WO1996039187A1 - Method for treating molluscum contagiosum resulting from hiv infection - Google Patents

Abstract

Methods for treating and inhibiting disease and symptoms associated with the human immunodeficiency virus (HIV) are provided. The method includes transforming the human immunodeficiency virus (HIV) infection into a nonserious disease through the infusion of monoclonal antibodies directed against particular antigens on anti-self, anti-CD4 cytotoxic T-lymphocytes. The method is further useful in the treatment of molluscum contagiosum arising due to human immunodeficiency virus (HIV) infection.

Description

"METHOD FOR TREATING MOLLUSCUM CONTAGIOSUM RESULTING FROM HIV INFECTION"

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending

application U.S. Serial No. 08/227,913, filed April 15, 1994, which

5 application is a continuation-in-part of prior application U.S. Serial No.

08/165,751 , filed December 13, 1993, which application is a

continuation-in-part application of prior application U.S. Serial No.

08/033,405, filed March 19, 1993, now abandoned.

TECHNICAL FIELD

10 The present invention relates generally to methods for treating

human disease conditions associated with the human

immunodeficiency virus (HIV) and more particularly to the use of

monoclonal antibodies directed against anti-self cytotoxic

T-lymphocytes or their lytics in order to inhibit or treat HIV and related

15 HIV diseases. The present invention also includes a method of

treatment for molluscum contagiosum arising due to human

immunodeficiency virus (HIV) infection. BACKGROUND OF THE INVENTION

Several viruses produce latent infection in humans and can

reactivate to produce recrudescent or persistent disease. One such

disease is the human immunodeficiency virus (HIV). HIV is associated

with a progressive catastrophic disease in certain primates, including

humans. Humans infected with HIV experience proliferation of a

certain class of white blood

cells known as cytotoxic T-lymphocytes (CTL). The final stage of this

disease is commonly known as acquired immune deficiency syndrome

(AIDS).

It is well known in the art that the clinical signs and symptoms

of AIDS are primarily due to a profound loss of all lymphocytes marked

with the CD3 and CD4 antigens (CD4+ T-lymphocytes). It is also

generally accepted that the infectious agent in AIDS is the human

immunodeficiency virus (HIV). Although HIV infects and destroys

CD4 + cells, the number of cells infected is inadequate to account for

the profound and indiscriminate loss of these cells that occurs in

individuals infected with HIV. It has been suggested by those in the

field that autoimmunity may play a role in the pathogenesis of AIDS.

However, few have suspected a pathogenic cytotoxic T-lymphocyte

(CTL). Rather, it is generally accepted by those skilled in the art that

CTL's are beneficial for those infected with HIV since it is believed

CTL's help control the infection, i.e., CTL's are believed to be

prognosticators that delay the progression of AIDS. Kilmas, et al,

"Phase I Trial of Adoptive Therapy with Purified CD8 Cells in HIV

Infection", Int. Conf. AIDS, July 1 9-24, 1 992; Abstract No. POB

3446, for example, have described infusion of CTL's into the

bloodstream of HIV-infected patients as an experimental method of

treatment. This particular type of infusion was directed to the

mitogen-expanded colonies of the host patient's autologous CD8 +

cells, a lymphocyte population that includes CTL's.

However, Zarling, et al, "HIV-lnfected Humans, But Not

Chimpanzees, Have Circulating Cytotoxic T-Lymphocytes That Lyse

Uninfected CD4 + Cells", J. Immunol. 1 990; 144: 2992-98 have

shown that HIV-infected humans have an anti-self, anti-CD4 CTL in

their circulating blood that lyses healthy, uninfected CD4 + cells. No

such CTL was found in the blood of HIV-seronegative humans.

Moreover, no such CTL or suicide cell was found in the blood of

HIV-infected chimpanzees. This is significant because HIV infection

manifests as a nonpathogenic colonization in the blood and tissue of

chimpanzees. T cell-monocyte adhesion pathways are important in HIV

replication. Diegel. et al, "Regulation of HIV Production by Blood

Mononuclear Cells from HIV Infected Donors: II. HIV-1 Production

Depends on T Cell-Monocyte Interaction", AIDS Res. Hum.

Retroviruses. 1993; 9:465-73 teach that blocking of either CD2-LFA-3

or CD18-ICAM-1 results in greater than 90% inhibition of HIV-1

production stimulated by anti-CD3 or staphylococcal

enterotoxin/superantigen. Inhibition of HIV production, but not

inhibition of CD4 + T lymphocyte proliferation, was observed when

either the T cell or monocyte coreceptor was bound by monoclonal

antibodies to these adhesion molecules. It is known that adhesion

molecules are essential for an interaction between cytotoxic T

lymphocytes (CTL) and their target cells. As mentioned above,

Zarling, et al. have shown that HIV-infected humans, but not

HIV-infected chimpanzees, have circulating CTL that lyse uninfected

CD4+ T cells. Because HIV-infected chimpanzees do not develop HIV

disease, autoreactive CTL directed against healthy CD4+ cells, and

other adverse CTL effects, may account for the emergence of disease

in HIV-infected humans.

BRIEF SUMMARY OF THE INVENTION

HIV vaccine studies have shown that reducing CTL's causes the

host's CD4 count to go up. The present invention is based on the deduction that the reason CD4 counts go down in the first place as a

result of HIV infection is because among the various types of CTL's,

there must be an anti-self, anti-CD4 CTL. Thus, the maladaptive CTL

synthesized by humans is the factor that transforms HIV infection into

a catastrophic disease. This is confirmed by the work of Zarling et al,

who found that because HIV infection does not lead to any serious

disease in chimpanzees, it is the anti-self, anti-CD4 suicide cell, rather

than HIV itself, that is directly responsible for the disease associated

with HIV infection in humans.

The destructive role of the anti-self, anti-CD4 cytotoxic

T-lymphocyte is overcome according to the teachings of the present

invention through the use of monoclonal antibodies directed against

one or more particular antigens on the anti-self, anti-CD4 killer cell or

antigens on the lytics produced by such killer cell. Through infusion of

particular monoclonal antibodies directed against such antigens, the

anti-self, anti-CD4 cytotoxic T-lymphocytes or their lytics as the case

may be are neutralized to prevent an HIV positive patient from

developing AIDS or to cure the disease itself if the disease has

sufficiently advanced into AIDS. In addition, use of adhesion

antibodies neutralizes cells producing HIV to improve the health of

infected patients. More specifically, one embodiment of the present invention

utilizes monoclonal S6F1 mouse antibodies (S6F1 mAb) directed

against an adhesion epitope of LFA-1 . An infusion of S6F1 mAb

elicits an immune response that is believed to remove HIV-producing

CD4+ T lymphocytes from the peripheral blood of some adults with

HIV disease. Lymphocyte trafficking into tissue has been eliminated

based on mathematical statistics. Four individuals with early disease

were treated in accordance with the present invention. HIV-producing

cells were removed by antibody infusion and replaced by single marked

(CD4 + CD8-)CD4 + T lymphocytes in all four individuals. The

replacement cells circulated while a decrease in serum levels of HIV

RNA persisted, thereby indicating that the newly circulating cells are

uninfected. These single-marked cells are functional as evidenced by

an improvement in delayed cutaneous hypersensitivity reaction.

Individuals with more advanced stages of HIV disease respond

to S6F1 mAb infusion with only double-marked (CD4 + CD8 + )CD4 +

T lymphocytes. However, double-marked cells, which had not

previously improved immune status, were replaced with single-marked

CD4 + CD8- T cells in two patients which were additionally treated

with thymic humoral factor (THF) following infusion of S6F1 mAb in

accordance with another embodiment of the invention. Accordingly,

immunodeficiency in early HIV disease is likely to occur because of the impaired functioning of HIV-transformed CD4 + cells. In later stages

of HIV disease, it is believed that the follicular dendritic network has

been compromised. However, it has now been found that treatment

with a combination of S6F1 mAb with a thymic hormone produces an

immune response which has not previously been obtained in

individuals with advanced stages of HIV disease.

The infusion of monoclonal antibodies has also proven effective

in the treatment of molluscum contagiosum arising as a result of HIV

infection. Treatment of individuals having this skin condition, by

infusion of monoclonal antibodies, has resulted in the improvement

and disappearance of lesions caused by molluscum contagiosum.

It is thus a primary objective of the present invention to provide

a method for preventing and/or curing HIV disease by eliminating or

neutralizing anti-self, anti-CD4 CTL's or their lytics from the circulating

blood of an HIV-infected patient through the infusion of monoclonal

antibodies directed against the antigens presented by such cells or

their lytics. Another primary objective is to neutralize HIV-producing

cells by infusing adhesion antibodies to improve the health of HIV

infected individuals.

It is another object of this invention to reduce or remove lesions

caused by molluscum contagiosum arising as a result of HIV infection. These and other objects of the invention are provided in a

method which transforms HIV into a nonserious infection. This is

accomplished by neutralizing or removing the anti-self, anti-CD4

suicide cell from the circulating blood of an individual infected with

HIV or who is at risk of such infection, and by neutralizing or removing

HIV-producing cells.

The foregoing has outlined some of the more pertinent objects

of the present invention. These objects should be construed to be

merely illustrative of some of the more prominent features and

applications of the invention. Many other beneficial results can be

attained by applying the disclosed invention in a different manner or

modifying the invention as will be described. Accordingly, other

objects and a fuller understanding of the invention may be had by

referring to the following Detailed Description of the preferred

embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention and

the advantages thereof, reference should be made to the following

Detailed Description taken in connection with the accompanying

drawings in which: FIGURE 1 is a schematic representation of AIDS pathogenesis

showing the role of the anti-self, anti-CD4 CTL in the progression of

HIV disease into AIDS;

FIGURE 2 is a schematic representation of a S6F1 monoclonal

antibody attached to the S6F1 antigen on the anti-self CTL according

to the teachings of the present invention.;

FIGURE 3 illustrates the placement of antigen and control on a

forearm for a skin test to measure delayed hypersensitivity reaction in

accordance with the present invention;

FIGURE 4 shows mean T cell/mm³ v. weeks since infusion of

S6F1 mAb alone according to the present invention;

FIGURES 5(a)-(d) show the results of several patients treated in

accordance with the method of the present invention who had a

baseline absolute CD4 count greater than 200 cells/mm³;

FIGURE 6 shows the results of a patient with advanced HIV

disease treated with IL-2 and S6F1 and subsequently with THF in

addition to IL-2 and S6F1 ;

FIGURE 7 compares the results of treatment of S6F1 mAb

infusion and infusion with S6F1 mAb, IL-2 and THF;

FIGURE 8 illustrates response to second infusion in accordance

with the present invention; and FIGURE 9 shows the arithmetic mean of CD8% and CTL% of T

cells for five patients who had single-marked CD8 + T cells.

DETAILED DESCRIPTION

By way of brief background, it is well known that cytotoxic

T-lymphocytes ("CTL's") are white blood cells that kill other cells. If a

CTL kills foreign cells (such as bacteria, fungus, viruses, cancer or the

like), it is deemed a normal cytotoxic T-lymphocyte. On the other

hand, if the CTL kills healthy cells of the body that the cell belongs to,

it is deemed an "anti-self" cytotoxic T-lymphocyte. In either case,

such cells typically function by destroying the cell membrane of the

target cell using one or more "lytics", which are known chemical

compounds. The process of breaking apart the target cell is referred

to as lysis.

CTL's belong to a group of lymphocytes that carry a CD8

antigen. HIV vaccine studies have shown that reducing CTL's causes

a host patient's CD4 count to go up. From this evidence, it has now

been recognized that the reason CD4 counts go down in the first place

as a result of HIV infection is because among the CTL's, there must be

an anti-self, anti-CD4 CTL. Thus, AIDS is caused not by the infection

itself, but by a white blood cell made in response to the infection.

In accordance with one embodiment of the present invention,

S6F1 mouse antibodies (S6F1 mAb) are directed against an epitope of LFA-1 . As discussed in Morimoto, et al., "A Novel Epitope of the

LFA-1 Antigen which can Distinguish Killer Effector and Suppressor

Cells in Human CD8 Cells", Nature. 1 987; 330:479-82, this epitope is

a ubiquitous human adhesion molecule. It marks cytotoxic CD8 + T

lymphocytes, as contrasted with suppressor CD8 + T cells.

Proliferation of CD8 + S6F1 + T cells is known to be characteristic of

progressing HIV disease. It should be noted, however, that the

present invention is not limited to the use of mouse antibodies. In

accordance with another embodiment of the present invention, LFA-1

and ICAM monoclonal antibodies are also directed against the

intercellular adhesion molecules. As discussed herein, other antibodies

are also suitable for use in accordance with the present invention.

FIGURE 1 is a schematic representation of what is believed to

be the AIDS pathogenesis. As seen in this figure, the HIV infection

leads to the destruction of CD4 cells through infection and budding of

new HIV virions. This process generates an immunologic signal that

causes the proliferation of anti-self, anti-CD4 cytotoxic T-lymphocytes.

As shown in FIGURE 1 , these cells carry various known antigens

including, without limitation, DR, CD8, LFA-1 , ICAM and TCR-1 . The

cells also include one or more lytics which are chemical compounds

used to attack the target cell; such lytics also include antigens. The

anti-self, anti-CD4 CTL's or their lytics then destroy healthy activated CD4 cells. Thus AIDS is probably caused not by the infection itself

but by the white blood cells made in response to the infection.

The present invention overcomes the destructive action of the

anti-self, anti-CD4 CTL's or their lytics by infusion of monoclonal

antibodies into the bloodstream of the host patient. It also overcomes

the deleterious effects of HIV replication. As is known in the art, a

monoclonal antibody is an antibody that is made from one cell so that

all resulting antibodies are the same. The standard process of making

monoclonal antibodies is described in, for example, Immunology III, by

Joseph A. Bellanti (W.B. Sanders, 1985) at pages 99-100, which

teachings are incorporated herein by reference. Of course, the

particular method for making the monoclonal antibodies is not limited

to such technique and it is envisioned that any technique for making

such antibodies is within the practice of the invention. The antibodies

are designed to be directed toward a particular antigen on the anti-self,

anti-CD4 CTL or an antigen on lytics produced by such CTL.

Referring now to FIGURE 2, a representation is shown of the

particular treatment method. As seen, monoclonal antibodies directed

against a specific antigen, in this case the S6F1 antigen on the

anti-self, anti-CD4 CTL, are produced in the conventional manner and

infused into the bloodstream of the host patient. The particular

monoclonal antibody is shown attached to the antigen. Such mating flags the immune system and triggers a known immunological

response to cause the body to attempt to neutralize the cells. In this

manner, the anti-self, anti-CD4 CTL cell is neutralized. A similar

mechanism would be used if the particular monoclonal antibodies were

directed to an antigen on a lytic produced by the CTL cell.

According to the invention, monoclonal antibodies are directed

to one or more of the antigens on the CTL cell or its lytics. Under

some circumstances, it may be desirable to limit the type of

monoclonal antibodies to certain specific antigens. Or, it may be

desirable to treat the patient first with a particular monoclonal

antibody and then use another monoclonal antibody later, or to use

multiple antibodies simultaneously. Thus, for example, since many

cells (besides the CTL) carry the CD8 antigen, it may be desirable to

limit use of the CD8 monoclonal antibodies until an initial improvement

in the patient's immune system is established through some other

antigen target. The present invention is intended to cover all such

variations on the sequence and scope of how the particular

monoclonal antibodies are infused.

Although not meant to be limiting, the monoclonal antibodies

are preferably infused once per month over a period of between 10

minutes in accordance with one embodiment of the invention. The

amount of antibodies should typically be about 0.1 milligrams per kilogram of the patient's body weight. The daily regimen is preferably

repeated as needed to maintain an effective immune response. As

used herein, an effective immune response will typically mean that the

patient's CD4/CD8 ratio is returning to normal, accepted levels, that

the patient's skin has an improved delayed cutaneous hypersensitivity

reaction and/or there is an improvement in HlV-related signs and

symptoms. Thereafter, maintenance treatments may be required

depending on the course of the infection or disease. Preferably, the

patient's blood should be measured on a monthly basis to track the

progress of the treatment. Although not meant to be limiting, the

monoclonal antibodies are typically supported in a suitable carrier such

as PBS. The infusion may be effected using a conventional syringe

and line.

As discussed above, the present invention thus exploits the

belief that it is the immunogenic component of the HIV infection that

results in the progression of HIV to a fatal disease. The significance of

the present invention is that it provides a method of neutralizing the

maladaptive CTL (or its lytics) that transform HIV infection into AIDS.

Thus according to the invention the HIV disease can be transformed

from a non-serious infection, and HIV infection can be prevented from

becoming a serious disease, if the suicide cell and/or its lytics are neutralized in, or removed from, an individual infected with HIV or at

risk of such infection.

Thus the method transforms HIV infection through the infusion

of monoclonal antibodies directed against anti-self CTL's or their lytics.

This approach recognizes that monoclonal antibodies have a direct

and specific effect against the body of specific antigens. As used

herein, S6F1 refers to a mouse antibody directed against an epitope of

the human LFA-1 antigen. LAF-1 and ICAM refer to monoclonal

antibodies directed against an epitope of the human LFA-1 , ICAM-1 ,

ICAM-2 and ICAM-3 antigens. According to the invention, a

necessary but sufficient dose of monoclonal antibodies is infused into

the bloodstream until anti-self CTL's have been eliminated or

neutralized and HIV disease cured, or anti-self CTL's are incapable of

proliferating and HIV disease has thereby been prevented, or the

replication of new HIV virions has been suppressed or reduced.

EXPERIMENTAL

EXAMPLE 1

A patient, infected with the HIV virus for about ten years, had

been receiving treatment by injection of his own T cells to achieve a

biphasic elevation of the CD4/CD8 ratio. The patient had been

responding to such injections for a period of about fifteen months of

treatment. At that time, the patient had also been on ddl for

approximately two years. However, given the advanced stage of the

patients' disease, both of these treatments were no longer providing

beneficial results. In fact, HIV could be cultured from his blood cells

even when the blood was diluted out to about one part per 3,120.

Even at such dilution, the p24 antigenemia, which is a measure of HIV

activity, was quite high at about 300 pg/ml.

The patient was then treated in accordance with the method of

the present invention. In particular, the patient was given about 68

mg of S6F1 antibodies over a period of 14 days. The 68 mg

corresponded to 1 mg/kg of the patient's body weight. A few days

after completing the treatment, the AIDS virus could no longer be

cultured from the circulating blood cells of the patient. Thus,

treatment in accordance with the present invention reduces viral load

in the circulating blood of patients with long-term HIV infection. The speed with which infectious cells disappeared from the

patient's circulating blood suggests that some phagocyte may have

destroyed the infected cells. In fact, the patient experienced a

marked increase in monocytes during treatment, and the treating

physician believed at the time, that these monocytes could be

destroying the infected cells. However, the antiviral effect could also

be due to the blockage of the adhesion pathway needed for

communication between antigen presenting cells, thus rendering

HIV-infected cells noninfectious.

Depending on the progression of disease in the individual and

other factors, the dosage range varies from about 0.01 to about 1 .0

mg/kg body weight for a patient treated in accordance with the

present invention. EXAMPLE 2

Written consent was obtained from six white, male young adult

homosexuals with stable HIV disease to participate in the following

treatment program. At baseline, the absolute CD4 counts in the six

participants ranged from 1 -681 cells/mm³. All patients were advised

to take acyclovir in the event that treatment might suppress peripheral

immunity. Those patients with absolute CD4 counts of less than 500

cells/mm³ were receiving prophylactic treatments to prevent

pneumocystis carinii pneumonia (PCP). The patients were receiving a variety of anti-HIV treatments. One patient, however, was naive to all

therapies. Two patients were receiving standard antiretroviral drugs,

and the three remaining patients were taking investigational drugs.

Antibody Production

One suitable S6F1 clone, S6F1 LDB1 1 LDH10, is disclosed in

U.S. Patent No. 5,002,869 to Dana Faber Cancer Institute. The clone

is derived from the hybridation of mouse NS/1 -AG4 myeloma cells

with spleen cells from BALB/cj mice immunized with the cell line

1670, an immortalized splenocyte population derived from a Herpes

virus saimiri strain II infected whitelip tamarin. Monoclonal S6F1 mAb

were produced by GMP ascites in BALB/c mice and purified by the

aseptic method using a protein G column. Purity was greater than

90% by SDS-PAGE and endotoxin levels were a remarkably low 3

EU/ml by Limulus Amebocyte Lysate (LAL) Pyrotellό test. Purified

product was provided in aliquots containing 1 mg/ml S6F1 mAb in PBS

without preservatives. Boluses of up to about 10 mg/kg produced no

evidence of toxicity in Sprague Dawley rats and all organ systems

were grossly normal on postmortem examination.

Laboratory Tests

T Lymphocyte Phenotype Enumeration

T cell phenotypes were enumerated by a 3-color flow cytometry

gating on CD3 + lymphocytes for accuracy. Some confirmation tests utilized the 2-color method gating on lymphocytes. While not meant

to be limiting, flow cytometers such as the EPICS^® Profile II available

from Coulter Corporation and FACscan^® available from

Becton-Dickinson are suitable for use in accordance with the method

of treatment of the present invention.

HIV RNA

HIV RNA was measured by polymerase chain reaction (PCR) in

accordance with Muul, "Current Status of Polymerase Chain Reaction

Assays in Clinical Research of Human Immunodeficiency Virus

Infection", AIDS Updates. 1 990; 3: 1 -1 9. Viral RNA was extracted

from the serum specimen by a modified Chromszinsky method using

guanidine/plant chloroform. The purified RNA was divided into two

portions and each was randomly mixed with hexamer primers.

Separate but identical reverse transcriptase reactions were then carried

out for each portion. Each resultant cDNA portion was subsequently

amplified by multiple Hot Start Polymerase Chain Reaction

amplification cycles using two separate primer sets. The post

amplification reaction material then underwent agarose gel

electrophoresis and

Southern blotting using nylon membranes and specific transfer buffers.

These were UV crosslinked, prehybridized, probed with

fragment-specific dUTP digoxigenin probes, stringency washed, and immunostained using alkaline phosphatase conjugated

digoxigene-specific antibody, X-phosphatase (an insoluble colorogenic

alkaline phosphatase substrate) and nitroblue tetrazolium (NBT). This

resulted in quantification of the RNA in copies per milliliter. Duplicate

samples were run as one batch in parallel with 50% positive and

negative controls. Negative controls were of two types: blanks with

no genetic material and cells which were known to be HIV-1 negative.

Discrete RNA and DNA material from HIV-1 served as the positive

controls. Negative and positive controls were interposed between

every sample.

Laboratory Controls

Critical data points were confirmed by parallel testing in 2 or 3

independent blinded laboratories.

Delayed Cutaneous Hypersensitivity

Cell mediated immunity was evaluated with the Multitest CMI^®

skin test for delayed cutaneous hypersensitivity reaction in accordance

with the procedure and materials of Connaught Laboratories, Inc. This

test is utilized to challenge the skin of the anterior forearm with 7

antigens and a glycerin control as shown in FIGURE 3. According to

the package insert, the response to an antigen is positive if, and only

if, it produces an induration with a mean axis of greater than or equal

to 2 mm. Among normal controls, the mean number of positive responses is 4.5. This, and similar tests, have been found reliable in

evaluating cell-mediated immunity in lung cancer patients treated with

radiotherapy, patients admitted to intensive care units, patients

undergoing gastrointestinal surgery and debulking of kidney tumors,

patients with HIV infection, and patients with diabetes, bronchial

asthma, chronic hepatitis, and other diseases. Reliability has been

assessed for both inter-readers and intra-readers. Significantly, the

use of this test has been shown to have no effect on blood

lymphocyte counts or functions. Although repeated testing such as

once every month or two for six months, can produce detectable

changes in the results, these changes are insignificant and minimal and

do not preclude paired testing to follow immunocompetence. It is

standard practice to use serial tests to follow changes in cell-mediated

immune function.

Infusion

Each patient was infused with about 7 mg (approximately 0.1

mg/kg) of S6F1 mAb over a period of about ten minutes. Ibuprofen,

400 mg every 4 hours for 12 hours, was prescribed the day of

infusion to prevent serum sickness (cytokine release syndrome).

Subjects were followed for the first six weeks and continue to be

monitored monthly. Reinfusions are administered as needed, which is

generally about every 1-2 months. Results

Within two weeks following infusion of S6F1 mAb, three out of

five patients or 60% of those treated with S6F1 mAb, but not II-2,

experienced a drop in HIV RNA greater than or equal to an order of

magnitude (log₁₀ 1 st RNA- log₁₀ 2nd RNA greater than or equal to 1 )

as measured by PCR. In all three patients, this decrease in viral RNA

was correlated with a transient drop in CD4 cells, which was observed

in 4 of the 5 patients or 80% of those treated. This is to be expected

as HIV is intracellular. Thus, a reduction in viral burden should involve

clearing of some CD4 cells. In all cases, there was a subsequent

rebound of circulating CD4+ T lymphocytes by the third week

following infusion of S6F1 mAb. As illustrated in FIGURE 4, there

was a correlated improvement in the skin test for the delayed

hypersensitivity reaction by the fifth week. Suppression of viral RNA

persisted well beyond the rebound of CD4 cells. While the mechanism

is not yet certain, the results indicate that HIV-producing cells are

replaced by uninfected cells.

Patients with Early HIV Disease

FIGURES 5(a)-(c) show the results for three patients with early

disease whose rebound of CD4 cells included only mature,

single-marked CD4 + CD8- T lymphocytes. FIGURE 5(d) illustrates the

results of another patient with early HIV disease whose rebound included both single-marked CD4 cells and double-marked CD4 + CD +

T lymphocytes. Five weeks after infusion of S6F1 mAb, this patient's

double-marked cells were replaced by single-marked cells. One month

after infusion, this patient had a transient increase in HIV RNA

because he was vaccinated with inactivated HIV virions while

participating in another study. Although immunization may generally

raise viral burden, HIV RNA will necessarily increase when a patient is

infused with HIV virions. All four of these patients exhibited an

improvement in the skin test for delayed hypersensitivity reaction. As

shown for example by FIGURE 5(a), this improvement in delayed

cutaneous hypersensitivity is sometimes dramatic.

Patients with Advanced HIV Disease

FIGURE 6 illustrates the results for a patient with more

advanced HIV disease. This patient's rebound included only

double-marked CD4 + CD8 + T lymphocytes. Unexpectedly, these

cells replaced all other T cell phenotypes. As shown by the

histograms in FIGURE 6, it is believed that the double-marked cells

were early thymocytes. This is supported by the finding that they

were also double-marked for CD1 and 3A1 . HIV infection has shown

to inhibit IL-2 production, in part because of cross-reacting antibodies.

Deficient IL-2 has been implicated in causing HIV-associated CD4 + T

lymphocytopenia and impaired T helper function. Thymic humoral factor (THF) has been reported to increase CD4 count and improve

skin reactivity. This patient was subsequently treated with twice daily

subcutaneous injections of 180,000 units of IL-2 for 14 weeks,

together with intramuscular injections of 2.17 micrograms THF for the

first 2 weeks, and then three times per week for twelve weeks. As

illustrated in FIGURE 6, this adjunctive therapy caused his

double-marked cells to be replaced by single-marked CD4 + CD8- T

cells.

FIGURE 7 shows the results of a patient who had no detectable

single-marked CD4 + CD8- T lymphocytes. This patient was treated

from the beginning with an infusion of S6F1 mAb and a regimen of

rlL-2 plus THF. By the fourth week following initiation of treatment, a

modicum of single-marked CD4 + CD8- cells could be detected in this

patient's peripheral blood, although he primarily proliferated

double-negative CD4-CD8- T lymphocytes. By the fifth week, these

double-negative cells amounted to a significant population. Despite

prophylaxis with aerosol pentamidine, this patient developed a mild

case of PCP at week 5, thereby indicating that double-negative

CD3 + CD4-CD8- T lymphocytes may offer little protection against

opportunistic infections. This patient had a 2-log increase in HIV RNA,

a known effect of IL-2 in HIV infected individuals. Reproducibilty

In order to determine whether the effects of S6F1 mAb are

reproduced by reinfusion, the patient illustrated in FIGURE 5(a) was

reinfused with 7 mg of S6F1 mAb 9 weeks after receiving the first

infusion. As shown in FIGURE 8, this patient's response to the

second infusion included a robust rebound of CD4 cells, and the brief

appearance of double-marked cells as compared to FIGURE 5(d). It

should be noted that the initial drop in CD4 cells did not occur since

his HIV RNA did not decrease and continued to be suppressed as

compared to baseline.

CD8 + Cells and CTL

As shown in FIGURE 9, S6F1 mAb produced only a transient

drop in the percentage of CD8 + cells that were cytotoxic

(CD3 + CD8 + S6F1 + ) during week 2.

Safety

Antibody infusion was well tolerated and no adverse reactions

were observed, except a mild allergic reaction in one patient.

However, transient mood changes were observed in some patients and

could be attributed to cytokine release.

It is currently uncertain whether immunocompromised HIV

patients may develop human anti-mouse antibodies (HAMA), which

can limit the efficacy of anti-CD3 antibodies used in renal transplantation. However, in order to test whether such patients may

develop HAMA, a custom ELISA plate was used to test a patient for

HAMA. The patient was not one of the six patients discussed in

Example 2 above, but had previously received 68 mg S6F1 mAb over

1 4 days as discussed above in Example 1 . No HAMA was detected.

The patient was reinfused using a single dose of 7 mg as described in

Example 2. At the time of reinfusion, the patient had a marked CD4 +

T lymphocytopenia and CD8 + T lymphocytosis. Because of this, the

patient was also treated with THF. IL-2 was not used because the

patient discussed above in Example 2 and whose results are shown in

FIGURE 6 did not exhibit replacement of double-marked T cells when

he was administered S6F1 mAb followed by IL-2 alone. Also, IL-2

appeared to increase viral load in another patient as illustrated in

FIGURE 7. Within two weeks of being reinfused, the patient being

evaluated for HAMA had 95% of his circulating T cells double-marked

for CD4 and CD8, and the remaining 5% were single-marked as

compared with the results in FIGURE 6, thereby indicating a response

to mAb infusion uninhibited by HAMA.

Nonetheless, there are known techniques to eliminate the heavy

chains in antibodies which are known to be responsible for causing

HAMA to develop. Moreover, these techniques leave the light chains,

which produce the benefit of treatment with antibodies, intact. Accordingly, HAMA may also avoided by removing the heavy chains in

antibodies.

Other antibodies are suitable for use in the present invention.

For example, it is known now to produce artificial antibodies from

peptides and the like. These antibodies may be used in the present

invention so long as the intended results are obtained. Additionally,

human antibodies are suitable for use in accordance with the

invention.

It is known that CD8 + T lymphocytes have been shown to

suppress HIV replication in vitro. The ability of ex vivo blood to

demonstrate this effect correlates with the clinical status of HIV

patients. On the other hand, the selective depletion of CD8 + T cells

from the circulating blood of HIV patients has a beneficial effect.

General evidence in this regard has led to the advent of the

"homeostasis" hypothesis of HIV disease. The results shown in

FIGURE 6 suggest that the CD8 + T cell significance is not yet

completely understood. This patient had no circulating CD8 + CD4- T

lymphocytes at all for greater than three months and there were no

apparent clinical consequences.

CD4+ T Lymphocyte Counts

The results shown in FIGURE 5(b) are particularly important

since a significant increase in skin reactivity occurred without a decrease in HIV RNA and without an increase in CD4 count above

baseline. The drop in CD4 count over the first two weeks suggests

that HIV-transformed cells were cleared despite the lack of decrease in

HIV RNA. The latter may have been due to an unrelated HIV vaccine

the patient received two weeks into treatment in accordance with the

present invention. As noted above, an infusion of HIV virions may

increase HIV RNA. In any event, these results demonstrate that the

cell mediated immunity improves significantly without an increase in

CD4 count. It is well known that CD4 cell function, and not simply

CD4 count, plays an important role in immunocompetence.

Double-Marked CD4 + CD8 + T Lymphocytes

A flow cytometer will count any lymphocyte that bears the CD3

(T cell) and CD4 markers as a CD4+ T lymphocyte. This includes

both mature, single-marked, CD4 + CD8- T lymphocytes and immature

double-marked, CD4 + CD8 + T lymphocytes. The patient whose

results are shown in FIGURE 6 for example, could thus be interpreted

to have had 900 CD4 cells early in treatment. Although this was true,

the patient also had 900 CD8 cells and a total of 900 T cells, meaning

that all of his T cells were both CD4 cells and CD8 cells. It should be

noted that double-marked CD4 + CD8 + T lymphocytes proliferate in

the natural course of HIV disease. To differentiate this phenomenon, a 3-color flow cytometry should be used rather than the 2-color flow

cytometry used in routine clinical practice.

Immature CD4 + CD8 + thymocytes express only a few

molecules serving as T cell receptors (TCR), and they have minimal

capacity for transducing intracellular signals. It is therefore not

surprising that improvements were not observed in delayed cutaneous

hypersensitivity in patients circulating only double-marked

CD4 + CD8 + T cells. The inhibition of TCR on these double-marked

cells appears to be mediated by mature CD4 + T cells. This may

explain a dim fluorescence of CD3 receptors that are sometimes

observed as a transient effect when mature CD4 cells rebounded.

Double-Negative CD4-CD8- T Lymphocytes

The double negative T cells shown in FIGURES 5(c) and 7 are

important because the role of double-negative TcRgd⁺ lymphocytes in

HIV disease has been the subject of considerable debate and

speculation. For example, DePaoli, et al., "A Subset of Gamma Delta

Lymphocytes is Increased During HIV-lnfection", Clin Exp Immunol..

1 991 ; 83: 1 97-91 ; Margolic, et al., "Flow Cytometric Analysis of

Gamma-Delta T Cells and Natural Killer Cells in HIV-1 Infection", CJin

Exp Immunol.. 1 991 ; 58: 1 26-38; and Autran, et al., "T Cell Receptor

Gamma/Delta Lymphocyte Subsets During HIV-1 Infection", Clin Exp

Immunol.. 1 989; 72: 206-10 report an increase in these cells while Hermier, et al., "Decreased Blood TcRgd⁺ Lymphocytes in AIDS and

p2Y-Antigenemic HIV-1 Infected Patients, Clin. Immunol.

Immunopathol.. 1 993; 69: 248-250 report a decrease. These cells

sometimes reflect secondary infections. Even in immunocompetent

patients, TcRgd ⁺ lymphocytes were found to be increased in the

course of several infections such as toxoplasmosis (See. Scalise, et

al., "Lymphocytes Bearing the Gamma-Delta T-Cell Receptor in Acute

Toxoplasmosis", Immunology. 1 992; 76: 668-70). This may explain

the double-negative cells illustrated in FIGURE 7 because the patient

had evidence of opportunistic infection. However, the double-negative

cells shown in FIGURE 5(c) are unclear at this time. In one study, high

percentages of TcRgd ⁺ cells were found in HIV-infected patients for

whom secondary infections appear to have been eliminated. Thus,

these cells may be related to peculiar immunopathologic processes

associated with HIV infection.

The patient whose results are shown in FIGURE 5(c) may have

chrpnic, undiagnosed bartonellosis. For about twenty years, he had

the characteristic skin lesions and hemolytic anemia, and spent

considerable time in Peru and other South American countries where

the infection is endemic. Because this history predates his HIV

infection, his skin lesions were never mistaken for Kaposi's sarcoma.

However, this is not an uncommon misdiagnosis. It should be noted that the flow cytometry may produce

spurious reports of double-negative cells on blood specimens collected

within a few days of S6F1 mAB infusion. Apparently this is due to

competitive interference between freely circulating S6F1 mAb and the

diagnostic mAb used for flow cytometry.

Pathogenesis of HIV Disease

The present invention establishes that there are two distinct

pathogenic elements in HIV disease. Initially, cell-mediated immune

function is probably degraded by the colonization of CD4 cells by HIV

with a resulting impairment of cell function. At this early stage,

immunodeficiency may be reversible if the HIV-producing cells are

neutralized. The infected cells that have been neutralized are then

replaced by mature, healthy uninfected CD4 cells. At a later stage of

the disease, however, the host is unable to replace infected cells with

mature, single-marked CD4 + CD8- T lymphocytes, as illustrated in

FIGURES 6 and 7. This is probably due to lymphatic architecture,

whjch becomes damaged as HIV disease progresses. As further

shown in FIGURES 6 and 7, this patient had a significant increase in

mature circulating CD4 cells after receiving rlL-2 and THF.

Anti-adhesion antibodies can be used to neutralize HIV-producing cells

from the lymph nodes, thereby preventing or retarding damage to the

follicular dendritic architecture. Furthermore, this may retard the spread of HIV infection since cell-to-cell infection occurs primarily in

the lymph nodes during the clinically latent period of infection.

Other Monoclonal Antibodies

The effectiveness of Applicant's method using LFA-1 , ICAM-1 ,

ICAM-2 AND ICAM-3 monoclonal antibodies on CD4 + T lymphocyte

depletion is further illustrated in Butini, et al, "Intercellular adhesion

molecules (ICAM)-1 , ICAM-2 and ICAM-3 function as counter-

receptors for lymphocyte function-associated molecule 1 in human

immunodeficiency virus-mediated syncytia formation", Journal of

Immunology. 1 994, Vol. 24, pp. 21 91 -21 95. The results of these

experimentations are incorporated herein by reference. The

monoclonal antibodies utilized within the experiements discussed in

the Butini article included the hybridoma cell lines for TS1 /22 and

TS1 /1 8 monoclonal antibodies, directed against LFA-1 antigens.

RR1 /1 , CBR-IC2/1 and CBR-IC2/2 monoclonal antibodies were directed

against ICAM-1 and ICAM-2 antigens. CBR-IC3/1 and CBR-IC3/2

monoclonal antibodies were directed against two different isotopes of

ICAM-3 antigens.

Particularly with respect to CD4 + T lymphocyte depletion, the

treatment of cultures with LFA-1 monoclonal antibodies significantly

reduced the amount of CD4 + cell depletion with respect to the treated

cultures. The CD4 + T cells remain completely viable until day ten after infection and showed only a minor depletion of CD4 + T cells

(approximately 20%) at day 1 9. In untreated cultures, a decrease in

CD4 + T cell viability was evident at day 1 0, and by day 1 6 the

depletion of CD4 + T cells was 80%. Similar results were obtained in

cultures treated with ICAM-1 , ICAM-2, and ICAM-3 monoclonal

antibodies. However, the depletion protection of day 1 9 was not as

high as that achieved with LFA-1 monoclonal antibodies and the

depletion was in the 50% range.

Molluscum Contagiosum

In yet another embodiment of the present invention, the method

for infusion of monoclonal antibodies may be used to treat molluscum

contagiosum arising as a result of human immunodeficiency virus (HIV)

infection. Molluscum contagiosum is a skin disease that is known to

be a sign of immune deficiency within individuals inflicted with the

human immunodeficiency virus (HIV) and causes lesions on the skin of

the infected patient. The lesions caused by the disease may be excised

but will only reappear. Standard HIV treatments do not help with this

condition. It is primarily a cosmetic problem because the lesions can

become disfiguring, especially when appearing on the face. By

utilizing the infusion of monoclonal antibodies into patients as

previously described, patients will enjoy a reduction or complete

remission of the lesions caused by molluscum contagiosum. EXPERIMENTAL

EXAMPLE 1

For three years, a 45 year old HIV positive male, had suffered

from molluscum contagiosum. The patient normally had two to four

lesions on his arms, shoulders, chest, abdomen, back, thighs, and/or

buttocks at any particular time. The patient was treated with

infusions of S6F1 antibodies as taught previously. Five weeks after

the first infusion with S6F1 antibodies the mulluscum lesions had

cleared. The patient has remained free from mulluscum lesions for a

period of 20 months.

EXAMPLE 2

A 41 year old HIV positive male who had suffered for 8 months

with confluent molluscum lesions on his forehead and 6 discrete

papular lesions on his malar cheeks. The patient was treated with

infusions of S6F1 monoclonal antibodies for alternating months. After

the second treatment, the patient's forehead was completely clear of

any molluscum lesions and there remained only one area of

hypopigmentation on the patient's cheek. EXAMPLE 3

A 37 year old HIV positive male had suffered from molluscum

contagiosum for a period of four years. The patient had two lesions

on his groin and two on his penis. The patient was treated with

infusions of S6F1 monoclonal antibodies every other month beginning

in January of 1995. Shortly after the second treatment, the

molluscum lesions had solidified and then fell off leaving only a scab.

EXAMPLE 4

The patient, a 35 year old HIV male, had suffered for two years

from approximately 20 molluscum lesions, both confluent and discrete

raised papular lesions, on his face and neck. The patient was treated

with infusions of S6F1 monoclonal antibodies every other month.

Subsequent to the second treatment, the patient had only

hypopigmentation on his neck and the raised lesions had shrunk.

EXAMPLE 5

The patient was a 45 year old male with advanced AIDS. The

patient had PCP five times, CMV retinitis, histoplasmosis, Kaposi's

sarcoma, etc. For the past two years, he has had 100-some

molluscum lesions on his face, neck, shoulders, upper arms and

anterior chest. He was first treated with S6F1 antibodies in March of

1995 and ten days later the molluscum lesions had completely cleared. An alternative embodiment of the present invention also serves

as a preventative measure for health care workers. In particular, an

HIV-infected individual requiring invasive medical or dental procedures,

undergoes treatment in accordance with the present invention prior to

such surgery or procedures. In this manner, infectious cells in the

circulating blood of the HIV-infected individual are reduced, thereby

protecting health care workers involved with the surgical procedures

by reducing the possibility of HIV exposure.

It should be appreciated by those skilled in the art that the

specific embodiments disclosed above may be readily utilized as a

basis for modifying or designing other techniques or processes for

carrying out the same purposes of the present invention. Thus, for

example, other delivery vehicles or techniques may be used for

delivering the monoclonal antibodies to the bloodstream. It should

also be realized by those skilled in the art that such equivalent

processes do not depart from the spirit and scope of the invention as

set forth in the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method for treating a patient having molluscum

contagiosum resulting from human immunodeficiency virus infection to

reduce molluscum lesions on a patient's body, comprising the steps of:

(a) infusing a dose of monoclonal antibodies, said dose

being between about 0.1 -1 .0 milligrams of said monoclonal antibody

per kilogram of the patient's weight; and

(b) repeating said infusion as necessary.

2. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC 9579.

3. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC TS1 /22.

4. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC TS1 /18.

5. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC RR1 /1 .

6. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC CBR-IC2/1.

7. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC CBR-IC2/2.

8. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line

ATCC CBR-IC3/1 .

9. The method of claim one wherein the monoclonal

antibodies comprise antibodies produced by the hybridoma cell line