2.1 Construct design
Replication-competent recombinant SeVs were rescued using a reverse genetics system, described previously [22
]. The full-length cDNA of SeV (Enders strain) was first cloned. To this end, Enders SeV RNA was extracted from purified stock virus and reverse transcription (RT)-PCR was performed. PCR products of each gene were cloned into pTF1 and then cloned into pUC19 to construct the full genome SeV Enders cDNA (pSV(E)). The SeV genome in this clone was straddled by a T7 promoter and a hepatitis delta virus ribozyme sequence. As shown in , a unique NotI
site was positioned in the non-coding region between the F and HN genes of SeV.
Production and testing of recombinant Sendai viruses
For cloning of the hPIV-3 F and HN genes, LLC-MK2 cells were infected with the C243 strain of hPIV-3 (VR-93, American Type Culture Collection [ATCC], Rockland, MD) and viral RNA was extracted. The hPIV-3 F and HN genes were amplified by RT-PCR (Titan One Tube System; Roche). The PCR forward primer included a NotI site and the reverse primer included an SeV transcription termination signal, an intergenic (IG) sequence CTT, a transcription initiation signal, and a second NotI site (). The hPIV-3 F and HN cDNAs were digested with NotI and ligated into the NotI site of pSV(E).
2.2 Virus Rescue
To rescue the recombinant viruses, we infected 293T cells with a UV-inactivated, T7 RNA polymerase-expressing recombinant vaccinia virus (vTF7.3 [26
]) for 1 h at 37 °C at an MOI of 3. Cells were then co-transfected with cDNA plasmids containing either the hPIV-3 F or HN gene (1 μg) and three supporting T7-driven plasmids expressing the NP, P, or L gene of SeV (1 μg pTF1SVNP, 1 μg pTF1SVP, and 0.1 μg pTF1SVL, respectively) in the presence of Lipofectamine (Life Technologies, Grand Island, NY). Cells were then incubated for 40 h. Cell lysates were prepared and inoculated into 10-day-old embryonated chicken eggs. Allantoic fluids were harvested after 72 h and viruses were plaque purified on LLC-MK2
cells. Recombinant SeV clones were amplified once more in embryonated eggs to prepare vaccine stocks. Recovered viruses were designated rSeV-hPIV3-F (SeV expressing hPIV-3 F protein) or rSeV-hPIV3-HN (SeV expressing hPIV-3 HN protein).
2.3 Immunoprecipitation of F and HN proteins from infected cells
To confirm that the recombinant vectors expressed hPIV-3 F or HN proteins, we examined lysates of rSeV-hPIV3-F or rSeV-hPIV3-HN infected Hep-2 cells by radio-immunoprecipitation. Briefly, Hep-2 cells were infected at an MOI of 5 with rSeV-hPIV3-F, rSeV-hPIV3-HN or wild-type SeV, and incubated at 33°C in DMEM, 10% FCS and 1% l-glutamine. Sixteen hours post-infection, the cells were washed twice with PBS containing 0.1 g/liter calcium and magnesium (PBS+). Cells were maintained in culture for 30 min in methionine- and cysteine-free medium and then labeled for 15 min with 100 μCi [35S]Promix (Amersham Pharmacia Biotech) in 1 ml of DMEM lacking methionine and cysteine and containing 20 mM HEPES buffer (pH 7.3). The cells were washed once with PBS+ and chased with 3 ml of DMEM containing 2 mM methionine, 2 mM cysteine, and 20 mM HEPES buffer (pH7.3) for 180 min. Samples were lysed with ice-cold radio-immunoprecipitation assay (RIPA) buffer containing 0.15 M NaCl, 9.25 mg/ml iodoacetamide, 1.7 mg/ml aprotinin, 10 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged at 67,000 × g in an Optima TLX ultracentrifuge (Beckman Coulter). The supernatant was incubated overnight (18-22h) at 4°C with 25 μl rabbit anti-hPIV-3-F or anti-hPIV-3-HN tail peptide polyclonal antibody (1:40 dilution of rabbit sera, Harlan Bioproducts for Science, Madison WI). Immune complexes were adsorbed to protein A-Sepharose Cl-4B (GE Healthcare) for 1 h at 4°C. Samples were washed three times with RIPA buffer containing 0.3 M NaCl, three times with RIPA buffer containing 0.15 M NaCl, and once with 50 mM Tris buffer (0.25 mM EDTA, 0.15 M NaCl, pH 7.4). The samples were then fractionated on 12% NuPAGE Bis-Tris polyacrylamide–SDS gels (Invitrogen). Protein bands were visualized using a Typhoon 9200 phosphoimager (GE Healthcare).
2.4 Animals, Inoculations and challenges
Groups of 5 adult female cotton rats (Sigmodon hispidus
; Harlan Sprague Dawley, Indianapolis, IN) were intranasally inoculated with rSeV-hPIV3-F (2×106
plaque-forming units [PFU]), rSeV-hPIV3-HN (2×106
PFU), a mixture of 1×106
rSeV-hPIV3-F and 1×106
rSeV-hPIV3-HN, or a mixture of 2×106
rSeV-hPIV3-HN and 2×106
rSeV-RSV-F (a previously described recombinant SeV expressing the full-length RSV F protein [22
]). Control animal groups received wild-type SeV (2 × 106
PFU/cotton rat) or PBS. After 5-10 days, mediastinal lymph nodes were collected for T-cell assays. Serum samples were taken four weeks post-inoculation and challenges were performed on week five. The intranasal challenge doses were 2×106
PFU/cotton rat of homologous hPIV-3 (strain C243) or 1.5×106
PFU/cotton rat of heterologous hPIV-3 (8-94, kindly provided by Dr. R. Hayden, Clinical Virology, St. Jude Children's Research Hospital). Lungs were harvested 3 days post-challenge for virus measurements. In some experiments, animals were challenged intranasally with either hPIV-1(C35 from ATCC, 2×106
PFU/cotton rat) or RSV (strain A2, 1.5×106
2.5 Enzyme-linked immunosorbent assay (ELISA)
For studies to detect anti-hPIV-3 F- or HN-specific antibodies, an hPIV-3 stock was prepared from culture supernatants by concentration with a Millipore Amicon filter unit. Concentrates were lysed in disruption buffer (0.5% TritonX-100, 0.6M KCl, 0.05M Tris pH7.8), diluted with PBS (1:3000) and coated on 96-well ELISA plates. Lysates of wild-type SeV were plated as controls. After overnight incubation, plates were blocked with PBS containing 3% bovine serum albumin (BSA, Sigma, St Louis, MO). Serum samples from vaccinated and control animals were serially diluted and incubated on plates for 2 h at 37°C. Plates were then washed and incubated with rabbit anti–cotton rat antibody (kindly provided by Dr. Greg Prince, Virion Systems, Rockville, MD) for 30 min at room temperature. After further washing, plates were incubated with anti-rabbit IgG-horseradish peroxidase conjugate (diluted 1:3,000 in PBS/1% BSA, Bio-Rad, Hercules, CA, Cat# 170-6515) for 30 min at room temperature, washed again, and incubated with 2,2′-azino-bis-(3-ethylbenzthiazolinesulfonic acid) (ABTS, Southern Biotechnology Associates, Inc, Birmingham, AL). Absorbance was read at 405 nm.
2.6 Neutralization assays
To conduct neutralization assays, we mixed serially diluted sera with approximately 10 TCID50 hPIV-3 per well in DMEM (Cambrex Bio Science Walkersville, Inc, Walkersville, MD) for 1 h at 37°C. Viruses were either homologous (C243) or heterologous (St. Jude Children's Research Hospital isolates 4-04, 5-97 and 8-94, named by the month and year of isolation) to the vaccine. The virus-serum mixtures were then added to wells (6 wells per sample in 24-well plates) of LLC-MK2 cell monolayers, which were incubated for 1 h (33°C, 5% CO2) and then fed with DMEM supplemented with glutamine, antibiotics and 5% fetal calf serum (FCS). After 4 days of culture (33°C, 5% CO2), supernatants (100 μl) from test wells were mixed with 100 μl of 0.5% fresh turkey red blood cells in round-bottomed 96-well plates and incubated at 4°C for 30 min. Hemagglutination was scored as positive or negative for each well and the percent neutralization was calculated as the reduction in frequency of positive wells for test versus control samples.
2.7 IFN-γ ELISPOT assays
For analyses of hPIV-3-specific T-cell responses, overlapping peptides (derived from the hPIV-3 F and HN sequences) were prepared by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. Peptides were generally 15 amino acids in length and were initiated at intervals of 10 amino acids to cover the length of the hPIV-3 F and HN proteins. Pools were prepared with 10 peptides per pool for use in the ELISPOT assay.
The ELISPOT assay was conducted by incubating 3.3 μg/ml goat anti-cotton rat IFN-γ antibody (R & D Systems, Minneapolis, MN) in multiscreen-hemagglutinin filtration plates (Millipore, Bedford, MA) overnight at 4°C. After washing, the plates were blocked for at least 1 h at 37°C with complete tumor medium (CTM [27
], a modified Eagle's medium [Invitrogen, Grand Island, NY] supplemented with 10% FCS, dextrose [500 μg/ml], glutamine [2 mM], 2-mercaptoethanol [3 × 10-5
M], essential and non-essential amino acids, sodium pyruvate, sodium bicarbonate, and antibiotics). Mediastinal lymph node cells were harvested from cotton rats 5-10 days after vaccination. Fresh cells were suspended in CTM and added to plates at 0.25-1×106
cells per well containing individual peptide pools. The final concentration of each peptide was approximately 10 μM. Positive control wells received 4 μg/ml Con A (Sigma-Aldrich, St. Louis, MO) rather than peptides. The plates were incubated for 48h at 37°C and washed four times with PBS and four times with PBS wash buffer (PBS with 0.05% Tween 20). Biotinylated goat anti-cotton rat IFN-γ antibody (R & D Systems, Minneapolis, MN) was diluted in PBS (containing 0.05% Tween 20 and 1% FCS) and was added to wells (100 μl aliquots, 0.5 μg/ml antibody). Plates were incubated at 37°C for at least 2 h. After additional washing, streptavidin-conjugated alkaline phosphatase (Cat# D0396, DAKO, Copenhagen, Denmark) diluted 1:500 in PBS wash buffer was added. One hour later, plates were rinsed with wash buffer and water. The IFN-γ spots were developed by adding 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium alkaline phosphatase substrate (Sigma-Aldrich). Spots were counted with an Axioplan 2 microscope and software (Carl Zeiss, Munich-Hallbergmoos, Germany).
2.8 Virus challenge assay
Three days after intranasal viral challenge, cotton rats were sacrificed and the lungs were harvested for measurement of virus titer. Briefly, lungs were homogenized on ice with a mechanical Dounce homogenizer (PowerGen125 PCR Tissue Homogenizing kit; Fisher Scientific) to yield 5 ml of homogenate in PBS. Homogenates were centrifuged (1500 × g, 10 min) and supernatants were collected.
For hPIV-1 detection, LLC-MK2 cells were grown to confluency in 6-well plates in complete medium (MEM, 0.2% NaHCO3, 2 mM glutamine, and 50 μg /ml gentamicin) with 5% FCS. Plates were washed once with PBS/calcium/magnesium.100 μl of serially diluted supernatants were inoculated into wells. After 1 hr at 33°C, 5% CO2, the cells were overlaid with 4 ml per well of complete medium supplemented with vitamins, amino acids, 0.15% BSA, 5 μg/ml acetylated trypsin (Sigma), and 0.9% agarose (electrophoresis grade, BRL, Gaithersberg, MD). After the agarose was set, plates were inverted and incubated at 33°C in a 5% CO2 incubator. 5 days later, plates received a second overlay (3 ml), similar to the first, but with 5% FCS instead of BSA, 0.0035% neutral red, and no trypsin supplement. Plates were incubated for one more day and plaques were counted.
For hPIV-3 detection, 100 μl of serially diluted lung supernatants were added to wells (in 24-well plates) of LLC-MK2 cell monolayers. Cultures were incubated for 1 hr (33°C, 5% CO2) and then fed with DMEM supplemented with glutamine, antibiotics and 5% FCS. After 4 days incubation (33°C, 5% CO2), 100 μl supernatants were removed from hPIV-3-infected wells for hemagglutination assays with turkey red blood cells. TCID50 were calculated using the Reed-Meunch formula.
For RSV measurements, serially diluted supernatants from lung homogenates were inoculated on Hep-2 cell monolayers in 12-well plates; after 1 h at 37 °C and 5% CO2, the wells were overlaid with EMEM medium supplemented with glutamine, antibiotics, 10% fetal calf serum and 0.75% methylcellulose. After incubation for 5 to 6 days at 37°C and 5% CO2, the methylcellulose was removed, cells were fixed with formalin phosphate, and the plates were stained with hematoxylin and eosin for enumeration of plaques. For each virus, the total pulmonary burden per cotton rat was scored.