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Previous Article | Next Article 
Infection and Immunity, July 2002, p. 3681-3688, Vol. 70, No. 7
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.7.3681-3688.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Mycobacterial Immunology, Pasteur Institute of Brussels, B1180 Brussels, Belgium, and,1 Vical, Inc., San Diego, California 921212
Received 26 December 2001/ Returned for modification 29 January 2002/ Accepted 3 April 2002
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ABSTRACT |
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INTRODUCTION |
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Extracellular and surface-exposed cell wall proteins from the pathogen are thought to be important for the elicitation of protective immune responses against TB. A major fraction of the secreted proteins in M. tuberculosis and BCG culture filtrates is formed by the antigen 85 (Ag85) complex (43), a 30- to 32-kDa family of three proteins (Ag85A, Ag85B, and Ag85C) which all possess enzymatic mycolyltransferase enzyme activity involved in the attachment of mycolic acids to the arabinogalactan of the cell wall and in the biogenesis of cord factor (33). The Ag85 complex is a promising vaccine candidate, as it sensitizes the immune system for strong T-cell proliferative responses and gamma interferon (IFN-
) production in most healthy individuals infected with M. tuberculosis or M. leprae (25) and in BCG-vaccinated mice (18) but not in TB or lepromatous leprosy patients (21, 26). It has been reported that immunization with naked plasmid DNA (pDNA) encoding Ag85A and Ag85B can stimulate strong humoral and cell-mediated immune responses and confer significant protection to C57BL/6 mice challenged by the aerosol or intravenous route with live M. tuberculosis H37Rv (1, 19, 22). Recently, priming with Ag85B DNA was shown to augment the protective efficacy of M. bovis BCG (10), and recombinant BCG overexpressing Ag85B was found to have increased immunogenicity and efficacy in guinea pigs (16). Finally, a fusion protein consisting of Ag85B and ESAT-6 is a very promising protein-subunit vaccine candidate for TB (41).
Another promising TB DNA vaccine consists of DNA encoding the 40-kDa protein PstS-3 (38). PstS-3, PstS-1 (also called the 38-kDa antigen), and PstS-2 are surface-exposed lipoproteins that are putative mycobacterial phosphate transport proteins, homologous to phosphate-binding protein PstS of Escherichia coli (2, 27).
Although the immunogenicity of DNA vaccines in humans is promising, increasing the potency of DNA vaccines is a clear necessity (40). Increased pDNA-induced antibody responses can be obtained, among others, by complexation with conventional adjuvants, such as monophosphoryl lipid A (34), alum (40), and QS-21 saponin (35). Priming with DNA followed by boosting with either purified protein (37) or recombinant modified vaccinia virus Ankara (29) was also shown to increase the immunogenicity and protective efficacy of DNA vaccines consisting of DNA encoding Ag85A. Here we report on an approach for improving TB DNA vaccines by formulation in two novel cationic and neutral colipid formulations, GAP-DLRIE:DOPE (aminopropyl-dimethyl-bis-dodecyloxy-propanaminium bromide-dioleoylphosphatidyl-ethanolamine) and VC1052:DPyPE (aminopropyl-dimethyl-myristoleyloxy-propanaminium bromide-diphytanoylphosphatidyl-ethanolamine), the latter also called Vaxfectin (Vical, Inc., San Diego, Calif.). Both formulations were previously demonstrated to enhance antibody responses to pDNA given by the intranasal and intramuscular routes, respectively (13, 32; L. Sukhu, M. Wloch, M. Sawdey, C. Wheeler, and M. Manthorpe, Abstr. 2nd Annu. Meet. Am. Soc. Gene Ther., abstr. 530, 1999). We show that these lipids can be used as adjuvants for DNA-based vaccination with Ag85 and PstS-3 from M. tuberculosis, resulting in significantly increased antibody titers and levels of Th1-type cytokine production in the spleen and lungs and more sustained cytolytic T-lymphocyte (CTL) responses as well as increased protective efficacy against an intravenous challenge with bioluminescent M. tuberculosis H37Rv.
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MATERIALS AND METHODS |
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Mice. C.D2 mice (BALB/c background, Bcgr allele) and C57BL/10 (B10) mice were bred in the animal facilities of the Pasteur Institute of Brussels from breeding pairs originally obtained from E. Skamene (McGill University, Montreal, Quebec, Canada) and R. ten Berg (Netherlands Cancer Institute), respectively. C57BL/6 (B6) mice were obtained from Bantin and Kingman (Grimston, United Kingdom). Only female mice, 6 to 8 weeks old at the start of vaccination, were used.
DNA immunizations. Mice were anesthetized by intraperitoneal injections of ketamine-xylazine. For intramuscular immunizations, mice were injected in both quadriceps or tibialis anterior muscles with two 50-µl volumes (total dose, 50 µg) of empty vector (control DNA) or plasmid vector containing Ag85A, Ag85B, or PstS-3 DNA in saline or formulated in Vaxfectin at a pDNA/cationic lipid molar ratio of 2:1. For intranasal immunizations, two 10-µl volumes of pDNA (total dose, 20 µg of DNA) in saline or formulated in GAP-DLRIE:DOPE (molar ratio, 4:1) were deposited in the left and right nares with a resting time of 60 s between the two instillations. Mice were immunized by the intramuscular, intranasal, or combined routes three or four times at 3-week intervals (as detailed further below).
For BCG vaccination, mice were injected intravenously with 0.1 mg (about 5 x 105 CFU) of M. bovis BCG (strain GL2), freshly prepared from surface-grown pellicles on synthetic Sauton medium.
ELISA. Sera from pDNA-immunized mice were collected by retro-orbital bleeding 3 weeks after the last immunization. For collection of bronchoalveolar fluid, mice were sacrificed by cervical dislocation and lungs were gently rinsed with 1 ml of phosphate-buffered saline injected with an 18-gauge needle-syringe through a narrow split in the trachea. Levels of total anti-Ag85 immunoglobulin antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) with sera from individual mice (three to five per group). The serum titer was converted to antibody concentration (nanograms per milliliter) by comparison with a standard monoclonal antibody with known potency, and the mean antibody concentration was calculated from at least three points of the linear portion of the titration curve. Concentrations were converted to log10 values. For immunoglobulin isotype analysis, equal volumes of sera were pooled per group and examined with peroxidase-labeled rat anti-mouse IgG1, IgG2a, IgG2b, and IgA (Experimental Immunology Unit, Université Catholique de Louvain, Brussels, Belgium). Immunoglobulin isotype titers were converted to arbitrary units by comparison with a standard serum pool from Ag85 DNA-immunized mice, arbitrarily assigned a titer of 1,000 for all isotypes.
Antigens. Native 32-kDa Ag85A and 30-kDa Ag85B were purified from 2-week-old culture filtrates of M. bovis BCG (strain GL2) grown as a surface pellicle on synthetic Sauton medium by sequential chromatography on phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-75 (4). Synthetic 20-mer peptides (overlapping by 10 amino acids) covering the entire mature Ag85A sequence were synthesized as described before (20). M. tuberculosis culture filtrates were prepared from 2-week-old cultures of M. tuberculosis H37Rv grown as a surface pellicle on Sauton medium and concentrated by 70% (NH4)2SO4 precipitation (18).
Cytokine production.
Vaccinated mice were sacrificed 3 weeks after the last immunization, and spleens and lungs were removed aseptically. Organs were homogenized by gentle disruption in a loosely fitting Dounce homogenizer, and lung cell suspensions were passed through a nylon-wool column to eliminate debris. Spleen cells from three or four mice per group were tested individually, whereas lung cells were pooled for each group. Cells were tested at 4 x 106 white blood cells/ml for cytokine production in response to purified Ag85A or Ag85B (5 µg/ml), culture filtrates from M. tuberculosis (25 µg/ml), or synthetic Ag85A peptides (10 µg/ml). Supernatants were harvested after 24 h (interleukin-2 [IL-2]) and 72 h (IFN-), when peak values of the respective cytokines can be measured. Supernatants from at least three separate wells were pooled and stored frozen at -20°C until assayed.
IL-2 assay. IL-2 activity was measured by using a bioassay with IL-2-dependent CTLL-2 cells as described before (18). Each sample was tested in duplicate. IL-2 levels were expressed as mean counts per minute of incorporated [3H]thymidine. The standard deviation was below 10%. In this assay, a standard preparation of IL-2 at 600 pg/ml corresponded to about 15,000 cpm, and the detection limit was 30 pg/ml.
IFN- assay.
IFN- activity was quantified by a sandwich ELISA with coating antibody R4-6A2 and biotinylated detection antibody XMG1.2 (both from Pharmingen). The sensitivity of the ELISA was 10 pg/ml.
Cytolytic assay. PstS-3-specific cytotoxic T-cell activity was determined with B6 mice according to a protocol previously described for Ag85A in BALB/c mice (6). Briefly, splenic lymphocytes from B6 mice vaccinated with PstS-3 DNA 1 or 5 months previously were stimulated for 1 week with Db-restricted peptide SGVGNDLVL (amino acids 291 to 299 of the mature PstS-3 sequence), purified on a Ficoll gradient, and tested for cytolytic activity in a standard 4-h 51Cr release assay; this assay was done with 104 51Cr-labeled RMA cells as targets (5), unpulsed or pulsed with the same peptide (5 µg/ml), at various effector/target ratios. Spontaneous or total release samples were prepared by adding targets to wells containing medium only or medium plus 2 M H2SO4, respectively. After 4 h of incubation at 37°C, the plates were centrifuged and 150 µl of supernatant was collected and counted in a gamma counter (LKB). Data were expressed as percent specific lysis. Spontaneous release was generally 10 to 15% of total release.
Intravenous M. tuberculosis H37Rv challenge. B10 mice were vaccinated intramuscularly with control DNA (empty vector V1J.ns) or Ag85B DNA in saline or in Vaxfectin or intravenously with M. bovis BCG. Mice were rested for 6 weeks after the third immunization and then challenged intravenously in a lateral tail vein with 106 CFU of luminescent, recombinant luciferase reporter M. tuberculosis H37Rv (36). Mice were sacrificed 14, 28, and 56 days after challenge, and the number of CFU in the lungs was enumerated by plating on 7H11 Middlebrook agar (37). The number of bioluminescent organisms (determined as relative light units [RLU]) in lung and spleen homogenates was also determined by using a bioluminescence assay with a Turner Design 20/20 luminometer and 1% n-decyl-aldehyde in ethanol as a substrate (36). It was shown previously that RLU counting is an easy and reliable alternative for labor-intensive CFU enumeration (37). For statistical analysis (Student's t test), CFU and milli-RLU (mRLU) values were converted to log10 values per organ per mouse; mean and standard deviation log10 CFU or mRLU values were calculated for each experimental group, which consisted of four to seven animals tested individually (see Table 5).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production.
IL-2 and IFN- production in response to purified native Ag85 and to culture filtrates from M. tuberculosis was increased by the Vaxfectin formulation two- to fourfold in cultures of spleen cells from C.D2 and B10 mice immunized with DNA encoding Ag85A or Ag85B (Table 2). IL-2 and IFN- production in response to synthetic 20-mer peptides spanning the mature Ag85A sequence was also two-to fourfold higher in C.D2 mice vaccinated with Ag85A DNA complexed in Vaxfectin than in mice immunized with Ag85A DNA in saline (Fig. 1). Interestingly, Vaxfectin seemed to increase preferentially CD4+-T-cell-mediated IFN- responses, whereas IFN- production in response to the previously defined major histocompatibility complex (MHC) class I-restricted peptides p7 and p15 (6) appeared not to be influenced by Vaxfectin.
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response in the draining mediastinal and cervical lymph nodes (data not shown).
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DISCUSSION |
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Here we have shown that formulation of pDNA vaccines consisting of DNA encoding three M. tuberculosis antigens with novel cationic lipid formulations is another effective means for increasing their immunogenicity and protective efficacy. Cationic lipid formulations have been reported to enhance antibody responses induced by pDNAs given by the intranasal route (Sukhu et al., Abstr. 2nd Annu. Meet. Am. Soc. Gene Ther.) and the intramuscular route (13, 32), but little is known so far about their effect on Th1-type cytokine responses. From the results presented here, it is clear that these cationic lipids can function as strong adjuvants for CD4+-T-cell-mediated IL-2 and IFN- production as well. Moreover, and somewhat in contradiction with previous reports (13, 32), we found that Vaxfectin also had a favorable effect on CD8+-T-cell-mediated responses. Vaxfectin had no effect on CTL responses measured shortly after immunization, but CTL memory was more sustained in the Vaxfectin-treated animals. It can be speculated that this increased CTL memory was the consequence of a higher level of IL-2 production in Vaxfectin-treated animals, which might have had a direct effect on the generation of CTL memory precursors during immunization. The fact that we used higher doses of DNA and three immunizations instead of two might explain the discrepancy between our results and those of other published reports.
It is not clear at the moment how Vaxfectin exerts its adjuvant effect. The lipid does not facilitate plasmid transfection of myocytes, nor does it increase the transcription or translation of a ß-galactosidase reporter plasmid in muscle tissue (13). On the other hand, Vaxfectin may increase the plasmid transfection of other cells, such as lung epithelial cells (42), macrophages, and dendritic cells (DCs). DCs are known to migrate from the tissues to the lymph nodes within 24 h when stimulated to maturity by exposure to lipopolysaccharide via Toll-like receptor 4 (TLR4) signaling. The migration and maturation of DCs may be enhanced by Vaxfectin through a similar stimulation of TLR4; moreover, this process could take place in concert with the stimulation of TLR9, which recognizes specifically immunostimulatory CpG motifs in bacterial pDNA (3, 14). Also, the adjuvant effect may involve increased antigen presentation, as cationic lipids related to Vaxfectin (specifically DMRIE:DOPE) are known to upregulate MHC class I molecules on tumor cells in tissue cultures (12). Moreover, cationic lipids certainly protect pDNA from nuclease degradation, and this effect may be of particular importance for mucosal immunizations. Finally, lipid-DNA complexes may have inherent stimulatory properties for Th1 and B cells through the induction of IL-12 or IFN- (8) and IL-6 (32), respectively.
Confirming previous findings with Ag85A DNA (9, 38), the protective efficacy of the DNA was only transient, and mice vaccinated with Ag85B DNA in saline were protected only during the first weeks after challenge. In contrast, mice vaccinated with the Vaxfectin DNA formulation showed a higher and more sustained reduction in CFU and RLU in the lungs. A number of factors, such as impaired signal transduction in IFN--activated lung macrophages and increased secretion of suppressive factors, such as transforming growth factor ß, may be involved in the waning of protection (11). It can be speculated that in DNA-Vaxfectin-immunized mice, larger numbers of Ag85-specific precursor and effector T cells might result in more strongly activated macrophages in which mycobacteria would be more efficiently eliminated, in turn resulting in a lower bacterial burden in macrophages, less impaired signal transduction, and a lower level of secretion of suppressive factors.
TB infection has been reported to specifically downregulate MHC class II expression (15); hence, Ag85-specific CD4+ T cells may become ineffective at some point in time because the relevant epitopes are no longer presented on infected cells. As neither Ag85A nor Ag85B contains Kb- or Db-restricted epitopes in its sequence (9), the totality of the immune response induced by Ag85 DNA vaccines in H-2b mice is mediated by CD4+ T cells. With the progression of infection, certain proteins of M. tuberculosis can escape from phagosomal containment and reach the cytoplasm, where they become accessible to MHC class I-restricted presentation (31). For Ag85 and B6 mice, this rescuing MHC class I pathway may be completely lacking. A combination of the Ag85 DNA vaccine, which stimulates strong CD4+-T-cell responses, with CTL epitopes, such as the Db-restricted epitope of the PstS-3 lipoprotein, described in this report, may help to overcome this problem (M. Romano, unpublished data). In summary, we have shown that DNA vaccines consisting of DNA encoding three M. tuberculosis antigens can be formulated in cationic lipids, resulting in increased antibody production and Th1-type cytokine secretion in the spleen and more importantly in the lungs, more sustained CD8+-T-cell-mediated CTL memory, and prolonged protection against M. tuberculosis challenge. Cationic lipids can be easily manufactured and have been found safe and well tolerated in animal and clinical trials. Our results may therefore be of importance for the future clinical use of TB DNA vaccines in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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, P < 0.01; °, P < 0.025 (compared to value for mice vaccinated with Ag85B DNA in Vaxfectin).