WO2019021305A1

WO2019021305A1 - Vaccine against foot-and-mouth disease

Info

Publication number: WO2019021305A1
Application number: PCT/IN2018/050479
Authority: WO
Inventors: Nagendra Ramachandra HEGDE; Pavuluri Panduranga RAO; Umakantha MADALA
Original assignee: Ella Foundation
Priority date: 2017-07-24
Filing date: 2018-07-24
Publication date: 2019-01-31

Abstract

Disclosed are a vector based foot and mouth disease virus vaccine formulation for prevention against foot and mouth disease infections in domestic as well as free-ranging wild or captive or wild cloven footed animals selected from cattle, buffaloes, sheep, goats, pigs, deer, camel, elephants and wild ruminants and boar including humans. The vaccine formulation of the present invention are made through recombinant techniques using a foreign vector selected from members of families such as Poxviridae and Herpesviridae, rhabdoviridae that caries the potentially immunogenic gene sequences of foot and mouth disease virus. Methods of making the said vaccine formulations are also accordingly disclosed.

Description

VACCINE AGAINST FOOT-AND-MOUTH DISEASE

FIELD OF INVENTION The invention relates to the field of vaccine manufacture. More particularly the present invention relates to the field of vaccine production using vector based vaccine antigens. The present invention relates to recombinant engineering techniques to generate vector based vaccine antigens against foot and mouth disease infections.

BACKGROUND OF THE INVENTION Foot-and-mouth disease (FMD): Foot-and-mouth disease (FMD) is a highly contagious viral disease of wild and domestic cloven-footed animals, including cattle, buffaloes, sheep, goats, pigs and deer. The disease is characterized by high morbidity and rapid spread. Clinical symptoms include fever followed by vesicular lesions in and around the mouth, hooves and teats. The animals show severe ulcerations in the affected regions, leading to their inability to eat and move. Most of the animals recover from the disease with very low mortality rates. However, high mortality can be observed in young animals as well as adult animals which are simultaneously infected with certain viral and bacterial infections. Consequent to infection and recovery, the productivity of the animals is reduced drastically through reduction in milk or meat production and draft power. Recovered animals may remain debilitated for several months to years due to persistent infection. Since presence of FMD imposes restrictions on trade of animals and animal products, the losses to livestock production are compounded. Thus the disease is economically devastating, and can therefore be an agent for biowarfare (Grubman and Baxt 2004; Mahy 2004; Parida 2009; Rodriguez and Grubman 2009; Rodriguez and Gay 2011; Ludi and Rodriguez 2013).

Foot-and-mouth disease virus (FMDV): FMD is caused by FMDV, which is a non- enveloped virus belonging to the Aphthovirus Genus of Picornaviridae Family. Its genome contains a single-stranded positive sense non-segmented RNA. The open reading frame is bound by 5' and 3' un-translated regions which play critical roles in replication, transcription, translation and packaging of the genome. The genome is transcribed and translated as a single polyprotein which is then cleaved by viral proteases to release the 12 individual proteins. Based on their participation in the formation of the virion, the proteins and the genome encoding them have been divided into structural and non- structural regions. Accordingly, the left one third of the genome encodes structural proteins, and the rest of the genome encodes the non- structural proteins.

The protein coding sequence reads L^pro-lA-lB-lC-lD-2A-2B-2C-3A-3B-3C-3D. Viral proteins (VP) VP1, VP2, VP3 and VP4 are encoded by ID, IB, 1C and 1A, respectively. The polyprotein is cleaved by L^pro and 2A, both of which cleave at their respective C-termini, and 3C, which cleaves at all the other junctions except VP4/VP2. The VP4/VP2 junction remains uncleaved until the genomic RNA is encapsidated, but this cleavage is required for infectivity of the released virus. 3B encodes a protein which is covalently linked to the 5' end of the genome and is required for priming replication. Whereas 3D functions as the polymerase, the function of 2A, 2B, 2C, and 3 A are poorly understood. L^pro also plays an important role in shut-off of host macromolecular synthesis, facilitating the production of large amount of virus in infected cells (Grubman and Baxt 2004).

Structurally, FMDV is an icosahedron consisting of just four proteins encasing the genome. The proteins VP1, VP2 and VP3 form an inter- linked cage-like outer structure which folds upon the slightly internal scaffold of VP4, which packages the RNA. The structure is highly labile to heat and acid pH (Grubman and Baxt 2004). A small region in VP1 acts both to mediate entry of the virus into cells and in stimulating a protective antibody responses (Borrego et al. 1995; Mateu et al. 1995). This region and VP1 are therefore the target of strategies for designing new and novel vaccines. FMD has been eradicated in several countries through extensive control and prevention strategies. However, the disease is still endemic to many Asian and African countries. Globally, FMDV exists as seven different serotypes which are defined by the property of antisera to one serotype being unable to neutralize another serotype. The serotypes are named A, O, C, Asia-1 and South African Territories (SAT) -1, -2 and -3. Whereas serotype C has not been reported anywhere for several years now, the SAT serotypes are mostly restricted to African continent, and the most common serotype throughout the world is O (Parida 2009; Zhang et al. 2011). The serotypes are determined by the amino acid sequences in a region of VP1 protein which overlaps with its receptor binding and neutralizing epitope regions. Further, multiple subtypes exist in each serotype and in some cases, no protection is afforded between the subtypes even though they belong to the same serotype (Paton 2005).

Immune responses to FMDV: Immunity to FMDV encompasses both innate and adaptive responses. Recent research suggests that interferons may be crucial for inhibiting the virus during the early phase of infection and other cytokines may play a role in potentiating immune responses (Brown et al. 2000a; Brown et al. 2000b; Alexanders et al. 2002; Barnett et al. 2002; Rigden et al. 2003; Parida 2009). As far as humoral immunity is concerned, the mucosal B cell and antibody response occurs earlier than the systemic appearance of IgM followed by IgG (Pega et al. 2013). Whereas serum IgG levels are reflective of the elicitation of a protective response, mucosal IgA and IgG are likely to be critical for clearing infection (Mulcahy et al. 1990; Salt et al. 1993; Salt et al. 1996; Capozzo et al. 1997). Neutralizing antibodies to VP1 are the basis of protection against FMD, but VP3 and VP2 may also be the targets (McMollough et al. 1992; Salt et al. 1993; Grubman and Baxt 2004; Mahapatra et al. 2012). Compared to humoral immunity, the role of cell-mediated immunity, specifically the importance of T-helper and cytotoxic T lymphocyte responses, is less well understood. Both whole virus vaccines and virus-like particles elicit T cell proliferation, and stimulating CD4+ T cells may be critical for the induction of optimum antibody responses (Juleff et al. 2009; Carr et al. 2013; Sun et al. 2013). T cell responses have been proposed to contribute to long- term (memory) immunity but there is no concrete evidence to support this premise. FMDV- specific CD8+ cytotoxic T cell proliferation and IFN-γ production has also been demonstrated, and has been shown to protect animals in the absence of significant antibody response (Childersone et al. 1999; Patch et al. 2013). Thus it appears that innate responses and cell-mediated immunity contribute to early as well as long-term protective responses. It has been recently shown that besides antibodies, the ability of CD4+ T cells to secrete interferon-γ when re-stimulated in vitro can be correlated with protection (Oh et al. 2012).

Infection with one serotype of FMDV results in fairly long lasting protective immunity against the homotype virus in the wild (Alexandersen et al. 2003). Partial protection against heterotypic viruses may be observed, but is never complete. Hence it is essential to develop immunity to different serotypes from strains or subtypes circulating in a particular geographical area. Vaccines against FMD: The only vaccine which is employed worldwide for prevention, control and eradication of FMD is the binary ethylenimine inactivated whole virus antigen. Both monovalent and polyvalent vaccines are used based on whether the situation calls for outbreak control or routine vaccination. The killed vaccines have been effective in the eradication of FMD in several countries. However, the killed vaccine fails to elicit adequate mucosal immunity, does not provide immunity lasting more than 4-6 months, and requires large doses and several boosters. Moreover, vaccination does not prevent infection, potentially resulting in persistent and carrier status among vaccinated animals (Grubman and Baxt 2004; Doel 2009; Rodriguez and Gay 2011). In addition, preparation of the antigen requires that live virus be propagated in large scale during production, and this can only be done under the stringent biosafety level 3 conditions. The maintenance of any bio- containment facility plays a critical role in biosafety. For example, the lack of proper maintenance increases the risk of virus escape; causes vaccine-related outbreaks linked to improper inactivation of virus and this has been demonstrated in several incidents which occurred worldwide. High-containment laboratories are sophisticated facilities and require specialized expertise to design, construct, operate, and maintain. Because they are intended to contain dangerous micro-organisms, usually in liquid or aerosol form, even minor structural defects - such as cracks in the wall, leaky pipes, or improper sealing around doors - may have severe consequences. Even with a proper biosafety measure human errors can never be completely eliminated. The human component accounts for the majority of accidents in high- containment laboratories. This risk persists, even in the most modern facilities and with the latest technology and highlights the importance of standard operational procedures and training of staff. Furthermore, many of the developing countries which produce such killed vaccines do not purify the virus particle, leading to the vaccine containing non-structural proteins. These NSPs also elicit an antibody response which is indistinguishable from those of infected animals. Therefore, it is difficult to differentiate vaccinated and infected animals.

Several alternative approaches have been attempted for the development of vaccines to combat FMD. These include peptides, inactivated antigen obtained from modified virus, DNA vaccines, virus-like particles, modified live virus vaccines and viral vectored vaccines.

Based on the identification of the neutralizing antibody and T-helper cell epitopes, peptides were designed to serve as vaccines either alone or in combination, in some cases along with other proteins which provided adjuvanticity. Purified whole proteins or fusion proteins expressed through bacteria, yeast, silkworm or plants have also been tested as vaccines (Bachrach et al. 1975; Kleid et al. 1981; DiMarchi et al. 1986; Mulcahy et al. 1990; Kit et al. 1991; Kitson et al. 1991; Mulcahy et al. 1991; Mulcahy et al. 1992; Grubman et al. 1993; Krebs et al. 1993; Meloen et al. 1995; Taboga et al. 1997; Wigdorovitz 1999a; Wigdorovitz 1999b; Blanco et al. 2001; Dus Santos et al. 2002; Wang et al. 2002; Rodriguez et al. 2003; Tami et al. 2003; Wang et al. 2003; Beignon et al. 2005; Challa et al. 2007; Cubillos et al. 2008; Pan et al. 2008; Shao et al. 2011). However, neither peptides nor proteins can protect animals completely against challenge or for longer duration, despite inducing good neutralizing antibodies, and the virus could easily produce escape mutants. Virus-like particles, which contain a proteinaceous virion structure devoid of viral genome, have also been explored as vaccines, but have shown inconsistent results or partial effectiveness in protecting animals even under experimental conditions (Rowlands et al. 1975; Rweyemamu et al. 1979; Roosien et al. 1990; Lewis et al. 1991; Grubman et al. 1993; Li et al. 2008; Pena et al. 2008; Li et al. 2011).

Live attenuated vaccines were attempted initially by passaging wild virus in unnatural hosts or cells, but these have not been successful because of unstable phenotype, differences in attenuation among different species, inconsistency in inducing protective immunity, and concerns of reversion to virulence (Bachrach 1968; Brooksby 1982; Saiz et al. 2002). Other live attenuated vaccines have been derived based on the understanding of the obligate role of certain proteins or sequences in virus biology or virulence. A genetically modified virus deleted of L^pro or its functional domains were shown to be attenuated, stable, avirulent and able to produce protective immunity, albeit incomplete; this virus was avirulent to cattle but could cause mild disease and spread to in-contact pigs (Piccone et al. 1995; Mason et al. 1997; Almeida et al. 1998 ; Chinsangaram et al. 1998; Chinsangaram et al. 2003; Segundo et al. 2011). Because of the concern about reversion to virulence, such vaccines have also been proposed to be used as killed vaccines, thus producing inactivated vaccine from an attenuated strain, which may be handled in lower containment than biosafety level 3 (Grubman and Baxt 2004; Uddowla et al. 2012). A second genetically modified live attenuated virus was produced by deleting the essential receptor binding sequence of the virus. Although this virus failed to produce disease in pigs, elicited neutralizing antibodies, and was able to completely protect the animals (McKenna et al. 1995), evolution of the virus to obviate this receptor mediated entry could not be ruled out (Grubman and Baxt 2004). Vaccine compositions containing segments of viral genome have also been proposed to function similar to live attenuated vaccines (Rodriguez-Calvo et al. 2010). DNA vaccines containing the structural proteins, either all the four or just VPl, with or without 3C protease, and with or without genes encoding proteins which boost immune responses, have been tested in both laboratory animals and in large animals. They have been shown to elicit both humoral and cell-mediated immunity, but their poor immunogenicity has led to the requirement of large and repeated doses; added to this is the concern about integration of the DNA into host chromosome, possibly leading to unintended consequences, as well as partial effectiveness or sometimes, negative effects (Benvenisti et al. 2001; Cedillo-Baron et al. 2001; Wong et al. 2002; Li et al. 2006; Park et al. 2006; Fan et al. 2007; Mingxiao et al. 2007; Xiao et al. 2007; Li et al. 2008; Zhang et al. 2008; Borrego et al. 2011; Ganges et al. 2011; Fowler et al. 2012).

Viral vectored vaccines have been the best vaccines among the novel approaches vectors expressing foot and mouth disease virus proteins is selected from members of families Adenoviridae, Poxviridae and Herpesviridae, rhabdoviridae.. Adeno, pox and herpes viruses expressing FMDV structural proteins, with or without 3C, and with or without cytokine genes, have been shown to induce protective responses (Pacheco et al. 2005; Mayr et al. 1999; Sanz-Parra et al. 1999a; Sanz-Parra et al. 1999b; Berenstein et al. 2000; Mayr et al. 2001; Moraes et al. 2002; Moraes et al. 2003; Wu et al. 2003; Qian et al. 2004; Grubman et al. 2005; Zheng et al. 2006; Hong et al. 2007; He et al. 2008; Yao et al. 2008; Moraes et al. 2011). The best among the vectored vaccines have been the adenovirus vectors, mostly on the background of human adenovirus type 5 (Ad5). Adenoviruses present several advantages: (a) high expression of the transgenes, (b) multiple species tropism (of Ad5), (c) easy construction and manipulation, (d) capacity to clone up to 8 kbp of DNA, (e) their natural tropism to mucosal surfaces (similar to FMDV), (f) mild, if any, infection, even in the natural host, (g) compatibility with tests to differentiate vaccinated and infected animals (because non- structural genes can be excluded), (h) ability to induce both humoral and cell- mediated immunity, and (i) compatibility with other vaccines, including classical killed vaccines, in prime boost strategies (Rodriguez and Grubman 2009). Recombinant Ad5- based FMDV vaccines have shown great promise in immunogenicity and protection studies in both laboratory animals and natural hosts under experimental conditions (Grubman et al. 2010). Importantly, they can be used to induce early protective immunity mediated by both CD4+ and CD8+ T cells (Sanz-Parra et al. 1999; Guzman et al. 2010; Moraes et al. 2011; Patch et al. 2011).

The below invention deals with a novel adenovirus vectored vaccine construct for use as vaccine against FMDV in all target animals.

Brief Description of Figures: Figure 1: Diagrammatic representation of FMDV genome (Example- 1): The genome is a linear, positive sense single-stranded polyadenylated RNA of about 7500 nucleotides, and is composed of a single ORF encoding a polyprotein. The viral polyprotein is cleaved into individual structural (PI) and non- structural (P2 and P3) proteins by its own proteases (L, 2A and3C). Figure 2: Schematic representation of the FMDV genes incorporated in the adenovirus constructs (Example-3): P12A region of serotypes A and O were synthetically obtained along with 3C and cloned into shuttle plasmid (pDC516). This constructs will represent the elements which are used for controlled expression and recombination during recombinant adenovirus generation. Figure 3: Schematic representation of the control elements to regulate protein expression via the recombinant adenovirus (Example-4): AdMax Hi-IQ system was obtained from Microbix Bioscience Inc., Canada for generation of replication-defective recombinant adenovirus. The transgenes are under the control of the bacterial lac operator and the 293 IQ cells express the lac repressor so that the recombinant gene is not active during the generation of Ad vectors as the repressor binds to the operator and does not allow transcription. On the other hand, in any other cells where the repressor is absent, the proteins are expressed. This avoids cytopathogenicity due to potentially toxic viral proteins (e.g., 3C), and allows the generation of higher titers of high transgene expressing adenoviruses.

Figure 4: Expression of FMDV proteins through the recombinant Ad vectors (Example-6): U373 cells were infected with recombinant adeno viruses. Cells were harvested 24 h post- infection and cell lysates were subjected to SDS-PAGE, blotted, and the expressed proteins were detected using rabbit polyclonal antibodies. Uninfected U373 cells were used as negative control. The location of the proteins is indicated.

Figure 5: Purification of recombinant adenoviruses (Example-7): Recombinant adenovirus infected 293IQ cell extracts were loaded onto Q-Sepharose columns and eluted using buffers containing various concentrations of sodium chloride. The top line graph shows OD280 values (y-axis) of the fractions eluted (x-axis) in various elution buffers. In this illustration, the virus eluted in fractions 5 and 6 of elutin buffer 3.

Figure 6: Immunogenicity testing of adenovirus vectored vaccines in laboratory animals: Serum Neutralization Test (SNT) (Example-9): The sera from the animals injected with recombinant adenoviruses were tested by SNT for the presence of FMDV neutralizing antibodies using respective serotypes.

OBJECTIVES OF THE INVENTION:

One objective of the invention is to provide a vector based vaccine against foot and mouth disease virus that minimizes the risk of foot and mouth disease outbreak due to escape of virus from the vaccine production unit. One more objective of the invention is to provide a novel vaccine formulation against foot and mouth disease virus, which can be produced in a cost-effective BSL-2 facility as against the present practices wherein the production of commercial inactivated whole FMD virus vaccine needs stringent BSL-3 facility. Another objective of the invention is to provide a cost-effective and affordable foot and mouth disease vaccine formulation.

One another objective of the invention is to provide a method of making a vector based vaccine against foot and mouth disease using a controlled expression system to increase high expressing adenovirus particles containing the desired immunogenic sequences against foot and mouth disease infections. One further objective of the invention is to provide a method of differentiating infected from vaccinated animals (DIVA) against foot and mouth disease infections using the vector based vaccine formulation of the present invention, the said vaccine formulation being an adenovirus vector based vaccine formulation against FMD.

SUMMARY OF THE INVENTION

According to the invention, the recombinant adenovirus is produced using a specialized system, where

a. The FMDV genes are expressed under controlled conditions.

b. The sequence of FMDV encompasses the region containing genes for the proteins VP4, VP2, VP3, VPl and 3C, as shown in Example 1, and where the sequences of the genes encoding the proteins are shown in Example 2, and the genes are incorporated into adenovirus vector as shown in Example 3.

c. The FMDV genes are under the control of murine cytomegalovirus immediately early promoter, which is immediately downstream of the lac operator, and an intron is included between the operator and the promoter, as shown in Example 4, in order to enhance stability of the RNA which produces the FMDV protein(s). d. A special kind of cell line, which stably expresses the bacterial lac repressor protein, is used for the production of the recombinant adenoviruses expressing FMDV proteins, so that (i) the lac repressor expressed in these cells suppresses the expression of FMDV genes whose promoter is under the lac operator, and (ii) the FMDV proteins can be expressed in any other cell lacking the ability to produce the lac repressor protein, as shown in Example 4.

e. The adenovirus carrying FMDV genes is generated by recombination between an adenovirus genomic plasmid and the shuttle plasmid containing the FMDV genes, and recombination between the plasmids is mediated by a recombinase, which is expressed from the genomic plasmid, as shown in Example 5.

2. The nucleic acid sequence encoding FMDV proteins can be

a. Derived from any of the many strains of FMDV belonging to any of the serotypes, particularly A, O, Asia-1 and C, but also the South African Territory (SAT) strains.

b. Full length or parts thereof, especially, truncated at the N-terminus of the genome. c. Any combination of structural genes from any FMDV strain belonging to any serotype.

d. With or without the 3C protease. Since, the 3C protease can cleave the PI into 1A, IB, 1C and ID structural proteins individually, therefore in absence of 3C, the structural genes are cloned and expressed using individual promoters. Therefore it is also possible to practice the present invention with individual structural proteins separated by individual promoters inserted into the Ad5 without presence of the 3C gene as well.

e. Obtained entirely synthetically to correspond to the amino acid sequence of the relevant protein or region of FMDV.

f. Modified so as to increase the amount or the stability of either the resultant RNA or protein.

The recombinant adenovirus is produced in large-scale in cells grown in adherent or suspension systems, or any sequential combination.

The recombinant adenovirus produced through the above methods, produces FMDV proteins, as shown in Example 6, and is an immunogenic composition, where

a. The composition is purified by chromatography techniques, as shown in Example 7.

b. The composition can induce antibodies against FMDV, as shown in Example 8, and these antibodies have the ability to neutralize FMDV as shown in Example 9. c. The composition can be used to prevent disease and/or infection with FMDV. The immunogenic composition as described above can be used to immunize domestic animals, including but not limited to cattle, sheep, goats, and pigs, as well as free-ranging wild or captive wild cloven-footed animals, including but not limited to deer, camel, elephants etc. against FMD.

The immunogenic composition as described above can be injected intramuscularly, subcutaneously, intradermal, intravenously, orally, nasally, vaginally or any other route, either by a single route or any of the combinations.

The immunogenic composition as described above can be administered in a prime -boost regimen where any vaccine against FMD, including live attenuated, killed, vectored, recombinant, DNA, subunit or other vaccine can be administered first followed by booster immunizations with the claimed composition, or vice versa, and repeated in any sequence for as long as required to develop robust immunity in the host species being targeted.

8. The immunogenic composition as described above can be mixed with other similar vaccines which contain antigenic components from various serotypes or subtypes of FMDV so that all genotypes prevailing in the targeted geographic area are represented in the vaccine mix, or with other vaccines for infectious diseases in the targeted animal species.

9. The immunogenic composition as described above does not induce antibodies to some non-structural proteins of FMDV, so that tests designed to differentiate vaccinated and infected animals based on the detection of antibodies to certain non-structural proteins can be employed in combination with immunization with the claimed composition.

10. The viral vector can be an adenovirus from other species, including but not limited to bovine, ovine, caprine, porcine, rhesus, chimpanzee or avian adenoviruses, or any other viral vector, in combination with the controlled system of expression as outlined above.

11. The immunogenic composition as described above but with a specific truncation/deletion in the N-terminus of the antigenic component.

DETAILED DESCRIPTION OF THE INVENTION

The invention describes preparation of an adenovirus vectored vaccine for FMD. Specifically, the invention describes a human adenovirus type 5 which is engineered to express FMDV proteins. The vaccine can be used to elicit immune responses against FMDV in animals. The vaccine is prepared by propagating the recombinant adenovirus in 293IQ human embryonic kidney cells stably expressing lac repressor. The vaccine virus can be clarified and concentrated or purified, and if required, mixed with a suitable adjuvant. Further, the vaccine can be administered either parenterally (intramuscular, subcutaneous, intravenous, intraperitoneal or other routes) or mucosally (oral, mucosal, vaginal or other routes) to species susceptible to infection with FMDV. The preparation can be used to protect against FMD in various species. Unlike the other adenovirus vectored vaccines, this invention does not use a constitutive gene expression system, but employs a controlled gene expression system. The controlled expression system leads to the production of stable high expressing adenoviruses. Unlike the conventional inactivated whole FMDV vaccine which has been made commercially available till date only in a BSL-3 facility, the adenovirus- vectored FMD vaccine can be produced in BSL-2 facility, which reduces the cost of production. This vaccine can also be used in FMD-free countries without the fear of FMD outbreaks. Differentiating the infected from vaccinated animals is crucial for the FMD control program. In this regard, the adenovirus-vectored FMD vaccine can be effectively used to determine the infected animals.

EXAMPLES

1. Diagrammatic representation of FMDV genome: The FMDV genome is transcribed as a single RNA and translated into a single polyprotein which is then cleaved by the viral proteases L, 2A (both at each of their C termini) and 3C (at all other junctions as indicated by the arrow). VP1, VP2, VP3 and VP4 constitute the structural proteins and the rest or the proteins are non-structural. The individual proteins are shown as blocks, and the structural proteins and the 3C protease have been circled. VP4-VP2-VP3- VP1-2A is called the P12A region, 2B and 2C are named the P2 region, and 3A-3B- 3C-3D form the P3 region.

2. Representative nucleotide sequence of P12A region of FMDV serotypes A and O of 3C gene: Complete nucleotide sequence of the five proteins (1A, IB, 1C, ID and 2A) encoded in the P12A region are shown. The constructs of the sequences are shown in Example 3. A short stretch of 2B (6 nucleotides, also shown separately) is included in order to provide the cleavage site for 2A. The nucleotide sequence of 3C gene is the same irrespective of the serotype.

Sequence ID No.-l (P12A3C-Awt) i.e. Serotype-A wildtype nucleic acid sequence

GGAGCT GGGC AATCC AGTCC GGC GACC GGGT CGCAGAACC AGTC AGGT AACACT GGAAGC ATCATTAACAACTACTACAT GCAACAATACCAGAATTCTATGGACACACAACTT GGGGAC AACGCCATTAGCGGAGGTTCCAACGAGGGTTCCACAGACACCACTTCCACCCACACAACC AACACCCAAAACAACGATTGGTTCTCAAAATTGGCCAGCTCTGCCTTTAGCGGACTTTTC GGCGCTCTTCTCGCCGACAAGAAGACCGAGGAGACTACTCTCCT GGAGGACCGCATTCTT ACCACCCGCAACGGACACACTACCTCCACAACTCAATCGAGTGTAGGAGTCACCTACGGG TACTCCACTGGAGAGGACCACGTTTCCGGACCCAACACATCTGGTTTGGAAACGCGGGT G GT GCAGGCAGAAAGATTCTTTAAGAAGCACCTGTTT GACT GGACAGCGGACAAGGCATTT GGACACTTGGAAAAGCTGGAACTCCCCACTGACCACAAGGGCGTCTACGGACACCT GGT G GACTCTTTTGCTTACATGAGGAATGGCTGGGACGTGGAGGTGTCTGCCGTTGGCAACCAG TTCAACGGCGGATGTCTCCTCGT GGCCATGGTTCCT GAGT GGAAGRAGTTCACCACGCGT GAGAAGTACCAGCTCACCTT GTTCCCCCACCAATTCATTAACCCCAGAACCAACACGACC GCTCACATCACGGTCCCCTACCTTGGT GTGAACCGGTACGACCAGTACAAGAAGCACAAA CCATGGACGTTGGTT GTAAT GGT GGTTTCGCCGCTTACCAATGCCGGCATTGGTGCCACT CAAATCAAGGTCTACGCCAACATCGCCCCGACCTACGTCCACGT GGCCGGTGAGCTCCCG TCGAAAGAGGGGATCGTACCGGTTGCGTGTGCGGACGGTTATGGCGGTTTGGTGACCACG GACCCGAAAACAGCT GACCCTGTTTAT GGCATGGTGTACAACCCCCCTCGGACAAATTTT CCTGGGCGGTTTACAAACCT GTT GGACGTGGCGGAGGCCT GCCCCACCTTCCTCTGTTTC GACAACGGGAAACCGTACGTT GAGACAAGAACGGATGAGCAACGTCTTCTGGCCAAATTC GAC GT T T CAT TGGCT GC AAAAC AC AT GT C AAAC ACC T AT C T T T C AGGGAT AGC AC AGT AC TACGCACAGTACTCT GGCACCATCAACCTCCACTTCATGTTTACTGGCTCCACTGACTCA AAGGCCCGCTACATGGTGGCGTACGTCCCGCCTGGT GTGGAAACACCGCCGGACACGCCT GAGAAAGCTGCACACTGCATCCACGCT GAGT GGGACACAGGGTTGAACTCCAAATTCACC TTTTCTATCCCGTACGTGTCTGCTGCAGACTACGCGTACACCGCGTCT GACAAGGCAGAA ACAACAAACGTACAGGGATGGGTCTGCATTTACCAGATCACCCACGGGAAGGCTGAAAAT GACACTCTGGTCGTGTCGGCCAGCGCCGGCAAAGACTTTGAGTT GCGTCTCCCGATTGAC CCCCGCGCACAAACCACCACGGCCGGGGAATCTGCAGACCCAGTCACCACCACT GTTGAG AACTACGGCGGTGAGACACAAGTCCAGCGGCGTCACCACACTGACGTCAGCTTCATAAT G GACAGATTTGT GAAG AT T GG AAC C AC T AAC CCCACACATGTCATT GAC C T C AT GC AAAC C CACCAACACGGGCTGGTGGGTGCCCTGTTGCGTGCT GCCACGTACTACTTCTCCGACTT G GAGATCGTGGTTCGTCACAGCGGCAACCTGACATGGGTACCCAATGGAGCACCTGAGGCA GCCCTGTCCAACACAGGAAACCCTACCGCCTACAACAAAGCGCCGTTCACGAGACTTGCG CTCCCCTACACTGCGCCACACCGCGTGTTGGCAACAGTGTACAACGGGACGAACAAGTAC TCCGCGGCCAGTGGGCGCACACGGGGT GACCTGGGGCAGCTCGCAGCGCGAATTGCCGCC CAACTTCCTGCCTCTTTCAATTTTGGT GCAATCAAGGCTGACGCCATCCACGAGCTTCTC GT GCGCATGAAGCGTGCCGAACTCTACTGCCCCAGACCACTGTT GGCGGTGAAGGTCTCG TCTCAAGACAGACACAAACAGAAGATCATTGCACCAGCAAAACAGCTCTTGAACTTTGAC CT GCTCAAGTTGGCGGGAGACGTTGAGTCCAACCCCGGGCCCTTCAGT GGAGCCCCACCG ACTGACCTGCAAAAGATGGTCAT GGGCAACACGAAGCCTGTTGAGCTCATCCTCGACGGG AAGACAGTAGCTATCTGCTGT GCTACTGGAGTGTTCGGCACT GCCTACCTCGTGCCTCGT C AT CT T T T C GC AGAGAAGT AT GAC AAGAT CAT GT T GGAC GGT AGAGCC AT GAC AGAC AGT GACTACAGAGTGTTT GAGTTTGAGATAAAAGTAAAAGGACAAGACATGCTCTCAGACGCC GCGCTCATGGTGCTTCACCGTGGGAATCGCGTGCGGGACATCACGAAGCACTTCCGTGAT GT GGCAAAAATGAAGAAAGGCACCCCCGTCGTTGGCGTGATTAACAACGCCGATGTCGGG AGACTGATTTTCTCT GGTGAGGCCCTTACCTACAAGGACATTGTAGTGTGCATGGACGGA GACACCATGCCTGGCCTCTTTGCCTACAAAGCCGCCACCAAGGCTGGCTACT GTGGAGGA GCCGTTCTTGCCAAGGACGGTGCCGAGACATTCATCGTTGGCACTCACTCCGCAGGTGGC AATGGAGTTGGATACTGCTCCTGTGTTTCCAGGTCCATGCTCAT GAAAAT GAAGGCACAC AT C GAC C C T G AAC CACACCACGAG

Sequence ID-2 (P12A3C-Atd) i.e. Serotype-A truncated nucleic acid sequence

GAATTCTATGGACACACAACTTGGGGACAACGCCATTAGCGGAGGTTCCAACGAGGGTTC CACAGACACCACTTCCACCCACACAACCAACACCCAAAACAACGATTGGTTCTCAAAATT GGCCAGCTCT GCCTTTAGCGGACTTTTCGGCGCTCTTCTCGCCGACAAGAAGACCGAGGA GACTACTCTCCT GGAGGACC GCATTCT T ACC ACCCGCAAC GGAC ACACTACCTCCACAAC TCAATCGAGT GTAGGAGTCACCTACGGGTACTCCACTGGAGAGGACCACGTTTCCGGACC CAACACATCT GGTTT GGAAACGCGGGT GGTGCAGGCAGAAAGATTCTTTAAGAAGCACCT GTTTGACTGGACAGCGGACAAGGCATTTGGACACTTGGAAAAGCTGGAACTCCCCACTGA CCACAAGGGCGTCTACGGACACCTGGT GGACTCTTTTGCTTACATGAGGAAT GGCT GGGA CGTGGAGGTGTCTGCCGTTGGCAACCAGTTCAACGGCGGATGTCTCCTCGTGGCCATGGT TCCTGAGTGGAAGRAGTTCACCACGCGTGAGAAGTACCAGCTCACCTT GTTCCCCCACCA AT T CAT T AAC C C C AG AAC C AAC AC GAC CGCTCACATCACGGTCCCCTACCTTGGTGT G AA CCGGTACGACCAGTACAAGAAGCACAAACCATGGACGTTGGTTGTAAT GGTGGTTTCGCC GCTTACCAAT GCCGGCATTGGTGCCACTCAAATCAAGGTCTACGCCAACATCGCCCCGAC CTACGTCCACGTGGCCGGTGAGCTCCCGTCGAAAGAGGGGATCGTACCGGTT GCGT GTGC GGACGGTTAT GGCGGTTTGGTGACCACGGACCCGAAAACAGCTGACCCTGTTTATGGCAT GGTGTACAACCCCCCTCGGACAAATTTTCCT GGGCGGTTTACAAACCT GTTGGACGTGGC GGAGGCCTGCCCCACCTTCCTCT GTTTCGACAACGGGAAACCGTACGTTGAGACAAGAAC GGATGAGCAACGTCTTCTGGCCAAATTCGACGTTTCATTGGCTGCAAAACACATGTCAAA CACCTATCTTTCAGGGATAGCACAGTACTACGCACAGTACTCTGGCACCATCAACCTCCA CTTCAT GTTTACTGGCTCCACTGACTCAAAGGCCCGCTACATGGTGGCGTACGTCCCGCC T GGT GT GGAAACACC GCCGGACACGCCT GAGAAAGCT GCACACT GCATCCAC GCTGAGT G GGACACAGGGTTGAACTCCAAATTCACCTTTTCTATCCCGTACGTGTCTGCT GCAGACTA C GC GT AC ACC GC GT C T GAC AAGGC AGAAAC AAC AAAC GT AC AGGGAT GGGT C T GC AT T T A CCAGATCACCCACGGGAAGGCTGAAAATGACACTCTGGTCGT GTCGGCCAGCGCCGGCAA AGACTTTGAGTTGCGTCTCCCGATTGACCCCCGCGCACAAACCACCACGGCCGGGGAATC T GC AGACCC AGT C AC C ACC ACT GT T GAGAAC T AC GGC GGT GAGAC AC AAGT C CAGC GGCG TCACCACACT GACGTCAGCTTCATAAT GGACAGATTTGTGAAGATTGGAACCACTAACCC CACACATGTCATTGACCTCATGCAAACCCACCAACACGGGCT GGT GGGT GCCCTGTTGCG TGCTGCCACGTACTACTTCTCCGACTT GGAGATCGT GGTTCGTCACAGCGGCAACCTGAC AT GGGTACCCAATGGAGCACCTGAGGCAGCCCTGTCCAACACAGGAAACCCTACCGCCTA CAACAAAGCGCCGTTCACGAGACTTGCGCTCCCCTACACT GCGCCACACCGCGTGTTGGC AACAGT GTACAACGGGACGAACAAGTACTCCGCGGCCAGTGGGCGCACACGGGGTGACCT GGGGCAGCTCGCAGCGCGAATTGCCGCCCAACTTCCTGCCTCTTTCAATTTT GGTGCAAT CAAGGCTGACGCCATCCACGAGCTTCTCGTGCGCAT GAAGCGTGCCGAACTCTACT GCCC C AGACC ACT GT T GGC GGT GAAGGTCTC GT CT C AAGAC AGAC AC AAAC AGAAGAT CAT T GC ACCAGCAAAACAGCTCTTGAACTTTGACCTGCTCAAGTTGGCGGGAGACGTTGAGTCCAA CCCCGGGCCCTTCAGTGGAGCCCCACCGACT GACCT GCAAAAGATGGTCATGGGCAACAC GAAGCCTGTT GAGCTCATCCTCGACGGGAAGACAGTAGCTATCT GCTGTGCTACTGGAGT GTTCGGCACT GCCTACCTCGTGCCTCGTCATCTTTTCGCAGAGAAGTATGACAAGATCAT GT T GGAC GGT AGAGC CAT GAC AGAC AGT GAC T AC AGAGT GT T T GAGT T T GAGAT AAAAGT AAAAGGACAAGACAT GCTCTCAGACGCCGCGCTCATGGTGCTTCACCGTGGGAATCGCGT GCGGGACATCACGAAGCACTTCCGTGATGTGGCAAAAAT GAAGAAAGGCACCCCCGTCGT TGGCGT GATTAACAACGCCGATGTCGGGAGACTGATTTTCTCTGGTGAGGCCCTTACCTA CAAGGACATT GTAGT GTGCATGGACGGAGACACCAT GCCT GGCCTCTTTGCCTACAAAGC CGCCACCAAGGCTGGCTACTGTGGAGGAGCCGTTCTTGCCAAGGACGGTGCCGAGACATT CATCGTTGGCACTCACTCCGCAGGTGGCAAT GGAGTTGGATACT GCTCCTGT GTTTCCAG GTCCAT GCTCAT GAAAAT GAAGGC AC AC AT C GAC C C T GAAC C AC AC C AC GAG

Sequence ID-3 (P12A3C-Q) i.e. Serotype-O wildtype nucleic acid sequence

GGAGCCGGGCAATCCAGTCCGGCGACTGGGTCGCAGAATCAGTCAGGCAACACTGGAAGC AT C AT C AAC AACT AC T AC AT GC AAC AGT ACC AGAAC T CC AT GGAC AC AC AAC T T GGT GAC AATGCTATAAGCGGAGGCTCCAACGAAGGGTCCACGGACACAACTTCCACCCACACAACC AGCACTCAGAACAACGACTGGTTTTCAAAGCTAGCCAGCTCTGCTTTTAGCGGTCTTTTC GGCGCCCTTCTCGCCGACAAGAAGACT GAGGAGACCACCCTCCTCGAGGACCGCATCCTC ACCACCCGCAACGGCCACACCACCTCGACAACCCAGTCGAGTGTTGGGGTCACGTACGGG TATGCAACAGCTGAGGATTTTGTAAGCGGACCGAACACCTCTGGTCTCGAGACCAGGGTT GTCCAGGCAGAGCRCTTCTTCAAAACCCACCTGTTT GACT GGGT CACC AGT GACTC ATT C GGAC GCTGCCACCT ACT GGAACTTCCAACTGACCACAAAGGTGTCTACGGCAGCCTGACT GACTCGTATGCTTATATGAGAAACGGTTGGGACGTCGAAGTCACTGCAGTGGGAAATCAG TTCAACGGAGGATGCCTGTT GGTAGCTATGGTGCCAGAGCTTCGTTCCCTTCAAAAGAGA GAGCTGTACCAGCTCACGCTCTTCCCCCACCAGTTCATCAACCCCCGGACGAACAT GACG GCACACATCACTGTGCCCTTTGTTGGCGTCAACCGCTACGACCAGTACAAGGTACACAAG CCCTGGACCCTCGTGGTTAT GGTTGTAGCTCCTTTGACTGTCAACACT GAAGGTGCCCCA CAAATAAAGGTATACGCCAACATCGCCCCTACCAACGTGCACGTTGCGGGTGAGTTCCCT TCCAAAGAGGGAATCTTTCCTGT GGCTTGTAGCGACGGTTACGGCGGTTTGGTGACCACG GACCCGAAGACGGCT GACCCCGCCTACGGGAAAGTGTTCAATCCCCCTCGCAACAT GTT G CCGGGGCGGTTCACCAACTTCCTTGAT GTGGCTGAGGCGT GCCCTACGTTTCTGCACTTC GAGGGT GAC GT GCC AT AC GT GAC C AC G AAGAC AGAT T C GGAC AGGGT GCTT GCTCAAT T T GACTTGTCTTTGGCAGCAAAGCACATGTCGAACACCTTCCTTGCAGGTCTCGCCCAGTAC TACACACAGTACAGCGGTACCATCAACCTGCACTTCATGTTCACAGGCCCCACCGACGCG AAGGCGCGGTACAT GATTGCATATGCCCCCCCTGGCATGGAGCCGCCCAAAACACCTGAG GCGGCT GCCCACTGCATTCATGCTGAATGGGACACAGGGTTGAACTCAAAGTTCACATTT TCAATTCCCTACCTCTCGGCAGCTGACTACGCGTACACCGCGTCTGACACCGCCGAGACC AC AAAT GT GC AGGGAT GGGT CT GCTT GTTCC AGAT AACAC ACGGGAAAGCT GAAGGT GAT GCCTTGGTTGTGTTGGCCAGTGCCGGCAAGGACTTTGAGTTGCGCCTACCAGTGGACGCC CGTAGAGAGACCACCTCCCC GGGT GAGTCAGCTGACCCCGTGACCGCC ACT GTTGAGAAC TACGGCGGTGAGACACAGGTCCAGAGGCGCCAACACACGGACGTCTCATTCATTTT GGAC AGATTT GTAAAAGTGACGCCAAAAGACCAAATTAAT GTACTGGACCTGATGCAAACCCCT GCTCACACTCTGGTGGGAGCGCTCCTTCGTACTGCCACTTACTATTTCGCTGACTTAGAA GT GGCAGTGAAACACGAGGGGAACCTCACTT GGGTCCCGAATGGGGCGCCTGAAGCGGCG TT GGCTAACACCACCAACCCAACGGCATACCGCAAGGCACCACTCACCCGGCTTGCATT G CCGTACACGGCACCACACCGTGT GTTGGCAACTGTTTACAACGGGAACTGCAAGTACGGT GAT GGTTCGGTGACCAACACAAGAGGT GACCTACAAGTGTTGGCCCAGAAGGCGGC GAGA GCGCTGCCTACCTCCTTCAACTACGGT GCCATCAAAGCTACTCGGGTGACTGAACT GCTT TACCGCATGAAGAGGGCTGAGACGTACTGCCCCCGGCCTCTTTT GGCCATTCACCCGAAC GAGGCCAGACACAAACAGAAGATTGTGGCACCTGTGAAGCAGCTCCTGAACTTTGAACT G CTCAAGTTGGCGGGAGACGTAGAGTCCAACCCTGGGCCCTTCAGTGGAGCCCCACCGACT GACCTGCAAAAGAT GGTCATGGGCAACACGAAGCCTGTTGAGCTCATCCTCGACGGGAAG ACAGTAGCTATCTGCTGTGCTACTGGAGTGTTCGGCACTGCCTACCTCGTGCCTCGTCAT CT T T T C GC AGAGAAGT AT GAC AAGAT C AT GT T GGAC GGTAGAGC CAT GACAGACAGT GAC T AC AGAGT GT T T GAGTT T GAGAT AAAAGT AAAAGGAC AAGAC AT GCTCT CAGAC GC C GC G CT C AT GGT GC T T C AC C GT GGGAAT C GC GT GC GGGAC AT C AC GAAGC ACT T C C GT GAT GT G GCAAAAATGAAGAAAGGCACCCCCGTCGTTGGCGTGATTAACAACGCCGATGTCGGGAGA CT GATTTTCTCTGGT GAGGCCCTTACCTACAAGGACATTGTAGT GTGCATGGACGGAGAC ACCATGCCTGGCCTCTTTGCCTACAAAGCCGCCACCAAGGCTGGCTACTGTGGAGGAGCC GTTCTT GCCAAGGACGGTGCCGAGACATTCATCGTTGGCACTCACTCCGCAGGTGGCAAT GGAGTT GGATACTGCTCCTGTGTTTCCAGGTCCATGCTCATGAAAAT GAAGGCACACATC GAC C C T GAAC C AC AC C AC GAG

Schematic representation of the FMDV genes incorporated in the adenovirus constructs: P12A region of serotypes A and O were synthetically obtained along with 3C in the same reading frame as shown, and cloned into the adenovirus shuttle plasmid (see Example 5). In another construct, 1A region was truncated at N-terminus. Only shuttle plasmid used as control.

Schematic representation of the control elements to regulate protein expression via the recombinant adenovirus: AdMax Hi-IQ system was obtained from Microbix Bioscience Inc., Canada for generation of replication-defective recombinant adenovirus. This system comprise of a shuttle plasmid [pDC516(io)] and adenovirus genomic plasmid [pBHGfrt(A)El,3 FLP].

The pDC515(io) has a pBR322 bacterial origin of replication and ampicillin resistance marker. Its expression cassette consists of mouse cytomegalovirus promoter (mCMV), /ac-operator, a hybrid intron, multiple cloning site for insertion of foreign genes, simian virus 40 (SV40) polyadenylation signal and a flippase recombinase recognition target (fit) for site-specific recombination with the adenovirus genomic plasmid. The human adenovirus 5 genome components such as the left and right inverted terminal repeats (ITR) and the packaging signal (ψ) are also present in pDC516(io).

The adenovirus genomic plasmid consists of the entire genome of adenovirus 5 (Ad 5), with deletions in the El and E3 regions, and the packaging signal (ψ). When co-transfected in El complementing 293IQ cells, the adenovirus genomic plasmid encoded flippase (FLP) recombinase mediates the frt site- specific recombination between the pBHGfrt(A)El,3 FLP and pDC515(io), leading to the generation of the recombinant adenovirus expressing the transgene.

The FMDV genes are under the control of the bacterial lac operator and the 293IQ cells express the lac repressor so that the recombinant gene is not active during the generation of Ad vectors as the repressor binds to the operator and does not allow transcription. On the other hand, in any other cells where the repressor is absent, the FMD proteins are expressed from the recombinant gene of FMDV present in the genome of Ad5 vector. This avoids cytopathogenicity due to potentially toxic viral proteins (e.g., 3C), and allows the generation of higher titers of high transgene expressing adenoviruses. Expression of recombinant FMDV proteins during replication of recombinant Ad5 vectors is undesirable because the protease 3C is toxic to Ad5. Therefore it decreases the total count of recombinant Ad5 particles thereby reducing the potency of the intended vector based vaccine antigen of FMDV with Ad5. As per the methods of this invention, the expression of recombinant FMDV proteins in recombinant Ad5 vector is almost negligible during replication of adeno virus. Therefore, it provides the advantage of getting very high count of recombinant adeno virus particles containing the FMDV genes of P12A3C or the truncated P12A3C.

Generation of replication-defective recombinant adenovirus based vaccine antigen for FMD: The FMDV genes (P12A3C) were cloned into the shuttle plasmid, which is recombined with the genomic plasmid of Ad5 in human kidney 293IQ cells or any other cell line which complement adenovirus El region and express lac repressor through co-transfection. The plasmids contain frt sites for recombination mediated by the flippase enzyme, which is expressed from the genomic plasmid. Recombination produces a full-length adenovirus with lac operator controlled expression cassette containing FMDV P12A3C inserted in place of the El region. Proteins encoded in the El region are essential for replication of adenoviruses, and this function is provided in trans by 293IQ cells, which carry a part of adenoviral genome including the El region. Four different adenoviruses were generated AdP12A3C-Awt (expressing P12A3C of FMDV 'A' serotype), AdP12A3C-Atd (expressing N-terminus truncated P12A3C of FMDV 'A' serotype), AdP12A3C-0 (expressing P12A3C of FMDV Ό' serotype) and AdpDC516 (control adenovirus without the transgene). For generating the recombinant adeno virus other cell lines complementing El region like 293, 293T, 293TT, 911, pTG6559, PER.C6, GH329, N52.E6, HeLa-El, UR, VLI-293^b can be used. . Expression of FMDV proteins through the recombinant Ad vectors: Any cell lines (Vero, 293, BHK, U373, Hela) that will not express lac repressor can be used to check the expression of transgene. The human astroglioma cells, U373 were infected with recombinant Ad viruses at 100 plaque forming units per cell, and cell lysates were subjected to western blotting using rabbit anti-VPl serum or rabbit anti-lAB serum. Uninfected (U373 lysate) and control Ad (AdpDC516) infected cells were used as negative controls. Detection of VP1 expression from recombinant ad viruses was used to confirm the P12A3C expression, cleavage of VP1 was observed due to 3C protease activity (AdP12A3C-Awt, AdP12A3C-Atd and AdP12A3C-0). The size of the 1AB protein from wild type Ad virus (AdP12A3C- Awt) is 33 kDa, whereas with truncated Ad virus (AdP12A3C-Atd) the size of 1AB protein is 29 kDa was observed when blotted with anti-lAB rabbit serum, It can be noted that, the size of 1AB protein from truncated Ad virus was smaller compared to wild type Ad virus due to deletion at N-terminus region. . Purification of recombinant adenoviruses: Recombinant adenovirus infected 293IQ cell extracts were loaded onto an anion-exchange column (particularly Q- Sepharose) and eluted using buffers containing various concentrations of sodium chloride. The top line graph shows OD280 values (y-axis) of the fractions eluted (x- axis) in various elution buffers. In this illustration, the virus eluted in fractions 5 and 6 of buffer 3. The thick band in these fractions represents the adenovirus hexon protein (-110 kD), which accounts for >60% of the virion mass.

Any anion column chromatography method selected from the following but not limited to: gel filtration, any ion exchange column chromatography, affinity matrix chromatography, hydrophobic interaction chromatography etc., preferably a column chromatographic method that elutes majority of the virus antigen. Ultracentrifugation, Density gradient centrifugation using sucrose or cesium chloride, tangential flow filtration using membranes with cut off from 100 kDa to 300 kDa may be used alternatively for purification of the viruses. Vaccine formulation:

Vaccine formulations with the recombinant Ad5 vector containing the desired gene of Interest i.e. SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 of FMDV serotypes A, O, and Asia-1 were made with suitable buffers, stabilizers such as sugars or sugar alcohols, and other excipients such as magnesium chloride and polysorbates.

The buffers in the vaccine formulation of this present invention include any physiologically acceptable buffer, can be selected from a list comprising of any one or more of the following but not limited to: phosphate buffer; citrate buffer; phosphate citrate buffer; borate buffer; tris (hydroxymethyl) aminomethane (Tris) containing buffer; succinate buffer; buffers containing glycine or histidine as one of the buffering agents.

The sugars and sugar alcohols can be selected from the list comprising sucrose, lactose, fructose, dextrose, fucose, trehalose, maltose or combination thereof with sorbitol, mannitol or glycerol etc.

The vaccine formulations may further include proteins or protein hydrolysates to impart stability to the vaccine. Suitable proteins can be human serum albumin, lactalbumin hydrolysates or amino acids but not limited to glycine, arginine, glutamate.

The vaccine formulation of the present invention optionally further comprising an adjuvant selected from any one or more of the following, but not limited to: a) aluminium salts, b) aluminium hydroxide with MPL, c) any water in oil emulsion, d) any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween, alpha-tocopherol, Sorbitol monooleate or simply sugar esters like mannitol oleates, sorbitol oleates or derivatives thereof any of the analogues and derivatives of the molecules thereof a) calcium phosphate b) combination of any of the aforementioned adjuvants.

Particularly, formulation of the recombinant adenovirus vector based vaccine against FMDV according to the methods prescribed under this invention includes purified recombinant adenovirus with the concentration of at least IX 10⁶ virus particles (VP)/dose that contains SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 integrated into the genome of the said recombinant Ad5 vector as the vaccine antigen. The said recombinant purified Ad5 vaccine antigen containing the FMDV genes of P12A3C was buffered with 10 mM Tris-HCl, at pH 8.0; 5% glycerol, 1 mM Mgck, 100 mM NaCl and 0.001% Polysorbate-80. This formulation was used for immunogenicity testing for pre-clinical studies as described in the below mentioned example.

9. Immunogenicity testing of adenovirus vectored vaccines in laboratory animals: Serum Neutralization Test (SNT). Guinea pigs were injected subcutaneously with candidate vaccines containing P12A3C as well as a control Ad virus (AdpDC516) separately, and boosted for 3 times. The animals were bled and sera were tested by SNT for the presence of FMDV neutralizing antibodies using respective serotypes. Sera from the all the animals immunized with recombinant Ad viruses were able to neutralize the FMD viruses with respective serotypes. Sera from the animals immunized with P12A3C-Atd adenoviruse gave more neutralizing antibody titres compared to sera from the animals immunized with P12A3C-Awt adenoviruses.

Claims

We claim:

1. A vaccine formulation for prevention against foot and mouth disease infections in mammals comprising:

(a) a vaccine antigen, wherein the said vaccine antigen is a recombinant vector based virus particle, containing the genes of foot and mouth disease virus (FMDV), the said gene selected from as disclosed in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3;

(b) a physiologically acceptable buffer, the said buffer selected from phosphate buffer; citrate buffer; phosphate citrate buffer; borate buffer; tris(hydroxymethyl)aminomethane (Tris) containing buffer; succinate buffer; buffers containing glycine or histidine as one of the buffering agents;

(c) sugars and sugar alcohols selected from the list comprising sucrose, lactose, fructose, dextrose, fucose, trehalose, maltose or combination thereof with sorbitol, mannitol or glycerol including combinations thereof;

(d) protein or amino acids selected from serum albumin, lactalbumin hydrolysates, glycine, arginine, glutamate including combination thereof;

(e) Magnesium chloride and Sodium Chloride;

(f) a polysorbate;

2. The vaccine formulation of claim 1, optionally further comprising an adjuvant selected from any one or more of the following, but not limited to: a) aluminium salts, b) aluminium hydroxide with MPL, c) any water in oil emulsion, d) any oil in water emulsion that contains one or more of the following constituents: squalene or its analogues or any pharmaceutically acceptable oil, tween, alpha-tocopherol, sorbitol mono-leate or any of the analogues and derivatives of the molecules thereof a) calcium phosphate b) combination of any of the aforementioned adjuvants.

3. The vaccine formulation of claim 1, wherein (a) the vaccine antigen is a purified recombinant adenovirus (Ad5) vector with the concentration of at least 1X10⁶ virus particles (VP)/dose that contains foot and mouth disease nucleotide sequences as disclosed in SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 of P12A3C;

(b) a buffer 5-50 mM Tris-HCl, at pH 7.5-8.5;

(c) 5% glycerol;

(d) 1 mM-10 mM Mgch;

(e) 10-500 mM NaCl;

(f) 0.001% to 0.1% Polysorbate-80.

4. The vaccine formulation of claim 1, wherein the said nucleotide sequence of SEQ ID No. 2 includes truncated nucleotide sequence of PI gene of FMDV serotype A from the N -terminus.

5. The vaccine antigen of claim 3, is prepared by a method comprising the steps:

(a) cloning of the FMDV gene sequences SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 into a shuttle plasmid, wherein the said FMDV genes are expressed through murine cytomegalovirus immediately early promoter under the control of lac operator, and an intron is included between the operator and the promoter, in order to enhance stability of the RNA which produces the FMDV protein(s);

(b) recombination of the shuttle plasmid of step (a) with a genomic plasmid of Ad5 in human kidney cells, through co-transfection, thereby producing a full-length adenovirus vector with a lac operator controlled expression cassette containing FMDV P12A3C (SEQ ID No. 1 or SEQ ID N. 2 or SEQ ID No. 3) inserted in the El region;

(c) purifying the said recombinant adenovirus vector using column chromatography method selected from the following but not limited to gel filtration, any ion-exchange column chromatography, affinity matrix chromatography, hydrophobic interaction chromatography, ultracentrifugation, density gradient centrifugation, tangential flow filtration using membranes with cut off from 100 kDa to 300 kDa wherein the said column chromatographic method elutes the virus antigen;

(d) formulating the recombinant adenovirus vector with buffers, sugars, sodium chloride, magnesium chloride, polysorbates to get the vaccine formulation of claim 1.

6. The vaccine antigen of claim 5, wherein the recombinant Ad5 vector is produced under a suitable transcriptional control system in competent cell lines that contain a lac repressor system to inhibit expression of SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 during virus replication to reduce the expression of foot and mouth disease virus protein during generation and propagation of adeno virus vectors expressing foot and mouth disease virus proteins.

7. The vaccine formulation of claim 1 wherein the said recombinant virus vector expressing foot and mouth disease virus proteins is selected from members of families Adenoviridae, Poxviridae and Herpesviridae, rhabdoviridae .

8. The vaccine formulation of claim 1, wherein the mammal is a domestic as well as free- ranging wild or captive or wild cloven footed animals selected from cattle, buffaloes, sheep, goats, pigs, deer, camel, elephants and wild ruminants and boar including humans.

9. The vaccine formulation of claim 1, capable for administration either parenterally intramuscular, subcutaneous, intravenous, intraperitoneal, intradermal or oral, mucosal, nasal, vaginal or any of the combinations of any of the above routes to species susceptible to infection with FMDV.

10. The vaccine formulation of claim 5, wherein the nucleic acid sequence encoding FMDV proteins are either full length sequences, or parts thereof derived from any of the many strains of FMDV belonging to any of the serotypes, particularly A, O, Asia-1 and C, the South African Territory (SAT) strains.

11. The vaccine formulation of claim 1. wherein the nucleic acid sequence SEQ ID No. 2 encoding the said FMDV gene is truncated at its N-terminus genome to increase the immunogenicity of the vaccine formulation against serotype A as compared to vaccine antigen using wildtype vaccine antigen of FMDV serotype A.

12. The vaccine formulation of claim, wherein the nucleic acid sequence encoding FMDV proteins are either full length sequences, or parts thereof derived from any of the many strains of FMDV belonging to any of the serotypes, particularly A, O, Asia-1 and C, the South African Territory (SAT) strains with or without the 3C protease.

13. The vaccine formulation of claim 5, wherein the recombinant adenovirus is produced in large-scale in cells grown in adherent or suspension systems, or any sequential combination thereof.

14. A method of making a vector based vaccine antigen against foot and mouth disease infections in mammals comprising the steps:

(c) purifying the said recombinant adenovirus vector using column chromatography method selected from the following but not limited to gel filtration, any ion-exchange column chromatography, affinity matrix chromatography, hydrophobic interaction chromatography, ultracentrifugation, density gradient centrifugation, tangential flow filtration using membranes with cut off from 100 kDa to 300 kDa wherein the said column chromatographic method elutes the purified virus antigen.

15. The method of claim 14, wherein the the recombinant Ad5 vector is produced under a suitable transcriptional control system in competent cell lines that contain a lac repressor system to inhibit expression of SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3 during virus replication to reduce the expression of foot and mouth disease virus protein during generation and propagation of adeno virus vectors expressing foot and mouth disease virus proteins.

16. The method of claim 14, wherein the said recombinant virus vector is selected from members of families Adenoviridae, Poxviridae and Herpesviridae, rhabdoviridae.

17. The method of claim 15, wherein the recombinant adenovirus is produced in large- scale in cells grown in adherent or suspension systems, or any sequential combination thereof.

18. A method of to differentiate infected subjects by screening antibodies against foot and mouth disease virus non- structural proteins except 3C protease from vaccinated subjects wherein the said subject is a mammal being a domestic as well as free-ranging wild or captive or wild cloven footed animals selected from cattle, buffaloes, sheep, goats, pigs, deer, camel, elephants and wild ruminants and boar including humans.