CA2007499C

CA2007499C - Treatment methods and vaccines for stimulating an immune response

Info

Publication number: CA2007499C
Application number: CA002007499A
Authority: CA
Inventors: Emil Bisaccia; Albert S. Klainer
Original assignee: Individual
Current assignee: Individual
Priority date: 1989-01-10
Filing date: 1990-01-10
Publication date: 2001-03-27
Anticipated expiration: 2010-01-10
Also published as: EP0453497A1; HUT62487A; JPH04502621A; EP0453497A4; AU638693B2; WO1990007952A1; BR9007016A; IL92996A; FI913318A0; FI105453B; CA2007499A1; AU4954590A

Abstract

A method is provided for treating patients who are infected with an HIV retrovirus using a photoactive compound that, upon activation by exposure to electromagnetic radiation of a prescribed spectrum, such as ultraviolet light, inactivates and/or attenuates the virus and permit the so treated virus and/or virus infected cells to be presented to the immune system of the patient.

Description

~~~'~499 TREATMENT METHODS AND VACCINES FOR
STIMULATING AN IMMUNE RESPONSE
The present invention relates to the field of immunology, particularly methods for inactivating and/or attenuating viruses and/or virus infected cells, particularly virus infected CD4 cells, using photopheresis and employing said inactivated and/or attenuated viruses and/or virus infected cells to engender an immune~response. A particular aspect of the invention relates to the treatment of patients who are infected with a virus, particularly an HIV retrovirus, and who have an abnormally low white blood cell count, by using photopheresis in combination with the administration of a photoactive compound such as 8-methoxypsoralen. The invention also relates to vaccines against viruses, particularly HIV retroviruses, and methods for producing said vaccines.

~~C3'~49~
There are many debilitating intectioua diseases which have been attributed to viral infections. There are also many different types of viruses including DNA viruses and RNA viruses.
Retroviruse fore a sub-group of RNA viruses which, in order to replicate, must first employ reverse transcription of the RNA of their genome into DNA ("transcription" conventionally describes the synthesis of RNA from DNA). once in the form of DNA, the viral genome is incorporated into the host cell genome, allowing it to take full advantage of the host cells transcription/translation machinery for the purpose of replication. once incorporated into the host cells DN~1, the virus may persist for as long a~ the cell live.
Certain retroviruse are known to cause a depression in an infected patients white blood cell count. This sub-set of ratroviruses which reduce white blood cell count are known as Human Immunodeficiency Viruses (HIV).
Particular species of HIV retroviruses have been isolated from patients who suffer from Acquired Immune Deficiency Syndrome (AIDS) and have been given the designations HIV 1 and HIV 2, sometimes collectively referred to as "HTLV III", "LAV" or simply "HIV". These retroviruses will infect cells expressing the CD4 marker, such as human T-lymphocytes and monocytes. These calls are involved in the functioning of the immune system. This infection, in turn, results in the progressive loss of the CD4 T-i ~~C~'~4J~
cell population and disturbs the function of other CD4 cells, such as aonoeyt~~ hereby reducing the patients ability to combat other infections, and predisposing the patient to opportunistic infections which frequently prove fatal.
There are at least three clinical manifestations of AIDS infection. In the initial "carries state, the only indication of infection is either the presence of IiIV antibodies in the blood-stream or the ability to culture the~virus. The next stage is known as MAIDS related complex~ (ARC) and the physical symptom associated therewith may include persistent general lymphadenopathy, general malaise, increased temperature and chronic infections. This condition usually progresses to the final, fatal AIDS condition, when the patient loses the ability to tight infection.
A particularly troublesome problem associated with combatting HIV retrovirus infections is that the RNA to DNA
reverse transcription process is fraught with repeated mutation which makes it extremely difficult for the body~s immune system to recognize and attack infected cells along with the virus itself.
It is therefore, an object of this invention to provide a method of treating virus infections in patients having an abnormally low white blood cell count.
It is a further object of the invention to provide a method of treating patients infected with an HIV retrovirus.
A still further object of the invention is to provide a vaccine against virus intoctiona, particularly wh~rt the inl.ction is causb by a ratrovirua such as an tilt/ r~trovirua.
Others objects o! the invention will b~ apparent from the d~acription o! the invention which follows.

~~~''~4~~
In accordance with the present invention a method has been found for treating patients who are infected with a virus, particularly an ~IIV retrovirus, using a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e. g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface (e. g., to a receptor and/or to a nucleic acid tragaent on the virus surface) and/or to nucleic acid (e.g. , DNlI or RNI~) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum, such as ultraviolet light, for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient for the purpose of engendering an immune response to the viral infection. Psoralen compounds are particularly preferred for this purpose, especially the compound 8-methoxypsoralen -- in which, case WA radiation is preferred for activating said compound.
An especially important feature of the present invention is that the treatment methods described herein are used in patients having an abnormally low number of circulating ~~~'~4~~
lymphocytes or white blood cells. In addition, it has been found by the imrentors that the treatment methods according to the invention can ~~ used to reconstitute the immune aystam~s !unction in such patients. This is of tremendous importance in the treatment of ARC/AIDS patients who, in addition to having an HIV infection (which itself has heretofore been extremely ditticult to treat), also have a depruead immune syetea. such patients generally cannot tolerate a reduction in the number e!
their~lymphocytes or white blood cells without succumbing to opportunistic infections.
It has thus been found by the inventors that the treatment methods in accordance with the present invention err useful to control HIV and other retrovirus intection~ in patients having an abnormally low white blood cell level, without causing a harmful depression of the patients immune systea.
Accordingly, the methods of the invention can be employed in the treatment of conditions such as ARC/AIDS without subjecting the treated patient to a risk of opportunistic infection. The treatment methods in accordance with the invention may also be used in such patients to improve the functioning of their immune systems. In addition, HIV retroviruses may also be responsible for causing diseases other than AIDS. The inventors believe that the inventive methods should be useful for treating such other diseases as well.
In accordance with the invention, a photoactive compound such as 8-methoxypsoralen is administered to the patients ~~3U'~4~9 nlood, or aom~ traction thereol, in vitro or in vivo using conventional administration routes. A portion o! the patient's bloody is tben treated (preferably, extracorporeally) using photopheruia, which comprise subjecting the blood to electromagnetic radiation in a wavelength suitable !or activating the photoactive compound, such as ultraviolet light, preferably long wavelength ultraviolet light in the wavelength range of 320 to 400 nm, commonly called WJ1 light. The treated blood, or a traction thereol, is returned to the patient (in the case of extracorporeal photopheresis) or remains in the patient (following in vivo photopheresia).
Vaccines against viruses and methods of making same are also provided according to the invention. According to they invention, a photoactive compound (as described herein) is administered to the blood or some fraction thereof of a donor who is infected with a virus, such as an HIV retrovirus and/or who is suffering Eros AIDS or AIDS Related Complex. At least a portion of the donor's blood is then treated using photophsreais, as described above. The treated blood or soma fraction thereof (eg., treated free isolated virus) may be used as a vaccine. In the case of treating an HIV infection it is preferred to use treated virus infected cells along with the treated virus in order to obtain the desired immune response.
Optionally, the treated blood is processed by conventional techniques to substantially remove the erythrocytes. The resulting processed fraction is then used as s . 2007499 a vaccines which can be administered to a patient.
A further aspect of the invention is as follows:
A method for producing a vaccine against an HIV
retrovirus infection in an infected donor, said method comprising the steps o!:
a. administering to at least a portion of the donor's blood a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane (e.g., by binding to a receptor and/or a nucleic acid fragment on the cell membrane) and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case o! either free virus or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e. g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation o! s prescribed apect~ for the purpose o! inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system o! the patient for the purpose o! engendering an immune response to the ~ira~ inlectiont and b. then treating the portion o! the donor's blood to which the photoactive compound has been adainisterad, said treatment comprising subjecting at least a traction o! said portion o! blood to photopheresis using said electro-magnetic radiation, said photoactive compound and said electro-magnetic A

radiation being administered in amounts which are pharmaceutically effective for attenuating and/or inactivating at least some o! the HIV retrovirus which is present in the treated traction.
Fig. 1 is a block diagram of a photopheresis apparatus which can be used to practice the inventive method:=
Fig. is a graph of changes in CD4 cells and GPZ4 and GPiZO
antibody levels of patient No. 3 in Example Z;
Fig. 3 is a graph of changes in GP24 antibody lever of the five patients treated in Example 2:
Fig. 4 is a graph of changes in GPiZO antibody levels of the five patients treated in Example Z; and Fig. 5 is a graph of changes in CD4 helper cell percentages o~
the Live patients treated in Example Z.

A

~..
While it is not intended that the scope of the present invention be limited by any specific theory of operation, it is believed that viral infections, particularly those which are not controlled by the normal immunological response of a patient, can be treated using a photopheresis treatment according to the invention. It is believed by the inventors that the photopheresis treatment according to the invention not only treats the viral infection, but is believed by the inventors to (i) restore the ability of a treated patient~a immunm systes (which has been weakened by the viral infection) to coabat othir infections, including non-viral infections, and (ii) restore the immune system's anamnestic response to previous infections.
The photopheresis treatment method according to the invention is of particular value in the treatment of frequently mutating viral infections, such as ratroviruses, for instance HIv retroviruses. In accordance with the photopheresis methods of the invention, treated infected cells as well as killed and/or attenuated virus, peptides, native sub-units of the virus itself (which are released upon cell break-up and/or shed into the blood) and/or pathogenic noninfectious viruses may be used.
Mutation of the viral antigen does not shield it from attenuation/inactivation during photopheresis and consequent generation of an immune response to the mutant forma of the viral '~~~''~49~
antigen. Thus, the treatment m~thod~ according to the invention provide s dyna,ia autogenous vaccine against viral infections.
The inventive methods haw been found by the inventors to bs useful in the treatment of patients having a virus infection and who have an abnormally low white blood cell count and are particullarly useful in treating HIV retrovirus infections. The inventive methods are also particullarly useful for treating patients who are AIDS Carriers or who have AIDS or AIDS Related Complex.
According to the claimed methods, a photoactivs compound is first administered to the blood of a patient who is infected with a virus. The photoactivs compound may bs administered in vivo (..g. orally os intravenously os may ber-administered in vitro to a portion of the patisnt~s blood which has been removed from tha patient by~employing conventional blood withdrawal techniques.
Alternatively, free virus is isolated from infected cells using conventional virus isolation methods which are known in the art. The photoactivs compound can be administered to the infected cells prior to virus isolation or can be administered to the free isolated virus. In the case of treating HIV infection, however, it is presently preferred to use both treated virus and treated virus infected cells in the methods described hersinbelow.
In accordance with the present invention, the photoactive compound selected should preferably be one that ' ~~~'~499 rinds, in the case of a virus infected cell, to the cell membrane (e~g~. ~ biding to a receptor and/or a nucleic acid fragment on the cell meabrantf and/or to nucleic acid in the cell nucleus or cell cytoplasts, or, in the case o! either tree.viru~ or cell associated virus, that binds to the virus surface (e.g., to a receptor and/or to a nucleic acid fragment on the virus surface) and/or to nucleic acid (e. g., DNA or RNA) which is incorporated in the virus, upon activation by exposure to electromagnetic radiation o! a prescribed spectrum, such as ultraviolet light, for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system o! the patient. Psoralen compounds are particularly preferred for this purpose especially the compound 8-methoxypsoralen -- in which case WA radiation is preferred. for activating said compound.
Next, the portion of the patient's blood, or the free isolated virus, to which the photoactive compound has been administered is treated by subjecting the portion of the blood, or the fre. isolated virus, to photopheresis using said electromagnetic radiation -- for example, ultraviolet light. The photopheresis step is preferably carried out in vitro using an extracorporeal photopheresis apparatus.
The photopheresis step in accordance with the present invention may also be carried out in vivo.
A presently preferred extracorporeal photopheresis apparatus for use in the methods according to the invention is .. 2007499 currently manufactured by Therakos, Inc., Westchester, Pennsylvania under the name UVAR. A description of the Tharakos WAR photopheresis apparatus may be found in U.S. Patent No.
4,683,889, granted to R.L. Edelson on August 14, 1987, As illustrated diagramatically in Pig. 1, the apparatus includes a pump 10 for removing blood from the patient via a donor needle placed in an appropriate vein of the patient: an irradiation chamber 2o; a radiation source 30 in close proximity to the irradiation chamber and a centrifuge 40, preferably of the continuous type. The various parts of the apparatus, such as tubing collection bags for the blood and the like, which corns in contact with the patient's blood or some fraction thereof, are preferably replaceable so that they may be disposed of after each use to prevent the possibility of transmitting blood-borne infections from one patient to others who are subsequently treated with the apparatus.
The exposure of blood, or free isolated virus, to ultraviolet light in a photopheresis apparatus is within the ability of persons having ordinary skill in the art.
When the photopheresis step is carried out in vitro, at least a fraction of the treated blood, or the treated free isolated virus, is returned to the patient following the photopheresis treatment. Preferably, the treatment method described hereinabove is repeated at an interval of about once A

par week to about once every four weeks. Most preferably, in the treatsant~o! MST infection, the treatment methods described herein arm adsinistered on two successive days and repeated approximately once per month (ie, the patient preferably receives two treatments every month).
In view of th~ disclosure contained herein, those persons who are skilled in the art will be able to adjust the treatment parameters -- ie, dosage o! the photoactive compound and electromagnetic radiation, periodicity of treatment (a. g., monthly, weekly, etc.) and the number of treatments administered in each period ( a.g., twice per month on two successive days) -, depending on the condition of the patient and the patiant~s response to the treatment.
Preferred photoactive compounds for use in accordance with the present invention are compounds known as psoralens (or furocoumarins) which are described in U.S. Patent No. 4,321,919.
The preferred photoactive compounds for use in accordance with the present invention include the following:
psoralen:

8-methoxypsoralen;
4,58-trimethylpsoralen:
5-methoxypsoralen;
4-msthylpsoralen:
4,4-dimethylpsoralen;

A

~Q(~ :49~
4-5~-dimethylpsoralent and 4~,8-mathoxypsoralen Tha most particularly preterrad photoactive compound for use in accordance with the invention is 8-methoxypsoralen.
The determination of an elective dosage for in vitro virus inactivation of free isolated virus is within the ability o! persons having ordinary skill in the art.
The photoactive compound, when administered to the patient's blood in vivo is preferably administered orally, but also can ba administered intravenously and/or by other conventional administration routes.
The preferred dosage o! the photoactivs compound is in the range o! about 0.3 to about 0.7 mg/kg of body weight although larger or smaller doses may be employed. when the photoactiva compound is administered in vitro to only a portion o! the patient's blood or fraction thereof, it is within the ability of those skilled in the art to calculate a dosage which is equivalent to said range based upon the volume of treated blood or fraction thereof.
When administered orally, the photoactive compound should preferably be administered at least about one hour prior to the photopheresis treatment and no more than about three hours prior to the photopheresis treatment. The timing of administration may be adjusted up or down as needed depending on tha bioavailability of the photoactive compound, its expected half-life, etc. If administered intravenously, the times would ~~f~'74~9 generally be shorter.
The photopheresis treatment in the treatment methods according to the invention is pr~terably carried out using long wavelength ultraviolet light (UVA) at a wavelength within the range of 3a0 to 400 nm. The exposure to ultraviolet light during the photopheresis treatment preferably has a duration of about three to tour hours, although shorter or longer treatment periods may be used it desired.
Whatever the spectrum of electromagnetic radiation, the exposure of virus infected cells and/or virus thereto, following administration of the photoactive compound, should be of sufficient intensity/duration to effectively inactivate and/or attenuate the virus. The selection of an appropriate wavelength for photopheresis as wall as the exposure, depending upon the photoactive compound being employed and the conditions of treatment (e.g., in vivo exposure or in vitro exposure), is within the ability of those skilled in the art in view of the present disclosure.
When the photoactive compound is 8-methoxypsoralen, it is preferred in accordance with the invention to utilize an exposure to WA radiation of about 2 Joules/meter2 based upon the surface area of the virus and virus infected cells undergoing treatment.
When the photopheresis treatment according to the invention is carried out in vivo, careful attention should be paid to controlling the maximum radiant exposure so as to avoid ' ~~~'~49~
,.nnecessary injury to the patient. Methods for calculating maximum radiant exposure to ultraviolet light are known in the art and,_ ttterelore, shall not be described herein.
In summary, the invention provides a.novel treatment for patients who are infected by a virus and who have depressed immune systems as a result of such infection, as well as for patients who are infected with an HIV retrovirus or who are AIDS
Carriers or who have AIDS or AIDS Related Complex. Such patients cannot tolerate a treatment that would depress their immune system .
The treatment methods according to the invention have beers loured by the inventors to be sale in this latter regard while also being ettective in combatting HIV infection fn h~ans.
The invention also provides methods !or making vaccines. According to the invention, a donor who is infected with a virus, such as an HIV retrovirus, may be utilized to produce a vaccine against his infection as follows.
First, a photoactive compound as described hereinabove is administered to at least a portion of the donor's blood containing free virus and/or virus infected cells either prior to removal of the blood, either orally or intravenously, or after removal from the donor in which case it is administered in vitro. Optionally, a portion of the donor's blood could first be processed using known methods to substantially remove the erythrocytes and the photoactive compound is then administered to the resulting fraction.

~~3C~'~4~~
In any caws, the portion o! blood (eg., an enriched lsukoayt~ !:'action thsrso!) and/or !ru isolated virus to which the photoactivs compound has been administered i~ subjected to a photophersais treatment using slsctromagnstia radiation o! a prescribed opsctrum, s.g.,ultraviolst light, preferably WA, in the manner previously described. Ths treated blood, the treated portion thsrso! or the treated tree isolated virus (as the case may bs) is then administered back to the donor as an autogsnous vaccine. It will be understood that in accordance with the present invention the treated virus can also bs isolated from the treated blood or portion thsrso! following photophsrsais traatmsnt !or use as a vaccine.
Additionally, in accordance with the prusnt invention, the treated blood, which is itssl! a mixture o!
various blood components including peptides or polypsptidss, eg., cytokinss, lymphokinss, monokinsa, stc., and/or the treated portion of blood may bs processed, as is within the ability o!
parsons having ordinary skill in the art, to isolate a particular component or components which may be used in the treatment of the virus infection of the donor and/or may be used as a vaccine against the virus.
A male patient, 39 years of age, weighing approximately 70 kg and who had bean diagnosed as having AIDS Related Complex was treated in accordance with the present invention as follows:
8-methoxypsoralen was administered orally to the patient during the afternoon of the first day of treatment at a dosage! o! 3a mg (i.a. about 0.4 ~/kg). Approximately one hour after the 8-methoxypeoralen had been administered to the pats~nt h~ was prepared for withdrawal of a portion of his blood for photopheresis treatment using said Therakos WJ,R photopheresis machine.
A centrifuge which is integral with the photopheresis machine was used to spin oft substantially all of the orythrocytu from the withdrawn blood and these were subsequently returned to the patient. Next, approximately 30occ of plasma and 240ca of butty coat (which includes the T-lymphocytea) were removed in six cycles (40cc o! bu!!y coat pegs cycle). The buoy coat and plasma were subjected to W1~ light axposur~ beginning after 40cc of bully coat had been collected in the first cycle. Tha irradiation was continued through all six cycles and then for an additional one and one-halt hours for a total irradiation time of approximately tour hours. The irradiated bully coat and plasma wire then returned to the patient. This process was repeated in the morning of the following day.
The blood parameters of the patient before receiving the photopheresis treatment according to the invention and five w~eks after receiving the treatment are set forth in Table 1.

~~U~'~4~:~
Hlood Paramatera o! ARC Patient Traatad ~ Photooherae ~ s Mst_hod Accords nc To Th! Twvs.,~. a ,:
Blood Belora Altar Normal Parameter Treatmg~
HEMOGLOHIZ1 11.7 G/DL 10.8 G/DL --HEMACRIT 33.9% 33.6% -_ 5.2 /UL 4.7 /UL -_ Hands 16% 16~
-.

Sag. Neut. 29%
23% -.

Lymphocytes 43% 46~
-.

Atyp. Lymph. 7% 7; -.

Monocytss 5% 6% --P~~~TS 208,000 241,000 --LYMPHOCYTE9s CD3 (T3) 17% 85% 56-78%

CD4 (T4) 4% 22% 32-50%

CD8 (T8) 26% 62$ 13-38%

T4/T8 RATIO 0.15 0.4 0.8-1.9 In addition, the patient, who had a negative mumps skin test (which was carried out in the usual manner known in the art) prior to receiving the treatment in accordance with the present ~:~C~'r~~~
invention a: described hereinabove, developed a positive mumps skin teat after six monthly treatments in accordance with the present invention-- this positive mumps skin test being documented by skin biopsy demonstrating the characteristic histologic changes of delayed hypersensitivity. The above results demonstrate a reconstitution o! normal immune function as a result o! the treatment method according to the invention.
This patient continued to receive the described treatment on two successive days on a monthly basis as part of a l0 clinical study involving five patients, the results o! which are discussed in Example 2 below in which thin patient is identified as patient No. 1.
Five patients with ARC characterized by fatigue, lymphadenopathy, fever, absent akin-test reactivity, and decreased CD4 cells volunteered and ent.red into a clinical study that was conducted in accordance with the provisions of the Institutional Review Board for the treatment of human subjects at Morristown Memorial Hospital, Morristown, New Jersey. Three homosexual males ages 27 to 39, one 42-year-old male reformed intravenous drug abuser, and one 27-year-old female reformed intravenous drug abuser comprised the study group. The rangs of time from the first known HIV antibody positivity until enrollment for the HIV positive patients was one to four years.

' W~~'~4 ~;~
inclusion criteria included only patients not having undergone any previous therapy against HIV infection.
Each patient received a monthly regimen o! the treatment substantially as described in.Example 1 repeated on two succesive days. The dosage o! 8-methoxypsoralen employed varied between O.t and 0.6 mg/kg and was administered orally.
Four of the patients received a total o! twelve treatments each over the six month course o! the study, that is, on two successive days at monthly intervals for six months. The to filth patient voluntarily discontinued his treatment alter the filth month and only received ten treatments. On the basis o!
the physical examination, monthly antibody and antigen studies, and CDR percentage, all five patients had either a stabilization~
of their disease or a positive response to the treatment. None of the patients experienced damage to their immune systems during the course of the treatment.
By strictly clinical criteria, all five patients demonstrated improvement with respect to lymph node size. Energy index and general state of ~~well being" improved in four. At the start of treatment, the increase in energy level lasted for approximately one week post-treatment. By the third month of treatment, a more consistent rise in energy level persisted.
Most notably, the first patient treated (the patient in Example l, above), a homosexual male who before treatment was able to walk up a flight of stairs only with difficulty, is now capable of jogging three and one-half miles per day and of lifting weights.
~ onm patient who did not sees to have a persistent rise in energy level was the feaale reformed intravenous drug abuser with three small children. Although her fatigue had been chronic prior to her entry into the study, no persistent change in energy level was observed after therapy.
Additional clinical observations include increased appetite and weight stability in four of the patients, the exception being the female who lost 3.3 kg over the =ix month treatment period. Lymphadenopathy disappeared in all live patients by the third month and has remained absent. The four male patient: also regained delayed cutaneous hypersensitivity sites six months of therapy. During the six-month period of treatment all patients were able to manage common community-acquired upper respiratory tract infections without difficulty.
Other than normal variations, hemoglobin and hamatocrit, reticulocyte count, and platelet count remained stable. There were minor variations in the white blood count, and circulating total lymphocytes remained stable in the four male patients: however, the single female patient showed a decrease in white blood count (4,000 prior to treatment to 2,000 after six months of therapy) and a parallel decrease in lymphocytes. In all cases, ESR, urinalysis, and SMAC-20 remained unchanged with the exception of minor rises in serum SGOT and SGPT. EBV titers remained constant in all but one patient in whom the IgG capsid antigen decreased from a titer of 1:1280 to a 2a IGr~~ l ~~~
~citsr o! 1:160 paralleling an increse in HIV antibody titers.
CMV titers regained unchanged in all patients. Those patients who wets PZ4 antigen negative resained negstivet those who were positive remained positive, and no changes in titer occured~by the end o! the six-month period. Viral cultures o! blood for IiIV
turned negative in one patient (Patient No. 3) but regained positive in the others aft~r the reported therapy.
The course of the patient whose HIV culture turned negative (Patient No. 3) is outlined in Fig. 2. The Gp 24 antibody livels o! all five patients over the course o! treatment are plotted in Pig. 3. The Gp 120 antibody levels o! all five patients over the course of treatment are plotted in Fig. 4.
Finally, the CD4 helper cell percentages o! all five patients over the course o! tr~atment are plotted in Fig. 5.
A summary of the clinical characteristics and laboratory results is provided in Table 2 below.

~~3~1'~4~~
Summary O! Clinical characteristics And Laboratory Resulta Patient No. 1 Age: 38 sex: Male Predisposing Factor: Homosexual Known Duration O! HIV Antibody Poaitivity: 3 years r~

Weight(kg) 71.3 70.5 CD4 Cells(8) 4 34 GP24 Antibody Level 2.5Z

(absorbance) GP120 Antibody Level 2.2Z 1.68 (absorbance) Delayed Skin Test neg. pos.

Hypersensitivity Virus Culture (blood) pos. pos.

Patient No. 2 Age: 30 Sex: Male Predisposing Factor: Homosexual Known Duration o! HIV Antibody Positivity: 2 years - PRE-TREATMENT POST-TREATMENT
Weight(kg) 64.5 66.0 CD4 Calls() 29 31 GP24 Antibody Level O.iZ 0.74 (ab~orbance) GPiZO Antibody I,ewel 0.03 0.14 (abaorbance) Delayed Skin Teat neg. . pos.
Hypersensitivity Virus Culture (blood) poi, poi.
Patient No. 3 Ages Z7 Seu: Male Predisposing Factor: Homosexual Known Duration of H=V Antibody Positivity: 4 years r i5 Weight(kg) 63.6 64.7 CD4 Cells(%) 34* 33**

GP24 Antibody Level 0.7Z
1.69 (absorbance) GP120 Antibody Level 1.04 2.07 (absorbance) Delayed Skin Test neg. pos.

Hypersensitivity Virus Culture (blood) pos. neg.

* Data for this patient's pre-treatment CD4 Cells(%) is not available. The value shown in the table was determined for blood drawn one month after the patient received his first treatment.
** This patient received only ten treatments over a five month period at which point he voluntarily opted to discontinue receiving treatment. Three months after voluntarily discontinuing treatment this patient's CD4 Cells(%) had risen to 40. However, five months after discontinuing treatment the CD4 Cells(%) fell to 24.

Patient No. a Age: 4Z

Sax: Mal~

Predisposing Factor: Reformed iVDA

Known Duration of Hiv Antibody Positivity: Z years -P

~ - ~T
Weight(kg) TBE~ 108 CD4 Calls () 11 33 GPZ4 Antibody Lwel 0.6Z
1.76 (absorbancej _ GP120 Antibody Level 0.21 1. Z3 (absorbanc~) D~layed Skin Test neg. pos, Hyp~rs~nsitivity Virus Culture (blood) pos. poe.

Patient No. 5 Age: 27 Sex: Female Predisposing Factor: Reformed IVDA
Known Duration Ot HIV Antibody Positivity: 2 years PRE-TREATMENT POST-TREATMENT
Waight(kgj 61.0 57,7 CD4 C~lls(~) 12 13 GP24 Antibody Level 1.41 2, g9 (absorbance) GP120 l~ntibody Level 0.46 1.95 (absorbancej Delayed Skin Teat nag. nag.
Hypersensitivity Virus Culture (blood) pos. pos.
Upon completion of the six month course of therapy described in Example 2, wherein patient Nos. 1,2,4 and 5 received treatment on two succusiVe days at monthly intervals, the course of therapy was modified with each patient receiving only one treatment per month instead of two. It was found by~th~
inventors that each patients CD4 Cell(%) tell oft markedly. The original course of therapy, wherein the patients received treatment on two successive days once per month was reinstituted with the result that in patients 1 and 2 the CD4 Cell(%) rose.
Data for CD4 Cell(%) in patients 4 and 5 is not yet available.
The individual results were as follows:
At the conclusion of the original six month course of therapy at two treatments per month, this patient's CD4 Cell(%) was 34. Attar receiving only one treatment per month for five months, the CD4 Cell(%) fell to 18. The original course of therapy was reinstituted and attar two months the CD4 Cell(%) rose to 39.

A~~~ l ~~~
At the conclusion of the original six month course of therapy at two treatments per month, this patient s CD,~ Call(;) was 31. Attar receiving only one traataant par month for tour months, the CD4 Call(%) fell to 10. The original course of therapy way reinatituted and after two months the CD4 Cell(%) rose to 27.
Patient No. 4 At the conclusion of the original six month course of therapy at two treatments per month, this patiant~~ CD4 Cell(%) was 33. After receiving only one treataant per month for four months, the CD4 Cell(%) fell to 7. The original course c!
therapy has been reinstituted but data on changes in the CD4 Cell(%) is not yet available.
Patient No. 5 At the conclusion of the original six month course of therapy at two treatments par month, this patient s CD4 Cell() was 13. After receiving only one treatment per month for four months, the CD4 Cell(%) fell to 8. The original course of therapy has bean reinstituted but data on changes in the CD4 Cell() ii not yet available.
It should be understood that while the foregoing description has been provided to illustrate the present inventions, it is not intended to limit the scope of the inventions as various modifications to the inventions described i~~~ i~~~
herein may be made by p~rson~ having ordinary skill in the art without departing lroa the spirit and scope thereo! as dslinsd in the lolloving claims.

Claims

1. A method for producing a vaccine against an HIV
retrovirus infection in an infected donor, said method comprising the steps of:
a. administering to at least a portion of the donor's blood a photoactive compound that binds, in the case of a virus infected cell, to the cell membrane and/or to nucleic acid in the cell nucleus or cell cytoplasm, or, in the case of either free virus or cell associated virus, that binds to the virus surface and/or to nucleic acid which is incorporated in the virus, upon activation by exposure to electromagnetic radiation of a prescribed spectrum for the purpose of inactivating and/or attenuating the virus and permitting the so treated virus and/or virus infected cells to be presented to the immune system of the patient for the purpose of engendering an immune response to the viral infection; and b. then treating the portion of the donor's blood to which the photoactive compound has been administered, said treatment comprising subjecting at least a fraction of said portion of blood to photopheresis using said electro-magnetic radiation, said photoactive compound and said electro-magnetic radiation being administered in amounts which are pharmaceutically effective for attenuating and/or inactivating at least some of the HIV retrovirus which is present in the treated fraction.

2. The method of claim 1, wherein the fraction of blood is processed to substantially remove erythrocytes.

3. The method of claim 2, wherein the fraction of blood is processed to substantially remove erythrocytes prior to performing step (b).

4. The method of claim 2, wherein the fraction of blood is processed to substantially remove erythrocytes after performing step (b).

5. The method of any one of claims 1 to 4, wherein the fraction which is treated in step (b) consists essentially of free isolated HIV retrovirus.

6. The method of any one of claims 1 to 5, further comprising processing the treated fraction of blood following step (b) to isolate treated HIV retrovirus for use as a vaccine against an HIV retrovirus infection.

7. The method of any one of claims 1 to 6 wherein said photoactive compound binds to the cell membrane by binding to a receptor and/or a nucleic acid fragment on the cell membrane.

8. The method of any one of claims 1 to 6 wherein said photoactive compounds binds to the virus surface by binding to a receptor and/or a nucleic acid fragment on the virus surface.

9. The method of any one of claims 1 to 6 wherein said photoactive compounds binds to the nucleic acid by binding to DNA or RNA.