Intravenous injection of allogenic canine mesenchymal stem cells in 40 client-owned dogs: a safety assessment in veterinary clinical trials.

BACKGROUND: The aim of this study was to evaluate the adverse effects of allogeneic mesenchymal stem cells (MSCs) transplanted via intravenous infusion in dogs and examine their safety. We performed a retrospective analysis of various clinical assessments, including physical examination, blood tests, and radiographs, and monitored the formation of neoplasms during a 6-month follow-up period in 40 client-owned dogs that received intravenous infusion of adipose tissue-derived MSCs (AT-MSCs) for the treatment of various underlying diseases between 2012 and 2018. RESULTS: No significant adverse effects of MSC therapy were detected by clinical assessment, blood tests, or radiographic examination in the 6-month follow-up period after the first MSC treatment. Additionally no new neoplasms were observed during this period. CONCLUSIONS: To our knowledge, this study is the first to evaluate the safety aspects (≥ 6 months) associated with intravenous allogeneic AT-MSC infusion. These results suggest that allogenic AT-MSC infusion could be a useful and relatively safe therapeutic approach in canines.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

Background: It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Methods: Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. Results: E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. Conclusion: E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Development and evaluation of two rapid lateral flow assays for on-site detection of African swine fever virus.

Introduction: African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. In the absence of a widely available and definitively proven vaccine, rapid and early detection is critical for ASF control. The quick and user-friendly lateral flow assay (LFA) can easily be performed by following simple instructions and is ideal for on-site use. This study describes the development and validation of two LFAs for the rapid detection of ASF virus (ASFV) in pig serum. Methods: The highly immunogenic antigens (p30 and p72) of ASFV Georgia 2007/1 (genotype II) were expressed in plants (Nicotiana benthamiana) and were used to immunize BALB/c mice to generate specific monoclonal antibodies (mAbs) against the p30 and p72 proteins. mAbs with the strongest binding ability to each protein were used to develop p30_LFA and p72_LFA for detecting the respective ASFV antigens. The assays were first evaluated using a spike-in test by adding the purified p30 or p72 protein to a serum sample from a healthy donor pig. Further validation of the tests was carried out using serum samples derived from experimentally infected domestic pigs, field domestic pigs, and feral pigs, and the results were compared with those of ASFV real-time PCR. Results: p30_LFA and p72_LFA showed no cross-reaction with common swine viruses and delivered visual results in 15 min. When testing with serially diluted proteins in swine serum samples, analytical sensitivity reached 10 ng/test for p30_LFA and 20 ng/test for p72_LFA. Using real-time PCR as a reference, both assays demonstrated high sensitivity (84.21% for p30_LFA and 100% for p72_LFA) with experimentally ASFV-infected pig sera. Specificity was 100% for both LFAs using a panel of PBS-inoculated domestic pig sera. Excellent specificity was also shown for field domestic pig sera (100% for p30_LFA and 93% for p72_LFA) and feral pig sera (100% for both LFAs). Conclusion: The results obtained in this study suggest that p30_LFA and p72_LFA hold promise as rapid, sensitive, user-friendly, and field-deployable tools for ASF control, particularly in settings with limited laboratory resources.

Protection of SPF Chickens by H9N2 Y439 and G1 Lineage Vaccine against Homologous and Heterologous Viruses.

Prior to the identification of low pathogenic avian influenza H9N2 viruses belonging to the Y280 lineage in 2020, Y439 lineage viruses had been circulating in the Republic of Korea since 1996. Here, we developed a whole inactivated vaccine (vac564) by multiple passage of Y439 lineage viruses and then evaluated immunogenicity and protective efficacy in specific-pathogen-free chickens. We found that LBM564 could be produced at high yield in eggs (108.4EID50/0.1 mL; 1024 hemagglutinin units) and was immunogenic (8.0 ± 1.2 log2) in chickens. The vaccine showed 100% inhibition of virus in the cecal tonsil with no viral shedding detected in either oropharyngeal or cloacal swabs after challenge with homologous virus. However, it did not induce effective protection against challenge with heterologous virus. An imported commercial G1 lineage vaccine inhibited viral replication against Y280 and Y439 lineage viruses in major tissues, although viral shedding in oropharyngeal and cloacal swabs was observed up until 5 dpi after exposure to both challenge viruses. These results suggest that a single vaccination with vac564 could elicit immune responses, showing it to be capable of protecting chickens against the Y439 lineage virus. Thus, our results suggest the need to prepare suitable vaccines for use against newly emerging and re-emerging H9N2 viruses.

A Plant-Derived Maternal Vaccine against Porcine Epidemic Diarrhea Protects Piglets through Maternally Derived Immunity.

Newborn piglets are susceptible to a highly contagious enteritis caused by the porcine epidemic diarrhea virus (PEDV), associated with high levels of mortality worldwide. There is pressing need for a rapid, safe, and cost-effective vaccine to safeguard pigs from getting infected by PEDV. PEDV belongs to the coronavirus family and is characterized by high levels of mutability. The primary goal of a PEDV vaccine is to provide immunity to newborn piglets through vaccination of sows. Plant-based vaccines are becoming more popular because they have low manufacturing costs, are easily scalable, have high thermostability, and a long shelf life. This is in contrast to conventional vaccines which include inactivated, live, and/or recombinant types that can be expensive and have limited ability to respond to rapidly mutating viruses. The binding of the virus to host cell receptors is primarily facilitated by the N-terminal subunit of the viral spike protein (S1), which also contains several epitopes that are recognized by virus-neutralizing antibodies. As a result, we generated a recombinant S1 protein using a plant-based vaccine platform. We found that the recombinant protein was highly glycosylated, comparable to the native viral antigen. Vaccination of pregnant sows at four and two weeks before farrowing led to the development of humoral immunity specific to S1 in the suckling piglets. In addition, we noted significant viral neutralization titers in both vaccinated sows and piglets. When challenged with PEDV, piglets born from vaccinated sows displayed less severe clinical symptoms and significantly lower mortality rates compared to piglets born from non-vaccinated sows.

Immunization with porcine epidemic diarrhea virus harbouring Fc domain of IgG enhances antibody production in pigs.

Background: Outbreaks of porcine epidemic diarrhea virus (PEDV) infection have re-emerged and spread rapidly worldwide, resulting in significant economic losses. Vaccination is the best way to prevent PEDV infection in young piglets.Objective: To enhance the efficacy of an inactivated vaccine against PEDV, we evaluated the adjuvant properties of Fc domain of IgG.Methods: Fifteen crossbred gilts (180 ∼ 210 days old) were used. Five pigs in group 1 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 106 TCID50 of inactivated PEDV. Five pigs in group 2 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 106 TCID50 of inactivated PEDV-sFc. Five pigs in group 3 were not vaccinated and served as negative controls. Serum samples were collected at farrowing and subjected to ELISA, a serum neutralizing (SN) test, and a cytokine assay. Statistical analysis was performed by a two-tailed unpaired t-test.Results: Vero cells expressing swine IgG Fc on its surface was established. When PEDV was propagated in the cells expressing the swine Fc, PEDV virion incorporated the Fc. Immunization of pigs with inactivated PEDV harbouring Fc induced significantly higher antibody production against PEDV, comparing to the immunization with normal inactivated PEDV. In addition, we observed significantly increased IFN-γ levels in sera.Conclusion: Our results indicate that Fc molecule facilitate immune responses and PEDV harbouring Fc molecule could be a possible vaccine candidate. However, a challenge experiment would be needed to investigate the protective efficacy of PEDV harbouring Fc.