Evaluation Of Protection Efficiency Of An Inactived Immune Complex Vaccine Based On A PRRSV-specific IgM

Porcine reproductive and respiratory syndrome（PRRS）is an infectious disease caused by Porcine reproductive and respiratory syndrome virus（PRRSV）characterized by reproductive disorders during pregnancy,the respiratory symptom in newborn piglets,which seriously threatens the sustainable and healthy development of the global pig industry.PRRS is mainly prevented and controlled through vaccine immunization,and both inactivated and modified live vaccines are used for clinical application.Immunization with an inactivated vaccine induces neutralizing antibodies,but has poor cellular immune response capacity.Meanwhile,immunization with a modified live vaccines has humoral and cellular immunity,limited protection against heterologous virus,and there is a potential risk of virulence reversion and recombination of vaccine strains with field strains.Therefore,it is necessary to develop a new PRRS vaccine that is safe,effective,and reliable to control PRRS as well as achieve epidemic decontamination.It has been reported that antigen-specific IgM as an adjuvant can enhance the immune effect of the vaccine.In our previous study,a hybridoma cell was obtained that stably secreted PRRSV-specific IgM monoclonal antibody,which was named Mab-PR5nf1.In vitro assays revealed Mab-PR5nf1 broadly recognized and neutralized heterologous PRRSV strains tested.In this study,an immune complex（IC）vaccine prepared by Mab-PR5nf1 and inactivated PRRSV in a certain proportion was tested and produced a strong cellular immune response after immunization in mice.The results of the post-immunization porcine challenge showed that the protection rate of IC vaccine was 100%against challenge of heterologous highly pathogenic PRRSV（HP-PRRSV）.It laid a basis for the development of a novel PRRS vaccine.The main studies are as follows.1.Preparation of Mab-PR5nf1 antibody immune complex vaccine and immunization experiments in mice.The Mab-PR5nf1 hybridoma cell was cultured in vitro.The cell culture supernatant was purified by Protein L affinity chromatography to obtain the highly purified Mab-PR5nf1.The Mab-PR5nf1 and inactivated purified PRRSV-SD16 viral protein were mixed in 5:1（W/W）and incubated for 2 h to form an immune complex（IC）.Subsequently,IC was emulsified with ISA 206 adjuvant in the ratio of 1:1（W/W）to prepare IgM-IC vaccine.A IgM specificly recognize Hepatitis E virus ORF3 protein（IgM-VP13）was included and prepared in the same way as the IgM isotype control.Meanwhile,inactivated vaccine was prepared by emulsifying inactivated purified PRRSV-SD16 with ISA206adjuvant in the ratio of 1:1（W/W）.The three vaccines,PBS,and inactivated PRRSV-SD16virus only were used to immunize in BALB/c mice at a dose of 100μL each,and secondary immunization was performed at an interval of 2 weeks.Fourteen days after the secondary immunization,blood and spleen were collected from mice for experiments.Meanwhile,CD8⁺T cells were isolated from spleen cells.ELISA results showed that PRRSV-specific antibodies were produced in all mice except the PBS group.After co-incubation of PRRSV-stimulated dendritic cells from unimmunized mice and CD8⁺T cells from vaccine-immunized mice,the number of IFN-γ-secreting CD8⁺T cells was detected by ELISpot.The results showed that the number of CD8⁺T cells secreting IFN-γin the IgM-IC vaccine group was 32,which was significantly higher than that in the inactivated vaccine group,indicating that the Mab-PR5nf1antibody immune complex can stimulate cellular immune response and enhance CTL response.2.Immunoprotective evaluation of Mab-PR5nf1 antibody immune complex vaccine on piglets.The Mab-PR5nf1 and inactivated purified PRRSV-VR2332 were mixed in ratio of10:1（W/W）and incubated for 2 h to form an immune complex（IC）.Subsequently,IC was emulsified with ISA 206 adjuvant in the ratio of 1:1（W/W）to prepare IgM-IC vaccine.Additionally,the inactivated purified PRRSV-VR2332 was emulsified with ISA 206 adjuvant in the ratio of 1:1（W/W）to prepare inactivated vaccine.These two vaccines and the commercial modified live vaccines（Inglvac MLV）,and PBS were immunized in 4-week-old piglets,and secondary immunization of the IgM-IC vaccine group and the inactivated vaccine group was performed at an interval of 2 weeks.On day 21 after the second immunization,a challenge experiment was performed with HP-PRRSV XJA1.The results showed that the protection ratio of IgM-IC vaccine group was 100%,which was higher than that of MLV group and inactivated vaccine group（both 80%）.The results of ELISA for PRRSV-specific antibody showed that the antibody titers of the IgM-IC vaccine and modified live vaccine groups were not significantly different on day 14 day after the first immunization,but were significantly higher than inactivated vaccine group.After 7 days of secondary immunization,the antibody levels of all groups were similar.Pathological autopsy showed that pigs in the unimmunized group exhibited severe hemorrhagic pneumonia with widened lung interstitium and massive inflammatory cells infiltration.Furthermore,pigs in the live attenuated and inactivated vaccine groups exhibited a small amount of inflammatory cell infiltration and hemorrhage in the lungs with slightly widened interstitial lung spaces.Moreover,the pigs in the IgM-IC vaccine group had the mildest lung lesions,with only partial widening of the interstitium was observed.The qPCR results of nasal swabs showed that PRRSV-RNA was detectable in all piglets in the inactivated vaccine and unimmunized groups at 3 dpi;PRRSV-RNA was detectable in all piglets in the IgM-IC vaccine and modified live vaccines groups at 7 dpi;all experimental groups were negative at 21 dpi,except for one piglet in the modified live vaccines group.The qPCR results of blood showed that the level of viremia in the IgM-IC vaccine group was similar to that in the modified live vaccines group and lower than that in the inactivated vaccine group at 3 dpi,and the viremia in the unimmunized groups was the highest.At 7 dpi,the level of viremia in the IgM-IC group was similar to that of the inactivated vaccine group and higher than that of the modified live vaccines group;at 14 dpi,there was a trend of reduction in viremia in the IgM-IC vaccine group compared with the inactivated vaccine group.At 21 dpi,the level of viremia in all piglets in the IgM-IC vaccine group was similar to inactivated vaccine group,while 2 of the surviving piglets in the modified live vaccines group were negative for viremia.In conclusion,IgM Mab-PR5nf1,as an immune adjuvant,can effectively enhance the immune effect of inactivated vaccine of PRRS,rapidly induce PRRSV-specific antibodies production,reduce the pathological damage of PRRSV to the lungs,and effectively reduce the time of viral shedding of pig nasal.This indicated that it was superior to inactivated vaccines in improving viremia,but slightly less effective than modified live vaccines.3.Immune response of swine to Mab-PR5nf1 immune complex vaccine.After immunization of piglets with IgM-IC vaccine,tests of humoral and cellular immune functions,detection of serum antibodies,virus levels and gene expression in porcine alveolar macrophages（PAMs）,and studies of the relationship between viremia and cellular and humoral immunity were performed.The results showed that before the challenge,the immune-induced antibodies of inactivated vaccine group and IgM-IC vaccine group were mainly against GP5,N and M proteins of PRRSV,whereas antibodies of modified live vaccines group was mainly against GP3 proteins.After the challenge,the IgM-IC vaccine group and the modified live vaccines group produced antibodies specific for the GP4 protein,and one piglet in modified live vaccines group produced antibodies for GP2a protein.The higher levels of GP5 and M specific antibodies in the IgM-IC and inactivated vaccine groups did not reduce viremia levels,and the presence of GP3 and GP4 specific antibodies was related to decreased levels of viremia.The levels of viremia in the modified live vaccines group decreased after the appearance of GP3 and GP4 specific antibodies.The result suggested that GP5 and M proteins of PRRSV may not be the only determinants of PRRSV antibodies neutralization,and the GP3 and GP4 proteins may also contribute to the reduction of viremia levels.Serum from vaccine-immunized pigs was collected and virus neutralization assays were performed with CRL-2843^CD163/CD169cells and MARC-145 cells respectively.The results showed that the CRL-2843^CD163/CD169 was more sensitive in reflecting the relationship between neutralizing antibodies and viremia.After the challenge,VN titers in serum increased in both the IgM-IC and modified live vaccines groups.At the end of the challenge,the VN titers of80%（4/5）surviving piglets in the IgM-IC and modified live vaccines groups were higher than1:16,of which one piglet in the IgM-IC group and 2 piglets in the modified live vaccines group were 1:32;the VN titers of 40%（2/5）surviving piglets in the inactivated vaccine group was1:16.It shows that immunizing animals with IgM immune complex vaccine could increase the level of neutralizing antibodies.ELISA and ELISpot were used to detect IFN-γin blood and peripheral blood mononuclear cells（PBMC）.The results showed that the level of IFN-γin the IgM-IC group was higher than that in other groups.Moreover,high level of IFN-γwas produced after HP-PRRSV XJA1 challenge,suggesting that IgM immune complex vaccines may contribute to the Th1 response,improving the cellular immunity level and facilitating the reduction of viremia.Detection of macrophage-related functional genes revealed that most functional genes of M1 and M2 macrophages（IL-1β,IL-4,IL-6,IL-8,IL-10,IL-13,CCL-2,CCL-17,ISG15,GM-CSF）were significantly decreased after HP-PRRSV XJA1 challenge compared to the blank group,except for CD163,IFN-γand MGL-1.There were no significant differences in the expression levels of these genes observed between the groups other than the blank control group.At the end of the experiment,MGL-1 expression was significantly increased in all surviving piglets,suggesting a potential role of MGL-1 in PRRSV infection,which is still replicating in the PAMs of surviving piglets and may regulate the expression of host genes in PAMs.In summary,an immune complex vaccine was prepared using Mab-PR5nf1 after purification and PRRSV.The immunization test of mice showed that the immune complex vaccines induced production of specific antibodies,better cellular immune response and enhanced CTL.The post-immunization challenge protection test of target animals（pigs）showed that the immune complex vaccine induced high levels of neutralizing antibodies and cellular immune responses,reduced the pathological damage to the lungs,decreased the time of viral shedding,reduced viremia,and the protection rate of IC vaccine was 100%against challenge of HP-PRRSV.