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National AIDS Center, Department of Drugs and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy
Correspondence
Andrea Cara
acara{at}iss.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Relph et al., 2004). In this context, if compared with the administration of protein antigens or plasmid DNA, a transducing vector can be more effective in eliciting humoral and cellular responses towards a transgene product (Liu et al., 2004; Tatsis & Ertl, 2004; Truckenmiller & Norbury, 2004).
In the case of simian immunodeficiency virus (SIV), chimeric simian/human immunodeficiency virus and human immunodeficiency virus 1 (HIV-1) infections, several viral vectors have been shown to induce strong immune responses towards viral antigens after immunization (Voltan & Robert-Guroff, 2003; Im & Hanke, 2004; Tatsis & Ertl, 2004). In addition, humoral and cellular immune responses towards the antigen have been shown to play an essential role in controlling viral infection and replication (Ogg et al., 1998; Goulder et al., 1999; Schmitz et al., 1999; Barouch et al., 2000; Kaul et al., 2001). Therefore, appropriate induction of both the humoral and cellular responses may have a significant impact on disease outcome and both should be elicited after immunization.
Among viral vectors, retroviral vectors represent a useful tool for gene-delivery purposes in that they are capable of efficient transduction into and intracellular expression of foreign genes in mammalian cells (Chang & He, 2001; Barquinero et al., 2004). Therefore, they provide a useful means for the introduction of potential immunogens into the endogenous antigen-presentation pathway. In particular, Moloney murine leukemia virus (Mo-MLV)-based vectors have already been used to deliver HIV-1 antigens and they proved efficacious in eliciting both humoral and cellular immune responses in animal models (Warner et al., 1991; Irwin et al., 1994; Laube et al., 1994; Kamantigue et al., 1996). Among retroviruses, lentiviral vectors, based on either primate or non-primate lentiviruses, have gained much interest because of their versatility. Lentiviral vectors are easy to engineer in order to infect a wide variety of cell types regardless of the replication status of the cell, both in vitro and in vivo, whereas Mo-MLV-based vectors have been shown to infect only actively replicating cells. This is of great importance as terminally differentiated dendritic cells (DCs), one of the major players for immune-response induction (Pope, 2003), have been shown to be transduced by lentiviral vectors and to elicit immune responses against the antigen (Koya et al., 2003; Palmowski et al., 2004; Rohrlich et al., 2004).
Concerning safety issues, the use of Mo-MLV-based vectors has recently been debated, as they have been seriously implicated in the development of a leukaemia-like condition in children receiving a retroviral vector as treatment for X-linked severe combined immunodeficiency disease (X-SCID) (Hacein-Bey-Abina et al., 2003; Couzin & Kaiser, 2005). With regard to lentiviral vectors, however, no cases of adverse effects have yet been reported (Manilla et al., 2005; http://www.wiley.co.uk/genmed/clinical/). Nevertheless, due to all the concerns inherent in their use, during the past several years, improved vector design and optimization have contributed to the generation of novel vectors almost completely devoid of viral wild-type sequences, packaging vectors with reduced homology with the parental virus, integration-defective vectors and novel envelopes for expanded tropism (Iwakuma et al., 1999; Schnell et al., 2000; Firat et al., 2002; Zaiss et al., 2002; Cockrell & Kafri, 2003; Vargas et al., 2004). Therefore, they represent a tool to be exploited to deliver foreign genes in vivo for immunization purposes.
In order to evaluate the efficacy of these vectors for the induction of immune responses against HIV-1 antigens, we tested two self-inactivating lentiviral vectors based on HIV-1, carrying the full-length HIV-1HXB2 Rev/Env or the codon-optimized HIV-1JR-FL gp120 coding sequence, in the BALB/c mouse model. Here, we report that a single intramuscular administration of these vectors separately is able to elicit a strong cell-mediated immune response, evaluated as both gamma interferon (IFN-
) production and cytotoxic activity. Conversely, specific anti-gp120 antibody production was observed only in the mice immunized with the codon-optimized HIV-1JR-FL gp120.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Vector construction.
Plasmid pTY-EFeGFP was obtained from NIH Repository Reagents (Bethesda, MD, USA) (Zolotukhin et al., 1996; Chang et al., 1999; Cui et al., 1999; Iwakuma et al., 1999). This plasmid was digested with ClaI/KpnI to eliminate the portion including the elongation factor 1
promoter and the green fluorescent protein (GFP) coding sequence and replaced with the cytomegalovirus (CMV) promoter, thus creating the pTY-CMVempty vector. A BssHII–ClaI fragment from the pHR central polypurine tract plasmid, containing the HIV-1 central polypurine tract and a shorter fragment of gag sequence including the packaging signal (
), was inserted in the corresponding sites of pTY-CMVempty, generating the pTY2-CMVempty vector. For construction of the pTY2-IIIBEnv vector, the full-length HXB2 Env/Rev coding sequence was obtained from the pSVIIIBEnv plasmid (kindly provided by Dr J. Sodroski, Dana-Farber Cancer Institute, Boston, MA, USA) after digestion with SalI/EcoRI and gel purification of the corresponding 3.0 kb band. A DNA fragment of 224 bp, containing the cytoplasmic transport element (CTE) sequence from the Mason–Pfizer virus, was obtained from the plasmid pBKCMV
Gag-CTE (kindly provided by Dr S. Indraccolo, University of Padua, Italy) after digestion with EcoRI/KpnI. Purified DNA fragments were inserted into the SalI/KpnI-digested pTY2-CMVempty vector. The pTY2-IIIBEnv-expressing cassette contains the rev gene and the CTE sequence, in addition to the HIV-1HXB2 env gene.
The codon-optimized gp120JR-FL coding sequence was obtained from the pSyngp120JR-FL plasmid (NIH Repository Reagents) (Haas et al., 1996; André et al., 1998). For construction of the lentiviral vector expressing gp120JR-FL, pSyngp120JR-FL plasmid was digested with SalI/KpnI and, after gel purification, the corresponding 1.6 kb DNA fragment was inserted into the pTY2-CMVempty vector digested with SalI/KpnI, generating the pTY2-JREnv lentiviral vector. For the construction of the retroviral vector expressing gp120JR-FL, pSyngp120JR-FL plasmid was digested with XhoI/BamHI and the corresponding 1.6 kb DNA fragment was inserted into the pFB-Neo vector (Stratagene).
Plasmid pCMVR8.2 (Naldini et al., 1996), obtained from Dr I. Verma (Salk Institute, La Jolla, CA, USA), produces all HIV-1 viral proteins with the exception of Env. Plasmid pMD.G (Naldini et al., 1996), obtained from Dr D. Trono (University of Lausanne, Switzerland), produces the vesicular stomatitis virus (vsV) envelope glycoprotein G.
Production of recombinant vectors.
The human epithelium kidney 293 cell line was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Invitrogen) supplemented with 10 % fetal calf serum (Euroclone) and 100 units penicillin/streptomycin/glutamine ml–1 (Gibco Invitrogen). For production of recombinant lentiviral vectors, cells were transiently transfected using the calcium phosphate-based Profection Mammalian Transfection system (Promega) as described by Zufferey et al. (1998). Briefly, the day before transfection, cells were plated at a density of 3x106 cells in a 100 mm diameter Petri dish at 80 % confluence. A total of 28 µg plasmid DNA was used for each plate in the ratio 3 : 2 : 0.7 (transfer vector : packaging vector : vsV envelope glycoprotein G vector) for the preparation of the TY2-IIIBEnv and TY2-JREnv vectors. Concerning the preparation of the control TY2-CMVempty vector, a fourth plasmid, pSVIIIBEnv, encoding the HIV-1 envelope, was added to the system in a ratio of 3 : 2 : 0.7 : 0.7 (transfer vector : packaging vector : vsV envelope vector : IIIBEnv vector) in order to produce control viral particles carrying on their surface both vsV-G and gp120/gp41 proteins. After 48 h, culture supernatants were cleared from cellular debris and passed through a 0.45 µm pore-size filter (Millipore). Filtered supernatants were concentrated by ultracentrifugation (Beckman Coulter) for 2 h at 27 000 r.p.m. on a 20 % sucrose gradient (Sigma). Finally, the viral pellets were resuspended in 1x PBS and stored at –80 °C for further analyses.
Viral titres were normalized by the reverse transcriptase (RT) (Weiss et al., 1982) and p24 ELISA (Innotest; Innogenetics) assays. Expression of each envelope was verified by either Western blot (data not shown) or ELISA, using the HIV-1 gp120 antigen-capture assay (Advanced BioScience Laboratories).
For the production of the recombinant retroviral vectors FB-Neo and FB-JREnv, the Phoenix cell line was used (Grignani et al., 1998). Cells were maintained in DMEM (Gibco Invitrogen) supplemented with 10 % fetal calf serum (Euroclone) and 100 units penicillin/streptomycin/glutamine ml–1 (Gibco Invitrogen). Either the pFB-Neo or the pFB-JREnv plasmid was transfected into the Phoenix cells along with the pMD.G plasmid with the calcium phosphate-based Profection Mammalian Transfection system (Promega) as described by Grignani et al. (1998). The retroviral vector-containing supernatants were then collected after 48 h and analysed for RT activity by using standard methodologies (Weiss et al., 1982).
In order to produce stimulator/target cells expressing the HIV-1JR-FL gp120 protein, the H2d-restricted BALB/c-derived fibrosarcoma WEHI 164 cell line (ATCC CRL 1751) was infected for 2 h at 37 °C with either the FB-Neo- or FB-JREnv-containing supernatants. After infection, cells were washed and cultured in RPMI 1640 (Gibco Invitrogen) containing 100 units penicillin/streptomycin/glutamine ml–1 (Gibco Invitrogen), 1 mM sodium pyruvate (Gibco Invitrogen) and 10 % fetal calf serum (Euroclone). After 48 h culture, transduced cells were selected by using 100 µg Geneticin ml–1 (Gibco Invitrogen) to obtain the stably transduced WEHI FB-Neo and WEHI FB-JREnv cell lines. Stable expression of HIV-1JR-FL gp120 was verified by Western blot (data not shown).
Mouse immunization.
Mice from all groups were injected intramuscularly (right thigh) with 0.2 ml viral preparation containing 1x107 RT units of each vector formulated in 1x PBS. For immunization with the TY2-IIIBEnv and TY2-CMVempty vectors, nine mice were injected for each group in order to perform analyses at days 15, 30 and 90 after injection. For immunization with the TY2-JREnv vector, three mice were immunized and analyses were performed at day 30 after injection. Naïve, non-immunized mice were kept for parallel analysis.
On days 15, 30 and 90, depending on the experimental analyses, mice were bled orbitally under metaphane-induced anaesthesia and sera were collected and kept at –80 °C for further analyses. After mouse euthanasia, the muscle-injection site was removed for DNA analysis and spleens were taken by using sterile forceps and tweezers and transferred onto a dish containing RPMI 1640 medium (Gibco Invitrogen) supplemented with 10 % fetal calf serum (Euroclone), 100 units penicillin/streptomycin/glutamine ml–1 (Gibco Invitrogen) and 1x10–5 M 2-mercaptoethanol (Sigma). Spleens were homogenized gently by using a Cytomation homogenizer (DakoCytomation) to obtain a single-cell suspension. For analyses, splenocytes were maintained in the cell-culture medium described above; alternatively, cells were stored in liquid nitrogen for further analyses.
In vitro stimulation of effector cells.
Splenocytes from immunized mice were cultured in 24-well plates at a cell density of 2x106 cells ml–1 in the presence of syngeneic cells as stimulators. In particular, splenocytes from naïve BALB/c mice were used. Cells were pulsed for 1 h with the appropriate peptides at a concentration of 5 µg ml–1 in serum-free medium, irradiated with 3000 rad (30 Gy) from a 60Co source, washed once and then added to splenocytes at an effector : stimulator ratio of 10 : 1. After 1 week culture, effector cells were tested by IFN- ELISPOT or 51Cr-release assays for the analysis of antigen-specific T cells.
For the studies with the TY2-JREnv vector, the WEHI FB-JREnv cells were used as both stimulators and target cells in the cytotoxic T-lymphocyte (CTL) assay as described previously (Negri et al., 2004a).
IFN- ELISPOT assay.
The IFN- ELISPOT assay was performed by using reagents from Mabtech. Splenocytes, ex vivo or 1-week-cultured cells, were resuspended in 100 µl to a concentration of 2x106 or 0.5x105 cells ml–1, respectively, and seeded in a microtitre plate (MultiScreen-IP plate; Millipore) coated with a mAb against mouse IFN- AN18. In the case of TY2-IIIBEnv, two different peptides, a 10mer (RGPGRAFVTI) and a 15mer (RIQRGPGRAFVTIGK), both containing an HIV-1HXB2 H2d-restricted epitope described by Alexander-Miller et al. (1996), were added separately to the cells to a final concentration of 5 µg ml–1. In the case of TY2-JREnv, a 15mer (SIHIGPGRAFYTTGE) containing the JR-FL V3-loop epitope was added to the splenocytes to a final concentration of 5 µg ml–1. After overnight incubation at 37 °C, cells were removed and a biotinylated detector antibody (R4-6A2) was added to the wells, followed by the addition of streptavidin–alkaline phosphatase and then of a chromogenic substrate (BCIP/NBT; Sigma). After development, spot-forming cells (SFCs) were analysed by an ELISPOT reader (AID; Amplimedical Bioline) and expressed as SCFs per 106 cells. Medium alone or an unrelated peptide pool (HIV-Tat 15mers, each overlapping by 10 aa) (UFPeptides s.r.l.) were used as negative controls, whilst concanavalin A (Sigma) (5 µg ml–1) was used as a positive control. Samples were scored positive when present at threefold or higher than the medium alone, with a minimum of 50 spots per 106 cells.
51Cr-release assay.
CTL cultures were prepared from splenocytes of mice sacrificed at the different time points. After 1 week stimulation as described above, cytolytic activity was measured with a standard 51Cr-release assay as described by Negri et al. (2004a). In brief, target WEHI 164 cells were incubated with Na251CrO4 for 2 h at 37 °C. In the case of the TY2-IIIBEnv experiment, after labelling, a fraction of cells were pulsed with the appropriate 10mer and 15mer peptides for 1 h at 37 °C and then washed once. In the case of the TY2-JREnv experiment, both the WEHI 164 cells transduced with FB-JREnv and those transduced with FB-Neo were used. Subsequently, 1x104 target cells were added to the effectors at different effector : target (E : T) ratios and incubated for 4 h at 37 °C in 96-well plates. Finally, 40 µl supernatant per well was harvested on a LumaPlate96 (Perkin-Elmer) and emitted radioactivity was counted by using a MicroBeta counter (Wallac Oy). Spontaneous release and total release were evaluated from those wells containing target cells with either medium or 0.1 % Triton X-100 (Sigma), respectively. The percentage of specific lysis was calculated as [(test release–spontaneous release)/(total release–spontaneous release)] x100. Spontaneous release of target cells was <10 % in all assays. The intra-assay variability was determined on duplicate wells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The full-length HIV-1HXB2 Rev/Env and the codon-optimized HIV-1JR-FL Env gene sequences were cloned into either the self-inactivating pTY2-CMVempty vector or the pFB-Neo retroviral vector as described in Methods. Expression of each envelope sequence from the vectors was verified by Western blot analysis in human epithelium kidney 293, murine WEHI 164 and HIV-1JR-FL Env-stably transduced WEHI 164 cell lines (data not shown). Expression of each envelope was also evaluated by gp120 ELISA after infection of murine cells with normalized amounts (1x105 RT counts in 3x105 cells) of TY2-IIIBEnv and TY2-JREnv recombinant viruses. Results indicated that a higher amount of envelope protein was produced after transduction with the codon-optimized HIV-1JR-FL envelope-expressing vector (88.8 ng per 106 cells) than with the HIV-1HXB2 envelope-expressing vector (3.8 ng per 106 cells), as expected (Haas et al., 1996; André et al., 1998).
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production (ELISPOT) and 51Cr-release (CTL) assays, and humoral responses, measured by anti-gp120 antibody production, were evaluated.
In the TY2-IIIBEnv-immunized mice, IFN- production was analysed at three different time points, i.e. 15, 30 and 90 days after inoculation, whilst 51Cr release was evaluated at 30 days after inoculation, by using two peptides, a 10mer and a 15mer, both containing an HIV-1HXB2 H2d-restricted epitope (Alexander-Miller et al., 1996), as described in Methods. In the TY2-JREnv-immunized mice, IFN- production and 51Cr release were evaluated at 30 days after inoculation by using a 15mer peptide and the WEHI FB-JREnv cell line stably expressing the HIV-1JR-FL gp120 envelope protein.
Persistence of lentiviral-vector sequences in immunized mice
All mice were injected intramuscularly once at week 0 and sacrificed by euthanasia at 15, 30 or 90 days after vector administration. Following mouse euthanasia, the presence of the vector at the injection site was evaluated in all muscle samples by PCR amplification of the long terminal repeat (LTR) sequences. A validated PCR assay for detection of viral DNA sequences was used (Zack et al., 1990). The results following intramuscular lentiviral-vector injection indicated that, at all time points analysed, i.e. 15, 30 and 90 days, all of the genomic DNA samples were positive for LTR amplification using the AA55/M667 primer pair (data not shown).
A single immunization with TY2-IIIBEnv induces durable and specific T-cell responses
At day 15 after inoculation, an Env-specific response was present in the fresh splenocytes stimulated with either peptide, as measured by IFN- ELISPOT assay on cells derived from the mice immunized with TY2-IIIBEnv. As shown in Fig. 2(a), 222 SFCs per 106 cells were found in the 10mer- and 15mer-stimulated splenocytes, corresponding to a 4.4-fold increase over the medium in both the 10mer- and 15mer-stimulated splenocytes. Conversely, no specific responses were detected in the splenocytes derived from the TY2-CMVempty-immunized and the naïve mice pulsed with the same peptides (Fig. 2b). Under all conditions tested, the use of an unrelated peptide pool did not reveal any response, indicating that the immune response recovered in the immunized mice was specific (Fig. 2a, b). Splenocytes were then cultured for 1 week in the presence of naïve splenocytes pulsed with the two peptides separately. As shown in Fig. 2(a), the specific-responder population was further expanded in the cell culture from the TY2-IIIBEnv-immunized mice, producing 520 SFCs per 106 cells with a 6.9-fold increase over the medium in the 10mer-stimulated splenocytes and 500 SFCs per 106 cells with a 4.5-fold increase over the medium in the 15mer-stimulated splenocytes. These data confirm the specificity of the immune response detected on fresh splenocytes. Conversely, no specific induction was observed in the splenocytes derived from the mice immunized with the TY2-CMVempty vector or from the naïve controls (Fig. 2b).
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ELISPOT assay performed on fresh cells resulted in a mean peptide-specific IFN- production of 132 SFCs per 106 cells and 102 SFCs per 106 cells for the 10mer- and 15mer-stimulated splenocytes, respectively, with 13.2- and 10.2-fold increases with respect to the medium (Fig. 2a
). Splenocytes derived from the TY2-CMVempty-immunized mice and the naïve mice, both pulsed with the same peptides, did not show any specific response (Fig. 2b).
After 1 week stimulation with irradiated naïve splenocytes pulsed separately with the two peptides, expansion of specific cells was observed only in the TY2-IIIBEnv-immunized mice, producing 540 and 900 SFCs per 106 cells for the 10mer and the 15mer, respectively, corresponding to 13.5- and 5.1-fold increases over the medium, respectively. Conversely, in the case of TY2-CMVempty, there was a non-specific cell expansion (Fig. 2a). Culturing cells for another week led to a further expansion of the specific response only in the cells from the TY2-IIIBEnv-immunized mice (data not shown).
Analysis of IFN- production in response to specific stimuli does not provide evidence for the cytotoxic activity of antigen-specific CD8+ cells. Therefore, the presence of specific CTLs was also investigated in all groups of mice at day 30 after immunization.
In the TY2-IIIBEnv-immunized mice, CTL activity was shown to be present by using both the 10mer- and the 15mer-pulsed target cells. In particular, specific lysis of target cells was high, at an E : T ratio of 25 : 1 both in the 10mer (30 %)- and in the 15mer (24 %)-pulsed cells. Conversely, specific lysis was low (10 %) in the cell cultures derived from the TY2-CMVempty mice and absent in the cells from the naïve controls at the same E : T ratio (Fig. 3).
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ELISPOT assay performed on fresh splenocytes did not show a positive result (Fig. 2a). However, after 1 week stimulation with irradiated naïve splenocytes pulsed separately with the two peptides, expansion of specific cells was observed, producing 890 and 280 SFCs per 106 cells for the 10mer and the 15mer, respectively, corresponding to 4.2- and 4.0-fold increases over the medium for each peptide, respectively (Fig. 2a). Again, in the case of TY2-CMVempty and naïve splenocytes, we did not observe any specific cell expansion (Fig. 2b). Concerning the humoral responses, analyses of sera of all mice injected with the TY2-IIIBEnv vector did not reveal the presence of anti-gp120 IgG antibodies (data not shown) at any of the analysed time points. Furthermore, in the naïve and in the control mice inoculated with the TY2-CMVempty vector, antibodies against gp120 were not detected, as expected.
A single injection with the codon-optimized TY2-JREnv induces both cellular and humoral responses
Concerning the TY2-JREnv-immunized mice, ELISPOT and CTL analyses were performed at 30 days after injection. As shown in Fig. 4(a), ex vivo peptide-specific IFN--producing cells (1472.5 SFCs per 106 cells) showed a 17.8-fold increase over the medium. Conversely, no specific responses were detected in the splenocytes derived from the TY2-CMVempty-immunized and the naïve mice pulsed with the same peptide (Fig. 4a). In addition, the use of an unrelated peptide pool did not reveal any response, indicating that the immune response recovered in the immunized mice was specific (Fig. 4a). For CTL analysis, splenocytes were stimulated for 1 week with the WEHI FB-JREnv cell line, stably producing the HIV-1JR-FL gp120 envelope protein, as described in Methods. The day of the assay, stimulated splenocytes were incubated with either the WEHI FB-JREnv or the WEHI FB-Neo cell line. Results indicated that specific lysis was observed in the presence of the WEHI FB-JREnv at two different E : T ratios (Fig. 4b), confirming the data obtained for the TY2-IIIBEnv-immunized mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Letvin, 2002; McMichael & Hanke, 2002; Heeney, 2004). In this context, many strategies are currently under investigation, both in animal models and in clinical trials (Berzofsky et al., 2004; Franchini et al., 2004; Im & Hanke, 2004; Vanniasinkam & Ertl, 2005). The most promising studies involve either repeated or prolonged stimulation with viral antigens delivered by plasmid DNAs and viral vectors, which have been shown to induce both humoral and cellular responses to the encoded immunogens, particularly when used in different combination approaches (Barouch et al., 2000; Amara et al., 2001; Shiver et al., 2002; Wang et al., 2003; Gómez et al., 2004; Negri et al., 2004b; Someya et al., 2004). However, the degree of vaccine efficacy remains poor when compared with the full protection mediated by the live-attenuated virus vaccine approach (reviewed by Whitney & Ruprecht, 2004). In this regard, we evaluated the use of replication-incompetent lentiviral vectors for the delivery of viral antigens in mice for genetic-immunization purposes. Our reasoning stemmed from prior work performed with attenuated strains of SIV in which chronic presentation of low levels of viral antigens in the setting of a viral infection provided protection, leading to several important observations concerning vaccine efficacy in the primate model (Heeney et al., 1994; Almond et al., 1995; Cranage et al., 1997; Titti et al., 1997; Johnson & Desrosiers, 1998; Letvin, 1998; Sernicola et al., 1999; Kumar & Narayan, 2001). However, the subsequent demonstration that the majority of neonates and some adult monkeys develop AIDS as a result of infection with attenuated SIV renders this strategy unacceptable for use in humans (Norley et al., 1996; Baba et al., 1999).
Recently, several groups have presented promising data showing that lentiviral vectors are very efficient for tumour-vaccine strategies. In particular, several studies have shown that DCs can be transduced efficiently with HIV-based lentiviral vectors (Chinnasamy et al., 2005; Kung et al., 2005) and that DCs transduced ex vivo can be injected into mice in order to deliver antigenic peptides or proteins to generate specific anti-tumour immune responses (He et al., 2005). In addition, lentiviral vectors have been used for direct injection in mice for in vivo cancer-vaccine studies (Esslinger et al., 2003; Palmowski et al., 2004; Morizono et al., 2005). In both cases, they proved able to induce a potent anti-tumour immunity in the mouse model, suggesting that the exploitation of lentiviral vectors for the induction of immune responses against the transgene can be applied not only to cancer, but also to infectious diseases.
In the present study, we evaluated the cellular and humoral immune responses elicited by the administration of two lentiviral vectors expressing, respectively, the full-length HIV-1IIIB and the codon-optimized HIV-1JR-FL envelope proteins. We hypothesized that the lentiviral vector-transduced cells may be an effective means for the induction of cellular responses against the introduced antigen and, considering the capability of these vectors to integrate into the host genome, the immunization schedule consisted of a single administration of the recombinant particles. After evaluating the presence of the lentiviral genome in the muscle-injection site, our study focused on the evaluation and persistence of the immune responses induced by the vector up to 90 days after injection.
Overall, our study establishes that both lentiviral vectors TY2-IIIBEnv and TY2-JREnv are able to elicit specific and durable cellular immune responses efficiently after a single intramuscular administration of either vector, as measured by IFN- ELISPOT and chromium-release assays, up to 30 days after injection. In addition, the presence of antigen-specific memory T cells was also detected 90 days after injection in the TY2-IIIBEnv-immunized mice. However, concerning the humoral response, only the sera derived from the TY2-JREnv-immunized mice were reactive for anti-gp120 IgG antibodies, whereas the mice immunized with the TY2-IIIBEnv vector and the control mice were negative at all time points analysed. This suggests that codon optimization and/or Rev independence may be important features in order to increase the efficiency of gene expression and thus the induction of specific humoral responses. This was expected, as codon usage has been considered as one of the critical factors limiting the rate of protein expression from heterologous genes and, therefore, codon optimization might contribute to higher expression levels, as has been described in other systems (Crameri et al., 1996; Kim et al., 1997; Nagata et al., 1999; Ramakrishna et al., 2004). In particular, in protocols using DNA vaccination in mice, an optimized gp120 coding sequence resulted in a significant increase in antibody titres compared with mice injected with a plasmid DNA containing a wild-type gp120 (André et al., 1998; Gao et al., 2003; Young et al., 2004). These results, together with ours, further recommend codon optimization as a useful means to improve the ability of the transducing vector to induce broader immune responses. In this context, we recently completed an immunization protocol in mice by using a lentiviral vector expressing a codon-optimized SIV-Gag and found results comparable to those described in this report (data not shown).
Besides all of the ethical concerns involved in the use of an HIV-based integrating vector (Connolly, 2002; Romano et al., 2003; Anson, 2004; Lever et al., 2004), the immunization protocol described here provides the first evidence of the immunizing efficacy of a single administration of a lentiviral vector expressing an HIV-1 antigen. In this context, the integration of the transducing vector may represent an advantage, as it would allow prolonged expression and increased immune responses against the delivered antigen. In this regard, Johnson et al. (2005) have recently shown that a single administration of vectors based on recombinant adeno-associated virus expressing several SIV genes separately was able to contain virus replication after challenge.
In addition, this approach could be improved further by the addition of an adjuvant, such as a cytokine, in the transducing vector (reviewed by Barouch et al., 2004) or by increasing its efficacy in a prime–boost regimen, for example by immunizing first with DNA (prime) and then with the lentiviral vector expressing the antigen of interest (boost), as has been shown in other studies (Pinto et al., 2003; Jaffray et al., 2004; Leung et al., 2004; Mäkitalo et al., 2004; Woodland, 2004).
Finally, in order to assess the efficacy of these vectors in the context of a disease, it will be important to evaluate the protection conferred by the introduced viral antigen by using either the vaccine-challenge model after vaccination or protection against a murine tumour cell line stably expressing the env gene.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Almond, N., Kent, K., Cranage, M., Rud, E., Clarke, B. & Stott, E. J. (1995). Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 345, 1342–1344.[CrossRef][Medline]
Amara, R. R., Villinger, F., Altman, J. D. & 19 other authors (2001). Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292, 69–74.[CrossRef][Medline]
André, S., Seed, B., Eberle, J., Schraut, W., Bültmann, A. & Haas, J. (1998). Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol 72, 1497–1503.
Anson, D. S. (2004). The use of retroviral vectors for gene therapy – what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genet Vaccines Ther 2, 9.[CrossRef][Medline]
Baba, T. W., Liska, V., Khimani, A. H. & 8 other authors (1999). Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nat Med 5, 194–203.[CrossRef][Medline]
Barouch, D. H., Santra, S., Schmitz, J. E. & 26 other authors (2000). Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science 290, 486–492.
Barouch, D. H., Letvin, N. L. & Seder, R. A. (2004). The role of cytokine DNAs as vaccine adjuvants for optimizing cellular immune responses. Immunol Rev 202, 266–274.[CrossRef][Medline]
Barquinero, J., Eixarch, H. & Pérez-Melgosa, M. (2004). Retroviral vectors: new applications for an old tool. Gene Ther 11, S3–S9.
Berzofsky, J. A., Ahlers, J. D., Janik, J., Morris, J., Oh, S., Terabe, M. & Belyakov, I. M. (2004). Progress on new vaccine strategies against chronic viral infections. J Clin Invest 114, 450–462.[CrossRef][Medline]
Chang, L.-J. & He, J. (2001). Retroviral vectors for gene therapy of AIDS and cancer. Curr Opin Mol Ther 3, 468–475.[Medline]
Chang, L.-J., Urlacher, V., Iwakuma, T., Cui, Y. & Zucali, J. (1999). Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system. Gene Ther 6, 715–728.[CrossRef][Medline]
Chinnasamy, N., Treisman, J. S., Oaks, M. K., Hanson, J. P., Jr & Chinnasamy, D. (2005). Ex vivo generation of genetically modified dendritic cells for immunotherapy: implications of lymphocyte contamination. Gene Ther 12, 259–271.[CrossRef][Medline]
Cockrell, A. S. & Kafri, T. (2003). HIV-1 vectors: fulfillment of expectations, further advancements, and still a way to go. Curr HIV Res 1, 419–439.[CrossRef][Medline]
Connolly, J. B. (2002). Lentiviruses in gene therapy clinical research. Gene Ther 9, 1730–1734.[CrossRef][Medline]
Couzin, J. & Kaiser, J. (2005). As Gelsinger case ends, gene therapy suffers another blow. Science 307, 1028.
Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. C. (1996). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14, 315–319.[CrossRef][Medline]
Cranage, M. P., Whatmore, A. M., Sharpe, S. A. & 7 other authors (1997). Macaques infected with live attenuated SIVmac are protected against superinfection via the rectal mucosa. Virology 229, 143–154.[CrossRef][Medline]
Cui, Y., Iwakuma, T. & Chang, L. J. (1999). Contributions of viral splice sites and cis-regulatory elements to lentivirus vector function. J Virol 73, 6171–6176.
Esslinger, C., Chapatte, L., Finke, D., Miconnet, I., Guillaume, P., Lévy, F. & MacDonald, H. R. (2003). In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8+ T cell responses. J Clin Invest 111, 1673–1681.[CrossRef][Medline]
Firat, H., Zennou, V., Garcia-Pons, F., Ginhoux, F., Cochet, M., Danos, O., Lemonnier, F. A., Langlade-Demoyen, P. & Charneau, P. (2002). Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy. J Gene Med 4, 38–45.[CrossRef][Medline]
Franchini, G., Gurunathan, S., Baglyos, L., Plotkin, S. & Tartaglia, J. (2004). Poxvirus-based vaccine candidates for HIV: two decades of experience with special emphasis on canarypox vectors. Expert Rev Vaccines 3, S75–S88.[CrossRef][Medline]
Gao, F., Li, Y., Decker, J. M. & 11 other authors (2003). Codon usage optimization of HIV type 1 subtype C gag, pol, env, and nef genes: in vitro expression and immune responses in DNA-vaccinated mice. AIDS Res Hum Retroviruses 19, 817–823.[CrossRef][Medline]
Gómez, C. E., Abaitua, F., Rodríguez, D. & Esteban, M. (2004). Efficient CD8+ T cell response to the HIV-env V3 loop epitope from multiple virus isolates by a DNA prime/vaccinia virus boost (rWR and rMVA strains) immunization regime and enhancement by the cytokine IFN-. Virus Res 105, 11–22.[CrossRef][Medline]
Goulder, P. J., Rowland-Jones, S. L., McMichael, A. J. & Walker, B. D. (1999). Anti-HIV cellular immunity: recent advances towards vaccine design. AIDS 13 (Suppl. A), S121–S136.
Grignani, F., Kinsella, T., Mencarelli, A. & 7 other authors (1998). High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein. Cancer Res 58, 14–19.
Haas, J., Park, E.-C. & Seed, B. (1996). Codon usage limitation in the expression of HIV-1 envelope glycoprotein. Curr Biol 6, 315–324.[CrossRef][Medline]
Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M. & 32 other authors (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419.
He, Y., Zhang, J., Mi, Z., Robbins, P. & Falo, L. D., Jr (2005). Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity. J Immunol 174, 3808–3817.
Heeney, J. L. (2004). Requirement of diverse T-helper responses elicited by HIV vaccines: induction of highly targeted humoral and CTL responses. Expert Rev Vaccines 3, S53–S64.[CrossRef][Medline]
Heeney, J. L., Holterman, L., ten Haaft, P., Dubbes, R., Koornstra, W., Teeuwsen, V., Bourquin, P., Norley, S. & Niphuis, H. (1994). Vaccine protection and reduced virus load from heterologous macaque-propagated SIV challenge. AIDS Res Hum Retroviruses 10, S117–S121.
Im, E. J. & Hanke, T. (2004). MVA as a vector for vaccines against HIV-1. Expert Rev Vaccines 3, S89–S97.[CrossRef][Medline]
Irwin, M. J., Laube, L. S., Lee, V., Austin, M., Chada, S., Anderson, C.-G., Townsend, K., Jolly, D. J. & Warner, J. F. (1994). Direct injection of a recombinant retroviral vector induces human immunodeficency virus-specific immune responses in mice and nonhuman primates. J Virol 68, 5036–5044.
Iwakuma, T., Cui, Y. & Chang, L.-J. (1999). Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology 261, 120–132.[CrossRef][Medline]
Jaffray, A., Shephard, E., van Harmelen, J., Williamson, C., Williamson, A. L. & Rybicki, E. P. (2004). Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice. J Gen Virol 85, 409–413.
Johnson, R. P. & Desrosiers, R. C. (1998). Protective immunity induced by live attenuated simian immunodeficiency virus. Curr Opin Immunol 10, 436–443.[CrossRef][Medline]
Johnson, P. R., Schnepp, B. C., Connell, M. J. & 8 other authors (2005). Novel adeno-associated virus vector vaccine restricts replication of simian immunodeficiency virus in macaques. J Virol 79, 955–965.
Kamantigue, E., Edwards, W., III, Chada, S., Brumm, D., Austin, M., Irwin, M., Mento, S., Kowal, K. & Sajjadi, N. (1996). Evidence for localization of biologically active recombinant retroviral vector to lymph nodes in mice injected intramuscularly. Gene Ther 3, 128–136.[Medline]
Kaul, R., Rowland-Jones, S. L., Kimani, J. & 12 other authors (2001). New insights into HIV-1 specific cytotoxic T-lymphocyte responses in exposed, persistently seronegative Kenyan sex workers. Immunol Lett 79, 3–13.[CrossRef][Medline]
Kim, C. H., Oh, Y. & Lee, T. H. (1997). Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells. Gene 199, 293–301.[CrossRef][Medline]
Koya, R. C., Kasahara, N., Favaro, P. M. B., Lau, R., Ta, H. Q., Weber, J. S. & Stripecke, R. (2003). Potent maturation of monocyte-derived dendritic cells after CD40L lentiviral gene delivery. J Immunother 26, 451–460.
Kumar, A. & Narayan, O. (2001). Immunization for long-term protection against AIDS using the macaque model. Virology 285, 1–5.[CrossRef][Medline]
Kung, S. K. P., Bonifacino, A., Metzger, M. E., Ringpis, G.-E., Donahue, R. E. & Chen, I. S. Y. (2005). Lentiviral vector-transduced dendritic cells induce specific T cell response in a nonhuman primate model. Hum Gene Ther 16, 527–532.[CrossRef][Medline]
Laube, L. S., Burrascano, M., Dejesus, C. E. & 11 other authors (1994). Cytotoxic T lymphocyte and antibody responses generated in rhesus monkeys immunized with retroviral vector-transduced fibroblasts expressing human immunodeficiency virus type-1 IIIB ENV/REV proteins. Hum Gene Ther 5, 853–862.[Medline]
Letvin, N. L. (1998). What immunity can protect against HIV infection? J Clin Invest 102, 1643–1644.[Medline]
Letvin, N. L. (2002). Strategies for an HIV vaccine. J Clin Invest 110, 15–27.[CrossRef][Medline]
Leung, L., Srivastava, I. K., Kan, E., Legg, H., Sun, Y., Greer, C., Montefiori, D. C., zur Megede, J. & Barnett, S. W. (2004). Immunogenicity of HIV-1 Env and Gag in baboons using a DNA prime/protein boost regimen. AIDS 18, 991–1001.[CrossRef][Medline]
Lever, A. M., Strappe, P. M. & Zhao, J. (2004). Lentiviral vectors. J Biomed Sci 11, 439–449.[CrossRef][Medline]
Liu, M., Acres, B., Balloul, J.-M., Bizouarne, N., Paul, S., Slos, P. & Squiban, P. (2004). Gene-based vaccines and immunotherapeutics. Proc Natl Acad Sci U S A 101, 14567–14571.
Mäkitalo, B., Lundholm, P., Hinkula, J. & 9 other authors (2004). Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen. J Gen Virol 85, 2407–2419.
Manilla, P., Rebello, T., Afable, C. & 10 other authors (2005). Regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector. Hum Gene Ther 16, 17–25.[CrossRef][Medline]
McMichael, A. & Hanke, T. (2002). The quest for an AIDS vaccine: is the CD8+ T-cell approach feasible? Nat Rev Immunol 2, 283–291.[CrossRef][Medline]
Morizono, K., Xie, Y., Ringpis, G.-E., Johnson, M., Nassanian, H., Lee, B., Wu, L. & Chen, I. S. Y. (2005). Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection. Nat Med 11, 346–352.[CrossRef][Medline]
Nagata, T., Uchijima, M., Yoshida, A., Kawashima, M. & Koide, Y. (1999). Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms. Biochem Biophys Res Commun 261, 445–451.[CrossRef][Medline]
Naldini, L., Blömer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M. & Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267.[Abstract]
Negri, D. R. M., Baroncelli, S., Catone, S. & 15 other authors (2004a). Protective efficacy of a multicomponent vector vaccine in cynomolgus monkeys after intrarectal simian immunodeficiency virus challenge. J Gen Virol 85, 1191–1201.
Negri, D. R. M., Buffa, V., Leone, P. & 7 other authors (2004b). Use of retroviral vectors for the analysis of SIV/HIV-specific CD8 T cell responses. J Immunol Methods 291, 153–163.[CrossRef][Medline]
Norley, S., Beer, B., Binninger-Schinzel, D., Vogel, T., Siegel, F., Cosma, C., Konig, H., Megede, J. Z. & Kurth, R. (1996). Simian immunodeficiency virus live and inactivated experimental vaccines. AIDS Res Hum Retroviruses 12, 447–449.[Medline]
Ogg, G. S., Jin, X., Bonhoeffer, S. & 12 other authors (1998). Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279, 2103–2106.
Palmowski, M. J., Lopes, L., Ikeda, Y., Salio, M., Cerundolo, V. & Collins, M. K. (2004). Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response. J Immunol 172, 1582–1587.
Pinto, A. R., Fitzgerald, J. C., Giles-Davis, W., Gao, G. P., Wilson, J. M. & Ertl, H. C. J. (2003). Induction of CD8+ T cells to an HIV-1 antigen through a prime boost regimen with heterologous E1-deleted adenoviral vaccine carriers. J Immunol 171, 6774–6779.
Pope, M. (2003). Dendritic cells as a conduit to improve HIV vaccines. Curr Mol Med 3, 229–242.[CrossRef][Medline]
Ramakrishna, L., Anand, K. K., Mohankumar, K. M. & Ranga, U. (2004). Codon optimization of the Tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol 78, 9174–9189.
Relph, K., Harrington, K. & Pandha, H. (2004). Recent developments and current status of gene therapy using viral vectors in the United Kingdom. BMJ 329, 839–842.
Rohrlich, P. S., Cardinaud, S., Lulè, J. & 11 other authors (2004). Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-transgenic mice and for ex vivo stimulation and expansion of CD8+ cytotoxic T cells from human peripheral blood cells. Hum Immunol 65, 514–522.[CrossRef][Medline]
Romano, G., Claudio, P. P., Tonini, T. & Giordano, A. (2003). Human immunodeficiency virus type 1 (HIV-1) derived vectors: safety considerations and controversy over therapeutic applications. Eur J Dermatol 13, 424–429.[Medline]
Schmitz, J. E., Kuroda, M. J., Santra, S. & 13 other authors (1999). Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283, 857–860.
Schnell, T., Foley, P., Wirth, M., Munch, J. & Uberla, K. (2000). Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus. Hum Gene Ther 11, 439–447.[CrossRef][Medline]
Sernicola, L., Corrias, F., Koanga-Mogtomo, M. L. & 10 other authors (1999). Long-lasting protection by live attenuated simian immunodeficiency virus in cynomolgus monkeys: no detection of reactivation after stimulation with a recall antigen. Virology 256, 291–302.[CrossRef][Medline]
Shiver, J. W., Fu, T.-M., Chen, L. & 49 other authors (2002). Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 415, 331–335.[CrossRef][Medline]
Someya, K., Xin, K.-Q., Matsuo, K., Okuda, K., Yamamoto, N. & Honda, M. (2004). A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity. J Virol 78, 9842–9853.
Tatsis, N. & Ertl, H. C. J. (2004). Adenoviruses as vaccine vectors. Mol Ther 10, 616–629.[CrossRef][Medline]
Titti, F., Sernicola, L., Geraci, A. & 12 other authors (1997). Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys. J Gen Virol 78, 2529–2539.[Abstract]
Truckenmiller, M. E. & Norbury, C. C. (2004). Viral vectors for inducing CD8+ T cell responses. Expert Opin Biol Ther 4, 861–868.[CrossRef][Medline]
Vanniasinkam, T. & Ertl, H. C. J. (2005). Adenoviral gene delivery for HIV-1 vaccination. Curr Gene Ther 5, 203–212.[CrossRef][Medline]
Vargas, J., Jr, Gusella, G. L., Najfeld, V., Klotman, M. E. & Cara, A. (2004). Novel integrase-defective lentiviral episomal vectors for gene transfer. Hum Gene Ther 15, 361–372.[CrossRef][Medline]
Voltan, R. & Robert-Guroff, M. (2003). Live recombinant vectors for AIDS vaccine development. Curr Mol Med 3, 273–284.[CrossRef][Medline]
Wang, X., Wiley, R. D., Evans, T. G., Bowers, W. J., Federoff, H. J. & Dewhurst, S. (2003). Cellular immune responses to helper-free HSV-1 amplicon particles encoding HIV-1 gp120 are enhanced by DNA priming. Vaccine 21, 2288–2297.[CrossRef]
) or the TY2-CMVempty (
) vectors and in naïve mice (
). Analysis was performed on 1-week-cultured splenocytes pulsed respectively with the 10mer and the 15mer as described in Methods. 51Cr-release assay was performed at three different E : T ratios: 50 : 1, 25 : 1 and 12.5 : 1. The percentage of specific lysis is shown (with background subtracted).