A CpG DNA adjuvant in avian vaccines is disclosed, which includes an immunostimulatory oligodeoxynucleotide (ODN) having a sequence of SEQ ID NO: 2. The CpG DNA adjuvant in avian vaccines is advantageous to carry out large-scale production, specifically enhance avian innate and adaptive immune responses, and the CpG DNA adjuvant is hardly to be digested by DNase due to its particular structures.

Description of the Invention

RELATED APPLICATIONS

This application claims priority to Taiwan Application Serial Number 97125312, filed Jul. 4, 2008, which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to a vaccine adjuvant, and more particularly, to a CpG DNA adjuvant in avian vaccines.

BACKGROUND OF THE INVENTION

Adjuvant is a key component in vaccine development. In current avian vaccines, there are some disadvantages existing in the conventional adjuvant such as aluminum hydroxide gel adjuvant and oil adjuvant. For example, the above two adjuvants are chemical adjuvants that cannot enhance specific Th1 cell immune response. Especially, the conventional adjuvant cannot stimulate enough innate and adaptive immune responses against infections.

Therefore, Krieg A. M. et al. found that unmethylated dinucleotide CpG motifs in bacterial deoxyribonucleic acid (DNA) has advantages on stimulating several immune cells to secrete cytokines for enhancements of innate and adaptive immunity. See Krieg A. M. et al., Nature 374:546-549, 1995. The effectiveness of the DNA containing CpG motifs has been confirmed in 1998. The DNA containing CpG motifs has been used as a vaccine adjuvant in 2003. Moreover, the oligodeoxynucleotides (ODN) containing CpG motif can enhance the activities of lymphocytes and antigen-presenting cells (APC), trigger dendritic cell (DC) maturation and antigen-presenting function, and drive immune systems toward Th1 cell immune response against specific antigens, in several large domestic animals such as cattle, pigs, sheep etc. For example, one of the present inventors has provided an ODN containing porcine-specific phosphorothioate (PTO)-modified CpG motif, as well as an immunostimulatory plasmid containing various sets of CpG motifs specifically to pigs.

In general, the mechanisms involved in enhancing immune responses by the ODNs containing CpG motifs (CpG ODN) can be as follows. At first, CpG ODN enhances DC activation, maturation and antigen-presenting function. Secondly, CpG ODN increases DC migration. Thirdly, CpG ODN significantly elevates expression of DC cell markers such as MHC-II, CD40, CD80, CD86, and IL-12 in mice and human. Fourthly, CpG ODN induces priming DC more responsive to antigen-specific Th1 cells. Fifthly, CpG ODN increases CD8.sup..quadrature. T cell cytotoxicity activity (CTL) to specific viruses or tumor cells. With application of CpG ODN, CpG ODN itself induces innate immune response to confer protective immune response against infection by viruses, bacteria and extracellular parasites, and B cells are activated by CpG ODN and vaccines to produce antibodies, to activate APC for secreting cytokines such as interferon-.gamma. (IFN-.gamma.), so as to enhance the immune response to vaccines.

However, the prior ODN containing PTO-modified CpG motif DNA synthesized by chemical processes is time-consuming, costs expensively and cannot be produced in mass. The CpG ODN is suitable for an adjuvant instead of PTO-modified CpG ODN. Typically, the CpG ODN contains unmethylated CpG motif, but without replacement of a phosphor atom of a phosphodiester bond in the PTO-modified CpG ODN with a sulfur atom, by which the phosphothioate reduces the degradation rate of the PTO-modified CpG ODN digested by deoxyribonuclease (DNase). In addition, the sequence of CpG ODN is species-specific; the CpG structures between different species are different in immunostimulatory activity. Currently, the sequence of most effective immunoregulatory CpG motif is different between human and mice, and thus specific CpG motifs effective in various species are necessarily confirmed by experiments. The CpG ODN was applied to enhance poultry immunity since 2002. Related researches on the effectiveness of CpG ODN in poultry are mostly involved in evaluation of chemically synthesized CpG ODN in vitro, or focused on antibodies-related humoral immune response in vivo; investigation on the adjuvanicity of CpG ODN on the cell-mediated immune response effectively against viral infection in avians is very limited.

Hence, it is necessary to provide an effective avian immunostimulatory CpG ODN as the DNA adjuvant in avian vaccines, thereby overcoming the disadvantages of the prior DNA adjuvant modified by chemical processes complicatedly.

SUMMARY OF THE INVENTION

Accordingly, an aspect of the present invention provides a CpG DNA adjuvant in avian vaccines, which includes an immunostimulatory oligodeoxynucleotide (ODN) having a plurality of TCG tandem repeats at a 5' end, a poly-G structure at a 3' end, and at least one CpG motif with avian specific flanking sequences at two ends thereof between the 5' end and the 3' end. The CpG DNA adjuvant in avian vaccines is advantageous to carry out large-scale production, specifically to enhance avian innate and adaptive immune responses, and to facilitate cellular uptake of the CpG DNA adjuvant.

According to the aforementioned aspect of the present invention, a CpG DNA adjuvant in avian vaccines is provided, which includes an immunostimulatory ODN having a plurality of TCG tandem repeats at a 5' end, a poly-G structure essentially consisted of 4 to 6 guanines at a 3' end, and at least one unmethylated CpG motif with avian specific flanking sequences at two ends thereof between the 5' end and the 3' end thereof.

In a preferred embodiment, the immunostimulatory ODN may include but be not limited in a first ODN (CTAGTGTCCA TGACGTTATG GGGGGGT, denoted as SEQ ID NO:1), a second ODN (CTAGTTCGTC GAAGTCGTTT TGGGGGGT, denoted as SEQ ID NO:2), a third ODN (CTAGTTCGAT CATCGTTGAG GGGGGT, denoted as SEQ ID NO:3) or any combination thereof.

In a preferred embodiment, the immunostimulatory ODN may further include a first restriction enzyme site at the 5' end.

In a preferred embodiment, the immunostimulatory ODN may further include a second restriction enzyme site in a different sequence from the first restriction enzyme site at the 3' end.

In a preferred embodiment, the avian vaccines may be, for example, Newcastle Disease (ND) live vaccine, ND inactivated vaccine, avian influenza inactivated vaccine, or fowl cholera inactivated bacterin.

With application to the aforementioned CpG DNA adjuvant in avian vaccines of the present invention, it includes an immunostimulatory oligodeoxynucleotide (ODN) having a plurality of TCG tandem repeats at a 5' end, a poly-G structure at a 3' end, and at least one CpG motif with avian specific flanking sequences at two ends thereof between the 5' end and the 3' end. The CpG DNA adjuvant in avian vaccines is advantageous to carry out large-scale production, specifically to enhance avian innate and adaptive immune responses, and to facilitate cellular uptake of the CpG DNA adjuvant, instead of the prior DNA adjuvant modified by chemical processes complicatedly.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Accordingly, the present invention provides a CpG DNA adjuvant in avian vaccines, which includes an immunostimulatory oligodeoxynucleotide (ODN) having a plurality of TCG tandem repeats at a 5' end, a poly-G structure at a 3' end, and at least one unmethylated CpG motif with avian specific flanking sequences at two ends thereof between the 5' end and the 3' end.

Specifically, the CpG DNA adjuvant in avian vaccines consists of an immunostimulatory oligodeoxynucleotide (ODN) having a sequence including the following formula: 5'TrBbPg3'

in which Tr denotes a plurality of TCG tandem repeats at a 5' end of the immunostimulatory ODN, Pg denotes a poly-G structure essentially consisted of 4 to 6 guanines at a 3' end of the immunostimulatory ODN, and Bb denotes a backbone sequence between the 5' end and the 3' end of the immunostimulatory CpG motif. The Bb has at least one CpG motif with avian specific flanking sequences at two ends of the CpG motif. The aforementioned "CpG motif" is referred as an unmethylated dinucleotide sequence containing a cytosine followed by guanine and linked by a phosphodiester bond.

It is worth mentioning that the CpG DNA adjuvant in avian vaccines has the TCG tandem repeats at a 5' end to specifically enhance avian innate and adaptive immune responses, the flanking sequences at two ends of the CpG motif to provide species specificity, the CpG motif itself being the avian immunostimulatory sequence, and the poly-G structure essentially consisted of 4 to 6 guanines at a 3' end to facilitate cellular uptake of the CpG DNA adjuvant. The specific DNA structure of the DNA sequence of the present invention possesses the function of DNA adjuvant in avian vaccines and the risk of the present CpG DNA adjuvant to be digested by DNase is effectively reduced due to its particular structures, rather than that the prior PTO-modified DNA adjuvant by using complicatedly chemical processes.

In a preferred embodiment, the immunostimulatory ODN of the CpG DNA adjuvant in avian vaccines may include but be not limited in a first ODN (CTAGTGTCCA TGACGTTATG GGGGGGT, denoted as SEQ ID NO:1), a second ODN (CTAGTTCGTC GAAGTCGTTT TGGGGGGT, denoted as SEQ ID NO:2), a third ODN (CTAGTTCGAT CATCGTTGAG GGGGGT, denoted as SEQ ID NO:3) or any combination thereof. Those immunostimulatory ODNs are listed as Table 1 -- see Original Patent.

As shown in Table 1, the first ODN, the second ODN and the third ODN may be useful examples as the immunostimulatory ODN of the CpG DNA adjuvant in avian vaccines. The species-specific CpG motif has exemplary sequences listed as the boxed letters in Table 1, including but not being limited to, for example, GACGTT, GTCGTT, or ATCGTT from 5' end to 3' end, however, GTCGTT is preferred. Secondly, according to a preferred embodiment of the present invention, the immunostimulatory ODN may optionally include a first restriction enzyme site at the 5' end and a second restriction enzyme site at the 3' end, and the second restriction enzyme site is different from the first restriction enzyme site in sequence. However, it is necessarily mentioned that, the first restriction enzyme site at the 5' end and the second restriction enzyme site at the 3' end of the immunostimulatory ODN of the present invention are designed to ligate into a vector to form a recombinant plasmid, so as to transform a competent cell (for example, E. coli) with the recombinant plasmid for large-scale production. Hence, the sequences of the first restriction enzyme site and the second restriction enzyme site are dependent on the desired vector rather than limiting to the aforementioned sequences. In addition, the sequences of the first restriction enzyme site and the second restriction enzyme site can also be applied to another recombinant recombinant plasmid for producing multiple CpG motifs. In a preferred embodiment, the first restriction enzyme site at the 5' end of the immunostimulatory ODN may be, for example, SpeI site as the single-underlined sequence of CTAGT (i.e. SpeI recognizes and cuts the 6 bp sequence of 5'-ACTAGT-3' to leave five overhanging residues CTAGT); besides, the second restriction enzyme site at the 3' end of the immunostimulatory ODN may be, for example, XbaI site as the double-underlined sequence of T (i.e. XbaI recognizes and cuts the 6 hp sequence of 5'-TCTAGA-3' to leave one overhanging residue T).

Additionally, the CpG DNA adjuvant in avian vaccines can be ligated into vectors to form recombinant plasmids, followed by transforming the competent cells (for example, E. coli) for large-scale production. Since the DNA ligation with the vector, transformation, large-scale production are subjected to the conventional methods understood by a person skilled in the art, the related details are unnecessary to be addressed herein.

It is understood that the CpG DNA adjuvant in avian vaccines produced in large scale and subjected to purification can indeed enhance the expression of interferon-.gamma. (IFN-.gamma.) gene, demonstrated by in vitro co-culture with avian peripheral blood mononucleocytes and splenocytes, as well as in vivo immune tests on experimental animals.

Thereinafter, various applications of the CpG DNA adjuvant in avian vaccines will be described in more details referring to several exemplary embodiments below, while not intended to be limiting. Thus, one skilled in the art can easily ascertain the essential characteristics of the present invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

EXAMPLE 1

Construction of Recombinant Plasmids Containing the CpG DNA Adjuvant in Avian Vaccines, Transformation and Large-Scale Production Thereof

EXAMPLE 1 is related to construct recombinant plasmids containing the immunostimulatory ODNs of the CpG DNA adjuvant in avian vaccines, transformation and large-scale production thereof, in which the immunostimulatory ODNs are designed to be the first ODN shown as SEQ ID NO:1 (referred as ODN-D1), the second ODN shown as SEQ ID NO:2 (referred as ODN-D2), or the third ODN shown as SEQ ID NO:3 (referred as ODN-D3).

Further, three comparative ODNs designed to be the fourth ODN lack of CpG motif and shown as SEQ ID NO:4 (referred as ODN-C), the fifth ODN containing the CpG motif and shown as SEQ ID NO:5 (referred as ODN-S1), or the sixth ODN containing the CpG motif and shown as SEQ ID NO:6 (referred as ODN-S2) are also utilized in EXAMPLE 1. With respect to the fifth ODN, see Wang X. et al., Avian Disease 47(9):5-12 (February 2003), the title as "Efficacy of DNA vaccines against infectious bursal disease virus in chickens enhanced by coadministration with CpG oligodeoxynucleotide". With respect to the sixth ODN, see Gomis S. et al., Infection and Immunity 71(2):857-863 (February 2003), the title as "Protection of Chickens against Escherichia coli Infections by DNA Containing CpG Motifs". Those immunostimulatory ODNs are listed as Table 2 -- see Original Patent.

Next, the first to sixth ODNs are optionally constructed into commercial vectors for large-scale production of the recombinant plasmids containing the first to sixth ODNs. In a preferred embodiment of the present invention, the DNA fragments of the first to sixth ODNs amplified by primers can be subjected to following evaluation of the avian immunostimulating activity in vitro and/or in vivo.

However, in another embodiment, the amplified DNA fragments are further constructed into the commercial vectors, for example, pGEM.RTM.T-Easy vector (Promega Corp., Madison, Wis., USA), respectively, to form recombinant plasmids. And then, suitable host cells, for example, competent cells of E. coli strain DH5.alpha., are transformed with the recombinant plasmids. In this embodiment, at least one set of the amplified DNA fragments having CpG motif can be constructed into the commercial vectors, for example, pGEM.RTM.T-Easy vector (Promega Corp., Madison, Wis., USA), in which the amplified DNA fragments in each recombinant plasmid may be 1 or up to 20 sets arranged in a sequence or with spacer there between. The amplified DNA fragments having CpG motif can be also more than 20 sets constructed in each recombinant plasmid, rather than being limited to the aforementioned quantity, even up to 32 or more sets therein.

After antibiotic selection and sequencing confirmation, the selected clone can be subjected to large-scale production. That is to say, the immunostimulatory ODNs utilized as CpG DNA adjuvant in avian vaccines can be produced in large scale by using bacteria. The construction of recombinant plasmids, transformation of the bacteria, large-scale production, purification of plasmid DNA, measurement of optical density (OD) of DNA at 260 nm, establishment of the standard curve and the like are known by a person skilled in the art, the related details are unnecessary to be addressed herein.

EXAMPLE 2

Construction of Recombinant Plasmids Containing Avian .beta.-Actin Gene and Interferon-.gamma. Gene, Transformation and Large-Scale Production Thereof

EXAMPLE 2 is related to construction of recombinant plasmids containing avian .beta.-actin gene and interferon-.gamma. gene, respectively, transformation and large-scale production thereof, in which the avian .beta.-actin gene and interferon-.gamma. gene are obtained from chicken peripheral blood mononuclear cells (PBMC) and splenocytes, by using the method disclosed by Haiqi H. et al., Developmental and Comparative Immunology 27(6-7):621-627 (2003) titled as "Identification of CpG oligodeoxynucleotide motifs that stimulate nitric oxide and cytokine production in avian macrophage and peripheral blood mononuclear cells". RNA isolation, synthesis of complementary DNA (cDNA), PCR reaction and the primers for cloning chicken .beta.-actin gene and interferon-.gamma. gene herein are referred to the research paper published by Haiqi H. et al. The chicken .beta.-actin gene is cloned by a PCR reaction with a first upstream primer shown as SEQ ID NO:7 and a first downstream primer shown as SEQ ID NO:8, and the chicken interferon-.gamma. gene is cloned by a second upstream primer shown as SEQ ID NO:9 and a second downstream primer shown as SEQ ID NO:10. The first upstream primer and the first downstream primer referred to the sequence of GenBank Accession No. NM.sub.--205518, and the second upstream primer and the second downstream primer referred to the sequence of GenBank Accession No. X99774, are listed below as Table 3 -- see Original Patent.

While performing the PCR reaction, the reaction solution is prepared by using the commercial product, for example, of Invitrogen Corp., Carlsbad, Calif., without being recited in detail herein. Next, the first upstream and downstream primer pair, and the second upstream and downstream primer pair, are added into the PCR reaction solution respectively to carry out the PCR reaction, in which the reaction condition may be exemplified as the following conditions but not limited thereto. For example, the PCR reaction mixture is firstly heated on 94.degree. C. for 5 minutes for denaturing double-stranded DNA (dsDNA) template to single-stranded DNA (ssDNA) templates. Next, the PCR reaction mixture is repeated for 30 cycles, and each cycle comprises a denaturation step on 94.degree. C. for 1 minute, an annealing step for annealing the primer pair to the ssDNA template on 60-70.degree. C. for 30 seconds to 1 minute, and an extension step on 68.degree. C. for 1 minute. Preferably, the annealing step is performed on 65.degree. C. for 45 seconds. After repeating the 30 cycles, the PCR reaction mixture is ended with the prolonged last extension step on 68.degree. C. for 5 minutes for ensuring the PCR reaction more completely.

Besides, the second upstream and downstream primer pair is added into the PCR reaction solution to carry out the PCR reaction, in which the reaction condition may be exemplified as the following conditions but not limited thereto. For example, the PCR reaction mixture is firstly heated on 94.degree. C. for 5 minutes for denaturing dsDNA template to ssDNA templates. Next, the PCR reaction mixture is repeated for 30 cycles, and each cycle comprises a denaturation step on 94.degree. C. for 30 seconds, an annealing step for annealing the primer pair to the ssDNA template on 60-70.degree. C. for 30 seconds to 1 minute, and an extension step on 68.degree. C. for 1 minute. Preferably, the annealing step is performed on 65.degree. C. for 45 seconds. After repeating the 30 cycles, the PCR reaction mixture is ended with the prolonged last extension step on 68.degree. C. for 5 minutes for ensuring the PCR reaction more completely.

After the aforementioned PCR reactions, a DNA fragment of .beta.-actin gene amplified by the first upstream and downstream primer pair has a full length of 408 base pairs (bp) approximately, another DNA fragment of interferon-.gamma. gene amplified by the second upstream and downstream primer pair has a full length of 571 bp approximately. The two DNA fragments are purified and further constructed into pGEM.RTM.T-Easy vector (Promega Corp., Madison, Wis., USA) with the same as EXAMPLE 1, respectively, to form recombinant plasmids. And then, suitable host cells, for example, competent cells of E. coli strain DH5.alpha., are transformed with the recombinant plasmids. After antibiotic selection and sequencing confirmation, the restriction enzyme E.coR.quadrature. digestion and the analysis by DNA electrophoresis are employed to check that the target gene fragments are inserted in the recombinant plasmids and their sequences are correct. Results of DNA electrophoresis of PCR reactions are shown as FIGS. 1A and 1B (see Original Patent).

Reference is made to FIGS. 1A and 1B, in which FIG. 1A is an electrophoresis image of PCR reaction product for analyzing the avian .beta.-actin gene according a preferred embodiment of the present invention, FIG. 1B is an electrophoresis image of PCR reaction product for analyzing the avian interferon-.gamma. gene according a preferred embodiment of the present invention, and FIGS. 1A and 1B are employed to determine that the DNA fragment inserted in the recombinant plasmids can be amplified in the correct size. In FIGS. 1A and 1B, the lane M is referred as DNA marker of 100 bp DNA ladder, and the "500 bp" labeled on the left of the lane M is referred as a site of the DNA ladder with 500 bp. In FIG. 1A, the lane 1 is referred as the inserted DNA fragment of the avian .beta.-actin gene with 408 bp approximately as indicated by Arrow 101. In FIG. 1B, the lane 1 is referred as the inserted DNA fragment of the avian interferon-.gamma. gene with 571 bp approximately as indicated by Arrow 103.

According to the results of FIGS. 1A and 1B (see Original Patent), the recombinant plasmids containing avian .beta.-actin gene and interferon-.gamma. gene constructed by the present invention can be amplified to the target DNA fragments in the correct size.

Claim 1 of 3 Claims

1. A CpG DNA adjuvant in avian vaccines, the CpG DNA adjuvant in avian vaccines has an immunostimulatory oligodeoxynucleotide (ODN) having a sequence of SEQ ID NO: 2.

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