Vaccine 22 (2004) 2586–2598

Codon optimization and ubiquitin conjugation of humanimmunodeficiency virus-1 Tat lead to enhanced

cell-mediated immune responses

Lakshmi Ramakrishnaa, Krishnamurthy Kumar Ananda,Marthandan Mahalingama, Kumarasamypet M. Mohankumara,

Shilpa Ramanib, Nagadenahalli B. Siddappaa, Udaykumar Rangaa,∗a Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research,

Jakkur (PO), Bangalore 560064, Indiab Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

Received 5 August 2003; received in revised form 13 November 2003; accepted 11 December 2003Available online 20 January 2004

Abstract

The transactivator protein, Tat, is a potential candidate for developing a vaccine against human immunodeficiency virus (HIV-1). SinceTat is not immunodominant, especially when delivered as a genetic vaccine, we expressed codon-optimized subtype-C Tat as a molecularconjugate of ubiquitin, to elicit antigen-specific cell-mediated immune responses. Immunization of mice with different ubiquitin-Tatconstructs elicited a strong cellular, but not a humoral, immune response. The combination of codon-optimization and ubiquitin-mediatedprocessing of Tat induced a Th-1 type cellular immune response that was detectable without in vitro stimulation, suggesting its potentialutility for destruction of virus-infected cells via CTL-mediated lysis. Preliminary attempts at characterizing the immunodominant regionsidentified a novel T-helper epitope within the core domain of Tat.© 2004 Published by Elsevier Ltd.

Keywords: HIV-1 subtype-C Tat; Molecular adjuvant; DNA vaccine

1. Introduction

A wide variety of attempts at developing a vaccine for hu-man immunodeficiency virus type 1 (HIV-1) focused mainlyon the envelope (env) gene of the virus. Although env vac-cines conferred protection against autologous viral strains,antigenic variation is a challenge for vaccine design[1].A need for developing multi-component vaccines is beingincreasingly realized, to induce broader immune responsesagainst the viral infection, by incorporating multiple viralantigens[2]. Extensive work from various laboratories hasidentified the viral structural proteins, gag and pol, and vi-ral regulatory proteins Nef, transactivator protein (Tat) andRev, as potential candidates for vaccine development[3–5].

The regulatory genes of the virus that are expressed earlyduring the viral life cycle have the advantage of stimu-lating immune responses with faster kinetics. Additionally,

∗ Corresponding author. Tel.:+91-80-2362-2750x2241;fax: +91-80-2362-2766.

E-mail address: udaykumar@jncasr.ac.in (U. Ranga).

their sequences are also conserved to a greater extent. Theviral transactivator protein has been delivered as a candi-date antigen in several vaccination formats including as apeptide [6,7], a protein [8], a toxoid [6,9], a DNA vac-cine [10–13], and a recombinant virus[13,14]. Althoughthe results of these studies have been conflicting, the im-portance of anti-Tat immune responses as correlates of pro-tection has been established. Cellular immune responses tothe regulatory proteins of HIV-1, especially Tat, are con-sidered important for protection against disease progression[15,16].

Strategies employing Tat as a vaccine must, however,overcome the problem of poor immunogenicity of this viralantigen. Immune responses to Tat usually are absent in themajority of the seropositive subjects and when present are oflow magnitude[17]. Especially when Tat is delivered in theform of a DNA vaccine, strategies to enhance the host im-mune response are essential, as this strategy of immunizationis not efficient. Vaccination using DNA induces weak hu-moral and cell-mediated responses even when administeredin multiple doses and requires adjuvants or boosting with

0264-410X/$ – see front matter © 2004 Published by Elsevier Ltd.doi:10.1016/j.vaccine.2003.12.007

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a heterologous vector expressing the same protein to elicitsignificant levels of B/T-cells. Tat delivered as a DNA vac-cine elicited cell-mediated and humoral immune responsesin mice, primates. and humans. In a human clinical trial, cel-lular immune responses to three viral regulatory genes weregenerated, in seropositive subjects, using DNA expression

Fig. 1. Ubiquitin tagging of Tat. (A) Schematic diagrams of two different ubiquitin-Tat expression vectors. Restriction enzymes at the junction of ubiquitinand Tat introduce Gly or Leu at the amino terminus of Tat, exposing them after intracellular processing; stb, stably expressed; rp, rapidly processed. (B)Schematic diagrams of various Tat expression vectors used in this study; ub, ubiquitin; wt, wildtype; co, codon-optimized. (C) Western blot of wildtypeand codon-optimized Ub-Tat expression constructs. 293 cells were transiently transfected with 10�g of the plasmid DNA. Cells were harvested 48 hafter the transfection, lysed in RIPA buffer and the cell extract was resolved on a 12% SDS-PAGE gel. Protein was transferred to a PVDF membrane(Bio-Rad) using a semi-dry transfer apparatus (Trans-Blot SD, Bio-Rad). Tat was detected with the monoclonal antibody 2D1.1 (#4138, NIH AIDSReference and Reagent Program) and a commercial chemiluminescent detection kit; -ve (−) mock transfection with parental vector. (D) Reporter SEAPactivity of ubiquitin-tagged Tat vectors in transiently transfected 293 cells. Different Ub-Tat expression vectors (10�g) and LTR-SEAP (1�g) reporterplasmid were transfected by the CaCl2 method. Following transfection, SEAP activity in 10�l of the sample at each time point was assayed in triplicate;pv, parental vector.

vectors, indicating that DNA vaccination could be a thera-peutic option[3,18]. Several molecular strategies have beenevaluated to enhance immune responses to Tat administeredas a genetic vaccine, including co-expression of cytokines[19], incorporation of CpG motifs[12], co-administration ofcationic block polymers[20], and others.

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Experimental ubiquitination of the antigens, as a meansof manipulating the N-terminal amino acid, targeted themfor rapid intracellular processing by the proteasome ma-chinery, in accordance with the ‘N-end rule’[21]. A directcorrelation between rapid antigen-processing and enhancedcell-mediated immune responses has been established[22,23]. In fact, expression of the DNA-encoded antigen asa molecularly conjugated chimera with the cellular ubiqui-tin protein has been one of the strategies extensively used toenhance antigen-specific CTL responses in the experimen-tal animals. Experimental ubiquitination of�-galactosidase[22] viral epitopes[24], viral antigens[25], mycobacterialantigens[26], and other pathogens stimulated enhancedantigen-specific cell-mediated immune responses. HIVgenes, gag and env, used as ubiquitin-fused constructs gen-erated significantly higher cellular immune responses[27]in mice and in an HLA-A∗0201 transgenic mouse model[28]. The ‘HIV-1 ubiquitin expression library’ vaccination,generated genome wide CD8 T-cell responses, assayed forthe gag, pol, env, and Nef regions[28].

We sought to investigate whether the strategy ofubiquitin-conjugation to Tat could enhance cell-mediatedimmune responses against this otherwise poor immunogen.We used a novel restriction enzyme strategy to place thewildtype or a codon-optimized synthetic Tat-expression cas-settes, corresponding to the first exon of HIV-1 C-Tat froman Indian isolate, downstream of ubiquitin (seeFig. 1 fora schematic representation). The ubiquitin-Tat expressionvectors were evaluated for elicitation of cellular immuneresponses in a murine model; two different mouse strains(BALB/c and C57BL/6) were used to account for haplotypedifference.

2. Materials and methods

2.1. Construction of the ubiquitin-Tat expressionvectors

Ubiquitin was amplified from the genomic DNA extractedfrom mouse liver, using the following primers that weredesigned based on the GenBank sequence X51703: forwardprimer N174, AAT GAA TTCGCC GCC GCC ATG CAGATT TTC GTG AAG ACC CTG AC; reverse primer N175,TAA GGT ACC GCC ACC TCT CAG GCG AAG GACC; reverse primer N176, TAA GGT ACC CTT AAGGCCACC TCT CAG GCG AAG GAC C. The hot-start PCRconditions were as follows: 941′

–6430′′–7240′′

for 2 cyclesand 941

′–7240′′

for 35 cycles. The resulting amplicons of228 bp were cloned directionally betweenEcoRI andKpnIsites into the mammalian expression vector, pUMVC3(http://www.med.umich.edu/vcore/Plasmids/pUMVC3.htm)under the control of a CMV promoter.

Exon-1 of HIV-1 Tat was amplified from p95IN21301(a molecular clone of Indian origin, GenBank accn. no.AF067156) as a template and using the following primers:

forward primer N177, TAA GGT ACCATG GAG CCAGTA GAT CCT AAC CTA; forward primer N178, AAAATA CTT AAG ATG GAG CCA GTA GAT CCT AAC CTA;reverse primer N180, TTT TCT AGACTA TTG CTT TGATAT AAG ATT TTG ATG. PCR conditions were as follows:941′

–5830′′–7230′′

for 3 cycles and 941′–6030′′

–7230′′for 32

cycles. All the PCR reactions of 50�l contained 1.25 U Taqpolymerase, 100�M dNTPs, and 500 nM primers. The re-sulting 240 bp amplicons were cloned directionally into theabove ubiquitin vectors betweenKpnI and XbaI (stb-Tatwt)and AflII and XbaI sites (rp-Tatwt), respectively. stb-Tatcowas obtained by amplifying synthetic HIV-1 Tat with theforward primer N297: TAA GGT ACCATG GAG CCAGTA GAT CCT AAC CT and reverse primer N180 usingthe above conditions. rp-Tatco was obtained by amplifyingcodon-optimized HIV-1 Tat that was synthetically assem-bled, with the primer pair, N178 and N180 using the aboveconditions. The codon-optimized sequence was derived fromthe consensus subtype-C sequence of the exon-1 of HIV-1Tat (Lakshmi R. and Udaykumar R., manuscript submitted).The gene was optimized for codons most frequently em-ployed in mammals[29–31] in an effort to overcome thecodon-bias of the AT-rich viral gene. All the expression cas-settes were confirmed by restriction enzyme analysis andsequencing.

2.2. Secreted alkaline phosphatase (SEAP) assay

HIV-1 Tat constructs were co-transfected with HIV-SEAP(a gift from Dr. Bryan Cullen) into 293 cells by the CaCl2method. The culture supernatant (200�l) was sampled atregular intervals and stored at−20◦C until use. The reporterSEAP activity was measured using pNPP as the substrate at405 nM as described previously[32].

2.3. Pulse-chase analysis of the ubiquitin-tagged Tatproteins

Human embryonic kidney (293) cells and HeLa cells weretransiently transfected with Tat-expression vectors using theCaCl2 method in 100 mm dishes with 10�g plasmid DNA.All the transfections also contained 1�g each of LTR-GFPand CMV-�-gal plasmids to serve as transfection and nor-malization controls, respectively. Prior to labeling, the cellswere starved for 1 h in culture media lacking the amino acidsMet and Cys (MEM Selectamine, GIBCO). The cells weremetabolically labeled for 3 h at 37◦C by adding 100�Ci/mlof 35S labeling mix (NEZ772, NEN Life Science Products,Inc.) containing labeled Met and Cys. Following labeling,complete medium enriched with cold Met and Cys (finalconcentration of 3.75�g/ml) was added and incubation con-tinued for different chase periods. Cells were harvested,lysed in 1 ml of RIPA buffer, and clear supernatants werestored frozen until use.

Protein A/G beads (#sc-2003, Protein A/G PLUS-agarose,Santa Cruz) were incubated overnight at 4◦C with rabbit

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anti-Tat antibodies raised in the laboratory against C-Tat, ata 1:250 dilution. Normal rabbit serum at the same dilutionwas included as a negative control. Antibody-bound beadswere added to the cell lysate to immunoprecipitate Tat andthe samples were resolved on an 18% SDS polyacrylamidegel. The gels were dried, exposed to an imaging plate andthe image was analyzed and quantitated using thel-processsoftware (FUJI, Japan). One-third of the cell lysate of eachexperiment was used for the quantitation of�-galactosidaseactivity for the purpose of experimental normalization[33].

2.4. DNA immunization

The plasmids intended for immunizations were preparedusing Qiagen endofree Giga kits as per the manufacturer’sinstructions. The DNA was resuspended in endofree PBS(Manukirti Biogems, India, endotoxin<0.06 EU), the endo-toxin concentration was analyzed by a standard LAL assay(QCL-1000, Biowhittaker) and found to be within recom-mended limits (<0.1 EU/�g DNA). One hundred microgramof the DNA was injected into the tibialis anterior (TA) mus-cle of mice that were 8–12-week-old. Each immunizationconsisted of four or five mice per group. The immuniza-tion schedule involved one primary immunization followedby three boosters, with each injection spaced 2 weeks apart.Animals were housed and maintained in a facility adheringto the recommendations of the Committee for the Purpose ofControl and Supervision of Experiments on Animals (CPC-SEA), India.

2.5. Lymphoproliferation assay

Five million splenocytes from the primed mice wereincubated with 5�g/ml of recombinantly expressed anti-gen for 5 days in a CO2 incubator. Tat-1 was purifiedusing the HIS-tag by the Ni-NTA (Qiagen) affinity chro-matography, while Tat-2 was purified using the GST-tag byglutathione–sepharose (Amersham) affinity chromatogra-phy. Purity of the proteins was confirmed using SDS-PAGE.Control proteins containing similar tags (HIS-p24 andGST-PC4) were also purified by similar means and used ascontrols for non-specific proliferation. Following incuba-tion, the extent of cell proliferation was measured by addi-tion of 3H-thymidine (5�Ci/ml, NET520A, Perkin-ElmerLife Sciences, Inc.) to the cells and the cultures were incu-bated for 3 h at 37◦C for incorporation of the label. The cellswere then harvested, washed, resuspended in 50�l PBSand deposited on filter paper discs (Whatman #3). This wasfollowed by cell lysis, filter drying, and radioactivity count-ing using a�-scintillation counter (Wallac 1409). Con-A(5�g/ml) was used as a positive control for cell proliferation.

2.6. CTL assay

Splenocytes obtained from primed mice were incubatedwith the irradiated stimulators (P815-Tat or EL-4-Tat) for

5 days in RPMI-1640 supplemented with 10% FBS. P815and EL-4 cells stably expressing the Tat protein under thecontrol of a CMV promoter (pcDNA 3.1) were used in theexperiments. Following the in vitro stimulation, the effectorcells were incubated at differentE:T ratios with Cr51-labeledtarget cells (104 cells/ reaction volume of 200�l) that wereMHC-restricted. Non-transfected parental cell lines (P815or EL-4) served as negative controls. The effector and tar-get cells were co-incubated for 5 h at 37◦C and 100�l ofthe supernatant was assayed using a gamma counter (LKBRack gamma, Wallac). Spontaneous lysis counts usually re-mained below 30% of the maximal lysis. Maximal lysis val-ues were obtained by incubating the labeled target cells inthe presence of 2% Triton-X 100. Percent specific lysis wascalculated using the following formula: specific lysis (%)= (experimental lysis− spontaneous lysis)/ (maximal lysis− spontaneous lysis).

2.7. ELISPOT assay

ELISPOT assays using commercial kits were performedfor the Th-1 cytokine IFN-� (m IFN� Eli-spot, DIACLONEResearch) and the Th-2 cytokine IL-4 (#551017, MouseIL-4 ELISPOT Set, BD Pharmingen) before and after invitro stimulation. Briefly, the cytokine specific capture anti-body (1�g/100�l PBS) was adsorbed on the PVDF backed96-well plates by incubating overnight at 4◦C. The plateswere blocked and the primed splenocytes (0.5 × 106 cells)were added along with the stimulator cells (P815-Tat andEL-4-Tat cells for BALB/c and C57BL/c, respectively) ata 1:3 ratio in a final volume of 200�l to the wells andincubated for 24–36 h. The cells were then lysed, celldebris removed by extensive washing and a biotinylatedanti-cytokine antibody (0.5�g/ml) was added for 2 h. Theplates were washed extensively. Enzyme-conjugated avidin(HRP for IL-4 and ALP for IFN-�) was added for detec-tion and incubated for 1 h. Spots were developed usingappropriate substrate (AEC and NBT-BCIP, respectively)and incubating the plates for 20 min at room temperature.Con-A (5�g/ml) was used as a positive control for IL-4secretion and PMA (1�g/ml) plus Ionomycin (0.5�g/ml)for IFN-� secretion. The spots were enumerated using astereo microscope (MZ6, Leica) at 16× magnification.

2.8. Epitope mapping

Six peptides spanning the consensus subtype-C sequenceof the HIV-1 Tat exon-1 were synthesized (Peptron, Korea)as 20 mers that overlapped with each other by 10 aminoacids. The peptides were reconstituted in DMSO and used ata final concentration of 2�g/ml. Pepscan analysis was per-formed on an IFN-� ELISPOT format by incubating the pep-tides with 5× 105 cells (splenocytes from primed BALB/cmice) per well for 36 h at 37◦C. The controls were normal-ized for DMSO concentrations. The responses were presen-ted as the number of spots per sample against each peptide.

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2.9. Statistical analysis

Results of lymphoproliferative assays are represented asSI values, mean± S.D. of at least three independent exper-iments, each of which was performed in triplicate. Resultsof ELISPOT assays are expressed as spots per million cells,means of triplicate wells. Comparisons between the Tat con-structs were made using the Studentt-test.P values less than0.05 were considered statistically significant.

3. Results

To enhance cell-mediated immune responses to Tat, weexpressed this viral antigen as a fusion protein of ubiquitin.Using a restriction enzyme-mediated approach, we placedTat downstream of ubiquitin in two different expression vec-tors. Cytoplasmic proteases capable of recognizing the ubiq-uitin fusion protein cleave the protein at the C-terminal ofubiquitin, thereby exposing the N-terminal amino acid ofthe tagged antigen. The strategy employed here to engineerN-terminal amino acid residues of an antigen is differentfrom that of the previous reports where an overlap-PCR ap-proach was used to generate the ubiquitin-antigen chimeras.Our strategy offers the flexibility of expressing any antigenas an ubiquitin chimera by simply placing the gene into thespecific restriction enzyme sites. The restriction enzymes se-lected for this purpose wereKpnI andAflII. The first tripletsof the enzyme recognition sites code for Gly and Leu, respec-tively. After ubiquitin is cleaved off intracellularly, the anti-gen will be released with one of these amino acid residuesat the N-terminal (Fig. 1A). While Gly at the N-terminal isexpected to stabilize the protein intracellularly, Leu is ex-pected to target the same protein for rapid processing in amanner similar to Met and Arg, respectively[34]. Our studyis the first attempt to use Gly and Leu for the N-end ruleapplication in the context of a DNA vaccine.

3.1. Functional evaluation of the Ub-Tat expression vectors

The functional integrity of the Tat protein expressedfrom various Ub-Tat expression vectors was confirmedprior to immunologic evaluation in 293 cells. RT-PCRanalysis showed that the cassettes of the Ub-Tat constructswere efficiently transcribed. Western blot analysis using aTat-specific monoclonal antibody NT3 2D1.1 (NIH AIDSResearch and Reference Reagent Program) identified ef-ficient translation of Tat from all the expression vectors(Fig. 1C).

To study the transactivation property of Tat, 293 cells wereco-transfected with various Tat-expression vectors and withone of the two reporter plasmids (LTR-SEAP or LTR-GFP).Culture supernatant was sampled at different time pointsand the enzyme activity was quantitated as reported previ-ously [32]. SEAP was identified in all the wells transfectedwith the Tat-expression vectors, but not in the wells with the

control vector (Fig. 1C). SEAP accumulated progressivelyin the spent-media as a function of time. Codon-optimizedTat vectors produced significantly higher levels of SEAPthan the wildtype counterparts suggesting efficient geneexpression. In the wells transfected with the synthetic-Tatgenes, enzyme levels peaked at a faster rate and the geneexpression appeared to be prolonged. Interestingly, SEAPsecretion from the untagged synthetic Tat construct wassignificantly higher than other synthetic chimera Tat genes.In contrast, SEAP secretion from rp-Tatco was the lowest,possibly representing the rapid intracellular degradationof the Tat protein. We observed identical results using anindependent reporter, GFP, for Tat-transactivation, with allthe expression vectors (data not shown).

3.2. The rp-Tat construct has a lower half-life

Having confirmed the functional integrity of differ-ent Ub-Tat constructs, we sought to evaluate whetherUb-tagging and engineering different amino acids at theN-terminal influenced the intracellular stability of the Tatproteins expressed from these vectors. Pulse-chase analysisof mammalian cell lines HeLa and 293 transfected withUb-Tatco vectors followed by immunoprecipitation using aspecific rabbit antiserum and electrophoresis was carriedout and the half-life values of different Tat proteins weredetermined by linear regression analysis (Table 1).

Using the pulse-chase strategy, we observed identical pat-tern of intracellular stability of different Tat proteins in thetwo cell lines. Only codon-optimized Tat vectors were usedin these experiments. The untagged Tat protein demonstratedgreater intracellular stability, whereas the Ub-conjugatedrp-Tatco was the least stable protein (Table 1). As expected,the calculated half-life for the rp-Tatco protein was nearlythree-fold less than that of the untagged Tat protein, sug-gesting rapid processing of the former. Tat protein encodedby the stb-Tatco appears to be less stable than the proteinencoded by the untagged Tat vector in both the cell lines.The Tat protein containing Leu at the N-terminus possiblyis less stable than the one carrying a natural Met residue atthis position. Nevertheless, there was a significant differencebetween the half-life values of Tat proteins encoded by thestb-Tatco and rp-Tatco vectors.

Table 1Intracellular stability of different Tat proteins

Cell line Mean half-life (h)

Tat stb-Tat rp-Tat

Hela 24.1± 12.7 15.0± 4.0 7.4± 2.1293 30.1± 8.6 18.7± 5.7 9.6± 2.9

HeLa and 293 cells were transiently transfected with differentTat-expression vectors as described inSection 2. We used the followingformula to calculate the approximate half-life of Tat, whereNt is theamount recovered at time ‘t’ and N0 is the amount present att = 0:T1/2 = (ln 2/ln (Nt/N0))t.

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3.3. Splenocytes from rp-Tat immunized mice showsignificant proliferation in response to the antigen

We next evaluated the immunogenic potential of theTat constructs in two different mouse strains, BALB/c andC57BL/6. In both the strains we observed identical patternof immune response. Splenocytes harvested from immu-nized mice were incubated in vitro with recombinantlyexpressed Tat antigen and the extent of antigen-specific cellproliferation was measured using3H-thymidine incorpora-tion (Fig. 2A and B). Both wildtype and codon-optimizedTat constructs elicited a response that was absent in controlmice administered with the parental vector. The responsewas not significantly different between untagged Tat andstb-Tat constructs for both the wildtype and synthetic

Fig. 2. Lymphoproliferative immune responses in mice genetically immu-nized with Ub-tagged Tat constructs. (A) C57BL/6 and (B) BALB/c mice(five animals per group) were immunized with 100�g of plasmid DNAintramuscularly following the immunization schedule depicted in the linediagram above. Splenocytes (5×106) were incubated with recombinantlyexpressed antigen (HIS-Tat-1, 5�g/ml) for 5 days. Following incubation,proliferation of lymphocytes in response to the Tat antigen was measuredby 3H-thymidine incorporation as described inSection 2; wt, wildtypeTat; co, codon-optimized Tat. A stimulation index above three was consid-ered as a positive immune response. An asterisk (*) represents statisticalcomparison between the wildtype and synthetic rp-Tat constructs, usingStudent’st-test. TheP values are shown.

versions. Lymphoproliferative responses to rp-Tat in bothwildtype and codon-optimized context were significantlyhigher than other formats indicating that the rp-Tat wasefficiently processed and presented to the T-cells to elicit ahigher cellular immune response. Between the rp-Tat genes,the codon-optimized Tat vector elicited significantly highercell proliferation than the wildtype counterpart, suggestingthat the codon-optimized Tat is a stronger immunogen.

3.4. rp-Tat constructs elicit enhanced cytolytic activity

Cytolytic activity is an important objective for a vaccine-induced cellular immune response. As anti-Tat CTLs havebeen shown to be the correlates of protection[16,35], wesought to measure the CTL activity of Tat-specific T cellselicited by various Ub-Tat constructs using a conventional51Cr-release assay. Primed splenocytes from DNA injectedmice (C57BL/6 and BALB/c, four or five mice per group)were incubated with labeled target cells that presented Tatpeptides in the appropriate MHC-1 context (EL-4-Tat andP815-Tat cells, respectively) at differentE:T ratio.

All the Tat-expression vectors generated moderate tostrong cytotoxic cell-mediated immune responses althoughthe magnitude of cell lysis was not significantly differentwhen the codon-optimized and wildtype Tat vectors werecompared (Fig. 3A and B). We, however, observed a signif-icantly enhanced antigen-specific cell lysis in mice immu-nized with the rp-Tat vector that was also codon-optimized(rp-Tatco). Importantly, the pattern of cell lysis detected inthis experiment was in agreement with that of the cell prolif-eration assay. Codon optimization, along with engineeringthe antigen for rapid processing, could work in concert toenhance the immunogenic potential of the DNA vaccine.The responses against the parental vector were minimal andremained below the spontaneous lysis values.

3.5. Ub-Tat constructs promote Th-1 type cellular immuneresponse

A Th-1 cytokine profile is considered to be the optimalimmune response for protection against HIV[36–38]. Weused the ELISPOT technique to evaluate the cytokine profileelicited by the Ub-Tat expression DNA vectors for the Th-1cytokine IFN-� and the Th-2 cytokine IL-4, before and afterin vitro stimulation.

In C57BL/6 mice, DNA immunization with the Ub-Tatvectors induced antigen-specific cytokine response with amarked predominance of IFN-� secreting cells over IL-4 se-cretors, regardless of the vector used (Fig. 4). Immunizationwith untagged Tat vectors stimulated considerable numbersof IL-4 secreting cells, although these numbers were signif-icantly less compared to IFN-� secretors. IFN-� secretingcells were present in the primed splenocytes prior to invitro stimulation and their numbers increased significantlyfollowing the stimulation. In contrast, IL-4 producing cellswere mostly apparent only following the in vitro stimulation.

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Fig. 3. Cytotoxic immune responses in mice genetically immunized with Ub-tagged Tat constructs. (A) C57BL/6 and (B) BALB/c mice (four or fiveanimals per group) were immunized with 100�g of plasmid DNA intramuscularly following the immunization schedule depicted in the line diagram ofFig. 2. Splenocytes were stimulated in vitro with cells stably expressing Tat-1 (EL-4-Tat and P815-Tat cells for C57BL/6 and BALB/c, respectively) at a10:1 ratio for 5 days and were used in a conventional51Cr-release assay with labeled target cells (1×104 cells per assay). The reaction was performed for5 h at differentE:T ratios. Data are presented as the mean value of triplicate samples± S.D. The experiment was repeated two times; pv, parental vector.

The ratio between IFN-� and IL-4 secreting cells wasconsiderably greater in immunizations with Ub-tagged Tatantigens as compared to untagged Tat, suggesting thatUb-conjugation stimulated Th-1 responses more efficiently.Similar data were obtained from BALB/c mice (Fig. 5).The difference in IFN-� secreting cells between stb-Tat andrp-Tat immunizations was statistically significant, as evalu-ated by Student’st-test, supporting the experimental resultsof cell proliferation and51Cr-release assays. The numberof cytokine-secreting cells was insignificant in the controlgroup where the mice were immunized with the parentalvector confirming the specificity of the immune response.

3.6. Anti-Tat-1 cellular immune responses are poorlycross-reactive with HIV-2 Tat

Dual infections of HIV-1 and HIV-2 and single infectionwith HIV-2 are prevalent in Africa, India, and other partsof the world. As the transactivator proteins from both the

types share considerable identity at the protein level (∼36%in the first exon), we sought to examine the magnitude ofthe cross-reactivity manifested by Tat-1 primed splenocytesagainst cells stably expressing Tat-2 (EL-4-Tat-2). Results ofthis analysis demonstrated cross-reactive immune responses,however, at significantly inferior magnitude (Fig. 6A and B).The responses were not considerably different among thevarious Ub-Tat constructs. Low levels of cross-reactivity be-tween the anti-Tat-1 and anti-Tat-2 responses suggested thatthe immunodominant regions of these antigens are possiblydifferent. The low cross-reactive responses could readily becontrasted with the higher, Tat-1 specific immune response(Fig. 6C).

3.7. The Ub-Tat constructs primarily target the core regionof Tat

All the Ub-Tat vectors elicited potent immune responsesagainst Tat-1. Previous work in our laboratory using

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Fig. 4. ELISPOT response in C57BL/6 mice genetically immunized with different Ub-Tat constructs. Mice (four or five animals per group) wereimmunized with 100�g of plasmid DNA according to the schedule described in the line diagram ofFig. 2. The assay was performed on cells directly(A) without, or (B) after in vitro stimulation with Tat-expressing syngenic cells (EL-4-Tat). Each bar represents a mean of three individual wells± S.D.The experiment was performed three times; pv, parental vector. An asterisk (*) represents statistical comparison between the wildtype and synthetic Tatconstructs in each format, using Student’st-test. TheP values are shown.

synthetic Tat constructs demonstrated that the core regionof Tat-1 is immunodominant in BALB/c mice immunizedwith the untagged Tatco DNA expression vector (LakshmiR. and Udaykumar R., manuscript submitted). We soughtto evaluate which region(s) of Tat-1 were targeted whenthe Tat antigen was tagged with ubiquitin. The possibil-ity of a modulated immune recognition can not be ruledout as a result of the presence of an immuno-modulator.To answer this question, we employed a pepscan strategyusing a set of 20 mers overlapping Tat-peptides (Fig. 7A).Splenocytes from BALB/c mice primed with Tat-1 vectorsusing a one-prime–one-boost regime, were analyzed forcytokine production against individual peptides in an IFN-�ELISPOT assay.

The results of the pepscan analysis demonstrated a cleardemarcation between wildtype and synthetic expression vec-tors of Tat, regardless of ubiquitin-tagging. All the three ex-pression vectors of the wildtype Tat (Tatwt, stb-Tatwt, andrp-Tatwt) stimulated broadly uniform, but a low magnitudeimmune response against all the Tat peptides (Fig. 7B).In contrast, all the three codon-optimized Tat expression

vectors (Tatco, stb-Tatco, and rp-Tatco) elicited a strong im-mune response, predominantly reactive with peptides 4 and5, although significant immune responses were also notedagainst peptides 1, 2, and 3. Peptides 4 and 5 span betweenthem, the core region of Tat (aa #37–48). Further charac-terization (molecular nature and fine mapping) of the epi-tope identified within the core-region of the Tat antigen ispresently in progress.

4. Discussion

The viral protein Tat plays a critical role in viral pathogen-esis and infectivity. Tat is known to be secreted from produc-tively infected cells and is believed to govern a wide arrayof pathogenic effects on the host immune system. Inductionof specific cell-mediated and humoral immune response toTat has been shown to be essential for restricting the viralinfection [16,39–42]. However, the potential value of Tatas a vaccine candidate is controversial. Several approacheshave been used to immunize experimental animals with

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Fig. 5. ELISPOT response in BALB/c mice genetically immunized with different Ub-Tat constructs. Mice (four or five animals per group) were immunizedwith 100�g of plasmid DNA according to the schedule described in the line diagram ofFig. 2. The assay was performed on cells directly (A) without, or(B) after in vitro stimulation with Tat-expressing syngenic cells (P815-Tat). Each bar represents a mean of three individual wells± S.D. The experimentwas performed three times; pv, parental vector. An asterisk (*) represents statistical comparison between the wildtype and synthetic Tat constructs ineach format, using Student’st-test. TheP values are shown.

Tat peptides[6], biologically active protein[8,10,43–45],chemically modified toxoid[9,46,47], recombinant viruses[48], genetic vaccines[10,12,19,49], or prime-boost strate-gies employing naked DNA priming followed by protein[5,19] or recombinant vaccinia boosters[13,14]. Mixed re-sults have been obtained in these experiments where cer-tain immunization strategies attenuated the viral infectionand partially or completely protected the vaccinated pri-mates against viral challenge[9,12,43] while others failedto show such a protection[5,13]. A direct comparative eval-uation of these results is difficult, as several variables havebeen incorporated into the experimental design. Importantly,of all the variables, the biological activity of the Tat pro-tein appears to be critical for the nature of the immuneresponses generated. While the use of biologically activeTat protein elicited a broad range humoral and cellular im-mune responses[8,44], use of Tat toxoid, in contrast, gener-ated immune responses against restricted epitopes that werenot cross-reactive across viral subtypes[47]. We, therefore,used biologically functional Tat in our genetic expressionvectors.

Poor immunogenicity could be one of the reasons for thelow efficacy of Tat vaccines in viral challenge experiments.

Attempts have been made to enhance specific immune re-sponse to Tat, especially when delivered as a genetic vaccine,including engineering CpG motifs into the Tat-encodingvector [12], co-delivering cytokine genes[19], and others.We used a different approach of tagging Tat to ubiqui-tin in an effort to elicit potent cellular immune responses.Several studies demonstrated a direct correlation betweenubiquitin-mediated rapid processing of antigens and induc-tion of strong cellular immune responses[25,26,50]. Thepresent study is the first report of subtype-C Tat being usedas a codon-optimized and ubiquitin-tagged genetic vaccine.

We generated several Ub-tagged Tat vectors and eval-uated the intracellular stability of the proteins expressedfrom these vectors. The half-life of the wildtype Tat pro-tein expressed intracellularly was previously reported tobe greater than 6 h[51,52]. However, the duration of thepulse-chase experiments in the above reports did not extendbeyond 6 h. Using two different cell lines, we identifiedthat the half-life of the codon-optimized Tat protein wasapproximately 24 h (Table 1). Importantly, in contrast to theuntagged Tat protein, ubiquitin-tagged Tat proteins demon-strated significantly reduced half-life in both the cell linessuggesting that this Tat format was efficiently targeted for

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Fig. 6. Cross-reactivity of anti-Tat-1 immune responses with Tat-2 expressing cells. C57BL/6 mice were injected with Tat-1 DNA according to theimmunization schedule depicted in the line diagram ofFig. 2. Splenocytes from immunized mice were incubated with Tat-2 expressing stable transfectants(EL-4-Tat-2) after in vitro stimulation and evaluated by using (A) ELISPOT and (B) CTL assay. For the ELISPOT assay 0.5 × 106 splenocytes wereincubated with 0.16× 106 stimulator cells for 24 h. For the51Cr-release assay, primed splenocytes were stimulated with the stimulator cells at a 10:1ratio for 5 days and incubated at differentE:T ratio with 104 labeled EL-4-Tat-2 cells as targets. (C) Comparison of CTL recognition of Tat-1 (EL-4-Tat)and Tat-2 (El-4-Tat-2) targets. Splenocytes used for this comparison were primed by rp-Tatco.

protein processing. Of note, depending on the pattern ofimmune responses observed in mice in this study, it is pos-sible that the differences in the intracellular processing ratesof various Tat constructs in vivo could be more pronouncedthan what has been noted in vitro.

Variable results have been reported on the correlationbetween ubiquitin tagging of the antigens and the rate ofmetabolic processing in vivo. In addition to other proper-ties, the intrinsic nature of the antigen and the cell lineused for testing critically influenced the metabolic stabil-ity of the antigens[50,53,54]. In our study, rp-Tatco proteindemonstrated the lowest half-life in vivo. Since some re-ports previously identified a strong correlation between therate of antigen degradation and elicitation of cellular im-mune response[22,23], we sought to study whether rapidprocessing of the Tat antigen resulted in the elicitation ofstronger cellular immune responses. Indeed, both the wild-type and codon-optimized Tat vectors elicited potent cellu-lar immune responses, in two different mouse strains, whenconjugated to ubiquitin and targeted for rapid processing(Figs. 2–5). Importantly, the rp-antigens in both the wildtype

and codon-optimized format elicited the highest response,which was evident even at the level of the lymphoprolifera-tion assay (Fig. 2).

The previous attempts of ubiquitin tagging of the antigensplaced Arg at the N-terminal, to target them for rapid pro-cessing[25,26,50]. In our study, we used a different aminoacid, Leu, for the same purpose with success, although Leuis placed lower in the order of N-terminal substrates. Thegreat advantage of our strategy is the flexibility with whichan antigen could be expressed as a ubiquitin chimera. A re-striction enzyme mediated cloning is all that is needed toplace a candidate antigen in frame with ubiquitin upstream.

The immune responses elicited by the Ub-Tat vectorswere skewed predominantly towards a Th-1 type indicat-ing the generation of optimal cytokine profiles. Induction ofa preferential Th-1 type cytokine profile was reported pre-viously when several antigens were expressed as ubiquitinconjugates[26,55,56]. As most of these studies measuredonly IFN-� in an ELISPOT or an ELISA, the relative con-tribution of the Th-2 cytokine could not be discerned. Inour study, a Th-1 profile of the cellular immune response

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Fig. 7. Epitope mapping of the T-cell epitopes in mice immunized withUb-Tat vectors. (A) Schematic representation of the HIV-1 Tat proteinand the six peptides used in this study. (B) BALB/c mice were injectedwith 100�g (per animal) of Tat-1 DNA intramuscularly. The mice wereadministered with the same quantity of DNA 2 weeks later and thesplenocytes were harvested and used in the ELISPOT for IFN-� secretion.Splenocytes (0.5×106 per well) were incubated for 36 h with 2�g/ml ofthe individual Tat peptides without in vitro stimulation. The frequency ofthe cells secreting IFN-� in response to the Tat peptides was evaluatedas described inSection 2. The experiments were performed in triplicateand repeated twice for reproducibility. Data are presented as the mean ofthree wells.

might have been fortified by the dual approach of codon op-timization and ubiquitin-tagging of the Tat antigen. Such adual approach elicited protective immune responses in rab-bits against a papilloma viral challenge[55]. Overall, therp-Tatco vector emerged as a promising candidate for furtherevaluation.

We failed to observe humoral immune responses to Tat inour immunizations with any of the Tat-expression vectors.Induction of humoral immune responses to Tat genetic vacci-nation has been reported previously[10]. Unlike in our study,the researchers of this study used bupivacaine as a facilitatorand also administered a large number of boosters. Ubiquitin-tagging of Tat in our study might have retained the translatedproduct in the cytoplasm and targeted it for degradation bythe proteasome pathway. Non-availability of the translatedsoluble Tat protein in circulation could have resulted in theabsence of cross-priming and generation of anti-Tat humoralimmune response. A previous study using ubiquitin-taggedinfluenza nucleoprotein genetic vaccine reported a similarobservation, where ubiquitin conjugation abrogated the gen-eration of antibody against the tagged antigen[25].

Identification of immunodominant domains within Tat isof interest for vaccine development and for the study of hostimmune response. Several T- and B-cell epitopes and a fewCTL epitopes have been identified in Tat-1[10,57–59]. Con-sidering the small size of Tat, it is not surprising that sev-eral of the epitopes overlap with each other. Protein immu-nization studies indicate a narrow epitope recognition withinthe N-terminus of Tat[10]. In contrast, DNA immunizationleads to a broader epitope spreading, with several regions ofTat being co-immunodominant, including the amino termi-nal, cysteine-rich, and the core domains[10,11].

Using the pepscan strategy for IFN-� secreting spleno-cytes, we attempted to identify the immunodominant do-mains in Tat-1 (Fig. 7). In agreement with the previousreports, we observed that multiple regions of Tat weretargeted through the immunizations, including the aminoterminal, cysteine-rich, and the core domain. However, twoof the adjoining peptides (peptides 4 and 5, consisting ofthe aa 31–50 and 41–60, respectively) elicited the strongestimmune response. This observation suggested that the coreregion of the Tat antigen, encompassed between these twopeptides, contained a T-cell epitope, possibly spanning theamino acid residues 40–50. Previous work from our lab-oratory, using several DNA constructs of codon-optimizedand untagged C-Tat, identified a T-helper epitope in thecore region, in BALB/c mice (Lakshmi R. and UdaykumarR., manuscript submitted). In the present study, we iden-tified the same sequence as the immunodominant epitoperegardless of the fusion of Tat to ubiquitin. As expected,ubiquitin-tagging of Tat only enhanced processing of theantigen through the proteasome pathway, without possiblyaltering antigen processing and peptide presentation. As thecore region of Tat is conserved to a large extent across sub-types, using ubiquitin-tagged, codon-optimized Tat antigensis expected to augment desirable immune responses againstmultiple subtypes of HIV.

Acknowledgements

This work was supported by a grant (BT/MED/HIV/05/99)from The Department of BioTechnology, Government ofIndia to U.R. L.R. K.K.A., R.S. and N.B.S. are recipientsof research fellowships of the Council for Scientific and In-dustrial Research of The Government of India. We wish tothank professor Vijaya S. for helpful comments. A numberof reagents used in this study were obtained through theAIDS Research and Reference Reagent Program, Divisionof AIDS, NIAID, NIH, USA and The Centralized Facil-ity for AIDS Reagents, National Institute for BiologicalStandards and Control, UNAIDS.

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