Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus
© Liu et al; licensee BioMed Central Ltd. 2007
Received: 06 June 2007
Accepted: 10 July 2007
Published: 10 July 2007
To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19), level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies.
Summary of results for HRSV F mutations.
1.0 ± 0.0
2.3 ± 0.7
2.2 ± 0.5
2.6 ± 0.1
1.0 ± 0.1
1.6 ± 0.2
2.9 ± 0.5
1.6 ± 0.6
0.75 ± 0.02
1.5 ± 0.02
4.0 ± 0.2
1.1 ± 0.1
1.2 ± 0.2
1.6 ± 0.3
1.8 ± 0.1
1.5 ± 0.2
0.7 ± 0.07
1.4 ± 0.3
0.4 ± 0.01
0.5 ± 0.1
2.2 ± 0.5
1.0 ± 0.8
2.0 ± 0.6
0.6 ± 0.04
0.98 ± 0.19
Mutations of residues K272 and S275 (K272M, K272N, K272Q, K272T, and S275F) reduced palivizumab binding as expected based upon previous studies [11–14, 24], yet retained binding by MAb19 and ch101F confirming that MAb19 and ch101F recognize a different epitope (site IV/V/VI) than palivizumab (site II). These mutations had fusion activity similar to or greater than WT. It is tempting to speculate that this may partially explain the observed increase in replicative fitness of the palivizumab escape mutant MP4 (change of K272 to M)  as this mutation had increased fusion activity (2.3 fold relative to WT).
Mutation of R429 to S reduced MAb19 binding (3.9%) as expected based upon previous studies [15, 18], yet retained binding by palivizumab confirming that MAb19 and palivizumab recognize different epitopes. Mutation of R429 to K similarly reduced MAb19 binding (11.2%). Binding of ch101F to R429S was somewhat reduced (44.6%), while binding of ch101F to R429K was largely unaffected (72.5% relative to WT) suggesting that these two mAbs recognize somewhat distinct epitopes. Mutation of R429 to S had no effect upon fusion activity. However, interestingly, the structurally conservative mutation of R429 to K caused a 4 fold increase in the fusion activity of the F protein, although this mutant was not identified during a selection of MAb19 escape mutants .
Previous results had identified K433 as a critical residue for the binding of ch101F. Mutation of K433 to D, L, N, Q, R, or T abolished ch101F binding as previously reported , but, with the exception of K433D and K433R, modest to no effects upon palivizumab binding. These mutations also reduced the binding of MAb19 to various degrees highlighting the complexity of antigenic site IV/V/VI. It is interesting to note that while mutation of K433 to threonine had such a marked effect upon ch101F binding, mutation of K433 to the structurally similar serine had little to no effect. Mutation of K433 either increased fusion activity (K433R, K433N), reduced it by 50% (K433D, K433L, K433T), or had little to no effect (K433Q, K433S). Mutation K433D appeared to reduce binding of all three mAbs suggesting that this mutation was not efficiently expressed on the cell surface as the other mutations, which may account for its reduced fusion activity. Mutations T400A, C422S , N428D, and N428Q around the site IV/V/VI region had no effect upon mAb binding or fusion activity which show this is not a region of F protein hypersensitive to mutations.
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. Antibody escape mutants selected with ch101F revealed only a single change in lysine residue 433 to threonine suggesting that there are additional constraints on this region of the protein that limit which amino acids changes are tolerated in this region. Furthermore, the only change identified for antibody escape mutant viruses selected with MAb19 was a single change at R429 to S. Interestingly, it appears that resistance to mAbs which map to antigenic site IV/V/VI requires more passages in vitro for selection ; however, the relative fitness of site II and site IV/V/VI escape mutants would need to be assessed in parallel to determine if this is true. The F protein shows a high degree of conservation both within and between subgroup A and B, as well as with bovine RSV. Given this high degree of conservation, it was somewhat surprising that the functional analysis of the fusion activity of the panel of mutations described here revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region. These changes included those identified from the selection of mAb escape mutants as well as other residue changes not identified in escape mutants. These results suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein. Although the F protein is the only virion surface protein required for fusion and viral entry [25–27], the F protein is also essential for the formation of mature virion particles [26, 28, 29]. It is possible that the RNA sequence of this region contains some critical function, and/or that mutations in this region of the F protein impact some other unknown function(s) essential for virus growth. If these mutation affect the F protein, they would also suggest that antibodies such as MAb19 and ch101F may not neutralize virus solely by inhibition of fusion. Additional studies may provide direct information on the mechanism of action of HRSV neutralizing antibodies directed against the F protein such as ch101F.
A thorough characterization of the epitopes on the F protein for HRSV neutralizing mAbs may provide insights into the mechanisms of action by which mAbs against the HRSV F protein neutralize virus as well as a better understanding of the mechanism by which the F protein mediates cell fusion. Such studies may provide additional insights into the function of the F protein, and help in the selection and development of clinical candidates, such as ch101F, for next generation antibody-based prophylaxis and therapy for HRSV infections.
Human respiratory syncytial virus
protein fusion protein
We thank Geraldine Taylor and Jose Melero for generously providing mAb19 hybridoma supernatant, and 101F antibody, as well as helpful discussions and comments.
- Stensballe LG, Devasundaram JK, Simoes EA: Respiratory syncytial virus epidemics: the ups and downs of a seasonal virus. Pediatr Infect Dis J. 2003, 22: S21-32. 10.1097/00006454-200302001-00004.PubMedGoogle Scholar
- Anderson LJ, Bingham P, Hierholzer JC: Neutralization of respiratory syncytial virus by individual and mixtures of F and G protein monoclonal antibodies. J Virol. 1988, 62: 4232-4238.PubMedPubMed CentralGoogle Scholar
- Beeler JA, van Wyke Coelingh K: Neutralization epitopes of the F glycoprotein of respiratory syncytial virus: effect of mutation upon fusion function. J Virol. 1989, 63: 2941-2950.PubMedPubMed CentralGoogle Scholar
- Garcia-Barreno B, Palomo C, Penas C, Delgado T, Perez-Brena P, Melero JA: Marked differences in the antigenic structure of human respiratory syncytial virus F and G glycoproteins. J Virol. 1989, 63: 925-932.PubMedPubMed CentralGoogle Scholar
- Trudel M, Nadon F, Seguin C, Payment P, Talbot PJ: Respiratory syncytial virus fusion glycoprotein: further characterization of a major epitope involved in virus neutralization. Can J Microbiol. 1987, 33: 933-938.PubMedView ArticleGoogle Scholar
- Johnson S, Oliver C, Prince GA, Hemming VG, Pfarr DS, Wang SC, Dormitzer M, O'Grady J, Koenig S, Tamura JK, Woods R, Bansal G, Couchenour D, Tsao E, Hall WC, Young JF: Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J Infect Dis. 1997, 176: 1215-1224.PubMedView ArticleGoogle Scholar
- Feltes TF, Cabalka AK, Meissner HC, Piazza FM, Carlin DA, Top FH, Connor EM, Sondheimer HM: Palivizumab prophylaxis reduces hospitalization due to respiratory syncytial virus in young children with hemodynamically significant congenital heart disease. J Pediatr. 2003, 143: 532-540. 10.1067/S0022-3476(03)00454-2.PubMedView ArticleGoogle Scholar
- DeVincenzo JP, Hall CB, Kimberlin DW, Sanchez PJ, Rodriguez WJ, Jantausch BA, Corey L, Kahn JS, Englund JA, Suzich JA, Palmer-Hill FJ, Branco L, Johnson S, Patel NK, Piazza FM: Surveillance of clinical isolates of respiratory syncytial virus for palivizumab (Synagis)-resistant mutants. J Infect Dis. 2004, 190: 975-978. 10.1086/423213.PubMedView ArticleGoogle Scholar
- Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener PA: Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J Mol Biol. 2007, 368: 652-665. 10.1016/j.jmb.2007.02.024.PubMedView ArticleGoogle Scholar
- Young JF, Koenig S, Johnson LS: Anti-RSV Antibodies. 2004, USA, MedImmune, Inc.Google Scholar
- Zhao X, Liu E, Chen FP, Sullender WM: In vitro and in vivo fitness of respiratory syncytial virus monoclonal antibody escape mutants. J Virol. 2006, 80: 11651-11657. 10.1128/JVI.01387-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhao X, Sullender WM: In vivo selection of respiratory syncytial viruses resistant to palivizumab. J Virol. 2005, 79: 3962-3968. 10.1128/JVI.79.7.3962-3968.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhao X, Chen FP, Megaw AG, Sullender WM: Variable resistance to palivizumab in cotton rats by respiratory syncytial virus mutants. J Infect Dis. 2004, 190: 1941-1946. 10.1086/425515.PubMedView ArticleGoogle Scholar
- Zhao X, Chen FP, Sullender WM: Respiratory syncytial virus escape mutant derived in vitro resists palivizumab prophylaxis in cotton rats. Virology. 2004, 318: 608-612. 10.1016/j.virol.2003.10.018.PubMedView ArticleGoogle Scholar
- Taylor G, Stott EJ, Furze J, Ford J, Sopp P: Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies. J Gen Virol. 1992, 73 ( Pt 9): 2217-2223.View ArticleGoogle Scholar
- Everitt DE, Davis CB, Thompson K, DiCicco R, Ilson B, Demuth SG, Herzyk DJ, Jorkasky DK: The pharmacokinetics, antigenicity, and fusion-inhibition activity of RSHZ19, a humanized monoclonal antibody to respiratory syncytial virus, in healthy volunteers. J Infect Dis. 1996, 174: 463-469.PubMedView ArticleGoogle Scholar
- Wyde PR, Moore DK, Hepburn T, Silverman CL, Porter TG, Gross M, Taylor G, Demuth SG, Dillon SB: Evaluation of the protective efficacy of reshaped human monoclonal antibody RSHZ19 against respiratory syncytial virus in cotton rats. Pediatr Res. 1995, 38: 543-550. 10.1203/00006450-199510000-00012.PubMedView ArticleGoogle Scholar
- Arbiza J, Taylor G, Lopez JA, Furze J, Wyld S, Whyte P, Stott EJ, Wertz G, Sullender W, Trudel M, et al: Characterization of two antigenic sites recognized by neutralizing monoclonal antibodies directed against the fusion glycoprotein of human respiratory syncytial virus. J Gen Virol. 1992, 73 ( Pt 9): 2225-2234.View ArticleGoogle Scholar
- Lopez JA, Bustos R, Orvell C, Berois M, Arbiza J, Garcia-Barreno B, Melero JA: Antigenic structure of human respiratory syncytial virus fusion glycoprotein. J Virol. 1998, 72: 6922-6928.PubMedPubMed CentralGoogle Scholar
- Wu SJ, Schmidt A, Beil EJ, Day ND, Branigan PJ, Liu C, Gutshall LL, Palomod C, Furze J, Taylor G, Melero JA, Tsui P, Del Vecchio AM, Kruszynski M: Characterization of the epitope for anti human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches. J Gen Virol. submitted:
- Day ND, Branigan PJ, Liu C, Gutshall LL, Luo J, Melero JA, Sarisky RT, Del Vecchio AM: Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function. Virol J. 2006, 3: 34-10.1186/1743-422X-3-34.PubMedPubMed CentralView ArticleGoogle Scholar
- Cardenas S, Auais A, Piedimonte G: Palivizumab in the prophylaxis of respiratory syncytial virus infection. Expert Rev Anti Infect Ther. 2005, 3: 719-726. 10.1586/14787184.108.40.2069.PubMedView ArticleGoogle Scholar
- Johnson S, Griego SD, Pfarr DS, Doyle ML, Woods R, Carlin D, Prince GA, Koenig S, Young JF, Dillon SB: A direct comparison of the activities of two humanized respiratory syncytial virus monoclonal antibodies: MEDI-493 and RSHZl9. J Infect Dis. 1999, 180: 35-40. 10.1086/314846.PubMedView ArticleGoogle Scholar
- Crowe JE, Firestone CY, Crim R, Beeler JA, Coelingh KL, Barbas CF, Burton DR, Chanock RM, Murphy BR: Monoclonal antibody-resistant mutants selected with a respiratory syncytial virus-neutralizing human antibody fab fragment (Fab 19) define a unique epitope on the fusion (F) glycoprotein. Virology. 1998, 252: 373-375. 10.1006/viro.1998.9462.PubMedView ArticleGoogle Scholar
- Karron RA, Wright PF, Crowe JE, Clements-Mann ML, Thompson J, Makhene M, Casey R, Murphy BR: Evaluation of two live, cold-passaged, temperature-sensitive respiratory syncytial virus vaccines in chimpanzees and in human adults, infants, and children. J Infect Dis. 1997, 176: 1428-1436.PubMedView ArticleGoogle Scholar
- Teng MN, Collins PL: Identification of the respiratory syncytial virus proteins required for formation and passage of helper-dependent infectious particles. J Virol. 1998, 72: 5707-5716.PubMedPubMed CentralGoogle Scholar
- Teng MN, Whitehead SS, Collins PL: Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo. Virology. 2001, 289: 283-296. 10.1006/viro.2001.1138.PubMedView ArticleGoogle Scholar
- Techaarpornkul S, Barretto N, Peeples ME: Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol. 2001, 75: 6825-6834. 10.1128/JVI.75.15.6825-6834.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Techaarpornkul S, Collins PL, Peeples ME: Respiratory syncytial virus with the fusion protein as its only viral glycoprotein is less dependent on cellular glycosaminoglycans for attachment than complete virus. Virology. 2002, 294: 296-304. 10.1006/viro.2001.1340.PubMedView ArticleGoogle Scholar
- Branigan PJ, Liu C, Day ND, Gutshall LL, Sarisky RT, Del Vecchio AM: Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein. Virol J. 2005, 2: 54-10.1186/1743-422X-2-54.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.