| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Research Centre in Infectious Diseases, CHUL Research Centre of Laval University and Department of Medical Biology, Faculty of Medicine, Laval University, QC G1V 4G2, Canada
Correspondence
Barbara Papadopoulou
barbara.papadopoulou{at}crchul.ulaval.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The ability to elicit potent cellular immune responses has therefore become a priority for HIV-1 vaccine candidates. The goal of T-cell vaccines is to generate long-lived memory CD8 cells capable of recognizing and rapidly expanding to combat viral infection.
The problematic stimulation of neutralizing antibodies in HIV-1 infection (Moore et al., 1995), the recent failure of a recombinant gp120 glycoprotein to demonstrate vaccine efficacy in a phase III human clinical trial (Gilbert et al., 2005) and the inability of traditional vaccine approaches to generate a suitable and safe vaccine for HIV-1 (Baba et al., 1995; Murphey-Corb et al., 1989; Whitney & Ruprecht, 2004) have prompted the quest for the development of novel vaccine strategies. Among these, the most promising are the use of plasmid DNA and live recombinant vectors, either alone or as part of heterologous prime–boost combinations. Although plasmid DNAs have been less immunogenic in early-phase clinical testing in humans than in laboratory animals (MacGregor et al., 1998; McConkey et al., 2003), a number of changes in plasmid DNA constructs have been shown to increase their immunogenicity (Donnelly et al., 2005). Live recombinant vaccines that express HIV-1 immunogens have proven generally to stimulate strong CD4+ and CD8+ T-cell responses and neutralizing antibodies in non-human primates compared with whole killed vaccines, virus-like particles and subunit vaccines (Amara et al., 2001; Barouch et al., 2001; Horton et al., 2002; Seth et al., 2000; reviewed by Letvin et al., 2002; McMichael & Hanke, 2003). Although live recombinant vectors are central to the development of new vaccine strategies against HIV-1, their utilization as vaccine candidates in humans is hampered due to problems related to pre-existing immunity, inefficient antigen delivery or presentation and toxicity issues, especially for immunocompromised individuals (Redfield et al., 1987). Therefore, there is an urgent need to develop new live-vaccine vectors that are capable of enhancing antigen presentation and eliciting potent immune responses without the risk of developing disease in humans.
In this study, we describe a novel, non-pathogenic, protozoan parasitic vector, Leishmania tarentolae, as a recombinant HIV-1 vaccine candidate to improve the efficacy of HIV vaccine strategies. L. tarentolae has several features that make it very attractive as the basis for an effective recombinant HIV vaccine. We have shown recently that L. tarentolae efficiently targets antigen-presenting cells (APCs) (e.g. macrophages and dendritic cells) and lymphoid organs and that it activates the process of dendritic cell maturation (Breton et al., 2005). Unlike other pathogenic Leishmania strains, L. tarentolae lacks the potential to replicate within the targeted APCs and is eliminated after several days from the infected murine host (Breton et al., 2005). Nevertheless, L. tarentolae can elicit T-cell proliferation and the production of gamma interferon (IFN-
), skewing the T-cell response towards a Th1-cell phenotype, and it provides inflammatory responses for the APC and acts as an immunostimulatory adjuvant (Breton et al., 2005). In the present study, we developed a recombinant L. tarentolae strain expressing high levels of full-length HIV-1 Gag and evaluated its ability to elicit HIV-1 Gag-specific T-cell responses in mice upon HIV antigen stimulation. Moreover, we immunized human lymphoid tissue cultured ex vivo that can be infected by both Leishmania and HIV-1 (Zhao et al., 2004) to assess the ability of the recombinant L. tarentolae–Gag vaccine vector to protect against HIV-1 infection. Such a system has not been used before to evaluate the protective efficacy of vaccine candidates.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) (Multicell) and 5 µg haemin ml–1. Leishmania transfections were carried out by electroporation as described elsewhere (Papadopoulou et al., 1992). Approximately 10–15 µg DNA was used for transfections and cells were selected with G-418 (40 µg ml–1; Sigma).
Plasmid construction and DNA manipulations.
The pNEO-Gag expression vector was made as follows. First, the intergenic region of the 
-tubulin gene necessary for mRNA processing was inserted into the XhoI/XbaI sites of pSP72 (Promega). Then, the neomycin phosphotransferase (NEO) gene with an additional intergenic region of the -tubulin gene was cloned as a KpnI–EcoRI fragment into the respective sites of vector pSP. The 18S rRNA gene promoter sequence, described elsewhere (Yan et al., 1999), was cloned in the NdeI site of the above vector. Finally, a 1.6 kb fragment containing the HIV-1 gag gene was amplified using Pwo polymerase (Roche) from pNL4.3 (Adachi et al., 1986), a full-length HIV-1 proviral molecular clone, using the following set of primers 5'Gag (XbaI): 5'-CGTCTAGAAGGAGAGAGATGG-3' and 3'Gag (KpnI): 5'-GGGGTACCTTTATTGTGACG-3', and then introduced as an XbaI–KpnI insert between the two -tubulin intergenic regions to generate vector pNEO-Gag. Southern and Western blot analyses were performed to test for the presence of Gag-expressing vector and to evaluate Gag protein expression using standard procedures.
Mice and immunizations.
Groups of 8-week-old female BALB/c mice (Charles River Laboratories) were immunized intraperitoneally (i.p.) with 5x107 stationary-phase recombinant L. tarentolae promastigotes expressing the HIV-1 Gag protein. L. tarentolae wild-type promastigotes were also used for comparison. At 2, 4, 6, 8 and 12 weeks post-immunization, mice were sacrificed and blood samples and spleens were collected for immunological analyses. For evaluating T-cell memory responses, groups of five mice were primed with 5x107 L. tarentolae–Gag cells (i.p. injection) and boosted 1 month later with the same vaccine dose. Six months after the first immunization, splenocytes were isolated, stimulated with 25 pg p24 ml–1 for 24 h in RPMI 1640 and antigen-specific responses were measured by flow cytometry. Approximately 106 spleen cells were stained with FITC-labelled anti-CD44 (H-CAM; BD Biosciences), phycoerythrin (PE)-labelled anti-CD62L (BD Biosciences) or FITC-labelled anti-CD69 (BD Biosciences), PE-labelled anti-CD25 (BD Biosciences) and tri-colour (TC)-labelled anti-CD4 (BD Biosciences). Splenocytes were incubated for 30 min at 4 °C with 500 µl PBS containing 1 % FBS and 0.09 % NaN3, containing a saturating amount of each antibody. Cells were then washed and fixed with 2 % paraformaldehyde. Flow cytometry was performed using an EPICS Elite ESP (Coulter Electronics) and data were further analysed with WinMDI software.
Proliferation assays.
Spleen cells were harvested from individual vaccinated mice on day 0 (just prior to immunization) and at 2, 4, 8 and 12 weeks post-immunization and tested in a standard [3H]thymidine incorporation assay. Cells were homogenized, washed and resuspended in 200 µl RPMI 1640 supplemented with 10 % FBS (Hyclone), 100 U penicillin ml–1, 100 µg streptomycin ml–1, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol. Cells were cultured in a 96-well flat-bottomed microplate (Corning) (2.5x106 splenocytes per well) with different concentrations of p24 (0–50 µg ml–1) for 3 days at 37 °C and 5 % CO2 atmosphere and pulsed with 1 µCi [3H]thymidine (Amersham) per well for 24 h. Cells were harvested on day 3 with a Harvester 96 (Tomtec) and [3H]thymidine incorporation was measured with a 1205 BetaPlate Liquid Scintillation Counter (Amersham). The mean number of c.p.m. for triplicate wells was used to calculate the stimulation index (SI) as follows: SI=c.p.m. with antigen stimulation/c.p.m. with medium alone.
IFN- ELISPOT assay.
To evaluate the frequency of T cells producing IFN-, we used an enzyme-linked immunosorbent spot (ELISPOT) assay as described elsewhere (Mashishi & Gray, 2002). Briefly, 96-well multiscreen plates (Millipore) were coated by overnight incubation at 4 °C with rat anti-mouse IFN- capture monoclonal antibody (clone R4-6A2; BD Pharmingen) at a concentration of 10 µg ml–1 in PBS. Splenocytes were harvested from mice at 2, 4, 8 and 12 weeks after immunization with recombinant L. tarentolae. Cells (1x105 per well) were plated in triplicate in a 100 µl final volume with medium alone or stimulated with a pool of overlapping Gag peptides in complete RPMI 1640. HIV-1 Gag peptides of 9, 15 or 20 aa in length were obtained from the AIDS Research and Reference Reagent Program, NIH (Rockville, MD, USA), and used for stimulation at a final concentration of 10 µM. After a 24 h incubation at 37 °C in 5 % CO2, the plates were washed with PBS containing 0.05 % Tween (PBST) and incubated for 6 h at room temperature with a secondary biotinylated rat anti-mouse IFN- antibody (XMG1.2; BD Pharmingen). Plates were washed six times with PBS and streptavidin–alkaline phosphatase (diluted 1 : 100 000; Sigma) was added. After incubation for 2 h, plates were washed and developed with NBT/BCIP (Bio-Rad). Plates were air-dried and the spots were counted using a stereomicroscope (Carl Zeiss Canada) at 40x magnification. An ELISPOT assay was also performed on CD4+ T-cell-depleted splenocytes using the panning method (Norton, 1997). The mean number of spots from triplicate wells was calculated for each animal and adjusted to represent the mean number of spots per 106 spleen cells. Data are presented as spot forming units (s.f.u.) per 106 spleen cells from five animals per group.
Anti-p24 antibody titres.
Serum was collected from individual mice at 8 weeks post-immunization and anti-p24 antibody titres were determined by ELISA. Ninety-six-well plates (ImmunoPlate Maxisorp; Nunc) were coated by overnight incubation at 4 °C with 50 ng p24 per well. Plates were blocked with PBS plus 5 % BSA for 2 h at room temperature and then washed with PBST. Sera were diluted in PBS from 1 : 100 to 1 : 500 and 100 µl peroxidase-conjugated rabbit anti-mouse IgG antibody (Sigma) was added and incubated for 1 h at room temperature. After at least five washes, plates were developed with 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Research Diagnostics). Reactions were stopped with 2 M H2SO4 and the absorbance was measured at 450 nm using an Organon Teknika Microwell system.
Delivery of L. tarentolae and NL4.3 HIV-1 strain to human tonsillar tissue blocks.
Human tonsils were obtained from patients who underwent routine tonsillectomy. Tonsils were washed thoroughly with PBS containing antibiotics, dissected into small pieces of 
2–3 mm3 and cultured in RPMI 1640 supplemented with FBS and antibiotics on collagen sponge gels at the air–liquid interface at 37 °C in a 5 % CO2 atmosphere as described previously (Zhao et al., 2004). After 24 h, different concentrations (1x103, 1x104, 5x104, 1x105 and 5x105 cells) of recombinant L. tarentolae expressing HIV-1 Gag were added on top of the tissue blocks and 3 h later the tonsillar pieces were washed with PBS and kept in culture for up to 2 weeks. At 3 weeks after immunization with recombinant L. tarentolae, 5 ng NL4.3 HIV-1 strain was applied on top of the tissue blocks. Aliquots of supernatants were harvested 6 days after infective HIV-1 challenge and a sandwich ELISA was performed on supernatants to quantify p24 production and monitor HIV replication. The p24 antibody capture assay has been described previously (Bounou et al., 2002). It should be noted that separate blocks of human tonsillar tissue from six different donors were tested (i.e. a total of eight tissue blocks in four different wells) to normalize for variation in cell number and composition and to allow comparison of immunized and non-immunized tissue.
Statistical analyses.
Data were expressed as means±SEM. The statistical significance of differences between groups was analysed using a paired Student's t-test. A P value of less than 0.05 was considered significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
), which allows high levels of expression. Western blot analysis using an anti-p24 antibody showed that recombinant L. tarentolae transformed with the HIV-1 Gag expression vector showed high levels of expression of the full-length 55 kDa Gag protein (Fig. 1b). Remarkably, the HIV-1 Gag polyprotein was processed into its other proteolytic products in Leishmania, such as p24, in a similar fashion to HIV-1 Gag from H9 HIV-1-infected cells. These results showed the successful expression of the full-length HIV-1 Gag protein in the non-pathogenic L. tarentolae and suggested that Gag polyprotein can be processed by proteases within the parasite.
|
12-fold in response to the highest p24 dose (50 pg ml–1) (Fig. 2a). No p24-specific proliferative responses were observed in splenocytes of mice immunized with wild-type L. tarentolae (Fig. 2a). Interestingly, antigen-stimulated proliferation was still observed at 12 weeks post-immunization and at slightly increased levels (Fig. 2b).
|
ELISPOT assay. Splenocytes were harvested from mice at weeks 2, 4, 8 and 12 after immunization with recombinant L. tarentolae and then stimulated with a pool of overlapping peptides representing a processed form of the HIV-1 Gag protein. Our results on total splenocytes indicated an important increase in the number of IFN--producing T cells following stimulation with the Gag peptide pool compared with unstimulated splenocytes (Fig. 3a). The peak response was elicited at 2 weeks post-immunization (386±52.3 s.f.c. per 106 splenocytes). However, the number of IFN--producing T cells remained relatively high (293±41.4 s.f.c. per 106 splenocytes), even 12 weeks after immunization with recombinant L. tarentolae (Fig. 3a).
|
, we used five different pools (P1–P5) of 15 overlapping HIV-1 Gag peptides each. These peptides were 9–20 aa long and overlapped by 8–11 residues. Peptide pool 4 (P4) was capable of inducing a greater stimulation of IFN--producing T cells in splenocytes isolated from BALB/c immunized mice at week 12 post-immunization (Fig. 3b
). Peptides in pool P4 cover a region of the HIV-1 Gag protein between aa 281 and 430. This region includes the end of the capsid (p24) and part of the nucleocapsid (p7).
The functional capacity of recombinant L. tarentolae to induce HIV-1 Gag-specific CD8+ T-cell responses was also determined. CD4+ T cells were selectively depleted from the splenocyte T-cell population by the panning approach (Norton, 1997) prior to assaying these cells in an IFN- ELISPOT assay. Depletion of CD4+ T cells had no significant effect on the Gag-specific-induced IFN- response (Fig. 3c), suggesting that recombinant L. tarentolae-elicited HIV-1-specific CD8+ T cells were capable of secreting IFN- in response to HIV-1 Gag peptide stimulation.
A booster immunization with recombinant L. tarentolae stimulates the development of long-lasting immune responses
Protection following viral clearance or successful immunization requires the generation and maintenance of long-lived, antigen-specific CD4+ Th cells (reviewed by Klenerman & Hill, 2005). CD4+ Th cells are also critical for the development of CD8+ T-cell memory (Janssen et al., 2003; Shedlock & Shen, 2003). We first measured the CD4+ effector memory response to HIV-1 Gag in mice immunized once with the recombinant L. tarentolae–Gag vaccine vector. Six months after immunization, bulk splenocytes were isolated, stimulated with HIV-1 p24 protein and stained with TC-conjugated monoclonal antibodies to CD4, FITC-conjugated antibodies to CD69 and CD44, and PE-labelled antibodies to CD25 and CD62L, and analysed by flow cytometry. CD25 and CD69 are cell-surface markers that are expressed in activated Th1 subpopulations. CD44 is a surface protein required for lymphocyte extravasation to inflammatory sites and its upregulation is a marker for all memory T cells (DeGrendele et al., 1997). CD62L is a lymph node homing receptor that is downregulated upon activation of T-cell populations (Andersson et al., 1994). Upon antigen stimulation, approximately 22 % of the CD4+ cells were effector cells, CD44High/CD62LLow, compared with 14 % in naive mice (Table 1). A significant proportion of the CD4+ T cells in vaccinated mice expressed the activation markers CD25 and CD69 (15 % and 11 %, respectively) (Table 1).
|
). The expression of CD25 and CD69 activation markers on the surface of CD4+ T cells was also increased by approximately 2-fold in boosted animals following stimulation with p24 Gag (Table 1). Thus, our data indicated that a second encounter with the HIV-1 Gag antigen stimulates the proliferation of effector memory CD4+ T cells.
Immunization with recombinant L. tarentolae expressing HIV-1 Gag elicits low antibody responses against HIV-1 Gag
We evaluated the production of anti-Gag-specific antibodies in the serum of L. tarentolae–Gag-immunized mice. Various dilutions (1 : 100, 1 : 250, 1 : 500) were used of sera obtained at 4 and 8 weeks following i.p. immunization with recombinant L. tarentolae expressing HIV-1 Gag protein. A p24 ELISA was used to measure the IgG p24-specific immune responses. No antibodies were found in sera after only a single injection with recombinant L. tarentolae (Fig. 4 and data not shown). However, we observed a low antibody response in the 1 : 100 dilution of sera collected 4 weeks after a booster immunization with recombinant L. tarentolae (Fig. 4). Thus, to trigger a specific humoral response against the HIV-1 Gag protein, a secondary encounter with the antigen following a booster immunization with recombinant L. tarentolae is required.
|
). We recently demonstrated that Leishmania donovani can infect human lymphoid tissue (Zhao et al., 2004). Here, we showed by both microscopic examination and FACS analysis that non-pathogenic L. tarentolae could be delivered to human lymphoid tissue (Fig. 5a, b). Indeed, L. tarentolae was found in approximately 40 % of the CD14+ cells (i.e. monocytes and macrophages) within the human tonsils (Fig. 5a). Eight separate blocks of human tonsillar tissue derived from each donor were used to allow comparison of immunized tissue with Gag-expressing L. tarentolae and unimmunized tissue (L. tarentolae wild-type). Three weeks after immunization, tonsillar tissues were infected with the NL4.3 HIV-1 strain. Aliquots of supernatants were harvested 6 days after HIV-1 infection and an ELISA sandwich was performed to quantify p24 production and to monitor HIV-1 replication. Immunization of human tonsillar tissues with recombinant L. tarentolae resulted in an 75 % decrease in virus replication compared with unimmunized tissue (Fig. 5c). Immunization with an increased dose of Gag-expressing L. tarentolae (105–106) resulted in a higher decrease in virus replication (Fig. 5c). Interestingly, highly reproducible results were obtained with blocks of tonsillar tissue from six different donors where a 66–75 % decrease in virus replication was observed following immunization of tonsillar tissue blocks with 106 L. tarentolae cells and subsequent exposure to the NL4.3 HIV-1 strain at 3 weeks post-immunization (data not shown). To rule out the possibility that the parasite could make the tonsils less suitable host cells for HIV replication, by stimulating innate immunity for instance, we incubated the tonsillar material in parallel with wild-type L. tarentolae and a recombinant L. tarentolae expressing an irrelevant antigen, i.e. green fluorescent protein (GFP). These experiments showed that HIV-1 replication was not affected by the presence of the wild-type parasite (Fig. 5c) or the parasite expressing GFP (data not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Furthermore, recombinant L. tarentolae as a non-replicating, live-vector vaccine achieves only one round of infection, which makes it even safer for human use. In addition, L. tarentolae has several other features that may make it an effective HIV vaccine vector. L. tarentolae is an intracellular parasite that shares common target cells with HIV-1, e.g. macrophages and dendritic cells and secondary lymphoid organs (Breton et al., 2005). Therefore, a recombinant L. tarentolae vaccine vector should mimic the processing, maturation and presentation of viral antigens seen during natural infection. A more efficient targeting of HIV-1 immunogens to APCs could enhance MHC/antigen-presentation functions and elicit a more potent stimulation of antigen-specific CD4+ Th cell and CD8+ CTL responses, which are critical for the control of HIV replication (Letvin, 2005). In addition, L. tarentolae induces dendritic cell maturation and elicits CD4+ T-cell proliferation and the production of IFN-, thereby driving Th1 cell polarization, which can be critical for the secondary expansion of memory CTLs (Breton et al., 2005). Finally, L. tarentolae demonstrates a high cloning capacity, is capable of expressing a wide range of multiple foreign antigens and grows rapidly in a cell-free medium at low cost.
The optimal vaccine vector would produce the vaccine antigen in excess of its own proteins (so that the immune response focuses on the target antigen) and would primarily be produced in APCs for induction of CTL responses. The presence of the ribosomal promoter greatly enhances the expression of HIV-1 Gag in L. tarentolae. Recombinant L. tarentolae elicited HIV-specific CD8+ T-cell responses that were maximal 2 weeks after immunization. This peak immune response has also been described in mice immunized with adenoviral and vaccinia vectors (Barouch et al., 2003; Seaman et al., 2004). CD8+ T-cell activation and IFN- production was higher in response to a specific pool of Gag peptides spanning the aa 281–430 region of the Gag protein, which corresponds to the end of the capsid (p24) and part of the nucleocapsid (p7). CD4+ T-cell depletion studies indicated that IFN--producing T cells were mainly of CD8+ type. Diverse evidence supports the importance of the cellular immune response in HIV containment. CTLs have been shown to play a particularly important role in the early control of HIV infection (Betts et al., 2001; Goulder et al., 2001; Jin et al., 1999; Schmitz et al., 1999). Long-term non-progressors also have consistently higher levels of HIV CTLs than progressors (Harrer et al., 1996). In addition, rhesus monkeys lacking CD8+ T cells fail to control simian immunodeficiency virus infection (Schmitz et al., 1999). The ability of L. tarentolae to target macrophages and dendritic cells may explain the induction of T-cell activation. In recent years, it has become increasingly apparent that dendritic cells potentially link the innate and adaptive immune systems, coordinating the activation of strong cellular and humoral immunity (Granucci et al., 2003; Melief, 2003).
Interestingly, in mice that have been immunized twice with recombinant L. tarentolae, 50 % of the HIV-1 Gag-specific CD4+ T cells within spleen cells were effector cells (CD44High/CD62LLow). Thus, these data suggest that recombinant L. tarentolae is able to elicit HIV-1-specific effector memory in vivo after re-encountering the antigen. Memory CD4+ T cells are critical for the development of CD8+ T-cell memory (Janssen et al., 2003; Shedlock & Shen, 2003). In addition to the induction of effector T-cell responses by the recombinant L. tarentolae vaccine vector, a booster immunization with this live-vector vaccine elicited Gag-specific humoral responses. The production of antibodies against p24 is probably due to the activation of CD4+ T cells. It is likely that the use of recombinant L. tarentolae expressing HIV-1 Gag as part of a heterologous prime–boost strategy may be superior for eliciting memory T-cell and recall responses, as all of the secondary responses will focus on the viral immunogen alone and not on internal Leishmania proteins shared between the priming and boosting vectors. Heterologous prime–boost vaccine regimens have been reported to induce more potent T-cell and antibody responses against HIV-1 (Amara et al., 2001).
An important finding of our studies is that immunization of human tonsillar tissue blocks with recombinant L. tarentolae expressing HIV-1 Gag elicited more than a 75 % decrease in HIV-1 replication following exposure to HIV-1 infection. The highest decrease in virus replication was observed when an increased inoculum of recombinant L. tarentolae was used for immunization. This can be explained, as non-replicating, live-vector vaccines typically require high doses and booster immunizations to achieve sufficient antigen to drive immune responses. Although further experiments are required to establish the link between the important decrease in virus replication seen only in immunized tonsillar tissue and the development of protective immunity, preliminary data indicated that there was no difference in the number of CD4 T cells between immunized and unimmunized tissue blocks, which suggests that elicitation of HIV-1 Gag-specific immune responses might be responsible for the observed phenotype. Our data support the possibility of using human tonsillar tissue as an ex vivo system to evaluate the protective efficacy of candidate vaccines towards HIV-1 infection. Previous reports have indicated that secondary lymphoid organs constitute preferred anatomical sites for HIV replication and propagation (Grivel et al., 2003). Moreover, the human lymphoid tissue cultured ex vivo has been shown to preserve the general cytoarchitecture found in normal human lymphoid tissue, including a network of follicular dendritic cells, macrophages, CD4+ and CD8+ T lymphocytes and dendritic cells (Glushakova et al., 1995; Margolis et al., 1997), hence permitting evaluation of cellular and humoral immune responses.
In summary, we have described a novel, live-vector vaccine, a recombinant L. tarentolae expressing HIV-1 Gag protein, which is capable of efficient delivery of HIV-1 immunogens to APCs and to lymphoid organs. We showed that this recombinant vaccine vector could induce HIV-1-specific CD8+ T-cell responses and that it stimulated long-lasting immunity by the production of effector memory CD4+ T cells. Moreover, this live-vector vaccine is safe for human use, which makes it an attractive candidate for a vaccination strategy not only against HIV-1 but also against other intracellular pathogens for which T-cell-mediated immunity is required for protection.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Amara, R. R., Villinger, F., Altman, J. D., Lydy, S. L., O'Neil, S. P., Staprans, S. I., Montefiori, D. C., Xu, Y., Herndon, J. G. & other authors (2001). Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292, 69–74.[CrossRef][Medline]
Andersson, E. C., Christensen, J. P., Marker, O. & Thomsen, A. R. (1994). Changes in cell adhesion molecule expression on T cells associated with systemic virus infection. J Immunol 152, 1237–1245.[Abstract]
Baba, T. W., Jeong, Y. S., Pennick, D., Bronson, R., Greene, M. F. & Ruprecht, R. M. (1995). Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science 267, 1820–1825.
Barouch, D. H., Santra, S., Kuroda, M. J., Schmitz, J. E., Plishka, R., Buckler-White, A., Gaitan, A. E., Zin, R., Nam, J.-H. & other authors (2001). Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination. J Virol 75, 5151–5158.
Barouch, D. H., McKay, P. F., Sumida, S. M., Santra, S., Jackson, S. S., Gorgone, D. A., Lifton, M. A., Chakrabarti, B. K., Xu, L. & other authors (2003). Plasmid chemokines and colony-stimulating factors enhance the immunogenicity of DNA priming-viral vector boosting human immunodeficiency virus type 1 vaccines. J Virol 77, 8729–8735.
Betts, M. R., Ambrozak, D. R., Douek, D. C., Bonhoeffer, S., Brenchley, J. M., Casazza, J. P., Koup, R. A. & Picker, L. J. (2001). Analysis of total human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell responses: relationship to viral load in untreated HIV infection. J Virol 75, 11983–11991.
Bounou, S., Leclerc, J. E. & Tremblay, M. J. (2002). Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4+-T-cell depletion in human lymphoid tissue, a major site of replication in vivo. J Virol 76, 1004–1014.
Breton, M., Tremblay, M. J., Ouellette, M. & Papadopoulou, B. (2005). Live nonpathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis. Infect Immun 73, 6372–6382.
Brun, R. & Schönenberger, M. (1979). Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop 36, 289–292.[Medline]
Clayton, C. E. (2002). Life without transcriptional control? From fly to man and back again. EMBO J 21, 1881–1888.[CrossRef][Medline]
DeGrendele, H. C., Estess, P. & Siegelman, M. H. (1997). Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science 278, 672–675.
Donnelly, J. J., Wahren, B. & Liu, M. A. (2005). DNA vaccines: progress and challenges. J Immunol 175, 633–639.
Gilbert, P. B., Peterson, M. L., Follmann, D., Hudgens, M. G., Francis, D. P., Gurwith, M., Heyward, W. L., Jobes, D. V., Popovic, V. & other authors (2005). Correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of HIV-1 infection in a phase 3 HIV-1 preventive vaccine trial. J Infect Dis 191, 666–677.[CrossRef][Medline]
Glushakova, S., Baibakov, B., Margolis, L. B. & Zimmerberg, J. (1995). Infection of human tonsil histocultures: a model for HIV pathogenesis. Nat Med 1, 1320–1322.[CrossRef][Medline]
Goulder, P. J. R., Altfeld, M. A., Rosenberg, E. S., Nguyen, T., Tang, Y., Eldridge, R. L., Addo, M. M., He, S., Mukherjee, J. S. & other authors (2001). Substantial differences in specificity of HIV-specific cytotoxic T cells in acute and chronic HIV infection. J Exp Med 193, 181–194.
Granucci, F., Feau, S., Zanoni, I., Pavelka, N., Vizzardelli, C., Raimondi, G. & Ricciardi-Castagnoli, P. (2003). The immune response is initiated by dendritic cells via interaction with microorganisms and interleukin-2 production. J Infect Dis 187 (Suppl. 2), S346–S350.
Grivel, J.-C., Biancotto, A., Ito, Y., Lima, R. G. & Margolis, L. B. (2003). Bystander CD4+ T lymphocytes survive in HIV-infected human lymphoid tissue. AIDS Res Hum Retroviruses 19, 211–216.[CrossRef][Medline]
Harrer, T., Harrer, E., Kalams, S. A., Elbeik, T., Staprans, S. I., Feinberg, M. B., Cao, Y., Ho, D. D., Yilma, T. & other authors (1996). Strong cytotoxic T cell and weak neutralizing antibody responses in a subset of persons with stable nonprogressing HIV type 1 infection. AIDS Res Hum Retroviruses 12, 585–592.[Medline]
Horton, H., Vogel, T. U., Carter, D. K., Vielhuber, K., Fuller, D. H., Shipley, T., Fuller, J. T., Kunstman, K. J., Sutter, G. & other authors (2002). Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239. J Virol 76, 7187–7202.
Janssen, E. M., Lemmens, E. E., Wolfe, T., Christen, U., von Herrath, M. G. & Schoenberger, S. P. (2003). CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421, 852–856.[CrossRef][Medline]
Jin, X., Bauer, D. E., Tuttleton, S. E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C. E., Safrit, J. T., Mittler, J. & other authors (1999). Dramatic rise in plasma viremia after CD8+ T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med 189, 991–998.
Klenerman, P. & Hill, A. (2005). T cells and viral persistence: lessons from diverse infections. Nat Immunol 6, 873–879.[CrossRef][Medline]
Letvin, N. L. (2005). Progress toward an HIV vaccine. Annu Rev Med 56, 213–223.[CrossRef][Medline]
Letvin, N. L., Barouch, D. H. & Montefiori, D. C. (2002). Prospects for vaccine protection against HIV-1 infection and AIDS. Annu Rev Immunol 20, 73–99.[CrossRef][Medline]
MacGregor, R. R., Boyer, J. D., Ugen, K. E., Lacy, K. E., Gluckman, S. J., Bagarazzi, M. L., Chattergoon, M. A., Baine, Y., Higgins, T. J. & other authors (1998). First human trial of a DNA-based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J Infect Dis 178, 92–100.[Medline]
Margolis, L. B., Fitzgerald, W., Glushakova, S., Hatfill, S., Amichay, N., Baibakov, B. & Zimmerberg, J. (1997). Lymphocyte trafficking and HIV infection of human lymphoid tissue in a rotating wall vessel bioreactor. AIDS Res Hum Retroviruses 13, 1411–1420.[Medline]
Mashishi, T. & Gray, C. M. (2002). The ELISPOT assay: an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin Chem Lab Med 40, 903–910.[CrossRef][Medline]
McConkey, S. J., Reece, W. H. H., Moorthy, V. S., Webster, D., Dunachie, S., Butcher, G., Vuola, J. M., Blanchard, T. J., Gothard, P. & other authors (2003). Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med 9, 729–735.[CrossRef][Medline]
McMichael, A. J. & Hanke, T. (2003). HIV vaccines 1983–2003. Nat Med 9, 874–880.[CrossRef][Medline]
Melief, C. J. M. (2003). Mini-review: regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming? Eur J Immunol 33, 2645–2654.[CrossRef][Medline]
Moore, J. P., Trkola, A., Korber, B., Boots, L. J., Kessler, J. A., II, McCutchan, F. E., Mascola, J., Ho, D. D., Robinson, J. & Conley, A. J. (1995). A human monoclonal antibody to a complex epitope in the V3 region of gp120 of human immunodeficiency virus type 1 has broad reactivity within and outside clade B. J Virol 69, 122–130.[Abstract]
Murphey-Corb, M., Martin, L. N., Davison-Fairburn, B., Montelaro, R. C., Miller, M., West, M., Ohkawa, S., Baskin, G. B., Zhang, J. Y. & other authors (1989). A formalin-inactivated whole SIV vaccine confers protection in macaques. Science 246, 1293–1297.
Norton, J. D. (1997). Technical tips online. Simple and efficient panning of transfected cell populations expressing a cell surface marker. Trends Genet 13, 41.
Papadopoulou, B., Roy, G. & Ouellette, M. (1992). A novel antifolate resistance gene on the amplified H circle of Leishmania. EMBO J 11, 3601–3608.[Medline]
Redfield, R. R., Wright, D. C., James, W. D., Jones, T. S., Brown, C. & Burke, D. S. (1987). Disseminated vaccinia in a military recruit with human immunodeficiency virus (HIV) disease. N Engl J Med 316, 673–676.[Medline]
Schmitz, J. E., Kuroda, M. J., Santra, S., Sasseville, V. G., Simon, M. A., Lifton, M. A., Racz, P., Tenner-Racz, K., Dalesandro, M. & other authors (1999). Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283, 857–860.
Seaman, M. S., Peyerl, F. W., Jackson, S. S., Lifton, M. A., Gorgone, D. A., Schmitz, J. E. & Letvin, N. L. (2004). Subsets of memory cytotoxic T lymphocytes elicited by vaccination influence the efficiency of secondary expansion in vivo. J Virol 78, 206–215.
Seth, A., Ourmanov, I., Schmitz, J. E., Kuroda, M. J., Lifton, M. A., Nickerson, C. E., Wyatt, L., Carroll, M., Moss, B. & other authors (2000). Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge. J Virol 74, 2502–2509.
Shedlock, D. J. & Shen, H. (2003). Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300, 337–339.
Whitney, J. B. & Ruprecht, R. M. (2004). Live attenuated HIV vaccines: pitfalls and prospects. Curr Opin Infect Dis 17, 17–26.[CrossRef][Medline]
Yan, S., Lodes, M. J., Fox, M., Myler, P. J. & Stuart, K. (1999). Characterization of the Leishmania donovani ribosomal RNA promoter. Mol Biochem Parasitol 103, 197–210.[CrossRef][Medline]
Zhao, C., Papadopoulou, B. & Tremblay, M. J. (2004). Leishmania infantum promotes replication of HIV type 1 in human lymphoid tissue cultured ex vivo by inducing secretion of the proinflammatory cytokines TNF- and IL-1. J Immunol 172, 3086–3093.
Received 1 March 2006;
accepted 7 September 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
) or 5x107 recombinant L. tarentolae–Gag cells (
). Two weeks after immunization, splenocytes were harvested and stimulated for 3 days with increasing concentrations of p24 ranging from 0 to 50 pg ml–1. Cells were then pulsed with [3H]thymidine for 24 h and proliferative responses were measured by the amount of incorporated radioactivity. (b) Proliferation of splenic cells from vaccinated BALB/c mice at 4, 8, 12 weeks after i.p. injection of 5x107 recombinant L. tarentolae cells was measured following stimulation with p24 (25 µg ml–1) for 3 days in culture. The stimulation index (SI) was determined as described in Methods. Values represent means±SEM of triplicate cultures and are representative of three independent experiments with five mice per group. *, P<0.05. 
, Medium alone; 
), primed with 5x107 recombinant L. tarentolae cells (
, HIV-1.