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Short Communication |
1 Protein Engineering, Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087, USA
2 Immunobiology, Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087, USA
3 Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain
4 Institute for Animal Health, Compton, Newbury, Berkshire RG20 7NN, UK
5 Molecular Discovery Technologies, Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087, USA
Correspondence
Marian Kruszynski
mkruszy2{at}cntus.jnj.com
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), causing up to 126 000 hospitalizations annually for bronchiolitis in the USA, with an estimated 500 associated deaths (Shay et al., 1999, 2001). HRSV is a cause of severe respiratory illness in the elderly (reviewed by Dowell et al., 1996; Han et al., 1999) and haematopoietic stem-cell and solid organ transplant recipients (Ebbert & Limper, 2005; Machado et al., 2003; Whimbey & Ghosh, 2000).
Three viral transmembrane glycoproteins (F, G and SH) are found on the surface of the virus particle in the viral envelope (Huang et al., 1985). Protective and neutralizing antibodies to HRSV are directed against the viral fusion (F) and G glycoproteins, with F representing the major protective antigen (Anderson et al., 1988; Beeler & van Wyke Coelingh, 1989; Garcia-Barreno et al., 1989; Trudel et al., 1987). The F glycoprotein is highly conserved (89 % amino acid identity) between the A and B subgroups of HRSV and, in contrast to the G glycoprotein, protective antibody responses against the F protein are cross-reactive between subgroups.
Currently, there is no prophylactic vaccine available for HRSV, and ribavirin, the only approved antiviral for treatment of serious HRSV infection, has minimal efficacy and safety concerns (Ventre & Randolph, 2004). Palivizumab (Synagis), an HRSV-neutralizing humanized monoclonal antibody (mAb) directed against the HRSV F protein, has been approved for immunoprophylaxis of infants of less than 35 weeks gestational age and those with bronchopulmonary dysplasia or congenital heart disease, and reduces hospitalization rates due to HRSV in these groups by 78, 39 and 45 %, respectively (Feltes et al., 2003; Johnson et al., 1997). An affinity-matured version of palivizumab (motavizumab, Numax) is currently in clinical development (Wu et al., 2005, 2007). Although resistance to palivizumab does not appear to be a clinical issue as yet (DeVincenzo et al., 2004), wider use of antibody prophylaxis may increase this potential. As motavizumab was derived from palivizumab, viral mutant resistance patterns are expected to be similar. As such, the development of other potent antibodies directed to other epitopes may be clinically useful.
Murine mAb 101F is a potent, neutralizing anti-HRSV murine antibody. Chimeric 101F (ch101F) was generated recombinantly by grafting the variable regions of the parental murine mAb (101F) onto human IgG1
constant frameworks. Competition binding of the parental murine antibody to other previously described escape mutants (Arbiza et al., 1992) suggested that, unlike palivizumab and motivizumab, the ch101F epitope was contained within the antigenic site IV, V, VI (approx. aa 422–438) of the F glycoprotein (data not shown) (Arbiza et al., 1992; Lopez et al., 1998). As further affinity maturation of ch101F would generate a potential next-generation antibody-based prophylaxis and therapy for HRSV infections, a detailed characterization of the ch101F epitope would be useful. To characterize the epitope of ch101F, we determined the binding of ch101F to a panel of synthetic peptides, as well as a panel of point mutations in the context of the full-length F protein expressed transiently on the cell surface. These results were confirmed by selecting escape mutants resistant to ch101F neutralization and mapping the changes associated with ch101F escape.
As the use of synthetic peptides to examine the binding of mAbs has been successful in determining the location of specific epitopes on the F protein (Arbiza et al., 1992; Beeler & van Wyke Coelingh, 1989; Bourgeois et al., 1991; Crowe et al., 1998; Martin-Gallardo et al., 1991), a series of peptides derived from antigenic site IV, V, VI [residues 422–438 of the F protein (HRSV, A2 strain)] were synthesized as described previously (Kruszynski et al., 2006). The peptide 
-amino group was selected as a site for biotinylation, with a hydrophilic spacer of four ethylenoxy units (PEG4) inserted between the biotin and the -amino group of the peptide. All peptides contained free C-terminal carboxylic groups. Analytical HPLC and capillary electrophoresis showed a high degree (>97 %) of peptide homogeneity (data not shown), and surface-enhanced laser desorption–ionization (SELDI) and matrix-assisted laser desorption–ionization (MALDI) mass spectrometry gave the expected molecular masses. The sequences of the peptides used in this study are shown in Table 1. The synthetic peptides were coated on ELISA plates (Meso Scale Discovery Inc.) and incubated with serial dilutions of either ch101F or palivizumab, which recognizes site II of the F glycoprotein (Arbiza et al., 1992; Beeler & van Wyke Coelingh, 1989), as a control. The binding results for the peptides are shown in Fig. 1. Palivizumab bound these peptides with levels similar to background, as expected (data not shown). The relative binding activities for palivizumab to synthetic peptides were <0.7 %, compared with the binding activity of ch101F to peptide 5. Peptide 1, comprising HRSV F protein residues 422–438, was bound strongly by ch101F, as shown in Fig. 1(a). Truncation of residues from the N terminus of peptide 1 (peptides 2, 3 and 4) reduced binding to ch101F significantly, indicating that these residues contributed to the mAb binding. Removal of the C-terminal asparaginyl–glycine from peptide 1 generated peptide 5, which bound ch101F with a signal comparable to that for peptide 1, suggesting that the asparagine and glycine residues at positions 437 and 438 are not critical for ch101F binding. However, further truncation from the C terminus significantly reduced (peptide 6) or abolished (peptide 7) binding to ch101F. Truncations of peptide 1 from both N and C termini (peptide 8, comprising HRSV F protein sequence 423–436) shows moderately reduced, but still significant, ch101F binding, compared with peptides 5 and 1 (Fig. 1), defining the minimal amino acid sequence required for antibody recognition. Further, to identify specific residues within residues 423–436 that contribute to the binding of ch101F, a series of substitution peptides were created and evaluated for ch101F binding by ELISA (Fig. 1b). Mutation of basic residues R429 and K433 reduced binding significantly, as shown by substitution of R429 to S or E and substitution of K433 to T or E. Another positively charged residue, K427, makes a minor contribution to binding, as demonstrated by the substitution of K427 to E (Fig. 1b).
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; Day et al., 2006). The relative expression levels of all mutant constructs that were assayed appeared to be similar to those of wild-type F protein, indicating that the specific point mutations introduced in the predicted ch101F antibody-binding region did not create gross perturbations in expression, processing or folding (data not shown). The list of expressed full-length HRSV F protein mutants is K427D, K427Q, R429S, R429K, K433D, K433T, K433S, K433L, K433N, K433Q and K433R. The binding of ch101F was assayed by ELISA using human 293T cells transiently transfected with plasmids expressing either the wild type or the panel of individual HRSV F point mutations. Palivizumab was used as a positive binding control, as this mAb recognizes an epitope outside the region being mutated (Arbiza et al., 1992; Beeler & van Wyke Coelingh, 1989). As ch101F and palivizumab are both human IgG1 antibodies, we were able to use an ELISA with the same secondary antibody and detection reagents to allow a direct comparison of binding. Values were calculated as percentages relative to wild-type HRSV F after adjusting for background signal from the vector-only control. Fig. 2(a) shows binding of ch101F and palivizumab to wild type, K427, R429 and K433 mutants. The binding of ch101F to R429S was reduced by approximately 50 % in comparison with palivizumab. The binding of ch101F was eliminated by mutations K433L, K433N and K433T and reduced by mutations K433Q, K433R and K433S, whilst the binding of palivizumab to all of the mutants, except K433D, was unaffected, indicating that these mutant proteins were expressed. Binding for both ch101F and palivizumab at K433D was dramatically reduced, suggesting that this protein was not expressed, in agreement with the metabolic labelling and immunoprecipitation data, which confirmed lack of expression of the K433D mutant (data not shown). In addition, ch101F binds well to the cell lysate that contain mutations at site II (data not shown). Together, the results confirm that ch101F and palivizumab have different epitope specificity and that residues K429 and K433 are key for ch101F recognition.
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), was classified as a mutant and used to produce ELISA antigen after a further round of plaque purification. Infected cells derived from either wild-type HRSV or two independently isolated ch101F escape virus mutants (RS/837 and RS/838) were treated with 0.5 % (w/v) NP-40 detergent to yield cell lysates, which were used as an ELISA antigens and tested for their ability to react with a panel of anti-HRSV F protein mAbs (Garcia-Barreno et al., 1989; Taylor et al., 1984; Trudel et al., 1987). The results are presented in Fig. 2(b)
. Lysates prepared from wild-type HRSV-infected cells reacted with all antibodies tested, whereas lysates prepared from escape mutant RS/837- and RS/838-infected cells reacted with all antibodies tested except ch101F. To determine the F protein sequence of the mutants, cDNA was prepared from the total cytoplasmic RNA of infected cell lysates. PCR products were generated by using Pfu proofreading thermostable polymerase (Promega) and two synthetic oligonucleotides: HRSVA2for (5'-GGGGCAAATAACAATGGAGTTGC-3') and HRSVA2rev (5'-GACAGATGGGTTGTCTATGAGCAG-3'). PCR products of the expected size were isolated after electrophoresis and subjected to sequence determination. Analysis of the sequence of the F protein coding regions of the two escape mutants and the parental A2 strain indicated that both mutants contained a single nucleotide change of A to C at nt 1311, resulting in an amino acid substitution of lysine 433 to threonine (K433T) compared with the parental wild-type A2 strain. This finding confirms the results obtained from the other methodologies.
In summary, the epitope on the HRSV F glycoprotein recognized by a neutralizing mAb, ch101F, was mapped by using three complementary approaches: (i) ELISA binding of the synthetic peptides covering the aa 422–438 sequence region of the antigenic site IV, V, VI of the F protein; (ii) ELISA binding to recombinant F proteins expressed at the cell surface and containing mutations at positions 427, 429 and 433; and (iii) selection of antibody-escape mutant viruses. ELISA assays of ch101F binding showed identical specificity for the synthetic peptides and recombinant HRSV F protein mutants expressed on the surface of transfected cells. These assays demonstrated that the linear peptides designed from the sequence of domain IV, V, VI reproduced the epitope of that antibody. We showed that synthetic peptide 5 (aa 422–436; CTASNKNRGIIKTFS) gave the highest binding signal to ch101F, and a minimum peptide sequence (aa 423–436) was sufficient for recognition. As demonstrated, R429 and K433 were critical for binding ch101F, whilst another basic residue, K427, showed a minor contribution to binding. The critical role of K433 was confirmed by using ELISA, demonstrating reduced or abolished binding of ch101F to recombinantly expressed F protein mutants containing single amino acid substitutions at positions 427, 429 and 433 expressed on cell surfaces, and loss of binding to ch101F antibody-escape mutants containing an amino acid substitution at position 433. These data also confirmed that ch101F recognizes an epitope distinct from that recognized by palivizumab, and appears to bind an epitope that overlaps with that of mAb 7.936, as an identical sequence change was found in a neutralization-escape mutant selected with this antibody (Lopez et al., 1998). The classical approach of selecting antibody-escape mutant viruses is limited by the impact of such mutations upon viral growth fitness and, in general, provides a much more limited spectrum of residue changes. The methods described here provide a rapid means of determining residues critical for antibody binding, may provide additional insights into the function of the F protein and should help in the selection and development of clinical candidates, such as ch101F, for next-generation antibody-based prophylaxis and therapy for HRSV infections.
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REFERENCES |
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Received 1 December 2006;
accepted 21 June 2007.
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