RNA based vaccines

The recognition that CD8(+) T-cell mediated Th1 immune responses were necessary to produce immunity to intracellular and transformed self pathogens led to intense interest in the delivery of nucleic acids, DNA, or RNA encoding candidate antigens, as vaccines. Antigen presenting cells (APC) encounter most protein and vaccine immunogens as extracellular proteins and, thus, present them on major histocompatibility complex (MHC) class II molecules leading to the activation of CD4(+) T cells. Protein antigens encoded by nucleic acids delivered to dendritic cell (DC) are produced inside the cell and, thus, can stimulate MHC class I mediated activation of CD8(+) T-cell immune responses. Unfortunately, DCs are not readily transfected with DNA (Akbari et al., 1999) resulting in the requirement for high concentrations of DNA and repeated immunizations to achieved immune responses. RNA, on the other hand, is readily taken up and expressed by DC, making it an alternative vaccine candidate. In this article, we will discuss immune responses developed, interactions between APC and RNA that activate and dictate DC activation, and preliminary studies using RNA in vivo and in vitro to develop protective immunity.     

Cytosolic DNA-activated human dendritic cells are potent activators of the adaptive immune response

Recent studies in cell lines and genetically engineered mice have demonstrated that cytosolic dsDNA could activate dendritic cells (DCs) to become effector APCs. Recognition of DNA might be a major factor in antimicrobial immune responses against cytosolic pathogens and also in human autoimmune diseases such as systemic lupus erythematosus. However, the role of cytosolic dsDNA in human DC activation and its effects on effector T and B cells are still elusive. In this study, we demonstrate that intracellular dsDNA is a potent activator of human monocyte-derived DCs as well as primary DCs. Activation by dsDNA depends on NF-κB activation, partially on the adaptor molecule IFN-promoter stimulator-1 and the novel cytosolic dsDNA receptor IFI16, but not on the previously recognized dsDNA sentinels absent in melanoma 2, DNA-dependent activator of IFN regulatory factor 3, RNA polymerase III, or high-mobility group boxes. More importantly, we report for the first time, to our knowledge, that human dsDNA-activated DCs, rather than LPS- or inflammatory cytokine mixture-activated DCs, represent the most potent inducers of naive CD4(+) T cells to promote Th1-type cytokine production and generate CD4(+) and CD8(+) cytotoxic T cells. dsDNA-DCs, but not LPS- or mixture-activated DCs, induce B cells to produce complement-fixing IgG1 and IgG3 Abs. We propose that cytosolic dsDNA represents a novel, more effective approach to generate DCs to enhance vaccine effectiveness in reprogramming the adaptive immune system to eradicate infectious agents, autoimmunity, allergy, and cancer.     

Potent CD4+ T cell responses elicited by a bicistronic HIV-1 DNA vaccine expressing gp120 and GM-CSF.

Virus-specific CD4(+) T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4(+) as well as CD8(+) T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4(+) T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4(+) T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4(+) T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4(+) T cell responses was selective, since vaccine-elicited Ab and CD8(+) T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4(+) T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

Lung cancer patients’ CD4<Superscript>+</Superscript> T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients' T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4(+) T lymphocytes in the activation of cytotoxic CD8(+) T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4(+) T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4(+) T cells. METHODS: Potential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4(+) T cell activation was quantified by measuring Type 1 and Type 2 cytokine release. RESULTS: The vaccines activated CD4(+) T cells from all ten patients, despite the presence of CD33(+)CD11b(+) MDSC. Activated CD4(+) T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts. CONCLUSIONS: The NSCLC vaccines activate tumor-specific CD4(+) T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.     

B lymphocytes participate in cross-presentation of antigen following gene gun vaccination

Although endocytosed proteins are commonly presented via the class II MHC pathway to stimulate CD4(+) T cells, professional APCs can also cross-present Ags, whereby these exogenous peptides can be complexed with class I MHC for cross-priming of CD8(+) T cells. Whereas the ability of dendritic cells (DCs) to cross-present Ags is well documented, it is not known whether other APCs may also play a role, or what is the relative contribution of cross-priming to the induction of acquired immunity after DNA immunization. In this study, we compared immune responses generated after gene gun vaccination of mice with DNA vaccine plasmids driven by the conventional CMV promoter, the DC-specific CD11c promoter, or the keratinocyte-specific K14 promoter. The CD11c promoter achieved equivalent expression in CD11c(+) DCs in draining lymph nodes over time, as did a conventional CMV-driven plasmid. However, immunization with DC-restricted DNA vaccines failed to generate protective humoral or cellular immunity to model Ags influenza hemagglutinin and OVA, despite the ability of CD11c(+) cells isolated from lymph nodes to stimulate proliferation of Ag-specific T cells directly ex vivo. In contrast, keratinocyte-restricted vaccines elicited comparable T and B cell activity as conventional CMV promoter-driven vaccines, indicating that cross-priming plays a major role in the generation of immune responses after gene gun immunization. Furthermore, parallel studies in B cell-deficient mu-MT mice demonstrated that B lymphocytes, in addition to DCs, mediate cross-priming of Ag-specific T cells. Collectively, these data indicate that broad expression of the immunogen is required for optimal induction of protective acquired immunity.     

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).     

Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8(+) T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8(+) T cell proliferation and IFN-gamma secretion, and strong induction of long-lived CD8(+) T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8(+) T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8(+) T cell function, we show that in the partial, but not total, absence of CD4(+) T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8(+) T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.     

T cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic cells

We have developed an individualized melanoma vaccine based on autologous dendritic cells (DCs) transfected with autologous tumor-mRNA. The vaccine targets the unique spectrum of tumor antigens in each patient and may recruit multiple T cell clones. In a recent phase I/II trial, we demonstrated T cell responses against vaccine antigens in 9/19 patients evaluable by T cell assays. Here, we report a follow-up study that was conducted to characterize interesting T cell responses and to investigate the effects of long-term booster vaccination. Two patients were selected for continued vaccine therapy. The clinical follow-up suggested a favorable clinical development in both patients. The immunological data (T cell proliferation/IFNgamma ELISPOT/Bioplex cytokine assays) indicated sustained T cell responses and suggested an enhancing effect of booster vaccinations. Both CD4(+) and CD8(+) T cell responses were demonstrated. From post-vaccination samples, we generated 39 T cell clones that responded specifically to stimulation by mRNA-transfected DCs and 12 clones that responded to mock-transfected DCs. These data clearly indicate a two-component vaccine response, against transfected and non-transfected antigens. T cell receptor (TCR) clonotype mapping, performed on 11 tDC-specific clones, demonstrated that 10/11 clones had different TCRs. The results thus indicate a broad spectrum T cell response against antigens encoded by the transfected tumor-mRNA. We generally observed mixed Th1/Th2 cytokine profiles, even in T cell clones that were confirmed to be derived from a single cell. This finding suggests that cytokine patterns after cancer vaccination may be more complex than indicated by the classic Th1/Th2 dichotomy.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake

Dendritic cell (DC) Ag cross-presentation is generally associated with immune responses to tumors and viral Ags, and enhancement of this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8(+) murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA Ag, although development, maturation, and Ag processing and presentation of DCs are normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of Ags such as OVA and transferrin but not fluid-phase or phagocytic Ag uptake. CD13 and Ag are cointernalized in DCs, but CD13 did not coimmunoprecipitate with Ag receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs, and blocking of these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance Ag uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.     

Immune evasion by Yersinia enterocolitica: differential targeting of dendritic cell subpopulations in vivo

CD4(+) T cells are essential for the control of Yersinia enterocolitica (Ye) infection in mice. Ye can inhibit dendritic cell (DC) antigen uptake and degradation, maturation and subsequently T-cell activation in vitro. Here we investigated the effects of Ye infection on splenic DCs and T-cell proliferation in an experimental mouse infection model. We found that OVA-specific CD4(+) T cells had a reduced potential to proliferate when stimulated with OVA after infection with Ye compared to control mice. Additionally, proliferation of OVA-specific CD4(+) T cells was markedly reduced when cultured with splenic CD8α(+) DCs from Ye infected mice in the presence of OVA. In contrast, T-cell proliferation was not impaired in cultures with CD4(+) or CD4(-)CD8α(-) DCs isolated from Ye infected mice. However, OVA uptake and degradation as well as cytokine production were impaired in CD8α(+) DCs, but not in CD4(+) and CD4(-)CD8α(-) DCs after Ye infection. Pathogenicity factors (Yops) from Ye were most frequently injected into CD8α(+) DCs, resulting in less MHC class II and CD86 expression than on non-injected CD8α(+) DCs. Three days post infection with Ye the number of splenic CD8α(+) and CD4(+) DCs was reduced by 50% and 90%, respectively. The decreased number of DC subsets, which was dependent on TLR4 and TRIF signaling, was the result of a faster proliferation and suppressed de novo DC generation. Together, we show that Ye infection negatively regulates the stimulatory capacity of some but not all splenic DC subpopulations in vivo. This leads to differential antigen uptake and degradation, cytokine production, cell loss, and cell death rates in various DC subpopulations. The data suggest that these effects might be caused directly by injection of Yops into DCs and indirectly by affecting the homeostasis of CD4(+) and CD8α(+) DCs. These events may contribute to reduced T-cell proliferation and immune evasion of Ye.     

Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.

The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

Mucosally delivered dendritic cells activate T cells independently of IL-12 and endogenous APCs

In vitro manipulated dendritic cells (DC) have increasingly been used as a promising vaccine formulation against cancer and infectious disease. However, improved understanding of the immune mechanisms is needed for the development of safe and efficacious mucosal DC immunization. We have developed a murine model of respiratory mucosal immunization by using a genetically manipulated DC vaccine. Within 24 h of intranasal delivery, the majority of vaccine DCs migrated to the lung mucosa and draining lymph nodes and elicited a significant level of T cells capable of IFN-gamma secretion and CTL in the airway lumen as well as substantial T cell responses in the spleen. And such T cell responses were associated with enhanced protection against respiratory mucosal intracellular bacterial challenge. In comparison, parenteral i.m. DC immunization did not elicit marked airway luminal T cell responses and immune protection regardless of strong systemic T cell activation. Although repeated mucosal DC delivery boosted Ag-specific T cells in the airway lumen, added benefits to CD8 T cell activation and immune protection were not observed. By using MHC-deficient vaccine DCs, we further demonstrated that mucosal DC immunization-mediated CD8 and CD4 T cell activation does not require endogenous DCs. By using IL-12-deficient vaccine DCs, we also observed that IL-12(-/-) DCs failed to migrate to the lymph nodes but remained capable of T cell activation. Our observations indicate that mucosal delivery of vaccine DCs represents an effective approach to enhance mucosal T cell immunity, which may operate independent of vaccine IL-12 and endogenous DCs.     

The role of human T-lymphotropic virus type 1 (HTLV-1)-infected dendritic cells in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis

The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4(+) T cells and CD8(+) T cells in a viral dose-dependent manner. However, the proliferation level of CD4(+) T cells was five- to sixfold higher than that of CD8(+) T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4(+) T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4(+) and CD8(+) T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP.     

Processing and presentation of exogenous HLA class I peptides by dendritic cells from human immunodeficiency virus type 1-infected persons

Dendritic cells (DCs) loaded with viral peptides are a potential form of immunotherapy of human immunodeficiency virus type 1 (HIV-1) infection. We show that DCs derived from blood monocytes of subjects with chronic HIV-1 infection on combination antiretroviral drug therapy have increases in expression of HLA, T-cell coreceptor, and T-cell activation molecules in response to the DC maturation factor CD40L comparable to those from uninfected persons. Mature DCs (mDCs) loaded with HLA A*0201-restricted viral peptides of the optimal length (9-mer) were more efficient at activating antiviral CD8(+) T cells than were immature DCs or peptide alone. Optimal presentation of these exogenous peptides required uptake and vesicular trafficking and was comparable in DCs derived from HIV-1-infected and uninfected persons. Furthermore, DCs from HIV-1-infected and uninfected persons had similar capacities to process viral peptides with C-terminal and N-terminal extensions through their proteasomal and cytosolic pathways, respectively. We conclude that DCs derived from HIV-1-infected persons have similar abilities to process exogenous peptides for presentation to CD8(+) T cells as those from uninfected persons. This conclusion supports the use of DCs loaded with synthetic peptides in immunotherapy of HIV-1 infection.     

CD8 T-cell activation in mice injected with a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8(+) T-cells and CD4(+) T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8(+) cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.     

Quantitation of CD8+ T cell expansion,memory, and protective immunity after immunization with peptide-coated dendritic cells

Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells.     

Induction of alpha-fetoprotein-specific CD4- and CD8-mediated T-cell response using RNA-transfected dendritic cells

alpha-Fetoprotein (AFP) may be a possible target for a hepatocellular carcinoma (HCC)-specific vaccination. But some studies have demonstrated that dendritic cells (DCs) treated with AFP become dysfunctional. So in this study, we try to transfect AFP mRNA into DCs and observe the ability of DCs to induce AFP-specific CD4(+) and CD8(+) T cells. We hope that AFP can be processed and presented by DCs directly, rather than released to the cultures. So there will be no AFP negative effect on the function of DCs. In the study, immature DCs generated from peripheral blood mononuclear cells (PBMCs) of HLA-A2(+) HCC patients were transfected with AFP mRNA. Then the transfected, matured DCs were used to stimulate autologous T cells. The results showed that the expressions of membrane molecules of DCs after transfection were increased dramatically, and interleukin-12 (IL-12) p70 release in the supernatant was elevated significantly. There was only a minority of AFP release in the supernatants of transfected DCs. CTLs induced by the transfected DCs recognized HLA-matched AFP positive HepG2 cell line specifically and the AFP-specific proliferative T-cell responses could also be induced. These findings indicate that this AFP mRNA transfection strategy could generate fully functional DCs, which could induce specific T cells to recognize AFP(+) HCC cells.     

Direct ex vivo kinetic and phenotypic analyses of CD8(+) T-cell responses induced by DNA immunization

CD8(+) T-cell responses can be induced by DNA immunization, but little is known about the kinetics of these responses in vivo in the absence of restimulation or how soon protective immunity is conferred by a DNA vaccine. It is also unclear if CD8(+) T cells primed by DNA vaccines express the vigorous effector functions characteristic of cells primed by natural infection or by immunization with a recombinant live virus vaccine. To address these issues, we have used the sensitive technique of intracellular cytokine staining to carry out direct ex vivo kinetic and phenotypic analyses of antigen-specific CD8(+) T cells present in the spleens of mice at various times after (i) a single intramuscular administration of a plasmid expressing the nucleoprotein (NP) gene from lymphocytic choriomeningitis virus (LCMV), (ii) infection by a recombinant vaccinia virus carrying the same protein (vvNP), or (iii) LCMV infection. In addition, we have evaluated the rapidity with which protective immunity against both lethal and sublethal LCMV infections is achieved following DNA vaccination. The CD8(+) T-cell response in DNA-vaccinated mice was slightly delayed compared to LCMV or vvNP vaccinees, peaking at 15 days postimmunization. Interestingly, the percentage of antigen-specific CD8(+) T cells present in the spleen at day 15 and later time points was similar to that observed following vvNP infection. T cells primed by DNA vaccination or by infection exhibited similar cytokine expression profiles and had similar avidities for an immunodominant cytotoxic T lymphocyte epitope peptide, implying that the responses induced by DNA vaccination differ quantitatively but not qualitatively from those induced by live virus infection. Surprisingly, protection from both lethal and sublethal LCMV infections was conferred within 1 week of DNA vaccination, well before the peak of the CD8(+) T-cell response.