CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.     

Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.     

Identification of a ras oncogene peptide that contains both CD4(+) and CD8(+) T cell epitopes in a nested configuration and elicits both T cell subset responses by peptide or DNA immunization

Mutations in ras proto-oncogenes are commonly found in a diversity of malignancies and may encode unique, non-self epitopes for T cell-mediated antitumor activity. In a BALB/c (H-2(d)) murine model, we have identified a single peptide sequence derived from the ras oncogenes that contained both CD8(+) and CD4(+) T cell epitopes in a nested configuration. This peptide reflected ras sequence 4-16, and contained the substitution of Gly to Val at position 12 ?i.e., 4-16(Val12)?. Mice immunized with this 13-mer peptide induced a strong antigen (Ag)-specific CD4(+) proliferative response in vitro. In contrast, mice inoculated with the wild-type ras sequence failed to generate a peptide-specific T cell response. Additionally, mice immunized with the ras 4-16(Val12) peptide concomitantly displayed an Ag-specific CD8(+) cytotoxic T lymphocyte (CTL) response, as determined by lysis of syngeneic tumor target cells incubated with the nominal 9-mer nested epitope peptide ?i.e., 4-12(Val12)?, as well as lysis of tumor target cells expressing the corresponding ras codon 12 mutation. Analysis of the Valpha- and Vbeta-chains of the T cell receptor (TCR) expressed by these CTL revealed usage of the Valpha1 and Vbeta9 subunits, consistent with the TCR phenotype of anti-ras Val12 CTL lines produced by in vivo immunization with the nominal peptide epitope alone. Moreover, immunization with the nested epitope peptide, as compared to immunization with either the 9-mer CTL peptide alone or an admixture of the 9-mer CTL peptide with an overlapping 13-mer CD4(+) T cell helper peptide ?i.e., 5-17(Val12)? lacking the class I N-terminus anchor site, enhanced the production of the CD8(+) T cell response. Finally, immunization with plasmid DNA encoding the ras 4-16(Val12) sequence led to the induction of both Ag-specific proliferative and cytotoxic responses. Overall, these results suggested that a single peptide immunogen containing nested mutant ras-specific CD4(+) and CD8(+) T cell epitopes: (1) can be processed in vivo to induce both subset-specific T lymphocyte responses; and (2) leads to the generation of a quantitatively enhanced CD8(+) CTL response, likely due to the intimate coexistence of CD4(+) help, which may have implications in peptide- or DNA-based immunotherapies.     

Pharmacokinetic differences between a T cell-tolerizing and a T cell-activating peptide

Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.     

Cancer vaccine design: a novel bacterial adjuvant for peptide-specific CTL induction

The recent identification of tumor Ags as potential vaccines has prompted the search for efficient adjuvants and delivery systems, especially in the case of peptide-based vaccination protocols. Here, we investigated the adjuvant potential of the recombinant 40-kDa outer membrane protein of Klebsiella pneumoniae (P40) for specific CTL induction. We studied the CTL response induced in HLA-A*0201/K(b) transgenic mice immunized with peptides derived from two melanoma-associated differentiation Ags, the HLA-A*0201-restricted decapeptide Melan-A(26--35) substituted at position 2 and the K(b)-restricted tyrosinase-related protein 2(181--188) T cell epitope. We found that both peptides are able to generate a specific CTL response when mixed with the protein in the absence of conventional adjuvant. This CTL response is a function of the amount of P40 used for immunization. Moreover, the CTL response generated against the tyrosinase-related protein 2(181-188) peptide in presence of P40 is associated with tumor protection in two different experimental models and is independent of the presence of CD4(+) T lymphocytes. Thus, the recombinant bacterial protein P40 functions as a potent immunological adjuvant for specific CTL induction.     

Depletion of CD4+ T cells precipitates immunopathology in immunodeficient mice infected with a noncytocidal virus

IFN-gamma-deficient (IFN-gamma(-/-)) mice inoculated with intermediate doses of a slowly replicating strain of lymphocytic choriomeningitis virus become chronically infected. In such mice a hypercompensated CTL response is observed that partially controls virus replication. Here we have investigated whether CD4(+) Th cells are required to establish and maintain this new equilibrium. The absence of IFN-gamma does not impair the generation of IL-2-producing CD4(+) cells, and depletion of these cells precipitates severe CD8(+) T cell-mediated immunopathology in IFN-gamma(-/-) mice, indicating an important role of CD4(+) T cells in preventing this syndrome. Analysis of organ virus levels revealed a further impairment of virus control in IFN-gamma(-/-) mice following CD4(+) cell depletion. Initially the antiviral CTL response did not require CD4(+) cells, but with time an impaired reactivity toward especially the glycoprotein 33--41 epitope was noted. Enumeration of epitope-specific (glycoprotein 33--41 and nucleoprotein 396--404) CD8(+) T cells by use of tetramers gave similar results. Finally, limiting dilution analysis of CTL precursors reveal an impaired capacity to sustain this population in CD4(+)-depleted mice, especially in mice also deficient in IFN-gamma. Thus, our findings disclose that T cell help is required to sustain the expanded CTL precursor pool required in IFN-gamma(-/-) mice. This interpretation is supported by mathematical modeling that predicts an increased requirement for help in IFN-gamma(-/-) hosts similar to what is found with fast replicating virus strains in normal hosts. Thus, the functional integrity of CD8(+) effector T cells is one important factor influencing the requirement for T cell help during viral infection.     

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

Mucosal delivery of a respiratory syncytial virus CTL peptide with enterotoxin-based adjuvants elicits protective, immunopathogenic, and immunoregulatory antiviral CD8+ T cell responses

In an effort to develop a safe and effective vaccine against respiratory syncytial virus (RSV), we used Escherichia coli heat-labile toxin (LT), and LTK63 (an LT mutant devoid of ADP-ribosyltransferase activity) to elicit murine CD8(+) CTL responses to an intranasally codelivered CTL peptide from the second matrix protein (M2) of RSV. M2(82-90)-specific CD8(+) T cells were detected by IFN-gamma enzyme-linked immunospot and (51)Cr release assay in local and systemic lymph nodes, and their induction was dependent on the use of a mucosal adjuvant. CTL elicited by peptide immunization afforded protection against RSV challenge, but also enhanced weight loss. CTL-mediated viral clearance was not dependent on IFN-gamma since depletion using specific mAb during RSV challenge did not affect cellular recruitment or viral clearance. Depletion of IFN-gamma did, however, reduce the concentration of TNF detected in lung homogenates of challenged mice and largely prevented the weight loss associated with CTL-mediated viral clearance. Mice primed with the attachment glycoprotein (G) develop lung eosinophilia after intranasal RSV challenge. Mucosal peptide vaccination reduced pulmonary eosinophilia in mice subsequently immunized with G and challenged with RSV. These studies emphasize that protective and immunoregulatory CD8(+) CTL responses can be mucosally elicited using enterotoxin-based mucosal adjuvants but that resistance against viral infection may be accompanied by enhanced disease.     

Synergism between CpG-containing oligodeoxynucleotides and IL-2 causes dramatic enhancement of vaccine-elicited CD8+ T cell responses

Novel anticancer vaccination regimens that can elicit large numbers of Ag-specific T cells are needed. When we administered therapeutic vaccines containing the MHC class I-presented self-peptide tyrosinase-related protein (TRP)-2(180-188) and CpG-containing oligodeoxynucleotides (CpG ODN) to mice, growth of the TRP-2-expressing B16F1 melanoma was not inhibited compared with growth in mice that received control vaccinations. When we added systemic IL-2 to the TRP-2(180-188) plus CpG ODN vaccines, growth of B16F1 was inhibited in a CD8-dependent, epitope-specific manner. Vaccines containing TRP-2(180-188) without CpG ODN did not cause epitope-specific tumor growth inhibition when administered with IL-2. The antitumor efficacy of the different regimens correlated with their ability to elicit TRP-2(180-188)-specific CD8+ T cell responses. When we administered TRP-2(180-188) plus CpG ODN-containing vaccines with systemic IL-2, 18.2% of CD8+ T cells were specific for TRP-2(180-188). Identical TRP-2(180-188) plus CpG ODN vaccines given without IL-2 elicited a TRP-2(180-188)-specific CD8+ T cell response of only 1.1% of CD8+ T cells. Vaccines containing TRP-2(180-188) without CpG ODN elicited TRP-2(180-188)-specific responses of 2.8% of CD8+ T cells when administered with IL-2. There was up to a 221-fold increase in the absolute number of TRP-2(180-188)-specific CD8+ T cells when IL-2 was added to TRP-2(180-188) plus CpG ODN-containing vaccines. Peptide plus CpG ODN vaccines administered with IL-2 generated epitope-specific CD8+ T cells by a mechanism that depended on endogenous IL-6. This is the first report of synergism between CpG ODN and IL-2. This synergism caused a striking increase in vaccine-elicited CD8+ T cells and led to epitope-specific antitumor immunity.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

CpG oligodeoxynucleotide induction of antiviral effector molecules in sheep

Immunostimulatory CpG oligodeoxynucleotide (ODN) can protect mice against infection by many pathogens but the mechanisms mediating disease protection are not well defined. Furthermore, the mechanisms of CpG ODN induced disease protection in vivo have not been investigated in other species. We investigated the induction of antiviral effector molecules in sheep treated with a class B CpG ODN (2007). Subcutaneous injection of ODN 2007 induced a dose-dependent increase in serum levels of the antiviral effector molecule, 2'5'-A synthetase. Peak levels of enzyme were observed 4 days following ODN injection and enzyme levels remained elevated for the following 3-5 days. Repeated ODN injections induced a more sustained elevation of serum 2'5'-A synthetase activity. Finally, formulation of ODN 2007 in emulsigen increased the level of serum 2'5'-A synthetase activity and this response was CpG-specific. Elevated serum 2'5'-A synthetase activity suggested that CpG ODN acted through the induction of either interferon (IFN)-alpha or IFN-gamma. ODN 2007 did not induce detectable levels of IFN-alpha or IFN-gamma when incubated with peripheral blood mononuclear cells, but both IFN-alpha and IFN-gamma were detected following stimulation of lymph node cells with ODN 2007. CpG ODN induction of 2'5'-A synthetase in vitro correlated with the secretion of both IFN-alpha and IFN-gamma. Furthermore, immunohistochemical staining of skin revealed a marked cellular infiltration at the site of ODN 2007 injection. This cellular infiltration was CpG-specific and consisted of primarily CD172(+) myeloid cells. Many of the cells recruited to the site of ODN 2007 injection expressed IFN-alpha and some IFN-gamma. These observations support the conclusion that localized cell recruitment and activation contribute to CpG ODN induction of antiviral effector molecules, such as interferon and 2'5'-A synthetase.     

Vaccination with metastasis-related tumor associated antigen TPD52 and CpG/ODN induces protective tumor immunity

Tumor protein D52 (TPD52) is involved in transformation and metastasis and has been shown to be over-expressed in tumor cells compared to normal cells and tissues. Murine TPD52 (mD52) shares 86% protein identity with the human TPD52 orthologue (hD52). To study TPD52 protein as a target for active vaccination recombinant, mD52 was administered as a protein-based vaccine. Naïve mice were immunized with either mD52 protein and CpG/ODN as a molecular adjuvant or CpG/ODN alone. Two weeks following the final immunization, mice were challenged s.c. with syngeneic tumor cells that over-express mD52. Two distinct murine tumor cell lines were used for challenge in this model, mKSA and 3T3.mD52. Half of the mice immunized with mD52 and CpG/ODN rejected or delayed onset of mKSA s.c. tumor cell growth, and 40% of mice challenged with 3T3.mD52 rejected s.c. tumor growth, as well as the formation of spontaneous lethal lung metastases. Mice immunized with mD52 and CpG/ODN generated detectable mD52-specific IgG antibody responses indicating that mD52 protein vaccination induced an adaptive immune response. In addition, mice that rejected tumor challenge generated tumor-specific cytotoxic T lymphocytes’ responses. Importantly, microscopic and gross evaluation of organs from mD52 immunized mice revealed no evidence of autoimmunity as assessed by absence of T cell infiltration and absence of microscopic pathology. Together, these data demonstrate that mD52 vaccination induces an immune response that is capable of rejecting tumors that over-express mD52 without the induction of harmful autoimmunity.     

Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma

Recent studies have shown that CTL epitopes derived from tumor-associated Ags can be encoded by both primary and nonprimary open reading frames (ORF). In this study we have analyzed the HLA-A2-restricted CD8(+) T cell response to a recently identified CTL epitope derived from an alternative ORF product of gene LAGE-1 (named CAMEL), and the highly homologous gene NY-ESO-1 in melanoma patients. Using MHC/peptide tetramers we detected CAMEL(1-11)-specific CD8(+) T cells in peptide-stimulated PBMC as well as among tumor-infiltrated lymph node cells from several patients. Sorting and expansion of tetramer(+) CD8(+) T cells allowed the isolation of tetramer(bright) and tetramer(dull) populations that specifically recognized the peptide Ag with high and low avidity, respectively. Remarkably, only high avidity CAMEL-specific CTL were able to recognize Ag-expressing tumor cells. A large series of HLA-A2-positive melanoma cell lines was characterized for the expression of LAGE-1 and NY-ESO-1 mRNA and protein and tested for recognition by CAMEL-specific CTL as well as CTL that recognize a peptide (NY-ESO-1(157-165)) encoded by the primary ORF products of the LAGE-1 and NY-ESO-1 genes. This analysis revealed that tumor-associated CD8(+) T cell epitopes are simultaneously and efficiently generated from both primary and nonprimary ORF products of LAGE-1 and NY-ESO-1 genes and, importantly, that this occurs in the majority of melanoma tumors. These findings underscore the in vivo immunological relevance of CTL epitopes derived from nonprimary ORF products and support their use as candidate vaccines for inducing tumor specific cell-mediated immunity against cancer.     

Immunostimulatory CpG motifs induce CTL responses to HIV type I oligomeric gp140 envelope protein

In the present study we investigated the immunomodulatory effects of two adjuvants, liposomal lipid A [L(LA)] and CpG-containing oligodeoxynucleotides (CpG ODN), to the HIV-1 ogp140 envelope protein. Administration of each of these adjuvants separately with unencapsulated ogp140 resulted in low antibody titres. Encapsulation of ogp140 in liposomes containing lipid A resulted in a sixfold increase in anti-ogp140 antibodies. The antibody titres were further enhanced threefold by the addition of CpG ODN. Priming and boosting BALB/c mice with unencapsulated ogp140 with L(LA) or encapsulation in liposomes containing lipid A induced a mixed Th1/Th2 type of immune response. In contrast, immunization with L(ogp140 + LA) plus CpG ODN switched the immune response to a Th-1 response with elevated anti-ogp140 IgG2a antibodies and IFN-gamma levels. Both adjuvants induced excellent ogp140-specific proliferative and CTL responses. Therefore, for the induction of high titre antibodies, but not for cellular responses, the antigen and lipid A have to be present in the same liposomes. These results can have significant implications in directing the Th1 or Th2 differentiation of antigen-specific immune responses in the context of vaccine development.     

Repeated administration of cytosine-phosphorothiolated guanine-containing oligonucleotides together with peptide/protein immunization results in enhanced CTL responses with anti-tumor activity

The development of therapeutic anti-cancer vaccines designed to elicit CTL responses with anti-tumor activity has become a reality thanks to the identification of several tumor-associated Ags and their corresponding peptide T cell epitopes. However, peptide-based vaccines, in general, fail to elicit sufficiently strong CTL responses capable of producing therapeutic anti-tumor effects (i.e., prolongation of survival, tumor reduction). Here we report that repeated administration of synthetic oligonucleotides containing foreign cytosine-phosphorothiolated guanine (CpG) motifs increased 10- to 100-fold the CTL response to immunization with various synthetic peptides corresponding to well-known T cell epitopes. Moreover, repeated CpG administration allowed the induction of CTL to soluble protein even in the absence of additional adjuvant. Our results indicate that the potentiating effect of CpG in CTL responses required the participation of Th lymphocytes. Repeated CpG administration resulted in overt splenomegaly and lymphadenopathy with a significant increase in the numbers of CTL precursors and dendritic cells. Protein vaccination in combination with repeated CpG therapy was effective in delaying tumor cell growth and extending survival in mice bearing melanoma tumors. These findings support the contention that repeated administration of CpG-oligonucleotides enhances the effect of peptide and protein vaccines leading to potent anti-tumor responses, presumably through the induction of Th1 and dendritic cells, which are essential for optimal CTL responses. The immunostimulatory properties of CpG motifs may be key in inducing a consistent long term immunity to tumor-associated Ags when using peptides or proteins as T cell-inducing vaccines.     

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.     

Trans-sialidase recombinant protein mixed with CpG motif-containing oligodeoxynucleotide induces protective mucosal and systemic trypanosoma cruzi immunity involving CD8+ CTL and B cell-mediated cross-priming

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4(+) and CD8(+) T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8(+) T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8(+) CTL responses.     

In vivo impact of CpG1826 oligodeoxynucleotide on CD8 T cell primary responses and survival

CpG oligodeoxynucleotide (ODN) promotes maturation of APCs in vivo and induces strong type 1 T cell responses in mice. In this study, we have investigated the ability of CpG1826 to modulate peptide-specific CD8 T cell responses in a context where CD4 T cells are likely to play a minor role. The effects of CpG1826 were evaluated in a system where a population of NP68-specific F5 TCR transgenic CD8 T cells is diluted into a polyclonal host following adoptive transfer into C57BL/10 syngeneic recipients. Using this approach, we found that CpG1826 enhanced the ability of F5 CD8 T cells to undergo multiple divisions in vivo, to express IFN-gamma ex vivo, and to up-regulate memory-associated cell surface markers such as CD122 (IL-2Rbeta) and Ly-6C. Moreover, CpG1826 greatly increased in vivo cytotoxic activity. Using tetramer detection, we found that CpG1826 promoted long-term survival of Ag-specific CD8 T cells after immunization while no NP68-specific cells were detected when the cognate peptide was injected alone. These results indicate that CpG1826 acts as an adjuvant which increases CD8 T cell effector responses and promotes long-term survival of NP68 peptide-specific cells in vivo. They also suggest that this adjuvant can modulate CD8 T cell responses in a system which is likely to be independent of CD4 T cell help.