Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice.
ABSTRACT: Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-?GP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.
Project description:Porcine reproductive and respiratory syndrome (PRRS) is characterized by a delayed and defective adaptive immune response. The viral nonstructural protein 1 (NSP1) of the PRRS virus (PRRSV) is able to suppress the type I interferon (IFN) response in vitro. In this study, recombinant adenoviruses (rAds) expressing NSP1 (rAd-NSP1), glycoprotein 5 (GP5) (rAd-GP5), and the NSP1-GP5 fusion protein (rAd-NSP1-GP5) were constructed, and the effect of NSP1 on immune responses was investigated in pigs. Pigs inoculated with rAd-NSP1 or rAd-NSP1-GP5 had significantly lower levels of IFN-? and higher levels of the immunosuppressive cytokine IL-10 than pigs inoculated with rAd-GP5, wild-type adenovirus, or cell culture medium alone. The antibody response to vaccination against classic swine fever virus (CSFV) was significantly decreased by inoculation of NSP1 7 d after CSFV vaccination in pigs. Thus, NSP1-mediated immune suppression may play an important role in PRRSV pathogenesis.
Project description:Passive administration of porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing antibodies (NAbs) can effectively protect pigs against PRRSV infection. However, after PRRSV infection, pigs typically develop a weak and deferred NAb response. One major reason for such a meager NAb response is the phenomenon of glycan shielding involving GP5, a major glycoprotein carrying one major neutralizing epitope. We describe here a type II PRRSV field isolate (PRRSV-01) that is highly susceptible to neutralization and induces an atypically rapid, robust NAb response in vivo. Sequence analysis shows that PRRSV-01 lacks two N-glycosylation sites, normally present in wild-type (wt) PRRSV strains, in two of its envelope glycoproteins, one in GP3 (position 131) and the other in GP5 (position 51). To determine the influence of these missing N-glycosylation sites on the distinct neutralization phenotype of PRRSV-01, a chimeric virus (FL01) was generated by replacing the structural genes of type II PRRSV strain FL12 cDNA infectious clone with those from PRRSV-01. N-glycosylation sites were reintroduced into GP3 and GP5 of FL01, separately or in combination, by site-directed mutagenesis. Reintroduction of the N-glycosylation site in either GP3 or GP5 allowed recovery of in vivo and in vitro glycan shielding capacity, with an additive effect when these sites were reintroduced into both glycoproteins simultaneously. Although the loss of these glycosylation sites has seemingly occurred naturally (presumably by passage through cell cultures), PRRSV-01 virus quickly regains these glycosylation sites through replication in vivo, suggesting that a strong selective pressure is exerted at these sites. Collectively, our data demonstrate the involvement of an N-glycan moiety located in GP3 in glycan shield interference.
Project description:After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.
Project description:Classical swine fever (CSF) caused by the CSF virus (CSFV) is one of the most important swine diseases, resulting in huge economic losses to the pig industry worldwide. Systematic vaccination is one of the most effective strategies for the prevention and control of this disease. Two main CSFV vaccines, the modified live vaccine (MLV) and the subunit E2 vaccine, are recommended. In Taiwan, CSF cases have not been reported since 2006, although systemic vaccination has been practiced for 70 years. Here, we examined the sero-dynamics of the piglets born from sows that received either the CSFV MLV or the E2 vaccine and investigated in the field the correlation between the porcine reproductive and respiratory syndrome virus (PRRSV) loads and levels of CSFV antibody. A total of 1398 serum samples from 42 PRRSV-positive farms were evaluated to determine the PRRSV loads by real-time PCR and to detect CSFV antibody levels by commercial ELISA. Upon comparing the two sow vaccination protocols (CSFV MLV vaccination at 4 weeks post-farrowing versus E2 vaccination at 4-5 weeks pre-farrowing), the lowest levels of CSFV antibody were found in piglets at 5-8 and 9-12 weeks of age for the MLV and E2 groups, respectively. Meanwhile, the appropriate time window for CSFV vaccination of offspring was at 5-8 and 9-12 weeks of age in the MLV and E2 groups, respectively. There was a very highly significant negative correlation between the PRRSV load and the level of CSFV antibody in the CSFV MLV vaccination group (P < 0.0001). The PRRSV detection rate in the pigs from the MLV group (27.78%) was significantly higher than that in pigs from the E2 group (21.32%) (P = 0.011). In addition, there was a significant difference (P = 0.019) in the PRRSV detection rate at 5-8 weeks of age between the MLV (42.15%) and E2 groups (29.79%). Our findings indicate that the vaccination of CSFV MLV in piglets during the PRRSV susceptibility period at 5-8 weeks of age may be overloading the piglet's immune system and should be a critical concern for industrial pork production in the field.
Project description:BACKGROUND:Outbreaks of Classical swine fever virus (CSFV) cause significant economic losses in the swine industry. Vaccination is the major method to prevent and control the disease. As live attenuated vaccines fail to elicit differentiable immunity between infected and vaccinated animals, subunit vaccine was considered as an alternative candidate to prevent and eradicate CSFV. Subunit vaccines present advantages in DIVA immunogenicity and safety. The technology was limited due to the low yield and the high cost with multiple and large doses. The native E2 signal peptide has not been well defined before. Here, the aim of this study is to develop a cost-effective and efficacious E2 vaccine candidate against CSFV with signal peptide and E2 sequence selection. RESULTS:A novel CSFV E2 sequence (E2ZJ) was identified from an epidemic strain of Zhejiang for outstanding secretion in baculovirus and enhanced immunogenicity. E2 secretion induced with the selected signal peptide, SPZJ (SP23), increase at least 50% as compared to any other signal peptides tested. Besides, unique antigenic features were identified in E2ZJ. As indicated with immunized sera in IFA against CSFV infection, E2ZJ elicited CSFV antibodies at the earlier stage than other E2 types tested in mice. Moreover, higher level of neutralizing and CSFV antibodies against CSFV with E2ZJ was detected than other E2s with the same dosage at 28 dpi. Further, E2ZJ successfully elicited neutralizing immunity in piglets. A single dose of 5??g of E2ZJ was sufficient to induce protective antibodies against CSFV in piglets and provided 100% protection against lethal virus challenge. CONCLUSIONS:Our studies provide evidence that E2ZJ guided by a novel E2 signal peptide (SPZJ) was efficiently secreted and presented significantly improved immunogenicity than conventional E2 vaccines. Moreover, a single dose of 5??g E2ZJ is efficacious against CSFV in piglets.
Project description:E2 is one of the envelope glycoproteins of pestiviruses, including classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV). E2 is involved in several critical functions, including virus entry into target cells, induction of a protective immune response and virulence in swine. However, there is no information regarding any host binding partners for the E2 proteins. Here, we utilized the yeast two-hybrid system and identified fifty-seven host proteins as positive binding partners which bound E2 from both CSFV and BVDV with the exception of two proteins that were found to be positive for binding only to CSFV E2. Alanine scanning of CSFV E2 demonstrated that the binding sites for these cellular proteins on E2 are likely non-linear binding sites. The possible roles of the identified host proteins are discussed as the results presented here will be important for future studies to elucidate mechanisms of host protein-virus interactions during pestivirus infection. However, due to the limitations of the yeast two hybrid system, the proteins identified is not exhaustive and each interaction identified needs to be confirmed by independent experimental approaches in the context of virus-infected cells before any definitive conclusion can be drawn on relevance for the virus life cycle.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by its genetic variation and limited cross protection among heterologous strains. Even though several viral structural proteins have been regarded as inducers of neutralizing antibodies (NAs) against PRRSV, the mechanism underlying limited cross-neutralization among heterologous strains is still controversial. In the present study, examinations of NA cross reaction between a highly pathogenic PRRSV (HP-PRRSV) strain, JXwn06, and a low pathogenic PRRSV (LP-PRRSV) strain, HB-1/3.9, were conducted with viral neutralization assays in MARC-145 cells. None of the JXwn06-hyperimmuned pigs' sera could neutralize HB-1/3.9 in vitro and vice versa. To address the genetic variation between these two viruses that are associated with limited cross-neutralization, chimeric viruses with coding regions swapped between these two strains were constructed. Viral neutralization assays indicated that variations in nonstructural protein 2 (nsp2) and structural proteins together contribute to weak cross-neutralization activity between JXwn06 and HB-1/3.9. Furthermore, we substituted the nsp2-, glycoprotein2 (GP2)-, GP3-, and GP4-coding regions together, or nsp2-, GP5-, and membrane (M) protein-coding regions simultaneously between these two viruses to construct chimeric viruses to test cross-neutralization reactivity with hyperimmunized sera induced by their parental viruses. The results indicated that the swapped nsp2 and GP5-M viruses increased the neutralization reactivity with the donor strain antisera in MARC-145 cells. Taken together, these results show that variations in nsp2 and GP5-M correlate with the limited neutralization reactivity between the heterologous strains HP-PRRSV JXwn06 and LP-PRRSV HB-1/3.9.
Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) is the major pathogen in the pig industry. Variability of the antigens and persistence are the biggest challenges for successful control and elimination of the disease. GP5, the major glycoprotein of PRRSV, is considered an important target of neutralizing antibodies, which however appear only late in infection. This was attributed to the presence of a "decoy epitope" located near a hypervariable region of GP5. This region also harbors the predicted signal peptide cleavage sites and (dependent on the virus strain) a variable number of potential N-glycosylation sites. Molecular processing of GP5 has not been addressed experimentally so far: whether and where the signal peptide is cleaved and (as a consequence) whether the "decoy epitope" is present in virus particles. We show that the signal peptide of GP5 from the American type 2 reference strain VR-2332 is cleaved, both during in vitro translation in the presence of microsomes and in transfected cells. This was found to be independent of neighboring glycosylation sites and occurred in a variety of porcine cells for GP5 sequences derived from various type 2 strains. The exact signal peptide cleavage site was elucidated by mass spectrometry of virus-derived and recombinant GP5. The results revealed that the signal peptide of GP5 is cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus particles, some of which still contain the "decoy epitope" sequence. Heterogeneity was also observed for the use of glycosylation sites in the hypervariable region. Lastly, GP5 mutants were engineered where one of the signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This indicates that the overwhelming majority of all GP5 molecules does not contain the "decoy epitope".
Project description:The porcine reproductive and respiratory syndrome virus (PRRSV) has three major structural proteins which designated as GP5, M, and N. Protein GP5 and M have been considered very important to arouse the humoral and cellular immune responses against PRRSV infection and proposed to be the excellent candidate proteins in the design of PRRS bioengineering vaccine. There were some attempts on expressing GP5 or M in DNA vaccine and adenovirus to arouse humoral and cellular immune responses, but few papers have been reported on that the immune response can be difference because of the expression patterns of GP5 and M proteins in the recombinant virus. In this article, four recombinant viruses that expressed GP5 and M proteins of PRRSV in the modified vaccinia virus ankara (MVA) with different expression patterns were made. In these recombinant virus (rMVAs), GP5 and M proteins were expressed in MVA in the same virus but under the control of two promoters (rMVA-GP5/M), or as a fusion protein under one promoter (rMVA-GP5-M), or separately (rMVA-GP5 and rMVA-M). The humoral and cellular immune responses for the four recombinant viruses were evaluated with mouse model. Every mouse was inoculated with 5 x 10(5) TCID50 of the different rMVAs and boosted 3 weeks later. Neutralizing antibody titers for each group were detected with virus neutralization test assay weekly after the primary inoculation for 13 weeks to evaluate the humoral immune response. The production of gamma interferon (IFN-gamma), interleukin-2 (IL-2), and interleukin-4 (IL-4) was detected in splenocytes of rMVA-inoculated mice at 30, 60, and 90 days post inoculation to evaluate the cellular immune response. Results showed that rMVA-GP5 and rMVA-M cannot induce obvious humoral and cellular immune responses; rMVA-GP5-M inoculated group developed better immune responses than rMVA-GP5 and rMVA-M inoculated groups; however, mice inoculated with rMVA-GP5/M maintained the strongest cellular response against PRRS and consistently enhanced the anti-PRRSV humoral responses. The strategy of co-expressing PRRSV GP5 and M protein in MVA under the control of different promoters might be an attractive method for future PRRSV vaccine design.
Project description:Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34?×?10-7 and 1.35?×?10-8?M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.