Specific antihepatocellular carcinoma T cells generated by dendritic cells pulsed with hepatocellular carcinoma cell line HepG2 total RNA

Dendritic cell (DC) vaccination with the use of total tumor RNA provides the potential to generate a polyclonal immune response to multiple known and unknown tumor antigens without HLA restriction. Our study evaluated this approach as potential immunotherapy for patients with hepatocellular carcinoma (HCC). Immature DCs generated from peripheral blood mononuclear cells of patients with HCC were transfected with HepG2-GFP (HepG2 cells transfected stably with plasmid pEGFP-C3) cells total RNA. Transfected, matured DCs were used to stimulate autologous T cells. Results revealed that DCs transfected with HepG2-GFP cells total RNA expressed EGFP when observed by flow cytometry. Compared with those before transfection, the expressions of membrane molecules were increased dramatically, and interleukin-12p70 release in the supernatant was elevated significantly. Specific T cells generated by DCs transfected with HepG2-GFP total RNA recognized HLA-matched HepG2 cell lines specifically. These findings indicate that these RNA-transfected DCs successfully generate specific T cells that specifically recognize HCC cells. Total tumor RNA-pulsed DCs may have potential as an adjuvant immunotherapy for patients with HCC.     

Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells loaded with hepatocellular carcinoma -derived RNA or cell lysate

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.     

Comparison of cytotoxic T lymphocyte responses against pancreatic cancer induced by dendritic cells transfected with total tumor RNA and fusion hybrided with tumor cell

Pancreatic cancer (PC) is a deadly human malignancy. Dendritic cell (DC)-based immunotherapy with whole tumor antigens demonstrates potential efficiency in cancer treatment. Tumor RNA and tumor fusion hybrid cells are sources of whole tumor antigens for preparing DC tumor vaccines. However, the efficacy of these sources in eliciting immune responses against PC has not yet to be directly compared. In the present study, patient-derived PC cells and DCs were fused (DC–tumor hybrids) and primary cultured PC cell-derived total RNA was electroporated into autologous DCs (DC–tumor RNA). The antitumor immune responses induced by DC–tumor hybrids and DC–tumor RNA were compared directly. The results showed that both RNA and hybrid methodologies could induce tumor-specific cytotoxic T lymphocyte (CTL) responses, but pulsing DCs with total tumor RNA could induce a higher frequency of activated CTLs and T-helper cells than fusing DCs with autologous tumor cells. In addition, DC–tumor RNA triggered stronger autologous tumor cell lysis than DC–tumor hybrids. It could be concluded that DCs pulsed with whole tumor RNA are superior to those fused with tumor cells in priming anti-PC CTL responses. Electroporation with total tumor RNA may be more suitable for DC-based PC vaccination.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4⁺ and CD8⁺ T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

Synergistic induction of antigen-specific CTL by fusions of TLR-stimulated dendritic cells and heat-stressed tumor cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- gamma at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+ IFN-gamma+ CD8+ T cells and CD154+ IFN-gamma+ CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.     

Peptides identified through phage display direct immunogenic antigen to dendritic cells

Dendritic cells (DC) play a critical role in adaptive immunity by presenting Ag, thereby priming naive T cells. Specific DC-binding peptides were identified using a phage display peptide library. DC-peptides were fused to hepatitis C virus nonstructural protein 3 (NS3) while preserving DC targeting selectivity and Ag immunogenicity. The NS3-DC-peptide fusion protein was efficiently presented to CD4+ and CD8+ T cells derived from hepatitis C virus-positive blood cells, inducing their activation and proliferation. This immunogenic fusion protein was significantly more potent than NS3 control fusion protein or NS3 alone. In chimeric NOD-SCID mice transplanted with human cells, DC-targeted NS3 primed naive CD4+ and CD8+ T cells for potent NS3-specific proliferation and cytokine secretion. The capacity of peptides to specifically target immunogenic Ags to DC may establish a novel strategy for vaccine development.     

Electroporation as a "prime/boost" strategy for naked DNA vaccination against a tumor antigen

We have developed novel DNA fusion vaccines encoding tumor Ags fused to pathogen-derived sequences. This strategy activates linked T cell help and, using fragment C of tetanus toxin, amplification of anti-tumor Ab, CD4(+), and CD8(+) T cell responses is achievable in mice. However, there is concern that simple DNA vaccine injection may produce inadequate responses in larger humans. To overcome this, we tested electroporation as a method to increase the transfection efficiency and immune responses by these tumor vaccines in vivo in mice. Using a DNA vaccine expressing the CTL epitope AH1 from colon carcinoma CT26, we confirmed that effective priming and tumor protection in mice are highly dependent on vaccine dose and volume. However, suboptimal vaccination was rendered effective by electroporation, priming higher levels of AH1-specific CD8(+) T cells able to protect mice from tumor growth. Electroporation during priming with our optimal vaccination protocol did not improve CD8(+) T cell responses. In contrast, electroporation during boosting strikingly improved vaccine performance. The prime/boost strategy was also effective if electroporation was used at both priming and boosting. For Ab induction, DNA vaccination is generally less effective than protein. However, prime/boost with naked DNA followed by electroporation dramatically increased Ab levels. Thus, the priming qualities of DNA fusion vaccines, integrated with the improved Ag expression offered by electroporation, can be combined in a novel homologous prime/boost approach, to generate superior antitumor immune responses. Therefore, boosting may not require viral vectors, but simply a physical change in delivery, facilitating application to the cancer clinic.     

mRNA transfection of DC in the immature or mature state: comparable in vitro priming of Th and cytotoxic T lymphocytes against DC electroporated with tumor cell line-derived mRNA

BACKGROUND: The use of mRNA in vaccine studies has generally been through loading or transfection of immature DC followed by a maturation step. A recent study has suggested that this strategy may result in inferior priming of cytotoxic T lymphocytes (CTL). Furthermore the study did not address any possible effects on the priming of CD4(+) T-cell responses. METHODS: We compared mRNA transfection of mature DC with that of immature DC, using as a read-out their capacity to prime autologous T cells during one cycle of in vitro stimulation. In this model system we used mRNA from the tumor cell line Jurkat E6. DC transfected at either the immature stage (day 5) or mature stage (day 7) displayed a similar phenotype. RESULTS: Interestingly, no major differences in their ability to prime CD4 and CD8 T-cell responses were observed. As in vitro priming to some extent may reflect the capacity of these DC to prime T cells in vivo after vaccination, these studies support the use of mRNA-transfected mature DC in clinical protocols. DISCUSSION: Transfection of DC at the end of the maturation process represents a logistical improvement in the GMP production of mRNA-transfected DC for clinical protocols.     

Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12-dependent mechanism

Myeloid-origin dendritic cells (DCs) can develop into IL-12-secreting DC1 or non-IL-12-secreting DC2 depending on signals received during maturation. Through rapid culture techniques that prepared either mature, CD83+ DC1 or DC2 from CD14+ monocytes in only 2 days followed by a single 6-7 day DC-T cell coculture, we sensitized normal donor CD8+ T cells to tumor Ags (HER-2/neu, MART-1, and gp100) such that peptide Ag-specific lymphocytes constituted up to 16% of the total CD8+ population. Both DC1 and DC2 could sensitize CD8+ T cells that recognized peptide-pulsed target cells. However, with DC2, a general decoupling was observed between recognition of peptide-pulsed T2 target cells and recognition of Ag-expressing tumor cells, with peptide-sensitized T cells responding to tumor only about 15% of the time. In contrast, direct recognition of tumor by T cells was dramatically increased (to 85%) when DC1 were used for sensitization. Enhanced tumor recognition was accompanied by 10- to 100-fold increases in peptide sensitivity and elevated expression of CD8beta, characteristic of high functional avidity T cells. Both of these properties were IL-12-dependent. These results demonstrate the utility of rapid DC culture methods for high efficiency in vitro T cell sensitization that achieves robust priming and expansion of Ag-specific populations in 6 days. They also demonstrate a novel function of IL-12, which is enhancement of CD8+ T cell functional avidity. A new approach to DC-based vaccines that emphasizes IL-12 secretion to enhance functional avidity and concomitant tumor recognition by CD8+ T cells is indicated.     

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.     

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.     

Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection

Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8(+) T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.     

Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells

The activation of naive CD8+ T cells has been attributed to two mechanisms: cross-priming and direct priming. Cross-priming and direct priming differ in the source of Ag and in the cell that presents the Ag to the responding CD8+ T cells. In cross-priming, exogenous Ag is acquired by professional APCs, such as dendritic cells (DC), which process the Ag into peptides that are subsequently presented. In direct priming, the APCs, which may or may not be DC, synthesize and process the Ag and present it themselves to CD8+ T cells. In this study, we demonstrate that naive CD8+ T cells are activated by a third mechanism, called cross-dressing. In cross-dressing, DC directly acquire MHC class I-peptide complexes from dead, but not live, donor cells by a cell contact-mediated mechanism, and present the intact complexes to naive CD8+ T cells. Such DC are cross-dressed because they are wearing peptide-MHC complexes generated by other cells. CD8+ T cells activated by cross-dressing are restricted to the MHC class I genotype of the donor cells and are specific for peptides generated by the donor cells. In vivo studies demonstrate that optimal priming of CD8+ T cells requires both cross-priming and cross-dressing. Thus, cross-dressing may be an important mechanism by which DC prime naive CD8+ T cells and may explain how CD8+ T cells are primed to Ags that are inefficiently cross-presented.     

Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.     

Production of myeloid dendritic cells (DC) pulsed with tumor-specific idiotype protein for vaccination of patients with multiple myeloma

BACKGROUND: Immunotherapy of cancer with DC vaccines has produced encouraging results in clinical trials. Antigen (Ag)-pulsed DC have elicited CD4+ and CD8+ T-cell immunity and tumor regression in humans. However, there is no standard method of DC production. The DC phenotype, number and Ag-loading process used in these studies have varied, making comparisons between trials difficult. METHODS: In the present report a reproducible method was developed for the production of a DC-based vaccine. Monocytes were enriched by adhesion from healthy donor apheresis products and cultured with growth factors for maturation into DC. The cells were loaded with the tumor Ag idiotype proteins from patients with multiple myeloma. DC culture and Ag loading were performed in an automated and closed system. The DC product was characterized for phenotype by flow cytometry and for function in Ag uptake and Ag presentation. RESULTS: These monocyte-derived DC expressed high levels of costimulatory molecules (CD80/86). Ag-pulsed DC functioned to induce allogeneic proliferative lymphocyte responses and Ag-specific cytotoxic T lymphocyte (CTL) responses. The DC viability, phenotype and function were well preserved following prolonged frozen storage. Aliquots from the product of a single DC preparation could be used for sequential vaccinations without batch to batch variability. DISCUSSION: Ag-pulsed DC can be reproducibly generated for clinical use. These standardized methods are now being employed for a clinical trial to evaluate idiotype-pulsed DC vaccine therapy following non-myeloablative transplant for the treatment of multiple myeloma.     

Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-gamma-producing CD4(+) donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.     

Prime-boost with alternating DNA vaccines designed to engage different antigen presentation pathways generates high frequencies of peptide-specific CD8+ T cells

The route for presentation of Ag to CD8+ or CD4+ T cells following DNA vaccination is critical for determining outcome, but the pathways involved are unclear. In this study, we compare two different DNA vaccine designs aimed to elicit CD8+ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4+ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of Th responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide. This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated Th-dependent priming of CD8+ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8+ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization.     

Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8(+) T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.