Title:  Compositions and methods for preventing transepithelial transmission of HIV

United States Patent:  6,566,095

Issued:  May 20, 2003

Inventors:  Markham; Richard B. (Columbia, MD); Khanna; Kristen V. (Columbia, MD)

Assignee:  Johns Hopkins University (Baltimore, MD)

Appl. No.:  599555

Filed:  June 23, 2000

Methods, compositions and articles for blocking transepithelial transmission of HIV with ICAM-1 angonist/antagonists are provided. The ICAM-1 agonist/antagonists block viral transmission at the site of exposure to the virus. Blocking viral transmission does not require blocking the binding of ICAM-1 to LFA-1. The ICAM-1 agonist/antagonists may be applied to mucosal surfaces, on or in contraceptive devices or in oral solutions such as breast milk supplements and infant formula.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Most newly acquired cases of HIV-1 infection result from sexual, primarily heterosexual, transmission. While mechanical barriers, such as condoms, can be very effective in preventing sexual transmission, this male-dependent method of protection is frequently not accepted or is impractical for use by the women at greatest risk. In most cases, women at highest risk for new HIV infections are from developing countries. The rapid spread of HIV-1 infection in Africa and Southeast Asia, however, has added new urgency to the need for prophylactic intervention to prevent STD transmission.

In accordance with the present invention, compositions and methods for blocking the transepithelial transmission of the human immunodeficiency virus in an animal using an ICAM-1 agonist/antagonist, at the site of exposure to the virus, are provided. The term "ICAM-1 agonist/antagonist" refers to a compound or bio-molecule capable of blocking or stimulating events associated with the normal function of ICAM-1. The term "site of exposure" refers to the portion of a body of an animal where exposure to HIV occurs, e.g., genital tract, oral cavity, rectum, ocular tissue, auricular tissue, skin etc. The blocking of HIV transmission according to the present invention does not require blocking the binding of ICAM-1 to LFA-1.

In a preferred embodiment, the ICAM antagonist is provided to a mucosal surface of an animal including, but not limited to, the genital, vaginal, rectal, oral or gastrointestinal surfaces. Mucosal surfaces represent potential sites of exposure for HIV through sexual contact, from mother-to-infant, and other mechanisms. The ICAM agonist/antagonist may be applied to a substrate such as a condom or intrauterine device or provided in a variety of forms including, e.g., vaginal wash, lubricant, spermicide, douche, suppository, breast milk supplement, or infant formula.

Antibodies are a versatile prophylactic intervention particularly suited for application to a variety of sites in the body, and in particular, to mucosal surfaces. The antibodies of the invention are directed to modulating the function of ICAM-1 in the etiology of an HIV infection. Other agonists/antagonists, e.g., antisense nucleotide sequences, and ribozymes, may also be used to modulate the function of ICAM-1. Antibodies are an example of a highly specific microbicide capable of prophylactic use in disease prevention. Mucosal antibodies are of particular interest because they generally function by excluding pathogens from the epithelium. Moreover, it is the versatility of the antibody structure to create a virtually inexhaustible repertoire of antigen binding specificities that is important for producing specific microbicides directed to a particular pathogens.

The term "antibodies" herein refers to the spectrum of human, humanized animal, or plant-derived humanized immunoglobulins ("Ig") including, but not limited to, IgG, IgM, IgA, IgE, and fragments, e.g., monovalent, bivalent, and multivalent fragments. Preferred antibodies include bivalent IgG and multivalent IgA human monoclonal antibodies produced in plants ("plantibodies"). Another preferred antibody is an anti-human ICAM-1 IgG1. The plantibody system can readily be used to generate large quantities of antibodies of a given specificity. Antibodies of specific valency, class, and, in the case of IgA, containing secretory component, can be produced in corn plants.

Expression of polymeric and secretory IgA in plants is preferred to more conventional hybridoma techniques for several reasons. The production of sIgA in plants is a single cell system, whereas two different cell types are required in mammalian production systems. The plantibody system has a polymeric IgA scaffold for inserting the cDNA from existing well-characterized antibodies. Plantibodies also have greater stability and are less subject to mutagenesis than antibodies generated and maintained in cell culture. Plants capable of producing plantibodies have a greater long term storage capacity than conventional hybridoma cell lines and can reduce the cost of manufacturing by three to six orders of magnitude. In addition, plantibodies are infinitely scalable, i.e., from a single plant to thousands of acres and avoid safety concerns arising from potential viral contamination of cell cultures.

Another preferred antibody type is sIgA, a common antibody at mucosal surfaces, including the vagina and cervix. This antibody is distinguished functionally from antibodies of the IgG class by the absence of Fc receptors for IgA on most phagocytic cells and by the inability of sIgA to fix complement by the classical pathway. These properties reduce the pro-inflammatory potential of sIgA, a useful characteristic at sites, such as the gastrointestinal tract or bronchioles, where inflammation may result in functional impairment. The reduced Fc receptor and complement binding capacity of sIgA render this class of immunoglobutin less likely to induce the enhancement of infection that has been associated with some HIV-1 specific IgGs. IgA is also distinguished from IgG by its unique reactivity with epithelial cells, which is mediated by the polymeric Ig receptor(s) expressed on the basolateral surface of epithelial cells. In its dimeric form, IgA attaches to these receptors on epithelial cells which permits the antibody molecule, and any antigen attached to it, to trancytose the epithelial cell without undergoing degradation. A portion of the receptor remains attached to the IgA molecule after it leaves the epithelial cell, giving rise to the secretory piece of IgA. The functional component of the polymeric Ig receptors is highly conserved among different species, including mice and humans.

In another preferred embodiment, a prophylactic pharmaceutical composition comprising an ICAM-1 agonist/antagonist and a pharmaceutically acceptable carrier is provided. A "pharmaceutically acceptable carrier" refers to a carrier or vehicle suitable for application to a mucosal surface. The ICAM-1 agonist/antagonist may be formulated in any appropriate carrier, provided that the antagonist and carrier are compatible. The activity of the antagonist should not be diminished by the carrier to the extent it no longer blocks transmission of HIV. The carrier can be aqueous, or non-aqueous, for example, alcoholic or oleaginous, or a mixture thereof, and may additionally contain other surfactants, emollients, lubricants, stabilizers, dyes and active ingredients such as preservatives, antibiotics, contraceptives, spermicides or pharmaceutical agents. The compositions can be in the form of, e.g., creams, foams, lotions, ointments, solutions, solids and sprays.

Preferred carriers are suitable for application to the vaginal tract, oral cavity, gastrointenstinal tract, and rectum. Particularly preferred carriers are mucoadhesive gels. Suitable carriers may comprise organic solvents, emulsifiers, gelling agents, moisturizers, stabilizers, wetting agents, time release agents, sequestering agents, dyes, perfumes and other components commonly employed in pharmaceutical compositions for administration to mucous membranes.

The compositions of the invention may be provided in vaginal washes or douches for vaginal application. Breast milk supplements and infant formula containing ICAM-1 agonist/antagonists are preferred for providing the ICAM-antagonists to infants through the oral mucosal surface.

Solid dosage forms include suppositories, powders, and granules. In solid dosage forms, the compositions may be admixed with at least one inert diluent such as sucrose, lactose, or starch and may additionally comprise lubricating agents, buffering agents, and other components well known to those skilled in the art.

Actual dosage levels of the ICAM-1 agonist/antagonist in the methods, compositions, and articles of the invention may be varied to obtain amounts at the site of exposure to the virus or point of entry to obtain the desired prophylactic response. Accordingly, the selected dosage level will depend on the nature and site of exposure to the virus or point of entry, the desired prophylactic response, the route of administration, the desired duration of the prophylaxis and other factors.

The preferred agonist/antagonist for the compositions, articles and methods of the present invention is an anti-ICAM-1 antibody. Effective concentrations of antibodies are generally in the range of about 20 to about 1000 .mu.g/ml, although the concentration may be varied depending on the particular circumstances. The dosage level of anti-ICAM-1 plantibodies may extend to about to 10 mg/ml.

The ICAM-1 agonist/antagonists of the invention are administered by any suitable method. Preferable, the ICAM-1 agonist/antagonists are topically applied prophylactically to mucosal surfaces for the prevention of HIV transmission. Preferably, the anti-ICAM-1 antibodies are delivered in a mucoadhesive gel vehicle that retains immunoglobulin activity for a period of one to several days.

The agonist/antagonists of the invention may also be delivered via a genetically-modified bacteria capable of producing an anti-ICAM-1 agonist/antagonist, e.g., anti-ICAM-1 antibody, anti-ICAM-1 antisense, ICAM-1 antigen, or ribozyme. A "genetically-modified" bacteria contains or is transfected with a plasmid DNA vector or other vector, e.g. viral, naked DNA or RNA, comprising nucleotides encoding an anti-ICAM antibody, anti-ICAM antisense nucleotide sequence, or ribozyme. Preferred genetically-modified bacteria are species that are part of the natural flora of the mucosal surface of interest, i.e. genital tract, oral cavity etc. The genetically-modified bacteria may be created using any suitable recombinant DNA or other method known to those of skill in the art.

In another preferred embodiment, an article of manufacture comprising a substrate and an ICAM-1 agonist/antagonist is provided. The term "support" or "support" refers to an object, e.g., sponges, condoms, diaphragms, intrauterine devices, or cervical rings capable of being impregnated with or coated with an ICAM-1 agonist/antagonist and delivering the ICAM-1 agonist/antagonist to mucosal surfaces of the body. Condoms, for example, may be coated by spraying the antagonist onto the surface of the condom, or by impregnating the antagonist into the condom during manufacture by processes known in the art. Baby bottle nipples may also be coated in a similar fashion. In another preferred embodiment, the anti-ICAM-1 antibodies are delivered in a slow-release product such as a cervical ring impregnated with the antibodies.

Because HIV is primarily transmitted through sexual activity, a preferred method of the invention comprises contacting the agonist/antagonistic compositions of the invention with vaginal epithelium or other mucosal surfaces during or prior to sexual activity. Preferably, a contraceptive device, such as a condom coated or impregnated with the ICAM-1 agonist/antagonist, or a topical composition such as a gel or foam is used in sufficient quantity to prevent HIV transmission and infection. The compositions may be combined with a spermicide when the added benefit of the prevention of pregnancy is desired.

In yet another embodiment of the invention, the ICAM-1 agonist/antagonists are affixed to a solid surface, e.g., plastic, or a similar material for delivery to the site of exposure to HIV. Suitable plastics include, for example, polyvinylchloride, polyethylene, polyurethane or silicon and other common substances for containers and baby bottles. Baby bottles, including the nipple assembly, or other containers and conduits for biological fluids may be coated with ICAM-1 agonist/antagonists in sufficient quantity to prevent or block cell-associated HIV transmission. The agonist/antagonist may also be incorporated during the polymerization of the plastic polymer and leach out of the surface in a time-release fashion.

The ability of particular ICAM-1 agonist/antagonists and compositions to block transepithelial transmission of HIV may be evaluated, for example, in a transwell culture system. A preferred transwell system contains an insert which divides wells from 24-well plates into upper and lower chambers separated by a removable mesh containing pores that are 5 m in diameter. A human cervical epithelial cell line such as ME180 (American Type Culture Collection, Rockville, Md.) is grown to confluence on the removable mesh. The confluent epithelial cells form a barrier which is impermeable to C¹⁴ -inulin and, selectively favors transmission of macrophage-, as opposed to peripheral blood lymphocyte (PBL)-associated virus. HIV transmitted across the epithelial barrier will appear in the lower chamber of the transwell system. Thus, an ICAM-1 agonist/antagonist can be evaluated for its ability to block the transmission of HIV across the mesh supporting the confluent epithelial cell barrier. The transwell system permits rapid and direct examination of the ability of Mabs of different specificities to block transmission of cell-associated HIV-1 across epithelial cell monolayers. Such a system can be used to compare the relative efficacy of antibody classes and isotypes, as well.

ICAM-1 agonist/antagonists may also be evaluated in an in vivo animal model that closely resembles the setting in which transmission actually occurs. For example, the Hu/PBL-SCID mouse vaginal transmission model is an excellent model for studying HIV-1 transmission. The activation state of transplanted human PMBC cells within the mouse at the time of infection (8-10 days post human cell implantation) is similar to that observed in human peripheral blood mononuclear cells (PBMC) and markedly different from that observed in tissue culture typically used for growing HIV. Furthermore, phylogenetic analysis of variants transmitted to Hu-PBL-SCID mice indicate that viral transmission is not random and that subsets of macrophage tropic viral variants are preferentially transmitted. This transmission pattern duplicates the human setting in which a very restricted range of macrophage-tropic viruses from the donor are transmitted to the recipient. ICAM-1 agonist/antagonists may be applied, for example, to the vaginal tract of Hu-PBL-SCID mice exposed to HIV to evaluate the ability of the ICAM-1 agonist/antagonist to block HIV transmission in vivo.

Claim 1 of 33 Claims

What is claimed as new and desired to be protected by Letters Patent of the United States is:

1. A method of blocking transepithelial transmission of HIV into an animal comprising providing an anti-ICAM-1 antibody to said animal at the site of exposure to said HIV.

 

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