Factors Involved in Removing the Non-Structural Protein of Foot-and-Mouth Disease Virus by Chloroform and Scale-Up Production of High-Purity Vaccine Antigens.

Foot-and-mouth disease (FMD) is an economically important and highly infectious viral disease, predominantly controlled by vaccination. The removal of non-structural proteins (NSPs) is very important in the process of FMD vaccine production, because vaccinated and naturally infected animals can be distinguished by the presence of NSP antibodies in the FMD serological surveillance. A previous study reported that 3AB protein, a representative of NSPs, was removed by chloroform treatment. Therefore, in this study, the causes of 3AB removal and factors affecting the effect of chloroform were investigated. As a result, the effectiveness of chloroform differed depending on the virus production medium and was eliminated by detergents. In addition, it was found that 3AB protein removal by chloroform is due to the transmembrane domain of the N-terminal region (59-76 amino acid domain). Further, industrial applicability was verified by applying the chloroform treatment process to scale-up FMD vaccine antigen production. A novel downstream process using ultrafiltration instead of polyethylene glycol precipitation for high-purity FMD vaccine antigen production was established. This result will contribute toward simplifying the conventional process of manufacturing FMD vaccine antigens and ultimately reducing the time and cost of vaccine production.

Systematic review and network meta-analysis to compare vaccine effectiveness against porcine edema disease caused by Shiga toxin-producing Escherichia coli.

The comprehensive effect size of several commercial vaccines and vaccine candidates against edema disease (ED) has not been evaluated to date. To integrate the effectiveness of ED vaccines reported so far and to compare and evaluate the posterior-effect estimates of each vaccine type with network models, we identified eligible studies (n = 12) from the electronic databases using specified search strings. Data for dichotomous outcomes (i.e., mortality and clinical symptoms) and continuous outcomes (i.e., fecal shedding and average daily gain) were extracted and analyzed. Conventional meta-analysis shows that, compared with that in non-vaccinated pigs, vaccinated animals are likely to show reduced mortality (OR = 0.07) and clinical signs of ED (OR = 0.11), and increased productivity (SMD = 0.73). Although reduced fecal shedding (SMD = - 1.29) was observed in vaccinated pigs, this could not be fully determined on insufficient grounds. In contrast to mortality and clinical symptoms, fecal shedding (I2 = 88%) and average daily gain (I2 = 85%) showed immense heterogeneity, which was attributed to the small sample size and vaccination route, respectively. According to the Bayesian network meta-analysis, the plasmid-based DNA vaccine demonstrated a better effect for all outcomes compared to other types of vaccines. However, these findings should be carefully interpreted with consideration to potential mediators, insufficient data, and inconsistent network models.

Comparison of the pathogenicity of porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 in pregnant sows.

To date, few studies related to the evaluation of the pathogenicity of different PRRSV isolates using a reproductive model have been undertaken, and the main focus has remained on respiratory models using young pigs. This study aimed to evaluate the pathogenicity of two PRRSV-1 isolates (D40 and CBNU0495) and two PRRSV-2 isolates (K07-2273 and K08-1054) in a reproductive model. Pregnant sows were experimentally infected with PRRSV at gestational day 93 or used as an uninfected negative control. Sera were collected at 0, 3, 7, 14, and 19 days post-challenge (dpc) for virological and serological assays. At 19 dpc, all sows were euthanized, and their fetuses were recovered by performing cesarean section and immediately euthanized for sample collection. Here, compared to the other isolates, the CBNU0495 isolate replicated most efficiently in the pregnant sows, and K07-2273 produced the highest rate of reproductive failure even though it did not replicate as efficiently as the other isolates in sows and fetuses, indicating that vertical transmission and reproductive failure due to PRRSV infection do not have any significant correlation with the viral loads in samples from sows and fetuses. Similarly, the viral loads and the histopathological lesions did not show any correlation with each other, as the PRRSV-2-infected groups displayed more prominent and frequent histopathological lesions with lower viral loads than the PRRSV-1-infected groups. However, viral loads in the myometrium/endometrium might be related to the spreading of PRRSV in the fetuses, which affected the birth weight of live fetuses. This study contributes to a better understanding of the pathogenicity of the most prevalent Korean PRRSVs in a reproductive model.

Protective immunity induced by concurrent intradermal injection of porcine circovirus type 2 and Mycoplasma hyopneumoniae inactivated vaccines in pigs.

Vaccines against porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (Mhp) are routinely used by intramuscular injection. However, since intramuscular vaccination causes stress and increases the risk of cross-contamination among pigs, research on intradermal vaccination is currently being actively conducted. This study was designed to evaluate the efficacy of intradermally administered inactivated vaccines against PCV2 and Mhp in pigs. Three-week-old specific pathogen-free pigs were divided into three groups (5 pigs per group). Pigs in the two groups were intradermally vaccinated with the PCV2 or Mhp vaccine using a needle-free injector. Pigs in the third group were kept as nonvaccinated controls. At 21 days post-vaccination, pigs in one of these vaccinated groups and the nonvaccinated group were intranasally challenged with PCV2b and Mhp, while the other vaccinated group pigs were maintained as vaccine controls. Vaccine efficacy was evaluated by observing weight gain, pathogen load, pathological changes, and humoral or cellular immune responses. As a result, vaccinated pigs revealed significantly higher body weight gain, with lower clinical scores. Vaccinated pigs also showed higher antibody responses but lower PCV2b or Mhp loads in sera, nasal swabs, or lungs than nonvaccinated pigs. Intriguingly, vaccinated pigs upregulated cytotoxic T cells (CTLs), helper T type 1 cells (Th1 cells), and helper T type 17 cells (Th17 cells) after immunization and showed significantly higher levels of CTLs, Th1 and Th17 cells at 14 days post-challenge than nonvaccinated and challenged pigs. This study demonstrated that protective immune responses against PCV2 and Mhp could be efficiently induced in pigs using a relatively small volume of intradermal vaccines, probably due to effective antigen delivery to antigen-presenting cells in the dermis.

Scale-Up Production of Type O and A Foot-and-Mouth Disease Bivalent Vaccine and Its Protective Efficacy in Pigs.

South Korea has experienced FMD outbreaks almost every year since 2014. Therefore, a novel local vaccine that can cover various topotypes of viruses is required. Two virus strains, O/Boeun/SKR/2017 and A/Yeoncheon/SKR/2017, were cultured up to the pilot scale based on the optimized conditions set up on the flask scale. FMDV particles (146S) of 2 µg/mL or more were obtained from the virus culture supernatant using a 100 L bioreactor. The viruses were fully inactivated using binary ethylenimine within 16 h through two inactivation cycles and mixed with an adjuvant into a bivalent vaccine (types O and A) consisting of 15 µg viruses per strain. The experimental bivalent vaccine showed a broad spectrum of high neutralizing antibody titers against heterologous viruses, including type O Cathay strain and type A Asia topotypes, except for GVII. The 50% protective dose was determined as 12.5 for O/Boeun/SKR/2017 and 15.6 for A/Yeoncheon/SKR/2017. Collectively, we expect that the bivalent vaccine could protect against FMDV types O and A circulating in South Korea and neighboring countries. To our knowledge, this is the first report demonstrating that the vaccine strains could be successfully scaled-up to a 100 L bioreactor, with the determination of its protective efficacy in pigs.

Foot-and-Mouth Disease Virus Evades Innate Immune Response by 3C-Targeting of MDA5.

Foot-and-mouth disease (FMD) is a highly contagious disease caused by FMD virus (FMDV) in cloven-hoofed animals. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are representative receptors in the cytoplasm for the detection of viral RNA and trigger antiviral responses, leading to the production of type I interferon. Although MDA5 is a crucial receptor for sensing picornavirus RNA, the interplay between MDA5 and FMDV is relatively unknown compared to the interplay between RIG-I and FMDV. Here, we observed that the FMDV infection inhibits MDA5 protein expression. Of the non-structural proteins, the Lb and 3C proteinases (Lbpro and 3Cpro) were identified to be primarily responsible for this inhibition. However, the inhibition by 3Cpro was independent of proteasome, lysosome and caspase-dependent pathway and was by 3C protease activity. A direct interaction between 3Cpro and MDA5 protein was observed. In conclusion, this is the first report that 3Cpro inhibits MDA5 protein expression as a mechanism to evade the innate immune response during FMDV infection. These results elucidate the pathogenesis of FMDV and provide fundamental insights for the development of a novel vaccine or therapeutic agent.

Evaluation of a multiplex PCR method for the detection of porcine parvovirus types 1 through 7 using various field samples.

Porcine parvoviruses (PPVs) are small, nonenveloped DNA viruses that are widespread in the global pig population. PPV type 1 (PPV1) is a major causative agent of reproductive failure and has been recognized since the 1960s. In recent decades, novel PPVs have been identified and designated as PPVs 2 through 7 (PPV2~PPV7). Although the epidemiological impacts of these newly recognized parvoviruses on pigs are largely unknown, continuous surveillance of these PPVs is needed. The aim of this study was to develop an improved and efficient detection tool for these PPVs and to assess the developed method with field samples. Using 7 sets of newly designed primers, a multiplex polymerase chain reaction (mPCR) protocol was developed for the simultaneous detection of the seven genotypes of PPV (PPV1~PPV7). The sensitivity of the mPCR assay was analyzed, and the detection limit was determined to be 3×103 viral copies. The assay was highly specific in detecting one or more of the viruses in various combinations in specimens. The mPCR method was evaluated with 80 serum samples, 40 lung or lymph node samples and 40 intestine or fecal samples. When applied to these samples, the mPCR method could detect the 7 viruses simultaneously, providing rapid results regarding infection and coinfection status. In conclusion, the developed mPCR assay can be utilized as an effective and accurate diagnostic tool for rapid differential detection and epidemiological surveillance of various PPVs in numerous types of field samples.

Efficient Removal of Non-Structural Protein Using Chloroform for Foot-and-Mouth Disease Vaccine Production.

To differentiate foot-and-mouth disease (FMD)-infected animals from vaccinated livestock, non-structural proteins (NSPs) must be removed during the FMD vaccine manufacturing process. Currently, NSPs cannot be selectively removed from FMD virus (FMDV) culture supernatant. Therefore, polyethylene glycol (PEG) is utilized to partially separate FMDV from NSPs. However, some NSPs remain in the FMD vaccine, which after repeated immunization, may elicit NSP antibodies in some livestock. To address this drawback, chloroform at a concentration of more than 2% (v/v) was found to remove NSP efficiently without damaging the FMDV particles. Contrary to the PEG-treated vaccine that showed positive NSP antibody responses after the third immunization in goats, the chloroform-treated vaccine did not induce NSP antibodies. In addition to this enhanced vaccine purity, this new method using chloroform could maximize antigen recovery and the vaccine production time could be shortened by two days due to omission of the PEG processing phase. To our knowledge, this is the first report to remove NSPs from FMDV culture supernatant by chemical addition. This novel method could revolutionize the conventional processes of FMD vaccine production.

Evaluation of local and systemic immune responses in pigs experimentally challenged with porcine reproductive and respiratory syndrome virus.

The host-associated defence system responsible for the clearance of porcine reproductive and respiratory syndrome virus (PRRSV) from infected pigs is currently poorly understood. To better understand the dynamics of host-pathogen interactions, seventy-five of 100 pigs infected with PRRSV-JA142 and 25 control pigs were euthanized at 3, 10, 21, 28 and 35 days post-challenge (dpc). Blood, lung, bronchoalveolar lavage (BAL) and bronchial lymph node (BLN) samples were collected to evaluate the cellular immune responses. The humoral responses were evaluated by measuring the levels of anti-PRRSV IgG and serum virus-neutralizing (SVN) antibodies. Consequently, the highest viral loads in the sera and lungs of the infected pigs were detected between 3 and 10 dpc, and these resulted in moderate to mild interstitial pneumonia, which resolved accompanied by the clearance of most of the virus by 28 dpc. At peak viremia, the frequencies of alveolar macrophages in infected pigs were significantly decreased, whereas the monocyte-derived DC/macrophage and conventional DC frequencies were increased, and these effects coincided with the early induction of local T-cell responses and the presence of proinflammatory cytokines/chemokines in the lungs, BAL, and BLN as early as 10 dpc. Conversely, the systemic T-cell responses measured in the peripheral blood mononuclear cells were delayed and significantly induced only after the peak viremic stage between 3 and 10 dpc. Taken together, our results suggest that activation of immune responses in the lung could be the key elements for restraining PRRSV through the early induction of T-cell responses at the sites of virus replication.

Effect of polymorphisms in porcine guanylate-binding proteins on host resistance to PRRSV infection in experimentally challenged pigs.

Guanylate-binding proteins (GBP1 and GBP5) are known to be important for host resistance against porcine reproductive and respiratory syndrome virus (PRRSV) infection. In this study, the effects of polymorphisms in GBP1 (GBP1E2 and WUR) and GBP5 on host immune responses against PRRSV were investigated to elucidate the mechanisms governing increased resistance to this disease. Seventy-one pigs [pre-genotyped based on three SNP markers (GBP1E2, WUR, and GBP5)] were assigned to homozygous (n = 36) and heterozygous (n = 35) groups and challenged with the JA142 PRRSV strain. Another group of nineteen pigs was kept separately as a negative control group. Serum and peripheral blood mononuclear cells (PBMCs) were collected at 0, 3, 7, 14, 21 and 28 days post-challenge (dpc). Viremia and weight gain were measured in all pigs at each time point, and a flow cytometry analysis of PBMCs was performed to evaluate T cell activation. In addition, 15 pigs (5 pigs per homozygous, heterozygous and negative groups) were sacrificed at 3, 14 and 28 dpc, and the local T cell responses were evaluated in the lungs, bronchoalveolar lavage cells (BALc), lymph nodes and tonsils. The heterozygous pigs showed lower viral loads in the serum and lungs and higher weight gains than the homozygous pigs based on the area under the curve calculation. Consistently, compared with the homozygous pigs, the heterozygous pigs exhibited significantly higher levels of IFN-α in the serum, proliferation of various T cells (γδT, Th1, and Th17) in PBMCs and tissues, and cytotoxic T cells in the lungs and BALc. These results indicate that the higher resistance in the pigs heterozygous for the GBP1E2, WUR and GBP5 markers could be mediated by increased antiviral cytokine (IFN-α) production and T cell activation.