;Heine and Boyle 1993;Paoletti 1996;Heine, Foord et al. 1997) and more recently it was shown in mice that if a DNA vaccine priming immunisation was administered shortly prior to rVV immunisation the magnitude of CD4 + and CD8 + CTL responses increased above those animals that received either the DNA vaccine or rVV alone (Ramsay, Kent et al. 1999). Further to this finding was the encouraging result where a chronically infected chimpanzee resolved chronic HBV infection shortly after being prime-boosted with DNA vaccines and recombinant canarypox vaccines expressing HBsAg (Pancholi, Lee et al. 2001). ...
... The adverse effect of maternal antibodies on the efficacy of rFPV-gB 1 has also been reported by Heine et al. (1997). However, in their study, the vaccinated chickens had maternal antibodies against FPV as well as against MDV. ...
... Fowlpox virus recombinants expressing a number of structural and non-structural MDV1 antigens have also been constructed but have failed to protect (Heine et al, 1997;Nazerian et al, 1996). These include gC, gD, UL47 (modulator of UL48), UL48 (trans-inducing factor VP16) and pp38 which is known to induce an MHC class 1restricted cell-mediated immune (CMI) response in chickens inoculated with MDV1 or SB1 (Omar & Schat 1997). ...
... In contrast to the situation with HSV and pseudorabies virus, gD or gC of MDV1 were not protective. Moreover, vaccination with a mixture of FPV recombinants expressing gC, gB and gD of MDV1 did not improve protection compared with gBl alone (Heine et al, 1997). Before it can be concluded that these antigens have no role in protective immunity, it will be necessary to determine whether the recombinants are capable of inducing an immune response at least against FPV. ...
Novel approaches to vaccination against very virulent (vv) strains of Marek's disease virus (MDV) are discussed. Fowlpox virus (FPV) and herpesvirus of turkeys (HVT) recombinants expressing MDV antigens have been constructed. It has been shown that glycoprotein B of MDV serotype 1 (gB1) is an effective immunogen which is particularly important for conferring protective immunity in genetically susceptible chickens. However, maternal antibodies against MDV diminished the efficacy of vaccination with recombinant FPV‐gB1. HVT recombinants expressing antigens of MDV and Newcastle disease virus have been constructed and have been shown to be effective in preventing Marek's disease as well as systemic Newcastle disease. Maternal antibodies against MDV and Newcastle disease virus did not affect significantly the efficacy of vaccination with cell‐associated HVT recombinants. The potential of retroviral insertion mutagenesis and other means of delivering MDV antigens for immunization are discussed.
... on Taxonomy of Viruses [1]. As FPV replication is limited to avian species [2], FPV is used as a vaccine vector to prevent various diseases in poultry [3][4][5][6][7] and has been exploited as a safe non-replicating vector for vaccination in mammals [8][9][10]. Future vaccine strategies will likely require simultaneous expression of different antigens or the expression of antigens with immune co-stimulatory molecules [11][12][13][14]. ...
... FPV has been used as a live vector to construct recombinant vaccines for both poultry and mammals [3][4][5][6][7][8][9]. The FPV genome encodes approximately 260 ORFs, which contain many non-essential genes. ...
Fowlpox virus (FPV) is used as a vaccine vector to prevent diseases in poultry and mammals. The insertion site is considered as one of the main factors influencing foreign gene expression. Therefore, the identification of insertion sites that can stably and efficiently express foreign genes is crucial for the construction of recombinant vaccines. In this study, we found that the insertion of foreign genes into ORF054 and the ORF161/ORF162 intergenic region of the FPV genome did not affect replication, and that the foreign genes inserted into the intergenic region were more efficiently expressed than when they were inserted into a gene. Based on these results, the recombinant virus rFPVNX10-NDV F–E was constructed and immune protection against virulent FPV and Newcastle disease virus (NDV) was evaluated. Tests for anti-FPV antibodies in the vaccinated chickens were positive within 14 days post-vaccination. After challenge with FPV102, no clinical signs of FP were observed in vaccinated chickens, as compared to that in the control group (unvaccinated), which showed 100% morbidity. Low levels of NDV-specific neutralizing antibodies were detected in vaccinated chickens before challenge. After challenge with NDV ck/CH/LHLJ/01/06, all control chickens died within 4 days post-challenge, whereas 5/15 vaccinated chickens died between 4 and 12 days post-challenge. Vaccination provided an immune protection rate of 66.7%, whereas the control group showed 100% mortality. These results indicate that the ORF161/ORF162 intergenic region of FPVNX10 can be used as a recombination site for foreign gene expression in vivo and in vitro.
... The sequence of the endocytosis signal in gD shows some conservation among alphaherpesviruses, albeit to a lesser extent than the endocytosis signal in gB. The gD orthologues of bovine herpesvirus 1, equine herpesvirus 1, and Marek's disease virus contain a YXXL motif (2,18,38) similar to that of the PRV gD sequence, but HSV type 1 (HSV-1) gD does not seem to contain a potential endocytosis motif (46). Future research will show whether these YXXL motifs in other gD orthologues also constitute functional endocytosis motifs. ...
... This supports the idea that HIV employs this endocytosis motif to evade the immune system and, importantly, this shows that mutation of these motifs may be a useful strategy to improve immunogenicity of vaccines. Therefore, our current finding that PRV gD encodes a functional endocytosis motif that, together with an endocytosis motif in gB, may reduce antibody-mediated elimination of the virus, may also be of significant importance for the construction of DNA vaccines encoding either gB or gD, which are generally considered to be potentially important vaccination strategies for many alphaherpesviruses, including PRV, bovine herpesvirus 1, equine herpesvirus 1, Marek's disease virus, and HSV (8,15,17,18,30,35,44,49). ...
Viral glycoproteins gB and gD of the swine alphaherpesvirus pseudorabies virus (PRV), which is closely related to human herpes
simplex virus and varicella-zoster virus, are able to drive internalization of antibody-antigen complexes that may form at
the cell surface of infected monocytes, thereby protecting these cells from efficient antibody-mediated lysis. We found earlier
that gB relies on an endocytosis motif in its cytoplasmic domain for its function during this internalization process. Here,
we report that the PRV gD protein also contains a functional endocytosis motif (YRLL) in its cytoplasmic domain that drives
spontaneous endocytosis of gD from the cell surface early in infection and that acts in concert with the endocytosis motif
in gB to contribute to efficient internalization of antibody-antigen complexes in PRV-infected monocytes.
... Recombinant vaccine is prepared by using HVT [68], Fowlpox [69,70] and Newcastle disease viruses as vectors [71][72][73][74]. ...
... Many factors affect genetic resistance to MD and vaccine protective efficacy. Some of these have been well investigated and reported [23][24][25][26][54][55][56]. We recently showed non-MHC genetic variation, in addition to MHC B haplotype that was primarily reported by Bacon and Witter in the 1990s [23][24][25][26]57], also plays an important role in modulating MD vaccine efficacy [31][32][33]. ...
... Most VE studies are conducted in SPF White Leghorn chickens which tend to have stronger immune response than field birds [7,30,38]. Group 2, included studies comparable to those in Group 1 but conducted in commercial chickens. Again, LPAI formulations neither had efficacy preventing respiratory viral shedding 2dpc nor preventing death 14dpc with a fourth order clade virus. ...
Indonesia has implemented multiple strategies to control Highly Pathogenic Avian Influenza H5N1 (HPAI/H5N1), including the licensure and use of multiple vaccine formulations. The continuous drift of Indonesian HPAI/H5N1 viruses and emergence of a new clade in 2012 that became dominant in 2016, demands the assessment of commercial vaccine formulations against Indonesian field viruses.
... Targets for vaccination included: highly pathogenic avian influenza (HPAI) virus, Newcastle disease virus [5,6,9,44,[56][57][58][59][60][61][62][63][64], infectious bronchitis virus, avian haemorrhagic enteritis virus [65,66], Marek's disease virus [67][68][69][70][71], turkey rhinotracheitis virus [48], infectious laryngotracheitis virus [32,62,72], REV [73], infectious bursal disease virus [28,[74][75][76] and duck hepatitis virus [77], as well as Mycoplasma gallisepticum [78] and eimeria species [39]. ...
Fowlpox virus (FWPV) is widespread, causing disease of mature poultry with significant social or economic impact and some mortality, which is higher in the diphtheritic form of the disease. Vaccines against the disease were some of the earliest developed for livestock. They are live vaccines based on attenuated isolates or passaged derivatives of FWPV, or the serologically closely related pigeonpox virus. Relying heavily on approaches developed for vaccinia virus, FWPV was developed as a live recombinant vaccine vector for use in poultry. Licensed, commercial, recombinant FWPV vaccines are available against avian influenza, Newcastle disease and infectious laryngotracheitis. Causing disease only in birds, FWPV is nevertheless able to infect mammalian cells, express proteins and induce immune responses, both humoral and cellular, which may in some circumstances be protective (a feature shared with another member of the genus, canarypox virus). Blocked during morphogenesis (or before) in mammalian cells, it has an extremely high safety profile as a live recombinant vaccine vector for use in humans and other mammals, as demonstrated in numerous clinical trials. Relatively low potency in mammals has been addressed by using prime-boost regimes and by co-expression of host cytokines (an approach also trialled in poultry) or co-stimulators. New generation vectors may involve deletion of FWPV immunomodulators to improve efficacy.
... This vaccination scheme was never tested before in ducks and looks promising. Field trials in 1-day-old ducklings will be essential to confirm induction of an earlier onset of immunity and of a broader cross-reactivity to various antigens, and to evaluate the duration of protection induced by such prime-boost vaccination scheme compared to two shots of inactivated vaccines in field conditions [36,59]. ...
The efficacy of different vaccination schedules was evaluated in 17-day-old Pekin ducks using an experimental inactivated whole virus vaccine based on the H5N9 A/chicken/Italy/22A/98 isolate (H5N9-It) and/or a fowlpox recombinant (vFP-H5) expressing a synthetic HA gene from an Asian H5N1 isolate (A/chicken/Indonesia/7/2003). Full protection against clinical signs and shedding was induced by the different vaccination schemes. However, the broadest antibody response and the lowest antibody increase after challenge were observed in the group of ducks whose immune system was primed with the fowlpox vectored vaccine and boosted with the inactivated vaccine, suggesting that this prime-boost strategy induced optimal immunity against H5N1 and minimal viral replication after challenge in ducks. In addition, this prime-boost vaccination scheme was shown to be immunogenic in 1-day-old ducklings.
... There have been a number of published trials reporting on vaccines that provide protective immunity against HPAI. However, many of those trials have been conducted in SPF poultry and past experience has shown that vaccine immunogenicity , and therefore efficacy, is often lower in conventional animals than in SPF animals (Heine et al., 1997). While SPF animals are valuable in eliminating variables during early studies, it is important to test vaccines intended for eventual field use, in animals with similar genetics and rearing conditions. ...
The current Asian H5N1 highly pathogenic avian influenza virus has spread over much of Asia and into Europe and Africa. As well as affecting village and commercial chicken operations in many South East Asian countries, it differs from past H5 avian influenza viruses in that it causes morbidity and mortalities in other domesticated birds, such as ducks and turkeys and in wild water birds. Effective vaccines that can prevent infection, as well as disease, and be used in a variety of avian species are needed for field use. In this report, a bivalent H5N9+H7N1 oil emulsion vaccine is compared, in ducks, to a monovalent H5N3 oil emulsion vaccine that has been derived by reverse genetics with an H5 from A/chicken/Vietnam/C58/04. While both vaccines protected against morbidity, the monovalent vaccine provided effective protection, with no evidence of shedding of the challenge virus and no serological response to the H5N1 challenge virus.
... Fowlpox virus (FPV) is the type member of the Avipoxvirus genus that causes disease in chickens (Gallus domesticus). "Shuttle vectors" enabling insertion of foreign genes into the non-essential thymidine kinase gene of the vaccine strain of FPV (FPV-M3) have previously been constructed (Boyle et al., 2004;Boyle and Coupar, 1988;Coupar et al., 2006) and used to derive recombinant FPV-M3 (rFPV) vaccine strains that induce vaccine specific immune responses in mice (Ramsay et al., 1999), chickens Heine and Boyle, 1993;Heine et al., 1997), macaques (Dale et al., 2004Kent et al., 2000) and humans (Coupar et al., 2006;Dale et al., 2006;De Rose et al., 2007, 2006Kelleher et al., 2006). Furthermore, "priming" with a DNA vaccine followed shortly afterwards with a rFPV "boost" was shown to enhance both CD4+ and CD8+ immune responses when compared to a dual rFPV "prime" and "boost" approach (Dale et al., 2004De Rose et al., 2006;Kent et al., 2005;Ramsay et al., 1999). ...
Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.
Low pathogenic avian influenza virus (LPAIV) usually causes mild disease or asymptomatic infection in poultry. LPAIV has, however, become a great threat to poultry industry due to mixed infections with other pathogens. Coinfections do frequently occur in the field but are not easily detected, and their impact on pathobiology is not clearly defined due to their complicated nature, but it is well known that there is an impact. One way to increase our knowledge of coinfections in poultry is to challenge birds in experimental and controlled conditions. While many articles report in vivo experiments with LPAIV in avian models, only a few have studied coinfections. Moreover, researchers tend to choose different bird types, ages, inoculation routes, and doses for their experiments, making it difficult to compare between studies. This review describes the state of the art for experimental infections with LPAIV alone or associated with coinfecting pathogens in avian models. It also discusses how best to mimic field infections in laboratory settings. In the field of avian diseases, experimental design is obviously directly linked with the research question addressed, but there is a gap between field and experimental data, and further studies are warranted to better understand how to bring laboratory settings closer to field situations.
The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems (ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.
Poxviruses identified in skin lesions of domestic, pet or wild birds are assigned largely by default to the Avipoxvirus genus within the subfamily Chordopoxvirinae of the family Poxviridae. Avipoxviruses have been identified as the causative agent of disease in at least 232 species in 23 orders of birds. Vaccines
based upon attenuated avipoxvirus strains provide good disease control in production poultry, although with the large and
intensive production systems there are suggestions and real risks of emergence of strains against which current vaccines might
be ineffective. Sequence analysis of the whole genome has revealed overall genome structure and function resemblance to the
Chordopoxvirinae; however, avipoxvirus genomes exhibit large-scale genomic rearrangements with more extensive gene families and novel host
range gene in comparison with the other Chordopoxvirinae. Phylogenetic analysis places the avipoxviruses externally to the Chorodopoxvirinae to such an extent that in the future
it might be appropriate to consider the Avipoxviruses as a separate subfamily within the Poxviridae. A unique relationship exists between Fowlpox virus (FWPV) and reticuloendothelosis viruses. All FWPV strains carry a remnant
long terminal repeat, while field strains carry a near full-length provirus integrated at the same location in the FWPV genome.
With the development of techniques to construct poxviruses expressing foreign vaccine antigens, the avipoxviruses have gone
from neglected obscurity to important vaccine vectors in the past 20 years. The seminal observation of their utility for delivery
of vaccine antigens to non-avian species has driven much of the interest in this group of viruses. In the veterinary area,
several recombinant avipoxviruses are commercially licensed vaccines. The most successful have been those expressing glycoprotein
antigens of enveloped viruses, e.g. avian influenza, Newcastle diseases and West Nile viruses. Several recombinants have undergone
extensive human clinical trials as experimental vaccines against HIV/AIDS and malaria or as treatment regimens in cancer patients.
The safety profile of avipoxvirus recombinants for use as veterinary and human vaccines or therapeutics is now well established.
Recombinant fowlpox virus (rFPV) was constructed to express glycoprotein B (gB) gene from CVI988/Rispens strain of Marek's disease virus (MDV). The rFPV-gB/R alone and in combination with herpesvirus of turkey (HVT) preparations were evaluated for their protective efficacy against challenge with very virulent MDV strains Md5 and RB1B in different chickens. The rFPV-gB/R alone induced protection comparable to that by HVT vaccines in both Ab- SPF chickens and Ab+ production chickens. Significant protective synergism was observed in one of these two types of commercial production chickens when rFPV-gB/R was combined with HVT of either cell-associated or cell-free preparations. Immunogenesis studies showed that rFPV-gB/R, just like conventional vaccines, significantly reduced the level of viremia, splenocytes infection and feather follicle shedding of challenge virus in vaccinated chickens.
Inactivated whole avian influenza virus (AIV) vaccine provides protection against homologous haemagglutinin (HA) subtype virus, but poor protection against a heterologous HA virus. Moreover, it induces chickens to produce antibodies to cross-reactive antigens, especially nucleoprotein, which is limits AIV serological surveillance. In this study, a recombinant fowlpox virus co-expressing HA (H5 subtype) and NA (NI subtype)genes of AIV was evaluated for its ability to protect chickens against intramuscular challenge with a lethal dose of highly pathogenic (HP) AIV. Susceptible chickens were also vaccinated by wing-web puncture with the parent fowlpox vaccine virus. Following challenge 4 weeks later with HPAIV, all chickens vaccinated with recombinant virus were protected, while the chickens vaccinated with either the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 100% mortality following challenge. This protection was accompanied by the high levels of specific antibody to the respective components of the recombinant vaccine. The above results showed that rFPV-HA-NA could be a potential vaccine to replace current inactivated vaccines for preventing AI.
The use of potency control testing is a valuable tool for testing the actual relative strength of manufactured assembly lots of vaccine. Biological-based manufacturing methods are inherently variable and potency testing is a tool to ensure lot-to-lot consistency of commercial vaccines. A strong historical link to clinical efficacy has been established where correlation to efficacy and adequate test validation have been achieved. The link to immunogenicity and efficacy has traditionally been strongest with attenuated vaccines and toxoids. Control potency test failure does predict that a serial or batch of vaccine would most likely provide insufficient immunogenicity in typical field applications. Because of the complexity of pathogenic processes and associated immune responses, potency tests may not always directly predict the effectiveness of a vaccine. Thus, vaccines that pass control potency testing may not always provide adequate efficacy. This is particularly true of adjuvanted, inactivated vaccines. In the development of vaccine formulations and control tests for vaccines, the nature of the desired protective immune responses to the targeted pathogen (when known) should be considered. These considerations could provide better alternatives in the assays chosen as correlates of immunity and may more accurately predict efficacy and assure batch-to-batch consistency. Also, the effects of the dose and duration of antigen exposure as well as the nature of antigen presentation and generation of extrinsic cytokines could be characterised and correlated to vaccine potency as additional indicators of vaccine efficacy.
Humans have a sophisticated immune system that functions to clear invading organisms and abnormal cells. However, cancers are able to arise despite this immune system. Vaccines have the potential of benefiting cancer patients by stimulating an immune response against tumor-associated antigens (TAA). Our enhanced understanding of how the immune system processes and presents antigens has allowed an array of vaccine modalities to be developed and tested. The TAA with the greatest number of vaccine platforms tested in colorectal cancer is carcinoembyronic antigen (CEA). Trials to date have demonstrated safety and evidence for the induction of an immune response against CEA. This article will review trials conducted with a variety of CEA vaccines. Most studies conducted are phase I or II in the metastatic disease setting, limiting our understanding of the role of the immune response in controlling colon cancers. Phase III trials conducted to date have conflicting data with respect to improvements in disease-free and overall survival. It is our challenge to determine if and which vaccines have sufficient benefit to warrant large-scale trials in the adjuvant and prevention settings.
Marek's disease (MD) is an economically important neoplastic disease of poultry. MD almost devastated the poultry industry in the 1960s but the disease was brought under control after Marek's disease herpesvirus (MDV) was identified and vaccines were developed. This is the first effective use of an antiviral vaccination to prevent a naturally occurring cancer in any species. MDV infection has many effects. Initially causing a cytolytic infection in B-lymphocytes, MDV infects activated T-lymphocytes where it becomes latent. In susceptible chicken genotypes MDV transforms CD4+ lymphocytes, causing visceral lymphomas and/or neural lesions and paralysis. Fully productive infection and shedding of infectious virus only occurs in the feather-follicle epithelium. Vaccination of newly-hatched chicks with live vaccines has been widely used to successfully control MD since the early 1970s. However, vaccinated chickens still become infected and shed MDV. Vaccine breaks have occurred with regularity and there is evidence that the use of MD vaccines could be driving MDV to greater virulence. MD continues to be a threat and a number of strategies have been adopted such as the use of more potent vaccines and vaccination of the embryonic stage to provide earlier protection. Recombinant MD vaccines are useful vectors and are being exploited to carry both viral and host genes to enhance protective immune responses. The future aim must be to develop a sustainable vaccine strategy that does not drive MDV to increased virulence.
Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.
Two Marek's disease virus (MDV) genes, one homologous to the glycoprotein B gene of herpes simplex virus and encoding the B antigen complex and the other encoding a 38-kDa phosphorylated protein (pp38), were inserted into the fowlpox virus (FPV) genome under the control of poxvirus promoters. Randomly selected nonessential regions of FPV were used for insertion, and the vaccinia virus 7.5 kDa polypeptide gene promoter or a poxvirus synthetic promoter was used for expression of MDV genes. Gene expression in cells infected with these recombinants was highly influenced by the promoter (the synthetic promoter being more effective) but was only slightly influenced by the insertion site and by the transcription direction of the insert relative to the direction of the flanking FPV sequences. Cells infected with an FPV recombinant expressing the MDV gB gene reacted positively with a monoclonal antibody specific to this glycoprotein in an immunofluorescence assay. Immunoprecipitation of infected cell lysates showed three glycoproteins identical to those associated with the B antigen complex of MDV (100, 60, and 49 kDa). Cells infected with a recombinant expressing the pp38 gene reacted positively with an anti-pp38 monoclonal antibody in an immunofluorescence assay. The generated protein was phosphorylated and had a molecular weight similar to that of the native pp38 protein. Sera from chickens immunized with an FPV recombinant expressing the MDV glycoprotein B gene reacted with MDV-infected cells.
Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.
The Marek's disease virus (MDV) homologue of the herpes simplex virus (HSV) gene encoding glycoprotein B (gB) has been identified within BamHI fragments I3 and K3 of the 'highly oncogenic' strain RB1B of MDV. The entire nucleotide sequence of the gene has been determined and its predicted amino acid sequence shown to share gross overall structural features with the gB genes of HSV, varicella-zoster virus (VZV) and other mammalian herpesviruses. In particular, all 10 cysteine residues were conserved in MDV gB and there was extensive homology throughout the gene with VZV, HSV and pseudorabies virus except for the N and C termini. The overall percentage amino acid identity between MDV gB and gB of the alphaherpesviruses had a mean of 50% which was almost twice that between cytomegalovirus and Epstein-Barr virus. Northern blot analysis showed that the main RNA transcribed from this gene is approx. 2.7 kb in size. Antibodies raised against synthetic peptides (residues 250 to 271 and 304 to 330) allowed the identification of a family of serologically related glycoproteins of Mr 110K, 64K and 48K in extracts of MDV-infected cells using immunoblots. Furthermore, the antisera were able to differentiate between the antigens of MDV and herpesvirus of turkeys in immunoblots. Immunofluorescence tests indicated that MDV gB is associated with granules in the cytoplasm and is present at the surface of MDV-infected cells.
Purified DNAs from Marek's disease virus (MDV) and the herpesvirus of turkeys (HVT) were randomly sheared and cloned into the M13 bacteriophage. Two-hundred and ten MDV and 130 HVT clones were sequenced to give representative samples of the genome sequences. The predicted amino acid sequences from these gammaherpes-viruses were compared to known sequences from other herpesviruses using computer analysis. Thirty-five MDV and 24 HVT genes were identified by comparison with varicella-zoster virus (VZV), an alphaherpesvirus. However, only 14 MDV and seven HVT genes, giving generally lower homology scores, were found by comparison with Epstein-Barr virus (EBV), a gammaherpesvirus, indicating that MDV and HVT sequences bear greater similarity to VZV than to EBV sequences. A number of sequences were mapped by hybridizing labelled M13 clones to Southern blots of restriction fragments of MDV or HVT DNA. The results were consistent with the MDV and HVT genomes being collinear with VZV.
The intensive poultry industries rely heavily upon the use of vaccines for disease control. Viral vector based vaccines offer new avenues for the development of vaccines for effective disease control in poultry. Techniques developed for the construction of recombinant vaccinia viruses have been readily adapted to the construction of recombinant viruses based on fowlpox virus (rFPV). The ability to insert several genes into the large genome of fowlpox may enable the development of multivalent vaccines and vaccines incorporating immune response modifiers such as lymphokines. Newcastle disease, avian influenza, infectious bursal disease and Marek's disease antigens expressed by rFPV have been shown to be effective vaccines in poultry. None appear, however, to provide a substantial improvement in vaccine efficacy. Recombinant FPV will be a valuable adjunct to conventional vaccines currently in widespread use. Whether rFPV or other vector based vaccines can circumvent the problems of vaccination in the presence of high maternally derived antibodies is yet to be resolved. The observation that avipoxvirus recombinants may be suitable for the vaccination of non-avian species provides an added dimension to vaccines based on FPV or other avipoxviruses. Recombinant FPV will find a useful role in poultry disease control when used in conjunction with conventional vaccines.
Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP4-VP3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP2 and VP3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP2 contained only the VP2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of the TK gene. The expression level of VP2 from FPV-VP2 was approximately 5 times higher than from FPV-VP2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.
Our previous studies have shown that of seven HSV-1 glycoproteins (gB, gC, gD, gE, gG, gH and gI) individually expressed in baculovirus, vaccination with gD provides the best protection against HSV-1 challenge. To establish whether vaccination with a mixture of these seven expressed glycoproteins would provide better protection against HSV-1 challenge than vaccination with gD alone, we determined the level of protection afforded by vaccination with a cocktail of the seven expressed glycoproteins. The amount of each of the seven expressed glycoproteins in the mixture was equivalent to one-seventh the amount of gD used in the gD alone vaccination. Thus, the total amount of glycoprotein was the same for the cocktail and gD alone vaccine.
Integration of two different avian retroviruses, reticuloendotheliosis virus (REV) and avian leukosis virus (ALV), into the genome of two different avian herpesviruses, the herpesvirus of turkeys (HVT) and Marek's disease virus (MDV), was investigated. Integration events occurred by the fourth and sixth in vitro passage of cells coinfected with REV/HVT and ALV/MDV, respectively. In order to further characterize the integration events, integrated proviruses and surrounding herpesviral genetic material were cloned and analyzed. In the REV/HVT coinfection experiment, one of the three unique integrated proviruses was found to have integrated into the HVT gD gene, resulting in disruption of the coding region of this gene. The two additional unique integrations were localized to the UL and IRL border regions of HVT, two previously described common sites of REV integration into MDV. Interestingly, one of the integrated proviruses in the HVT genome appeared to be full length, was infectious when transfected into CEF cells, and therefore could potentially function to produce infectious REV from an HVT infectious platform. In the ALV/MDV coinfection experiment, one of two unique integrated proviruses was found to have integrated into the gD gene, resulting in disruption of the coding region of this gene. The second unique integration site was in the polyadenylation site of the SORF2 gene at the boundary of the IRs and Us, once again a common site of REV integration into MDV. These results demonstrate that the U/IR-TR border regions of herpesviruses are common sites of retroviral integration. In addition gD, in the Us region of the herpesvirus, is a common site of retroviral integration in multiple herpesvirus, indicating a possible selective advantage for disruption of this gene in the in vitro growth of a herpesvirus. Finally, this is the first instance of a full-length provirus found integrated into a herpesvirus genome, indicating that a retrovirus could alter its route of infection by being carried in a herpesvirus genome.
Glycoproteins B (MDV1-gB) and C (MDV1-gC) of Marek's disease virus type 1 (MDV 1) expressed by baculovirus recombinants [rAcNPV(s)] were investigated for their ability to confer protective immunity in chickens against virulent MDV1 challenge. Immunization of chickens with rAcNPV-expressed MDV1-gB induced antibody against MDV1 and resulted in strong protection against the lethal challenge with MDV1. In contrast, immunization of chickens with rAcNPV-expressed MDV1-gC induced antibody against MDV1, but failed to protect against challenge with the virulent MDV1. Co-immunization with both rAcNPV-expressed MDV1-gB and MDV1-gC seemed to show no synergistic effect.
Lines of chickens selected from a common ancestral population for either resistance or susceptibility to Marek's disease developed
contrasting frequencies of particular B alloalleles. Comparison of inoculated sibs in backcross-families revealed that the
B alloalleles characterizing the two lines accounted for an eightfold difference in tumor incidence. This genetic difference
in tumorigenesis associated with the alloalleles of the major histocompatibility complex is probably expressed through the
cell-mediated immune system.
The DNA sequence of a 5.5 kbp EcoRI fragment located in the short unique region (US) of the 'highly oncogenic' strain RB1B of Marek's disease virus (MDV) was determined. The sequence contained six open reading frames (ORFs), four of which were homologous to proteins mapping in the US region of herpes simplex virus type 1 (HSV-1). These include the homologues of HSV-1 protein kinase, glycoprotein D (gD), glycoprotein I (gI) and US2 which is of unknown function. The MDV ORFs had a marked bias for A or T in the third codon position and analysis of the dinucleotide frequencies showed a marginal deficit in ApG/CpT but no overall deviation of CpG from random expectations. Comparison of genes in the US region of MDV to herpesvirus proteins confirmed and extended our previous observation that MDV is more closely related to alphaherpesviruses than to gamma-herpesviruses. We also showed that MDV possessed a homologue of HSV-1 gD which is lacking in varicella-zoster virus (VZV) but that MDV probably lacked homologues of US4 and US5 of HSV-1. These results show that in contrast to the genes in the long unique region which were grossly collinear in HSV, VZV and MDV, those mapping in US show greater diversity.
Very virulent Marek's disease viruses (vvMDV), defined as isolates against which the herpesvirus of turkey (HVT) vaccine provide poor protection, have been isolated from poultry flocks in both the United States and Europe. Twenty-one samples from vaccinated Australian flocks, experiencing problems with excessive Marek's disease (MD), were tested for the presence of transmissible MD viruses (MDV). Of the 16 samples which contained a transmissible agent, 14 were pathogenic in chickens, based on the development of MD lesions or depression of the bursa/body weight ratio. Of the pathogenic isolates which have been successfully typed 10 were serotype 1, and one was serotype 2 MDV. Pathogenicity of isolates varied. Several isolates caused tumours in 20-30% of both vaccinated and unvaccinated chickens. Two isolates, MPF6 and MPF23, caused tumours in more than 50% of chickens. When MPF6 and MPF23 were tested in vaccine trials bivalent vaccine gave no better protection against development of MD lesions than a monovalent vaccine. Isolate MPF23 was so pathogenic that lesions were produced in all chickens, regardless of the vaccine protocol used. Therefore vvMDV have been isolated in Australia, and unlike the vaccines tested overseas, bivalent Australian vaccines do not appear to provide greater protection against these vvMDV.
The A antigen gene from a very virulent strain of Marek's disease virus, RB-1B, has been cloned and the nucleotide sequence determined. The predicted amino acid sequence showed 99% identity to that determined for the MDV GA A antigen (Coussens and Velicer, J. Virol. 62, 2373-2379, 1988) over all but the carboxy-terminal region where the sequence diverged extensively. The divergence results from three nucleotide frameshifts in the reported sequence of the MDV GA gene which are not present in a cloned copy of the MDV GA A antigen gene sequenced by us. The MDV A antigen shows significant homology to a number of herpes virus gC homologues, the homology being most extensive in the carboxy-halves of the proteins.
DNA fragments containing the secretory glycoprotein A (gA) gene of Marek's disease virus type 1 (MDV1) were cloned from the DNA libraries of very virulent Md5 and virulent BC-1 strains and sequenced. Two open reading frames (ORF1 and ORF2) were identified for both strains. The ORF1 has the potential to code for a protein of 501 amino acids with a molecular weight of 56 kD that contains strong hydrophobic regions in both the amino and carboxyl termini, and nine potential N-linked glycosylation sites, while the ORF2 is capable of coding for a 24-kD protein. These results indicate that the ORF1 codes for the unprocessed form of gA. Between the Md5 and BC-1 strains, only two sequence mismatches exist in the DNA fragment. More differences appear to exist in the gA sequence of the MDV1 GA strain (12), which lacks a strong hydrophobic anchor sequence. Similarities between the predicted amino acid sequences of the MDV1 gA and the proteins of the other herpesviruses such as herpes simplex type I gC, pseudorabies virus gIII, and varicella zoster virus gpV were noted.
The thymidine kinase (TK) gene of fowlpox virus (FPV) is located in a 2.2-kb HindIII-ClaI fragment derived from a 5.5-kb EcoR1 fragment of the FPV genome. The TK gene was mapped to the region of a 700-bp XbaI fragment contained within this HindIII-ClaI fragment. Nucleotide sequence analysis of this region revealed an open reading frame of 183 codons. Identification of this region as the FPV TK gene was confirmed by its homology with the vaccinia virus TK at both the nucleotide and amino acid levels. The derived FPV TK polypeptide has a calculated molecular weight of 20,380 and is six amino acids larger than the vaccinia virus TK gene product. We have reported previously that the FPV TK gene operates in vaccinia virus without the requirement for a vaccinia virus promoter. The sequence homologies between the two TK promoters substantiated this observation. Northern blot analysis of RNAs from cells infected with a vaccinia virus recombinant expressing the FPV TK gene showed major (700 nucleotide) and minor (1000 nucleotide) transcripts from the FPV TK gene. The deduced amino acid sequence of the FPV TK has significant homology with the TKs from chicken, man, and three other poxviruses, but shows no homology with herpes simplex virus TK. Comparisons of the homologous sequences indicated that the "core" of the enzyme has probably evolved in poxviruses four times as quickly as in vertebrates. Characterization of the FPV TK gene may facilitate the construction of recombinant FPVs as vehicles for the delivery of vaccine antigens to poultry and other avian species.
The major immunogenic viral proteins of Marek's disease virus (MDV) and turkey herpesvirus (HVT) share antigenic determinants. The polyacrylamide gel electrophoresis pattern of five viral polypeptides immunoprecipitated with homologous convalescent chicken plasma was identical with the pattern obtained after immunoprecipitation with heterologous convalescent chicken plasma. A panel of monoclonal antibodies was used to identify MDV and HVT polypeptides. Sixteen monoclonal antibodies were positive in an immunofluorescence assay. However, only eight monoclonal antibodies immunoprecipitated a total of seven distinct viral proteins from MDV- and HVT-infected cells. Six of these monoclonals immunoprecipitated multiple viral proteins. None of the monoclonal antibodies recognized the common A antigen of MDV or HVT. Monoclonal antibodies immunoprecipitated three glycoproteins (100,000, 60,000, and 49,000 Da) that comigrate in polyacrylamide gels with three of the five common polypeptides obtained with the convalescent chicken plasma. In addition, a 79,000-Da protein was common to all MDV- and HVT-infected cells. Competition immunoprecipitation and peptide mapping by limited proteolysis confirmed that the three glycoproteins and the 79,000-Da protein contain MDV-HVT common epitopes. MDV-specific antigenic determinants were detected on the three remaining viral proteins (41,000, 38,000, and 24,000 Da).
The biological complexities of the human herpesviruses and the wide range of diseases that they cause present many difficulties for vaccine development. Until recently, progress towards this aim has been slow; however, advances in immunology and molecular biology have yielded an exciting array of new approaches for vaccination that have shown promise in model systems. This explosion in technology, together with renewed appreciation of the public-health benefits of vaccination, has sparked a resurgence of interest in the development of new vaccines and several are in, or near, clinical trials in humans. These look set to have a major impact on the incidence of herpesvirus diseases in the future.
The influence of dose and route of inoculation on responses of chickens to vaccination with recombinant fowlpox viruses (rFPVs) expressing an influenza haemagglutinin (HA) (FPV-HA) and the infectious bursal disease virus (IBDV) VP2 antigen (FPV-VP2) has been evaluated. Antibody responses to influenza and fowlpox virus were generated following vaccination via the wing web by subcutaneous inoculation or skin scarification. Intranasal and conjunctival inoculation failed to induce antibodies to FPV or influenza. Following direct intratracheal inoculation antibodies developed to influenza but not FPV. Dose response studies with the FPV-HA and FPV-VP2 recombinants showed that good responses to FPV and the vaccine antigen could be generated over a wide (10000 fold) dose range following wing web inoculation. The responses generated by the FPV-VP2 recombinant over this vaccine dose range protected against IBDV infection of the bursae following challenge with the Australian IBDV 002/73 isolate. These data suggest that effective application of rFPVs for poultry vaccination may be restricted to wing web and parenteral routes of inoculation.
Despite its previous classification as a gammaherpesvirus, primarily due to its lymphotropism, Marek's disease virus (MDV), an oncogenic avian herpesvirus, is phylogenetically more related to the "neurotropic" alphaherpesviruses, characterized by its prototype, herpes simplex virus (HSV) (Buckmaster et al., 1988, J. Gen. Virol. 69, 2033-2042). In this report we present the DNA sequence of an 11,286-bp DNA segment encompassing the entire 11,160-bp-long Us region of the oncogenic avian herpesvirus, Marek's disease virus. Eleven open reading frames (ORFs) likely to code for proteins were identified; of these, 7 represent homologs exclusive to alphaherpesvirus S component genes. These include MDV counterparts of HSV US1 (ICP22), US2, US3 (a serine-threonine protein kinase), US6, US7, and US8 (HSV glycoproteins gD, gI, and gE, respectively), and US10. Three additional ORFs were identified with no apparent relation to any sequences currently present in the SwissProt or GenBank/EMBL databases, while a fourth was found to exhibit significant homology to an uncharacterized fowlpox virus (FPV) ORF. Having precisely identified the IRs-U(s) and U(s)-TRs junctions, we have corrected and clarified their previously reported locations. By characterizing genes encoding three new alphaherpesvirus-related homologs (US1, US8, and US10), completing the sequence for a fourth (US7), and identifying 2 new MDV-specific ORFs (SORF1 and SORF3) and a fowlpox homolog (SORF2), our sequence analysis of the "virulent" GA strain of MDV (vMDV) extends upon that of a 5255-bp segment located in the U(s) region of the "very virulent" RB1B strain of MDV (vvMDV) (Ross et al., 1991, J. Gen. Virol. 72, 939-947; 949-954). These two sequences were found to exhibit 99% identity at both nucleotide and predicted amino acid levels. Combined with the fact that MDV U(s) sequences failed to show statistically significant CpG deficiencies, our analysis is consistent with MDV bearing a closer phylogenetic relation to alphaherpesviruses than to gammaherpesviruses. Because alphaherpesvirus-specific U(s) region genes are primarily nonessential for virus replication, they are thought to be important biological property determinants. Thus, our sequence provides a foundation for further MDV studies aimed at resolving the apparent discrepancy between MDV's genetic and biologic properties.
We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100% survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.
To determine if B-haplotype differentially influences vaccinal immunity to very virulent Marek's disease (MD) virus challenge, chickens of five 15.B-congenic lines were vaccinated with vaccines representing serotypes 1, 2, and 3. B-haplotype differentially influenced vaccinal immunity to very virulent MD virus challenge using vaccines of all three serotypes, and different MD vaccines were optimal for some B-haplotypes. Regarding specific haplotypes, the 15.B-congenic chickens with B2 and B13 developed less protection against MD than chickens with B15 following vaccination with all three serotypes of MD vaccine, whereas the chickens with B5 and B21 developed variable protection with different MD vaccines. Thus, for induction of maximum MD resistance, it may be necessary to select a vaccine appropriate for the predominant B-haplotypes of the chicken flock.
A fowl pox-based recombinant virus TROVAC-NDV (vFP96.5) was developed expressing the fusion and hemagglutinin-neuraminidase glycoproteins from a velogenic strain of Newcastle disease virus (NDV). Studies in specific-pathogen-free birds indicated that inoculation of a single dose of the recombinant led to the induction of significant levels of hemagglutination-inhibiting antibody that were maintained to 8 wk postinoculation. Further, the recombinant induced protective immunity against a combined intramuscular velogenic NDV challenge and respiratory NDV challenge. In commercial broiler chickens that were inoculated in the presence of maternally derived NDV immunity, the level of the NDV-specific humoral response was dampened, but significant levels of protection against both a lethal intramuscular NDV challenge and a fowl poxvirus challenge were obtained.
Recombinant fowl poxviruses (rFPV) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B (gB1), C (gC), and D (gD) and tegument proteins UL47 and UL48, as well as genes from serotypes 2 and 3 MDV coding for glycoprotein B (gB2 and gB3). These rFPVs, alone and in various combinations, including combinations of fowl poxvirus (FPV)/gBs with turkey herpesvirus (HVT), were evaluated for ability to protect maternal antibody-positive (ab+) and -negative (ab-) chickens against challenge with highly virulent MDV isolates. The protective efficacy was also compared with that of prototype Marek's disease (MD) vaccines. No protection was induced in ab+ chickens by rFPV expressing gC, gD, UL47, or UL48. In contrast, the rFPV/gB1 construct protected about 23% of ab+ chickens against MDV challenge compared with 26% for cell-associated HVT. Levels of protection by rFPV/gBs of different MDV serotypes was highest for gB1, intermediate for gB2, and lowest for gB3. When rFPV/gB1 was combined with cell-associated HVT, protection was enhanced by an average of 138% compared with the best component monovalent vaccine, and the mean level of protection was 59% compared with 67% for the HVT+SB-1 bivalent vaccine. Relatively high protection (50%) and enhancement (200%) were also observed between rFPV/gB1 and cell-free HVT. These results suggest a specific synergistic interaction between rFPV/gB1 and HVT, possibly analogous to that previously described between serotypes 2 and 3 viruses. Levels of protection by rFPV/ gB1 alone or by bivalent rFPV/gB1+cell-associated HVT were similar to those of conventional cell-associated MD vaccines. However, the bivalent rFPV/gB1+cell-free HVT vaccine was clearly more protective than cell-free HVT alone and, thus, may be the most protective, entirely cell-free MD vaccine thus far described.
Two inbred lines of White Leghorn chickens which differ in B-haplotype were immunized at 2 days of age with a thymidine kinase negative (tk-ve) herpesvirus of turkeys (HVT) recombinant expressing the glycoprotein B (gB) gene of Marek's disease virus (MDV) and were challenged 6 days later with 1000 p.f.u. of the highly virulent RB1B strain of MDV. Mock-vaccinated chickens and chickens immunized with a spontaneous tk-ve HVT mutant served as controls. Genetically resistant B21 chickens were protected by immunization with the recombinant as well as by the tk-ve HVT, whereas highly susceptible B13 chickens were partially protected by the recombinant but were not protected by the tk-ve HVT. Rhode Island Red chickens (HPRS RIR), which differ from the White Leghorns at the B locus, were protected by both vaccines but the recombinant conferred a significantly higher level of protection than the tk-ve HVT. The results suggest that the gB gene of MDV serotype 1 has an important role in the induction of protective immunity against highly virulent MDV in genetically susceptible lines of chickens and that vaccinal immunity in White Leghorns might be influenced by the B haplotype.
Birds, like mammals, have highly a polymorphic MHC that determines strong allograft rejection. However, chickens have a much smaller, more compact and simpler MHC than mammals, as though the MHC has been stripped down to the essentials during evolution. The selection pressure on a single MHC gene should be much stronger than on a large multigene family, and, in contrast to mammals, there are a number of viral diseases for which resistance and susceptibility are determined by particular chicken MHC haplotypes. We have determined the peptide motifs for the dominant class I molecules from a number of chicken MHC haplotypes, which may explain some disease associations quite simply. Other disease associations, like the famous examples with Marek's disease, may be due to polymorphism in the level of expression of MHC class I molecules. We believe that the compact and simple nature of the MHC is due to the presence of microchromosomes in birds and suggest that the evolutionary origin of birds has been strongly influenced by the emergence of microchromosomes.
Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.
Chicken MHC molecules, disease resistance and the evolutionary origin of birds Current Topics in Microbi-ology and Immunology Isolation of very virulent Marek's disease viruses from vaccinated chickens in Australia
- J Kaufman
- H J Wallny
- Springer
- Berlin
- J L Mckimm-Breschkin
- J T Faragher
- J Withell
- W M Forsyth
Kaufman, J., Wallny, H.J., 1996. Chicken MHC molecules, disease resistance and the evolutionary origin of birds. In: Vainio, O., Imhof, B.A. (Eds.), Current Topics in Microbi-ology and Immunology, Vol. 212. Immunology and Devel-opmental Biology of the Chicken. Springer, Berlin. McKimm-Breschkin, J.L., Faragher, J.T., Withell, J., Forsyth, W.M., 1990. Isolation of very virulent Marek's disease viruses from vaccinated chickens in Australia. Aust. Vet. J 67, 205 – 209.