Pseudorabies gD protein protects mice and piglets against lethal doses of pseudorabies virus.

Introduction: Pseudorabies (PR) is a highly contagious viral disease caused by the pseudorabies virus (PRV), which can cause disease in a wide range of domestic and wild animals. Studies have shown that new mutant strains have emerged in pig farms in many regions and that commercial inactivated and live attenuated vaccines are becoming less effective at protecting pigs. Methods: Porcine pseudorabies glycoprotein D (gD) gene (GenBank: QEY95774.1) with hexa-His tag to the C terminus for further purification processes was cloned into the lentiviral expression plasmid pLV-CMV-eGFP by restriction enzyme, the resulting plasmid was designated as pLV-CMV-gD. HEK-293T cells with robust and stable expression of recombinant gD protein was established by infection with recombinant lentivirus vector pLV-CMV-gD. We expressed porcine pseudorabies virus gD protein using HEK-293T cells. Results: We describe in this study that individual gD proteins produced by a mammalian cell expression system are well immunogenic and stimulate high levels of PRV-specific and neutralizing antibodies in mice and piglets. All mice and piglets survived lethal doses of PRV, significantly reducing the amount of PRV virus in piglets' lymph nodes, lungs, spleen, and other tissues. It also significantly reduced the time cycle and amount of viral excretion from piglets to the environment through the nasal and anal cavities. Discussion: The results suggest that PRV gD protein is expected to be a potential candidate for the preparation of genetically engineered PR vaccines for the prevention of PRV infection and the control of PR epidemics.

Immunogenicity and Antigenicity of the Ectodomain of Rabies Virus Glycoprotein Stably Expressed in HEK293T Cells.

Rabies continues to be a huge threat to public health. The rabies virus envelope glycoprotein (RABV G) is a major rabies virus antigen and contains neutralizing epitopes, which are primary candidates for subunit vaccines and diagnostic antigens. However, the production and purification of rRABV G while retaining its antigenic and immunogenic remains to be a challenge. Here, we aimed to establish a platform for rRABV G production and purification, and determine the immunogenicity and antigenicity of rRABV G. The cDNA fragment encoding the soluble form of RABV G was synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-RABV G-eGFP was packaged, titered, and then transduced into HEK 293T cells. The cell culture supernatant was purified using nickel affinity chromatography and subsequently confirmed through Western Blot analysis and indirect enzyme-linked immunosorbent assay (ELISA). The ELISA utilized human sera obtained from individuals who had been vaccinated with the human commercial Purified Vero Cells Rabies Vaccine (PVRV). Notably, we observed a neutralizing antibody response in immunized pigs rather than in mice. This discrepancy could potentially be attributed to factors such as the instability of the rRABV G protein, variations in host responses, and variances in the adjuvant used. Taking all these findings into account, the rRABV G protein generated in this study exhibits promise as a potential vaccine candidate for the prevention of rabies.

A Multi-institutional Analysis of the Combined Effect of Micropapillary Component and Consolidation-to-Tumor Ratio >0.5 on the Prognosis of Pathological, Stage IA3, Lung Adenocarcinoma.

INTRODUCTION: The study investigated the synergistic effect of the micropapillary (MIP) component and consolidation-to-tumor ratio (CTR) on the recurrence and survival of patients with pathologic stage IA3 lung adenocarcinoma. METHODS: We enrolled 419 patients confirmed pathological stage IA3 adenocarcinoma from four institutions. Kaplan-Meier analysis was performed to examine the value of the MIP component and CTR on relapse-free survival (RFS) and overall survival (OS). The cumulative recurrence between different stages was analyzed by using cumulative event curves. RESULTS: RFS (P < 0.0001) and OS (P = 0.008) in the presence of the MIP group were significantly lower than those in the absence of the MIP group, and CTR > 5 only reduced RFS (P = 0.0004), but not OS (P = 0.063), in the patients. In addition, the prognosis of patients with both the MIP component and CTR > 5 was worse than that of those without the MIP component or CTR ≤ 5. Therefore, we established new subtypes of the stage IA3: IA3a, IA3b, and IA3c. RFS and OS for IA3c staging were significantly lower than those for IA3a and IA3b. For IA3c, the cumulative incidence of local recurrence (P < 0.001) and that of distant metastasis (P = 0.004) were significantly higher than those for IA3a and IA3b. CONCLUSIONS: The MIP component combined with CTR > 0.5 can effectively predict the prognosis of patients with pathological stage IA3 lung adenocarcinoma and may offer more detailed recurrence and survival information according to the established subtype stage of IA3.

Establishment and validation of a nomogram model for predicting postoperative recurrence-free survival in stage IA3 lung adenocarcinoma: a retrospective cohort study.

Background: The increased use of computed tomography has brought a corresponding increase in the numbers of early-stage lung cancer patients receiving treatment. However, even for stage IA3 lung adenocarcinoma, many patients experience postoperative recurrence and metastasis. The existing TNM staging system for lung cancer does not take many clinical and pathological factors into consideration, resulting in the failure to detect and intervene as soon as possible in those with high recurrence risk. The purpose of this study was to explore the risk factors for postoperative recurrence-free survival (RFS) in patients with stage IA3 lung adenocarcinoma, and to construct and verify a nomogram model for predicting RFS in patients with the disease. Methods: This study analyzed patients with stage IA3 lung adenocarcinoma who underwent surgical treatment. Univariate and multivariate analysis were used to analyze the independent risk factors for postoperative RFS and establish a nomogram model. Concordance index (C-index), receiver operating characteristic curve, clinical decision analysis, and calibration curve were used to evaluate the discrimination and calibration of the nomogram model. Data from two other institutions were used for external validation, and the nomogram scores were combined with X-tile software to screen high-risk groups of recurrence. Results: The internal cohort included 235 eligible patients with stage IA3 lung adenocarcinoma from 7,235 lung cancer. Multivariate analysis showed smoking, solid nodules, mucinous lung adenocarcinoma, and micropapillary component ≥5% were independent risk factors for RFS. A nomogram model was constructed based on the above results and the bootstrap method was used for internal validation. The internal and external validation C-indexes of the nomogram were 0.822 (95% CI: 0.751-0.891) and 0.812, respectively, indicating the obvious prediction performance was good. The X-tile software combined with nomogram scores showed the low-risk group (5-RFS rate, 0.65-0.99) had better RFS than the high-risk group (5-RFS rate, 0.20-0.65) (P<0.0001). Conclusions: We constructed a nomogram model for predicting postoperative RFS in patients with stage IA3 lung adenocarcinoma which can individually evaluate the risk of postoperative recurrence, screen high-risk groups, and develop individualized follow-up and intervention strategies to improve the survival rate of the patients.

Characterization of monoclonal antibodies that specifically differentiate field isolates from vaccine strains of classical swine fever virus.

Classical swine fever virus (CSFV) is a major animal pathogen threatening the global pork industry. To date, numerous anti-CSFV monoclonal antibodies (mAbs) and their recognizing epitopes have been reported. However, few mAbs were systematically characterized for the capacity to differentiate field CSFV isolates from CSF vaccine strains, and the molecular basis associated with antigenic differences between vaccines and field isolates is still largely unknown. In the present study, recombinant CSFV structural glycoproteins E2 of both virulent and vaccine strains and Erns of vaccine strain were expressed using eukaryotic cells and murine mAbs generated against E2 and Erns. After serial screening and cloning of the hybridomas, the viral spectra of mAbs were respectively determined by indirect fluorescent antibody assay (IFA) using 108 CSFVs, followed by Western blot analysis using expressed glycoproteins of all CSFV sub-genotypes including vaccine strains. The antigenic structures recognized by these mAbs were characterized by epitope mapping using truncated, chimeric, and site-directed mutated E2 and Erns proteins. We have identified two vaccine-specific, one field isolate-specific, and two universal CSFV-specific mAbs and five novel conformational epitopes with critical amino acid (aa) motifs that are associated with these five mAbs: 213EPD215, 271RXGP274, and 37LXLNDG42 on E2 and 38CKGVP42, W81, and D100/V107 on Erns. Particularly, E213 of E2 is field isolate-specific, while N40 of E2 and D100/V107 of Erns are vaccine strain-specific. Results from our study further indicate that N40D of E2 mutation in field strains was likely produced under positive selection associated with long-term mass vaccination, leading to CSFV evasion of host immune response. Taking together, this study provides new insights into the antigenic structure of CSFV E2 and Erns and the differentiating mAbs will contribute to the development of a diagnostic strategy to differentiate C-strain vaccination from natural infection (DIVA) of CSFV in terms of elimination of CSF in China.

Efficient Gene Transfer to Kidney Using a Lentiviral Vector Pseudotyped with Zika Virus Envelope Glycoprotein.

Gene therapy's entrance into clinical settings has made it an ever more attractive field of study for various diseases. However, relatively little progress has been made in targeting kidney diseases due to poor gene delivery efficiency in renal cells. The development of novel gene therapy vectors for medical intervention to treat kidney diseases is needed. In this study, we designed and produced a pseudotyped lentiviral vector with envelope glycoproteins of Zika virus (ZIKV), and evaluated its potential use in viral vector entry, neutralization assay, and gene delivery especially in the renal context. The lentiviral vector, simplified as ZIKV-E, is pseudotyped with Env/G-TC representing the transmembrane (TM) and cytoplasmic (CY) domains of Env replaced with the TM and CY domains of the glycoprotein (G) of the vesicular stomatitis virus. In vivo results show that ZIKV-E induced efficient transduction in tubular epithelial cells in mouse kidneys, demonstrating >100-fold higher expression of exogenous green fluorescent protein gene compared with that achieved by vesicular stomatitis virus G (VSV-G) protein pseudotyped lentiviral vector. The results also showed that the vector ZIKV-E transduced cells in a pH-independent manner and the transduction was inhibited by anti-ZIKV Env domain III antibodies. Results also show that ZIKV-E can be used as a surrogate for studies of ZIKV entry mechanisms and neutralization antibody assay. In all, this study successfully demonstrated a novel pseudotyped lentiviral vector ZIKV-E for inducing high transduction efficiency in renal tubular epithelial cells that could serve as a foundation for gene therapy for the treatment of inherited renal diseases in humans.

Long-term and efficient inhibition of hepatitis B virus replication by AAV8-delivered artificial microRNAs.

Chronic hepatitis B virus (HBV) infection remains a global health problem and current treatments are insufficient due to immune tolerance to hepatitis B surface antigen (HBsAg). RNA interference (RNAi) is a more promising approach for antiviral therapy. Here, 17 single artificial microRNAs (amiRNAs) targeting the highly conserved regions of HBV genome were screened to inhibit HBV replication. In addition, we compared three tandem amiRNAs, each containing 3 different amiRNAs, out of which amiRNA135 was selected to be studied in detail. In vitro data showed that amiRNA135 significantly inhibited the replication of different HBV genotypes (including resistant and mutant). In vivo study was carried out by adeno-associated virus 8-mediated gene delivery, we found that the anti-HBV effects of AAV8-amiRNA135 were time and dose-dependent. Serum HBsAg and HBeAg in high dose groups were significantly reduced at 7 days after a single intravenous vector injection, and maintained at low levels throughout a 15-month experiment. Immunohistochemical staining and HBV core particle DNA analysis confirmed that HBV replication in the liver was strongly inhibited by AAV8-amiRNA135. Taken together, our data suggest that AAV8-mediated trimeric amiRNA expression is a promising therapeutic approach for chronic HBV infection.

Construction of a eukaryotic expression system with stable and secretory expression of mycobacterium tuberculosis 38 kDa protein.

The 38 kDa protein is a major antigen of mycobacterium tuberculosis and has been widely used in TB serodiagnosis, due to its highly sensitivity and specificity. Here we attempt to establish a production platform of recombinant 38 kDa protein in mammalian cells and to evaluate the potential value of 38 kDa protein in TB serodiagnosis. The 38 kDa gene is synthesized and cloned into a lentiviral expressing vector. Recombinant lentiviral vector LV-CMV-38 kDa-eGFP was packaged, titered, and then transduced into HEK 293 T cells. Recombinant cell lines were selected by limiting dilution. Supernatants were collected and purified by HisTrapTM HP column. Western blot showed a molecular weight of approximate 38 kDa in cell supernatants as expected. ELISA assay confirmed the immunological specificity of the obtained protein in the presence of MTB-infected human serum samples. In all, we have obtained a stable cell line with long-term and robust expression of secretory MTB 38 kDa protein, which may provide a promising candidate antigen for the development of TB serological diagnosis.

Angiotensin 1-7 Overexpression Mediated by a Capsid-optimized AAV8 Vector Leads to Significant Growth Inhibition of Hepatocellular Carcinoma In vivo.

Background: Angiotensin-(1-7) [Ang-(1-7)] has been identified to inhibit the growth of many types of tumor cells both in vitro and in vivo. However, the rapid degradation of Ang-(1-7) in vivo limits its clinical application. Adeno-associated virus (AAV) serotype-8 is a remarkable vector for long-term in vivo gene delivery. Method: This study was designed to investigate the effects of AAV-mediated Ang-(1-7) overexpression on hepatocellular carcinoma. We first generated three different tyrosine (Y) to phenylalanine (F) mutants of AAV8 (Y447F, Y703F, Y708F) and evaluated their in vivo transduction efficiencies. Results: The data indicated that the Y703F mutant elicited a significant enhancement of liver gene delivery when compared with wild-type AAV8 (wtAAV8). The anti-tumor effect of Ang-(1-7) mediated by this optimized vector was evaluated in H22 hepatoma-bearing mice. Our results demonstrated that AAV-Ang-(1-7) persistently inhibited the growth of hepatocellular carcinoma by significantly downregulating angiogenesis. This was confirmed by observed decreases in the levels of the proangiogenic factors VEGF and PIGF. Conclusion: Collectively, these data suggest that Ang-(1-7) overexpression mediated by the optimized vector may be an effective alternative for hepatocellular carcinoma therapy due to its long-term and significant anti-tumor activity.

AAV-Mediated angiotensin 1-7 overexpression inhibits tumor growth of lung cancer in vitro and in vivo.

Ang-(1-7) inhibits lung cancer cell growth both in vitro and in vivo. However, the molecular mechanism of action is unclear and also the rapid degradation of Ang-(1-7) in vivo limits its clinical application. Here, we have demonstrated that Ang- (1-7) inhibits lung cancer cell growth by interrupting pre-replicative complex assembly and restrains epithelial-mesenchymal transition via Cdc6 inhibition. Furthermore, we constructed a mutant adeno-associated viral vector AAV8 (Y733F) that produced stable and high efficient Ang-(1-7) expression in a xenograft tumor model. The results show that AAV8-mediated Ang-(1-7) over-expression can remarkably suppress tumor growth in vivo by down-regulating Cdc6 and anti-angiogenesis. Ang-(1-7) over-expression via the AAV8 method may be a promising strategy for lung cancer treatment.

Single point mutation in adeno-associated viral vectors -DJ capsid leads to improvement for gene delivery in vivo.

BACKGROUND: Rational design of AAV capsids is a simple method for enhancing AAV transduction efficiency. AAV-DJ is a highly recombinogenic hybrid vector created from DNA shuffling of eight AAV serotypes, which mediates efficient gene expression both in vitro and in vivo. AAV2 and AAV8 are the closest parental vectors of AAV-DJ and it has been reported that mutations on the 137/251/503 ubiquitination or phosphorylation sites of the AAV2 or AAV8 capsid lead to dramatic enhancement of gene delivery. Here, we aimed to find out whether the same point mutations on the AAV-DJ capsid could lead to significant improvement for gene delivery both in vitro and in vivo. RESULTS: We constructed three single point mutants (K137R/T251A/S503A) of AAV-DJ and the transduction efficiency of these mutants and AAV-DJ were investigated using two reporter gene systems including green fluorescent protein (GFP) and dual-luciferase (Gaussia luciferase and Firefly luciferase). Data indicated that single point mutations T251A/S503A lead to significant improvement of dual-luciferase expression in vivo after tail vein (TV) injection in mice respectively, despite limited enhancement of GFP expression in 293 T, Hela and HepG2 cells in vitro. Moreover, in vivo bioluminescence image and viral genome DNA copy number in tissue analysis showed that these mutants reserved the liver tropism characteristics, consistent with AAV-DJ. CONCLUSION: Single point mutations on the 251/503 sites of AAV-DJ capsid can lead to a significant improvement for in vivo gene expression. These enhanced AAV vectors have great potential in gene therapy applications.

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells.

OBJECTIVES: Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors. METHODS: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p'HR.cppt.3'1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(ß-globin intron)-eGFP containing CMV promoter and ß-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution. RESULTS: GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (ß-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells. CONCLUSION: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (ß-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.

Diverse manganese(II) coordination polymers derived from achiral/chiral imidazolium-carboxylate zwitterions and azide: structure and magnetic properties.

Five Mn(II) coordination polymers containing azide and carboxylate as simultaneous bridges have been derived from different imidazolium-carboxylate zwitterionic ligands: 1-methyl-3-(carboxylatomethyl)imidazolium (L(1)), 1,3-bis(carboxylatomethyl)imidazolium (L(2)), (S,S)-, (R,R)-, and (R,S)-1,3-bis(1-carboxylatoethyl)imidazolium (S,S-L(3), R,R-L(3) and R,S-L(3)). The compounds are formulated as [Mn(L(1))(N3)2] (1), [Mn(L(2))(N3)] (2), [Mn(R,R-L(3))(N3)]·0.5CH3OH (3-R), [Mn(S,S-L(3))(N3)]·0.5CH3OH (3-S), [Mn(R,S-L(3))(N3)] (4). In compound 1, the neutral monocarboxylate zwitterion ligand (L(1)) leads to uniform chains with bis(azide)(carboxylate) bridges. For compounds 2-4, the anionic dicarboxylate zwitterions L(2) and L(3) lead to (azide)bis(carboxylate) bridges, but the overall coordination networks are different. In 2 and 3-S (or 3-R), chains with the (azide)bis(carboxylate) bridges are connected by L(2) and S,S-L(3) (or R,R-L(3)), respectively, to give achiral and chiral 2D coordination networks with different connecting topologies. In compounds 4, which is derived from the mesomeric ligand R,S-L(3), linear trinuclear units with the (azide)bis(carboxylate) bridges are linked by µ-1,3 azides to give 2D layers, and the layers are pillared into a 3D framework by the 1,3-dimethyleneimidazolium tethers. Magnetic analyses suggested that compounds 1-3 behave as 1D antiferromagnetic systems, while 4 shows canted antiferromagnetism with weak ferromagnetic ordering below T(C) = 12.4 K.

[Construction of a recombinant lentiviral vector of p38 MAPK and establishment of a human prostatic carcinoma cell line stably expressing p38 MAPK].

OBJECTIVE: To construct a recombinant lentiviral vector for p38 MAPK and establish a human prostatic carcinoma cell line that stably expresses p38 MAPK. METHODS: EGFP/p38 fusion gene was subcloned into the lentiviral vector pTYF- EF1α-IRES-EGFP. The recombinant lentiviral vector pTYF-EF1α-EGFP/p38 was indentified by restriction enzyme digestion, and packaged in HEK 293T cells using lipofectamintm2000 with the packaging plasmid psPAX2 and envelope plasmid pMD2.G. The viral titer was tested according to the expression level of GFP. The resulting recombinant lentiviral vector was transduced into human prostatic carcinoma DU145 cells, and stably transduced cells were selected by limiting dilution analysis. The intracellular expression level of total p38 was detected by Western blotting and the cell growth curve was drawn. RESULTS: DNA restriction enzyme digestion demonstrated that the recombinant lentiviral vector of the fusion gene EGFP/p38 (pTYF-EF1α-EGFP/p38) was constructed successfully. The recombinant lentiviral vector was packaged in 293T with a viral titer of 4.7×10(6) TU/ml. A stable cell line, EGFP/p38-DU145, was established, which stably expressed exogenous EGFP/p38 MAPK fusion protein as detected by Western blotting and showed a lowered growth rate compared to the control cells. CONCLUSION: We have successfully constructed a recombinant lentiviral vector of the fusion gene EGFP/p38 and established a stable cell line EGFP/p38-DU145. Overexpression of p38 has a significant inhibitory effect on the proliferation of DU145 cells in vitro.