Co-expressing GP5 and M proteins under different promoters in recombinant modified vaccinia virus ankara (rMVA)-based vaccine vector enhanced the humoral and cellular immune responses of porcine reproductive and respiratory syndrome virus (PRRSV)

Abstract

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.

Fig. 1

Design of the recombinant plasmids. (A) The basic structure of plasmid pLRII. (B) ORF5 gene was cloned into pLRII (named as pLR-ORF5) that was used to express GP5 of PRRSV. (C) ORF6 gene was cloned into pLRII (pLR-ORF6) that were used to express M of PRRSV. (D) GP5 and M were expressed as a fusion protein under the control of the same promoter (Psyn). (E) GP5 and M were expressed separately under the control of two different promoters (Psyn and VP7.5). Psyn, the synthesized strong early/late compound promoter; VP7.5, the late/early promoter of vaccine virus; TTTTTAT, the transcription termination signal sequence; ORF5, ORF5 gene of PRRSV; ORF6, ORF6 gene of PRRSV; DeletionII, the deletion II region of MVA genome

Fig. 2

PCR identification of ORF5 and ORF6 in rMVAs. 1, DNA molecular Marker (DL2000); 2, rMVA-GP5; 3, rMVA-M; 4, wtMVA: 5, rMVA-GP5/M; 6, rMVA-GP5-M; 7, DNA molecular Marker (DL15000). Also, the results were double checked by DNA sequencing (data not shown)

Fig. 3

Western-blots identify the expression of GP5 and M proteins in different recombinant rMVAs. 1, rMVA-GP5-M; 2, rMVA-GP5/M; 3, rMVA-M; 4, rMVA-GP; 5, wtMVA. The arrows indicate the expected molecular sizes of GP5+M, GP5, and M

Fig. 4

The IFA identification of the GP5 and M expression. BHK-21 cells were infected with rMVA-GP5, rMVA-M, rMVA-GP5-M, and rMVA-GP5/M at 2.0 multiplicities of infection (m.o.i.), respectively. Figure 4A–D represented the IFA identification of the cells infected with rMVA-GP5, rMVA-M, rMVA-GP5-M, and rMVA-GP5/M, respectively. Figure 4E was the picture of negative control that was infected with wtMVA

Fig. 5

One-step and multicycle growth curves. BHK-21 cells were infected at 2.0 m.o.i (one-step growth curve, A) and 0.1 (multicycle growth, B) with rMVA-GP5-M, rMVA-GP5/M, and wild type MVA. rMVAs growth rate at m.o.i 2.0 almost had the same growth curves with that of at 0.1 m.o.i. The insertion of PRRSV GP5 and M into MVA by the forms of single protein, fusion protein and co-expressed together, did not effect the growth of the recombinant rMVAS. The rMVAs have the similar growth ability with wild type MVA. Experiments were triplicate

Fig. 6

Serum neutralization assay (NA) of the mice immunized with rMVAs of GP5 and M proteins of PRRSV. Each group of mice (n = 5) were immunized (i.m.) with 5 × 105 TCID50 of rMVAs at 0 week and boosted at 3 weeks. Sera were collected weekly after inoculation. The neutralizing antibody titers of each group were measured immediately after bleeding from tail. As shown upon, the neutralizing antibody titers of rMVA-GP5-M inoculation mouse group are significantly less than rMVA-GP5/M inoculation group but higher than that of rMVA-GP5 and rMVA-M inoculation mouse groups (p < 0.05). The efficacy of humoral immunity to PRRSV, co-expressing GP5 and M under the control of different promoters in the same virus is the best. The ranking is rMVA-GP5/M, rMVA-GP5-M, rMVA-GP5, and rMVA according to the order, high to low. No production of PRRSV NA titers was detected in wtMVA and PBS injected mice. Experiments were triplicated. Data are presented as the mean ± standard error

Fig. 7

Cellular immune responses in mice immunized with rMVAs. Each group of mice (n = 27) was immunized (i.m.) with 5 × 105 TCID50 of rMVAs at 0 week and boosted at 3 weeks. The IFN-γ and IL-2 level of rMVA-GP5-M inoculation mouse group are significantly less than rMVA-GP5/M inoculation group but higher than that of rMVA-GP5 and rMVA-M inoculation mouse group (p < 0.05). No production of IL-4 was detected in all rMVAs inoculated mice. The efficacy of cellular immunity of PRRSV, co-expressing GP5 and M under the control of different promoters in the same virus is the best ranking in rMVA-GP5/M, rMVA-GP5-M, rMVA-GP5, and rMVA according to the order, high to low. No significant production of IFN-γ, IL-2, and IL-4 were detected in wtMVA and PBS injection mice. Data were triplicated and presented as the mean ± standard error