WO1990013281A2 - Method of suppressing hiv infection - Google Patents

Abstract

A therapeutic method for suppressing or preventing the infection of leukocytes with HIV in an individual exposed to or effected by HIV. The invention may be used in the treatment of diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by HIV.

Description

TITLE OF THE INVENTION

METHOD OF SUPPRESSING HIV INFECTION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. Patent Application Serial No. 07/344,868 (filed April 28, 1989), herein incorporated by reference.

FIELD OF THE INVENTION

The invention concerns therapeutic and prophylactic methods for suppressing the infection of leukocytes with HIV, and particularly _* with HIV-1, in an individual who is exposed to HIV or effected by HIV, and is thus in need of such suppression. It therefore provides a therapy for diseases, such as AIDS (Acquired Immunodeficiency Syndrome) which are caused by the HIV virus. This invention was supported in part by the U.S. Government. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

It has been determined that in order to properly defend a host against foreign invaders such as bacteria or viruses, leukocytes must be able to migrate from circulation to sites of infection and inflammation. Leukocytes must also be able to attach to antigen- presenting cells so that a normal specific immune response can occur, and finally, they must attach to appropriate target cells so that lysis of virally-infected or tumor cells can occur. Each of these processes requires that leukocytes have the ability to adhere to other cells, especially endothelial cells. An excellent review of the properties and characteristics of leukocytes is provided by Eisen, H.W., fin: Microbiology. 3rd Ed., Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418).

Recently, a family of leukocyte surface molecules involved in mediating such adhesion have been identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T- cells (Davignon, D. et a!.. Proc. Nat! . Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells (Springer, T. et al . Eur. J. Immunol. 9:301-306 (1979)) were identified which bound to leukocyte surfaces and inhibited the attachment related functions described above (Springer, T. et al.. Fed. Proc. 44:2660-2663 (1985)).

The family of receptor molecules identified by the above- described method has been termed the "CD11/CD18 family of receptor molecules." The receptor molecules of the CD11/CD18 family are heterodimers containing an alpha subunit (CD11) and a beta subunit (CD18) (Sanchez-Madrid, F. et al .. J. Exoer. Med. 158:1785-1803 (1983); Keizer, G.D. et al.. Eur. J. Immunol. 15:1142-1147 (1985)).

Three different alpha subunits have been characterized: CDlla (equivalently referred to as the LFA-1 alpha subunit), CDllb (equivalently referred to as the Mac-1 alpha subunit) and CDllc (equivalently referred to as the pl50,95 alpha subunit). The CDlla/CD18 heterodimer is found on most lymphocytes (Springer, T.A., et al. Immunol. Rev. 68:111-135 (1982)). The CDllb/CD18 and CDllc/CD18 heterodimers are found on macrophages, granulocytes and large granular lymphocytes. These three molecules play a role in cellular adhesion (Keizer, G. et al.. Eur. J. Immunol. 15:1142-1147 (1985)). The natural binding ligand for the CD11/CD18 receptor molecules is ICAM-1 (Rothlein et al .. J> Immunol. 137:1270 (1986)), European Patent Application Publication No. 289,949, which references are incorporated herein by reference).

The beta chains of the heterodimers share extensive ho ology. The CD18 molecules were found to have a molecular weight of 95 kd whereas the molecular weights of the alpha chains were found to vary from 150 kd to 180 kd (Springer, T., Fed. Proc. 44:2660-2663 (1985)). Although the alpha subunits of the membrane proteins do not share the extensive homology shared by the beta subunits, close analysis of the alpha subunits of the glycoproteins has revealed that there are substantial similarities between them. Reviews of the similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by Sanchez-Madrid, F. et al .. (J. Exoer. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983)).

A group of individuals has been identified who are unable to express normal amounts of any member of this adhesion protein family on their leukocyte cell surface (Anderson, D.C., et al .. Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et al.. J. Infect. Pis. 152:668- 689 (1985)). This condition has been termed "LAD" ("leukocyte adherence deficiency disease"). Leukocytes from LAD patients displayed in vitro defects similar to normal counterparts whose LFA-1 family of molecules had been antagonized by antibodies. Furthermore, these individuals were unable to mount a normal immune response due to an inability of their cells to adhere to cellular substrates (Anderson, D.C., et al.. Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et al.. J. Infect. Pis. 152:668-689 (1985)). These data show that immune reactions are mitigated when leukocytes are unable to adhere in a normal fashion due to the lack of functional adhesion molecules of the LFA-1 family.

Thus, in summary, the ability of leukocytes to maintain the health and viability of an animal requires that they be capable of adhering to other cells (such as endothelial cells). This adherence has been found to require cell-cell contacts which involve specific receptor molecules present on the cell surface of the leukocytes. These receptors enable a leukocyte to adhere to other leukocytes or to endothelial, and other non-vascular cells. The cell surface receptor molecules have been found to be highly related to one another. Humans whose leukocytes lack these cell surface receptor molecules exhibit chronic and recurring infections, as well as other clinical symptoms including defective antibody responses.

HIV (human immunodeficiency virus also known as HTLV-III and LAV) is a double stranded RNA retrovirus. HIV is the causal agent of AIPS (Acquired Immunodeficiency Syndrome). The virus is believed to cause AIPS by binding to, and infecting, the T cells of a susceptible host.

SUMMARY OF THE INVENTION

The invention concerns the influence of LFA-1 and ICAM-1- dependent interactions in the spread of HIV infection, and particularly HIV-1 infection, to leukocytes. To illustrate this interaction, peripheral blood mononuclear cells from CP18 deficient donors were challenged with cell-free HIV-1. Accrual of cells containing HIV-1 was found to be significantly delayed among CP18 deficient lymphoblastoid cells.

The role of CPlla/CP18-ICAM-l dependent interactions in the replication of HIV-1 was further illustrated by studies of MT4 cells inoculated with cell-free HIV-1 and cultured with monoclonal antibodies that react with CD18, CDlla and ICAM-1. HIV specific mRNA in the monoclonal antibody treated cultures was found to be substantially suppressed relative to control cultures.

The specific impact of CPlla/CP18-ICAM-l interactions in the spread of HIV from infected monocytoid cells to susceptible T cells was also evaluated for the presence of the HIV p24 antigen and for HIV-associated cytopathic effects (CPE). Treatment with monoclonal antibodies specific for CP18, CPlla or ICAM-1 significantly suppressed the emergence of CPE and p24 antigen in vitro. These findings indicate that molecular interactions mediated by LFA-1 (CPlla/CD18), particularly with structural motifs expressed by the amino-terminal domain of ICAM-1, play an important role in cellular interactions which permit HIV to infect susceptible cells.

In detail, the invention comprises a method for suppressing the infection of leukocytes with HIV, which comprises administering to a patient exposed to or effected by HIV, an effective amount of an HIV-1 infection suppression agent, the agent being capable of binding to ICAM-1, ICAM-2, CD11, CD18 or to a CD11/CD18 heterodimer.

The invention further concerns the embodiment of the above method wherein the HIV is HIV-1.

The invention further concerns the embodiment of the above method wherein the agent is an immunoglobulin (such as a polyclonal antibody, a monoclonal antibody, or a humanized antibody (either chi eric or CDR-grafted) , or an antigen binding fragment of such an immunoglobulin.

The invention further concerns the embodiment of the above method wherein the immunoglobulin is an antibody capable of binding to a CD11 molecule (especially, a CD11 molecule selected from the group consisting of CDlla, CDllb, and CDllc).

The invention further concerns the embodiment of the above method wherein the immunoglobulin is an antibody capable of binding to a CD18 molecule.

The invention further concerns the embodiment of the above method wherein the immunoglobulin is an antibody capable of binding to ICAM-1 (especially the antibody R6.5) or ICAM-2.

The invention further concerns the embodiment of the above method wherein the agent is a soluble derivative of ICAM-1, ICAM-2 or CD11 (especially wherein the soluble derivative of CD11 is selected from the group consisting of: a soluble derivative of CDlla, a soluble derivative of CDllb, and a soluble derivative of CDllc).

The invention further concerns the embodiment of the above method wherein the agent is a soluble derivative of CD18 or a soluble derivative of CD11/CD18 (especially, wherein the soluble derivative of CD11/CD18 is selected from the group consisting of: a soluble derivative of CDlla/CD18, a soluble derivative of CDllb/CD18 and a soluble derivative of CDllc/CD18.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a comparison of the p24 antigen content of culture supernatants collected daily from PHA stimulated mononuclear leukocytes following inoculation with HIV-1 at multiplicities of infection of .01 and .001, from a donor with the leukocyte deficiency syndrome (LADS) and from a normal donor (control) whose peripheral blood cells expressed normal amount of CD4*+ cells.

Figure 2 shows the relative quantities of HIV specific mRNA in 2 x 10⁵ MT4 T cells inoculated with HIV-1 72 hr earlier and treated with irrelevant monoclonal antibodies (anti- BC monoclonal antibodies), a mixture of monoclonal antibodies specific for CDlla, CD18 and ICAM-1 or nothing.

Figure 3 shows the kinetics of p24 antigen release from well- washed HIV-1 infected U937 cells cultured at a 1:50 ratio with uninfected MT4 cells in the presence of 30 μg/ 10⁶ cells monoclonal antibodies specific for ICAM-1 (RR1.1 or R6.5), CD18 (R15.7), CDlla (R7.1) and CD4 (leu3a).

Figure 4 shows the p24 antigen content after 7 days of co- cultures containing, initially, 50,000 uninfected MT4 cells, and 10,000 well washed HIV-1 infected U937 cells. Values shown are the geometric mean and the standard error of the mean for cultures treated with monoclonal antibodies specific for CDlla, CD18, and ICAM-1. Controls were treated with irrelevant anti-WBC monoclonal antibodies, or left untreated. N = 8 experiments.

Figure 5 shows the p24 antigen content of co-cultures containing HIV-1 infected U937 cells and uninfected MT4 cells established using the same conditions described in Figure 4 and treated with monoclonal antibodies specific for the first extr& ellular domain (RR1.1 and R6.1), or for the second (R6.5) or fifth (CL203) domain of ICAM-1.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The CD18 Family of Receptor Molecules

As discussed above, the C011/CP18 family is comprised of three heterodimers which share a common beta subunit, (CP18) and a distinct a chain: CPlla, (LFA-1 alpha or at, a 177,000 dalton molecule), CDllb, (Mac-1 alpha, αM, 165,000 daltons) and COllc (αX or P150,95 alpha, a 150,000 dalton molecule) (E. Ruoslahti et al .. Science 238:491 (1987); P.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)).

As used herein, the term "CP11/CP18" is intended to generally refer to any and all members of the family of receptor molecules contain CP18. The term "CPU" is intended to refer to any alpha subunit of the CP11/CP18 family of receptor molecules. In contrast, the terms "CPlla," "CDllb," and "CDllc" are intended to refer to the specific alpha subunit molecule designated (i.e. the LFA-1, Mac-1 and pl50,95 alpha subunits, respectively). The term "CD18" is intended to refer to the beta subunit of the CD11/CD18 family. The terms "CDlla/CD18," "CDllb/CDlδ," and "CDllc/CD18" are intended to refer to the specific receptor molecule designated (i.e. the LFA-1, Mac-1 and pl50,95 receptor molecules, respectively).

CDllb/CD18 and CDllc/CD18 are found, in various quantities on monocytes, macrophages, and granulocytes (E. Ruoslahti et al.. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)); CDlla/CD18 is expressed on lymphocytes, monocytes, granulocytes and large granular lymphocytes (E. Ruoslahti et al .. Science 238:491 (1987); P.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)). The CP11/CP18 complex is also involved in other cell-cell interactions involved in host defence against infection, including binding and phagocytosis of iC3b-opsonized particles, a property of CPllb/CP18 on granulocytes and monocytoid cells, and Mg²⁺-dependent adhesion and killing of target cells by T cells and killer cells, a property of the CDlla/CD18 heteroduplex (E. Ruoslahti et al .. Science 218:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)).

ICAM-1

ICAM-1 is a cell surface glycoprotein expressed on non- hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen- sti ulated T lymphocyte blasts, and germinal centered B cells and dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 is highly expressed on vascular endothelial cells in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood lymphocytes. Phorbol ester-stimulated differentiation of some yelomonocytic cell lines greatly increases ICAM-1 expression. Thus, ICAM-1 is preferentially expressed at sites of inflammation, and is not generally expressed by quiescent cells. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either inter!eukin 1 or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible.

ICAM-1 displays molecular weight heterogeneity in different cell types with a molecular weight of 97 kd on fibroblasts, 114 kd on the myelomonocytic cell line U937 [ATCC CRL 1593] (Miller, L.J., et al.. J. Immunol . 137:2891 (1986)), and 90 kd on the B lymphoblastoid cell JY. ICAM-1 biosynthesis has been found to involve an approximately 73 kd intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment (which inhibits glycosylation) has a molecular weight of 55 kd.

ICAM-1 isolated from phorbol ester stimulated U937 cells or from fibroblast cells yields an identical major product having a molecular weight of 60 kd after chemical deglycosylation. ICAM-1 monoclonal antibodies interfere with the adhesion of phytohemagglutinin blasts to LFA-1 deficient cell lines. Pretreatment of fibroblasts, but not lymphocytes, with monoclonal antibodies capable of binding ICAM-1 inhibits lymphocyte-fibroblast adhesion. Pretreatment of lymphocytes, but not fibroblasts, with antibodies against LFA-1 has also been found to inhibit lymphocyte-fibroblast adhesion.

ICAM-1 is, thus, the binding ligand of the CD 18 complex on leukocytes. It is inducible on fibroblasts and endothelial cells jn vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor in a time frame consistent with the infiltration of lymphocytes into inflammatory lesions in vivo (Dustin, M.L., et. al.. J. Immunol 137:245-254. (1986); Prober, J.S., et. al.. J. Immunol 137:1893-1896, (1986)). Further ICAM-1 is expressed on non-hematopoietic cells such as vascular endothelial cells, thy ic epithelial cells, other epithelial cells, and fibroblasts and on hematopoietic cells such as tissue macophages, mitogen-stimulated T lymphocyte blasts, and germinal center B-cells and dendritic cells in tonsils, lymph nodes and Peyer's patches (Dustin, M.L., et. al . , J . Immunol 137:245-254, (1986)). ICAM-1 is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen planus, exanthema, urticaria and bullous diseases. Allergic skin reactions provoked by the application of a hapten on the skin to which the patient is allergic also revealed a heavy ICAM-1 expression on the keratinocytes. On the other hand toxic patches on the skin did not reveal ICAM-1 expression on the keratinocytes. ICAM-1 is present on keratinocytes from biopsies of skin lesions from various dermatological disorders and ICAM-1 expression is induced on lesions from allergic patch tests while keratinocytes from toxic patch test lesions failed to express ICAM-1.

ICAM-1 is, therefore, a cellular substrate to which lymphocytes can attach, so that the lymphocytes may migrate to sites of infection or inflammation.

ICAM-2

A second LFA-1 ligand, distinct from ICAM-1, has been postulated (Rothlein, R. et al .. J. Immunol. 137:1270-1274 (1986); Makgoba, M.W. et al.. Eur. J. Immunol. 18:637-640 (1988); Dustin, M.L. et al.. _JL Cell. Biol. 107:321-331 (1988)). This second ligand has been designated ICAM-2 (Staunton, D.M. et al.. FASEB J. 3:a446 (1989)). ICAM-2 binds to the CD11/CD18 receptor.

Leukocyte Adherence Deficiency Disease

"Leukocyte adherence deficiency disease" ("LAD") (Anderson, D.C., et al.. Fed. Proc. 44:2671-2677 (1985); Anderson, D.C., et al.. J. Infect. Pis. 152:668-689 (1985)) is a hereditable, autoso al recessive, disease caused by a mutation in the gene encoding the CD18 subunit of the CD11/CD18 family of leukocyte adhesion molecules. The leukocytes of afflicted individuals lack CD11/CD18 adhesion molecules on their cell surfaces.

Characteristic features of LAD patients include necrotic soft tissue lesions, impaired pus formation and wound healing, as well as abnormalities of adhesion-dependent leukocyte functions in vitro. Granulocytes from these LAD patients behave in the same defective manner in vitro as do their normal counterparts in the presence of anti-CD18 monoclonal antibody. That is, they are unable to perform adhesion related functions such as aggregation or attachment to endo¬ thelial cells. More importantly, however, is the observation that these patients are unable to mount a normal inflammatory response because of the inability of their granulocytes to attach to cellular substrates. Most remarkable is the observation that granulocytes from these LAD patients are unable to get to sites of inflammation such as skin infections due to their inability to attach to the endothelial cells in the blood vessels near the inflammation lesions. Such attachment is a necessary step for extravasation.

The importance of the CDlla/CD18 complex in host defense has thus been illuminated by identification of disorder characterized by recurrent, severe bacterial infections in which affected individuals are unable to synthesize normal CD18 molecules (E. Ruoslahti et al .. Science 238:491 (1987); D.C. Anderson et al.. Ann. Rev. Med. 38:175 (1987)). Leukocytes from such individuals are unresponsive to stimuli which induce leukocytes to adhere to and move across vascular endothelial cells (C.W. Smith et al .. J. Clin. Invest. 82:1746 (1988)).

Characteristics of HIV Infection

HIV infection is the cause of AIDS. Two major variants of HIV have been described: HIV-1 and HIV-2. HIV-1 is prevalent in North America and Europe, in contrast to HIV-2 which is prevalent only in Africa. The viruses have similar structures and encode proteins having similar function. The nucleotide and protein sequences of the genes and gene products of the two variants have been found to have about 40% homology with one another.

HIV infection is believed to occur via the binding of a viral protein (termed "gpl20") to a receptor molecule (termed "CD4") present on the surface of T4 ("T helper") lymphocytes (Schnittman, S. M. et al .. J. Immunol. 141:4181-4186 (1988), which reference is incorporated herein by reference). The virus then enters the cell and proceeds to replicate, in a process which ultimately results in the death of the T cell. The destruction of an individual's T4 population is a direct result of HIV infection. HIV can be recovered from peripheral blood mononuclear cells and human plasma (J. Clin. Microbiol. 26:2371-2376 (1988); N. Enol . J. Med. 321:1621-1625 (1989)). Results suggest more viremia than had been previously estimated and a T-cell infection frequencyas high as 1%.

The destruction of the T cells results in an impairment in the ability of the infected patient to combat opportunistic infections. Although individuals afflicted with AIDS often develop cancers, the relationship between these cancers and HIV infection is, in most cases, uncertain.

Although the mere replication of the HIV virus is lethal to infected cells, such replication is typically detected in only a small fraction of the T4 cells of an infected individual. Several lines of research have elucidated other mechanisms through which the HIV virus mediates the destruction of the T4 population.

Apart from through HIV replication, HIV infected cells can be destroyed through the action of cytotoxic, killer cells. Killer cells are normally present in humans, and serve to monitor the host and destroy any foreign cells (such as in mismatched blood transfusions or organ transplants, etc.) which may be encountered. Upon infection with HIV, T4 cells display the gpl20 molecule on their cell surfaces. Killer cells recognize such T4 cells as foreign (rather than native cells), and accordingly, mediate their destruction.

HIV infection can also lead to the destruction of non-infected healthy cells. Infected cells can secrete the gpl20 protein into the blood system. The free gpl20 molecules can then bind to the CD4 receptors of healthy, uninfected cells. Such binding causes the cells to take on the appearance of HIV infected cells. Cytotoxic, killer cells recognize the gpl20 bound to the uninfected T4 cells, conclude that the cell is foreign, and mediate the destruction of the T4 cells.

An additional mechanism, and one of special interest to the present invention, with which HIV can cause T4 death is through the formation of "syncytia." A "syncytium" is a ultinucleated giant cell, formed from the fusion of as many as several hundred T4 cells. Infection with HIV causes the infected cell to become able to fuse with other T4 cells. Such fusion partners may themselves be HIV infected, or they may be uninfected healthy cells. The syncytium cannot function and soon dies. Its death accomplishes the destruction of both HIV infected and HIV uninfected T4 cells. This process is of special interest to the present invention since it entails the direct cell-cell contact of T4 cells.

Infection with HIV is normally followed by a substantial and vigorous immune response. The sera of healthy HIV-infected individuals has been found to contain virus neutralizing antibody, and to have a high titer of antiviral antibodies (Karpas, A. et al .. Lancet ϋ:695-697 (1985)). In contrast, the sera of AIDS patients does not contain HIV neutralizing antibodies (Karpas, A. et al ., Proc. Natl. Acad. Sci . (U.S.A.) 85:9234-9237 (1988)). Thus, anti-HIV antibodies may delay the onset of clinical symptoms of AIDS, however, ultimately, the virus is capable of proliferating, and advancing the disease, even in the face of the initial antibody response.

The apparent ability of HIV to evade the host's antibody response suggests that the virus may proliferate in a process which renders it inaccessible to antibody. The ability of HIV-infected cells to form syncytia indicates that such cells acquire a means for fusing with healthy cells. Thus, cell-cell contacts may be of fundamental importance in the process through which HIV infection is transmitted from one cell to another within an individual.

The Symptoms of AIDS

The first symptom of AIDS is typically chronically swollen lymph nodes. This stage of the disease may last from 3-5 years. During this period the HIV virus replicates and gradually reduces the number of T4 cells which are available to prevent disease. Eventually, the number of T4 cells falls to less than 400 cells/μl.

Generally, within 18 months after an individuals T cells have fallen below the 400 cells//.! level, the disease can be shown to have impaired the patient's ability to mount and sustain an immune response against specific proteins which are injected under the skin (i.e. impairment of the patient's ability to mount a delayed hypersensitivity response). This conditions rapidly worsens until the patient is completely unable to mount a systemic delayed hypersensitivity response. At this stage, patients frequently develop opportunistic infections, such as oral thrush (candidiasis), herpes simplex, cytomegalovirus and molluscu contaoiosum infections. AIDS patients generally succumb to the disease within 10 years from infection. The disease and its treatment are reviewed in Scientific American 259:40-134 (1988); Clumeck, N. et al .. Eur. J. Clin. Microbiol. Infect. Pis. 7:2-10 (1988); Waldmann, T.A. et al.. Blood 72:1805-1816 (1988); Sarin, P.S., Ann. Rev. Pharmacol. 28:411-428 (1988); which references are incorporated herein by reference).

Although immunodeficiency associated with C04+ T cell depletion remains the most obvious consequence of HIV infection, it is more and more evident that organs outside the hematopoietic and immune systems are affected by this virus in man, and by viruses with similar characteristics in other primates (R.T. Johnson, et al. FASEB J. 2:2970 (1988); M.H. Stoler et al.. J. Amer. Med. Assn. 256:2360 (1986); S. Gartner et al.. J. Amer. Med. Assn. 256:2365 (1986); S. Gartner et al.. Science 233:215 (1986); S.P. Raffanti et al.. Chest 93:592, (1988); M.C. Dalakas et al .. Neurology 36:569 (1986); M.C.Dalakas et al .. J. Amer. Med. Assn. 256:2381 (1986); R.D. Bailey et al .. Human Pathology 18:749 (1987); C. Cammarosano, et al .. jh. Amer. Coll. Cardiol. 5:703 (1985); B.K. Valle et al.. Heart-Luno 16:584 (1987); T.K. Rao et al.. N. Enol . J. Med. 310:669 (1984); N.W. King et al.. Amer. J. Pathol . 113:382 (1983); P. Solal-Celigy et al.. Amer. Rev. Respir. Pis. 131:956 (1985); C.C. Tsai et al .. Amer. J. Pathol . 120:30 (1985); L.J. Couderc et al.. Arch Intern. Med. 147:898 (1987)).

Oysfunctions of the central and peripheral nervous systems in HIV infected individuals are particularly striking (R.T. Johnson, et al . FASEB J. 2:2970 (1988); M.H. Stoler et al.. J. Amer. Med. Assn. 256:2360 (1986); S. Gartner et al . J. Amer. Med. Assn. 256:2365 (1986); S. Gartner et al . Science 233:215 (1986)). The heart (S.P. Raffanti et al. Chest 93:592. (1988); C. Cammarosano, et al .. J. Amer. Coll. Cardiol. 5:703 (1985); B.K. Valle et al .. Heart-Lung 16:584 (1987)) and skeletal muscle (M.C. Oalakas et al .. Neurology 36:569 (1986); M.C.Palakas et al.. J. Amer. Med. Assn. 256:2381 (1986); R.P. Bailey et al.. Human Pathology 18:749 (1987); N.W. King et al .. Amer. J. Pathol. 113:382 (1983), the kidney (T.K. Rao, et al.. N. Enol. J. Med. 310:669 (1984)), the lung (P. Solal-Celigy et al.. Amer. Rev. Respir. Pis. 111:956 (1985)) and other organs (N.W. King et al.. Amer. J. Pathol. 113:382 (1983); C.C. Tsai et al.. Amer. J. Pathol. 120:30 (1985); L.J. Couderc et al.. Arch Intern. Med. 147:898 (1987)) also may be involved.

The Relationship Between HIV Infection and the Expression of the CD18/CD11 Family of Molecules and Their Binding Ligands

HIV infection, and especially HIV-1 infection, appears to influence cell surface expression of the leukocyte integrins and cellular adherence reactions mediated by these heterodimers (Petit, A.J., et al.. J. Clin. Invest. 79:188 (1987); Hildreth, J.E.K., et al.. Science 244:1075 (1989); Valentin, A., et al .. J. Immunology 144:934-937 (1990); Rossen, R.P., et al .. Trans. Assoc. American Phvsicians 102:117-130 (1989), all of which references are incorporated herein by reference). Following infection with HIV-1, homotypic aggregation of U937 cells is increased, as is cell surface expression of C018, CDllb (Petit, A.J., et al .. J. Clin. Invest. 79:188 (1987)). HIV-1 infected U937 cells adhere to IL-1 stimulated endothelium in greater frequency than uninfected U937 cells; this behavior can be suppressed by treating the infected cells with anti- CD18 or anti-CDlla monoclonal antibodies or by treating endothelial substrates with anti-ICAM-1 (Rossen, R.D., et al .. Trans. Assoc. American Physicians 102:117-130 (1989)). Monoclonal antibodies to CD18 or CDlla have also been found to be able to inhibit formation of syncytia involving phytohemagglutinin (PHA)-stimulated lymphoblastoid cells and constitutively infected, CD4-negative T cells (Hildreth, J.E.K., et al .. Science 244:1075 (1989)). Treatment of only the virus infected cells with anti-CD18, or anti-CDlla monoclonal antibodies was found to have little effect on syncytium formation, suggesting that these antibodies principally protect uninfected target cells from infection (Hildreth, J.E.K., et al .. Science 244:1075 (1989); Valentin, A., et al.. J. Immunology 144:934-937 (1990)). Valentin et al. (Valentin, A., et al.. J. Immunology 144:934-937 (1990)) have recently confirmed these observations by demonstrating that monoclonal antibodies specific for CD18 inhibit syncytia formed when continuous T cell lines are co-cultured with HIV-1 infected U937 cells.

Although the mechanism through which monoclonal antibodies specific for CD18 or CDlla protect susceptible cells from fusing with HIV infected cells remains unknown, and is not necessary to an appreciation of the present invention, studies with radio!abeled gpl20 suggest that heterodimers containing CD18 do not provide a binding site for the virus (Valentin, A., et al.. J. Immunology 144:934-937 (1990)). Thus, HIV infection involves cell-cell interactions, and/or viral-cell interactions which mimic such cell-cell interactions. The cell-cell interactions may result in the transport of cell-free virus or the transport of virus across endothelial barriers within the cytoplasm of infected mononuclear cells. Viral-cell interactions which mimic the cell-cell interactions may facilitate or enable free virus to attach to and/or infect healthy cells.

The present invention thus derives, in part, from the discovery that HIV infection, and particularly HIV-1, infection results in increased expression of the CDlla/CD18 heterodi er, and its binding ligand, ICAM-1. This increased expression is significant in that it enhances the ability of HIV-infected T cells to adhere or aggregate with one another (i.e. to undergo "homotypic aggregation"). Since such homotypic aggregation is not observed to occur among quiescent normal leukocytes, this discovery indicates that the expression of the CD11/CD18 receptors and/or ICAM-1 is required for such aggregation. Such adhesion permits HIV-1 to be transmitted from an infected cell to a healthy cell of an individual, and also permits or facilitates infection of healthy cells with free virus.

Thus, the present invention provides a method for suppressing the infection of HIV, which comprises administering to an HIV-infected individual an effective amount of an HIV infection suppression agent. Although the invention is particularly concerned with a method for the suppression of HIV-1 infection, it is to be understood that the method may be applied to any HIV-1 variant (such as, for example, HIV-2) which may infect cells in a way which may be suppressed by the agents of the present invention. Such variants are the equivalents of HIV-1 for the purposes of the present invention.

The HIV Infection Suppression Agents of the Present Invention

One aspect of the present invention derives from the recognition that expression of LFA-1 and, in some cases, ICAM-1, stimulated by HIV infection, promotes cell-to-cell adherence reactions that can increase the contact time of infected with uninfected cells, facilitating transfer of virus from infected to uninfected cells. Thus, monoclonal antibodies specific for CD18, CDlla or their equivalents (such as soluble ICAM-1), or monoclonal antibodies specific for ICAM-1, or their equivalents (such as soluble CD18/CD11 heterodimer, etc.) are able to suppress infection by Hl f and, in particular, by HIV-1.

Similarly, the use of such agents will cause HIV infection to spread more slowly among phytohemagglutinin stimulated peripheral blood cells of CD18 deficient as compared to normal individuals, challenged with the same inoculum of this virus.

Thus, the HIV infection suppression agents of the present invention include any agent capable of impairing the ability of an HIV-infected cell to bind to CDlla, CD18, CDlla/CD18, or their ligands such as ICAM-1 or ICAM-2. One means through which molecules which bind to CDlla, CD18, or CDlla/CD18 may suppress HIV infection is by impairing the ability of the CDlla/CD18 or ICAM-1 expressed by HIV- infected cells to bind to the ICAM-1, ICAM-2 or CDlla/Cdl8 of a healthy T cell. One means through which molecules which bind to ICAM- 1 or ICAM-2 may suppress HIV infection is by impairing the ability of the ICAM-1 or ICAM-2 expressed by HIV-infected cells to bind to the CD11/CD18 receptors of a healthy T cell. In order to impair the ability of a cell to bind to the CDIla/CD18 receptor, or to the ICAM-1 or ICAM-2 ligand molecule, it is possible to employ either immunoglobulin or non-immunoglobulin antagonists of these molecules.

Examples of immunoglobulin antagonists include monoclonal or polyclonal antibodies which are capable of binding to either the CDIla/CD18 molecule, or either of its subunits, or ICAM-1 or ICAM-2. Suitable antagonists also include the antigen-binding fragments of such antibody molecules (for example F(ab) or F(ab)2 fragments. Such antibodies can be derived from mouse, or other mammalian cells (including human).

As indicated above, both polyclonal and monoclonal antibodies may be employed in accordance with the present invention. Of special interest to the present invention are antibodies to ICAM-1 (or their functional derivatives), or to members of the CD18 family (or their functional derivatives), iich are produced in humans, or are "humanized" (i.e. non-immunogenic in a human) by recombinant or other technology. Such antibodies are the equivalents of the monoclonal and polyclonal antibodies disclosed herein, but are less i munogenic, and are better tolerated by the patient.

Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non- immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al.. International Patent Publication PCT/US86/02269; Akira, K. et al.. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S.L. et al .. European Patent Application 173,494; Neuberger, M.S. et al .. PCT Application WO 86/01533; Cabilly, S. et al.. European Patent Application 125,023; Better, M. et al .. Science 240:1041-1043 (1988); Liu, A.Y. et al.. Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A.Y. et al.. J Immunol . 139:3521-3526 (1987); Sun, L.K. et al.. Proc. Natl. Acad. Sci . USA 84:214-218 (1987); Nishimura, Y. et al.. Cane. Res. 4Z.-999-1005 (1987); Wood, C.R. et al.. Nature 314:446-449 (1985)); Shaw et al.. J. Natl.Cancer Inst. 80:1553-1559 (1988); all of which references are incorporated herein by reference). General reviews of "humanized" chimeric antibodies are provided by Morrison, S.L. (Science. 229:1202-1207 (1985)) and by Oi, V.T. et al.. BioTechnioues 4:214 (1986); which references are incorporated herein by reference).

Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones, P.T. et al.. Nature 321:552-525 (1986); Verhoeyan et al.. Science 239:1534 (1988); Beidler, C.B. et al.. J. Immunol. 141:4053-4060 (1988); all of which references are incorporated herein by reference).

Examples of preferred immunoglobulin antagonists of the CDlla/CD18 receptor include monoclonal antibody TS 1/18 (anti-CD18) or monoclonal antibody TS 1/22 (anti-CDlla). Examples of immunoglobulin antagonists of the ICAM-1 ligand include RR 1/1 (Rothlein et al.. jh Immunol . 137:1270 (1986)), and R6.5 (disclosed in European Patent Application Publication No. 289,949, which application is incorporated herein by reference). Examples of immunoglobulin antagonists of the CD11/CD18 receptor include R15.7 (anti-CD18) and R7.1 (anti-CDlla).

The non-immunoglobulin antagonists which may be employed in accordance with the present invention as HIV infection suppression agents include soluble derivatives of the CDlla/CD18, CDllb/CD18, or CDllc/CD18 receptor molecules, as well as soluble derivatives of each of the subunits of the receptor molecule (i.e. CDlla, CDllb, CPllc or CP18). Such molecules may suppress HIV infection by binding to the ICAM-1 or ICAM-2 expressed by HIV-infected T cells. Alternatively, soluble derivatives of ICAM-1 or ICAM-2 may be employed. Such molecules may suppress HIV infection by binding to the CDlla/CP18 receptors expressed by HIV-infected T cells.

The soluble derivatives referred to above are derivatives which are not bound to a membrane of a cell. Such derivatives may comprise truncated molecules which lack a transmembrane domain. Alternatively, they may comprise mutant forms of the natural molecules which lack the capacity to be bound (or stably bound) to the membrane of a cell even though they contain a transmembrane domain. Soluble derivatives of ICAM-1 and their preparation are disclosed by Marlin, S.P. et al .. Nature 344:70-72 (1990), which reference is incorporated herein by reference).

The therapeutic effects of the present invention may be obtained by providing to a patient any of the above-described HIV infection suppression agents. Such agents may be obtained either synthetically, through the use of recombinant DNA technology, or by proteolysis, or by a combination of such methods.

The agents of the present invention are said to be "substantially free of natural contaminants" if preparations which contain them are substantially free of materials with which these products are normally and naturally found. Administration of the Agents of the Present Invention

In providing a patient with antibodies, or fragments thereof, capable of binding to any of the CD11, C018 or ICAM-1 molecules, or when providing CD11, CD18, CD11/CD18 or ICAM-1 or ICAM-2 (or a fragment, variant, or derivative of any of these molecules) to a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient), although a lower or higher dosage may be administered. When providing the agents of the present invention, or their functional derivatives to a patient, it is preferable to administer such molecules in a dosage which also ranges from about 1 pg/kg to 10 mg/kg (body weight of patient) although a lower or higher dosage may also be administered. The therapeutically effective dose can be lowered if anti-CPlla or anti-CP18 antibody is additionally administered with a soluble form of CPU, CP18, CP11/CP18, ICAM-1 or ICAM-2. As used herein, one compound or agent is said to be additionally administered (or co-administered) with a second compound or agent, when the administration of the two compounds or agents is in such proximity of time that both compounds or agents can be detected at the same time in the patient's serum.

The agents of the present invention may be administered to patients intravenously, intranasally, intramuscularly, subcutaneously, enterally, or parenterally. When administering such agents by injection, the administration may be by continuous infusion, or by single or multiple boluses.

The agents of the present invention are intended to be provided to recipient subjects in an amount sufficient to achieve a suppression of HIV infection. An amount is said to be sufficient to "suppress" HIV infection if the dosage, route of administration, etc. of the agent are sufficient to attenuate or prevent such HIV infection. The agents are to be provided to patients who are exposed to, or effected by HIV infection.

Soluble CPU, CD18, CD11/CD18, ICAM-1, or ICAM-2, or a fragment or derivative of any of these molecules, may be administered either alone or in combination with one or more additional agents (such as antibody to CDlla or CD18). The administration of such compound(s) may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compound(s) are provided in advance of any symptom of viral infection (for example, prior to, at, or shortly after) the time of such infection, but in advance of any symptoms of such infection). The prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent HIV infection. When provided therapeutically, the compound(s) is provided at (or shortly after) the detection of virally infected cells. The therapeutic administration of the compound(s) serves to attenuate any additional HIV infection.

The agents of the present invention may, thus, be provided either prior to the onset of viral infection (so as to suppress the anticipated HIV infection) or after the actual detection of such virally infected cells (to suppress further infection).

In particular, the invention provides an improved therapy for AIDS, and an enhanced means for suppressing HIV infection, and particularly HIV-1 infection, which comprises the co-administration of:

(I) ICAM-1, a soluble ICAM-1 derivative, CD11 (either CDlla, CDllb, or CDllc), a soluble CD11 derivative, CD18, a soluble CD18 derivative, or a CD11/CD18 heterodimer, or a soluble derivative of a CD11/CD18 heterodimer and/or

(II) a molecule (preferably an antibody or antibody fragment) capable of binding to ICAM-1, CD11 (either CDlla, CDllb, or CDllc), CD18, or a CD11/CD18 heterodimer with (III) cell or particle associated CD4 or a soluble derivative of CD4 and/or

(IV) a molecule (preferably an antibody or antibody fragment) capable of binding to CD4.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

The agents of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)). In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the agents of the invention, or their functional derivatives, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the agents of the present invention, or their functional derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxy ethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the agents of the present invention, or their functional derivatives, into particles of a poly¬ meric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980).

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1 EFFECT OF HIV-1 ON THE HOMOTYPIC AGGREGATION OF LEUKOCYTES

HIV-1 infection increased homotypic aggregation of leukocytes in the presence of phorbol myristate acetate. Preincubating monocytoid cells with anti-ICAM-1 antibodies suppressed these cell to cell interactions. Adherence of monocytoid cells to cultured human umbilical vein endothelial cells was also increased by infection with HIV-1. Preincubating T cells with anti-LFA-1 alpha subunit or anti- LFA-1 beta subunit antibodies suppressed the ability of such cells to adhere to endothelial cells. Similarly, cellular adhesion could be impaired by preincubating HUVEC with anti-ICAM-1 antibody.

Thus, HIV-1 infection stimulates expression of molecules involved in leukocyte adherence in vitro. In vivo, these effects can facilitate the cell-cell adhesion of HIV-1 infected cells. Treatment with either anti-ICAM-1 antibodies, or antibodies to either the CDll alpha or CD18 beta subunit can suppress the cell to cell interactions, and thereby prevent such adhesion.

EXAMPLE 2

COMPARISON OF INFECTABILITY WITH HIV-1 OF PERIPHERAL

BLOOD MONONUCLEAR CELLS FROM LAO AND A NORMAL INDIVIDUAL

Since the leukocytes of LAD patients lack the CD11/CD18 recep¬ tors, the use of such leukocytes is analogous to the use of normal leukocytes incubated in the presence of antibody to CDll, CD18 or ICAM-1 or soluble derivatives of CDll, CD18, ICAM-1 or ICAM-2.

In order to determine the role of the CD11/CD18 - ICAM-1 interaction in HIV-1 infection, the ability of mononuclear cells which congenitally lack CD18/CD11 surface molecules (i.e. derived from an LAD patient) to propagate HIV-1 was, thus, compared with that of normal cells.

For this comparison, mononuclear cells were harvested by ficoll- /hypaque from dextran sedimented blood. Cells were counted and a differential count was performed in order to adjust cell suspensions to equal numbers (2 x 10^) of mononuclear cells/ml. These cells were cultured in 20% fetal calf sera (FCS) in RPMI 1640 culture medium and stimulated with human interleukin 2 (IL-2) (from Cellular Products) and 3 .g/ml of PHA in 20 ml Teflon coated jars (to facilitate recovery of monocytes).

Viability was found to be greater than 95% by trypan blue ex¬ clusion. The LAD patients' cells and the control cell normal cells were analyzed by flow cytometry to assure that both populations contained equivalent concentrations of CD4⁺ cells. After 3 days' stimulation, the cells were harvested and a viability of greater than 95% was confirmed.

The cultures were then inoculated with 1.8 x 10¹⁰ particles/ml of HTLV IIIB strain (HIV-1). The HIV-1 preparation contained 1 x 10⁶ infectious particles per ml. Cells were challenged at a multiplicity of infection (MOI) of 0.1, 0.01 and 0.001 infectious virions per cell, Cells were incubated for 1.5 hrs in 5% CO² humidified air, the cultures were diluted with 12 ml RPMI 1640 culture medium containing 10% FCS and washed 4 times to remove cell free virus.

Subsequently, the cells were cultured in the same medium containing 20% FCS, 5% v/v IL-2 and antibiotics (penicillin, etc.) in fresh Teflon vials. HIV-1 proliferation was determined by assaying for the p24 protein of HIV.

The results of this experiment are shown in Table 1. They reveal that the leukocytes of LAD patients were substantially less capable of propagating HIV-1 than were the leukocytes of normal individuals. They, therefore, indicate that agents which are capable of blocking cellular adhesion are able to suppress HIV-1 infection.

TABLE 1 ASSAY FOR HIV-1 PROLIFERATION

EXPERIMENT 1 P24 Assays on Culture Supernatants (.g/ml) Serum Day MOI Undiluted 1:10 1:100 1:1000

Normal .1 34 8 0 ND

.01 0 0 0 ND

.001 0 0 0 ND

LAD .1 33 0 0 ND

.01 0 0 0 ND

.001 11 0 0 ND

Normal .1 >200 66 14 ND

.01 >200 >200 20 ND

.001 40 0 0 ND

LAD .1 ND 74 13 ND

.01 ND 25 0 ND

.001 ND 0 0 ND

Normal .1 >200 13 5 NP

.01 >200 >200 149 NP

.001 >200 54 5 NP

LAD .1 >200 23 5 NP

.01 >200 59 5 NP

.001 6 0 0 NP

TABLE 1 (continued)

EXPERIMENT 2 P24 Assays on Culture Supe

Pay MOI P24 (pg/rnl) Normal LAD

2 0.01 61 21 0.001 54 17

3 0.01 480 114 0.001 310 37

4 0.01 1350 420 0.001 1060 250

5 0.01 2900 840 0.001 2200 460

6 0.01 5700 1480 0.001 5000 790

7 0.01 14900 2200 0.001 13000 1400

EXAMPLE 3

DISSEMINATION OF HIV-1 IN PHYTOHEMAGGLUTININ-STIMULATED PERIPHERAL BLOOD MONONUCLEAR CELLS FROM CD18 DEFICIENT DONORS

In order to investigate the dissemination of HIV-1 in phyytohemagglutinin-stimulated peripheral blood mononuclear cells from CD18 deficient donors, 20 ml of heparinized peripheral blood were collected from CD18 deficient donors 1 and 10 (Anderson, D.C., et al .. Ann. Rev. Med. 38:175 (1987)) and from normal controls. The blood was mixed 10:1 (v/v) with 6% dextran and allowed to sediment for 1 hr. Mononuclear cells were harvested from the dextran sedimented plasma by Ficoll/Hypaque gradient centrifugation (Rossen, R.D., et al.. ι Immunol. 135:3289 (1985)). Mononuclear cells were then washed in RPMI 1640 containing 10% FCS and cultured in 60 ml Teflon jars (van der Meer, J.W.M., et al.. J. EXP. Med. 147:271 (1978)) at a concentration of 2 x 10⁶ cells/ml in RPMI 1640 supplemented with 20% FCS, penicillin, streptomycin, gluta ine and interleukin-2 (Collaborative Research, Bedford, MA 01730). Cultured cells were stimulated with 3 mg/ml of phytohemagglutinin (PHA) for 3 days, analyzed by flow cytometry in order to adjust the concentrations of the cells in these cultures to contain equal numbers of CD4+ cells.

Flow cytometric analysis was performed as described by Rossen, R.D., et al. (Trans. Assoc. American Physicians 102:117-130 (1989)); and Smith, C.W., et al. (J. Clin. Invest. 82:1746 (1988)). Fluorescein-conjugated F(ab')2 fragments of rabbit anti-mouse Ig were used as the second antibody. Monoclonal antibodies used include the anti-CD4 IgGl, leu 3a (Becton, Dickinson, Mountain View, CA 94039), and 4A5, an IgGl made against isolated human polymorphonuclear leukocytes. Flow cytometric analyses of the reactions of this monoclonal antibody with cells in unfractionated human blood indicate that it does not react with mononuclear cells; nor does it affect adherence reactions of activated leukocytes. The leukocyte cell surface antigen(s) with which it reacts have not been characterized. In addition, the following monoclonal antibodies were used: W6/32, an IgG2a which reacts with class I major histocompatibility framework antigens (Barnstable, C.J., et al.. Cell 14:9 (1978)); R3.1, an IgGl anti-CDlla (Smith, C.W., et al.. J. Clin. Invest. 82:1746 (1988)), R15.7, an IgGl anti-CD18 monoclonal antibody (Entman, M.L., et al .. J. Clin Invest. (1990), which reference is incorporated herein by reference) and the following anti-ICAM-1 monoclonal antibodies, R6.5D6, an IgG2a antibody reactive with the second immunoglobulin-like domain, RR1.1 and R6.1, IgGl monoclonal antibodies which react with the first, amino-terminal domain (Rothlein, R., et al .. J. Immunol. 13_7_:1270 (1986)) and CL203.4 (Matsui, M., et al.. J. Immunol. 139:2088 (1987)) which reacts with the fifth extracellular ICAM-1 domain ((Staunton, D.E. et al.. Cell 56:849-853 (1989)), which reference is incorporated herein by reference).

The cultures were then inoculated with cell-free HIV-1 at multiplicities of infection (MOI) = .001 and .01. After incubation for 90 min the cells were centrifuged at 450 xg for 5 min at 8°C and washed 4 times with complete media. This treatment was sufficient to render p24 antigen undetectable in the last wash. The cells were then returned to the culture conditions described above.

Portions of the cells and cell-free supernatant were collected daily thereafter to enumerate HIV-1 positive cells by in situ hybridization and measure p24 antigen.

Figure 1 and Table 2 show the results of one of 3 experiments in which the dissemination of HIV-1 among PHA stimulated peripheral blood mononuclear cells from normal and CD18 deficient donors was studied. After stimulation, the cells were analyzed by flow cytometry and adjusted to contain equal numbers of CD4+ cells. HIV-1 was then added in the quantities indicated and the cells were cultured for 7 more days. At both MOI = .01 and .001, the p24 antigen levels in the cultured cells from the normal donor were significantly higher than in cultured cells from the CD18 deficient donor, challenged with the same quantities of virus (p<.014, Wilcoxam, signed rank test). Using >5 silver grains per cell as the indication that a cell was infected (Table 2) in situ hybridization analyses also provided evidence that the infection spread significantly more slowly among the CD18 deficient donor's cells, especially prior to day 6; by day 7 the cultures of the CD18 deficient and the normal donor inoculated at MOI = .001 contained equal numbers of HIV-1 infected cells as measured by in situ hybridization but, as indicated in Figure 1, the quantities of p24 antigen in cultures containing CD18 deficient cells were still significantly reduced.

TABLE 2 PERCENT OF HIV-1 INFECTED CELLS AS AN ESTIMATE BY IN SITU

HYBRIDIZATION WITH ^3ϊ •S-RNA FOR HIV-1 LTR*

MOI = .01 MOI = .001

CD18 CD18

Dav Normal Deficient Normal Deficient

2 ND 2.3 (0.2) 5.4 (0.6) 3.2 (0.8) 3 6.2 (0.6) 0.8 (0.2) 7.4 (1.0) 3.0 (0.0) 4 9.2 (1.0) 0.8 (0.0) 8.0 (1.4) 3.4 (0.4) 5 27.6 (11.8) 15.0 (4.8) 8.6 (1.6) 4.4 (0.2) 6 36.0 (6.6) 18.2 (1.4) 18.8 (3.8) 15.2 (1.8) 7 28.2 (2.8) 24.8 (0.8) 30.6 (1.6) 30.6 (3.0)

^♦Numbers indicate percent of cells with >5 silver grains (percent with >25 silver grains in parentheses); p < .03 by paired t test, comparing numbers of HIV-1 infected cells in normal vs. CD18 deficient donor.

Thus, phytohemagglutinin-stimulated peripheral blood mononuclear cells from CD18 deficient donors were challenged with cell-free HIV-1, at multiplicities of infection = 0.01 and 0.001. Accrual of cells containing HIV-1, as demonstrated by in situ hybridization, was significantly delayed among CD18 deficient lymphoblastoid cells. By 7 days after virus inoculation, the HIV p24 antigen content of culture supernatants was only 18% of that achieved with normal donor cells, cultured under identical conditions. The studies of PHA-activated peripheral blood lymphocytes from CDI8 deficient donors thus extends evidence which suggests that cellular interactions dependent on the leukocyte integrins can affect dissemination of HIV (Hildreth, J.E.K., et al .. Science 244:1075 (1989); Valentin, A., et al.. J. Immunology 144:934-937 (1990)). But in view of the fact that responses of CD18 deficient lymphocytes to mitogenic stimuli are relatively poor (Miedema, F., et al .. J^. Immunol. 134:3075 (1985); Springer, T.A., et al .. Ann. Rev. Immunology .5:223 (1987)), the failure of virus to disseminate among these cells may be specifically related to their failure to express CD18, or to their failure to respond adequately to activating stimuli provided by PHA.

EXAMPLE 4

THE ROLE OF CD11A/CD18-ICAM-1 DEPENDENT INTERACTIONS IN THE DISSEMINATION OF HIV-1

The role of CDlla/CD18-ICAM-l interactions on the dissemination of HIV-1 among T cells inoculated with cell-free virus was studied using a combination of monoclonal antibodies specific for the heterodimer, CDlla/CD18, and ICAM-1. Specifically, the ability of these antibodies to suppress virus replication in cultures of MT4 T cells inoculated with cell-free HIV-1 was investigated. The MT4 cell line is described by Rey, F. et al. (J. Virol. Meth. 16:239-250 (1987)); Gogu, S.R. et al. (Biochem. Biophvs. Res. Commun. 165:401-407 (1989)); Nakashima, H. et al . (Virol. 159:169-173 (1987)); all of which references are incorporated herein by reference.

To accomplish this objective, 2 x 10⁷ MT4 cells (6.3 x 10°/¥I) were challenged with HTLV-IIIB at MOI = .001. The cells were incubated with HIV-1 for 2 hrs at 37^βC in 5% CO2 and humidified air. The cell suspension was then diluted to 13 ml, and washed 4-6 times with interval centrifugations at 500 x g at 8^βC to remove virus which had not attached to the MT4 cells during the incubation period. Thereafter, the cells were then resuspended in fresh medium, and aliquots containing 10*> cells were then cultured in the presence of (a) no mononuclear antibodies, (b) a mixture of each of the W6/32 and 4A5 monoclonal antibodies, as a non-specific control or (c) a mixture of equal quantities of R15.7 (anti-CD18), R3.1 (anti-CDlla) and R6.5D6 (anti-ICAM-1). Each antibody was added at a concentration of 10 μg/10⁶ cells.

After 3 days in culture, 48 hrs before cytopathic effect was evident by the reclustering assay, aliquots of each culture were harvested for the extraction of total cellular RNA. RNA was extracted with RNAzol B (Cho czynski, P., et al.. Anal. Biochem. 162:156 (1987)) (Cinna-Biotecx Labs, Friendswood, TX 77546). Serial dilutions of the RNA were applied to nylon membranes, using a slot blot apparatus (Biorad Corp., Richmond, CA 94804) and probed with ³²P-labeled ARV-2 cDNA from the pARV/7A plasmid (Chiron Corp., Emeryville, CA 94608). These membranes were subsequently reprobed, after decay of the 32 associated with ARV-2 with labeled cDNA for 0-actin. Subsequent autoradiographs of these blots were analyzed by laser densitometry (LKB Ultroscan XL, LKB Biochrom, Cambridge CB44BH, England). Results were expressed as the ratio of relative densities achieved at the same dilution of RNA with cDNA probes for HIV and }-actin (Figure 2).

The ratio of the two mRNAs indicated that there were considerable quantities of viral message in the MT4 cells cultured either with no monoclonal antibodies or the mixture of the 4A5 and W6/32 monoclonal antibodies, even before cytopathic effects were evident. In contrast, production of viral message was significantly suppressed in the cultures treated with the mixture of monoclonal antibodies specific for CDlla, CD18 and ICAM-1 (p<.001, paired t test, comparing density ratios of HIV to J-actin, using the ARV-2 probe for HIV).

Thus, the role of CDlla/CD18-ICAM-l dependent interactions in the replication of HIV was investigated using MT4 cells inoculated with cell-free HIV-1 and cultured with monoclonal antibodies that react with CD18, CDlla and ICAM-1. After 72 hrs, HIV specific mRNA in the monoclonal antibody treated cultures was 25% of that achieved in MT4 cells treated with irrelevant immunoglobulin.

The existence of a direct role of the CD18-dependent integrins and ICAM-1 in HIV replication is thus demonstrated by the above- described measurements of viral mRNA in virus inoculated MT4 cells, treated with monoclonal antibodies that block functions of the leukocyte integrins and ICAM-1, and by studies of the dissemination of virus by infected U937 cells. These findings confirmed a role for CDlla/CD18 (LFA-1) and demonstrated the crucial role of ICAM-1 in cellular interactions which facilitate spread of this virus.

EXAMPLE 5

THE SPECIFIC IMPACT OF CD11A/CD18-ICAM-1 INTERACTIONS

IN THE SPREAD OF HIV FROM INFECTED MONOCYTOID CELLS

TO SUSCEPTIBLE T CELLS

In order to further evaluate the role of CDlla/CD18-ICAM-l interactions on the dissemination of HIV, the effects of specific antibodies on the rate of transfer of HIV-1 from infected cells to uninfected, healthy cells was studied.

For this purpose, a CD4+, continuous T cell line (MT4) and a monocytoid line (U937) which had been constitutively infected with HIV-1 were employed. U937 cells [ATCC CRL 1593] (Miller, L.J., et al.. J. Immunol. 137:2891 (1986)) were obtained from the American Type Culture Collection. Cells were cultured in RPMI 1640 with 10% mycoplasma free fetal calf serum and passaged serially at intervals sufficient to maintain >90% viability, as estimated by trypan blue dye exclusion. Cell cultures, media and virus stocks were monitored for mycoplasma infection by the Hoescht stain and by mycoplasma culture.

The prototype HTLV IIIB strain was used as a source of HIV-1; the virus was propagated from a clone of constitutively infected U937 cells, from cells inoculated with this prototype. Infectious particles were estimated by measuring the dilution of virus stock which caused multinucleated giant cell formation and cytopathic effect within 5-7 days following inoculation of MT4 cells, cultured at a density of 5 x 10 cells per 200 μ\ in 96-well microtiter plates (Pauwels, R., et al.. J. Virolooic Methods 16:171 (1987)). HIV-1 infected cells were also demonstrated by in situ hybridization (Rossen, R.D., et al.. Trans. Assoc. American Physicians 102:117-130 (1989)). Briefly, cells were cytocentrifuged onto glass slides, air dried, fixed in 5% paraformaldehyde and processed for hybridization with a ³⁵S-labeled RNA probe specific for the H1V-1 LTR (NEP 200, DuPont Co. Biotechnology Systems, Wilmington, DE 19898). At least 500 cells were counted to determine the percent of cells infected in each sample. A cell was considered positive for HIV-1 if it had >5 overlying silver grains, and strongly positive if it had >25 overlying silver grains. HIV p24 core antigen in culture supernatants was measured by a commercial ELISA kit (DuPont Biotechnology Systems Div., Wilmington, DE 19880).

To study the dose requirements and time-dependent effects of monoclonal antibodies specific for CD4, CDlla, CP18 and ICAM-1 on the infection caused by the transmission of cell-associated HIV-1 from U937 to MT4 cells, 1.5 x 10⁴ constitutively infected U937 cells were washed 4 times and co-cultured with 7.5 x 10⁵ uninfected MT4 cells in 1 ml complete medium. Four washes was sufficient to render p24 antigen undetectable in the last washing. The U937 cells used as a source of cell-associated virus in these experiments were treated with 25 μg of mitomycin-C per 10⁷ cells for 1 hr at 37^βC to suppress their replication. Monoclonal antibodies were added at doses of 10, 20 or 30 μg/10^ cells on day 0 and again on day 1. These quantities were selected on the basis of preliminary flow cytometric studies to assure that the culture medium contained approximately 2, 4 and 6 times the quantity of monoclonal antibodies required to saturate the surfaces of the cultured cells for the duration of the experiment. Figure 3 shows the quantities of p24 antigen detected over a nine-day period in culture supernatants when the 750,000 initially uninfected MT4 cells were cultured with 15,000 mitomycin-C treated HIV-1 infected U937 cells in 1 ml medium containing 6 times the dose required to saturate the monoclonal antibody cell surface receptors. The suppressive effects of these same monoclonal antibodies on p24 antigen release was not as evident when tested at 2 times or 4 times the saturating dose. The quantity of monoclonal antibodies used in the experiment shown in Figure 3 was 30 μg per 10*> cells, equivalent, approximately, to 1.2 x 10⁸ antibody molecules per cell. By day 7, a significant increase in p24 antigen was detected in all cultures except those which contained the RR1.1 anti-ICAM-1 monoclonal antibody and those which contained the anti-C04 monoclonal antibody, leu3a. Comparison of the mean p24 antigen concentrations achieved between days 1 through 9 demonstrated significant suppression in cultures treated with RR1.1, anti-ICAM-1, the R7.1 anti-CPlla and leu 3a (p<.04, paired t test and p < .03, Wilcoxan signed rank test).

To evaluate the effects of monoclonal antibodies on cytopathic effects produced in MT4 cells by HIV-1 infection, MT4 cells were cultured at a density of 50,000 cells per 200 /.l in 96-well microtiter plates. To these were added 10,000 constitutively infected U937 cells, previously treated with mitomycin C and the monoclonal antibody as described above. Cultures were monitored daily for the appearance of multinucleated giant cells and characteristic cytopathic effects (Pauwels, R., et al.. J. Virolooic Methods 16:171 (1987)), e.g., the cells failed to recluster when drawn through a micropipette and became permeable to trypan blue. The failure to recluster was taken as an endpoint and culture supernatants were harvested to assay p24 antigen.

Significant suppression of cytopathic effects was demonstrated with monoclonal antibodies specific for CPlla, CP18 and ICAM-1 (Table 3). Analysis of the geometric mean values for p24 antigen in these cocultures, by Student's t test, demonstrated that p24 release was significantly suppressed by addition of 0.5 or 1.0 μg anti-CBlla (p<.04), anti-C018 (p<.05), and the RR1.1 anti-ICAM-1 monoclonal antibody (p<.003) but not by the R6.5 anti-ICAM-1 (p>0.2) or the irrelevant anti-leukocyte antibody, 4A5 (p>.12) (Figure 4).

TABLE 3

EFFECT OF MONOCLONAL ANTIBODY TREATMENT ON CYTOPATHIC

EFFECT IN MT4 CELLS CO-CULTURED WITH

HIV-1-INFECTED U937 CELLS

A: Expt. 1: Monoclonal Antibody Treatment with Anti-

None WBC CDlla CD18 ICAM-1¹ ICAM-1² Pool

CPE* 8 6 0 4 2 1 2 No CPE* 0 2 8 4 6 7 6 p value >.05 <.005 .05 .01 .005 .01

B: Expt. 2: Monoclonal Antibody Treatment with Anti-

None ICAM-1¹ ICAM-1² ICAM-1³ ICAM-1⁴

CPE* 8 0 0 1 2 No CPE* 0 8 8 7 6 p values <.005 <.005 <.005 <.01

Footnotes:

^♦Number of cultures with this treatment showing CPE or lack of CPE.

CPE = failure of cultured cells to recluster when resuspended. No CPE = cells reclustered normally when resuspended. anti-WBC = 4A5 Mab anti-ICAM-1¹ = R6.5D6 anti-CD18 = R15.7 anti-ICAM-1² = RR1.1 anti-CDlla = R3.1 anti-ICAM-1³, = R6.1

Pool = R15.7 + R6.5D6 + R3.1 Mabs anti-ICAM-1⁴ = CL203.4 p values representing statistical significance of differences in CPE in monoclonal antibody treated cultures versus untreated controls were determined by Fisher extact test (Siegel, S., Non Parametric Statistics for the Behavioral Sciences. McGraw Hill Book Company, New York, p. 310 (1956)). Since RR1.1 and R6.5 react with non-overlapping epitopes in the first and second immunoglobulin-like domains of ICAM-1, respectively (Staunton, D.E. et al.. Cell 56:849-853 (1989)), the effects of adding 1.0 μg of other monoclonal antibodies specific for ICAM-1 to 10,000 infected U937 cells co-cultured with 50,000 uninfected MT4 cells (Figure 5) were studied under the same conditions used in Figure 4. The R6.1 monoclonal antibody, also reactive with the first amino- terminal domain of ICAM-1, was equivalent to RR1.1 in its ability to suppress CPE (p<.005, Fisher Exact test) and p24 antigen release (p<.002, Student's t test).

While the R6.5 and the CL203.4 monoclonal antibodies, which bind respectively to the second and the fifth extracellular domains of ICAM-1 suppressed CPE associated with HIV-1 infection of MT4 cells (Table 3), they did not significantly suppress release of p24 antigen (R6.5 vs. no treatment, p = .269; CL203.4 vs. no treatment, p = .355, Student's t test) (Figure 4).

Thus, to evaluate the specific impact of CDlla/CD18-ICAM-l interactions in the spread of HIV-1 from infected monocytoid cells to susceptible T cells, constitutively infected U937 monocytoid cells were repeatedly washed to remove free virus and cultured in the presence of uninfected MT4 T cells.

Cultures were monitored subsequently for p24 antigen and cytopathic effect (CPE). Treatment with monoclonal antibodies specific for CD18, CDlla or ICAM-1 significantly suppressed the emergence of CPE and p24 antigen in vitro.

The suppression of cytopathic effect (CPE) produced by anti- ICAM-1 monoclonal antibodies was similar to that achieved with anti- CD4 (p<.01, with respect to untreated samples). Monoclonal antibodies RR1.1 and R6.1, which react with epitopes in the first amino-terminal domain of ICAM-1 suppressed p24 production by 95% (p<.001); R6.5D6, a monoclonal antibody which reacts with the second immunoglobulin-like domain, and CL203.4, a monoclonal antibody which binds to the 5th domain, suppressed CPE but did not significantly suppress release of p24 antigen. These findings indicate that molecular interactions mediated by LFA-1 (CDlla/CD18), particularly with structural motifs expressed by the amino-terminal domain of ICAM-1, play an important role in cellular interactions which disseminate HIV among susceptible eel1s.

EXAMPLE 6

ABILITY OF FUNCTIONAL DERIVATIVES OF ICAM-1 TO

SUPPRESS HIV DISSEMINATION

In order to determine the ability of functional derivatives of ICAM-1 to suppress the dissemination of HIV, a soluble derivative of ICAM-1 was prepared and incubated with a mixture of MT4 T cells and HIV-infected U937 monocytoid cells.

Specifically, MT4 cells were exposed to mitomycin-C treated HIV- infected U937 cells at a ratio of 50,000 uninfected to 10,000 infected cells in 200 μl volumes in flat bottom microtiter wells with antibodies to the CD4 molecule (αCD4) as a positive control and soluble ICAM-1 at various doses.

The anti-CP4 as Leu3a was obtained from the Becton Pickinson Corporation. The preparation was dialyzed overnight resulting in 1:2 dilution of the original concentration, such that 40 μl/10⁶ cells would be required for saturation for FACS or 80 μl/10⁶ cells for 2x saturation; 4 μl/5xl04 cells is equivalent to 80 μl/106 cells.

Soluble ICAM-1 was created by deletion of the transmembrane domain of ICAM-1. The apparent molecular weight of the material was 84,000, thus, 2.5 μg is eqiuvalent to 0.3 x 10^"10M. The material had a protein concentration of approximately 3 mg/ml. A solution of 27 μl sICAM, or 81 μg, was made to 1600 μl with medium to give the following equivalents: 40 μl = 2 μg/50,000 cells or 40 μg/10⁶ cells; 20 μl = 20 μg/10⁶ cells; 10 μl = 10 μg/10⁶ cells; 5 μl = 5 μg/10⁶ cells; 2.5 μl = 2.5 μg/10⁶ cells. Approximately 10 ml of harvested MT4 cells were collected by centrifugation (5', 8^βC, 500 x g) and resuspended in 10 ml of RPMI 1640 medium (supplemented to contain 10% fetal calf serum) to a final cell concentration of 0.5 x 10⁶ v/ml (91%/v) (16 ml).

HIV-infected U937 monocytoid cells were prepared by harvesting approximately 9 ml of cells by centrifugation (5', 8'C 500 x g). The cells were re-suspended in 2 ml RPMI 1640 medium supplemented with 10% fetal calf serum and 50 μg/ml of Mitomycin C (Sigma Chemical Corp.). The cells were incubated at 37'C in a 5% CO2 atmosphere for 1 hour with mixing at 30 minutes. The cells were then diluted with 8 ml of medium, centrifuged (5', 8^βC 500 xg), and then washed 4 times with 10 ml medium. The cells were then suspended in 8 ml RPMI 1640 (supplemented with 10% fetal calf serum) to a final concentration of 1.7 x 10⁶ v/ml (99%v). Thus, 8 ml at 0.1 x 10⁶ v/ml = 0.8 x 10⁶ cells = 0.47 ml; 100 μl/well = 10,000 cells.

MT4 cells were introduced into wells, and then anti-CD4 monoclonal antibodies or soluble ICAM-1 were added according to the protocol shown in the legend of Table 4 (8 wells were used per experiment). Cells were then incubated (37^βC, 5% CO2 atmosphere) in the presence of either fresh medium or HIV-infected U937 cells. Additional monoclonal antibody was added on Day 3, to those samples receiving anti-CD4. Beginning on day 5, each day, through day 9, the cells were pipetted x 3 with 200 μl pipette to suspend the cells. The cells were permitted to "recluster" for 3 hours (under culture conditions) at which time the wells were scored microscopically for "reclustering" using the Nikon inverted microscope with 20x objective.

On day 5, the cells appeared very healthy. Cells appeared more crowded on day 6, and showed some CPE of HIV-1 infection by day 7. CPE were more apparent on days 7 and 8. On day 9, CPE were clearly evident in HIV-1 infected cultures; plates were centrifuged 5' 1000 rpm and supernatant were harvested for p24 core antigen quantification, as described above. The results of this experiment are shown in Table 4. TABLE 4 ABILITY OF SOLUBLE ICAM-1 TO SUPPRESS HIV DISSEMINATION

A. Legend

1. MT4 + U937/HIV_m

2. MT4 + U927/HIV_m + 4 μl αCD4 (αteu 3a)

3. MT4 + U937/HIV_m + 40 μl sol ICAM-1 (= 40 μG/10⁶ cells)

4. MT4 + U937/HIV_m + 20 μl sol ICAM-1 (= 20 μG/10⁶ cells)

5. MT4 + U937/HIV_m + 10 μl sol ICAM-1 (= 10 μG/10⁶ cells)

6. MT4 + U937/HIV_m + 5 μl sol ICAM-1 (= 5 μG/10⁶ cells)

7. MT4 + U937/HIV_m + 2.5 μl sol ICAM-1 (= 2.5 μG/10⁶ cells)

8. MT4 + U927/HIV_m

9. MT4 + Medium

10. MT4 + medium + 4 μl αCD4

11. MT4 + medium + 40 μl sol ICAM-1

12. MT4 + medium + 20 μl sol ICAM-1

13. MT4 + medium + 10 μl sol ICAM-1

14. MT4 + medium + 5 μl sol ICAM-1

15. MT4 + medium + 2.5 μl sol ICAM-1

16. MT4 + medium + nothing

B. CPE per Well of Cells Treated with Soluble ICAM-1

Sample # Day 5 Day 6 Day 7 Day 8 Day 9

1. 0/8 0/8 0/8 1/8 8/8 2. 0/8 0/8 0/8 0/8 0/8 3. 0/8 0/8 0/8 0/8 0/8 4. 0/8 0/8 0/8 0/8 0/8 5. 0/8 0/8 0/8 0/8 0/8 6. 0/8 0/8 0/8 0/8 0/8 7. 0/8 0/8 0/8 0/8 0/8 8. 0/8 0/8 0/8 0/8 4/8 9. - 16. 0/8 FOR ALL DAYS

B. Nanograms of p24 Antigen per ml of Culture Fluid

Sample # A B C D E F G H

1 22 38 31 141 10 6 8 8

2. 2 4 3 3 8 5 4 5

3. >400 61 10 64 29 16 9 93

4. 15 54 82 >400 62 10 6 6

5. 9 11 7 106 5 3 4 71

6. 6 379 81 >400 21 18 39 16

7. 6 39 13 7 >400 77 67 358

8. 8 149 >400 49 25 9 54 16

9. - 16. <.001 FOR ALL WELLS Although soluble ICAM-1 was, thus, effective in suppressing the dissemination of HIV, it did not result in a decrease in p24 production. The results of this experiment thus indicate that soluble ICAM-1 was as effective as anti-CD4 antibody in suppressing the cytotoxic effects of HIV infection, and indicate that ICAM-1, CDll, CD18, or the CD11/CD18 heterodimer (or molecules such as antibody which bind to these molecules) may be used in combination with cell or particle associated CD4, soluble CD4, or antibody to CD4 to treat AIDS, or to suppress the dissemination of HIV.

EXAMPLE 7

THE INTERACTION OF ICAM-1 AND CD11/CD18 WITH HIV DISSEMINATION

Each of the four antibodies to ICAM-I, used to study the influence of this molecule in HIV infection, were capable of suppressing the cytopathic effects of the virus in co-cultures involving HIV-1 infected U937 cells and susceptible MT4 cells. Only monoclonal antibodies reactive with the first amino-terminal domain of ICAM-1 significantly suppressed release of p24 antigen. This observation indicates that the sites of interaction of the ICAM-1 specific antibodies with ICAM-1 are particularly important. RR1.1 and R6.5 are equally effective at inhibiting homotypic aggregation of activated lymphocytes. On the other hand, CL203.4, which reacts with the domain of ICAM-1 most closely associated with the cell membrane, has no effect on physiological adherence reactions involving activated leukocytes, even though it can precipitate ICAM-1 from detergent lysates of cells expressing this molecule (Staunton, D.E. et al .. Cell 56:849-853 (1989))). The different effects of these monoclonal antibodies on replication of HIV-1 in vitro may be determined solely by their effects on adherence reactions involving ICAM-1 and CDlla/CD18. The ability of anti-ICAM-1 and anti-CDll/CD18 antibodies to suppress the dissemination of HIV may indicate that interactions among ICAM-1 and CDlla/CD18 (LFA-1) promotes and prolongs the apposition of the membranes of contiguous cells so as to facilitate transfer of budding virus from infected to susceptible cells (Hildreth, J.E.K., et al.. Science 244:1075 (1989)).

This mechanism is supported by studies which demonstrated that HIV-1 infection increases monocytoid cell expression of LFA-1 and, concomitantly, the tendency of HIV-1 infected cells to aggregate homotypically or adhere to endothelial monolayers expressing high levels of ICAM-1 (Rossen, R.D., et al .. Trans. Assoc. American Physicians 102:117-130 (1989)). CDlla/CD18 molecules participate in cellular adherence reactions involving class II major histocompatibility antigens and CD4 that present antigen to CD4+ T lymphocytes (Springer, T.A., et al .. Ann. Rev. Immunology 5:223 (1987)). The increased expression of these integrins or of ICAM-1 by HIV-1 infected cells is likely to prolong surface contact between cells engaged in physiological interactions such as antigen presentation. This, in turn, increases the opportunity to transfer infectious virus.

The possibility that CDlla/CD18 or ICAM-1 themselves provide attachment sites for HIV provides a second possible mechanism through which anti-ICAM-1 and anti-CDll/CD18 antibodies may suppress infection by HIV.

Evidence against this second alternative has recently been provided by Valentin et al. (Valentin, A., et al .. J. Immunology 144:934-937 (1990), which reference is incorporated herein by reference) who showed that pre-treatment of cells susceptible to HIV-1 infection with antibodies to CD18 did not interfere with cell surface binding of radiolabeled GP120. However, their data did not exclude the possibility that ICAM-1, ICAM-2 or other molecules which interact with heterodimers containing CD18 may provide alternative sites for attachment of HIV-1. ICAM-1 is widely distributed along with CD18 dependent heterodimers on cells of the lymphoreticular system (Springer, T.A., et al .. Ann. Rev. Immunology 5:223 (1987); Kishimoto, T.K., et al .. Adv. Immunol. 5_:223 (1987); Staunton, D.E., et al .. Nature 339:61 (1989)); increased ICAM-1 expression is observed on the surfaces of some HIV-1 infected H9 T cells (Rossen, R.D., et al.. Trans. Assoc. American Physicians 102:117-130 (1989)).

While not diminishing the role of CD4 in mediating cell surface attachment and intercellular transport of HIV-1, recent studies have demonstrated that other cell surface structures, most notably immunoglobulin Fc (Takeda, A., et al.. Science 242:580 (1988); Homsy, J., et al.. Science 244:1357 (1989); Robinson, W.E., Jr., et al.. Proc. Natl. Acad. Sci. USA 86:4710 (1989); Montefiori, D.C., et al.. J. Virol. 64:113 (1990)) and complement receptors (Montefiori, D.C., et al ., Antiviral Research 11:137 (1989)) can facilitate attachment and intercellular transport of this virus. In the presence of transactivating factors provided by cytomegalovirus or other agents, replication of HIV-1 can proceed even in cells which do not express CD4 (McKeating, J.A., et al.. Nature 343:657 (1990)). Thus, ICAM-1, ICAM-2 or possibly other molecules that bind LFA-1 can potentially provide a cell-surface attachment site for this virus.

Alternatively the ability of anti-ICAM-1 and anti-CDll/CD18 antibodies to suppress the HIV infection may indicate that short segments of cell membrane, released from disintegrating syncytia, which contain CD4, the CDlIa/CD18 heterodimers and/or ICAM-1, as integrally embedded membrane proteins, are physically associated in some way with the virus envelope and provide "molecular handles" to attach the virus to uninfected cells.

This mechanism is supported by studies which show that the surface of T cells infected with HIV-1 that fuse and form multi- nucleated giant cells are unstable (Sodroski, J., et al .. Nature 322:470 (1986); Lifson, J.D., et al.. Nature 232:725 (1986)); a few hours after forming these syncytia, the cells lyse, releasing their intracellular contents. Under these circumstances it is likely that fragments of cell membrane containing CD4 and other transmembrane proteins, including CDll, CD18 and ICAM-1, may remain associated with the envelope of some HIV-1 particles as they leave the cell (Clayton, L.K., et al.. Nature 339:548 (1989); Schols, D., et al.. J. Gen. Virol. 70:2397 (1989); Habeshaw, J.A., et al .. J. Acαuir. Immune Defic. Svndr. 2:457 (1989)). Alternatively, fragments of cell membrane from syncytia of HIV-1 infected cells, incorporating these molecules, may attach to cell-free virus by virtue of the affinity of the viral GP120 for CD4. In either case, the virus particle may carry host cell surface molecules which facilitate attachment to ICAM-1 or to CDlla/CD18, expressed by uninfected cells. The effect in either case would enhance the likelihood of virus infection.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for suppressing the infection of leukocytes with HIV, which comprises administering to a patient exposed to or effected by HIV, an effective amount of an HIV-1 infection suppression agent, said agent being capable of binding to ICAM-1, ICAM-2, CDll, CD18 or to a CD11/CD18 heterodimer.

2. The method of claim 1 wherein said HIV is HIV-I.

3. The method of any one of claims 1 or 2, wherein said agent is an immunoglobulin, or an antigen binding fragment of an immunoglobulin.

4. The method of claim 3, wherein said immunoglobulin is a monoclonal antibody.

5. The method of claim 3, wherein said immunoglobulin is an antibody capable of binding to a CDll molecule.

6. The method of claim 5, wherein said antibody is capable of binding to a CDll molecule selected from the group consisting of CDlla, CDllb, and CDllc.

7. The method of claim 3, wherein said immunoglobulin is an antibody capable of binding to a CDI8 molecule.

8. The method of claim 3, wherein said immunoglobulin is an antibody capable of binding to ICAM-1 or ICAM-2.

9. The method of claim 8, wherein said antibody is capable of binding to ICAM-1, and wherein said antibody is the antibody R6.5.

10. The method of any one of claims 1 or 2, wherein said agent is a soluble derivative of ICAM-1 or ICAM-2.

11. The method of any one of claims 1 or 2, wherein said agent is a soluble derivative of CDll.

12. The method of claim 11, wherein said soluble derivative of CDll is selected from the group consisting of: a soluble derivative of CDlla, a soluble derivative of CDllb, and a soluble derivative of CDllc.

13. The method of any one of claims 1 or 2, wherein said agent is a soluble derivative of CD18.

14. The method of any one of claims 1 or 2, wherein said agent is a soluble derivative of CD11/CD18.

15. The method of claim 14, wherein said soluble derivative of CD11/CD18 is selected from the group consisting of: a soluble derivative of CDlla/CD18, a soluble derivative of CDllb/CD18 and a soluble derivative of CDllc/CD18.