WO2000025820A1

WO2000025820A1 - Compounds and methods for genetic immunization

Info

Publication number: WO2000025820A1
Application number: PCT/US1999/025979
Authority: WO
Inventors: John Gebhard; Barbara A. Araneo
Original assignee: Pharmadigm, Inc.
Priority date: 1998-11-04
Filing date: 1999-11-03
Publication date: 2000-05-11
Also published as: AU1606100A

Abstract

The present invention relates to compounds and methods for genetic immunization of, for example, an elderly patient. The compounds and methods of the invention provide for efficient immunization of elderly patients, a population of patients normally refractile to immunization, as well as more efficient immunization of non-elderly patients as compared with prior technique and compositions. Also provided are compounds and methods useful for screening potential antigens for their ability to elicit an immune response.

Description

COMPOUNDS AND METHODS FOR GENETIC IMMUNIZATION

1. INTRODUCTION

The present invention relates to compounds and methods for genetic immunization of, for example, an elderly patient. The compounds and methods of the invention provide for efficient immunization of elderly patients, a population of patients normally refractile to immunization, as well as more efficient immunization of non-elderly patients as compared with prior technique and compositions. Also provided are compounds and methods useful for screening potential antigens for their ability to elicit an immune response. Antigens able to elicit an immune response can be further tested for their ability to elicit a protective immune response. Antigens able to elicit a protective immune response are useful for the development of vaccines .

2. BACKGROUND OF THE INVENTION

Vaccination and immunization generally refer to the administration to an individual of a non-virulent agent against which the individual's immune system can initiate an immune response which will then be available to defend against challenge by the pathogen from which the non-virulent agent is derived. The agent, usually a protein, represents a target against which the immune response is made. The immune system can provide multiple means for eliminating such targets, including humoral and cellular responses. Briefly, the humoral response involves B cells which produce antibodies that specifically bind to antigens. —The cellular immune response involves helper T cells, which produce cytokines and elicit participation of additional immune cells in the immune response, and killer T cells, also known as cytotoxic T lymphocytes (CTLs) , which are immune cells capable of recognizing antigens on host cells and attacking the cell or particle displaying the antigens to rid the host of the pathogen. It is important to understand the type of helper T lymphocyte response induced by immunization since the type of helper T lymphocyte stimulated by an antigen is an important factor for defining which type of immune response will follow. Mosmann and colleagues (Cherwinski et al., 1987, Journal of Experimental Medicine 166:1229-1244; Mosmann and Coffman, 1989, Annual Reviews of Immunology 7.: 145-173) discovered that there are at least two different types of helper T lymphocytes (Th) which can be identified based on cytokine secretion. Thl lymphocytes secrete substantial amounts of IL-2 and INF-gamma and execute cell-mediated immune responses, whereas Th2 lymphocytes secrete IL-4, IL-5, IL-6 and IL-10 and assist in antibody production for humoral immunity. Theoretically then, antigenic stimulation of one T helper cell subset and not the other would result in production of a particular set of cytokines which would define the resulting immune response.

There are several vaccine strategies for presenting pathogen proteins, including the administration of (1) killed or attenuated pathogens that are unable to pathogenically infect the host, (2) isolated pathogen proteins and (3) recombinantly inactivated pathogens. One of the more recently developed strategies is genetic immunization.

Genetic immunization is a method of using an expressible DNA encoding an antigen to immunize against the infectious agent from which the antigen is derived (U.S. Patent No. 5,589,466; U.S. Patent No. 5,679,647, U.S. Patent No. 5,593,972; and U.S. Patent No. 5,703,057, each of which is incorporated by reference herein in its entirety) . The antigen-encoding gene's expression is driven by_^a strong promoter when intramuscularly, intradermally, orally, or otherwise delivered. While the mechanisms of DNA uptake and expression by the vaccinated host are unclear, it has been shown that antigens coded for by such DNA are expressed in the context of the major histocompatibility complex (MHC) of the host cells. Depending on the attached targeting signals, the antigen can be directed toward major histocompatibility complex (MHC) class I or II presentation (Ulmer et al , 1993, Science 259:1745-1749; Wang et al . , 1993, Proc . Natl . Acad . Sci . USA 90_.:4156-4160) ^'. This results in the stimulation of both a humoral and cellular immune response to the antigen coded for by the injected DNA. DNA vaccines are considerably more stable, safe, and inexpensive to produce, than proteinaceous or live/attenuated vaccines, and do not require refrigeration. Genetic immunization (a.k.a. DNA, polynucleotide etc. immunization) with specific genes has shown promise in several model systems of pathogenic disease (Davis et al, 1993, Human Molecular Genetics 2 : (11) 1847-1851 ; Conry et al, 1994, Cancer Research 54_.: 1164-1168 ; Xiang et al , 1994, Virolo y 199:132-140), and a few natural systems (Cox et al, 1993, Journal of Virology 67(9) : 5664-5667; Fynan et al, 1993, Proc . Natl . Acad. Sci . USA 90:11478-11482). One problem associated with genetic immunization has been that, depending upon the method of DNA delivery, either a Thl or a Th2 immune respone, but not both, is elicited. Injection of DNA causes a Th2 response, while delivery via gene gun causes a Thl response (Feltquate, et al, 1997, J. Immunology, 158 (5) : 2278-84. It is believed that the generation of both responses is important for protection from most, if not all, pathogens. Thus, the polarized immune response elicited by prior genetic immunization methods and compositions is not ideal for the generation of a protective immune response.

Increased age is associated with a decline in both the cell-mediated and humoral immune response (Klinman et al, 1997, Springer Semin Immunopathol 19.:245-256) . T cell responses are thought to be more severely effected by aging than are B cell responses (Weigle et al . , 1994, Immunology and Infectious Disease 5.(2) :133-146) . Various factors have been identified as potentially contributing to age-associated reductions in immune response, including involution of the thymus, loss of T effector cell precursors, diminished antigen-presenting cell activity, and changes in the number, frequency and activation state of type 1 and type 2 cytokine secreting cells (Klinman et al, 1997, Springer Semin Immunopathol 19:245-256) .

Paradoxically, the age-associated reduction in immune response is accompanied by an age-associated increase in 5 autoimmune response. The presence of autoantibodies is positively correlated with age, as is autoreactivity of T cells, thus both the humoral and cellular components of the immune response are effected.

A vaccine for the elderly patient population must be

10 able to compensate for age-associated impairment of the humoral and cellular immune responses.

Prior attempts at genetic immunization of aged mice have been unable to overcome the age-associated decrease in immune response (Klinman et al, 1997, Springer Semin Immunopathol

15.19:245-256) . While an immune response is elicited in the aged mice, it is significantly less than the immune response elicited using the same vaccination protocol in young, adult mice. Hence, in a mouse model, genetic immunization has been unable to provide for equally efficient immunization of the

20 elderly as compared to the young adult.

3. SUMMARY OF THE INVENTION

The present invention relates to compositions and methods useful for immunization of mammals, preferably

25 humans. Additionally, the present invention relates to compositions and methods useful for the identification of antigens which may prove to be effective at eliciting a protective immune response in a mammal, preferably a human. The invention is based, in part, on the Applicants'

30 discovery that a DNA sequence encoding an antigen operably linked to a promoter or control element can be used as a DNA or genetic vaccine which elicits an immune response in aged mice which is nearly identical to the immune response elicited in young mice. While not being bound to any theory,

35 it appears that the promoter should be tightly regulated, relatively weak (as compared, for example, to a strong promoter that results in high levels of expression) and that it is beneficial to remove all unnecessary bacterial sequences from the genetic vaccine DNA construct. The principle of the invention is demonstrated hereinbelow by working examples. Surprisingly, the immune response 5 generated is equally protective of both aged and young adult mice against the pathogen from which the antigen is derived.

The invention is also based, in part, on the Applicants' discovery that a DNA sequence encoding an antigen operably linked to a promoter or control element can be used as a DNA

10 or genetic vaccine which elicits both a Thl type and Th2 type immune response. While not being bound to any theory, it appears that the promoter must be tightly regulated, and that it is beneficial to remove all unnecessary bacterial sequences from the genetic vaccine DNA construct. The

15 principle of the invention is demonstrated hereinbelow by working examples. Surprisingly, both a Thl and a Th2 type immune response is generated from intramuscular injection of DNA. Preferably, the Thl and Th2 responses are approximately equivalent. In other embodiments, the Th2 response is within

20 50%, 40%, 30%, 20%, or 10% of the Thl response.

Accordingly, the present invention provides a DNA immunogen comprising a DNA coding for an antigen operably linked to a promoter, such that, when administered to an aged mammal intramuscularly, the DNA immunogen is able to elicit

25 an immune response in the aged mammal. Preferably, the immune response elicited in the aged mammal is substantially equal to that elicited by the same treatment in an adult mammal of the same species. In a preferred embodiment, the immune response is a protective immune response, i.e., able

30 to prevent disease or ameliorate subsequent infection by or symptoms associated with the pathogen from which the antigen was derived. Preferably, the protective effect elicited in the aged mammal is substantially equal to that elicited by the same treatment in an adult mammal of the same species.

35 The promoter can be any promoter capable of directing the expression of the DNA coding for the antigen. In a preferred embodiment, the promoter is any promoter other than a viral promoter. In another preferred embodiment, the promoter is a muscle specific promoter.

The present invention also provides a DNA immunogen comprising a DNA coding for an antigen operably linked to a promoter, such that, when administered to a mammal intramuscularly, the DNA immunogen is able to elicit both a Thl and a Th2 type immune response . In a preferred embodiment, the immune response is a protective immune response, i.e., able to prevent disease or ameliorate subsequent infection by or symptoms associated with the pathogen from which the antigen was derived. The promoter can be any promoter capable of directing the expression of the DNA coding for the antigen. In a preferred embodiment, the promoter is any promoter other than a viral promoter. In another preferred embodiment, the promoter is a muscle specific promoter.

The present invention also provides methods of immunizing mammals, either aged or not, with the DNA immunogens of the invention. Such methods comprise the steps of obtaining an expressible DNA coding for an antigen and administering the DNA to a mammal .

The present invention also provides methods of screening for DNA immunogens. Such methods comprise the steps of obtaining an expressible DNA coding for a potential antigen, administering the DNA to a mammal, either aged or not, and testing for an immune response to the antigen in the mammal. DNA able to elicit an immune response to the potential antigen is identified as a DNA immunogen.

The present invention also provides methods of screening for DNA vaccines. Such methods comprise the steps of obtaining an expressible DNA coding for a potential antigen, administering the DNA to an mammal, either aged or not, and testing for a protective immune response in the mammal . DNA able to elicit a protective immune response is identified as a DNA vaccine.

The constructs of the invention can also be used for expression library immunization strategies, which involve immunizations against a pathogen when the identity of one or more particular antigens able to elicit an immune response is unknown (Barry and Johnston, 1997, Vaccine 15(8) : 788-91; Ulmer et al . , 1996, Trends Microbiol . 4 (5) : 169-70 ; Barry et al., 1995, Nature 377_.: 637-35) . The entire expression library itself, or portions thereof, can be used as an immunogen in aged individuals, or the technique can be used to identify the particular antigen or antigens effective at eliciting an immune response. Preferably, the immune response is a protective immune response.

4. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 graphically illustrates the survival rates of DNA immunized adult and aged mice after challenge with HSV-2. In the legend, MCKv.2 refers to mock immunized mice, i.e., mice immunized with the plasmid pMCKv.2 without the gD2 DNA insert. gD2 refers to mice immunized with the plasmid pMCKv.2 with the gD2 insert.

Figure 2 graphically illustrates the occurrence of genital herpes in DNA immunized adult and aged mice after challenge with HSV-2. In the legend, pMCKv.2 refers to mock immunized mice, i.e., mice immunized with the plasmid pMCKv.2 without the gD2 DNA insert. pMCKv.2-gD2 refers to mice immunized with the plasmid pMCKv.2 with the gD2 insert. Figure 3 graphically illustrates the immune response generated in DNA immunized aged and adult mice before and after challenge with HSV-2. In the legend, MCKv.2 refers to mock immunized mice, i.e., mice immunized with the plasmid pMCKv.2 without the gD2 DNA insert. MCKv.2-gD2_ refers to mice immunized with the plasmid pMCKv.2 with the gD2 insert. On the X axis, primary, secondary and tertiary refer to the first, second, and third immunizations with the indicated plasmid prior to virus infection or challenge. Also on the X axis, "PID" refers to days post-infection with HSV-2. Figure 4 graphically illustrates the immune response generated in DNA immunized adult mice before and after challenge with HSV-2. On the X axis, first, second and third refer to the first, second, and third immunizations with the plasmid pMCKv.2 with the gD2 insert prior to virus infection or challenge. Also on the X axis, "PID" refers to days post- infection with HSV-2. The bars represent the number of mice generating an IgGl (Th2) or an IgG2a (Thl) response. Also indicated are the number of mice in which equivalent Thl and Th2 responses were generated, and the number of mice in which neither a Thl nor a Th2 immune response was generated.

5. DETAILED DESCRIPTION OF THE INVENTION

The DNA constructs of the invention comprise a promoter element and one or more DNA sequences coding for an antigen operably linked to the promoter element. By "operably linked", it is meant that the promoter is able to drive the expression of the DNA sequence coding for the antigen. The DNA sequence encoding the antigen is preferably heterologous .

5.1. Promoter Elements

The DNA constructs of the present invention comprise a promoter, and may include one or more enhancer elements. Any promoter capable of directing the expression of DNA coding for an antigen can be used. In a preferred embodiment, the promoter is any promoter other than a viral promoter. The promoter may be constitutive, inducible, or tissue specific. Examples of promoters include, but are not limited to, promoters associated with the genes for: skeletal actin, myosin, phospholipase C-γ-1, interferon , p-phosphoenol pyruvate carboxykinase, keratinocyte 14, keratin 5, and phosphoglycerate kinase . __ Inducible promoters may also be utilized in the DNA construct of the invention. The strength, i.e., the ability to direct expression of downstream DNA sequences, of an inducible promoter varies in response to an environmental factor, termed an inducer. For example, the metallothionein promoter is more active in the presence of a metal ion, such as Zn²⁺ . The use of an inducible promoter is advantageous in situations where precise control of the timing of antigen expression is required. An example of such a situation would be an immunization protocol that consists of a primary immunization followed by one or more secondary (i.e., "booster") immunizations. After injection of the DNA immunogen, the timing of the exposure to the antigen can be controlled by administration of an inducer for the desired period of time. The antigen will only be expressed in the presence of the inducer. Subsequent immunizations can then be carried out by administering the inducer alone, with no further injection of DNA immunogen necessary. Expression of DNA injected into muscle can persist for at least 60 days (U.S. Patent No. 5,589,466).

Inducible promoters useful for the constructs and methods of the present invention include the tet on/off switch (Dhawan et al . , 1995, Somat. Cell . Mol . Genet . 2i(4) :233-40) , heat shock promoters, the interferon γ promoter, and the metallothionein promoter. Vitamin D3 regions and glucocorticoid elements may also be used to confer, respectively, vitamin D3 and glucocorticoid inducibility.

The use of tissue specific promoters, in particular, muscle specific promoters, is also contemplated. Examples of muscle specific regulatory elements include those which are isolated from muscle specific genes, such as the muscle isozyme of creatine kinase (MCK) (Sternberg et al . , 1988, Mol. Cell . Biol . 8_.:2896) , myosin light kinase (Merlie 1992, Cell 69:67; Merlie 1992, Cell 69:79), muscle-specific aldolase (Concordet et al . , 1993, Mol . Cell . Biol . 13:9), muscle specific enolase (Gaillongo et al . , 1993_, Virology 70:71), troponin C (Prigozy et al . , 1993, Somatic Cell Mol . Genet . .19:111), myosin (Kitsis et al . , 1991, Proc. Natl . Acad. Sci . USA 8j3:4138; Takeda et al . , 1992, J. Biol . Chem. 267:16957; von Harsdorf et al . , 1993, Circulat . Res . 72:689). These elements can be modified, by, for example, the removal of unnecessary or interfering sequences. Preferably, the promoter element substantially retains muscle specificity of transcription. In a preferred embodiment, the muscle specific promoter is the MCK promoter. In a further preferred embodiment, the DNA construct of the invention comprises the pMCK plasmid, or portions thereof . The pMCK plasmid contains the MCK promoter, and is the subject of United States Patent

Application No. 08/530,529, allowed in January of 1998, which is incorporated herein in its entirety.

The use of chimeric promoters is also contemplated. A chimeric promoter is produced by placing tissue specific enhancer elements in proximity to a constitutive promoter, thus conferring tissue specificity on the construct. For example, MCK enhancer elements are able to confer muscle specificity on a normally non-specific, constitutive promoter .

5.2. Antigenic Coding Regions

The DNA constructs of the invention also comprise DNA sequence coding for an antigen or potential antigen. An antigen is any protein which is capable of eliciting an immune response to the protein in a host. In the. following table, sources of antigens or potential antigens, and known or hypothesized mediators of cellular and humoral immunity are listed. Under each of the mediators, in parenthesis, a particular antigen expected to elicit that mediator is provided, where known. In general, antigens that are expressed and remain within or on the cell are expected to be presented to the immune system in association with major histocompatibility complex class I proteins, thereby eliciting a cellular immune response. Antigens, which are secreted and not associated with a cell are expected to elicit a humoral immune response (U.S. Patent No. 5,589,466, incorporated herein in its entirety) . This table is primarily useful for a listing of potential antigen sources; the listed particular mediators of the immune response are not intended to limit the invention to embodiments that elicit the listed immune response for each pathogen. Methods and compositions useful for the elicitation of any immune response to any antigen in the elderly through genetic immunization are within the scope of the invention.

List of Candidate Pathogens for DNA Vaccines Bacterial

**Bold shading indicates a prominent role in immunity

Fungi

Viruses

Parasitic

Other Viruses DNA

Other Microorganisms

Fungi

Cryptococcus neoformans Aspergi11us His topi asma Coccidioides immitia Pneximocystis carnii Bacterial

Hemophilus influenza Campy 1 oba cter

Bordatella pertussis (Whooping cough pathogen)

Hyperproliferative Disorders

In addition, antigens may be those associated with hyperproliferative disorders. Non-limiting examples of such antigens include protein products of oncogenes myb, myc, fyn, ras, src, neu and trk; protein products of translocation gene bcr/abl; P53; variable regions of antibodies made by B cell lymphomas; and variable regions of T cell receptors of T cell lymphomas. xxx add tumor antigens

5.3 Identification Of Antigens

In cases where a pathogen has been identified and isolated, but an antigen capable of eliciting an immune response to that pathogen has not been identified and isolated, there are several methods of eliciting an immune response to the pathogen, and of identifying particular antigens and epitopes capable of eliciting such a response.

One such method is expression library injection (ELI) . Briefly, an expression library of the pathogen's genome is constructed, and the entire library, or a portion of the library, is injected into the host. The subsequent expression of many of the peptides coded for in the pathogen's genome results in the development of an immune response to such peptides . A fraction of the peptides are expected to elicit a protective immune response. This has successfully been used and is completely described in U.S. Patent No. 5,703,057, which is incorporated by reference herein in its entirety. Consecutive sub-libraries composed of subsets of the library's members can be used to identify the particular clone or clones capable of eliciting an immune response to the pathogen.

In another approach, when a particular protein is expected to be immunogenic, but the particular epitope is unknown, vaccinia virus recombinants can be used to identify the epitope. The gene coding for the protein of interest is truncated in both the 5' and 3' directions using modifying endonucleases such that a series of overlapping truncations and deletions are obtained. The gene fragments are cloned into a vaccinia virus transfer plasmid, which contains vaccinia sequences to facilitate homologous recombination. The transfer plasmid harboring the gene fragments and a thymidine kinase deleted vaccinia virus are picked and plaque purified three times. The resultant recombinants are utilized as a source of epitopes representing portions of the corrupted gene of interest. The aforementioned technique could be applied to genomic DNA also, in cases where the identification of a suspected antigenic protein is not possible. To identify the epitopes, mice are infected with the pathogen from which the suspected antigenic protein was derived. At an appropriate time (approximately 7 days) after infection to allow for the generation of cytotoxic T lymphocytes (CTLs) , the spleens are harvested and splenocytes prepared. MHC restricted fibroblasts infected with the targets in ratios of 10:1 to 100:1 and the amount of cytolysis of the fibroblast observed. In such a way it is possible to identify MHC-restricted CTL epitopes that are immunodominant or immunorecessive. Recently, Rodriguez and Whitton described a technique whereby it is possible to force a protein into the MHC class I pathway, and thus generate CTLs. Their technique is based on ubiquitination of the desired antigen. These CTLs could be tested as described above (Rodriguez et al . , 1998, J. Virol . 72 (6) : 5174-81) . Candidate antigens could also be acquired by differential display or expressed sequence tags. For instance, the pattern of RNA expression in a cell in the presence and absence of a pathogen may serve to identify potential upregulated viral proteins . Such RNAs could be reverse transcribed into cDNA, subcloned into a plasmid, and mice immunized to determine the immunogenicity of the selected RNA (Fung et al . , 1998, Int. J. Oncol . 13 (1) :85-89; Frank et al . , 1998, Cancer Lett. 123 (1) : 7-14) .

5.4. Methods of Immunization and Screening The present invention also provides methods of immunizing aged mammals with the DNA immunogens of the invention. Such methods comprise the steps of: a) obtaining a functional recombinant plasmid suitable for use as a DNA vaccine by operably linking DNA which codes for an antigen to a promoter such that the expression of the inserted DNA is controlled by the promoter; and b) administering the functional recombinant plasmid by

7intramuscular injection to an aged mammal. The present invention also provides methods of identifying DNA immunogens. Such methods comprise the steps of: a) obtaining a functional recombinant plasmid suitable for use as a DNA vaccine, the plasmid having a DNA which codes for a potential antigen operably linked to a promoter such that the expression of the inserted DNA is controlled by the promoter; and b) administering the functional recombinant plasmid by intramuscular injection to an aged mammal; c) testing for an immune response to the antigen in the aged mammal ; wherein recombinant plasmids which are able to elicit an immune response to the potential antigen are identified as DNA immunogens . _ The present invention also provides methods of identifying DNA vaccines which are useful for immunizing aged mammals. Such methods comprise the steps of: a) obtaining a functional recombinant plasmid suitable for use as a DNA vaccine, the plasmid having a DNA which codes for a potential antigen operably linked to a promoter such that the expression of the inserted DNA is controlled by the promoter; and b) administering the functional recombinant plasmid by intramuscular injection to an aged mammal; c) testing for a protective immune response in the aged mammal ; wherein recombinant plasmids which are able to elicit a protective immune response are identified as DNA vaccines useful for immunizing aged mammals.

The present invention also provides methods of immunizing mammals with the DNA immunogens of the invention, methods of identifying DNA immunogens, and methods of identifying DNA vaccines which are useful for immunizing mammals, regardless of the age of the mammal to be vaccinated, treated, or injected with the immunogen. For such methods, the above described methods relating to aged mammals are modified such that any mammal, preferably a non- aged mammal, is utilized instead of an aged mammal. For methods directed to mammals regardless of age, the immunization preferably elicits both a Thl and a Th2 immune response. Accordingly, the methods of identifying antigens can include the additional step of determining whether both a Thl response and a Th2 response is elicited. Those antigens which elicit both responses may be desireable antigens. In a preferred embodiment, the Thl and Th2 responses are approximately equivalent. In other embodiments, the Th2 response is within 50%, 40%, 30%, 20%, or 10% of the Thl response. The Thl and Th2 responses may be measured by any method known to those of skill in the art. For example, the Thl response may be measured by detecting the level of IgG2a antibodies specific for the antigen, while the Th2 response may be measured by detecting the level of IgGi antibodies specific for the antigen.

The above methods can be repeated at predefined intervals in order to potentiate the immune response, e.g., by administering "booster" immunizations. When booster immunizations are to be given, the first administration is sometimes referred to as the "primary" immunization, and subsequent immunizations termed "secondary", "tertiary", and so on. In a preferred embodiment, a primary immunization is followed by a secondary and a tertiary immunization, for a total of three immunizations, however, any number of immunizations can be performed. It is preferable that, before exposure to the pathogen from which protection is sought by immunization, sufficient time for the development if an immune response to the immunization antigen is allowed to pass.

For each of the above methods, in preferred embodiments, the promoter is a muscle specific promoter. In further preferred embodiments, the promoter is the MCK promoter. In other embodiments, the constructs are made by inserting the DNA encoding the antigen into the plasmid pMCK, which is fully described in U.S. Application No. 08/530,529. The DNA encoding the antigen can also be inserted into the plasmid pMCKv.2, the construction of which is described in the examples below.

Expression library injection (ELI) is a method of immunizing against a pathogen when the particular antigen or antigens capable of inducing a protective immune response are unknown. To summarize the technique, an expression library of the pathogen's genome is constructed, and the entire library, or a portion of the library, is injected into the host. The subsequent expression of many of the peptides coded for in the pathogen's genome results in the development of an immune response to such peptides. A fraction of the peptides are expected to elicit a protective immune response. This has successfully been used and is completely described in U.S. Patent No. 5,703,057, which is incorporated by reference herein in its entirety. Contemplated in the present invention is the use of the expression library injection method, wherein the host is an aged mammal. The promoter used for construction of the expression library can be any promoter capable of directing expression of downstream DNA sequences, and in a particular embodiment, is a muscle specific promoter. In a further embodiment, the promoter is the MCK promoter. In another embodiment, the plasmid used to construct and express the expression library is the plasmid pMCK.

5.5. Modes of Administration Genetic constructs may be administered by means including, but not limited to, syringes, needleless injection devices, or "microprojectile bombardment gene guns". Alternatively, the genetic vaccine may be introduced by various means into cells that are removed from the subject. Such means include, for example, ex vivo transfection, electroporation, microinjection and microprojectile bombardment. After the genetic construct is taken up by the cells, they are reimplanted into the subject. It is contemplated that otherwise non-immunogenic cells that have genetic constructs incorporated therein can be implanted into the subject even if the vaccinated cells were originally taken from another subject.

The genetic constructs can be "naked", i.e., free from any delivery vehicle that can act to facilitate entry into the cell, for example, the polynucleotide sequences are free of viral particles which may carry genetic information. They can be similarly free from, or naked with respect to, any material which promotes transfection, such as liposomal formulations, charged lipids such as Lipofectin (tm) or precipitating agents such as CaP0₄.

Alternatively, the genetic construct may be associated with any delivery vehicle that can act to facilitate entry into the cell. They may also be co-administered with any substance capable of enhancing the immune response to the peptide product of the construct, such as co-administration with an adjuvant.

The genetic vaccines according to the present invention comprise about 1 nanogram to about 1000 micrograms of DNA. In some preferred embodiments, the vaccines contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the vaccines contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the vaccines contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the vaccines contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the vaccines contain about 100 micrograms DNA. The genetic vaccines according to the present invention are formulated according to the mode of administration to be used. One having ordinary skill in the art can readily formulate a genetic vaccine that comprises a genetic construct. In cases where intramuscular injection is the chosen mode of administration, an isotonic formulation is preferably used.

Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. The pharmaceutical preparations according to the present invention are preferably provided sterile and pyrogen free.

5.6. Pharmaceutical Compositions The present invention also includes pharmaceutical products for all of the uses contemplated in the methods described herein. For example, the invention provides a pharmaceutical product for use in immunizing a mammal, comprising a DNA operatively coding for an antigen in solution in a pharmaceutically acceptable carrier suitable for intramuscular injection to cause expression of the antigen in the muscle, a container enclosing the solution, and a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals.

6. EXAMPLES

6.1. Genetic Immunization Strategy for Aged Mice Mice. Female mice were approximately 30 months of age at the time of vaccination. Virus and Cell Lines. In all experiments, herpes simplex virus type 2 (HSV-2) strain 333 was utilized. Vero cells used in HSV-2 plaque assays were obtained from Robert Fujinami (University of Utah) . Vero cells were maintained in DMEM supplemented with 10% fetal calf serum (Gibco-LTI) . C2C12 murine myoblasts were purchased from the American Type Culture Collection (ATCC) , and maintained in the undifferentiated state through passage at low density in DMEM supplemented with 20% fetal calf serum and 0.5% chicken embryo extract. To induce formation of myofibers or the differentiated state, C2C12 myoblasts were allowed to reach 80-90% confluency, at which time the medium was changed to DMEM supplemented with 2% horse serum (Hyclone) .

Virus Plaque Assay. HSV-2 was diluted serially ten-fold in DMEM without serum. The virus dilutions were applied to Vero cells at 80% confluency. After gentle rocking at 37°C, 5% C0₂ for 1 hour, the virus inoculum was aspirated, fresh media applied, and virally infected cells were incubated 48 hours at 37°C, 5% C0₂, at which time cytopathic effect (CPE) was noted. Viral plaques were visualized with a stain that consisted of 0.1% crystal violet (Sigma) in 20% ethanol .

Plasmid DNA. Parental plasmid pMCKv.2 derivatized from pcDNA3.1 (Invitrogen) was constructed as follows. The CMV promoter, the neomycin gene, and the SV40 origin and polyadenylation signal was excised from pCDNA3.1 to prepare for insertion of the modified MCK promoter. The MCK minimal enhancer/promoter was obtained by polymerase cha.in reaction (PCR) from BALB/c genomic DNA. The 1.3 kb MCK minimal enhancer/promoter was subcloned into the derivatized pcDNA3.1 vector to yield pMCKv.2. Into this plasmid, the full-length glycoprotein D gene from HSV-2 (a kind gift from Gary Cohen, University of Pennsylvania) was inserted to yield the plasmid, pMCKv.2-gD2. All plasmids were propagated in XL- Blue (Stratagene) Escherichia coli bacteria according to standard methods and endotoxin DNA prepared through the use of affinity chromatography for the preparation of endotoxin free DNA (Qiagen, Inc.).

Plasmid immunizations and challenge. Aged (30 month old) adult female mice were genetically immunized with 100 μg of DNA (either pMCKv.2 or pMCKv.2-gD2) dissolved in 50 μl of sterile phosphate buffered saline (PBS) . Injections were given at three-week intervals for a total of three injections. All injections were given into the left anterior tibeal muscle through a 28 gauge needle.

Four weeks after the third DNA immunization, the mice were readied for infection by HSV-2. A 2 mg/ml injection of Depo-Provera (UpJohn) was administered subcutaneously, the mice rested for seven days, and infected with HSV-2. To infect the mice intra-vaginally, the mice were anesthetized with an i.p. injection of 2 mg of ketamine, and then infected intra-vaginally with 1 x 10⁵ PFU/ l soaked dacron plugs for thirty minutes at which time the plugs were removed. Animals were monitored daily for both survival and for the physical manifestations of acute disease (genital herpes) . The following numeric score was applied: 0 = no visible redness or lesions, 1 = redness or mild swelling, 2 = erosions, vesicles, or moderate swelling, 3 = several large vesicles, 4 = large ulcers with severe maceration and/or urinary retention.

As can be seen in figure 1, nearly identical survival rates were observed in immunized aged mice and immunized young mice. 100% survival was seen in both groups up to 10 days after infection, and greater than 75% of the animals in both groups survived for two weeks after infection (80% survival for aged mice, and 89% survival for adult mice) . Mock immunized mice (i.e., immunized with plasmid DNA without the gD2 insert) of both the aged and young groups exhibited substantial reductions in survival rates at 10 days, and at 14 days, there was less than a 10% survival rate.

Similar results were obtained when assaying for the physical manifestations of genital herpes. As seen in figure 2, immunized mice of either group (aged or young) exhibited substantially reduced symptoms of infection as compared to mock immunized mice of either group. There was virtually no difference observed between the young and aged mice. These results illustrate that aged mice immunized with pMCKv.2-gD2 plasmid DNA and subsequently challenged with HSV-2 possessed protective immunity nearly identical to that of similarly treated young mice.

Serology. At the indicated times in the text, sera was collected into Microtainer tubes from mice by retroorbital puncture, and clarified by centrifugation. The sera was assayed for HSV-2 gD specific antibody by enzyme-linked immunosorbent assay (ELISA) through the use of a recombinant baculovirus that secreted HSV-2 glycoprotein D. Ninety-six well flat bottomed plates (Corning) were coated overnight at 4°C with a dilution of supernatant obtained from the recombinant baculovirus in phosphate buffered saline (PBS; the exact dilution was determined empirically) . The next day, the plates were washed 4 times with distilled water followed by 3 washes with PBS/0.5% Tween 20. The plates were blocked for 2 hours at 37°C. 5% C02, with a buffer that consisted of 10% FCS/PBS. Clarified sera diluted two- fold was applied to blocked plates and incubated overnight at 4°C. After primary antibody treatment, unreacted antibody was removed by washes of distilled water and PBS/0.5% Tween 20 at which point rabbit anti-mouse IgG (or in isotype profiles IgGi or IgG2a) conjugated to horseradish peroxidase was applied and allowed to incubate for 2 hours at room temperature . Thg plates were washed, developed with a citrate buffer that contained the chromagen 2 , 2 ' -Azino-bis-3-ethylbenthiazoline-6-sulfonic acid (ABTS) . After color development, the reaction was stopped by addition of a solution of SDS/DMF. The absorbances were determined at 405 nm in a Molecular Devices v-Max kinetics microplate reader (Molecular Devices, Inc.). The geometric endpoint mean titer (GMT) of each mouse was determined through the use of the statistical software program, Prizm 2.01.

The results are illustrated in figure 3. Aged and young mice administered the genetic vaccine pMCKv.2-gD2 generated very similar immune responses to gD at every time point assayed. There was a non-specific response observed in mock immunized aged mice alone that disappeared after 4 days post- infection with HSV-2. As demonstrated above, this response was not protective against HSV-2 infection. This demonstrates that aged mice generate a gD2 specific antibody response similar to that of 12 week old mice.

6.2. Genetic Immunization Strategy for Adult Mice Mice. Female mice were approximately 12 weeks of age at the time of vaccination.

Virus and Cell Lines. In all experiments, herpes simplex virus type 2 (HSV-2) strain 333 was utilized. Vero cells used in HSV-2 plaque assays were obtained from Robert

Fujinami (University of Utah) . Vero cells were maintained in DMEM supplemented with 10% fetal calf serum (Gibco-LTI) . C2C12 urine myoblasts were purchased from the American Type Culture Collection (ATCC) , and maintained in the undifferentiated state through passage at low density in DMEM supplemented with 20% fetal calf serum and 0.5% chicken embryo extract. To induce formation of myofibers or the differentiated state, C2C12 myoblasts were allowed to reach 80-90% confluency, at which time the medium wag changed to DMEM supplemented with 2% horse serum (Hyclone) .

Virus Plague Assay. HSV-2 was diluted serially ten-fold in DMEM without serum. The virus dilutions were applied to Vero cells at 80% confluency. After gentle rocking at 37°C, 5% C0₂ for 1 hour, the virus inoculum was aspirated, fresh media applied, and virally infected cells were incubated 48 hours at 37°C, 5% C0₂, at which time cytopathic effect (CPE) was noted. Viral plaques were visualized with a stain that consisted of 0.1% crystal violet (Sigma) in 20% ethanol .

Plasmid DNA. Parental plasmid pMCKv.2 derivatized from pcDNA3.1 (Invitrogen) was constructed as follows. The CMV promoter, the neomycin gene, and the SV40 origin and polyadenylation signal was excised from pCDNA3.1 to prepare for insertion of the modified MCK promoter. The MCK minimal enhancer/promoter was obtained by polymerase chain reaction (PCR) from BALB/c genomic DNA. The 1.3 kb MCK minimal enhancer/promoter was subcloned into the derivatized pcDNA3.1 vector to yield pMCKv.2. Into this plasmid, the full-length glycoprotein D gene from HSV-2 (a kind gift from Gary Cohen, University of Pennsylvania) was inserted to yield the plasmid, pMCKv.2-gD2. All plasmids were propagated in XL- Blue (Stratagene) Escherichia coli bacteria according to standard methods and endotoxin DNA prepared through the use of affinity chromatography for the preparation of endotoxin free DNA (Qiagen, Inc.).

Plasmid immunizations and challenge. Adult (12 month old) adult female mice were genetically immunized with 100 μg of DNA (either pMCKv.2 or pMCKv.2-gD2) dissolved in 50 μl of sterile phosphate buffered saline (PBS) . Injections were given at three-week intervals for a total of three injections. All injections were given into the left anterior tibeal muscle through a 28 gauge needle.

Four weeks after the third DNA immunization, the mice were readied for infection by HSV-2. A 2 mg/ml injection of Depo-Provera (UpJohn) was administered subcutaneously, the mice rested for seven days, and infected with HSV-2. To infect the mice intra-vaginally, the mice were anesthetized with an i.p. injection of 2 mg of ketamine, and then infected intra-vaginally with 1 x 10⁵ PFU/ml soaked dacron plugs for thirty minutes at which time the plugs were removed. Animals were monitored daily for both survival and for the physical manifestations of acute disease (genital herpes, data not shown) .

Serology. At the indicated times in the text, sera was collected into Microtainer tubes from mice by retroorbital puncture, and clarified by centrifugation. The sera was assayed for HSV-2 gD specific antibody by enzyme-linked immunosorbent assay (ELISA) through the use of a recombinant baculovirus that secreted HSV-2 glycoprotein D. Ninety-six well flat bottomed plates (Corning) were coated overnight at 4°C with a dilution of supernatant obtained from the recombinant baculovirus in phosphate buffered saline (PBS; the exact dilution was determined empirically) . The next day, the plates were washed 4 times with distilled water followed by 3 washes with PBS/0.5% Tween 20. The plates were blocked for 2 hours at 37°C. 5% C02, with a buffer that consisted of 10% FCS/PBS. Clarified sera diluted two-fold was applied to blocked plates and incubated overnight at 4°C. After primary antibody treatment, unreacted antibody was removed by washes of distilled water and PBS/0.5% Tween 20 at which point rabbit anti -mouse IgG (or in isotype profiles IgGi or IgG2a) conjugated to horseradish peroxidase was applied and allowed to incubate for 2 hours at room temperature. The plates were washed, developed with a citrate buffer that contained the chromagen 2, 2 ' -Azino-bis-3-ethylbenthiazoline-6-sulfonic acid (ABTS) . After color development, the reaction was stopped by addition of a solution of SDS/DMF. The absorbances were determined at 405 nm in a Molecular Devices v-Max kinetics microplate reader (Molecular Devices, Inc.).

The geometric endpoint mean titer (GMT) of jeach mouse was determined through the use of the statistical software program, Prizm 2.01.

As can be seen in figure 4, both a Thl (IgG2a measurements) and a Th2 (IgGi measurements) response are elicited in some adult mice by genetic immunization with pMCKv.2-gD2. In some mice, the Thl and Th2 responses were equivalent . These results illustrate that adult mice immunized with pMCKv.2-gD2 plasmid DNA generate both a Thl and a Th2 immune response.

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.

The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings . Such modifications are intended to fall within the scope of the appended claims .

Claims

CLAIMSWe claim:

1. A method of immunizing a mammal comprising:, injecting into skeletal muscle tissue of the mammal a DNA molecule that comprises a DNA sequence that encodes an antigen from a pathogen, the DNA sequence operatively linked to a promoter; wherein the DNA molecule is taken up by cells in the skeletal muscle tissue, the DNA sequence is expressed in the cells and both a Thl and a Th2 immune response is generated against the antigen.

2. The method of claim 1 wherein the promoter is a muscle specific promoter.

3. The method of claim 2 wherein the muscle specific promoter is the muscle creatine kinase promoter.

4. The method of claim 1 wherein the pathogen is selected from the group consisting of: bacterial pathogens; viral pathogens; parasitic pathogens; and fungal pathogens.

5. The method of claim 1 wherein the pathogen is a virus selected from the group consisting of : human immunodeficiency virus, HIV; human T cell leukemia virus, HTLV; influenza virus; hepatitis A virus; hepatitis B virus; hepatitis C virus; human papilloma virus, HPV; Herpes simplex 1 virus, HSV1; Herpes simplex 2 virus, HSV2 ; Cytomegalovirus, CMV; Epstein-Barr virus, EBR; rhinovirus; coronavirus and respiratory syncytial virus. —

6. The method of claim 1 wherein the mammal is a human.

7. The method of claim 1 wherein the Thl and Th2 immune responses are approximately equivalent.

8. The method of claim 1 wherein the Th2 immune response is within 20% of the Thl immune response.

9. The method of claim 4 wherein the Th2 immune response is within 20% of the Thl immune response.

10. A method of immunizing a mammal comprising: injecting

5 into skeletal muscle tissue of the mammal a DNA molecule that comprises a DNA sequence that encodes a hyperproliferative disease-associated protein operatively linked to a promoter; wherein the DNA molecule is taken up by cells in the skeletal muscle tissue, the DNA sequence is expressed in the cells, an 10 immune response is generated against the hyperproliferative disease-associated protein, and both a Thl and a Th2 immune response are generated.

11. The method of claim 10 wherein the promoter is a muscle 15 specific promoter.

12. The method of claim 11 wherein the muscle specific promoter is the muscle creatine kinase promoter.

20 13. The method of claim 10 wherein the pathogen is selected from the group consisting of: bacterial pathogens; viral pathogens; parasitic pathogens; and fungal pathogens.

14. The method of claim 10 wherein the DNA molecule

25 comprises a DNA sequence encoding a target protein selected from the group consisting of : protein products of oncogenes myb, myc, fyn, ras, src, neu and trk; protein products of translocation gene bcr/abl; P53; variable regions of antibodies made by B cell lymphomas; and variahle regions of

30 T cell receptors of T cell lymphomas.

15. A method of immunizing a mammal comprising: injecting into skeletal muscle tissue of the mammal a DNA molecule that comprises a DNA sequence that encodes an autoimmune disease-

35 associated protein operatively linked to a promoter; wherein the DNA molecule is taken up by cells in the skeletal muscle tissue, the DNA sequence is expressed in the cells, an immune response is generated against the autoimmune disease- associated protein, and both a Thl and a Th2 immune response are generated.

16. The method of claim 15 wherein the promoter is a muscle specific promoter.

17. The method of claim 16 wherein the muscle specific promoter is the muscle creatine kinase promoter.

18. The method of claim 15 wherein the DNA molecule comprises a DNA sequence encoding a target protein selected from the group consisting of: variable regions of antibodies involved in B cell mediated autoimmune disease; and variable regions of T cell receptors involved in T cell mediated autoimmune disease.

19. A method of identifying a DNA immunogens comprising the steps of: a) obtaining a functional recombinant plasmid suitable for use as a DNA vaccine, the plasmid having a DNA which codes for a potential antigen operably linked to a promoter such that the expression of the DNA is controlled by the promoter; and b) administering the functional recombinant plasmid by intramuscular injection to a mammal; c) testing for an immune response to the antigen in the mammal ; wherein recombinant plasmids which are able tO elicit both a Thl and a Th2 immune response to the potential antigen are identified as DNA immunogens.

20. The method of claim 19 wherein the promoter is a muscle specific promoter.

21. The method of claim 20 wherein the muscle specific promoter is the muscle creatine kinase promoter.

22. A method of identifying a DNA vaccine comprising the steps of : a) obtaining a functional recombinant plasmid suitable for use as a DNA vaccine, the plasmid having a DNA which codes for a potential antigen operably linked to a muscle specific promoter such that the expression of the DNA is controlled by the promoter; and b) administering the functional recombinant plasmid by intramuscular injection to a mammal; c) testing for a protective immune response in the mammal ; wherein recombinant plasmids which are able to elicit a protective immune response comprising both a Thl and a Th2 response are identified as DNA vaccines useful for immunizing mammals .

23. The method of claim 22 wherein the promoter is a muscle specific promoter.

24. The method of claim 23 wherein the muscle specific promoter is the muscle creatine kinase promoter.