IE59814B1 - Method and composition for the prophylaxis and treatment of retroviral infections - Google Patents

Abstract

Retroviral infections e.g. Aids or Aids related complex, are prevented or treated by administration of tumor necrosis factor, optionally with an inteferon; and preferably by continuous infusion of tumor necrosis factor and interferon.

Description

METHOD ass composition fob, Tbe s&epsaaw AND 3SSA3SEST OF SETRSVXOL INFECTIONS 1$ ‘Shis Isventloa relates to the prevention and treatment of infections fey retroviruses. is particular, It Is oaacesned with the therapy of retroviral infection* leading to IsKsaslsgical defects, Is particular these infections thoeghs te fee responsible 2Θ for acquired bunas deficiency syndrome AIM is a traasaissifele deficiency of cellular Isssunity characterised by opportunistic infection* «ad certain malignancies „ notably gjaetawcvstl* carlaii pneusoaia and Kaposif« sareo:aa, ia patients without «aether reeegsaised cause for contracting these rare disease* ¢1-3). AIDS is manifested fey * profound lysqpfeopenia, a generalised cutaneous energy «nd a markedly seduced proliferative response to Mitogens „ antigen* and allogeneic cell*, seeaing to result freaa depletion of the CKM* T~lymgto»cyte subset ¢4). While huaoral iasstaity i* relatively unaffected, there 1* Increasing 3® evidence for a hyperactive B-cell proliferative response which say fee related causally to the high Incidence of E-lymphoma in AIDS patients (5,6). la addition to the fully developed syndrome, an epidemic ef a related disease, AIDS-related complex (ARC), has X£8xOS9.bk3& -2- . appeared, characterized by generalised chronic lyqphadenopathy. This syndrome shares many of the epidemiological features and isnune abnormalities and often precedes the clinical manifestations of AIDS.

Recent evidence has implicated strongly a lymphocytetropic retrovirus as the primary aetiological agent of AIDS and the AIDS-related complex. Lysaphadenopathy-assoclated virus (IAV) was isolated initially from cultured lysaph-node T cells of patients with lynphadenopathy and AIDS as well as an AIDS patient and· an asymptomatic sibling, both with haemophilia 3 (7-9). A similar virus, designated human T-lyaphotrophic virus type III (HTLV-III), has bees isolated from a large nsaafeer ©f AIDS and ARC patient blood samples by co-cultivation with the permissive T-cell line 99 (10, 11). LAV and HTLV-III, as well as related retroviruses isolated recently from AIDS patients (12,13), share several important characteristics. Viral replication ©eeurs In the 0^14* T-lyaphocyte population in yiyo .and in vitro and is associated with impaired cell proliferation and the appearance ef cytopathie effects (8,10,14). The virus has a Mg^*-dependent reverse transcriptase, exhibits a dense cylindrical core aorphology sinilar to type D retroviruses (8,13,15) and is recognized by antibodies found ia the sera of virtually all AIDS and ARC patients (8,13,15-21). ETLV-IH and LAV now are believed t© be strains of the same virus, which has been given the name human iaaunodef iciency virus (sIV) .

Issssae function in AIDS or ARC afflicted patients is severely coaprooised. Sowever, the precise nature of she abnormalities induced fey HIV continue to be under study. Of particular interest was the effect of HIV infection ©n the production of lysphokines by the iassune cells of such patients as well as their responses to exogenously adainistered lynphokines.

Of 16 AIDS patients tested for T lymphocyte capacity to secrete naerophage-activating products, including ganaa interferon, 14 failed to generate active lysphokines and 13-14 coapletely failed to secrete gamma interferon. Furthermore, aacrophages from 1X8x089 .mdh -3IS patients with AIDS showed enhanced aatinlcrobial activity when Incubated in vitro with gasm© Interferon, thus raising the possibility that such cells aay respond ja viyp to exogenously administered gsosaa interferon (22, 2d).

Lymphocytes fre© AIDS patients also have been reported to fee deficient In their ability to produce interleukin* 2, a lymphokine having a role in the proliferation and differentiation of X lymphocytes and which stimulates geass Interferon production (23).

Finally, gasasa interferon was know® to exert a direct antiviral effect oa vesicular stomatitis virus (VSV) Infected cells and to potentiate the effect of a cytokine on VSV infected cells (25) . · This cytokine «as tentatively identified by these authors as the polypeptide referred to herein as tumor necrosis £actor-o. See also Slfel et si..., Cell. Itmun. &7: 197-203 (1979). Issunosuppleseatatlon therapy for AIDS therefore appeared to be a promising opportunity, and clinical studies with patients were instituted.

Despite the expectations for lysphokine therapy of AIDS patients, In yiw results have been disappointing. Contrary to the results encountered in ip vitro experiments, a prolonged course of intravenous recoabinaat gaassa interferon treatment appeared to inhibit rather than enhance csonocyte respiratory burst function (26), sad intravenous treatment of AIDS patients with varying doses of interleukin* 2 or gaama interferon led t© the conclusion that in vivo lympheklna therapy was not of significant value In the treatment of patients with AIDS (27). In fact, fey the tine of the invention herein, * school of thought had developed that the IsaEune stimulation or reconstruction approach t® AIDS therapy was actually dangerous because sell activation by lynphokines was thought to contribute to virus spreading and TA cell depletion, and thus would accelerate progression of the d!sease(23). In Jus©, 1985, laaeasto al. (29) disclosed that human alpha and beta (but not gsoaa) interferons suppressed the in vitro replication of ΗΓ9 strains, but when exposure to the LC8x089 .Eidh <*£*,*> isxsrfexoris ceased ch® viral production of infected cells was enhanced. This further suggested that issaune therapy of AIDS would he counterproductive.

Finally, it has been suggested that lyaphotoxis* is produced hy HIV-infected X cells by virally-induced eramsactivstism of the T cells1' endogenous lyaphotoxin gene, thus leading to secretion of autotoxic quantities of lymphotoxin (35) .

Ttsaor necrosis factors are polypeptides produced lay aitogen-stianslaced aacrophages (INF-a) or lymphocytes (TSF-0) which are cytotoxic for certain malignantly transformed cells. TNF-a has been suggested co be responsible for wasting and cachexia in patients with cancer of severe infections (34), .and passive lammisatlon against T8iF-.« was reported to project alee against the lethal effect ©f endotoxin (30). Th.® antl-tunor effect of TNF-a is known to he syaergistlcally potentiated by interferons, and the aati-tuaor effect of Έ8Ρ~/3 Is similarly potentiated by interferon gessaa. The anti-tumor effect of TNF=a and TNF~$ in admixture, however, are only additive, as axe the antiviral effects of interferons alpha and beta.

It is aa object of this invention to employ iseunotherapy in the successful treatment of persons Infected with retroviruses, in particular lyaphoeytotropie viruses such as BIv.

It is a further object herein to treat those at risk for active retroviral infection, e.g. persons harboring latent retrovirus .

Aa additional object ' is. to provide iaauaotherapeutic prophylaxis against retroviral infections.

These end other objects of the invention will fee apparent from consideration of the specification as a whole. g-W>asv Unexpectedly, ϊ have discovered that administration of a therapeutically effective amount of a tumor necrosis factor alone or, preferably, in eonbinatlom with as* interferon, confers 1X3x089 .mdh «5«* protection on persons at risk to retroviral infection and kills retrcvirally infected cells, while gamma·interferon alone exerts little or ss protective activity «gainst retroviral infection nor is gfisms interferon significantly cytotoxic tc retrovirally infected cells s and INF alone is only modestly active st high concentrations , the combination. of these two agents is dramatically potent. This phenomenon ie observed in xisn, despite the deranged condition of the Issauas systems of retrovlr ally -infected patients. 'This result «as particularly surprising because THF was not known to fee deficient is such patients. gsia£Jtea-s^s^9U-9XJ^-fjUs«r.gg.

Figure 1 Is a tferthera gel shewing the dranatlc reduction In HIV sSKA la HIV-infected HuT73 cells after pretreatnent with, INF-e* and IFH-y as csrapared to control cells that «ere not pretreated .

Figure 2 deswsnstrates the antiviral protective effect of catalase ia combination with HIF-e ead/or ISB-y.

Retroviruses are defined as viruses containing single or double stranded EHA. These viruses replicate by harnessing th® cellular metabolism &£ permissive hosts to reverse transcribe the 3H& genetic naterial of the virus. The large eaounts of DHA so produced are trans lated int© HIV protein and ERA for the assembly of progeny vlrlsrs, Examples of such viruses Include the so-called slow er lentlviruses «ad the I-cell leukemia viruses such as HTLV-I and HTL.V-IX, but most preferably ere eh® HIV strains associated with AIDS.

Th® tumor necrosis factors useful herein Include TNF-e and TNF-,0. The former is described in copending U.S.S.R. 881,311, filed July 2, 1988, together with methods, for its synthesis in reecmbinant cell culture. Similarly, the latter (previously called lysrphotoxin) and suitable recombinant synthesis methods are LC8xO8S.sadh =6 described in sopending U.S.S.N. Sla,502, filed Hay 31, 1984 and U.S.S.H. 732,312, filed May 9, 1985. The TMF-β and THF-/? described in these applications include cytotoxic eaino acid sequence and glycosylation variants which also ar® used herein. Of course, TSfFa ©r THF-/? from non-recoabinant sources are also useful in the saethod of this itventim. * TMlF~« or TNF-^ are used alone or ia admixture with ose another in proportions empirically determined to exert th® .most effective clinical response. TNF is sot species specific, s© THFs from other aniiaal species, e.g. porcine or bovias, are useful herein. The preferred TN? is satur® human THF-β fron recombinant aierohial cell culture. The TS? ordinarily will have a cytolytic activity en susceptible L-929 ssarine cells ef greater than about 1x10® wnlts/sg, wherein a wait is defined as set forth in the above-described patent applications, the disclosures of which are incorporated by reference.

Interferons are well Ssaowa. In nature they comprise che γ and about 20 different interferon-» subtypes. Their assess relevant characteristic for the purposes herein is that they b® capable ©f protecting cells in vitro and its vivo from viral infection. The interferons used ia the process or eosposicioa ©f this invention typically are interferons-», and/er γ. Interferons produced in recombinant cell culture, free natural isolates or fey stable untransforaed cell lines are satisfactory for use herein, as are Interferon anino acid sequence or glycosylation v&risats (including unglycosylated ferns) so long as they exhibit anti-viral activity, Interferon-γ should be of Che sane animal species for which therapy is Intended because interferon-y is known to be species specific. The interferons desirably are substantially homogeneous asd will have a specific activity la excess of about 1x10® International Unics/ng.

The compositions herein include a. pharmaceutically acceptable vehicle such as those heretofore used ia the therapeutic adalnistratlon of interferons, TS? ©r IT, e.g. physiological saline LC8x0S9.sdh -710 or 5% dextrose, together with conventional stabilizers and/or excipients such *s husa&n serum albcmi® or aamaitol. The compositions are provided lyophilized ex in the fossa ef sterile aqueous solutions.

Z&veTal variables will fee taken Into account by th® ordinary artisan in determining the proportions of interferons, es, β or y and ©f TKF-α or TWF-^, the net proportion of interferon to TNF. the concentration of interferon and TNF in the therapeutic compositions and she dasages co be administered on a Kg basis. Therapeutic variables .also include the aniasal species to fee treated, the adaisd.ssraci.es route, asd the clinical condition of the patient (including the stage and degree of retroviral and/or adventitious organise» infection„ if any, preheat at the ccraaeaceB©nt of treatment) . Doses of Interfere® ranging about froa 1 to 50 p:g/sa~ are suitable initial dosing levels. The tolerated dose aay not exceed about 25 .

The INF and Interferon doses are adainlstared together or separately. If the latter, the interferon should be administered first and the T331F thereafter within 24 hours. Is Is within the scope ef this invention so administer the ZHF and interferon In multiple cycles, 'depending upon the clinical response of the patient. This approach to the therapy will be effective la attacking . latently-Infected cells entering the active phase of infection, whether due to exogenously administered T cell cuLtogens ©s adventitious infections that lead to 1 cell activation.

Since the treatment with TST end Interferes will lyse vitally-Infected cells asd oay result In tin© release of infective virus, it Is advantageous in the course a£ tikusns&y to odainister substances: capable of neutralizing further viral Infectlvlty. This can be accomplished by several methods. One can administer antibodies such && aoaoclonal cr polyclonal anti-retroviral antibodies during the course of therapy, preferably at the ssa® time as TKF Is edsiinistered. Alternatively, isswaaaologically competent patients con be vaccinated against «he retrovirus in order to actively LCSxO89.tadh -810 induce neutralizing antibody. A suitable vaccine for this purpose eoatalas the HIV gpl20 ewv and is described in U.S.S.M. 861,016, filed May 8, 1986. This vaccine is disclosed to induce HIV neutralizing antibodies, which in turn can be -used in the passive isaunlzation strategy described above. Other agents that interdict the potential Inxeccivlty of released viruses also can b© administered together with the TMF, for exsusple gp!20 eny or fragments thereof which hind to the cell surface receptor ordinarily recognized by the retrovirus In question (in the case ox HIV, the ©ΚΪ4* cell surface Barker present oa helper T cells) and thus competitively inhibit viral adhesion te target cell surfaces.

The synergistic antiviral and aati-cussor activity ©f T®i? and/or aa interferon is further potentiated by including in the treatment regimen and/or compositions a therapeutically effective amount of aa oxygen free-radical scavenger (including oxygen protective enzymes and peroxidatively active substance). Such substances, including catalase, superoxide demutase, peroxidase ©r chloroperoxidase , greatly enhance the activity of TKF and interferes. Such enzymes are respectively hoesa to catalyze the breakdown of EgO? to water asd oxygen SHgO * O—E-O, although at present the Bs&daasaisia The TfiiF and Interferons are administered by the same ©r separate routes, for example fey intravenous, intranasal or Intramuscular actelnis trat ion. Either ©r both components ear fee administered fees sustained release co^ositiosis, for esasr>le as LC8xO89.Bdh -910 IS polylactide ©r polyhydroxylfeutyrate Implant® off liposomes such as are described in S? 17.2007A, or by continuous Infusion. At the present time it is preferred to inf we the TNF and interferon intravenously is the dosages described above.

In order to avoid side effects froa the combined therapy «men high doses are used, the TRS* and interferon can be ««ployed in an extracorporeal treatment regimen heretofore referred t© as adoptive ixanwotherapy (U.S.S.N.s 763,637 «ad 763,570, filed August 8, 1985 and June 11, 1985, respectively). In these methods the peripheral blood mononuclear or lymphocytic cells ©f a patient are separated from the blocd in «a extracorporeal plasmapheresis cycle, treated wish interleukin-2 ©r interferon, and chess reinfused into the patient. The patient"s iasacae responses against malignancies were greatly stimulated by this procedure. For the treatment of retroviral infections, the same general procedure is employed except chat the peripheral taonocytes and/or lymphocytes are incubated in the presence of TNF or TNF and interferon. Vlrallyinfected cells are killed by TSF or the combination of agents. The extracorporeal cells also can fee incubated with a killer sell activating agent such as a lymph cell mitogen (e.g., phytohesaaggluitinin) and/or interleukin-2. The cells then are washed and resuspended in «a infusion medium for reinfusion into the seae patient from wfaoa the cells were first obtained. Th© reinfusion can be followed* up fey administration of TMF end interferon as is otherwise provided herein. The optimal amounts of TJsT or T^F and Interferon, and the optimum conditions for extracorporeal treatment of the patients® lymph cells are routinely determined fey assaying the patients1 viral titer and assessing Improvements in the patients" clinical condition.

Persons that are candidates for treatment la accordance with Sals Invention - are those at risk for exposure c© retroviral Infection or who exhibit signs of actual exposure to such an infection. Those at risk Include members'-©£ high risk groups such as homosexuals, Intravenous drug ’users and those who haw® received LC8x089.radh -ιο10 transfusions sr other product:® made fra® blood not «screened for HIV antibodies. Evidence of exposure te retrovirus includes seroconversion e© antibodies against HIV, positive serua, assays for B1V„ symptoms associated with AIDS-related coaplex (AHC) or frank AIDS. AIDS patients may or may not be diagnosed for Kaposi's sareooa or other malignancies, adventitious microbial infections such as £n.eu»acystls_- .carinil or thrush, neural impairment or cachexia (wasting) .

The invention will be sore fully understand its. the light of the following examples. The interferons were the products of recombinant bacterial cell culture and were purified to a specific activity? of about 10® usits/ag. ISF-e «red ΐΒΕ-β also 'were recced»inant, and had a Specific activity of about 5x10? uaits/ag. All literature citations are hereby incorporated by reference.

Example 1 JEagfelaKH &S. .W-J^gyl^as The CKT4*-positive feuss® T-cell leuSceaia cell lines HS, BuT/B and '0937 were cultured in suspension with SEMI-1640 sediua at 37*S. Cultures were centrifuged to separate cells froa thi® aediwa and the cells tfo.es resuspended la fresh H2MI-1640 at a density of lxl0®/WL. Sufficient 3NF-e and gaaeaa interferon were added to the resuspended culture to produce 0.1 ,^g/«l TNF-a and 0.1 ^g/al gas=a interferon. The combination of TNF-« and gamma interferon was not toxic tc uninfected BaT78 and H9 cells. Sue culture then was incubated at 37*0 for 24 feours, after waiea 1x10^ qpa milts of HlV/ml was added. The cpm units were determined by reverse transcriptase activity (31); each unit is calibrated to represent the reverse transcriptase activity of one virion. After 3 days of further Incubation at 37* C the cells were screened for viral antigens hy indirect Immunofluorescence using a murine monoclonal antibody against the p2A (core) protein of ΒΓΡ and labelled goat aatl-csaus.® ismsa&globulln (32). The result* of replicate experiments are shown below as the percentage of cells which LCSxO8©.sadh -ΛΛcontain HIV core protein.

Table 1 HuT78 S9 U937 1 . 2. τ . ..2 . . 1 ?. Control 54 S3 48 36 11 14 TNF-e 27 34 32 18 7 8 garsva interferon 63 51 42 41 12 11 TNF-s and gasses®. Interferon 1 8 4 3 2 1 These results 'demonstrate that combination therapy with TNF interferon is highly effective In preventing HIV infection of otherwise susceptible T cells.

Example 2 3tesaCT8&^^af^gE^x3^£gS£gCJSsilA 1x3.0^ H9> and RFMI-1788 lyssphoblastoid cells/aal of ΒΒίίϊ1640 aediwa were exposed to IxlCp eps of BlV/sal la the presence of 1 pg/ssl poly&rene la order to enhance the percentage of cells infected by HIV. These cells were cultured for about 30 days, during which the culture aedlua was replaced every 3-5 days and cells subcultiared approximately ©nee a week. Sufficient TNF-a and gamsaa interferon were substantially simultaneously added to cultures containing 1x10® eells/ssl in order t© establish concentrations as shown in Table 2 below. Gaabinations of TNF-β and garasa Interferon at these concentrations were aot cytotoxic towards uninfeeted S3? and Rj?MI»178S cells. The cultures were Incubated at 37" C for 3 'days and then assayed for the percentage of viable cells by trypan blue staining. The results of replicate experiments are shows in Table 2 (ND — net done). 1X8x089 .sadh -12IS Table 2 ggasgnaca&tgEL .,.& -ViableC<11 & H9 1788 1 -2 1 Cenex©! -- 89 76 95 86 THF-a 1.0 ng/ml MD HD 90 81 0.1 ^g/sl 85 74 SI 76 iGasssa 1.0 ng/ml HD ND 83 84 Isterferon 0.1 Mg/ml S5 74 SO 82 THF-e and Gam 1.0 ng/ml HD HD SS 72 Interferon 0.1 Mg/ml 50 42 34 53 These results demonstrate concentration - dependent synergistic killing o£ vitally-Infected sells By combinations of THF asd Interferon sad, co a lesser 'degree, THF-sx alone. This, seisblsed with the protective effect of THF or the combinations fsx «alafected sells, demonstrates the value of THF sx combination therapy la the treatment ©f retroviral infections.

SxisspXe 3 aa3 JSgsfefeHg teJSsmgy HuT7S cells were treated with ©.1 ^sg/al each of TNF-e and gaana interferon and then, infected with ΗΓ7 as described in Sasample 1. After 2 days ©f incubation ac 37" C total SNA. was extracted fro® the HIV-Infected cells as follows. The cells were washed 'with EDS and resuspended in 0.35 al TSH buffer (10 aii Tris pH 7.5, 0.15 H 2?&C1, 2cM HgClg) containing 0.5% HE-40 and. 17 μ! VSC (BSX> at about O'"S. After 3-5 min. nuclei were centrifuged free of the lysed cells:. The supernatant containing sSHA was added te 0.35 al ef TSE buffer (10 s« Tris pH 7.5, 0.15 M NaCl, 5 sii EDTA) containing It LC8X089 .ssdh -13SDS and extracted 3x with 0.7 si of phenol: chloro form: iso-emylalcohol (24:24:1). The phenol was equilibrated with water and 0.1% 8-OE-quinoline. HHA was precipitated fro» the aqueous layer with ETCH and sodium acetate. Foly(A) BM& was prepared by '®lxgo(dT) cellulose electrophoresis (35). Sorthem hybridization using 1 tyg RHA/lane was performed as described (37). The results, shown in Fig. 1, demonsstate that THF-» pretreataaent was able co suppress the appearance of HIV SNA in host cells, but that tax less KJSA appeared «ήβη THF and interferon were used together. These results 2.Q are consistent with the Infectivity data shown in Table 1. Ho change in actin &3HA (a structural protein) «as observed with or without THF and interferon treatment,. demons crating that the effect of TMF~« and interferes^ was targeted to the virus rather than cell growth in general.

Stamp!® 4 Hales seropositive for HIV antibody and having blood which is ip ZlXSS cell culture positive for demonstrable HIV titer are enrolled. These patients generally present with synptotaology cons xs tent with AIDS or ARG, and were serocoixwerted for HIV antibody. The treatment group is divided into cohorts based on the β « following treatment variables: Siswaltaneows or sequential THF and interferon treatment.

Intravenous cs Intramuscular Injection asd, in the ease ef intravenous injection, by bolus or continuously by infusion (1,, 2, 5 «md 10 days). Dosage regimen ever hoars, starting at pg/m^ of THF, and ^5g/s- ef Interferon, and increasing te S, 10, 25, 30 ... gssg/sr of each agent as tolerated.

Ratios of THF to interferon: 1:100 ts» 100:1 Repeat cycles cf treatments: I to 5, with Inter25 1. 2. 4. . 1X3x089.mdh -1410 mediate treatment suspensions af 1, 2, 5 ax 10 days. 6. Selection and proportions of interferon types: se and y„ 1:10 -10:1; β and γ, 1:10 - 10:1; ys a or β alone, 1:1:1 to 10:1:1 to 1:5:5.

A suitable starting regimen will comprise treating ASC patients with a ceebinacion of THF-β, ganeaa interferon and alpha Interferon. The combination Is administered by coatimous Infusion using an intravenous infusion pump calibrated to deliver & dosage of 1-10 pg/a2/24 hours TNF-ae 1-10 pg/m"/24 hours for gamma interferon and 1-10 pg/a^/24 hours for alpha interferon. Fatieats may fee escalated to higher doses as tolerated. This infusion is continued for ©a® week. patients are rested for another week and the infusion repeated. Side effects such as fever, chills and the like ar© treated fey conventional means sr fey reducing the dosage. When enti-3XV antibody or gpl20 eaty are employed as antiviral agents in coofoination with the above, the antibody or env polypeptide are present in a dssage calculated to fee capable of sequestering virions released fey the cssbined therapy. This will depend ©a the affinity of antibody for virion and. its neutralising titer as well as Che viral titer of the patient. Deteselnatlon of suitable dosages will fee within the skill of the routineer.

The clinical condition aad viral lafecvivity titers ©£ the patients are monitored during asd after the treatment protocol. Consistent wits, the la vitro studies Is Esanples 1-3s the issaste competence asd viral titer of a statistically significant proportion of the patients improved as a result of the treatment.

Example 5 Cstaj^sejegtanEiat^^lieL^aEiMSiy^fiSlEiSZ a£.Jte^grfi?sgssdX.aBdZgs. IRKra A549 cells were seeded at 2xl0&/»ell In 96-well flatbottes trays (Falcon Plastics) for 24 hr prior to incubation with The test samples were as shown in Fig. 2 (CAT Is The samples otherwise contained Dulbeeeo modified test sacples, catalase).

W8x089 .sadfe -15Saglees (DKS) sedlta isupplssaeated with 5 percent beat-Inactivated fetal calf serum (FCS), glutasine (2 cM) „ penicillin (100 U/sal) „ and streptomycin (100 pg/al). After 18 hr of incubation at 37C, culture supernatants were replaced with fresh BHB eedl^ss free of Interferon, TKF ©r catalase but containing 2 percent fetal calf setts and EMC virus at a multiplicity ©f Infection (MOI",, the ratio of infectious virus/cell) of 1. The cytopathic effect (CFE) was determined fey staining the viable cells with crystal violet and the titer vas. quantitatively monitored using a siereeliss autol0 ««der (HSS80. By^eech) «=S further eotfiarf vitally. Th. CHS antiviral titer is expressed as the reciprocal ©f the dilution found to inhibit 50 percent of the cell eytopathy and was standardized against the international reference sample of hsaaaa Interfere®—y (Ho. f3g 23-901-530). " " Ώ«· 2· ««!»«> -SaMXKaaly *=synergistlcally enhanced the anti-viral effect of interfere®—? and TMF~e„ This effect is not .unique t© EHC virus. It has bees observed with a variety of permissive cells and viruses. 1438x089 .ndh -15« ΒΤ3Ε.Ϊ OGR a owv Gottlieb, Μ. et al.. New Sagl. J. Bed. 305:16251631(1981).

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Claims (25)

CLAIMS:

1. The use of a tumor necrosis factor alone, or a tumor necrosis factor and an interferon, in the manufacture of an anti-retroviral or prophylactic medicament for administration to a patient previously infected or at risk for such an infection., with the proviso that when tumor necrosis factor is the sole active ingredient the medicament is for the treatment or prophylaxis of HIV viral infection.

2. The use of claim 1 wherein the tumor necrosis factor is tumor necrosis factor-c or tumor necrosis factor-8.

3. The use of claim 2 wherein the tumor necrosis factor is tumor necrosis factor-cu

4. The use of claim 1 wherein the interferon is interferon a, β, or γ.

5. The use of claim 1 wherein the interferon is interferon-γ.

6. The use of claim 1 wherein the medicament is to he administered to a patient infected with HIV.

7. The use of claim 6 wherein the patient is afflicted with AIDS-Related Complex.

8. The use of claim 1 wherein the medicament is to he administered to a patient free of any detectable malignancy.

9. The use of claim 1 wherein the medicament is for administration ex vivo in. the course of adoptive immunotherapy. -2010.

10.The use of claim 9 wherein the administration of either the tumor necrosis factor alone or the tumor necrosis factor and interferon occurs intravenously or intramuscularly after the course of the ex vivo adoptive immunotherapy.

11. The use of claim 1 wherein the medicament comprises both tumor necrosis factor and interferon.

12. The use of claim 11 wherein the interferon is to be administered prior to administration of the tumor nacrosis factor.

13. The use of claim 11 wherein the interferon is a mixture of gamma interferon plus alpha or beta interferon.

14. The use of claim 12 wherein the interferon is gamma interferon.

15. The use of claim 11 wherein the dosages of tumor necrosis factor and interferon are each about from 1 to 25 pg/m2/24 hours by continuous intravenous infusion. 1 δ.

16.The use of claim 11 wherein the medicament further comprises antibody capable of neutralising viral infectivity.

17. The use of claim 11 wherein the administration of tumor necrosis factor and interferon is to Poe repeated through about from 1 to 5 cycles. -2118.

18.The use of claim 6 wherein the medicament comprises means for immunising the animal against Ηϊν prior to administration of either the tumor necrosis factor alone or the tumor necrosis factor and interferon.

19. The use of claim 11 wherein the medicament is administered by continuous intravenous infusion for about from 1 to 5 days.

20. 20, The use of claim 1 wherein the medicament further comprises a physiologically acceptable oxygen free-radical scavenger substance.

21. The use of claim 20 wherein the scavenger substance is 15 catalase.

22. The use of claim 21 wherein the scavenger substance is human erythrocyte catalase. 20

23. A composition comprising a tumor necrosis factor, an interferon and an oxygen free-radical scavenger substance.

24. A composition of claim 23 wherein the substance is a 25 peroxidatively active enzyme.

25. A composition of claim 23 wherein the enzyme Is human erythrocyte catalase. 25» A composition of claim 23 further comprising a substance capable of preventing (a) replication of a retrovirus or (b) retroviral binding to a cell surface receptor. -2227. A composition comprising a tumor necrosis factor, an interferon and a substance capable of preventing the (a) replication of a retrovirus or ib) retroviral binding to a cell surface receptor. 28. The composition of claim 27 wherein the substance capable of preventing retroviral binding to a cell surface receptor is an antibody directed against an envelope polypeptide of the retrovirus. 29. A composition according to claim 23, substantially as hereinbefore described. 30. A composition according to claim 27, substantially as 15 hereinbefore described.