CONDITIONALLY CONTROLLED, ATTENUATED HIV VACCINE

Abstract: The present invention provides an attenuated HIV vaccine comprising an HIV virus modified to replicate only in the presence of at least one tetracycline analogue. Additionally, the present invention provides a method for immunization of humans against HIV which comprises administering to a human a vaccine including an HIV virus modified to replicate only in the presence of at least one tetracycline analogue. Simultaneously, at least one tetracycline analogue is administered for a period of time to allow replication of the modified HIV virus in vivo sufficient to produce immunity. Preferably, the tetracycline analogue is doxycycline. A replication competent HIV-DoxT virus genome which can be controlled by the presence or absence of doxycycline is produced by preparing a promoter, TetopTCAT; producing a provirus, pHIV-DoxT, using the TetopCAT promoter, and transfecting the pHIV-DoxT in cell lines in the presence of doxycycline. A replication competent HIV-DoxSp virus genome which can be controlled by the presence or absence of doxycycline is produced by preparing a promoter, TetopSpCAT; producing a provirus, pHIV-DoxSp, using the TetopCAT promoter, and transfecting the pHIV-DoxSp in cell lines in the presence of doxycycline. Anti-HIV vaccines are prepared using the HIV-DoxT and HIV-DoxSp virus genomes. These viruses and doxycycline are administered to human hosts. The doxycycline is administered for a time sufficient to build up immunity and then the administration of the drug is stopped so that the doxycycline-dependent viruses will no longer replicate.

Description: TITLE OF THE INVENTION: Conditionally Controlled, Attenuated HIV Vaccine RELATED APPLICATION This application claims pπonty of the application entitled Conditionally Controlled, Attenuated HIV-1 Vaccine Candidate, havmg a senal number of 60/146,085 and filed on 07/28/99 BACKGROUND OF THE INVENTION This invention relates to the field of vaccmes More particularly, this invention is directed to a process for controlling the expression of an HIV provirus to produce a doxycycline-inducible HIV genome The genome may be used in attenuated HIV vaccmes Vaccination and immumzation generally refer to the introduction of a non- virulent agent against which an individual's immune system can initiate an immune response which will then be available to defend against challenge by a pathogen The immune system identifies mvadmg "foreign" compositions and agents pnmaπly by identifying proteins and other large molecules which are not normally present m the individual The foreign pro tern represents a target agamst which the immune response is made The immune system can provides multiple means for eliminating targets that are identified as foreign These means include humoral and cellular responses which participate in antigen recogmtion and elimination Bnefly, the humoral response involves B cells which produce antibodies that specifically bmd to antigens There are two arms of the cellular immune response The first mvolves helper T cells which produce cytokines and elicit participation of additional immune cells in the immune response The second involves killer T cells, also known as cytotoxic T lymphocytes (CTLs), which are cells capable of recognizing antigens and attacking the antigen including the cell or particle it is attached to Vaccination has been singularly responsible for conferring immune protection against several human pathogens In the search for safe and effective vaccmes for immunizing individuals against infective pathogemc agents such as viruses, bactena, and infective eukaryotic orgamsms, several strategies have been employed thus far Each strategy aims to achieve the goal of protectmg the individual agamst pathogen mfection by administering to the individual, a target protein associated with the pathogen which can elicit an immune response Thus, when the individual is challenged by an mfective pathogen, the individual's immune system can recognize the protein and mount an effective defense agamst mfection There are several vaccme strategies for presentmg pathogen proteins which include presentmg the protein as part of a non-infective or less infective agent or as a discreet protein composition One strategy for immunizing agamst infection uses killed or mactivated vaccines to present pathogen protems to an individual's immune system In such vaccmes, the pathogen is either killed or otherwise mactivated using means such as, for example, heat or chemicals The administration of killed or inactivated pathogen mto an individual presents the pathogen to the individual's immune system m a nonmfective form and the individual can thereby mount an immune response agamst it Killed or mactivated pathogen vaccmes provide protection by directly generating T-helper and humoral immune responses agamst the pathogenic lmrnunogens Because the pathogen is killed or otherwise mactivated, there is little threat of mfection Another method of vaccinatmg against pathogens is to provide an attenuated vaccme Attenuated vaccines are essentially live vaccmes which exhibit a reduced infectivity Attenuated vaccmes are often produced by passagmg several generations of the pathogen through a permissive host until the progeny agents are no longer virulent By usmg an attenuated vaccme, an agent that displays limited infectivity may be employed to elicit an immune response agamst the pathogen By maintaining a certain level of infectivity, the attenuated vaccine produces a low level mfection and elicits a stronger immune response than killed or mactivated vaccmes For example, live attenuated vaccines, such as the pohovirus and smallpox vaccmes, stimulate protective T-helper, T-cytotoxic, and humoral immunities during their nonpathogenic infection of the host Another means of immunizing against pathogens is provided by recombinant vaccines There are two types of recombmant vaccmes one is a pathogen m which specific genes are deleted in order to render the resultmg agent non-virulent Essentially, this type of recombinant vaccme is attenuated by design and requires the administration of an active, non- virulent infective agent which, upon establishing itself m a host, produces or causes to be produced antigens used to elicit the immune response The second type of recombmant vaccine employs non-virulent vectors which carry genetic matenal that encode target antigens This type of recombmant vaccme similarly requires the administration of an active mfective non- virulent agent which, upon establishing itself m a host, produces or causes to be produced, the antigen used to elicit the immune response Such vaccines essentially employ non-virulent agents to present pathogen antigens that can then serve as targets for an anti- pathogen immune response For example, the development of vaccinia as an expression system for vaccination has theoretically simplified the safety and development of mfectious vaccination strategies with broader T-cell immune responses Another method of immunizing agamst mfection uses subunit vaccmes Subumt vaccines generally consist of one or more isolated proteins denved from the pathogen These protems act as target antigens against which an immune response may be mounted by an individual The protems selected for subumt vaccme are displayed by the pathogen so that upon mfection of an individual by the pathogen, the individuals immune system recognizes the pathogen and mounts a defense agamst it Because subumt vaccmes are not whole mfective agents, they are incapable of becommg infective Thus, they present no nsk of undesirable virulent infectivity that is associated with other types of vaccmes It has been reported that recombinant subumt vaccines such as the hepatitis B surface antigen vaccme (HBsAg) stimulate a more specific protective T-helper and humoral immune response agamst a single antigen However, the use of this technology to stimulate broad protection agamst diverse pathogens remains to be confirmed Each of these types of vaccmes carry severe drawbacks which render them less than optimally desirable for immunizing individuals against a particular pathogen It has been observed that absent an active infection, a complete immune response is not elicited Killed and mactivated vaccmes, because they do not reproduce or otherwise undergo an infective cycle, do not elicit the CTL arm of the cellular immune response m most cases Additionally, killed and mactivated vaccmes are sometimes altered by the means used to render them inactivated These changes can sometimes affect the lmmunogemcity of the antigens Subunit vaccines, which are merely discreet components of a pathogen, do not undergo any sort of mfective cycle and often do not elicit the CTL arm of the cellular immune response Absent the CTL arm, the immune response elicited by either vaccme is often insufficient to adequately protect an individual In addition, subunit vaccmes have the additional drawback of being both expensive to produce and puπfy Attenuated vaccmes, on the other hand, often make very effective vaccines because they are capable of a limited, non-virulent mfection and result in immune responses involving a humoral response and both arms of the cellular immune response However, there are several problems associated with attenuated vaccines First, it is difficult to test attenuated vaccmes to determine when they are no longer pathogemc The nsk of the vaccme being virulent is often too great to properly test for effective attenuation. For example, it is not practically possible to test an attenuated form of Human Immunodeficiency virus (HIV) to determine if it is sufficiently attenuated to be a safe vaccme Secondly, attenuated vaccmes carry the nsk of reverting mto a virulent form of the pathogen There is a nsk of mfectmg individuals with a virulent form of the pathogen when using an attenuated vaccine Recombinant vaccmes require the mtroduction of an active mfective agent which, m many cases, is undesirable Furthermore, in cases where the recombinant vaccine is the result of deletion of genes essential for virulence, such genes must exist and be identified In vaccines in which pathogen genes are inserted into non-virulent vectors, many problems exist related to the immune response elicited agamst the vector antigens which negatively impact the immune response elicited against the target antigen First, the recombinant vaccme introduces a great number of vector antigens against which the immune system also responds Secondly, the vector can be used only once per individual since, after the first exposure, the individual will develop immunity to the vector These problems are both present, for example, in recombmant vaccines that employ vaccima vectors such as those disclosed m U S Pat No 5,017,487 issued May 21, 1991 to Stunnenberg et al This technology has not been universally successful agamst diverse pathogemc orgamsms and it is also complicated by the large amount of excess vaccima antigens presented m the vaccmee Once vaccmated with the vaccmia vector, the vaccinee cannot be effectively vaccmated again using the vaccima vector Accordingly, the most effective vaccines for invoking a strong and complete immune response carry the most nsk of harming the individual while the safer alternatives induce an mcomplete, and are therefore, less effective immune response Furthermore, many subumt vaccines and recombinant vaccines usmg non-virulent vectors to produce target protems are most useful if a smgle antigemc component can be identified which is singularly protective agamst live challenge by a pathogen However, both technologies require that the protective component be identified Such identification is often both labonous and time-consummg A distinct advantage would exist if there were a rapid system for directly testing subumt vaccmation strategies without tissue culture and m the absence of excess vector antigens Furthermore, it would be particularly advantageous if such a system could deliver an antigen that could be presented for development of both T cell immune arms There is a need for a means to immunize individuals agamst pathogen mfection which can elicit a broad, biologically active protective immune response without nsk of mfecting the individual Administration of a protem or peptide does not elicit a CTL response HIV mfection represents a great threat to the human population today Despite the mtense resources expended and efforts made to develop an effective vaccme, the problem remains intractable No vaccme is currently available that protects an individual agamst HIV mfection There is a great need for a method of immunizing an individual agamst HIV mfection There is a great need for an effective lmmunotherapy method to combat the development of AIDS in HIV infected individuals Most of the successful viral vaccmes are live, attenuated versions of the wild-type virus These attenuated viruses replicate to a lower level than their wild-type counterparts This low level of replication is minimally deletenous to the host, but can mduce a very strong immune response For example, the initial polio vaccine was an inactive form of the pohovirus, which did not rephcate The immune response to this vaccine was much less than that to the later version, which was an attenuated, replicating form of pohovirus Consequently, the hve- attenuated polio viral vaccine induced protective immunity in a much higher percentage of recipients than did the mactivated polio vaccme Other live-attenuated viral vaccmes m clinical use include the measles, mumps, rubella, and chicken pox vaccmes Each of these vaccines replicates to some degree m the host A disadvantage of live-replicating vaccmes is that they can, in certain circumstances, cause diseases that the inactivated vaccmes could not In immunocompromised hosts, the live vaccines can sometimes rephcate more robustly than expected and could consequently be harmful to the host For instance, the live-attenuated polio virus vaccine caused paralytic polio at a rate of 1 m l million hosts While a sub-umt based non-rephcatmg viral vaccine, which is incapable of infection, has been successful for hepatitis B virus (containing only the surface protem of the virus), this approach has not been successful for most viral diseases Hence, despite the predictable but small amount of disease-inducing potential of live-attenuated vaccmes, these vaccines remain the vaccmes of choice Acquired Immune Deficiency Syndrome (AIDS) is a devastating and deadly condition that has affected millions worldwide The condition is clinically charactenzed by a set of typical syndromes which manifests itself by the development of opportumstic mfections such as pneumocystic cairnn pneumonia, toxoplasmosis, and cytomegalovirus Additional characteπstics of the AIDS-associated syndromes are the clinical manifestation of neuropsychiatπc abnormalities, AIDS encephalopathy, kidney failure of AIDS nephropathy, heart failure of AIDS cardiomyopathy and certain malignancies such as Kaposi's sarcoma or B-cell lymphoma The etiological agent for this condition has been identified as a virus, the human immunodeficiency virus (HIV) HIV is a retrovirus, that is, it is an RNA virus that replicates by transcnbing genetic information from RNA to DNA, msertmg this DNA mto the host genome, and ultimately forming new RNA from the pro-viral DNA template For HIV- 1 , many attempts have been made at producmg a sub-umt based, particle vaccine However, it is unclear whether this type of vaccme will generate adequate protective immunity In the macaque model of AIDS, the sub-particle approach has consistently failed to induce protective immunity By contrast, m the same macaque model, the live-attenuated virus approach has worked with great efficacy Several investigators have shown that by moculatmg macaques with attenuated viruses that contam large deletions in the viral genome, the hosts developed an immune response over time and became protected from subsequent challenges with wild- type viruses This approach has not been extended to HIV-1 m humans for several reasons The primary reason for this is that attenuated HIV vaccmes could still rephcate chronically Chrome replication of the attenuated HIV could lead to the disease itself. Alternatively, the attenuated HIV could mutate over time and develop the ability to rephcate to higher levels The reverted vaccine could then mduce disease Finally, the reverted pathogemc vaccme could then be transmitted to other people In short, the host would not eliminate the attenuated HIV, and, thus the potential for senous side effects is unknown This problem was illustrated by a senes of cases in Australia wherein individuals were mfected via blood products transfused from a single donor The blood contained HIV- 1 virus, which was found to have deletions in the U3 region of the LTR and in the nef-codmg region Initially, these patients, despite bemg mfected for several years, maintained stable CD4 lymphocyte counts Their lack of clinical progression suggested that mutations, found in this naturally defective strain of HIV-1, could be used to design an attenuated HIV-1 vaccme However, longer follow-up of these individuals has revealed a clear declme in CD4 lymphocytes in some, accompamed by detectable viral loads This example crystallizes the central issue confronting live-attenuated HIV-1, which is the potential of an innocuous form evolving over time into a different form that could rephcate to higher levels The evolved virus then has the potential to mduce disease, which could then be transmitted to others PCT International Application Number PCT US90/01515 published Oct 4, 1990 discloses methods of immunizing an individual agamst pathogen infection by directly injecting polynucleotides mto the individual's cells m a smgle step procedure. The stimulation of inoculated cells is neither disclosed nor suggested. An HIV vaccme is disclosed which consists of the mtroduction of polynucleotides that encode the viral protem gpl20. The operabihty of this vaccine is not evidenced U. S Pat Nos. 5, 153,202. 5,278,173 and 5,318,979 to Davis disclose the treatment of HIV with antimalanal drugs in combination with antimalanal antibiotics such as doxycyclme. These combmations that mclude tetracyclme analogs are disclosed to inhibit replication of U.S. Pat. Nos. 5,242,820 to Lo discloses that M fermentans is associated with HIV infection and is sensitive to doxycyclme U.S. Pat No. 5,534,413 to Lo, et al. teaches that the mycoplasma, M. penetrans is associated with HIV mfection and is sensitive to doxycycline. U.S. Pat No 5,830,876 to Werner, et al teaches a method for immunizing a human agamst HIV by administering two different DNA molecules to different cells of the human The different DNA molecules encode different HIV structural proteins which are selected from the group consisting of gag, pol and env. U.S. Pat No. 5,994,108 discloses the utilization of transdominant HIV tat substitution and truncated gene mutants of ammo acid residues as pharmaceutical agents. The disclosure teaches the removal of at least 72 ammo acids from the HIV virus m order to utilize the mutant virus as a possible vaccme U.S. Pat No. 6,015,661 is directed toward immunologic and nucleic acid based methodologies for the detection of non-pathogenic human immunodeficiency virus type I(HΓV-I ) strains m the body fluids of HlV-infected individuals. SUMMARY OF THE INVENTION An object of the invention is to provide an attenuated HIV vaccine with minimal nsk of uncontrolled replication Another object of the invention is to engmeer an HIV pro viral plasmid cloned to allow the production of the controlled virus m the presence of a tetracyclme analogue such as doxycyclme The vaccination process compnses the co-administration of an attenuated provirus and a tetracyclme analogue, particularly doxycycline, to form a controlled attenuated virus in the body The attenuated virus of the present invention is removed from the body after stoppmg the availability of the tetracyclme analogue. Another object of the invention is to administer doxycycline with an attenuated provirus for a limited penod of time sufficient to mduce an immune response The attenuated provirus is capable of forming the virus, and thus eliciting an immune response agamst infection by HIV, only when administered with tetracyclme analogs such as doxycyclme There exists a senous need for an attenuate HIV vaccme that would provide mduced protective immunity agamst human immunodeficiency virus while reducing and or eliminating the nsks of long term side effects and transmittal One embodiment of the present invention provides an attenuated HIV vaccme composing an HIV provirus modified to produce the corresponding virus only m the presence of at least one tetracyclme analogue In another embodiment, the present invention provides a method for immumzation of humans agamst HIV which compπses administering to a human a vaccme including an HIV proviral plasmid modified to produce the HIV virus only m the presence of at least one tetracychne analogue Simultaneously, at least one tetracyclme analogue is administered for a penod of time to allow production of the modified HIV virus m vivo sufficient to produce immunity Preferably, the tetracyclme analogue is doxycyclme Amongst the structural and regulatory protems encoded by HIV is a transacting polypeptide termed the Trans-Activator of Transcnption or TAT, which acts by binding to a specific region of the genomic RNA near to the long terminal repeat (LTR) termed the TAR (trans-activation response region) The action of TAT, a polypeptide of some 86-101 ammo acid residues, promotes viral RNA synthesis, so that blockmg of its action presents a potential therapeutic target We and others have previously introduced the concept of controlling simian immunodeficiency virus (SIV) and HIV-1 replication through a gain-of-function approach Through the addition to the proviral genome of the herpes simplex virus type 1 enzyme, thymidine kmase, HIV-1 and SIV can be made sensitive to the drug, ganciclovir In vitro infection with HIV- 1 -TK and SIV-TK can be eliminated by ganciclovir However, the gene for thymidine kmase was quickly deleted during reverse transcnption HIV-Dox will produce virus in the presence of doxycyclme When doxycyclme is removed, the HIV-Dox stops producmg virus The present mvention is designed to provide an HIV vaccme This is achieved by providing a means for the control of the expression of an HIV provirus in producing a doxycyclme-mducible HIV-1 genome Usmg the present invention, a vaccme is provided which contains an attenuated HIV-1 provirus wherein the production of the virus m the host is under the control of another drug, namely tetracychne analogues such as doxycyclme Specifically, the virus is produced only in the presence of the drug When the drug is no longer administered, the virus m no longer produced Further, the produced virus does not uncontrollably rephcate m the body Replication is inhibited m the absence of the tetracyclme analogue In the wild-type HIV, the replication process is dependent on the interaction of the protem. TAT, and on the TAR RNA region In the HIV used in the present mvention, the Tar region has been mutated such that TAR no longer interacts with TAT This mutation essentially kills the HIV In accordance with one embodiment of the present invention, the first portion (U3 region) of the HIV is modified to contain a sequence that allows binding of reverse tetracyclme transactivator (RTTA) This protein will bind to the specific DNA sequence only in the presence of doxycyclme When the RTTA is bound to the DNA sequence, it promotes transcnption of production of the HIV RNA, which gives nse to all the HIV protems and genome The gene of the RTTA is placed within the HIV genome forming HIV-Dox HIV- Dox will produce virus in the presence of doxycyclme When doxycyclme is removed, the HIV-Dox stops producing virus In accordance with one embodiment, the HIV-Dox and the drug, doxycyclme, are given simultaneously to the host The administration of doxycyclme is for the penod of time needed to mduce an immune response and is then discontmued After the discontinuation of the drug, the HIV-Dox stops replicating and the HIV-Dox is then eliminated from the host, drastically reducing the possibility of long-term effects In accordance with another embodiment of the present mvention, cells are removed from the host body and transfected with the provirus to produce the controlled virus The transfected cells are remtroduced to the body mducmg an immune response to the viral disease BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic illustration of TetopT promoter with mutation in Tar m accordance with one embodiment of the present mvention Fig. 2 is a schematic illustration of TetopSp promoter with mutation m Tar m accordance with one embodiment of the present mvention Fig. 3 is photograph showing CAT production from TetopT caused by a plasmid expressing RTTA m the absence and presence of doxycyclme m accordance with one embodiment of the present mvention Fig. 4 is a photograph showing CAT production from TetopSp caused by a plasmid expressing RTTA in the absence and presence of doxycycline in accordance with one embodiment of the present invention. Fig. 5 is a map of pHIV-Dox in accordance with one embodiment of the present invention. Fig. 6 is a chart showing the effect of doxycycline on the expression of HIV-Dox by HIV-DoxT in accordance with one embodiment of the present invention. Fig. 7 shows the electron microscope images of an HIV virus produced in accordance with one embodiment of the present invention. DESCRIPTION OF THE PREFERRED EMBODIMENTS For the purposes of this application, a "tetracyclme analogue" is any one of a number of compounds that are closely related to tetracyclme (Tc) and which bind to the tet repressor with a Ka of at least about 10 6 M "1 . Preferably, the tetracychne analogue binds with an affinity of about 10 9 M "1 or greater. Examples of such tetracychne analogues include, but are not limited to those disclosed by Hlavka and Boothe, "The Tetracyclines," in Handbook of Experimental Pharmacology 78, R. K. Blackwood et al. (eds.), Springer Verlag, Berlin-New York, 1985; L. A. Mitscher "The Chemistry of the Tetracychne Antibiotics, Medicinal Research 9, Dekker, New York, 1978; Noyee Development Corporation, "Tetracyclme Manufacturing Processes," Chemical Process Reviews, Park Ridge, N.J., 2 volumes, 1969; R. C. Evans, "The Technology of the Tetracyclines," Biochemical Reference Series 1, Quadrangle Press, New York, 1968; and H. F. Dowling, "Tetracyclme," Antibiotics Monographs, no. 3, Medical Encyclopedia, New York. 1955; the contents of each of which are fully incorporated by reference herein. Examples of tetracyclme analogues include anhydrotetracycline, doxycycline, chlorotetracycline, epioxytetracycline, and the like. Certain tetracyclme analogues, such as anhydrotetracycline and epioxytetracycline, have reduced antibiotic activity compared to tetracychne. One embodiment of the present invention provides an attenuated HIV vaccine comprising an HIV plasmid modified to produce a controlled virus only in the presence of at least one tetracyclme analogue. In another embodiment, the present invention provides a method for immunization of humans against HIV which comprises administering to a human a vaccine including an HIV plasmid modified to produce the virus only in the presence of at least one tetracyclme analogue. Simultaneously, at least one tetracyclme analogue is administered for a penod of time to allow the production of the modified HIV virus m vivo sufficient to produce immunity Preferably, the tetracyclme analogue is doxycyclme The introduction of the provirus of the present mvention into body cells with the tetracyclme analogue leads to the controlled production of the corresponding virus Production of the virus is stopped m the absence of the tetracyclme analogue Replication of the virus, and the associated nsks are inhibited m the absence of the tetracychne analogue The replication is either completely eliminated or limited to degrees below the levels of generating a nsk of infection Additionally, the present invention provides a process for the production of the promoter, TetopTCAT, which compπses providmg a HIV-1 LTR Cat construct contammg a Tar sequence, tetO sequences, and having only the HIV-1 TATAA box of the U3 region The Tar sequence is mutated to change nucleotides +24 to +32 from TGAGCCTGG to CCTCGGACC The tetO sequences are positioned upstream of the TATAA box to produce TetopTCAT The production of the promoter, TetopSpCAT compnses addmg three Sp 1 - cognate motifs between the tetO sequences and the TATAA box m the TetopTCAT The preparation of a doxycyclme-regulated HIV-1 provirus, pHIV-DoxT, compnses cloning the TetopTCAT promoter prepared above m the 3'LTR of pNL4-3 to substitute the TetopTCAT promoter for the wild-type LTRs from U3 through R The codmg sequence for RTTA was introduced into the nef reading frame to produce pHIV-DoxT The preparation of a doxycyclme-regulated HIV-1 provirus, pHTV-DoxSp, compnses cloning the TetopSpCAT promoter prepared above in the 5'LTR or the 3'LTR of pNL4-3 to substitute the TetopSpCAT promoter for the wild-type LTRs from U3 through R The coding sequence for RTTA was introduced mto the nef reading frame to produce pHIV-DoxSp The process for the production of doxycycline-inducible HIV-1 genome compnses transfectrng the doxycyclme-regulated proviruses pHIV-DoxT in cells which support the production of the controlled virus of these proviruses m the presence of doxycyclme to produce HIV-DoxT The cells are 293-T or HeLa cells The process for the production of a doxycycline-inducible HIV-1 genome compnses transfectrng the doxycyclme-regulated proviruses pHIV-DoxSp m cells which support the production of the controlled virus of these proviruses m the presence of doxycyclme to produce HTV-DoxSp The cells are agam the 293-T or HeLa cells An mjectable pharmaceutical earner is utilized to admimster an immunizing effective amount of the virus The method of preventing HIV-1 mfection compnses administering a combmation of an immunizing effective amount of the vaccme in combination with an amount of doxycyclme (a tetracyclme analogue) to cause the replication of the virus genome present m the vaccme. The introduction of doxycyclme is continued for a time sufficient to produce immunity m the host Subsequently, the administration of doxycyclme is discontinued. It is expected that standard dosages of the tetracyclme analogues is applicable m the present invention. For example, the normal dose for doxycycline of 100 mg by mouth twice per day for a penod of approximately 3-6 months is applicable. The proviral plasmids, pHIVDoxT andpHIVDoxSp, were constructed usmg clomng techniques similar to those descnbed in Huang et al. Huang, L. M., A. Joshi, R. Willey, J. Orenstem, and K. T. Jeang. 1994. Human immunodeficiency viruses regulated by alternative trans- activators: genetic evidence for a novel non-transcnptional function of Tat m vinon infectivity. Embo J 13:2886-96 Preparation of doxycyclme-regulated HIV- 1 promoters. In the wild-type HIV operon, the tetracyclme repressor (tetR) binds to the tetracyclme operator (tetO) sequences m the absence of tetracyclme and blocks transcnption from the tetracyclme promoter. In the presence of tetracyclme, the tetR does not bind to the tetO sequences and transcnption from the tet operon promoter can occur In the past, tetR has been fused with a trans-activatmg protein from herpes simplex virus (VP-16). This fusion protem, called tetracycline-controlled transactivator (TTA), bmds specifically and conditionally to the tetO sequences. When bound to the operator DNA, TTA activates the adjacent promoters. This system permits the conditional expression of proteins, based on the presence or absence of tetracyclme. A mutated version of the TTA has been created which has an opposite phenotype with respect to tetracychne-induced tetO bmdmg. This modified transactivator is called RTTA for reverse tetracyclme-controlled transactivator. RTTA binds to the tetO sequences only m the presence of tetracyclme. In the absence of tetracychne, RTTA does not bmd to tetO sequences and the adjacent promoter is not activated. The RTTA system can also be used to tightly and to conditionally regulate protem expression based on the presence or absence of tetracyclme or doxycyclme, a tetracyclme denvative. Two versions of doxycyclme-regulated HIV-1 proviruses (HTV-Dox) were prepared. As shown below, and as seen from Figs. 2 and 3, it has been unexpectedly found that RTTA can slightly activate tetO-contaimng promoters even in the absence of doxycyclme. Furthermore, because most promoters are less activatable after integration mto cellular chromosomes, two promoter formats were tested The first format, tetopT, was designed to be a less potent promoter which might be activated to a lower extent, but would theoretically be more tightly regulated by doxycycline The second, tetopSp, would have higher promoter activity that TetopT, but would be less tightly regulated by doxycyclme. Both were prepared in order to empmcally assess which of the two alternatives might be best for an HIV-provιrus The first hybnd promoter was developed from the HIV-1 long terminal repeat (LTR) and the tetO sequences. Starting with an HIV- 1 LTR Cat construct, which contained only the HIV-1 TATAA box of the U3 region, the Tar sequence was mutated in a manner previously shown to abrogate Tat-responsiveness, i.e., nucleotides +24 to +32 were changed from TGAGCCTGG to CCTCGGACC The tetO sequences were then positioned upstream of the TATAA box This construct is called TetopCAT, and can be seen pictonally in Fig. 1 The second hybnd promoter was formed by addmg three Spl -cognate motifs between the tetO sequences and the HIP-1 TATAA box This construct is called TetopSpCAT, and can be seen pictonally in Fig. 2. EXAMPLE I Transactivation of TetopT with RTTA With reference to Fig. 3, TetopTCAT was co-transfected mto HeLa cells with either pUC (lanes 1 and 4), a Tat-expression vector (lanes 2 and 5), or an RTTA-expression vector (lanes 3 and 6) in duplicate wells. Doxycyclme was added to one well (lanes 4, 5 and 6) of each co- transfection at 24 hours. At 48 hours, a CAT assay on protein extracts from each transfection was performed. Tat had no effect on CAT production RTTA co-transfection resulted in acetylation of 6% of the chloramphenicol in the absence of doxycycline. This level of activation increased to 38% (7.7 fold) with doxycycline induction TetopT had very low basal activity EXAMPLE II Transactivation of TetopSp with RTTA With reference to Fig. 4, TetopSpCAT was co-transfected with pUC (lanes 1 and 4), an RTTA-expression vector (lanes 2 and 5), and a Tat-expression vector (lanes 3 and 6). Half of the wells were treated with doxycyclme (lanes 4, 5 and 6). Tat and the pUC control had no effect on CAT acetylation RTTA in co-transfection resulted m nearly 100% acetylation, regardless of doxycyclme induction TetopSp had very low basal activity Construction of doxycyclme-regulated HIV-1 proviruses With reference to Fig 5, full-length HIV-1 genomes containing the tetopT and tetopSp promoters were created The tetopT on the tetopSp were placed in the 3'LTR The tetopSp promoter was cloned in the 5'LTR of pNL4-3 In both cloning formats, the wild-type LTRs from U3 through R were substituted with the novel promoters Additionally, the coding sequence for RTTA was placed mto the nef reading frame The resultmg proviruses were called pHIV-DoxT and pHIV-DoxSp Figure 5 shows a schematic illustration of these constructs, which differ only m their 3'LTRs Smce the 5' U3 region is not maintained after reverse transcnption, HIV-DoxT would, after the first round of reverse transcnption, have no Spl boxes in either LTR The LTRs have been altered and TAR has been mutated The gene for RTTA is inserted into the nef reading frame The other viral reading frames remain unchanged pHIV-DoxT and pHIV-DoxSp differ only in the 3'LTR where pHIV-DoxSp has 3 Spl boxes Each of the proviruses produced as above were transfected into 293-T and HeLa cells by known methods Following transfection into 293-T or HeLa cells, both proviruses released gag (as measured by a CA-p24 ELISA) and reverse transcnptase (as measured by an enzymatic RT assay) into the supernatant Gag and RT production were dependent upon the presence of doxycyclme EXAMPLE IE pHIV-DoxT was transfected mto 293-T cells in duplicate plates on DayO Doxycyclme (final concentration 1 meg/ml) was added to one half of the plates on Day 1 On Day 2, p24 levels in each supernatant were determined The supernatant from the doxycyclme-treated cells had 50 ng/ml of p24 Supernatant from the untreated cells contained 500 pg/ml of p24 As shown in Fig 6, the level of HIV-Dox produced by doxycyclme induction after transient transfection was exceeded 100-fold However, preliminary results mdicate that neither HIV-DoxT nor HIV-Dox Sp replicated efficiently in CD4+ T cell lmes (either m the presence or absence of doxycycline) Currently, a spreadmg virus mfection could not be reliably measured by p24 or RT assays Vaccines containing HIV-Dox are prepared m accordance with the above SIV-Dox from the corresponding simian immunodeficiency virus (SIV) can also be prepared in a like manner An immunizing amount of the HIV-Dox (or SIV-Dox) is added to a pharmaceutically mjectable earner and administered to a host The HIV-Dox vaccine and the drug, doxycycline, are given simultaneously The doxycycline is given for a penod necessary to mduce immunity After this time, the doxycyclme is discontinued After the doxycycline is discontinued, the HIV-Dox stops replicating The HIV-Dox is then eliminated from the host, reducing the possibility of long term effects To assess release and maturation of viral particles, we used electron microscopy (EM) 293T cells were transfected with pNL4-3 (positive control), pHIV-DoxT, and pHIV-DoxSp Following transfection, the HIV-Dox transfected cells were exposed to doxycycline (2 μg/ml) All transfected wells produced Gag as confirmed by p24 Ehsa on the culture supernatant The cells were fixed with glutaraldehyde and EM was performed and mterpreted As shown m Figure 7, HIV-Dox proviruses produced mature virus particles with a normal release pattern pHIV-DoxT produced similar mature and immature virus particles Figure 7 shows that HIV-Dox produces normal viral particles On Day 2 after transfection the cells were fixed with glutaraldehyde Electron microscopy was performed Panel A shows buddmg and immature vmon Panel B shows mature and immature vmon The data confirm that normal, mature virus particles are produced by pHIVDox and pHIVDoxT In other words, these data prove that virus particles, not only viral proteins, are produced by pHIVDoxT and pHIVDoxSp and that this production is conditionally regulated by doxycyclme This observation is important in vaccine development HIV vaccmes may need to produce many or all of the viral protems m a natural form m order to elicit protective immunity, as is achieved with pHIVDoxT and pHIVDoxSp In accordance with another embodiment of the present invention, the HIV virus replication in the host is controlled in the presence of the tetracyclme analogue Although the invention has been descnbed and illustrated in detail, it is to be clearly understood that the same is by way of illustration and example, and is not to be taken by way of limitation The spint and scope of the present mvention are to be limited only by the terms of the appended claims For example, vanous proviruses may be introduced mto the host body to produce the different corresponding viruses This expands the generated immune response Additionally, cells are optionally removed from the host body The removed cells are transfected with the process of the present mvention The transfected cells are reintroduced mto the host to induce the immune response The removed cells are optionally transfected with more than one provirus thus inducing an immune response to more than one virus. Production of the virus and any associated replication potential are inhibited in the absence of the tetracychne analogues.

Claims: WE CLAIM 1. A process for the production of the promoter, TetopTCAT, which compnses providmg a HIV-1 LTR Cat construct contammg a Tar sequence, tetO sequences, and having only the HIV-1 TATAA box of the U3 region, mutatmg the Tar sequence to change nucleotides +24 to +32 from TGAGCCTGG to CCTCGGACC, and positioning the tetO sequences upstream of the TATAA box to produce TetopTCAT 2. A process for the production of the promoter, TetopSpCAT which compnses: adding three Spl -cognate motifs between the tetO sequences and the TATAA box m the TetopTCAT produced according to the process of claim 1. 3. A process for the preparation of a doxycyclme-regulated HIV-1 provirus, pHIV-DoxT, compnsing cloning the TetopTCAT promoter prepared accordmg to the process of claim 1 in the 3'LTR of pNL4-3 to substitute the TetopTCAT promoter for the wild-type LTRs from U3 through R and introducing the codmg sequence for RTTA into the nef reading frame to produce pHIV-DoxT 4 A process for the preparation of a doxycyclme-regulated HIV-1 provirus, pHIV¬ DoxSp, compnsing. clomng the TetopSpCAT promoter prepared according to the process of claim 2 in the 5'LTR or the 3'LTR of pNL4-3 to substitute the TetopSpCAT promoter for the wild- type LTRs from U3 through R and introducing the codmg sequence for RTTA into the nef readmg frame to produce pHIV-DoxSp 5. A process for the production of a doxycycline-inducible HIV-1 genome compnsing: transfectrng a doxycyclme-regulated provirus pHIV-DoxT prepared according to the process of claim 3 m cells which support the production of corresponding virus m the presence of doxycyclme. 6. The process of claim 5 wherem the cells are 293-T or HeLa cells. 7. A process for the production of a doxycycline-inducible HIV- 1 genome compnsing transfectrng a doxycyclme-regulated proviruses pHIV-DoxSp prepared accordmg to the process of claim 4 in cells which support the production of corresponding virus m the presence of doxycyclme 8. The process of claim 7 wherem the cells are 293-T or HeLa cells. 9. A genome HIV-DoxT as prepared by the process of claim 5. 10. A genome HIV-DoxSp as prepared by the process of claim 7 11 A method of preventing HIV-1 mfection which compnses: admimstenng a combination of the product of claim 9 m combmation with an amount of doxycyclme which is effective to cause the production of the virus genome present in the product, continuing to admimster doxycyclme for a time sufficient to mduce an immune response in the host, and discontinuing the administration of doxycyclme 12 A method of preventmg HIV-1 mfection which compnses" admimstenng a combination of the product of claim 10 m combmation with an amount of doxycyclme which is effective to cause the production of the virus genome present in the product, continuing to admimster doxycyclme for a time sufficient to produce immunity in the host, and discontinuing the administration of doxycyclme 13. A composition compnsmg an HIV provirus modified to produce a correspondmg HIV virus only m the presence of at least one tetracychne analogue 14 The composition of claim 13 wherem the tetracychne analogue is doxycyclme. 15 A method compnsing: admmistenng to a human a composition compnsmg an HIV provirus modified to produce a corresponding HIV virus only m the presence of at least one tetracychne analogue, and admimstenng to said human the at least one tetracyclme analogue for a penod of time to allow production of the corresponding HIV virus m vivo sufficient to induce an immune response 16. The method of claim 15 wherem the tetracychne analogue is doxycyclme 17 The method of claim 15 wherem a number of HIV proviruses are administered. 18 A method compnsing- admimstenng to a human a composition compnsmg more than one HIV plasmids modified to produce conesponding HTV viruses only in the presence of at least one tetracyclme analogue; and admimstenng to said human the at least one tetracyclme analogue for a penod of time to allow the production of conespondmg HIV viruses m vivo sufficient to induce an immune response A method compnsing admimstenng to a human an injection compnsing at least one plasmid of a virus causmg viral disease wherein the at least one plasmid has been modified to produce a corresponding virus only m the presence of at least one tetracyclme analogue, and admimstenng to said human the at least one tetracychne analogue for a penod of time to allow the production of the corresponding virus in vivo sufficient to induce an immune response A method of transfectrng human cells with a controlled virus compnsing introducing into the cells a plasmid of the virus wherem the plasmid has been modified to produce a corresponding virus only m the presence of at least one tetracyclme analogue, and mtroducing mto the cells the at least one tetracychne analogue for a penod of time to allow the production of the conespondmg virus m vivo sufficient to mduce an immune response The method of claim 20 wherem the cells are removed from a human host, transfected with the virus, and reintroduced to the human host to induce an immune response

Inventors: SMITH STEPHEN, SMITH, STEPHEN
Applicants: SMITH STEPHEN, SMITH, STEPHEN
Application year: 2000
Main classification: C12N15
IPC codes: A61K31, A61K35, A61K39, A61K48, A61P31, A61P43, C12N5, C12N7, C12N15