RESUMO
Toll-like receptors (TLRs) are critical components to stimulate immune responses against various infections. Recently, TLR agonists have emerged as a promising way to activate anti-tumor immunity. L-pampo, a TLR1/2 and TLR3 agonist, induces humoral and cellular immune responses and also causes cancer cell death. In this study, we investigated the L-pampo-induced signals and delineated their interactions with molecular signaling pathways using RNA-seq in immune cells and colon and prostate cancer cells. We first constructed a template network with differentially expressed genes and influential genes from network propagation using the weighted gene co-expression network analysis. Next, we obtained perturbed modules using the above method and extracted core submodules from them by conducting Walktrap. Finally, we reconstructed the subnetworks of major molecular signals utilizing a shortest path-finding algorithm, TOPAS. Our analysis suggests that TLR signaling activated by L-pampo is transmitted to oxidative phosphorylation (OXPHOS) with reactive oxygen species (ROS) through PI3K-AKT and JAK-STAT only in immune and prostate cancer cells that highly express TLRs. This signal flow may further sensitize prostate cancer to L-pampo due to its high basal expression level of OXPHOS and ROS. Our computational approaches can be applied for inferring underlying molecular mechanisms from complex gene expression profiles.
Assuntos
Redes Reguladoras de Genes , Transdução de Sinais , Receptores Toll-Like , Humanos , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Linhagem Celular Tumoral , Espécies Reativas de Oxigênio/metabolismo , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Fosforilação Oxidativa , Agonistas do Receptor Semelhante a TollRESUMO
Herpes zoster (HZ), also known as shingles, is caused by the reactivation of latent varicella-zoster virus (VZV). Decreased VZV-specific T-cell immune responses significantly contribute to the development of HZ. Shingrix is a recombinant zoster vaccine that is currently used to prevent HZ. However, Shingrix has high reactogenicity and pain at the injection site due to QS21, one of the adjuvant components. In this study, we developed a new herpes zoster vaccine formulation called CVI-VZV-001, containing gE protein and a novel liposome-based adjuvant Lipo-pam™, which consists of two TLR agonists. We evaluated the immunogenicity of CVI-VZV-001 in mouse and rabbit models. CVI-VZV-001 elicited robust gE-specific T-cell immune responses and gE-specific antibody production. Specifically, CVI-VZV-001 induced polyfunctional CD4+ T cell populations that secrete multiple cytokines. Furthermore, CVI-VZV-001 sustained the gE-specific immune responses for up to six months after immunization. To ensure CVI-VZV-001's safety for further development, we conducted a good laboratory practice (GLP) toxicity test, which confirmed that CVI-VZV-001 is safe for use. At present, CVI-VZV-001 is undergoing phase I clinical trials. This study suggests that CVI-VZV-001 can be a potent candidate for the HZ vaccine with high immunogenicity and safety.
RESUMO
Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.
Assuntos
Lectina de Ligação a Manose/biossíntese , Lectina de Ligação a Manose/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Animais , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Humanos , Lectina de Ligação a Manose/química , Proteínas Recombinantes/químicaRESUMO
TLR agonists have emerged as an efficient cancer vaccine adjuvant system that induces robust immune responses. L-pampo™, a proprietary vaccine adjuvant of TLR2 and TLR3 agonists, promotes strong humoral and cellular immune responses against infectious diseases. In this study, we demonstrate that vaccines formulated with L-pampo™ affect the recruitment and activation of dendritic cells (DCs) in draining lymph nodes (dLNs) and leading to antigen-specific T-cell responses and anti-tumor efficacy. We analyzed DC maturation and T-cell proliferation using flow cytometry and ELISA. We determined the effect of L-pampo™ on DCs in dLNs and antigen-specific T-cell responses using flow cytometric analysis and the ELISPOT assay. We employed murine tumor models and analyzed the anti-tumor effect of L-pampo™. We found that L-pampo™ directly enhanced the maturation and cytokine production of DCs and, consequently, T-cell proliferation. OVA or OVA peptide formulated with L-pampo™ promoted DC migration into dLNs and increased activation markers and specific DC subsets within dLNs. In addition, vaccines admixed with L-pampo™ promoted antigen-specific T-cell responses and anti-tumor efficacy. Moreover, the combination of L-pampo™ with an immune checkpoint inhibitor synergistically improved the anti-tumor effect. This study suggests that L-pampo™ can be a potent cancer vaccine adjuvant and a suitable candidate for combination immunotherapy.
RESUMO
BACKGROUND: Toll-like receptors (TLRs) are critical innate immune sensors that elicit antitumor immune responses in cancer immunotherapy. Although a few TLR agonists have been approved for the treatment of patients with early-stage superficial cancers, their therapeutic efficacy is limited in patient with advanced invasive cancers. Here, we identified the therapeutic role of a TLR2/3 agonist, L-pampo (LP), which promotes antitumor immunity and enhances the immune checkpoint blockade. METHODS: We generated LP by combining a TLR2 agonist, Pam3CSK4, with a TLR3 agonist, Poly (I:C). Immune responses to stimulation with various TLR agonists were compared. Tumor-bearing mice were intratumorally treated with LP, and their tumor sizes were measured. The antitumor effects of LP treatment were determined using flow cytometry, multiplexed imaging, and NanoString nCounter immune profiling. The immunotherapeutic potential of LP in combination with α-programmed cell death protein-1 (PD-1) or α-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) was evaluated in syngeneic MC38 colon cancer and B16F10 melanoma. RESULTS: The LP treatment induced a potent activation of T helper 1 (Th1) and 2 (Th2)-mediated immunity, tumor cell apoptosis, and immunogenic tumor cell death. Intratumoral LP treatment effectively inhibited tumor progression by activating tumor-specific T cell immunity. LP-induced immune responses were mediated by CD8+ T cells and interferon-γ, but not by CD4+ T cells and CD25+ T cells. LP simultaneously activated TLR2 and TLR3 signaling, thereby extensively changing the immune-related gene signatures within the tumor microenvironment (TME). Moreover, intratumoral LP treatment led to systemic abscopal antitumor effects in non-injected distant tumors. Notably, LP treatment combined with ÉPD-1 and ÉCTLA-4 further enhanced the efficacy of monotherapy, resulting in complete tumor regression and prolonged overall survival. Furthermore, LP-based combination immunotherapy elicited durable antitumor immunity with tumor-specific immune memory in colon cancer and melanoma. CONCLUSIONS: Our study demonstrated that intratumoral LP treatment improves the innate and adaptive antitumor immunity within the TME and enhances the efficacy of αPD-1 and αCTLA-4 immune checkpoint blockade.
Assuntos
Neoplasias do Colo , Melanoma , Adjuvantes Imunológicos , Animais , Linfócitos T CD8-Positivos , Inibidores de Checkpoint Imunológico , Imunidade , Fatores Imunológicos , Imunoterapia , Camundongos , Receptor 2 Toll-Like , Receptor 3 Toll-Like , Microambiente TumoralRESUMO
The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.
RESUMO
INTRODUCTION: Individuals deficient in mannose-binding lectin (MBL), an important component of the innate immune system, show increased susceptibility to infection. We investigated whether polymorphisms in the MBL2 gene and the serum level are associated with the severity and prognosis of sepsis. METHODS: A total of 266 patients with sepsis and 398 healthy controls were enrolled. We analyzed the three single nucleotide polymorphisms (Gly54Asp, -550, and +4) in the MBL2 gene. Serum samples collected on day 1 were analyzed for the levels of MBL. RESULTS: Patients who were heterozygous (A/B) or homozygous (B/B) at codon 54 (adjusted odds ratio (OR), 0.370; 95% confidence interval (CI), 0.207-0.661, P = 0.001) and who were heterozygous (H/L) or homozygous (L/L) at -550 (adjusted OR, 0.476; 95% CI, 0.249-0.910, P = 0.025) were less likely to have septic shock in the sepsis group. Using Cox regression analysis for 28-day mortality, an MBL level > or = 1.3 microg/mL showed significantly lower 28-day mortality (P = 0.020; hazard ratio, 0.571; 95% CI, 0.355-0.916) in the septic shock group. CONCLUSIONS: Homozygosity at codons 54 (A/A) and -550 (H/H) appears to be associated with the severity, but not the outcome, of sepsis, whereas a low MBL level may be an independent risk factor for mortality. These findings suggest that the genotype and serum level for MBL2 may have different clinical implications.
Assuntos
Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/genética , Polimorfismo de Nucleotídeo Único , Sepse/sangue , Sepse/genética , Idoso , Substituição de Aminoácidos , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Lectina de Ligação a Manose/deficiência , Pessoa de Meia-Idade , Prognóstico , Valores de Referência , Estudos Retrospectivos , Choque Séptico/sangue , Choque Séptico/genética , Resultado do TratamentoRESUMO
BACKGROUND: Mannose-binding lectin (MBL) deficiency leads to increased susceptibility to infection. We investigated whether serial changes in MBL levels are associated with the prognosis of patients diagnosed with septic shock, and correlated with cytokine levels. METHODS: We enrolled 131 patients with septic shock in the study. We analyzed the serum samples for MBL and cytokine levels at baseline and 7 days later. Samples on day 7 were available in 73 patients. RESULTS: We divided the patients with septic shock into four groups according to serum MBL levels (<1.3 µg/mL or ≥1.3 µg/mL) on days 1 and 7. Patients with low MBL levels on day 1 and high MBL levels on day 7 showed a favorable prognosis for 28-day survival (odds ratio, 1.96, 95% confidence interval, 1.10-2.87; p=0.087). The high MBL group on day 7 showed a significant decrease in monocyte chemoattractant protein 1, interleukin (IL)-1ß, IL-6, IL-8, interferon-γ, and granulocyte macrophage colony-stimulating factor levels compared with the low MBL group on day 7. CONCLUSION: The increase in MBL levels of patients with septic shock may suggest a favorable prognosis and attenuate pro-inflammatory and anti-inflammatory responses.
RESUMO
Mannose-binding lectin (MBL) is a C-type lectin produced by the liver and involved in the innate immune response. We have analyzed six SNPs of MBL2 gene--three at promoter (-550, -435, and -221), one at 5'-untranslational region (UTR) (+4), and two at coding (Gly54Asp and Leu126Leu) regions--in the Korean population (N=129), and have correlated genotypes with the serum concentration and functional characteristics. Of those, the Asp54 allele (P<10(-15)), L allele at -550 (P<10(-7)), and P allele at +4 (P=0.012) were correlated with low MBL levels. The effect of the X allele at -221 on MBL levels in the Korean population appeared to be less profound than that of other populations. The highest MBL producing promoter haplotype in the Korean population was HYP, followed by LYQ and LYP, and then LXP. From functional analysis of MBL, low MBL levels were correlated with low mannan-binding, low C4 complement activation, and lack of high ordered oligomers. Our results support that the promoter and coding polymorphisms of MBL are correlated with its functional activity as well as circulating levels, and the association patterns are quite similar to those of other populations.
Assuntos
Lectina de Ligação a Manose/análogos & derivados , Lectina de Ligação a Manose/genética , Western Blotting , Complemento C4/metabolismo , Frequência do Gene , Genótipo , Haplótipos , Humanos , Coreia (Geográfico) , Mananas/metabolismo , Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/fisiologia , Polimorfismo de Nucleotídeo Único , Soro/metabolismoRESUMO
Despite the possibility of combining Toll-like receptor (TLR) ligands as adjuvants to improve vaccine efficacy, it remains unclear which combinations of TLR ligands are effective or what their underlying mechanisms may be. Here, we investigated the mechanism of action of L-pampo, a proprietary adjuvant composed of TLR1/2 and TLR3 ligands. L-pampo dramatically increased humoral immune responses against the tested target antigens, which was correlated with an increase in follicular helper T cells and the maintenance of antigen-specific CD4(+) T cells. During the initial priming phase, in contrast to the induction of type I interferon (IFN) and pro-inflammatory cytokines stimulated by polyI:C, L-pampo showed a greatly diminished induction of type I IFN, but not of other cytokines, and remarkably attenuated IRF3 signaling, which appeared to be critical to L-pampo-mediated adjuvanticity. Collectively, our results demonstrate that the adjuvant L-pampo contributes to the promotion of antigen-specific antibodies and CD4(+) T cell responses via a fine regulation of the TLR1/2 and TLR3 signaling pathways, which may be helpful in the design of improved vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Imunidade Humoral/efeitos dos fármacos , Interferon Tipo I/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 3 Toll-Like/imunologia , Transferência Adotiva , Animais , Antígenos/administração & dosagem , Regulação da Expressão Gênica , Imunização , Interferon Tipo I/genética , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Poli I-C/administração & dosagem , Células RAW 264.7 , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/transplante , Receptor 1 Toll-Like/genética , Receptor 2 Toll-Like/genética , Receptor 3 Toll-Like/genéticaRESUMO
A hepatitis B virus (HBV) vaccine has been developed using a new adjuvant and HBV surface antigens produced from a CHO cell line. The purified HBV surface antigens are composed of L protein, M protein, and S protein in a mixture of 20- and 40-nm-diameter particles and filamentous forms. This HBV surface antigen, formulated with L-pampo, a proprietary adjuvant, induced 10 times more antibody than the same antigen with alum and was capable of inducing strong immune responses in three different HBV transgenic mice. In spite of the presence of a large amount of HBV antigen in the blood, no antibody against HBV surface antigen was normally detected in these transgenic mice. After immunization, the HBV antigen was also cleared from the blood.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Precursores de Proteínas/imunologia , Alanina Transaminase/sangue , Animais , Anticorpos Antivirais/sangue , Células CHO , Cricetinae , Feminino , Anticorpos Anti-Hepatite B/sangue , Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/metabolismo , Hepatite B Crônica/prevenção & controle , Hepatite B Crônica/terapia , Imunoterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Precursores de Proteínas/metabolismo , Vacinação , Proteínas do Envelope Viral/imunologiaRESUMO
Coupling between certain pathogen-associated molecular patterns and corresponding pattern recognition receptors of endothelial cells is important for the mediation of vascular inflammatory responses. Mannose-binding lectin (MBL) recognizes certain carbohydrate structures of microbes and subsequently activates the complement system as well as facilitates the phagocytosis of targets. We investigated whether MBL can intervene in the interaction between bacterial lipopolysaccharide (LPS) and endothelial cells to modulate subsequent inflammatory responses. In response to LPS, human umbilical vein endothelial cells (HUVEC) produced various cytokines/chemokines. Addition of the purified human MBL/MBL-associated serine proteases (MASP) complex or recombinant human MBL enhanced LPS-mediated cytokine/chemokine secretion by HUVEC, including interleukin-8 (IL-8), IL-6 and monocyte chemoattractant protein-1 in a dose-dependent manner. This enhancing effect was ameliorated by the addition of anti-MBL antibody or mannan. Among the cytokines/chemokines we analysed, IL-6 showed the greatest increase of secretion in the presence of native MBL/MASP complex or recombinant MBL. MBL, regardless of its association with MASP, alters LPS-mediated cytokine/chemokine secretion of HUVEC. Besides the well-known functions of MBL, to activate the lectin-complement pathway and to facilitate clearance of targets, alteration of cytokine/chemokine secretion may provide an additional role for MBL in modulating vascular inflammation.
Assuntos
Quimiocinas/metabolismo , Células Endoteliais/imunologia , Endotélio Vascular/imunologia , Lectina de Ligação a Manose/imunologia , Células Cultivadas , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta Imunológica , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/imunologia , Serina Proteases Associadas a Proteína de Ligação a Manose/imunologia , Proteínas Recombinantes/imunologiaRESUMO
In Saccharomyces cerevisiae, we synthesized and secreted L-HBVsAg (named as pre-S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre-S degree degree::S (Asn15Gln and Asn123Gln), pre-S degree degree::S degree (Asn15Gln, Asn123Gln, and Asn320Gln), and pre-S degree degree::S degree degree (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre-S::S was N-glycosylated, i.e., hyper-mannosylated. In the secretion of pre-S degree degree::S and pre-S degree degree::S degree, besides the hyper-mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre-S degree degree::S and pre-S degree degree::S degree. Only a part of the secreted pre-S degree degree::S or pre-S degree degree::S degree molecules was N-glycosylated, and the site for the partial N-glycosylation seems to be Asn233 in S-antigen region. Compared to the N-glycosylated pre-S degree degree::S and pre-S degree degree::S degree, pre-S degree degree::S degree degree (non-N-glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti-S antigen monoclonal Ab. Interestingly, unlike pre-S degree degree::S degree degree with authentic C-terminus, the recombinant pre-S degree degree::S degree degree with C-terminal myc or poly-histidine tag (pre-S degree degree::S degree degree::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C-terminal sequence and glycosylation in S-antigen region seem to be of crucial importance in determining the secretion efficiency of L-HBVsAg in S. cerevisiae.
Assuntos
Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Glicosilação , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.