Antigenic structures stably expressed by recombinant TGEV-derived vectors

Abstract

Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate.

Keywords: Coronavirus derived vectors; PRRSV; Positive-strand RNA viruses; RNA vector stability; TGEV.

Fig. 1

Expression of PRRSV M protein by rTGEV-derived vectors. (A) Schematic structure of the rTGEV cDNA encoding the PRRSV M gene. The numbers and letters inside the rectangles indicate the viral genes. TRS, transcription-regulating sequence. (B) RT-PCR analysis of ten clones from plaque-purified passage 16 rTGEV-S_7.1-TRS_3a-M virus. Genomic RNA (gRNA) and subgenomic mRNA (mRNA) encoding PRRSV M protein were detected. The arrow indicates the expected size of the corresponding PCR product. Numbers on the left indicate the molecular weight markers (Mw) size in base pairs. (C) Immunofluorescence analysis of ST cells infected with the passage 16 rTGEV-S_7.1-TRS_3a-M at 8 hpi. A polyclonal antibody specific for TGEV and a secondary antibody staining red were used to identify virus-infected cells. Expression of PRRSV M protein was detected with a monoclonal antibody and a secondary antibody staining green.

Fig. 2

Stability of PRRSV GP5 domains in rTGEV vectors. (A) Schematic representation of PRRSV GP5 constructs: full-length GP5 (GP5), GP5 ectodomain (GP5ecto), and GP5 fragment (GP5fr) that comprises the ectodomain lacking the signal peptide (SP). Immunodominant epitope (IDE) and epitope critical in neutralization (ECN), N-glycosylation sites (yellow), and the cysteine involved in GP5-M heterodimer formation (red) are also shown. GP5ecto and GP5fr included an HA or FLAG tag, respectively, for their detection (TG, blue). (B) RT-PCR analysis of ten clones from plaque-purified passage 8 rTGEV-S_7.1-TRS_3a-GP5-TRS_22N-M (GP5), rTGEV-S_7.1-TRS_3a-GP5ecto-TRS_22N-M (GP5ecto) and rTGEV-S_7.1-TRS_3a-GP5fr-TRS_22N-M (GP5fr) viruses. The arrow indicates the expected size of the corresponding PCR product. Numbers on the left indicate the molecular weight markers (Mw) size in base pairs. Lower size bands (indicated by red asterisks) correspond to deletion products from heterologous gene, meaning genomic instability. Numbers on the right indicate the overall stability of each construct.

Fig. 3

Expression of PRRSV minor envelope protein domains by rTGEV vectors. (A) Schematic representation of PRRSV GP3 and GP4 proteins, and corresponding GP3fr and GP4fr expressed by rTGEV. Several motifs are indicated, such as signal peptide (SP), the epitope critical in neutralization (ECN), the transmembrane domain (TM), the glycosylation sites (yellow), and immunodominant epitope (IDE). GP3fr and GP4fr included a FLAG tag (FG) for their detection. (B) RT-PCR analysis of ten clones from plaque-purified passage 16 rTGEV-S_7.1-TRS_3a-GP3fr (GP3fr) and rTGEV-S_7.1-TRS_3a-GP4fr (GP4fr) viruses. Genomic RNA (gRNA) and GP3fr and GP4fr subgenomic mRNAs (mRNA) were detected. The arrow indicates the expected size of the corresponding PCR product. Numbers on the left indicate the molecular weight markers (Mw) size in base pairs.

Fig. 4

PRRSV M protein as an scaffold for antigenic domain expression. (A) Chimeric proteins expressed by rTGEV. Two M protein locations were chosen for the expression of GP3 epitope involved in neutralization (GP3ECN) fused to a FLAG tag (blue), leading respectively to the chimeras GP3ep-NtermM and GP3ep-Mloop. M protein contains in its C-terminal an endoplasmic reticulum retention signal (green). (B) RT-PCR analysis of ten clones from plaque-purified passage 16 rTGEV-S_7.1-TRS_3a-GP3ep-NtermM (GP3ep-NtermM) and rTGEV-S_7.1-TRS_3a-GP3ep-Mloop (GP3ep-Mloop) viruses. Genomic RNA (gRNA) and PRRSV M protein subgenomic mRNA (mRNA) were detected. The arrow indicates the expected size of the corresponding PCR product. Numbers on the left indicate the molecular weight markers (Mw) size in base pairs. Lower size bands (indicated by red asterisks) correspond to deletion products from heterologous gene, meaning genomic instability. (C) Immunofluorescence analysis of ST cells infected with passage 16 rTGEV-S_7.1-TRS_3a-GP3ep-NtermM (GP3ep-NtermM) and rTGEV-S_7.1-TRS_3a-GP3ep-Mloop (GP3ep-Mloop) at 8 hpi. TGEV specific polyclonal antiserum and a secondary antibody staining red were used to identify infected cells. Expression of chimeric proteins was detected using a monoclonal antibody specific for PRRSV M protein (αM) or the FLAG tag (αFLAG) and a secondary antibody staining green.

Fig. 5

Protection conferred by rTGEVs expressing PRRSV antigens. (A) Percentage of animals showing respiratory symptoms. Observations were made daily during a time frame of 28 days post-challenge. ^⁎⁎, p-value<0.05, ^⁎, p-value<0.1. (B) Lung samples collected at 28 days post-challenge were stained with hematoxylin-eosin. Representative pictures obtained with a 10x objective are shown (left panels). Lung damage was scored by observation of 50 random fields per animal (right panel). (C) IL-8 levels in serum. Serum samples were collected at the indicated times post-immunization, mixed in pools of three animals per sample, and cytokine levels were analyzed by a 7-plex fluorescent microsphere immunoassay. Dots represent individual samples and solid lines represent mean titer for each group. ^⁎, p-value<0.1.

Fig. 6

PRRSV titer in serum. PRRSV RNA was isolated from sera at the indicated times post-immunization, and viremia in serum was quantified by RT-qPCR. Dots represent individual animals and solid lines represent mean titer for each group. ^**, p-value<0.05.

Fig. 7

Humoral immune response elicited by rTGEV expressing PRRSV antigens. (A) Humoral response specific for GP5. Serum samples were collected at the indicated times post-immunization and analyzed by ELISA using purified full-length GP5. Dots represent individual animals and solid lines represent mean titer of each group. The arrow indicates the time when challenge was performed. ^⁎⁎⁎, p-value<0.01; ^⁎⁎, p-value<0.05. (B) Neutralizing antibodies induced at the indicated times post-challenge. Neutralization assays were performed with PRRSV Olot91 strain infecting MARC-145 cells. Dots represent individual animals and solid lines represent mean titer of each group. ^⁎, p-value<0.1.