RSV (Long strain) was grown on HEp-2 cells and harvested at an early stage of the cytopathic effect. Supernatant was centrifuged (15 min, 1,500 × g) to remove cellular debris, and subsequently the virus was pelleted by ultracentrifugation (Beckman; SW28; 4 h at 25,000 rpm). The pellet was resuspended in TEN buffer (20 mM Tris-HCl, 1 mM EDTA, 0.15 M NaCl [pH 7.8]). MV antigen (Edmonston strain; produced on Vero cells) was kindly provided by R. S. van Binnendijk (Bilthoven, The Netherlands). Both virus preparations were dialyzed against TEN buffer, adjusted to a protein concentration of 1 mg/ml, and inactivated by addition of 37% formalin (1:4,000; 3 days at 37°C). The final preparations, which did not contain infectious virus as shown by cell culture, were adjusted to a protein concentration of 0.2 mg/ml and frozen in aliquots at −70°C.
A vaccine dose was prepared by mixing FI-RSV or FI-MV (10 μg of total protein) with 1 ml of Adju-Phos (kindly provided by Superfos Biosector, Frederikssund, Denmark) and incubating the mixture for 30 min at room temperature. The vaccine was administered intramuscularly in the gluteal muscle in two 0.5-ml doses divided over the two legs.
In a first experiment, eight infant cynomolgus macaques were vaccinated with either FI-RSV (animals RS1 to -4) or FI-MV (animals MV1 to -4). All animals received three vaccinations with 4-week intervals. EDTA-blood samples were collected every 2 weeks. Approximately 3 months after the third vaccination, all animals were challenged with RSV. Subsequently, five more infant cynomolgus macaques were vaccinated by using the same materials and experimental design; three received FI-RSV (animals RS5 to -7) and two received FI-MV (animals MV5 and -6). During this last experiment, one animal of each group was sacrificed for histopathological analysis at days 7 (animals RS5 and MV5) and 13 (animals RS7 and MV6) after challenge.
All animals were between 5 and 12 months of age at the moment of first vaccination, with the exception of MV1, which was 19 months old. During the vaccination periods the animals were housed in groups with their mothers. During the challenge period the animals were housed in smaller standard cages (three or four animals per cage). The animal study was approved by the Local Animal Ethics Committee of the Erasmus MC, Rotterdam, The Netherlands, and was carried out in accordance with animal experimentation guidelines.
Virus neutralization assays were carried out as previously described (31
). Briefly, twofold serial dilutions of heat-inactivated (30 min, 56°C) macaque EDTA-plasma samples were tested for their ability to neutralize 100 50% tissue culture infectious doses (TCID50
) of RSV (Long strain) or MV (Edmonston strain). The VN titer was defined as the reciprocal of the plasma dilution at which 50% of the wells showed cytopathic effect, as calculated from triplicate measurements by the method of Reed and Muench (34
For production of antigens for in vitro stimulations, RSV (Long strain) and MV (Edmonston strain) were grown in Vero cells. After clarification the virus was pelleted by ultracentrifugation and resuspended in RPMI 1640 medium and inactivated with β-propiolactone (BPL; Sigma-Aldrich, St. Louis, Mo.). A cell control antigen was produced by processing freeze-thawed Vero cells in an identical manner.
Macaque peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation and resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 1% heat-inactivated pooled macaque serum. PBMC were cultured in 96-well round-bottom plates (2 × 105 cells in 200 μl per well) in the presence of BPL-RSV, BPL-MV, or BPL-Vero antigen at previously determined optimal concentrations. Each lymphoproliferation assay was carried out in duplicate, and the resulting culture supernatants (125 μl per well) were harvested after 3 and 5 days, respectively, and frozen. The supernatant was replaced with 75 μl of culture medium, 3H-labeled thymidine (0.5 μCi per well) was added, and cell-associated radioactivity was measured the following day.
Macaque cytokines (interleukin-2 [IL-2], IL-4, IL-5, IL-10, IL-13, and gamma interferon [IFN-γ]) were measured in culture supernatants by macaque-specific enzyme-linked immunosorbent assay (ELISA) systems (U-Cytech, Utrecht, The Netherlands) in accordance with the manufacturer's instructions. IL-2 and IFN-γ were measured in supernatants harvested after 3 days of culture, while IL-4, IL-5, IL-10, and IL-13 were measured in supernatants harvested after 5 days of culture.
A non-tissue-culture-adapted wild-type RSV A strain (nasal lavage of an infant hospitalized with RSV in 1996) was passaged in an RSV-seronegative cynomolgus macaque. Virus could be reisolated from throat swab samples between days 4 and 9 after infection and from a bronchoalveolar lavage (BAL) sample collected 7 days after challenge, but viral titers remained low. In an attempt to achieve higher titers, a second macaque was first immunocompromised by intramuscular administration of rabbit anti-human thymocyte globulin (ATG; National Institute of Public Health and the Environment, Bilthoven, The Netherlands; 80 mg/kg of body weight). Two days after ATG administration, almost all lymphocytes had been depleted from peripheral blood, as measured by a routine hematology analyzer (Sysmex; Myco Instrumentation Source, Renton, Wash.). At this point the animal was inoculated with a positive throat swab sample and a BAL sample from the first animal (intranasally and intratracheally, respectively). The total challenge dose as estimated from isolations on HEp-2 cells was 2 × 103 TCID50. ATG administration was continued at days 0, 2, and 6 after infection. RSV could be reisolated over a prolonged period of time (until day 21 after infection), but virus titers still remained relatively low. Pooled throat swab samples of this animal collected 6, 9, and 12 days after infection were used to inoculate another ATG-treated cynomolgus macaque with virtually identical results. Virus could be reisolated until day 26 after infection, but titers remained below 104 TCID50 per ml. An isolate from BAL cells of the third monkey was passaged three times in vitro in tertiary cynomolgus macaque kidney cells and subsequently aliquotted and stored at −135°C. The titer of this challenge stock as measured on HEp-2 cells was 2.4 × 106 TCID50 per ml. The challenge virus stock (as well as the FI-RSV and FI-MV preparations) was checked by reverse transcription PCR (RT-PCR) for the presence of coronavirus, enterovirus, human metapneumovirus, influenza A virus, influenza B virus, and rhinovirus genomic sequences, which could not be demonstrated.
The RSV challenge virus was thawed, sonicated in a cup sonicator for 3 min, and diluted in phosphate-buffered saline (PBS; 1 in 7.5). Subsequently, 3 ml (approximately 106 TCID50) was inoculated intratracheally, just below the larynx. EDTA-blood samples were collected at days 0, 6, 13, 17, and 22 after challenge for collection of plasma and PBMC. BAL samples were collected 6 days before challenge and at days 3, 6, 9, and 13 after challenge. Chest X-ray photographs were made at days 0, 3, 6, and 9 after challenge, and animals RS1 and RS2 were given chest X rays at day 12 after challenge.
BAL samples were collected by intratracheal infusion and subsequent recovery of 10 ml of PBS with a flexible catheter (Cordis). BAL samples were pelleted, resuspended in PBS, and counted. For RT-PCR, 5 × 105 BAL cells were frozen as a dry pellet at −70°C. Cytospin slides were later stained (May-Grünwald-Giemsa), and differential cell counts were obtained by light microscopy, with 1,000 cells per slide counted.
RNA from 5 × 105 BAL cells was isolated with the High Pure viral RNA kit (Roche Diagnostics, Almere, The Netherlands). Tenfold dilutions (100 to 10−4) were prepared in RNase-free water, and RT-PCR was performed using a single-tube reaction. The forward primer was 5′-TTAACCAGCAAAGTGTTA-3′ (RSV fusion protein gene positions 567 to 584), and the reverse primer was 5′-TTTGTTATAGGCATATCATTG-3′ (complementary to positions 808 to 788). PCR products were hybridized with a 32P-labeled oligonucleotide probe (5′-GACTACTAGAGATTACCAGGG-3′; F gene positions 697 to 711). A positive RT-PCR was defined as an amplified fragment of the correct size (243 bp) which hybridized with the specific probe. Results are shown as the highest RNA dilution at which the RT-PCR was still found positive.
Necropsy samples were stored in 10% neutral-buffered formalin (lungs after inflation with formalin), embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. For quantification of eosinophils in different parts of the respiratory tract, cells in 10 arbitrarily chosen high power fields (40× objective) were counted.
Data were analyzed by using a mixed-model analysis of variance after log transformation of the dependent variable (see Fig. ) or by using the Wilcoxon matched-pair signed-rank test (see Fig. and ).
FIG. 4. Semiquantitative RSV-specific RT-PCR signals (A and B) and percentages of eosinophils (C and D) in BAL samples collected at different time points after RSV challenge. Symbols represent the measurements of the individual animals (the correlation between (more ...)
FIG. 2. Proliferative PBMC responses of macaques vaccinated with FI-RSV (A to C) or FI-MV (D to F) after in vitro stimulation with BPL-RSV (A and D), BPL-MV (B and E), or BPL-Vero antigen (C and F). Arrows, times of vaccination; asterisks, times of RSV challenge. (more ...)
FIG. 5. IL-13 (A and B), IL-5 (C and D), and IFN-γ (E and F) levels in culture supernatants of PBMC collected from macaques vaccinated with FI-RSV (A, C, and E) or FI-MV (B, D, and F) at days 0 and 6 after RSV challenge (indicated on the x axes). PBMC (more ...)