Lentiviral Vectors Encoding HIV-1 Polyepitopes Induce Broad CTL Responses In Vivo

Abstract

Lentiviral vectors have been tested as vaccination vectors in anti-tumoral and anti-viral models. They efficiently transduce dendritic cells and stimulate strong T-cell responses against the encoded antigen. However, their capacity to stimulate a cytotoxic T-lymphocyte (CTL) response against several antigens has not been evaluated. Broad anti-human immunodeficiency virus 1 (HIV-1) T-cell immune responses are important for the control of HIV replication. We evaluated the potential of polyepitope-encoding lentiviral vectors to induce broad anti-HIV CTL responses. We constructed two lentiviral vectors coding for an HLA-A2- or HLA-B7-restricted polyepitope and evaluated their immunogenicity by direct injection of vector particles in HLA-A2 or HLA-B7 transgenic mice. In vitro cytotoxicity assays showed that a single immunization induces a strong, diversified, and long-lasting CTL response in both mouse models. CTL responses were directed against all 13 epitopes in the HLA-A2 system and 8 out of 12 in the HLA-B7 system. A second immunization augmented the number of responding mice in the HLA-A2 system but not in the HLA-B7 system. HLA-B7-immunized mice mounted strong interferon-γ (IFN-γ)-secreting T-cell responses against a majority of the epitopes and lysed peptide-loaded target cells in vivo. CTL responses in HLA-B7 mice were only partially dependent on CD4 T-cell help. This work underlines the potential of lentiviral vectors as candidates for therapeutic vaccination against acquired immunodeficiency syndrome. Lentiviral vectors have been tested as vaccination vectors in anti-tumoral and anti-viral models. They efficiently transduce dendritic cells and stimulate strong T-cell responses against the encoded antigen. However, their capacity to stimulate a cytotoxic T-lymphocyte (CTL) response against several antigens has not been evaluated. Broad anti-human immunodeficiency virus 1 (HIV-1) T-cell immune responses are important for the control of HIV replication. We evaluated the potential of polyepitope-encoding lentiviral vectors to induce broad anti-HIV CTL responses. We constructed two lentiviral vectors coding for an HLA-A2- or HLA-B7-restricted polyepitope and evaluated their immunogenicity by direct injection of vector particles in HLA-A2 or HLA-B7 transgenic mice. In vitro cytotoxicity assays showed that a single immunization induces a strong, diversified, and long-lasting CTL response in both mouse models. CTL responses were directed against all 13 epitopes in the HLA-A2 system and 8 out of 12 in the HLA-B7 system. A second immunization augmented the number of responding mice in the HLA-A2 system but not in the HLA-B7 system. HLA-B7-immunized mice mounted strong interferon-γ (IFN-γ)-secreting T-cell responses against a majority of the epitopes and lysed peptide-loaded target cells in vivo. CTL responses in HLA-B7 mice were only partially dependent on CD4 T-cell help. This work underlines the potential of lentiviral vectors as candidates for therapeutic vaccination against acquired immunodeficiency syndrome. Lentiviral vectors are widely used gene-transfer vectors that achieve high transduction efficiency irrespective of the proliferative status of the target cells. Recently, their capacity to transduce dendritic cells (DCs) efficiently ex vivo1Breckpot K Dullaers M Bonehill A van Meirvenne S Heirman C de Greef C et al.Lentivirally transduced dendritic cells as a tool for cancer immunotherapy.J Gene Med. 2003; 5: 654-667Crossref PubMed Scopus (145) Google Scholar,2Dyall J Latouche JB Schnell S Sadelain M Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes.Blood. 2001; 97: 114-121Crossref PubMed Scopus (127) Google Scholar,3Esslinger C Romero P MacDonald HR Efficient transduction of dendritic cells and induction of a T-cell response by third-generation lentivectors.Hum Gene Ther. 2002; 13: 1091-1100Crossref PubMed Scopus (61) Google Scholar,4Firat H Zennou V Garcia-Pons F Ginhoux F Cochet M Danos O et al.Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy.J Gene Med. 2002; 4: 38-45Crossref PubMed Scopus (61) Google Scholar and in vivo5Esslinger C Chapatte L Finke D Miconnet I Guillaume P Levy F et al.In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8+ T cell responses.J Clin Invest. 2003; 111: 1673-1681Crossref PubMed Scopus (134) Google Scholar,6Palmowski MJ Lopes L Ikeda Y Salio M Cerundolo V Collins MK Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response.J Immunol. 2004; 172: 1582-1587Crossref PubMed Scopus (92) Google Scholar,7VandenDriessche T Thorrez L Naldini L Follenzi A Moons L Berneman Z et al.Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo.Blood. 2002; 100: 813-822Crossref PubMed Scopus (202) Google Scholar has prompted their evaluation as vaccination vectors, with promising results. 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Anti-HIV-1 T-cell immune responses have been shown to play a key role in the control of HIV replication. The onset of HIV-specific CTLs is temporally associated with the viral load reduction in acute infection.17Borrow P Lewicki H Hahn BH Shaw GM Oldstone MB Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection.J Virol. 1994; 68: 6103-6110Crossref PubMed Google Scholar,18Koup RA Safrit JT Cao Y Andrews CA McLeod G Borkowsky W et al.Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.J Virol. 1994; 68: 4650-4655Crossref PubMed Google Scholar In long-term non-progressors, vigorous HIV-specific CD4+ Th1 cells and CTLs are associated with control of viremia.19Candotti D Costagliola D Joberty C Bonduelle O Rouzioux C Autran B et al.Status of long-term asymptomatic HIV-1 infection correlates with viral load but not with virus replication properties and cell tropism. 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An HIV recombinant canarypox viral vaccine induced cellular immune responses and provided a delay in viral rebound when administered in combination with lipopeptides and interleukin-2.25Levy Y Gahery-Segard H Durier C Lascaux AS Goujard C Meiffredy V et al.Immunological and virological efficacy of a therapeutic immunization combined with interleukin-2 in chronically HIV-1 infected patients.AIDS. 2005; 19: 279-286Crossref PubMed Scopus (1) Google Scholar,26Tubiana R Carcelain G Vray M Gourlain K Dalban C Chermak A et al.Therapeutic immunization with a human immunodeficiency virus (HIV) type 1-recombinant canarypox vaccine in chronically HIV-infected patients: the Vacciter Study (ANRS 094).Vaccine. 2005; 23: 4292-4301Crossref PubMed Scopus (45) Google Scholar Immunization with autologous ex vivo antigen-pulsed DCs allowed for delayed viral rebounds and lower viral loads.27Garcia F Lejeune M Climent N Gil C Alcami J Morente V et al.Therapeutic immunization with dendritic cells loaded with heat-inactivated autologous HIV-1 in patients with chronic HIV-1 infection.J Infect Dis. 2005; 191: 1680-1685Crossref PubMed Scopus (137) Google Scholar,28Lu W Arraes LC Ferreira WT Andrieu JM Therapeutic dendritic-cell vaccine for chronic HIV-1 infection.Nat Med. 2004; 10: 1359-1365Crossref PubMed Scopus (371) Google Scholar These findings are encouraging, and the search for more efficient therapeutic vaccination approaches that could grant individuals longer—or ideally definitive—antiretroviral therapy interruption must continue. Candidate vaccines should not only stimulate strong, broad, and long-lasting cellular immune responses but also be easy to produce and administer, thus making them accessible. To assess the potential of lentiviral vectors as candidates for therapeutic vaccination against acquired immunodeficiency syndrome, we have constructed and evaluated two lentiviral vector vaccines, derived from HIV-1, coding for several HIV-1 CD8 epitopes restricted by the two most common HLA class I alleles in the Caucasian population: HLA-A2 and HLA-B7. The lentiviral vector coding for the HLA-A2-restricted polyepitope (hereafter called TRIP/A2-HIV) was evaluated in HHD mice, which are transgenic for human HLA-A*0201 and are double knockout for the H-2Db and mouse β2m genes.29Firat H Garcia-Pons F Tourdot S Pascolo S Scardino A Garcia Z et al.H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies.Eur J Immunol. 1999; 29: 3112-3121Crossref PubMed Scopus (158) Google Scholar,30Firat H Tourdot S Ureta-Vidal A Scardino A Suhrbier A Buseyne F et al.Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class I KO mice.Eur J Immunol. 2001; 31: 3064-3074Crossref PubMed Scopus (39) Google Scholar,31Pascolo S Bervas N Ure JM Smith AG Lemonnier FA Perarnau B HLA-A2.1-restricted education and cytolytic activity of CD8+ T lymphocytes from beta2 microglobulin (beta2m) HLA-A2.1 monochain transgenic H-2Db beta2m double knockout mice.J Exp Med. 1997; 185: 2043-2051Crossref PubMed Scopus (422) Google Scholar HLA-B7 Tg mice, transgenic for human HLA-B*0702 and double knockout for H-2 KbDb,32Rohrlich PS Cardinaud S Firat H Lamari M Briand P Escriou N et al.HLA-B*0702 transgenic, H-2KbDb double-knockout mice: phenotypical and functional characterization in response to influenza virus.Int Immunol. 2003; 15: 765-772Crossref PubMed Scopus (53) Google Scholar were used to evaluate the lentiviral vector coding for the HLA-B7-restricted polyepitope (hereafter called TRIP/B7-HIV). Here we show that a single injection with HLA-A2 or HLA-B7 polyepitope–encoding lentiviral vector particles elicits strong, diverse, and long-lasting CTL responses. The TRIP/A2-HIV codes for 13 HLA-A2-restricted HIV epitopes reported to be immunogenic. Eight of the epitopes have been modified by a tyrosine substitution in the N-terminal residue that enhances their affinity and stabilizing capacity.30Firat H Tourdot S Ureta-Vidal A Scardino A Suhrbier A Buseyne F et al.Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class I KO mice.Eur J Immunol. 2001; 31: 3064-3074Crossref PubMed Scopus (39) Google Scholar The TRIP/B7-HIV encodes 12 HLA-B7-restricted epitopes; 9 have already been described as immunogenic,33Altfeld M Addo MM Eldridge RL Yu XG Thomas S Khatri A et al.Vpr is preferentially targeted by CTL during HIV-1 infection.J Immunol. 2001; 167: 2743-2752Crossref PubMed Scopus (98) Google Scholar,34Jin X Roberts CG Nixon DF Safrit JT Zhang LQ Huang YX et al.Identification of subdominant cytotoxic T lymphocyte epitopes encoded by autologous HIV type 1 sequences, using dendritic cell stimulation and computer-driven algorithm.AIDS Res Hum Retroviruses. 2000; 16: 67-76Crossref PubMed Scopus (30) Google Scholar,35Brander C Goulder PJ The evolving field of HIV CTL epitope mapping: new approaches to the identification of novel epitopes.HIV Molecular Immunology. Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, NM2000Google Scholar and the other 3 (S10EV, R10LL, and I10VI) were obtained with the SYFPEITHI epitope prediction program (http://www.syfpeithi.de36Rammensee H Bachmann J Emmerich NP Bachor OA Stevanovic S SYFPEITHI: database for MHC ligands and peptide motifs.Immunogenetics. 1999; 50: 213-219Crossref PubMed Scopus (1946) Google Scholar) (Figure 1). To assess whether immunization with these HIV polyepitope–encoding lentiviral vectors could stimulate a broad CTL response, adult HHD or HLA-B7 Tg mice were intraperitoneally immunized with a single dose of 108 transduction units (TU) of TRIP/A2-HIV or 2 × 107 TU of TRIP/B7-HIV (the same doses were used throughout the study). Ten days after immunization, the spleens were recovered, splenocytes were restimulated in vitro, and 51Cr-release cytotoxicity assays were performed. Results show that HHD mice injected with TRIP/A2-HIV developed CTL responses against the majority (11 out of 13) of the epitopes encoded by the vector in at least one immunized mouse, using a cutoff value of 10% specific lysis (Table 1, primary response), and 3 of the epitopes (T9V, P10L, and S9L) stimulated strong responses in all immunized mice. For the two “missing” epitopes, A9M and E9V, specific lysis under 10% was detected (data not shown) and further experiments confirmed immunogenicity (Table 1, memory and prime–boost responses). In TRIP/B7-HIV-immunized HLA-B7 Tg mice, primary CTL responses were developed against 6 out of 12 encoded epitopes (Table 2, primary response), with 3 of them (K10LC, H10HI, and S10EV) being strongly immunogenic in the majority of immunized mice.Table 1In vitro cytotoxic response of HHD mice immunized with the TRIP/A2-HIV lentiviral vectorPrimary responsebMice were immunized intraperitoneally (i.p.) with 108 transduction units (TU) of TRIP/A2-HIV vector. Ten (primary) or forty-five days later (memory), splenocytes were restimulated twice in vitro over a 10-day period and tested in a 51Cr-release assay against peptide-pulsed HHD transfected RMA-S cells.Memory responsecMice were immunized i.p. with two doses of 108 TU of TRIP/A2-HIV vector with 45 days of interval. Ten days later, splenocytes were twice restimulated in vitro over a 10- day period and tested in a 51Cr-release assay against peptide pulsed HHD transfected RMA-S cells.Prime-boost responsedSpecific lysis usually at 50:1 ratio. Specific lysis is considered positive if > 10%.PeptidesProtein of orginaNumbering is based on the amino acid sequence of the human immunodeficiency virus 1 (HIV 1) WEAU clone 1.60 (GenBank accession no. U21135). Note that the WEAU sequence may not be identical to that of the reactive peptide and simply indicates its location in the viral proteins.SequenceR/T% specific lysiseR/T = number of responders/total number of immunized mice.R/T% specific lysiseR/T = number of responders/total number of immunized mice.R/T% specific lysiseR/T = number of responders/total number of immunized mice.A9MPol (188–196)ALVEICTEM0/61/3393/339, 28, 5519VPol (464–472)YLKEPVHGV2/630, 542/340, 513/344, 24, 100K9LEnv gp120 (120–128)YLTPLCVSL3/625, 25, 592/394, 872/317, 90T9VGag p24 (19–27)YLNAWVKVV6/616, 76, 45, 70, 73, 74NDNDV9LPol (334–342)YIYQYMDDL2/622,54NDNDP10LNef (134–143)YLTFGWCFKL6/682, 70, 33, 26, 39, 523/380, 70, 433/356, 75, 47V11VPol (263–273)VLDVGDAYFSV4/681, 63, 27, 563/363, 59, 603/3100, 100, 64P9LPol (576–584)YLVKLWYQL1/6132/321, 332/337, 27S9LGag p17 (77–85)SLYNTVATL6/615, 17, 20, 28, 16, 192/326, 482/318, 63E9VGag p24 (212–221)YMMTACQGV0/61/3253/340, 21, 51L10VPol (79–88)LLDTGADDTV1/6142/339, 203/330, 46, 25L9VPol (956–964)LLWKGEGAV1/6183/3100, 100, 483/3100, 57, 63K9L (T)Env gp120 (120–128)YLTPLCVTL2/637342/320, 500/3a Numbering is based on the amino acid sequence of the human immunodeficiency virus 1 (HIV 1) WEAU clone 1.60 (GenBank accession no. U21135). Note that the WEAU sequence may not be identical to that of the reactive peptide and simply indicates its location in the viral proteins.b Mice were immunized intraperitoneally (i.p.) with 108 transduction units (TU) of TRIP/A2-HIV vector. Ten (primary) or forty-five days later (memory), splenocytes were restimulated twice in vitro over a 10-day period and tested in a 51Cr-release assay against peptide-pulsed HHD transfected RMA-S cells.c Mice were immunized i.p. with two doses of 108 TU of TRIP/A2-HIV vector with 45 days of interval. Ten days later, splenocytes were twice restimulated in vitro over a 10- day period and tested in a 51Cr-release assay against peptide pulsed HHD transfected RMA-S cells.d Specific lysis usually at 50:1 ratio. Specific lysis is considered positive if > 10%.e R/T = number of responders/total number of immunized mice. Open table in a new tab Table 2In vitro cytotoxic response of HLA-B7 mice immunized with the TRIP/B7-HIV lentiviral vectorPrimary responsebMice were immunized intraperitoneally (i.p.) with 108 transduction units (TU) of TRIP/B7-HIV vector. Ten (primary) or forty-five days later (memory), splenocytes were restimulated once in vitro for 5 days and tested in a 51Cr-release assay against peptide-pulsed HLA-B7 transfected RMA cells.Memory responsebMice were immunized intraperitoneally (i.p.) with 108 transduction units (TU) of TRIP/B7-HIV vector. Ten (primary) or forty-five days later (memory), splenocytes were restimulated once in vitro for 5 days and tested in a 51Cr-release assay against peptide-pulsed HLA-B7 transfected RMA cells.Prime-boost responsecMice were immunized i.p. with two doses of 108 TU of TRIP/B7-HIV vector with 45 days of interval. Ten days later, splenocytes were restimulated in vitro for 5 days and tested in a 51Cr-release assay against peptide pulsed HLA-B7 transfected RMA cells.PeptidesProtein of originaNumbering is based on the amino acid sequence of the human immunodeficiency virus (HIV 1) WEAU clone 1.60 (GenBank accession no. U21135). Note that the WEAU sequence may not be identical to that of the reactive peptide and simply indicates its location in the viral proteins.SequenceR/T% specific lysiscSpecific lysis usually at 20:1 ratio. Specific lysis is considered positive if > 10% after one in vitro restimualtion. R/T = number of responders/total number of immunized mice.R/T% specific lysiscSpecific lysis usually at 20:1 ratio. Specific lysis is considered positive if > 10% after one in vitro restimualtion. R/T = number of responders/total number of immunized mice.R/T% specific lysiscSpecific lysis usually at 20:1 ratio. Specific lysis is considered positive if > 10% after one in vitro restimualtion. R/T = number of responders/total number of immunized mice.S9WVGagSPRTLNAWV0/60/30/3K10LCEnvKPCVKLTPLC6/630, 49, 34, 41, 43, 273/315, 51, 471/322F10LRNefFPVRPQVPLR1/6103/314, 52, 340/3T10PLNefTPGPGIRYPL0/62/339, 440/3I9GLEnvIPRRIRQGL0/61/3250/3H10HIVifHPRISSEVHI6/650, 56, 54, 52, 46, 533/359, 61, 593/351, 42, 53S10EVVifSPHPRISSEV6/621, 53, 26, 45, 36, 393/342, 26, 400/3R10SIEnvRPNNNTRKSI0/61/3340/3R10LLEnvRPVVSTQLLL2/614, 141/3351/338I10VIVifIPLGDARLVI0/60/30/3R9ALNefRPMTYKAAL0/60/30/3F9GLNefFPRIWLHGL3/610, 18, 180/30/3a Numbering is based on the amino acid sequence of the human immunodeficiency virus (HIV 1) WEAU clone 1.60 (GenBank accession no. U21135). Note that the WEAU sequence may not be identical to that of the reactive peptide and simply indicates its location in the viral proteins.b Mice were immunized intraperitoneally (i.p.) with 108 transduction units (TU) of TRIP/B7-HIV vector. Ten (primary) or forty-five days later (memory), splenocytes were restimulated once in vitro for 5 days and tested in a 51Cr-release assay against peptide-pulsed HLA-B7 transfected RMA cells.c Mice were immunized i.p. with two doses of 108 TU of TRIP/B7-HIV vector with 45 days of interval. Ten days later, splenocytes were restimulated in vitro for 5 days and tested in a 51Cr-release assay against peptide pulsed HLA-B7 transfected RMA cells.d Specific lysis usually at 20:1 ratio. Specific lysis is considered positive if > 10% after one in vitro restimualtion. R/T = number of responders/total number of immunized mice. Open table in a new tab Thus, in vivo immunization with the HIV polyepitope–encoding lentiviral vectors induces a diversified primary CTL response in transgenic mice. We next determined whether a single immunization with these lentiviral vectors was sufficient to stimulate long-lasting CTL responses in transgenic mice. In vitro cytotoxicity tests showed that 45 days after the single immunization, in HHD as well as in HLA-B7 transgenic mice, CTL responses against the majority of the epitopes coded by the vectors could still be found (Tables 1 and 2, long-term response). In HHD mice the long-term response was as diversified as the primary response, and in HLA-B7 mice it was slightly more diversified than the primary response, with 8 out of 12 epitopes generating a response in at least one mouse, compared with 6 out of 12 in the primary response. The differences seen in both mouse models between the epitopes recognized in the primary and the long-term response could be due to individual mouse variations in T-cell repertoire mobilization. However, the most immunogenic epitopes from each polyepitope (S9L, V11V, and P10L for HLA-A2-restricted and K10LC, H10HI, and S10EV for HLA-B7-restricted polyepitope) still stimulated strong responses in the majority of immunized mice. The lentiviral vectors coding for polyepitopes are capable of stimulating a long-lasting CTL response that 45 days after a single immunization is as broad as it was at the peak of the primary response. We wanted to know whether we could improve these CTL responses by performing a homologous prime–boost using the polyepitope-encoding lentiviral vectors. Mice received two lentiviral vector immunizations 45 days apart. Ten days after the second immunization, splenocytes were stimulated in vitro to be used in 51Cr-release cytotoxicity assays. In HHD mice we found that the second immunization generally increased the number of responding mice (Table 1, prime–boost response). Responses to epitopes such as I9V and L10V were found in one or two of three singly immunized mice, and in 100% of doubly immunized ones. However, in HLA-B7 Tg mice, the second immunization seemed to focus the CTL response against the same immunogenic epitopes (Table 2, prime–boost response). The responses in HLA-B7 mice were further confirmed by enzyme-linked immunosorbent spot (Elispot) assays (data not shown). Having established that our polyepitope-encoding lentiviral vectors efficiently stimulate a diversified CTL repertoire, we set out to determine the strength of the immune response that can be triggered by these vectors using an ex vivo Elispot assay to quantify the number of interferon-γ (IFN-γ)-secreting T cells. Ten days after a single immunization, splenocytes from HHD or HLA-B7 Tg mice were plated and stimulated for 40 hours with individual peptides in an IFN-γ Elispot assay. In HLA-B7 Tg mice we found IFN-γ-secreting responses to more or