Mark
1,2
Mogler ;
1Harrisvaccines
Kurt
1,3
Kamrud ;
DL Hank
1,3,4
Harris
Inc d/b/a Sirrah-Bios, Ames, Iowa; Department of 2VMPM, 3Animal Science, 4VDPAM, Iowa State University, Ames, Iowa
Expression and characterization of PRRSV ORF5a protein using an alphavirus-derived replicon vector system
5’-
Non-structural genes
Replicon RNA only is
introduced into cells
by electroporation
Protein encoded by the gene(s)
of interest is expressed at high
levels and harvested for use as
the vaccine antigen.
Subunit protein production
IRES
ORF5a gene - 3’
Helper mRNA
-3’
TC-83structural genes
Replicon RNA and
helper RNA are
introduced into cells
by electroporation
Primer
Sequence
ORF5a-AscI-F
ORF5a-PacI-R
5’-GATTGGCGCGCCGCACCATGTTCAAGTATGTTGGGGAGATGC-3’
1
ATGTTCAAGT ATGTTGGGGA GATGCTTGAC CGCGGGCTGT TGCTCGCGAT TGCTTTCTTT
61 GTGGTGTATC GTGCCATTTT GTTTTGTTGC GCTCGTCAAC GCCAACAGCA ACAGCGGCTC
121 TCATCTTCAG TTAATTTACA ACTTGACGCT ATGTGA
Single-cycle vaccine particles,
containing the replicon RNA
expressing gene(s) of interest
are harvested and used to
formulate the vaccine.
replicon particle production process
Figure 1. Process description of both subunit protein and replicon particles using the alphavirus-derived
replicon vector system.
Results
Construction of alphavirus vector
The ORF5a gene from a Type 2 PRRSV strain was successfully cloned into an
alphavirus replicon vector. The gene does not encode any marker epitopes, such as
6xHis. This construct can be used to produce either recombinant protein or replicon
particles (Figure 1).
Expression of ORF5a protein
In Western blot analysis, recombinant ORF5a protein migrated and reacted similarly
to ORF5a found in purified PRRSV preparations. The ORF5a protein migrates
slowly compared to its predicted mass, confirming what has been described by other
investigators. Both the native and recombinant ORF5a proteins were detected using
sera taken from infected pigs (Figure 2).
Anti-peptide antibody purification
Antibodies from convalescent sera were purified using synthetic peptides.
Antibodies purified using the peptide corresponding to the N-terminal twelve
residues of ORF5a protein reacted with recombinant ORF5a protein on Western blot
(Figure 3 & 4). Antibodies purified using two other peptides did not detect the
antigen.
Immunization of pigs with ORF5a replicon particles
Young pig vaccination trials evaluating RP expressing ORF5a protein are ongoing.
MFKYVGEMLD RGLLLAIAFF VVYRAILFCC ARQRQQQQRL SSSVNLQLDA M
Peptide
N12
Table 2. Nucleotide sequence of ORF5a derived from PRRSV strain HLV013, containing 156 nucleotides
including stop (TGA). The underlined sequence represents the start codon of the GP5 coding region.
Expression analysis
Replicon RNA was transcribed in vitro using linearized pVEK-ORF5a plasmid as
template and RiboMax T7 transcription reagents (Promega). The purified replicon
RNA was electroporated into Vero cells, which were seeded into a roller bottle and
incubated overnight. Cells were collected, washed with PBS and lysed with RIPA
buffer (Thermo). Cell lysates were analyzed for ORF5a protein expression by
Western blot.
Briefly, prepared cell lysates and control antigens were run on a 12% SDS-PAGE gel
(Invitrogen). The proteins were then transferred to a PVDF membrane, blocked with
5% non-fat dry milk, then incubated with primary antibody at 4°C overnight. The
next day the blots were washed and incubated with secondary antibody for 1 hour at
room temp. Blots were then washed and developed with TMB substrate (KPL). The
primary antibodies were swine origin (convalescent sera, described in Figure 2) and
the secondary antibody was goat-anti-swine IgG-HRP (Fitzgerald).
Lane
1
Contents
Purified PRRSV antigen (strain HLV013)
2
pVEK-ORF5a-transfected Vero cell lysate
3
Non-transfected control Vero cell lysate
Figure 2. Western blot of native and recombinant ORF5a protein. The primary antibody is convalescent sera
from a pig hyperimmunized by two successive PRRSV challenges (strain HLV013).
Peptide
RQ12
Peptide
C13
Figure 3. Amino acid sequence of ORF5a protein. Underlined amino acids are represented in the synthetic peptide
of the indicated name.
Anti-peptide antibody purification
Synthetic peptides (GenScript) corresponding to various amino acid residues of ORF5a
protein (Figure 3) were used to purify antibodies from convalescent sera. Briefly,
synthetic peptide was coupled to NHS-activated agarose (Thermo Pierce) and
incubated with sera overnight at 4°C. After washing with PBS, the bound antibodies
were eluted with 0.1M glycine·HCl (pH 3.0) and neutralized with 1M phosphate.
Purified antibodies were used in Western blot to determine reactivity with ORF5a
protein (Figure 4). Antibodies purified against the peptide “N12” reacted with
recombinant ORF5a protein. Antibodies purified against peptide “RQ12” and peptide
“C13” did not detect recombinant ORF5a protein.
Lane Contents
5’-GCACTTAATTAATCACATAGCGTCAAGTTGTAAATTAACTGAAGATG-3’
Table 1. Primer sequences used to amplify ORF5a from PRRSV RNA using RT-PCR. Underlined sequence
denotes restriction site used in later cloning steps. Bolded sequence represents either the start or stop codon.
kDa
Self-replicating mRNA or “replicon”
Cloning and sequencing
Native ORF5a sequence from PRRSV genomic RNA was amplified using primers
“ORF5a-AscI-F” and “ORF5a-PacI-R” with OneStep RT-PCR (Qiagen) reagents.
These primers (Table 1) include restriction sites to facilitate further cloning steps.
The RT-PCR product was confirmed to have the correct-sized band by agarose gel
electrophoresis, then subcloned into pCR2.1-TOPO and transformed into TOP-10 E.
coli (Invitrogen). Colonies were screened for inserts and sequenced to confirm that
native nucleotide sequence had been maintained (Table 2).
Replicon construction
The alphavirus-derived replicon plasmid (pVEK) contains TC-83 non-structural
protein 1-4 genes, an IRES element derived from enterovirus 71, a multiple cloning
site and selection markers. The replicon plasmid and TOPO-ORF5a plasmid were
digested with AscI and PacI, and ORF5a DNA fragment was ligated into the
multiple cloning site. Assembled replicon clones were screened for inserts and
confirmed by sequencing. A replicon clone containing the ORF5a gene, termed
“pVEK-ORF5a” was selected for further study.
kDa
Introduction
Porcine reproductive and respiratory syndrome virus (PRRSV) remains a very
important disease of swine worldwide. Recent investigations in both PRRSV and
equine arteritis virus have identified a previously unknown viral protein produced
from the subgenomic mRNA5, designated ORF5a protein. In PRRSV, this protein
consists of 51 amino acids, and possesses a conserved RQ-rich domain on the
carboxyl terminus. The protein has been demonstrated in highly purified virus
preparations, and has been successfully expressed in bacterial cells.
Our group has been evaluating vaccine candidates by expressing various PRRSV
structural proteins using an alphavirus-derived replicon vector system. This
approach utilizes an expression vector derived from Venezuelan equine encephalitis
virus (VEEV) strain TC-83. The desired protein can be expressed in eukaryotic
cells and harvested for use as a vaccine antigen. Alternatively, the RNA replicon
can be packaged into a VEEV capsid and envelope for use as a single-cycle viral
vector, termed a replicon particle (RP) (Figure 1).
Detection antibody
1
Recombinant ORF5a protein
Convalescent sera
2
Recombinant ORF5a protein
Anti-ORF5a-N12
3
Recombinant ORF5a protein
Anti-ORF5a-RQ12
4
Recombinant ORF5a protein
Anti-ORF5a-C13
Figure 4. Western blot of recombinant ORF5a protein detected by either convalescent sera or anti-peptide
purified antibodies. The convalescent sera is the same source as Figure 2.
RP production
Replicon RNA (pVEK-ORF5a) and helper RNA (TC-83 glyoprotein and capsid) were
co-electroporated into Vero cells (Figure 1). After overnight incubation, RP were
harvested by affinity chromatography using cellufine sulfate (Chisso). Titration of RP
was accomplished by IFA targeting the nsp2 of TC-83 (Figure 5). Aliquots of RP
were frozen at -80°C prior to use.
Figure 5. Immunofluorescent straining of RPinfected Vero cells using anti-nsp2 antibody
(left).
Roller bottle apparatus used in both recombinant
protein and RP production (right).
Young pig immunization
Weaned pigs were obtained at three weeks of age from a PRRSV-negative source farm.
Pigs were randomized into two groups. One group received RP expressing ORF5a
protein, and the other group received a sham RP vaccine. This study is currently in
progress. Sera from this study will be tested for anti-PRRSV activity using Western
blot, ELISA, and virus neutralization assays.
Selected references
Firth et al (2011). Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production. J Gen
Virol 92: 1097-106.
Johnson et al (2011). Novel structural protein in porcine reproductive and respiratory syndrome virus encoded by a alternative ORF5
present in all arteriviruses. J Gen Virol 92:1107-16.
Kamrud et al (2010) Development and characterization of promoterless helper RNAs for the production of alphavirus replicon particle. J
Gen Virol 91:1723-7.
The authors would like to thank Jill Gander, Ryan Vander Veen, Kara Burrack, Kay Kimpston-Burkgren, Pam Whitson, and Ashley Baert.