Patent Application
20110014232
The invention relates to chimeric foot and mouth disease viruses and to a method of constructing the chimeric virus. The method further relates to a method of eliciting an immune response to FMDV using the chimeric virus.
Foot-and-mouth disease (FMD) ranks as one of the most economically important infectious diseases of cloven-hoofed animals, affecting cattle, pigs, sheep, goats and other artiodactyl species. FMD is not only a disease affecting national and international trade, but impacts on the whole livestock industry with damaging consequences for the local farmer with invariable loss of income. Although eradicated from most parts of the world, FMD continues to occur in many developing countries where it severely constrains the livelihoods of poor livestock keepers. It seems unlikely that FMD will be eradicated from sub-Saharan Africa in the near future, because of the presence of large numbers of free-living maintenance hosts, particularly African buffalo. Livestock farming forms the backbone of rural economies for most of the SADC member countries. More than 75% of livestock is raised under the communal smallholder systems where it represents a multi-functional resource for the poor, providing meat, milk and fiber for household consumption or sale, traction for ploughing and transport, manure as fertiliser or fuel. Although mortality is usually low (less than 5%), FMD severely affects all of these functions as painful blisters in the mouth, feet and udder reduce livestock productivity, and the presence of the disease limits access to markets.
In southern Africa, the disease is essentially controlled through the separation of domestic and wildlife animals using fences, strategic vaccination of susceptible farm animals, restriction of animal movement and frequent inspections of animals and vaccination in controlled areas.
Movement restrictions and quarantine on animals and animal products during and after an outbreak severely impede trade, which is an important source of revenue for all income groups. Despite the fact that farmers are compensated if a stamping out policy is adopted, in many cases people are discouraged to continue producing livestock. Therefore, regular immunisation and improved vaccines, in terms of antigen yield, stability and protection against emerging FMD viruses (FMDV) are essential for disease control and maintaining the FMD-free status of South Africa.
FMDV is a naked icosahedral virus of about 25 nm in diameter, containing a single-stranded RNA molecule consisting of about 8500 nucleotides, with a positive polarity. FMDV exists as seven serologically distinct serotypes A, C, O, SAT (Southern African Territories) 1, 2, 3 and Asia 1. Although generally referred to as a single disease and clinically indistinguishable, the seven viral serotypes, distributed globally, have different geographical distributions and epidemiological profiles. The practical implication is that an animal infected with one serotype is not cross-protected and thus fully susceptible to infection by other FMDV serotypes. Six of the seven types of FMD virus, viz. SAT1, SAT2, SAT3, A, O and C, occur in sub-Saharan Africa. The fact that the SAT types are unique to Africa and have appreciably greater intratypic genomic and antigenic variation than the traditional “European” types, complicates FMD control in the subcontinent. SAT2 has the highest incidence in domestic animals in Africa causing more frequent outbreaks, while SAT1 viruses are recovered more frequently from carrier buffalo.
Vaccines are the most effective means of controlling and perhaps eventually eliminating infectious diseases, but existing FMD vaccines are not ideal. The effective administration and optimal induction of protective immunity against clinical disease are hampered by several factors. Vaccination against a specific serotype does not protect against the others. Even within a serotype distinct genetic and antigenic variants exist in different geographical regions with serious implications for the control of the disease by vaccination since it may render available vaccines inadequate. As an inactivated vaccine, it induces a short-lived immunity and animals have to be vaccinated twice annually. Vaccination does not prevent infection, it only delays the onset/progress of the disease and animals could become persistently infected, and in turn may be able to infect non-vaccinated animals. As it is problematic to distinguish between vaccinated and convalescent animals, the export market is lost for farmers in the FMD-controlled zone.
Commercial FMD vaccines are still classically produced by infection of cell culture by the virus followed by inactivation of the virus, usually by chemical treatment, e.g. with binary-ethylenimine (BEI). In order for FMD-virus vaccine production to be economically feasible, the FMD virus must be grown on cells in suspension, rather than cells attached to a monolayer. Therefore, classical FMD vaccines are limited to virus strains that are adapted to growth in cell cultures, most preferably suspension cell cultures.
Adaptation of new vaccine strains of FMDV up till now requires repeated passaging in cell cultures and depends on the acquisition of the capacity to bind cell-surface heparan sulphate, an alternative receptor for FMDV cell-entry. The acquisition of this capacity is totally dependent on random mutations and can therefore in no way be influenced. During the adaptation process, a virus isolate is first grown on, for example, primary pig or bovine epithelium cells, followed by adaptation on, for example, immortal pig kidney (IB-RS-2) and/or baby hamster kidney (BHK-21; ATCC-CCL-10) cells.
Cells grown in suspenson, e.g. suspension BHK-21 cells, are often insensitive to infection with wild-type FMDV for vaccine production, and thus the viruses have to be adapted to such cells before large scale production can commence.
This adaptation process for FMDV has two severe drawbacks:
The outcome may thus be an adapted vaccine strain that does not elicit a protective immune response against the parental virus or a vaccine strain that results in low or unstable antigen yield in large scale production.
There is therefore a need to provide new FMD viruses which are more easily adapted to grow on BHK-21 cells, more specifically on BHK-21 cells in suspension, and are therefore ready to use in large scale production, allowing for fast and effective production of new vaccine strains.
According to a first embodiment of the invention, there is provided a chimeric foot and mouth disease virus (FMDV) nucleic acid molecule encoding a first FMDV strain, wherein nucleotides encoding an outer capsid region have been replaced with nucleotides encoding an outer capsid region of a second FMDV strain which includes or has been modified so as to introduce a heparan sulphate proteoglycan binding site.
The first and second FMDV strains may be the same or different serotypes, independently selected from SAT1, SAT2, SAT3, A, C, O and Asia 1 serotypes.
The first FMDV strain is typically a strain which is able to grow in vitro on a commercial scale, and the second strain is typically a wild-type strain in current circulation.
The heparan sulphate proteoglycan binding site may be introduced by modifying one or more nucleotides of the outer capsid region of the second FMDV strain to encode:
The nucleotides encoding amino acid residues at positions 110 and 112 of VP1 (SEQ ID NO: 22) or at positions 135 and 175 of VP3 (SEQ ID NO: 24) may be additionally modified to encode a lysine or arginine residue if the second FMDV is a SAT1 serotype.
The nucleotides encoding amino acid residues at positions 83 and 85 of VP1 or at position 161 of VP1 (SEQ ID NO: 25) may be additionally modified to encode a lysine or arginine residue if the second FMDV is a SAT2 serotype.
The first FMDV strain may have at least 70%, 80%, 90% or 95% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, or an RNA equivalent thereof.
The capsid encoding region of the second FMDV strain may be a DNA or RNA sequence encoding the amino acid sequence of SEQ ID NOs: 3, 4 or 5, or a sequence which has at least 70%, 80%, 90% or 95% sequence identity thereto.
According to a further embodiment of the invention, there is provided a vector comprising a nucleic acid molecule described above.
According to a further embodiment of the invention, there is provided a host cell comprising a nucleic acid molecule described above. The host cell may be a BHK-21 cell.
According to a further embodiment of the invention, there is provided a virus expressed by or comprising the nucleic acid molecule described above. The virus may be inactivated.
According to a further embodiment of the invention, there is provided a composition comprising the virus or nucleic acid molecule described above. The composition may include a suitable adjuvant.
The composition may be used for eliciting an immune response against FMDV in a subject.
According to a further embodiment of the invention, there is provided a method of eliciting an immune response to FMDV in a subject, the method comprising administering the virus or the composition described above to the subject.
According to a further embodiment of the invention, there is provided a method of producing a chimeric FMDV nucleic acid molecule, the method comprising the steps of:
The modified capsid-coding nucleotide sequence of the first FMDV strain may replace nucleotides encoding the external capsid protein of the second FMDV strain.
Another nucleotide sequence encoding another capsid protein of the first FMDV strain may be additionally inserted into the second FMDV strain.
The first and second FMDV strains may be the same or different serotypes and may be independently selected from serotypes SAT1, SAT2, SAT3, A, C, O and Asia 1.
The heparan sulphate proteoglycan binding site may be introduced by modifying one or more nucleotides of the outer capsid region of the second FMDV strain to encode:
The nucleotides encoding amino acid residues at positions 110 and 112 of VP1 (SEQ ID NO: 22) or at positions 135 and 175 of VP3 (SEQ ID NO: 24) may be additionally modified to encode a lysine or arginine residue if the first FMDV strain is a SAT1 serotype.
The nucleotides encoding amino acid residues at positions 83 and 85 of VP1 or at position 161 of VP1 (SEQ ID NO: 25) may be additionally modified to encode a lysine or arginine residue if the first FMDV strain is a SAT2 serotype.
New foot and mouth disease (FMV) viruses which are able to grow on BHK-21 cells in suspension (and which therefore do not need to undergo the time-consuming and possibly immunogenicity-destroying adaptation process) are described herein. As they are immediately able to grow on BHK-21 cells in suspension, they are ready for use in the large scale production of vaccines.
The new viruses are recombinant chimeric viruses formed by replacing the outer capsid coding region of a first FMDV strain which has previously been shown to be an effective vaccine strain with the outer capsid coding region of a second FMDV strain. The outer capsid coding region of the second FMDV strain is also modified to introduce a heparan sulphate proteoglycan (HSPG) binding site. The chimeric viruses are then used as seed viruses in the production of inactivated vaccine antigens which have been tailored for specific outbreak situations or locality.
These chimeric viruses, which contain the antigenic determinants of a field virus, do not need to undergo the time consuming and expensive adaptation process in order to be grown in vitro to large scale. Also, as the virus does not need to undergo numerous passages, uncertainty about final antigen yields and characteristics can be avoided.
The invention also relates to the product of expression of the chimeric FMD viruses and to uses therefor, such as to form antigenic, immunological or vaccine compositions for prevention of FMD.
The chimeric viruses, vectors containing them or expression products, such as antigens, can be administered to a subject to prevent FMD. The subject can be any animal which can become infected with FMDV, in particular, bovine, ovine, porcine or caprine species.
The chimeric viruses, vectors or expression products thereof, or immunological, antigenic or vaccine compositions containing them, are typically administered via a parenteral route (intradermal, intramuscular or subcutaneous). Such an administration enables a systemic immune response, or humoral or cell-mediated responses.
The compositions contemplated by the invention can also contain an adjuvant. Suitable adjuvants are well-known in the art.
The use of infectious cDNA technology in synthesising vaccines for specific geographic localities or an outbreak situation against emerging or contemporary virus strains has previously been described (Rieder et al., 1993; Zibert et al., 1990; Almeida et al., 1998; Beard and Mason, 2000; van Rensburg et al., 2004; Fowler et al., 2008). Viable genome-length cDNA clones have been applied successfully in recent years in studying the biological properties of FMDV. The cDNA clones can be manipulated by genetic engineering techniques, exchanging genome segments or introducing single nucleotide changes and still rendering viable chimeric viruses following transfection and successive passages in vitro.
Infectious genome-length cDNA clones of SAT1 and SAT2 strains were constructed with the desirable biological properties of good vaccine strains (van Rensburg et al., 2004). The antigenic characteristics of such a clone can then be manipulated by merely exchanging the determinants of antigenicity, i.e. the structural proteins or parts of it (Rieder et al., 1993; Sa-Carvalho et al., 1997; Almeida et al., 1998; Beard and Mason, 2000; van Rensburg et al., 2004). The fact that the viral RNA can be made infectious in the absence of other components of the virion allows the recovery of genetically engineered new viruses from in vitro-generated RNA molecules (Zibert et al., 1990).
The chimera technology can be applied in the development of custom-made vaccines specific for a geographical region. The applicants used a chimera virus containing the outer capsid proteins of a SAT1 virus, cloned into a SAT2 genetic background, to vaccinate animals in a full potency trial and observed similar protection compared to the parental SAT1 vaccine. In the construction of a chimera, the cell-entry determinants, like the antigenicity, of the field isolate are transferred to the derived chimeric virus.
A major factor that is likely to contribute to the poor growth of field viruses in cell culture is the lack of appropriate host-specific integrin receptors for virus cell-attachment. Cultivation of FMDV in cultured BHK-21 cells leads to the adaption of FMDV for growth in cell culture and can select for variants with a high affinity for HSPGs (glycosaminoglycans or GAG's). This phenotype, and consequently an ability to use HSPGs as alternative receptors to initiate infection, is associated with the accumulation of positively charged residues in surface-exposed loops of the outer capsid proteins. HSPG receptors are found on most cell surfaces. This is a major advantage for vaccine manufactures, as HSPG-binding results in an expanded host range for cultured cells and permits the use of established cell lines, like BHK-21 cells, in suspension in fermentors. Heparan sulphate binding sites are described in more detail in Fry et al. (Embo J., Vol 18, pp 543-554, 1999). The downside of this adaptation process has been discussed above, specifically in relation to the disadvantages of random mutagenesis affecting the antigenic characteristics. These disadvantages could, however, not be avoided until now.
The applicants have now identified unique HSPG-binding sites (amino acid residues) located on the outer capsid proteins of SAT1 and SAT2 FMDV. The sites are exposed on the surface of the virion and are structurally accessible for binding to the alternative HSPG receptors.
These binding sites were identified on FMDV isolates (vaccine strains) that have adapted to growth on BHK-21 cells (ATCC-CCL-10) in suspension, a cell used in the production of FMDV vaccines, by comparing the amino acid sequences of current SAT1 and SAT2 vaccine strains with the corresponding primary isolates, available at Transboundary Animal Diseases Program (TADP) of the ARC-OVI (Onderstepoort Veterinary Institute, South Africa). The vaccine strains also have the ability to infect and replicate in Chinese hamster ovary cells strain K1 (CHO—K1 ATCC CCL-61) cells, a feature characteristic of viruses that use HSPG receptors for cell entry. The residue substitutions were located on surface-exposed loops and included a change to a positive charge residue. These binding sites were shown to simultaneously affect plaque phenotype, virus host range in cell culture and the ability to infect cells in culture via HSPG.
The invention is illustrated in more detail in the Example section, below, for two of the most distantly related FMDV-viruses; SAT 1 and SAT 2. However, it is emphasized that the same approach is perfectly and without undue burden applicable to SAT 3, A, O, C and Asia I serotypes.
The eight novel amino acid positions/sites on the outer capsid proteins of SAT1 and SAT2 viruses identified by the applicants are typically associated with changes observed in the plaque morphology on BHK21 cells, infection and replication of CHO—K1 cells and the ability to utilise HSPG for cell entry. CHO—K1 cells do not express the integrins that facilitate cell entry of FMDV and infection is via HSPG receptors. This characteristic is also associated with the ability of FMDV to infect BHK-21 cells in suspension. Five of the eight amino acid positions were specifically identified on SAT1 isolates and the remaining three on the SAT2 serotype.
The sites in SAT1 viruses included a (1) lysine or arginine at residue 110 of VP1, (2) lysine or arginine at residue 112 of SAT1 VP1, (3) lysine or arginine at residue 135 of VP3, (4) lysine or arginine at residue 175 of VP3, (5) lysine or arginine at residue 74 of VP2. The position of the sites prone to change during adaptation of SAT2 viruses was a (6) lysine or arginine at residue 83 of VP1, (7) lysine or arginine at residue 85 of VP1, (8) lysine or arginine at residue 161 of VP1. Residues 110-112 of VP1 seem to be a “hotspot” for change in SAT1 viruses during cell culture adaptation, since three viruses with substitutions at this position were identified, i.e. SAR/9/81, KNP/196/91 and ZAM/2/93. Similarly, the residues 83 and 85 were prone to change during adaptation of two SAT2 viruses, i.e. KNP/19/89 and ZAM/7/96. These novel HSPG-binding sites have been shown to improve the cell-entry and replication ability of SAT1 and SAT2 isolates in BHK-21 monolayers or suspension cultures, which are characteristics sought after in a good vaccine strain.
The novel amino acid substitutions identified by the applicants during adaptation of SAT viruses (like vaccine strains) on BHK-21 cells can be engineered into new vaccine strains using recombinant DNA technology. Introducing the identified HSPG-binding sites when constructing a chimeric virus from a field isolate can similarly improve the cell-entry mechanism and result in a virus that is immediately adapted for large scale production in suspension cells. This allows for fast and effective adaptation of recombinantly generated new vaccine strains from an isolate in an outbreak situation or specific geographic location. The engineered HSPG-binding virus can be amplified within a few passages directly on BHK-21 to create a master seed stock, without prior isolation on primary cell lines and further adaptation.
The HSPG binding regions can be used in combination with recombinant chimeric technology. In particular, the outer capsid-coding region from a genome-length cDNA clone can be exchanged with the corresponding region of a field isolate. The virus recovered from such a chimeric DNA construct will have features from both the recombinant genetic backbone and the field isolate. For SAT1 serotypes, a lysine or arginine can be simultaneously introduced at positions 110 and 112 of VP1 or a lysine or arginine can be simultaneously introduced at positions 135 and 175 of VP3 of the wild-type sequence via site-directed mutagenesis. The new SAT1 recombinant chimeric virus can be multiplied to generate vaccine seed virus for large scale production of the chimeric SAT1 inactivated vaccines. Similarly, the HSPG-binding sites, a lysine or arginine can be simultaneously inserted at positions 83 and 85 of VP1 or at position 161 of VP1 in a wild-type SAT2sequence. The SAT2 chimeric virus can be used to generate vaccine seed virus. Custom-made vaccines from isolates from a specific outbreak situation or geographic region can be produced according to this method.
The present invention is further described by the following examples. Such examples, however, are not to be construed as limiting in any way either the spirit or scope of the invention.
Infectious Genome-Length cDNA Technology
A genome-length cDNA copy (pSAT2) of the SAT2 vaccine strain, ZIM/7/83, was constructed following an exchange-cassette strategy using an Al 2 genome-length clone as template (Rieder et al., 1993; van Rensburg et al. 2004). The SAT2/ZIM/7/83 virus contained all the characteristics of an ideal vaccine candidate, including fast growth properties, high antigen yields and a broad antigenic coverage. This initial construct was used for the transfection of in vitro synthesized RNA transcripts, followed by the recovery of infectious viral particles. Through manipulation of this clone, in particular the inclusion of hammerhead and hepatitis delta virus ribozymes, a pSAT2r+ clone was generated that could be utilised for the production of viable viruses by direct transfection of baby hamster kidney (BHK-21) cells with DNA, eliminating the time consuming process of RNA synthesis in vitro.
Using similar cloning methodology previously described (van Rensburg et al. 2004), a genome-length cDNA copy of the SAT1 vaccine strain, SAR/9/81, was also constructed (designated pSAT1). A SAT1 strain (SAR/9/81) isolated from impala epithelium (SAR/9/81imp) and the tissue culture adapted version (SAR/9/8lvacc; PK1RS4-BHK5) were selected to facilitate the construction of the clones. The SAR/9/81 virus was selected for its favourable growth properties, easy adaptation on tissue culture cells, i.e. IB-RS2 and BHK-21 cell lines, and excellent vaccine strain properties.
The importance of the pSAT1 clone stems from the fact that the FMDV serotypes are antigenically diverse and very little or no cross-protection exists between serotypes. Also, the outer capsid proteins of SAT1 viruses are seven amino acids longer than for SAT2. Both the pSAT1 and pSAT2 vectors can be used to prepare synthetic RNA, which in turn is used to transfect BHK-21 cells. The general vector map for pSAT1 and pSAT2 is depicted in
Both the pSAT1 and pSAT2 clones were modified by introduction of novel restriction enzyme (RE) sites to allow the exchange of the outer capsid-coding region with the corresponding region of contemporary viruses. The RE sites for sspI (ATTAAT (SEQ ID NO: 17)) and xmaI (CCCGGG (SEQ ID NO: 18)) were introduced, while natural occurring xmaI sites in the pSAT1 were removed, by standard site-directed mutagenesis protocols (Sambrook and Russel., 2001).
The method of the present invention has been shown to work equally well for divergent serotypes of FMDV, and is easily applicable to other serotypes not specifically exemplified herein.
Application of Infectious cDNA Clones and the Construction of Chimeric Viruses
The outer capsid-coding regions of pSAT1 and pSAT2 were replaced with that of SAT1, SAT2 and SAT3 field and vaccine strains. Basic cloning methodology as described in Sambrook and Russel, 2001 was used. The genome-replacement strategy is illustrated in
The applicants were able to construct a panel of viable chimeric viruses from the pSAT2 and pSAT1 genome-length cDNA clones by replacing the external capsid-coding region with the corresponding region from SAT1, 2 and 3 vaccines strains and/or field isolates. The resulting chimeras showed growth characteristics and immune profiles comparable to the parental viruses used for the cloning process, indicating that the derived chimeras were similar to the field strains. In many instances, the chimeras represented a subpopulation of the field strains as a result of the quasispecies nature of FMDV, and in at least one instance the biological properties of the field isolate were improved by the presence of the encoded replication determinants of the genome-length backbone. The cell-receptor binding preference of the field isolates was retained in the chimeric viruses.
The SAT field strains that were selected for the chimeras are summarised in Table 1 and included three SAT2 strains from the southern African topotype (ZIM/17/90, ZIM/14/91 and ZAM/07/96), two SAT1 viruses (NAM/307/98 and ZAM/02/93) and a SAT3 virus (ZAM/04/96). The external capsid-coding regions of 6 field strains were recovered via PCR amplification, introducing unique restriction enzyme sites to facilitate cloning (
The applicants have shown that the antigenic determinants of the field isolates are transferrable to the recombinant synthesized chimeric virus. Similarly, the receptor preference and inability to enter cultured cells via HSPG receptors of the field isolates was also transferred to the chimera viruses. The chimera technology for the production of vaccines specific for geographic locality or outbreak situation can be refined by introducing HSPG-binding sites during the construction of the chimera.
aTopotypes refers to genotypes distributed to specific geographic regions and the topotypes for the SAT serotypes are described by Bastos et al., 2001 and Bastos et al., 2003a, b.
bViruses recovered by transfection of BHK-21 cells are designated “v” followed by the parental isolate number and the SAT2 plasmid used for cloning purposes.
cThe genome-length clone used for the construction of the chimera is indicated after “/” in the designated name.
Mapping of Heparan Sulfate Binding Regions of SAT1 and SAT2 Virions
At least four SAT1 and four SAT2 viruses, grown to high passage in BHK-21 cells, were used in the study. The viruses include isolates that are currently in use in the preparation of inactivated vaccines at ARC-OVI, and these were compared to the parental isolates (low passage) from which they were derived. The virus isolates included SAT1/SAR/9/81, SAT1/KNP/196/91, SAT1/ZAM/2/93 and SAT1/NAM/307/98 from the SAT1 serotype and SAT2/KNP/19/89, SAT2/ZIM/7/83 (parental is labeled SAT2/ZIM/5/83), SAT2/ZIM/14/90 and SAT2/ZAM/7/96 of SAT2 serotype. The plaque phenotypes and cell culture host range of the high and low passage isolates of the abovementioned viruses were compared (
In
The nucleotide sequences of the outer capsid-coding regions were determined and the deduced amino acid sequences were compared (
Asn-
Arg-
Gly
Lys-
Arg-
Arg
Lys-
Gly-
Gly
Lys-
Gly-
Arg
His-
Asn-
Gly
Lys-
Asn-
Arg
Binding of viruses to HSPG or other glycosaminoglycans (GAG) occurs mainly through electrostatic interactions between positively-charged Lys and Arg groups on the virus surface and the negatively-charged N and O sulphated groups of the GAG molecules (Gromm et al., 1995; Byrnes and Griffin, 1998 and 2000). The accumulated positively-charged residues and increased affinity to HS probably lead to direct interaction between the Arg or Lys and heparin. The selection of positively-charged residues was previously reported for type O viruses (Sa-Carvalho et al., 1997; Jackson et al., 1996; Zhao et al., 2003). Adaptation of O1 Campos to cell culture selected viruses with an H→R change at position 56 of VP3 (Jackson et al., 1996; Sa-Carvalho et al., 1997).
Therefore, in the investigation of residues or sites involved in HSPG binding, attention was placed on where the adaptation-to-suspension-cell-culture phenotype was accompanied by the acquisition of positive charged amino acid residues on surface exposed loops in the VP1, VP2 and VP3-capsid proteins (
Gln
Arg
Glu
Lys
Glu
Glu
Lys
Lys
Gln
Thr
Arg
Lys
Evaluation of the recombinant vSAT1 revealed the accumulation of positively-charged residues Lys110 and Arg111 surrounding the five-fold axis of the virion, responsible for the acquisition of the ability to interact with HSPG receptors and replicate in CHO—K1 cells. An in-depth look at the residues present in this position of the SAR/9/81wt impala isolate (low passage) and BHK-21 adapted isolate (tc) showed that the cell culture adaptation of the SAT1 virus was accompanied by amino acid changes at positions 110 and 112 of the VP1 capsid protein. The 110NRG112 motif of the impala isolate, in this short βF-βG loop, changed to a mixture of Asn, His or Lys residues at location 110 and Arg, Lys or Asp at position 112 in the adapted strain. The amino acid variation correlated also with the mixed plaque phenotypes observed. The Arg111 in the 110NRG112 motif, in the absence of other positively-charged residues, was not sufficient for the acquisition of SAR/9/81wt to bind to HSPG and growth in CHO—K1 cells. The progeny viruses within the SAR/9/81tc population, on the other hand, were equipped with an altered surface-exposed positive patch neighboring the five-fold pore (
Similarly to SAR/9/81, the SAT1/KNP196/91P isolate (P; wild-type isolated on primary pig kidney cells) revealed mainly large plaques with turbid edges on BHK-21 cells (
The SAT1/NAM/307/98 virus was previously isolated from buffalo (Syncerus caffer) in the West Caprivi Game Reserve, Namibia, in 1998 (Bastos et al., 2001; Storey et al., 2007). The primary isolate of this virus (SAT1/NAM/307/98P) had difficulty to adapt to BHK-21 cells, and only after repeated cultivation in BHK-21 cells, it finally resulted in a variant (SAT1/NAM/307/98BHK) revealing medium sized and small plaques with well-defined edges on BHK-21 cells. This variant was able to grow in CHO—K1 cells, as evident by the small plaques observed (
Two vaccine strains belonging to the SAT2 serotype were investigated for disparate plaque morphologies during adaptation on BHK-21 to create vaccine master seed virus stocks (Table 1). The SAT2/KNP/19/89P was isolated from buffalo in the Kruger National Park. This low passage isolate produced a mixture of medium to large sized plaques on BHK-21 cells with opaque edges, but CHO—K1 were unable to sustain growth of this isolate as observed by the absence of plaques. However, passaging four times on BHK-21 cells (SAT2/KNP/19/89Vac) consequently revealed mostly medium-sized plaques with well-defined edges as well as growth on CHO—K1 cells (
The amino acid substitutions in the SAT2 vaccine strains (Table 2), only Gln85→Arg and Glu161→Lys in VP1, showed significant charge difference on the surface of the virion (
Confirmation of the role of the three residue motif in VP1 at position 83-85 of SAT2 viruses came from the adaptation of the field isolate SAT2/ZAM/7/96 (Table 2), isolated from buffalo when the large plaques changed to a mixture of plaques and growth in CHO—K1 cells (
In summary, the VP1 residues at position 110-112 of SAT1 viruses appear to be a “hotspot” to change during cell culture adaptation, while other distantly located residues in the capsid proteins may also be involved (74 of VP2, 135 and 175 of VP3). This site is unique to SAT1 viruses. Similarly, the residues 83-85 (noteworthy residue 86 is also a positive charge residue) of VP1 are prone to change during adaptation of SAT2 viruses.
Introduction of HSPG Binding Sites Into Chimeric Viruses
The application of the novel SAT HSPG-binding regions was investigated by introducing the positive charge amino acids into chimeric viruses that do not have this characteristic, using standard site-directed mutagenesis techniques. The two chimeric viruses chosen for this purpose included pNAM/SAT and pSAU/SAT, containing the outer capsid-coding region of the SAT1/NAM/307/98 and SAT2/SAU/6/00 cloned into the pSAT2 genetic backbone. The two chimeric viruses were selected for lacking the HSPG phenotype as measured by the inability to infect CHO—K1 cells. Neither of the two viruses was able to acquire this phenotype with repeated cultivation in BHK-21 cells. The putative HSPG-binding residues located adjacent to the 5-fold axes of the virion were introduced into the pNAM/SAT and pSAU/SAT. The most prominent and significant site observed for SAT1 viruses was the residues 110-112, where accumulation of positive charge residues was observed for three SAT1 isolates. The sequence of KRR was therefore introduced into the corresponding region of pNAM/SAT, which contained the sequence RGG. A site prone to accumulation of positive charge residues, during adaptation of SAT2 viruses in cell culture, was residues 1083 to 1085 of the VP1 protein. This motif surrounds the 5-fold axis of the virion. The KRK motif was located at the base of the GH-loop and was chosen as the second site to be introduced into pSAU/SAT.
The vNAM/SAT chimeric virus, containing the outer capsid proteins of the NAM/307/98 virus produced large, opaque plaques of BHK-21 cells, similar to the wild-type virus. The vNAMΔKRR mutant with the KRR motif introduced at residues 110-112 revealed plaque morphology similar to that of the recombinant vSAT1 virus.
A Full Potency Protection Experiment of a Chimera Vaccine in Pigs
An alternative approach in the development of inactivated vaccines involves the engineering of chimeric FMD viruses of which the antigenic properties can be readily manipulated. This recombinant DNA technology is unique in its application in FMD vaccines, as it allows for rapid alteration of the external capsid-coding region of a stable infectious clone to that of a current outbreak strain. In the present study, by engineering such a chimeric virus, a possible alternative to the conventional inactivated vaccine production of the SAT type viruses was investigated for the development of custom-engineered inactivated FMD vaccines. A cross-serotype chimeric virus, vKNP/SAT2, was constructed consisting of the external capsid-coding region of a SAT1 virus in the genetic backbone of a SAT2 virus. The viral progeny replicated stably in both FMD host and non-host species derived cell lines and the infective titres and ability to produce plaques were similar for the chimera and parental virus, from which it was derived. The efficient cell-entry ability of vKNP/SAT2 and high infectious particle production rates render chimeras that can be inactivated and utilised for vaccine manufacturing purposes.
Two separate double oil emulsions incorporating inactivated 146S antigens of the chimera, vKNP/SAT2, and parental, KNP/196/91, were prepared and used for vaccination in a full potency trial (European Pharmacopoeia, 1997; OIE Manual of Standards, 2004). In order to monitor the antibody response elicited in pigs by the full (6.0 μg), quarter (1.5 μg) and one-sixteenth (0.375 μg) doses of the vaccines, sera samples collected were tested in a KNP/196/91-specific SPCE and the average titres for each vaccine dose were determined at weekly intervals (
Serum neutralising antibody responses were measured by the VNT at the day of challenge for the vaccinated and control animals. All of the pigs were negative for FMDV-specific neutralising antibody at the onset of the study. At four weeks post-vaccination, 86.7% and 53.3% of the KNP/196/91 and vKNP/SAT2 vaccinated pigs were sero-positive on the VNT, respectively, especially those animals that received higher antigen doses. The chimeric vaccine induced high levels of homologous antibodies that cross reacted with the KNP/196/91 parental viruses; BHK-21 cell line-adapted and PK1RS4 isolates. Positive neutralising antibody titres were induced for the full doses of both vaccines. For the quarter dose of the chimera and parental vaccines, three and four animals, respectively, were positive for neutralisation of the KNP/196/91 virus. Whilst four pigs vaccinated with the parental one-sixteenth dose had positive neutralising antibody titres, the entire chimera one-sixteenth group was negative. Similar antibody response profiles were observed in both the SPCE and VNT for animals from all the groups and vaccinated with both antigens. Following challenge, none of the animals vaccinated with the KNP/196/91 vaccine developed lesions, while 60% of the animals receiving the chimeric vaccine were fully protected against disease. The onset of FMD lesions in animals with clinical disease was delayed and restricted in distribution, indicative of partial protection in these animals. By contrast, the onset of lesions in the control animals was faster than for those vaccinated with the chimera vaccine.
The vKNP/SAT2 displayed promising potential as a recombinant vaccine in its ability to retain phenotypic properties of the parental KNP/196/91 and the high titres achieved during infection resulted in high antigen yields that can readily be formulated as inactivated vaccine. In addition, the chimera and parental vaccines, elicited good humeral immune responses in pigs. The antibody titre increased more rapidly for the groups that received the higher antigen payloads of both vaccines. The onset of disease was delayed for the majority of the chimera vaccinated pigs when compared to the control animals and the clinical signs were less severe. Moreover, the majority of the pigs vaccinated with the chimera were protected against live virus challenge. This is indeed promising as the antigen range of up to 6 μg per dose is typically used in commercially available FMD vaccines.
Thus, more effective new generation inactivated vaccines for this highly infectious and economically important disease, which are custom-engineered and specifically produced for certain geographic localities, can be generated.
Methods
Cells, Viruses and Plasmids
Baby hamster kidney (BHK-21) cells, strain 21, clone 13 (ATCC CCL-10) were maintained as previously described (Rieder et al., 1993) and were used during transfection, virus recovery and plaque assays. Plaque assays were also performed using Chinese hamster ovary (CHO) cells strain K1 (ATCC CCL-61) maintained in Ham's F-12 medium (Invitrogen), supplemented with 10% FCS (Delta Bioproducts). Plaque assays were performed using a tragacanth overlay method and 1% methylene blue staining (Grubman et al., 1979; Rieder et al., 1993). Two SAT1 viruses i.e. a buffalo isolate, ZAM/2/93, and the vaccine strain, KNP/196/91; four SAT2 strains isolated from buffalo, i.e. ZIM/17/91, ZIM/14/90, ZIM/5/83 and ZAM/7/96, as well as the vaccine strain ZIM/7/83 and a SAT3 virus, KNP/19/89, utilized in vaccine manufacture were used in this study. Plasmids pSAT2, pNAM/SAT2 and pSAU/SAT2 have been described elsewhere (van Rensburg et al., 2004; Storey et al., 2007; Bohmer, MSc thesis 2004). The pSAT2 contains the genome-length cDNA of the wild-type FMDV SAT2 strain, ZIM/7/83, and was used in the construction of chimeric clones. Two SAT1 genome-length clones; constructed from a cell culture-adapted strain, (SAT1/SAR/9/81tc) and virus that was previously isolated from impala epithelium (SAR/9/81wt; wild-type), respectively, were also included for comparison.
Plasmid pSAT2 and derivatives of this plasmid have been described elsewhere (van Rensburg et al., 2004). The pSAT2 contains the genome-length cDNA of the wild-type FMDV SAT2 strain, ZIM/7/83, and unique SspI and XmaI cloning sites for the removal of the outer capsid and 2A-coding region.
RNA Extraction, cDNA Synthesis and Construction of Infectious Genome-Length cDNA and Chimeras
RNA was extracted from infected cell lysates using either a guanidium-based nucleic acid extraction method (Bastos, 1998) or TRizol® reagent (Life Technologies) according to the manufacturer's specifications and used as template for cDNA synthesis. Viral cDNA was synthesised with SuperScript III™ (Life Technologies) and oligonucleotide 2B208R (Knowles et al., 2009). The ca. 2.2 kb external capsid-coding regions of the viral isolates were obtained by PCR amplification with specific ologonucleotides to facilitate cloning or nucleotide sequence determination. Sequencing of the amplicons was performed using the ABI PRISM™ BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.0 (Perkin Elmer Applied Biosystems).
pSAT1 plasmid carrying the genomic sequence of SAT1/SAR/9/81tc was constructed using a similar cloning strategy to the one employed by Rieder et al. (1993) and van Rensburg et al. (2004). The nucleotide sequence of the cloned regions was subsequently determined.
Construction of Mutant cDNA Clones
Site-directed mutagenesis of SAT capsid residues was carried out on plasmids pSAT1, pNAM/SAT2 and pSAU/SAT2 using an overlap extension mutagenesis method. The resulting ca. 2.2 kb DNA fragment was digested with SspI and XmaI (pNAM/SAT2 and pSAU/SAT2 mutations) and the external capsid-coding region (1B-1D/2A) was used to replace the corresponding region that was excised from the pSAT2. Alternatively, a ca. 3.2 kb fragment of the pSAT1 mutagenesis amplicon was digested with SnaB1 and Bln1 and cloned into the corresponding region of pSAT1. The mutations were verified by automated sequencing as described in section 2.2 and no second/other site mutations were found
In Vitro RNA Synthesis, Transfection and Virus Recovery
Plasmids containing genome-length cDNAs, chimeric cDNA or site-directed mutated cDNA clones were linearised at the SwaI site downstream of the poly-A tract and used as templates for RNA synthesis, using the MEGAscript™ T7 kit (Ambion). BHK-21 cell monolayers, in 35 mm diameter cell culture plates, were transfected with the in vitro-generated RNA using Lipofectamine2000™ (Life Technologies). Transfected monolayers were incubated at 37° C. with 5% CO2 up to 48 hours in BME containing 1% FCS and 25 mM HEPES. The supernatants were used to infect BHK-21 monolayers and incubated for up to 48 hours at 37° C. Viruses were subsequently harvested by a freeze-thaw cycle and passaged four times in BHK-21 cells, using 10% of the supernatant of the previous passage, as described before (van Rensburg et al., 2004). Following the recovery of viable viruses the presence of the mutations were verified once more with automated sequencing.
Analysis of HSPG Utilization During Cell Entry of SAT Types of FMDV
The utilization of HSPG for cell entry was analyzed in CHO—K1 (positive for HSPG) cells which were infected with the specified viral strains and incubated for 1 hour and 24 hours, respectively, washed with MES-buffer (pH 4.0) to remove residual extracellular virus and frozen at −70° C. Virus titres were determined in BHK-21 cells and viral growth was calculated by subtracting the 24 hour titre results from the 1 hour titre results.
Amplification of High Passage Isolates in CHO—K1 Cells
BHK cell-adapted viruses were used to infect CHO—K1 cells for 1 hour, followed by an acidic wash step as described before, prior to incubation at 37° C. The viruses were harvested at greater than 90% CPE or at 48 hours and frozen at −80° C. The nucleotide sequences of the isolates with the ability to infect and produce greater than 90% CPE within 24 hours were determined and compared to those of the parental/original viruses.
Antigen Preparation and Vaccine Formulation
Following the original isolation (PK1RS4) from buffalo in the Kruger National Park, South Africa, the KNP/196/91 virus was passaged in cattle and BHK-21 cells (passage history: PK1RS4B1BHK4), prior to its application in engineering a genome-length construct by replacing the external capsid-coding region (1B-1D/2A) of the infectious cDNA clone SAT2/ZIM/7/83, pSAT2 (van Rensburg et al., 2004), with that of KNP/196/91 (pKNP/SAT2). The chimera, vKNP/SAT2, and parental, KNP/196/91, viruses harvested from infected BHK-21 monolayers were inactivated with 5 mM BEI for 26 h at 26° C., concentrated and purified as above. The genetic integrity of the viruses used for infection (passage 5) and vaccine formulation (passage 6) were verified. Two separate vaccine formulations, incorporating vKNP/SAT2 and KNP/196/91 inactivated 146S antigens as double oil emulsion (water-in-oil-in-water (WOW)) with Montanide ISA 206 (Seppic, Paris) were prepared. The appropriate antigen concentration was diluted in Tris-KCl buffer (0.1 M Tris, 0.3 M KCl, pH 7.5), followed by the addition of chloroform to a final concentration of 0.3% v/v. The oil adjuvant was mixed into the aqueous antigen phase (50:50) at 30° C. for 15 minutes and stored at 4° C. for 24 hours, followed by another brief mixing cycle for 5 minutes. A placebo vaccine was prepared for the control animals containing all the components except antigen.
Vaccination and Challenge of Pigs
Thirty-four, FMD-free female pigs, 3-4 months of age and weighing 25-30 kg were housed separately in six groups of five animals each (Groups 1-6) and one group of four controls (Group 7). Subsequent to an initial acclimation period, the pigs were vaccinated by the intramuscular route immediately caudal to the ear with 2 ml, 0.5 ml and 0.125 ml of 3 μg/ml of either vKNP/SAT2 (groups 1-3) or KNP/196/91 (groups 4-6) 146S antigen. The control group was administered a placebo formulation without antigen. Rectal temperatures and clinical signs were recorded daily. At 28 dpv the animals were inoculated intra-epidermally in the coronary band of the left hind heel bulb with 0.1 ml of 105 TCID50/ml challenge virus and examined daily for lesions, whereupon pigs were removed from the experiment. At day 10 post-infection the remainder of the animals were terminated. A body temperature equal to or greater than 39.6° C. was considered as mild fever, whereas temperatures equal to or greater than 40° C. were considered as severe fever. Serum samples were taken at 0, 7, 14, 21, 28 dpv and on the day of termination for serology.
Homologous challenge virus was prepared by three passages of KNP/196/91 (PK1RS4B1BHK4) in pigs. The pig adapted virus, designated PK1RS4B1BHK4P3, was titrated in pigs, primary pig kidney (PK) cells and IB-RS-2 cells and the titre expressed as pig infective doses per ml (PID50/ml) or tissue culture infective doses per ml (TCID50/ml).
Pig Antibody Response Detected by Solid-Phase Competition ELISA
The presence of antibodies directed to SAT1 146S particles in sera was detected by a KNP/196/91-specific solid-phase competition ELISA (SPCE) that has been developed for this investigation. Trapping antibody and KNP/196/91 virus were added to the plates as above. Of each sample, 100 μl of an 1/20 dilution was added in triplicate and diluted 1:1 in 50 μl 0.5% (w/v) casein across the plate. Guinea pig anti-KNP/196/91 serum diluted 1/6000 in casein (50 μl) was added to the wells incubated and washed. The addition of antispecies conjugate and subsequent detection steps were as described before.
Specific Neutralising Antibody Against FMDV Detected by Virus Neutralisation Assay
Serum samples collected at 0 and 28 days post-vaccination were tested in the virus neutralisation test (VNT) for the presence of neutralising antibodies against FMDV. The VNT was carried out in micro-titre plates as described in the OIE Manual of Standards (2004). The serum samples were initially diluted 1/8, followed by a 1:1 dilution across the plate and the virus neutralising ability was tested against four dilutions of the homologous viruses (Esterhuysen et al., 1985). A regression line was calculated from the results and the 50% serum end-point titre at the log102.0TCID50 level established (Esterhuysen et al., 1988). Serum titres were expressed as the logarithm of the reciprocal of the final serum dilution to neutralise 100 TCID50 of homologous FMDV in 50% of the wells, as calculated by the method of Kärber (1931).
While the invention has been described in detail with respect to specific embodiments thereof, it will be appreciated by those skilled in the art that various alterations, modifications and other changes may be made to the invention without departing from the spirit and scope of the present invention. It is therefore intended that the claims cover or encompass all such modifications, alterations and/or changes.
Almeida, M. R., Rieder, E., Chinsangaram, J., Ward, G., Beard, C., Grubman, M. J., Mason, P. W. 1998. Construction and evaluation of an attenuated vaccine for foot-and-mouth disease: difficulty adapting the leader proteinase-deleted strategy to the serotype O1 virus. Virus Res. 55, 49-60.
Bastos, A. D. S., Haydon, D. T., Forsberg, R., Knowles, N. J., Anderson, E. C., Bengis, R. G., Nel, L. H., Thomson, G. R. 2001. Genetic heterogeneity of SAT-1 type foot-and-mouth disease viruses in southern Africa. Arch. Virol. 146, 1537-1551.
Beard, C. W., Mason, P. W. 2000. Genetic determinants of altered virulence of Taiwanese foot-and-mouth disease virus. J. Virol. 74, 987-991.
Böhmer, MSc thesis 2004, Engineering of a chimeric SAT2 foot-and-mouth disease virus for vaccine production. Submitted in partial fulfilment of the requirements for the degree Master of Science in the Faculty of Natural and Agricultural Sciences Department of Microbiology and Plant Pathology University of Pretoria, South Africa
Esterhuysen, J. J., Thomson, G. R., Ashford, W. A., Lentz, D. W., Gainaru, M. D., Sayer, A. J., Meredith, C. D., Janse van Rensburg, D., Pini, A. 1988. The suitability of a rolled BHK21 monolayer system for the production of vaccines against the SAT types of foot-and-mouth disease virus. I. Adaptation of virus isolates to the system, immunogen yields achieved and assessment of subtype cross reactivity. Onderstepoort J. Vet. Res. 55, 77-84.
Esterhuysen, J. J., Thomson, G. R., Flammand, J. R. B., Bengis, R. G. 1985. Buffalo in the northern Natal Game parks show no serological evidence of infection with foot-and-mouth disease virus. Onderstepoort J. Vet. Res. 52, 63-66.
European Pharmacopoeia, 5th Ed. 1997. European Directorate for the Quality of Medicines, Strasbourg.
Fowler V. L., Paton D. J., Rieder E. and Barnett P. V. 2008. Chimeric foot-and-mouth disease viruses: evaluation of their efficacy as potential marker vaccines in cattle. Vaccine. 26(16):1982-9.
Grubman, M. J., Baxt, B., Bachrach, H. L. 1979. Foot-and-mouth disease virion RNA: studies on the relation between the lengths of its 3′-poly(A) segment and infectivity. Virol. 97, 22-31.
Jackson, T., Ellard, F. M., Abu-Ghazalch, R., Brooks, S. M., Blakemore, W. E., Corteyn, A. H., Stuart, D. L., Newman, J. W. I., King, A. M. Q. 1996. Efficient infection of cells in culture by type O foot-and-mouth disease virus requires binding to cell surface heparan sulfate. J. Virol. 70, 5282-5287.
Kärber, G. 1931. Beitrag zur kolletiven Behandlung pharmakologischer Reihenversuche. Archiv. Exp. Path. Pharm. 162, 480-483.
Cottam E M, Wadsworth J, Knowles N J, King D P. 2009. Full sequencing of viral genomes: practical strategies used for the amplification and characterization of foot-and-mouth disease virus. Methods Mol Biol., 551:217-30.
Sambrook, J. and Russel, D. W. Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.
OIE manual of standards for diagnostic tests and vaccines. 5th Ed 2004 Office International des Epizooties. Paris, France, 2004
Rieder, E., Bunch, T., Brown, F., Mason, P. W. 1993. Genetically engineered foot-and-mouth disease viruses with poly(C) tracts of two nucleotides are virulent in mice. J. Virol. 67, 5139-5145.
Sa-Carvalho, D., Rieder, E., Baxt, B., Rodarte, R., Tanuri, A., Mason, P. W. 1997. Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle. J. Virol. 71, 5115-5123.
Storey P., Theron J., Maree F. F., O'Neill H. G. 2007. A second RGD motif in the 1D capsid protein of a SAT1 type foot-and-mouth disease virus field isolate is not essential for attachment to target cells. Virus Res. 124(1-2), 184-92.
Van Rensburg, H. G., Henry, T. M., Mason, P. W. 2004. Studies of genetically defined chimeras of a European type A virus and a South African Territories type 2 virus reveal growth determinants for the foot-and-mouth disease virus. J. Gen. Virol. 85, 61-68.
Zhao, Q., Pacheco, J. M., Mason, P. W. 2003. Evaluation of genetically engineered derivatives of a Chinese strain of foot-and-mouth disease virus reveals a novel cell-binding site which functions in cell culture and in animals. J. Virol. 77, 3269-3280.
Zibert, A., Maass, G., Strebel, K., Falk, M. M., Beck, E. 1990. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA. J. Virol. 64, 2467-2473.
