Background cf the Invention

Throughout this invention, various publications are referenced by Arabic numerals. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.

l. Clinical Background

The risk of becoming infected with a communicable blood-borne pathogen such as human immunodeficiency virus (HIV) poses a serious occupational hazard for all health-care workers (physicians and surgeons, dentists, nurses, etc.). Among resident physicians, operating room personnel have the greatest risk of blood exposure (1, 2) . In 1988, the Centers for Disease Control (CDC) recommended universal precautions to prevent occupational HIV transmission in the health care setting (3) . The fundamental premise of the CDC recommendation is that blood from an HIV-seropositive individual is a toxic substance (4) . Unfortunately, efforts to diminish the risk of occupational HIV transmission have met with limited success.

Several important factors affect the risk of occupational HIV transmission. These include i) the HIV seroprevalence of the patient population; ii) the risk of occupational blood exposure; and iii) the risk of seroconversion after blood exposure.

i) HIV-Seroprevalence

The HIV seroprevalence of a given patient population influences the risk of occupationally acguired HIV transmission. In many urban hospital centers, the HIV seroprevalence is significant. For example, at a trauma center in Hannover, Germany, 0.1% of surgical patients were HIV-seropositive over an 18-month period (5) . In contrast, 13.6% of all patients admitted with penetrating traumas at the Johns Hopkins Hospital in Baltimore, Maryland were HIV- seropositive (6) . In the Johns Hopkins study, the HIV- seroprevalence of 2,302 adult patients screened in the emergency room was 5.2%. Furthermore, 29% of all surgical patients at San Francisco General Hospital had risk factors for HIV infection (7).

ii) Occupational Exposure

Several recent studies have examined the risk of blood exposure to operating room personnel. Hussain et al. (8) determined that perioperative exposure rates may be as high as 5.6 sharp injuries per 100 procedures, and Panlilio et al. (9) found that the total exposure rates (mucocutaneous exposure and sharp injury) may be as high as 30.1 exposures per 100 procedures. In the San Francisco General Hospital study, accidental parenteral and mucocutaneous blood exposures occurred in 6.4% of 1307 consecutive operative procedures (7) . The parenteral blood exposure rate was 1.7% (7) . The highest risk of perioperative blood exposure occurred during long procedures (more than 3 hours) , when blood loss exceeded 300 ml, and during major vascular and intra-abdominal gynecological surgery.

In a study at Yale-New Haven Hospital during a 3-month period in 1990, there were 249 glove tears and 70 sharp injuries during 2,292 operative procedures (10). The rate

of perioperative glove tears was 10.9%, and the rate of perioperative sharp injuries was 3.1%. Visible skin contact with patient's blood occurred in 156 glove tears (63%) . Of the 70 sharp injuries, 47 (67%) were caused by needles and usually occurred during suturing.

The Centers for Disease Control conducted a prospective study at four U.S. teaching hospitals in two cities with a high incidence of HIV infection (11) . There were 95 perioperative sharp injuries during 1,382 procedures on the hospital's general surgical, orthopedic, gynecological, cardiac and trauma services. Injury rates were as follows: 2.5% for house officers; 2.1% for attending surgeons; 1.8% for physicians' assistants; 0.5% for medical students; and 0.2% for scrub nurses. The highest frequency of injuries (10 out of 47 cases) occurred during vaginal hysterectomies. Most perioperative injuries involved the health care workers' hands, and suture needles accounted for a majority of the sharp objects causing injuries. Scalpels, bovies, wire and scissors caused the remainder of injuries.

In addition to percutaneous injury, cutaneous exposure to infected blood may result in the transmission of blood-borne agents through breaks in the skin (12). During surgical procedures, glove tears create a significant risk of cutaneous transmission because they are likely to result in exposure to blood. Although the rate of transmission from cutaneous exposure is probably much lower than that from needle sticks (12, 13) , glove tears nevertheless may present a significant risk because of their high frequency. Moreover, operating room personnel are especially susceptible to skin breaks on the finger tips, the location of most glove tears, due to routine hand scrubbing (14) . Unfortunately, operating room personnel frequently fail to notice glove tears, and such failure leads to prolonged

cutaneous exposure to the patient's blood.

ϋi) Seroconversion After Exposure

After a single injury with a hollow needle contaminated with HIV-infected blood, the risk of seroconversion is approximately 0.4% (15-18). In other words, one in 250 needle stick injuries from a seropositive patient will lead to infection with HIV. This risk probably varies with the depth of the injury, the volume of blood injected, and the immune status of the injured health care worker. The risk of HIV seroconversion after injury with a solid suture needle is currently unknown. Although anecdotal cases of seroconversion after mucocutaneous exposure to HIV-infected blood have been reported (19) , sharp injuries pose the most serious risk of occupational HIV transmission. In the Yale study, sharp injuries resulted in bleeding in 85% of reported incidents, and this result suggests inoculation of the patient's blood (10).

Surgeons are subjected to numerous blood exposures over a career that extends several decades. The cumulative lifetime risk of HIV infection to a surgeon can be estimated by probability models. Using two recent models, it has been estimated that the cumulative lifetime risk of seroconversion for the average surgeon is between 1% and 20% (20, 21) . The San Francisco General Hospital study estimates that every eight years, one of their staff surgeons will become infected with HIV as a result of occupational transmission (7) .

Concern about occupational HIV transmission has begun to influence the types of practices that surgeons choose and the kind of care that HIV-infected patients receive, and it may eventually affect the career decisions of medical students. It is therefore important to generate specific

protective measures to diminish the risk of occupational HIV transmission. Several authors have recommended that surgical equipment and techniques be modified to reduce the risk of perioperative blood exposure. However, because surgical instruments are designed to cut tissue, simple barrier precautions are not likely to be effective in substantially reducing perioperative blood exposures. Since suturing and cutting tissue are essential surgical procedures, there are few preventive strategies available. In addition, medical authorities have made nonspecific recommendations such as "do not hurry," "be aware and cautious," and "personnel must use extraordinary care to prevent injuries to hands caused by sharp instruments." Although strict infection-control precautions, modifications in surgical technique and the above recommendations are effective in reducing the risk of blood exposure to some extent, they do not eliminate the risk of occupational HIV transmission. In addition, adherence to these infection-control precautions is generally incomplete in the health care setting.

Currently available measures to manage health care workers after exposure to HIV-infected blood are quite limited and have questionable efficacy. Zidovudine (AZT) , a nucleoside analogue which blocks HIV reverse transcriptase and viral replication, is available for prophylactic therapy of exposed individuals. However, in early 1990, the CDC published a detailed report which concludes that "data from (both) animal and human studies are inadequate to establish the efficacy or safety of AZT for prophylaxis after occupational exposure to HIV" (22) . Recently, two cases of failed prophylactic therapy with AZT after occupational exposure were published (23, 24).

2- The Rationale for CD4-Based Prophylaxis

HIV infects primarily helper T lymphocytes and monocytes/macrophages—cells that express surface CD4— leading to a gradual loss of immune function which results in the development of the human acquired immune deficiency syndrome (AIDS) . The initial phase of the HIV replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gpl20 and the cellular receptor CD4 (25) . Following the attachment of HIV to the cell surface, viral and target cell membranes fuse, resulting in the introduction of the viral genome into the cytoplasm. Several lines of evidence demonstrate the requirement of this interaction for viral infectivity. In vitro, the introduction of a functional cDNA encoding CD4 into human cells which do not express CD4 is sufficient to render otherwise resistant cells susceptible to HIV infection (26) .

Characterization of the interaction between HIV gpl20 and CD4 has been facilitated by the isolation of cDNA clones encoding both molecules (27, 28). CD4 is a nonpolymorphic, lineage-restricted cell surface glycoprotein that is a member of the immunoglobulin gene superfa ily. High-level expression of both full-length and truncated, soluble versions of CD4 (sCD4) have been described in stable expression systems. The availability of large quantities of purified sCD4 has permitted a detailed understanding of the structure of this complex glycoprotein. Mature CD4 has a relative molecular mass (Mr) of 55 kilodaltons and consists of an amino-terminal 372 amino acid extracellular domain containing four tandem im unoglobulin-like regions denoted V1-V4, followed by a 23 amino acid transmembrane domain and a 38 amino acid cytoplasmic segment. Experiments using truncated sCD4 proteins demonstrate that the determinants of high-affinity binding to HIV gpl20 lie within the

amino-terminal immunoglobulin-like domain VI (29) . Mutational analysis of VI has defined a discrete gpl20 binding site (residues 38-52 of the mature CD4 protein) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulins (29).

Since the discovery of CD4's role as the cellular receptor for HIV, researchers have explored its potential uses as a therapeutic agent to treat patients with HIV disease. A water-soluble version of the entire extracellular segment of CD4 (V1-V4, termed sCD4) has been described (30). In vitro experiments with sCD4 demonstrate three important antiviral properties: 1) sCD4 acts as a "molecular decoy" by binding to HIV gpl20 and competitively inhibiting viral attachment to and subsequent infection of human cells; 2) sCD4 "strips" the viral envelope glycoprotein gpl20 from the viral surface, thereby inactivating HIV particles directly; and 3) sCD4 blocks the intercellular spread of virus from HIV-infected cells to uninfected cells by inhibiting virus-mediated cell fusion (25, 31). In vivo pretreatment of chimpanzees with a CD4-based molecule prior to challenge with HIV protects them from infection (32) .

Phase I human clinical trials with AIDS patients demonstrated that there is no significant toxicity or immunogenicity associated with the administration of sCD4 at doses as high as 30 mg/day (33) . Preliminary antiviral studies were inconclusive with respect to CD4 cell count and level of HIV antigen. Because the maximum tolerated dose was not reached, the antiviral effect of sCD4 may have been underestimated, especially in light of recent data concerning differences in sCD4 concentrations required to inhibit laboratory strains of HIV compared to primary viral isolates (34) . Although these in vitro, primate and human

clinical studies have generated some encouraging results, they have also defined several limitations. For example, the measured serum half-life of sCD4 is relatively short and therefore may prohibit its use in a chronic setting. Although CD4-based fusion proteins have been described which have significantly longer serum half-lives (35) , chronic administration of these chimeric molecules may not be feasible.

In addition to therapeutically treating patients infected with HIV, substantially reducing the risk of occupational HIV transmission is an important goal in the health care setting. As discussed above, there is currently no specific protective measure that can substantially reduce the risk of occupational transmission of HIV.

The subject invention provides a method of substantially reducing the risk of occupational transmission of HIV comprising administering to a patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of a non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's bodily fluid during a bodily fluid-exposing medical procedure.

The method of the subject invention does not involve therapeutically treating an HIV-infected individual. Accordingly, the potential problems of short serum half-life and limited in vivo therapeutic efficacy during chronic use of certain CD4-containing molecules, discussed supra, are not a barrier to practicing this invention.

The in vivo efficacy of this method is demonstrated by recent single and chronic therapy studies, which show that reco binant sCD4 can abrogate plasma viremia in persons with

advanced HIV disease (38) . Specifically, when high doses of sCD4 are given to HIV-infected individuals, infectious HIV cannot be detected in the individuals' plasma for six hours.

summary of the Invention

This invention provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure. This invention also provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood-exposing medical procedure involving a patient which further comprises administering to the patient during a suitable time period a suitable dose of a mixture comprising an HIV-neutralizing antibody and a CD4-containing molecule, such mixture being effective to substantially reduce the likelihood of the non- infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure. Finally, this invention provides a method of substantially reducing the likelihood of a non- infected medical practitioner becoming infected with HIV during a bodily fluid-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with

HIV by virtue of contact with the patient's bodily fluid during the medical procedure.

Brief Description of the Figures

Figure 1 Neutralization assay. Soluble CD4 (Δ Δ) , anti-HIV IgG

(■ ■) , or a mixture of the two (t •) were tested for inhibition of infectivity. The plot shows fraction inhibited (percent inhibition/100) versus dose (D) .

Figure 2

Combination Index. The data in Figure 1 were transformed to the median effect plot, and combination indices were calculated for multiple levels of inhibition. The plot shows combination index versus fraction inhibited.

Figure 3

Inactivation of HIV by sCD4. Anti-HIV Serum, or a Mixture of the Two. An HIV inoculum was preincubated with 50 micrograms/milliliter sCD4 (Δ Δ) , a 1:4 dilution of anti-HIV serum (t §) , or a mixture of the two (■ ■) at

37°C for 2 hours. Serial 10-fold dilutions were made, added to cells, and the infectivity titer determined in microculture. A control experiment with HIV alone is shown (O 0) ; the controls described in the text approximate this curve.

Detailed Description of the Invention

The plasmidε CD4-IgG ₁ HC-pRcCMV, CD4-kLC-pRcCMV, CD4IgG ₂ - pcDNAl and CD4-IgG ₂ HC-pRcCMV were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 75192, 75194, 40952 and 75193, respectively.

More specifically, this invention provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure. In one embodiment of this invention, the patient is HIV-infected, i.e., possesses HIV particles in his bloodstream.

As used in this invention, substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV means reducing the likelihood by at least two-fold. For example, if the likelihood of infection were 1 infection occurring for every 1,000,000 blood-exposing medical procedures performed, a two-fold reduction in the likelihood of infection would result in a likelihood of 1 infection occurring for every 2,000,000 blood-exposing medical procedures performed. In a preferred embodiment of this invention, substantially reducing the likelihood of a

non-infected medical practitioner becoming infected with HIV means reducing the likelihood by at least ten-fold.

As used in this invention, a blood-exposing medical procedure is any procedure which places the medical practitioner at risk of contacting the patient's blood in a manner sufficient to cause HIV in the patient's blood to be transmitted to the medical practitioner's body, and thereby infect the medical practitioner with HIV.

In the practice of this invention, the medical procedure may comprise, but is not limited to, a dental procedure such as oral surgery or dental cleaning, a surgical procedure such as an emergency care surgical procedure or a surgical transplant procedure, a dialysis procedure, a catheterization procedure, an obstetric procedure or a gynecological procedure.

Further in the practice of this invention, the administering of the CD4-containing molecule may be effected or performed using any of the various methods known to those skilled in the art. In one embodiment, the administering comprises administering intravenously. In another embodiment, the administering comprises administering intramuscularly. In yet another embodiment, the administering comprises administering subcutaneously.

In the practice of this invention, the suitable time period during which a CD4-containing molecule is administered can be any time period such that, when the CD4-containing molecule is administered within this time period, an optimal prophylactic concentration of CD4-containing molecule is maintained in the patient's bloodstream throughout the medical procedure.

In one embodiment, the suitable time period comprises a time period sufficiently prior to the medical procedure. An example of administration within this time period would be a single injection of a CD4-containing molecule prior to a medical procedure.

In another embodiment, the suitable time period comprises a continuous time period spanning the duration of the procedure. An example of administration within this time period would be a continual IV infusion of a CD4-containing molecule throughout the duration of a medical procedure.

In a further embodiment, the suitable time period comprises intermittent time periods spanning the duration of the procedure. An example of administration within this time period would be an intermittent series of injections of a CD4-containing molecule throughout the duration of the medical procedure.

In the subject invention, the amount of the CD4-containing molecule is sufficient to maintain the concentration of circulating CD4-containing molecule in the patient's blood during the medical procedure above the minimum amount necessary to substantially reduce the likelihood of the non- infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.

In the subject invention, the amount of the CD4-containing molecule sufficient to maintain the necessary concentration of circulating CD4-containing molecule depends on the molecule's weight and the number of gpl20-binding sites it possesses. For example, assume that it requires amount X of sCD4 , having a molecular weight of A and 1 gpl20-binding site, to achieve a sufficient serum concentration of CD4-

containing molecule. For a CD4-containing molecule of molecular weight 4A having 1 gpl20-binding site, it would require 4X of this molecule to achieve the same concentration. However, for a CD4-containing molecule of molecular weight 4A having 2 gpl20-binding sites, it would only require 2X of this molecule to achieve the same concentration. This illustration does not take into account any non-linear relationships which may actually exist between the amount of CD4-containing molecule administered and the resulting serum concentration of the molecule. One of ordinary skill in the art would know to consider such non-linear relationships when calculating the amount of CD4- containing molecule to administer.

As used herein, a CD4-containing molecule may be any molecule comprising a portion of the CD4 protein capable of forming a complex with the HIV-1 gpl20 envelope glycoprotein. Examples of CD4-containing molecules include, but are in no way limited to, the CD4-containing molecules discussed infra.

The CD4-containing molecule may be soluble CD4 (sCD4) , which comprises the extracellular domain of CD4. In one embodiment, the amount is greater than lμg of sCD4 per ml of serum. In another embodiment, the amount is greater than lOμg of sCD4 per ml of serum. In yet another embodiment, the amount is greater than lOOμg of sCD4 per ml of serum.

The CD4-containing molecule may comprise a gpl20-binding fragment of soluble CD4 (sCD4) . The CD4-containing molecule may also be a CD4-immunoconjugate.

The CD4-immunoconjugate may be a CD4-gammal chimeric heavy chain homodimer.

The CD4-immunoconjugate may also be a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either a) IgGl heavy chains or b) chimeric CD4- IgGl heavy chains, and both light chains being either a) kappa light chains or b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

The heterotetramer may be a heterotetramer wherein the chimeric CD4-IgGl heavy chains are encoded by the expression vector designated CD4-IgGlHC-pRcCMV (ATCC No. 75192) , and the chimeric CD4-kappa light chains are encoded by the expression vector designated CD4-kLC-pRcCMV (ATCC No. 75194) .

The CD4-immunoconjugate may be a CD4-gamma2 chimeric heavy chain homodimer. In one embodiment, the CD4-gamma2 chimeric heavy chain homodimer is the CD4-gamma2 chimeric heavy chain homodimer whose chains are encoded by the expression vector designated CD4IgG ₂ -pcDNAl (ATCC No. 40952) .

The CD4-immunoconjugate may also be a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either a) IgG2 heavy chains or b) chimeric CD4- IgG2 heavy chains, and both light chains being either a) kappa light chains or b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.

The heterotetramer may be a heterotetramer wherein the chimeric CD4-IgG2 heavy chains are encoded by the expression vector designated CD4-IgG2HC-pRcCMV (ATCC No. 75193) , and the chimeric CD4-kappa light chains are encoded by the expression vector designated CD4-kLC-pRcCMV . (ATCC No. 75194) .

This invention also provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood- exposing medical procedure involving a patient which further comprises administering to the patient during a suitable time period a suitable dose of a mixture comprising an HIV- neutralizing antibody and a CD4-containing molecule, such mixture being effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.

After infection with HIV, individuals develop specific anti- HIV humoral and cellular immune responses. The humoral immune response to HIV is characterized by neutralizing antibodies. Neutralizing antibodies directly neutralize HIV particles by preventing the initiation of infection and are believed to be one of the most important component of the immune response against HIV and many other viruses.

HIV neutralizing antibodies are directed against the envelope glycoprotein (gpl20/gp41) of the virus. There are at least two major classifications of HIV neutralizing antibodies. These two classifications are the type-specific HIV neutralizing antibodies and the group-common HIV neutralizing antibodies. Type-specific HIV neutralizing antibodies primarily recognize the third variable domain (V3) of gpl20, a highly variable loop structure termed the principal neutralizing determinant. Because of the extreme variability in amino acid sequence, type-specific HIV neutralizing antibodies generally neutralize only a particular isolate or closely related strains. Type- specific HIV neutralizing antibodies generally act by inhibiting HIV membrane fusion with the target cell. In contrast, group-common HIV neutralizing antibodies possess

the ability to neutralize a broader set of isolates. Many group-common neutralizing antibodies recognize a site on gpl20 at or near the CD4-binding site, although antibodies directed against other regions of gpl20 may have broadly neutralizing activity as well. Consequently, many group- common HIV-neutralizing antibodies inhibit viral attachment to the target cell by blocking the interaction between gpl20 and surface CD4. Both type-specific and group-common neutralizing activity have been detected in HIV antibody- positive human sera. In addition, both type-specific and group-common HIV monoclonal neutralizing antibodies have been generated in several species.

A third kind of HIV-neutralizing antibody is directed toward the gp41 component of the HIV envelope glycoprotein. Anti- gp41 neutralizing antibodies recognize an extracellular component of this trans embrane glycoprotein. Anti-gp41 antibodies have been generated which possess either strain- specific reactivity (like the type-specific neutralizing antibody) or more broad reactivity (like the group-common neutralizing antibody) .

In this invention, the neutralizing antibody may be a group- common neutralizing antibody. The neutralizing antibody may also be a type-specific neutralizing antibody. In one embodiment, the type-specific neutralizing antibody is an anti-V3 loop antibody. The neutralizing antibody may also be an anti-gp41 antibody.

Finally, this invention provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a bodily fluid-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective

to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's bodily fluid during the medical procedure.

As used in this invention, a bodily fluid is any fluid which is present in the human body and is capable of containing infectious HIV in an HIV-infected patient. Bodily fluids include, but are not limited to, saliva, cerebrospinal fluid, tears, vaginal secretions, urine, alveolar fluid, synovial fluid and pleural fluid.

As used in this invention, a bodily fluid-exposing medical procedure is any procedure which places the medical practitioner at risk of contacting the patient's bodily fluid in a manner sufficient to cause HIV in the patient's bodily fluid to be transmitted to the medical practitioner's -body, and thereby infect the medical practitioner with .HIV.

This invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art to which this invention pertains will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details

1- Materials and Methods

A. Reagents

i) General

Sera were obtained from HIV-seropositive men who were asymptomatic or had chronic lymphadenopathy. Serum from one patient with clinical AIDS was also tested. Sera were stored at -70°C and were heat inactivated at 56°C for 30 minutes before use. The IgG fraction was purified from some sera by ammonium sulfate precipitation and DEAE chro atography as previously described (36) .

The lymphadenopathy virus (LAV-i) prototype strain of HIV-i was used in the infectivity assays. In some infectivity experiments, clinical isolates or first passage isolates were obtained by coculture of patient lymphocytes with phytohemagglutinin (PHA)-stimulated normal peripheral blood lymphocytes.

ii) sCD4

Soluble CD4 (a genetically-engineered, water-soluble extracellular fragment of human CD4) is disclosed, for example, in Patent Cooperation Treaty International Publication No. WO 88/01304. Soluble CD4 is also commercially available.

Soluble CD4, also designated sCD4 or sCD4 fragments, may be produced by truncating pT4B (ATCC No. 68389) after the V4J4 domain. Such DNA fragments terminate before the transmembrane segment, which begins at approximately

nucleotide position 1264.

Purification and characterization of soluble CD4 fragments is greatly enhanced by constructing a cell line (preferably mammalian) which overexpresses the secreted protein fragment. Strategies which allow the overexpression of proteins may be employed in bacteria, yeast, insect and mammalian systems. Inducible expression systems may also be employed in bacteria and yeast to overproduce proteins which may be toxic if constitutively expressed. Overexpression of soluble CD4 fragments may be accomplished by amplifying a soluble CD4 expression vector, resulting in constitutive overexpression. The amplification of dihydrofolate reductase (dhfr) genes by growth in progressively increased concentrations of the drug methotrexate, an antagonist of dhfr, is widely employed. Since the amplified unit is not limited to dhfr coding sequences adjacent to them, this approach results in the coamplification of sequences adjacent to them. Therefore, dhfr may be used as a selectable marker and as a means of coamplifying newly introduced sequences. This strategy may be successfully employed to increase the expression of several different genes cotransformed with dhfr plasmids.

Using recombinant DNA technology, a vector expressing a secreted, soluble, extracellular fragment of CD4 encoded by the human cDNA clone pT4B may be generated. Base pairs 1- 1252 of pT4B encode the leader peptide of CD4 needed for the synthesis of secreted protein, as well as the extracellular portion of CD4 encompassing the four VJ-like domains (V1J1- V4J4) , but not the transmembrane and cytoplasmic regions which anchor the protein in the membrane. This vector contains sequences encoding the extracellular portion of the CD4 protein which contains the HIV binding domain. These sequences are placed downstream from the SV40 early region

promoter. In addition, a TAA termination codon followed by the polyadenylation region of the bovine growth hormone gene is placed downstream from the truncated CD4 cDNA to provide the signals necessary for the termination of protein synthesis, transcription termination, and polyadenylation of the RNA transcript. The resulting soluble CD4 minigene is then ligated to the mouse dihydrofolate reductase (dhfr) gene to generate a plasmid capable of being amplified after introduction into dhfr-deficient (dhfr-) Chinese hamster ovary (CHO) cells.

For example, the 1.8 kb EcoRI-BamHI fragment of pT4B, which contains the entire CD4 coding sequence, is inserted between the StuI and Bell sites of the mammalian expression vector DSP modified to contain the SV-40 early promoter and the bovine growth hormone polyadenylation sequence. Through the use of synthetic linkers, the Haell (bp 124) - Hpall (bp 1252) fragment of pT4B is inserted between the Kpnl and Xbal sites of the plasmid pUClδ. A soluble CD4 expression vector is created by ligating:

1. a 0.95 kb Bglll - Sad fragment of modified DSP which contains the 1.8 kb EcoRI-BamHI fragment of pT4B (this segment contains the SV40 early promoter, the CD4 leader sequence, and the amino terminal portion of the extracellular CD4 sequence) ;

2. the 0.66 kb Sad - Xbal fragment of the pUClδ plasmid containing the Haell-Hpall fragment of pT4B (this segment contains the carboxy terminal portion of the extracellular CD4 sequence followed by a TAA termination codon inserted after valine 371) ; and

3. the 2.48 kb Bglll - Xbal fragment of modified DSP which contains the bovine growth hormone polyadenylation sequence.

Finally, the 2.2 kb Bglll - BamHI fragment from another modified DSP containing a mouse dhfr expression cassette (β- globin promoter - mouse dhfr coding region - SV40 polyadenylation region) flanked by Bglll and BamHI sites, is inserted into the BamHI site of a plasmid to create a soluble CD4 expression plasmid.

DXB-11, a clone of Chinese hamster ovary cells deficient in dhfr, is transfected with the soluble CD4 expression plasmid. The DXB-11 transformants are then grown in F12 medium, without hypoxanthine or thymidine, containing 10% dialyzed fetal bovine serum. Clones are selected and subjected to stepwise increasing concentrations of methotrexate (mtx) , an antagonist of dhfr, to select for stable transformants which have amplified the newly introduced dhfr gene and adjacent soluble CD4 sequences.

iii) CD4-IgGl Chimeras

Transient expression CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence. On the following day, the cells were transfected for 16-20 hours with 10 micrograms of a CsCl-purified plasmid encoding a chain of a CD4-IgGl heavy chain homodimer by the standard CaPO(4) precipitation technique. After transfection, fresh medium was added to the cells. Analysis of the products synthesized 48-72 hours post-transfection was performed by radiolabelling of transfectants with ³⁵ S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads

alone followed by SDS-PAGE under reducing or non-reducing conditions. In addition, analysis of media and cell lysates was performed 48-72 hours post-transfection by standard Western blotting procedures.

Stable expression

Dhfr- Chinese hamster ovary cells (CHO) were transfected with 20 microgra s of CsCl-purified DNA in a 1000:1 molar ratio of CD4IgGl-pcDNAl:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) . Approximately 10-15 days post-selection, individual cell clones were picked and analyzed for stable expression of CD4-gammal chimeric heavy chain homodimer by several screening techniques, such as ELISA and precipitation with Protein A- sepharose beads followed by SDS-PAGE under reducing and non- reducing conditions. Clones expressing the highest levels were subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete between 10-100 micrograms/ illiliter of CD4- gammal chimeric heavy chain homodimer.

Purification of CD4-qammal chimeric heavy chain homodimer from CHO conditioned media

CD4-gammal chimeric heavy chain homodimer was purified by column chromatography. CHO cells secreting CD4-gammal chimeric heavy chain homodimer were grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media was then applied to

a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the specifically bound material was eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. The fractions were then analyzed by SDS- PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

The pooled fractions were then applied to a 10 ml column of S-sepharose fast flow previously equilibrated with 50mM BES pH 7.0 at a flow rate of 120ml/hr. After application of the sample, a step elution gradient (consisting of the following 4 steps: 5 column volumes of 50mM BES pH 7.0, 4 column volumes of 50mM BES pH 7.0, lOOmM NaCl, 6 column volumes of 50mM BES pH 7.0 225mM NaCl, followed by 8 column volumes of 50mM BES pH 7.0, 500mM NaCl) was employed for specific elution of the CD4-gammal chimeric heavy chain homodimer. The CD4-gammal chimeric heavy chain homodimer was eluted from the column in 50mM BES pH 7.0, 500mM NaCl. The peak fractions were then pooled and concentrated to yield a final protein concentration of at least lmg/ml.

Co-expression of CD4-IgGlHC-pRcCMV and CD4-kLC-pRcCMV in mammalian cells to produce CD4-IqGl chimeric heterotetramer

Transient expression

CosM5 cells grown in DMEM containing 10% fetal calf serum are split to 75% confluence. On the following day, the cells are transfected for 16-20 hours with 5 micrograms of

CsCl-purified CD4-IgGlHC-pRcCMV DNA and 5 micrograms of CsCl-purified CD4-kLC-pRcCMV plasmid DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium is added to the cells. Analysis of the products synthesized 48-72 hours post-transfection is performed by

radiolabelling of transfectants with ³⁵ S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads alone followed by SDS-PAGE under reducing or non-reducing conditions. In addition, analysis of media and cell lysates is performed 48-72 hours post- transfection by standard Western blotting procedures.

Stable expression Dhfr- Chinese hamster ovary cells (CHO) are transfected with 20 micrograms of CsCl purified DNA in a ratio of 1000:1000:1 CD4-IgGlHC-pRcCMV:CD4-kLC-pRcCMV:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. At approximately 3-5 days post-transfection, cells are placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) . At approximately 10-15 days post-selection, individual cell clones are picked. The clones are then analyzed for stable expression of CD4-IgGl chimeric heterotetramers by several screening techniques, such as ELISA and precipitation with

Protein A-sepharose beads followed by SDS-PAGE under reducing or non-reducing conditions. Clones expressing the highest levels are subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines are thus generated which secrete high levels of CD4- IgGl chimeric heterotetramer.

Purification of CD4-IqGl chimeric heterotetramers from CHO conditioned media

CD4-IgGl chimeric heterotetramers are purified using Protein A-Sepharose column chromatography. CHO cells secreting CD4- IgGl chimeric heterotetramers are grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media is collected,

clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent bufferε. The diluted media is then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the bound material is eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. Fractions are then analyzed by SDS-PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

iv) CD4-IgG2 Chimeras

Expression of CD4-IgG2-pcDNAl in mammalian cells Transient expression

CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence. On the following day, the cells were transfected for 16-20 hours with 10 micrograms of CsCl-purified plasmid CD4IgG2-pcDNAl DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium was added to the cells. Analysis of the products synthesized 48-72 hours post-transfection was performed by radiolabelling of transfectants with ³⁵ S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads alone followed by SDS-PAGE under reducing or non-reducing conditions. In addition, analysis of media and cell lysates was performed 48-72 hours post- transfection by standard Western blotting procedures.

Stable expression

Dhfr- Chinese hamster ovary cells (CHO) were transfected with 20 micrograms of CsCl-purified DNA in a 1000:1 molar ratio of CD4IgG2-pcDNAl:p410 (p410 is an expression plasmid

containing the dhfr gene) , although other ratios may also be used. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) . Approximately 10-15 days post-selection, individual cell clones were picked and analyzed for stable expression of CD4-gamma2 chimeric heavy chain homodimer by several screening techniques, such as ELISA and precipitation with Protein A- sepharose beads followed by SDS-PAGE under reducing and non- reducing conditions. Clones expressing the highest levels were subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete between 10-100 micrograms/milliliter of CD4- gamma2 chimeric heavy chain homodimer.

Purification of CD4-gamma2 chimeric heavy chain homodimer from CHO conditioned media

CD4-gamma2 chimeric heavy chain homodimer was purified by column chromatography. CHO cells secreting CD4-gamma2 chimeric heavy chain homodimer were grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media was then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the specifically bound material was eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. The fractions were then analyzed by SDS- PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

The pooled fractions were then applied to a 10 ml column of S-sepharose fast flow previously equilibrated with 50mM BES pH 7.0 at a flow rate of 120ml/hr. After application of the sample, a step elution gradient (consisting of the following 4 steps: 5 column volumes of 50mM BES pH 7.0, 4 column volumes of 50mM BES pH 7.0, lOOmM NaCl, 6 column volumes of 50mM BES pH 7.0 225mM NaCl, followed by 8 column volumes of 50mM BES pH 7.0, 500mM NaCl) was employed for specific elution of the CD4-gamma2 chimeric heavy chain homodimer. The CD4-gamma2 chimeric heavy chain homodimer was eluted from the column in 50mM BES pH 7.0, 500mM NaCl. The peak fractions were then pooled and concentrated to yield a final protein concentration of at least 1 mg/ml. The pooled and concentrated fractions were then applied to a 120 ml column of Sephacryl S-300HR previously equilibrated with PBS, at a flow rate of 8ml/hr. The CD4-gamma2 chimeric heavy chain homodimer fraction was specifically eluted in PBS, and concentrated to at least lmg/ml.

Co-expression of CD4-IgG2HC-pRcCMV and CD4-kLC-pRcCMV in mammalian cells to produce CD4-IgG2 chimeric heterotetramer

Transient expression

CosM5 cells grown in DMEM containing 10% fetal calf serum are split to 75% confluence. On the following day, the cells are transfected for 16-20 hours with 5 micrograms of CsCl-purified CD4-IgG2HC-pRcCMV DNA and 5 micrograms of CsCl-purified CD4-kLC-pRcCMV plasmid DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium is added to the cells. Analysis of the products synthesized 48-72 hours post-transfection is performed by radiolabelling of transfectantε with ³⁵ S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads alone followed by SDS-PAGE under

reducing or non-reducing conditions. In addition, analysis of media and cell lysates is performed 48-72 hours post- transfection by standard Western blotting procedures.

Stable expresεion

Dhfr- Chinese hamster ovary cells (CHO) are transfected with 20 micrograms of CsCl-purified DNA in a ratio of 1000:1000:1 CD4-IgG2HC-pRcCMV:CD4-kLC-pRcCMV:p 10 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. At approximately 3-5 days post-transfection, cells are placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) . At approximately 10-15 days post-selection, individual cell clones are picked. The clones are then analyzed for stable expression of CD4-IgG2 chimeric heterotetramers by several screening techniques, such as ELISA and precipitation with Protein A-sepharose beads followed by SDS-PAGE under reducing or non-reducing conditions. Clones expressing the highest levels are subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines are thus generated which secrete high levels of CD4- IgG2 chimeric heterotetramer.

Purification of CD4-IqG2 chimeric heterotetramers from CHO conditioned media

CD4-IgG2 chimeric heterotetramers are purified using Protein A-Sepharose column chro atography. CHO cells secreting CD4- IgG2 chimeric heterotetramers are grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media is collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent bufferε. The diluted media is then applied to a 5ml column of Protein A-Sepharose fast flow previouεly

equilibrated with PBS, at a flow rate of 60ml/hour. After extensive washing, the bound material is eluted with lOOmM glycine/HCl, pH 3.5, directly into an aliquot of 1M Tris.HCl pH 8.0 to immediately neutralize the eluted fractions. Fractions are then analyzed by SDS-PAGE under reducing and non-reducing conditions followed by silver staining and pooled.

v) Antibodies

Group-common and type-specific neutralizing antibodies are commercially available. Specifically, anti-gpl20 antibodies, anti-V3 loop antibodies and anti-gp41 antibodies are commercially available.

It is also possible for one skilled in the art to make human or urine anti-gpl20 or anti-gp41 antibodies. For example,

•human monoclonal anti-gp41 antibodies may be made as described, infra. Peripheral blood mononuclear cells (PBMCs) are isolated from the blood of HIV-l-infected individuals who exhibit anti-gp41 antibodies in their serum. Epstein Barr Virus (EBV) (obtained, for example, from B95-8 cell supernatants) is added to the PBMC preparation which iε then plated out in 96-well tissue culture plates at limiting dilution. Colonies of EBV-immortalized B lymphocytes grow out and those colonies producing anti-gp41 antibodies are identified by immunoprecipitation of gp41 from metabolically radiolabelled cells expreεεing gpl20/gp41, or by western blotting. These colonies are expanded and fused with a suitable partner cell line, for example, a mouse/human heteromyeloma. Hybrids are selected by culture in selective medium in the presence of feeder cells, and stable antibody- secreting hybrids are cloned and expanded.

B. HIV Neutralization Assay

The microculture asεay for productive viral replication was used (36). Briefly, sCD4, antibody, or a mixture was preincubated for 30 minutes at room temperature with an HIV-1 inoculum (100 TCID-50) . The mixtures were added to PHA-stimulated lymphocytes and incubated at 37°C overnight. The cells were washed by centrifugation, plated in microculture (1 x 10 ⁴ cells/culture; 10 cultures/dilution) , and monitored for reproductive viral replication by detection of HIV antigen in culture supernates 8 and 12 days later. In this assay, antibody and sCD4 are washed away after initial exposure of virus to cells and before plating in microculture. They are not replaced in the plating and feeding media. Residual antibody or sCD4 does not interfere with the detection of supernate viral antigen as determined by testing mixtureε of εupernateε from cultures incubated with sCD4/antibody alone and from cultures with HIV-1 alone.

C. HIV Inactivation Assay

An HIV preparation was combined with sCD4, anti-HIV serum, or a mixture of the two in a 100 microliter volume. Final concentrations were 50 micrograms/milliliter for sCD4 and a 1:4 dilution for serum. The preparations were incubated for

2 hours at 37°C; serial 10-fold dilutions were made and added to PHA-stimulated ly phocyteε for an overnight incubation at 37 ^C C. The cellε were waεhed, plated in microculture, and monitored for viral replication as deεcribed above. In the control titration, HIV and εCD4/εerum were preincubated at 37°C separately, dilutions were made, and the dilutions were combined just before addition to cellε. Thuε the experimental and control cultureε have identical amountε of HIV, sCD4, and serum but differ in the opportunity for interaction at 37°C before

exposure to cells.

2. Experimental Discussion

HIV Neutralization Assay

In order to determine whether sCD4 would neutralize HIV in the presence of HIV-1 antibody-positive human sera, the following assay was performed. Purified IgG or anti-HIV human sera and sCD4, alone and in combination, were tested at multiple dilutions in a microculture assay for neutralization of productive HIV infection. A representative plot of fraction inhibited (% inhibition/100) versus dose of antibody/sCD4 is shown in Figure 1. The results demonstrate that sCD4 or HIV antibody neutralize HIV infection, and inhibition by sCD4 is more potent than inhibition by HIV antibody. Moreover, the reduction in HIV infectivity was greatest with mixtures of sCD4 and HIV antibody.

These data were then mathematically transformed using a multiple drug effect analysis based on the median effect principle described by Chou and Talalay (37) . This involves a log-log dose-response plot (the median effect plot) from which the slopes and intercepts are used for computer-assisted calculation of a combination index (Cl) . The Cl reflects the relative potency of the agents when used alone versus when used together for any given level of inhibition. A Cl value of greater than 1 indicates antagonism. A value of 1 indicates an additive effect, and a value of less than 1 indicates synergy. A plot of the combination index versus level of effect is shown in Figure 2. The results demonstrate a general additive effect or slight synergism between HIV antibody and sCD4, In other words, the reduction in HIV infectivity mediated by mixtures

of sCD4 and antibody was always equal to or greater than the arithmetic sum of the reductions by either agent alone. Similar additive effects were obtained with a fresh clinical HIV isolate that requires higher concentrations of sCD4 for inhibition in the neutralization assay.

HIV Inactivation Assay

In addition to competitive inhibition of receptor binding, sCD4 causes shedding of gpl20 from virions resulting in irreversible inactivation of HIV infectivity (30, 40) . A model syεtem waε designed to measure this effect in the presence of HIV antibody-positive human sera. An HIV inoculum was mixed with εCD4, sera, or a mixture of the two, and incubated for 2 hours at 37°C. Serial 10-fold dilutions were made, added to cells, and viral replication was monitored. In a control titration, HIV, sCD4, and sera were preincubated separately at 37°C, dilutions were made, and the dilutions were combined just before addition to cells. Thus, the inactivation and control cultureε are identical with reεpect to HIV, εCD4, and εera concentrationε, but differ in the opportunity for interaction at 37°C before addition to cellε. It should be noted that with dilution, the concentration of sCD4/εerum in the cultureε is orders of magnitude lower than that required for inhibition in the neutralization assay where graded doses of sCD4/serum are mixed with a constant amount of HIV just before addition to cells (see supra .

As shown in Figure 3 and Table 1, considerable reduction in titer occurs under these conditions. Inactivation by the mixtures waε alwayε greater than the inactivation cauεed by either agent alone. In many experiments, the mixture resulted in complete inactivation or inactivation below the threshold of the assay. Therefore, the reported log titer

reduction is a minimal estimate. In those experiments where an actual measurement of titer reduction was obtained with the mixture, the ID-50 (50% inhibitory dose) unit reduction (anti-log ID-50 titer reduction) by the mixtures was greater than the arithmetic sum of the inactivation due to each agent alone.

Table 1

Inactivation of HIV Infectivity by Anti-HIV Serum, sCD4, and Serum/sCD4 Mixtures.

Reduction in log ID-50 titer

HIV was expoεed to anti-HIV εerum (final concentration 1:4) and/or sCD4 (50 microgramε/milliliter in Expoεures 1 and 3; 25 micrograms/milliliter in Exposure 2) in a 100 microliter volume for 2 hours at 37°C before dilution and microculture infectivity titration. Titer reduction is the difference between thiε titration and the reεpective control titration. In Expoεure 3, titer reduction is the difference between the experimental titration and the titration of HIV in the absence of sCD4 or εerum.

Examples

1. Emergency Room Trauma Patient Because of penetrating wounds and the nature of surgical treatment, the treatment of trauma patients constitutes one of the most significant risks of blood exposure to health care workers. In many urban hospital centers, the HIV- seroprevalence of patients is high. For example, at the Johns Hopkins Hospital emergency department in Baltimore, Maryland, 13.6% of all patients admitted with penetrating trauma were HIV-seropositive (6) .

In order to substantially reduce the risk of occupational HIV transmission in this high risk setting, a CD4-containing molecule would be administered to trauma patients either before or during treatment in the emergency room and operating room. The CD4-containing molecule might be administered by IV (intravenous) bolus or continual IV infusion, IM (intramuscular) injection, or SC (subcutaneous) injection. Depending upon the route of administration and the nature of the treatment, the CD4-containing molecule might be given continuously or intermittently. The dose of the CD4-containing molecule administered would depend on the serum concentration required to substantially reduce the infectivity of HIV in the patient's blood. It is presumed that the above options will vary depending on the type and pharmacokinetics of the CD4-containing molecule administered.

For example, upon arrival at an emergency room, a patient with penetrating trauma would receive an IV bolus of a CD4-immunoglobulin chimeric molecule in order to reach a serum concentration of greater than 10 micrograms/milliliter. IV administration is preferred in

order to achieve peak concentrations rapidly. For a CD4-immunoglobulin chimeric molecule, this might require a dose equal to or greater than 1.0 milligram/kilogram of body weight. Since the serum half-life of CD4-immunoglobulin chimeric molecules is on the order of several days, additional administration of the CD4-containing molecule might not be required during the initial treatment. Periodic blood sampleε would be assayed for the serum concentrations of the CD4-containing molecule in order to ascertain whether the desired blood levels have been achieved. Additional doseε of the CD4-containing molecule might be adminiεtered during the courεe of hospitalization or additional operative procedures.

2. Surgical Patient

Knowledge of a patient's HIV statuε might have significant implications in the type of treatment offered. In patients who are known to be HIV-seropoεitive, or who have riεk factorε for HIV infection, nonoperative treatment of εurgically correctable illneεε to εubεtantially reduce the riεk of occupational HIV tranεmiεεion iε a controverεial ethical iεεue. As an alternative, a CD4-containing molecule would be administered during the operative procedure to εubεtantially reduce the riεk of occupational HIV tranεmiεεion.

For example, a patient would receive a CD4-containing molecule prior to an operative procedure. Such procedures include, but are not limited to, obεtetric and gynecologic procedures, general εurgical procedureε, cardiac and vaεcular procedureε, orthopedic procedures, and urological procedures. The CD -containing molecule might be administered by IV bolus or continual IV infusion, IM injection or SC injection. Depending on the route of adminiεtration and the nature of the treatment, the

CD4-containing molecule might be given continuously or intermittently. The dose of the CD4-containing molecule administered would depend on the serum concentration required to substantially reduce the infectivity of HIV in the patient's blood. It is presumed that the above options will vary depending on the type and phar acokinetics of the CD4-containing molecule administered.

For example, a CD4-immunoglobulin chimeric molecule might be delivered by IV bolus in order to rapidly achieve serum concentrations of greater than 10 micrograms/milliliter. For a CD4-immunoglobulin chimeric molecule, this might require a dose equal to or greater than 1.0 milligram/kilogram of body weight. Since the serum half-life of CD4-immunoglobulin molecules is several days, additional administration of the CD4-containing molecule might not be required during the initial procedure. Periodic blood samples would be assayed for the serum concentrations of the CD4-containing molecule in order to ascertain whether the desired blood levels have been achieved. Additional doseε of the CD4-containing molecule might be administered during the course of hospitalization or additional operative procedures.

3. Hospitalized Patient:

In many urban medical centers, the HIV-seroprevalence of hospitalized patients iε εignificant. In addition to operating room perεonnel, other health care workerε such as physicians, dentists, nurses, medical students, and phlebotomistε perform procedureε which put them at riεk of expoεure to patientε' blood. In order to substantially reduce the risk of HIV transmission during the course of hospitalization, patients who are known to be HIV seropositive or who have risk factors for HIV infection would receive a CD4-containing molecule.

For example, a patient would receive a CD4-containing molecule during the course of hospitalization. The CD4- containing molecule might be adminiεtered by IV boluε or continuouε IV infuεion, intramuεcular injection, or εubcutaneous injection. Depending on the route of administration and the nature of the treatment, the CD4- containing molecule might be given continuously or intermittently. The dose of the CD4-containing molecule administered would depend on the serum concentration required to εubεtantially reduce the infectivity of HIV in the patient'ε blood. It iε preεumed that the above options will vary depending on the type and pharmacokineticε of the CD4-containing molecule administered.

For example, εoon after admiεsion of a patient, a CD4- immunoconjugate might be delivered by IV bolus in order to rapidly achieve εerum concentrationε of greater than 10 micrograms/milliliter. For a CD4-immunoconjugate, this might require a dose equal to or greater than 1.0 milligram/kilogram of body weight. Since the serum half- life of CD4-immunoglobulin molecules is on the order of several days, additional administration of the CD4- containing molecule might not be required during the hospitalization. Periodic blood εampleε would be assayed for the εerum concentrationε of the CD4-containing molecule in order to aεcertain whether the desired blood levelε have been achieved. Additional doseε of the CD4-containing molecule might be administered during the course of hospitalization.

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