Clinical application of a new warfarin-dosing regimen based on the CYP2C9 and VKORC1 genotypes in atrial fibrillation patients.

The polymorphisms of cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) are important genetic factors for warfarin dose determinations. The present study aimed to investigate the contribution of the CYP2C9 and VKORC1 genotypes to warfarin dose requirement in atrial fibrillation (AF) patients, and to evaluate the clinical application of a warfarin-dosing algorithm. A total of 122 AF patients with a target international normalized ratio of 2.0 to 3.0 were included to determine the genotypes of CYP2C9 (rs1057910) and VKORC1 (rs9923231). A warfarin-dosing algorithm was developed based on age, height, and the CYP2C9 and VKORC1 genotypes of AF patients. The results indicated that the mean warfarin daily dose requirement was lower in the CYP2C9*1/*3 genotype compared with those in the homozygous wild-type CYP2C9*1/*1 patients (P<0.05), and was higher in patients with the VKORC1 AG and GG genotypes compared with those with the AA genotype (P<0.05). The multivariate regression model showed that age, height, and the CYP2C9 and VKORC1 genotypes were the best variables for estimating warfarin dose (R2=56.4%). A new warfarin-dosing algorithm was developed and its validity was confirmed in a second cohort of AF patients. During the 50-day follow-up, 63.3% (19/30) of control group patients and 86.7% (26/30) of patients in the experimental group acquired the warfarin maintenance dose. Among all the patients who acquired the warfarin maintenance dose, the mean time elapse from initiation until warfarin maintenance dose was significantly less in the experimental group (25.8±1.7 day) compared to the control group (33.1±1.9 day) (P<0.05). There was significant linear correlation between predicted warfarin maintenance dose and actual dose (r=0.822, P<0.01). In conclusion, a new warfarin-dosing algorithm was developed based on the CYP2C9 and VKORC1 genotypes, and it can shorten the time elapse from initiation until warfarin maintenance dose in AF patients with warfarin therapy.

Activation of B cells by a dendritic cell-targeted oral vaccine.

Production of long-lived, high affinity humoral immunity is an essential characteristic of successful vaccination and requires cognate interactions between T and B cells in germinal centers. Within germinal centers, specialized T follicular helper cells assist B cells and regulate the antibody response by mediating the differentiation of B cells into memory or plasma cells after exposure to T cell-dependent antigens. It is now appreciated that local immune responses are also essential for protection against infectious diseases that gain entry to the host by the mucosal route; therefore, targeting the mucosal compartments is the optimum strategy to induce protective immunity. However, because the gastrointestinal mucosae are exposed to large amounts of environmental and dietary antigens on a daily basis, immune regulatory mechanisms exist to favor tolerance and discourage autoimmunity at these sites. Thus, mucosal vaccination strategies must ensure that the immunogen is efficiently taken up by the antigen presenting cells, and that the vaccine is capable of activating humoral and cellular immunity, while avoiding the induction of tolerance. Despite significant progress in mucosal vaccination, this potent platform for immunotherapy and disease prevention must be further explored and refined. Here we discuss recent progress in the understanding of the role of different phenotypes of B cells in the development of an efficacious mucosal vaccine against infectious disease.

Development of a loop-mediated isothermal amplification assay for rapid detection of bovine parvovirus.

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of bovine parvovirus (BPV) DNA. Four primers were designed to recognize six distinct regions on the target DNA based on a highly conserved sequence in the VP2 region of the BPV genome. The optimized LAMP reaction conditions were 8 mM Mg²âº, 1.2 mM betaine, and an incubation at 63°C for 45 min. After amplification the products were detected either by observing a ladder pattern following gel electrophoresis, observation of turbidity, or a color change with the addition of SYBR Green I to the reaction tube. The detection limit of the LAMP assay was 9 copies of BPV-DNA and was 100 times more sensitive than conventional PCR. A ladder pattern of bands after gel electrophoresis was observed for only BPV isolates and showed that the BPV LAMP assay was highly specific without any cross-reactivity with other related viruses. The LAMP assay was evaluated further using 59 field samples and the results were comparable to conventional PCR. The LAMP assay is a simple, rapid and economic detection method; it can provide a useful technique suitable for detection of BPV infection in both field conditions and laboratory settings.

Construction of recombinant lactobacilli expressing the core neutralizing epitope (COE) of porcine epidemic diarrhea virus and a fusion protein consisting of COE and Escherichia coli heat-labile enterotoxin B, and comparison of the immune responses by orogastric immunization.

The core neutralizing epitope (COE) region of porcine epidemic diarrhea virus (PEDV) plays an important role in the development of the subunit vaccine against PEDV infection. To enhance the vaccine's immunogenicity, Escherichia coli heat-labile enterotoxin B (LTB) has usually been adopted as a molecular adjuvant. In this study, the COE and LTB-COE genes were engineered into the Lactobacillus -Escherichia coli shuttle vectors pSAPG1 (surface-displaying) and pSAPG2 (secreting) followed by electrotransformation into Lactobacillus casei (Lc) to yield the following recombinant strains: Lc:PG1-LTB-COE, Lc:PG2-LTB-COE, Lc:PG1-COE, and Lc:PG2-COE. Our results showed that mice immunized orogastrically with L. casei expressing COE or LTB-COE produced secretory immunoglobulin A and immunoglobulin G with the ability to neutralize PEDV in sera and mucus. Moreover, higher levels of interleukin-4 and gamma interferon were also exhibited compared with negative control. These data displayed the tendency of Lc:PG2-LTB-COE > Lc:PG1-LTB-COE > Lc:PG2-COE > Lc:PG1-COE at the same time point. Taken together, LTB-COE is more suitable for Lactobacillus expressing system to engineer mucosal vaccine against PEDV infection.

Expression of infectious pancreatic necrosis virus (IPNV) VP2-VP3 fusion protein in Lactobacillus casei and immunogenicity in rainbow trouts.

Infectious pancreatic necrosis virus (IPNV) infects wild and cultured salmonids, causing high mortality in juvenile trouts and salmons. IPNV VP2-VP3 fusion gene was constructed by splicing overlap extension (SOE) PCR and inserted into Lactobacillus/Escherichia coli shuttle vectors (pPG1and pPG2) followed by transformation of Lactobacillus casei competent cell to yield two recombinant strains: Lc:PG1-VP2-VP3 (surface-displayed) and Lc:PG2-VP2-VP3 (secretory). Subsequently, juvenile rainbow trouts were inoculated with the recombinant strains via orogastric route. Our results demonstrated that Lactobacillus-derived VP2-VP3 fusion protein could induce production of serum IgM specific for IPNV with neutralizing activity in rainbow trouts. Statistical analyses of IgM levels showed that immunogenicity of Lc:PG1-VP2-VP3 was more powerful than that of Lc:PG2-VP2-VP3 (P<0.001) in rainbow trouts. This result has been confirmed by viral loads reduction analyzed by real-time RT-PCR in orogastrically immunized rainbow trouts after virus challenging. Comparing to trouts received Lactobacillus (control), rainbow trouts orogastrically dosed with Lc:PG1-VP2-VP3 resulted in ∼10-fold reduction in viral loads on day 10 post-virus challenging, and ∼4-fold did by Lc:PG2-VP2-VP3. Taken together, Lc:PG1-VP2-VP3 functions as novel mucosal vaccine against IPNV infection in rainbow trouts, which most likely come true.

High-level mucosal and systemic immune responses induced by oral administration with Lactobacillus-expressed porcine epidemic diarrhea virus (PEDV) S1 region combined with Lactobacillus-expressed N protein.

To develop effective mucosal vaccine formulation against porcine epidemic diarrhea virus (PEDV) infection, the DNA fragments encoding spike protein immunodominant region S1 and nucleocapsid N of PEDV were inserted into pPG1 (surface-displayed) or pPG2 (secretory) plasmids followed by electrotransformation into Lactobacillus casei (Lc) to yield four recombinant strains: PG1-S1, PG2-S1, PG1-N, and PG2-N. After intragastric administration, it was observed that live Lc-expressing S1 protein combined with Lc-expressing N protein could elicit much more potent mucosal and systemic immune responses than the former alone (P < 0.001), however slightly inferior to the latter alone (P > 0.05). Furthermore, the surface-displayed mixture (PG1-S1+ PG1-N) revealed stronger immunogenicity than the secretory mixture (PG2-S1+ PG2-N) as well as PEDV-neutralizing potency in vitro (P < 0.001). On 49th day after the last immunization, splenocytes were prepared from mice immunized with surface-displayed mixture, secretory mixture and negative control to be stimulated by purified N and S protein, respectively. The results of ELISA analysis showed that N protein was capable of inducing a higher level of IL-4 (P < 0.001) and IFN-γ (P < 0.001) than S1 protein in the immunized mice. Taken together, Lc-expressed N protein as molecular adjuvant or immunoenhancer was able to effectively facilitate the induction of mucosal and systemic immune responses by Lc-expressing S1 region.

Oral vaccination with the porcine rotavirus VP4 outer capsid protein expressed by Lactococcus lactis induces specific antibody production.

The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

[Isolation and characteristics of virus culture of porcine epidemic diarrhea virus LJB/03].

Porcine epidemic diarrhea virus (PEDV) LJB/03 strain was isolated from the feces of piglets suspected to be suffering from a severe diarrhea in Heilongjiang Province, and was identified by immunofluorescence test, immunelectronmicroscopy, RT-PCR and indirect ELISA assay. Characteristics of the virus culture and the methods of improvement of virus titer were explored. The results showed that the virus had the typical appearance of the coronavirus. Analysis of the nucleotide sequences of RT-PCR products revealed 98% homology with the reference strains. Indirect immunofluorescence assay showed a significant presence of green fluorescence, and an average P/N ratio of 7.6 by indirect ELISA assay. Taken together, these tests showed positive isolation of PEDV. Using the virus plaque purification cloning methods established in the test, the purified PEDV large plaque and small plaque were obtained, and the large plaque and small plaque titers were measured with significant difference. These results provide potential for the application of PEDV on the basis of the biological features of isolated virus.

[Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus].

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets that leads to great economic losses in East Asia. It was reported that aminopeptidase N (APN) was the receptor for Transmissible gastroenteritis virus (TGEV), Human coronavirus 229E (HCoV-229E) and Feline coronavirus (FeCoV) which all belonged to group I coronavirus including PEDV. It was also confirmed previously that porcine aminopeptidase N (pAPN) could bind to PEDV, and anti-pAPN antibodies could inhibit the combination. To investigate whether pAPN was a receptor for PEDV, we transfected MDCK cells with porcine aminopeptidase (pAPN) cDNA and this enabled non-susceptible cells to support PEDV replication and serial viral propagation. Moreover, the infection was blocked by antibodies against pAPN, implying the critical role of pAPN during virus entry. In addition, immunofluorescence assays for detection of pAPN and PEDV antigens, together with neutralization assays using antibodies against pAPN, further confirmed the correlation between pAPN expression and viral replication in pAPN-transfected MDCK cells. These results indicated that pAPN is a functional receptor for PEDV.

[The oral immune efficacy of recombinant lactobacillus casei expressing CSFV E290 peptide and it elicited specific CTL response].

UNLABELLED: The gene encoding classical swine fever virus (CSFV) T cell epitope E290 peptide was synthesized by PCR, cloned into the expression vector pPG-VP2 and named pPG-VP2-E290. The recombinant plasmid was electrotransformed into Lactobacillus casei 393 generating pPG-VP2-E290/L. casei 393. Specific anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of BALB/c mice and rabbits immunized with recombinant strain by oral administration. The CTL of E290 was analyzed with lymphocytes taken from the immunized mice, and the immunized rabbits were attacked with CSFV to validate the protective function of E290 antibody induced. RESULT: The recombinant expression system constructed with L. casei 393 in this study show a good immunization property and could elicit the mice and rabbits to produce high anti-E290 antibody levels. Furthermore, E290 peptide antibody could elicit specific CTL response, and restrain attack of CSFV to rabbits.

[Co-expression of CSFV T cell epitope E290 peptide and PPV VP2 protein in Lactobacillus casei and determination of specific antibodies in immunized mice].

Lactobacillus casei strain 393 was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant classical swine fever virus (CSFV) T cell epitope E290 peptide and porcine parvovirus (PPV) VP2 protein. The recombinant genes encoding CSFV T cell epitope E290 peptide and PPV VP2 protein, respectively, were cloned into the secretion expression vector pPG, and then the pPG-VP2-E290 was electrotransformed into L. casei 393 giving rise to recombinant strain pPG-VP2-E290/L. casei 393. The recombinant L. casei 393 was induced by 2% lactose in MRS and about 70kDa protein was detected with SDS-PAGE in induced recombinant strain and culture supernatants. The result of Western blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The indirect ELISA test also indicated that the interest protein was expressed and secreted from the recombinant strain. Specific anti-PPV VP2 secret immunoglobulin A (sIgA) antibody was detected by indirect ELISA in the feces, anti-PPV VP2 and anti-CSFV E290 peptide immunoglobulin G (IgG) antibody was detected by indirect ELISA in the serum of immunized mice after intragastric administration. The results indicated that the mice immunized with recombinant strain pPG-VP2-E290/L. casei 393 could produce clear antibody level, which establish important material basement for the development of lactic acid bacteria oral vaccine of recombinant CSFV and PPV.

[Construction of recombinant Lactococcus lactis expressing porcine transmissible gastroenteritis spike glycoprotein and analysis of immunogenicity].

Transmissible gastroenteritis virus (TGEV), is an enteropathogenic coronavirus that causes a highly fatal acute diarrhea in newborn pigs. It's typically clinical manifestations consist of omitting, severe diarrhea, loss water and highly infectious disease. All kinds and ages of pigs can be infected. Particular, the mortality piglets under 3 weeks may reach 100% . The effective protection against TGEV requires the development of vaccines that are able to induce local mucosal immunization. Lactococcus lactis was selected as a bacterial carrier for the expression of TGEV spike glycoprotein. The gene of S glycoprotein was cloned into the Lactococcus lactis vectors pNZ8112. An approximately 2000 bps fragments of TGEV gene S that encompasses all the four major antigenic domains critical for neutralization was transformed into Lactococcus lactis NZ9000 by electroporation, resulting in the recombinant strain pNZ8112-Sa/NZ9000. The recombinant glycoprotein S was detected by SDS-PAGE and Western blot after induced by 1ng/mL nisin. The result indicated that the expressed product maintain the antigenicity of TGEV as expected. In order to detect the location of expressed protein, the yellow and green fluorescence of the recombinant strain pNZ8112-Sa/NZ9000 was detected by the IFA experiments, which indicated that the expressed recombinant protein was secreted and located on the surface of the bacterium cell. Oral immunization of BALB/c mice with recombinant strain that constitutively express the 66kDa fragment of the glycoprotein S, Specific anti-TGEV glycoprotein S secret immunoglobulin A (sIgA) antibodies were detected by indirect enzyme linked immunosorbent assay (ELISA) in the feces after immunization. It was showed that the mice immunized with pNZ8112-Sa/NZ9000 recombinant strain had produced clear antibody level anti TGEV, and which had provided important substance foundation and explored the feasibility of Lactobacillus as oral vaccine.

[The surface display of porcine parvovirus VP2 protein in Lactobacillus casei].

Lactobacillus casei 393 was selected as a bacterial carrier for the expression of Porcine Parvovirus (PPV) protective antigen VP2 protein. The gene encoding PPV VP2 protein was cloned into the Lactobacillus casei surface expression vector pPG, and then the constructed recombinant vector pPG-VP2 was electrotransformed into Lactobacillus casei 393 generating the recombinant system pPG-VP2/L. casei393 expressing PPV VP2 protein. The recombinant strain was induced by 2% Lactose in MRS and about 74kD protein was detected with SDS-PAGE. The result of Western-blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescent test showed that the expressed protein was secreted on the cell surface Lactobacillus casei.