Studies On Candidate Molecules Of Recombinant Adenovirus Vaccines Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is one of the mosteconomically significant viral diseases in the swine industry. It is characterized byreproductive problems in sows such as poor farrowing rates, premature farrowings, andincreased stillbirths, as well as respiratory problems in piglets such as pneumonia andatrophic rhinitis. Current commercial PRRS vaccines, including both live attenuated andkilled vaccines, have been wildly used, but they cannot provide effective protection againstPRRS. The causative agent, PRRS virus (PRRSV), belongs to the family Arteriviridaewithin the genus Arterivirus, order Nodovirales. Its genome is a non-segmentedsingle-stranded positive-sense RNA molecule of approximately 15 kb in length, whichcontains nine open reading frames (ORFs). GP5 and M proteins are known to have goodimmunogenicity and associate with protection against PRRSV infection, but little is knownabout that of the minor envelope proteins, such GP3 and GP4.In this study, several recombinant adenoviruses expressing GP3, GP4, GP5, and Mproteins of PRRSV and GP5 mutants and their immunogenicity were determined in amouse model. The contents of the paper contain six parts as following:1. Establishment of PRRSV-specific cytotoxic T lymphocytes assayTo investigate the cell-mediated immunity against PRRSV, a non-radioactivecytotoxicity assay by lactate dehydrogenase (LDH) system was established. The GP3, GP4,GP5, and M genes of PRRSV were amplified with RT-PCR and cloned into the plasmidpcDNA3, respectively. The constructed plasmids pcDNA3-GP3, pcDNA3-GP4,pcDNA3-GP5, and pcDNA3-M were transfected into NIH3T3 cells, respectively, andscreened by G418 for 14 d. Anti-G418 NIH3T3 cells were obtained. The expression of GP3,GP4, GP5, and M proteins in anti-G418 NIH3T3 cells was proved by indirectimmunofluorescence assay (IFA) using hyperimmune sera against PRRSV. It suggested thatfour cell clones NIH3T3/GP3,NIH3T3/GP4,NIH3T3/GP5 and NIH3T3/M were successfully constructed and can be used as target cells for detection of cytotoxic Tlymphocytcs of PRRSV. Subsequently, the optimal cell concentration for the assay wasdetermined and the method of LDH assay used to detect cytotoxic T lymphocytes inducedby PRRSV was established.2. The immunogenicity of M-GP5 fusion protein expressed by recombinant adenovirusTo enhance the immunogenicity of GP5, the recombinant adenovirus expressing M andGP5 fusion protein was generated, confirmed by indirect immunofluoresccnce assay usingmonoclonal antibodies against GP5 and M proteins and Westem blot using hypcrimmuneserum against PRRSV. BALB/c mice were inoculated subcutaneously twice at 2-weekintervals with the recombinants expressing PRRSV GP5 (rAd-GPS), M (tAd-M), andM-GP5 fusion protein (rAd-M-GPS). Groups were injected with wild type adcnovirus(wtAd) or PBS as control. The results showed that the mice immunized with recombinantadenovirus rAd-M-GP5 developed significantly higher titcrs of neutralizing antibodies toPRRSV (1:102 at 55dpi) compared to mice irnmunized with rAd-M or rAd-GP5 alone. Itwas also found that mice immunized with rAd-M-GP5 were primed for significant higherlevels of anti-PRRSV CTL responses 05.2% of lysis at 42dpi) than mice immunized withrAd-M (28％of lysis at 42dpi). Mice receiving rAd-GP5 mounted lower level ofPRRSV-spccific response(18％). It suggested that M-GP5 fusion protein expressing byrecombinant adcnovirus could enhance the ability of GP5 to elicit neutralizing antibodiesand cell-mediated immunity.3. Effect of the signal peptide and hydrophobic region in its N-terminus on theimmunogenicity of GP3Though the minor envelope protein GP3 is associated with protective immunity, itsimmunogenieity and protective mechanism are poorly known. Two recombinantadenoviruses rAd-GP3 expressing complete GP3 and rAd-tGP3 expressing truncated GP3in which aa2-64 were deleted were constructed and the immunogenicity was tested in mice.BALB/c mice were immunized subcutaneously twice at 2-week internals with therecombinants rAd-GP3 and rAd-tGP3 or with wtAd and PBS as control. The resultsshowed that the mice immunized with recombinant adenoviruses developedPRRSV-specifie cellular immune response, including T-cell proliferation responses andcytotoxic T responses, by 2 weeks post boost-immunization. Moreover, the levels ofimmune responses of mice immunized with rAd-tGP3 (1:14 of the titer of neutralizingantibody,4.5 of SI, 36％of lysis at 56dpi) were significantly higher than that of mice with rAd-GP3 (1:10 of the titer of NA,3.3 of SI, 28％of lysis at 56dpi) (P＜0.05). It indicatedthat the first 64aa fragment of GP3 might affect the conformation of the antigen structuresof GP3 protein.4. The immunogenicity of GP5 enhanced by fusion with GP3 or GP3 and GP4To determine whether the immunogenicity of GP5 can be enhanced by GP3, GP4, orGP3 and GP4, three recombinant adenoviruses expressing GP3 and GP5, GP4 and GPS, orGP3, GP4 and GP5 fusion protein were constructed and confirmed by indirectimmunofluorescence assay using monospecific antibodies against GP3, GP4, and (3P5proteins and Western blot using hypedmmune serum against PRRSV. In addition, tworecombinants expressing (3P3 or GP4 alone were constructed. BALB/c mice wereinoculated subcutaneously twice at 2-week internals with above recombinants and otheradenoviruses expressing single GP3, GP4, or GP5 protein. Groups were injected with wtAdor PBS as control. The results showed that the mice inoculated with recombinantadenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weekspost boost-immunization. However, only mice immunized with recombinant adenovirusesrAd-GP3-GPS, rAd-GP4-GP5, and rAd-GP3-GP4-GP5 developed significantly higher titersof neutralizing antibodies to PRRSV (1:82,1 : 96,1:120 at 56dpi, respectively) andproduced stronger lymphocyte proliferation responses compared to mice immunized withtAd-tiP3, rAd-GP4 or rAd-GP5 alone. It was also found that mice immunized withrAd-GP3-GP5 and rAd-GP3-GP4-GP5 were primed for significant higher levels ofanti-PRRSV CTL responses (37.5％and 44.7％at 42dpi) than mice immunized withrAd-GP3 (21.4％) and rAd-GP5 (18％). It suggested that the GP3 or GP3 and GP4 canenhance the immunogenicity of GP5.5. Effect of its signal peptide and nonneutralizing epitope on the ability of GP5 toinduce neutralizing antibodiesTo determine whether the immunogenicity of GP5 can be affected by its signal peptideand nonneutralizing epitope (A), three recombinant adenoviruses were constructed:rAd-GP5△27/31 (mutant A epitope, aa27-31), rAd-GPS△27/36 (mutant A epitope andsequences between A and B epitopes, aa27-36), and rAd-GPS△2/36 (mutant sequencesbetween ATG and B epitopes, aa2-36). Their abilities were evaluated in BALB/c mice toinduce neutralizing antibody responses. The results showed that mice inoculated withadenovirus mutant recombinants developed a significant anti-PRRSV IgG response.Significant neutralizing antibody titers were detected following immunized with all three recombinants (1:30, 1:19, 1:14 at 56dpi), which were significantly higher than thatinduced by rAd-GP5 expressing wild type GP5 (1:8). It indicated that mutation of Aepitope could increase the level of neutralizing antibodies induced by GP5 protein.Moreover, the signal peptide and sequences near the A epitope can affect the ability toinduce neutralizing antibodies.6. Effect of its N-glycans on the ability of GP5 to induce neutralizing antibodies"Glycan shielding" is postulated to be a primary mechanism to explain evasion fromneutralizing immune response, ensuring in vivo persistence of virus, such as HIV, SIV, andHBV. The objective of this study was to construct recombinant adenoviruses expressingsingle or multiple N-glycosylation-site mutant PRRSV GP5 proteins, and evaluate theexpression of GP5 in cell culture and potential to induce immune responses in BALB/cmice. Six recombinant adenoviruses were constructed each expressing wild-type GP5 and1-4 N-glycosylation-site mutants: N44S, N44/51S, N30/44/51S, N30/33/44/51S, andN30/33S. Inoculation of BALB/e mice with adenovirus mutant recombinants resulted in asignificant anti-PRRSV IgG response. Significant neutralizing antibody titers were detectedfollowing immunized with all five recombinants expressing N-glyeosylation-site mutantGP5 proteins. It indicated that all four N-glycans can affect the ability of GP5 to induceneutralizing antibodies. The titer of neutralizing antibody developed by mice inoculatedwith recombinant adenovirus rAd-GP5N30/33S was higher than those elicited byrAd-GP5N44S, rAd-GP5N44/51S, and rAd-GP5N30/44/51S, but lower than that ofrAd-GP5N30/33/44/51S, which suggested that glycosylations of GP5 protein at residuesN30 and N33 which are near the non-neutralizing epitope (aa27-30) are critical forinduction of neutralizing antibodies. There were no significant difference in lymphocyteproliferation responses induced by wild-type and N-glyeosylation-site mutant GP5 proteins.In summary, fusion proteins M-GP5, GP3-GP5, and GP3-GP4-GP5 expressed byrecombinant adenoviruses could significantly improve both PRRSV-speeifie neutralizingantibodies and cell-mediated immune responses. It suggested that the recombinantadenoviruses expressing above fusion proteins might be attractive candidate vaccines to betested for preventing PRRSV infection. Moreover, the effect of N-glyeosylation-sites andnonneutralizing epitope of GP5 on the ability to elicit neutralizing antibodies and highimmunogenicity of GP3 protein may lead to a new approach for the generation of PRRSVvaccines.