ADAM9 drives the immunosuppressive microenvironment by cholesterol biosynthesis-mediated activation of IL6-STAT3 signaling for lung tumor progression.

Chronic inflammation associated with lung cancers contributes to immunosuppressive tumor microenvironments, reducing CD8+ T-cell function and leading to poor patient outcomes. A disintegrin and metalloprotease domain 9 (ADAM9) promotes cancer progression. Here, we aim to elucidate the role of ADAM9 in the immunosuppressive tumor microenvironment. A bioinformatic analysis of TIMER2.0 was used to investigate the correlation of ADAM9 and to infiltrate immune cells in the human lung cancer database and mouse lung tumor samples. Flow cytometry, immunohistochemistry, and RNA sequencing (RNA-seq) were performed to investigate the ADAM9-mediated immunosuppressive microenvironment. The coculture system of lung cancer cells with immune cells, cytokine array assays, and proteomic approach was used to investigate the mechanism. By analyzing the human LUAD database and the mouse lung cancer models, we showed that ADAM9 was associated with the immunosuppressive microenvironment. Additionally, ADAM9 released IL6 protein from cancer cells to inhibit IL12p40 secretion from dendritic cells, therefore leading to dendritic cell dysfunction and further affecting T-cell functions. Proteomic analysis indicated that ADAM9 promoted cholesterol biosynthesis and increased IL6-STAT3 signaling. Mechanistically, ADAM9 reduced the protein stability of LDLR, resulting in reduced cholesterol uptake and induced cholesterol biosynthesis. Moreover, LDLR reduction enhanced IL6-STAT3 activation. We reveal that ADAM9 has a novel biological function that drives the immunosuppressive tumor microenvironment by linking lung cancer's metabolic and signaling axes. Thus, by targeting ADAM9 an innovative and promising therapeutic opportunity was indicated for regulating the immunosuppression of lung cancer.

Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters.

DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the coronavirus disease 2019 (COVID-19) pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared with TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant T helper 1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and the Omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.

Correction: DNA vaccination induced protective immunity against SARS CoV-2 infection in hamsterss.

[This corrects the article DOI: 10.1371/journal.pntd.0009374.].

Omicron-specific mRNA vaccine induced cross-protective immunity against ancestral SARS-CoV-2 infection with low neutralizing antibodies.

The major challenge in COVID-19 vaccine effectiveness is immune escape by SARS-CoV-2 variants. To overcome this, an Omicron-specific messenger RNA (mRNA) vaccine was designed. The extracellular domain of the spike of the Omicron variant was fused with a modified GCN4 trimerization domain with low immunogenicity (TSomi). After immunization with TSomi mRNA in hamsters, animals were challenged with SARS-CoV-2 virus. The raised nonneutralizing antibodies or cytokine secretion responses can recognize both Wuhan S and Omicron S. However, the raised antibodies neutralized SARS-CoV-2 Omicron virus infection but failed to generate Wuhan virus neutralizing antibodies. Surprisingly, TSomi mRNA immunization protected animals from Wuhan virus challenge. These data indicated that non-neutralizing antibodies or cellular immunity may play a more important role in vaccine-induced protection than previously believed. Next-generation COVID-19 vaccines using the Omicron S antigen may provide sufficient protection against ancestral or current SARS-CoV-2 variants.

A DNA vaccine candidate delivered by an electroacupuncture machine provides protective immunity against SARS-CoV-2 infection.

A major challenge in the use of DNA vaccines is efficient DNA delivery in vivo. Establishing a safe and efficient electric transfer method is the key to developing rapid DNA vaccines against emerging infectious diseases. To overcome the complexity of designing new electric transfer machines for DNA delivery, a clinically approved electric transfer machine could be considered as an alternative. Here, we report an electroacupuncture machine-based method for DNA vaccine delivery after intramuscular injection of the COVID-19 DNA vaccine. The S gene of SARS-CoV-2 in the pVAX1 plasmid (pSARS2-S) was used as an antigen in this study. We optimized the clinically used electroacupuncture machine settings for efficient induction of the neutralizing antibody titer after intramuscular injection of pSARS2-S in mice. We found that pSARS2-S immunization at 40 Vpp for 3-5 s could induce high neutralizing antibody titers and Th1-biased immune responses. IFN-γ/TNF-α-secreting CD4+ and CD8+ T cells were also observed in the DNA vaccination group but not in the recombinant protein vaccination group. T-cell epitope mapping shows that the major reactive epitopes were located in the N-terminal domain (a.a. 261-285) and receptor-binding domain (a.a. 352-363). Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. In the preclinical toxicology study, blood biochemistry, hematology, and DNA persistence analysis reveal that the DNA delivery method is safe. Furthermore, the raised antisera could also cross-neutralize different variants of concern. These findings suggest that DNA vaccination using an electroacupuncture machine is feasible for use in humans in the future.

Low-Dose SARS-CoV-2 S-Trimer with an Emulsion Adjuvant Induced Th1-Biased Protective Immunity.

During the sustained COVID-19 pandemic, global mass vaccination to achieve herd immunity can prevent further viral spread and mutation. A protein subunit vaccine that is safe, effective, stable, has few storage restrictions, and involves a liable manufacturing process would be advantageous to distribute around the world. Here, we designed and produced a recombinant spike (S)-Trimer that is maintained in a prefusion state and exhibits a high ACE2 binding affinity. Rodents received different doses of S-Trimer (0.5, 5, or 20 µg) antigen formulated with aluminum hydroxide (Alum) or an emulsion-type adjuvant (SWE), or no adjuvant. After two vaccinations, the antibody response, T-cell responses, and number of follicular helper T-cells (Tfh) or germinal center (GC) B cells were assessed in mice; the protective efficacy was evaluated on a Syrian hamster infection model. The mouse studies demonstrated that adjuvating the S-Trimer with SWE induced a potent humoral immune response and Th1-biased cellular immune responses (in low dose) that were superior to those induced by Alum. In the Syrian hamster studies, when S-Trimer was adjuvanted with SWE, higher levels of neutralizing antibodies were induced against live SARS-CoV-2 from the original lineage and against the emergence of variants (Beta or Delta) with a slightly decreased potency. In addition, the SWE adjuvant demonstrated a dose-sparing effect; thus, a lower dose of S-Trimer as an antigen (0.5 µg) can induce comparable antisera and provide complete protection from viral infection. These data support the utility of SWE as an adjuvant to enhance the immunogenicity of the S-Trimer vaccine, which is feasible for further clinical testing.

Activation of GM-CSF and TLR2 signaling synergistically enhances antigen-specific antitumor immunity and modulates the tumor microenvironment.

BACKGROUND: The major challenge of antitumor immunotherapy is dealing with the immunosuppressive tumor microenvironment, which involves immature myeloid cell accumulation that results in T cell dysfunction. Myeloid cell activation is induced by Toll-like receptor agonists. Additionally, granulocyte/macrophage colony stimulating factor (GM-CSF) promotes myelopoiesis and recruits myeloid cells. Here, we combined the Toll-like receptor 2 (TLR2) agonist lipoprotein and GM-CSF to assess whether this bifunctional immunotherapy has synergistic effects on myeloid cells and could be further developed as a therapeutic intervention that enhances the antitumor response. METHODS: We investigated the synergistic effects of biadjuvanted tumor antigen on antigen-presenting cell (APC) activation in bone marrow-derived dendritic cells. Furthermore, therapeutic efficacy was monitored in different tumor models treated via intratumoral or subcutaneous administration routes. The immune effects of the bifunctional fusion protein on myeloid cells in the tumor mass and draining lymph nodes were analyzed by flow cytometry. The induction of cytotoxic T lymphocytes was evaluated via intracellular cytokine levels, perforin/granzyme B staining and an in vivo killing assay. RESULTS: The TLR2 agonist lipoprotein combined with GM-CSF synergistically induced DC maturation, which subsequently enhanced antitumor immunity. In addition, rlipoE7m-MoGM modulated tumor-infiltrating myeloid cell populations. Vaccination with rlipoE7m-MoGM therapy increased the number of CCR7+CD103+ cDC1s, whereas the number of suppressive tumor-associated macrophages was reduced in the tumor lesions. Consistent with this observation, proliferating antigen-specific CD8+ T cells are highly infiltrated within the tumor, and the expression of IFN-r and perforin was most pronounced within antigen-specific CD8+ T cells in mice administered rlipoE7m-MoGM therapy. This finding corresponded with observation that the combination of a TLR2 agonist and GM-CSF provides increased antitumor activity by inhibiting established tumor outgrowth and protecting against metastatic cancer compared with a TLR2 agonist alone. Importantly, tumor growth inhibition was not due to the direct effects of the TLR2 agonist or GM-CSF but was instead due to the induction of antigen-specific immunity. CONCLUSIONS: The combination of a TLR2 agonist and GM-CSF has synergistic effects that inhibit tumor growth and modulate tumor-infiltrating APCs. This therapeutic approach could be applied to other tumor antigens to treat different cancers.

Assessment of adjuvantation strategy of lipid squalene nanoparticles for enhancing the immunogenicity of a SARS-CoV-2 spike subunit protein against COVID-19.

Vaccination is regarded as the most effective intervention for controlling the coronavirus disease 2019 (COVID-19) pandemic. The objective of this study is to provide comprehensive information on lipid squalene nanoparticle (SQ@NP)-adjuvanted COVID-19 vaccines regarding modulating immune response and enhancing vaccine efficacy. After being adjuvanted with SQ@NP, the SARS-CoV-2 spike (S) subunit protein was intramuscularly (i.m.) administered to mice. Serum samples investigated by ELISA and virus neutralizing assay showed that a single-dose SQ@NP-adjuvanted S-protein vaccine can induce antigen-specific IgG and protective antibodies comparable with those induced by two doses of nonadjuvanted protein vaccine. When the mice received a boosting vaccine injection, anamnestic response was observed in the groups of adjuvanted vaccine. Furthermore, the secretion of cytokines in splenocytes, such as interferon (IFN)-γ, interleukin (IL)-5 and IL-10, was significantly enhanced after adjuvantation of S-protein vaccine with SQ@NP; however, this was not the case for the vaccine adjuvanted with conventional aluminum mineral salts. Histological examination of injection sites showed that the SQ@NP-adjuvanted vaccine was considerably well tolerated following i.m. injection in mice. These results pave the way for the performance tuning of optimal vaccine formulations against COVID-19.

DNA vaccination induced protective immunity against SARS CoV-2 infection in hamsterss.

The development of efficient vaccines against COVID-19 is an emergent need for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccine. To quickly respond to the outbreak of the SARS-CoV-2 pandemic, a nucleic acid-based vaccine is a novel option, beyond the traditional inactivated virus vaccine or recombinant protein vaccine. Here, we report a DNA vaccine containing the spike gene for delivery via electroporation. The spike genes of SARS-CoV and SARS-CoV-2 were codon optimized for mammalian cell expression and then cloned into mammalian cell expression vectors, called pSARS-S and pSARS2-S, respectively. Spike protein expression was confirmed by immunoblotting after transient expression in HEK293T cells. After immunization, sera were collected for antigen-specific antibody and neutralizing antibody titer analyses. We found that both pSARS-S and pSARS2-S immunization induced similar levels of antibodies against S2 of SARS-CoV-2. In contrast, only pSARS2-S immunization induced antibodies against the receptor-binding domain of SARS-CoV-2. We further found that pSARS2-S immunization, but not pSARS-S immunization, could induce very high titers of neutralizing antibodies against SARS-CoV-2. We further analyzed SARS-CoV-2 S protein-specific T cell responses and found that the immune responses were biased toward Th1. Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. These data suggest that DNA vaccination could be a promising approach for protecting against COVID-19.

A Polypeptide of Tumor-Associated Antigen L6 with Intrinsic Adjuvant Activity Enhances Antitumor Immunity.

Peptide vaccines are safe, and aim to elicit and expand tumor-specific immunity so as to eradicate tumors. However, achieving strong and long-lasting anti-tumor immunity with peptide vaccines for the antigen-specific treatment of cancer is challenging, in part because their efficacy depends on strong adjuvants or immunomodulators. We approached this problem by conjugating an epitope-based cancer vaccine with a lipidated sequence (an immunomodulator) to elicit a strong immune response. Lipidated and non-lipidated polyepitope proteins were generated that contained the universal T helper cell epitope (pan-DR), B cell epitopes, and the extended loop sequence of extracellular domain 2 of tumor-associated antigen L6 (TAL6). We show that the lipidated polyepitope cancer vaccine can activate bone marrow-derived dendritic cells, and trigger effective antigen-specific antibody and T helper cell responses, more effectively than the non-lipidated vaccine. Moreover, potent T cell immune responses were elicited in mice inoculated with the lipidated polyepitope cancer vaccine, providing protective antitumor immunity in mice bearing TAL6 tumors. Our study demonstrates that a lipidated polyepitope cancer vaccine could be employed to generate potent anti-tumor immune responses, including humoral and cellular immunity, which could be beneficial in the treatment of TAL6+ cancer.

Liposomal TLR9 Agonist Combined with TLR2 Agonist-Fused Antigen Can Modulate Tumor Microenvironment through Dendritic Cells.

Dendritic cells (DCs) are antigen-presenting cells involved in T cell activation and differentiation to regulate immune responses. Lipoimmunogens can be developed as pharmaceutical lipoproteins for cancer immunotherapy to target DCs via toll-like receptor 2 (TLR2) signaling. Previously, we constructed a lipoimmunogen, a lipidated human papillomavirus (HPV) E7 inactive mutant (rlipoE7m), to inhibit the growth of HPV16 E7-expressing tumor cells in a murine model. Moreover, this antitumor effect could be enhanced by a combinatory treatment with CpG oligodeoxynucleotides (ODN). To improve safety, we developed a rlipoE7m plus DOTAP liposome-encapsulated native phosphodiester CpG (POCpG/DOTAP) treatment to target DCs to enhance antitumor immunity. We optimized the formulation of rlipoE7m and POCpG/DOTAP liposomes to promote conventional DC and plasmacytoid DC maturation in vitro and in vivo. Combination of rlipoE7m plus POCpG/DOTAP could activate conventional DCs and plasmacytoid DCs to augment IL-12 production to promote antitumor responses by intravenous injection. In addition, the combination of rlipoE7m plus POCpG/DOTAP could elicit robust cytotoxic T lymphocytes (CTLs) by intravenous immunization. Interestingly, the combination of rlipoE7m plus POCpG/DOTAP could efficiently inhibit tumor growth via intravenous immunization. Moreover, rlipoE7m plus POCpG/DOTAP combined reduced the number of tumor-infiltrating regulatory T cells dramatically due to downregulation of IL-10 production by DCs. These results showed that the combination of rlipoE7m plus POCpG/DOTAP could target DCs via intravenous delivery to enhance antitumor immunity and reduce the number of immunosuppressive cells in the tumor microenvironment.

Immunological evaluation of a novel HLA-A2 restricted phosphopeptide of tumor associated Antigen, TRAP1, on cancer therapy.

The tumor necrosis factor receptor associated protein 1 (TRAP1) is a mitochondria chaperon protein that has been previously implicated as a target for cancer therapy due to its expression level is linked to tumor progression. In this study, an immunodominant phosphopeptide of TRAP1 was identified from an HLA-A2 gene transfected mouse cancer cell line using mass spectrometry, and a synthetic phosphopeptide was generated to evaluate the potency on cancer immunotherapy. In the transporter associated with antigen processing (TAP) deficient cell, the conjugated phosphate group plays a critical role to enhance the binding affinity of phosphopeptide with HLA-A2 molecule. On the basis of immunological assay, immunization of synthetic phosphopeptide could induce a high frequency of IFN-γ-secreting CD8+ T cells in HLA-A2 transgenic mice, and the stimulated cytotoxic T lymphocytes showed a high target specificity to lysis the epitope-pulsed splenocytes in vivo and the human lung cancer cell in vitro. In a tumor challenge assay, vaccination of the HLA-A2 restricted phosphopeptide appeared to suppress the tumor growth and prolong the survival period of tumor-bearing mice. These results suggest that novel phosphopeptide is naturally presented as a HLA-A2-restricted CTL epitope and capable of being a potential candidate for the development of therapeutic vaccine against high TRAP1-expressing cancers.

Recent progress in GM-CSF-based cancer immunotherapy.

Cancer immunotherapy is a growing field. GM-CSF, a potent cytokine promoting the differentiation of myeloid cells, can also be used as an immunostimulatory adjuvant to elicit antitumor immunity. Additionally, GM-CSF is essential for the differentiation of dendritic cells, which are responsible for processing and presenting tumor antigens for the priming of antitumor cytotoxic T lymphocytes. Some strategies have been developed for GM-CSF-based cancer immunotherapy in clinical practice: GM-CSF monotherapy, GM-CSF-secreting cancer cell vaccines, GM-CSF-fused tumor-associated antigen protein-based vaccines, GM-CSF-based DNA vaccines and GM-CSF combination therapy. GM-CSF also contributes to the regulation of immunosuppression in the tumor microenvironment. This review provides recommendations regarding GM-CSF-based cancer immunotherapy.

A novel liposomal recombinant lipoimmunogen enhances anti-tumor immunity.

Synthetic liposomes provide a biocompatible and biodegradable approach for delivering drugs and antigens. In addition, self-adjuvanting recombinant lipoproteins (rlipoproteins) can enhance Th1 anti-tumor immune responses via the TLR2 signaling pathway. To generate a liposomal rlipoprotein for a cancer immunotherapeutic vaccine, we assessed 3 types of synthetic liposomes for use with the rlipoproteins rlipoE7m and rlipoOVA. We determined that the cationic liposome DOTAP could stabilize anionic rlipoproteins and delay rlipoprotein release. Surprisingly, rlipoproteins and DOTAP could synergistically up-regulate CD83 expression in bone marrow-derived dendritic cells (BMDCs). Compared with other liposome formulations, the rlipoprotein/DOTAP formulation elicited higher cytotoxic T-lymphocyte (CTL) responses. To explore the mechanism of BMDC activation by rlipoprotein/DOTAP, we assessed the production of reactive oxygen species (ROS) and the TNF-α secretion of BMDCs. We observed that rlipoprotein/DOTAP induced ROS to the same extent as DOTAP did. In addition, TLR2 signaling was also required for the TNF-α secretion of rlipoprotein/DOTAP-treated BMDCs. Moreover, compared with rlipoOVA-treated BMDCs, rlipoOVA/DOTAP-treated BMDCs increased the levels of IFN-γ produced by OVA-specific T cells. We also observed that rlipoE7m/DOTAP treatment but not rlipoE7m treatment delayed tumor growth. These results indicate that the rlipoprotein/DOTAP formulation can synergistically activate BMDCs via ROS and the TLR2 signaling pathway. In summary, rlipoprotein/DOTAP is a novel and stable formulation for cancer immunotherapy.

A Toll-like receptor 2 agonist-fused antigen enhanced antitumor immunity by increasing antigen presentation and the CD8 memory T cells population.

The induction of long-lived effector CD8+ T cells is key to the development of efficient cancer vaccines. In this study, we demonstrated that a Toll-like receptor 2 (TLR2) agonist-fused antigen increased antigen presentation via TLR2 signaling and induced effector memory-like CD8+ T cells against cancer after immunization. The N-terminus of ovalbumin (OVA) was biologically fused with a bacterial lipid moiety TLR2 agonist to produce a recombinant lipidated ovalbumin (rlipo-OVA). We demonstrated that rlipo-OVA activated bone marrow-derived dendritic cells (BM-DCs) maturation and increased antigen presentation by major histocompatibility complex (MHC) class I via TLR2. After immunization, rlipo-OVA skewed the immune response towards T helper (Th) 1 and induced OVA-specific cytotoxic T lymphocyte (CTL) responses. Moreover, immunization with rlipo-OVA induced higher numbers of effector memory (CD44+CD62L-) CD8+ T cells compared with recombinant ovalbumin (rOVA) alone or rOVA mixed with the TLR2 agonist Pam3CSK4. Accordingly, the CD27+CD43+ effector memory CD8+ T cells expressed high levels of the long-lived CD127 marker. The administration of rlipo-OVA could inhibit tumor growth, but the anti-tumor effects were lost after the depletion of CD8 or CD127 cells in vivo. These findings suggested that the TLR2 agonist-fused antigen induced long-lived memory CD8+ T cells for efficient cancer therapy.

Self-adjuvanting lipoimmunogens for therapeutic HPV vaccine development: potential clinical impact.

The goal of therapeutic HPV vaccines is the induction of cytotoxic T lymphocyte immunity against HPV-associated cancers. Recombinant proteins and synthetic peptides have high safety profiles but low immunogenicity, which limits their efficacy when used in a vaccine. Self-adjuvanting lipid moieties have been conjugated to synthetic peptides or expressed as lipoproteins to enhance the immunogenicity of vaccine candidates. Mono-, di- and tri-palmitoylated peptides have been demonstrated to activate dendritic cells and induce robust cellular immunity against infectious diseases and cancer. Recently, a platform technology using the high-yield production of recombinant lipoproteins with Toll-like receptor 2 agonist activity was established for the development of novel subunit vaccines. This technology represents a novel strategy for the development of therapeutic HPV vaccines. In this review, we describe recent progress in the design of therapeutic HPV vaccines using lipoimmunogens.

Depletion of tumor-associated macrophages enhances the anti-tumor immunity induced by a Toll-like receptor agonist-conjugated peptide.

It has been reported that lipopeptides can be used to elicit cytotoxic T lymphocyte (CTL) responses against viral diseases and cancer. In our previous study, we determined that mono-palmitoylated peptides can enhance anti-tumor responses in the absence of adjuvant activity. To investigate whether di-palmitoylated peptides with TLR2 agonist activity are able to induce anti-tumor immunity, we synthesized a di-palmitic acid-conjugated long peptide that contains a murine CTL epitope of HPV E749-57 (Pam2IDG). Pam2IDG stimulated the maturation of bone marrow-derived dendritic cells (BMDCs) through TLR2/6. After immunization, Pam2IDG induced higher levels of T cell responses than those obtained with its non-lipidated counterpart (IDG). In the prophylactic model, Pam2IDG immunization completely inhibited tumor growth, whereas IDG immunization was unable to inhibit tumor growth. However, Pam2IDG immunization could not effectively inhibit the growth of established tumors. Therefore, we further investigated whether the depletion of immunosuppressive factors could improve the therapeutic effects of Pam2IDG. Our data indicate that treatment with Pam2IDG combined with clodronate/liposome delays tumor growth and increases the survival rate. We also observed that the therapeutic effects of Pam2IDG are improved by diminishing the function of tumor-associate macrophages (TAMs) and through the use of an IL10 receptor blocking antibody or a Cyclooxygenase 2 (Cox-2) inhibitor. In conclusion, the depletion of TAMs may enhance the anti-tumor immunity of a TLR2 agonist-conjugated peptide.

Molecular mechanisms of TLR2-mediated antigen cross-presentation in dendritic cells.

Cross-presentation is a key function of dendritic cells (DCs), which present exogenous Ags on MHC class I molecules to prime CTL responses. The effects of TLR triggering on the cross-presentation of exogenous Ags by DCs remain unclear. In this study, we used synthetic dipalmitoylated peptides and TLR2 agonist-conjugated peptides as models to elucidate the mechanisms of TLR2-mediated cross-presentation. We observed that the internalization of dipalmitoylated peptides by bone marrow-derived DCs was facilitated by TLR2 via clathrin-mediated endocytosis. The administration of these dipalmitoylated peptide-pulsed bone marrow-derived DCs eliminated established tumors through TLR2 signaling. We further demonstrated that the induction of Ag-specific CTL responses and tumor regression by dipalmitoylated peptides was TAP independent. In addition, presentation of dipalmitoylated peptides by MHC class I molecules was blocked in the presence of an endosomal acidification inhibitor (chloroquine) or a lysosomal degradation inhibitor (Z-FL-COCHO). The endocytosed dipalmitoylated peptide also passed rapidly from early endosome Ag-1-positive endosomes to RAS-related GTP-binding protein 7 (Rab7)-associated late endosomes compared with their nonlipidated counterparts. Furthermore, we found that dipalmitoylated peptide-upregulated Rab7 expression correlated with Ag presentation via the TLR2/MyD88 pathway. Both JNK and ERK signaling pathways are required for upregulation of Rab7. In summary, our data suggest that TLR2-mediated cross-presentation occurs through the upregulation of Rab7 and a TAP-independent pathway that prime CTL responses.

Recombinant lipidated HPV E7 induces a Th-1-biased immune response and protective immunity against cervical cancer in a mouse model.

The E7 oncoprotein of human papillomavirus (HPV) is an ideal target for developing immunotherapeutic strategies against HPV-associated tumors. However, because protein-based immunogens alone are poor elicitors of the cytotoxic T-lymphocyte (CTL) responses, they have been difficult to exploit for therapeutic purposes. In this study, we report that a recombinant lipoprotein consisting of inactive E7 (E7m) biologically linked to a bacterial lipid moiety (rlipo-E7m) induces the maturation of mouse bone marrow-derived dendritic cells through toll-like receptor 2 (TLR2), skews the immune responses toward the Th1 responses and induces E7-specific CTL responses. We further studied the ability of rlipo-E7m to provide protection against a TC-1 tumor cell challenge in an animal model. Mice prophylactically immunized with two 10-µg doses of rlipo-E7m were found to be free of TC-1 tumor growth. Experiments in a therapeutic immunization model showed that the tumor volume in mice receiving a single dose of rlipo-E7m was less than 0.01 cm(3) on day 40, whereas the tumor volume in mice treated with rE7m was 2.28±1.21 cm(3). The tumor volume of the entire control group was over 3 cm(3). In addition, we demonstrated that the CD8+ T cells play a major role in anti-tumor immunity when administration of rlipo-E7m. These results demonstrate that rlipo-E7m could be a promising candidate for treating HPV-associated tumors.

Presentation of lipopeptide by dendritic cells induces anti-tumor responses via an endocytosis-independent pathway in vivo.

Cross-presentation by DCs is the major mechanism by which exogenous antigens activate CTLs. However, the mechanisms of entry and presentation of vaccine peptides by DCs remain unclear. In this study, we determined that the mechanisms of antigen presentation differed between nonlipidated and monopalmitoylated peptide antigens. We found that a nonlipidated long peptide could be taken up by DCs and that the peptide could be colocalized with early endosomes. The uptake of nonlipidated peptides by DCs was inhibited at low temperatures or by the depolymerization of actin filaments or microtubules. In contrast, lipidated peptides were internalized by DCs at low temperatures, and internalization was not inhibited when actin filaments or microtubules were depolymerized. Moreover, lipidated peptide, but not nonlipidated peptide, was internalized by nonphagocytic Jurkat cells. The endosomal/lysosomal and proteasomal degradation pathways were necessary for nonlipidated presentation leading to the activation of CD8(+) T cells, but the proteasomal degradation pathway alone was sufficient to process lipidated peptides for MHC class I presentation. We further found that lipidated peptides could enhance peptide-specific T cell responses in vitro and in vivo and induced stronger antitumor responses than nonlipidated peptides. Taken together, our results demonstrate that DCs present lipidated peptides through an endocytosis-independent pathway to promote strong anti-tumor effects in vivo.