Identification of multiple protective epitopes (protectopes) in the central conserved domain of a prototype human respiratory syncytial virus G protein

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously
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  J OURNAL OF  V IROLOGY ,0022-538X/99/$04.00  0July 1999, p. 5637–5645 Vol. 73, No. 7Copyright © 1999, American Society for Microbiology. All Rights Reserved. Identification of Multiple Protective Epitopes (Protectopes) inthe Central Conserved Domain of a Prototype HumanRespiratory Syncytial Virus G Protein HE´LE`NE PLOTNICKY-GILQUIN, LILIANE GOETSCH, THIERRY HUSS, THIERRY CHAMPION, ALAIN BECK, JEAN-FRANC¸OIS HAEUW, THIEN NGOC NGUYEN, JEAN-YVES BONNEFOY,NATHALIE CORVAI¨ A,  AND  ULTAN F. POWER* Centre d’Immunologie Pierre Fabre, 74164 Saint-Julien-en-Genevois Cedex, France Received 22 December 1998/Accepted 14 April 1999  A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratorysyncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domainof streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratorytracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S.Stahl, M. Uhle´n, T. N. Nguyen, and H. Binz, Virology 230:155–166, 1997). Panels of monoclonal antibodies(MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domainimplicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptideimmunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely theURT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal serarevealed lower-level epitope usage within the central conserved region in the former, suggesting diminishedimmunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) thatmapped to the central conserved domain were recognized in man. Should these murine protectopes also beimplicated in human LRT protection, their clustering around the highly conserved cysteine noose region willhave important implications for the development of RSV vaccines. Respiratory syncytial virus (RSV) is a member of the genus  Pneumovirus  and the family  Paramyxoviridae . It is the principleetiologic agent of serious respiratory disease in infants and young children (reviewed in references 11 and 25). RSV ischaracterized by the ability to repeatedly infect the upper re-spiratory tracts (URTs) of humans throughout their lives, andan important role for this organism in severe respiratory illnessof immunocompromised adults and the elderly is becomingincreasingly evident (14–17, 19, 34). Furthermore, a primaryRSV infection in infants which results in serious lower respi-ratory tract (LRT) pathology does not necessarily prevent asecond serious infection (26). However, the frequency of seri-ous LRT disease in infants progressively diminishes upon sub-sequent infections, suggesting accumulation of LRT-protectiveimmunity upon repeated virus exposure (for a review, seereference 13). Despite the medical importance of this virus,many aspects of RSV-induced immunity remain unresolved.RSV encodes two major surface glycoproteins, G and F, which are incorporated into the viral envelope. The G proteinis a highly glycosylated type II glycoprotein that functions in viral attachment to an unknown cell receptor (31). The fusion(F) protein is a type I glycoprotein that mediates virus and cellmembrane fusion and syncytium formation (52). Both proteinsinduce neutralizing antibodies and protection against RSVchallenge in animal models (reviewed in reference 11). Despitethe high degree of conservation of the F protein (29), RSVclinical isolates were classified into two subgroups, A and B,based srcinally on the antigenic diversity of the G protein (2,35). Indeed, there is 47% amino acid sequence diversity be-tween prototype RSV subgroup A (RSV-A) and RSV-B Gproteins (28). Furthermore, up to 20% amino acid differencesin the G protein have been reported within the subgroups (7,48), with corresponding antigenic divergence also being evi-dent (20). This antigenic and genetic diversity among the RSVG proteins, together with the generally low-level immunoge-nicity of F and G proteins in infants less than 8 months old, ishypothesized to play an important role in repeat infections (3,8, 9, 20, 37, 46).Sequence analyses of RSV clinical isolates (both subgroups)revealed that the G protein contains two hypervariable regionsseparated by a central conserved domain (7, 20, 48). Whilemonoclonal antibody (MAb) epitopes have been mapped tomany regions of the G protein (reviewed in reference 33),several lines of evidence indicate that the C-terminal hyper- variable region is immunogenic and may even be immunodom-inant in the context of the whole G protein: (i) convalescent-phase human infant sera reacted with recombinant fragmentsof the C terminus in a strain-specific manner (10); (ii) themajority of reactivity escape mutants derived from a panel of Gprotein-specific murine MAbs were found to have mutations inthe variable C terminus (43); (iii) one escape mutant containeda frameshift mutation which resulted in a completely alteredC-terminal one-third of the molecule, and this mutant lostreactivity to many MAbs and, importantly, a polyclonal anti-serum raised against affinity-purified G protein (22); (iv) thegreatest differences between infecting and heterologous RSVgroups in terms of antibody titers are for anti-G protein anti- * Corresponding author. Mailing address: Centre d’ImmunologiePierre Fabre, 5 Ave. Napolean II, BP 497, 74164 Saint-Julien-en-Genevois Cedex, France. Phone: (33) 450.35.35.49. Fax: (33)450.35.35.90. E-mail:   onF  e b r  u ar  y  8  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   bodies (27); and (v) the native G protein induces subgroup-specific protection (41, 47). Immunodominance of the hyper- variable C terminus would concur with the hypothesis that Gprotein variability is implicated in repeated RSV infections. A corollary of this is that a single full-length native G protein would be of limited value for vaccine purposes.We (42) and others (45) have previously demonstrated thatrecombinant fragments containing G protein residues 130 to230 and 124 to 204, respectively, which contain the centralconserved domain, are sufficient to elicit protective immuneresponses in rodent models. Indeed, in contrast to the native Gprotein, the former induced protection against both RSV-A and -B prototype strains when injected in the context of therecombinant fusion protein BBG2Na. Trudel et al. (51)mapped a subgroup-specific protective epitope (protectope) tothe cysteine noose region (residues 174 to 187) by MAb pas-sive-transfer experiments and active immunization with a syn-thetic peptide (coupled to keyhole limpet hemocyanin [KLH]).Interestingly, an equivalent peptide derived from the bovineRSV G protein reduced the incidence of bovine RSV-associ-ated pneumonia in calves (5). An anti-G protein MAb thatreacts with both RSV-A and -B G proteins was shown to becross-protective after passive transfer (53) and mapped to res-idues 165 to 174 based on sequencing of neutralization escapemutants (54). Other groups have also mapped murine MAbepitopes to this central conserved region, although the role of these epitopes in protection is not yet known (for a review, seereference 33). Finally, human sera were found to react withsynthetic peptides derived from this region, indicating immu-nogenicity in humans (39). Therefore, direction of primaryimmune responses to the central conserved region, which maybe poorly immunogenic in the context of the whole G protein,is conceivable and may be important and sufficient for theinduction of LRT protection that is not RSV strain restricted.The goal of the present study was to dissect the structuralelements implicated in inducing protective immune responsesagainst RSV-A challenge. To do so, we generated a panel of MAbs and RSV-A G protein-derived synthetic peptides. MAbepitope mapping was undertaken by Pepscan analyses and/orpeptide reactivity. Protectopes were identified by MAb pas-sive-transfer studies and/or active immunization with carrier-protein-conjugated synthetic peptides in mice. Finally, mouseand human polyclonal sera were subjected to Pepscan analysesto determine epitope usage following BBG2Na immunizationand RSV infection in mice and humans, respectively. Interest-ingly, they were found to recognize common epitopes, some of  which were protective in mice.(This work was presented in part at the 16th Annual Meet-ing of the American Society for Virology, Bozeman, Mont., 19to 23 July 1997. MATERIALS AND METHODS Viruses, cells, and ELISA antigens.  Propagation of RSV-A (Long strain; ATCC VR-26 [American Type Culture Collection, Rockville, Md.]) in HEp-2cells (ECACC 86030501; European Collection of Animal Cell Cultures, PortonDown, Salisbury, United Kingdom), as well as the production of viral protein anduninfected-cell enzyme-linked immunosorbent assay (ELISA) antigens, was per-formed as previously described (42). Recombinant protein G2  Ca was producedby expressing and purifying BBG2  Ca as previously described (36), cleavingBBG2  Ca by cyanogen bromide hydrolysis into BB and G2  Ca components,and purifying the products by reverse-phase high-performance liquid chroma-tography (RP-HPLC). Proteins BBG2Na and G2Na were produced and purifiedas previously described (12). Expression and purification of the carrier proteins P40 and BB.  Gene assem-bly, vector construction, and P40 and BB protein expression in  Escherichia coli  were undertaken as previously described (38, 40). P40, an outer membraneprotein A from  Klebsiella pneumonia  with carrier-protein properties, was purifiedto homogeneity by two ion-exchange chromatography steps (24). BB, the albu-min binding domain of streptococcal protein G, was purified by affinity chroma-tography on albumin-Sepharose followed by cation-exchange chromatographyand RP-HPLC. Protein purity was  95% as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% homogeneous gels under reducingconditions. Synthetic peptides.  Synthesis of individual peptides was performed by a solid-phase method on an Applied Biosystems 433A synthesizer, using fluorenylme-thoxycarbonyl/  t -butyl (FMOC/tBu) chemistry. The synthesized peptides, cleavedfrom resin by trifluoroacetic acid in the presence of scavengers, were lyophilizedand purified by preparative RP-HPLC. The purity of the peptides was greaterthan 90% according to both RP-HPLC and free-zone capillary electrophoresisanalyses. Peptides G 174–187  C , G 172–187 , G 171–187 , G 144–159 Cys and CysG 144–159 ,G 190–204 Cys and CysG 190–204 , and G 164–176  and G 164–176  C  corresponded toresidues 174 to 187, 172 to 187, 171 to 187, 144 to 159, 190 to 204, and 164 to 176,respectively, of the RSV-A (Long) G protein (Fig. 1). Peptides G 144–159 Cys,CysG 144–159 , G 190–204 Cys, and CysG 190–204  each contain an extra cysteine residueadded either N terminally or C terminally to facilitate orientation of peptidecoupling to carrier proteins. Peptides G 172–187  and G 171–187 , each of whichcontains 4 cysteines, were obtained after oxidation of the crude mercaptoetha-nol-reduced derivatives with dimethyl sulfoxide (49). All three possible oxidizedisomers of the latter peptides were synthesized under various oxidation condi-tions, resulting in different ratios of isomers. Each isolated isomer was charac-terized by RP-HPLC, free-zone capillary electrophoresis, and electrospray massspectrometry after purification by preparative RP-HPLC. The different cysteinepairings were unambiguously established by enzymatic digestion, liquid chroma-tography-mass spectrometry analysis, and peptide microsequencing (data notFIG. 1. Schematic representation of synthetic peptides used to characterize murine MAbs and polyclonal sera and to identify protectopes in the central conservedregion of the RSV-A G protein. The asterisks denote the locations of the conserved Cys residues. The S in peptide G 174–187  C  signifies a Cys-to-Ser substitution atposition 186. 5638 PLOTNICKY-GILQUIN ET AL. J. V IROL  .   onF  e b r  u ar  y  8  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   shown). Peptides G 174–187  C  and G 164–176  C  contained Cys-Ser substitutions atresidues 186 and 173, respectively. G 174–187  C  corresponds precisely to the pro-tective peptide identified by Trudel et al. (51), with the substitution inserted tofacilitate formation of the native Cys176-Cys182 disulfide bond (32). The sub-stitution in G 164–176  C  (a Ser at Cys173 [Cys173Ser]) was inserted to preventnonnative Cys173-Cys186 disulfide bridge formation and to facilitate orientationof peptide coupling to the carrier protein. Peptide-carrier protein coupling.  Peptides G 174–187  C , G 172–187 , G 171–187 , andG 164–176  were coupled to P40 by treatment with glutaraldehyde as previouslydescribed (24). The amino acid sequences of these peptides indicated that glu-taraldehyde coupling would result in a population of both N- and C-terminus-coupled G 174–187  C  and G 171–187  while G 172–187  and G 164–176  would be coupleduniquely by their C and N termini, respectively. Peptides CysG 144–159 , G 144–159 Cys, CysG 190–204 , and G 164–176  C  were conjugated by using  N  -hydroxysuccin-imidyl bromoacetate as a coupling reagent (6, 24), thereby specifically controllingtheir coupling orientation. For hybridoma screening and immunization purposes,all peptides were also coupled to BB, KLH (both from Calbiochem, Meudon,France), and/or bovine serum albumin (Sigma, Saint Quentin Fallavier, France)by the same methods. P40 and BB conjugates were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis on 12% homogeneous gels under re-ducing conditions.  Animals.  Female BALB/c mice, aged 6 to 9 weeks, were purchased from IffaCredo (L’Arbresle, France) and kept under specific-pathogen-free conditions.They were confirmed as seronegative vis-a`-vis RSV before inclusion in thestudies. All animals were fed rat and mouse maintenance diet A04 (UAR,Villemoissin-sur-Orge, France) and water ad libitum and were housed and ma-nipulated according to French and European guidelines. MAbs and polyclonal sera.  MAbs 11F7, 5B7, 6H66, and 6H69 were producedby immunizing BALB/c mice twice, subcutaneously (s.c.), with 30  g of BBG2Nain phosphate-buffered saline (PBS) containing 20% (vol/vol) Alhydrogel[Al(OH) 3 ]; Superfos BioSector a/s, Vedbaek, Denmark). Four days after a fur-ther, intravenous inoculation of BBG2Na, the mice were killed and spleen cells were fused with SP2/0-Ag14 myeloma cells (American Type Culture Collection)by using polyethylene glycol (Serva, Heidelberg, Germany). Resultant hybrid-omas were initially screened for secretion of G2Na- and RSV-A-specific anti-bodies by ELISA. Positive reactors were subsequently screened for reactivityagainst KLH-conjugated G 172–187 , G 144–159 , G 190–204 , and G 164–176  C . A panel of hybridomas was selected on the basis of their peptide reactivity patterns andcloned three times by serial limiting dilution. Four independent clones wereexpanded and injected into pristane (Sigma) primed mice to induce the produc-tion of ascitic fluid. Ascites fluids were purified, isotyped, and used in subsequentstudies.MAbs 18D1 and 5C2 were produced by immunizing BALB/c mice intraperi-toneally (i.p.) with 50   g of KLH-G 174–187  C  or G2  Ca in complete Freund’sadjuvant. A second immunization was performed with incomplete Freund’s ad- juvant. Four days after a further, intravenous inoculation of 10   g of eachpeptide, the mice were killed and spleen cells were fused with SP2/0-Ag14myeloma cells as described above. The resulting hybridomas were screened forantibody secretion by ELISA with bovine serum albumin-G 174–187  C  or G2  Ca,respectively, as the coating antigen. Positive reactors were cloned, expanded, andused for ascites fluid production, as described above.Murine polyclonal serum against G 190–204  was produced by immunizing 25BALB/c mice i.p. three times at 2-week intervals with 20  g of BB-CysG 190–204 .Three weeks after the last immunization, the mice were sacrificed and exsangui-nated by cardiac puncture. Blood samples were collected in serum separationtubes (Beckton Dickinson, Meylan, France) and centrifuged at 1,850   g   for 10min, and pooled sera were stored at  80°C until used for titration in ELISAs andneutralization assays or in passive-transfer studies. Production of murine anti-BBG2Na and BB polyclonal sera was previously described (42), while anti-RSV-A polyclonal sera were prepared by immunizing mice three times i.p. (in Alhydrogel) or five times intranasally (i.n.) (in 50-  l volumes) with 10 5 50%tissue culture infectious doses (TCID 50 ) at 2-week intervals. The human RSV-positive sera (kindly provided by Michel Segondy, Centre Hospitalier Universi-taire, Montpellier, France) were derived from two individuals demonstratinghigh anti-RSV-A ELISA titers (log 10  3.8 and 4, respectively). Pepscan analysis.  Ninety-four overlapping 8-mer peptides or 90 overlapping12-mer peptides spanning residues 130 to 230 (G2Na) of the human RSV-A Gprotein were synthesized on noncleavable derivatized rods (Chiron Technolo-gies, Emeryville, Calif.) according to established procedures. The peptides weretested for their reactivities with MAbs or sera by ELISA. Briefly, nonspecificbinding was blocked by incubation for 1 h at 37°C with PBS containing 0.1%Tween (Sigma) and 1% gelatin. The rods were subsequently incubated at 4°Covernight with the sera or MAbs, washed three times, and incubated 1 h at roomtemperature with a horseradish peroxidase-conjugated goat anti-mouse antibody(1/5,000; Southern Biotechnology Associates, Birmingham, Ala.). After being washed four times, the rods were transferred to a microtiter plate (Nunc, Rosk-ilde, Denmark) containing 100  l of tetramethyl benzidine (Dynatech, Chantilly,Va.). The reaction was terminated with 100  l of 1 M H 2 SO 4  per well. Opticaldensities were measured at 450 nm. ELISA titrations and neutralization assays.  MAb characterization and RSV- A-specific serum immunoglobulin G (IgG) determinations were accomplished byELISA as previously described (42), except that for MAb titrations, blocking and washing solutions consisted of PBS–0.1% (wt/vol) gelatin and PBS–0.05% Tween20 (Sigma)–0.01% (wt/vol) gelatin, respectively. MAb isotyping was undertaken with an ImmunoPure MAb isotyping kit (Pierce, Rockford, Ill.). ELISA titers were expressed as the reciprocal of the last dilution with an optical density of   0.15 and at least twofold higher than that of the negative control. Because the virus stocks contained cellular antigen, RSV-A-specific antibody titers were cal-culated by subtracting anti-HEp-2 titers from those of anti-RSV-A. Neutraliza-tion assays were undertaken in triplicate as previously described (42). Neutral-ization titers were expressed as the reciprocal of the highest dilution that reducedpositive-control syncytium numbers by at least 60%.  Active and passive immunization and challenge procedures.  Mice were im-munized i.p. with 200-  l volumes of peptide-carrier protein conjugate solutionscontaining 20% (vol/vol) Alhydrogel in PBS. Second and subsequent immuniza-tions were given at 2-week intervals. Animals were bled 2 weeks after the lastimmunization to determine RSV-A-specific serum antibody titers and neutral-izing activity. They were challenged 3 weeks postimmunization with 10 5 TCID 50 of RSV-A i.n. after anesthetization with 2.5 ml of a 4/1 mixture of ketamine(Imalge`ne 500; Rhoˆne Me´rieux, Lyon, France) and xylazine (Rompun at 2%;Bayer, Puteaux, France) per kilogram of body weight. Immunoprophylaxis stud-ies were undertaken by passively transferring various dilutions of MAbs orpolyclonal sera in 200-  l volumes by the i.p. route to mice 1 day before chal-lenging with RSV-A as described above. Mice were sacrificed 5 days afterchallenge.  Animal sample preparation and virus titration.  Animals were anesthetized asdescribed above and exsanguinated by cardiac puncture. Lung removal, lunghomogenate preparation, nasal tract lavage (NTL), and virus titrations wereundertaken as previously described, except that NTL fluids were snap frozen without being subjected to centrifugation (42). The limit of detection for lungtissues was   1.45 log 10  TCID 50  /g of lung tissue, except when insufficient lunghomogenate was available. The limit of detection for NTL samples was invariablylog 10  0.45/ml. When no virus was detected, actual detection limits were used forstatistical analyses. Thus, standard deviations of   0 were occasionally recordedfor lung titers of some virus-free animal groups. Animal organs were consideredprotected when virus titers were reduced by at least 2 log 10  relative to PBS-immunized control mouse levels. Statistics.  Statistical analyses were done with the  t  test or Kolmogorov-Smir-nov nonparametric test (for small sample sizes) of the Statigraphic softwareprogram (Manugistics, Rockville, Md.). Probability values of greater than 0.05 were considered insignificant. RESULTSIsolation and characterization of RSV-A G protein-specificMAbs.  To localize B-cell determinants on the central con-served domain of the RSV (Long) G protein, MAbs weregenerated by immunizing mice with BBG2Na (MAb 5B7,6H66, 6H69, and 11F7), G2  Ca (MAb 5C2), KLH-G 174–187  C (MAb 18D1), or KLH-G 190–204  (MAb 8A3). Because of diffi-culties in establishing hybridomas that secrete MAbs specificfor the G 190–204  region (residues 190 to 204), a polyclonalantiserum was also produced in parallel by immunizing mice with BB-G 190–204 . All isolated MAbs and the polyclonal anti-serum recognized both BBG2Na and RSV-A, while the MAbs were exclusively of the IgG1 isotype (Table 1). With the ex-ception of MAb 8A3, all had anti-RSV-A ELISA titers of   4log 10 . Pepscan analyses identified three independent linearepitopes (detailed in Table 1) recognized by MAbs 5B7, 5C2,11F7, and 18D1, with the last two MAbs showing reactivities tothe same region (Table 1). These results were confirmed byELISA titrations against relevant synthetic peptides. MAb 8A3and anti-BB-G 190–204  recognized a fourth, independent linearepitope as determined by peptide ELISA reactivities. Surpris-ingly, anti-BB-G 190–204  also reacted with P40-G 144–159 Cys, al-though the ELISA titer was considerably lower than thoseagainst P40-G 190–204 Cys, BBG2Na, and RSV-A. The nature of this cross-reactivity is under investigation. Interestingly, MAb6H66 and 6H69 did not show any reactivity in Pepscan analysis,but both demonstrated high peptide G 171–187 -specific ELISA titers. This peptide is conformational in that it contains twodisulfide bridges. Since none of the Pepscan peptides containall 4 Cys residues, and therefore the disulfide bridges, theseresults indicate that MAbs 6H66 and 6H69 recognize contig-uous residues in the residue 171 to 187 region in a conforma-tion-dependent manner. Furthermore, MAbs 6H66 and 6H69 V OL  . 73, 1999 RSV G PROTEIN PROTECTOPES 5639   onF  e b r  u ar  y  8  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   reacted very weakly with peptide G 172–187 , indicating that res-idue 171V is a critical component and hence identifying a fifthepitope. Competition ELISAs demonstrated that these MAbsrecognized the same epitope while MAbs 11F7 and 18D1 alsorecognized a single epitope that overlapped with the 6H66/ 6H69 epitope (data not shown). Thus, five independent B-cellepitopes were mapped on the G2Na fragment of the RSV-A Gprotein. None of the MAbs was capable of neutralizing RSV-A in vitro, as demonstrated in microneutralization assays (Table1). Protective efficacy of RSV-A G protein specific polyclonalantibodies and MAbs.  Passive-transfer experiments with theMAbs and anti-BB-G 190–204 Cys polyclonal serum were under-taken with groups of six to seven mice to determine if theidentified B-cell epitopes were protective (protectopes). Asindicated in Fig. 2A, MAbs 5B7, 5C2, 6H66, 6H69, and 18D1protected the lungs from RSV-A challenge, although most of the mice contained infectious virus. MAb 11F7 was less pro-tective under these conditions, since only three of six animals were considered protected while a fourth animal demonstrateda virus titer reduction of log 10  1.7 relative to control animals.Nonetheless, MAb 11F7 has the capacity to protect mouselungs from RSV-A challenge. Like MAb 11F7, anti-BB-G 190–204  passive transfer resulted in significant virus titer re-ductions in the lungs of recipient mice (  P   0.001). However,only one of six mice was considered protected. MAb 8A3, which maps to the G 190–204  peptide, had little impact on theprevention of lung infection in most animals under these ex-perimental conditions, although one of six mice was protected.Interestingly, both anti-BB-G 190–204  and MAb 8A3 were foundto be highly labile, demonstrating diminished reactivitiesagainst both BBG2Na and RSV-A with time in ELISAs (datanot shown). These data therefore demonstrate that all fiveB-cell epitopes identified above are LRT protectopes, albeit with considerably different efficacies under these experimentalconditions. Unlike lung protection, and consistent with previ-ous observations (42), none of the MAbs or polyclonal seraprotected URTs from RSV-A challenge (Fig. 2B). Immunogenicity and protective efficacy of RSV-A G protein-derived synthetic peptides.  To confirm the results of the anti-body passive-transfer studies, a series of peptides was synthe-sized and coupled to either P40 or BB by their N termini(CysG 144–159 , CysG 190–204 , and G 164–176 ), C termini (G 144–159 Cysand G 190–204 Cys), or both (G 174–187  C  and G 171–187 ) and in- jected into BALB/c mice. Mice primed with 20  g of P40-G 174–187  C  and subsequently given two boosters of 100   g eachdeveloped elevated RSV-A serum antibody titers, and theirlungs were protected from challenge (Fig. 3A and B). In ourhands, P40-G 171–187  was less immunogenic than P40-G 174–187  C  under similar immunization conditions (  P     0.02). Thedifferential immunogenicity was reflected in the respective pro-tective efficacies of the peptides; the P40-G 171–187  was slightlyless efficacious in reducing virus titers in the lungs followingRSV-A challenge. However, differences in protective efficacy were not statistically significant. As indicated in Fig. 3, peptide G 144–159  was immunogenicand protected the LRT from RSV-A challenge. However,these properties depended entirely on the orientation of pep-tide coupling to the carrier protein. Mice immunized twice with 20   g of P40-G 144–159 Cys (C-terminal coupling) devel-oped moderate RSV-A serum antibody titers, and their lungs were protected from RSV-A challenge; most lacked detectable virus. In contrast, 2 doses of 20   g of P40-CysG 144–159  (N-terminal coupling) were poorly immunogenic, and no lungprotection was observed. Similar results were obtained whenBB was used (data not shown), indicating that the results wereindependent of the carrier protein. In addition to the G 174–187  C  /G 171–187  and G 144–159  regions, G 190–204  was also found tobe immunogenic and protective. Mice immunized three times with 20   g of conjugate demonstrated potent lung protection(Fig. 3B) that was independent of peptide orientation (datanot shown). These results are consistent with and confirm theantibody passive-transfer study results described above. In con-trast to the MAb passive-transfer results, peptide G 164–176  waspoorly immunogenic in BALB/c mice that were primed with 20  g and boosted twice with 100   g of conjugate. Their poorimmunogenicity was reflected in the lack of significant lung virus titer reductions in mice immunized with either P40-G 164–176 (Fig. 3B) or P40-G 164–176  C  (data not shown).In contrast to the lung-protective efficacy observed for mostof the peptides, none induced URT protection (Fig. 3C). Thisis also in agreement with the antibody passive-transfer studydata. The combined results suggest that serum antibodies andlinear RSV-A-specific B-cell epitopes in the G2Na fragmentare insufficient for URT protection. Reactivities of murine polyclonal sera with G protein-de-rived synthetic peptides.  To determine and compare theepitope usage in mice following BBG2Na or RSV-A immuni-zations or infections, relevant sera were screened in a Pepscanassay using a series of overlapping dodecapeptides thatspanned the central conserved domain (residues 130 to 230). As evidenced in Fig. 4A, sera derived from mice immunizedi.p. with BBG2Na demonstrated six independent major peakreactivities, including peptides 140 to 151, 145 to 156, 152 to TABLE 1. Characterization of the anti-RSV-A monoclonal antibodies and anti-BB-G 190–204  polyclonal serum MAb or polyclonalserum  a IgisotypePepscanreactivities(amino acidresidues)  b ELISA titer (log 10 ) vs: In vitroneutralizingactivity/25  lRSV-A BBG2Na P40-G 172–187  P40-G 171–187  P40-G 144–159 Cys P40-G 190–204 Cys P40-G 164–176 5B7 (a) IgG1 163–174 4.58 6.25   1.95   1.95   1.95   1.95 5.53   85C2 (b) IgG1 150–157 5.77 5.77 1.95   1.95 5.77   1.95   1.95   86H66 (a) IgG1 NR  c 4.82 6.25 2.90 5.53   1.95   1.95   1.95   86H69 (a) IgG1 NR 4.10 5.77 2.90 4.58   1.95   1.95   1.95   811F7 (a) IgG1 176–187 5.29 6.73 6.25 5.29 2.43 2.19   1.95   818D1 (c) IgG1 178–185 4.58 6.73 6.73 6.01 2.90 2.43 2.90   88A3 (d) IgG1 ND  d 3.38 4.82   1.95   1.95   1.95 4.34   1.95   8 Anti-BB-G 190–204  ND ND 5.22 6.25 1.95   1.95 3.38 5.59 2.90   8  a MAbs produced by immunizing BALB/c mice with BBG2Na (a), G2  C (b), KLH-G1  Ca (c), or P40-G 190–204  (d).  b Reactivities against overlapping 8-mer peptides spanning the region containing residues 130 to 230 of the RSV-A(Long) G protein.  c NR, no reactivity.  d ND, not determined. 5640 PLOTNICKY-GILQUIN ET AL. J. V IROL  .   onF  e b r  u ar  y  8  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   163, 157 to 168, 164 to 175, and 195 to 206. Four independentminor reactivity peaks were also evident, including peptides161 to 172, 177 to 188, 188 to 199, and 199 to 210. There wasa noticeable absence of reactivity with peptides spanning theconserved cysteine-rich region. However, as indicated above toexplain the lack of Pepscan reactivity of MAbs 6H66 and 6H69,this may be an artifact of the Pepscan assay. Therefore, withthe exception of this region, and as expected, the protectopesidentified above are clearly immunogenic after immunization with BBG2Na.Sera from mice immunized i.p. (Fig. 4B) or infected i.n. (Fig.4C) with RSV-A demonstrated far less epitope usage withinthe G2Na fragment than the anti-BBG2Na serum. Nonethe-less, common reactivities with anti-BBG2Na serum were iden-tified and mapped to the regions 152 to 163, 157 to 168, 164 to175, and 176 to 187. Two other common reactivities with anti-BBG2Na serum were evident only in sera from mice infectedi.n. with RSV-A; these mapped to residues 161 to 172 and 199to 210 and coincided with three of the protectopes describedabove. Mapping of B-cell epitopes in RSV-convalescent-phase hu-man sera.  Pepscan analyses were undertaken with sera fromhumans with RSV infections to determine whether the humanimmune responses following infection result in antibodies re- FIG. 2. LRT (A) and URT (B) prophylactic efficacies of murine anti-RSV-A MAbs and polyclonal sera. Groups of six or seven mice received a single doseeach of 10 4 anti-RSV-A ELISA titer equivalents of antibody 24 h before chal-lenge with 10 5 TCID 50  of RSV-A by i.n. instillation. Control mice receivedmurine anti-BB polyclonal serum (at an anti-BB ELISA titer equivalent of 10 4 ).Mice were sacrificed 5 days later, at which point the lungs were removed and10% lung homogenates were prepared. Nasal tracts were rinsed by forciblyinjecting 1.5 ml of virus transport medium through the nasopharyngeal cavity andrecovering exudate from the nares. Virus titers were determined as described inMaterials and Methods.FIG. 3. Humoral immune responses (A) and LRT- (B) and URT- (C) pro-tective efficacies in mice following immunization with carrier-protein-coupledsynthetic peptides. Groups of 7 to 12 mice were immunized i.p. at 2-weekintervals two or three times with peptide coupled to P40 carrier protein in 200-  l volumes containing 20% (vol/vol) Alhydrogel. Mice immunized with P40-G 174–187  C , P40- G172–187 , or P40-G 164–176  were primed with 20   g of proteinfollowed by two boosters of 100  g each. Those immunized with P40-CysG 190–204 received three doses of 20   g of protein. Finally, mice immunized with P40-CysG 144–159  or P40-G 144–159 Cys were immunized twice with 20   g of protein.Controls included animals immunized with PBS. All mice were bled 2 weeks afterthe last immunization, challenged 1 week later with 10 5 TCID 50  of RSV-A, andsacrificed 5 days postchallenge. Sera were tested in ELISAs against RSV-A. Tenpercent lung homogenates were prepared. Nasal tracts were rinsed by forciblyinjecting 1.5 ml of virus transport medium through the nasopharyngeal cavity andrecovering exudate from the nares. LRT and URT virus titers were determinedas described in Materials and Methods. V OL  . 73, 1999 RSV G PROTEIN PROTECTOPES 5641   onF  e b r  u ar  y  8  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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