Reverse transcriptase-based DNA vaccines against drug-resistant HIV-1 tested in a mouse model

Vaccine 22 (2004) 1810–1819

Reverse transcriptase-based DNA vaccines against drug-resistantHIV-1 tested in a mouse model

Maria G. Isaguliantsa,b,∗, Bartek Zubera, Andreas Boberga, Dan Sjöstranda,Sergey V. Belikovb,c, Erik Rollmana, Anne Kjerrström Zubera, Vladimir O. Rechinskyd,

Ann-Sofie Ryttinge, Clas F.R. Källandere, Jorma Hinkulaa, Sergey N. Kochetkovd,Margaret Liuf,g, Britta Wahrena,g

a Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Swedenb D.I. Ivanovsky Institute of Virology, Gamaleja str. 16, 123098 Moscow, Russia

c Department of Cell Molecular Biology, Karolinska Institute, SE-171 77 Stockholm, Swedend W.A. Engelhardt Institute of Molecular Biology, Vavilova str. 32, 117984 Moscow, Russia

e Cavidi Tech AB, Uppsala Science Park, Uppsala, Swedenf Transgene, 76082 Strasburg Cedex, France

g Microbiology and Tumor Biology Center, Karolinska Institutet, 17177 Stockholm, Sweden

Abstract

Drug resistance is becoming a problem in the treatment of the human immunodeficiency virus type one (HIV-1). To obtain therapeuticDNA vaccines that would target multiple drug-resistance (DR) mutations, we cloned genes for DR HIV-1 reverse transcriptase (RT) andcodon-optimized synthetic genes encoding clusters of human CTL epitopes located at the sites of DR-mutations (RT minigenes) and antibodyand CTL-epitope tags. Expression of RT genes/minigenes in eukaryotic cells was confirmed by Western blotting and immunofluoresencestaining with RT- or tag-specific antibodies. Immunization of mice with DR–RT gene induced no RT-specific antibodies. Immunizationof HLA-A ∗0201-transgenic mice with RT minigenes induced RT-specific cellular responses detected by interferon-� secretion. Thisdocuments first steps in creating therapeutic vaccine against drug-resistant HIV strains.© 2004 Elsevier Ltd. All rights reserved.

Keywords:HIV-1 reverse transcriptase; Drug-resistance mutations; DNA-immunization

1. Introduction

The essential HIV-1 enzyme reverse transcriptase (RT)has been one of the major targets for anti-HIV-1 chemother-apy. Both nucleoside RT inhibitors (NRTIs)[1,2] andnon-nucleoside RT inhibitors (NNRTIs)[3,4] have been de-veloped. Treatment with just one of these drugs gives rise toresistant viral strains in a very short time. Patients receivingmonotherapy with the NNRTI Nevirapine, had mutant virusafter already 14 days[5]. The more recent combinations oftwo anti-RT drugs and a protease inhibitor, or three or moreRT inhibitors appear to be more promising. Still, the num-ber of treatment failures due to emergence of drug-resistantmutants and toxicity is increasing[6]. HIV-1 can acquiremutations in RT and protease that confer resistance to allcurrently available RT and protease inhibitors[7,8]. High

∗ Corresponding author. Tel.:+46-8-4572609; fax:+46-8-337272.E-mail address:maria.isaguliants@smi.ki.se (M.G. Isaguliants).

levels of HIV-1 resistance to multiple inhibitors leading totreatment failures correlate with the emergence of combina-tions of mutations called “multiple drug-resistance patterns”[6,9]. Advanced stages of HIV-1 infection, high viral loadand poor compliance are risk factors for the developmentof resistance[10]. Primary infection with drug-resistantvirus is also on the rise[11]. Little is known about the in-fluence of the immune system on the development of drugresistance and the efficacy of drug therapy.

CD8+ cytotoxic T lymphocytes (CTLs) appear to beimportant in containing the spread of HIV-1 in infectedindividuals[12]. The replication of HIV-1 in CD4+ T-cellscan be inhibited by autologous CD8+ CTLs [13–15]. Theviral containment in early HIV-1 infection coincides intime with the emergence of a virus specific CTL response[16]. In HIV-1 infected individuals, high frequency of CTLis correlated with the maintenance of low viral load andstable clinical status[14,17], while loss of HIV CTLs isthe major immune correlate of disease progression[14,18].

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.vaccine.2003.10.052

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Possible mechanisms for CTL effects involve both lysis ofinfected cells and the release of chemokines and cytokines[19,20].

Recent data have shown that mutations of RT conferringdrug resistance are immunogenic[21,22]. Some of thesemutations appear to have even increased immunogenicity ascompared to the wild-type sequences, although the speci-ficity of the response appears to be changed[22]. The latterdata suggest that mutant escaping from drug pressure wouldnot escape from the immune response when it is established.Pre-existing immune response against mutations conferringdrug-resistance might, therefore, hinder generation, selec-tion and amplification of drug-resistant virus populations.

HIV DNA vaccines aided with live viral vectors showedpromising results in human clinical trials as safe to useand immunogenic (see[23–26] for reviews). Today, thesevaccines do not prevent the infection, but rather con-trol virus replication [26]. Therefore, the induction ofimmune responses against drug-resistant (DR) RT byDNA-immunization seemed to be a feasible approach. Thewild-type RT gene is immunogenic in rodents, rabbits, andnonhuman primates, both alone[27–29]and as a constituentof the humanised Pol gene[30,31]. In the present study, wehave achieved eukaryotic expression of DR HIV-1 RTs andRT epitopes from DR mutation sites and characterised theirimmunogenicity in an experimental model.

2. Materials and methods

2.1. DR–RT genes

Genes for multi-drug-resistant RTs were derived fromHIV-1 MN isolated from patients J14562 and J14007. Pa-tient J14562 was treated with 3TC, d4T, saquinavir andritonavir. When all available (1998) treatment failed for pa-tient J14007, he was treated with saquinavir, ritonavir andabacavir. Virus was isolated from peripheral blood cells af-ter 15 month of treatment. Preparation of viral RNA, cDNAsynthesis, PCR and sequencing were done as was describedpreviously[32]. RT cDNA was re-amplified using a forwardprimer carrying theSalGI site, consensus ribosomal bindingsite with start codon, and a reverse primer carrying theEcoRIsite and stop codon TGA. TheSalGI/EcoRI-digested PCRproduct was ligated intoSalGI/EcoRI-digested pKCMV[27] to generate pKCMVRT1.14multi-mut (J14562) andpKCMVRT2.20multi-mut (J14007) (Fig. 1A). The presenceof the inserts with respective mutations was confirmed bysequencing.

2.2. Synthetic multiepitope encoding genes(RT minigenes)

HLA-A ∗0201 restricted CTL epitopes in HIV-1 RT andprotease overlapping sites of DR mutations were selectedfrom the database of the National Institutes of Health (NIH)

(http://hiv-web.lanl.gov/content/index). HIV-1 RT and pro-tease epitopes, the B-cell epitope YPYDVPDYA from in-fluenza haemagglutinin (HA), the H2-Dd restricted mouseepitope RGPGRAFVTI derived from HIV-1 gp120[33] anda promiscuous T-helper cell epitope QYIKANSKFIGITEL[34] were combined into synthetic multipepitope constructsNCVT and KNCVT (Fig. 1B). Synthetic genes for multiepi-tope constructs were designed based on the codons optimalfor expression in eukaryotic cells. A consensus ribosomalbinding site with a start codon GCCACCATG was includedfor efficient initiation of translation. Genes were synthesizedusing overlapping primers and several rounds of PCR ac-cording to a modified version of a two-step method of genesynthesis,[35] and cloned into pKCMV[27] to generatepNCVT and pKNCVT.

2.3. Immunofluoresence analysis of DR–RT and RTminigene expression in transfected cells

Human HeLa cells were cultured on cover-slips andtransfected with polyethyleneimine[36]. At 36–40 h post-transfection, cells were washed three times in PBS, air-dried, and fixed for 30 min at−20◦C in acetone–methanol(80:20). RT-expressing cells were immunostained by dou-ble antibody technique using supernatant of mouse anti-RThybridoma 3F6[37] and FITC-conjugated goat anti-mouseantibodies (DAKOPatts AB, Denmark) diluted 1/50 in PBScontaining 0.5% BSA and 2% normal goat serum. Multi-peitope expressing cells were immunostained with anti-HApolyclonal mouse monoclonal antibody (#H9658, Sigma,Deisenhofen, Germany) and anti-mouse FITC-conjugatedsecondary antibodies. Cells were counter-stained for 5 minwith Hoechst 33258 (25 ng/ml). Staining was detected witha Nicon Eclipse 400 Fluorescence Microscope connectedto an Orca II Digital Camera (Hamatsu, Japan).

2.4. Immunoblot analysis of DR–RT expression intransfected cells

Human HeLa cells were transfected with polyethylene-imine as in[36]. After 48 h, cells were washed with PBS,collected by centrifugation, re-suspended (106 cells/ml) andlysed in the buffer containing 50 mM Tris–HCl pH 7.5,150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP40, 0.25%DOC, 0.1% SDS and protease inhibitor cocktail for generaluse (Sigma). Cell debris was sedimented by centrifugation.Supernatants from cell lysates were analyzed by gradient4–15% SDS-PAGE using a Modular Mini-PROTEAN IICellTM (BioRad Laboratories Inc., Hercules, CA). The gelwas blotted onto a nitrocellulose membrane (BioRad). Im-munoblots were analyzed by standard methods using rabbitanti-RT polyclonal antibodies[28], or mouse monoclonal3F6 [37] and horseradish peroxidase (HRP)-conjugatedspeci-specific antibodies (DAKOPatts AB, Denmark). Theperoxidase activity was detected using the ECLTM system(Amersham Pharmacia Biotech, Freiburg, Germany).

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Fig. 1. (A) Multiple alignment of amino acid sequences of wild-type and drug-resistant reverse transcriptase (RT). RT of consensus clade B; HXB2RT; wild-type RT encoded by pCMVRT with R172K/Q207R, wtRT; RT of HIV-1 MN isolate from patient J14007 with multiple resistance mutationsencoded by pKCMVRT1.14multi-mut, RT1.14multi-mut; RT of HIV-1 MN isolate from patient J14562 with multiple drug-resistance mutations encodedby pKCMVRT2.20multi-mut, RT2.20multi-mut. All aa residues different from clade B consensus and HXB2 sequence are given in bold. Mutationsconferring drug-resistance are underlined. (B) Multiepitope constructs with wild-type RT and protease (PR) CTL epitopes, the marker H2-Dd mouseepitope from HIV-1 gp160, the promiscuous T-helper cell epitope from tetanus toxoid, and an B-cell epitope tag from influenza HA. Lys residues flankingthe gp160 CTL-epitope in KNCVT are given in bold.

2.5. Oocyte maintenance, microinjection and lysis

FemaleXenopus laeviswere purchased from Xenopus I(Ann Arbor, MI) and manipulated as described[38]. De-folliculated stage VIXenopus laevisoocytes were preparedby collagenase treatment[38]. Oocytes were incubated

overnight at 18–19◦C in OR2 medium containing 1 mMCaCl2. The next day, healthy oocytes from one frog wereinjected directly into the nucleus with 2 ng of a plasmid dis-solved in 20 nl of water using a Nanoliter 2000TM (WorldPrecision Instruments Inc.). A pool of 10 oocytes was left un-treated. Oocytes were kept for 20 h at 19.5◦C, then frozen in

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liquid nitrogen. Ten oocytes injected with either pCMVRT,or pKCMVRT1.14multi-mut, or pKCMVRT2.20multi-mut,or empty vector were pooled (one pool per plasmid). Eachoocyte pool was immersed in 100�l of sterile ice-cold PBScontaining 0.05% Tween 20 and protease inhibitor cocktailfor general use (Sigma). Homogenization of oocytes wasdone by pipeting at 0◦C until an even homogenate wasobtained (∼15–20 times). The lysate was centrifuged for10 min at 4◦C at 40,000 rpm. The supernatant was collectedand stored on ice. Cell and nuclear debris was washed with50�l of ice-cold lysis buffer, and centrifuged for 5 min at4◦C at 40000 rpm. Supernatants were pooled and assayedfor RT expression immediately.

2.6. Quantification of RT expression by sandwich ELISA

Supernatant from anti-RT hybridoma 3F6[37] was coatedon a 96-well ELISA plate (MaxiSorb, Nunc, Denmark) incarbonate buffer, pH 9.6, at dilution 30 corresponding to theantibody concentration of 0.5�g/ml. Plates were blocked for1 h at 37◦C with PBS containing 0.5% BSA, 5% goat serumand 0.01% Tween 20. Wild-type (wt) and 67N/215F-RTwere prepared as described[39] and diluted in the rangeof 40–5000 pg/ml. These standards and aliquots of oocytelysates were applied on the 3F6-coated plates, and incubatedfor 1.5 h at 37◦C. Captured RTs were detected by anti-RTrabbit antibodies[28]. Rabbit antibody binding was regis-tered by HRP-conjugated goat anti-rabbit immunoglobulins,and TMB (both DAKO) as a substrate. Detection antibodywas incubated overnight at 4◦C; secondary antibody, for1.5 h at 37◦C; and substrate, for 10–15 min at 20◦C. Spe-cific absorbance at each of the lysate dilutions was deter-mined as [OD450 of RT-containing sample]− [OD450 oflysate of non-injected oocytes]. RT concentration in the sam-ple was evaluated by comparing with the linear part of thecalibration curves. Calibration curves made with wtRT and67N/215F-RT gave concordant results. The final RT concen-tration in the sample was evaluated as an arithmetic mean ofRT concentrations calculated for two closest lysate dilutionsin two independent ELISA runs.

2.7. Immunization of mice

HLA-A ∗0201/Kb transgenic (A2/Kb tg)[40,41] andC57BL/6 mice were bred at the Karolinska Institutet, andBALB/c mice were purchased. Animal care was in accor-dance with institutional guidelines. Mice were used at anage of 10± 2 weeks. For immunization, plasmid DNA wasamplified in E. coli/DH5alphaF1′, extracted and purifiedusing Qiagen Endo Free plasmid Maxi kit (Qiagen GmbH,Germany). In gene-gun immunization, plasmids were coatedonto 0.95-mm gold particles (Aldrich, Milwaukee, WI) anddelivered in the abdominal skin by a helium-pulse Accelldevice (courtesy of J. Haynes, Auragen, Madison, WI).

In the first experiment, three groups of C57BL/6 mice(five mice/group) received totally 4�g of pCMVRT, or

pKCMVRT2.20multi-mut, or vector pKCMV per mouse,and a group of BALB/c mice (n = 4) received 4�g ofpCMVRT per mouse, divided into two gene-gun admin-istrations. Administrations of pKCMVRT2.20multi-mutand pKCMV were preceded by the injection of 1�g ofrGM-CSF (R&D). A group of C57BL/6 mice (n = 5)had received intramuscularly totally 100�g of pCMVRTdivided into two injections at weeks 0 and 4. Blood wascollected two weeks after each injection.

In the second experiment, four groups of BALB/c (H2-Dd)mice (five mice/group) were immunized intramuscularlywith 150�g of pNCVT, or pKNCVT, or pKCMVgp160together with pKCMVrev to potentiate gp160 expression[42]. A group of BALB/c mice (n = 4) was immunizedsubcutaneously with the total of 300�g HIV-1 gp120peptide RGPGRAFVTI in incomplete Freund’s adjuvant,administered in three injections. DNA-immunogens wereinoculated in tibialis anterior, and peptides, in the base ofthe tail, at weeks 0, 4 and 11.

In the third experiment, two groups of A2/Kb tg mice (fivemice/group) were immunized with either 100�g pCMVRT,or pKNCVT intramuscularly in tibialis anterior. Plasmidswere administered in three injections at weeks 0, 4, and 10.One group of A2/Kb tg mice (n = 5) was immunized witha mixture of 100�g of each of the RT peptides formingKNCVT (Fig. 1B), given subcutaneously at weeks 0, 4, and10 in the base of the tail.

2.8. Immune assays

Mice were bled 10–12 days after every immunization.Mouse sera were assessed for antibodies against RT byindirect ELISA on MaxiSorb (Nunc) 96-well plates coatedwith wtRT as was described[28]. Heparinized blood frommice belonging to each group was pooled and periph-eral blood mononuclear cells (PBMCs) were separatedby Lymphoprep/Isopaque-Ficoll (Gibco BRL, Paisley,Scotland) gradient centrifugation. At the end of immu-nization, mice were anesthetized, sacrificed, and spleenswere collected. Spleens were homogenized in RPMI-1640supplemented with 2 mMl-glutamine and 10% fetal calfserum (Gibco BRL), and splenic T-cells were separated bygradient centrifugation. Individual splenocytes or pooledPBMCs were assayed for interferon-� (IFN-�) productionby ELISPOT.

IFN-� ELISPOT (Diaclone Research, Cedex, France;Mabtech, Sweden in parallel) was performed on PVDFplates (MAIPS45-10, Millipore). Plates were activatedwith 70% ethanol, washed with PBS and incubated witha mouse IFN-� specific monoclonal antibody at 4◦C overnight. Plates were then washed six times with RPMI andblocked with RPMI containing 10% FCS at 37◦C for1 h. Individual mouse splenocytes or pooled PBMC werere-suspended in RPMI-1640 at 2× 106/ml and distributed100�l per well. Cells were stimulated with RGPGRAFVTI,or pools of 15-mer peptides covering RT amino acids 1–127,

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or 138–282, or 273–417 (Centralized Facility for AIDSReagents, Medical Research Council, UK), or RT-derivedpeptides ALVEICTEL, VIYQYMDDL, LLRWGLTTPDKKand ILKEPVHGV. Peptide SLYNTVATL (from the HIVp24 protein) was used as a negative, and PHA, as positivecontrol. Antigens were resuspended in RPMI-1640 con-taining 20% FCS, 1% PEST, and 1%l-glutamine, and dis-pensed on the plates in a total amount of 0.5�g/well. Stim-ulation with each antigen was done in triplicate. Cells werestimulated for 24 h at 37◦C. After that, plates were rinsedand developed according to the manufacturers instructions.The number of spots in each well was counted automat-ically using the AID EliSpot Reader System. RT-specificIFN-� spots detected in PBMCs were each blanked forunspecific responses produced by immune mouse PBMCsstimulated with SLYNTVATL, or PBMCs of naıve micestimulated with RT peptides, the highest of the valuesselected.

2.9. Computing and statistical analysis

Consensus amino acid sequences of RT of HIV-1 sub-types were derived fromhttp://hiv-web.lanl.gov/PEPTGEN,the reference HXB2 RT access number #K03455. Sequenceswere aligned using a multiple alignment program MUL-TALIN at Pole Bio-Informatique Lyonnais (http://npsa-pbil.ibcp.fr). Modeling of RT tertiary structure was doneusing software Rasmol athttp://www.umass.edu/microbio/rasmol. Codon usage was selected from the database of theKazusa DNA Research Institute, Japan (http://www.kazusa.or.jp/codon). The levels of RT expression, antibody titers,and spots in the IFN-� ELISPOT assay represent arith-metic mean values. Standard deviation was calculated usingbiased orn-method.

3. Results

3.1. Drug-resistant RT genes

Genes encoding multiple drug-resistant RT mutants(DR–RTs: RT1.14multi-mut, RT2.20multi-mut;Fig. 1A)were derived from HIV-1 MN-infected patients treatedwith different drug combinations. RT1.14multi-mut ex-hibited multi-NRTI-resistant mutation pattern[9,43].RT2.20multi-mut carried mutations that confer high-levelviral resistance to AZT (zidovudine), 3TC (lamivudine), anda clinically significant loss of resistance to ddI (didanosine)and d4T (stavudine)[43]. RT2.20multi-mut also had themutation H208Y that was shown to be involved in the resis-tance to the pyrophosphate analogue foscarnet[44]. Othermutations of interest included S322T, M357T, A360T andAT400S in the connection domain; numerous mutations inthe RNAse H and deletion of 416F with insertion of an extraF at 426, in RT2.20multi-mut. In addition, RT2.20multi-muthad mutations localized at aa 114, 117 of the dNTP binding

pocket and at 218 of the “palm” subdomain (Fig. 1A). Mod-eling of RT tertiary structure demonstrated that positions114, 117, and 218 belong to the region in direct contactwith incoming dNTP/NRTI (data not shown).

3.2. Synthetic genes encoding HIV multiepitopes

We used the literature and the NIH HIV database to findHLA-A ∗0201-restricted CTL epitopes at the sites of DRmutations in RT and protease. CTL HLA-A∗0201 epitopeswere joined into a multipeitope construct NCVT (Fig. 1B).A B-cell epitope from influenza haemagglutinin (HA) wasincluded as a tag to detect the expression of the gene productby HA-specific antibodies. Sequence encoding the H2-Dd

restricted epitope from HIV-1 gp120[33] was introducedas a CTL-tag to detect immunogenicity of the multiepitopegene in H2-Dd BALB/C mice. A promiscuous T-helper cellepitope from Tetanus toxoid[34] was included to provideT-cell help. In a multipitope variant KNCVT, the HIV-1gp120 CTL-epitope tag was flanked with lysin residues forbetter processing[45] (Fig. 1B).

3.3. Transient expression of DR–RTs and multiepitopegenes

To achieve eukaryotic expression, drug-resistant RTgenes and synthetic codon-optimized genes for multiepi-tope constructs (RT minigenes) were cloned into a CMVIE-driven vector. Plasmids were transiently transfected intoHeLa cells. SDS-PAGE with subsequent Western blot-ting demonstrated that DR–RT genes expressed proteinswith the expected molecular weight of 66 kDa specificallystained by anti-RT antibodies (Fig. 2). A weak processingof the 66 kDa subunit into p51 was observed even in the

Fig. 2. SDS-PAGE with subsequent Western blotting of the lysatesfrom HeLa cells transiently transfected with pCMVRT (lane 1),pKCMVRT2.20multi-mut (lane 2), and pKCMVRT1.14multi-mut (lane3). Immunostaining was performed with polyclonal rabbit sera against RT[28]. Positions of molecular mass markers (kDa) are given to the left.

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Fig. 3. Immunofluorescence staining of HeLa cells transfected withpKCMVRT1.14multi-mut (A); pKNCVT (B); and pKCMV vector (C).RT-expressing cells are stained with anti-RT monoclonal antibody 3F6(A); and cells expressing multipepitope KNCVT, with HA-specific anti-body (B). Cells transfected with empty vector and stained with anti-RTmonoclonal antibody 3F6 are shown for comparison (C).

presence of protease inhibitors (Fig. 2, and data not shown).Eukaryotic expression of RT minigenes could be detectedby immunofluoresence staining only. Both DR–RTs andmultiepitope constructs were localized in the cytoplasm ofexpressing cells (Fig. 3A and B).

3.4. Immunization with DR–RT gene

C57BL/6 mice were immunized with DR–RT and RTgenes delivered as pKCMVRT2.20multi-mut and pCMVRTplasmids. Anti-RT antibody response was assessed by in-direct ELISA. At a plasmid dose of 50�g per intramuscu-lar injection, the average titer of anti-RT antibodies in thepCMVRT-immunized mice was 35,000± 27,000; and at2�g delivered by gene gun, it was reduced to 400 (400±300) (Fig. 4). C57BL/6 mice immunized with the same2�g dose of pKCMVRT2.20multi-mut produced no anti-bodies (Fig. 4). Gene-gun injections of BALB/c mice with2�g dose pKCMVRT2.20multi-mut generated low titers ofanti-RT antibodies (Fig. 4).

3.5. Quantification of DR–RT expression

To find reasons for poor immunogenic performance ofDR–RT genes, we analysed the level of DR–RT expression.A sandwich ELISA for RT was designed enabling DR–RTquantification. The assay was based on the use of mono-clonal anti-RT MAb3F6[37] as a capture, and rabbit poly-clonal anti-RT[28] as detection antibody. Rytting et al. hadshown that conformational MAb3F6 can bind and neutral-ize RTs of different HIV-1 clades, and even SIV RT, beinginsensitive to RT mutations[37]. Both MAb3F6 and rabbitpolyclonal anti-RT antibodies equally well recognized re-combinant AZT-resistant[39] and wild-type RTs (data forindirect ELISA not shown). The sandwich ELISA alloweddetection of RT variants in a concentration of≥40 pg/ml.

Direct quantitative comparison of the expression efficacyof RT-encoding plasmids was done by microinjection ofRT-encoding plasmids into the nucleus of oocytes of theAfrican frog Xenopus laevis. Results of a typical injec-tion experiment are given inFig. 5. Wild-type RT was ex-pressed at the level of 10–30 pg per oocyte depending on thefrog. DR–RTs were expressed at considerably lower levels(Fig. 5). Independent injection experiments gave the sameproportion between wtRT and DR–RT expression levels.

3.6. Immunization with RT minigenes: response against aCTL epitope tag

In parallel, BALB/c mice were immunized with minigenesfor NCVT and KNCVT (Fig. 1B). Cellular response againstthe H2-Dd-restricted CTL epitope tag, a gp120-derived se-quence RGPGRAFVTI served as a marker of multiepitopeimmunogenicity. To define the immunogenicity of a CTLtag in the context of the native protein, a group of mice wasimmunized with the gene for HIV-1 gp160. Control micewere immunized with the RGPGRAFVTI peptide. Strongand specific IFN-� responses were induced in the controlRGPGRAFVTI-immunized mice (Fig. 6). The NCVT geneinduced no significant IFN-� response (Fig. 6). In con-trast, mice immunized with the optimised KNCVT genedemonstrated RGPGRAFVTI-specific IFN-� responses of

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Fig. 4. Antibody reactivity against RT in mice immunized with genes of wtRT (pCMVRT) and drug-resistant RT2.20multi-mut (pKCMVRT2.20multi-mut).

both splenocytes (three of five mice, data not shown) andPBMCs (Fig. 6). No IFN-� response was observed againstthe CTL tag in the context of native gp160 (Fig. 6).

3.7. Immunization with RT multiepitope genes: RT-specificresponse

HLA-A ∗0201/Kb transgenic (A2/Kb tg) mice were im-munized with either a plasmid encoding KNCVRT, or plas-mid encoding wtRT, or a mixture of RT-derived peptidesconstituting KNCVT (Fig. 1B). The dynamics of specificIFN-� production in response to stimulation with RT-derivedpeptides is given inFig. 7. Mice immunized with the wtRTgene failed to induce specific IFN-� production. Weak,but sustained IFN-� production was detected in PBMCs

Fig. 5. Quantification of the expression of wild-type and drug-resistantRTs in the oocytes ofXenopus laevis(n = 10).

of A2/Kb tg mice immunized with a plasmid encodingKNCVT, or a mixture of KNCVT-constituting RT peptides(Fig. 7).

4. Discussion

We succeeded in cloning and expressing RTs from DRHIV-1 primary isolates. These RT genes carried mutationpatterns characteristic of multiple nucleoside resistance,and mutations of nonpolymorphic residues in close proxim-ity to the known DR-clusters at aa 113–118 and 210–219[9,43,44,46,47]. In parallel, we constructed synthetic genescontaining multiple HIV-1 specific CTL epitopes locatedat the sites of DR mutations (RT minigenes). RT genesand minigenes were used to induce immune response indiverse mouse strains, including mice transgenic for humanHLA-A ∗0201.

A standard dose of the wtRT gene induced good antibodyproduction, in contrast to the same dose of the DR–RT gene.The level of antibodies is proportional to the level of antigenexpression[48]. Poor humoral response of the DR–RT generecipients pointed at a significant decrease of the in vivolevel of DR–RT expression, as compared to the wild-typeprotein. To find out whether this was the case, we quantifiedthe DR–RT expression. Enzymatic RT assays seemed to beinadequate, since mutations are known to modulate the RTactivity [49,50]. Instead, we elaborated a quantitative RT im-munoassay. Quantification of RT expression in the oocytes ofXenopus laevisdemonstrated that DR–RTs were expressedat a 10-fold lower level than the wild-type enzyme. We hadrecently demonstrated a decreased proteolytic stability ofdrug-resistant RT[51]. DR–RTs immunogenic performance

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Fig. 6. IFN-� response against the H2-Dd restricted HIV-1 gp120-derived epitope RGPGRAFVTI, a tag introduced to trace cellular response in BALB/cmice.

could result from both low DR–RT levels, and unfavorableprocessing.

Decreased expression efficacy of multi-mutated RTs hasa direct impact on the strategy of immune therapy aimedat prevention of drug resistance. We addressed this prob-lem by substituting full-length DR–RT genes with the“string-of-beads” constructs[24,52] composed of linked

Fig. 7. Dynamics of IFN-� response in HLA-A∗0201/Kb transgenic miceimmunized with the RT gene (pCMVRT), the multipepitope encodingminigene pKNCVT, or the RT-peptides of the KNCVT multipepitopicconstruct. Data is presented as total RT-specific spots in PBMCs. Controlvalues have been subtracted.

minigenes for CTL-epitopes located at the sites of DRmutations. Cellular immune response to these constructsin mouse PBMC and splenocytes was assessed by anIFN-�-based ELISPOT assay. HLA-A∗0201/Kb transgenicmice immunized with the RT minigene, but not with thewhole RT gene, produced a sustained RT-specific IFN-�response. The results from such IFN-� assays have beenshown to correlate strongly with results of the51Cr-releaseassays of CD8+-cells [24]. In a parallel experiment, immu-nization of BALB/c mice with the minigene induced potentIFN-� response against H2-Dd restricted CTL-epitope tag.Data on the mouse CTL-epitope tag recognition demon-strated that, in concordance with the data of Livingstonet al. [45], introduction of flanking lysins enhances cellularimmunogenicity of CTL epitopes within the minigene. Thisapproach would be further used to optimise multiepitopeimmunogenicity.

Epitopes with drug-escape mutations represent attractivenew targets for HIV-1 specific vaccines. In a recent paperit was demonstrated that mutations induced by mono- orbi-therapy with NRTI, namely M41L, L74V, M184V, andT215Y/F, did not preclude subsequent CTL recognitionof the mutated sites[21,22]. For example, the RT M184Vmutation within an HLA-A2 restricted HIV-1 CTL epitope(RT 179–187) induces escape from the NRTI drug lamivu-dine (3TC) and temporarily abolishes recognition by CTLin HIV-1 infected patients. However later, HIV-1 patientsreceiving lamivudine treatment were able to generated RTspecific CTL that targeted lamivudine-resistant viruses[21].Here, we demonstrated that DR mutation regions repre-sented by multiepitope “string-of-beads” constructs are im-munogenic. More studies are needed to examine whether the

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induction of CTL against drug-escape variants can help de-lay or prevent the emergence of drug-resistant HIV-1 strains.

Acknowledgements

The work was financed by the research grants from theSwedish Research Council, the Swedish Foundation for In-ternational Cooperation in Research and Higher Education(STINT), the Swedish Royal Academy of Science, Karolin-ska Institutet, and INTAS of the European Commission.

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