OA11528A

OA11528A - Self-replicating vector for DNA immunization against HIV.

Info

Publication number: OA11528A
Application number: OA1200000232A
Authority: OA
Inventors: Marja Tahtinen; Paert Peterson; Kai Krohn; Paeivi Annamari Ranki
Original assignee: Finnish Immunotechnology Ltd O
Priority date: 1998-02-27
Filing date: 1999-02-26
Publication date: 2004-05-07
Also published as: RU2232815C2; AP2000001892A0; FI105105B; FI980463A0; WO1999043841A1; CN1295623A; FI980463A; AU2626699A; EP1056879A1

Abstract

A nucleotide sequence encoding the HIV regulatory protein NEF, REV or TAT or an immunologically active fragment thereof is inserted into a vector comprising papilloma virus nucleotide sequences necessary and sufficient for long-term persistence. The resulting vectors are self-replicating and have a high copy number. They express the HIV genes in high amounts for a long period of time. The vectors elicit both a humoral and cell-mediated immune response and are therefore potential DNA immunization vaccines against HIV. The invention is directed to said vectors and vaccines and to a method for preparing the vectors. The invention is further directed to a host cell comprising the vector, to the use of the vector in the manufacture of a vaccine and to a method of preventing or treating HIV.

Description

01 1 528 1

Self-replicating vector for DNA immunization against HIV

Field of the invention

The invention is directed to a self-replicating recombinant vector5 usefui in DNA immunization against HIV. The invention is also directed to avaccine comprising said vector, a method for preparing the vector, and ahost cell comprising it. The invention further relates to the use of said vectorsfor the manufacture of a vaccine against HIV and to a method of treating or preventing HIV. w Backg round of the invention

The interaction of vertebrates and thus also human beings withpat’nogenic microbes, such as bacteria, fungi and viruses is regulated by thecapacity of the vertebrate organism to mount an immune response towardsthe invading microbe. This reaction is based on the capacity of immune 15 system to distinguish between self and non-self; in normal situation immuneresponse tolérâtes the own structures, ceils and antigenic molécules of theorganism while attacks foreign antigens, expressed by the invading microbes.When a vertebrate, such as man, is infected with microbes, the immune - response helps in clearing the infection by killing microbes or ceils infected 20 with microbes or by preventing the spread of infection through the action ofneutralizing antibodies. Secondly, and more importantly, immune responseonce elicited to an invading organism has an inborn mechanism of memoryand thus an individual who has once experienced infection with a particularmicrobe is often immune and can not be infected the second time. 25 This immunological memory, caused in natural situation by infection is the basis for vaccines that mimic natural infection in many ways. An idéalvaccine will cause no or only siight symptoms in the vaccinated individuals butstill resuit in induction of immunological memory with a capability to mount astrong préventive immune responsive in case the vaccine is encountered with 30 the microbe in question.

The design of a vaccine against a particulate microbe is dépendenton the mechanism by which organism in a natural infection may clearthis particular organism and prevent subséquent infections. There are severalways how immune response can elicit its favorable function. Firstiy, antibodies 35 synthesized and secreted by the B lymphocytes can bind to microbe and through complement-mediated lysis destroy it. Secondly, neutralizing 011 528 antibodies can- prevent a spreading of infection by inhibiting with thebinding of the microbe to its target cell. Thirdly, antibodies in conjunction withcomplément activation can destroy infected cells, and, finally, spécifie cytotoxicT lymphocytes (CTL) can kiil and destroy cells infected with the microbe'. Ali 5 these mechanisms hâve been thought to be involved in the immune responseelicited by the human immunodeficiency virus (HIV) type 1 and 2 (HIV-1 andHIV-2). However, the immune response in HIV-infected individuals is usuallycharacterized by a strong antibody response and less effective or even lackingT lymphocyte response (Gerstott, J. et al. Scand. J. Immunol. 22(5):463-470, 10 1985; Re, M.C. et al. J.CTmical Pathol. 42(5):282-283, 1989). This may be the reason why individuals, once infected with HIV developed a chronic infectiondespite the strong antibody-mediated immune response.

Préventive immune response towards viral infection in general canbe mediated by the four types of immune response described above, but it is 15 known that the CTL response, capable of killing viral infected cells is mosteffective. This is the reason why generally speaking live attenuâted viralvaccines hâve proven to be most effective. In fact, the first vaccine developedby Jenner more than 200 years ago, the live vaccinia virus that can preventthe infection by the small-pox virus is an example of this principle. When an 20 individual is vaccinated with a live attenuated, viral vaccine, host cells areinfected, viral proteins are. synthesized. Some of the viral protein moléculesare used to produce virus particles while others are proteolytically cleaved tosmall peptides that bind to the major histocompatibility (MHC) antigens (in.manHLA class l and II) and are presented to T lymphocytes on the surface of 25 infected cells. Subsequently, T lymphocytes carrying a proper T cell receptor(TCR) will recognize the foreign peptide in association with HLA and eithergive help to B cells for antibody production (helper/inducer T cells; Th) ordestroy the infected cell (cytotoxic T lymphocyte; CTL). • In spite of a decade of efforts, an effective vaccine against human 30 immunodeficiency virus (HIV) infection and AIDS is still lacking. Most earlierefforts hâve concentrated on obtaining sterilizing immunity with neutralizingantibodies against the outer envelope glycoprotein of HIV, gp120/gp160.Phase l/ll studies with gp160/gp120 demonstrate, however, the neutralizationagainst laboratory strains only but failure to neutralize field. isolâtes. 35 In contrast, experiments with attenuated virus hâve been successful

in the simian immunodeficiency (SIV) model. SIV deleted in the NEF or REV 011528 3 .-

gene behaves as an attenuated virus and protects the vaccinated animaisagainst disease development but not against infection by the wild-type- challenge virus. With a REV- defective virus, the only immunological correlatewith- protection was cell-medîated immune (CMl) response against SIV 5 regulatory proteins NEF and TAT. An important observation in this experimentwas, that vaccinated animais could even clear the infection caused by thewild-type challenge virus. It has recentiy been reported that also HIV-infectedpatients may occasionally clear an overt infection. The only relevant corréla-tion to protection in these animal and/or human studies seems to be a cell 10 mediated immune response towards HIV.

This type of immune response is generally not obtained with protein immunization. In terms of HIV, live attenuated vaccines could be effectiveto prevent infection but they are theoretically dangerous for several reasons.A recentiy described method, genetic immunization (synonyms: nucleic acid 15 immunization, DNA immunization) has several of the advantages of liveattenuated vaccines but not their potentialiy harmful adverse effects. A DNAvaccine, in a form of eukaryotic expression vector that carries the gene for oneor a few of the viral proteins can induce the synthesis of the viral protein, oncethe DNA'vector is transfected into host cell. Viral proteins synthesized in 20 the target cell will then be procêssed by proteolytic enzymes; the formedpeptides will be bound to MHC/HLA molécules and presented on the surfacetransfected cells. This will cause a CTL-mediated immunological memorythat in case the individual is subs.equently infected with the virulent wild-type virus will be effective in killing viral infected cells immediately upon 25 infection and thus preventing the infection. Direct intramuscular or intradermalinjection of cDNA in an eukaryotic expression plasmid has been shown toinduce an immune response (Wolff et al. Science 246:1465-1468, 1990). Theexpression of foreign antigens by such means results predominantly in helperT-cell subset 1 (TH1) type immune responses, with strong cytotoxic T- 30 lymphocyte (CTL) response and, occasionally, also high- titer antibodyresponse (Wang, B. et al, PNAS 90: 4156-4160. 1993; Wang et al. Ann. NYAcad. Sci 772: 186-197, 1995; Haynes et al. AIDS Res. Hum. Retroviruses10 (2)’43-45, 1994)· Moreover, using viral nucleoprotein antigen of influenzaA, antigen-specific CTL and protection has been reported (Ulmer. et al. 35 Science 259, 1745-1749, 1993). Furthermore, protection against infection us- ing DNA immunization has been obtained for mycoplasme in mice (Barry et al. 011528

Nature 377(6550):632-535, 1995; Lai et al. DNA Cell. Biol. 14(7):643-651,1995 ) and for human papillomavirus in a rabbît mode! (Donnelly et al. J. Inf.Dis. 173(2):314-320, 1996). DNA immunization has also been used to induceantitumor immunity mediatsd by cytotoxic lymphocytes (Bohm et al. Cancer 5 Immunoi. immunother. 44(4):230-238,1997). DNA immunization has severai advantages in comparison to live attenuated viral vaccines. As no infectious virus is formed, the viral genesinduced to the host organism stay only in those cells thaï are originallytransfected and no symptoms of virus infection occurs. In question of HiV, the 10 major theoretical harmful effect for a live attenuated virus would be réversion,by mutations to a virulent wild-type virus. Furthermore, with DNA immunizationonly those virai genes, or parts ' thereof that are known to be .effective ininducing préventive immune response can be used.

For an effective CTL response, it wouid be important that the 15 cytotoxic T-cells would destroy the infecied cells before structural proteins areformed and prior to the release of mature viral particles. Therefore, immuneresponse îowards the early proteins in virai cycle would be bénéficiai. Theréplication of HiV is reguiated by its own regulatory genes and proteins. TheHIV genome encodes three nonstructural regulatory proteins (NEF, TAT, REV) 20 which are indispensable for the réplication of the virus in vivo, REV is atransporter of genomic RNA into the cytoplasm, TAT upregulates viral tran-scription and NEF provides réplication in resting cells. Experiments with SIVindicate that viruses lacking the fonction of one of these regulatory genes maynot be abie to induce disease because of insufficient viral réplication. These 25 three proteins are expressed transiently and in smail quantifies during the firsthours of the viral infectious cycle (Ranki et al. Arch.Vîrol. 139:365-378, 1994).Only a smali proportion of HlV-infecied individuals shows humoral and/orcellular response to these proteins, and the response correlstes with afavorable clinîcal course. 30 ’· CTL responses against NEF, TAT and REV hâve been extensively studied. NEF-specific CTL and Th (T helper cell) responses correlate with afavorable clinicai prognosis. With a REV defective SlV-vaccine, immuneresponses to NEF and TAT were protective. Th and CTL epitopes in TAT andREV proteins, which are recognized by HiV-1 infecied individuals and which 35 show a clinicai corrélation, hâve been identifisd (Blazev.ic et al. J AIDS 6:881 -890, 1993; Blazevicei al. AIDS Res. Hum. Retroviruses 11:1335-1341, 1995). 01 1 528

Taken together, these results indicate that for protection against disease a moderate réplication of virus (attenuated growth) in combination with spécifie immune responses against the regulatory proteins involved in support of virus - réplication may be necessary. 5 Several eukaryotic expression vectors can be used in DNA immu- nization but their efficacy varies. Some of the parameters that regulâte theefficacy of a given expression vector in inducing the immune response areunknown but obviously high ievel of expression of the antigenic proteinwould be advantageous. The time period that the vector, introduced to the cell 10 can express the foreign antigenic viral'protein may also. be of importance.Finally, expression vectors that induce certain level of cell injury may aiso beadvantageous as il is known that tissue destruction will amplify immuneresponse through several biologically active'molécules, such as cytokines,lymphokines and chemokines, secreted by the cell expressing the antigenic 15 protein. This is probably one further reason why live attenuated virus thatcauses a certain level of tissue and cell destruction is so effective in inducingimmunity, and thus a DNA vector that in this respect mimics live attenuatedvaccine would be advantageous.

Nonspecific factors such as cytokines and lymphokines may also 20 regulâte the viral réplication and immune responses in HIV-1 infection.- Therôle of the helper cell Th1/Th2 balance, reflected by production of lymphokinesspécifie for the two helper T-cell populations, has been demonstrated byClerici and Shearer (Immunology Today 14(3):107-111, 1993) and others.Soluble factors, produced by CD8 cells and capable in suppressing the viral 25 production by HIV-1 infected CD4 cells, were recently identified as RANTES,MIP1-a and ΜΙΡ1-β (Cocchi et al. Science 270(5243):1811-1815, 1995). It ispossible, that cytokines whose production is either increased or decreased inHIV-1 infection will regulâte viral transcription.

Previous studies on DNA immunization using the gene encoding 30 the HIV regulatory protein NEF hâve demonstrated T-ceîl proliférativeresponses (Hinkula et al. Vaccine 15 (8):874-878, 1997 and Hinkula et al. J.Virol. Jul, 71(7):5528-5539, 1997). However, it is the CTL response that has apositive effect corrélation with a favorable clinical course.

One of the main objects of the présent invention is therefore to 35 provide a DNA immunization vaccine encoding an HIV regulatory protein, thevaccine being capable of eliciting a CTL response against HIV infected cells in 011 528 the early phase of the infectious cycle, before new mature infectious viralparticles are released.

Another object of the invention is to provide a vaccine, which furtherelicits a humoral response against HIV. 5 A further object of the invention is to provide an HIV vaccine, which is safe to use, because it does not expose the récipient to the structural genesor proteins of HIV.

Another object of the présent invention is to provide a self-repiicating vector that causes a prolonged and high level of HIV regulatory 10 protein expression and a certain degree of cell destruction, which will further'stimulate the immune response.

Still another object of the invention is to provide a seif-replicatingrecombinant vector expressing HIV regulatory proteins, which vector conferslong-term stable maintenance and a high copy number in .transfected cells 15 including mammalian cells. A further object of the invention is to provide a host cell comprisingsaid vector.

Yet another object of the invention is to provide a method forpreparing the above-mentioned self-replicating vector. 20 . The présent invention further provides a method of treating or preventing HIV.

Still another object of the invention is the use of said vector for themanufacture of a DNA immunization vaccine against HIV.

Summary of the Invention 25 The objects of the présent invention can be achieved by incorporating a heterologous nucléotide sequence encoding the HIV regulatoryprotein NEF, REV or TAT or an immunologically active fragment thereof into avector comprising a papilloma virus E1 gene and E2 gene, a minimal origin ofréplication of a papilloma virus and a minichromosomal maintenance element 30 of a papilloma virus.

In other words the invention is directed to a self-replicatingrecombinant vector comprising papilloma virus nucléotide sequencesconsisting essentially of (i) a papilloma E1 gene and E2 gene, 35 (ii) a minimal origin of réplication of a papilloma virus -011 528 (iii) a minichromosomal maintenance element of a papilloma virus,and a heterologous nucléotide sequence encoding the HIV regulatoryprotein NEF, REV or TAT or ah immunologically active fragment thereof.. ' 5 " The invention further provides a vaccine for DNA immunization against HIV comprising said vector, the use of said vector for the manufactureof a vaccine against HIV, and a method of treating or preventing HIVcomprising administering to a person in need thereof an effective amount ofthe self-replicatïng vector and expressing the NEF, REV or TAT protein or an 10 immunologically active fragment thereof in said person.

The invention still provides a method for preparing a self-replicating recombinant vector, said method comprising A) inserting a heterologous nucléotide sequence encoding the HIV-regulatory protein NEF, REV or TAT or an immunologically active fragment 15 thereof into a vector comprising papilloma virus nucléotide sequencesconsisting essentialiy of - (i) a papilloma E1 gene and E2 gene, - (ii) a minimal origin of réplication of a papilloma virus, and - (iii) a minichromosomal-maintenance. element of a papilloma virus, 20 and B) transforming a host cell with the resulting self-replicatingrecombinant vector, C) culturing the host cell, and D) recovering said vector. 25 The invention also provides a host cell comprising said vector.

Brief description of the DrawingsFigure 1A shows the shuttle vector pUE83.

Figure 1B shows the shuttle vector pNP177.

Figure 2 shows the pBNtkREV plasmid of the invention. 3θ Figure 3 shows the pBNsraTAT plasmid of the invention.

Figure 4 shows the pBNsraNEF plasmid of the invention.

Figure 5 shows the NEF expression in COS-7 cells transfected with pBNsraNEF. The Western blot samples are taken 72 h post transfection and visualized with ECL. 35 Figure 6 demonstrates anti-NEF antibodies in sera of mtce immunized with pBNsraNEF as detected in Western blot. Samples 1 - 4 were 8 011528 taken 2 weeks post last immunization and samples 5-8 were taken 4 weeks post last immunization.

Figure 7 shows CTL responses in mice immunized with the pBNsraNEF vector. Figure 7A shows CTL responses, expressed as % 5 spécifie lysis of the target cells, in the four mice tested two weeks after thelast immunization. Figure 7B shows the values at four weeks after the last immunization. Spécifie lysis > 4 % is considered positive.

Figure 8 shows the immunoglobulin subclass distribution in threemice immunized with pBN-Nef. 10 Detailed description of the invention

According to the invention the heterologous HIV nucléotidesequence is inserted into a vector comprising a papilloma virus E1 gene andE2 gene, a minimal origin of réplication of a papilloma virus (MO), and aminichromosomal maintenance element of a papilloma virus (MME). This 15 E1/E2/MO/MME comprising vector is hereinafter called pBN, and if has beendescribed in detail in WO 97/24451, which is incorporated by reference. Saidpatent publication is based on the discovery that DNA réplication in papillomaviruses from the MO per se is not sufficient for stable long-term persistence,but in addition another virai sequence MME is required and that the best 20 results are obtained when the vector further comprises the El and E2 genesof the papilloma virus. ‘Papilloma virus’ as used herein means any member of thepapilloma virus family. Preferably the papilloma virus used in the invention isbovine papilloma virus (BPV) or human papilloma virus (HPV). 25 ΈΓ and Έ2’ are regulatory proteins of papilloma viruses, which repticate via MO and which are necessary for réplication. ‘Minimal origin of réplication’ (MO) is a minimal sequence of apapilloma virus which is necessary for initiation of DNA synthesis. ‘Minichromosomal maintenance element (MME) refers to a région of30 the papilloma viral genome to which viral or human proteins essentialfor papilloma viral réplication bind. MME is essential for stable episomalmaintenance of the papilloma viral MO in a host. Preferably MME comprises multiple binding sites for the transcriptional activator protein E2. ‘Self-replicating vector’ as used in the présent application means a 35 vector plasmid capable of autonomous réplication in à eukaryotic host cell. 011528 ‘Heterologous’ means foreign. For .example with respect to thevectors of the invention a heterologous nucléotide sequence means anon-papilloma sequence. 'Immunologically active fragment' means a fragment capable of5 eliciting an immunological response in a récipient. 'Papilloma virus nucléotide sequences consisting essentially of"means that the vector comprises the papilloma nucléotide sequences whichare necessary and sufficient for long-term vector persistence and réplication.This means for example that superfluous sequences like ail papilloma- 10 encoded oncogenic sequences hâve been deleted from the pBN vectors usedin the présent invention.

In addition to the E1 and.E2 genes, MO, MME and the NEF, REV orTAT gene, the vectors of the invention comprise promotors for the encodedproteins as well as additional regulatory sequences, poly-adenylation 15 sequences and introns. Preferably the vectors also include a bacterial host ceilorigiri of réplication and one or more genes for selectable markers for thepréparation of the vector DNA in a bacterial host cell.

An essential feature of the pBN vectors is that they are not host cellspécifie. This is because the expression of the E1 and E2 proteins is controlled 20 by promotors which are non-native i.e. heterologous. Said promotors areeither functional in a broad range of mammaiian cells or tissues or are cell- ortissue-specific.

In the vectors of the présent invention the E1 gene is preferablyunder the control of the sr-α promotor or the thymidine kinase promotor (tk) 25 and the E2 gene is preferably under the control of the LTR gag promotor. TheNEF, reV or TAT gene can be under the control of a CMV promotor or anRSV LTR promotor. The vector can further comprise an SV40 early promotorto induce the expression of the gene for antibiotic sélection (neomycin orkanamycin). 3Q The host cell origin of réplication in the vectors of the invention is preferably pUC ORI and the sélective markers used are e.g. kanamycin and/orneomycin. Preferably the intron is the beta-globin IVS.

The octseq found in TK-promoter based plasmids is a non-coding sequence from octamer protein. It has no functional purpose in the plasmid, 35 but was needed for creating suitable restriction sites for the préparation of the final plasmids. 10 011528

The NEF, REV or TAT genes to be inserted into pBN can beobtained from several commercial sources such as the plasmïd pKP59, whichis available from the AIDS Reagent Project MHC repositary. Said genes arewell known and- hâve been fully sequenGed (Wain-Hobson, et al. Cell 40:9-17, 5 1985). Of course it is also possible to insert a sequence encoding only an immunologically active fragment of said HIV proteins.

The NEF, REV or TAT genes or their fragments are first insértedinto appropriate shuttle vectors. These vectors can either include or notinclude the MO région. Two shuttle vectors are illustrated in the examples: 10 pNp177, which does not include the MO, and pUE83, which includes the MO.

Of course it is possible to use other shuttle vectors too. Both the shuttlevectors and the resulting vectors of the invention are preferably multiplied inEscherichia coii. Examples of the resulting pBN-NEF, pBN-REV or pBN-TATvectors of the. présent invention are setforth in Figures 2, 3 and 4. The vectors 15 of the invention are stable and self-replicating in a large copy number. Upontransfection into a eukaryotic host cell, the vector (plasmid) will multiply andproduce 100 - 1000 fold amount of new plasmids, each capable of expressingthe HIV protein in demand.

The host cell claimed in the présent invention can be either a 20 eukaryotic cell transfected by the vector or a prokaryotic cell transformedby the- vector. The eukaryotic cell is preferably a mammalian cell and theprokaryotic cell is preferably a bacterial cell, especially E. coli.

The expression of HIV NEF, REV and TAT of the resulting plasmidvectors of the présent invention was tested both in transfected ÇOS-7 cells 25 and in mice immunized with said plasmids. A high expression of the HIVproteins could be demonstrated in the COS-7 cells and the immunized miceshowed a remarkable humoral and cell mediated (CTL) immune response. Asignificant CTL response was also demonstrated in monkeys. These resultsindicate that the vectors of the invention hâve a potential use as effective vac- 30 cines against HIV.

It was further demonstrated that a mixture of pBN-vectors encodingdifferent HIV reagulatory proteins mounted an immune response to severalregulatory genes. The présent invention thus includes vaccines comprising amixture of vectors encoding different HIV regulatory proteins or immun- 35 ologically active fragments thereof and the use of said mixture in the manu-facture of the vaccine and the treatment or prévention of HIV. The vaccine 11 011528 may Contain a mixture of vectors encoding ail three different regulatory 'pro-teins. The vaccines of the présent invention may also contain other genes or gene fragments e.g. selected from the group consisting of the HIV structuralgenes. 5 The présent invention is further illustratedin the following examples.

The examples describe in detail some embodiments of the invention, but theyshould not be interpreted to restrict the invention, which is defined by theattached daims.

Example 1 10 Cloning of H1V-1 genes REV and TAT into self-replicating pBNsr-α and pBNtk plasmids

Production of pBNtkREV and pBNsraTAT

Phase 1:

The HIV-1 REV and TAT genes from isolate BRU also called LAI 15 (Wain-Hobson et al. Cell 40:9-17, 1985) were amplified from the pcREV andpcTAT vectors (Arya et al. Science 229:69-73, 1985) using Dynazyme TaqDNA poiymerase (Finnzymes, Finland) and the following primers that hâverestriction enzyme sites for enzymes Xhol and Xbal: 20 For REV: - 5 '-TTTTTCTAGAACCATGGCAGGAAGAAGCGGA-3 ' ' 5'-TTTTCTCGAGCTATTCTTTAGTTCCTGG-3'

For TAT: 25 5'-TTTTTCTAGAACCATGGAGCCAGTAGATCCT-3' 5 '-TTTTCTCGAGCTAATCGAACGGATCTGC-3 '

The amplified genes and pUE83 shuttle vector (Figure 1) weredigested at +37 °C with Xbal and Xhol (New England BioLabs, USA) overnight 30 in order to get compatible ends. The digested DNA-fragments were analyzedon 1.5 % agarose gels, and further purified using Band Prep Kit (PharmaciaBiotech, Sweden). Each gene was ligated into the vector separately using T4DNA ligase (New England BioLabs, USA) in an overnight incubation at +16 °C.The ligation products were transformed into One Shot Kit (Invitrogen, 35 The Netherlands) competent E. coli cells, which were plated on LB-platescontaining kanamycin for sélection. Minipreps were prepared from the growing 12 011528 clones, and the presence of cloned genes was analyzed by digestion withXhol and Xbal. The presence of the cloned genes was also confirmed by PCRfrom miniprep préparation using the above mentioned primers. Clones 'containing the right gene were mass cültivated' and plasmids were purified 5 using Megaprep columns (Qiagen, Germany).

Phase 2: A DNA fragment containing BPVori, RSV LTR promoter, REV- orTAT-gene and b-globin IVS poly(A).was digested from the shuttle vector byHindlll (New England BioLabs, USA), and purified using 1 % agarose gel and10 Band Prep Kit. Ligation to Hindlll digested and dephosporylated (alkalinephosphatase, CIP, Promega, USA) pBNsra or pBNtk, transformation of cells,vérification of the presence of cloned gene and purification of the plasmid were done as in phase 1.

The resulting plasmids are called pBNtkREV and pBNsraTAT and15 are set forth in Figures 2 and 3.

Example 2

Cloning of HIV-1 NEF into self-replicating pBNsra plasmid

Production of pBNsraNEF

Phase 1: 20 The HIV-1 NEF gene was obtained from a -plasmid pcNEF vector, which contained the LAI isolate NEF gene inserted into a pcTAT vector lackingthe TAT gene. The NEF gene used for further cloning was achieved as a 1.3 kb fragment by Spe I and Hind III digestion from pcNEF. To eliminate thereformation of the Hind III site on ligation, after Hind III digestion the fragment 25 was treated with Klenow enzyme and a mix of dATP, dCTP, dGTP nucléotidesafter which the Spe l digestion was performed. The fragments obtained wereseparated by electrophoresis on a 1% agarose gel alongside standard sizemarkers. Bands of correct size were eut out and the DNA recovered using theSephaglas Bandprep Kit (Pharmacia Biotech), following the manufacturées 30 protocol.

The shuttle vector pNP177 of Figure 1B was first digested with XhoI, then treated with Klenow enzyme and dNTP mix, and, finaliy, digested withXba I; The vector was also treated with calf intestinal alkaline phosphatase(CIP). 35 The fragment containing the NEF gene was ligated with cleaved pNP177 vector by using T4 ligase in +14°C ovemight. One Shot competent E. 13 011528 coli kit (Invitrogen) was used for transformation. Positive clones were identifiedby using restriction enzyme digestions and electrophoresis. Plasmid DNA wasfurther amplified in E. coli and purified in a large scale with Qiag.en columns.The resulting final plasmid was called pNP177cHIVNEF. 5 Phase 2:

The shuttle vector pNP177 is designed to hâve only two Hind IIIsites between which an insert can be cloned. A Hind lll digest of the plasmidthus gives a fragment which can be cloned further. The Hind lll fragment ofpNP177cHIVNEF was cloned into pBNsra. The vector was digested with Hind10 lll and treated with CIP. The same methods. of band séparation, ligation,transformation were used as in the first phase and correct orientation of theinsert was confirmed by restriction analysis. The final plasmid was called pBNsraNEF and is shown i Figure-4.

Example 3 15 Démonstration of expression of HIV-Nef in vitro 3A.Transféctions

To test the expression of the pBN-constructs of examples 1 and 2,they were transfected by electroporation into COS-7 cells. 10 pg of ρΒΝβ-Galas a control, and 10 pg of pBN-NEF cotransfected with 1pg pCMVp-Gal, were 20 electroporated each into three million cells. Salmon sperm carrier DNA wasused. The electroporation was made at 960 mF capacitance and 260 Vvoltage. Protein concentration and β-gal activity measurements were made tocontrol the efficiency of transfection and to caiibrate the amount of the lysâtesin Western blot assay. 25 3B. Immunohistochemistry and Western blotting

The harvested cells transfected with the pBN-constructs were lysed for use in Western blotting. After lysis, protein samples were boiled in samplebuffer and run in a 12% SDS polyacrylamide gel, then transferred onto a2 pm nitrocellulose filter which was blocked with a solution of 5% milk in TBS. 30 As a primary antibody a mixture of mouse anti-NEF monoclonals (Ovod V. etal. AIDS 6:25-34, 1992) diluted to 1:1000 each was used. The secondaryantibody was a biotinylated anti-mouse in a 1:500 dilution.

After transfection to COS-7 cells, the vectors produced a strongtransient HIV regulatory protein expression, as detected by Western blotting of 35 the lysed cells at 72 hours. The results obtained with pBNsraNEF are shownin Figure 5. In long-term cultures of the transfected cells, NEF expression 14 o-11 528 sustained up to 7 weeks in the cells transfected with the self-replicating pBNvector.

The NEF-transfected cells were also used to préparé cytospin-préparâtes and they were stained with haematoxyJin and a monoclonai5 antibody against NEF followed by a secondary biotinyiated anti- mouse wereused in immunohistochemistry as described in Ovod et al. supra. The cytospinslides indicated expression as positive staining was seen in a large numberof cells as granules occupying the cell cytoplasm. A portion of the NEFexpressing cells showed morphological signs of cell destruction, indicating 10 apoptosis. Still the level of expression was high though the condition of thecells was getting worse.

Example 4. Démonstration of immunogenicity of pBNsra and pBNtk vectorexpression of HIV-Nef, HIV-Tat or HIV-Rev in vivo 15 4A. GeneGun DNA was precipitated onto 1 pm gold particles using spermidineand CaCI2 following the procedure in the Helios Gene Gun Instruction Manual(Bio-Rad Laboratories). Cartridges were made to carry 0.5 mg gold and 1 pgDNA each. The amount of- DNA was controlled spectophotometrically as 20 instructed in the manual. inoculations were performed using the Helios GeneGun System (Bio-Rad Laboratories). Hélium discharge pressure for DNAdelivery was set to 300 psi. In our optimization of thé bombardment conditionswe found 300 psi to be sufficient to propel the gold particles into the dermis. 4B. lmmunizations 25 Female 6-8 week-old balb/c mice were used. Before immunizations the mice were anesthetized and the abdominal fur was removed.

Inoculations on the abdominal skin of 8 mice were done on days 1,2 3 10, 11 and 12 using the gene gun described above and following theinstruction of the manufacturer. A total of 6 pg of pBN-NEF was administered 30 per mouse. Four mice from both groups were sacrificed two weeks post last immunization and the remaining four mice four weeks post last immunization. Sérum samples for Western blotting were taken and splénocytes harvested for a CTL assay. Ail eight mice immunized with the pBN-NEF- vector, showed an antibody response at 2 weeks and 4 weeks (Figure 6). The intensity of the 35 reaction in the Western blotting varied. 15 011528 4C. Measurement of cytotoxic T-cell activity in the immunized mice 4C1. Stimuiation of effector cells

Spleens were removed aseptically from the immunized mice îwo 5 (18 mice) and four weeks (16 mice) after immunization. They were disrupted in

Hanks, fiitered through gauzê and the érythrocytes were removed. Cells werethen suspended 5 x 10e ! mi in culture medium: RPMI 1640 medium cc.ntaining10 % fêtai caîf sérum (FCS; GibcoBRL), 1 % giutamin, 100 U of penicillin perml, 100 pg of streptomycin per mi and 5 x 10'5 M 2-mercaptoethanol. The re- 1.0 sponding celis (5 x 10e) were co-cuitured in 25 ml ceïl culture fiask in 5 ml ofculture medium with 4x10® antigen presenting cells (APCs; see below) forfive days. 10 U / ml of recombinant interleukin - 2 (rlL-2) was added at the firstday to the cells (Hiserodt J. et al. J. immunol. Jul, 135(1):53-59, 1985;Lagranderie M. et al. J. Virol. Mar, 71(3):2303-2309, 1997; Tsuji T. et al. 15 Immunology Jan. 90(1):1-6, 1997; Vahlsing H.L. et ai. Journal ofImmunological Methods 175:11-22, 1994; Varkila K. étal. Acta path. Micorbiol.Immunol. Scand. Sect. C 95:141-148, 1987). 4C2. Antigen presenting celle

Syngeneic P815 mastocytoma (H-2d) cells were infected with 20 modified vaccinia virus Ankara (MVA) engineered to express the HIV-1 LAINEF gene (MVA-HIVNEF). MVA is a highiy attenuated replicatioh-deficientvaccinia virus, which can serve as an efficient vector for expression ofheterologous genes providing an exceptionally high level of bioiogical safety(Sutter G. et al. J. Virol. Jul, 68(7);4109-4116, 1994; Sutter et al. Vaccine 25 12(11):1032-1039, 1994: Drexler l. et al. J. Gen. Virol. 79:347-352, 1998; . Sutter G. et ai. Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992). Infectionswith MVA-HIVNEF were performed at a multiplicity of infection (MOI) of 5 in 24- well plates (1 x 105 cells per weli). After 1h virus absorption at +37ÔC, thecells were incubated for 15 h in +37°C (Carmichael A. et al. Journal of Virology 30 70:8468-8476, 1996). After infection the cells were washed twice with PBS(phosphate bufrered saline) containing 10 % FCS and suspended in thissolution 5x10® ceils/ml. Cells were then γ-irradiated at 5000 rad and washedWith culture medium before adding to responder celis. 4C3. Cytotoxicity assays 35 CTL activity was tested by the slCr-release assay (Hiserodt J. et al. J; Immunol. Jul, 135(1):53-59, 1985; Lagranderie M. et ai. J. Virol. Mar, 16 011528 71(3):2303-2309, 1997; Varkila K. si al. Acta path. Micorbiol. Immunol. Scand.

Sect C 95:141-143, 1937; vsn Saalen C. et al. AIDS 7:781-736, 1SS3). Briefly, 2.x 10e P-815 ceils were infected with MVA-HIVNEF as described above for antigen presenting ceils. Afîer infection the ceils were washed once in sérum 5 free culture medium. Target ceils then were suspended in 200 μί of sérum freeculture medium and 100 μ€ί of 51Cr (Amersham) ! 1 x 10s ceils was added for1 h aî 37°C. Target ceils were then washed four times in medium andsuspended in concentration 5 x 10* / ml. The stimulated effector ceils werewashed once in culture medium before adding to the target ceils. Target ceils 10 were plated in u-bottom 96- well plate 100 μΙ (5 x 1Q3) per well and effectorcelis were added in triplicates in 100 μΙ at effector. target ratios 50, 25 and12.5. For spontaneous release, target celis were plated in six welis with 100 μΙof culture medium and for maximum release in six welis with 2.5 % Triton-X-100. The plates were spun briefly, incubated for 4 hours in 37°C and 15 the supernatants were counted ïn à gamma-cou nier. The percent spécifielysis of target eeîis was caicuiated as (test 51Cr release - spontaneousrelease)/(maximum release - spontaneous release) x 100. The percent spécifielysis à 6 % was considered to be positive.

An example showing CTL activity in 6 of the 8 mice irrimunized 20 with pSNsraNEF is shown in figure 7. D. Humoral immune response in immunized mice

To test the occurrence of antibodies against. HIV-1 NEF in immunized mice sera NEF protein was electrcphoresed on PAGE, transferredto nitroceiiulose filters and the antibody reactivity was detected as described 25 above.

Summary of the results

The results of the transfection and immunization tests are summs-rized in Table 1. The immune response in the immunized mice was assessedby immunoblotiing (W.E) for humoral and by cytotoxic t-lymphocyte (CTL) 30 assay for the cell mea'iated immunity. As seen in the table, ali ejght miceimmunized with the NEF expressing vector showed both humorai and celimediated immune response. 17 011528

Table 1. Démonstration by immunoblotting (Western blotting, WB) or byimmunohistocbemistry of the expression of the HJV-1 NEF, TAT and REVproteins in COS-7 cells transfected with said vectors and démonstrationof Induction of humoral and cell mediated immune response in .mice im-

5 munized with one of the vectors, pBNsraNEF

Transfection WB Immunohisto- chemistry Immunization WB CTL pBNsraTAT ND ++ 6/8 6/8 pBNtkREV ++ 7/8 . 7/8 pBNsraNEF ++ ND 6/8 6/8

In this study we demonstrate that DNA immunization using a self-replicating expression vector as described can induce a clearly détectable CTL 10 response in mice. In addition a humoral immune response was achieved. Inview of the above results it is feasible to assume that the pBN-NEF, pBN-REVand pBN-TAT plasmids do express NEF, REV and TAT in vivo in an amountsufficient to induce both the humoral and the cell-mediated immune responsenecessary for preventing or treating HIV. 15 Example 5

Measurement of Th1/Th2 type response in intramusculariyimmunized mice

The humoral immune response seen in mice immunized. with pBNconstructs expressing HIV regulatory proteins was tested for immunoglobulin 20 subclass specificity. It is well known that antibody response dominated bylgG2a subclasses of immunoglobulins is a characteristic of a Th1 type cell-mediated immune response while lgG1, lgG2b and lgG3 are characteristic fora Th2 cellular response. Furthermore, Th1 type responses are known to indu-ce and help cell-mediated cytotoxic immune responses (CTL response) while 25 Th2 response will induce antibody response but less active CTL responses.

The immunization schedüle was as follows:

Four Balb/c mice were immunized six times with 24 micrograms of 30 pBN-Nef of Example 2 in two weeks, the total amount of DNA being thus 144 18 011 528 and the sera analyzed for antibodies in Western biot. Three out of four micehad antibodies against H1V-1 Nef and the subclass of these antibodies wasmeasured in an ELISA assay as follows:

The antigen was pipetted’ on Nunc Maxi Sorb plates for overnight 5 incubation in +4 °C; the antigen used was HIV-1 Nef protein (NIH, AIDSResearch and Reference Reagent Program) in PBS (50 ng/well). The plateswere blocked in an overnight incubation with 1 % BSA (Sigma), and thereafterincubated with the mice sera (diluted 1:100 in blocking solution) for 4 hours atroom température. Plates were washed with PBS-0.1 % Tween 20 three times 10 and thereafter with PBS two times. As secondary antibodies peroxidase con-- jugated anti-mouse lgG1, lgG2a,-lgG2b, lgG3 and IgM (Calbiochem) dilutedT1000 in blocking solution were used and the plates were incubated 2 hoursat room température. After washing steps performed as earlier described, sub-strate ABTS (Sigma) and H2O2 in citrate buffer was added for 10 minutes and 15 the photometric détermination was carried out in an ELISA-reader at 405 nm.The results are shown in Figure 8. The highest response in the three mice wasdetected with lgG2a secondary antibody (Absorbance at405 nm 0.117, 0.262,0 743 respectively), the response with lgG1 antibody being much lower(A(405) 0.004, 0.020, 0.038). This indicated that the type of response in these 20 mice after intramuscular immunization is merely Th1-type leading to cell medi-ated immune response.

The results demonstrate that practically ail mice had a strong lgG2atype response toward recombinant Nef while antibodies representing otherIgG subclasses were very low. The results further prove that the pBN Nef con- 25 strucf is able to mount a Th1 type response and subsequently strong cell- mediated immune response capable of destroying HIV-infected cells in the early phase of viral infectious cycle. 19 011528

Example 6 Génération of cell-mediated immune response inMacaca fascicularis monkeys by pBN constructs expressingHIV-1 regulatory proteins

5 Expérimente with mice clearly indicated that the pBN Nef, pBN

Rev and pBN Tat constructs were able to mount a CTL response in immunizedmice. Further experiment was performed with a non-human primate to provethat the constructs could be used as préventive vaccines in human beings. !t isimportant to demonstrate that the immune response can also be generated in 10 non-human primates that are genetically doser to man and that can be in-fected with a corresponding primate retrovirus SIV that is closely related toHIV-1 and HlV-2, infecting man. We therefore performed an experiment whereMacaca fascicularis monkeys were immunized with the pBN constructs ex-pressing HIV-1 regulatory proteins Nef, Rev and Tat. These were prepared as 15 described in Examples 1 and 2. Three Macaca fascicularis monkeys were im-munized with a mixture of pBN Nef, pBN Rev and pBN Tat. Three monkeysserved as Controls. The immunization schedule was as follows:

Monkeys were immunized with a total amount of 300 micrograms ofpBN-Nef, pBN-Rev and pBN-Tat (100 micrograms of each) twice. The first 20 immunization was given into deltoid muscle and the second (2 weeks later)was given intradermally. Cytotoxic T-lymphocyte assays was performed twomonths after the last immunization as described in Example 4C3. The resultswere as follows:

Target cell: Immunized monkey Control monkey Nef 10.2* 0* Tat Ί 10.3* 0* Rev 0* 0* * = Percent spécifie lysis of the target cell expressing correspondingHIV-1 antigen 20 01 1 528

One of the three monkeys had a demonstrable CTL responseagainst autologous B cells expressing H1V-1 Nef and Tat. The results demon-strate that not only mice but also primates can be immunized with the pBNconstructs expressing the HIV regulatory proteins and the immunized animais5 will mount cell-mediated T cell response charaçterized by the presence of cy-totoxic T lymphocytes that are capable of destroying HIV infected cells in theearly phase of viral infectious cycle. .Furthermore, the results show that theconstructs can be given simultaneously as a mixture and that the presence ofone construct in the mixture does not interféré with the immune response ge- 10 nerated with another one.

Claims

21 01 1 528 Claims

1. A self-rëplicating recombinant vector comprising papilloma virusnucléotide sequences consisting essentially of 5 (i) a papilloma E1 gene and E2 gene, (ii) a minimal origin of réplication of a papiiloma virus (iii) a minichromosomal maintenance element of a papilloma virus,and a heterologous nucléotide sequence encoding the HIV regulatory 10 protein NEF, REV or TAT or an immunologicatly active fragment thereof.
2. A self-replicating vector of claim 1 wherein the papilloma virus isbovine papilloma virus (BPV).
3. A self-replicating vector of claim 1 or 2 wherein the heterologousnucléotide sequence encodes the HIV-1 NEF protein. 15 4. A self-replicating vector of any of the preceding claims wherein E1 is under the control of the sra promotor or the thymidine kinase promotor.
5. A self-replicating vector of claim 4 which is pBNtkREV,pBNsraTAT or pBNsraNEF as shown i Figure 2, 3 or 4. _6. A vaccine for DNA immunization against HIV comprising a self- 20 replicating vector of any of claims 1-5. .7. A vaccine of claim 6 comprising a mixture of vectors encoding different HIV regulatory proteins or immunologically active fragments thereof.
8. Method for preparing a self-replicating recombinant vector of any of claims 1 - 5, said method comprising 25 A) inserting a heterologous nucléotide sequence encoding the HIV regulatory protein NEF, REV or TAT or an immunologically active fragmentthereof into a vector comprising papilloma virus nucléotide sequencesconsisting essentially of (i) a papilloma E1 gene and E2 gene, 30 (ü) a minimal origin of réplication of a papilloma virus, and (iii) a minichromosomal maintenance element of a papilloma virus, and B) transforming a host cell with the resulting self-replicatingrecombinant vector, 35 C) culturing the host cell, and D) recovering said vector. 22 011528
9. The method of claim 8 wherein the host cell is an E. coli cell.
10. Use of a self-replicating vector of any of daims 1 - 5 for themanufacture of a DNA immunization vaccine against HIV. -
11. The use of claim 9 in the manufacture of a vaccine comprising a5 mixture of vectors encoding different HIV regulatory proteins or immunologi- cally active fragments thereof.
12. Method of treating or preventing HIV comprising administeringto a person in need thereof an effective amount of a self-replicating vector ofany of daims 1 - 5, and expressing the NEF,' REV or TAT protein or an 10 immunologically active fragment thereof in said person.
13. The method of daim 12 comprising administering a mixture ofvectors encoding different HIV regulatory proteins or immunologically activefragments thereof.
14. A host cell comprising the self-replicating vector of any of daims 15 1-5. ’
15. The host cell of claim 14, which is a bacterial cell or amammaiian cell.