Designing a conjugate vaccine targeting Klebsiella pneumoniae ST258 and ST11.

Klebsiella pneumoniae (K. pneumoniae) is a common bacterium that can cause iatrogenic infection. Recently, the rise of antibiotic resistance among K. pneumoniae strains is one key factor associated with antibiotic treatment failure. Hencefore, there is an urgent need for effective K. pneumoniae vaccines. This study aimed to design a multi-epitope vaccine (MEV) candidate against K. pneumonia by utilizing an immunoinformatics method. In this study, we obtained 15 cytotoxic T lymphocyte epitopes, 10 helper T lymphocyte epitopes, 6 linear B-cell epitopes, and 2 conformational B-cell epitopes for further research. Then, we designed a multi-epitope vaccine composed of a total of 743 amino acids, containing the epitopes linked by GPGPG flexible links and an EAAAK linker to the Cholera Toxin Subunit B coadjuvant. The observed properties of the MEV, including non-allergenicity, high antigenicity, and hydrophilicity, are noteworthy. The improvements in the tertiary structure through structural refinement and disulfide bonding, coupled with promising molecular interactions revealed by molecular dynamics simulations with TLR4, position the MEV as a strong candidate for further investigation against K. pneumoniae.

Serum TNF-α level and probing depth as a combined indicator for peri-implant disease.

Peri-implant disease (PID) is a general term for inflammatory diseases of soft and hard tissues that occur around implants, including peri-implant mucositis and peri-implantitis. Cytokines are a class of small molecule proteins, which have various functions such as regulating innate immunity, adaptive immunity, and repairing damaged tissues. In order to explore the characteristics and clinical significance of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and tumor growth factor (TGF)-ß1 expression levels in serum of patients with peri-implant disease, 31 patients with PID and 31 patients without PID were enrolled. The modified plaque index (mPLI), modified sulcus bleeding index (mSBI), and peri-implant probing depth (PD) were recorded. The levels of serum TNF-α, IL-6, IL-10, and TGF-ß1 were detected by ELISA. TNF-α, mPLI, mSBI, and PD levels were significantly higher in the PID group. TGF-ß1 levels were significantly higher in the control group. There was a significant positive correlation between TNF-α and mPLI, mSBI, and PD. TGF-ß1 was negatively associated with TNF-α, mPLI, mSBI, and PD. Multiple logistic regression analysis showed that TNF-α and PD were risk factors for the severity of PID. The receiver operating curve analysis showed that high TNF-α levels (cut-off value of 140 pg/mL) and greater PD values (cut-off value of 4 mm) were good predictors of PID severity with an area under the curve of 0.922. These results indicated that TNF-α and PD can be used as a biological indicator for diagnosing the occurrence and progression of PID.

Serum TNF-α level and probing depth as a combined indicator for peri-implant disease

Peri-implant disease (PID) is a general term for inflammatory diseases of soft and hard tissues that occur around implants, including peri-implant mucositis and peri-implantitis. Cytokines are a class of small molecule proteins, which have various functions such as regulating innate immunity, adaptive immunity, and repairing damaged tissues. In order to explore the characteristics and clinical significance of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and tumor growth factor (TGF)-β1 expression levels in serum of patients with peri-implant disease, 31 patients with PID and 31 patients without PID were enrolled. The modified plaque index (mPLI), modified sulcus bleeding index (mSBI), and peri-implant probing depth (PD) were recorded. The levels of serum TNF-α, IL-6, IL-10, and TGF-β1 were detected by ELISA. TNF-α, mPLI, mSBI, and PD levels were significantly higher in the PID group. TGF-β1 levels were significantly higher in the control group. There was a significant positive correlation between TNF-α and mPLI, mSBI, and PD. TGF-β1 was negatively associated with TNF-α, mPLI, mSBI, and PD. Multiple logistic regression analysis showed that TNF-α and PD were risk factors for the severity of PID. The receiver operating curve analysis showed that high TNF-α levels (cut-off value of 140 pg/mL) and greater PD values (cut-off value of 4 mm) were good predictors of PID severity with an area under the curve of 0.922. These results indicated that TNF-α and PD can be used as a biological indicator for diagnosing the occurrence and progression of PID.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT.

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Design of multi-epitope vaccine candidate against Brucella type IV secretion system (T4SS).

Brucellosis is a common zoonosis, which is caused by Brucella infection, and Brucella often infects livestock, leading to abortion and infertility. At present, human brucellosis remains one of the major public health problems in China. According to previous research, most areas in northwest China, including Xinjiang, Tibet, and other regions, are severely affected by Brucella. Although there are vaccines against animal Brucellosis, the effect is often poor. In addition, there is no corresponding vaccine for human Brucellosis infection. Therefore, a new strategy for early prevention and treatment of Brucella is needed. A multi-epitope vaccine should be developed. In this study, we identified the antigenic epitopes of the Brucella type IV secretion system VirB8 and Virb10 using an immunoinformatics approach, and screened out 2 cytotoxic T lymphocyte (CTL) epitopes, 9 helper T lymphocyte (HTL) epitopes, 6 linear B cell epitopes, and 6 conformational B cell epitopes. These advantageous epitopes are spliced together through different linkers to construct a multi-epitope vaccine. The silico tests showed that the multi-epitope vaccine was non-allergenic and had a strong interaction with TLR4 molecular docking. In immune simulation results, the vaccine construct may be useful in helping brucellosis patients to initiate cellular and humoral immunity. Overall, our findings indicated that the multi-epitope vaccine construct has a high-quality structure and suitable characteristics, which may provide a theoretical basis for the development of a Brucella vaccine.

Contriving a novel of CHB therapeutic vaccine based on IgV_CTLA-4 and L protein via immunoinformatics approach.

Chronic infection induced by immune tolerance to hepatitis B virus (HBV) is one of the most common causes of hepatic cirrhosis and hepatoma. Fortunately, the application of therapeutic vaccine can not only reverse HBV-tolerance, but also serve a potentially effective therapeutic strategy for treating chronic hepatitis B (CHB). However, the clinical effect of the currently developed CHB therapeutic vaccine is not optimistic due to the weak immunogenicity. Given that the human leukocyte antigen CTLA-4 owns strong binding ability to the surface B7 molecules (CD80 and CD86) of antigen presenting cell (APCs), the immunoglobulin variable region of CTLA-4 (IgV_CTLA-4) was fused with the L protein of HBV to contrive a novel therapeutic vaccine (V_C4HBL) for CHB in this study. We found that the addition of IgV_CTLA-4 did not interfere with the formation of L protein T cell and B cell epitopes after analysis by means of immunoinformatics approaches. Meanwhile, we also found that the IgV_CTLA-4 had strong binding force to B7 molecules through molecular docking and molecular dynamics (MD) simulation. Notably, our vaccine V_C4HBL showed good immunogenicity and antigenicity by in vitro and in vivo experiments. Therefore, the V_C4HBL is promising to again effectively activate the cellular and humoral immunity of CHB patients, and provides a potentially effective therapeutic strategy for the treatment of CHB in the future.Communicated by Ramaswamy H. Sarma.

Design and functional preliminary investigation of recombinant antigen EgG1Y162-EgG1Y162 against Echinococcus granulosus.

In the early stage, our research group cloned Echinococcus granulosus-specific antigen, EgG1Y162, from protoscolex and adult worms of E. granulosus. In order to enhance the immunogenicity of the vaccine, we prepared a recombinant vaccine by tandemly linking EgG1Y162, splicing the protein and linker at the gene level. This approach is expected to improve the immunogenicity of the vaccine by enhancing the molecular weight of the protein and increasing the antigenic epitopes. Bioinformatics was used to predict the physicochemical properties, transmembrane domain, protein structure, and T-/B-cell antigenic epitope of different recombinant proteins, EgG1Y162-linker-EgG1Y162. Finally, the linker sequence, "GGGGSGGG," which had the least influence on the migration of recombinant protein T/B epitope and can fold normally in series with EgG1Y162, was selected to design the recombinant vaccine. The plasmid was produced using genetic engineering techniques, and the recombinant protein, EGG1Y162-GGGGSGGG-EgG1Y162, was induced to be expressed and purified. EgG1Y162-GGGGSGGG-EgG1Y162 was identified to be correctly expressed with 100% specificity. Compared with EgG1Y162, EgG1Y162-GGGGSGGG-EgG1Y162 was more likely to promote dendritic cell maturation. EgG1Y162-GGGGSGGG-EgG1Y162 was speculated to have the potential to improve antigen immunogenicity by increasing the molecular weight and antigenic epitope.

The level of Tim-3+CD8+ T cells can serve as a potential marker for evaluating the severity of acute graft-versus-host disease after haplo-PBSCT

Early and accurate diagnosis of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation is crucial for the prognosis of patients. This study identified a potential biomarker for the severity of aGVHD after human leukocyte antigen (HLA)-haploidentical peripheral blood hematopoietic stem cell transplantation (haplo-PBSCT). We included 20 healthy subjects and 57 patients who underwent haplo-PBSCT. Of these patients, 22 developed aGVHD after haplo-PBSCT. The results showed that patients with aGVHD had significantly increased levels of Tim-3+/Perforin+/Granzyme B+CD8+ T cells, but significantly decreased Galectin-9. The differences in Galectin-9 and Tim-3+/Granzyme B+CD8+ T cells between grade I-II aGVHD and III-IV aGVHD were also significant. In vitro, the apoptosis of CD8+ T cells from aGVHD patients was significantly increased after Tim-3/Galectin-9 pathway activation, which decreased Granzyme B secretion. As revealed by univariate analysis, the level of Tim-3+CD8+ T cells was a risk factor for severe aGVHD. ROC analysis demonstrated that high levels of Tim-3+CD8+ T cells had a significant diagnostic value for severe aGVHD, with an area under the curve of 0.854 and cut-off value of 14.155%. In conclusion, the binding of Tim-3 with exogenous Galectin-9 can promote apoptosis of CD8+ T cells and affect the secretion of Granzyme B. Tim-3+CD8+ T cells have the potential to serve as immunological markers for assessing the severity of aGVHD after haplo-PBSCT and identifying patients at a higher risk for severe aGVHD.

Design of a multi-epitope vaccine candidate against Brucella melitensis.

Brucella is a typical facultative intracellular bacterium that can cause zoonotic infections. For Brucella, it is difficult to eliminate with current medical treatment. Therefore, a multi-epitope vaccine (MEV) should be designed to prevent Brucella infection. For this purpose, we applied the reverse vaccinology approach from Omp10, Omp25, Omp31 and BtpB. Finally, we obtained 13 cytotoxic T lymphocyte (CTL) epitopes, 17 helper T lymphocyte (HTL) epitopes, 9 linear B cell epitopes, and 2 conformational B cell epitopes for further study. To keep the protein folded normally, we linked AAY, GPGPG, and KK to CTL epitopes, HTL epitopes, and B cell epitopes, respectively. The N-terminal of the vaccine peptide is supplemented with appropriate adjuvants to enhance immunogenicity. To evaluate its immunogenicity, stability, safety, and feasibility, a final MEV containing 806 amino acids was constructed by linking linkers and adjuvants. In addition, molecular docking and molecular dynamics simulations were performed to verify the affinity and stability of the MEV-TLR4. Then, codon adaptation and in silico cloning studies were carried out to identify the possible codons for expressing the MEV. In animal experiments, the results demonstrated that the MEV had high immunogenicity. Collectively, this study provided a theoretical basis for the development of a Brucella vaccine.

Design of a Recombinant Multivalent Epitope Vaccine Based on SARS-CoV-2 and Its Variants in Immunoinformatics Approaches.

The development of an effective multivalent vaccine against SARS-CoV-2 variants is an important means to improve the global public health situation caused by COVID-19. In this study, we identified the antigen epitopes of the main global epidemic SARS-CoV-2 and mutated virus strains using immunoinformatics approach, and screened out 8 cytotoxic T lymphocyte epitopes (CTLEs), 17 helper T lymphocyte epitopes (HTLEs), 9 linear B-cell epitopes (LBEs) and 4 conformational B-cell epitopes (CBEs). The global population coverage of CTLEs and HTLEs was 93.16% and 99.9% respectively. These epitopes were spliced together by corresponding linkers and recombined into multivalent vaccine. In silico tests, the vaccine protein was a non-allergen and the docking with TLR-3 molecule showed a strong interaction. The results of immune simulation showed that the vaccine may be helpful to initiate both cellular and humoral immunity against all VOC. The optimistic immunogenicity of the vaccine was confirmed in vivo and in vitro finally. Therefore, our vaccine may have potential protection against SARS-CoV-2 and its variants.

Immmunoinformatics-based design of a multi-epitope vaccine with CTLA-4 extracellular domain to combat Helicobacter pylori.

In view of the high infection rate of Helicobacter pylori, a safe and effective vaccine is urgently needed. Recent trends in vaccine design have shifted toward safe and specific epitope-based vaccines. In this study, by using different immunoinformatics approaches, a total of eight linear B cell epitopes, four HTL and three CTL epitopes of FlaA and UreB proteins of H. pylori G27 strain were screened out, we also predicted the conformational epitopes of the two proteins. Then, the dominant epitopes were sequentially linked by appropriate linkers, and the cytotoxic T lymphocyte-associated antigen 4 extracellular domain was attached to the N-terminal of the epitope sequence. Meanwhile, molecular docking, molecular dynamics simulations and principal component analysis were performed to show that the multi-epitope vaccine structure had strong interactions with B7 (B7-1, B7-2) and Toll-like receptors (TLR-2, -4). Eventually, the effectiveness of the vaccine was validated using in silico cloning. These analyses suggested that the designed vaccine could target antigen-presenting cells and had high potency against H. pylori, which could provide a reference for the future development of efficient H. pylori vaccines.

Design of a Novel Multi-Epitope Vaccine Against Echinococcus granulosus in Immunoinformatics.

All the time, echinococcosis is a global zoonotic disease which seriously endangers public health all over the world. In order to speed up the development process of anti-Echinococcus granulosus vaccine, at the same time, it can also save economic cost. In this study, immunoinformatics tools and molecular docking methods were used to predict and screen the antigen epitopes of Echinococcus granulosus, to design a multi-epitope vaccine containing B- and T-cell epitopes. The multi-epitope vaccine could activate B lymphocytes to produce specific antibodies theoretically, which could protect the human body against Echinococcus granulosus infection. It also could activate T lymphocytes and clear the infected parasites in the body. In this study, four CD8+ T-cell epitopes, three CD4+ T-cell epitopes and four B-cell epitopes of Protein EgTeg were identified by immunoinformatics methods. Meanwhile, three CD8+ T-cell epitopes, two CD4+ T-cell epitopes and four B-cell epitopes of Protein EgFABP1 were identified. We constructed the multi-epitope vaccine using linker proteins. The study based on the traditional methods of antigen epitope prediction, further optimized the prediction results combined with molecular docking technology and improved the precision and accuracy of the results. Finally, in vivo and in vitro experiments had verified that the vaccine designed in this study had good antigenicity and immunogenicity.

A Multi-Epitope Chitosan Nanoparticles Vaccine of Canine Against Echinococcus granulosus.

Cystic Echinococcosis (CE) is caused by Echinococcus granulosus (Eg), which endangers the health of the intermediate host. Therefore, effective canid vaccines against Eg infection are urgently needed to reduce the incidence of this disease. In the present work, the aim was to predict epitopes in four vaccine candidate antigens (VCAs) in Eg as a basis to design a multi-epitope canine-directed vaccine. This vaccine is based on chitosan nanoparticles (CS-NPs) and is directed against Eg infection in the definitive host. The canine-directed vaccine was designed based on Eg antigens EgM9, Eg_10196, EgA31 and EgG1Y162. Several tools in online servers were used to predict VCAs information, which was combined with B cell, CTL and Th epitopes. Considering that acquiring experimental information in canids is difficult, and that it may be possible to perform future experiments in mice, we predicted both canine and murine T cell epitopes. The multi-epitope vaccine was synthetically prepared by ionic crosslinking method, and CS-NPs was used as adjuvant. The mice were immunized by oral gavage and laser scanning confocal microscopy was used to localize the fluorescein- labeled multi-epitope peptide in the intestinal tract. The final multi-epitope vaccine was construct consist of Co1 targeting peptide, four B-cell epitopes, four canine-directed CTL epitopes and four murine-directed Th epitopes. It has been proven experimentally by this research that multi-epitope antigen concentration merged with microfold cells was high in the CS-NPs vaccine group. The present bioinformatics study is a first step towards the construction of a canine-specific multiepitope vaccine against Eg with twelve predicted epitopes. CS-NPs is a potential adjuvant with relatively safe penetration enhancement delivery and a potent immunostimulant.

State and parameter estimation from exact partial state observation in stochastic reaction networks.

We consider chemical reaction networks modeled by a discrete state and continuous in time Markov process for the vector copy number of the species and provide a novel particle filter method for state and parameter estimation based on exact observation of some of the species in continuous time. The conditional probability distribution of the unobserved states is shown to satisfy a system of differential equations with jumps. We provide a method of simulating a process that is a proxy for the vector copy number of the unobserved species along with a weight. The resulting weighted Monte Carlo simulation is then used to compute the conditional probability distribution of the unobserved species. We also show how our algorithm can be adapted for a Bayesian estimation of parameters and for the estimation of a past state value based on observations up to a future time.

The Corrosion Probability and Flexural Strength of an RC Beam under Chloride Ingress Considering the Randomness of Temperature and Humidity.

The load capacity of reinforced concrete structure will decrease by chloride ingress in coastal region. In this paper, the corrosion probability and flexural strength of a typical reinforced concrete beam design under the influence of temperature and humidity was obtained by the Monte Carlo method. The relationship between flexural strength, temperature, relative humidity, and geometric parameters was established. The annual average temperature and relative humidity were treated as random variables together with the geometric size and concrete compressive strength. The corrosion probability and flexural strength in a wave splashing zone, coastal atmospheric zone, and offshore atmospheric zone were calculated. The results show that the corrosion probabilities of the three regions are obviously different. When the standard deviation of temperature is less than 1.5 °C, the temperature can be treated as a constant in the calculation of the concrete cracking probability in the wave splashing zone. A binary logistic regression formula was given to predict whether the randomness of temperature and humidity should be considered in the offshore atmospheric zone. When the standard deviation of the temperature is less than 1 °C, the temperature randomness has no significant effect on the flexural strength of beams in the wave splashing zone. The flexural strength distribution conforms to the normal distribution in the early stage of service and the Weibull distribution after concrete cracking.

Influence of guide wire removal on tip location in peripherally inserted central catheters (PICCs): a retrospective cross-sectional study.

OBJECTIVES: The aim of this study was to identify the prevalence of peripherally inserted central catheter (PICC) malposition and the influence of guide wire removal on tip location in PICCs and determine whether related factors, including age, sex, side of insertion and brand of catheter, influence the PICC tip location. SETTING: Single-centre research institute in China recruiting patients from the hospital. PARTICIPANTS: A total of 837 adult patients with inserted PICCs were recruited from October 2016 to May 2017. INTERVENTIONS: This was a cross-sectional study aiming to identify the prevalence of PICC malposition and the influence of guide wire removal on tip location in PICCs. A linear regression model and a variance of factorial design analysis were performed. The PICC tip location was documented on a postinsertion chest X-ray. Multivariable analyses were performed based on the following related factors: age, sex, side of insertion and brand of catheter. RESULTS: The tip location moved a mean of 17.4 mm among the 837 included patients. The prevalence of PICC malposition was 83.6% (700/837), while 16.4% (137/837) of PICCs remained in correct location. The mean movement caused by guide wire removal without an adjusted tail end was (-1.95±26.90) mm. The difference between tail end adjustment movement and actual tip position movement in each PICC was (33.0±17.1) mm in type C, which was significantly higher than the findings for type A (12.8±13.3) mm and type B (12.9±12.7) mm. CONCLUSIONS: PICC malposition is a frequent event. Different catheter brands were associated with different ranges of movement in tip location after guide wire removal. The age and sex of the patients and the insertion side did not influence the extent of movement.