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1.
J Chromatogr A ; : 460962, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-32067760

RESUMO

Human papillomavirus (HPV) vaccination is the most effective mean to prevent HPV infection and cervical carcinoma. Licensed HPV prophylactic vaccines are formulated to contain a defined amount of different major capsid protein (L1), the critical antigen to elicit protection. No method is currently available to simultaneously quantify individual L1s in multivalent vaccines, presenting a daunting challenge for the quality control of HPV vaccines. Here, HPV16 and HPV18 L1 can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using tryptic digestion without pre-digestion reduction and alkylation in multiple reaction monitoring (MRM) mode. Two signature peptides were selected to be the markers of the two L1s and can be well separated within 5.1 min. Their linear calibration curves were both obtained in the range of 20-500 nmol/L (R2 > 0.990). To HPV16 L1, intra/inter assay precisions and accuracies of the assay were below 11% and between 83.96-113.57%. While for HPV18 L1, they were below 12% and between 81.40-103.49%. In addition, the limits of quantitation (LOQ) were as low as 2.8 nmol/L for HPV16 L1 and 1.7 nmol/L for HPV18 L1, respectively, representing about 68 and 112 times more sensitive than those obtained with Smith Bicinchoninic Acid (BCA) assay. This LC-MS/MS method can be applied to the quantification of both bulk products and the final multivalent vaccines. This method is superior to the current assays in terms of sensitivity, specificity, precision, accuracy and throughput; it could become the method of choice for absolute quantification of proteins in multivalent vaccines.

2.
J Cell Mol Med ; 24(1): 862-874, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31749335

RESUMO

Growing evidence has well established the protective effects of CYP2J2/EET on the cardiovascular system. The aim of the present study was to determine whether CYP2J2/EET has a preventive effect on atrial fibrillation (AF) and to investigate the underlying mechanisms. Wild-type mice were injected with or without AAV9-CYP2J2 before abdominal aortic constriction (AAC) operation. After 8 weeks, compared with wild-type mice, AAC mice display higher AF inducibility and longer AF durations, which were remarkably attenuated with AAV9-CYP2J2. Also, AAV9-CYP2J2 reduced atrial fibrosis area and the deposit of collagen-I/III in AAC mice, accompanied by the blockade of TGF-ß/Smad-2/3 signalling pathways, as well as the recovery in Smad-7 expression. In vitro, isolated atrial fibroblasts were administrated with TGF-ß1, EET, EEZE, GW9662, SiRNA Smad-7 and pre-MiR-21, and EET was demonstrated to restrain the differentiation of atrial fibroblasts largely dependent on Smad-7, due to the inhibition of EET on MiR-21. In addition, increased inflammatory cytokines, as well as activated NF-κB pathways induced by AAC surgery, were also significantly blunted by AAV9-CYP2J2 treatment. These effects of CYP2J2/EET were partially blocked by GW9662, the antagonist of PPAR-γ. In conclusion, this study revealed that CYP2J2/EET ameliorates atrial fibrosis through modulating atrial fibroblasts activation by disinhibition of MiR-21 on Smad-7, and attenuates atrial inflammatory response by repressing NF-κB pathways, reducing the vulnerability to AF, and CYP2J2/EET exerts its role at least partially through PPAR-γ activation. Our findings might provide a novel upstream therapeutic strategy for AF.

3.
Exp Cell Res ; 386(1): 111716, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734152

RESUMO

BACKGROUND: Emerging evidence demonstrates that epoxyeicosatrienoic acids (EETs) as important active eicosanoids that regulate cardiovascular homeostasis, but the mechanisms underlying its favorable anti-hypertrophic benefits in overpressure model remain obscure. METHODS AND RESULTS: Four weeks after transverse aortic constriction (TAC), TAC mice developed maladaptive cardiac hypertrophy and consequent cardiac failure. Conversely, a cardiotropic adeno-associated viral vector (AAV9) encoding CYP2J2 prevented transverse aortic constriction-induced cardiac hypertrophy with preserved ejection fraction. EET also conferred protection against phenylephrine-induced hypertrophy in H9c2 cardiomyoblasts. Further investigations indicate CYP2J2/EET exerts protection against cardiac hypertrophy through opposing the increase of intracellular Ca2+ level and Ca2+-mediated calcineurin/NFATc3 signaling. Meanwhile, extended myocardial fibrosis in TAC mice was also effectively abolished with the administration of AAV9-2J2. Intriguingly, TAC mice display activated TGF-ß/Samd-3 signaling with decreased Smad-7 expression, whereas AAV9-2J2 attenuated the phosphorylation of Smad-3 without altering TGF-ß expression, whilst preservation of Smad-7. Subsequently, the differentiation of cardiac fibroblasts into myofibroblasts in the presence of TGF-ß1 stimulation was significantly disrupted with EET treatment, accompanied by declined Smad-3 activation and collagen production, whereas inhibition of Smad-7 with SiRNA Smad-7 substantially abrogated these effects of EET on cardiac fibroblasts. CONCLUSIONS: EET has synergistic actions on cardiomyocytes and cardiac fibroblasts, preventing cardiac hypertrophy through inhibition of Ca2+-mediated calcineurin/NFATc3 signaling cascades, and ameliorating myocardial fibrosis dependent on Smad-7. This work further extends the potential mechanisms of EET, providing a novel therapeutic approach for the treatment of pathological remodeling and heart failure.

4.
Emerg Microbes Infect ; 8(1): 1584-1592, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31682199

RESUMO

The genetic and/or antigenic differences between street rabies virus (RABV) and vaccine strains could potentially affect effectiveness of rabies vaccines. As such, it is important to continue monitoring the glycoprotein (G) of the street isolates. All RABVG sequences in public database were retrieved and analysed. Using a pseudovirus system, we investigated 99 naturally occurring mutants for their reactivities to well-characterized neutralizing monoclonal antibodies (mAbs) and vaccine-induced antisera. A divergence in G sequences was found between vaccine strains and recent street isolates, with mutants demonstrating resistance to neutralizing mAbs and vaccine-induced antibodies. Moreover, antigenic variants were observed in a wide range of animal hosts and geographic locations, with most of them emerging since 2010. As the number of antigenic variants has increased in recent years, close monitoring on street isolates should be strengthened.

5.
Regen Biomater ; 6(5): 259-267, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31616563

RESUMO

Cell migration in three-dimensional environment is extremely important for tissue regeneration and other biological processes. In this work, a model system was developed to study how endothelial cells (ECs) migrate into photo-responsive hydrogels under the presence of pro-inflammatory macrophages. The hydrogel was synthesized from hyaluronic acid grafted with coumarin and methacrylate moieties by both carbon-carbon covalent linking and coumarin dimerization under UV irradiation at 365 nm. The structure of the hydrogel was conveniently modulated by UV irradiation at 254 nm to decompose the coumarin dimers, leading to the significant decrease of modulus and increase of swelling ratio and mesh size. Under the presence of M1 macrophages, ECs were induced to migrate into the hydrogels with a different degree. A significant larger net displacement of ECs was found in the softer hydrogel obtained by irradiation with UV at 254 nm than in the stiffer original one at day 7.

6.
Biosci Rep ; 39(10)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31481526

RESUMO

Olive oil could attenuate carbon tetrachloride (CCl4) induced liver fibrosis (LF) in mouse model. The present study aimed to evaluate the effects of other common oils on CCl4 induced LF. Healthy male ICR mice were administered with CCl4 intraperitoneally at 2.5 ml/kg twice a week for total 3 weeks. Mice were pre-treated with olive oil, soybean oil, corn oil or lard oil. After treatment, histopathological changes were observed using Masson trichrome staining, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA), hydroxyproline (HYP) and triglyceride (TG) were measured by commercial kits. The expression of LF related genes was detected by quantitative real-time PCR. We found that soybean oil or olive oil significantly reduced ALT and AST levels in serum, and MDA, HYP and TG levels in the liver, compared with corn oil or lard oil. Moreover, Masson trichrome staining and real-time PCR showed that the mice treated with CCl4 dissolved in soybean oil or olive oil had less fibrosis and apoptosis in the liver comparted to the mice treated with CCl4 dissolved in corn oil or lard oil. In conclusion, soybean oil but not corn or lard oil exerts protective effects against CCl4 induced LF in mice, possibly due to its antioxidant activity.

7.
J Mater Chem B ; 7(36): 5478-5489, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31415053

RESUMO

The design of hyaluronic acid (HA)-based and stimuli-responsive hydrogels to elicit highly controlled and tunable cell response and behaviors is a major field of interest in tissue engineering and regenerative medicine. The pH-responsive hydrogel can respond to pH variation during wound healing, which may in turn regulate the tissue regeneration process. In this study, a double-network hydrogel cross-linked with vinyl double bonds and Schiff base was prepared, whose properties were further adjusted by incubation in pH 7.4 and pH 5 buffers. The endothelial cells (ECs) migrated much deeper into the softer HA hydrogel pre-treated with pH 5 buffer than the stiffer hydrogel. By contrast, the mesenchymal stem cells (MSCs) migrated easily into the stiffer hydrogel. The ECs highly expressed RhoA and non-muscle myosin (NM) II genes in the softer hydrogel, which may facilitate amoeboid migration. Meanwhile, the MSCs were stiffer than the ECs, and highly expressed Rac1, RhoA, vinculin, NM II, hyaluronidase (HYAL) 2 and CD44 genes in the stiffer hydrogel, which facilitate mesenchymal migration. These results provide important clues for revealing the different migration strategies of the ECs and MSCs in HA hydrogels with different stiffness, and suggest that the mechanical properties and the network structure of hydrogels play an important role in regulating the three-dimensional migration process of these cells.

8.
Vaccine ; 37(47): 7108-7116, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31383490

RESUMO

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strainA/PuertoRico/8/1934H1N1)homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6'-sulfate-ß-D-Galp-(1,4)-ß-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.

9.
Sci Rep ; 9(1): 11638, 2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406266

RESUMO

Sigmodon hispidus or cotton rat is an excellent animal model for studying human infections of respiratory viruses including respiratory syncytial virus (RSV), which is the leading cause of hospitalization in infants and causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research, no vaccine has been licensed whereas inactivated vaccines have been shown to induce severe adverse reaction in a clinical trial, with other forms of RSV vaccine also found to induce enhanced disease in preclinical animal studies. While arguably the cotton rat is the best small animal model for evaluation of RSV vaccines and antivirals, many important genes of the immune system remain to be isolated. Programmed cell death-1 (PD-1) plays an integral role in regulating many aspects of immunity by inducing suppressive signals. In this study, we report the isolation of mRNA encoding the cotton rat PD-1 (crPD-1) and characterization of the PD-1 protein. crPD-1 bound to its cognate ligand on dendritic cells and effectively suppressed cytokine secretion. Moreover, using the newly acquired gene sequence, we observed a decreased level of crPD-1 levels in cotton rats with enhanced respiratory disease induced by inactivated RSV vaccine, unraveling a new facet of vaccine-induced disease.

10.
Virology ; 535: 179-188, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31310875

RESUMO

Neuraminidase is the second major surface antigen on influenza virus. We investigated the immunogenicity and cross protective efficacy of virus-like particle containing neuraminidase derived from 2009 pandemic H1N1 influenza virus (N1 VLP) in comparison with inactivated split influenza vaccine. Immunization of mice with N1 VLP induced antibody responses specific for virus and cross-reactive neuraminidase inhibition activity whereas an inactivated split vaccine induced strain-specific hemagglutination inhibition activity. N1 VLP-immunized mice developed cross protective immunity against antigenically different influenza viruses, as determined by body weight changes, lung viral titers, infiltrating innate immune cells, and cytokines, and antibody secreting cells, and germinal center B cells. Also, N1 VLP-immune sera provided cross-protection in naïve mice. Immunity by N1 VLP vaccination was not compromised in Fc receptor γ-chain deficient mice. These results suggest that neuraminidase-presenting VLP can be developed as an effective cross-protective vaccine candidate along with current influenza vaccination.

11.
Vaccine ; 37(32): 4477-4484, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31266673

RESUMO

The high global burden of tuberculosis (TB) underscores the urgent need for an effective TB vaccine since the only licensed Bacillus Calmette-Guérin (BCG) vaccine is ineffective in preventing adult pulmonary TB and affords no protection against latent TB infection (LTBI). Herein we investigated the potential of Mycobacterium tuberculosis (Mtb) antigen proteins AEC comprised of Ag85b and ESAT6-CFP10 proteins in conjunction with aluminum (Al) and polyriboinosinic-polyribocytidylic acid (poly-IC) as a novel subunit vaccine against TB. The immunogenicity and protection induced by the adjuvanted vaccine were evaluated in two animal models. Mice vaccinated with AEC/Al/poly-IC exhibited significant antigen-specific humoral immune responses and cell-mediated immunity as determined by immunoassay and multicolor flow cytometric assay, and the protective effect of the vaccine was demonstrated in a guinea pig model of latent Mtb infection. Compared to the control group, the mean pathological scores and bacterial loads in lungs and spleens of AEC/Al/poly-IC-immunized guinea pigs were significantly reduced. These data indicate that the AEC/Al/poly-IC is highly immunogenic in mice and can effectively protect guinea pigs against latent Mtb infection; it may represent a promising candidate vaccine for the control of latent TB.

12.
Vaccine ; 37(30): 4031-4039, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31186190

RESUMO

Chitosan is a polysaccharide capable of augmenting immune responses with a proven safety record in animals and humans. These properties make it a potentially attractive agent for the prevention and treatment of infectious disease. Infection by respiratory syncytial virus (RSV) is the leading cause of serious lower respiratory disease in young children throughout the world. There is no licensed vaccine available against RSV whereas inactivated vaccine is known to cause enhanced respiratory disease instead of protection. Here, we investigated whether chitosan administered one or three days post-infection could protect animals against RSV infection and whether it could alter immune responses or immunopathology induced by inactivated RSV vaccine when administered twice before RSV infection. We found chitosan could modestly protect animals against RSV infection when given post-infection, while, in conjunction with inactivated RSV vaccine when given pre-infection, it could significantly reduce RSV infection in mice. Further mechanistic investigation revealed that chitosan enhanced antigen-specific immune responses through augmenting the induction of regulatory T cells, lung resident T cells and neutralizing antibodies while reversing Th2-skewed immune responses induced by inactivated RSV vaccine but, surprisingly, failing to reverse lung histopathology. Overall, this study sheds more light on the molecular mechanisms underlying inactivated RSV vaccine-induced disease.

13.
Anal Chem ; 91(14): 8908-8917, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31251585

RESUMO

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

14.
Front Immunol ; 10: 597, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984178

RESUMO

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

15.
Anal Chem ; 91(8): 5083-5090, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30908021

RESUMO

Sulfated N-glycans are biologically important structures derived from enzymatically post-glycosylational modifications of glycoproteins in many therapeutic biologics. The high-throughput analysis of sulfated N-glycomes remains a daunting technical challenge, because of negatively charged heterogeneous composition, large molecular structures, lability of sulfate attachments, and a lack of highly selective enrichment methods. Using liquid chromatography-mass spectrometry, we have analyzed the N-glycans of influenza viral hemagglutinin and neuraminidase from several subtypes of influenza vaccines, and utilized the existing resource to establish an N-glycan library consisting of 927 N-glycan structures and 387 sulfated N-glycan compositions. With the aid of database for data mining, 1380 unique N-glycopeptides were identified and manually validated by de novo glycopeptide sequencing, of which 514 were sulfated at the site-specific locations. We report here a mass spectrometric method that is able to identify and distinguish the isobaric structures of complex and hybrid N-glycans flanked by a terminal sulfation sequon on Gal-GlcNAc and GalNAc-GlcNAc of sulfated-3-Gal, sulfated-6-GlcNAc, and sulfated-4-GalNAc. The database-aided glycoproteomic analyses enable rapid determination of new sulfated-N-glycan structures in large sets of influenza vaccines, including those highly branched nonsialyl sulfo-N-glycans bearing lactosaminic extensions in both complex and hybrid N-glycans that especially interact with sulfotransferases. The novel findings highlight the tremendous structural diversity of sulfated N-glycans and strongly suggest potential functional importance of N-glycan sulfation of influenza glycoproteins.

16.
J Infect Dis ; 220(10): 1558-1567, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-30911758

RESUMO

BACKGROUND: Infection control measures have played a major role in limiting human/camel-to-human transmission of Middle East respiratory syndrome coronavirus (MERS-CoV); however, development of effective and safe human or camel vaccines is warranted. METHODS: We extended and optimized our previous recombinant adenovirus 5 (rAd5)-based vaccine platform characterized by in vivo amplified and CD40-mediated specific responses to generate MERS-CoV S1 subunit-based vaccine. We generated rAd5 constructs expressing CD40-targeted S1 fusion protein (rAd5-S1/F/CD40L), untargeted S1 (rAd5-S1), and Green Fluorescent Protein (rAd5-GFP), and evaluated their efficacy and safety in human dipeptidyl peptidase 4 transgenic (hDPP4 Tg+) mice. RESULTS: Immunization of hDPP4 Tg+ mice with a single dose of rAd5-S1/F/CD40L elicited as robust and significant specific immunoglobulin G and neutralizing antibodies as those induced with 2 doses of rAd5-S1. After MERS-CoV challenge, both vaccines conferred complete protection against morbidity and mortality, as evidenced by significantly undetectable/reduced pulmonary viral loads compared to the control group. However, rAd5-S1- but not rAd5-S1/F/CD40L-immunized mice exhibited marked pulmonary perivascular hemorrhage post-MERS-CoV challenge despite the observed protection. CONCLUSIONS: Incorporation of CD40L into rAd5-based MERS-CoV S1 vaccine targeting molecule and molecular adjuvants not only enhances immunogenicity and efficacy but also prevents inadvertent pulmonary pathology after viral challenge, thereby offering a promising strategy to enhance safety and potency of vaccines.

17.
Bioresour Technol ; 284: 65-71, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30925424

RESUMO

Two biochars, from Chinese cabbage (Cc, Brassica rapa pekinensis) and rape (Ra, Brassia campestris L.), were used to prepare biochar/Mg-Al layered double oxides (LDOs) as adsorbents for phosphate removal from aqueous solution. The biochar/LDOs were horizontally alternated lamellar particles and had abundant groups of oxides and biochars. The phosphate removal percentage remained above 92% at a pH range of 2-10, and above 95% during the first 5 min for 50 mg/L phosphate by 0.05 g biochar/LDOs. The adsorption kinetics and isotherms data were well fitted by the pseudo-second-order kinetic equation, as well as by the Freundlich and Langmuir models. Based on FTIR, XPS, and zeta potential analysis, the interaction mechanisms were defined as "memory effect", electrostatic attraction, surface complexation, and anion exchange. The results indicate that vegetable biochar/LDOs can be considered a novel and efficient sorbent for phosphate removal from water or wastewater.


Assuntos
Carvão Vegetal/química , Óxidos/química , Fosfatos/química , Verduras/química , Adsorção , Cinética , Soluções/química , Água , Purificação da Água/métodos
18.
Fish Shellfish Immunol ; 86: 571-580, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30529463

RESUMO

Peptidoglycan (PGN) is an important target of recognition in invertebrate innate immunity. PGN recognition proteins (PGRPs) are responsible for PGN recognition. In this study, we cloned and functionally analyzed a short PGRP (HcPGRP2) from the triangle-shell pearl mussel Hyriopsis cumingii. The full-length cDNA sequence of HcPGRP2 gene was 1185 bp containing an open reading frame of 882 bp encoding a 293 amino acid protein. HcPGRP2 was predicted to have two SH3b domains and a conserved C-terminal PGRP domain. Quantitative real-time RT-PCR showed that HcPGRP2 was expressed in all examined tissues and its expression was induced most significantly by Staphylococcus aureus and Vibrio parahaemolyticus in the hepatopancreas and gills. RNA interference by siRNA results revealed that HcPGRP2 was involved in the regulation of whey acidic protein, theromacin, and defensin expression. As a pattern-recognition receptor, recombinant HcPGRP2 (rHcPGRP2) protein can bind and agglutinate (Ca2+ dependent) all tested bacteria. rHcPGRP2 exhibited specific binding to PGN but not to lipopolysaccharide. Moreover, rHcPGRP2 inhibited the growth activities of S. aureus and V. parahaemolyticus in vitro and accelerated the clearance of V. parahaemolyticus in vivo. Overall, our results indicated that HcPGRP2 may play an important role in the antibacterial immune mechanisms of H. cumingii.


Assuntos
Bivalves/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Animais , Bivalves/genética , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica , Brânquias/imunologia , Brânquias/microbiologia , Hepatopâncreas/imunologia , Hepatopâncreas/microbiologia , Imunidade Inata , Lipopolissacarídeos/metabolismo , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Alinhamento de Sequência , Staphylococcus aureus , Vibrio parahaemolyticus
19.
Hum Vaccin Immunother ; 15(2): 349-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30273512

RESUMO

The evaluation of the immunogenicity of Sabin strain based Inactivated Poliovirus Vaccines (sIPV) necessitates the use of wild strains in neutralization assays to assess the potential cross-reactivity of antibodies. The live virus strains including wild and Sabin strains must be handled in level 3 biocontainment laboratories. To develop an alternative assay without the use of a live virus, we constructed Mahoney, MEF-1, and Saukett pseudovirions by inserting luciferase reporter genes into intact capsid proteins. Afterward, we developed a pseudovirus-based neutralization test (pNT) and evaluated for the specificity and reproducibility. We tested serum samples from a clinical trial on sIPV vaccines by pNT and compared the results with those obtained from conventional neutralization tests (cNT). A strong correlation was observed between two methods, with the correlation coefficients of all three types of IPV vaccines being greater than 0.82 (p < 0.0001). The Geometric Mean Titer (GMT) values obtained by pNT were approximately four times higher than that by cNT, revealing the better sensitivity of pNT. In conclusion, pNT is a safe, rapid and sensitive quantitative assay with the potential of being an alternative for the evaluation of the potency of polio vaccines.

20.
J Infect Dis ; 219(1): 19-25, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29982588

RESUMO

Background: Since the emergence of influenza A(H7N9) virus in 2013, there have been 5 waves of influenza A(H7N9) epidemics in China. However, evolution of the hemagglutinin (HA) protein antigenicity has not been systematically investigated. Methods: To better understand how antigenic drift in HA proteins of influenza (A)H7N9 virus occurs, 902 influenza A(H7N9) virus HA protein sequences from a public database were retrieved and analyzed. Fifty-three mutants with single amino acid substitutions in HA protein were introduced into pseudoviruses, and their antigenic characteristics were analyzed using pseudovirus-based assays. Results: The frequencies of 9 mutations incrementally increased over the past 5 years, with mutations identified at multiple sites. While mean neutralization titers of most variants remained unchanged, 3 mutations, A143V, A143T, and R148K, displayed a median 4-fold lower susceptibility to neutralization by antisera against influenza A/Anhui/1/2013(H7N9) virus. Notably, A143V and A143T were located outside the previously reported antigenic sites. The most dominant variant (A143V/R148K) in the most recent season constituted 74.11% of all mutations and demonstrated a 10-fold reduction in its reactivity to influenza A/Anhui/1/2013(H7N9) virus antisera. Importantly, compared with the DNA construct without the corresponding HA protein mutation, DNA vaccine encoding the A143V/R148K mutant induced a 5-fold increase in the neutralizing activity against this circulating virus. Conclusions: An appropriate vaccine strain should be considered in response to increasing antigenic drift in influenza A(H7N9) virus HA protein.


Assuntos
Substituição de Aminoácidos/imunologia , Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Substituição de Aminoácidos/genética , Animais , China/epidemiologia , Modelos Animais de Doenças , Cães , Feminino , Cobaias , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunização , Subtipo H7N9 do Vírus da Influenza A/genética , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Mutação , Neuraminidase/genética , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Vacinas de DNA/imunologia
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