WO1989002918A1

WO1989002918A1 - Process for selectively modulating the expression and function of a cell-surface determinant and production of novel human cells produced thereby

Info

Publication number: WO1989002918A1
Application number: PCT/US1988/003219
Authority: WO
Inventors: Arthur Allen Vandenbark; Halina Offner
Original assignee: Arthur Allen Vandenbark; Halina Offner
Priority date: 1987-09-22
Filing date: 1988-09-16
Publication date: 1989-04-06
Also published as: JPH02501534A; EP0331716A4; AU2523888A; EP0331716A1

Abstract

This invention is directed to a process for inhibiting infection in humans by selectively modulating the expression of a specific cell-surface determinant, namely, the CD4 molecule on human cells, with a chemical agent. The chemical agent is a ganglioside composition which induces the rapid and selective disappearance of CD4 cell-surface determinants from human cells. By selectively modulating the expression and function of the CD4 cell-surface determinant, employing a ganglioside composition which induces the rapid and selective disappearance of the CD4 cell-surface determinants from human cells, the point of attachment and entry into the T-helper cell for viruses which infect the human cells through the CD4 cell-surface determinants, such as HIV, is eliminated, and the receptivity of these human cells is inhibited, without inhibiting other cell-surface determinants.

Description

PROCESS FOR SELECTIVELY MODULATING THE EXPRESSION

AND FUNCTION OF A CELL-SURFACE DETERMINANT AND PRODUCTION OF NOVEL HUMAN CELLS PRODUCED THEREBY

BACKGROUND OF THE INVENTION This invention generally relates to a process in which the expression and function of a human cell- surface determinant on human cells is selectively modulated and to the novel selectively modulated human cells produced by that process. By selectively modulating the expression and function of specific human cell-surface determinants, certain human cells can in turn be protected from debilitants and thereby can be maintained intact and available to function.

The human immune system, when functional, acts to protect the body against infectious agents, including viruses, bacteria, parasites, and the like. An individual's ability to combat these agents, or other substances (antigens) recognized as foreign by^'the body's immune system, is critical to health and survival. Under normal circumstances, the immune system is able to contain most infections, thus allowing complete recovery. When the immune system is inadequate, however, severe infections may result.

Two classes of white blood cells, macrophages and lymphocytes, are primarily responsible for immunity. Macrophages digest and destroy infectious agents, and then present structural information about these agents to the lymphocytes. Lymphocytes are divided into two general categories. One class of lymphocytes, B cells, produce antibodies in response to infectious agents. Antibodies have unique combining sites that recognize the distinguishing structural features of individual antigens. When antibody molecules bind to antigen, the interaction sets in motion a chain of events which may neutralize the effects of the foreign substance. The other group of lymphocytes, T cells, produce unique membrane receptors- which recognize the individual antigen structures presented by the macrophage. When T cells interact with antigen, a variety of hormones (lymphokines) are released which may induce inflammation, amplify or suppress antibody formation by B cells, or destroy directly foreign cells such as tumors or transplanted organs.

The interplay amongst T cells, B cells, and macrophages determines the strength and depth of a person's response to infection. It is generally believed that the activities of these immune cells are controlled, to a large extent, by lymphokines and monokines (macrophage hormones). There are many different lymphokines and monokines, each of which has distinctive chemical and functional properties.

In an article entitled, "The CD4 Molecule: „ Function and Structure," by Dalgleish, A.G., in the journal entitled Immunology Today, Vol. 7, p. 142 (1986), it was generally stated that a specific cell- surface determinant, i.e., the CD4 molecule, defines a subpopulation of mature T lymphocytes which, after antigen recognition restricted by Class II major histocompatibility (MHC) antigens, carries out a variety of regulatory and effector functions. The perturbation of the CD4 cell-surface determinant also apparently transmits a negative signal to the T cells and may act to switch off T cell function. See Bank, I., et al., "Perturbation of the T-4 Molecule Transmits a Negative Signal to T-cells," Journal of Experimental Medicine, Vol. 162, p. 1294 (1985).

Gangliosides are a diverse group of glycosphingolipids characterized by the presence of one or more sialic acid units on the oligosaccharide chain. Gangliosides have been found to inhibit T cell proliferation and function. This has been suggested in the following_.articles: Lengle, E.E., et al., "Inhibition of Lectin-Induced Mitogenic Response of Thymocytes By Glycolipids," in Cancer Research, Vol. 39, p. 817 (1979), and Whisler, R.L., et al., "Regulation of Lymphocyte Responses by Human Gangliosides: Characteristics of Inhibitory Effects and the Induction of Impaired Activation," in the Journal of Immunology, Vol. 125, p. 2106 (1980). It has also been reported that gangliosides inhibit the encephalitogeniσ activity and effect the expression of the T-helper cell phenotype. Gangliosides were found to inhibit the fluorescent staining of the W3/25 monoclonal antibody on BP-1 rat T-helper cell line in vitro. This inhibitory effect was not exhibited by cells stained with W3/13 (T-total), OX-8 (T-non-helper) or OX-6(Ia) antibodies. 'See Offner, H. and A.A. Vandenbark, "Gangliosides Inhibit Phenotypic and

Functional Properties of an Encephalitogenic T-helper Lymphocyte Line," Cellular Immunology, Vol. 95, p. 84 (1985).

Soluble columns of polysaccharide derivatives comprising covalently bonded gangliosides have been employed in U.S. 4,125,492 to quantitatively adsorb cholera toxin of chromatographed samples. In U.S.

4,464,360 certain glycosphingolipids have been used in pharmaceutical preparations to inhibit the adherence of bacteria to human urinary tract epithelial cells. Finally, U.S. 4,476,119; U.S. 4,593,091 and U.S. 4,639,437 relate to methods for preparing ganglioside inner ester derivatives, the use thereof in pharmaceutical compositions, as well as providing inhalation kits containing such ganglioside derivatives, for promoting nerve regeneration by stimulating nerve sprouting. An extremely important property of a biologically active material in humans is its ability to discriminate, i.e., to modulate selectively the expression and function of a given cell-surface determinant. This property thus allows all of the unmodulated human cells associated with immune protection to proliferate and function in their normal requisite manner. Antibodies are discriminatory agents which selectively bind to specific cell-surface determinants on particular human cells. However, the functional effects on a given cell of this discriminatory, selective binding are unpredictable and must be determined through experimentation on an ad hoc basis. Chemical compositions such as drugs and the like, on the other han ~_*d, are non-discrim_^inatory and may affect many types of hutan cellis. For example, a chemical composition such as methyltrexate* a chemotherapeutic agent for treatment of cancer, functions by inhibiting the proliferation of all actively dividing human cells. This chemical is non- selective and, therefore, does not discriminate between the destruction of malignant and non-malignant cells. Thus, many necessary human functions involving rapidly dividing cells (e.g., the immune system) are disabled as a result of treatment with this chemotherapeutic agent.

Accordingly, there is a need for chemical compositions which are discriminatory and selective in their ability to modulate specific cell-surface determinants of particular human cells to prevent infection of such human cells without destroying their functional abilities. SUMMARY OF THE INVENTION This invention is directed to a process for inhibiting infection in humans by selectively modulating the expression of a specific cell-surface determinant, namely, the CD4 molecule on human cells, with a chemical agent. The CD4 (T4) antigen of the helper T-lymphocyte is thought to be an essential component of the receptor for the AIDS retrovirus. See Dalgleish, A.G., et al., "The CD4 (T4) Antigen of the Receptor For the AIDS Retrovirus," Nature, Vol. 312, p. 763 (1984).

The chemical agent is a ganglioside composition which induces the rapid and selective disappearance of CD4 cell-surface determinants from human cells. Human cells after treatment with this ganglioside composition, no longer express the cell-surface CD4 molecules, and are thus selectively inhibited from receptivity (infection) by viruses, including the human immunodeficiency virus ("HIV") which causes acquired immunodeficiency syndrome, commonly know as AIDS, or other viruses such as retroviruses, which infect target cells through the CD4 cell-surface molecules.

Immune T lymphocytes and macrophages play an essential rule in protecting against infectious diseases. Certain viruses, such as HIV, selectively attack both of these cell types which possess the CD4 molecule on the cell surface. When these T lymphocytes and macrophages are attacked and destroyed after infection with HIV, global immunodeficiency results, leading to severe complications and death due to secondary infections. Thus, prevention of HIV infections by gangliosides and the maintenance of effective T lymphocyte responses have widespread prophylactic and therapeutic potential in AIDS. Attempts have been made by researchers to overcome the AIDS health hazard by inhibiting HIV prior to its interaction with the CD4 cell-surface determinant. Moreover, other scientists have tried to solve the problem by blocking the CD4 reception mechanism so that HIV will not combine therewith. To date, these undertakings have been unproductive and the epidemic continues unchecked.

It has now been discovered that by selectively modulating the expression and function of the CD4 cell- surface determinant, employing a ganglioside composition which induces the rapid and selective disappearance of the CD4 cell-surface determinants from human cells, the point of attachment and entry into the T-helper cell for viruses which infect the human cells through the CD4 cell-surface determinants, such as HIV, is eliminated, an.d the receptivity of these human cells is-inhibited, without inhibiting other cell-surface determinants. Human cells which include selectively modulated CD4 cell-surface determinants comprise human cells modulated with a ganglioside composition. They are readily identifiable by the absence of the CD4 cell- surface determinant from the cell surface. These selectively modulated human cells are effective in inhibiting infection in humans, particularly infection by HIV.

For purposes of this invention, the term "ganglioside" shall be defined as any compound including a lipid (ceramide), or a chemically-modified lipid, and a sialated oligosaccharide, or a chemically- modified sialated ogliosaccharide, respectively. The ganglioside compositions employed in producing the CD4- modulated human cells are preferably selected from a group consisting of mono-sialated gangliosides, di- sialated gangliosides, tri-sialated gangliosides, tetra-sialated gangliosides, modified gangliosides, and mixtures thereof.

Selective modulation of the human cells is preferably conducted in an environment of substantially low blood serum concentration in order to avoid the neutralizing effects of serum on the ganglioside composition. Thus, an effective concentration of the ganglioside composition is added to overcome such serum neutralization.

The selectivity of modulation of the CD4 cell- surface determinants is carried out without effecting the expression and function of other cell-surface determinants. In this way, one can inhibit the receptivity the CD4 cell-surface determinants to viruses without endangering the desired effective functioning of any other determinants.

The selective modulation of this invention can be maintained by providing effective amounts of said ganglioside composition at a level sufficient to prevent re-expression of said CD4 cell-surface determinants on the human cells. Thus, a user can continue the ganglioside addition step as long as they deem it necessary. However, if the user so desires, the addition of said ganglioside composition can be terminated thereby allowing the re-expression of said CD4 cell-surface determinants on said human cells. This allows the user to re-express the receptive attributes of the CD4 molecule when needed and to modulate it when needed to prevent such receptivity by viruses (e.g., HIV). This causes the recovery of cell function which is dependent upon CD4 expression. If the addition of the ganglioside is terminated, re- expression of said CD4 cell-surface determinants on 8 said human cells will typically be substantially completed with.in 24 hours after terminating said ganglioside composition addition.

In a preferred form of this invention, the ganglioside composition is topically applied to the skin of a human, particularly the vaginal and rectal areas, to prevent infection of CD4 positive epithelial cells by a virus. Typically such topical application is provided prophylatically to the human cells thereby preventing viral infection.

The foregoing and other objects, features and advantages of the invention will become more readily apparent from the following detailed description of a preferred embodiment which proceeds with reference to the examples.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT This invention relates to humantcells which include selectively modulated CD4 cell-surface determinants, and to a process for inhibiting infection in humans by the human immunodeficiency virus (HIV) . The subject human cells are selectively modulated with a ganglioside composition capable of inducing the rapid and selective disappearance of CD4 cell-surface determinants from human cells. In this way, the receptivity of the modulated human cells to the human immunodeficiency virus is selectively inhibited.

EXAMPLE 1 The selective inhibition and disappearance of the expression and function of the CD4 cell-surface determinants on human cells employing a chemical agent comprising a ganglioside composition has been experimentally demonstrated.

Human T lymphocyte cells were incubated with varying concentrations of mixed gangliosides. The mixed ganglioside composition comprised 21% GM. (mono- sialated ganglioside), 40% GD, (di-sialated ganglioside),- 16% CD,, (tri-sialated ganglioside), and 19% GT,. (tetra-sialated ganglioside), having an average molecular weight of 1756 Daltons. The anti CD4 antibody used for this experiment was Leu 3a. The CD3 antibody (Leu 4) was also tested. By evaluating the mean fluorescence intensity (MFI) of the stained cell population at each concentration of the ganglioside composition used, it was possible to quantitate the amount of gangliosides required to produce 50% inhibition of the fluorescence (I₅₀ value). The I_5Q value for human T-lymphocytes was 5 uM. The concentration of the ganglioside composition to reduce the MFI to 0%, i.e., to cause complete selective modulation and disappearance of the CD4 cell-surface determinants, was also experimentally resolved. More specifically, human blood cells were incubated, with saline or varying concentrations of mixed gangliosides in the absence of blood serum at 37°C for one hour. The cells were washed twice, stained with optimal concentrations (determined previously) of the various monoclonal antibodies and fluorescein-labeled goat anti-mouse second antibody, washed again, fixed in 1% formalin, and analyzed for MFI after excitation with a 488nm laser light using an Ortho Systems 50 Cytofluorograf. The mononuclear cells were gated on the basis of forward angle and right angle scatter, and the mean fluorescence intensity (MFI) of the stained cell population was determined from a cytogram of 10,000 human cells. The MFI of the ganglioside treated cells were compared to the MFI of untreated cells and the percent inhibition of CD4 expression was established after subtracting MFI of cells stained only with second antibody. Ganglioside inhibition of cell-surface antibody staining was calculated by the following formula: 10

MFI channel (ganglioside-treated cells)- MFI^" channel (IgG-background) xlOO MFI channel (saline treated)- MFI channel (IgG-background). The value above was calculated for the mixed

^•gangliosides at concentrations of 2ug, 5ug, lOug, 20ug and 50ug, respectively. ^■ As set forth in TABLE 1, the values of concentration were then plotted and the I_gQ values were interpolated from the line of best fit. I₅₀ is defined as the ganglioside concentration which reduced MFI the channel by 50%. Based on this titration curve, the I_gQ=7.3 ug/ml (4.9 uM) .

TABLE 1 Ganglioside Concentration Mean Fluorescene Intensity (ug/ml) (% Positive Control)

2 ' Leu 3a (CD4) 100

Leu 4 (CD3) 100

5 Leu 3a (CD4) 80 Leu 4 (CD3) 100

10 Leu 3a (CD4) 28

Leu 4 (CD3) 100

20 Leu 3a (CD4) 5

Leu 4 (CD3) 100 50 Leu 3a (CD4) 0

Leu 4 (CD3) 100

It is, therefore, quite clear from examining TABLE 1 that by adding ganglioside to a concentration of 50 ug that the MFI indicated that the CD4 cell-surface determinants on the hugan T-helper cells tested have disappeared and, therefore, have been modulated. The site for the receptivity of the HIV (AIDS virus) has therefore been inhibited.

In order for the expression of the CD4 cell- surface determinants on the hugan cells to be selectively modulated, the CD4 is induced to disappear 11 as previously described, without effecting the expression and function of the CD3 cell-surface determinants on the same human cells. This is demonstrated in TABLE 1 where cells treated with gangliosides do not change their fluorescence intensity when stained with an antibody compared to another T cell member (defined by the Leu 4 monoclonal antibody).

EXAMPLE 2 The high selectivity of the modulation of the expression of CD4 cell-surface determinants by treatment with a ganglioside composition has been experimentally demonstrated.

As shown in TABLE 2 below, a ganglioside composition induced selective loss of the expression of CD4 cell-surface determinants on human T cells, but did not effect the phenotype of any other human cell- surface determinants as recognized By the sp cific monoclonal antibodies listed in that TABLE. Ganglioside pretreatment effected almost equally the staining detected by nine different monoclonal antibodies specific to the CD4 molecule on human T cells.

Experimentally, human blood cells were incubated in saline or in varying concentrations of mixed gangliosides in a similar manner to the procedure described in EXAMPLE 1. Antibody specificities in which the MFI were reduced by 95% after ganglioside pretreatment relative to saline pretreatment controls were considered as effective ganglioside pretreatment. AS before, I_ςf. values, which are in parentheses in TABLE 2, represent the concentration of gangliosides which reduced the MFI by 50%. Antibody specificities which were uneffected by ganglioside pretreatment had MFI reduction of less than 10% when pre-incubated with 300uM of the gangliosides. TABLE 2

Antibodies Effected Antibodies Not Effected By By Gangliosides (I Gangliosides (all I_c._f)>300 uM)

5J

For T-helper cells: For Total T Cells:

Leu 3a (4.9) Leu 4, OKT3, 533, T28

Leu 3b (6.0) For E Rosette Receptor:

OKT4 (4.8) Leu 5

OKT4a (4.9) For T-Suppressor Cells:

OKT4b (5.3) Leu 2, OKT8

OKT4c (4.2) For K/NK Cells:

OKT4d (4.9) Leu 7, Leu 11

OKT4e (4.9) For B-Cells:

OKT4f (4.6) OKB7, K+L, Ig For HLA-DR, HLA-DQ: For Macrophages:

OKM1

Based on the data in TABLE 2, it is clear that the CD4 cell-surf ce determinants have been removed from the human T-helper cells, since all of the monoclonal antibodies which specifically detect, the CD4 determinant on human cells have been substantially inhibited. On the other hand, cell-surface determinants different from CD4 were not effected by treatment with ganglioside. Therefore, gangliosides selectively modulate the expression of the CD4 molecule while having no effect on the expression of other cell- surface molecules. Moreover, ganglioside treatment of T-helper cells induced the selective loss of the CD4 cell-surface determinants without effecting the expression of other cell-surface molecules (see Table 1) . In a similar manner, a variety of other determinants on T cells and other cell types identified by specific antibodies to cell-surface determinants other than CD4, were not effected by ganglioside treatment (see Table 2). Thus gangliosides induced the selective modulation of the CD4 molecule on human cells.

Selective modulation of the expression and function of the CD4 cell-surface determinants on human 13 cells is conducted in the presence of sufficient ganglioside composition to overcome the neutralizing effects of the serum present. One approach is to add an amount of ganglioside which will first neutralize the serum and then will also selectively modulate CD4 cell-surface determinants. Another approach is for the selective modulation of the human cells to be conducted in an area of substantially low blood serum concentration. For example, when this blood serum level was maintained at not more than about 5% by volume, selective modulation was effected. Clearly, when the selective modulation process was conducted in the presence of substantially no blood serum there was a 100% inhibition of the CD4 expression when a lower concentration of gangliosides were used to treat the human cells.

Although the process for^" selectively modulating the expression and function of CD4 cell-surface determinants extends generally to all human cells which express CD4, the preferred human cells for selective^' modulation comprise T-helper cells, epithelial cells, and macrophages and brain glial cells, respectively.

EXAMPLE 3 The use of ganglioside compositions, but not the individual components of such ganglioside compositions, to selectively modulate CD4 cell-surface determinants has been experimentally demonstrated.

To evaluate which components of the ganglioside structure were required in order to induce the loss of CD4 expression, T-helper cells were incubated with different concentrations of mixed gangliosides, purified individual gangliosides, modified gangliosides, and ganglioside components, namely, the individual lipid, oligosaccharide, and sialic acid moieties. The activity of gangliosides and ganglioside components on-the expression of CD4 cell-surface determinants was determined. The results are summarized in TABLE 3 below. Lymphocyte populations (10 /ml) were pretreated for one hour at 37°C with varying dilutions of ganglioside compositions, or ganglioside components, prior to staining with anti-CD4 or control antibodies and a fluorascein-labeled second antibody. The staining procedure employed was similar to that used in EXAMPLE 1.

TABLE 3 Inhibitor l,-_n Value (uM)

Mixed Gangliosides* 5 GM_. (mono-sialated) 6

GD^~T (di-sialated) 7

GT:\ (tri-sialated) 4

GQ^r (tetra-sialated) 6

Galactocerebroside No Inhibition

Ceramide • No Inhibition

Asialogangliosides ^ . No Inhibition

Sialic acid No Inhibition oligosaccharides

(Ganglioside Derived) No Inhibition Modified Gangliosides

(Esterification between sialic acids and 2nd sugar) 100

^♦contained 21% G ₁, 40% CD. , 16% GD_χb, and 19% GT_lt).

in TABLE 3 above, mixed gangliosides and individual ganglioside compositions with different sialic acid moieties had similar I-_Q values. The ganglioside modulation of the CD4 expression of human T cells required respective ceramide, oligosaccharide and sialic acid moieties. Modification of gangliosides by internal esterification of the carbohydrate moiety reduced the ganglioside effect on human T cells. Isolated individual ceramide, oligosaccharide, and/or sialic acid compounds had no inhibitory activities. The sialic acids which distinguish gangliosides from other glycosphingolipids, were also necessary 15 components for CD4 modulation, since asialogangliosides and galactocerebrosides, which contained no sialic acids, had no inhibitory activity.

The process of this invention also includes the step of maintaining the selected modulation described above by providing continued use in the form of additional amounts of the ganglioside composition provided at a level sufficient to prevent re-expression of: the CD4 cell-surface determinants, i.e., reappearance of the CD4 cell-surface determinants on the human cells.

EXAMPLE 4

(a) The use of continued treatment of human T cells with a ganglioside composition to prevent the re- expression of jthe CD4 cell determinants on human cells was demonstrated.

In an experiment designed to examine the prevention of such re-expression, T-helper cells were washed at the end of four hours, twenty-four hours, and forty-eight hours and, after aliquots were removed for staining, the remaining cells were re-suspended in 67uM of gangliosides. The result was 100% inhibition of CD4 expression upon continued use and presence of gangliosides.

(b) It was also experimentally demonstrated that ganglioside pretreatment was not toxic to the cells with the CD4 cell determinants, since, after modulation by the gangliosides, the CD4 cell-surface determinants reappeared on the cell surface within twenty-four hours after ganglioside removal.

The continued addition of the ganglioside composition was terminated which caused re-expression of the CD4 cell determinants on the human cells. In a series of experiments, human cells were treated according to the procedure set forth in EXAMPLE 1 and the percent inhibition measured at various time periods. The.results of that"testing as shown in TABLE 4 below were that after twenty-four hours from ganglioside removal the percent inhibition dropped from 100% to 9%, respectively.

One way in which a patient who is infected with the HIV virus could be medically treated is by in vitro modulation of the CD4 cell-surface determinants on the patient's cells according the teachings of the present invention, followed by reinfusing the modulated cells into the patient. For example, in a special report in The New England Journal Of Medicine, Vol. 313, No. 23, pages 1485-1492, by Dr. Steven A. Rosenberg, et al, entitled "Observations On The Systemic Administration Of Autologous Lymphokine-Activated Killer Cells And Recombinant Interleukin-2 To Patients With Metastatic Cancer," a method including leukapheresis, lymphocyte harvest and culture, interleukin-2 treatment, and admininstration of LAK cells and recombinant interleukin-2, is described. This method, which is particularly described on pages 1486-7, could be applicable in conducting the in vitro modulation set forth above.

For example, a process for treating a patient infected with human immunodeficiency virus would comprise the initial step of isolating human blood cells in vitro from the patient as in leukaperesis and lymphocyte harvest and culture, respectively. The isolated human blood cells would then be treated with a 17 chemical agent comprising a ganglioside composition capable of inducing the selective disappearance of said CD4 cell-surface determinants from the human cells by selectively modulating the expression and function of said CD4 cell-surface determinants, and thereby selectively inhibiting the receptivity of the human blood cells by the virus, using the leukapheresis procedure described by Rosenberg, et al. Finally, the CD4-modulated human blood cells would be reinfused into the patient.

More particularly, a typical leukapheresis procedure can be conducted as follows:

Large numbers of lymphocytes and macrophages

(between 5 x 10 9 and 5 x 1010 per procedure) are collected by repeated lymphocytaphereses with a continuous-flow cell separator. Acid-citrate-dextrose is used' as anticoagulant during the collection procedure, and additional heparin was added to the collection bags at the time of apheresis. The final , volume of each leukapheresis pack is 300 to 400 ml, which is collected in a Fenwal transfer bag. Mononuclear cells are then separated from neutraphils and erythrocytes using Ficoll-Hypaque density gradients. The separated lymphocytes are harvested, washed twice and resuspended at a concentration of 1- 1.5 x 10 cells/ml in Hank's balanced salt solution containing 75 uM mixed gangliosides, and penicillin, streptomycin sulfate, glutamine, and gentamicin sulfate without additional serum in 1 liter roller bottles. The cells are incubated for 1 hour with continuous rotation at 0.5-1 revolution/min. Aliquots of cells are removed for staining with antibodies to CD3 and CD4 to ascertain the ganglioside effect on CD4 expression. The ganglioside treated cells are centrifuged at 510 x g for 15 minutes in 1 liter bottles, the pellets are pooled in 250 ml centrifuge tubes, and washed twice more in Hank's balanced salt solution without calcium, magnesium, or. phenol red, and the cells are resuspended in infusion medium consisting of 200 ml of 0.9% sodium chloride containing 1% human serum albumen. The final cell suspension is filtered through sterile Nytex and transferred to a Fenwal transer pack. The ganglioside treated cells are administered intravenously through a central venous catheter or into a large peripheral vein. Approximately 10 cells are infused initially, and the remainder are infused 5 minutes later over approximately 20 minutes, with gentle mixing every 5 minutes. No filters are used in the infusion line.

EXAMPLE 5 The use of gangliosides to inhibit the infection of CD4 positive target cells by the Human ^* Immunodeficiency Virus (HIV)-has been demonstrated experimentally.

To demonstrate that gangliosides could inhibit HIV infection, H9 or CEM target cells were incubated with either 10% HIV infected cells or supernatants from HIV infected cultures in the presence and absence of gangliosides. To follow the extent of HIV infection, aliquots of the infected cells and control cells were counted daily and stained with monoclonal antibodies specific for the gag antigen, which is expressed as a consequence of HIV infection. Other aliquots of cells were stained with anti-CD4 antibody to determine if this molecule had been modulated by the gangliosides. Ten thousand cells from each aliquot were evaluated by flow cytometry as described in EXAMPLE 1 to determine the level of fluorescence intensity of both the gag antigen and CD4.

TABLE 5 below is representative of six similar experiments. Gangiosides inhibited unequivocally both the expression of CD4 and the infection of H9 target 19 cells_ by HIV, added as infected culture supernatant. In the absence of gangliosides, expression of HIV gag antigen increased detectably from 7% to 11% by day 4 after infection, reached a maximal percentage of 64% on day 6, and decreased to 38% by day 8. In the presence of 50 uM mixed gangliosides, HIV expression was only 3% on day 3, 11% on day 4, 27% on day 6 (58% lower than the control), and 13% on day 8 (. 66% lower than control). In the absence of the infectious dose of HIV, the percentage of gag antigen detected remained at approximately 3%, with or without gangliosides (data not shown) .

Thus, it has been demonstrated that the treatment of HIV susceptible target cells with gangliosides not only reduced their expression of CD4, but also inhibited significantly their infection by HIV.

Having illustrated and described the principles of our invention in a preferred embodiment thereof, it should be readily apparent to those skilled in the art that the invention can be modified in arrangement and detail without departing from' such principles. We claim all modifications coming within the spirit and scope of the accompanying claims.

Claims

21CLAIMS

1. A p-rocess for inhibiting infection in humans by a virus which infects human cells through attachment to CD4 cell-surface determinants, which comprises selectively modulating the expression and function of said CD4 cell-surface determinants on human cells with a chemical agent comprising a ganglioside composition capable of inducing the rapid and selective substantial disappearance of said CD4 cell-surface determinants from said human cells, and thereby selectively inhibiting the receptivity of said human cells by said virus.

2. The process of claim 1, wherein said virus comprises human immunodeficiency virus.

3. The process of claim 1, wherein said human cells comprise brain glial cells.

4.^" The process of claim 1, wherein said human -cells comprise human T-helper*cells.

5. The process of claim 1, wherein said human cells comprise human epithelial cells.

6. The process of claim 1, wherein said human cells comprise human macrophages.

7. The process of claim 1, wherein said selective modulation of said CD4 cell-surface determinants is carried out without affecting the expression of other cell-surface determinants.

8. The process of claim 1, wherein said ganglioside composition is selected from a group consisting of mono-sialated gangliosides, di-sialated gangliosides, tri-sialated gangliosides, tetra-sialated gangliosides, modified gangliosides, and mixtures thereof.

9. The process of claim 1, which further includes the step of maintaining said selective modulation by providing said ganglioside composition at a level sufficient to prevent re-expression of said CD4. cell-surface determinants on said human cells.

10. The process of claim 9, which further includes the step of terminating the providing of said ganglioside composition thereby causing the re- expressing on said CD4 cell-surface determinants on said human cells with recovery of cell function which is dependent upon CD4 expression.

11. The process of claim 10, wherein the step of re-expressing said CD4 cell-surface determinants on said human cells is substantially completed within 24 hours after terminating said addition of the ganglioside composition.

12. Modified human cells which include CD4 cell- surface determinants normally susceptible to infection by a virus which infects human cells through attachment to the CD4 cell-surface determinants, which comprise „ ^• human cells selectively modulated with a ganglioside composition capable of inducing the selective modulation of the expression of said CD4 cell-surface determinants on said human cells, thereby selectively inhibiting the receptivity of said modulated human cells by said virus.

13. The human cells of claim 12, wherein said virus comprises human immunodeficiency virus.

14. The human cells of claim 12, wherein said ganglioside composition is selected from the group consisting of mono-sialated gangliosides, di-sialated gangliosides, tri-sialated gangliosides, tetra-sialated gangliosides, modified gangliosides, and mixtures thereof ,

15. The human cells of claim 12, wherein said CD4 cell-surface determinants is selectively modulated without affecting the expression of other cell-surface determinants. 23

16. The human cells of claim 12, which comprise T-helper cells.

17. The human cells of claim 12, which comprise human epithelial cells.

18. The human cells of claim 12, which comprise macrophages.

19. The human cells of claim 12, which comprises brain glial cells.

20. A process for using a chemical agent comprising a ganglioside composition for inhibiting infection in humans by a virus which infects human cells through attachment to CD4 cell-surface determinants.

21. The process of claim 20, wherein said virus is human immunodeficiency virus.

22. The process of claim 20, wherein said ganglioside composition is -selected from the group consisting of mono-sialated gangliosides, di-sialated gangliosides, tri-sialated gangliosides, tetra-sialated gangliosides, modified gangliosides, and mixtures thereof.

23. The process of claim 20, wherein said ganglioside composition is topically applied to the skin of a human to inhibit further infection by said virus.

24. The process of claim 20, wherein said ganglioside composition is topically applied to human cells to prophylatically inhibit infection by said virus.

25. The process of claim 20, wherein said human cells comprise human epithelial cells.

26. The process of claim 20, wherein said selective modulation of said CD4 cell-surface determinants is carried out without affecting the expression of other cell-surface determinants.

27. The process of claim 20, which further includes the step of maintaining said selective modulation by providing said ganglioside composition at a level sufficient to prevent re-expression of said CD4 cell-surface determinants on said human cells.

28. The process of claim 20, which further includes the step of terminating the providing of said ganglioside composition thereby causing the re- expressing on said CD4 cell-surface determinants on said human cells with recovery of cell function which is dependent upon CD4 expression.

29. The process .of claim 20, wherein the step of re-expressing said CD4 cell-surface determinants on said human cells is substantially completed within 24 hours after terminating said addition of the ganglioside composition.

^'30. A process "for inhibiting viral infection of human cells having CD4 cell-surface determinants , -which comprises: providing a ganglioside composition; and treating said human cells with said ganglioside composition in an amount sufficient to selectively modulate the expression of said CD4 cell-surface determinants thereby inhibiting the receptivity of said modulated human cells for said virus.

31. The process of claim 30, which further includes the step of maintaining said selective modulation by providing said ganglioside composition at a level sufficient to prevent re-expression of said CD4 cell-surface determinants on said human cells.

32. The process of claim 30, which further includes the step of terminating the providing of said ganglioside composition thereby causing the re- expressing on said CD4 cell-surface determinants on said human cells with recovery of cell function which is dependent upon CD4 expression.

33. The process of claim 30, wherein the step of re-expressing, said CD4 cell-surface determinants on said human cells is substantially completed within 24 hours after terminating said addition of the ganglioside composition.

34. The process of claim 30, wherein said selective modulation of said CD4 cell-surface determinants is carried out without affecting the expression of other cell-surface determinants.

35. The process of claim 30, wherein said virus comprises human immunodeficiency virus.

36. A process for treating an infection by a virus in humans which infects human cells through attachment to CD4 cell-surface determinants, which comprises: isolating human blood cells in vitro from a patient; . - treating said isolated human blood cells with a chemical agent comprising a ganglioside composition capable of inducing the selective disappearance of said CD4 cell-surface determinants from said human cells by selectively modulating the expression and function of said CD4 cell-surface determinants, and thereby selectively inhibiting the receptivity of said human blood cells by said virus; and reinfusing said CD4-modulated human blood cells into said patient.