CA2045583A1 - Compositions and methods for treating or preventing aids, arc and hiv infection - Google Patents

Abstract

This invention relates to vaccines, compositions, and methods useful for the treatment and prevention of acquired immunodeficiency syndrome, AIDS related complex, and HIV infection. More particularly, this invention relates to pharmaceutically effective compositions for treating or preventing AIDS, ARC, and HIV infection that are characterized by an immunologically effective amount of a soluble T4 protein which elicits in a treated patient the formation of antibodies to the soluble T4 protein or, alternatively, the release of other proteins which, in turn, are effective to protect against or to lessen the spread, severity or immunocompromising effects of AIDS, ARC or HIV infection.

Description

PCT/~Ss0/003 woso/o8198 2 0 ~ ~ ~ 8 :3 ' ~
. :.

COMPOSIT~ONS AND METHODS FOR 7 TREATING OR PREVENTING
AI~S. ARC AND HIV INFECTION
. .
5TECHNLCAh FIELD OF I~VENTION
This invention relates to vaccines, compo~itions, and methods use~ul ~or the treatment and prevention o~ ac~uired lmmunod~iciency ~yndromQ, ~IDS
related complex, and H~V in~ection. More p~rticularly, this invention r~lat~s to pharmac~uticall~ er~ectiv~
composi~ions ~or ~reating or preventing AIDS, ARC, and HIV in~ection that are characterized by an .
immunologically e~ctive amount o~ a soluble T4 protein which elicits in a treated patient the formation of antibodies to soluble T4 protQin or, alternatively, the release of other proteins which, in turn, are e~ective to protect against or to lessen the spr~ad, ~eve~ity or immunocompromising effects of AIDS, ~-A~C or HIV infection. ~ -B~CRGROUND OF THE INVENTION - .
The class of immune regulatory cells known as T~lymphocytes ca~ be divided into two broad ~unctional olasses, the first class comprising T helper or inducer cells -- which mediate T cell proliferation, lymphokine release and helper cell interactions for Ig release;
and the second clasi3 comprising T cytotoxic or suppressor cells -- which participate in T cell-.

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. ~ PCT/I S90/003'N
2~ a~5~3 2 - :~

mediated killing and immune response supp~ession, In general, ~hese two classes of lymphocytes are distinguished by expressio~ of one of two surface glycoproteins: T4 or CD4 (m.w. 5S,000-62,ooo daltons) 5 which is expressed on T helper or inducer cells, probably as a monomeric p~otein, or T3 or CD8 (m.w.
32,000 daltons) which is exptressed on T cytatoxic or suppressc~r cells as a dimeric pro~tsin.
In immunocompetent individuals, T4 10 lymphocytes interact with other specialized cell types of the im~une system to con~er immunity to or defense against in~ection rE. L. Reinherz and S. F. Schlossman, "The Differentiation Function or Human T-Cells", Qll, 19, pp. 821-27 (19~0)~. More speci~ically, T4 15 lymphocytes ~timulate production o~ growth ~actors which are critical to a ~unctionlng immune system. For example, they act to ~timulate ~ cell~, ~he d~sct~ndant3 o~ h~matopoiatic ~tam cells, which promo~a the production o~ dQrensi~e antibodies. They also activate 20 macrophages ~"Xiller cells") to attack infected or o~herwise abnormal host cells and they induce ~onocytes ("scavenger ctells") to encompags and destroy invading micxobes.
It has been found that tha primary target o~
25 certain infectiv~t agents is the T4 surface protein.
ThR-<~e agents include, for example, some viruses and retroviruses. When T4 1ymphocytes are exp~osed to such agen~s, they are rendered non-functional. As a result, the host's complex immune defense syste~ is destroyed 3 0 and the host becom~s susceptible to a wide range of t opportunistic in~ections. , Such immunosuppression is seen in patients -~u~ering fro~ acquired immuno~eficiency syndro~e ("AIDS'~). AIDS is a disease characterized by s~vere 35 or, typically, complete immunosuppression and a~tendant ,"

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W~ "0/08198 PCT/I_;S90/003~8 2 ~ g 3 - 3 ~

host susceptibility to a wide range of opportunistic infections and malignancies. In some cases, AIDS
infection is accompanied by central nervous system disorders. Complete clinical manifestation of AIDS is usually preceded by AIDS related complex ("ARc'l), a syndrome accompanied by symptoms such as persistent generalizad lymphadenopathy, ~ever and weight 105s.
The human immunode~iciency virug ("HIV") retrovirus is thought to be the etiological agent responsible for AIDS infection and its precursor, ARC ~M. G.
Sarngadharan et al., "Detection, Isolation And Continuous Production Of Cytopathic Retroviruses (HTLV-III) From ~atiQnts With AIDS And Pre-AIDS", Sci~nçe, 224, pp. 497 508 (1984)].* Between a5 and 100% o~ the AIDS/ARCS population test seropositive ~or HIV ~G. N.
Shaw ~t al., "Molacular Characte~ization 0~ Human ~-CQ11 L~ukemia (Lymphotropic) Virus ~ype I~I In ~he Acquir~d Immune Da~ioiency Syndrome'l, ~ , 226, pp. 1165-70 ~lg84)~.
The ho~t range of the HIU virus is associated with cells which bear the T4 sur~ace glycoprotein.
Such cells include T4 lymphocyte~ and brain cells ~P. J. Maddon et al., "The T4 Gene Encodes The AIDS
Virus Receptor And Is Expressed In The Immune System And The.~ai~ ç~j 47, pp. 333-48 ~1986)~. Upon ~-~
infaction o~.a host by HIV virus, the T4 ly~phocytes -~
are r~ndered non-~unctional. The progression o~
~IDS/ARC syndromes can be correlated with the depletion o~ T4~ lymphocytes, which display the T4 surface * In this application, human immunodeficiency virus ("HIV"~, tha generic term adopted by the human retro~irus subcommittee of the International Commi~tee on Taxonomy Of Viruses refers to independent isolates ~ro~ AIDS pati~nts, includin~ human ~ cell lymphotxopic virus typa III ~"HTLV-III"), l~mphadenopathy-as~ociated virus ~I'LAV"),.human immunode~iciency vixus type 1 ~"HIV-l") and AIDS-associated retrovirus ("ARV").

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PCT/~S90/003~8 ~,o ~5S~3 glycoprotein. This T cell depletion, with ensuing immunological compromise, may be attributable to both recurrent cycles of infection and lytic growth and from cell-mediated spread of the virus. In addition, :~
clinical observations suggest that the HIV virus is directly responsible for the central nervous system disorders ~een in many AIDS patients.
The tropism o~ th~ HIV virus ~or T4+ cells is believed to be attributed to the role o~ the T4 cell lo sur~ace glycoprotein as the membrane-anchored virus receptor. ~ecause T4 behaves as the HIV virus receptor, its extracellular sequence probably plays a direct role in binding HIV. More ~p~ci~ically, it is believed that RIV envelope protein selectively ~inds to the T4 ~pitopo~s), using thii~ intexactlon to initiate entry lnto the host cell ~A. G. DalgQlii~h et al., "Th~
CD4 (T4) ~ntigen ~3 An Essential Component 0~ rhe Receptor For The ATDS Rat~o~lrus~ , 312, pp. 763-67 (lg84); D. Klat~mann et al., "T-Lymphocyte T4 Molecule Behaves A3 The Receptor For Human ~etrovirus LAV," ~ , 312, pp. 767-68 (1984)~.
Accordingly, cellular expression o~ T4 is believed to ~Q su~ficient ~or HIV binding, with thQ T4 protein ser~ing as a receptor for the HIV virus.
Therapeutics based upon soluble T4 pr~tein -have been proposed for the treatment and prevention of the HIV-rela~ed infections AIDS and ARC. The nucleotide equience and a deduced a~ino acid ~eguence ~or a DNA that purportedly encodes the entire human T4 protein have been reported ~P. J. Maddon et al., "The Isolation And Nucleotide Sequence Of A cDNA Enc~ding ;
The T Cell Surface Protein T4: A New Member O~ The Immunoglobulin Gene Family," 5~ll, 42, pp. 93-104 (1985)~. 8ased upon i~s deduc~d primary structUre, the ?4 protein is divided into the ~ollowing domains:
. .

, . ~ ' ' .. . ~ ' ' ' ~ . .. . ' ' . ' , .

W~ /0~l98 P~T/~S~ 0 Amino Acid S~ruçture/P~osed Location 5s9~5Lh~S~
Hydrophobic/Secretory Signal -23 to -1 Ho~ology to V-Regions/ +1 to ~94 Extracellular Homology to J-Regions/ ~95 to ~109 Extracellular Gl~cosylated Region/ ~110 to ~314 Extracellular Hydrophobic/Transmembrane +375 to *395 Sequence Very Hydrophilic/ +396 to 1435 Intracytoplasmic Soluble T4 protQins hava been constructed by truncating thQ ~ull l~ngth T4 protein at amino acld 375, to eliminate the transmembran~ and cy~oplA~m~c domains. Such protelns hava been produc~d by recombinant technlque~ ~. A. Fi~her ~t al., "HIV
Infection Is Blocked 1~ o ~y Recombinant Soluble CD4," ~ , 331, pp. 76-78 (1988)~. Soluble T4 proteins advantageou~ly interrcro with the T4/HIV
interaction by blocking or competitive bin~ing mechanisms which inhibit HIV infection o~ cells expresslng the T4 surface protein. And soluble T4 proteins inhibit interaction between T4~ lymphocytes and antigen presenting cells and targets of T4' . lymphocyte ~ediated killinq. By acting as soluble : virus receptors, soluble T4 proteins are usef~l as anti-viral therapeutics to in~ibit HIV binding to T4t ~ells and virally induced syncytiu~ for~ation.
Proposed methods for ~reating or preventing AIDS and ~RC have also focused on the deve~opment of anti-retroviral agents which target the reverse transcriptase enzyme o~ HIV as a uni~uo ~ep in ~he li~e cycle of the viru5. Such agents utilize HIV

"uoI~ PCT/~S90/003 reverse transcriptase inhibition as the mechanism of action. These agents include, for example, suramin, azidothymidine t"AZT") and dideoxycytidine [H. Mitsuya et al., "3'-Azido-3' -Deoxythymidine (BW A509U): An Antiviral Ag~nt That Inhibits The Infectivity And Cytopathic E~ect O~ Human T-Lymphotropic Virus Type III/~mphadenopathy-A3sociated Virus In Vit~Q," P~oc.
Natl. ~ 5~, 82, pp. 70~6-7100 (1985);
~. Mitsuya and S. Broder, "Inhibition O~ The 1~ vitro In~ectivity And Cytopathic Effect Of Human T-~ymphotropic Virus Type III/Lymphodenopathy-Associated Virus (HTLV-III/LAV) By 2',3'-Dideoxynucleosides," Pr~çs Na~. Ac~ , 83, pp. 1911-lS ~1986); R. Yarchoan ~t al., "Administration Of 3'-Azido~3'-Deoxythymidin~, An Inhibitor 0~ HTLV-III/LAV Rapl~catio~, To Patiant~ Wi~h ~I~5 or ~X~S-~elat~d Complax," h~n~Q~ pp. 575-80 (March 15, 1986)].
Although aach o~ ~he~a agen~ has axhibited activi~y agains~ HIV 1~ Yi~Q, only AZT has . ~0 demonstratsd clinical bane~it5 in properly designed placebo controll~d clinical trials. An increasing number o~ patients receiving AZT, however, tolerate only low doses of the drug. Certain dosage regimens of AZT have ~ en reported to be lymphotoxic [Y~rchoan ç~_al~, supra.]. AZT administration in effective amount- has be~n accompanied by undesirable and debilitating side effects, such as gr~nulocytopenia and anemia. Over t~e long. term, therefore, hPmatologic toxic~ty appQars to be a significant limiting factor in the use of AZT in the treatment of AIDS and ARC ~D. D.
Richman et al., "The Toxicity Of Azidothymidine (AZT) In The Treatment Of Patients With AIDS And AIDS-Related Complex: A Double-Blind, Placebo-Controllqd Trial," N.
Enq. ~LL~d.~, 317, pp. 192-97 (1987)].
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~o ~OtOB198 PCT/~590/00~'~
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Other prophylactic and therapeutic regimens are based on agents exhibiting anti-retroviral activity against st~ps in the viral replicative cycle other than reverse transcription [PCT patent application Wo 87/03903]. Such methods include the ad~inistration o~ glucosidase inhibitors, such as the plant alkaloid castanospermine, which modi~y glycosylation of envelope glycoproteins o~ ~IV in~ected cells by interfering with the normal processing Oe N-linked oligosaccharide chains on those glycoproteins, leading to reduced expression of a functional envelope protein at the cell surface and inhibition of productio~ o~ infectious virus particles. Such anti-retroviral agents, however, may exert toxic e~ects on cellular metabolism at higher doses when given as monotherapy.
To dake, th~re~ore, the nc~d exists ~or the develop~ent o~ octlve immuno~herapeutic ag~nts, methods, and str~tegies ~or thQ treat~ant or prevention o~ AIDS, ARC, and HIV ln~ection which avoid the disadvantagQs o~ conventional agents.
DISC~OSURE 0~ ~EE_IEyENTION
~ he present invention solves the problems re~erred to ~bove by providing pharmaceutically e~ectiY~ composi.tions and methods for the treatment and prevention of acquired immunodeficiency syndrome, AIDS related complex and HIV infection. The composi~ions!,and.methods of this invention are ch~racterized by a soluble T4 protein which elicits in a treated patient the formation of antibodies to ~oluble T~ protein or, alternatively, the release of other protein~ which, in turn, are e~fective to protect again~t or-to lessen the spread, severity or im~unocompromising e~ects o~ AIDS, ARC, and HIV
inPection.

J~J ~ 7~ PC~/~ S90/~103'~

2~ 3 - 8 -~RIE~E. I:)ESCRIPTION OF THE DRAWINGS
Figure 1 depicts the nucleotide sequence and the derived amino acid se~uence of T4 cDNA of plasmid pl70-2.
Figure 2 depicts the nucleotide sequence and the derived amino acid sequence o~ T4 cDNA
o~ plasmid p~G381.
In Flgures 1 and 2, the amino acids are represented by singlQ letter codes as follows:
lo Phe: F Leu: L Ile: I Met: M
Val: V Ser: S Pro: P Thr: T
Ala: A Tyr: Y His: H Gln: Q
Asn: N Lys: X Asp: D Glu: E
Cys: C Trp: W Arg: ~ Gly: G
5 b ~ position a~ which a stop codon is present.
In Figure 1, the T4 proteln translation start (~A-23) i~ loaa~Qd at tha meth~onine ~t nucl~otide~
1199-1201 and the mature N-~erminu3 is located at the asparagine ~AA3) at nucleot1des 1274-1276.
In Figure 2, the T4 protein translation start (AA 23) iæ located at the m~thionine at nucleotides 1207-1209 and the mature N-terminus is located at the asparagine (AA3) at nucleotides 1282-1285. . .
Pigure 3A depicts the reverse transcriptase 25 activ~ties of bone marrow cells o~ normal monkeys :-subj~cted in vitro to exogenous SIVm~ prior to, during :
and a~ter rsT4 treatment.
~:Figure jB depicts in tabular form the reverse transcriptase activities generated in P3L from a normal monkey exposed .in vitro to exogenous SIVmaC in th~
presence of plasma of an rsT4-treated monkey~
Figure 4 depicts, in tabular form, the reverse transcriptase activities of bone marrow cell cultures from SIV-infected monkeys and o~ periph~xal ~(~) "0/08198 PCT/~S90/003'~
? ~ S ~ J
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blood lymphocyte (SIV-infected monkeys)/H9 co-cultures before, during and after those monkeys were treated with rsT4.
Figure 5 depicts, in graphic form, the effects of rsT4 treatment on functional abnormalities caused by 5IVmac-induced disease activity in rsT4-treated SIVmac-infected monkeys.
FigurQ 6 depicts, in graphic form, the effects oP rsT4 retreatment on ~unctional abnormalities lo caused by SIVmac-induc~d disease activity in A monkey previou~ly treated with rsT4.
Figure 7 depicts, in tabular form, the increase in CFU-GM and BFU-E colony counts in r~T4-treated SIVmac-in~ected monkeys.
Figur~ 8 depicts, in tabular Porm, the ~n er~ect of either pla~ma ~rom rsT4-kreated monkays or rsT4 on colony ~orming cell~ ~rom bon~ ma~row of SIVmac-infected monk~y~.
Figure 9 depic~s, in tabular ~orm, the ~a vitro ~f~ect of plasma from rsT4-treated monkeys on colony ~orming calls ~rom bone marrow of SIV~C-~nfected monkeys harvested during rsT~ treatm~nt.
Figure lO depicts, in tabular form, the }n vi~o effect of plasma ~rom rsT4-treated monkeys on colony for~ing cells from bone marrow of SIVmaC-in~ected monkeys after completion of rsT4 treatment.
Figure 11 depicts, in tabular form, the in vi~ro augmentation of CFU-GM in bone marrow of SIV aC~
infected monkeys following addition of rsT4 or plasma 3 0 f ro~ an rsT4 -treated monkey.
Figure 12 depicts, in tabular form, the augmentation of CFU-GM by IgG fractions and non-IgG
fractions af plasma o~ an rsT4-~reated monkey.

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PCT/~S90/003~x ;~
~,~ 4~ o Figure 13 depicts the surface bound immunoglobulin on circulating lymphocytes in an rsT4-treated monkey.
Figure 14 depicts the binding o~
immunoglobulin in plasma of an rsT4-treated monkey to CD4~ but not C~8+ human lymphocytes.
Figure 15 depicts the binding o~ ' immunoglobulin in plasma o~ an rsT4-treated monkey to CD4~ ~ut not CD8~ rh~sus monkey lymphocytes.
Figure 16 depicts the binding of plasma immunoglobulin ~rom an rsT4 immunized monkey to monkey PBLs.
Figure 17 depicts, in graphic ~orm, inhibition o~ reverse transcriptase activity in SIVmdC ~`
inrected monkey PBLs, by plasma o~ an rsT4-immunized monkey.
~ lgure lB depicts, in tabul~r ~or~, inhibition o~ rQ~Qrso tran w riptasa actlvity in SIVm~c 1n~ect~d bona marrow macraphag~s, by plasma o~ an rsT4-immunized monkey.
Figure 19 depict~, in tabular ~orm, CFU-GM
~rom bone marrow cells o~ three SIVm~c-in~ected monkeys in the presence o~ plasma ~rom an rsT4-immunized monkey.
Figu~e 20 depicts, in tabular form, that bone :-m~xrow mac~ophages from normal rhesus monkeys become ';
resi~tant to STVmaC infection following rs~4/CF~
i~wtization.
Figure 21 depicts in tabular ~orm, that PBLs fro~ no~al rhesus monkeys become resistant to SIV
infection following -sT4 immunization. mac .
Figure 22 depicts, in tabular form, that viru5 isolations ~rom PBLs and bone marrow cells become negative ~ollowing immunization o~ SIVmaC in~ected monkeys with rsT4 in adjuvant.
.

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~ 90/0~ 1 9~ PCr/ ~ S90/003'~

~J4` :J ~ 3 Fi~ure 23 depicts, in graphic form, the increase of bone marrow CFU-GM and BFU-E in SIVmaC-in~ected monkeys following immunization with rsT4 in adjuvant.
~ETAILED DESCRIPTION OF THE ~NV~N~ION
This invention relates to pharmaceutically aec~ptabl~ composition and methods ~or treating or pr~venting AIDS, ARC, and HIV in~ection. More particularly, thls invention relates to pharmaceuti-lo cally e~ective compositions comprising animmunologically effective amount of a soluble T4 protein which elicits in a treated patient the ~orma~ion o~ antibodies to soluble T4 protein or, alternatively, the ralease o~ o~her protelns which, in turn, are ~ective to protect against or to le~sen ~he spr~ad, 5~varity or lmmunocompromi~in~ a~ct~ Oe ~DS, ARC or HIV in~ection. Acaordin~ to one ~mbodiment, the method o~ this invention comprises the step o~ treating or immunizing a patient in a pharmaceutically acceptable manner with an immunologically e~ecti~e amount of a soluble T4 protein, for a period o~ time suf~icient to lessen the immunocompromising effects of HIV infection, to prevent HIV infection, or to prevent intxacellular spread of HIV infection.
The vaccines, compositions and methods of this inventio~ may be used to treat or prevent AIDS, ARC, or ~IV infection in mammals, including humans.
These vaccines, compositions and methods may also be : used for treating or preventing AIDS-like diseases caus~d by retroviruses, such as simian immunodeficiency viruses, in mammals including humans.
As used.in this application, the term nimmunologically e~fective" denotes tha ability tQ
elicit in a treated patient the ~o~mation o~ antibodies ; : '` : ` . ` ' ' . :. , . : ~. . . : . - ' ' ' '` . . ': ' ': . ' ' .

PCT/~SsO/003~

~ $ - 12 -to soluble T4 protein which are effective to protect the patient for some period of time against AI~S, ARC
or HIv infection or to lessen the spread, severity or immunocompromising effects of AIDS, ARC or HIV
infection. The term "immunologically effective" also denotes the ability to elicit in a treated patient the production or release of ~actors such as lymphokines, colony stimulating ~actori~ or oth~r proteins which enhance the immune response in a patient to a degr~e which is e~ective to protect against AIDS, ARC or HIV
infection or to lessen the severity, spread or immunocompromising effects of AIDS, ARC or H~V
infection.
The compositions and methods o~ this invention are characteriæed by an indirect mechanism o~
activity o~ the 801ubl~ T4 protein which i~
prophylactic or therapeutia. Without being bound by theory, we b~lieve that the indirec~ ~ch~nl~m o~
actlon o~ ~oluble ~ protein which ch~racterizes the methods and compo~i~ions o~ this invention may be due to the generation of antibodies to soluble T4 protein which themselves protect against or are e~ecti~e in treating HIV infection. Alternatively, this indirect mechani~ o~ -~oluble T4 protein activity may be 25 mediated by the generation of lymphokines, such as :;
int~rleukins, colony stimulating factors, or other proteins which enhance i~mune responses in a patient .~
which, in turn, are ef~ective in protecting against or . :: -treating ~IV infection. . .
As used in thi~ application, "soluble T4 protein" includes all proteins, po}ypeptides and peptides which are natural or recombinant soluble T4 .
proteins, or soluble derivatives thereof, and which are :
. characterized by the immunotherapeutic (anti-retroviral) activity o~ soluble T4 protein. They ~0~0~08198 PCT/~S90/003~
~ u ~ ~ 1) (3 3 include soluble T4 like compounds from a variety of sources, such as soluble T4 protein derived ~rom natural sources, recombinant soluble T4 protei~ and synthet~~ or semi-synthetic soluble T4 protein. Such s soluble T4-like compounds ~dvantageously interfere with the T4 /HIV interaction by blocking or competitive binding mechanisms which inhibit HIV inrection o~ cells expr~sslng th~ T4 sur~ace protein.
Soluble T4 proteins include polypeptides selected from the group consisting o~ a polypeptide of the formula AA-23-AA362 f Figure 1, a polypeptide of 1 AA362 of Figure 1, a polypeptida o~ th formula Net-AAl-AA362 o~ Figure 1, a polypeptide o~ the ~ormula AA1-AA374 o~ Fi~ure 1, a polypeptide o~ the 15 ~ormula Met-~A1_374 o~ Figur~ 1, a polyp~ptlde o~ the ~ormula AA1-AA377 o~ Fi~ure 1, a polypeptid~ o~ tho ~A1-377 ~ Figure ~, a polypeptide o~ the ~ormula ~A 23-AA374 o~ Figure 1, a polypeptide o~ the -23 AA377 of Figure 1, or portions ther~o~
Additionally, soluble T4 proteins include polypeptides selected ~rom the group consisting of a polypeptide of the formula AA_23-AA362 of m prote~n, a polypeptide of the formula AAl 362 ~ mature T4 protein, a:polypeptide of the formuIa Met-AAl 362 f mat~re T4 pro~ein, a polypeptide of ~he formula AAl 374 o~ ~ature T4 protein, a polypeptide of the formula Met-AAl 37i of-m~t~re T4 prot~in, a polypeptide of the formula AA1 7~ of mature T4 protein, a polypeptide of the for~ula Met-AAl_377 of mature T4 protein, a polypeptide of the formula AA 23-AA374 o~ mature T4 protein, a polypeptide of the formula AA 23-AA377 o~
mature T4 protein, or portions thereo~
~ he amino terminal amino acid o~ mature ~4 protein isolated ~rom T cells begins at lysine, the . PCT/LS90/003~
~o~ 3 third amino acid of the sequence depicted in Figure l.
Ascordingly, soluble T4 proteins also include polypeptides of the formula AA3-AA377 0~ Figure 1, or portions thereof. Such polypeptides include polypeptides selected ~rom the group consistng of a polypeptide o~ the ~ormula AA3 to AA362 ~ Figure 1, a polypeptidQ o~ the formula AA3 to AA374 o~ Figure 1.
Soluble T4 proteins al~o include the above-recited polypeptides preceded by an N-terminal methionine lo group.
So'::ble T4 proteins useful in the vaccines, compositions and methods of thii3 invention may be produced in ~ variety oP ways. We have depicted in FigurQ l the nucleotide sequence o~ ~ull-length T4 cDNA
obtalned ~ro~ deposited clone pl70-Z and the amlno acid sequence deduc~d therQ~rom. The T4 cDNA o~ p~70-2 is almo~t identical to the approximat~ly 1,700 bp ~quencc reported ~y ~ s~,g5~ upra. Th~ T4 cD~A o~ pl70-2, however, contains three nucleotide substitutions that, in the translation product o~ this cDNA, produce a protein containing three amino acid 3ubstitutions compared to the sequence reported by ~
~hes~ di~ferences are at amino acid position 3, where the asparagine of Maddon et al. is replaced with 25 lysine; posltion 64, where the tryptop~an of Maddon - ~:
et al, is replaced with arginine-and at position 231, where the phenylalanine of Maddon et al. is replaced .
with ~erine. The asparagine reported at position 3 of ~addon~@5_3Ll. instead of lysine was the result of a DNA
sequencing error ~.R. Littman et al., "Corrected CD4 : Sequence", SÇll, 55, p. 541 tl988)~.
Soluble T4 protein constructs may be produced by truncating the full length T4 sequence at various posltions to remove the coding regions ~or th~
transmembr~ne and lntracytoplasmic domains, while wo~oJo8ls8 PcT/~sso/oo3~i 2~

retaining the extracellular region believed to be responsible for HIV binding. More particularly, soluble T4 proteins may be produced by conventional techniques o~ oligonucleotide directad mutagenesis, restriction digestion, followed by insertion of linkers, or chewing ~ack full-length T4 protein with enzymes.
Prior to ~uch constructions, the cDNA coding sequence o~ a full length T4 clono, such as pl70-2, may be modi~ied in sequential steps of site-directed mutagenesis and restriotion fragment substitution to modi~y the amino acids at positions 64 and 231. For example, one may employ oligonucleotide-directed mutagenqsis to modi~y amino acid 64. Subsequently, restrlction ~ragment substitution w~th a ~ragm~nt including the serinR 231 codon o~ a piartial T4 cDNA
isolated ~rom a T~ pai3i~ive lymphoc~e cell lin~ ~0~
Acuto n~ al., ~ , 34, pp. 7~7-26 ~19~3)~ library in ~gt 11 may be used to modi~y the amino acid at position 231 ~R. A. Fisher et al., Nature, supra].
DNA saquence~ coding ~or soluble T4 proteins may be us¢d to trans~orm eukaryotic and prokaryotic host cells by conventional recombinant techniques to produc~ recombinant soluble T4 proteins in clinically and commercially useful amounts. Such soluble T4 proteins include those produced according to the processes set forth in United StAtes pat~nt application 094,322, ~iled Septem~er 4, 1987,.United States patent application 1~1,649, filed January 7, 1988 and PCT
patent application PCT/US88/02940, ~iled Septe~ber 1, 1988, the disclosures of which are hereby incorporated by r~f erence.
~ icroorganisms and recombinant DNA molecules characterized by DN~ sequences coding ~or ~oluble T4 proteins are examplified by cultures deposited in the ~ uo~o PCT/~S90/003~

2~ 3 - 16 - ~

In vitro International, Inc. culture collection, in Linthic~m, Maryland, on September 2, 1987 and identi~ied as: -;
EC100: E-coli JM83/pEC100 - IVI 10146 BG377: E.çQli MC1061/pBG377 - IVI 10147 3G380: E~Qli MC106}/pBG380 - IVI 10148 BG381: E.,~Ql~ MC1061/pBG381 - IVI 10149.

Alt~rnatively, soluble T4 proteins may be chemically synthesized by conventional peptide synthesis techniques, such as solid phase synthesis.
~R. B. Merrifield, "Solid Phase Peptide Synthesis. I.
The Synthesis O~ A Tetrapeptide", ~ L~ C~e~. So~., 83, pp. 2149-54 (1963)].
According to one embodiment o~ the present invention, the ~ioluble T4 prote~n igi pre~erably modi~ied ar sub~ct~d to traatm~nts ~o modl~y th~
protei~ to anhanco it~ lmmunogenic charac~ar in the intended racipient. For exampla, v~rious amino acid substitution~, modl~ications or d~letions may be :~ :
20 carri~d out during preparation o~ the soluble T4 protein. Altarnatively, a soluble T4 protein may be madi~i~d by the addition o~ various pharmaceutically acceptable adjuvants protein prior to administration.
Such an adjuvant may contain, for example, a muramyl dipeptid~ derivative and a carrier which includes a detergent and a combination oS free fatty acids. i~
Either type of such modification may be one that increases the immunogenicity o~ the soluble T4 protein ~ ~.
beyo~d that of a soluble T4 protein ~ndogenous to the patient or its speci~s.
The vac-ines and compositions of this invention may be `.n a variety of conventional depot forms. ~hese include, ~or example, solid~ semi-solid and liquid dosage forms, such ia~ tabl~ts, pills, powders, liquld solut1ons or suspensions, l1posomes, " ~ .

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:: . , : . . . .

~o/~ls~ PCT/~590/OQ3~
~ ~5~J~3 capsules, suppositories, injectable and infusable solutions. The preferred form depends upon the intended mode of administration and therapeutic or prophylactic application.
Such dosage forms may include pharmaceuti-cally acceptable carriers and adjuvants which are known to those o~ skill in the art. These carriers and adjuvants inclu~e, for example, ion exchangers, alumina, aluminum stearate, lecithin, sierum proteins, such as human serum albumin, bu~er substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes ~uch as protamine sul~ate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilica~e, polyvinyl pyrrolidone, cellulos~-based substan~es, and polyethylen~ glycal. ~d~uvanti~ ~or topic~l or g~l ba~e ~orms may bc s~l~cted ~rom ~he gro~p consi~t~ng o~
isodium carboxym~thyla~llulos~, polyacryl~es, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
The vaccines and compositions oS this invention may also include other components or be sub~ect to other treatments during preparation to ~ :
enhance th¢ir immunogenic character or to improve their tolerance in patients.
Ge~erally, the soluble T4 protein ~ay be -~
~or~ulated and administered to ~he patient using methods and co~positions similar to those employed for .
other phar~aceutically important polypeptides (e.g., ~-IFN). Any pha~maceutically acceptable dosage route, ..
including parenteral~ intravenous, intramuscular, intralesional or subcutaneous injection, may ~e used to administer the soluble T4 protein. -~l090/08198 PCT/~S90/003~8 ~ 3 18 -For example, solubl~ T4 protein may b~
administer~d to the patient in any pharmaceutically acceptable dosage form including those which may b~
administared to a patient intravenously as bolus or by ::~
continued infusion over a period of hours, days, weeks or months, intramuscularly -- including paravertebrally and periarticularly -- subcutaneously, intra-cutaneou~ly, intra-articularly, intrasynovially, intrathecally, intralesionally, p~rio~tally or by oral or topical routes. Pre~erably, the compositions o~ the invention are in the ~orm oP a unit doise and will usually be administered to the patient intramuscularly.
The soluble T4 protein may be administered to the patient at one time or over a series of treatments.
The most e~ectiv~ mode o~ adminlstration and do~age regimen o~ ~oluble T4 protein will d~pend upon th~
particular composit~on and/or ad~uvan~ used ~or treatment, the ~evority and course o~ in~e~t~on, previou~ therapy, the pa~ient's health ~tatu~ and response to treatment, and the judgment o~ the treating phy~ician. .
~ ccording to ono embodiment o~ this invention, a da1ly dose equal to or less than about 1 ~g/kg ~ody weight of a soluble T4 protein may be administer~d to the patient, via one or several ad~inistrations, or released from a depot form per day .
o~ treatm~nt over a treat~ent period of between about 1 to 30 days. Subseguent boosters may be administered as : needed to ~aintain ~he initial therape~tic or ~ :
prophylactic e~ect.
For example, a typical dosage regimen for tr~at~ent or provention of HIV infection using a soluble T4 protein which has been ~odified to enhance its immunogenic character in the int~nded patient would involve the ad~inistration o~ about 0~5 mg/kg body ,; ' ~ , : ' ' '" ' ' ` ' '` ' ' ' ~ ' ' . ' UO 90/0819X r~ `JUIUU~

2 ~
-- 19 -- ;

weight to the patient once a day for about 30 days.
Patients may re~uira intermittent boosters of about 1.0 mg/kg body weight daily, once a week, on a weekly basis.
In a preferred embodiment of this invention, the soluble T4 protein or modified i~oluble T4 protein is administered with an adjuvant, in order to increase lts immunogenicity. Use~ul adjuvants include si~ple metal salts such as aluminum hydroxid~, as well as oil based adjuvants such as complete and incomplete Freund's adjuvant. When an oil based adjuvant is used, the soluble T4 protein usually is administsred in an emulsion with the adjuvant. Most preferably, the soluble T4 protein is administered in an emulslon with incomplete Freund's adjuvant.
When the soluble T4 protein ls mixed with an adjuvant, the mixture usually would be administ~red at several site~ intramuscularly, intrader~ally or subcutaneously. In~ramuscular adminii~tration i mos~
preferred.
Where an adjuvant is added to the soluble T4 protein prior to administration about 1 mg/kg b~dy weight could be administered on one day, followed by b~osters o~ 1 mg/kg body weight once a week until the desired r~sponse is achieved. Thereafter, a booster of about l mgJkg body weight may be administered every one month.
Where the soluble T4 protein is admin~stered in an emulsion with incomplete Freund's adjuvant, about 1 mg per individua~ could be administered on one day, followed by boosters of 1 mg per individual about once a month for two months, and thereafter about once every three months.
It shall, of course, be un~erstood that the dosage and length of treatment will v~ry d~pending on WO90/08198 PCT/~S90/003~

~ 5~3 - 20 -s~ch ~actors as the level of immunogenicity of the soluble ~4 protein used, whether an adjuvant is administered with the soluble T4 protein, the nature of any adjuvant used, and the immune status of the individual being treated. For example, the more highly i~munogenic the soluble T4 protein, the lower the dosage and neceissary treatment time. Similarly, the dosage and nece~ ry treatment time will be lowered if the soluble T4 protein Ls administered with an adjuvant.
It should also be understood that dosage regimens according to this invention may include the administration o~ more than 1.0 mg/kg body weight/day ovQr a given treatment period.
15In order that the invention described herein may b~ more ~ully understood, th~ ~ollowing exampl~
ar~ ~t ~orth. It ~hould b~ und~rskood ~hat ~ha?3~
example?3 ara ~or illustrative purpo3~s only and are not ko be ~onstrued as limiting this invention in any manner.

Th~se examples demonstrate the effects of soluble T4 protein in inhibiting viral replication and - -in improving ~unctional abnormalities associated with 25 SIV~ac-induced disease activity. ~ ~;
In these examples, the soluble T4 protein j used was re~om~inant human soluble T4 protein ¢"rsT4") ¦
or ("rsCD~") supplied by Biogen Research Corp. tCambridge, Massachusetts). That racombinant soluble T4 protein was derived from a Chinese hamster ov~ry cell transfected with animal cell expression vector pBG381 tR. A. Fisher et al., "HIV Inrection Is Blocked InLYi~Q By ~ecombinant Soluble CD4," ~ X~, 33I, pp. 76-78 ~1988)]. pBG~81 is characterized by DNA

.' ~...

~O~o/~81s8 PCT/~sso/003~

--21 2 ~ , 3 ~, 3 coding for AA-23 to AA3~ of T4 protein, as depicted in Figure 2.
- we obtained two rhesus monkeys (Macaca mulatta) (colony born and raised at the New England Regional Primate Research Center, Southborough, Massachusetts) each weighing 5-7 kg, who were free of in~ectious di~ease and in good health. We also obtained ~our SIvm~c-ln~ectsd rhesus monkey~ (colony born and raised at the New England Regional Primate Research Center~ o~ similar weight. SIVmac-infection had been induced in these four animals by intravenous injection of tissue culture supernatant containing .
cell-~ree SXVmac virus. We also induced SIV
in~ection in three additional rhesus monkeys ~colony born and ral~ed at the New England R~gional Prima~e Rc~arch Center) o~ similar weight who were not part o~
the rsT4-tr~atment group.
Thrae o~ the viru~ln~cted monkey~ had relatively normal immune parameters at the start of treatment; the ~ourth had a circulating absolute T4 lymphocyte ~ount o~ less than 100 per mm3 ~nor~al rhesus monksys have >1,000 T4~ P9Ls per mm3). Each monkey had been housed in a captive primate colony for more than 6 ~onths prior to administration of the soluble T4 protein. Throughout the administration period, ~ach monkey was maintained on a conventional :
diet o~ monkey chow supplemented with fresh fr.uit.
The monkeys described in the treatment and non-treatment groups were designated as follows:
Normal/rsT4-treated Mm 156-84 Mm 158-84 WO 90/OK I 9X PCT/~!S90/003`~ ~ .
3 - ~ ~

nfected/rs~ -treated Mm 156-85 Mm 159-86 Mm 167-84 S Mm 202-84 ~-infectç~/n Q r9~4 t~ m&~
Mm 129-86 Mm 244-86 Mm 335-78 lo over a period of 50 days, each normal animal and four of the infected animals received recombinant~ -human soluble T4 protein once daily by lntramuscular injection to the large muscle3 of the thighs or buttocks, rOr a total dose o~ 2 mg/day/monkey o~ rsT4.3 (from th~ p~G381-trans~ormed cell lin~ BG 381). Serum sample~ ~or clearance d~termination and to a~3~ss liver and renal ~unction~ wer~ collacted on ~ay~ 8, 15, and 44 o~ treatment, as w~11 as on ~ays 8 and 18 ~ollowing treatment. on~ o~ the in~ected monkeys, Mm 156-85, was re-treated ~or an additional 50 day period commencing on the 96th day a~ter the end o~ the ~irst cour~e o~
treatment. ~Q ~ound that none of the animals experienced any significant adverse reactions to the rsT4.
The di~tribution of lymphocyte subsets in the ;~
peripheral blood o~ all the animals ~T2, T4+, T8+, IL-2 receptor~ and Bl') and the function of those cells (pro?iferation following stimulation with xenogenic cells and pokeweed mitogen) did not chang2 during the 30~ course of treatment. The serum chemistry studies to ass ss live~ and renal function were unchanged when compared to baseline values. Although the ani~als experienced a transient drop in the number ~ bone marrow granulocyte-macrophage (CFU-GMs~ pro~enitor col,onies w~th an associated transient neutropenia, this :
.

:
,~

W~ 9~/08198 PCT/I.S90/0~3 ~
2 9 ~ 3 transient bone marrow suppression never became clinically significant. During treatment, none of the normal or in~ected monkeys showed changes in the distribution of lymphocyte subsets in their peripheral blood, in PBL function or in serum chemistry assessment of liver and renal function. *
Prepar~tion o~ SIV~,aC~5~
We prepared an SIV~ac-containing culture by expanding SIVmaC isolate 251 (available ~rom the New lo England Regional Primate Research Center, Southborough, Massachusetts) in a culture of peripheral blood lymphocyt~s (PBLi3) obtain~d ~rom a healthy human donor.
Specifically, we isolaked PBLs ~rom a blood sample on a Ficoll-diatrizoate density gradient ~sp. gr. 1.077) and incubat~d them for 3 days in culture medium ~P~I
1640 ~ 10~ ~etal cal~ ~erum) supplementQd wlth 1 ~g/ml Concanavalin A. The stimulated PBLs were then washed in Hanks' bu~fered saline solution and 3 x 107 cells were brought up to a 30 ml volume in culture medium .. ;
.. ..
~ The infected monkey (Mm 167-84) that showed evidenc~ of severe immunological impairment at the start o~ treat~enS died, on the third day after the `~
co~ipl~tion of the 50 day treatment period, of severe Co~mb positive hemolytic anemia. In the last lO days bs~or~ death, the ani~al's hemoglobi~ dropped from 7.7 to 3 . 7 gra~s per deciliter. Lesions se2n at necropsy includ~d ~ large a~ount of clotted blood in the small i~testine, severe myocardial fibrosis and histiocytic - - ~ -infiltration of the splenic lymphoid follicles. These las~ two findings are not uncommon in rhesus monkeys chronically infecte~ with SIV. The presence of blood in the proximal small bowel indicated that the animal may have died from an acute gastrointestinal hemorrhage, possibly secondary to stress induced by the acute he~olytic process. Such signi~icant hemolytic ane~ias have been reported in AIDS patient~ ~Z~ A.
SchrQiber ~t al., ~loo~i, 5~, p. 117a (1983)].
Accordingly, d~ath may have been a natural conseguence of the SIV in~ection.
mao r~ YU/()U3~x 2~ 83 suppleme~ted with 2 units/ml IL-2 (IL-2 obtained from Du Pont). Following a 2 hour incubation at 37C, we washed the cells, added 3 ml SIVmac isolate 251 containing tissue ~ulture medium and incubated at 37C.
we harv~stad the supernatant at the time of peak reverse transcriptase tRT) activity (1.8 x 105 cpm/ml of RT activity) as described in M. Kannagi et al., "I~
Y1~LQ Growth CharacteristiCs o~ Simian T-Lymphotropic Virus Type III," Proc. ~tl. AC~ sci~-usA, 82, pp. 7053-57 (1985) and M. D. Daniel et al., Sci~e~çe, 228, pp. 1201-04 tl9a5)- The supernatant was passed through 0.45 ~m filters, divided into 1 ml aliquots and stored at -70C until use.
Pr~ar~ion o~ Bon~ M~rQw ~ tu~e~
HQparinized bone marrow ~ample~ were obtained ~rom ketamine ane~thetized monkays by posterior lliac cr~st aspiraticn. Mononu~lo~r c~lls w~r~ i~ol~tcd ~rom the bone ~arrow a~pirate5 by density gradient centrirugation, washed in ~anks' balanced salt solution and cultured in Iscove's modi~ied Dulbecco's MEM (IMDM) supplemented with 12.5~ FBS and 12.5% horse serum in 4 or 8 chamber tis~ue culture slides (~ab-Tek, Miles Scienti~ic, Naperville, Illinois) at a cell concentration of 1 x 105/ml. A~ter 7 days of culture 25 at 37C, nonadherent cells were removed. ~-~
PFeP~ration O~_~BL/H9 Co-Cultures Peripheral blood lymphocytes were isolated fro~ blood samples of monkeyc by Ficoll-diatrizoate density ~radient centrifugation (sp. grr 1.077) and sti~ulated with 1 ~g/ml Concanava}in A ~or 3 days as described 5~ - The stimulated PBLs wer~ waQhed in Hanks ' buffered saline solution and 1 x 106 cells were added to l x I0~ unin~ected H9 cells ~a gi~t ~r~m Wo~0/08198 PCT/~IS90tO03 2~ J

Dr. Robert C. Gallo, National Cancer Institute, Bethesda, Maryland) and the co-culture was maintained at 37C for a minim~lm of 3 weeks. We monitored reverse transcriptase activity in culture supernatants as an indication o~ virus replication.
A~i~avs 0~ An~i-Viral Activity In the~e examples, we evaluated the antiviral activities o~ the compositions o~ this invention using modifications oZ various 1~ vitro assays used to study anti-viral agents and neutralizing antibodies.
Rev~rse Transcri~t~e As~aY
Bone marrow cell samples, prepared as descrlbed ~a~, were incubated with a l:20 dilution o~
SIVm~C culture ~upernatant ~l.8 x 105 cpm/ml o~ RT
activlty) at a cqll concen~ra~ion o~ l x lO6 per ml ~or 2 hours at 37~C. ~he cells were then washed tw~ce in Hanks' balanced salt solution and placed in culture in complete medium. Alternatively, bone marrow cell ~amples obtained ~rom in~ected monkeys, or PB~/Hs co-cultures prepared as described supra, were placed inculture in complete medium. The culture medium was changed e~ery 3-4 days. Culture supernatant was harvested every 2-3 days and replaced with an equal volume of culture medium (RPMI 1640 ~ 10% fetal calf serum).
~ e then measured RT activities of harvested cultur~ supernatants as described below. Supernatants wi~h RT activity 5x backgrc~1nd or greater, i.e., greater than about lO00 cpm/ml, were scored as positive ;
~or in~ection with SIVmaC.
We measured reverqe transcriptas~ activi~y as an indlcator o~ the e~ect~ o~ the composition~ of this invention on HIV viral replication as described in ~090/oxls~ PCT/~590/~03~x 2 ~ 8 ~ - 2~

M. ~annagi et al., "In vitro Growth Characteristics Of simian T-Lymphotrophic Virus Type III," P~oc~_~atl.
Acad. sci. u~, 82, pp. 7053-57 (1985). Specifically, assay samples were prepared by placing 1.4 ml o~ test s sample i~ a 1.5 ml Eppendorf tube and centrifuging at 12,000 xg for ~0 minutes. we then removed the supernatant and incubated the pelleted virus on ice ~or 10 minutei~ with 20 ~1 of dissociation bu~fer [o.ol M
Tris-HC1 ~pH 7.~)/0.2% Triton X-100/o.001 M EDTA~0.05 M
dithiothr~itol/9.06 M Rcl]. We then mixed 15 ~1 of dissociated virus solution with 60 ~1 of RT cockta~l L0.05 M Tri~-HCl (pH 8.2)/0.007 M MgC12/0.06 M ~Cl/0.08 mg poly(rC).oligo(dG) primer per ml/0.007 M
dithiothreitol/3.3 ~Ci [a32P]dGTP ~3000Ci/mmol;
lCi-37GBg; Amersham)] and incubated at 37C ~or ~0 minutes. Wo then applied 60 ~1 o~ each sample on~o a Whatman 3 di~k and wa~hed each di5k in ~ beak~r with 5 tricholoroacQtic acid/2~ sodium pyrophoi~phat~. The disks were then drled, and the radioactivity o~ each disX was measured.
ColQn~ FQ~ti~n A~ays Bone marrow granulocyte-mo~ocyte (CFU-GM) and erythrocyte (~FU-E) progenitor cell growth of bone marrow cell sa~ples, prepared as described supra, were quantitated as fol}ows.
Ne established a two layer culture ~or tha quantitation of CFU-GM colonies. Th~ underlayer of 1 m} 0.5% Nobel agar contained 60 ng/ml ~c~mbinant human granulocyte-macrophage colony stimulating factor (a gi~t of Genetics Institute, Inc., Cambridge, ~assachusetts). The overlayer contained 105 bone marrow cells in 1 ml IMDM supplemented with 12.S~-FBS
and 12~5% horse serum in 0.3% Nobel agar. Wa layered the overlayer onto the underlayer in 35 X 10 mm Lux ,. ~ .. ,, - . . ..
: i. - : . : . . . . . . : : , . :

wf~90~08l98 P'CT/'~S90/00~Y
2 ~

tissue culture dishes ~Nunc, Ir.ffc., Naperville, Illinois) and the two layer cultures were maintained at 37OC in a 5~ CO2-humidified atmosphere.
BFU-E colonies were assessed in cultures of 5 105 bone marrow cells maintained in 0.9% w/v .
methylcff~ffllulose (Dow Chemical, Midland, Michif~an) in IM~M supplemented with 30% FBS, 0.9% deionized bovine serf~f albumin ~Fraction V, Sif~ma, St. Louis, Missouri~, 5 x 10 5 M 2-mercaptoethanol, containing 60 ng/ml I0 recombinant human granulocyte-macrophage colony stimulating factor, 1 unit/ml sheep erythropofietin (Step III, Connaught Laboratory, Willowdale, Ontario) and 5% phytohemaglutinin-stimulated rhesus monkey ccffnditioned medium. The phytohemaglutinin-stimulated rhesus monkf3y conf~fitioned medium wa~ prepareff~ as ~ollows. PBLfs weref isolatfefd ~xom heparinizf3fd blood o~
normal rhesufQf mfonkf~ys by Ficoll diatrizoatf~ dfefn~ity gradient centrifugatifcfn, a-~ defscribfsd ~ . The PBLfs were then incubated S days in culture medium (~PMI
1640 + 10% Pf~tal cal~ serum) supplemented with l0fugtml phytohemaglutinin. After incubation, the cells were .. `
pelleted, A'lld the supernatant was u~ed as the .;.
conditioned ~edium . :~
CFU-GM and BFU-E colonies of greater than 50 ~ -25 cells wf~fre then counted under an inverted microscope ~ :-12-14 days after the cultures were established.
Total-neutrophil counts in the peripheral blood were determined from complete blood co~nts and di~ferentials done on EDTA-anticoagulated blood ~:
samples.
Inhibition of SIVmaC Infection of Normal BQne Mar~o~æh~aefs ~__ f, We subjected bone marrfow cell cultures to SIV~aC and measured RT activities of culture .. ': ',: " :
' .'. , '' ', " ' , ' ' '' , ' ' , ` ' ' "" ';, ' ,. , ' ' ' .' "" ';'' ' . ' , . ' "i " . "' ' ' ` ' ' ' ~ , ' ' ' , ',"' '', ' ' . " ",, ' ,' ` '' ' ' " ' ' ' ": . '' ' ' ' ' '` ' ' ' ' ' , ,, ' ' ' , ~ ' " . ' ' ' ...... .,": ', ' . . . ''' '.;"' . . :. ', ,' . ,',, ' . ~' . ' ~ , ,:'.
, ,' ' ''' ' ''""''' "'' '':',' ' ,' . ., ' , ''' "' ' ' ' ;;. ' ' ' ' '', ;;'. ' ~ .~, ,'"; , ,' .' i ' ' . , ' , . ,. ,, " " . ' . " . ".. " . , .. .'. .. ' ' . ' ",. ' .,: ', .. , ' ~ ' ' ': . . . ' WO 90/08198 PCT/ ~ S9û/003~K
., 20 ~ 83 - 28 -supernatants as described supra. We found that more than 90% o~ bone marrow cultures established from non-infected normal monkeys could be reproducibly in~ected with exoge~ously introduced SIVmac (data not shown).
Bone marrow from both of the rsT4 treated normal monkeys was susceptible to infection ~ vi~
with exogenously introduced SIVmaC prior to and 4 weeks ~ollowing completion o~ treatment. However, three attempts at in~ection of bone marrow cell cultures from the rsT4 treated normal monkeys during the 50 day treatment period and a single attempt at 18 days after -treatment was completed did not yield positive cultures.
Speci~ically, w~ sampled bone marrow ~rom the rsT4-treated normal monkey~ on th~ day~ not~d b~low and introduced SIVm~a into cultures o~ th~s~ cclls lg day~
prior to treatm~nt, on d~ys 10, 30 and 40 during tr~atment, and on ~ays 13, 32, B7, 108, ~28, 156, 163, a~d 183 a~ter ~"po~t") treatmen~. RT activity was measured in supQrnatants oS thesa cultures. The results are shown in the table below and in Figure 3A.
REVERSE TRANSCRIPTlON ASSAY
OF SIVma~ REPLICATION INHIBITION
(Normal monkeY Mm 156-84) Day Of Culture 25 BonQ Marrow Peak RT Values On Which Peak Sa~ ay fcpm/ml~ RTI~asured -l9 2488 14 post-18 466 30 pos~-~2 1~36 50 post-87 10~7 15 W090/0~l98 PCTt~'S90/003 . Day Of Culture Bone Marrow Peak RT Values On Which Peak ~ample Day ~cpm/ml) RT Measured post-108 5R30 75 post-128 3621 50 post-163 7489 50 post-183 25~43 50 REVERSE TRANSCRIPTION ASSAY
OF SIVma~ REPLICATION INHIBITION
~Normal monkeY Mm 158-84~

Day o~ Culture 10 ~one Marrow Peak RT Values on Which Peak Sample Day (cpm/m~ RT ~ ç~ _ -19 182g ~4 .
1~ ~27 14 ~g0 30 post-18 419 30 post-32 754 50 post-87 3298 50 .
po~t-108 17~9 5~
post-128 1~757 50 post-156 ~ 22463 70 pos~-183 28163 50 ~ e also found that SIV~aC infection of normal monkey bone marrow macrophages was inhibited to a greater degree by plasma of rsT4-treated monkeys than by rsT4 itself. In this assay, we prepared bone marrow cell cultures a~ desicribed supra and added ei~her rsT~

.. . . . .. .... ....... .. . .. .. . .

wo 90/08198 PCT/~S90/003~
:, 2 ~^58~ - 30 -or plasma from an rsT4-treated monkey. We then subjected the cell cultures to exogenous SIVmaC as described ~YEE~-As shown in the table below, SIVmaG infection of normal bone marrow cell cultures was inhibited to agreater degree by plasma of the rsT4-treated monkey than by r~T4 itsel~, even at rsT4 concentrations exceeding the p~ak plasma levels o~ r~T4 achieved in the plasma o~ treated animals during the treatment period.*
RT ~cpm/ml~

Control 7489 rsT4 (200 ~g/ml) 6333 ~20 ~g/ml) S727 Plasma (~rsT4 Ab~ 5 331 (day 47 o~ treatmant (Mm 156-85)) 1:25 1284 As sho~n in Figure 3B, plasma ~rom day ~2 post-treatment o~ the rsT4-treated monkey Mm 156-84 also e~ficiently inhibited SIVmaC replication in PBLs of a normal rhesus monkey.
We also measured the reverse transcriptase activities o~ bone marrow cultures o~ rsT4-treated ~onkey~ or ~ro~ co-cultures o~ peripheral blood ly~phocytes of the treated ~onkeys and H9 cell~ before, ; during and~after the rs~4-treatment period.

* We observed that peak plasma levels of rs~4 in the treated animals were reaohed two hours after the first administration of rsT4 on d ay 1 o~ the treatment period. In normal monkey 158-84, a peak plasma level o~ ~457 ng/ml o~ rsT4 was pr~sen~ 2 hour~ after administration o~ rsT4 on treat~ent day l. In contrast, on day 47 of treatment, the peak plasma reaohed 2 hours a~ter administration was 243 ng/ml.

.. ,.. ...... , ., . ,., .,,,, " ,. .. .

WC~ 90/~)81 98 PCr/l 'S9OtO03'~;
f~ ?f ~f ~ 'r3 g As illustrated in Figure 4, co-cultivation of PBLs ofMm156-85, Mm 159-86 and Mm202-84 with H9 cells .
yielded SIV~ac prior to the start of treatment and up until approximately the end of the secon~ week o~ the rsT4 treatment period. Virus isolation attempts therea~ter were negative until, at th~ earliest, one month a~t~r completlon o~ treatment.
SIVm~C replication in bone marrow cultures, a re~lection o~ virus replication in macrophages rather than T lymphocytes, showed parallel changes. While virus was readily isolated from bone marrow prior to the initiation o~ treatment o~ Mm 156-85, Mm 159-86 and Mm 202-84, virus isolation att~mpts during the course o~ treatment were negatlve. Virui~ isolatlon~ ~rom bone marrow reverted to positive on day 18 ~t~r completion o~ treatment in Mm 1~6~85 and r~mained negatlve as lat~
as 45 days ~ollowing treatmant ln Mm 15g-B6 and Mm 202-8~.
In Mm 167-84, the monkey that was immunologically co~promised at the outi~et o~ treatment, virus was isolated only episodically ~rom PBLs ~ollowing co-cultivation with H9 cells both prior to and during rsT4 treatment. While isolation of virus from the bone marrow of Mm 167-84 was positive before , initiating treatment, three attempts at isolatin~ virus during treatmen~ were negative.
Thus, in the rsT4-treated SIVm~c-in~ected monkeys, viru5 was more di~ficult to isolate during period c~ rsT4 treatmen~ than before and after 30 treatment. We believe that these observations reflect `
a quantitative decrease in replication o~ virus in the infected monXeys due to the rsT4 treatment.

. . .

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- ~ 7~ U~ 1 70 ~CT/ ~S90~003~

20~8~ - 32 -Improvement in Functional Abnormalities Caused ~y SIV a ~Induced ~isease Activity _ _ we also found that functional abnormalitios reflecting SIVmac-induced disease activity in the rsT4-treated SIVma~-infected monkeys improved as a result of treatment with soluble T4 protein. Prior to rsT4 -treatment o~ the SIVmac-infected monkeys, the ln vit~o growth o~ both CFU-GM and BFU-E colonies from the bone marrow o~ these monkeys was depressed, as commonly found in SIVmac-infected monkeys and HIV-l-infected humans [C. C. Stella et al., J. Clin. Invest., 80, pp. 286-93 (1987)]. CFU-GM colonies in a series of assays of 10 normal anlmals were 2~2 ~ 52/5 x 104 cultured bone marrow cells and 3FU-E countq were 76 i 12/2 x 105 cultured bone marrow cell~. A5 a result o~ treatment, the depregsed CFU-GM ~nd BFU-E
colony count~ improved ~n the in~ct~d animals~ A~
shown in Figure~ 5 7, the number o~ colony ~orming and erythrocyte progenitor cells ~rom the bone marrow o~
the SIV-in~ected animals increased during the rsT4 treatment period.
A~ter treatment, a decline in bone marrow CPU-GM and ~FU-E colony counts to ~aseline values occurred in two of the three surviving SIV-infected 25 ani~als. The colony count of Mm 202-84 remained -~
elevated. A transient neutropenia occurred in two of the four ~onkeys during the first two weeks of treatment. As shown in Figure 5, however, we observed an eventual rise in total peripheral blood neutrophil counts in all four animals which accompanied the incrèased colony-forming cell counts fro~ their bone marrows.
Mn 156-85 was retreated in exactly t~e manner described su~ra a~ter a period of 85 days ~ollowing the first treatment. As shown in Figure 6, the same , " ', ': ' :', .'i . ' '. ' . . ' ' ", ' ' ', , ' ' "' ' ; ' , ` : . " ': ''`' " ,. ., ' . ' , ', .. . . .. . .... . .

WO~o/081~8 PCT/~SgO/003'~ ~
2 ~ ? ~ :

increases in CFU-GM and ~FU-E were seen associated with this treatment.
We also studied the ~n vitrQ effect of rsT4 or plasma from rsT4-treated monkeys on the colony forming cells from bone marrow o~ SIVma~-in~ected monkeys. The results are depicted in Figure 8, in which the "r~iT4 Added" control was no added rsT4 and the "Plasma Source'l control was plasma ~rom a normal monkey who had not been treated with rsT4. The "pre ~m" plasma source was plasma obtained from an infected monkey prior to rsT4 treatment. The plasma was maintained in the cultures at a 1:40 ~inal dilution. As shown in Fi~ure 8, the plasma o~ r~T4-treated monkays was ~ore e~ective in increa~ing colony ~or~ing cQlls than rsT4, even when high concentra~ions o~ rsT4 wcre addod.
Figur~s ~ and 10 depi~ th~ e~ect o~ plasma ~rom xsT4-treated monkeys on colony ~or~ing cells ~rom bone marrow Or S~Vmac-in~ected monkeys ZO harve5ted during rsT4 treatment (Figure 9) and harvested a~ter r~T4 treatment ~Figure lO). In those fi~ures, the "Plasma Source" controls and the "pre-Mm"
designations are the same as those re~erred to for Figure 8. A~ shown in Figure 9, colony forming cells in the in~ected animals increased over the course of treatment. Furthermore, upon re-treat~ent of an in~ected animal, CFU-GM cell counts again increased.
As shown in Figure 10, the incr~ase in CFU-G~ colonies of infected monkeysi caused by plasma fro~ rsT4 treated ~onkeys was still evident for some time after the end of the treat~ent period.
Figure 11 depicts the augmentation of CFU-GM
i~ yitro in bone marrow from SIVmac-infect~d ~nkeys ~ollowing addition o~ elther rsT4 or plasma ~rom rsT4-treated monkey. ~n Figure 11, the control used was ~JJu~ Pcr/~s9o/o~

2 0 ~ 34 -bone marrow without added rsT4 or added plasma. As shown in Figure 11, the plasma from an rsT4-treated monk~y caused a greater increase in CFU-GM colony counts of SIVmac-infected monkeys when compared with concentrations of rsT4 far exceeding those achieved in the plasma of the rsT4-treated monkey.
Figure 12 d~picts the rQsults of an assay carried out to determine what portion of the plasma was responsible for the colony formation augmenting lo activity observed. Speci~ically, we passed the plasma over a protein A Sepharose column. The flowthrough of the column was collected and retained as the "non-IgG"
fraction. The bound material was then eluted as follow~. The column was incubated ~or 30 minutei~ at room temperature in approximately 1 column volume o~
elution bu~er ~0.58 M acet~c acid, pH 2.~/0~15 M
NaC1). ~he column was then elutQd wi~h NaHC0~-neutralized elukion bu~cr, and the cluate wa~ d~alyzed overnight at room temperature against phosphate buf~ered saline. Thi5 dialyzed eluate was ratained as the "IgG fraction". We then te~ted those rractlons ~or their CFV-GM augmenting activity. In Figure 12, the control used was a bone marrow culture alone. As shown in Figure 12, the CFU-GM augmenting activity resides in the IgG fr~ction of the plasma of the rsT4-treated monkeys.
Charac~erization of the Antibody ~esponse Ind~ed y ~s~4 ~reatment _ _ We believe that the antibody response to soluble ~4 protein developed by the rsT4-treated monkeys was characteriæed by functional anti-viral properties responsible for the ~herapeutic e~ects observed. Accordingly, we next characterized the antibody response observed.

, , ,. : .. ,.,., . ,.: . , :,., .. : i, : : ' ' '' `

PC~/~S90/003~X

First, we determined whether peripheral blood lymphocytes circulating in the rs~4-treated rhesus monkeys had antibodies bound to their surface membranes. Moxe specifically, we evaluated two SIVmaC
infected r~T4-treated animals (Mm 202-84 and Mm 156-85) as ~ollows. PBLs ~rom heparinized blood were isolated 5 days after completion o~ a 50 day course o~ rsT4 treatment as described su ra. We incubated the PBLs in Yitro- with FITC-conjugated goat anti-human IgG (Tago) at a dilution o~ 1:40 in phosphate buf~ered saline for 30 minutes. ~he cells were then washed in Hanks' buffered salt solution (H8SS) and analyzed by flow cytometry ~Epics CS, Coulter Electronics, ~ialiah, Florida). ~S~e generally H. M. Shapiro, Cytomet~y, Alan R. Li5s, Inc., New York, New York ~1985)-]
The PBLs o~ Mm 202-84 and Mm 156-~5 e~hlbited sur~ace ~lu~r~saence o~ 31~ and 40%, respeatlvely.
PBLs o~ Mm 202-84 continued to demonstrate sur~ace staining as latQ as 71 days a~ter completion o~ rsT4 treatment. PBLs o~ Mm 156-85 exhibit~d less than 7~
cell sur~ace ~luorescence by 20 days after treatment was completed. However, upon retreatment of this animal with single intramuscular inoculations of 4 mg rsT4 in a 0.8 ml volume on day 20, and 5 mg in a 1.0 ml volume on day 34 ~ollowing completion of the first treat~ent course, an increase in the number of cells with bound surface Ig was observed. As shown in C
Figure 13, this level reached 50% of the cells by 42 days after completion of the 50 day treatment course.
Thus, the SIVmac-infected rsT4-treated rhesus monkeys clearly developed rsT4-treatment related immunoglobulin (Ig) coating of some of their circulating PBLs. This suggest~d that the treated monkeys developed anti~ody .

~, ~ ' '-' ' ,'' ' -'` :
,~ : ' "': . ~' ' ':. : . :',' ' :. , : . , ' ~ "

?????? PCT/US90/00358 responses with specificity for molecules expressed by some but not all circulating lymphocytes.
We then defined the specificity of the antibody response observed. Specifically, we tested plasma from Mm 156-84 for its ability to bind to purified CD8+ and CD4+ human PBLs. We has previously generated populations by antibody-mediated complement lysis utilizing monoclonal anti-CD4 (19Thy5D7) and anti-CD8 (7PT3F9) antibodies [Miskell and Shiigi, Selected Methods in Cellular Immunology, Freeman & Co.
1980, p. 211]. The anti-CD4 and anti-CD8 monoclonal antibodies used were gifts from Dr. S. F. Schlossman, Dana Farber Institute, Boston, Massachusetts. These antibodies are characterized in Leukocyte Typing II, Springer-Verlag, New York, New York, Reinherz et al., eds. (1986). First, we incubated the CD4+ and Cd8+ cell populations with the plasma of the rsT4-treated animal Mm 156-84 at a 1:40 dilution for 30 minutes at room temperature. Then, we washed the cells in HBSS and incubated for 30 minutes at room temperature in a 1:40 dilution of FITC-conjugated goat anti-human Ig. The cells were then washed in HBSS 3 times and analyzed by flow cytometry (Epics CS). As shown in Figure 14, approximately 94% of the CD4+ cells but less than 6% of the CD8+ cells exhibited fluorescence. Thus, Ig in the plasma of the rsT4-treated animal bound human CD4+ but not CD8+ cells.
We next assessed the plasma of an rsT4-treated monkey for binding to CD4+ and CD8+ rhesus monkey PBLs. PBLs from a normal rhesus monkey were isolated as described supra and reacted with 10 µl of either phycoerythrin conjugated anti-CD4 (OKT4, Ortho Diagnostics) or phycoerythrin conjugated anti-CD8 (OKTS, Ortho Diagnostics) for 30 minutes. We then washed the cells in HBSS and incubated them for 30 WC 90/08198 PCT/~:S9~/003'~;

2 0 ~ 5 ~ ~ 3 minutes in a 1:40 dilution of plasma from monXey ~m 156-84. The cells were washed in HBSS and incubated in phosphate bu~fered saline for 30 minutes at room temperature with a 1:40 dilution of FITC-conjugated ~oat anti-human Ig (Tago) and washed again with HBSS.
We ~ixed the cells in 0.1% paraformaldehyde-PBS, and analyzed them by two color ~low cyto~etry ~Epics CS).
As shown ln Figure 15, the plasma stalned CD4~ but did not stain CD8~ rhesus monkey PBLs. Thirty percent of the PBLs stained with both plasma and O~T4, 19% of the PBLs stai~ed with OKT4 and not plasma.
Im~unization O~ Normal Rhesus Monkeys Wi~h rsT4 In A~uvant A~ demon~trated above, the plasma o~ the rsT4-treated mon~eys ig highly e~ici~nt at blocking ln SIVmaC replication and at enhancin~ b~ne marrow hematopoietic ~unction. In additlon, the rsT4-treated monkeys developed an anti-CD4 antibody respons~. Based on these observations, we believed that very low doses o~ rsT4 doliv~red in adjuvant would provide a highly ef~icient way to generate an antibody response containing anti-SIVmac activity. To assess this belief, we obtained two normal rhesus monkeys, Mm 152-81 and ~m :
346-80. Both monkeys were colony born and raised at the New England Regional Primate Research Center, Sou~hborough, ~assachusetts. Each weighed 5~7 kg and was ~rse of infectious disease and in good health. we i~munized thase animals with 1 mg rsT4 in an emulsion with complete Freund's adjuvant (Sigma) on day O and another 1 mg in an emulsion with incomplete Freund's adjuvant (Sigma) on day 30. These immuniza~ions were each delivered subcutaneously with a total volume of 0.4 ml in 6 separate si~es on the back. T?~ ani~als were then bled twice monthly.

~ uJual~J~i PCT/~S90/003'~, 2~ 38 - `

we tested plasma from Mm 346-80 ~or Ig l:
binding to normal rhesus monkey PBLs.which had been con A activated and cultivated in IL-2 containing ~edium as d~scribed su~ra. As shown in Figure 16, binding to activated rhe~us PBLs was detectable by day 28 following initial immunization and was maximal on day 90, with blnding detected on 34~ o~ the cells.
Binding continued to be detected as late as 167 days ~ollowing initial immunization. ;
10The plasma of the r~iT4-immunized monkey Mm .
346 80 was then assesied for anti-SIV~jc activity in 'n Vit~Q cultures of PBLs and bone marrow. The techni~ues utilized ~or in~ection and determination o~ reverse transcriptase activity are de~cribed ~ . As shown in Figure 17, addition o~ Mm 346-80 ~lasma at a 1:40 ~inal dilution in RPMI 1640 plu8 10~ ~etal cal~ ~rum resulted in an almost ten-~old inh~bition o~ ~h~ RT
activity ganerated by th~ PBLs ~rom th~ SXVmac-in~ected rh2sus monkey Mm 351-85. As a positive control, Figure 17 also depicts the af~ect on reverse transcriptase activity of the anti-CD4 monQclonal antibody l9ThySD7 ~see 5up~a). As shown in Figure 18, the plas~a ~rom Mm 346-80 also caused a ten-~old inhibition o~ SIV~ac replication in bone marrow cells of -~
25 a normal rhesus mon~ey. :
We the~ tested the plasma of an rsT4- ~ -immunized rhesus monkey for its a~ect on he~atopoietic ~unction i~ itro using-methods described su~a. As : shown~in Pigure 19, a 1:25 and a 1:125 final dilution of plas~a of Mm 346-80 caused a significant increase in the CFU-GM from bone marrow of three SIVmac-infected rhesus monkeys (Mm 244-86, Mm 108-84 and Mm 169-79). 1 ;
We also tested the rsT4-immunized animals to determine whether any anti-viral activity wa~
detectable in their bone marrow cells or PBLs. These ~O~O/OXl98 PCT/~'S90/00 determinations ~ere carried out as described s~ra forassessm~nt of n vitro suscep~ibility of bone marrow cells to infection with SIVmaC. Following ~mmunlzation, both bone marrow cells and PBL-, from the rsT4-i~munized rhesus monkeys Mm 1~2-81 and Mm 346-80 were periodically placed in culture with various dilutions o~ cell-~r~e SIVmaC. The RT activity in these culture supernatants was determined as described ~ . As show~ in Fiyures 20 and 21, signi~icant resistance to SIVmaC rep}ication was detectable by day 62 a~ter the .
initial immunization and remained until approximately day 145. Thereafter, the PBLs and bone marrow cells o~
thes~ animals appQar~d to be as ~usceptible to SIV~
infcction as they were prior to immunization.
Immunii~ation o~ SIV -In~ected Monkeys ~i~h r~r4 ln_,~di~va ~
W~ next determin~d the th~rapeutic a~icacy rsT4 in adjuvant. The three SIVmac-in~ected monkeys utilized ~Mm 179-86, Mm 104-86 and Mm 388-87) were colony born and raised at the New England Regional Primate Research Center, Southborough, Massachu~etts.
Each weighed 5-7 kg and was infected experimentally with SIV~a~ isolate 251 4-12 months prior to initiating this study. Two of thP monkeys (Mm 179-86 and M~ 104-86) were i~unized with rsT4 in complete Freund's - adjuYant on day O and in incomplete Freund's adjuvant on day 30 as described supra. The third monkey (Mm 388-87) was used as a control and immunized with adjuvant as described supra, but with 1 mg human serum albumin (HSA) in place of rsT4. The presence o~ virus in the bone marrow and PBL of these animals was khen determined periodically, as described ~E~
As shown in Figure 23, viru9 isolations from ~one marrow cells and PBLs were positive prior to the wU ~/u~PCI /~S90/Oû3~
,~
2~4~5~3 - 40 -initial immunizations in all three monkeys. Virus isolations remained positive in the HSA-treated animal throughout the course of the assay. However ~ollowing .
the second rsT4 immunization on day 30, virus isolations from bone marrow and PB~ from Mm 179-86 became negative until day loO. Moreover, except for ona positive virus isolation ~rom PBLs and bone marrow o~ monkey Mm 104-86, virus lsolations ~rom Mm 104-86 also b~came negative ~rom th~ time of the second immunization on day 30 until approximately day lOo.
As shown in Figure 24, during this treatment, CFU-GM and BFU-E from bone marrow cells of the two rsT4-immunized monkeys but not ~rom bone marrow cells o~ the HSA-immunized monkey rose to the normal range.
This incrQase in hQmatopoietic pot~ntial indicates an improvement in an SIVmac-induced ~unctional abnormality.
Advantangeou~l~, th~re~ore, the lmprov~d cl~nical and virologic parameter~ ob5~rved Pollowing daily treatment with soluble ~4 protein may alternatively be achieved by immunizatio~ with low doses of soluble T4 protein in adjuvant.
Whlle we have hereinbe~ore descr~bQd a number of embodiments of this invention, it is apparent that our basic con~tructions can be altered to provide other e~bodim~nts.~.hich u.tilize the processes.and composi-tions of thi8 invention. Therefore, it will be appreciated that.-the scope of thiS invention i5 to be defi~ed by the claims appended hereto rather th~n by the specific e~bodiments which have been presented hereinbefore by way of example.

Claims (33)

1. A method for treating or preventing AIDS, ARC or HIV infection in a patient comprising the step of administering to the patient a pharmaceutically acceptable composition which comprises an immunologically effective amount of a soluble T4 protein.

2. The method according to claim 1, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.

3. The method according to claim 2, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.

4. The method according to claim 2, wherein the soluble T4 protein is modified by the addition of a pharmaceutically acceptable adjuvant.

5. The method according to claim 4, wherein the soluble T4 protein is administered to the patient at a dosage of about 1 mg/kg body weight/day.

6. The method according to claim 3 or 5, wherein the composition is administered to the patient for a period of time between about 1 and 30 days.

7. A pharmaceutically acceptable composition for treating or preventing AIDS, ARC or HIV
infection in a patient which comprises an immunologically effective amount of a soluble T4 protein.

8. The composition according to claim 7, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.

9. The composition according to claim 7, further comprising a pharmaceutically acceptable carrier.

10. The use of a pharmaceutically acceptable composition which comprises an immunologically effective amount of a soluble T4 protein for the treatment or prevention of AIDS, ARC or HIV infection.

11. The use according to claim 10, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.

12. The use according to claim 11, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight/day to 1.0 mg/kg body weight/day.

13. The use according to claim 11, wherein the soluble T4 protein is modified by the addition of a pharmaceutically acceptable adjuvant.

14. The use according the claim 13, wherein the soluble T4 protein is administered to the patient at a dosage of about 1 mg/kg body weight/day.

15. The use according to claim 12 or 14, wherein the soluble T4 protein is administered to the patient for a period of time between about 1 and 30 days.

16. The method according to claim 1 or 2, wherein the pharmaceutically acceptable composition further comprises an adjuvant.

17. The method according to claim 16, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight to 1.0 mg/kg body weight per administration.

18. The method according to claim 17, wherein the soluble T4 protein is administered once a week until the desired immunological effect is achieved.

19. The method according to claim 18, wherein after the desired immunological effect is achieved, the soluble T4 protein is administered once a month.

20. The method according to claim 1 or 2, wherein the pharmaceutically acceptable composition further comprises incomplete Freund's adjuvant.

21. The method according to claim 20, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg to to 1.0 mg per patient per administration.

22. The method according to claim 21, wherein the soluble T4 protein is administered about once a month for two months and is thereafter administered about once every three months.

23. A pharmaceutically acceptable composition for treating or preventing AIDS, ARC or HIV
infection which comprises an immunologically effective amount of a soluble T4 protein and an adjuvant.

24. A pharmaceutically acceptable composition for treating or preventing AIDS, ARC or HIV
infection in a patient which comprises an immunologically effective amount of an adjuvant and a soluble T4 protein that is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.

25. The composition according to claim 23 or 24, further comprising a pharmaceutically acceptable carrier.

26. The use of a pharmaceutically acceptable composition which comprises an immunologically effective amount of a soluble T4 protein and an adjuvant for the treatment or prevention of AIDS, ARC
or HIV infection in a patient.

27. The use according to claim 26, wherein the soluble T4 protein is modified to increase its immunogenicity beyond that of a soluble T4 protein endogenous to the patient.

28. The use according to claim 27, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg/kg body weight to 1.0 mg/kg body weight per administration.

29. The use according to claim 28, wherein the soluble T4 protein is administered once a week until the desired immunological effect is achieved.

30. The use according to claim 29, wherein after the desired immunological effect is achieved, the soluble T4 protein is administered once a month.

31. The use according to claim 24 or 25, wherein the pharmaceutically acceptable composition further comprises incomplete Freund's adjuvant.

32. The use according to claim 31, wherein the soluble T4 protein is administered to the patient at a dosage equal to or less than about 0.1 mg to to 1.0 mg per patient per administration.

33. The use according to claim 32, wherein the soluble T4 protein is administered about once a month for two months and is thereafter administered about once every three months.