Article Figures & Data
Figures
- Fig. 1.
Insertion of the F and G genes of RSV strain B1 into recombinant strain A2. (A) The G and F genes of RSV B1 (stippled rectangles) were amplified by PCR as a single cassette with oligonucleotide primers designed to create upstream PacI and downstream SphI restriction sites. Following an additional modification described below, this cassette was cloned into the naturally occurring PacI site and the previously introducedSphI site of the parental RSV A2 cDNA plasmid (6). The RSV genes are shown as open rectangles; the GS and GE transcription signals are shown as shaded and solid bars, respectively. (B) In order to stabilize the G gene end signal of the B1 cDNA, the sequence in this region was modified as shown (new): the third nucleotide in each of the last 11 codons of the G ORF was changed without altering the amino acid coding assignment; the termination codon of the G ORF was changed to an alternative termination codon; 4 nucleotides were introduced into the downstream nontranslated region of the G gene between the ORF and the gene end signal; the G gene end signal was made identical to that of the F gene of strain A2 by substituting 2 nucleotides and deleting a nucleotide in the A tract; and the G-F intergenic region was shortened by 47 nucleotides and anMfeI restriction site was introduced. The sequence is divided into triplets at coding nucleotides, with the amino acid assignment shown directly below each codon. Nucleotides which differ from the B1 wt sequence are underlined, and the introducedMfeI restriction site is shown in boldface italics.
- Fig. 2.
Kinetics of replication in the upper (A) and lower (B) respiratory tracts of chimpanzees inoculated intranasally and intratracheally with wt control viruses A2 and B1 and chimeric viruses rAB and rABcp248/404/1030. The number of animals in each group (n) is shown. The dose of virus administered intranasally and intratracheally to each group was 5.0 log10 PFU/ml, except for wt RSV A2, which was administered at 4.0 log10 PFU/ml (11). Nasopharyngeal-swab and tracheal-lavage samples were collected on the days shown and titered by plaque assay on HEp-2 cell monolayers. The limit of detection for this assay is 0.7 log10 PFU/ml of sample. The data for wt RSV A2 and wt RSV B1 are from Crowe et al. (11, 12).
Tables
- Table 1.
Temperature sensitivity and subgroup identity ofwt rAB chimeric RSV and derivatives containing attenuating mutationsa
↵a The A2-derived mutations contained in the various rAB derivatives have been described previously and were derived from two different biologically derived viruses,cpts248/404 (16) and cpts530/1030 (41). The mutations were as follows: the cpgenotype consists of five amino acid substitutions in the N (V267I), F (E218A, T523I), and L (C319Y, H1690Y) proteins, which are employed as a set and confer a non-ts attenuated phenotype (8, 42). Here, because of the replacement of the F gene,cp involves only the changes in N and L. The 248 and 1030 mutations involve amino acid substitution Q831L and Y1321N, respectively, in the L protein, each of which confers the tsand attenuation phenotypes (40, 41). The 404 genotype involves an amino acid substitution in L, D1183E, which is not associated with the ts or attenuation phenotype, and a nucleotide substitution in the GS signal of the M2 gene, with confers the ts and attenuation phenotypes (40). The ΔSH mutation involves deletion of the SH gene, which confers a modest attenuation phenotype in vivo (2).
b Shut-off temperature is defined as the lowest restrictive temperature at which a 100-fold or greater reduction of titer is observed. The virus titers at the shut-off temperature are boxed.
c Infected cell monolayers were immunostained with either RSV F monoclonal antibody (MAb) 92-11C (subgroup A specific) or 102-10B (subgroup B specific) and scored visually for staining. +, staining; −, nonstaining.
d Pinpoint plaque size.
- Table 2.
rAB chimeric RSV derivatives are attenuated in cotton rats
Virusa No. of rats Mean virus titer (log10PFU/g of tissue)b (Duncan grouping)c in: Nasal turbinates Lung A2wt 6 6.48 ± 0.03 (A) 6.08 ± 0.06 (A) B1 wt 6 6.43 ± 0.05 (A) 6.00 ± 0.05 (A) rAB 5 2.62 ± 0.11 (B) 2.50 ± 0.27 (B) rABΔSH 6 2.38 ± 0.24 (B) 2.21 ± 0.40 (B) rABcp 6 3.48 ± 0.56 (C) 3.45 ± 0.23 (C) rABcpΔSH 6 3.83 ± 0.11 (C) 4.00 ± 0.06 (C) ↵a Rats were administered 6.0 log10PFU intranasally under light anesthesia on day 0 and sacrificed on day 4.
↵b Virus titer was determined in the nasal turbinate and lung tissues and is shown as mean titer ± standard error.
↵c Mean virus titers were assigned to statistically similar groups (A to C) by Duncan’s multiple range test (α = 0.05). Therefore, the means in each column with different letters are significantly different.
- Table 3.
The wt rAB chimeric virus has awt-like phenotype in chimpanzees and can be attenuated by inclusion of mutations derived from RSV subgroup A vaccine candidates
Virusa No. of animals Doseb (log10PFU per site) Mean peak virus titer (log10PFU/ml) Rhinorrhea scoreb Mean serum neutralizing antibody titerc (reciprocal log2) Nasopharyngeal swab Tracheal lavage Peak Mean Day 0 Day 28 wt RSV B1 4d 4.0 2.98 ± 0.26 1.37 ± 0.67 3.0 2.5 <3.3 12.8 wt RSV B1 2d 5.0 2.75 ± 0.25 2.45 ± 1.45 2.0 1.5 <3.3 13.0 rAB 4 5.0 4.28 ± 0.12 4.05 ± 0.17 3.0 2.5 <3.3 12.3 rABcp248/404/1030 3 5.0 2.03 ± 0.19 <0.7 ± 0.00 0.0 0.0 <3.3 10.5 ↵a Chimpanzees were inoculated by the intranasal and intratracheal routes with the indicated amount of virus in a 1-ml inoculum per site. Nasopharyngeal-swab samples were collected daily for 12 days, and tracheal-lavage samples were collected on days 2, 5, 6, 8, and 12.
↵b The amount of rhinorrhea was estimated daily and assigned a score (0 to 4) that indicated extent and severity: 0, none; 1, trace; 2, mild; 3, moderate; 4, severe.
↵c Serum RSV-neutralizing antibody titers were determined by a complement-enhanced 60% plaque reduction assay withwt RSV B1 and HEp-2 cell monolayer cultures incubated at 37°C (4). RSV-seronegative chimpanzee serum used as a negative control had a neutralizing antibody titer of <3.3 log2. Seropositive adult human serum used as a positive control had a neutralizing antibody titer of 11.0 log2.
↵d Historic control animals from Crowe et al. (12).
- Table 4.
Chimeric virus rABcp248/404/1030 is protective against challenge with rAB in the upper and lower respiratory tracts of chimpanzees
Immunizing virusa Inoculum dose No. of chimpanzees Mean virus titer following RSV challenge (log10PFU/ml) Peak rhinorrhea scoreb Nasopharyngeal swab Tracheal lavage Day 3 Day 5 Day 7 Day 3 Day 5 Day 7 rABcp248/404/1030 5.0 2 <0.7 ± 0.00 1.00 ± 0.40 <0.7 ± 0.00 <0.7 ± 0.00 <0.7 ± 0.00 <0.7 ± 0.00 0 None 4 3.12 ± 0.16 3.72 ± 0.14 4.28 ± 0.12 <0.7 ± 0.00 3.78 ± 0.26 3.80 ± 0.15 3 ↵a Chimpanzees were inoculated by the intranasal and intratracheal routes with the indicated amount of virus in a 1-ml dose per site. After 28 days, the chimpanzees were administered 5.0 log10PFU of rAB by the intranasal and intratracheal routes. Nasopharyngeal-swab and tracheal-lavage samples were then collected after 3, 5, and 7 days.
↵b The amount of rhinorrhea was estimated daily and assigned a score (0 to 4) that indicated extent and severity: 0, none; 1, trace; 2, mild; 3, moderate; 4, severe.
