The Study Of Intracellular Expression Of An Anti-idiotypic Antibody Single-chain Variable Fragment Reduces Porcine Reproductive And Respiratory Syndrome Virus Infection In MARC-145 Cells

Porcine reproductive and respiratory syndrome virus(PRRSV) is an enveloped positive single-stranded RNA virus and belongs to the family of Arteriviridae, which also includes lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus. It is the causative agent of Porcine reproductive and respiratory syndrome(PRRS) and characterized by persistent infection, high mortality and respiratory distress in young swine and widespread reproductive failure in pregnant sows, including mummified, stillborn and aborted fetuses. Because the high frequency mutation and immunosuppression could be observed in PRRSV, neither live-attenuated nor inactivated PRRSV vaccines can provide sustainable disease control and this disease has become one of the most economically important viral diseases affecting the swine industry worldwide. Our previous studies have demonstrated that PRRSV infection can produce the auto-anti-idiotypic antibodies specific to the idiotypic antibodies against PRRSV GP5, which plays an important role in the host immune responses to PRRSV infection. Using the sequential immunization method, we generated a monoclonal anti-idiotypic antibody(Mab2-5G2) which mimicked the GP5 antigen. This antibody retains the characteristics of auto-anti-idiotypic antibodies and reduces the efficacy of attenuated vaccines against HP-PRRSV infection. These results suggested that the role of anti-idiotypic antibody is complicated and warranted more studies. Because the production of auto-Ab2 from swine sera is limited and the animal experiments are expensive and labor-intensive, the more feasible approach would be to express a single-chain variable fragment antibody in susceptible cell lines to overcome these limitations and explore its immunological properties against PRRSV infection.In the present study, a single-chain variable antibody fragments(scFv) from Mab2-5G2 was expressed in MARC-145 cells using the lentiviral vector system and explored whether this scFv could inhibit PRRSV replication through blocking virus attachment or other molecular mechanism. The results of this study were as follows:1. The cDNAs of the variable light chain and heavy chain regions of Mab2-5G2 monoclonal IgG transcripts were synthesized by reverse transcriptase-polymerase chain reaction(RT-PCR). Then the scFv was constructed with the insertion of(GGGGS)3 as a flexible linker between the VH and VL by overlap PCR. The 5G2 scFv EGFP genes were constructed with GPGP linker and cloned the fusion gene into the lentiviral vector. 1B5 scFv, generated from the monoclonal antibody to avian hepatitis E virus capsid antigen, was used as control. The integrity of 5G2 sc Fv or 1B5 scFv as well as linker and EGPF sequences were verified by DNA sequencing and the plasmid named pTRIP-CMV-5G2scFv-GPGP-EGFP-IRES-Puro or pTRIP-CMV-1B5scFv-GPGP-EGFP-IRES-Puro, respectively.2. Lentivirus was produced by transfection of 293 T packaging cells with recombinant lentivirus vector plasmids. The cell culture supernatants containing lentivirus were collected and used to infect MARC-145 cells. The GFP-positive and puromycin-resistant colonies were formed. MARC-145 cells transduced with the 1B5 scFv were utilized as the control. In order to confirm the expression of 5G2 sc Fv in MARC-145 cells, IFA and Western blot were employed. These results demonstrated the successful construction of MARC-145 cells expressing 5G2 scFv or 1B5 scFv and were named MARC-5G2 scFv or MARC-1B5 scFv, respectively.3. In order to check whether intracellular expression of 5G2 scFv could reduce PRRSV replication in MARC-145, Western blot and TCID50 assay were used to detect PRRSV N protein and viral particle production after SD16 and VR-2332 had been added to MARC-145, MARC-1B5 scFv and MARC-5G2 scFv. The quantity of N protein was reduced markedly in the lane of MARC-5G2 scFv cells while MARC-145 and MARC-1B5 scFv showed increases in quantity of N protein. Consistent with this finding, the virus titers in the supernatants of MARC-5G2 scFv were reduced significantly compared to the two control groups. Compared the titer of SD16 in these three cells at different time points, the value of TCID50 in the supernatants of MARC-5G2 scFv was significantly lower than that from the control groups, especially at 12 h post-infection(P<0.01).4. To examine whether virus attachment blocking or production of type I IFN would be involved in the inhibition of PRRSV infection in MARC-5G2 scFv cells, PRRSV attachment to cells and levels of IFN-α and IFN-β in MARC-5G2 scFv cells as well as the other two control groups were examined. The result of PRRSV attachment suggested that 5G2 scFv expression in MARC-145 cells may not affect PRRSV attachment to the cells. The result of semi-quantitative RT-PCR and ELISA assay showed the mRNA and protein levels of IFN-α in MARC-5G2 scFv cells increased significantly than that in all three cell lines without virus treatment or MARC-145 and MARC-1B5 scFv cells infected with PRRSV at 48 h post-infection(P<0.05). However, the mRNA or protein levels of IFN-β were similar in all three cell lines either infected or no infected with PRRSV(P>0.05).In conclusion, 5G2 scFv was successfully expressed in MARC-145 cells using the lentiviral vector system. The stable cell line expressing 5G2 scFv in cytoplasm showed obvious inhibition of PRRSV replication. The appearance of IFN-α may correlate with the resistance of MARC-5G2 scFv cells to PRRSV replication. The present study extended the use of intracellular expression of scFv and suggested that the scFv of anti-idiotypic antibody Mab2-5G2 is quite effective in inhibiting PRRSV replication in MARC-145 and should be useful in the future development of a novel antiviral therapy.