DNA-based plasmid formulations and vaccines and prophylactics containing the same

Abstract

The invention is a general method for improving the performance of the DNA-based vaccines. The method utilizes a complex DNA-generated profile of antigens to extend the effects of DNA-based vaccines and to broaden the immune response. This broadened immune response in turn improves the protection of the recipient from divergent (but related) strains of a pathogen. In addition, it effectively improves the efficacy of DNA-based vaccines used for treatment of viral diseases, including acquired immunity disorder (AIDS). One embodiment, where the target viral pathogen is HIV (the causative agent for aids), the method identifies an orderly set of plasmids of related sequences that may be used to prime a broad and strong immune response to HLA-restricted viral antigens. This mixture of plasmids is thus capable of priming an appropriate immune response to reduce the viral burden in HIV infected patients or to protect uninfected patients from HIV infection.

Claims

1. A polymorphic vaccine comprising a hypervalent mixture of nucleic acid sequences capable of expressing a mixture of distinct antigens within the host organism; the host immune response being broader than that invoked by any one member of the hypervalent mixture by itself.

2. The vaccine of claim 1, wherein the hypervalent mixture of nucleic acid sequences are self-replicating plasmids and virus vectors.

3. The vaccine of claim 2, wherein the self-replicating plasmids and virus vectors are adenovirus.

4. The vaccine of claim 1, wherein the hypervalent mixture of nucleic acid sequences are in a vector.

5. A DNA based vaccine or prophylactic comprising twenty or more distinct plasmids, each of the plasmids having a nucleotide-encoding region for a distinct antigen of a pathogen and furthermore wherein none of the plasmids is dominant and wherein said plasmids are capable of evoking a broad specificity toward the pathogen; the host immune response being broader than that invoked by any of the plasmids individually.

6. The vaccine of claim 5, wherein the pathogen is retroviral pathogen HIV.

7. The vaccine of claim 5, wherein at least one of the plasmids is a linear or circular DNA molecule from a prokaryotic cell or an eukaryotic cell.

8. The vaccine of claim 5, wherein the plasmids or variants thereof are capable of immunizing the host immune system in order to mount a response to a challenge by the target pathogen.

9. The vaccine of claim 5, wherein each of the plasmids contain epitopes capable of priming the host immune system.

10. The vaccine of claim 5, wherein the nucleic acid sequences are polynucleotides derived from at least one gene in the target pathogen such that multiple variants of the selected antigens are generated.

11. A polymorphic vaccine against at least one pathogen, wherein said vaccine comprises a mixture of distinct plasmids, each of the plasmids having a nucleotide-encoding or polypeptide-encoding region for expression of related but distinct antigens, wherein none of the plasmids is dominant and further wherein said plasmids are capable of evoking a broad specificity toward the pathogen and variants thereof; the host immune response being broader than that invoked by any of the plasmids singularly.

12. The polymorphic vaccine of claim 11, wherein the distinct plasmids are composed of variants of the parental plasmid or the combination of parental plasmid and variants thereof.

13. The polymorphic vaccine of claim 11, wherein the plasmids, when expressed, are capable of generating a polypeptide for either the parental epitope or a variant thereof.

14. The polymorphic vaccine of claim 11, wherein the vaccine is for HIV, rabies, plague, influenza, smallpox, Ebola, Marburg, West Nile virus, anthrax, resistant strains of staphylococcus and streptococcus, Salmonella or E. Coli or any combination thereof.

15. The polymorphic vaccine of claim 11, wherein the pathogen is a bacteria or virus.

16. The polymorphic vaccine of claim 11, wherein the vaccine is for the treatment of cancer or toxins.

17. The polymorphic vaccine of claim 11, wherein pathogen is retroviral pathogen HIV.

18. The polymorphic vaccine of claim 12, wherein the plasmids are capable of encoding for pathogenic variants by conservative substitution of the amino acid residues of the parental epitope.

19. The polymorphic vaccine of claim 1, wherein the nucleic acid sequences include residues 39 through 70 of SEQ ID NO. 1.

20. The polymorphic vaccine of claim 1, wherein the nucleic acid sequences include residues 130 through 159 of SEQ ID NO. 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more folly understand the drawings referred to in the detailed description of the present invention, a brief description of each drawing is presented, in which:

[0013] FIG. 1 depicts the nucleic acid sequence of the parental peptide from Pol HTLV-IIIb (SEQ ID: 1). Two sites of multivariance are shown in bold print (SEQ ID: 2 & 3).

[0014] FIG. 2A shows the amino acid sequence of the parental antigenic peptide from Pol HTLV-IIIb (SEQ ID: 4). The multivariant epitopes are shown in bold (SEQ ID: 5 & 6). In FIG. 2B, amino acid substitutions within the multivalent epitopes are shown (SEQ ID: 7-11). These are designed from known sequences of clinical isolates of HIV. All possible combinations of the substitutions in the two epitopes are used in the design of the vaccine. The actual antigen is fused to a peptide for expression of the antigen from a DNA vaccine.

[0015] FIG. 3 shows the results of immunization testing in HLA-A*0201 transgenic mice with wild type and mutant HIV peptide fragments.

[0016] FIG. 4 shows the experimental results of murine responses versus drug-resistant HIV protease mutants and effects of boosting on immune responses.

DETAILED DESCRIPTION OF THE INVENTION

[0017] As used herein, the following terms shall have the designated meanings:

[0018] Hyperpolyvalenta preparation where twenty or more distinct plasmids are used as components in a DNA vaccine, and are used to evoke a host immune response that, while effective against a target pathogen, is broader that can be invoked by any of the plasmids delivered singly.

[0019] Nucleic acid sequencea polynucleotide chain that consists of various combinations of four monomers: adenine, guanine, cytosine, and thymine.

[0020] Full-length nucleic acid sequencea nucleic acid sequence that encodes a protein.

[0021] Fragment of nucleic acid sequencea portion of a full-length nucleic acid sequence that only encodes a region of a protein.

[0022] Antigenthe substance that stimulates the immune system to produce antibodies, usually a protein.

[0023] Mutationa nucleotide within a wild-type gene was substituted with a different nucleotide.

[0024] Conservative substitutionConservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids having basic side chains is lysine, arginine and histidine; a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acid substitutions are: valine-isoleucine-leucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine and asparagine-glutamine.

[0025] Epitopethe portion of an antigen that is recognized by and combines with the antibody.

[0026] Dominant epitopean epitope that is critical to the interaction between antibody and antigen.

[0027] The improvement is a hyperpolyvalent mixture of nucleic acid sequences that results in the expression of a mixture of related but distinct antigens within the host organism. These can include DNA, RNA, nucleic acids other than these, and both full-length and partial nucleic acid sequences. The nucleic acids may be presented as naked nucleic acid, or in a vector. Such vectors would include but are not limited to self-replicating plasmids and virus vectors (e.g. adenovirus). The term plasmid is used to refer to any small, independently-replicating piece of DNA that can be transferred from one organism to another. These include linear or circular DNA molecules that are found in both pro- and eucaryotes. Plasmids are capable of being integrated into the hosts genome or remaining independent and can be used to transfer genes to a host. This improvement uses a large number of closely related plasmid variants to immunize the host immune system so it can mount a response to a challenge by a variety of related but distinct strains of the target pathogens. This set of plasmids has a systematic design that defines the range of epitopes used to prime the host immune system. The present method uses polynucleotides derived from one or a small number of genes in the target pathogen, and multiple variants of the selected antigen are generated.

[0028] DNA vaccines are particularly effective for evoking a cytolytic T lymphocyte (CTL) response. The specificity of CTL and other immune responses are dependent in detail on the types and amounts of antigen produced and on the presentation of antigens on cells that receive plasmids. Using a single plasmid encoding an antigen to immunize an animal will promote the maturation of T cells specific for that antigen. However, the overall response may be dominated by a highly specific reaction and many components of the overall immune response will be relatively intolerant of even minor antigenic variation. A mixture of plasmids that expresses a set of closely related polypeptides but no single dominant specific antigen would lead to an immune response that is broad in its specificity. The set of plasmids is developed to vary the nature of the epitopes presented and the relative efficiency of presentation results in a broad specificity toward the pathogen and variants. This allows the host to develop effective resistance to the pathogen, in the face of challenge by previously undescribed variant strains or to overcome rapid mutation of the pathogen in response to immune system elimination.

Development of an Orderly Set of Plasmids as Components of a Vaccine

[0029] In one aspect, for development of DNA vaccine, a polypeptide-encoding region of the pathogen genome is selected as the parental antigen. As an example, the nucleotide coding sequence for the parental antigen of the HIV gene pol is provided in FIG. 1. However, this is only given as an example and is not meant to limit the scope of this invention to HIV alone nor is it meant to limit this invention to the pol gene of HIV. For any particular antigenic polypeptide, typically one or two regions of the antigen (approximately nine amino acid long and called the dominant epitope) are presented and dominate the MHC mediated immune response. The DNA polynucleotide encoding each amino acid of the antigenic region can be fused to a polynucleotide that encodes an antigenic peptide of the type typically used to enhance the immunicity of the resulting fused polypeptide.

[0030] In the present invention, a set of plasmids are created containing nucleic acid base sequences that, when translated, systematically replace amino acid residues in the dominant epitope region, for example, by substituting with conservative amino acid replacements. Examples of conservative changes can be identified from Table 1 below.

TABLE-US-00001 TABLE 1 Amino Acid A G P C M K Conservative G P G M C A replacement(s) V A S T T E S S R D The amino acids are designated by their standard one letter code.
However, random mutations may also be incorporated. Furthermore, the amino acid sequence flanking the epitope can be varied to alter the strength and specificity of the MHC restriction system's presentation of the antigen.

[0031] An example of a translated polynucleotide sequence with dominant epitopes is shown in FIGS. 2A and 2B. An example of multivariant epitopes using a fragment of the HIV pol gene is shown in FIG. 2B. However, this is only given as an example and is not meant to limit the scope of this invention to HIV alone nor is it meant to limit this invention to the pol gene of HIV. Additionally, the hyperpolyvalent mixture can include the variants for one or several genes of the same organism.

[0032] One key feature of the system is to create a mixture of plasmids encoding a rich variety of related antigens, each capable of expressing significant amounts of antigen in the host cells that receive it. The variant dominant epitopes presented in each transfected host cell play a role in triggering the host organism's total immune response. T lymphocytes that can key in on a broader range of the available epitopes are favored in selection of those with restrictively narrow response specificity. The overall CTL response is thus dominated by T lymphocytes with immune response profiles that are broader than the response triggered by a single antigenic plasmid.

[0033] The compositions of the present invention can be used for vaccination of animals against a variety of diseases caused by bacterial and/or viral pathogens. For example, the compositions of the present invention can be used for vaccination against HIV, rabies, plague, influenza, smallpox, Ebola, Marburg, and West Nile viruses as well as treatment for infection with anthrax, resistant strains of staphylococcus and streptococcus, Salmonella, E. coli, and other bacterial agents. This technology can also be used in treatment of different cancers or toxins. Additionally it can be used to strengthen current antibiotics, vaccines, or prophylactics. In a current embodiment (example) the compositions of the present invention can be used to vaccinate animals against HIV.

Testing as a Part of Vaccine Development

[0034] This aspect of the invention is tested in a mouse model system, a step for testing any vaccine prior to assessment in human volunteers. The general concept of testing DNA based vaccines in mouse model systems is well known to those skilled in the art of vaccine development. In such a test, a suitable strain of mice is inoculated using the plasmid based vaccine formulation. This formulation is delivered using standard techniques of injecting naked DNA intramuscularly. The delivery system may be saline solution of a gel-based depo for gradual release of plasmid. Adjuvants may be used to enhance the immune response to the antigen expressed from the DNA plasmids. Cells that receive and express the plasmid mixture present processed a mixture of related antigens to the immune system. Supplying the cytokine IL6 along with the plasmid may further stimulate the immune system. While IL6 may be supplied as a protein, for purposes of this test the cytokine is encoded on as an expressible DNA sequence inserted into a plasmid, so that cells in the vicinity of injection site produce the cytokine. The immunization is boosted by periodic re-inoculation with the vaccine formulation to enhance the immune response. Control mice are injected with saline alone and additional control mice are inoculated with IL6 plus adjuvant only. A third control group consists of mice inoculated with a single species of antigen supplying plasmid, which stimulates a relatively narrow immune response to the specific HIV (pol) antigen presented. The mice are sacrificed periodically (after 2, 6 and 12 weeks) and spleens are collected to perform a test of the CTL response. The standard test is to look for the activation of the CTL responses using synthetic peptides (Wu L, Barry M A., Mol Ther 2000 September; 2(3): 288-297). In this test, one specific variety of dominant epitope is deliberately omitted from the plasmid mixture used to inoculate the test mice. The parental antigen is omitted so that the known dominant antigen may be used to assay the effectiveness of the vaccine in stimulating a broad CTL response. The reasoning is that the broad CTL response stimulated by the complex plasmid mixture will be able to recognize a related epitope not specifically supplied in the vaccine (in comparison to the control mice). Furthermore, the assay for the parental type is highly reliable. Additional antigens are also tested to verify the effectiveness of the immune response to a broad range of antigens.

[0035] Practical creation of large sets of sequence related DNA plasmids is possible using recently described automated DNA synthesis techniques (U.S. 60/276,161). These techniques use automated liquid handling equipment to generate tens to hundreds of sequence related polynucleotides for cloning. Such a system is used to create an orderly set of clones with the engineered changes to the antigen's dominant epitope and flanking regions.

[0036] An important aspect of this invention is a method for the vaccination against HIV, the causative agent for AIDS. The current treatments for HIV/AIDS are problematic due to the antigenic shift/mutation rate of the virus. By vaccination with variants of the genes/pathogenic epitopes contained in HIV, a broader immune response is triggered. This would help defend the host against the antigenic shift observed with HIV.

[0037] The present invention provides a pharmaceutical product, comprising of a hypervalent mixture of the gene or genes of interest as part of a plasmid. This hypervalent mixture includes epitope variants of each of the gene or genes of interest. The number of variants is at least nine; one per amino acid within the dominant epitope. Each polynucleotide operatively codes for a biologically active polypeptide. The hypervalent polynucleotide is administered in a physiologically acceptable form, in a container, and a notice associated with the container in form prescribed by a governmental regulatory agency regulating the manufacture, sale, and/or use of pharmaceuticals. Obviously many modifications and variations of this invention are possible in the light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.

[0038] The invention is further described by the following examples, which are provided for illustrative purposes only and are not intended nor should they be construed as limiting the invention in any manner. Those skilled in the art will appreciate that variations on the following examples can be made without deviating from the spirit or scope of the invention.

Example I

[0039] The mouse strain BALB/C (H-2d) is used to demonstrate the effect of the hyperpolyvalent vaccination on ct cell response. In brief, thirty mice are divided into three groups of ten animals each. The mice are bred and housed in a pathogen free environment. The three groups are: control group of mice injected only with saline, a group of mice injected with a single plasmid type with the parental sequence, encoding the target epitope and a third group injected with the hypervalent vaccine which is formulated to encode a mixture of sequence types but deliberately not containing the parental epitope. This formulation is used so that any response to the parental epitope is a direct indication of broad specificity of t-cell response. The animals are injected (50 microliters of saline solution) with purified DNA plasmid (vaccine or control) into the Quadriceps and tibialis muscles using a 0.5 cc syringe with a 28-gauge needle. Spleens and blood serum are collected from animals sacrificed at 2, 4 and 12-week intervals. Western Blot Assays are used to measure humeral (antibody response) against viral encoded antigens and CTL response is measured using the T-cell activation assays (Ulmer, J. B., et al., Science 259:1745-1749).

Example II

[0040] FIG. 7 shows the immunization results based on mutant and wild type HIV immunization. Plasmids encoding the wild type HIV protease and mutants were used for immunization into HLA-A*0201 transgenic mice. Percent CD69+ T cells were quantified using FACS sorting after stimulation of splenocytes with the wild-type epitope. The immune response for wild type and mutants was of similar potency measured for response versus wild type HIV protease, with the best response being for single mutants and a slight decrease with increasing number of mutations, as expected. From the foregoing, it will be observed that numerous variations and modifications may be effected without departing from the true spirit and scope of the novel concepts of the invention.

Example III

[0041] FIG. 8 shows the results of experimental responses versus drug-resistant HIV protease mutants and effect of boosting on immune responses. Transgenic mice (n=5/vaccine) were genetically immunized with 12.8 g of plasmid DNA encoding either ELI gag-pol, codon-optimized (CO)-gag-pol or, wt-gag-pol vaccine or with 0.8 g of plasmid DNA encoding CO-gag-pol or, wt-gag-pol vaccine plus 12 g pCMVi plasmid DNA without or with boosting. One (primed only) or two months later (primed and boosted) splenocytes (110.sup.6/sample) were stimulated for 6 h with irradiated (6000 rads) 10T/2 stimulator cell lines stably transfected with HLA-A*0201 molecule at responder: stimulator ratio of 10:1 in the presence of indicated peptide (5 gimp. Peptides used for stimulation are shown below with mutations in bold (Clade B reference). CD3/CD8 double positive cells (5-1010.sup.4/sample) were analyzed for intracellular IFN- production on a FACScan using CellQuest software. Data shown are mean+/standard deviation (n=5).

[0042] Splenocytes were tested for their ability to recognize wild-type (45-B and 75-B) or mutant peptides (45mt-B and 75mt-B) for pol.sub.45-54 and pol.sub.75-84. Boosting with both gag-pol plasmids drastically attenuated the repertoire of cross-reactive CD8 T cell responses against the sub-dominant cognate epitope and against the mutated epitopes from the drug-resistant virions, similar to the responses against clade mutants. The HIV-1 ELI vaccine generated stronger cross-reactive CD8 responses after priming and all of these responses were boosted after second immunization. These data indicate that wild-type gag-pol genetic vaccine drive focused CD8 T cell responses against immunodominant epitopes. In contrast, the ELI vaccine avoids immunofocusing, drives stronger responses against dominant and sub-dominant epitopes, and also maintains cross-reactive responses against mutated epitopes.