Th-Cytotoxic T-Lymphocyte Chimeric Epitopes Extend
http://www.100md.com
病菌学杂志 2005年第24期
The Cellular and Molecular Immunology Laboratory, The Eye Institute, University of California, Irvine, School of Medicine, Irvine, California 92697-4375
TwentyFirst Century Biochemicals Center for Immunology, University of California, Irvine, Irvine, California 92697-1450
ABSTRACT
Molecularly defined vaccine formulations capable of inducing antiviral CD8+ T-cell-specific immunity in a manner compatible with human delivery are limited. Few molecules achieve this target without the support of an appropriate immunological adjuvant. In this study, we investigate the potential of totally synthetic palmitoyl-tailed helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL chimeric lipopeptides) to induce herpes simplex virus type 1 (HSV-1)-specific CD8+ T-cell responses. As a model antigen, the HSV-1 glycoprotein B (498-505) (gB498-505) CD8+ CTL epitope was synthesized in line with the Pan DR peptide (PADRE), a universal CD4+ Th epitope. The peptide backbone, composed solely of both epitopes, was extended by N-terminal attachment of one (PAM-Th-CTL), two [(PAM)2-Th-CTL], or three [(PAM)3-Th-CTL] palmitoyl lysines and delivered to H2b mice in adjuvant-free saline. Potent HSV-1 gB498-505-specific antiviral CD8+ T-cell effector type 1 responses were induced by each of the palmitoyl-tailed Th-CTL chimeric epitopes, irrespective of the number of lipid moieties. The palmitoyl-tailed Th-CTL chimeric epitopes provoked cell surface expression of major histocompatibility complex and costimulatory molecules and production of interleukin-12 and tumor necrosis factor alpha proinflammatory cytokines by immature dendritic cells. Following ocular HSV-1 challenge, palmitoyl-tailed Th-CTL-immunized mice exhibited a decrease of virus replication in the eye and in the local trigeminal ganglion and reduced herpetic blepharitis and corneal scarring. The rational of the molecularly defined vaccine approach presented in this study may be applied to ocular herpes and other viral infections in humans, providing steps are taken to include appropriate Th and CTL epitopes and lipid groups.
INTRODUCTION
Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) are prevalent microbial pathogens, affecting up to 90% of the adult population throughout the world (3, 23, 43). HSV-1, the most prevalent cause of viral infection of the eye (3, 23, 34, 43), causes a spectrum of clinical manifestations ranging from blepharitis, conjunctivitis, and dendritic keratitis to disciform stromal edema and necrotizing stromal keratitis (3, 23, 34). The corneal scarring induced by HSV-1 often leads to corneal opacification, making this virus the leading cause of blindness by an infectious agent in developed countries (3, 23, 43). In the United States alone, every year more than 450,000 people exhibit recurrent ocular HSV episodes requiring doctor visits, drug treatments, and in severe cases, corneal transplants (15). Despite pharmaceutical approaches to therapy and prevention, ocular herpes infections are still prevalent and no vaccine has yet been approved for clinical use (3, 34). Developing an effective immunoprophylactic and/or immunotherapeutic vaccine would represent a powerful and cost-effective means of controlling ocular herpes infection (3, 30, 31, 34).
Direct and indirect evidence, in both animal models and humans, suggests that CD8+ T cells play a major role in antiherpesvirus immunity (11). While CD8+ T cells are important effectors responsible for the clearance of herpes infections, CD4+ T cells are required to help prime and sustain antiviral CD8+ T-cell immunity (3, 41, 43). Consequently, an immunogenic formulation should be targeted towards inducing both CD4+ and CD8+ T-cell populations (3, 11, 22, 27, 35, 43). Concomitant CD4+ Th-cell responses can be easily induced by targeting exogenous major histocompatibility complex (MHC) class II pathways of antigen (Ag) presentation (3, 41, 43). However, induction of CD8+ T-cell responses requires Ag synthesis or transport by an appropriate vector into the cytoplasm of antigen-presenting cells (APCs) (1, 14, 28, 41). Many strategies for CD8+ cytotoxic T-cell (CTL) activation are based on immunization with recombinant live attenuated viruses or bacteria that infect APCs and express the target epitope(s) (reviewed in references 28 and 41). Yet, beside the safety issues that still remain to be addressed before widespread clinical use of many live vectors, protective CTL responses are usually only obtained following a prime-boost regimen (28, 41). Approaches other than live vectors use administration of naked DNA plasmids, from which the target epitopes are directly synthesized within the APCs, or immunization with heat shock or bacterial proteins that channel selected epitopes into MHC class I Ag presentation pathways (14, 28, 41). Alternatively, directly "jump-starting" APCs and CD8+ T cells by loading selected minimal epitope peptides onto MHC molecules at the surfaces of APCs has been proposed (1, 14, 41). In vivo delivery of peptide epitopes either loaded on APCs or emulsified with strong immunological adjuvants induces specific CD8+ T-cell responses in murine models (9). Unfortunately, among the large number of adjuvants that have been tested in small laboratory animal models, many are toxic and have limited clinical usefulness (9).
To avoid toxic adjuvant effects, recent attempts have used chemical modifications of peptide epitopes that act as built-in adjuvants (9, 18, 37, 38). One of the most advanced modifications, which has reached phase II clinical trials in Europe, uses peptide epitopes extended by a lipid moiety (lipopeptides) (9, 16, 29, 33, 42). Lipopeptides have attracted much attention as potential safe vaccines for many microbial pathogens as well as cancers, with the capacity to promote potent CD8+ CTL responses when delivered in adjuvant-free saline (reviewed in reference 9).
In this study, we hypothesize that potent virus-specific CD8+ T-cell responses would be induced by optimal HSV-1 T-cell epitope(s) conjugated to a lipid moiety and delivered in adjuvant-free saline. We asked if palmitoyl-tailed chimeric CD4+ Th-CD8+ CTL epitopes would be sufficient to induce protective immunity in the mouse model of ocular herpes infection. We chose HSV-gB498-505, recognized by H2-Kb-restricted CTLs, as a target epitope (11, 19). We fused the HSV-gB498-505 epitope with the Pan HLA-DR-binding epitope (PADRE), a promiscuous CD4+ Th determinant that binds to most murine and human MHC class II molecules (5, 26, 32, 39). To assess the influence of the number of lipid units on the induction of protective CD8+ T cells, the resulting chimeric peptide backbone, solely composed of both Th and CTL epitopes, was extended by N-terminal addition of one, two, or three palmitic acid moieties. The palmitoyl-tailed HSV-gB498-505-PADRE chimeric epitopes delivered in adjuvant-free saline induced potent virus-specific gamma interferon (IFN-)-producing CD8+ CTLs in H2b mice. Interestingly, such immunization reduced acute infection in the eye, lessened ocular herpetic disease, and interfered with latent infection in trigeminal ganglia (TGs) following an ocular HSV-1 challenge. These results suggest that palmitoyl-tailed Th-CTL chimeric epitopes should be considered as a safe alternative to more complex vaccine strategies for human.
MATERIALS AND METHODS
Peptide and lipopeptides synthesis. The SSIEFARL CD8+ CTL peptide (HSV-gB498-505) (10) and the {dA}K{Cha}VAAWTLKAA{dA}{Ahx}C Pan DR peptide (PADRE) (32) were synthesized either individually or as PADRE-CTL chimeric epitopes using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, with PyBOP/HOBt (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate/N-hydroxybenzotriazole) activation. The parental peptides were washed three times with dimethylformamide (DMF) and treated twice with 2% hydrazine in DMF. After six additional washings with DMF and hydrazine acetic acid, peptides were cleaved using trifluoroacetic acid (TFA)-triisopropylsilane-water (95:2.5:2.5) and the resultant peptides precipitated in cold ether. The peptides were then washed (three times) with cold ether and analyzed by mass spectrometry (MS) and high-performance liquid chromatography (HPLC). Purification of the peptides was performed using Gilson HPLCs with Vydac C18 columns (2.2 by 25 cm or, for larger amounts of crude peptide, 5 by 25 cm). The analysis was performed using Vydac C18, 5-μm, 0.46- by 25-cm columns, with a gradient of 2% per minute of water, 0.1% TFA, 95% acetonitrile, and 0.1% TFA. Once peptides were purified to over 95%, they were lyophilized. Mass-spectrometric analysis was performed with an MDS/Sciex QStar XL mass spectrometer equipped with a nanospray source. Final quality control included collision-induced dissociation tandem MS experiment using nitrogen as the collision gas, which provided sequence confirmation of the peptides.
For the lipopeptide synthesis, one, two, or three N-terminal attachment points were created for subsequent attachments of glyoxylyl lipids following synthesis and purification. This was achieved by adding one, two, or three lysine residues whose side chains were selectively protected with 1-(4,4-dimethyl-2,6-dioxoxyxlohex-1-ylidene)-3-methylbutyl, a hydrazine-sensitive side chain protecting group. In order to allow maximal attachment of the lipid moieties, the lysine residues were interspaced with alanines. The synthesis of the glyoxylyl derivative of palmitate and ligation of peptides were performed using a modification of chemoselective ligation (18). Briefly, dimethyl-2,3-O-isopropylidene tartrate was added to a polyethylene glycol amino resin using PyBOP activation. The second ester was then displaced via the addition of 1,3-diaminopropane. Finally, palmitic acid was activated using PyBOP and used to acylate the amino terminus. Treatment with TFA followed by periodate oxidation generated the alpha-oxo-aldehyde moiety. Following lyophilization, the peptides were transferred in 50-ml round-bottom flasks, fitted with septa, and flushed with nitrogen. A minimal amount of degassed water was added until the peptides were solubilized and displayed as a gel. Stochiometric amounts of lipid were then added in 2-methyl-propan-2-ol dropwise with stirring. The final ratio of water to organic solvent was 95:5. To add the second and third lipids, an aliquot equal to 120% of the concentration of the peptide was added, with 10 to 20 min of stirring in between each addition. The reactions were monitored using the QStar XL mass spectrometer (see Fig. 1). The disappearance of the parent peptide was observed concomitantly with the appearance of the lipid-tailed peptide. In all cases, the parent peptide was not detectable at the end of the acylation process.
Virus and cell lines. Triple plaque-purified wild-type McKrae HSV-1 was prepared as we described previously (3). Rabbit skin (RS) cells used to prepare the virus stocks and culture the virus from eye swabs were grown in Eagle's minimum essential medium supplemented with 5% fetal calf serum (InvitrogenGibco, Grand Island, NY).
Mice. Female C57BL/6 (H2b) mice, ages 4 to 5 weeks, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were handled in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
Peptide and lipopeptide immunization. In an initial experiment, we determined the optimal dose response to the HSV-gB498-505 epitope following immunization with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL lipopeptides and found no differences between 50-, 100-, and 200-μg doses (not shown). Subsequent experiments were then carried out with a medium dose of 100 μg of each construct delivered in phosphate-buffered saline, injected subcutaneously (s.c.) on days 0 and 14. As a negative control, mice were injected s.c. with saline alone (mock-immunized mice).
Cytokine ELISAs. Two weeks after the second immunization, mice were euthanized and single suspensions of spleen (SP) cells were stimulated either with the target HSV-gB498-505 alone or with heat-inactivated HSV-infected stimulator cells. Cells were plated at a density of 1 x 106/well in 24-well plates in a humidified 5% CO2 atmosphere. Seventy-two hours later, the supernatants were collected and the concentrations of interleukin-2 (IL-2), IL-4, and IL-12, and IFN- were determined in a sandwich ELISA using kits specific for each cytokine according to the manufacturer's instructions (BD PharMingen, San Diego, CA).
T-cell proliferation. SP were removed and placed into ice-cold serum free HL-1 medium supplemented with 15 mM HEPES, 5 x 10–5 M ?-mercaptoethanol, 2 mM glutamine, 50 IU of penicillin, and 50 μg of streptomycin (GIBCO-BRL, Grand Island, NY) (CM) (2, 3). The cells were cultured in 96-well plates at 5 x 105 cells/well in CM, with recall PADRE peptide at a 1-μg/ml concentration, with heat-inactivated HSV-1 (0.3 multiplicity of infection), or with concanavalin A as a positive control. The SP cell suspensions were incubated for 72 h at 37°C in 5% CO2, and their proliferation was determined in an [3H]thymidine incorporation assay as we described previously (2, 4).
IFN- ELISpOT assay. SP cells were cultured in 24-well plates for 5 days in a humidified 5%-CO2 atmosphere with HSV-gB498-505 peptide alone (10 μg/ml), the irrelevant OVA257-264 CD8+ T-cell peptide (10 μg/ml), or autologous HSV-1-infected or mock-infected stimulator cells and subsequently analyzed in an IFN- enzyme-linked immunospot (ELISPOT) assay. Functional T-cell recognition IFN- ELISPOT assays were performed with the mouse IFN- ELISPOT monoclonal antibody (MAb) pair (BD PharMingen, San Diego, CA). Briefly, on day 4, 96-well multiscreen immunoprecipitation plates were coated overnight at 4°C with 100 μl (1:250) of anti-IFN- capture MAb. Plates were then blocked with RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) for 2 h at room temperature. Cells (5 x 104/well) were added in triplicate to MAb-coated plates and incubated for an additional 18 to 24 h at 37°C, 5% CO2. Plates were then washed with phosphate-buffered saline and supplemented with a detection peroxidase-labeled antibody followed by a substrate according to the manufacturer's instructions. The developed spots were counted under a light microscope.
Flow cytometry. Standard flow cytometry was employed, as we described previously (2, 9), to assess surface expression of various markers using the following MAbs directly conjugated with either phyocoerythrin (PE) or fluorescein isothiocyanate (FITC): FITC-CD4, FITC-CD8, PE HSV-gB tetramer, PE-CD80 (clone 10-10A1, immunoglobulin G [IgG]), PE-CD86 (clone GL1, IgG2a), FITC-MHC-I (clone TU36, IgG2b), and FITC-MHC-II (clone FLI8.26, IgG2b) (PharMingen (San Diego, CA). IgG isotype-matched control antibodies were used in all experiments. After staining, cells were washed and fixed in 1% buffered paraformaldehyde before being acquired on a Becton Dickinson (Mountain View, CA) FACSCalibur instrument. Gating was done on large granular cells, and for each sample, 20,000 events were acquired on a FACSCalibur instrument and analyzed with CellQuest software on an integrated Macintosh G4 (Becton Dickinson, San Jose, CA).
CTL activity. Spleen-derived immune CD8+ T cells were restimulated in vitro with HSV-gB498-505 target peptide (5 μg/ml)-pulsed syngeneic irradiated T-depleted splenocytes as we described previously (2, 8). Irradiated EL-4 cells (3,000 rad from a 137Cs source) were added to the coculture as APC. CTL activity was assessed by a standard 4-h 51Cr release assay against EL4 target cells loaded with 10 μM HSV-gB498-505 target peptide or infected with heat-inactivated HSV-1 (multiplicity of infection = 3) as described previously (6, 8). After 5 days of culture, effector CD8+ cells were mixed at 1, 3, 10, 30, and 100 effector/target (E/T) ratios with 51Cr-labeled EL4 cells for 4 h. Maximum release of 51Cr was determined by adding 5% Triton X-100 to 51Cr-labeled EL4 cells. Spontaneous release (<10% of total release) was determined by incubating target EL4 cells with medium alone. The percentage of specific lysis was calculated as follows: 100 x [(experimental release – spontaneous release)/(maximum release – spontaneous release)].
Generation and maturation of bone marrow-derived DCs. Murine bone marrow-derived immature dendritic cells (DCs) were generated using our previously described protocol (2, 9). Immature DCs were incubated in vitro for 72 h with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide and monitored for cell surface expression of MHC class I, CD80, and CD86 costimulatory molecules and for production of proinflammatory IL-12 and IFN- cytokines. Medium-treated immature DCs and LPS stimulated DCs were included as negative and positive controls, respectively.
Virus challenge and ocular disease reading. Ocular challenge was performed 2 weeks postimmunization. An inoculum of 4 x 105 PFU of HSV-1 (McKrae) in 4 μl tissue culture medium was placed on each eye, and the lids were gently rubbed against the eye for 30 s. The severity of blepharitis and corneal scarring in each group of 10 mice was assessed by examination with a slit-lamp biomicroscope after addition of 1% fluorescein dye as eye drops. The examination was performed by investigators masked to the treatment and scored according to a standard 0-to-4 scale.
Monitoring replication and clearance of HSV-1 from the eye. This procedure was performed by swabbing eyes of 10 mice once daily (1 to 10 days) with a Dacron swab and transferring each swab to a 75-mm culture tube containing 0.5 ml of medium. Aliquots (100 μl) of 10-fold serial dilutions were placed on confluent monolayer of RS cells in six-well plates, incubated at 37°C for 1 h, and overlaid with medium containing 1% methylcellulose. The plates were incubated at 37°C for 3 days and stained with 1% cresyl violet, and the viral plaques were counted.
Detection of latent virus in trigeminal ganglia. Equal numbers of mice in each group surviving 30 days postinfection were euthanized, their TGs removed, and individually explanted onto RS cell monolayers in RPMI medium. The culture was monitored for 10 days for the presence of infectious virus.
Statistical analysis. Protective parameters were analyzed by Student's t test and the Fisher exact test (Instat; GraphPad, San Diego, CA). Results were considered to be statistically significant at P values of <0.05.
RESULTS
(i) Construction and physicochemical characterization of palmitoyl-tailed Th-CTL chimeric epitopes. The immunodominant HSV-1-gB498-505 CD8+ T-cell target epitope (11, 35) was fused to the PADRE peptide (5, 26, 39). To optimize its immunogenic potential, the resulting Th-CTL chimeric peptide backbone was covalently linked to one (PAM-Th-CTL), two [PAM)2-Th-CTL], or three [(PAM)3-Th-CTL] N-palmitoyl-lysine residues via functional base lysine -amino groups. An illustration of the overall structure of each of the three palmitoyl-tailed chimeric Th-CTL constructs is presented in Fig. 1A and B. Construction of these chimeric lipopeptides was carried out using a modified version of the newly described chemoselective ligation method that allows synthesis of high-yield, molecularly pure, and water-soluble molecules (12, 18). Unlike the "first generation of lipopeptides" synthesized using classic solid-phase methods which introduce the fatty acyl moiety onto the crude peptide backbone before its purification (4, 7, 8, 13), the lipopeptides employed in this study were constructed in two steps (12, 18, 43). The first step of synthesis and purification of the peptide backbone was followed by a second step of ligation of the lipid moiety, site-specifically introduced in solution (12, 18, 43). This method is compatible with sequences that encompass hydrophobic residues: cysteines, l-alanines (dA), l-cyclohexyl alanines (Cha), or aminocaproic acids (Ahx), such as PADRE [(dA)K(Cha)VAAWTLKAA(dA) (Ahx)]. Indeed, no aggregation was observed when PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL were formulated in water or in saline solution. Physicochemical properties of these Th-CTL chimeric lipopeptides were compatible with multidimensional analysis. All constructs were eluted as a single peak in analytical reverse-phase HPLC with the expected mass and sequences when analyzed by mass spectrometry (Fig. 1C and D). The physicochemical characterization highlights the fine molecular definition of palmitoyl-tailed Th-CTL chimeric epitopes as totally synthetic vaccine constructs, which would probably make them suitable for potential future clinical application.
(ii) Palmitoyl-tailed Th-CTL chimeric epitopes trigger dendritic cell maturation regardless of the number of lipid moieties. We first examined the ability of Th-CTL chimeric lipopeptides to induce phenotypic maturation of DCs. Incubation of immature DCs with Th-CTL chimeric lipopeptides induced significant expression of MHC class I, CD80, and CD86 molecules compared to medium-treated immature DC controls (Fig. 2A) (P < 0.005). Maturation of DCs stimulated with Th-CTL chimeric lipopeptides occurred irrespective of the number of lipid moieties. As a second indicator of maturation, exposure of DCs to chimeric lipopeptides was associated with a lipopeptide dose-dependent increase in IL-12 and IFN- secretion (Fig. 2B). Under similar conditions, there was no up-regulation of MHC and costimulatory molecules or production of IL-12 or TNF- following incubation of immature DCs with the parental nonlipidated peptides alone or the palmitic acid alone (Fig. 2A and B) (P < 0.05). Together these results indicated that Th-CTL chimeric lipopeptides, but not the Th-CTL parental peptide, provoked DC maturation and that covalent linkage to the lipid moiety is required for DC maturation.
(iii) Palmitoyl-tailed PADRE-HSV-gB498-505 chimeric epitopes delivered in saline prime for antigen-specific CTL and Th responses. Next, we evaluated the ability of equimolar amounts of PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL chimeric lipopeptides to prime for gB498-505- and HSV-1-specific CTLs in H2b mice following a subcutaneous delivery in saline. Two weeks after the second injection, functional HSV-gB498-505-specific CTLs were evaluated in the SP by a standard 51Cr release assay. Irrespective of the number of lipid groups, mice immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide developed CTLs that lyse autologous H2b EL-4 cells loaded with target HSV-gB498-505 epitope but not EL-4 cells loaded with the irrelevant OVA257-264 CTL peptide (Fig. 3A and B) (P < 0.005). When the magnitude of CTL activity was compared at an equal E/T ratio of 30, each Th-CTL chimeric lipopeptide generated more than 50% of HSV-gB498-505-specific lysis, with no significant difference in the maximal specific lysis induced by the three Th-CTL chimeric lipopeptides (Fig. 3B). More important, CTLs induced by Th-CTL chimeric lipopeptides killed HSV-1-infected target cells but not mock-infected cells (Fig. 3C and D) (P < 0.005). At equal E/T ratios, there was no effect of the number of lipid units on the magnitude of virus-specific CTL activity elicited by each Th-CTL chimeric lipopeptides. The HSV-gB498-505-specific CTL activity induced by the Th-CTL chimeric lipopeptides was abrogated by CD8 MAb blockage (not shown). Effector T cells derived in parallel from SP of mock-immunized mice and restimulated in vitro with gB498-505 epitope did not lyse target EL-4 cells loaded with gB498-505 peptide or target cells infected with HSV-1 (Fig. 3A to D). This experiment provides a control to illustrate that in vitro amplification of effector T cells with gB498-505 epitope does not by itself induce primary CTLs in culture. There was a lack of specific CTLs in mice immunized with nonlipidated Th-CTL chimeric peptide, with PADRE peptide alone, with HSV-gB498-505 alone, or with palmitic acid alone (Fig. 3), indicating the requirement of the three components (i.e., Th and CTL epitopes and the lipid moiety) attached together for induction of efficient CTL responses.
We next determined the magnitude of concomitant PADRE-specific CD4+ T-cell responses induced by each palmitoyl-tailed Th-CTL chimeric epitope construct. Two weeks after the second immunization with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL, SP-derived CD4+ T cells were isolated in each group and restimulated in vitro with the PADRE peptide or with the irrelevant gD1-29 Th peptide. Gated CD4+ T-cell responses were then examined by fluorescence-activated cell sorting for cell surface expression of interleukin-2 receptor (CD25) as a marker of activation (Fig. 3E). Proliferating CD4+ T cells were also assessed in an [3H]thymidine incorporation assay (Fig. 3F). As shown in Fig. 3E and F, each chimeric lipopeptide generated substantial PADRE-specific CD4+ T-cell stimulation, regardless of the number of lipid moieties. The specificity of the Th response was ascertained with a lack of response to the gD1-29 peptide. Only insignificant T-cell responses were detected in mock-immunized mice or in mice immunized with Th-CTL parental peptide alone or with palmitic acid alone (Fig. 3E to F).
(iv) HSV-1-specific CD8+ CTL response induced by palmitoyl-tailed Th-CTL chimeric epitopes is accompanied by IFN- release. Twelve weeks after final immunization, we evaluated gB498-505- and HSV-1-specific IFN--producing memory CD8+ T cells induced by each lipopeptide construct. Significant numbers of spot-forming cells (SFCs) producing IFN- specific to gB498-505 were generated by each palmitoyl-tailed Th-CTL chimeric lipopeptide (Fig. 4A) (P < 0.005). There was a slight increase in IFN--producing memory CD8+ T cells with the increase in the number of lipid units. A range of 150 to 178 T cells per 10,000 SP cells produced IFN- in response to the gB498-505 peptide, but there was negligible stimulation by the control OVA257-264 peptide (5 to 10 T cells per 10,000 SP cells), indicating the specificity of the response.
The induced memory IFN--producing CD8+ T cells recognized a naturally processed epitope on HSV-1-infected cells, with only a slight increase of the number of SFCs induced by the (PAM)2-Th-CTL and (PAM)3-Th-CTL compared to results with the PAM-Th-CTL lipopeptide analogue (Fig. 4B). Ranges of 15 to 25, 28 to 32, and 34 to 39 T cells per 10,000 SP cells were induced by the PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL lipopeptides, respectively. No IFN--producing CD8+ T cells were detected against mock-infected cells, providing a control to illustrate the virus specificity of the induced T cells. Few SFCs producing IFN- were detected in mock-immunized mice following in vitro stimulation with either gB498-505 or HSV-1-infected stimulators (Fig. 4A and B). These results indicate that immunization with palmitoyl-tailed Th-CTL chimeric epitopes, without an external adjuvant, generated memory HSV-1-specific IFN--producing CD8+ T cells.
The CTL and IFN- ELISPOT results (Fig. 3, 4A, and 4B) were supported by subsequent intracellular cytokine experiments performed to enumerate the induced HSV-1-specific IFN--secreting CD8+ T cells. For each of the PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL groups of mice, 15.52%, 16.36%, and 18.20% of SP T cells were CD8+ and IFN-+, respectively (Fig. 4C). A nonsignificant percentage of CD8+ IFN-+ double-positive cells was detected for mock-immunized mice (0.98%). Neither the parental Th-CTL peptide alone (Fig. 4C) alone nor palmitic acid alone (not shown) generated IFN--producing CD8+ T cells.
(v) Induction of HSV-gB498-505-specific Tc1 effector cells by the palmitoyl-tailed Th-CTL chimeric epitopes. Cytolytic CD8+ effector T cells fall into two subpopulations based on cytokine profile (28, 40, 41). Type 1 CD8+ T cells (Tc1) produce IFN-, IL-2, and IL-12, whereas type 2 CD8+ T cells produce IL-4. In subsequent experiments, we verified whether immunization with the palmitoyl-tailed Th-CTL chimeric epitopes would induce a polarized memory effector Tc1 or type 2 cytotoxic T-cell response. Twelve weeks after the second immunization with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL lipopeptide, SP CD8+ T cells were isolated from each group and restimulated in vitro with HSV-1 gB498-505 target peptide-pulsed APCs. The amounts of IL-2, IL-4, IL-12 and IFN- released in the culture medium were determined in a sandwich ELISA assay. Each palmitoyl-tailed Th-CTL chimeric epitope induced high amounts of IL-2, IL-12, and IFN- but only a negligible amount of IL-4 (Fig. 5). The levels of IL-2 and IL-12 but not IL-4 and IFN- appeared to increase with the number of lipid units, with the highest amounts induced by the (PAM)3-Th-CTL construct. The levels of IFN-, IL-2, and IL-12 correlated with CTL activity shown in Fig. 3A to D. The specificity of cytokine responses was ascertained by the lack of response in mock-immunized mice. Collectively, these data show that palmitoyl-tailed Th-CTL chimeric epitopes delivered in saline not only elicited cytolytic CD8+ T cells (Fig. 3A to D) but also induced a pattern of cytokines (Fig. 5) that fits a polarized Tc1 response.
(vi) The strength of HSV-gB498-505-specific CD8+ T-cell immunity induced by palmitoyl-tailed Th-CTL chimeric epitopes confirmed by tetramer staining. The specificity of the CD8+ Tc1 response induced by the palmitoyl-tailed Th-CTL chimeric epitopes demonstrated using chromium release assay, IFN- ELISPOT, and intracellular cytokine assays prompted an objective enumeration of HSV-gB498-505-specific CD8+ T cells induced by each lipopeptide construct. We thus employed an MHC tetramer-staining assay which provides a quantitative measure of the frequency of epitope-specific CD8+ T cells. A number of investigators have established the specificity of the HSV-gB498-505 epitope in combination with H2-Kb MHC heavy chain modified for tetramer formation (24). HSV-gB498-505-specific CD8+ T cells were quantified in groups of H2b mice immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide and compared to cells from mock-immunized mice. After the second immunization, SP cells from each group were restimulated in vitro with heat-inactivated virus and stained with either HSV-gB498-505 H2-Kb tetramer (test) or OVA257-264/H2-Kb tetramer (control), followed by CD8 staining. As shown in Fig. 6, 4.53%, 5.63%, and 8.42% of total CD8+ T cells detected in PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL chimeric lipopeptide were HSV-gB498-505 H2-Kb tetramer positive, respectively. As expected, staining for the irrelevant OVA257-264/H2-Kb tetramer was at a nonsignificant level (0.01% to 0.025%). A strict correlation was found between the percentages of HSV-gB498-505 H2-Kb tetramer+ /CD8+ T cells, the magnitude of virus-specific cytolytic activities, and IFN- production (P < 0.05). It should be pointed out that while Th-CTL lipopeptide constructs induced HSV-1-specific CD8+ T-cell responses, they failed to induce neutralizing antibodies (not shown).
(vii) Immunization with palmitoyl-tailed Th-CTL chimeric epitopes protects against ocular herpes infection and reduces disease severity. Since immunization with palmitoyl-tailed Th-CTL chimeric epitopes elicited potent HSV-specific CD8+ CTLs that produced IFN-, it was of interest to determine if such effectors would be sufficient to control an ocular infection with HSV-1. To test this hypothesis, 4 groups of 20 age- and sex-matched H2b mice each were immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide or injected with saline alone (mock-immunized control). Two weeks after the second immunization, animals in each group were uniformly inoculated in both eyes with 4 x 105 PFU of HSV-1 (McKrae). Th-CTL chimeric lipopeptide- and mock-immunized mice were then monitored as follows: (i) for acute replication in the eye, (ii) for severity of herpetic blepharitis and corneal scaring, (iii) for latent infection in the TGs, and (iv) for protection against lethal infection.
HSV-1 ocular infection was monitored by titration of infectious virus in tear films collected in each eye from days 1 to 10 postchallenge. The virus titers were lower for Th-CTL chimeric lipopeptide-immunized groups, regardless of the number of lipid moieties, than for the mock-immunized group (Fig. 7A; Table 1) (P < 0.05). The duration of virus clearance was faster for Th-CTL chimeric lipopeptide-immunized groups than for the mock-immunized group (5 to 6 days versus 10 days, respectively) (Table 1) (P < 0.05). In addition, the range of virus titers was narrowed significantly for Th-CTL chimeric lipopeptides-immunized groups, regardless of the number of lipid moieties, compared to mock-immunized group (0 to 325 versus 0 to 5,500, respectively) (Table 1) (P < 0.05).
It has been established that with the mouse model of ocular herpes, the severity of herpetic blepharitis, an inflammation of the lid margin, correlates with increased HSV-1 replication (31). Th-CTL chimeric lipopeptide- and mock-immunized mice surviving the infection were monitored for 4 weeks postchallenge, with the disease scored on a scale of 0 (no disease) to 5 (very severe disease). On day 7 postchallenge, most mice in the mock-immunized group had severe blepharitis, defined as scores of 4+ (score, 4.0 ± 0.5), while only occasional mild blepharitis was observed for the groups of mice immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptides (score, 1 ± 0.5) (Fig. 7B) (P < 0.05). There was no effect of the number of lipid groups on the Th-CTL chimeric lipopeptide-induced blepharitis protection. In addition, there was a significant reduction in the severity of corneal scarring, scored on day 17 post-ocular challenge in Th-CTL lipopeptide-immunized groups, regardless of the number of lipid groups (score, 2 ± 0.5) (Fig. 7C) (P < 0.005). However, the mock-immunized mice had considerable corneal scarring (score, 4.0 ± 0.5). Thus, analysis of disease severity in these mice further highlighted the protective efficacy of Th-CTL chimeric lipopeptides.
Results of survival, determined at 4 weeks post-ocular challenge, collected from two separate experiments showed a 90 to 100% survival in PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL-immunized groups and only 50 to 55% survival in the mock-immunized control group (P < 0.05). To assess if there is an effect of immunization on latent infection, equal numbers (n = 10) of animals that survived on day 30 postinfection (e.g., during latency) were randomly selected in each of Th-CTL chimeric lipopeptide- and mock-immunized groups. The animals were then euthanized, and latent infection of the TGs was assessed by explant cocultivation assay. Results pooled from two experiments showed the percentage of TGs with latent virus was significantly reduced in Th-CTL chimeric lipopeptide-immunized groups (9 to 49%) from that in the mock-immunized group (88 to 100%) (Fig. 7D) (P < 0.05). The mock-immunized mice had the highest percentage of TGs with latent virus, followed by the PAM-Th-CTL group, the (PAM)2-Th-CTL group, and the (PAM)3-Th-CTL group. Immunization with the CTL epitope alone, the lipid alone, the Th epitope alone, or the nonlipidated parental Th-CTL chimeric peptide alone did not result in protection against infection or against disease.
DISCUSSION
We report on the ability of molecularly defined palmitoyl-tailed Th-CTL chimeric epitopes, delivered in an adjuvant-free saline, to induce antiherpesvirus CD8+ effector T cells. Th-CTL chimeric epitopes extended by a single palmitic acid were sufficient to generate potent antiviral CD8+ T cells and to protect against herpesvirus infection and disease in the mouse model of ocular infection. The protection against ocular HSV-1 replication and disease correlated with antiviral cytolytic T-cell activity and IFN- production. In addition, we found that the palmitoyl-tailed Th-CTL chimeric epitopes provoked DC maturation, as attested by up-regulation of cell surface MHC and costimulatory molecules as well as production of proinflammatory cytokines. Finally, possibly related to their effect on maturation of DCs, immunization with palmitoyl-tailed chimeric Th-CTL epitopes preferentially skewed the CD8+ T-cell responses toward a Tc1 pattern.
Several studies, both with experimental animal models and with humans, have supported a crucial role of CD8+ T cells in antiherpesvirus immunity (11, 17, 22-24, 38). (i) Results from genetically T-cell-deficient mice or from normal murine models adoptively transferred by or depleted of T-cell populations have demonstrated CD8+ T cells to control HSV primary infection (17). (ii) Hendricks and collaborators recently demonstrated with the mouse model of ocular herpes that HSV-1 gB498-505-specific memory CD8+ T cells are selectively activated and retained in latently infected sensory ganglia, pointing to their potential role in controlling virus reactivation (22). (iii) Finally, a study of HIV- and HSV-coinfected individuals showed a positive correlation between frequency of HSV-specific CD8+ T-cell precursors and resistance to herpes infection and recurrences (3, 23). The present study supports a role of CD8+ T cells in herpes immunity by demonstrating that selective induction of antiviral CD8+ T cells specific to the single HSV-gB498-505 epitope is effective both in inhibiting primary herpes infection in the eye and in reducing the percentage of latent infection in TGs.
Among the requirements that must be fulfilled by a clinical vaccine formulation is to be safe, even for immunocompromised individuals (3, 9, 37, 38). Several investigators have attempted to deliver peptides bearing minimal CTL epitopes which showed promise in inducing potent CD8+ T cells both in experimental animals and in phase I trials (28, 36). In those studies, enhanced immunogenicity of minimal CTL peptide epitopes was achieved using a wide range of traditional adjuvants, such as incomplete Freund’s adjuvant, Montanide, MF-59, monophosphoryl lipid A, QS-21, CpG DNA, or cholera and Escherichia coli subunit toxins, or molecular adjuvants, such as inflammatory cytokines and chemokines (reviewed in reference 10). Other approaches use incorporation or association of peptide epitopes into liposomes and immunostaining complexes or fusion with bacterial proteins that channel epitopes across cell membranes into cytosolic pathways (reviewed in references 9, 28, and 38). Because of the complexity of many of these constructs and the potential toxicity of the adjuvants, alternative pathways of delivering peptide epitopes in clinical settings have been extensively explored in the last two decades (9, 28, 36-38). These efforts have been tempered with the realization that other than the requirement for a safe formulation, the immunogenicity of minimal CD8+ CTL epitopes often relies on a concomitant stimulation of CD4+ Th-cell responses (6, 8, 41, 43). Recently, many studies, including ours, have shown the usefulness of lipopeptide-bearing CD4 and CD8+ T-cell epitopes in inducing potent immune responses in vivo (4, 9, 43). To our knowledge, the present report shows for the first time the success of herpesvirus lipid-tailed Th-CTL chimeric epitopes in inducing a potent virus-specific CD8+ T-cell response against an ocular infection. We propose that using this noninvasive and nontoxic strategy may help in evaluating the immunogenicity and protective efficacy of the growing number of human CD8+ T-cell epitopes recently identified from HSV proteins and glycoproteins (23).
It is encouraging that prophylactic immunization with Th-CTL lipopeptides employed in this study resulted not only in a decrease of primary infection of the eye but also in a reduction of the percentage of latently infected trigeminal ganglia (Fig. 7; Table 1). However, we should recognize the need to develop potent immunotherapeutic vaccine formulations that are more effective in preventing HSV reactivations, recurrences, and reinfections. Such formulation should enhance stronger immune responses than those induced by natural infection. In this regard, the lipopeptide immunization strategy employed in this study is currently being investigated as an immunotherapeutic vaccine with rabbit (ocular herpes) and guinea pig (genital herpes) models to determine the effect on HSV recurrences, and the results will be the subjects of future reports.
Individuals with severe recurrent ocular herpes tend to have higher antiherpesvirus immune responses than individuals with no history of recurrent ocular herpes. One possibility is that one or more of these immune responses is involved in the recurrent ocular pathology. Therefore, there is the concern that an immunotherapeutic vaccine against herpes simplex virus, even if it is efficacious against primary challenge, may induce an immunopathological response that in seropositive individuals could exacerbate the pathology associated with ocular recurrences. For this reason, all herpes simplex vaccines, including lipopeptide vaccines, should be tested with a recurrent-infection eye model, such as the rabbit model, and would need extensive human safety studies.
Mature DCs are the only APCs capable of priming naive T cells (2). We demonstrated that incubation of immature DCs in vitro with palmitoyl-tailed Th-CTL chimeric epitopes increased cell surface expression of MHC and costimulatory molecules and provoked the induction of proinflammatory cytokines, resulting in mature DCs, confirming earlier reports (9, 20, 25, 43). We should emphasize that the palmitoyl lysines used in the construction of PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL are synthetic versions of the lipid moiety from the 2-kDa macrophage-activating lipopeptide 2, derived from Mycoplasma fermentans, that binds to Toll-like receptors. We and others have recently reported that these synthetic palmitic acid moieties bind Toll-like receptor 2 present on DCs and induce their maturation (20, 25, 43). In addition, the present study confirmed previous reports of a strong immunogenicity of palmitoyl-tailed peptides (9, 25, 43) and extended those observations by showing that palmitoyl-tailed CTL epitopes preferentially induce an effector CD8+ Tc1 response. Additionally, palmitoyl-lipidated chimeric epitopes promote Tc1 responses, likely related to their differential effect on Ag uptake and maturation of DCs.
Considering the wealth of data addressing methods for CD8+ T-cell induction using epitope-based strategies, it is surprising how few reports exist describing effective T-cell-mediated viral clearing responses in vivo (reviewed in references 9, 23, and 28). To our knowledge, the data presented here represent a first demonstration of CD8+ T-cell-mediated immunity induced by lipid-tailed Th-CTL chimeric epitopes that enhances clearance of an ocular herpes infection, decreases latent virus in the TGs, and reduces both herpetic blepharitis and corneal scarring. The lack of protection against death seen in H2b mice suggests that there is a critical threshold of infection, which the HSV-gB498-505-specific CTLs can adequately control, and that higher levels of viral challenge may overwhelm the capacity of induced CTLs to control a lethal infection. This report represents one of the few describing protective responses induced by a single CD8+ T-cell-determinant-based lipopeptide vaccine (21).
Bearing in mind the particular constraints for a prospective human vaccine, the present study defines the minimal requirement in terms of the number of lipid groups and underlines the safety, immunogenicity, and protective efficacy with the mouse model of ocular infection of relatively easy-to-construct mono-palmitoyl-tailed Th-CTL chimeric lipopeptides. This finding is encouraging, since it indicates that simple and molecularly defined chimeric lipopeptides are immunogenic and can lead to high-yielding and immunogenic formulation for a prospective clinical delivery. What formulation will be both safe and effective for humans remains to be determined. Nonetheless, the qualities outlined in this report may render mono-palmitoyl-tailed Th-CTL chimeric lipopeptides an efficient delivery system for the growing number of recently discovered human CD8+ T-cell epitopes.
ACKNOWLEDGMENTS
Supported by Public Health Service research grants EY14900 and EY14017 to L.B.M. and EY015225 to A.B.N. from the National Eye Institute, National Institutes of Health, and by The Discovery Fund for Eye Research and Challenge Grant from Research to Prevent Blindness.
We thank the NIH Tetramer Facility for providing the tetramers used in this study and Pamela Crowley (TCB) and Xiaoming Zhu (UCIMC) for technical support.
REFERENCES
Banks, T. A., S. Nair, and B. T. Rouse. 1993. Recognition by and in vitro induction of cytotoxic T lymphocytes against predicted epitopes of the immediate-early protein ICP27 of herpes simplex virus. J. Virol. 67:613-616.
BenMohamed, L., Y. Belkaid, E. Loing, K. Brahimi, H. Gras-Masse, and P. Druilhe. 2002. Systemic immune responses induced by mucosal administration of lipopeptides without adjuvant. Eur. J. Immunol. 32:2274-2281.
BenMohamed, L., G. Bertrand, C. D. McNamara, H. Gras-Masse, J. Hammer, S. L. Wechsler, and A. B. Nesburn. 2003. Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity. J. Virol. 77:9463-9473.
BenMohamed, L., H. Gras-Masse, A. Tartar, P. Daubersies, K. Brahimi, M. Bossus, A. Thomas, and P. Druilhe. 1997. Lipopeptide immunization without adjuvant induces potent and long-lasting B, T helper, and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees. Eur. J. Immunol. 27:1242-1253.
BenMohamed, L., R. Krishnan, C. Auge, J. F. Primus, and D. J. Diamond. 2002. Intranasal administration of a synthetic lipopeptide without adjuvant induces systemic immune responses. Immunology 106:113-121.
BenMohamed, L., R. Krishnan, J. Longmate, C. Auge, L. Low, J. Primus, and D. J. Diamond. 2000. Induction of CTL response by a minimal epitope vaccine in HLA A0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response. Hum. Immunol. 61:764-779.
BenMohamed, L., A. Thomas, M. Bossus, K. Brahimi, J. Wubben, H. Gras-Masse, and P. Druilhe. 2000. High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules. Vaccine 18:2843-2855.
BenMohamed, L., A. Thomas, and P. Druilhe. 2004. Long-term multiepitopic cytotoxic T lymphocytes induced in chimpanzees by combinations of Plasmodium falciparum liver stage peptides and lipopeptides. Infect. Immun. 72:11-19.
BenMohamed, L., S. L. Wechsler, and A. B. Nesburn. 2002. Lipopeptide vaccines—yesterday, today, and tomorrow. Lancet Infect. Dis. 2:425-431.
Berzofsky, J. A., J. D. Ahlers, M. A. Derby, C. D. Pendleton, T. Arichi, and I. M. Belyakov. 1999. Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections. Immunol. Rev. 170:151-172.
Blaney, J. E., Jr., E. Nobusawa, M. A. Brehm, R. H. Bonneau, L. M. Mylin, T. M. Fu, Y. Kawaoka, and S. S. Tevethia. 1998. Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity. J. Virol. 72:9567-9574.
Bourel-Bonnet, L., D. Bonnet, F. Malingue, H. Gras-Masse, and O. Melnyk. 2003. Simultaneous lipidation of a characterized peptide mixture by chemoselective ligation. Bioconjug. Chem. 14:494-499.
Brynestad, K., B. Babbit, L. Huang, and B. T. Rouse. 1990. Influence of peptide acylation, liposome incorporation, and synthetic immunomodulators on the immunogenicity of a 1-23 peptide of glycoprotein D of herpes simplex virus: implications for subunit vaccines. J. Virol. 64:680-685.
Ciupitu, A. M., M. Petersson, C. L. O'Donnell, K. Williams, S. Jindal, R. Kiessling, and R. M. Welsh. 1998. Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes. J. Exp. Med. 187:685-691.
Dana, M. R., Y. Qian, and P. Hamrah. 2000. Twenty-five-year panorama of corneal immunology: emerging concepts in the immunopathogenesis of microbial keratitis, peripheral ulcerative keratitis, and corneal transplant rejection. Cornea 19:625-643.
Gahery-Segard, H., G. Pialoux, S. Figueiredo, C. Igea, M. Surenaud, J. Gaston, H. Gras-Masse, J. P. Levy, and J. G. Guillet. 2003. Long-term specific immune responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine: characterization of CD8+-T-cell epitopes recognized. J. Virol. 77:11220-11231.
Goldsmith, K., W. Chen, D. C. Johnson, and R. L. Hendricks. 1998. Infected cell protein (ICP)47 enhances herpes simplex virus neurovirulence by blocking the CD8+ T cell response. J. Exp. Med. 187:341-348.
Gras-Masse, H. 2001. Chemoselective ligation and antigen vectorization. Biologicals 29:183-188.
Hanke, T., F. L. Graham, K. L. Rosenthal, and D. C. Johnson. 1991. Identification of an immunodominant cytotoxic T-lymphocyte recognition site in glycoprotein B of herpes simplex virus by using recombinant adenovirus vectors and synthetic peptides. J. Virol. 65:1177-1186.
Hosmalin, A., M. Andrieu, E. Loing, J. F. Desoutter, D. Hanau, H. Gras-Masse, A. Dautry-Varsat, and J. G. Guillet. 2001. Lipopeptide presentation pathway in dendritic cells. Immunol. Lett. 79:97-100.
Jackson, D. C., Y. F. Lau, T. Le, A. Suhrbier, G. Deliyannis, C. Cheers, C. Smith, W. Zeng, and L. E. Brown. 2004. A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses. Proc. Natl. Acad. Sci. USA 101:15440-15445.
Khanna, K. M., A. J. Lepisto, and R. L. Hendricks. 2004. Immunity to latent viral infection: many skirmishes but few fatalities. Trends Immunol. 25:230-234.
Koelle, D. M., and L. Corey. 2003. Recent progress in herpes simplex virus immunobiology and vaccine research. Clin. Microbiol. Rev. 16:96-113.
Kumaraguru, U., M. Gierynska, S. Norman, B. D. Bruce, and B. T. Rouse. 2002. Immunization with chaperone-peptide complex induces low-avidity cytotoxic T lymphocytes providing transient protection against herpes simplex virus infection. J. Virol. 76:136-141.
Loing, E., M. Andrieu, K. Thiam, D. Schorner, K. H. Wiesmuller, A. Hosmalin, G. Jung, and H. Gras-Masse. 2000. Extension of HLA-A0201-restricted minimal epitope by N epsilon-palmitoyl-lysine increases the life span of functional presentation to cytotoxic T cells. J. Immunol. 164:900-907.
Lo-Man, R., S. Vichier-Guerre, R. Perraut, E. Deriaud, V. Huteau, L. BenMohamed, O. M. Diop, P. O. Livingston, S. Bay, and C. Leclerc. 2004. A fully synthetic therapeutic vaccine candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-specific antibodies in nonhuman primates. Cancer Res. 64:4987-4994.
Mikloska, Z., M. Ruckholdt, I. Ghadiminejad, H. Dunckley, M. Denis, and A. L. Cunningham. 2000. Monophosphoryl lipid A and QS21 increase CD8 T lymphocyte cytotoxicity to herpes simplex virus-2 infected cell proteins 4 and 27 through IFN-gamma and IL-12 production. J. Immunol. 164:5167-5176.
Moron, G., G. Dadaglio, and C. Leclerc. 2004. New tools for antigen delivery to the MHC class I pathway. Trends Immunol. 25:92-97.
Nardin, E. H., J. M. Calvo-Calle, G. A. Oliveira, R. S. Nussenzweig, M. Schneider, J. M. Tiercy, L. Loutan, D. Hochstrasser, and K. Rose. 2001. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types. J. Immunol. 166:481-489.
Nesburn, A., T. Ramos, X. Zhu, H. Asgarzadeh, V. Nguyen, and L. BenMohamed. 2005. Local and systemic B-cell and Th1 responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type 1 glycoprotein D together with cytosine-phosphate-guanine adjuvant. Vaccine 23:873-883.
Osorio, Y., J. Cohen, and H. Ghiasi. 2004. Improved protection from primary ocular HSV-1 infection and establishment of latency using multigenic DNA vaccines. Investig. Ophthalmol. Vis. Sci. 45:506-514.
Pamonsinlapatham, P., N. Decroix, L. Mihaila-Amrouche, A. Bouvet, and J. P. Bouvet. 2004. Induction of a mucosal immune response to the streptococcal M protein by intramuscular administration of a PADRE-ASREAK peptide. Scand. J. Immunol. 59:504-510.
Pialoux, G., H. Gahery-Segard, S. Sermet, H. Poncelet, S. Fournier, L. Gerard, A. Tartar, H. Gras-Masse, J. P. Levy, and J. G. Guillet. 2001. Lipopeptides induce cell-mediated anti-HIV immune responses in seronegative volunteers. AIDS 15:1239-1249.
Richards, C. M., R. Case, T. R. Hirst, T. J. Hill, and N. A. Williams. 2003. Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice. J. Virol. 77:6692-6699.
Rosenthal, K. S., H. Mao, W. I. Horne, C. Wright, and D. Zimmerman. 1999. Immunization with a LEAPS heteroconjugate containing a CTL epitope and a peptide from beta-2-microglobulin elicits a protective and DTH response to herpes simplex virus type 1. Vaccine 17:535-542.
Sette, A., B. Livingston, D. McKinney, E. Appella, J. Fikes, J. Sidney, M. Newman, and R. Chesnut. 2001. The development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation. Biologicals 29:271-276.
Stanberry, L. R. 2004. Clinical trials of prophylactic and therapeutic herpes simplex virus vaccines. Herpes 11(Suppl. 3):161A-169A.
Toka, F. N., C. D. Pack, and B. T. Rouse. 2004. Molecular adjuvants for mucosal immunity. Immunol. Rev. 199:100-112.
Vichier-Guerre, S., R. Lo-Man, L. BenMohamed, E. Deriaud, S. Kovats, C. Leclerc, and S. Bay. 2003. Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use. J. Pept. Res. 62:117-124.
Woodland, D. L., and R. W. Dutton. 2003. Heterogeneity of CD4(+) and CD8(+) T cells. Curr. Opin. Immunol. 15:336-342.
Zajac, A. J., K. Murali-Krishna, J. N. Blattman, and R. Ahmed. 1998. Therapeutic vaccination against chronic viral infection: the importance of cooperation between CD4+ and CD8+ T cells. Curr. Opin. Immunol. 10:444-449.
Zeng, W., S. Ghosh, Y. F. Lau, L. E. Brown, and D. C. Jackson. 2002. Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines. J. Immunol. 169:4905-4912.
Zhu, X., T. V. Ramos, H. Gras-Masse, B. E. Kaplan, and L. BenMohamed. 2004. Lipopeptide epitopes extended by Ne-palmitoyl lysine moiety increases uptake and maturation of dendritic cell through a Toll-like receptor 2 pathway and triggers a Th1-dependent protective immunity. Eur. J. Immunol. 34:1142-1149.(Xiuli Zhang, Annie Issagh)
TwentyFirst Century Biochemicals Center for Immunology, University of California, Irvine, Irvine, California 92697-1450
ABSTRACT
Molecularly defined vaccine formulations capable of inducing antiviral CD8+ T-cell-specific immunity in a manner compatible with human delivery are limited. Few molecules achieve this target without the support of an appropriate immunological adjuvant. In this study, we investigate the potential of totally synthetic palmitoyl-tailed helper-cytotoxic-T-lymphocyte chimeric epitopes (Th-CTL chimeric lipopeptides) to induce herpes simplex virus type 1 (HSV-1)-specific CD8+ T-cell responses. As a model antigen, the HSV-1 glycoprotein B (498-505) (gB498-505) CD8+ CTL epitope was synthesized in line with the Pan DR peptide (PADRE), a universal CD4+ Th epitope. The peptide backbone, composed solely of both epitopes, was extended by N-terminal attachment of one (PAM-Th-CTL), two [(PAM)2-Th-CTL], or three [(PAM)3-Th-CTL] palmitoyl lysines and delivered to H2b mice in adjuvant-free saline. Potent HSV-1 gB498-505-specific antiviral CD8+ T-cell effector type 1 responses were induced by each of the palmitoyl-tailed Th-CTL chimeric epitopes, irrespective of the number of lipid moieties. The palmitoyl-tailed Th-CTL chimeric epitopes provoked cell surface expression of major histocompatibility complex and costimulatory molecules and production of interleukin-12 and tumor necrosis factor alpha proinflammatory cytokines by immature dendritic cells. Following ocular HSV-1 challenge, palmitoyl-tailed Th-CTL-immunized mice exhibited a decrease of virus replication in the eye and in the local trigeminal ganglion and reduced herpetic blepharitis and corneal scarring. The rational of the molecularly defined vaccine approach presented in this study may be applied to ocular herpes and other viral infections in humans, providing steps are taken to include appropriate Th and CTL epitopes and lipid groups.
INTRODUCTION
Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) are prevalent microbial pathogens, affecting up to 90% of the adult population throughout the world (3, 23, 43). HSV-1, the most prevalent cause of viral infection of the eye (3, 23, 34, 43), causes a spectrum of clinical manifestations ranging from blepharitis, conjunctivitis, and dendritic keratitis to disciform stromal edema and necrotizing stromal keratitis (3, 23, 34). The corneal scarring induced by HSV-1 often leads to corneal opacification, making this virus the leading cause of blindness by an infectious agent in developed countries (3, 23, 43). In the United States alone, every year more than 450,000 people exhibit recurrent ocular HSV episodes requiring doctor visits, drug treatments, and in severe cases, corneal transplants (15). Despite pharmaceutical approaches to therapy and prevention, ocular herpes infections are still prevalent and no vaccine has yet been approved for clinical use (3, 34). Developing an effective immunoprophylactic and/or immunotherapeutic vaccine would represent a powerful and cost-effective means of controlling ocular herpes infection (3, 30, 31, 34).
Direct and indirect evidence, in both animal models and humans, suggests that CD8+ T cells play a major role in antiherpesvirus immunity (11). While CD8+ T cells are important effectors responsible for the clearance of herpes infections, CD4+ T cells are required to help prime and sustain antiviral CD8+ T-cell immunity (3, 41, 43). Consequently, an immunogenic formulation should be targeted towards inducing both CD4+ and CD8+ T-cell populations (3, 11, 22, 27, 35, 43). Concomitant CD4+ Th-cell responses can be easily induced by targeting exogenous major histocompatibility complex (MHC) class II pathways of antigen (Ag) presentation (3, 41, 43). However, induction of CD8+ T-cell responses requires Ag synthesis or transport by an appropriate vector into the cytoplasm of antigen-presenting cells (APCs) (1, 14, 28, 41). Many strategies for CD8+ cytotoxic T-cell (CTL) activation are based on immunization with recombinant live attenuated viruses or bacteria that infect APCs and express the target epitope(s) (reviewed in references 28 and 41). Yet, beside the safety issues that still remain to be addressed before widespread clinical use of many live vectors, protective CTL responses are usually only obtained following a prime-boost regimen (28, 41). Approaches other than live vectors use administration of naked DNA plasmids, from which the target epitopes are directly synthesized within the APCs, or immunization with heat shock or bacterial proteins that channel selected epitopes into MHC class I Ag presentation pathways (14, 28, 41). Alternatively, directly "jump-starting" APCs and CD8+ T cells by loading selected minimal epitope peptides onto MHC molecules at the surfaces of APCs has been proposed (1, 14, 41). In vivo delivery of peptide epitopes either loaded on APCs or emulsified with strong immunological adjuvants induces specific CD8+ T-cell responses in murine models (9). Unfortunately, among the large number of adjuvants that have been tested in small laboratory animal models, many are toxic and have limited clinical usefulness (9).
To avoid toxic adjuvant effects, recent attempts have used chemical modifications of peptide epitopes that act as built-in adjuvants (9, 18, 37, 38). One of the most advanced modifications, which has reached phase II clinical trials in Europe, uses peptide epitopes extended by a lipid moiety (lipopeptides) (9, 16, 29, 33, 42). Lipopeptides have attracted much attention as potential safe vaccines for many microbial pathogens as well as cancers, with the capacity to promote potent CD8+ CTL responses when delivered in adjuvant-free saline (reviewed in reference 9).
In this study, we hypothesize that potent virus-specific CD8+ T-cell responses would be induced by optimal HSV-1 T-cell epitope(s) conjugated to a lipid moiety and delivered in adjuvant-free saline. We asked if palmitoyl-tailed chimeric CD4+ Th-CD8+ CTL epitopes would be sufficient to induce protective immunity in the mouse model of ocular herpes infection. We chose HSV-gB498-505, recognized by H2-Kb-restricted CTLs, as a target epitope (11, 19). We fused the HSV-gB498-505 epitope with the Pan HLA-DR-binding epitope (PADRE), a promiscuous CD4+ Th determinant that binds to most murine and human MHC class II molecules (5, 26, 32, 39). To assess the influence of the number of lipid units on the induction of protective CD8+ T cells, the resulting chimeric peptide backbone, solely composed of both Th and CTL epitopes, was extended by N-terminal addition of one, two, or three palmitic acid moieties. The palmitoyl-tailed HSV-gB498-505-PADRE chimeric epitopes delivered in adjuvant-free saline induced potent virus-specific gamma interferon (IFN-)-producing CD8+ CTLs in H2b mice. Interestingly, such immunization reduced acute infection in the eye, lessened ocular herpetic disease, and interfered with latent infection in trigeminal ganglia (TGs) following an ocular HSV-1 challenge. These results suggest that palmitoyl-tailed Th-CTL chimeric epitopes should be considered as a safe alternative to more complex vaccine strategies for human.
MATERIALS AND METHODS
Peptide and lipopeptides synthesis. The SSIEFARL CD8+ CTL peptide (HSV-gB498-505) (10) and the {dA}K{Cha}VAAWTLKAA{dA}{Ahx}C Pan DR peptide (PADRE) (32) were synthesized either individually or as PADRE-CTL chimeric epitopes using Fmoc (9-fluorenylmethoxycarbonyl) chemistry, with PyBOP/HOBt (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate/N-hydroxybenzotriazole) activation. The parental peptides were washed three times with dimethylformamide (DMF) and treated twice with 2% hydrazine in DMF. After six additional washings with DMF and hydrazine acetic acid, peptides were cleaved using trifluoroacetic acid (TFA)-triisopropylsilane-water (95:2.5:2.5) and the resultant peptides precipitated in cold ether. The peptides were then washed (three times) with cold ether and analyzed by mass spectrometry (MS) and high-performance liquid chromatography (HPLC). Purification of the peptides was performed using Gilson HPLCs with Vydac C18 columns (2.2 by 25 cm or, for larger amounts of crude peptide, 5 by 25 cm). The analysis was performed using Vydac C18, 5-μm, 0.46- by 25-cm columns, with a gradient of 2% per minute of water, 0.1% TFA, 95% acetonitrile, and 0.1% TFA. Once peptides were purified to over 95%, they were lyophilized. Mass-spectrometric analysis was performed with an MDS/Sciex QStar XL mass spectrometer equipped with a nanospray source. Final quality control included collision-induced dissociation tandem MS experiment using nitrogen as the collision gas, which provided sequence confirmation of the peptides.
For the lipopeptide synthesis, one, two, or three N-terminal attachment points were created for subsequent attachments of glyoxylyl lipids following synthesis and purification. This was achieved by adding one, two, or three lysine residues whose side chains were selectively protected with 1-(4,4-dimethyl-2,6-dioxoxyxlohex-1-ylidene)-3-methylbutyl, a hydrazine-sensitive side chain protecting group. In order to allow maximal attachment of the lipid moieties, the lysine residues were interspaced with alanines. The synthesis of the glyoxylyl derivative of palmitate and ligation of peptides were performed using a modification of chemoselective ligation (18). Briefly, dimethyl-2,3-O-isopropylidene tartrate was added to a polyethylene glycol amino resin using PyBOP activation. The second ester was then displaced via the addition of 1,3-diaminopropane. Finally, palmitic acid was activated using PyBOP and used to acylate the amino terminus. Treatment with TFA followed by periodate oxidation generated the alpha-oxo-aldehyde moiety. Following lyophilization, the peptides were transferred in 50-ml round-bottom flasks, fitted with septa, and flushed with nitrogen. A minimal amount of degassed water was added until the peptides were solubilized and displayed as a gel. Stochiometric amounts of lipid were then added in 2-methyl-propan-2-ol dropwise with stirring. The final ratio of water to organic solvent was 95:5. To add the second and third lipids, an aliquot equal to 120% of the concentration of the peptide was added, with 10 to 20 min of stirring in between each addition. The reactions were monitored using the QStar XL mass spectrometer (see Fig. 1). The disappearance of the parent peptide was observed concomitantly with the appearance of the lipid-tailed peptide. In all cases, the parent peptide was not detectable at the end of the acylation process.
Virus and cell lines. Triple plaque-purified wild-type McKrae HSV-1 was prepared as we described previously (3). Rabbit skin (RS) cells used to prepare the virus stocks and culture the virus from eye swabs were grown in Eagle's minimum essential medium supplemented with 5% fetal calf serum (InvitrogenGibco, Grand Island, NY).
Mice. Female C57BL/6 (H2b) mice, ages 4 to 5 weeks, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were handled in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
Peptide and lipopeptide immunization. In an initial experiment, we determined the optimal dose response to the HSV-gB498-505 epitope following immunization with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL lipopeptides and found no differences between 50-, 100-, and 200-μg doses (not shown). Subsequent experiments were then carried out with a medium dose of 100 μg of each construct delivered in phosphate-buffered saline, injected subcutaneously (s.c.) on days 0 and 14. As a negative control, mice were injected s.c. with saline alone (mock-immunized mice).
Cytokine ELISAs. Two weeks after the second immunization, mice were euthanized and single suspensions of spleen (SP) cells were stimulated either with the target HSV-gB498-505 alone or with heat-inactivated HSV-infected stimulator cells. Cells were plated at a density of 1 x 106/well in 24-well plates in a humidified 5% CO2 atmosphere. Seventy-two hours later, the supernatants were collected and the concentrations of interleukin-2 (IL-2), IL-4, and IL-12, and IFN- were determined in a sandwich ELISA using kits specific for each cytokine according to the manufacturer's instructions (BD PharMingen, San Diego, CA).
T-cell proliferation. SP were removed and placed into ice-cold serum free HL-1 medium supplemented with 15 mM HEPES, 5 x 10–5 M ?-mercaptoethanol, 2 mM glutamine, 50 IU of penicillin, and 50 μg of streptomycin (GIBCO-BRL, Grand Island, NY) (CM) (2, 3). The cells were cultured in 96-well plates at 5 x 105 cells/well in CM, with recall PADRE peptide at a 1-μg/ml concentration, with heat-inactivated HSV-1 (0.3 multiplicity of infection), or with concanavalin A as a positive control. The SP cell suspensions were incubated for 72 h at 37°C in 5% CO2, and their proliferation was determined in an [3H]thymidine incorporation assay as we described previously (2, 4).
IFN- ELISpOT assay. SP cells were cultured in 24-well plates for 5 days in a humidified 5%-CO2 atmosphere with HSV-gB498-505 peptide alone (10 μg/ml), the irrelevant OVA257-264 CD8+ T-cell peptide (10 μg/ml), or autologous HSV-1-infected or mock-infected stimulator cells and subsequently analyzed in an IFN- enzyme-linked immunospot (ELISPOT) assay. Functional T-cell recognition IFN- ELISPOT assays were performed with the mouse IFN- ELISPOT monoclonal antibody (MAb) pair (BD PharMingen, San Diego, CA). Briefly, on day 4, 96-well multiscreen immunoprecipitation plates were coated overnight at 4°C with 100 μl (1:250) of anti-IFN- capture MAb. Plates were then blocked with RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) for 2 h at room temperature. Cells (5 x 104/well) were added in triplicate to MAb-coated plates and incubated for an additional 18 to 24 h at 37°C, 5% CO2. Plates were then washed with phosphate-buffered saline and supplemented with a detection peroxidase-labeled antibody followed by a substrate according to the manufacturer's instructions. The developed spots were counted under a light microscope.
Flow cytometry. Standard flow cytometry was employed, as we described previously (2, 9), to assess surface expression of various markers using the following MAbs directly conjugated with either phyocoerythrin (PE) or fluorescein isothiocyanate (FITC): FITC-CD4, FITC-CD8, PE HSV-gB tetramer, PE-CD80 (clone 10-10A1, immunoglobulin G [IgG]), PE-CD86 (clone GL1, IgG2a), FITC-MHC-I (clone TU36, IgG2b), and FITC-MHC-II (clone FLI8.26, IgG2b) (PharMingen (San Diego, CA). IgG isotype-matched control antibodies were used in all experiments. After staining, cells were washed and fixed in 1% buffered paraformaldehyde before being acquired on a Becton Dickinson (Mountain View, CA) FACSCalibur instrument. Gating was done on large granular cells, and for each sample, 20,000 events were acquired on a FACSCalibur instrument and analyzed with CellQuest software on an integrated Macintosh G4 (Becton Dickinson, San Jose, CA).
CTL activity. Spleen-derived immune CD8+ T cells were restimulated in vitro with HSV-gB498-505 target peptide (5 μg/ml)-pulsed syngeneic irradiated T-depleted splenocytes as we described previously (2, 8). Irradiated EL-4 cells (3,000 rad from a 137Cs source) were added to the coculture as APC. CTL activity was assessed by a standard 4-h 51Cr release assay against EL4 target cells loaded with 10 μM HSV-gB498-505 target peptide or infected with heat-inactivated HSV-1 (multiplicity of infection = 3) as described previously (6, 8). After 5 days of culture, effector CD8+ cells were mixed at 1, 3, 10, 30, and 100 effector/target (E/T) ratios with 51Cr-labeled EL4 cells for 4 h. Maximum release of 51Cr was determined by adding 5% Triton X-100 to 51Cr-labeled EL4 cells. Spontaneous release (<10% of total release) was determined by incubating target EL4 cells with medium alone. The percentage of specific lysis was calculated as follows: 100 x [(experimental release – spontaneous release)/(maximum release – spontaneous release)].
Generation and maturation of bone marrow-derived DCs. Murine bone marrow-derived immature dendritic cells (DCs) were generated using our previously described protocol (2, 9). Immature DCs were incubated in vitro for 72 h with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide and monitored for cell surface expression of MHC class I, CD80, and CD86 costimulatory molecules and for production of proinflammatory IL-12 and IFN- cytokines. Medium-treated immature DCs and LPS stimulated DCs were included as negative and positive controls, respectively.
Virus challenge and ocular disease reading. Ocular challenge was performed 2 weeks postimmunization. An inoculum of 4 x 105 PFU of HSV-1 (McKrae) in 4 μl tissue culture medium was placed on each eye, and the lids were gently rubbed against the eye for 30 s. The severity of blepharitis and corneal scarring in each group of 10 mice was assessed by examination with a slit-lamp biomicroscope after addition of 1% fluorescein dye as eye drops. The examination was performed by investigators masked to the treatment and scored according to a standard 0-to-4 scale.
Monitoring replication and clearance of HSV-1 from the eye. This procedure was performed by swabbing eyes of 10 mice once daily (1 to 10 days) with a Dacron swab and transferring each swab to a 75-mm culture tube containing 0.5 ml of medium. Aliquots (100 μl) of 10-fold serial dilutions were placed on confluent monolayer of RS cells in six-well plates, incubated at 37°C for 1 h, and overlaid with medium containing 1% methylcellulose. The plates were incubated at 37°C for 3 days and stained with 1% cresyl violet, and the viral plaques were counted.
Detection of latent virus in trigeminal ganglia. Equal numbers of mice in each group surviving 30 days postinfection were euthanized, their TGs removed, and individually explanted onto RS cell monolayers in RPMI medium. The culture was monitored for 10 days for the presence of infectious virus.
Statistical analysis. Protective parameters were analyzed by Student's t test and the Fisher exact test (Instat; GraphPad, San Diego, CA). Results were considered to be statistically significant at P values of <0.05.
RESULTS
(i) Construction and physicochemical characterization of palmitoyl-tailed Th-CTL chimeric epitopes. The immunodominant HSV-1-gB498-505 CD8+ T-cell target epitope (11, 35) was fused to the PADRE peptide (5, 26, 39). To optimize its immunogenic potential, the resulting Th-CTL chimeric peptide backbone was covalently linked to one (PAM-Th-CTL), two [PAM)2-Th-CTL], or three [(PAM)3-Th-CTL] N-palmitoyl-lysine residues via functional base lysine -amino groups. An illustration of the overall structure of each of the three palmitoyl-tailed chimeric Th-CTL constructs is presented in Fig. 1A and B. Construction of these chimeric lipopeptides was carried out using a modified version of the newly described chemoselective ligation method that allows synthesis of high-yield, molecularly pure, and water-soluble molecules (12, 18). Unlike the "first generation of lipopeptides" synthesized using classic solid-phase methods which introduce the fatty acyl moiety onto the crude peptide backbone before its purification (4, 7, 8, 13), the lipopeptides employed in this study were constructed in two steps (12, 18, 43). The first step of synthesis and purification of the peptide backbone was followed by a second step of ligation of the lipid moiety, site-specifically introduced in solution (12, 18, 43). This method is compatible with sequences that encompass hydrophobic residues: cysteines, l-alanines (dA), l-cyclohexyl alanines (Cha), or aminocaproic acids (Ahx), such as PADRE [(dA)K(Cha)VAAWTLKAA(dA) (Ahx)]. Indeed, no aggregation was observed when PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL were formulated in water or in saline solution. Physicochemical properties of these Th-CTL chimeric lipopeptides were compatible with multidimensional analysis. All constructs were eluted as a single peak in analytical reverse-phase HPLC with the expected mass and sequences when analyzed by mass spectrometry (Fig. 1C and D). The physicochemical characterization highlights the fine molecular definition of palmitoyl-tailed Th-CTL chimeric epitopes as totally synthetic vaccine constructs, which would probably make them suitable for potential future clinical application.
(ii) Palmitoyl-tailed Th-CTL chimeric epitopes trigger dendritic cell maturation regardless of the number of lipid moieties. We first examined the ability of Th-CTL chimeric lipopeptides to induce phenotypic maturation of DCs. Incubation of immature DCs with Th-CTL chimeric lipopeptides induced significant expression of MHC class I, CD80, and CD86 molecules compared to medium-treated immature DC controls (Fig. 2A) (P < 0.005). Maturation of DCs stimulated with Th-CTL chimeric lipopeptides occurred irrespective of the number of lipid moieties. As a second indicator of maturation, exposure of DCs to chimeric lipopeptides was associated with a lipopeptide dose-dependent increase in IL-12 and IFN- secretion (Fig. 2B). Under similar conditions, there was no up-regulation of MHC and costimulatory molecules or production of IL-12 or TNF- following incubation of immature DCs with the parental nonlipidated peptides alone or the palmitic acid alone (Fig. 2A and B) (P < 0.05). Together these results indicated that Th-CTL chimeric lipopeptides, but not the Th-CTL parental peptide, provoked DC maturation and that covalent linkage to the lipid moiety is required for DC maturation.
(iii) Palmitoyl-tailed PADRE-HSV-gB498-505 chimeric epitopes delivered in saline prime for antigen-specific CTL and Th responses. Next, we evaluated the ability of equimolar amounts of PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL chimeric lipopeptides to prime for gB498-505- and HSV-1-specific CTLs in H2b mice following a subcutaneous delivery in saline. Two weeks after the second injection, functional HSV-gB498-505-specific CTLs were evaluated in the SP by a standard 51Cr release assay. Irrespective of the number of lipid groups, mice immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide developed CTLs that lyse autologous H2b EL-4 cells loaded with target HSV-gB498-505 epitope but not EL-4 cells loaded with the irrelevant OVA257-264 CTL peptide (Fig. 3A and B) (P < 0.005). When the magnitude of CTL activity was compared at an equal E/T ratio of 30, each Th-CTL chimeric lipopeptide generated more than 50% of HSV-gB498-505-specific lysis, with no significant difference in the maximal specific lysis induced by the three Th-CTL chimeric lipopeptides (Fig. 3B). More important, CTLs induced by Th-CTL chimeric lipopeptides killed HSV-1-infected target cells but not mock-infected cells (Fig. 3C and D) (P < 0.005). At equal E/T ratios, there was no effect of the number of lipid units on the magnitude of virus-specific CTL activity elicited by each Th-CTL chimeric lipopeptides. The HSV-gB498-505-specific CTL activity induced by the Th-CTL chimeric lipopeptides was abrogated by CD8 MAb blockage (not shown). Effector T cells derived in parallel from SP of mock-immunized mice and restimulated in vitro with gB498-505 epitope did not lyse target EL-4 cells loaded with gB498-505 peptide or target cells infected with HSV-1 (Fig. 3A to D). This experiment provides a control to illustrate that in vitro amplification of effector T cells with gB498-505 epitope does not by itself induce primary CTLs in culture. There was a lack of specific CTLs in mice immunized with nonlipidated Th-CTL chimeric peptide, with PADRE peptide alone, with HSV-gB498-505 alone, or with palmitic acid alone (Fig. 3), indicating the requirement of the three components (i.e., Th and CTL epitopes and the lipid moiety) attached together for induction of efficient CTL responses.
We next determined the magnitude of concomitant PADRE-specific CD4+ T-cell responses induced by each palmitoyl-tailed Th-CTL chimeric epitope construct. Two weeks after the second immunization with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL, SP-derived CD4+ T cells were isolated in each group and restimulated in vitro with the PADRE peptide or with the irrelevant gD1-29 Th peptide. Gated CD4+ T-cell responses were then examined by fluorescence-activated cell sorting for cell surface expression of interleukin-2 receptor (CD25) as a marker of activation (Fig. 3E). Proliferating CD4+ T cells were also assessed in an [3H]thymidine incorporation assay (Fig. 3F). As shown in Fig. 3E and F, each chimeric lipopeptide generated substantial PADRE-specific CD4+ T-cell stimulation, regardless of the number of lipid moieties. The specificity of the Th response was ascertained with a lack of response to the gD1-29 peptide. Only insignificant T-cell responses were detected in mock-immunized mice or in mice immunized with Th-CTL parental peptide alone or with palmitic acid alone (Fig. 3E to F).
(iv) HSV-1-specific CD8+ CTL response induced by palmitoyl-tailed Th-CTL chimeric epitopes is accompanied by IFN- release. Twelve weeks after final immunization, we evaluated gB498-505- and HSV-1-specific IFN--producing memory CD8+ T cells induced by each lipopeptide construct. Significant numbers of spot-forming cells (SFCs) producing IFN- specific to gB498-505 were generated by each palmitoyl-tailed Th-CTL chimeric lipopeptide (Fig. 4A) (P < 0.005). There was a slight increase in IFN--producing memory CD8+ T cells with the increase in the number of lipid units. A range of 150 to 178 T cells per 10,000 SP cells produced IFN- in response to the gB498-505 peptide, but there was negligible stimulation by the control OVA257-264 peptide (5 to 10 T cells per 10,000 SP cells), indicating the specificity of the response.
The induced memory IFN--producing CD8+ T cells recognized a naturally processed epitope on HSV-1-infected cells, with only a slight increase of the number of SFCs induced by the (PAM)2-Th-CTL and (PAM)3-Th-CTL compared to results with the PAM-Th-CTL lipopeptide analogue (Fig. 4B). Ranges of 15 to 25, 28 to 32, and 34 to 39 T cells per 10,000 SP cells were induced by the PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL lipopeptides, respectively. No IFN--producing CD8+ T cells were detected against mock-infected cells, providing a control to illustrate the virus specificity of the induced T cells. Few SFCs producing IFN- were detected in mock-immunized mice following in vitro stimulation with either gB498-505 or HSV-1-infected stimulators (Fig. 4A and B). These results indicate that immunization with palmitoyl-tailed Th-CTL chimeric epitopes, without an external adjuvant, generated memory HSV-1-specific IFN--producing CD8+ T cells.
The CTL and IFN- ELISPOT results (Fig. 3, 4A, and 4B) were supported by subsequent intracellular cytokine experiments performed to enumerate the induced HSV-1-specific IFN--secreting CD8+ T cells. For each of the PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL groups of mice, 15.52%, 16.36%, and 18.20% of SP T cells were CD8+ and IFN-+, respectively (Fig. 4C). A nonsignificant percentage of CD8+ IFN-+ double-positive cells was detected for mock-immunized mice (0.98%). Neither the parental Th-CTL peptide alone (Fig. 4C) alone nor palmitic acid alone (not shown) generated IFN--producing CD8+ T cells.
(v) Induction of HSV-gB498-505-specific Tc1 effector cells by the palmitoyl-tailed Th-CTL chimeric epitopes. Cytolytic CD8+ effector T cells fall into two subpopulations based on cytokine profile (28, 40, 41). Type 1 CD8+ T cells (Tc1) produce IFN-, IL-2, and IL-12, whereas type 2 CD8+ T cells produce IL-4. In subsequent experiments, we verified whether immunization with the palmitoyl-tailed Th-CTL chimeric epitopes would induce a polarized memory effector Tc1 or type 2 cytotoxic T-cell response. Twelve weeks after the second immunization with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL lipopeptide, SP CD8+ T cells were isolated from each group and restimulated in vitro with HSV-1 gB498-505 target peptide-pulsed APCs. The amounts of IL-2, IL-4, IL-12 and IFN- released in the culture medium were determined in a sandwich ELISA assay. Each palmitoyl-tailed Th-CTL chimeric epitope induced high amounts of IL-2, IL-12, and IFN- but only a negligible amount of IL-4 (Fig. 5). The levels of IL-2 and IL-12 but not IL-4 and IFN- appeared to increase with the number of lipid units, with the highest amounts induced by the (PAM)3-Th-CTL construct. The levels of IFN-, IL-2, and IL-12 correlated with CTL activity shown in Fig. 3A to D. The specificity of cytokine responses was ascertained by the lack of response in mock-immunized mice. Collectively, these data show that palmitoyl-tailed Th-CTL chimeric epitopes delivered in saline not only elicited cytolytic CD8+ T cells (Fig. 3A to D) but also induced a pattern of cytokines (Fig. 5) that fits a polarized Tc1 response.
(vi) The strength of HSV-gB498-505-specific CD8+ T-cell immunity induced by palmitoyl-tailed Th-CTL chimeric epitopes confirmed by tetramer staining. The specificity of the CD8+ Tc1 response induced by the palmitoyl-tailed Th-CTL chimeric epitopes demonstrated using chromium release assay, IFN- ELISPOT, and intracellular cytokine assays prompted an objective enumeration of HSV-gB498-505-specific CD8+ T cells induced by each lipopeptide construct. We thus employed an MHC tetramer-staining assay which provides a quantitative measure of the frequency of epitope-specific CD8+ T cells. A number of investigators have established the specificity of the HSV-gB498-505 epitope in combination with H2-Kb MHC heavy chain modified for tetramer formation (24). HSV-gB498-505-specific CD8+ T cells were quantified in groups of H2b mice immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide and compared to cells from mock-immunized mice. After the second immunization, SP cells from each group were restimulated in vitro with heat-inactivated virus and stained with either HSV-gB498-505 H2-Kb tetramer (test) or OVA257-264/H2-Kb tetramer (control), followed by CD8 staining. As shown in Fig. 6, 4.53%, 5.63%, and 8.42% of total CD8+ T cells detected in PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL chimeric lipopeptide were HSV-gB498-505 H2-Kb tetramer positive, respectively. As expected, staining for the irrelevant OVA257-264/H2-Kb tetramer was at a nonsignificant level (0.01% to 0.025%). A strict correlation was found between the percentages of HSV-gB498-505 H2-Kb tetramer+ /CD8+ T cells, the magnitude of virus-specific cytolytic activities, and IFN- production (P < 0.05). It should be pointed out that while Th-CTL lipopeptide constructs induced HSV-1-specific CD8+ T-cell responses, they failed to induce neutralizing antibodies (not shown).
(vii) Immunization with palmitoyl-tailed Th-CTL chimeric epitopes protects against ocular herpes infection and reduces disease severity. Since immunization with palmitoyl-tailed Th-CTL chimeric epitopes elicited potent HSV-specific CD8+ CTLs that produced IFN-, it was of interest to determine if such effectors would be sufficient to control an ocular infection with HSV-1. To test this hypothesis, 4 groups of 20 age- and sex-matched H2b mice each were immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptide or injected with saline alone (mock-immunized control). Two weeks after the second immunization, animals in each group were uniformly inoculated in both eyes with 4 x 105 PFU of HSV-1 (McKrae). Th-CTL chimeric lipopeptide- and mock-immunized mice were then monitored as follows: (i) for acute replication in the eye, (ii) for severity of herpetic blepharitis and corneal scaring, (iii) for latent infection in the TGs, and (iv) for protection against lethal infection.
HSV-1 ocular infection was monitored by titration of infectious virus in tear films collected in each eye from days 1 to 10 postchallenge. The virus titers were lower for Th-CTL chimeric lipopeptide-immunized groups, regardless of the number of lipid moieties, than for the mock-immunized group (Fig. 7A; Table 1) (P < 0.05). The duration of virus clearance was faster for Th-CTL chimeric lipopeptide-immunized groups than for the mock-immunized group (5 to 6 days versus 10 days, respectively) (Table 1) (P < 0.05). In addition, the range of virus titers was narrowed significantly for Th-CTL chimeric lipopeptides-immunized groups, regardless of the number of lipid moieties, compared to mock-immunized group (0 to 325 versus 0 to 5,500, respectively) (Table 1) (P < 0.05).
It has been established that with the mouse model of ocular herpes, the severity of herpetic blepharitis, an inflammation of the lid margin, correlates with increased HSV-1 replication (31). Th-CTL chimeric lipopeptide- and mock-immunized mice surviving the infection were monitored for 4 weeks postchallenge, with the disease scored on a scale of 0 (no disease) to 5 (very severe disease). On day 7 postchallenge, most mice in the mock-immunized group had severe blepharitis, defined as scores of 4+ (score, 4.0 ± 0.5), while only occasional mild blepharitis was observed for the groups of mice immunized with PAM-Th-CTL, (PAM)2-Th-CTL, or (PAM)3-Th-CTL chimeric lipopeptides (score, 1 ± 0.5) (Fig. 7B) (P < 0.05). There was no effect of the number of lipid groups on the Th-CTL chimeric lipopeptide-induced blepharitis protection. In addition, there was a significant reduction in the severity of corneal scarring, scored on day 17 post-ocular challenge in Th-CTL lipopeptide-immunized groups, regardless of the number of lipid groups (score, 2 ± 0.5) (Fig. 7C) (P < 0.005). However, the mock-immunized mice had considerable corneal scarring (score, 4.0 ± 0.5). Thus, analysis of disease severity in these mice further highlighted the protective efficacy of Th-CTL chimeric lipopeptides.
Results of survival, determined at 4 weeks post-ocular challenge, collected from two separate experiments showed a 90 to 100% survival in PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL-immunized groups and only 50 to 55% survival in the mock-immunized control group (P < 0.05). To assess if there is an effect of immunization on latent infection, equal numbers (n = 10) of animals that survived on day 30 postinfection (e.g., during latency) were randomly selected in each of Th-CTL chimeric lipopeptide- and mock-immunized groups. The animals were then euthanized, and latent infection of the TGs was assessed by explant cocultivation assay. Results pooled from two experiments showed the percentage of TGs with latent virus was significantly reduced in Th-CTL chimeric lipopeptide-immunized groups (9 to 49%) from that in the mock-immunized group (88 to 100%) (Fig. 7D) (P < 0.05). The mock-immunized mice had the highest percentage of TGs with latent virus, followed by the PAM-Th-CTL group, the (PAM)2-Th-CTL group, and the (PAM)3-Th-CTL group. Immunization with the CTL epitope alone, the lipid alone, the Th epitope alone, or the nonlipidated parental Th-CTL chimeric peptide alone did not result in protection against infection or against disease.
DISCUSSION
We report on the ability of molecularly defined palmitoyl-tailed Th-CTL chimeric epitopes, delivered in an adjuvant-free saline, to induce antiherpesvirus CD8+ effector T cells. Th-CTL chimeric epitopes extended by a single palmitic acid were sufficient to generate potent antiviral CD8+ T cells and to protect against herpesvirus infection and disease in the mouse model of ocular infection. The protection against ocular HSV-1 replication and disease correlated with antiviral cytolytic T-cell activity and IFN- production. In addition, we found that the palmitoyl-tailed Th-CTL chimeric epitopes provoked DC maturation, as attested by up-regulation of cell surface MHC and costimulatory molecules as well as production of proinflammatory cytokines. Finally, possibly related to their effect on maturation of DCs, immunization with palmitoyl-tailed chimeric Th-CTL epitopes preferentially skewed the CD8+ T-cell responses toward a Tc1 pattern.
Several studies, both with experimental animal models and with humans, have supported a crucial role of CD8+ T cells in antiherpesvirus immunity (11, 17, 22-24, 38). (i) Results from genetically T-cell-deficient mice or from normal murine models adoptively transferred by or depleted of T-cell populations have demonstrated CD8+ T cells to control HSV primary infection (17). (ii) Hendricks and collaborators recently demonstrated with the mouse model of ocular herpes that HSV-1 gB498-505-specific memory CD8+ T cells are selectively activated and retained in latently infected sensory ganglia, pointing to their potential role in controlling virus reactivation (22). (iii) Finally, a study of HIV- and HSV-coinfected individuals showed a positive correlation between frequency of HSV-specific CD8+ T-cell precursors and resistance to herpes infection and recurrences (3, 23). The present study supports a role of CD8+ T cells in herpes immunity by demonstrating that selective induction of antiviral CD8+ T cells specific to the single HSV-gB498-505 epitope is effective both in inhibiting primary herpes infection in the eye and in reducing the percentage of latent infection in TGs.
Among the requirements that must be fulfilled by a clinical vaccine formulation is to be safe, even for immunocompromised individuals (3, 9, 37, 38). Several investigators have attempted to deliver peptides bearing minimal CTL epitopes which showed promise in inducing potent CD8+ T cells both in experimental animals and in phase I trials (28, 36). In those studies, enhanced immunogenicity of minimal CTL peptide epitopes was achieved using a wide range of traditional adjuvants, such as incomplete Freund’s adjuvant, Montanide, MF-59, monophosphoryl lipid A, QS-21, CpG DNA, or cholera and Escherichia coli subunit toxins, or molecular adjuvants, such as inflammatory cytokines and chemokines (reviewed in reference 10). Other approaches use incorporation or association of peptide epitopes into liposomes and immunostaining complexes or fusion with bacterial proteins that channel epitopes across cell membranes into cytosolic pathways (reviewed in references 9, 28, and 38). Because of the complexity of many of these constructs and the potential toxicity of the adjuvants, alternative pathways of delivering peptide epitopes in clinical settings have been extensively explored in the last two decades (9, 28, 36-38). These efforts have been tempered with the realization that other than the requirement for a safe formulation, the immunogenicity of minimal CD8+ CTL epitopes often relies on a concomitant stimulation of CD4+ Th-cell responses (6, 8, 41, 43). Recently, many studies, including ours, have shown the usefulness of lipopeptide-bearing CD4 and CD8+ T-cell epitopes in inducing potent immune responses in vivo (4, 9, 43). To our knowledge, the present report shows for the first time the success of herpesvirus lipid-tailed Th-CTL chimeric epitopes in inducing a potent virus-specific CD8+ T-cell response against an ocular infection. We propose that using this noninvasive and nontoxic strategy may help in evaluating the immunogenicity and protective efficacy of the growing number of human CD8+ T-cell epitopes recently identified from HSV proteins and glycoproteins (23).
It is encouraging that prophylactic immunization with Th-CTL lipopeptides employed in this study resulted not only in a decrease of primary infection of the eye but also in a reduction of the percentage of latently infected trigeminal ganglia (Fig. 7; Table 1). However, we should recognize the need to develop potent immunotherapeutic vaccine formulations that are more effective in preventing HSV reactivations, recurrences, and reinfections. Such formulation should enhance stronger immune responses than those induced by natural infection. In this regard, the lipopeptide immunization strategy employed in this study is currently being investigated as an immunotherapeutic vaccine with rabbit (ocular herpes) and guinea pig (genital herpes) models to determine the effect on HSV recurrences, and the results will be the subjects of future reports.
Individuals with severe recurrent ocular herpes tend to have higher antiherpesvirus immune responses than individuals with no history of recurrent ocular herpes. One possibility is that one or more of these immune responses is involved in the recurrent ocular pathology. Therefore, there is the concern that an immunotherapeutic vaccine against herpes simplex virus, even if it is efficacious against primary challenge, may induce an immunopathological response that in seropositive individuals could exacerbate the pathology associated with ocular recurrences. For this reason, all herpes simplex vaccines, including lipopeptide vaccines, should be tested with a recurrent-infection eye model, such as the rabbit model, and would need extensive human safety studies.
Mature DCs are the only APCs capable of priming naive T cells (2). We demonstrated that incubation of immature DCs in vitro with palmitoyl-tailed Th-CTL chimeric epitopes increased cell surface expression of MHC and costimulatory molecules and provoked the induction of proinflammatory cytokines, resulting in mature DCs, confirming earlier reports (9, 20, 25, 43). We should emphasize that the palmitoyl lysines used in the construction of PAM-Th-CTL, (PAM)2-Th-CTL, and (PAM)3-Th-CTL are synthetic versions of the lipid moiety from the 2-kDa macrophage-activating lipopeptide 2, derived from Mycoplasma fermentans, that binds to Toll-like receptors. We and others have recently reported that these synthetic palmitic acid moieties bind Toll-like receptor 2 present on DCs and induce their maturation (20, 25, 43). In addition, the present study confirmed previous reports of a strong immunogenicity of palmitoyl-tailed peptides (9, 25, 43) and extended those observations by showing that palmitoyl-tailed CTL epitopes preferentially induce an effector CD8+ Tc1 response. Additionally, palmitoyl-lipidated chimeric epitopes promote Tc1 responses, likely related to their differential effect on Ag uptake and maturation of DCs.
Considering the wealth of data addressing methods for CD8+ T-cell induction using epitope-based strategies, it is surprising how few reports exist describing effective T-cell-mediated viral clearing responses in vivo (reviewed in references 9, 23, and 28). To our knowledge, the data presented here represent a first demonstration of CD8+ T-cell-mediated immunity induced by lipid-tailed Th-CTL chimeric epitopes that enhances clearance of an ocular herpes infection, decreases latent virus in the TGs, and reduces both herpetic blepharitis and corneal scarring. The lack of protection against death seen in H2b mice suggests that there is a critical threshold of infection, which the HSV-gB498-505-specific CTLs can adequately control, and that higher levels of viral challenge may overwhelm the capacity of induced CTLs to control a lethal infection. This report represents one of the few describing protective responses induced by a single CD8+ T-cell-determinant-based lipopeptide vaccine (21).
Bearing in mind the particular constraints for a prospective human vaccine, the present study defines the minimal requirement in terms of the number of lipid groups and underlines the safety, immunogenicity, and protective efficacy with the mouse model of ocular infection of relatively easy-to-construct mono-palmitoyl-tailed Th-CTL chimeric lipopeptides. This finding is encouraging, since it indicates that simple and molecularly defined chimeric lipopeptides are immunogenic and can lead to high-yielding and immunogenic formulation for a prospective clinical delivery. What formulation will be both safe and effective for humans remains to be determined. Nonetheless, the qualities outlined in this report may render mono-palmitoyl-tailed Th-CTL chimeric lipopeptides an efficient delivery system for the growing number of recently discovered human CD8+ T-cell epitopes.
ACKNOWLEDGMENTS
Supported by Public Health Service research grants EY14900 and EY14017 to L.B.M. and EY015225 to A.B.N. from the National Eye Institute, National Institutes of Health, and by The Discovery Fund for Eye Research and Challenge Grant from Research to Prevent Blindness.
We thank the NIH Tetramer Facility for providing the tetramers used in this study and Pamela Crowley (TCB) and Xiaoming Zhu (UCIMC) for technical support.
REFERENCES
Banks, T. A., S. Nair, and B. T. Rouse. 1993. Recognition by and in vitro induction of cytotoxic T lymphocytes against predicted epitopes of the immediate-early protein ICP27 of herpes simplex virus. J. Virol. 67:613-616.
BenMohamed, L., Y. Belkaid, E. Loing, K. Brahimi, H. Gras-Masse, and P. Druilhe. 2002. Systemic immune responses induced by mucosal administration of lipopeptides without adjuvant. Eur. J. Immunol. 32:2274-2281.
BenMohamed, L., G. Bertrand, C. D. McNamara, H. Gras-Masse, J. Hammer, S. L. Wechsler, and A. B. Nesburn. 2003. Identification of novel immunodominant CD4+ Th1-type T-cell peptide epitopes from herpes simplex virus glycoprotein D that confer protective immunity. J. Virol. 77:9463-9473.
BenMohamed, L., H. Gras-Masse, A. Tartar, P. Daubersies, K. Brahimi, M. Bossus, A. Thomas, and P. Druilhe. 1997. Lipopeptide immunization without adjuvant induces potent and long-lasting B, T helper, and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees. Eur. J. Immunol. 27:1242-1253.
BenMohamed, L., R. Krishnan, C. Auge, J. F. Primus, and D. J. Diamond. 2002. Intranasal administration of a synthetic lipopeptide without adjuvant induces systemic immune responses. Immunology 106:113-121.
BenMohamed, L., R. Krishnan, J. Longmate, C. Auge, L. Low, J. Primus, and D. J. Diamond. 2000. Induction of CTL response by a minimal epitope vaccine in HLA A0201/DR1 transgenic mice: dependence on HLA class II restricted T(H) response. Hum. Immunol. 61:764-779.
BenMohamed, L., A. Thomas, M. Bossus, K. Brahimi, J. Wubben, H. Gras-Masse, and P. Druilhe. 2000. High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules. Vaccine 18:2843-2855.
BenMohamed, L., A. Thomas, and P. Druilhe. 2004. Long-term multiepitopic cytotoxic T lymphocytes induced in chimpanzees by combinations of Plasmodium falciparum liver stage peptides and lipopeptides. Infect. Immun. 72:11-19.
BenMohamed, L., S. L. Wechsler, and A. B. Nesburn. 2002. Lipopeptide vaccines—yesterday, today, and tomorrow. Lancet Infect. Dis. 2:425-431.
Berzofsky, J. A., J. D. Ahlers, M. A. Derby, C. D. Pendleton, T. Arichi, and I. M. Belyakov. 1999. Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections. Immunol. Rev. 170:151-172.
Blaney, J. E., Jr., E. Nobusawa, M. A. Brehm, R. H. Bonneau, L. M. Mylin, T. M. Fu, Y. Kawaoka, and S. S. Tevethia. 1998. Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity. J. Virol. 72:9567-9574.
Bourel-Bonnet, L., D. Bonnet, F. Malingue, H. Gras-Masse, and O. Melnyk. 2003. Simultaneous lipidation of a characterized peptide mixture by chemoselective ligation. Bioconjug. Chem. 14:494-499.
Brynestad, K., B. Babbit, L. Huang, and B. T. Rouse. 1990. Influence of peptide acylation, liposome incorporation, and synthetic immunomodulators on the immunogenicity of a 1-23 peptide of glycoprotein D of herpes simplex virus: implications for subunit vaccines. J. Virol. 64:680-685.
Ciupitu, A. M., M. Petersson, C. L. O'Donnell, K. Williams, S. Jindal, R. Kiessling, and R. M. Welsh. 1998. Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes. J. Exp. Med. 187:685-691.
Dana, M. R., Y. Qian, and P. Hamrah. 2000. Twenty-five-year panorama of corneal immunology: emerging concepts in the immunopathogenesis of microbial keratitis, peripheral ulcerative keratitis, and corneal transplant rejection. Cornea 19:625-643.
Gahery-Segard, H., G. Pialoux, S. Figueiredo, C. Igea, M. Surenaud, J. Gaston, H. Gras-Masse, J. P. Levy, and J. G. Guillet. 2003. Long-term specific immune responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine: characterization of CD8+-T-cell epitopes recognized. J. Virol. 77:11220-11231.
Goldsmith, K., W. Chen, D. C. Johnson, and R. L. Hendricks. 1998. Infected cell protein (ICP)47 enhances herpes simplex virus neurovirulence by blocking the CD8+ T cell response. J. Exp. Med. 187:341-348.
Gras-Masse, H. 2001. Chemoselective ligation and antigen vectorization. Biologicals 29:183-188.
Hanke, T., F. L. Graham, K. L. Rosenthal, and D. C. Johnson. 1991. Identification of an immunodominant cytotoxic T-lymphocyte recognition site in glycoprotein B of herpes simplex virus by using recombinant adenovirus vectors and synthetic peptides. J. Virol. 65:1177-1186.
Hosmalin, A., M. Andrieu, E. Loing, J. F. Desoutter, D. Hanau, H. Gras-Masse, A. Dautry-Varsat, and J. G. Guillet. 2001. Lipopeptide presentation pathway in dendritic cells. Immunol. Lett. 79:97-100.
Jackson, D. C., Y. F. Lau, T. Le, A. Suhrbier, G. Deliyannis, C. Cheers, C. Smith, W. Zeng, and L. E. Brown. 2004. A totally synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses. Proc. Natl. Acad. Sci. USA 101:15440-15445.
Khanna, K. M., A. J. Lepisto, and R. L. Hendricks. 2004. Immunity to latent viral infection: many skirmishes but few fatalities. Trends Immunol. 25:230-234.
Koelle, D. M., and L. Corey. 2003. Recent progress in herpes simplex virus immunobiology and vaccine research. Clin. Microbiol. Rev. 16:96-113.
Kumaraguru, U., M. Gierynska, S. Norman, B. D. Bruce, and B. T. Rouse. 2002. Immunization with chaperone-peptide complex induces low-avidity cytotoxic T lymphocytes providing transient protection against herpes simplex virus infection. J. Virol. 76:136-141.
Loing, E., M. Andrieu, K. Thiam, D. Schorner, K. H. Wiesmuller, A. Hosmalin, G. Jung, and H. Gras-Masse. 2000. Extension of HLA-A0201-restricted minimal epitope by N epsilon-palmitoyl-lysine increases the life span of functional presentation to cytotoxic T cells. J. Immunol. 164:900-907.
Lo-Man, R., S. Vichier-Guerre, R. Perraut, E. Deriaud, V. Huteau, L. BenMohamed, O. M. Diop, P. O. Livingston, S. Bay, and C. Leclerc. 2004. A fully synthetic therapeutic vaccine candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-specific antibodies in nonhuman primates. Cancer Res. 64:4987-4994.
Mikloska, Z., M. Ruckholdt, I. Ghadiminejad, H. Dunckley, M. Denis, and A. L. Cunningham. 2000. Monophosphoryl lipid A and QS21 increase CD8 T lymphocyte cytotoxicity to herpes simplex virus-2 infected cell proteins 4 and 27 through IFN-gamma and IL-12 production. J. Immunol. 164:5167-5176.
Moron, G., G. Dadaglio, and C. Leclerc. 2004. New tools for antigen delivery to the MHC class I pathway. Trends Immunol. 25:92-97.
Nardin, E. H., J. M. Calvo-Calle, G. A. Oliveira, R. S. Nussenzweig, M. Schneider, J. M. Tiercy, L. Loutan, D. Hochstrasser, and K. Rose. 2001. A totally synthetic polyoxime malaria vaccine containing Plasmodium falciparum B cell and universal T cell epitopes elicits immune responses in volunteers of diverse HLA types. J. Immunol. 166:481-489.
Nesburn, A., T. Ramos, X. Zhu, H. Asgarzadeh, V. Nguyen, and L. BenMohamed. 2005. Local and systemic B-cell and Th1 responses induced following ocular mucosal delivery of multiple epitopes of herpes simplex virus type 1 glycoprotein D together with cytosine-phosphate-guanine adjuvant. Vaccine 23:873-883.
Osorio, Y., J. Cohen, and H. Ghiasi. 2004. Improved protection from primary ocular HSV-1 infection and establishment of latency using multigenic DNA vaccines. Investig. Ophthalmol. Vis. Sci. 45:506-514.
Pamonsinlapatham, P., N. Decroix, L. Mihaila-Amrouche, A. Bouvet, and J. P. Bouvet. 2004. Induction of a mucosal immune response to the streptococcal M protein by intramuscular administration of a PADRE-ASREAK peptide. Scand. J. Immunol. 59:504-510.
Pialoux, G., H. Gahery-Segard, S. Sermet, H. Poncelet, S. Fournier, L. Gerard, A. Tartar, H. Gras-Masse, J. P. Levy, and J. G. Guillet. 2001. Lipopeptides induce cell-mediated anti-HIV immune responses in seronegative volunteers. AIDS 15:1239-1249.
Richards, C. M., R. Case, T. R. Hirst, T. J. Hill, and N. A. Williams. 2003. Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice. J. Virol. 77:6692-6699.
Rosenthal, K. S., H. Mao, W. I. Horne, C. Wright, and D. Zimmerman. 1999. Immunization with a LEAPS heteroconjugate containing a CTL epitope and a peptide from beta-2-microglobulin elicits a protective and DTH response to herpes simplex virus type 1. Vaccine 17:535-542.
Sette, A., B. Livingston, D. McKinney, E. Appella, J. Fikes, J. Sidney, M. Newman, and R. Chesnut. 2001. The development of multi-epitope vaccines: epitope identification, vaccine design and clinical evaluation. Biologicals 29:271-276.
Stanberry, L. R. 2004. Clinical trials of prophylactic and therapeutic herpes simplex virus vaccines. Herpes 11(Suppl. 3):161A-169A.
Toka, F. N., C. D. Pack, and B. T. Rouse. 2004. Molecular adjuvants for mucosal immunity. Immunol. Rev. 199:100-112.
Vichier-Guerre, S., R. Lo-Man, L. BenMohamed, E. Deriaud, S. Kovats, C. Leclerc, and S. Bay. 2003. Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use. J. Pept. Res. 62:117-124.
Woodland, D. L., and R. W. Dutton. 2003. Heterogeneity of CD4(+) and CD8(+) T cells. Curr. Opin. Immunol. 15:336-342.
Zajac, A. J., K. Murali-Krishna, J. N. Blattman, and R. Ahmed. 1998. Therapeutic vaccination against chronic viral infection: the importance of cooperation between CD4+ and CD8+ T cells. Curr. Opin. Immunol. 10:444-449.
Zeng, W., S. Ghosh, Y. F. Lau, L. E. Brown, and D. C. Jackson. 2002. Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines. J. Immunol. 169:4905-4912.
Zhu, X., T. V. Ramos, H. Gras-Masse, B. E. Kaplan, and L. BenMohamed. 2004. Lipopeptide epitopes extended by Ne-palmitoyl lysine moiety increases uptake and maturation of dendritic cell through a Toll-like receptor 2 pathway and triggers a Th1-dependent protective immunity. Eur. J. Immunol. 34:1142-1149.(Xiuli Zhang, Annie Issagh)