Title:  Methods for inducing mucosal immune responses

United States Patent:  6,630,455

Issued:  October 7, 2003

Inventors:  Mitchell; William M. (Nashville, TN)

Assignee:  Vanderbilt University (Nashville, TN)

Appl. No.:  372429

Filed:  January 13, 1995

Abstract

The invention provides a method of inducing a mucosal immune response in a subject, comprising administering to the mucosa of the subject an amount of antigen-encoding DNA effective to induce a mucosal immune response complexed to a transfection-facilitating lipospermine or a lipospermidine. In the method of inducing a mucosal immune response, the antigen-encoding DNA can encode an antigen that is expressed on the surface of infected cells during the course of infection. DNA encoding the envelope glycoproteins of viral pathogens is used in the present method. Lipospermines and lipospermidines are bifunctional molecules consisting of one or more hydrophobic chains covalently linked to a cationic grouping in which there is coordination of three or more amide hydrogens with a phosphate oxygen of the DNA chain forming an ionic charge complex. One preferred example of a lipospermine is DOGS (droctadecylamidoglycylspermine). The invention also provides a composition, comprising an amount of DNA encoding an envelope antigen or envelope-associated antigen of a pathogen complexed to a lipospermine. More specifically, the invention provides a composition, comprising an amount of DNA encoding an envelope antigen of HIV complexed to a lipospermine

SUMMARY OF THE INVENTION

The invention provides a method of inducing a mucosal immune response in a subject, comprising administering to the mucosa of the subject an amount of antigen-encoding DNA effective to induce a mucosal immune response complexed to a transfection-facilitating lipospermine or a lipospermidine. In the method of inducing a mucosal immune response, the antigen-encoding DNA can encode an antigen that is expressed on the surface of infected cells during the course of infection. The present method should apply to all mucosally acquired pathogens in which expression of antigen on the surface of a mucosal cell mimics natural infection. DNA encoding the envelope glycoproteins of viral pathogens is the rational choice for use in the present method.

Lipospermines and lipospermidines are bifunctional molecules consisting of one or more hydrophobic chains covalently linked to a cationic grouping in which there is coordination of three or more amide hydrogens with a phosphate oxygen of the DNA chain forming an ionic charge complex. One preferred example of a lipospermine is DOGS (dioctadecylamidoglycylspermine). Diotadecylamidoglycylspermidine is another likely candidate, because it has the same structure as DOGS, but lacks one of the two arms having two non-essential cationic charges.

The invention also provides a composition, comprising an amount of DNA encoding an envelope antigen or envelope-associated antigen of a pathogen complexed to a lipospermine. More specifically, the invention provides a composition, comprising an amount of DNA encoding an envelope antigen of HIV complexed to a lipospermine

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of inducing a mucosal immune response in a subject, comprising administering to the mucosa of the subject an amount of antigen-encoding DNA effective to induce a mucosal immune response complexed to a transfection-facilitating lipospermine or a lipospermidine.

The invention is applicable to pathogens generally, because expression of the pathogen antigen encoded by the antigen-encoding DNA results in exposure of the pathogen antigen on the surface of the cell, mimicking either a portion of the replicative cycle of the pathogen or the initial attachment of the pathogen to the cell surface. Examples of viral pathogens include, but are not limited to, retroviruses (human immunodeficiency viruses), herpesviruses (herpes simplex virus; Epstein Barr virus; varicella zoster virus), orthomyxoviruses (influenza), paramyxoviruses (measles virus; mumps virus; respiratory syncytial virus), picornaviruses (Coxsackie viruses; rhinoviruses), hepatitis viruses (hepatitis C), bunyaviruses (hantavirus; Rift Valley fever virus), arenaviruses (Lassa fever virus), flaviviruses (dengue fever virus; yellow fever virus; chikungunya virus) and coronaviruses, among others. Examples of bacterial pathogens include, but are not limited to, species of the following genera: Salmonella, Shigella, Chlamydia, Helicobacter, Yersinia, Bordatella, Pseudomonas, Neisseria, Vibrio and Haemophilus, among others.

Antigen-Encoding DNA

In the method of inducing, a mucosal immune response, the antigen-encoding DNA can encode an antigen that is expressed on the surface of infected cells during the course of infection. The present method should apply to all mucosally acquired pathogens in which expression of antigen on the surface of a mucosal cell mimics natural infection. Because the primary immune response to bacteria is to a relatively small number of cell surface antigens, the process for selecting antigen-encoding DNA for bacterial pathogens is similarly routine. For example, the major bacterial immunogens are epitopes on surface structures (191). There are numerous examples of viral antigens in which this is expected to be the case. It is expected that antigens of other microbiological pathogens will share this characteristic. As used herein, an antigen is a molecule that elicits an immune response.

DNA encoding the envelope glycoproteins (e.g., gp160 HIV or its cleaved derivative proteins, gp41 and gp120) of viral pathogens is the rational choice for use in the present method. Envelope-associated-proteins, such as gp17 are also reasonable choices, because of their presentation on the cell surface of infected cells. A reasonable terminology to define a subset of antigens that will be effective in this method is "envelope and envelope-associated proteins." Specific epitopes of these proteins that elicit an immune response in a subject can be selected by routine methods, including epitope mapping and analysis of conformational dependency. Particularly, epitopes that elicit neutralizing antibodies are important bases of the present method. DNA encoding these antigens can be obtained by cloning and synthesis methods known in the art and further described below.

For example, the antigen-encoding DNA can encode an antigen of a human immunodeficiency virus. As a more specific example, the antigen-encoding DNA can encode a human immunodeficiency virus envelope glycoprotein. Although the envelope antigens are expected to be the main inducers of antibodies and cytotoxic lymphocytes (CTLs), there is literature evidence of CTLs against the gag (i.e. internal antigen) of HIV. The preferred antigen-encoding DNAs include gp160, gp120 and gp41 separately expressed (i.e., gp160 is normally cleaved by a host protease to gp120 and gp41). DNA encoding gp17, which is one of the gag proteins that is attached by a myristylation link to: the envelope, and for which there is literature evidence for a neutralizing antibody epitope close to the myristylation site, can also be included. The antigen encoding DNA can encode antigenic fragments of the envelope and envelope-associated proteins, for example, the V3 loop of a human immunodeficiency virus envelope glycoprotein gene.

An antigen-encoding DNA will need to have a start codon, a stop codon and a membrane anchor. Thus, if these are not present, or in order to optimize the present method, it is,expected that the sequences of antigen encoding DNA will be mutated in one or more ways to preserve or enhance the antigenicity of the expressed antigen. For example, a mutation of the gp160 cleavage site can be made to keep the protein uncleaved. A stop signal has to be generated for gp120 as well as a membrane anchor. In addition, the known antibody enhancing domain of gp41 will be removed for both HIV and RSV as described in detail in the Examples. Numerous versions of the V3 region of the envelope glycoprotein can be made to reflect the major quasispecies found in viral isolates. These can then be administered in multiple genetic constructs, each containing a single transcribed ORF, or in a single or a few genetic constructs, each containing multiple transcribed ORFs. Genetic manipulations of this nature are known in the art (188) and specific examples described in the Examples.

Briefly, mutations are produced using the p-Alter-1 kit from Promega, which incorporates antibiotic selection for selection of the desired mutations. It necessary to use the ssDNA template procedure for reliable generation of desired mutations. A critical change from the kit protocol is the generation of our own helper phage ssDNA. The ss Phage DNA isolation kit and procedure from Biolabs, Inc. is used for the production of pure ssDNA. Another critical change is the substitution of the ES 1301 mutS E. coli supplied with the kit with XL mutS E. coli from Stratagene for transformation. DH5.alpha. a E. coli for which the subject mutS mutations have been generated have also been successfully used. The latter are devoid of repair enzymes. The components of the above method are generally applicable to DNA encoding other antigens.

Examples of gene engineering that are expected to be incorporated into a plasmid containing, for example, the HIV envelope for eukaryote cell transfection and antigen expression on the surface of the cell include: 1) elimination of the HIV LTR control elements and placement under a more powerful promoter such as CMV, 2) elimination of the gp160 proteolytic cleavage site so that gp120 does not disassociate from the membrane anchored gp41, 3); conversion of the RRE present in the 5' end of gp41 and which forms extensive secondary structure to a linear structure by the introducing of silent mutations with no changes in the amino acid sequences of the product and 4) the elimination of the primary enhancing domain by point or deletion mutations which destroy this capacity. Examples of these mutations are further described in the Examples. Although, specific mutations for HIV envelope glycoproteins are given, it is understood that the same considerations for the generation of an efficient immunization construct apply to the generation of a construct using an antigen-encoding DNA for a different antigen.

The vectors used in the present method can include promoters and regulatory sequences that are relevant to the antigen-encoding DNA. Typically, the vector must be a eukaryotic vector that is capable of replication in E. coli. The preferred vector contains a bacterial origin of replication, an antibiotic resistance selection gene, eukaryotic promoter and a polyadenylation gene. As clearly demonstrated in the Examples, other vectors can be designed by the skilled artisan that do not share all of the above characteristics, yet permit transfection.

Lipospermine/Lipospermidine

The transfection-facilitating lipospermines and lipospermidines used in the methods are bifunctional molecules consisting of one or more hydrophobic chains covalently linked to a cationic grouping in which there is coordination of three or more amide hydrogens with a phosphate oxygen of the DNA chain forming an ionic charge complex. To facilitate transfection, the lipospermines.backslash.lipospermidines can both protect the DNA and make it appear more hydrophobic to the cell membrane of the cell to be transfected. For example, the charge interaction positions the hydrophobic arms along the major or minor groove of DNA (see Examples) provide a hydrophobic covering for the highly charged DNA macromolecule and affords facilitated cellular entry by association of the hydrophobic surface covering DNA with the hydrophobic component of the plasma membrane of the cell. Based upon molecular modeling using DOGS, it appears that the amino hydrogen of the peptide bond and adjacent amide hydrogens all coordinate on one phosphate oxygen (i.e., 1.91 to 2.0 .ANG. distance). The other two amide hydrogens are useless. Thus, the use of spermidine in the construction of the complexing agent might be more effective.

One preferred example of a lipospermine is DOGS (dioctadecylamidoglycylspermine). Diotadecylamidoglycylspermidine is another likely candidate, because it has the same structure as DOGS, but lacks one of the two arms having two non-essential cationic charges. Additionally, lipospermines or lipospermidines having hydrophobic chains of 8 to 20 carbons could also be expected to interact similarly with the major and minor grooves of the DNA. Although less preferred, the lipospermine or lipospermidine could have a single hydrophobic side chain (e.g., monooctyl, monooctadecyl, monododecyl, etc.).

The preferred molar cationic ratio of the lipospermine to DNA is about 5:1. Alternatively, the lipospermine can be complexed to DNA in a molar cationic ratio ranging from about 2 to about 10. Because the ionic interactions between the lipospermine/lipospermidine and the DNA will be the same regardless of the antigen encoded, the present teaching with regard to the formulation of DNA-lipospermine/lipospermidine complexes is applicable to the antigen-encoding DNAs of the invention.

Thus, the invention also provides a composition, comprising an amount of DNA encoding an envelope antigen or envelope-associated antigen of a pathogen complexed to a lipospermine. More specifically, the invention provides a composition, comprising an amount of DNA encoding an envelope antigen of HIV complexed to a lipospermine. The DNA in the plasmid described in the Examples. As described above the antigen-encoding DNA of the invention can encode any antigen that is presented on the surface of host cells during infection or on the surface of the pathogen and exposed to the host immune system. Examples of such antigens are described in virology textbooks (see for example Fundamental Virology, 2nd. Ed., pp373-375 (189)).

In the method of inducing a mucosal immune response, the antigen-encoding DNA can also be administered without the aide of a complexing molecule. For example, the DNA can be bolistically administered, along with an activated form of vitamin D3 as described in the Examples. Briefly, the DNA is complexed to gold particles and delivered to skin cells by propelling them through plasma membranes using a helium propellant. The activated form of vitamin D3 can be delivered to the same cells by inclusion in the gold suspension containing the naked DNA and propelled into the cells by the same means. The activated vitamin D3 can, alternatively, be delivered to the transfected skin cells by topical application in a solvent carrier, such as dimethyl sulfoxide. In this manner, the skin can act as a mucosal surrogate in terms of the ability to induce mucosal immunity.

The Mucosal Immune System

Significant indirect evidence indicates the presence of a common mucosal immune system (47,50). Induction of mucosal immunity in bronchus-associated lymphoid tissues usually yields evidence of immunity in gut-associated lymphoid tissues. The common element is the generation of mobile IgA secreting plasma cells with an affinity for mucosal-associated lymphoid tissues of various types.

The mechanism of selective transport, of J-chain-containing polymeric IgA (pIgA) through an epithelial cell to the mucosal surface has been determined. Briefly, pIgA assembled in subepithelial plasma cells from monomeric IgA (mIgA) with the participation of J chain, interacts with the membrane form of secretory component (SC). The pIgA-SC complex is internalized in endoplasmic vesicles which fuse with the apical membrane, and S-IgA is released into the external secretion. See Reference 42.

Although IgG can be found on mucosal surfaces following mucosal immunizations, IgA is the predominant Ig in mucosal immunity. This is secondary to the presence of an Ig receptor with greatest affinity for pIgA. This receptor is expressed on the surface of mucosal epithelial cells and actively transports pIgA to the mucosal surface (47-49) through mucosal epithelial cells.

Mucosal Administration

In the method of inducing a mucosal immune response, the antigen-encoding DNA is administered to the mucosa of the subject. Thus, specific examples of the mucosal administration include nasal, oral, rectal and vaginal. Nasal administration can be by nasal aerosol spray (see Examples) or nebulizer among other well practiced methods. Rectal and vaginal administration can be by a variety of: methods, including lavage (douches, enemas, etc.), suppositories, creams, gels, etc. For nasal administration, an aerosol spray or nebulizer can be used.

Depending on the intended mode of administration, the compounds of the present invention can be in pharmaceutical compositions in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above, an effective amount of the DNA and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. By "pharmaceutically acceptable" is meant a material that is not biologically orotherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences (190).

In the present method, the DNA is complexed to lipospermine and is administered to the subject as a single primary vaccination followed by one or more booster vaccinations at three week to three month intervals. Routine optimization of this administration regimen can be made using routine optimization procedures.

The exact amount of DNA required will vary from subject to subject, depending on the age, weight and general condition of the subject, the particular formulation used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. Thus, the amount of DNA administered can be any effective amount. There should be little difference in a human immunizing dose vs. mouse dose, because there is no reason to expect that human cells are more or less susceptible to transfection than mouse cells. Typically, the preferred amount of DNA required for effective transfection is from about 10 ng to 10 .mu.g. Variations in the transfection efficiency between humans and mice can be accommodated by routine adjustments in the dosage. For example, the. amount can range from 1.0 ng to 1 mg. Anything over 10 .mu.g DNA becomes logistically difficult to handle and increases the risk of toxicity and is impractical.

Claim 1 of 7 Claims

What is claimed is:

1. A method of inducing a mucosal immune response to antigen in a mammal, comprising administering to the mucosa of said mammal antigen-encoding DNA, operably linked to a promoter for expression of said antigen and complexed to a transfection-facilitating lipospermine or lipospermidine, in an amount effective to induce a mucosal immune response to expressed antigen.

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