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Foot-and-mouth-disease is a highly contagious, economically significant disease of animals. The disease affects domestic cloven- hoofed animals, including cattle, swine, sheep, and goats, as well as more than 70 species of wild animals, including deer (Fenner, F. J., 1993). Although FMD does not result in high mortality in adult animals, the disease has debilitating effects, including weight loss, decreased milk production, and loss of draught power, resulting in a loss in productivity for a considerable time (Pereira H.G., 1981). Mortality, however, can be high in young animals, where the virus can affect the heart. FMD is on the A list of infectious diseases of animals of the Office International des E´pizooties (OIE) and has been recognized as the most important constraint to international trade in animals and animal products (Leforban. Y., 1999). The FMD outbreak in Taiwan which resulted in the slaughter of more than 4 million pigs at a cost of approximately U.S. $6 billion (Yang, P. C., 1999) and the United Kingdom Outbreak in 2001 resulted in the slaughter of 4 million animals, mainly sheep (Scudamore, J. M., 2002) at a cost of around U.S. $12.3 billion to $13.8 billion (Thompson, D., 2002) are examples of destructive economic impact of the FMD to livestock.
FMD is caused by foot and mouth disease virus (FMDV) belonging to the genus Aphthovirus in the family Picornaviridae. The virion is a non-enveloped 140S particle of about 28-30nm in diameter consisting of a single stranded RNA genome and 60 copies each of four structural proteins (VP1 [1D], VP2 [1B], VP3 [1C], and VP4 [1A]). The FMDV genome has a basic organization similar to those of other members of the Picornaviridae, and the nomenclature for the viral proteins was established by Rueckert and Wimmer (Rueckert, R. R., 1984). Seven serotypes (A, O, C, Asia 1, and South African Territories 1, 2, and 3) have been identified serologically, and multiple subtypes occur within each serotype (Bachrach, H. L., 1968).
The FMD viral particle contains a positive strand RNA genome of about 8500 nucleotides, enclosed within a protein capsid. The FMDV genome is composed of three parts, (i) the 5' untranslated region (5' UTR) consists of pseudoknots, poly-C tract and an internal ribosome entry site (IRES), (ii) the coding region which includes structural and non-structural genes and (iii) the 3' untranslated region (3' UTR) containing a heteropolymeric segment and a poly (A) tail. A small viral protein, VPg, is covalently linked to the 5' end of the viral RNA.
FMDV initiates infection in cultured cells by binding to any of four members of the αv subgroup of the integrin family of cellular receptors (αvβ1, αvβ3, αvβ6 and αvβ8) (Berinstein, A., 1995; Duque, H., 2003; Jackson, T., 2004; Jackson, T., 2002; Jackson, T., 2000) via a highly conserved arginine-glycine-aspartic (RGD) acid amino acid sequence motif located within the βG-βH loop of VP1 (Baxt, B., 1990; Fox, G., 1989; Leippert, M., 1997; Mason, P. W., 1994). After binding to the cell surface, the 140S virion breaks down into 12S pentameric subunits, releasing the RNA (Baxt, B. 1987; Baxt, B., 1982; Baxt, B., 1980). As soon as the RNA is released into the cytoplasm the protein synthesis begins internally at the IRES by a cap-independent mechanism (Belsham, G. J., 1990; Jang, S. K., 1988; Kuhn, R., 1990; Pelletier, J., 1988). Following initiation, translation results in the production of a single polypeptide. The polyprotein then undergoes a series of cleavages leading to the production of both structural and non-structural proteins. The primary cleavage reactions are performed by three different proteases. Lpro autocatalytically cleaves itself from polyprotein. 2A, (an 18-amino-acid peptide), autocatalytically removes itself from the P2 polyprotein, and remains associated with the P1 precursor (Grubman, M. J., 1995; Ryan, M. D., 1991; Vakharia, V. N., 1987). Number of studies have shown that 3C protease involves in majority of other cleavages of P1 (Bablanian, G. M., 1993; Clarke, B. E., 1988; Vakharia, V. N., 1987). 3C protease cleaves the structural protein precursor P1 which results in production of three structural proteins 1AB (VP0), 1C (VP3) and 1D (VP1) which assembles to form the empty capsid. At the same time the 3D, the RNA dependent RNA polymerase, makes several copies of genomic RNA by an yet unclear mechanisms. The empty capsid, following encapsidation of the RNA and a subsequent maturation cleavage of VP0 to VP2 and VP4, forms a mature virion. Picornaviruses encapsidates only plus-strand RNA, linked to VPg, to the exclusion of all other viral and cellular RNAs (Nomoto, A., 1977; Novak, J. E., 1991; Wimmer, E., 1982).
Infection in cattle generally occurs via the respiratory route by aerosolized virus. Infection can also occur through abrasions on the skin or mucous membranes, but is very inefficient, requiring almost 10,000 times more virus (Donaldson, A. I. 1987). The infected animals excretes virus into the milk (Burrows, R. 1968; Hyde, J. L., 1975; Ray, D. K., 1989) semen, urine as well as in feces (Kitching, P. 1992; Donaldson, A. I. 1987) which could become source for infection to other animals. Infected animals excrete large amount of aerosolized virus which spreads the disease more rapidly. Young calves can become infected by inhaling milk droplets. Pigs usually become infected either by eating FMDV-contaminated food, by direct contact with infected animals, or by being placed into areas that had once housed FMDV-infected animals. They are, however, much less susceptible to aerosol infection than cattle (Alexandersen, S., 2002a; Alexandersen, S., 2002b), yet they excrete far more aerosolized virus than cattle or sheep (Alexandersen, S., 2002b; Alexandersen, S., 2002c).
A number of studies have suggested that the lung or pharyngeal areas are the sites of initial virus replication with rapid dissemination of the virus to oral and pedal epithelial areas (Brown, C. C., 1992; Burrows, R., 1981; Sutmoller, P., 1976). Vesicles develop at multiple sites, generally on the feet and tongue, and are usually preceded by fever. Severe lesions often occur in areas subjected to trauma or physical stress, and most animals develop viremia. The incubation period can be between 2 and 14 days, depending on the infecting dose and route of infection (Gailiunas, P., 1966). In young piglets, the infection may be fatal due to myocarditis. Clinical disease in sheep is characterized by lesions on the feet and mouth, fever, and viremia.
The virus elicits a rapid humoral response in either infected or vaccinated animals. Virus-specific antibodies protect animals in a serotype-specific manner against reinfection, or against infection in the case of vaccination, and protection is generally correlated with high levels of neutralizing antibodies (McCullough, K. C., 1992; Salt, J. S. 1993). The role of cellular immunity in the protection of animals from FMD is yet not clearly understood but it has been suggested that cell-mediated immunity is involved in clearance of virus from persistently infected animals (Childerstone, A. J., 1999; Ilott, M. C., 1997). In addition to IFN (Alexandersen, S., 2002; Brown, C. C., 2000; Chinsangaram, J., 2001; Chinsangaram, J., 1999; Zhang, Z. D., 2002), other cytokines may also play a role in the host response.
The effective control of the disease is mainly attained by vaccination with inactivated whole virus preparation that is formulated with adjuvant prior to use in the field. The introduction of the killed FMD vaccine has been extremely successful in reducing the number of disease outbreaks in many parts of the world where the disease is enzootic. However, there are a number of concerns and limitations with its use in regular and emergency control programs, including the following. (i) High-containment facilities are required for the production of vaccine. (ii) There are chances for virus escaping inactivation which can potentially cause disease to vaccinated animals. (iii) Most virus preparations used for vaccines are concentrated cell culture supernatants from FMDV-infected cells and, depending on the manufacturer, contain various amounts of contaminating viral NS proteins. Vaccinated animals develop antibody responses against the contaminating proteins, in addition to the viral structural proteins, making it difficult to reliably distinguish vaccinated from infected or convalescent animals with currently approved diagnostic tests. (iv) The vaccine does not induce rapid protection against challenge by direct inoculation or direct contact. Thus, there is a window of susceptibility of vaccinated animals prior to the induction of the adaptive immune response. (v) Vaccinated animals can become long-term carriers following contact with FMDV (Marvin, J. G., 2004)
Some of these major concerns are being addressed by development of new marker vaccines that do not require infectious virus. Peptide vaccines comprising one or mixture of more than one epitopes from VP1 were studied extensively as vaccine candidates as the VP1 consists of the major immunogenic sites which are responsible for eliciting the neutralizing immune response against FMDV. Chemically synthesized VP1 peptides (64, 127, 160, 331, 352) containing either B or T cell epitopes, DNA vaccines expressing VP1 epitopes (478) with or without a co-administered IL-2 (479) and transgenic plants expressing immunogenic epitope of VP1 (MarÄ±a J. Dus Santos., 2002)(472, 473) have also been investigated. Although these strategies resulted in production of high titers of neutralizing immune response against FMDV, they present a limited subset of viral immunogens to the vaccinated animal as they do not always confer protection against virus challenge in livestock (127, 326-328). Apart from those offered limited protection, they pose a risk of antigenic variants being selected among animals vaccinated with these products (259, 313) or develop viral escape mutants that were antigenically variant (447, 448).
Subunit vaccines consist of complete VP1 either from the purified virus or produced by recombinant DNA techniques (23, 248), live viral vectors expressing VP1 fusion proteins with either interferon α or granulocyte macrophage colony-stimulating factor (242, 246) and transgenic plants expressing VP1 antigen (Carrillo, C., 1998) resulted in neutralizing antibody response in mice and guinea pigs and offered protection in guinea pigs. But the effect of these antigens in target animals may vary as they are not uniformly recognized in all host species.
Live attenuated strains of FMDV are other attractive vaccine candidates, produced either by passage in non-susceptible species, such as mice, rabbits, and embryonated eggs, until their virulence for cattle was weakened or by developing RGD mutant of FMDV utilizing recombinant DNA techniques. In some cases the attenuated vaccines resulted in a degree of protection, it was found that strains attenuated for a certain host were often virulent in other susceptible animals. Furthermore, it has been difficult to obtain viruses that are both attenuated and immunogenic and are often pose a risk of reversion to virulence. The deletion of non-structural protein coding regions that are not essential for virus replication in cell culture is an alternative method of creating live attenuated vaccines. However, to be useful as a vaccine, this deletion virus must still be able to replicate in susceptible animals. The advantage of this approach is that the risk of reversion to virulence is significantly reduced. A Leaderless (Lpro deleted) live attenuated FMD virus replicated in BHK cells but did not cause disease in cattle or swine (98, 296, 356) and delayed clinical disease compared to naive animals upon challenge yet were not fully protected (98, 296).
DNA based vaccine strategies designed to produce single or multiple immunogenic peptides, complete VP1 protein with or without IL-2 and IL-15, the complete structural protein precursor P1, have been investigated. DNA vaccines expressing P1-2A, 3C which forms empty capsids in inoculated animals (46, 52, 91, 95) or DNA vaccines carrying L and RGD receptor binding site deleted full length infectious clone have also been tried as a vaccine candidates. These vaccines often resulted in the stimulation of neutralizing antibody response specific for FMDV in mice, guinea pigs and pigs (46, 95), however, to induce low levels of FMDV-specific neutralizing antibody response, huge amounts of DNA and at least two or three inoculations were needed (46, 52, 91). Hence, in a large scale vaccination programs this would be difficult to vaccinate animals with DNA vaccine.
Empty capsid or virus like particles (VLP) that contain the entire structural antigens present on intact virus but lack infectious nucleic acid (50, 188, 190, 269, 396) is an attractive, safer yet efficient vaccine candidates. This method of producing vaccines is considered to be very safer as they do not involve the use of live virus and present less chance for reversion to virulence. The strategy of producing VLPs involves the molecular cloning of P1-2A and 3Cpro coding regions results in production of antigenically similar virus like particles which are as immunogenic as virions in animals (190, 400, 407). FMDV capsid structures expressed in Escherichia coli or in recombinant baculovirus-infected cells were used as vaccines. Although these products did offer some protection, they did not reach the efficacy of the current inactivated whole-virus vaccine because only small amounts of antigen were obtained (50, 188, 269, 396) which often results making the vaccine expensive
Live attenuated viral vectors expressing FMDV capsid antigen are promising vaccine candidates as they are known to elicit potentially high immune response against target antigens. Human adenovirus and Poxviruses are well-characterized viral vectors for foreign gene expression (182, 225, 284, 303, 349, 404) and have been used to deliver FMDV capsid proteins (1, 57, 301, 302, 320, 423, 424, 480). A replication defective adenovirus 5 expressing either epitopes of VP1 or complete VP1 was used successfully against FMDV with or without interferon alpha and GMCSF which elicited protective amount of immune response in mice and pigs. Recombinant adenoviruses expressing either capsid precursor P1-2A alone or together with 3C/3CD have also been used as a vaccine candidate conferred protection in swine. On the other hand poxviruses are also considered to be equally efficient yet safer viral vectors for delivering numerous heterologous antigens.
Poxviruses are the largest of all animal viruses and can be visualized by light microscopy. Poxvirus virions appear to be oval or brick-shaped structures of about 200 to 400 nm in length, with axial ratios of 1.2 to 1.7. The virion contains a noninfectious, linear; A+T-rich, double- stranded DNA genome than can vary from 130 to 300 kbp depending on the poxvirus species. Several characteristics of pox viruses contribute to its wide use as an expression system which includes (i) relatively simple methods of recombinant virus construction, (ii) a wide choice of cell types, (iii) cytoplasmic expression eliminating special requirements for nuclear processing and transport of RNA, (iv) relatively high expression levels and (v) controlled and synchronized expression by choice of promoters. Proteins synthesized by vaccinia virus vectors are processed and transported in accord with their primary structure and the inherent capabilities of the host cell. Generally, the expression level is considerably higher than that of conventional eukaryotic transfection systems.
The numerous examples, in which immunization of experimental animals (from mouse to chimpanzee) with recombinant vaccinia viruses that express one or more genes of a DNA or RNA virus have provided partial or complete protection against challenge (94-96). In many cases, protection was correlated with neutralizing antibody against viral envelope proteins expressed by the recombinant vector. In other cases, vaccination provided a priming effect and protection was associated with an anamnestic antibody response (97). The ability of poxviruses to induce strong CTL responses has led to consideration of their use as attenuated and non-replicating vectors. Some members of the poxvirus family have a naturally restricted host range, which can provide increased safety. Avian poxviruses were initially considered as vectors for birds (150-152) since they do not replicate in mammals. Subsequent studies, however, indicated that expression of recombinant genes occurs in mammalian cells which induced immune response in mammals (153).
Recombinant poxviruses are capable of protectively immunizing against diseases of human and veterinary importance. Examples include the use of vaccinia virus recombinants to protect cattle against vesicular stomatitis virus (159) and rinderpest (160, 161), chickens against influenza virus (162), raccoons and foxes against rabies virus (163-166), monkeys against simian immunodeficiency virus and mice against Japanese encephalitis virus. Examples of the use of other poxvirus vectors for veterinary purposes include a raccoon poxvirus vector to protect raccoons against rabies virus (156); a capripoxvirus vector to protect cattle against rinderpest (168), sheep against blue tongue virus, goats against peste des petits ruminants virus; swinepox vectors to protect pigs against Aujeszky disease (pseudorabies) (158); fowlpox vectors to protect chickens against influenza virus (169), infectious bronchitis, Newcastle disease virus (152, 170-172), and infectious bursal disease virus (173); canarypox virus to protect dogs against canine distemper virus (154), dogs and horse against west nile virus, ponies against equine herpes virus, horse against African horse sickness virus, rabbits against rabbit hemorrhagic disease virus, sheep against blue tongue virus ; and pigeonpox viral vectors to protect chickens against Newcastle disease virus (174).
Recombinant pox viruses expressing structural genes of FMDV have been studied extensively. Recombinant vaccinia virus has been shown to express cDNA cassettes encoding FMDV P1-2A3C which resulted in forming FMDV empty capsids in cultured cells. Recombinant vaccinia virus expressing FMDV capsid precursor conferred protection against FMDV in mice. Fowlpox virus expressing P1-2A3C induced protective immune response in swine and guinea pigs.
Buffalopox virus (BPV) is a close variant of vaccinia virus belongs to the genus Orthopoxvirus (OPV) family Poxviridae and subfamily Chordopoxvirinae (Murphy et al., 1999). Attenuation of the virulent buffalopox virus by continuous passaging (Mohanty et al., 1989) and its use as a vaccine candidate against pox has been demonstrated. However the applicability of BPV as a recombinant viral vector for expressing heterologous protein has not yet been investigated. Not much scientific information is available on the attenuation of buffalopox virus and development of recombinant virus. Being a closely related virus to vaccinia, buffalopox viruses would be also an attractive recombinant viral vector for the expression of heterologous antigens.
Thus the purpose of this study was aimed at following objectives.
To develop a transfer vector for the development of recombinant buffalopox virus
To clone the FMDV capsid precursor and 3C protease in to the transfer vector
To develop recombinant buffalopox virus expressing cloned FMDV capsid precursor and 3C in cultured cells
To study the immunogenecity of the recombinant buffalopox virus expressing FMDV capsid antigen and 3C
To determine the safety of recombinant buffalopox virus expressing FMDV capsid antigen and 3C
To determine the efficacy of recombinant buffalopox virus expressing FMDV capsid antigen and 3C in buffaloes.