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1.
Iran Biomed J ; 24(2): 81-8, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31677604

RESUMO

Background: Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct. Methods: In this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation. Results: Flow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA+), as compared with DU145 (PSMA-). Conclusion: Here, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.

2.
Iran Biomed J ; 24(1): 15-23, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454859

RESUMO

Background: Streptokinase (SK), a heterogeneous plasminogen activator (PA) protein from groups A, C, and G streptococci (GAS, GCS, GGS, respectively) contains three structural domains (SKα, SKß, and SK). Based on the variable region of SKß, GAS-SK (ska) are clustered as SK1 and SK2 (including cluster2-streptokinase (SK2a)/SK2b), which show low and high fibrinogen (FG)-dependent plasminogen (Plg) activation properties, respectively. Despite being co-clustered as SK2a, GCS/GGS-SK (skcg) variants display properties similar to SK1. Herein, by SKß exchange between GGS (G88) and GAS-SK2a (STAB902) variants, the potential roles of SK domains in amidolytic/proteolytic activity and FG-bound-Plg activation are represented. Methods: Two parental SKG88 and SKSTAB902 genes were cloned into the NdeI/XhoI site of pET26b expression vector. The two chimeric SKß-exchanged constructs (SKC1: αG88-ßSTAB-γG88 and SKC2; αSTAB-ßG88-γSTAB) were constructed by BstEII/BsiWI digestion/cross-ligation in parental plasmids. SK were expressed in E. coli and purified by nickel-nitriloacetic acid chromatography. PA potencies of SKs were measured by colorimetric assay. Results: SDS-PAGE and Western-blot analyses confirmed the proper expression of 47-kDa SK. Analyses indicated that the catalytic efficiency (Kcat/Km) for amidolytic and proteolytic activity were less and moderately dependent on SKß, respectively. The increase of FG-bound-Plg activation for SKSTAB902/SKC1 containing SK2aß was around six times, whereas for SKG88/SKC2 containing skcgß, it was four times. Conclusion: Although SKß has noticeable contribution in FG-bound-Plg activation activity, it had minor contribution in fibrin-independent, amidolytic activity. These data might be of interest for engineering fibrin-specific versions of SK.

3.
J Med Virol ; 2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31774174

RESUMO

The present study was conducted to analyze the genotypic diversity of circulating species A rotavirus (RVA) strains in Iran and also to investigate comparative analysis between the genotypes of VP4 and VP7 of cocirculating RVA and vaccine strains before the vaccine is introduced in the national immunization program. The G3-lineage I was found in this study as the most common G genotype which was followed by G9-lineage III, G1-lineages I, II, G12-lineage III, G2-lineage IV, and G4-lineage I. Also, P[8]-lineages III, IV was found as the predominant P genotype which was followed by P[4]-lineage V, and P[6]-lineage I. Overally, G3P[8] was determined as the most common combination. Moreover, the analysis of the VP7 antigenic epitopes showed that several amino acid differences existed between circulating Iranian and the vaccine strains. The comparison of genotype G1 of Iranian and vaccine strains (RotaTeq and Rotarix), and genotypes G2, G3, and G4 of Iranian and RotaTeq vaccine strains revealed three to five amino acids differences on the VP7 antigenic epitopes. Furthermore, analyzing of the VP8* epitopes of Iranian P[8] strains indicated that they contained up to 11 and 14 amino acid differences with Rotarix and RotaTeq, respectively. Based on different patterns of amino acid substitutions in circulating and vaccine strains, the emergence of antibody escaping mutants and potentially the decrease of immune protection might ensue in vaccinated children. However, considering the broad cross-protective activity of RVA vaccines, their efficacy should be monitored after the introduction in Iran.

5.
J Med Virol ; 91(11): 1930-1936, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31283018

RESUMO

Human adenoviruses (HAdVs), especially AdV-40 and 41, are common causes of nonbacterial sporadic and outbreak gastroenteritis in children. The present study aimed to describe the frequency and genetic analysis of HAdVs in hospitalized children <5 years old with acute gastroenteritis. A total of 376 stool samples obtained from June 2015 to December 2017 were investigated for the presence of HAdVs by polymerase chain reaction. The HAdV DNA was detected in 16 (4.3%) out of 376 stool samples. Based on the hexon hypervariable region (HVR), B, C, and F HADV species including five types HAdV-1, 2, 3, 6, and 41 were identified, among which enteric AdV species F (EAdV-41) was the most dominant. Moreover, our findings showed the presence of genomic type cluster 1 (GTC1) pattern in Iranian type 41 strains, which was closely similar to the D1 prototype strain (Tak) and D28. In this regard, a recombination was found in AdV-41 strains presenting the hexon sequence that belonged to GTC1, while fiber sequence clustered with GTC2.

6.
Eur J Pharmacol ; 854: 398-405, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31039344

RESUMO

Hemoglobinopathies, such as ß-thalassemia, and sickle cell disease (SCD) are caused by abnormal structure or reduced production of ß-chains and affect millions of people worldwide. Hereditary persistence of fetal hemoglobin (HPFH) is a condition which is naturally occurring and characterized by a considerable elevation of fetal hemoglobin (HbF) in adult red blood cells. Individuals with compound heterozygous ß-thalassemia or SCD and HPFH have milder clinical symptoms. So, HbF reactivation has long been sought as an approach to mitigate the clinical symptoms of ß-thalassemia and SCD. Using CRISPR-Cas9 genome-editing strategy, we deleted a 200bp genomic region within the human erythroid-specific BCL11A (B-cell lymphoma/leukemia 11A) enhancer in KU-812, KG-1, and K562 cell lines. In our study, deletion of 200bp of BCL11A erythroid enhancer including GATAA motif leads to strong induction of γ-hemoglobin expression in K562 cells, but not in KU-812 and KG-1 cells. Altogether, our findings highlight the therapeutic potential of CRISPR-Cas9 as a precision genome editing tool for treating ß-thalassemia. In addition, our data indicate that KU-812 and KG-1 cell lines are not good models for studying HbF reactivation through inactivation of BCL11A silencing pathway.


Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Transporte/genética , Hemoglobina Fetal/metabolismo , Deleção de Genes , Terapia Genética/métodos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Talassemia beta/terapia , Sequência de Bases , Edição de Genes , Humanos , Células K562 , Proteínas Repressoras , Talassemia beta/genética , Talassemia beta/metabolismo , gama-Globinas/genética
7.
FEBS Open Bio ; 9(7): 1259-1269, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31087538

RESUMO

Streptokinase (SK) is a plasminogen activator which converts inactive plasminogen (Pg) to active plasmin (Pm), which cleaves fibrin clots. SK secreted by groups A, C, and G Streptococcus (SKA/SKC/SKG) is composed of three domains: SKα, SKß and SKγ. Previous domain-swapping studies between SK1/SK2b-cluster variants revealed that SKß plays a major role in the activation of human Pg. Here, we carried out domain-swapping between skcg-SK/SK2-cluster variants to determine the involvement of SKß in several SK functionalities, including specific/proteolytic activity kinetics, fibrinogen-bound Pg activation and α2 -antiplasmin resistance. Our results indicate that SKß has a minor to determining role in these diverse functionalities for skcg-SK and SK2b variants, which might potentially be accompanied by few critical residues acting as hot spots. Our findings enhance our understanding of the roles of SKß and hot spots in different functional characteristics of SK clusters and may aid in the engineering of fibrin-specific variants of SK for breaking down blood clots with potentially higher efficacy and safety.

8.
IUBMB Life ; 71(9): 1259-1267, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30724452

RESUMO

Solid tumors that are responsible for more than 85% of cancer death cases need angiogenesis for their growth and metastasis. Among antiangiogenic therapies, targeting the vascular endothelial growth factor receptor 2 (VEGFR2) that is over-expressed on tumor vasculatures has been a promising strategy. In this study, we developed a second generation nanobody (VHH)-based CAR T cell targeting VEGFR2-expressing tumor cells. The CAR T cell was developed by linking the anti-VEGFR2 VHH to a spacer, and signaling domains of CD28 and CD3 ζ. The T cells were activated with anti-CD3 plus rIL-2 and electroporated with a plasmid encoding the CAR construct. The expression of activation markers, CD69 and CD25, on CAR T cells upon coculturing with VEGFR2-expressing cells were 41% and 48%, and the IL-2 and IFN-γ production were 470 pg/mL and 360 pg/mL, respectively. The expression of degranulation marker, CD107a, was 30% and the cytotoxic activity of the CAR T cells reached to more than 30% with E:T ratio of 9:1. The anti-VEGFR2 CAR but not mock T cells mediated specific lysis of 293-KDR cells expressing human VEGFR2 and might be considered as a candidate for adoptive T-cell immunotherapy of solid tumors. © 2019 IUBMB Life, 71(9):1259-1267, 2019.

9.
J Cell Biochem ; 120(6): 10787-10795, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30672018

RESUMO

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA+ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.

10.
J Cell Physiol ; 234(8): 12569-12580, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30628064

RESUMO

The epithelial cell adhesion molecule (EpCAM) is a Type I transmembrane superficial glycoprotein antigen that is expressed on the surface of basolateral membrane of multiple epithelial cells with some exceptions such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, squamous stratified epithelial cells, and myoepithelial cells that do not express the molecule. The molecule plays a pivotal role in the structural integrity, adhesion of the epithelial tissues and their interaction with the underlying layers. EpCAM prevents claudin-7 and claudin-1 molecules from degradation, thereby, decreasing the number of tight junctions and cellular interconnections, and promoting the cells toward carcinogenic transformation. Moreover, the mutations in the EpCAM gene lead to congenital tufting enteropathy, severe intestinal epithelium homeostasis disorders, and Lynch and Lynch syndrome. Overexpression of EpCAM on stem cells of some cancers and the presence of this molecule on circulating tumor cells (CTCs) makes it a promising candidate for cancer diagnosis as well as tracing and isolation of CTCs.

11.
J Cell Physiol ; 234(6): 8636-8646, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30515798

RESUMO

Cancer therapy using oncolytic viruses is an emerging area, in which viruses are engineered to selectively propagate in tumor tissues without affecting healthy cells. Because of the advantages that adenoviruses (Ads) have over other viruses, they are more considered. To achieve tumor selectivity, two main modifications on Ads genome have been applied: small deletions and insertion of tissue- or tumor-specific promoters. Despite oncolytic adenoviruses ability in tumor cell lysis and immune responses stimulation, to further increase their antitumor effects, genomic modifications have been carried out including insertion of checkpoint inhibitors and antigenic or immunostimulatory molecules into the adenovirus genome and combination with dendritic cells and chemotherapeutic agents. This study reviews oncolytic adenoviruses structures, their antitumor efficacy in combination with other therapeutic strategies, and finally challenges around this treatment approach.

12.
J Immunol Res ; 2018: 9464186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29854852

RESUMO

E. coli-derived concatenated, multitype L2-conserved epitopes of human papillomavirus (HPV) L2 protein might represent a less expensive and pan-type vaccine alternative (compared to type-specific HPV L1 virus-like particles), if stable protein expression and strong immunogenicity features could be met. Herein, three dual-type- (DT-) HPV L2 fusion peptides comprising the three head-to-tail tandem repeats (multimers) of either HPV 16 epitope "17-36" or "69-81" or one copy (monomer) of 11-88 fused to the same residues of HPV 18 were constructed and expressed in E. coli. SDS-PAGE and Western blot analyses indicated the proper expression and stability of the E. coli-derived DT peptides. Mice immunized by formulation of the purified DT peptides and Freund's adjuvant (CFA/IFA) raised neutralizing antibodies (NAbs; the highest for DT: 11-88 peptide) which showed proper cross-reactivity to HPV types: 18, 16, 31, and 45 and efficiently neutralized HPV 18/16 pseudoviruses in vitro. Immunization studies in mice by formulation of the DT: 11-88 × 1 peptide with various adjuvants (alum, MF59, and Montanides ISA 720 and 50) indicated that Montanide adjuvants elicited the highest cross-reactive titers of NAbs and similar levels of IgG1 and IgG2a (switching towards balanced Th1/Th2 responses). The results implied development of low-cost E. coli-derived DT: 11-88 peptide formulated in human compatible ISA 720 adjuvant as a HPV vaccine.


Assuntos
Proteínas do Capsídeo/imunologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Reações Cruzadas , Escherichia coli/genética , Feminino , Expressão Gênica , Humanos , Imunidade Celular , Imunidade Humoral , Manitol/análogos & derivados , Camundongos , Camundongos Endogâmicos BALB C , Ácidos Oleicos , Proteínas Oncogênicas Virais/genética , Fragmentos de Peptídeos/genética , Equilíbrio Th1-Th2 , Vacinação , Vacinas de Subunidades
13.
Med Microbiol Immunol ; 207(3-4): 201-210, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29619604

RESUMO

Viral gastroenteritis is a major public health problem worldwide. In Iran, very limited studies have been performed with regard to the epidemiology of noroviruses. This study aimed to evaluate the prevalence and molecular epidemiology of GII noroviruses in hospitalized children less than 5 years of age with acute gastroenteritis (AGE). A total of 210 stool specimens were collected from Ali Asghar Children's Hospital and Bahrami Children's Hospital in Tehran, from June 2015 to June 2016. The samples were screened by real-time RT-PCR for genogroup II (GII). Positive samples were genotyped by semi-nested PCR followed by Sanger sequencing and phylogenetic analysis. Norovirus was identified in 36 (17.1%) of 210 specimens. Based on genetic analysis of RdRp and capsid sequences, the strains were clustered into eight RdRp-capsid genotypes: GII.P4-GII.4 Sydney_2012 (41.7%), GII.Pe-GII.4 Sydney_2012 (30.6%), GII.P21-GII.3 (13.9%), GII.P16-GII.4 Sydney_2012 (2.8%), GII.P16-GII.12 (2.8%), GII.P2-GII.4 Sydney_2012 (2.8%), GII.P7-GII.7 (2.8%) and GII.P2-GII.2 (2.8%). We determined several different co-circulating norovirus genotypes in children < 5 years of age with AGE in our hospital in Tehran, Iran. Continued molecular surveillance of noroviruses, including typing of both RdRp and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.


Assuntos
Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genótipo , Norovirus/classificação , Norovirus/genética , Pré-Escolar , Fezes/virologia , Feminino , Variação Genética , Humanos , Lactente , Recém-Nascido , Irã (Geográfico)/epidemiologia , Masculino , Epidemiologia Molecular , Norovirus/isolamento & purificação , Prevalência , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
14.
Protein Pept Lett ; 25(3): 244-252, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29336241

RESUMO

BACKGROUND: Immunotherapies targeting the human papillomavirus (HPV) oncogenic proteins, E6 and E7, are effective to treat HPV-associated cervical malignancies. OBJECTIVE: The main objective of this study was to generate the fluorescent HPV16 E7 protein for detection of delivery in vitro. METHODS: Two types of the fusion E7-GFP proteins (i.e., with or without linker) were expressed in different E. coli strains. Then, the efficiency of GFP and E7-GFP transfection was compared with FITC-antibody protein control using TurboFect reagent in the HEK-293T cell line. RESULTS: Our data indicated that both E7-GFP fusion proteins were efficiently produced in M15 E. coli strain, but not in BL21 or Rosetta strains. The E7-GFP fusion showed a clear band of ~ 50 kDa in SDS-PAGE. Moreover, the E7-GFP protein maintained the fluorescent properties only when there was a distance between E7 and GFP genes, suggesting a promising potential to use GFP fusion protein in generating soluble form of protein. This fluorescent property was stable and could be detected in vitro. Moreover, the HEK-293T cells transfected by GFP/TurboFect and E7- GFP/TurboFect complexes demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the GFP fluorescence was stable even at 24 h post-transfection. CONCLUSION: Briefly, the E7-GFP fusion protein with linker can be useful for the development of protein vaccines against HPV16 infections and detection in vivo.


Assuntos
Proteínas de Fluorescência Verde/genética , Proteínas E7 de Papillomavirus/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Bases , Escherichia coli , Feminino , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas E7 de Papillomavirus/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
15.
IUBMB Life ; 70(3): 207-214, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29369472

RESUMO

Hepatitis E virus (HEV) infection remains a serious threat to life and productivity in developing world. Vaccine seems to be an effective, safe, and affordable approach to address HEV disease burden. The HEV genome consists of three open reading frames (ORFs). Of these, ORF2 encodes a single structural protein (pORF2) for the HEV capsid which has been studied extensively as vaccine candidates. Recently, it has been recognized that autophagy plays an important role in innate and adaptive immunity defense against intracellular pathogens. This mechanism could therefore promote a protective immune response by inducing CD4+ and CD8+ T cells. In this study, HEV 239 and Beclin1 proteins were expressed in prokaryotic host cell [Escherichia coli (BL21)]. HEV 239 protein with different formulations (+Alum, +Beclin1, and +Alum-Beclin1) were used as candidate vaccines and administrated subcutaneously in BALB/c mice on 0, 14, and 28 days. Finally, elicited cellular and humoral immunity were evaluated. Taken together, although our results indicated that mice immunized with HEV 239 protein formulated with Alum, Beclin1, and Alum + Beclin1 displayed humoral and cellular response that was not significant in comparison with each other (P > 0.05); whereas they were significant while compared with control groups (P < 0.05). A comprehensive understanding of the intricate interplay between autophagy and immune response remains to be unraveled. Further study will clear the detailed impact of autophagy manipulation to enhance vaccine efficacy and boost the immune responses against the disease. © 2018 IUBMB Life, 70(3):207-214, 2018.


Assuntos
Vírus da Hepatite E/imunologia , Hepatite E/imunologia , Imunidade Humoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas Virais/administração & dosagem , Imunidade Adaptativa/imunologia , Animais , Autofagia/imunologia , Proteína Beclina-1/administração & dosagem , Proteína Beclina-1/genética , Proteína Beclina-1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Escherichia coli/genética , Genoma Viral/imunologia , Hepatite E/prevenção & controle , Hepatite E/virologia , Vírus da Hepatite E/patogenicidade , Humanos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia
16.
Viral Immunol ; 31(3): 233-241, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29185875

RESUMO

Due to the limitations and safety issues of the two currently approved live attenuated rotavirus (RV) vaccines "RotaTeq and Rotarix," studies on nonreplicating sources of RV vaccines and search for proper RV antigens are actively carried out. The adjuvant activity of NSP4 and highly immunogenic properties of RV VP6 protein prompted us to consider the construction of a NSP4112-175-VP6 fusion protein and to assess the anti-VP6 IgG, IgA, and IgG subclass responses induced by Escherichia coli-derived NSP4-VP6 fusion protein compared to that of VP6 protein with/without formulation in Montanide ISA 50V2 (M50) in BALB/c mice. Results indicated to the proper expression of the fused NSP4-VP6 and VP6 proteins in E. coli. Intraperitoneal immunization by M50 formulated NSP4-VP6 fusion protein (M5+NSP4-VP6) induced the highest titration of VP6-specific IgG and IgA responses compared to the other groups. Indeed, the presence of NSP4 resulted to the induction of stronger humoral immune responses against the fused protein compared to that elicited by administration of VP6 protein alone (with/without M50 formulation), implying the adjuvant properties of NSP4 for the fused protein. Moreover, the "M50+NSP4-VP6" formulation induced higher serum IgG2a titers than IgG1 and increased Interferon-γ levels, despite unchanged interleukin-4 amounts compared to other groups, indicating Th1-oriented responses with a possible role of NSP4. In conclusion, this study further highlights the potentiality of NSP4-VP6 fusion protein as an efficient and cost-effective immunogen in the field of RV vaccine development.


Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Glicoproteínas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vacinas contra Rotavirus/imunologia , Rotavirus/imunologia , Toxinas Biológicas/imunologia , Proteínas não Estruturais Virais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Glicoproteínas/administração & dosagem , Glicoproteínas/genética , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Vacinas contra Rotavirus/administração & dosagem , Vacinas contra Rotavirus/genética , Toxinas Biológicas/administração & dosagem , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genética
17.
Res Pharm Sci ; 12(4): 265-273, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28855937

RESUMO

Current licensed and commercially available prophylactic human papillomavirus (HPV) vaccines (Cervarix and quadrivalent/nine valents Gardasil) are based on major capsid protein L1 virus-like particles (VLPs) production which are expensive and type specific. Minor capsid L2-RG1 linear epitope (17-36) is a known candidate for induction of cross-neutralizing antibodies to develop low-cost pan-HPV vaccines. Herein, we report construction and expression of a three tandem repeats of L2-RG1 derived from HPV16 and 18 fused with the same head to tail pattern (HPV16:17-36×3+ HPV18:17-36×3; hereafter termed dual-type fusion L2 peptide) in E. coli and provide the results of its immunogenicity in mice. SDS-PAGE and western blot analyses indicated proper expression of the peptide that could be further purified by Ni-NTA affinity chromatography via the located C-terminal 6xHis-tag. Mice immunized by formulation of the purified peptide and Freund adjuvant raised neutralizing antibodies which showed proper cross reactivity to HPV L2 (11-200) of types: 18, 16, 31 and 45 (which totally are responsible for 90% of cervical cancers) and efficiently neutralized HPV18/16 pseudoviruses in vitro. Our results imply the possibility of development of a simple, low-cost preventive HPV vaccine based on this dual-type fusion L2 peptide in bacterial expression system with broad spectrum.

18.
Appl Biochem Biotechnol ; 182(4): 1540-1547, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28247308

RESUMO

While some microRNAs are transcribed from a specific promoter, at least one third of human miRNA genes are clustered, wherein multiple miRNA genes are generated from a single primary transcript such as miR-17 ~ 92 cluster. Although six members of the cluster are generated from a single transcript, the mature level of each member may be diverse in various cell types. Here, we attempt to monitor the mature level of miR-17, miR-92a, and miR-20a from miR-17 ~ 92 cluster in blood (HL60 (human promyelocytic leukemia cells) and Jurkat) and breast (MDA-MB-231 and MCF-7) cancer cell lines. Interestingly, different mature levels of the miRNAs were observed in each cell line. While miR-20 was highly matured in HL60 and MDA-MB-231 cell lines, higher mature level of miR-92a was observed in Jurkat cell line compared to that of miR-20 and miR-17. Further, the mature level of miRNAs was also measured in normal and cancer cell lines. Although the mature level of miR-17 and miR-92a increased in HL60 and Jurkat cell lines, miR-20 expression showed an almost identical level in blood cancer cell lines compared to controls. Conversely, miR-20 mature level significantly increased in breast cancer cell lines whereas the expression level of miR-92a was comparable in MDA-MB-231, MCF-7, and MCF-10A cell lines.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Adolescente , Linhagem Celular Tumoral , Humanos , Masculino
19.
Protein Pept Lett ; 24(3): 190-196, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28000571

RESUMO

Up to now, different protein vaccine modalities against human papillomavirus (HPV) have been designed to control cervical cancer. The important issue is to increase their immunogenicity using appropriate adjuvants. Among heat shock proteins (HSPs), glycoprotein 96 (Gp96) and its Nterminal region (NT-gp96) have attracted a specific interest in stimulation of antigen-specific immune responses in vivo. Furthermore, the potency of high mobility group box 1 (HMGB1) protein and its fragment (Hp91) was reported to enhance the desired immune responses against various disorders. In this study, the recombinant (r) HPV16 E7 and rNT-gp96 proteins were generated in bacterial expression system. Mice were vaccinated three times with E7 antigen mixed with Montanide, Hp91, and NT-gp96 as the adjuvant and their preventive and therapeutic efficiencies were evaluated in a murine tumor model. Mice vaccinated with E7 co-delivered by Hp91 peptide induced higher IgG2a and IFN-γ responses in comparison with E7 co-injected with Montanide and NT-gp96 protein suggesting a strong Th1 cellular immune response. The data showed that vaccination with noncovalent rE7 + rNT-gp96 complex delayed the tumor growth as compared to control groups. Mice immunized with rE7 + Montanide and rE7 + Hp91 protected 100% of mice versus 75% survival in groups vaccinated with rE7 + rNT-gp96 after TC-1 tumor challenge. The percentage of tumor free mice was decreased in group immunized with rE7 + rNT-gp96 in therapeutic experiments (~ 50%). These results demonstrated that Hp91 peptide is a safe and strong adjuvant against rNT-gp96 with the potent anti-tumor effects similar to Montanide adjuvant.


Assuntos
Proteína HMGB1/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Vacinação , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antivirais/biossíntese , Feminino , Expressão Gênica , Proteína HMGB1/administração & dosagem , Proteína HMGB1/genética , Papillomavirus Humano 16/efeitos dos fármacos , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/patogenicidade , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Imunização Secundária , Imunoglobulina G/biossíntese , Interferon gama/biossíntese , Manitol/administração & dosagem , Manitol/análogos & derivados , Glicoproteínas de Membrana/administração & dosagem , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos/administração & dosagem , Proteínas E7 de Papillomavirus/administração & dosagem , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/mortalidade , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Peptídeos/administração & dosagem , Peptídeos/genética , Peptídeos/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Análise de Sobrevida , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/virologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Mol Immunol ; 75: 101-11, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27267270

RESUMO

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to high mortality worldwide. It is well-established that cellular immunity plays a critical role to control Mtb infection. Dendritic Cells (DCs) are potent antigen presenting cells, which play an important role to prime cell-mediated immune responses. In vivo targeting of DCs has been shown to induce both strong cellular immunity and protection against tumor challenges. The aim of the present study was not only to assess the immunizing potential of a novel DC-targeted recombinant lentivirus expressing fusion antigen Ag85A-ESAT6 of Mtb, but also to compare it with a recombinant lentivirus with broad cellular tropism expressing the same antigen in mice. The findings demonstrated that our novel recombinant DC-targeted lentivector was able to successfully transduce and express the fusion antigen Ag85A-E6 in vitro and in vivo. Moreover, a single footpad injection of targeted lentivectors could elicit strong T-helper 1 (Th1) immunity against the above mentioned antigen, as indicated by the specific high-level production of IFN-γ and IL-2 using spleen lymphocytes and lymphoproliferative responses. Despite of these promising results, more attempts are required to elucidate the protective and therapeutic efficacy of this approach in future.


Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Células Dendríticas/imunologia , Vacinas contra a Tuberculose/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Apresentação do Antígeno/imunologia , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Vetores Genéticos , Humanos , Imunidade Celular/imunologia , Lentivirus , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/imunologia , Transdução Genética , Tuberculose/imunologia , Tuberculose/prevenção & controle
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