Search

Mucosal immunization has potential benefits over conventional parenteral immunization, eliciting immune defense in both mucosal and systemic tissue for protecting from pathogen invasion at mucosal surfaces. To provide a first line of protection at these entry ports, mucosal vaccines have been developed and hold a significant promise for reducing the burden of infectious diseases. However, until very recently, only limited mucosal vaccines are available. This review summarizes recent advances in selected aspects regarding mucosal vaccination, including appropriate administration routes, reasonable formulations, antigen-sampling and immune responses of mucosal immunity, and the strategies used to improve mucosal vaccine efficacy. Finally, the challenges of developing successful mucosal vaccines and the potential solutions are discussed., (Copyright © 2019. Published by Elsevier B.V.)

Classical swine fever (CSF) and pseudorabies (PR) are both major infectious diseases of pigs, causing enormous economic losses to the swine industry in many countries. A marker vaccine that enables differentiation of infected from vaccinated animals (DIVA) is highly desirable for control and eradication of these two diseases in endemic areas. Since late 2011, PR outbreaks have been frequently reported in many Bartha-K61-vaccinated pig farms in China. It has been demonstrated that a pseudorabies virus (PRV) variant with altered antigenicity and increased pathogenicity was responsible for the outbreaks. Previously, we showed that rPRVTJ-delgE/gI/TK, a gE/gI/TK-deleted PRV variant, was safe for susceptible animals and provided a complete protection against lethal PRV variant challenge, indicating that rPRVTJ-delgE/gI/TK can be used as an attractive vaccine vector. To develop a safe bivalent vaccine against CSF and PR, we generated a recombinant virus rPRVTJ-delgE/gI/TK-E2 expressing the E2 protein of classical swine fever virus (CSFV) based on rPRVTJ-delgE/gI/TK and evaluated its safety and immunogenicity in pigs. The results indicated that pigs (n=5) immunized with rPRVTJ-delgE/gI/TK-E2 of different doses did not exhibit clinical signs or viral shedding following immunization, the immunized pigs produced anti-PRV or anti-CSFV neutralizing antibodies and the pigs immunized with 10(6) or 10(5) TCID50 rPRVTJ-delgE/gI/TK-E2 were completely protected against the lethal challenge with either CSFV Shimen strain or variant PRV TJ strain. These findings suggest that rPRVTJ-delgE/gI/TK-E2 is a promising bivalent DIVA vaccine candidate against CSFV and PRV coinfections., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a devastating disease of swine worldwide. Although a mandatory vaccination with the modified live vaccine C-strain has been implemented in China for decades, CSF remains a serious threat to the swine industry. To facilitate the control and eradication of CSF in China, the E2-based marker vaccine rAdV-SFV-E2, an adenovirus-delivered, alphavirus replicon-vectored vaccine, has been developed. Accordingly, an accompanying discriminatory test that allows differentiating infected from vaccinated animals (DIVA) is required. Here, the enhanced expression of E(rns) protein of CSFV was achieved in the methyltropic yeast Pichia pastoris by codon-optimization of the E(rns) gene, and an indirect enzyme-linked immunosorbent assay (iELISA) based on the yeast-expressed E(rns) (yE(rns)) was developed and evaluated. The optimized iELISA was able to detect CSFV-specific antibodies in the serum samples from the CSFV-infected pigs as early as 6 days post-infection, and discriminate the CSFV-infected pigs from those vaccinated with rAdV-SFV-E2. The iELISA was evaluated using a panel of swine sera, and showed comparable sensitivity (94.6%) and specificity (97.1%), and the consistence rates with the virus neutralization test were 96.8% for CSFV-infected swine sera, 83.3% for C-strain-vaccinated swine sera, and 95.0% for field swine sera. In addition, the iELISA showed higher sensitivity (90.4%) compared with PrioCHECK CSFV E(rns) (59.6%). Taken together, the yE(rns)-based iELISA is specific and sensitive, representing a promising DIVA test for E2-based marker vaccines against CSF., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) is a highly contagious viral disease and may cause heavy economic loss to farmers. The rapid, simple and accurate diagnosis of the disease at the frontline, for example on the farms of concern is crucial for disease control. This study describes the development and evaluation of a new loop-mediated isothermal amplification (LAMP) assay coupled with lateral flow dipstick (LFD) for the detection of classical swine fever virus (CSFV). This RT-LAMP-LFD assay combines the efficient one-step isothermal amplification of CSF viral RNA and the simplicity of the LFD to read the results within two to five minutes. Seven genotypes (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 and 3.1), but not genotype 3.4, were successfully detected by the RT-LAMP-LFD assay, indicating that the method has a broad range of detection and can be applied in different geographical areas where CSFV strains belonging to these genotypes are present. The performance of this RT-LAMP-LFD assay was similar to that of the real-time RT-PCR. The analytical sensitivity was about 100copies per reaction when testing two genotypes (1.1 and 2.3). No cross-reactivity to non-CSFV pestiviruses was observed. This RT-LAMP-LFD assay can be a useful novel tool for the rapid, simple and economic diagnosis of classical swine fever in the field., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N(pro) gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious and often fatal swine disease that is responsible for significant losses to the swine industry worldwide. Previously, we demonstrated that pigs immunized with a recombinant adenovirus (rAdV-E2) expressing the E2 glycoprotein of CSFV were protected against virulent CSFV; however, a few pigs showed a short-term fever and occasional pathological changes. To enhance the efficacy of the vaccine, we constructed two recombinant adenoviruses, namely, rAdV-E2UL49, which encodes the CSFV E2 gene fused with the UL49 gene from pseudorabies virus (PRV), and rAdV-optiE2, which expresses the codon-optimized CSFV E2 gene. With these viruses, we performed a comparative immunogenicity trial in rabbits and pigs and compared these recombinant adenovirus vaccines (rAdV-E2UL49 and rAdV-optiE2) with the one containing the wild-type E2 gene (rAdV-E2). In terms of antibody titers, IFN-γ production, lymphocyte proliferation, viral loads and clinical protection from the disease, rAdV-E2UL49 was more immunogenic and protective against C-strain CSFV in rabbits and Shimen strain CSFV in pigs than rAdV-optiE2 and rAdV-E2. Data from this study could assist in making decisions for further development of recombinant adenoviruses as vaccine candidates against CSF., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the rapid and specific detection of HCLV vaccine strain against classical swine fever. Four primers were designed for amplification of NS5B gene region with Bst DNA polymerase at a constant temperature of 65°C. The products showed ladder-like pattern on 2% agarose gel, and can be visualised after addition of SYBR Green I dye. The detection limit of the assay was 5 copies of the HCLV genome per reaction. No cross-reaction with other porcine viruses including different wild-type CSFV strains and the bovine viral diarrhoea virus was observed. The agreement between the LAMP and TaqMan real-time RT-PCR assays was 94.4% for the detection of 72 batches of HCLV vaccine. The assay provides a rapid tool for the control of vaccine quality and can be an accompanying assay of the LAMP for wild-type CSFV described previously for differential diagnosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

This study describes evaluation of a real-time PCR assay based on primer-probe energy transfer (PriProET) technology for detection of classical swine fever virus (CSFV). The PriProET technology allows melting curve analysis following PCR amplification and thus provides a higher specificity. The assay was compared with a TaqMan assay by testing a total of 203 samples including 175 clinical specimens and 28 batches of Hog Cholera Lapinized Virus (HCLV) vaccine. The two assays gave the same results for 184 (91%) samples. Compared with the TaqMan assay, 19 additional samples were found to be positive for CSFV using the PriProET assay. In an RNA mixture of both wild type CSFV and C-strain vaccine, the melting curves displayed only one curve: either a wild type-like or a vaccine-like depending on the dominating RNA. The PriProET assay can be a routine molecular tool or a confirmative tool for diagnosis of classical swine fever (CSF), especially in the case of samples that yield an inconclusive result by the TaqMan assay., (Copyright 2010 Elsevier B.V. All rights reserved.)

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method applied and adapted to the detection of a number of pathogens. A LAMP assay was developed for the specific detection of wild-type classical swine fever virus (CSFV). Based on an alignment of genomic sequences of pestiviruses available in GenBank, four primers were selected targeting six positions in the NS5B gene region of the viral genome. The assay was performed in a simple water bath at a constant temperature of 62 degrees C, and after adding SYBR Green I, the results were visualised by the naked eye. This assay allowed easy applicability in a laboratory that is equipped simply, or even on site, close to the outbreaks and under field conditions. For confirmation, the results could also be visualised under UV light or by separation of the amplified products on 2% agarose gels. The detection limit of the assay was 2.5 medium tissue culture infectious dose (TCID(50)). The assay was validated with 116 clinical samples from vaccinated swineherds and 53 blood samples from experimental infections, and the results were comparable to a real-time RT-PCR assay. In summary, the LAMP assay provides a simple, rapid, and sensitive tool for the detection of wild-type CSFV under field conditions., (Copyright 2010 Elsevier B.V. All rights reserved.)

Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections.

Classical swine fever and porcine reproductive and respiratory syndrome are both notifiable diseases of the World Organization for Animal Health (OIE). The two diseases exhibit indistinguishable clinical symptoms and sometimes co-exist in swine herds. In this study, a duplex real-time RT-PCR for simultaneous detection of Classical swine fever virus (CSFV) and North American (NA) genotype Porcine reproductive and respiratory syndrome virus (PRRSV) based on two differently labeled TaqMan probes was developed and evaluated. The detection limit of the assay was 3.2 TCID(50) or 13 RNA copies for CSFV and 1.8 TCID(50) or 10 RNA copies for PRRSV, about 50 times more sensitive than conventional RT-PCRs. The duplex real-time RT-PCR was capable of specifically detecting different subgroups of wild-type CSFV and different strains of NA-genotype PRRSV, whereas a number of non-CSFV/PRRSV porcine viruses and bovine pestivirus were tested negative. Out of 155 field samples, 16 were tested positive for CSFV, 73 were positive for PRRSV, and 13 were co-infected with the two viruses. These results were 99.4% in agreement with those using conventional RT-PCRs. Therefore, the assay provides sensitive and simultaneous detection and differentiation of CSFV and PRRSV.

In a previous study, it has been shown that a Semliki Forest virus (SFV) replicon vectored DNA vaccine (pSFV1CS-E2) expressing the E2 glycoprotein of classical swine fever virus (CSFV) conferred full protection for pigs immunized three times with 600 microg of the vaccine. This study was designed to evaluate further the efficacy of the vaccine with lower dosage and fewer inoculations. Pigs were immunized twice with 100 microg of pSFV1CS-E2 (n=5) or control plasmid pSFV1CS (n=3), respectively, and challenged with virulent Shimen strain 6 weeks following the booster immunization. Pigs immunized with pSFV1CS-E2 developed high titers of specific neutralizing antibodies against CSFV after the booster, and the antibody titers increased rapidly upon challenge. The immunized animals showed no clinical symptoms except short-term fever and low-level viremia, whereas, the control pigs immunized with the control plasmid produced no detectable antibody prior to challenge, and showed obvious clinical signs following challenge, and two pigs died of illness. All control animals developed extended viremia as detected by nested RT-PCR and real-time RT-PCR. Severe pathologic lesions typical of CSFV infection were observed at necropsy. It is concluded that the alphavirus replicon-vectored DNA-based vaccine can be a potential marker vaccine against CSFV.

A multiplex nested RT-PCR (RT-nPCR) was developed for the detection and differentiation of classical swine fever virus (CSFV). A fragment of 447 or 343 bp was amplified from the genomic RNA of C-strain or virulent Shimen strain, respectively, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed samples of C-strain and Shimen. When detecting several wild-type isolates representative of different subgroups (1.1, 2.1, 2.2, and 2.3) circulating in Mainland China and samples from pigs experimentally infected with Shimen strain, the RT-nPCR resulted in an amplification pattern similar to Shimen. No amplification was achieved for uninfected cells, or cells infected with bovine viral diarrhea virus (BVDV), and other viruses of porcine origin. The RT-nPCR was able to detect as little as 0.04 pg of CSFV RNA. The restrictive fragment length polymorphism (RFLP) demonstrated unique patterns of wild-type viruses and C-strain. Among the 133 field samples, 42 were tested to contain wild-type viruses and 18 showing presence of C-strain. The RT-nPCR can be used to detect and differentiate pigs infected with wild-type CSFV from those vaccinated with C-strain vaccine, thus minimizing the risk of culling vaccinates during outbreaks.