# Patent

## Title
Protein sequences containing antigenized antibodies for foot-and-mouth disease

## Abstract

Protein sequences encoding antigenized antibodies for treatment of foot-and-mouth disease (FMD) or foot-and-mouth disease virus (FMDV) of swine is provided. The antigenized antibodies are created from the grafting of peptide epitopes derived from FMDV into swine antibody CDR loops. FMDV peptide epitopes are cloned by PCR from VP1 gene of FMDV. The overlapping PCR method is used to insert the FMDV peptide epitopes into the CDR regions of swine immnuoglobulin heavy and light chains genes. The resulting antigenized antibody genes were cloned into mammalian expression vector. The plasmids are transfected into CHO or myeloma cells. The stable transfectant cell line was selected for high yield of the desired protein antibodies.

## Background
RELATED APPLICATIONS AND PRIORITY CLAIM
This is a continuation of U.S. application Ser. No. 09/291,299 filed Apr. 15, 1999, now issued as U.S. Pat. No. 6,774,225, entitled “Antigenized Antibody Vaccine For Foot-And-Mouth Disease” incorporated herein by reference.


BACKGROUND
1. Field
The present invention relates to protein sequences encoding antigenized swine antibodies for Foot-and-Mouth Disease (FMD), Foot-and-Mouth Disease Virus (FMDV), or their epitopes.
2. Background
Antibodies are proteins produced by the body's immune system in response to foreign elements known as antigens, which invade the body. Antibodies can elicit an immune response, which subsequently protects the host from infection by the disease-causing agents (antigens).
Foot-and-Mouth Disease, also known as FMD hereinafter, is a highly contagious, severely debilitating disease that infects all cloven-hoofed animals. It is endemic in many developing countries worldwide. In particular, swine in Asia have often been affected by FMD. FMD reduces livestock, productivity, incurs high vaccination costs, and restricts the international trade of livestock and livestock products. FMD is a viral infectious disease and the foot-and-mouth disease virus, also known as FMDV hereinafter, is a small animal virus having a single stranded positive sense RNA genome of about 8,000 nucleotides.
Two major types of vaccines have been produced against FMD. They are conventional vaccines and synthetic peptide vaccines. Conventional vaccines against FMDV use either inactivated FMD virus or a live attenuated FMD virus. The conventional vaccine approach, although generally effective, have several undesirable drawbacks associated with it.
Firstly, it is cost inefficient. This type of vaccine is made from large amounts of live, infectious virus. Maintaining and processing a large quantity of infectious virus is expensive, labor intensive and space inefficient.
Second and most importantly, these vaccines are potentially dangerous. Most of the outbreaks of FMD in recent years have been caused either by the escape of virus from vaccine production units or the use of incompletely inactivated or insufficiently attenuated virus. For example, during the 1980's, a number of outbreaks occurred in European countries including Italy, United Kingdom, France and in Taiwan in 1996. As the causative viruses of outbreaks were often found to be closely related or even identical to the strains that were used in Europe for manufacturing, it is possible that the primary outbreaks were caused by inadequately inactivated vaccines or by virus that escaped from vaccine production plants.
Another problem associated with producing conventional vaccine is that they are thermolabile. Conventional FMD vaccines are relatively unstable when exposed to elevated temperatures and they have to be stored at low temperatures. Constantly maintaining the required low temperatures is often not easily achievable, especially in tropical countries.
An addition potential problem in a virus culturing procedure of the conventional vaccine production vaccine production process is the use of fetal bovine serum virus culturing. It is possible that diseases can be introduced from the fetal bovine serum and affect the vaccinated animals.
Yet another major disadvantage of using conventional FMD vaccines is that most vaccines produced using the conventional method are relatively crude preparations of inactivated tissue culture grown virus. This tissue cultural mix may cause serious side effects such as allergic responses and abortions in susceptible stock.
There are newer forms of FMDV vaccines that do not use inactivated virus. They are synthetic peptide vaccines and recombinant protein vaccines. The identification of the immuno-dominant sites on viral protein 1 (VP1) of FMDV provided new ideas for designing synthetic peptide and recombinant protein FMD vaccines. Compared to conventional vaccines, these two types of vaccines are both safe in production and application. They are also very easy to handle, store, transport and can be designed to meet specific requirements.
The study of synthetic FMDV peptide vaccine was started by polymerizing the 141 a.a.-160a.a. peptide from VP1 with either glutaraldehyde or air-oxidized after a cysteine residue was added at each terminus. It was found that uncoupled peptides could be made immunogenic. In 1987, Francis and his colleagues reported that the presence of C-terminal cysteines with a free thiol group largely enhanced the immunogenicity of free 141-160 a.a. peptide. Similar results were also obtained when multiple cysteine residues were added. It was suggested that the presence of a free thiol cysteine residue would allow the formation of peptide dimers leading to a more ordered secondary structure causing immune complex formation in vivo (Francis, 1995). According to this idea, immunogenicity of tandem repeats (Cys 137-162(x2)) was compared to that of a single copy of Cys 137-163 peptide. It was found that tandem repeats of the FMDV peptide were generally more immunogenic than the single copy of disulphide dimers. The addition of a cysteine residue could result in the formation of disulphide tetramer structures, which improved the immune response further.
The concept of multiple copies synthetic peptides was further tested by using Tam's multiple antigenic peptide (MAP) system (Tam 1988). This system allows solid phase synthesis of a peptide antigen onto a branching lysine backbone to produce several polylysine octamer constructs. This system where there are multiple copies of the peptide resulted in greatly enhanced response.
In order to apply multiple copies of the FMDV peptide, recombinant DNA technology has been applied by fusing small peptide sequences to the gene coding for larger proteins. These larger proteins of recombinant vaccine have a number of characteristics. The goal of linking the peptide to the carrier is to provide a completely uniform and defined structure for the presentation of the immunogens as compared with those prepared by chemical cross-linking (Francis, 1991). This approach was first investigated by fusing single or multiple copies of the FMDV immunogenic peptides to the N-terminus of a bacterial protein, beta-galactosidase (Broekhuijsen et al., 1986; Winther et al., 1986). Beta-galatosidase was chosen because it has been shown that antibodies can be elicited against the epitopes from VP1 that are located at the N-terminus, and it also contains several T cell epitopes (Krzych et al., 1982; Manca et al., 1985). The immunogenicity of this multiple copy FMDV peptide-beta-galactosidase recombinant protein is found to be similar to that obtained from using the lysine background system (Broekhuijen et al., 1987).
Multiple peptide presentation was then further developed using FMDV peptide sequence fused to the N-terminus of the hepatitis B virus core antigen (HBcAg) to produce HBc fusion particles. It was reported that this 27 nm hybrid protein particle was able to give full protection to guinea pigs with results that were close to that elicited by inactivated FMDV VP1 142 a.a.-160 a.a. peptide and could protect animals against challenge infections.
Although initially promising, the synthetic peptide approach and recombinant protein vaccine approach appear to have shortcomings. Among these are poor predictability of the tertiary structure and weak immunogenicity. Peptides in solution exist in conformations that may not be always optimal for receptor binding (B-cell receptor and possibly T-cell receptor and major histocompatibility gene products) if specific conformation at the three-dimensional level is required for it to exhibit its intended functions.
In the case in which synthetic peptides that are relatively small in size, they tend to be easily degraded in the body after injection. Therefore, they may not be very effective in providing long term immune response probably because the recombinant protein vaccine fails to exhibit a proper conformation. Also, peptide synthesis is expensive which may lead to high production cost of the vaccine. As mentioned, presentation of the FMDV epitopes on peptide vaccines can be achieved by fusing them to the N-terminus of microbial proteins like beta-galactosidase or HBcAg. However, using beta-galactosidase may elicit a lot of additional and undesirable immune responses (Bona et al., 1994). After repetitive immunization of this recombinant protein vaccine, side effects may occur such as immediate hypersensitivity that can cause severe hay fever and asthma in the animal.
Nucleic acid vaccines or DNA vaccine represent a new approach to the control of infectious agents. These novel vaccines are easier to design and manufacture. Recombinant DNA technology is used to clone DNA sequences encoding the protein or proteins to be used as immunogens into an eukaryotic expression vector.
Antigenized antibodies are antibodies, which are genetically engineered in their variable domains to express epitopes of different antigens. Antigenized antibodies can be used as immunogens that focus the immune response on specific B- or T-cell epitopes. As such, antigenized antibodies can be used as an alternative approach to conventional or synthetic peptide vaccination.
Therefore, an effective kind of vaccine that offers both the possibilities of safety and efficiency is the antigenized antibody vaccine. The process of antibody antigenization consists of grafting peptide epitopes derived from antigens other than immunoglobulins into complementarity determining region (“CDR”) loops of an antibody molecule. Because the CDR loops are exposed at the surface of the antibody molecule, they provide the major contribution to antibody antigenicity. Unlike the synthetic vaccines described above, antigenized antibodies target antigen-presenting cells via the Fc receptor, thereby maximizing antigen presentation by class II major hisocompatibility (MHC) molecules. Also, antigenized antibodies provide B-cells with a continuous source of antigenic peptides for presentation in class I MHC molecules. In addition to immunogenicity at the B-cell level, antigenized antibodies act as processed peptide products to generate Th-cell immunogenicity.
SUMMARY OF INVENTION
The present invention can be used against FMD in swine, although by using FMDV viral epitopes for cows linked to cow IgG, this vaccine can be applied in other animals like cows as well.
The present invention may be used in a vaccine delivered in four different forms, namely two constructs of protein vaccine and two correlative constructs of DNA vaccine counterparts. All forms of this vaccine deliver the functions of immunization against FMD and FMDV in swine. To be specific, the vaccine in its protein forms are an antigenized antibody vaccine; this peptide sequence contains FMDV epitopes that replace CDR loops in swine IgG or a chimeric protein which FMDV single or tandem repeat epitopes carried by swine IgG heavy chain constant region protein.
As an example shown, the particular FMDV epitopes used for grafting into CDR was engineered. The first form of the DNA counterpart that corresponds to the first form of the protein vaccine utilizes FMDV epitope cDNA sequences as the carrier for single IgG in plasmid form. The second form of the DNA counterpart that corresponds to the second form of the protein vaccine utilizes FMDV epitope DNA sequences linked differently with the heavy chain constant region of swine Ig cDNA. Further, immunization methods of swine against FMD or FMDV are carried out by the use of this vaccine. There are different ways in which this vaccine can be administered. For the protein forms of the vaccine, it can be, for example, administered through conventional injection. In the case of administering the vaccine in its DNA forms, it can be carried out by using epidermis gene gun or injection.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the diagrammatic structure of the antigenized antibody vaccine using swine IgG;
FIG. 1B shows the diagrammatic structure of the antigenized chimeric vaccine using FMDV single or tandem repeat epitopes;
FIG. 2A shows the corresponding cDNA of the antigenized antibody vaccine, which uses IgG cDNA as a carrier in the plasmid;
FIG. 2B shows the corresponding cDNA of the antigenized chimeric vaccine using FMDV single or tandem repeat epitopes.
FIG. 3 shows the amino acid sequence (SEQ ID NO: 1) of the antigenized antibody heavy chain molecule in which the CDR3 region is replaced with FMDV VP1 aa 200 to 213 (SEQ ID NO: 19);
FIG. 4 shows the corresponding cDNA sequence (SEQ ID NO: 2) of the amino acid mentioned in FIG. 3;
FIG. 5 shows the amino acid sequence (SEQ ID NO: 3) of the antigenized antibody heavy chain molecule in which the CDR3 is replaced with FMDV VP1 aa 141 to 160 (SEQ ID NO: 23);
FIG. 6 shows the corresponding cDNA sequence (SEQ ID NO: 4) of the amino acid mentioned in FIG. 5;
FIG. 7 shows the cDNA sequence (SEQ ID NO: 5) of antigenized chimeric vaccine molecule. The bold part shows the nucleotide sequence encoding the epitopes of FMDV VP1 (aa 141 to 160, aa 200 to 213; SEQ ID NO: 22). The rest of the sequence belongs to PIG IgG heavy chain constant region.
FIG. 8 shows the protein sequence (SEQ ID NO: 6) of antigenized chimeric vaccine molecule. The bold part shows the epitopes of FMDV VP1 aa 141 to 160, aa 200 to 213 (SEQ ID NO: 24). The rest of the sequence belongs to PIG IgG heavy chain constant region.
FIG. 9 shows the four amino acid sequences (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10) of which the IgG light chain in FIG. 8 can be coded for. The bolded sequences are the framework regions. The underlined sequences are the CDR regions. The CDR2 or CDR3 sequence could be replaced by correspondence FMDV epitope sequences.
FIGS. 10A-10D show the four cDNA sequences (SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14) corresponding to the cDNA sequences of the IgG light chain in FIG. 8.
FIG. 11A shows the overlapping (extension) PCR taking place;
FIG. 11 B shows the overlapping (extension) PCR taking place.



DETAILED DESCRIPTION OF THE INVENTION
Embodiments illustrated the claims herein elicit an immune response against FMD. One embodiment contains an engineered cDNA sequence encoding for FMDV epitopes. The protein sequence also contains swine IgG cDNA construct as a carrier for the FMDV epitopes. The conjugation of the FMDV epitopes and swine IgG in its protein forms are carried out by grafting of FMDV peptide epitopes into swine IgG CDR loops or link FMDV epitopes with the swine IgG constant region as shown in FIG. 1B.
Therefore, the antigenized antibody molecules in its protein form are created from the grafting of peptide epitopes derived from FMDV into swine antibody CDR loops, as shown in FIGS. 1A and 1B. FMDV peptide epitopes were synthesized by PCR based on VP1 gene of FMDV. The overlapping PCR method was used to insert the FMDV peptide epitopes into the CDR regions of swine immunoglobulin heavy and light chain genes. The resulting antigenized antibody genes were cloned into mammalian expression vector. The plasmids were transfected into CHO or myeloma cells.
Four different embodiments are illustrated in FIGS. 1A, 1B, and FIGS. 2A, 2B. Two of them are protein forms (FIGS. 1A, 1B), known as antigenized antibody vaccines that can be administered by injection. The first type is an antigenized antibody vaccine that utilizes swine IgG protein as the carrier for FMDV epitopes and is injected into swine muscle tissue. The first form of this antigenized antibody vaccine (FIG. 1A) inserts the FMDV epitopes into the CDR2 and CDR3 regions of both heavy and light chains swine IgG. The second form of this antigenized antibody vaccine (FIG. 1B) utilizes single or tandem-repeat FMDV epitopes linked to only the heavy chain constant region of swine IgG, forming a chimeric protein. Hence, the second form of this antigenized antibody vaccine can also be called as antigenized chimeric vaccine.
Two other embodiments describe naked DNA vaccines, which can be administered through gene gun shooting. Each DNA form has a corresponding protein form counterpart. There are two forms of this naked DNA vaccine that corresponds to their protein counterparts as mentioned above. The two DNA forms will be expressed by the host's cell machinery after being administered. The corresponding protein equivalents will be directly administered into the animal, which have the same functions as the naked DNA vaccine forms. The first form of this naked DNA vaccine grafts FMDV epitope DNA sequences onto the CDR2 and CDR3 regions of both heavy and light chain swine IgG cDNA. The second form of this DNA vaccine utilizes FMDV epitope DNA sequences (FIG. 4) linked only with the heavy chain constant region of swine IgG cDNA. The two forms of naked DNA vaccine will exhibit the functions of the two forms of antigenized antibody vaccine respectively, when the transcription and translation process produce the products within the host cell machinery.
METHODS
The preparation of the claimed protein sequences involves three major steps, namely 1) cloning of the swine single IgG heavy chain constant region, and swine light chain 2) joining of the two FMDV immunogenic sequences and 3) joining of the swine IgG single heavy chain constant region with the FMDV immunogenic sequence, then inserting it into bacterial expression vector. Detailed procedures are explained as follow.
1) Cloning of the Swine Single IgG Heavy Chain Constant Region
The extraction and purification of mRNA from swine spleen was done by using a commercially available kit (mRNA Preparation kit, by Pharmacia). The procedures were followed as described by the manufacturer. In brief, 3 g of fresh swine spleen was homogenized in 1.2 ml Extraction Buffer. The homogenized tissue extract was diluted by 2.4 ml Elution Buffer and was mixed thoroughly. The homogenate was transferred to a sterile tube and centrifuged for one minute to obtain cleared homogenate. 1 ml of the cleared homogenate was placed on the top of the Oligo(dT)-Cellulose pellet. The Oligo(dT)-Cellulose was resuspended by inverting the tube for 3 minutes. The pellet was collected by centrifugation at 16,000×g for 10 seconds. For washing, the Oligo(dT)-Cellulose was washed five times with 1 ml High-Salt Buffer and centrifuged at 16,000×g for 10 seconds, followed with washing three times with 1 ml Low-Salt Buffer and centrifuged at 16,000×g for 10 seconds. Then the pellet was resuspended in 0.3 ml Low-Salt Buffer and transferred to a MicroSpinTM Column. The column was centrifuged at full speed for 5 seconds. The effluent was discarded and a new collection tube was put in place. This step was repeated twice. The column was placed in a sterile microcentrifuge tube and 0.2 ml pre-warmed Elution Buffer was added. The eluate containing mRNA was collected by centrifugation at full speed for 5 seconds. 10 ml of Glycogen Solution and 1/10 volume of Potassium Acetate Solution was added to the sample. The sample was mixed with 500 ml 100% ethanol and placed at −20° C. for at least 30 minutes. The precipitated mRNA was collected by centrifugation at 14,000 rpm at 4° C. for 5 minutes The supernatant was discarded and the precipitated mRNA was dissolved in DEPC-treated water. The quantity of RNAs was determined by UV absorbance at 260 nm.
RT-PCR Analysis
The cDNA fragments encoding the constant region of the heavy chain of the swine IgG were amplified by RT-PCR using a set of swine IgG 5′ and 3′ specific primers. Swine IgG 3′ specific primers were used to prime the first strand cDNA synthesis from total RNA. Reverse transcription was carried out at 37° C. for 60 minutes by MMLV reverse transcriptase and terminated at 70° C. for 15 minutes. The cDNA products were amplified by PCR in the presence of swine IgG 5′ specific primer. The PCR settings were as follows and ran for 30 cycles: denaturing at 94° C. for 1 min, annealing at 50° C. for 1 min and extension at 72° C. for 2 min, with a final extension at 72° C. for 6 min. The PCR products were fractionated on 1.0% low melting agarose gel and the band with sizes corresponding to the constant region of the IgG heavy chain was purified from the gel using phenol:chloroform extraction and ethanol precipitation. The DNA was then ligated to the FMDV immunogenic sequences (F1) later.
The PCR primers used were designed from swine IgG genes (Kacskovics et al., 1994) and the sequence of the primers are as listed below:









Swine IgG 3′ specific primer



(SEQ ID NO: 15)



5′GAC GCT CGA GTC ATC ATT TAC CCT GAG T 3′



 



Swine IgG 5′ specific primer



(SEQ ID NO: 16)



5′AGC TAA GCT TGC CCC CAA GAC GGC CCC A 3′






2) Joining of the two FMDV Immunogenic Sequences

Two oligonucleotide sequences were made corresponding to the residues 141-160 and 200-213 (two sequences were made with residues 155-160 sequence as overlapping region) on the VP1 of FMDV (Kurz et al., 1981). The two sequences were joined by overlapping PCR. The setting was as follows and ran for 5 cycles: 94° C. for 1 minute, 63° C. for 3 minutes. Two primers, VP1 3′ primer and VP1 5′primer, were used to add in one Hind III restriction site at the 3′ end and one Nde I restriction site at the 5′end. The PCR setting was as follow and ran for 24 cycles: denaturing at 94° C. for 1 minute, annealing at 50° C. for 1 minute, extension at 72° C. for 2 minutes and final extension at 72° C. for 6 minutes. The PCR product was analyzed on 1.5% low melting agarose gel. Product with correct size was cut and purified. The joined fragment was called FMDV-immuno-sequence. The VP1 3′ and 5′ primers used were designed from VP1 141a.a.-160a.a. and 200a.a.-213a.a. (Kurz et al., 1981). Sequences of the primers are as listed below:









VP1 5′ primer(SEQ ID NO: 17)



5′ ATG CCA TAT GGT ACC AAA C 3′



 



VP1 5′ primer(SEQ ID NO: 18)



5′ ATG CAA GCT TCA ACT TCT G 3′






FIG. 11A illustrates the overlap (extension) PCR taking place in two stages. First, the CDR1/CDR2 region is amplified, as is the CH/CDR3 region. The oligonucleotides at CDR3 are complementary; the longer primer 3 also contains VP1 141-160 or 200-213 residues. This permits fusion of these two products (a and b) in a subsequent PCR. FIG. 11B illustrates the overlap (extension) PCR takes place in two stages. First, the CDR1/CDR2 region is amplified, as is the CH/CDR3 region. The oligos at CDR3 are complementary; the longer primer 7 also contains VP1 141-160 or 200-213 residues. This permits fusion of these two products (c and d) in a subsequent PCR.
3) Linking of the Swine IgG Single Heavy Chain Constant Region, the FMDV-Immuno-Sequence Fragment and the Bacterial Expression Vector
The above fragment was digested with Nde I and Hind III; the swine single IgG heavy chain constant region was digested with Hind III and Xho I, and the bacterial expression vector was digested with Nde I and Xho I. The three digested fragments were purified by phenol/chloroform extraction and ethanol precipitation. They were then ligated by using T4 DNA ligase at 16° C. overnight. The ligation product was transformed into JM109 and colonies were screened by mini-plasmid isolation and then by restriction enzyme digestion. Plasmid from correct clone was isolated and checked by DNA sequencing. Finally, the correct plasmid was transformed into E. coli BL(21)DE3pLysE. The plasmid was named as pF1-IgG.
Thus, the present invention can provide an antigenized antibody vaccine against Foot-and-Mouth disease to provide a safer, more cost efficient and/or more effective vaccine product, which can overcome some of the disadvantages of the prior art.
The invention as described is deemed to incorporate equivalents to the integers recited where such equivalents would be apparent to those skilled in the art. The description is provided by way of example and is not to be considered limited to the scope of the invention which is defined in the appended claims.

## Claims

1. An antigenized swine antibody for Foot-and-Mouth Disease Virus (FMDV) comprising a swine antibody heavy chain, wherein said heavy chain comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 19; SEQ ID NO:23; and SEQ ID NO:24 in the variable region.

2. The antibody according to claim 1, wherein said swine antibody heavy chain consists of SEQ ID NO: 1.

3. The antibody according to claim 1, wherein said swine antibody heavy chain consists of SEQ ID NO: 3.

4. The antibody according to claim 1, wherein said swine antibody heavy chain consists of SEQ ID NO: 6.

5. The antibody according to claim 1, further comprising a modified light chain of swine IgG.

6. The antibody of claim 5, wherein the light chain comprises a complementarity determining region (CDR) sequence, wherein said light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

7. A method of preparing an antigenized swine antibody for Foot-and-Mouth Disease (FMD) comprising the steps:
a) cloning a first nucleotide sequence encoding the heavy chain constant region of swine IgG;
b) joining a second nucleotide sequence encoding one or more epitopes of Foot-and-Mouth Disease Virus (FMDV) to the first nucleotide sequence, whereby the resulting nucleotide sequence encodes a protein selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO:23 and SEQ ID NO:24; and
c) expressing said cloned nucleotide sequence to produce the swine antibody.

8. The method according to claim 7, wherein said swine antibody heavy chain consists of SEQ ID NO: 1.

9. The method according to claim 7, wherein said swine antibody heavy chain consists of SEQ ID NO: 3.

10. The method according to claim 7, wherein said swine antibody heavy chain consists of SEQ ID NO: 6.

11. The method according to claim 7, further comprising the step of expressing a third nucleotide sequence encoding a modified light chain.

12. The method of claim 11, whereby the third nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.