CA2180193A1 - Enhancing host immunity against viral infections - Google Patents
Enhancing host immunity against viral infectionsInfo
- Publication number
- CA2180193A1 CA2180193A1 CA002180193A CA2180193A CA2180193A1 CA 2180193 A1 CA2180193 A1 CA 2180193A1 CA 002180193 A CA002180193 A CA 002180193A CA 2180193 A CA2180193 A CA 2180193A CA 2180193 A1 CA2180193 A1 CA 2180193A1
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- CA
- Canada
- Prior art keywords
- antiestrogen
- interferon
- host
- virus
- tamoxifen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Cell mediated immunity against persistent viral infection in a host is potentiated by administering to the host an effective amount of at least one antiestrogen capable of enhancing cytotoxic T lymphocytes in the host, such as a triphenylethylene tamoxifen, particularly tamoxifen or toremifene. This administration leads to the elimination of all infected cells and to the complete clearance of virus and virus induced tumor cells from the body, leaving protective cell mediated immunity. This therapy requires MHC restricted virus specific CTLs which then will kill virus infected very specifically, which may be initially present or formed in vivo or in vitro.
The effect of the antiestrogen may be enhanced by the use of interferons optionally with indomethacin.
The effect of the antiestrogen may be enhanced by the use of interferons optionally with indomethacin.
Description
218~19~
.
'rITLE OF INVENTION
EN~IANCING ~IOST lM~lNl-l~ AGAINST VIRAI lN~i~;
FIBLD OF INVENTION
The present invention relates to immunology.
~3ACKGROUND OF INVENTION
Viru3es carry infectious genetic material (either DNA or RNA) with the capacity of entering susceptible host cells and redirecting the biosynthetic ~ ;nPry of the infected cells towards the multiplication of viral particles (virions) . The profound conversion of the host cell toward viral production results in cell lysis in most cases (cytotoxic viruses), whereas in other cases the virus has no cytolytic effect, but rather persists within infected cells in the absence of morphological alteration. However, such virus infected cells may show a severe impairment of functions that are not nf~C~ ry for cell survival (e.g. the secretion of hormones, antibodies, and other important molecules) (Fenner &
White, 1976; de la Torre et al., 1991).
The DNA viruses most frequently causing persistent infections in man are cytomegaloviruses, the Epstein-Barr virus, herpes viruses (types I and II), hepatitis viruses (B, D), papilloma viruses, and occasionally measles virus. Recently, an association has been found between type II diabetes and the persistence of cytomegalovirus in the pancreas. Herpes viruses persist in nerve cells and may cause severe ;nfl: tory disease in the nervous system and also in various other parts of the body after having travelled along the nerve f ibers and triggering inflammation in the area of innervation of the fibers infected. The Epstein-~garr virus is known to cause infectious ~n~n~ eosis~ and contributes to the pathogenesis of Bur]{itt's lymphoma, primarily in Africa, and nasopharyngeal carcinoma in the Orient. Hepatitis viruses cause ;nfl: tory liver disease which may become 218019~
~ .
persistent in some individuals leading to cirrhosis. In addition, the hepatitis B virus has been; ~l~nt; f i ed as a contributing agent to the genesis of hepatocellular carcinomas. The measles virus may also persist in the nervous system and :is known to cause the condition called subacute sclerosing rAnlon~rhA~7itis~ Papilloma viruses (Papova group) exist in many species, including man, and are known to cause common warts and genital tumors.
Progressive multifocal encephalop~thy i8 also caused by Papova virus (Fenner & White, 1976; ~;r^lr-n et al., 1985; Chow, 1993 ) .
The common RNA viruses with the potential of causing persistent infections in man are hepatitis A and C, rubella, the human T lymphocyte virus, types I and II
(HTLV), which are kl1own to cause lymphomas, and the human immunodeficiency virus (HIV) which is recognized as the causative agent of acciuired immunodeficiency syndrome (AIDS) . HTLV and HIV possess an enzyme, called reverse transcriptase, which i8 capable of transcribing the viral RNA to a DNA templa.te which in turn will permanently be inserted in the genome of the host cell. For this reason they are classified as retroviruses. Both DNA and RNA
tumor viruses carry genes that are capable of growth stimulation of the host cell (viral oncogenes) (Fenner &
White, 1976; Chow, 1993).
Persistent viral infections of farm animals that have economic signiEicance are: aviaiî leukosis caused by retroviruses (Okazaki et al., 1982); bovine leul~osis, another retroviral disease (Daniel et al., 1993); bovine viral diarrhea (RNA pestivirus) (Thiel et al., 1993);
bluetongue, a disease of rllr;n^ntA transmitted by insects and caused by RNA viruses (arboviruses) (Sellers, 1981);
equine infectious anemia caused by retroviruses (Sellon et al., 1994; Kennedy, 1992); retroviral disease of sheep caused by the Maidi-Visna virus (Kennedy, 1992); porcine ~ 21801~3 reproductive and respiratory syndrome caused by RNA
lentivirus (Thiel et al., 1993).
Virus infected cells and virus induced tumors carry specific antigens on their surface which are recognized 5 by the immune system of the host. These antigens may stimulate cell r---l;At~d and humoral immune reactions.
Major histocompatibility complex antigen (MXC) restricted cytotoxic T lymphocytes, which are capable of specif ically killing virus inf ected cells, are 10 f1ln~l; tAl to hogt defence against viruses. Xowever, it is recognized that natural killer cells and activated macrophages and the cytokines that are capable of activating these effector cells, namely IL-2, interferon-~ and ~y, and migration inhibitory factor, also contribute 15 significantly to host defence against viral infections.
Interf erons Cl~ and ~ have the capacity to inhibit the intracellular replication of viruses. Although some antibodies are capable of neutralizing viral particles, humoral immunity i8 considered to play a secondary role 20 in host defence against viral infections (Fenner h White, 1976; Rinaldo & Torpey, 1993).
Initially, cell mediated immunity is present in immunocompetent hosts infected with persistent or oncogenic viruses. Xowever, with time a gradual 25 reduction occurs in the cell mediated antiviral/antitumor response, which is followed by nonprotective humoral responses, and some of the antibodies produced may actually interfere ~lith cell ~--l;Ated immunity (~nhAnr;ng antibodies) . Virus infected non-neoplastic cells may be 3 0 similarly protected . In the case of tumors, immune derived growth factors may actually have a direct promoting effect on tumor growth which is known as immunostimulation. Apart from inducing a switch in the host immune response, which permits the persistence of 35 viral infections and the growth of virus induced tumors, numerous other ~ A-l I have been proposed by which n~ truction of infected cells can be avoided. Some of these are: molecular mimicry, growing in immunologically privileged sites (such as the central nervous system), rendering immunocytes nonfunctional by direct infection, triggering nonspecific cytotoxic and autoimmune reaction8 (Oldstone & Rall, 1993; Tishon et al., 1993; Roger et al., 1994; Seligmann, 1990). HIV
exposed individual~3 who are capable of resisting the establishment of persistent infection possess MHC-re3tricted cytotoxic T lymphocytes, but remain seronegative (Rowland-Jones et al., 1995) .
Recent evidence indicates that virus specif ic CTL
can be recovered from EIIV infected individuals and from AIDS patiellts with active disease by in vi tro stimulation with antigen and cytokines such as interleukin-2 or interleukin-12 (Clerici et al., 1993; Walker, 1989;
Whiteside, 1993). Similarly, progressing neoplasms contain tumor inf i].trating lymphocytes (TIL) which are inefficient in rejecting the tumor in vivo, but can be reactivated in vitro to exert tumor specific cytotoxicity (Berczi et al., 1973; Schendel & ~'.AnRhA~h~'r, 1993;
Ioannides et al., 1993). Such reactivated CTL is being tested at present for the immunotherapy of cancer and of some viral infections (Kradin et al., 1989; Topalian et al., 1988; Torpey et al., 1993; Whiteside et al., 1993;
Riddell et al., 1993).
SUM~ARY 0~ THE Il~VENTIO~
We have discovered that the treatment of Epstein-Barr virus (EBV) transformed human B lymphocytes with the antiestrogenic agents, tamoxifen (TX) or toremifene (TO), enhances the destruction of these cells when used as targets for specific autologous CTL and LAK cells (see Tables 1 and 2 below). Currently, tamoxifen and toremifene are used in medicine for the treatment of estrogen receptor positive breast carcinomas and some 2I 8~1 93 -other estrogen dependent tumors. Human s lymphocytes do not express classical estrogen receptors, which suggests that the sensitizillg effect is mediated by some other receptor .
Having regard to the new observations we have made, the present invention, in one aspect, provides a method for the potentiation of cell ~ terl immunity against persistent viral infections by administration to a host of at leagt one antiestrogenic agent capable of Pnh~nr; n~
cytotoxic T lymphocytes (CT~), which is expected to lead to the elimination of all infected cells and to the complete clearance of the virug and virus induced tumor cells from the body, leaving behind protective cell mediated immunity. The prerequisite for this adjuvant therapy iB the presence oi MHC restricted virus specific CTL which kill virus infected target cells very specifically. The elimination of virus producing cells leads to virus clearance and lasting cell-m~ ted immunity. ~e showed earlier that TX iB immunosuppressive (Nagy ~ Berczi, 1986; Baral et al., 1979) . Therefore, TX
and possibly T0, have a dual function during therapy with CT~, namely the sensitization of target cells and the inhibition of switch to humoral, nonprotective immune responses .
In accordance with one aspect of the invention, immunity in persistently infected hosts can be potentiated to cause viral clearance according to the following steps:
a. detecting MHC restricted cytotoxic T
lymphocytes in the llost, b. demonstrating ill vitro that antiestrogens enhance the killing of virus infected target cells, and c. treating the infected host with an efiective amount of at lea6t one triphenylethylene antiestrogen to potentiate the killing of virus infected cells in a synergistic manner by CTL
21801~
To fortify the immune response in hosts with advanced persistent viral infections in order to induce viral clearance and cure, the present invention, in another aspect, provides a method, as follows:
a. P~nrl;n~ killer cell~ in tissue culture and reinfusing to the host, b. testing that antiestrogens enhance the cytotoxic action of these cells on virus infected targets, and c. treating the host with an effective amount of at least one antiestrogen.
The l-nh:qn, t of viral immunity in chronically infected host3 may be e~fected, in accordance with a further aspect of the invention as follows:
a. treating the host with an effective amount of at least one cytokine, such as interleukin-2 (IL-2) or interleukin-12 (I~-12), to induce and/or enhance cell mediated immune responses in the infected host, b. demonstrating CTL activity and antiestrogen induced Pnl~nc -nt of virus infected target cell destruction, and c. applying antiestrogen therapy in order to potentiate the killing effect of cytotoxic cells induced.
The treatment of hosts infected with persistent viruses may additionally be carried out as follows:
a . inducing virus specif ic CTL in the host by vaccination, b. demonstrating CT~ activity and antiestrogen induced Pnh~n~e-^nt of cytotoxicity, and 3 0 c . initiatin~ antiestrogen therapy as soon as virus specific CTL is demonstrable in the immunized host.
Interferon-~ (IFNo!) is being used currently for the treatment of viral hepatitis and some other viral diseases. In order to take advantage of the anti-viral effect of interferon and the ~nll~n,- t of host immunity by antiestrogens, we te~ted whether or not interferons ~ 218~1 93 and antiestrogen~ can be applied jointly. As shown in Tables 3, 4 and 5 below, antiestrogens had a modest l.nh~n~-;n~ effect on the cytotoxic action of killer cells as did interferons. Joint application of interferons 5 with anti-estrogens resulted in a further increase in cytotoxicity. Interferons are known to induce pro3taglandins, whi ch in turn can inhibit cytotoxicity .
For this reason, we tested ;n~l th~f~in (INDO) in order to see whether or not prostaglandin mediated inhibition 10 exists in our system (Rossol et al., 1992) . As shown in Tables 4 and 5 below, the joint application of antiestrogen plus irlterferon-~y plus indomethacin resulted in the highest cytotoxicity. This was true regardless of whether the killer cells were pretreated or when the 15 drugs were applied during the cytotoxic reactions 80 that both target and effector cells were exposed to them. On the basis of these results, the following re, rli~t;ons are made f or treatment:
a. Use antiestrogen and interferon jointly to 20 enhance host immunity and virus clearance in all situations listed above.
b. Additional treatment with ; n~ h~cin, especially in combination with interferon-~y and antiestrogen, is rF-~ ~.od for best results.
DETAI~ED ~ESCRIPTION OF THE INVENTION
A. General treatment protocols 1. Treatment of virus infected hosts in an early stage .
a. Virus infection is determined by the demonstration of viremia with the aid of tests for viral nucleic acid and/or viral antigen. An alternate method may be the detectiol~ of virus specif ic antibodies in the serum .
b. The presence in the infected host of ma~or histocompatibility antigen restricted cytotoxic T
lymphocyte~ CR~CT~) using in vitro methods of cytotoxicity is ~1P~Prm; nPd . If killer cell activity is found, the effect of TX and T0 and other effective antiestrogens on the in vitro - cytotoxic reaction is 5 PYAm; nf~
c. If MHCR-CTL is present in the host in suf f icient level and TX and/or T0 or other antiestrogen is found to sensitize virus infected target cells in vitro for immune cytolysis, the host is treated with at 10 least one antiestro~en. Treatment with antiestrogen may be combined with treatment with at least one interferon and, optionally, ;n~ hA~;n and maintained until virus clearance is achieved.
d. If no significant decrease in viral titer is 15 observed in the blood during four weeks of treatment, additional treatment with interleukin-2 or interleukin-12, which are known to stimulate CT~ activity, may be ef f ected .
- e. An alternate solution to the problem of 20 insufficient viral clearance is the expansion of MHCR-CT~
in tissue culture and reinfusion of the PyrAn~lpd CTL
population to the host. Such treatment may be combined with cytokine treatlTlent, such as with I~-2 and/or I~-12.
f. Vaccination may be an additional way to 25 increase the level of MHCR-CT~ in the host which could then be followed by treatment with antiestrogens in order to promote the destruction of infected cells and viral clearance .
.
'rITLE OF INVENTION
EN~IANCING ~IOST lM~lNl-l~ AGAINST VIRAI lN~i~;
FIBLD OF INVENTION
The present invention relates to immunology.
~3ACKGROUND OF INVENTION
Viru3es carry infectious genetic material (either DNA or RNA) with the capacity of entering susceptible host cells and redirecting the biosynthetic ~ ;nPry of the infected cells towards the multiplication of viral particles (virions) . The profound conversion of the host cell toward viral production results in cell lysis in most cases (cytotoxic viruses), whereas in other cases the virus has no cytolytic effect, but rather persists within infected cells in the absence of morphological alteration. However, such virus infected cells may show a severe impairment of functions that are not nf~C~ ry for cell survival (e.g. the secretion of hormones, antibodies, and other important molecules) (Fenner &
White, 1976; de la Torre et al., 1991).
The DNA viruses most frequently causing persistent infections in man are cytomegaloviruses, the Epstein-Barr virus, herpes viruses (types I and II), hepatitis viruses (B, D), papilloma viruses, and occasionally measles virus. Recently, an association has been found between type II diabetes and the persistence of cytomegalovirus in the pancreas. Herpes viruses persist in nerve cells and may cause severe ;nfl: tory disease in the nervous system and also in various other parts of the body after having travelled along the nerve f ibers and triggering inflammation in the area of innervation of the fibers infected. The Epstein-~garr virus is known to cause infectious ~n~n~ eosis~ and contributes to the pathogenesis of Bur]{itt's lymphoma, primarily in Africa, and nasopharyngeal carcinoma in the Orient. Hepatitis viruses cause ;nfl: tory liver disease which may become 218019~
~ .
persistent in some individuals leading to cirrhosis. In addition, the hepatitis B virus has been; ~l~nt; f i ed as a contributing agent to the genesis of hepatocellular carcinomas. The measles virus may also persist in the nervous system and :is known to cause the condition called subacute sclerosing rAnlon~rhA~7itis~ Papilloma viruses (Papova group) exist in many species, including man, and are known to cause common warts and genital tumors.
Progressive multifocal encephalop~thy i8 also caused by Papova virus (Fenner & White, 1976; ~;r^lr-n et al., 1985; Chow, 1993 ) .
The common RNA viruses with the potential of causing persistent infections in man are hepatitis A and C, rubella, the human T lymphocyte virus, types I and II
(HTLV), which are kl1own to cause lymphomas, and the human immunodeficiency virus (HIV) which is recognized as the causative agent of acciuired immunodeficiency syndrome (AIDS) . HTLV and HIV possess an enzyme, called reverse transcriptase, which i8 capable of transcribing the viral RNA to a DNA templa.te which in turn will permanently be inserted in the genome of the host cell. For this reason they are classified as retroviruses. Both DNA and RNA
tumor viruses carry genes that are capable of growth stimulation of the host cell (viral oncogenes) (Fenner &
White, 1976; Chow, 1993).
Persistent viral infections of farm animals that have economic signiEicance are: aviaiî leukosis caused by retroviruses (Okazaki et al., 1982); bovine leul~osis, another retroviral disease (Daniel et al., 1993); bovine viral diarrhea (RNA pestivirus) (Thiel et al., 1993);
bluetongue, a disease of rllr;n^ntA transmitted by insects and caused by RNA viruses (arboviruses) (Sellers, 1981);
equine infectious anemia caused by retroviruses (Sellon et al., 1994; Kennedy, 1992); retroviral disease of sheep caused by the Maidi-Visna virus (Kennedy, 1992); porcine ~ 21801~3 reproductive and respiratory syndrome caused by RNA
lentivirus (Thiel et al., 1993).
Virus infected cells and virus induced tumors carry specific antigens on their surface which are recognized 5 by the immune system of the host. These antigens may stimulate cell r---l;At~d and humoral immune reactions.
Major histocompatibility complex antigen (MXC) restricted cytotoxic T lymphocytes, which are capable of specif ically killing virus inf ected cells, are 10 f1ln~l; tAl to hogt defence against viruses. Xowever, it is recognized that natural killer cells and activated macrophages and the cytokines that are capable of activating these effector cells, namely IL-2, interferon-~ and ~y, and migration inhibitory factor, also contribute 15 significantly to host defence against viral infections.
Interf erons Cl~ and ~ have the capacity to inhibit the intracellular replication of viruses. Although some antibodies are capable of neutralizing viral particles, humoral immunity i8 considered to play a secondary role 20 in host defence against viral infections (Fenner h White, 1976; Rinaldo & Torpey, 1993).
Initially, cell mediated immunity is present in immunocompetent hosts infected with persistent or oncogenic viruses. Xowever, with time a gradual 25 reduction occurs in the cell mediated antiviral/antitumor response, which is followed by nonprotective humoral responses, and some of the antibodies produced may actually interfere ~lith cell ~--l;Ated immunity (~nhAnr;ng antibodies) . Virus infected non-neoplastic cells may be 3 0 similarly protected . In the case of tumors, immune derived growth factors may actually have a direct promoting effect on tumor growth which is known as immunostimulation. Apart from inducing a switch in the host immune response, which permits the persistence of 35 viral infections and the growth of virus induced tumors, numerous other ~ A-l I have been proposed by which n~ truction of infected cells can be avoided. Some of these are: molecular mimicry, growing in immunologically privileged sites (such as the central nervous system), rendering immunocytes nonfunctional by direct infection, triggering nonspecific cytotoxic and autoimmune reaction8 (Oldstone & Rall, 1993; Tishon et al., 1993; Roger et al., 1994; Seligmann, 1990). HIV
exposed individual~3 who are capable of resisting the establishment of persistent infection possess MHC-re3tricted cytotoxic T lymphocytes, but remain seronegative (Rowland-Jones et al., 1995) .
Recent evidence indicates that virus specif ic CTL
can be recovered from EIIV infected individuals and from AIDS patiellts with active disease by in vi tro stimulation with antigen and cytokines such as interleukin-2 or interleukin-12 (Clerici et al., 1993; Walker, 1989;
Whiteside, 1993). Similarly, progressing neoplasms contain tumor inf i].trating lymphocytes (TIL) which are inefficient in rejecting the tumor in vivo, but can be reactivated in vitro to exert tumor specific cytotoxicity (Berczi et al., 1973; Schendel & ~'.AnRhA~h~'r, 1993;
Ioannides et al., 1993). Such reactivated CTL is being tested at present for the immunotherapy of cancer and of some viral infections (Kradin et al., 1989; Topalian et al., 1988; Torpey et al., 1993; Whiteside et al., 1993;
Riddell et al., 1993).
SUM~ARY 0~ THE Il~VENTIO~
We have discovered that the treatment of Epstein-Barr virus (EBV) transformed human B lymphocytes with the antiestrogenic agents, tamoxifen (TX) or toremifene (TO), enhances the destruction of these cells when used as targets for specific autologous CTL and LAK cells (see Tables 1 and 2 below). Currently, tamoxifen and toremifene are used in medicine for the treatment of estrogen receptor positive breast carcinomas and some 2I 8~1 93 -other estrogen dependent tumors. Human s lymphocytes do not express classical estrogen receptors, which suggests that the sensitizillg effect is mediated by some other receptor .
Having regard to the new observations we have made, the present invention, in one aspect, provides a method for the potentiation of cell ~ terl immunity against persistent viral infections by administration to a host of at leagt one antiestrogenic agent capable of Pnh~nr; n~
cytotoxic T lymphocytes (CT~), which is expected to lead to the elimination of all infected cells and to the complete clearance of the virug and virus induced tumor cells from the body, leaving behind protective cell mediated immunity. The prerequisite for this adjuvant therapy iB the presence oi MHC restricted virus specific CTL which kill virus infected target cells very specifically. The elimination of virus producing cells leads to virus clearance and lasting cell-m~ ted immunity. ~e showed earlier that TX iB immunosuppressive (Nagy ~ Berczi, 1986; Baral et al., 1979) . Therefore, TX
and possibly T0, have a dual function during therapy with CT~, namely the sensitization of target cells and the inhibition of switch to humoral, nonprotective immune responses .
In accordance with one aspect of the invention, immunity in persistently infected hosts can be potentiated to cause viral clearance according to the following steps:
a. detecting MHC restricted cytotoxic T
lymphocytes in the llost, b. demonstrating ill vitro that antiestrogens enhance the killing of virus infected target cells, and c. treating the infected host with an efiective amount of at lea6t one triphenylethylene antiestrogen to potentiate the killing of virus infected cells in a synergistic manner by CTL
21801~
To fortify the immune response in hosts with advanced persistent viral infections in order to induce viral clearance and cure, the present invention, in another aspect, provides a method, as follows:
a. P~nrl;n~ killer cell~ in tissue culture and reinfusing to the host, b. testing that antiestrogens enhance the cytotoxic action of these cells on virus infected targets, and c. treating the host with an effective amount of at least one antiestrogen.
The l-nh:qn, t of viral immunity in chronically infected host3 may be e~fected, in accordance with a further aspect of the invention as follows:
a. treating the host with an effective amount of at least one cytokine, such as interleukin-2 (IL-2) or interleukin-12 (I~-12), to induce and/or enhance cell mediated immune responses in the infected host, b. demonstrating CTL activity and antiestrogen induced Pnl~nc -nt of virus infected target cell destruction, and c. applying antiestrogen therapy in order to potentiate the killing effect of cytotoxic cells induced.
The treatment of hosts infected with persistent viruses may additionally be carried out as follows:
a . inducing virus specif ic CTL in the host by vaccination, b. demonstrating CT~ activity and antiestrogen induced Pnh~n~e-^nt of cytotoxicity, and 3 0 c . initiatin~ antiestrogen therapy as soon as virus specific CTL is demonstrable in the immunized host.
Interferon-~ (IFNo!) is being used currently for the treatment of viral hepatitis and some other viral diseases. In order to take advantage of the anti-viral effect of interferon and the ~nll~n,- t of host immunity by antiestrogens, we te~ted whether or not interferons ~ 218~1 93 and antiestrogen~ can be applied jointly. As shown in Tables 3, 4 and 5 below, antiestrogens had a modest l.nh~n~-;n~ effect on the cytotoxic action of killer cells as did interferons. Joint application of interferons 5 with anti-estrogens resulted in a further increase in cytotoxicity. Interferons are known to induce pro3taglandins, whi ch in turn can inhibit cytotoxicity .
For this reason, we tested ;n~l th~f~in (INDO) in order to see whether or not prostaglandin mediated inhibition 10 exists in our system (Rossol et al., 1992) . As shown in Tables 4 and 5 below, the joint application of antiestrogen plus irlterferon-~y plus indomethacin resulted in the highest cytotoxicity. This was true regardless of whether the killer cells were pretreated or when the 15 drugs were applied during the cytotoxic reactions 80 that both target and effector cells were exposed to them. On the basis of these results, the following re, rli~t;ons are made f or treatment:
a. Use antiestrogen and interferon jointly to 20 enhance host immunity and virus clearance in all situations listed above.
b. Additional treatment with ; n~ h~cin, especially in combination with interferon-~y and antiestrogen, is rF-~ ~.od for best results.
DETAI~ED ~ESCRIPTION OF THE INVENTION
A. General treatment protocols 1. Treatment of virus infected hosts in an early stage .
a. Virus infection is determined by the demonstration of viremia with the aid of tests for viral nucleic acid and/or viral antigen. An alternate method may be the detectiol~ of virus specif ic antibodies in the serum .
b. The presence in the infected host of ma~or histocompatibility antigen restricted cytotoxic T
lymphocyte~ CR~CT~) using in vitro methods of cytotoxicity is ~1P~Prm; nPd . If killer cell activity is found, the effect of TX and T0 and other effective antiestrogens on the in vitro - cytotoxic reaction is 5 PYAm; nf~
c. If MHCR-CTL is present in the host in suf f icient level and TX and/or T0 or other antiestrogen is found to sensitize virus infected target cells in vitro for immune cytolysis, the host is treated with at 10 least one antiestro~en. Treatment with antiestrogen may be combined with treatment with at least one interferon and, optionally, ;n~ hA~;n and maintained until virus clearance is achieved.
d. If no significant decrease in viral titer is 15 observed in the blood during four weeks of treatment, additional treatment with interleukin-2 or interleukin-12, which are known to stimulate CT~ activity, may be ef f ected .
- e. An alternate solution to the problem of 20 insufficient viral clearance is the expansion of MHCR-CT~
in tissue culture and reinfusion of the PyrAn~lpd CTL
population to the host. Such treatment may be combined with cytokine treatlTlent, such as with I~-2 and/or I~-12.
f. Vaccination may be an additional way to 25 increase the level of MHCR-CT~ in the host which could then be followed by treatment with antiestrogens in order to promote the destruction of infected cells and viral clearance .
2. Treatment of hosts with advanced infections but 3 0 no apparent disease .
a . The diagnosis of inf ection and the presence of ~CR-CTL is done as described under 1. In the present case, it is expected that antibodies are present and nonspecific cytotoxic ~^~hAn; ~mc also operate (for 35 example nonspecific CD8+ CTI" antibody dependent cellular cytotoxicity, and complement fixing An~iho~;es), 21~0193 .
g whereas MHCR-CTL may be weakly cytotoxie or completely paralyzed. Therapy is contraindieated with central nervous system involvement.
b. Reactivate MHCR-CTL il:L vitro using antigen 5 presenting cells and cytokines, such as Il,-2 and/or I~-12 .
e. Establish that antiestrogens enhance cytotoxieity .
d. Expand the eultures and reinfuse into the host 10 while antiestrogen treatment is applied.
e. Additional treatment with IL-2 and/or IL-12 in order to help ~';nt_;n eytotoxieity at a signifieant level and treatment with interferons is re~ n~_tl to limit virus multiplication.
3. Vaccination.
a. The difficulty of vaccination against persistent viral infections i8 that nonprotective or even _nh_n~-~;n~ immune reactions may also be induced, which would interfere with MHCR-CT~ that mediate host response.
I.ive virus vaecines that are capable of infecting the susceptible cell3 have been found to be far superior for vaccination than killed viruses. The apparent explanation f or this is that only virus inf ected cells are able to present processed antigen assoeiated with MHC-I which stimulate CTL specifically. Onee a sufficient level of MHCR-CT~ has been reached after the injection of the vaccine, and Pnhilnc t of cytotoxicity by antiestrogens es~-hl;-h--l, antiestrogen treatment may be initiated in order to prevent the switch to humoral immune r-~ t;~ n-~ The vaccination of infected animals in combination with antiestrogen treatment is expected to lead to viral clearance and complete recovery which may not happen if the vaccine is given alone.
B. Method-.
Ne use HIV infection and AIDS as an example for our methodology. However, it will be apparent to those skilled in the art that the methodology employed herein equally applies to other viral infections, including those described in the Background to the Invention.
1. D~ o inection. Quantitative 5 polymerase chain reaction f or the detection of proviral DNA of HIV, a reliable assay for the detection of p24 viral antigen in plasma and assays for the detection of virus specific ;Int;~ c for routine tests are available (Wood et al., 1993) . The DNA-PCR test is most useful for 10 the follow-up of viral burden in patients.
2. Detection of cytotoxic T lymphocyte~. MHCR-CTL
can be assayed by using EBV-transformed B lymphocytes as targets that have been infected with a vaccinia virus carrying HIV genes. Both autologous and allogeneic B
15 cell3 can readily he infected by such virus and by this means it is possible to test whether or not the CT~
detected is MHC restricted ~T~mll~m~fl;-cherradi et al., 1992; Grant et al., 1992; Ho et al., 1993) . This test is also suitable for the ~letf~rm; n~tion of MHC restriction 20 which is of utmost importance for our treatment protocol.
3. Drug treatment. For ~n vitro assays it is suf f icient to treat target cells with TX (up to 1 llM) and TO (up to 5 ~M) for 4 hours which is followed by 1 ~h~l 1; n~ with ~Cr and using the target cells in 25 cytotoxicity assays. The most important virus carrying target in HIV infections is the CD4+ helper T lymphocyte, although monocyte-macrophages, astrocytes, neurons and epithelial cells may also be infected. The F~nlmln~;n~
effect of antiestrogens on the lysis of autologous HIV
30 infected targets by MHCR-CT~ is to be established prior to the initiation of treatment.
For the treatment of patients, the doses of ~X and TO that are currently in use for cancer therapy may be applied. For TX, a suitable oral dose is about 20 to 40 35 mg given daily (Anderson et al., 1991). The therapeutic dose for TO is about 40 to 60 mg, but can be raised up to about 240 mg daily ~'Hietanen et al., lsso) . Antiestrogen treatment is to be started as soon as MXCR-CTL is present in the patient ~for example, endogenous killer cells, vaccine induced killer cells or in vitro activated killer cells reinfused to the patient). The antiestrogen therapy and killer cell therapy (if given) are then m-;n~A;n~d ~ointly, optimally until the complete clearance of the virus. Additional treatment with interf eron would help to control virus production and enhance further host immunity.
a . The diagnosis of inf ection and the presence of ~CR-CTL is done as described under 1. In the present case, it is expected that antibodies are present and nonspecific cytotoxic ~^~hAn; ~mc also operate (for 35 example nonspecific CD8+ CTI" antibody dependent cellular cytotoxicity, and complement fixing An~iho~;es), 21~0193 .
g whereas MHCR-CTL may be weakly cytotoxie or completely paralyzed. Therapy is contraindieated with central nervous system involvement.
b. Reactivate MHCR-CTL il:L vitro using antigen 5 presenting cells and cytokines, such as Il,-2 and/or I~-12 .
e. Establish that antiestrogens enhance cytotoxieity .
d. Expand the eultures and reinfuse into the host 10 while antiestrogen treatment is applied.
e. Additional treatment with IL-2 and/or IL-12 in order to help ~';nt_;n eytotoxieity at a signifieant level and treatment with interferons is re~ n~_tl to limit virus multiplication.
3. Vaccination.
a. The difficulty of vaccination against persistent viral infections i8 that nonprotective or even _nh_n~-~;n~ immune reactions may also be induced, which would interfere with MHCR-CT~ that mediate host response.
I.ive virus vaecines that are capable of infecting the susceptible cell3 have been found to be far superior for vaccination than killed viruses. The apparent explanation f or this is that only virus inf ected cells are able to present processed antigen assoeiated with MHC-I which stimulate CTL specifically. Onee a sufficient level of MHCR-CT~ has been reached after the injection of the vaccine, and Pnhilnc t of cytotoxicity by antiestrogens es~-hl;-h--l, antiestrogen treatment may be initiated in order to prevent the switch to humoral immune r-~ t;~ n-~ The vaccination of infected animals in combination with antiestrogen treatment is expected to lead to viral clearance and complete recovery which may not happen if the vaccine is given alone.
B. Method-.
Ne use HIV infection and AIDS as an example for our methodology. However, it will be apparent to those skilled in the art that the methodology employed herein equally applies to other viral infections, including those described in the Background to the Invention.
1. D~ o inection. Quantitative 5 polymerase chain reaction f or the detection of proviral DNA of HIV, a reliable assay for the detection of p24 viral antigen in plasma and assays for the detection of virus specific ;Int;~ c for routine tests are available (Wood et al., 1993) . The DNA-PCR test is most useful for 10 the follow-up of viral burden in patients.
2. Detection of cytotoxic T lymphocyte~. MHCR-CTL
can be assayed by using EBV-transformed B lymphocytes as targets that have been infected with a vaccinia virus carrying HIV genes. Both autologous and allogeneic B
15 cell3 can readily he infected by such virus and by this means it is possible to test whether or not the CT~
detected is MHC restricted ~T~mll~m~fl;-cherradi et al., 1992; Grant et al., 1992; Ho et al., 1993) . This test is also suitable for the ~letf~rm; n~tion of MHC restriction 20 which is of utmost importance for our treatment protocol.
3. Drug treatment. For ~n vitro assays it is suf f icient to treat target cells with TX (up to 1 llM) and TO (up to 5 ~M) for 4 hours which is followed by 1 ~h~l 1; n~ with ~Cr and using the target cells in 25 cytotoxicity assays. The most important virus carrying target in HIV infections is the CD4+ helper T lymphocyte, although monocyte-macrophages, astrocytes, neurons and epithelial cells may also be infected. The F~nlmln~;n~
effect of antiestrogens on the lysis of autologous HIV
30 infected targets by MHCR-CT~ is to be established prior to the initiation of treatment.
For the treatment of patients, the doses of ~X and TO that are currently in use for cancer therapy may be applied. For TX, a suitable oral dose is about 20 to 40 35 mg given daily (Anderson et al., 1991). The therapeutic dose for TO is about 40 to 60 mg, but can be raised up to about 240 mg daily ~'Hietanen et al., lsso) . Antiestrogen treatment is to be started as soon as MXCR-CTL is present in the patient ~for example, endogenous killer cells, vaccine induced killer cells or in vitro activated killer cells reinfused to the patient). The antiestrogen therapy and killer cell therapy (if given) are then m-;n~A;n~d ~ointly, optimally until the complete clearance of the virus. Additional treatment with interf eron would help to control virus production and enhance further host immunity.
4. Treatment with cytokines. Interleukin-2 has long been demonstrated to have a therapeutic effect on syngeneic animal tun~ors and some beneficial effects could also be demonstrated in certain human malignancies.
Recently treatment of AIDS patients with IL-2 also produced beneficia] effects, especially when used in combination with zidovudine (Schwartz et al., 1991; Mazza et al., 1992; McMahon et al., 1994). 0.2-12 x 106 IU/sq m doses were used and well tolerated when inj ected s . c .
Our survey of the cancer literature indicates that the dose of IL-2 which is well tolerated ranges from about 2 to 8 million IU (about 7.5 to 30,000 IU/kg) which is given subcutaneously (s . c) for about 4 to 5 days (often combined with killer cells). Fever, chills, skin rash, anxiety and dizziness may occur as complications of subcutaneous IL-2 treatment (Schoof et al ., 1988 ;
Mitchell et al., 1988; Topalian et al., 1988; Xancock, l991; Sosman et al., 1988). We recommend treatment with 5 million IU/day s.c., which is r~;nt~;n~l for 10 days while the patient is also treated with TX or TO or other antiestrogen. This treatment may be repeated several times in a week to 10 day intervals if it is deemed to be necessary while drug treatment is mA;n~A;n~l continuously .
3 5 Interleukin- 12 has been recognized recently as a cytokine with a powerful stimulatory effect of cell mediated immunity, especially of CTL and NK cells. IL-12 has suppressed tumor growth in animal model systems and exerted antimicrobial activity against bacterial, yeast, parasitic and viral models of infection (Chehimi &
Trinchieri, 1994; Wolf et al., 1994) . IL-12 has also been shown to stimulate CTL responses to viral antigens such as influenza virus and HIV in vitro, and is capable of reactivating anergic CTII from HIV infected patients during in vitro stiTn~ t;on (Clerici et al., 1993).
Currently, there i8 in6T1Tff;~ Tlt human experience with IL-12 treatment to recommend a schedule for dosage.
It is known, however, that IL-12 is less toxic than is IL-2 80 it is to be expected that the dosage would be more lenient Once a suitable dose has been established, we would recommend to use an effective daily dosage for 10 day intervals jointly with TX or TO treatment similar to our re~ tion for Ili-2.
The treatment of chronic hepatitis C in patie~ts with interferon-~Y is of current interest. The standard dose of IFN~Y 2b is 3 million units/week for 24 weeks.
High doses (15 ~nillion units or 22.5-30 million units/week) for 24 weeks have also been tested. Higher doses of IFN ameliorated the severity of hepatitis in patie~ts who did not respond to the standard dose;
however, a sustained response was not produced as a rule.
Side effects were increa6ed with high doses (Bonkovsky et al., 1996) . AIDS patients were treated with low dose IFNo~ (3 million units s.c., 3 x week). This dose was well tolerated and had a beneficial effect on HIv-related thrombocytopenic purpura (Northfelt et al., 1995).
Interferon-~ has been used experimentally on cancer patients at doses of 150-200 ILg s.c. (~ummen et al., 1996; Fiehn et al., 1995) .
5 F!~r~n~ n of CTL in culture. Peripheral blood lymphocytes are stimulated with autologous B
1~ h~hl ~toid cell lines derived by infection with the 21801~3 , --~pstein-Barr virus followed by infection with recombinant vaccinia virus ~--~nt~;n;n~ HIV genes. Such stimulator cells can be fixed with paraformaldehyde and used as antigen presenting cells to stimulate peripheral blood 5 m~n~nl1o-l ear cellg from asymptomatic HIV seropositive individuals . Specif ic cytotoxic activity can be determined by 5~Cr release from viable antigen bearing B
cell lines used for stimulation and from similar targets generated with B cell lines of unrelated subj ects .
10 Interleukin-2 is useful to aid expansion of CT~ in this system. HIV specific CTL may be maintained for as long as 11 months and may be reinfused to the donor patients without any significant complications (Walker et al., 1989; Grant et al., 1992; T.;~mh. i; -Cherradi et al., 1992; Ahmad et al., 1993; Torpey et al., 1993; van Baalen et al., 1993; Whiteside et al., 1993).
Recently treatment of AIDS patients with IL-2 also produced beneficia] effects, especially when used in combination with zidovudine (Schwartz et al., 1991; Mazza et al., 1992; McMahon et al., 1994). 0.2-12 x 106 IU/sq m doses were used and well tolerated when inj ected s . c .
Our survey of the cancer literature indicates that the dose of IL-2 which is well tolerated ranges from about 2 to 8 million IU (about 7.5 to 30,000 IU/kg) which is given subcutaneously (s . c) for about 4 to 5 days (often combined with killer cells). Fever, chills, skin rash, anxiety and dizziness may occur as complications of subcutaneous IL-2 treatment (Schoof et al ., 1988 ;
Mitchell et al., 1988; Topalian et al., 1988; Xancock, l991; Sosman et al., 1988). We recommend treatment with 5 million IU/day s.c., which is r~;nt~;n~l for 10 days while the patient is also treated with TX or TO or other antiestrogen. This treatment may be repeated several times in a week to 10 day intervals if it is deemed to be necessary while drug treatment is mA;n~A;n~l continuously .
3 5 Interleukin- 12 has been recognized recently as a cytokine with a powerful stimulatory effect of cell mediated immunity, especially of CTL and NK cells. IL-12 has suppressed tumor growth in animal model systems and exerted antimicrobial activity against bacterial, yeast, parasitic and viral models of infection (Chehimi &
Trinchieri, 1994; Wolf et al., 1994) . IL-12 has also been shown to stimulate CTL responses to viral antigens such as influenza virus and HIV in vitro, and is capable of reactivating anergic CTII from HIV infected patients during in vitro stiTn~ t;on (Clerici et al., 1993).
Currently, there i8 in6T1Tff;~ Tlt human experience with IL-12 treatment to recommend a schedule for dosage.
It is known, however, that IL-12 is less toxic than is IL-2 80 it is to be expected that the dosage would be more lenient Once a suitable dose has been established, we would recommend to use an effective daily dosage for 10 day intervals jointly with TX or TO treatment similar to our re~ tion for Ili-2.
The treatment of chronic hepatitis C in patie~ts with interferon-~Y is of current interest. The standard dose of IFN~Y 2b is 3 million units/week for 24 weeks.
High doses (15 ~nillion units or 22.5-30 million units/week) for 24 weeks have also been tested. Higher doses of IFN ameliorated the severity of hepatitis in patie~ts who did not respond to the standard dose;
however, a sustained response was not produced as a rule.
Side effects were increa6ed with high doses (Bonkovsky et al., 1996) . AIDS patients were treated with low dose IFNo~ (3 million units s.c., 3 x week). This dose was well tolerated and had a beneficial effect on HIv-related thrombocytopenic purpura (Northfelt et al., 1995).
Interferon-~ has been used experimentally on cancer patients at doses of 150-200 ILg s.c. (~ummen et al., 1996; Fiehn et al., 1995) .
5 F!~r~n~ n of CTL in culture. Peripheral blood lymphocytes are stimulated with autologous B
1~ h~hl ~toid cell lines derived by infection with the 21801~3 , --~pstein-Barr virus followed by infection with recombinant vaccinia virus ~--~nt~;n;n~ HIV genes. Such stimulator cells can be fixed with paraformaldehyde and used as antigen presenting cells to stimulate peripheral blood 5 m~n~nl1o-l ear cellg from asymptomatic HIV seropositive individuals . Specif ic cytotoxic activity can be determined by 5~Cr release from viable antigen bearing B
cell lines used for stimulation and from similar targets generated with B cell lines of unrelated subj ects .
10 Interleukin-2 is useful to aid expansion of CT~ in this system. HIV specific CTL may be maintained for as long as 11 months and may be reinfused to the donor patients without any significant complications (Walker et al., 1989; Grant et al., 1992; T.;~mh. i; -Cherradi et al., 1992; Ahmad et al., 1993; Torpey et al., 1993; van Baalen et al., 1993; Whiteside et al., 1993).
6. Adaptive immunotherapy o~ AIDS patient~ with in vitro ~An~d M~ICR-CTL. There is not enough experience with regard to the adaptive immunotherapy of AIDS
patients with i~l vitro generated CTL for the establishment of a clefinite treatment schedule. However, in cancer immunotherapy there is somewhat more experience and a cell dose of about 1 x 108 to 1 x 101 has been suggested as being ef f ective f or treatment with 1~ k; n~ activated killer cells . The cells may be infused every day or every other day for up to about 10 days. The patient may require about 1 to 4 such treatments seqllPnt; ~l 1 y before achieving tumor remission.
CTL may be administered in a similar fashion (Rosenberg et al., 1987; Fujimoto, 1992; Horvath et al., 1993). We recommend daily intravenous infusion with about 5 x 109 to 101 MHCR-CTL for 5 days, which is to be repeated in 10 day intervals, while the patient is ~-;ntA;n~tl on TX or T0 continuously. Additional treatment with interferon is desirable.
14 218~1~3 7. Vaccination. Hu et al. (1989) vaccinated macaSIues with recombinant vaccinia virus which expre3sed the envelope glycoprotein or simian type D retrovirus.
The vaccinated animals showed complete protection when 5 challenged with pathogenic doses of simian AIDS virus.
HIV infected subjec~ 8 have been vaccinated similarly with recombinant vaccinia virus expressing HIV glycoprotein 160. Such vaccination resulted in MHC restricted cytotoxic T lymphocytes showing specificity for gpl60 (Kundu et al., 1992; el-Daher et al., 1993) . In the anticipation that effective vaccine(s) will be developed against HIV in the future, we recommend the treatment schedule outlined below.
Once viral immLmity (mediated by MHCR-CTL) has been induced in HIV infected patients, we recommend immediately the initiation of TX or TO treatment as described above. Additional treatment with IL-2 or IL-12 is also rec~ nflF~d in order to support the long term "~intf-n~C~ of virus specific MHC restricted CTL. If the level of CTL induced is judged to be unsati3factory, one may consider the in vitro expansion of CTL and reinfusion as discussed above. Interferon treatment should be considered as an adjunct therapy, especially if the viral load is eignificant.
EXPMPLES ~
A series of experiments were carried out, the results of which are outlined in Tables 1 to 5 below.
Exam~le 1:
This Example shows the effect of tamoxifen (TX) and toremifene (TO) on lysis of Epstein-Barr virus (EBV) by autologous cytotoxic T lymphocytes.
In a first set of experiments, cytotoxic lymphocytes were generated by co-culturing Epstein-Barr virus (EBV) transformed B lymphocytes with autologous peripheral blood - ~n~nllc1 ear cells for 5 dayR or longer. Tamoxifen (TX) (1 IlM) or toremifene (TO) (5 ~M) treatment of the 21~193 target cells wa~ efi~ected for 4 hours, prior to labelling the target cells with 51Cr. The cytotoxicity reaction was carried out at a 1:25 target:effector ratio for four hours .
The results of this f irst set of experiments is set forth in Table 1 below. As may be seen from this Table, TX and TO potentiated the lysis of EBV-transformed B
lymphocytes by autologous cytotoxic T lymphocytes.
F:Y~?le 2:
This Example sllows the effects of TX and TO on lysis of Epstein-Barr virus (EBV) by autologous LAK cells.
In a second set of experiments, ly ~ k;n~ activated killer (LAK) cells were generated by the st; l~t;on of autologous peripheral blood lymphocytes (2 x 106/ml) with human recombinant interleukin-2 (500 IU/ml) for 6 days.
TX (1 ,uM) and TO (5 /lM) treatment of target cells was effected for 4 hours prior to l~h~ll;ng the cells with slCr. The cytotoxicity reaction (1:25 effector:target ratio) was terminated at 4 hours.
The results of this second set of experiments are set forth in Table 2 below. As may be seen from Table 2, TX and TO potentiated the lysis of EBV-transformed B
lymphocytes by autologous LAK cells.
ExamPle 3:
This Example shows the effect of interferon on the cytotoxic action of TX and TO.
Next we tested whether or not treatment of the cytotoxic cells with TX, TO, or with IF~a would affect their cytotoxic action. The treatment shown in Table 3 was applied to CTL for 4 hours (TX at 1 ~M, TO at 5 ~M, and IFNcl! at 500 IU/ml), which was followed by cytotoxicity assay (5~Cr release) for another 4 hours at 1:25 target:effector ratios. As is shown in Table 3, interferon did increase the cytotoxic action of CTL as did TX and TO, and treatment with both agents resulted in the highest cytotoxicity.
21801g3 ~xam~le 4:
This Example shows the effect of interferon-~, interf eron-~y and indomethacin in combination with TO on target cells.
In the next series of experiments, we extended our studies for the treatment of target cells with interferon-~, interferon-~ (100 IU/ml), and indomethacin (5 /lg/ml), in combi.nation with TO (5 /~M). The results are given in Table 4. Experiments with non-treated targets indicate that the treatment of effector cells with TO, IFN-~ plu8 INDO raised significantly the mean cytotoxicity from 9 . 6 :t 1. 596 to 22 . 5 1 0 . 9~ (PcO . 01) .
When cytotoxicity O~1 non-treated targets was compared to T0 treated target6, significant f~nh~n~ t occurred with CTL after TO treatment (P<0 . 05), after IFN-~ plus IND0 treatment (P<0.05) and with CT~ treated with T0 plus IFN-~y plus IND0 (P<0 . 01) . These experiments indicate that the .-nh~n~ t of cytotoxicity by TO will take place in the presence of interferon, and that interferon-~y especially in combination with indomethacin actually enhances significantly the cytotoxic action of killer cells (Table 4 ) .
Exam~le 5:
This ~xample shows the effect of IEN-~, IFN-~y, T0 and IND0 and combinations thereof.
Finally, we exposed both the target and the effector cells to IFN-~, IFN-l~, T0 and IND0, and to their various combinations (at concentrations given for Table 5) for the duration of the cytotoxic interaction (6 or 18 hrs).
In case of 6 hr treatment a significant elevation of cytotoxicity was observed with T0 treatment alone, and TO
plus INDO (P<0 . 05), IFN-~ plus TO plus INDO and IFN-1~
plus TO plus IND0 (P<o . 01) . However, no further enhancement of the cytotoxic reaction was achieved by the addition of IFN and IND0 compared to TO alone. The trend obtained in 18 hour cultures was similar. Elere target cell destruction reached its highest value in the culture~ treated with TO pluF~ IFN-~r plus INDO (P<o . 01) .
These experiments indicate that both the antiestrogens (in this case, TO) can enhance the cytotoxic effect of CTL and that additional treatment with interferons and 5 INDO does not interfere with this effect. Therefore, it seems feasible that one can enhance the destruction of virus infected target cells by antiestrogens while interfering with virus production using interferon-~ or -~ r- Taken together, these experiments indicate that the 10 treatment of viral diseases, such as HIV infection or hepatitis C, with interferons can be improved if additional treatment with antiestrogens is applied.
Indomethacin can be used to oppose the generation of immuno3uppressive prostaglandin E by interferon 15 treatment.
SI~MMARY OF DISC~OSIJRE
In summar,v of this disclosure, a method is provided for the pot~n~;At;~n of cell-mediated immunity in an 20 infected host against persistent viral infection by the administering to the host, which may be a human or animal host, an ef f ective amount of at least one antiestrogenic agent, which may be a triphenylethylene antiestrogen, including tamoxifen and toremifene. This treatment is 25 c~ompatible with interferon therapy, which is known to limit the production of viral particles. Combination treatment with antiestrogens and interf eron is expected to lead to better results than interferon treatment alone. Modifications are possible within the scope of 30 this invention.
21~01~3 . --TAB~E l The anti 6.~L~ ., tamoxifen (~rx) and toremifene (T0), enhance the killi of EBV infected target cell_ by Alltr~l A~Ollq cytotoxic T lymphocytes (CTL) .
I30tope release (~'Cr cpm i gE) Exp. Treatment Percent cr~n~An"~ Total lysis None 3$0 . 3 189il5 1925il4 TX 22il.2' 179i20 1889i94 T0 18i0.6b l90ill 1912i73 52 None 14il.1 177il2 1657illl TX 34i2' 184i25 1701i98 T0 32iO . 9 196~17 1715I135 3 None 4i0 279il2 2703i48 TX 15i0 . 6b 265ill 2561::71 T0 14iO, 3b 236il7 2447i94 4 None 3:1:0.3 289~20 2613i25 TX 12~1.1' 274il6 2648ill5 T0 11$0.3' 280il5 2697i98 5 None 3iO 284i7 2645il45 TX 14il .1 275i 11 2579i94 T0 12~0.3~ 291 13 2701illl Statistic3 (t test): Controls with no treatment were compar~d with groupq tr~ated with TX or T0.
a = P<O.os; b = P<o.01; c = P<0.001 --The antiestrogens, TX and TO, enhance the killing o~ E'3V in~ected target cells by a1l~nl o~o11a lymphokine activated killer (LA!~) cells .
Isotope release (5ICr, cpm ~ SE) ExpTreatment Percent lysis SrAn~:.n~.A~1a Total None 1~ 257ilS 1678it114 TX10~0 . 6b 284~19 1934:~198 T016~1. 5' 361+27 2067~157 2None 3~0.3 238~31 2001~113 TX1SI1, 3b 274~25 2484~177 TO17~0.5' 297i18 2613~194 Statistic3: Please see legends to Table 1.
218~193 The efect of TX, TO and IR~Y on the cytotoxic action of CTL.
Treatment of CTL Percent lysis none 1 + 0 I~ 7 _ 0 . 9~
TX 5 _ 0 . 7' TO 8 + 0.3~
IFN~Y + TX 18 _ 2 . 9' IFN~Y + TO 29 1 1.2' Please see legends to Ta~le 1.
~18019~
.
7'~i3I.3~ 4 The effect of T0, IFN(Y, -y and IND0 on the cytotoxic potential of CTL.
Treatment Percent lysis of CTL Exp 1 2 Mean_SEI 3 (T0) 4 (T0) Mean _SE2 None 6_0.8 13_0~6 9.6_1.5 13+0.6 23 _ 1.1 18_2.3 IFNcY 10_0 . 6 14_0 ~ 9 12il .1 16_0 . 7 26 i 9 21. 5_2 . 3 IFNy l1iO.9 16~0 3 13_5.1 18_1.1 27 ~ 0.3 19.3_3.1 T017_0.9 14_0~3 16_0.8 24 0.6 29 :: 1.2 26.5_1.2 IND0 8_0.3 13 1 1 10.6_1.1 14_1.1 20 :~: 0.6 17_1.4 IFN~ + IND0 10_1.1 15_0 6 12.3_1.Z 17::0.3 18 _ 1.1 17.3_0.6 Ir7Ny + IND0 13_0.9 20_0 6 16.62+1.5 24_0.9 30 _ 1.7 26.8_1.6 T0 + IND0 17_1.1 16_0 3 16.6_0.6 27il~5 31 _ 1.2 25.3+3.3 IFN~ + IND0 + T0 19_1.5 17_1 1 18_1 27_0.3 31 _ 1.7 24.1_4 IFNy+ IND0 + T0 22il.4 23_1.2 22.52:0.9b 32_1.1 39 _ 0.9 35.6_1.8b ~ Mean_SE was rAlrlllAter~ from exps 1 and 2, which were conducted with non-treated target cellli.
2 Mean+SE was calculated from exp,~ 3 and 4, which were done with T0 treated target cell3 .
Statistics (t teEit): Mean cytotox:icity of control groupA COmpared to all other groups using the same target cell~ - u~treated vs T0 + IFNy + IND0 (P~0 . 01) . Control~
compared to all other groupb~ Ot l~;rjn;f;rAn~. Mean cytotoxicity on untreated targets compared to T0 treated targets - CTL treated with T0 or IFNy + IND0 treatment (P~0 . 05); CTL treated with T0 + Ir7Ny + IND0 (P~0 . 01) .
2180~3 TAhLE S
The killing potential of CTL is increaAed by the presence duri the c totoxic reaction of TO, interferon-y (IFNy) and 1 hAr;n (INDO) . ng Y
Treatment Percent ly9i8 ~xp: 1 (6 h) a (6 h) MeaniSE~ 3 (18 h) 4 (18 h) MeaniSE
None 9i0 7 lOil.l 9.3iO.6 17il.5 20 i 1.1 18.6il IFN~ 7i0-3 lOi0.3 8.8+0,7 16iO.9 18 i 0 3 17i0 5 IFNy 8iO.3 llil.l 9.6iO.8 2liO.6 22 i 0.6 21.5iO.4 TO 20iO.8 l9iO.3 19.5iO.6' 25_2.9 29 i 2.6 27il.9 INDO 8il .1 9il .1 8 . SiO . 7 17iO . 3 17 i 1.1 17iO . 5 IFN~ f INDO 12iO.6 14il.2 13iO.6 18il.4 19 i 1.2 18iO.9 IFNy + INDO 14i0 3 l9iO.6 16.3il.2 18il.2 23 i 1.4 20.5il.5 TO +INDO 18iO.9 20iO.6 18.6iO.9 22il.1 22 i 1.1 22iO.7 IFN~+ INDO + TO 20il.1 20il.1 20ilb 21il.5 25 i 0.9 23il.2 IFNy+ INDO + TO 24iO.3 ~2il.Z 23iO.7b 29il.4 33 i 1.1 31il.1b ' Mean cytotoxicity i standard errors was rAlrl-lA~ from experiments l and 2, which were conducted for 6 hours.
2 Mean cytotoxicity _ standard errors was rAlrlllA~G~ from ~ rim~ntA 3 and 4, which were conducted for 18 hours.
Statistic (t test): Mean cytotoxicity of control groups with no treatment in ~he 6 hr experiment compared to all other groups - Aignificant increase in cytotoxicity occurred with TO and TO + INDO (P~O.os), and IFN~ + TO + INDO and IFNy + TO + INDO (P~O.Ol) .
ControlA compared to all other groupA - not A~n~f;~rAn~ In the 18 hr ~
cytotoxicity is ~rJn~f~rAn~ly higher than in controls in the IFNy + TO + INDO treated group (P~O.Ol). The other groups are not A~r~n;f~rAn~ly different.
.
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patients with i~l vitro generated CTL for the establishment of a clefinite treatment schedule. However, in cancer immunotherapy there is somewhat more experience and a cell dose of about 1 x 108 to 1 x 101 has been suggested as being ef f ective f or treatment with 1~ k; n~ activated killer cells . The cells may be infused every day or every other day for up to about 10 days. The patient may require about 1 to 4 such treatments seqllPnt; ~l 1 y before achieving tumor remission.
CTL may be administered in a similar fashion (Rosenberg et al., 1987; Fujimoto, 1992; Horvath et al., 1993). We recommend daily intravenous infusion with about 5 x 109 to 101 MHCR-CTL for 5 days, which is to be repeated in 10 day intervals, while the patient is ~-;ntA;n~tl on TX or T0 continuously. Additional treatment with interferon is desirable.
14 218~1~3 7. Vaccination. Hu et al. (1989) vaccinated macaSIues with recombinant vaccinia virus which expre3sed the envelope glycoprotein or simian type D retrovirus.
The vaccinated animals showed complete protection when 5 challenged with pathogenic doses of simian AIDS virus.
HIV infected subjec~ 8 have been vaccinated similarly with recombinant vaccinia virus expressing HIV glycoprotein 160. Such vaccination resulted in MHC restricted cytotoxic T lymphocytes showing specificity for gpl60 (Kundu et al., 1992; el-Daher et al., 1993) . In the anticipation that effective vaccine(s) will be developed against HIV in the future, we recommend the treatment schedule outlined below.
Once viral immLmity (mediated by MHCR-CTL) has been induced in HIV infected patients, we recommend immediately the initiation of TX or TO treatment as described above. Additional treatment with IL-2 or IL-12 is also rec~ nflF~d in order to support the long term "~intf-n~C~ of virus specific MHC restricted CTL. If the level of CTL induced is judged to be unsati3factory, one may consider the in vitro expansion of CTL and reinfusion as discussed above. Interferon treatment should be considered as an adjunct therapy, especially if the viral load is eignificant.
EXPMPLES ~
A series of experiments were carried out, the results of which are outlined in Tables 1 to 5 below.
Exam~le 1:
This Example shows the effect of tamoxifen (TX) and toremifene (TO) on lysis of Epstein-Barr virus (EBV) by autologous cytotoxic T lymphocytes.
In a first set of experiments, cytotoxic lymphocytes were generated by co-culturing Epstein-Barr virus (EBV) transformed B lymphocytes with autologous peripheral blood - ~n~nllc1 ear cells for 5 dayR or longer. Tamoxifen (TX) (1 IlM) or toremifene (TO) (5 ~M) treatment of the 21~193 target cells wa~ efi~ected for 4 hours, prior to labelling the target cells with 51Cr. The cytotoxicity reaction was carried out at a 1:25 target:effector ratio for four hours .
The results of this f irst set of experiments is set forth in Table 1 below. As may be seen from this Table, TX and TO potentiated the lysis of EBV-transformed B
lymphocytes by autologous cytotoxic T lymphocytes.
F:Y~?le 2:
This Example sllows the effects of TX and TO on lysis of Epstein-Barr virus (EBV) by autologous LAK cells.
In a second set of experiments, ly ~ k;n~ activated killer (LAK) cells were generated by the st; l~t;on of autologous peripheral blood lymphocytes (2 x 106/ml) with human recombinant interleukin-2 (500 IU/ml) for 6 days.
TX (1 ,uM) and TO (5 /lM) treatment of target cells was effected for 4 hours prior to l~h~ll;ng the cells with slCr. The cytotoxicity reaction (1:25 effector:target ratio) was terminated at 4 hours.
The results of this second set of experiments are set forth in Table 2 below. As may be seen from Table 2, TX and TO potentiated the lysis of EBV-transformed B
lymphocytes by autologous LAK cells.
ExamPle 3:
This Example shows the effect of interferon on the cytotoxic action of TX and TO.
Next we tested whether or not treatment of the cytotoxic cells with TX, TO, or with IF~a would affect their cytotoxic action. The treatment shown in Table 3 was applied to CTL for 4 hours (TX at 1 ~M, TO at 5 ~M, and IFNcl! at 500 IU/ml), which was followed by cytotoxicity assay (5~Cr release) for another 4 hours at 1:25 target:effector ratios. As is shown in Table 3, interferon did increase the cytotoxic action of CTL as did TX and TO, and treatment with both agents resulted in the highest cytotoxicity.
21801g3 ~xam~le 4:
This Example shows the effect of interferon-~, interf eron-~y and indomethacin in combination with TO on target cells.
In the next series of experiments, we extended our studies for the treatment of target cells with interferon-~, interferon-~ (100 IU/ml), and indomethacin (5 /lg/ml), in combi.nation with TO (5 /~M). The results are given in Table 4. Experiments with non-treated targets indicate that the treatment of effector cells with TO, IFN-~ plu8 INDO raised significantly the mean cytotoxicity from 9 . 6 :t 1. 596 to 22 . 5 1 0 . 9~ (PcO . 01) .
When cytotoxicity O~1 non-treated targets was compared to T0 treated target6, significant f~nh~n~ t occurred with CTL after TO treatment (P<0 . 05), after IFN-~ plus IND0 treatment (P<0.05) and with CT~ treated with T0 plus IFN-~y plus IND0 (P<0 . 01) . These experiments indicate that the .-nh~n~ t of cytotoxicity by TO will take place in the presence of interferon, and that interferon-~y especially in combination with indomethacin actually enhances significantly the cytotoxic action of killer cells (Table 4 ) .
Exam~le 5:
This ~xample shows the effect of IEN-~, IFN-~y, T0 and IND0 and combinations thereof.
Finally, we exposed both the target and the effector cells to IFN-~, IFN-l~, T0 and IND0, and to their various combinations (at concentrations given for Table 5) for the duration of the cytotoxic interaction (6 or 18 hrs).
In case of 6 hr treatment a significant elevation of cytotoxicity was observed with T0 treatment alone, and TO
plus INDO (P<0 . 05), IFN-~ plus TO plus INDO and IFN-1~
plus TO plus IND0 (P<o . 01) . However, no further enhancement of the cytotoxic reaction was achieved by the addition of IFN and IND0 compared to TO alone. The trend obtained in 18 hour cultures was similar. Elere target cell destruction reached its highest value in the culture~ treated with TO pluF~ IFN-~r plus INDO (P<o . 01) .
These experiments indicate that both the antiestrogens (in this case, TO) can enhance the cytotoxic effect of CTL and that additional treatment with interferons and 5 INDO does not interfere with this effect. Therefore, it seems feasible that one can enhance the destruction of virus infected target cells by antiestrogens while interfering with virus production using interferon-~ or -~ r- Taken together, these experiments indicate that the 10 treatment of viral diseases, such as HIV infection or hepatitis C, with interferons can be improved if additional treatment with antiestrogens is applied.
Indomethacin can be used to oppose the generation of immuno3uppressive prostaglandin E by interferon 15 treatment.
SI~MMARY OF DISC~OSIJRE
In summar,v of this disclosure, a method is provided for the pot~n~;At;~n of cell-mediated immunity in an 20 infected host against persistent viral infection by the administering to the host, which may be a human or animal host, an ef f ective amount of at least one antiestrogenic agent, which may be a triphenylethylene antiestrogen, including tamoxifen and toremifene. This treatment is 25 c~ompatible with interferon therapy, which is known to limit the production of viral particles. Combination treatment with antiestrogens and interf eron is expected to lead to better results than interferon treatment alone. Modifications are possible within the scope of 30 this invention.
21~01~3 . --TAB~E l The anti 6.~L~ ., tamoxifen (~rx) and toremifene (T0), enhance the killi of EBV infected target cell_ by Alltr~l A~Ollq cytotoxic T lymphocytes (CTL) .
I30tope release (~'Cr cpm i gE) Exp. Treatment Percent cr~n~An"~ Total lysis None 3$0 . 3 189il5 1925il4 TX 22il.2' 179i20 1889i94 T0 18i0.6b l90ill 1912i73 52 None 14il.1 177il2 1657illl TX 34i2' 184i25 1701i98 T0 32iO . 9 196~17 1715I135 3 None 4i0 279il2 2703i48 TX 15i0 . 6b 265ill 2561::71 T0 14iO, 3b 236il7 2447i94 4 None 3:1:0.3 289~20 2613i25 TX 12~1.1' 274il6 2648ill5 T0 11$0.3' 280il5 2697i98 5 None 3iO 284i7 2645il45 TX 14il .1 275i 11 2579i94 T0 12~0.3~ 291 13 2701illl Statistic3 (t test): Controls with no treatment were compar~d with groupq tr~ated with TX or T0.
a = P<O.os; b = P<o.01; c = P<0.001 --The antiestrogens, TX and TO, enhance the killing o~ E'3V in~ected target cells by a1l~nl o~o11a lymphokine activated killer (LA!~) cells .
Isotope release (5ICr, cpm ~ SE) ExpTreatment Percent lysis SrAn~:.n~.A~1a Total None 1~ 257ilS 1678it114 TX10~0 . 6b 284~19 1934:~198 T016~1. 5' 361+27 2067~157 2None 3~0.3 238~31 2001~113 TX1SI1, 3b 274~25 2484~177 TO17~0.5' 297i18 2613~194 Statistic3: Please see legends to Table 1.
218~193 The efect of TX, TO and IR~Y on the cytotoxic action of CTL.
Treatment of CTL Percent lysis none 1 + 0 I~ 7 _ 0 . 9~
TX 5 _ 0 . 7' TO 8 + 0.3~
IFN~Y + TX 18 _ 2 . 9' IFN~Y + TO 29 1 1.2' Please see legends to Ta~le 1.
~18019~
.
7'~i3I.3~ 4 The effect of T0, IFN(Y, -y and IND0 on the cytotoxic potential of CTL.
Treatment Percent lysis of CTL Exp 1 2 Mean_SEI 3 (T0) 4 (T0) Mean _SE2 None 6_0.8 13_0~6 9.6_1.5 13+0.6 23 _ 1.1 18_2.3 IFNcY 10_0 . 6 14_0 ~ 9 12il .1 16_0 . 7 26 i 9 21. 5_2 . 3 IFNy l1iO.9 16~0 3 13_5.1 18_1.1 27 ~ 0.3 19.3_3.1 T017_0.9 14_0~3 16_0.8 24 0.6 29 :: 1.2 26.5_1.2 IND0 8_0.3 13 1 1 10.6_1.1 14_1.1 20 :~: 0.6 17_1.4 IFN~ + IND0 10_1.1 15_0 6 12.3_1.Z 17::0.3 18 _ 1.1 17.3_0.6 Ir7Ny + IND0 13_0.9 20_0 6 16.62+1.5 24_0.9 30 _ 1.7 26.8_1.6 T0 + IND0 17_1.1 16_0 3 16.6_0.6 27il~5 31 _ 1.2 25.3+3.3 IFN~ + IND0 + T0 19_1.5 17_1 1 18_1 27_0.3 31 _ 1.7 24.1_4 IFNy+ IND0 + T0 22il.4 23_1.2 22.52:0.9b 32_1.1 39 _ 0.9 35.6_1.8b ~ Mean_SE was rAlrlllAter~ from exps 1 and 2, which were conducted with non-treated target cellli.
2 Mean+SE was calculated from exp,~ 3 and 4, which were done with T0 treated target cell3 .
Statistics (t teEit): Mean cytotox:icity of control groupA COmpared to all other groups using the same target cell~ - u~treated vs T0 + IFNy + IND0 (P~0 . 01) . Control~
compared to all other groupb~ Ot l~;rjn;f;rAn~. Mean cytotoxicity on untreated targets compared to T0 treated targets - CTL treated with T0 or IFNy + IND0 treatment (P~0 . 05); CTL treated with T0 + Ir7Ny + IND0 (P~0 . 01) .
2180~3 TAhLE S
The killing potential of CTL is increaAed by the presence duri the c totoxic reaction of TO, interferon-y (IFNy) and 1 hAr;n (INDO) . ng Y
Treatment Percent ly9i8 ~xp: 1 (6 h) a (6 h) MeaniSE~ 3 (18 h) 4 (18 h) MeaniSE
None 9i0 7 lOil.l 9.3iO.6 17il.5 20 i 1.1 18.6il IFN~ 7i0-3 lOi0.3 8.8+0,7 16iO.9 18 i 0 3 17i0 5 IFNy 8iO.3 llil.l 9.6iO.8 2liO.6 22 i 0.6 21.5iO.4 TO 20iO.8 l9iO.3 19.5iO.6' 25_2.9 29 i 2.6 27il.9 INDO 8il .1 9il .1 8 . SiO . 7 17iO . 3 17 i 1.1 17iO . 5 IFN~ f INDO 12iO.6 14il.2 13iO.6 18il.4 19 i 1.2 18iO.9 IFNy + INDO 14i0 3 l9iO.6 16.3il.2 18il.2 23 i 1.4 20.5il.5 TO +INDO 18iO.9 20iO.6 18.6iO.9 22il.1 22 i 1.1 22iO.7 IFN~+ INDO + TO 20il.1 20il.1 20ilb 21il.5 25 i 0.9 23il.2 IFNy+ INDO + TO 24iO.3 ~2il.Z 23iO.7b 29il.4 33 i 1.1 31il.1b ' Mean cytotoxicity i standard errors was rAlrl-lA~ from experiments l and 2, which were conducted for 6 hours.
2 Mean cytotoxicity _ standard errors was rAlrlllA~G~ from ~ rim~ntA 3 and 4, which were conducted for 18 hours.
Statistic (t test): Mean cytotoxicity of control groups with no treatment in ~he 6 hr experiment compared to all other groups - Aignificant increase in cytotoxicity occurred with TO and TO + INDO (P~O.os), and IFN~ + TO + INDO and IFNy + TO + INDO (P~O.Ol) .
ControlA compared to all other groupA - not A~n~f;~rAn~ In the 18 hr ~
cytotoxicity is ~rJn~f~rAn~ly higher than in controls in the IFNy + TO + INDO treated group (P~O.Ol). The other groups are not A~r~n;f~rAn~ly different.
.
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Claims (26)
1. A method for the potentiation of cell mediated immunity against persistent viral infection in a host, which comprises:
administering to a host an effective amount of at least one antiestrogen capable of enhancing cytotoxic T
lymphocytes (CTLs) in the host.
administering to a host an effective amount of at least one antiestrogen capable of enhancing cytotoxic T
lymphocytes (CTLs) in the host.
2. The method of claim 1 wherein said host harbours MHC-restricted CTLs and said at least one antiestrogen is initially tested in vitro to demonstrate enhancement of lysis of virus infected cells.
3. The method of claim 2 wherein the antiestrogen is a triphenylethylene antiestrogen.
4. The method of claim 2 wherein said antiestrogen is tamoxifen or toremifene.
5. The method of claim 2 wherein said at least one antiestrogen is administered along with at least one interferon.
6. The method of claim 5 wherein said antiestrogen is tamoxifen or toremifene and said at least one interferon is interferon-.alpha. or interferon-.gamma..
7. The method of claim 6 wherein said at least one antiestrogen and at said at least one interferon are administered along with indomethacin.
8. The method of claim 1 wherein said host does not harbour MHC-restricted cytotoxic T lymphocytes, and MHC-restricted cytotoxic lymphocytes are activated and expanded in tissue culture and reinfused into the host and treatment with at least one antiestrogen is initiated with the first infusion of CTL and maintained for the entire treatment period.
9. The method of claim 8 wherein the antiestrogen is a triphenylethylene antiestrogen.
10. The method of claim 8 wherein said antiestrogen is tamoxifen or toremifene.
11. The method of claim 8 wherein said at least one antiestrogen is administered along with at least one interferon.
12. The method of claim 11 wherein said antiestrogen is tamoxifen or toremifene and said at least one interferon is interferon-.alpha. or interferon-.gamma..
13. The method of claim 12 wherein said at least one antiestrogen and at said at least one interferon are administered along with indomethacin.
14. The method of claim 1 wherein said host is initially treated with at least one immunostimulatory cytokine to stimulate MHC-restricted CTLs.
15. The method of claim 14 wherein said immunostimulatory cytokine is interleukin-2 or interleukin-12.
16. The method of claim 15 wherein the antiestrogen is a triphenylethylene antiestrogen.
17. The method of claim 15 wherein said antiestrogen is tamoxifen or toremifene.
18. The method of claim 15 wherein said at least one antiestrogen is administered along with at least one interferon.
19. The method of claim 18 wherein said antiestrogen is tamoxifen or toremifene and said at least one interferon is interferon-.alpha. or interferon-.gamma..
20. The method of claim 19 wherein said at least one antiestrogen and at said at least one interferon are administered along with indomethacin.
21. The method of claim 1 wherein said host is virus infected but otherwise immunocompetent, said host is vaccinated to stimulate MHC-restricted CTLs, and said host is tested for the presence of MHC-restricted CTLs prior to administration of said at least one antiestrogen.
22. The method of claim 21 wherein the antiestrogen is a triphenylethylene antiestrogen.
23. The method of claim 21 wherein said antiestrogen is tamoxifen or toremifene.
24. The method of claim 21 wherein said at least one antiestrogen is administered along with at least one interferon.
25. The method of claim 24 wherein said antiestrogen is tamoxifen or toremifene and said at least one interferon is interferon-.alpha. or interferon-.gamma..
26. The method of claim 25 wherein said at least one antiestrogen and at said at least one interferon are administered along with indomethacin.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US57895P | 1995-06-28 | 1995-06-28 | |
| US60/000,578 | 1995-06-28 |
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| Publication Number | Publication Date |
|---|---|
| CA2180193A1 true CA2180193A1 (en) | 1996-12-29 |
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ID=21692112
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002180193A Abandoned CA2180193A1 (en) | 1995-06-28 | 1996-06-28 | Enhancing host immunity against viral infections |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997042969A1 (en) * | 1996-05-13 | 1997-11-20 | F. Hoffmann-La Roche Ag | USE OF IL-12 AND IFN-α FOR THE TREATMENT OF INFECTIOUS DISEASES |
| WO2001012215A3 (en) * | 1999-08-13 | 2001-08-23 | Ralph M Steinman | Protective antigen of Epstein barr virus |
| US7005131B1 (en) | 1999-08-13 | 2006-02-28 | The Rockefeller University | Protective antigen of Epstein Barr Virus |
-
1996
- 1996-06-28 CA CA002180193A patent/CA2180193A1/en not_active Abandoned
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997042969A1 (en) * | 1996-05-13 | 1997-11-20 | F. Hoffmann-La Roche Ag | USE OF IL-12 AND IFN-α FOR THE TREATMENT OF INFECTIOUS DISEASES |
| US5928636A (en) * | 1996-05-13 | 1999-07-27 | Hoffmann-La Roche Inc. | Use of IL-12 and IFNα for the treatment of infectious diseases |
| WO2001012215A3 (en) * | 1999-08-13 | 2001-08-23 | Ralph M Steinman | Protective antigen of Epstein barr virus |
| US7005131B1 (en) | 1999-08-13 | 2006-02-28 | The Rockefeller University | Protective antigen of Epstein Barr Virus |
| US7442377B2 (en) | 1999-08-13 | 2008-10-28 | The Rockefeller University | Protective antigen of epstein barr virus |
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