JOURNAL OF VIROLOGY, Oct. 1997, p. 7240–7245 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 71, No. 10
Identification of an Immunodominant Neutralizing and Protective Epitope from Measles Virus Fusion Protein by Using Human Sera from Acute Infection SOWSAN F. ATABANI,1 OBEID E. OBEID,1 DANIEL CHARGELEGUE,1 PETER AABY,2 HILTON WHITTLE,3 AND MICHAEL W. STEWARD1* Department of Clinical Sciences, London School of Hygiene and Tropical Medicine, London, United Kingdom1; Danish Epidemiology Science Centre, Statens Seruminstitut, Copenhagen, Denmark2; and MRC Laboratories, Fajara, The Gambia3 Received 3 March 1997/Accepted 20 June 1997
Polyclonal sera obtained from African children with acute measles were used to screen a panel of 15-mer overlapping peptides representing the sequence of measles virus (MV) fusion (F) protein. An immunodominant antigenic region from the F protein (p32; amino acids 388 to 402) was found to represent an amino acid sequence within the highly conserved cysteine-rich domain of the F protein of paramyxoviruses. Epitope mapping of this peptide indicated that the complete 15-amino-acid sequence was necessary for high-affinity interaction with anti-MV antibodies. Immunization of two strains of mice with the p32 peptide indicated that it was immunogenic and could induce antipeptide antibodies which cross-reacted with and neutralized MV infectivity in vitro. Moreover, passive transfer of antipeptide antibodies conferred significant protection against fatal rodent-adapted MV-induced encephalitis in susceptible mice. These results indicate that this epitope represents a candidate for inclusion in a future peptide vaccine for measles. protect against measles encephalitis in mice (13). Therefore, any new MV vaccines must also induce neutralizing and hemolysis-inhibiting antibodies to the F protein. As infants have a higher case fatality rate and suffer more from the delayed impact of MV infection (1), peptide-based vaccines against measles represent a stable alternative to live, attenuated vaccines which may be administered early in life, even in the presence of maternal immunity. Synthetic peptides representing specific linear epitopes of viral proteins have been shown to be immunogenic and to induce both antipeptide and neutralizing antiviral antibodies in vivo (8, 18). Identification of protective B- and T-cell epitopes on specific viral proteins is thus vital for the development of rationally designed synthetic peptide vaccines. Previous studies on the F protein of MV have identified an immunodominant helper T-cell epitope in mice and humans (19), while screening of a solid-phase peptide library with a mouse monoclonal anti-F antibody has allowed the identification of peptide mimics of conformational B-cell epitopes (23), one of which actively protected mice from fatal encephalitis. Mapping of linear F peptides by using serum from healthy human donors has previously identified 7 to 10 antigenic regions which were located along the whole F-protein sequence; however, the function of these antigenic regions remains unclear (28). This study describes the identification of a major antigenic epitope, recognized by acute postinfection sera, using overlapping synthetic F peptides. The immunogenicity of a synthetic peptide representing this epitope was assessed in mice, and the antipeptide antibodies generated cross-reacted with MV and could be shown to inhibit MV infectivity in vitro. Finally, these antipeptide antibodies were shown to passively protect mice against fatal encephalitis following challenge with a neuroadapted strain of MV.
Despite the widespread use of a live attenuated vaccine, measles remains the leading killer among vaccine preventable diseases of childhood, responsible for approximately 1 million deaths annually, particularly among infants less than 9 months of age (31). This mortality is in part due to the rapid decline of maternal antibodies among infants in developing countries (11) and the policy of the World Health Organization to recommend only immunization with standard dose measles virus (MV) vaccine from 9 months of age. Among the morbillivirus group of paramyxoviruses, the fusion (F) and hemagglutinin (HA) surface glycoproteins play a vital role in the induction of protective immunity. Fusion protein is synthesized as an inactive precursor, F0, that is subsequently cleaved by host cell proteases to yield two active disulfide-linked subunits, F1 and F2. The F protein is responsible for virus penetration through fusion of viral and host cell membranes, cell fusion, and hemolysis (4). Cell fusion mediated by F protein is responsible for the characteristic cytopathic effect seen in paramyxovirus infections. Vaccinia virus recombinants expressing either the H or F gene of rinderpest virus have been shown to induce virus-neutralizing antibodies and to completely protect against cattle plague (32). For MV, induction of antibody responses to F protein is essential for protection, as evidenced by the development of severe atypical disease in children years following immunization with inactivated MV vaccine, who developed adequate neutralizing and hemagglutination inhibiting antibodies but no antibodies to the F protein (15). Vaccinia virus recombinants encoding F protein have been shown to induce MV-neutralizing antibodies in mice and to protect them from fatal acute measles encephalitis (7). Furthermore, mouse monoclonal antibodies raised to recombinant F protein neutralize virus infectivity in vitro and * Corresponding author. Mailing address: Department of Clinical Sciences, London School of Hygiene & Tropical Medicine, Keppel St., London WC1E 7HT, United Kingdom. Phone: 44 171 927 2256. Fax: 44 171 637 4314. E-mail:
[email protected].
MATERIALS AND METHODS Peptide synthesis. Overlapping pentadecapeptides representing amino acids (aa) 33 to 550 (excluding the transmembrane regions) of the Edmonston strain
7240
VOL. 71, 1997
IMMUNODOMINANT EPITOPE FROM MEASLES VIRUS FUSION PROTEIN
of MV F glycoprotein (22) were synthesized manually by rapid multiple peptide synthesis (Du Pont) (17). Briefly, 44 overlapping peptides were synthesized by using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry with 4-(29,49-dimethyloxphenyl-Fmoc amino-methyl)-phenoxy resin. Each peptide was 15 aa long and had a five-residue overlap with the following peptide. Peptides were analyzed by high-performance liquid chromatography (Shimatzu, Tokyo, Japan). The molecular weights of peptides were analyzed by fast atom bombardment mass spectrometry at the London School of Pharmacy. Four additional 8-mer peptides and two 15-mer substitution peptides were later synthesized to delineate regions of interest on a peptide of the F protein, using an automated peptide synthesizer (PepSynthesizer; PerSeptive Biosystems, Watford, Hertfordshire, England). Acute MV sera. Acute MV sera were obtained from 4- to 36-month-old children in Guinea-Bissau who had measles during a severe measles epidemic in 1991. A clinical diagnosis of measles was reached, and sera were collected up to 4 weeks following the appearance of a maculopapular rash. Control sera were provided by infants enrolled in a high-dose Edmonston-Zagreb MV vaccine trial. Sera were obtained from infants at 5 or 9 months of age, immediately prior to vaccination. All sera were heat inactivated at 56°C for 0.5 h before use. Prevaccination anti-MV antibodies were measured by hemagglutination inhibition (27). Mice. Six- to eight-week-old inbred female BALB/c (H-2d) and CBA (H-2k) mice were purchased from the Medical Research Council, Mill Hill, London, England, and maintained at the Biological Services Unit, London School of Hygiene and Tropical Medicine. MV. The Edmonston strain of MV (107 PFU/ml), propagated in Vero cells in minimum essential medium (Gibco) supplemented with 5% (vol/vol) fetal calf serum, was used for neutralization assays. Rodent-neuroadapted MV (CAM/RB strain) was kindly provided by U. G. Liebert, Institut fu ¨r Virologie und Immunologie, Wurzburg, Germany. Mouse brain homogenates from infected suckling mice were used as the source of virus. Immunization and challenge of mice. Groups of four BALB/c (H-2d) and CBA (H-2k) mice were primed intraperitoneally with 100 mg of peptide in complete Freund’s adjuvant. Three weeks after priming, mice were boosted by the same route and with the same dose of peptide in incomplete Freund’s adjuvant. Serum samples were obtained weekly by tail bleeding from the time of the boost. For passive protection studies, offspring of female BALB/c mice (five and six per group) received 150 ml of BALB/c or CBA antipeptide serum or normal mouse serum (as controls) at 12 days of age. Mice were challenged intracranially with the neuroadapted MV (104 PFU/mouse) 24 h later. Mice were observed daily for signs of encephalitis, and survival was monitored for up to 1 month following challenge. ELISA. (i) Detection of anti-MV IgG antibodies. Immunoglobulin G (IgG) anti-MV antibody activity in human postmeasles sera and the ability of mouse antipeptide sera to cross-react with MV was assessed by enzyme-linked immunosorbent assay (ELISA), using sucrose density gradient-purified MV and control (uninfected Vero cell) antigens (5). Serial twofold dilutions of sera in blocking buffer were then added, and the plates were incubated for 45 min at 37°C. To each well was added 50 ml of a 1/5,000 dilution of peroxidase-conjugated goat anti-human IgG (heavy plus light chain; Jackson Immunochemicals) or 1/2,000 dilution of peroxidase-conjugated rabbit anti-mouse IgG (heavy plus light chain; Nordic), and the plates were incubated for a further 45 min at 37°C. Plates were washed, and bound enzyme was detected by the addition of enzyme substrate (0.04% o-phenylenediamine–0.004% hydrogen peroxide in phosphate-citrate buffer [pH 5.5]) at 50 ml/well. The reaction was stopped after 10 min with the addition of 2 M H2SO4 (50 ml/well), and the A490 values were read on a Titertek Multiskan (Dynatech MR5000). Second International Reference anti-MV human serum (66/202; National Institute of Biological Standards and Controls, Potters Bar, Hertfordshire, England) and pooled serum from MV-immunized BALB/c mice were included as positive human and mouse controls, respectively. A corrected optical density (OD) value at A490 was obtained for each sample by subtraction of the OD in control antigen wells from the OD in the protein antigen wells. (ii) Reactivity of human sera with F peptides. The antigenic profile of F protein was assessed by using 44 overlapping 15-mer peptides as solid-phase antigens on ELISA. Human sera were added at a dilution of 1/200 to all wells of the plate and incubated for 45 min at 37°C; the remainder of the assay was performed as described above. The reactivity of serum with each peptide, expressed as DOD at A490, was determined by subtraction of the OD of serum in empty blocked wells from the OD of serum reacting with the specified peptide antigen well. (iii) Detection of mouse IgG antipeptide antibodies. Mouse IgG antipeptide antibodies induced following peptide immunization were determined by enzyme immunoassay. Microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with the appropriate peptide in 0.1 M carbonate-bicarbonate buffer. Plates were washed, and remaining binding sites were blocked as described above. Twofold serial dilutions of mouse antipeptide serum were added at 50 ml/well and incubated for an hour at 37°C. After washing, the remaining steps of the assay were carried out as before. Antipeptide antibody titers were calculated as log10 of the reciprocal of the antibody dilution giving an OD greater than 0.2 at A490. Irrelevant peptides from the F protein were routinely included in the assays to ensure the specificity of the binding detected. (iv) Affinity assays. The relative affinities of human anti-MV antibodies to the relevant peptide and mouse antipeptide IgG antibodies to the homologous
7241
peptide were determined by a solid-phase enzyme inhibition assay (21). Appropriate dilutions of serum in blocking buffer were preincubated with 0.5 log10 dilutions of inhibiting peptide at 37°C for 1 h. The relative antipeptide antibody affinity was determined as the reciprocal of the molar concentration which gave 50% inhibition of the binding observed in the absence of inhibiting peptide. Virus neutralization. Plaque neutralization assays were performed as described previously (2). Vero cells were seeded overnight at 2 3 104 cells/well in 96-well flat-bottom culture plates (Nunc); 30 PFU of the Edmonston strain of MV was added to twofold dilutions of heat-inactivated mouse serum and incubated for 1 h at 37°C in 5% CO2. The virus-serum mixtures were added to wells containing Vero cells and incubated for a further hour at 37°C in 5% CO2, the medium was discarded, and a carboxymethylcellulose overlay was added. Plates were observed daily for plaque formation, and up to 4 days later, cell sheets were fixed with 20% formaldehyde in phosphate-buffered saline and stained with 0.1% crystal violet. Endpoint titration was determined as the dilution of serum which reduced the plaque number by 50% of the mean value observed in virus control wells. Normal mouse serum and pooled serum from MV-immunized BALB/c mice were included as negative and positive controls, respectively. Cell-cell fusion inhibition. MV was added to a monolayer of Vero cells at 0.1 PFU/cell. After 1 h, the cells were washed and prediluted test and control sera were added. Appropriate positive and negative controls were included. Plates were read 24 to 48 h later (29). Statistical analyses. Comparisons between antipeptide antibody affinities raised in the two groups of mice and among the polyclonal human sera were assessed by the Mann-Whitney U test (nonparametric method rank test). Comparisons between two groups of mice in protection studies were assessed by Fisher’s exact two-tailed test, comparing the frequency of survival and mortality in immunized mice versus control groups.
RESULTS Epitope mapping of F protein by using synthetic peptides and human serum. Following positive confirmation of a diagnosis of measles by anti-MV IgG ELISA, a panel of 18 acute postmeasles serum samples obtained from children 4 to 36 months of age was used to map linear B-cell epitopes of F protein. Forty-four overlapping F peptides, used as solid-phase antigens on ELISA, were screened with each serum at a dilution of 1/200. The reactivity of sera to purified MV on ELISA was used as a positive control. All of 18 human serum samples screened were found to react strongly with a single peptide, p32 (aa 388 to 402) (mean OD, 0.57 6 0.24; range, 0.28 to 1.20), while 50% recognized p35 (aa 418 to 432) to a lesser extent (mean OD, 0.22 6 0.16; range, 0.20 to 0.56) (Fig. 1a). Furthermore, none of 15 serum samples obtained prevaccination from infants with anti-MV antibodies ,1/20 (as measured by hemagglutination inhibition) reacted with any of the 44 peptides with an OD of .0.2 (Fig. 1b). The mean reactivities of human sera with p32 and p35 were determined by endpoint titration ELISA, which gave titers of 1/3,200 and 1/400, respectively. Immunogenicity of p32 and p35 in BALB/c and CBA mice. IgG antibodies to peptides p32 and p35 were demonstrable in BALB/c and CBA mice following intraperitoneal priming with peptide in Freund’s complete adjuvant and boosting with peptide in incomplete Freund’s adjuvant 3 weeks later. With p32 as an immunogen, antipeptide antibodies were detectable before the boost, and titers continued to increase for 5 weeks after priming. The maximum anti-p32 antibody titers in BALB/c mice (log10 4.1) were 1 log10 higher than those in CBA mice. When p35 was used as an immunogen in BALB/c mice, antipeptide antibodies were detectable after priming and reached a maximum titer of log10 4.75 after the boost. However, anti-p35 antibodies in CBA mice were detectable only following the boost (maximum titer, log10 1.9). A control 15mer peptide from the F protein, p17 (aa 238 to 252), which was not antigenic with human anti-MV antisera, failed to generate antipeptide antibodies when used as an immunogen in either BALB/c or CBA mice (data not shown). Reactivities of anti-p32 and anti-p35 antibodies with MV. Antipeptide antisera raised in BALB/c and CBA mice were
7242
ATABANI ET AL.
J. VIROL.
FIG. 2. Cross-reactivity of anti-p32 serum raised in BALB/c and CBA mice with purified MV as solid-phase antigen on ELISA. Values are means for groups of four mice, and results are expressed as the corrected OD value at A490 obtained for each sample by subtraction of the OD in control antigen wells from the OD obtained in the protein antigen wells. The OD at A490 for normal mouse sera did not exceed 0.05 at 1/10, the lowest dilution of serum tested.
FIG. 1. Reactivities of 18 acute human postmeasles sera (a) and 15 prevaccination sera with undetectable anti-MV antibody (b) with 44 overlapping F peptides on ELISA. Each sample was tested at 1/200 dilution. The reactivity of each of the 18 postinfection human sera (a) and each of the 15 prevaccination sera (b) with each peptide, expressed as mean OD at A490, was determined as the OD of serum in empty blocked wells subtracted from the OD of serum reacting with the specified peptide antigen well.
tested for cross-reactivity with MV in ELISA. Anti-p32 antibodies from both BALB/c and CBA mice were cross-reactive with MV at low dilutions (Fig. 2), neutralized MV infectivity in a plaque inhibition assay (Table 1), but did not inhibit MVinduced cell-cell fusion (fusion from within) (data not shown). Anti-p35 antibodies from both strains failed to cross-react with MV. Affinities of mouse antipeptide antibodies for p32. The relative affinities of antipeptide antibodies to p32 raised in BALB/c and CBA mice were assessed by an inhibition enzyme immunoassay. The relative affinities of antipeptide antibodies for the homologous peptide raised in CBA mice were significantly higher than those of antipeptide antibodies raised in BALB/c mice (P 5 0.03 [Table 1]). Characterization of the fine specificity of binding of p32 sequence by human sera. The 18 postinfection samples were tested for reactivity with overlapping 8-mer peptides representing the original 15-mer p32 peptide used as solid-phase antigens on ELISA. Table 2 (assay A) show that 33.3% of the human serum samples reacted with peptide p32-2. The relative antibody affinities of polyclonal human antisera for the 15-mer peptide and the overlapping 8-mer peptides were assessed by a solid-phase inhibition ELISA (Table 2, assay A). The polyclonal human antibodies had significantly higher relative affinities for the original 15-mer peptide p32 than the 8-mer peptides. To investigate further the role of cysteine residues and
possible disulfide bond formation in antigenic recognition, two additional 15-mer peptides containing conserved amino acid substitutions (serine for cysteine at positions 390, 395, and 397 and serine for cysteine at positions 390 and 397) were synthesized and used as solid-phase antigens on ELISA. None of the acute postinfection sera recognized the substituted peptides (Table 2, assay B). Passive protection studies. Only p32 induced mouse antipeptide antibodies which cross-reacted with MV on ELISA and also inhibited MV infectivity in a plaque assay. Therefore, each mouse in two groups of 12-day-old BALB/c offspring was passively immunized with 150 ml of anti-p32 antiserum raised in BALB/c mice (with an average antipeptide titer of log10 4.1) (6 mice) or CBA mice (with an average antipeptide titer of log10 3.1) (5 mice) or with normal mouse serum (10 mice), followed by intracranial inoculation of neuroadapted MV 1 day later. The passive transfer of either BALB/c or CBA immune serum conferred significant protection against fatal MV encephalitis (P 5 0.001 and P 5 0.022, respectively). No significant difference in protection was observed between mice receiving antipeptide serum from either BALB/c or CBA mice (P 5 0.545 [Fig. 3]).
TABLE 1. Functional antipeptide antibody responses in groups of four BALB/c and CBA mice 8 weeks following intraperitoneal priming with p32 Mouse strain
Mean antibody affinity for p32 (M21) 6 SE
Mean plaque inhibition titera 6 SE
BALB/c CBA
(6.98 6 1.9) 3 106 (1.75 6 1.3) 3 107
1.05 6 0.15 1.43 6 0.07
a
Log10 MV plaque inhibition titer of antipeptide antisera.
VOL. 71, 1997
IMMUNODOMINANT EPITOPE FROM MEASLES VIRUS FUSION PROTEIN
TABLE 2. Fine mapping of the structure of the peptide p32 antigenic determinant Assay
Sequence
No. of samples positive (%)a
Mean relative affinityb (M21) 6 SE
Ac p32 p32-1 p32-2 p32-3 p32-4
ANCAGILCKCYTTGT ANCAGILC CAGILCKC GILCKCYT LCKCYTTGT
100 11.1 33.3 11.1 16.7
(2.0 6 0.9) 3 107 (2.6 6 0.1) 3 105 (4.3 6 1.5) 3 105 (1.2 6 5.0) 3 106 (4.5 6 1.4) 3 105
ANCAGILCKCYTTGT ANSAGILSKSYTTGT ANSAGILCKSYTTGT
100 0 0
(2.0 6 0.9) 3 107 ND ND
Bd
a Peptides were used as solid-phase antigens in a direct ELISA. The cutoff for positive reactivity of each serum sample with the peptide is expressed as the mean optical density at A490 1 2 standard deviations for the negative control samples, at a serum dilution of 1/200. b Assessed by a solid-phase inhibition ELISA, using peptides as fluid phase inhibitors. *P , 0.001 significant difference of affinities between original 15-mer peptide and the 8-mer peptides. ND, not determined. c Percentage positivity and mean relative affinity of 18 human polyclonal anti-MV antibodies for the peptide p32 as a 15-mer and as overlapping 8-mers. d Recognition of 18 human anti-MV antisera with two further 15-mer peptides in which two or three of the cysteines were replaced by serines.
Sequence analysis of p32. The amino acid sequence of the 15-mer p32 (aa 388 to 402 [Table 3]) is conserved in all morbilliviruses (6), and the cysteine residues within the p32 sequence are conserved among other paramyxoviruses (3). Screening of the Swissprot database revealed no significant homology between p32 and any other human, animal, or plant viral proteins. DISCUSSION In spite of the availability of a live attenuated vaccine, more than 1 million childhood deaths from measles occur annually. This worrying statistic emphasizes the requirement for alter-
7243
native vaccine strategies. The potential use of synthetic peptides as a vaccine against MV has previously been studied in experimental animals (16, 17, 19, 24). In the work described here, polyclonal human anti-MV antisera were used to identify antigenic and immunogenic peptides which represent linear B-cell epitopes from the F protein of MV. Overlapping F peptides were screened with acute postmeasles sera, obtained from African children, to map linear B-cell epitopes. A single antigenic peptide, p32, representing aa 388 to 402 of the F protein, was recognized by all of the serum samples obtained from infected children but by none of the sera with undetectable anti-MV antibodies. Previous analysis of immunodominant recognition sites of the F protein with human sera by using overlapping peptides revealed 7 to 10 binding regions which were located along the whole F sequence (14, 28). In the absence of anti-MV-negative antisera in these studies, the heterogeneity of antigenic regions observed may represent nonspecific binding of convalescent sera to some of the peptides. Although no immune function for these antibody binding regions was shown, peptide p32 was included in one such antigenic region as being recognized by more than 50% of convalescent sera (28). Furthermore, these antibody binding regions were found to lie near experimentally defined helper T-cell epitopes, thus providing evidence for nonrandom spanning of T- and B-cell epitopes (13). The apparent discrepancy between the data in reference 28 and those in the present study may arise from the fact that Wiesmuller et al. used late convalescent European sera, whereas our sera were from African children in the acute stage of MV infection. Peptide p32 represents a sequence within the cysteine-rich region of the F1 subunit of the protein, which is known to be highly conserved among paramyxoviruses (22). Following further characterization of this sequence by using shorter versions of the original p32 peptide, approximately 35% of acute postmeasles polyclonal sera were shown to react with the shorter peptide containing a cysteine residue at each terminus. However, the sera bound with the highest affinity to the parent 15-mer peptide. Moreover, the polyclonal sera failed to recognize the substitution peptides with conserved replacements at
FIG. 3. Effect of passive immunization of BALB/c mice with anti-p32 serum raised in BALB/c (log10 4.1) and in CBA mice (log10 3.1) on the response to challenge with the neuroadapted strain of MV. Groups of 2-week-old BALB/c mice received 150 ml of either anti-p32 serum derived from BALB/c or CBA mice or normal mouse serum (NMS). Survival in mice receiving BALB/c serum (83%) was significantly different from that in control mice. Survival in mice receiving CBA serum (60%) was also significantly different from that in control mice.
7244
ATABANI ET AL.
J. VIROL.
TABLE 3. Comparison of the amino acid sequence of identified antigenic peptide p32 of the MV F glycoprotein (20) with sequences from other morbilliviruses (6) and other paramyxoviruses (3) Source of peptidea
Comparison with MV p32 (aa 388–402)b
A N C A S
Morbilliviruses PDV CDV RPV
* * *
* * *
* * *
Paramyxoviruses SNV PF3 NDV SV5
* * * *
* * * *
* * *
* * *
I
* * * * * *
*
L C K C Y T T G T * * *
* * *
*
* * * *
* * *
* * *
*
* * * *
* * *
a
PDV, phocine distemper virus; CDV, canine distemper virus; RPV, rinderpest virus; SNV, Sendai virus; PF3, parainfluenza type 3; NDV, Newcastle disease virus; SV5, simian virus 5. b Asterisks indicate conserved residues.
the site of the cysteine residues, thus establishing the requirement of cysteines for the antigenic conformation of the peptide. The cysteine-rich region of the MV F glycoprotein (aa 337 to 423), comprising eight cysteine residues, interacts specifically with MV H protein to induce cell fusion, and a limited region, Cys337 to Arg381, is all that is required for fusion (30). Furthermore, in an MV F-protein mutant in which two amino acid changes occurred within the cysteine-rich region (aa 330 to 390), replication could no longer be inhibited by a fusioninhibiting peptide (12). However, the lack of recognition of all peptides representing the regions within the cysteine-rich domain by polyclonal human serum samples in this study suggests that these peptides do not assume the correct conformation on a solid-phase support. Peptide p32 was shown to be immunogenic in two strains of mice and induced antipeptide antibodies which cross-reacted with MV and inhibited MV infectivity in vitro. The anti-p32 antibodies were effective in a plaque inhibition assay but did not inhibit MV-induced cell-cell fusion. It therefore appears that these antibodies inhibit fusion from without (23, 29) by binding to the appropriate epitope on the F protein of the virus. The ability of p32 peptide alone to induce an antibody response indicates the existence of both an antigenic B-cell epitope and a helper T-cell epitope within its sequence. Furthermore, a boosting effect observed 3 weeks following priming in the two strains of mice provides further evidence of T-cell help in the induction of memory B cells. The immunogenicity of this peptide in two inbred strains of mice (H-2d and H-2k) suggests that the helper T-cell epitope is non-major histocompatibility complex restricted. However, determining whether the amino acid sequence of p32 comprises a promiscuous helper T-cell epitope, capable of presentation by a wide range of major histocompatibility complex class II molecules, would require immunogenic studies on further inbred strains of experimental animals. Linear sequences which comprise both B- and T-cell epitopes have been described previously (9). Antiviral neutralizing monoclonal antibodies have generally been considered to be directed toward conformational epitopes, with the role played by linear B-cell epitopes being debatable. However, linear epitopes have been shown to induce neutralizing antibodies against foot-and-mouth disease virus (9) and human immunodeficiency virus (18).
In the present study, antibodies raised to the linear peptide p32 afforded passive protection to mice against a fatal encephalitis following challenge with a neuroadapted MV. As the peptide contained three cysteine residues within its sequences, the possibility exists that disulfide bonds can be formed to allow the peptide to adopt a cyclic secondary structure which mimics an external loop on the native protein. Antibodies to this structure would thus be able to cross-react with the virus and neutralize its infectivity. Passively transferred anti-p32 antibodies induced in both BALB/c and CBA mice were similarly protective, although the antipeptide antibody titers in BALB/c mice were higher, and the inhibition of MV infectivity in vitro was similar. However, the anti-p32 antibodies raised in CBA mice had a significantly higher relative antibody affinity for the homologous peptide. Earlier reports have indicated that little association exists between the level and affinity of antiprotein antibodies in different strains of inbred mice (25). In addition, the affinity of antibodies for synthetic peptides has previously been shown to affect the passive and active protection afforded in vivo (16). In the present study, mice receiving passively transferred BALB/c or CBA antipeptide sera were protected against a fatal encephalitis following challenge with neuroadapted MV, thus providing further evidence that the affinity of the antibodies raised is an important factor in their protective capacity. The results presented here illustrate the potential of the use of acute postmeasles human sera for the identification of peptide sequences representing single immunodominant regions within MV F protein which may be used as immunogens for the induction of neutralizing and protective antibodies. Although the sample numbers tested are small, the results of the work presented here clearly identify an important antigenic and immunogenic determinant from the conserved region of MV F protein which has potential for use in an immunodiagnostic assay for MV antibodies. Moreover, these results indicate that this epitope represents a candidate for inclusion, along with other protective epitopes, in a subunit vaccine for MV. ACKNOWLEDGMENT This work was supported by a grant from Action Research. REFERENCES 1. Aaby, P., J. Bukh, D. Kronborg, I. M. Lisse, and M. C. da Silva. 1990. Delayed excess mortality after exposure to measles during the first six months of life. Am. J. Epidemiol. 132:211–219. 2. Albrecht, P., K. Herrmann, and G. R. Burns. 1981. Role of virus strain in conventional and enhanced measles plaque neutralization test. J. Virol. Methods 3:251–260. 3. Buckland, R., C. Gerald, R. Barke, and T. F. Wild. 1987. Fusion glycoprotein of measles virus: nucleotide sequence of the gene and comparison with other paramyxoviruses. J. Gen. Virol. 6:1695–1703. 4. Choppin, P. W., C. D. Richardson, D. C. Merz, W. W. Hall, and A. Scheid. 1981. The functions and inhibition of the membrane glycoproteins of paramyxoviruses and myxoviruses and the role of the measles virus M protein in subacute sclerosing panencephalitis. J. Infect. Dis. 143:352–363. 5. Crowe, J. E. J., P. T. Bui, G. R. Siber, W. R. Elkin, R. M. Chanock, and B. R. Murphy. 1995. Cold-passaged, temperature sensitive mutants of human respiratory syncytial virus (RSV) are highly attenuated, immunogenic and protective in seronegative chimpanzees, even when RSV antibodies are infused shortly before immunization. Vaccine 13:847–855. 6. Curran, M. D., Y. J. Lu ¨, and B. K. Rima. 1992. The fusion protein gene of phocine distemper virus: nucleotide and deduced amino acid sequences and a comparison of morbillivirus fusion proteins. Arch. Virol. 126(1-4):159–169. 7. Drillien, R., D. Spehner, A. Kirn, P. Giraudon, R. Buckland, F. Wild, and J. P. Lecocq. 1988. Protection of mice from fatal measles encephalitis by vaccination with vaccinia virus recombinants encoding either the haemagglutinin or the fusion protein. Proc. Natl. Acad. Sci. USA 85:1252–1256. 8. Francis, M. J., G. Z. Hastings, D. V. Sangar, R. P. Clark, A. Syned, B. E. Clarke, D. J. Rowlands, and F. Brown. 1987. A synthetic peptide which elicits neutralizing antibody against human rhinovirus type 2. J. Gen. Virol. 68: 2687–2691.
VOL. 71, 1997
IMMUNODOMINANT EPITOPE FROM MEASLES VIRUS FUSION PROTEIN
9. Francis, M. J., C. M. Fry, D. J. Rowlands, J. L. Bittle, R. A. Houghten, R. A. Lerner, and F. Brown. 1987. Immune response to uncoupled peptides of foot-and-mouth disease virus. Immunology 61:1–6. 10. Gnann, J., J. A. Nelson, and M. B. A. Oldstone. 1987. Fine mapping of an immunodominant domain in the transmembrane glycoprotein of human immunodeficiency virus. J. Virol. 61:2639–2641. 11. Halsey, N. A. 1983. The optimal age for administering measles vaccine, p. 4–17. In N. A. Halsey and C. A. deQuadros (ed.), Recent advances in immunization. Pan-American Health Organization, Washington, D.C. 12. Hull, J. D., D. L. Krah, and P. W. Choppin. 1987. Resistance of a measles virus mutant to fusion inhibitory oligopeptides is not associated with mutations in the fusion peptide. Virology 159:368–372. 13. Malvoisin, E., and F. Wild. 1990. Contribution of measles virus fusion protein in protective immunity: anti-F monoclonal antibodies neutralize virus infectivity and protect mice against challenge. J. Virol. 64:5160–5162. 14. Muller, C. P., D. Handtmann, N. H. Brons, M. Weinmann, K. H. Wiesmuller, G. Spahn, M. Wiesneth, F. Schneider, and G. Jung. 1993. Analysis of antibody responses to the measles virus using synthetic peptides of the fusion protein: evidence of non-random pairing of T and B cell epitopes. Virus Res. 30:271–280. 15. Norrby, F., and Y. Gollmar. 1972. Appearance and persistence of antibodies against different virus components after regular measles infections. Infect. Immun. 6:240–247. 16. Obeid, O. E., C. D. Partidos, C. R. Howard, and M. W. Steward. 1995. Protection against morbillivirus-induced encephalitis by immunization with a rationally designed synthetic peptide vaccine containing B and T-cell epitopes from the fusion protein of measles virus. J. Virol. 69:1420–1428. 17. Obeid, O. E., C. D. Partidos, and M. W. Steward. 1994. Analysis of the antigenic profile of measles virus haemagglutinin in mice and humans using overlapping synthetic peptides. Virus Res. 32:69–84. 18. Palker, T. J., T. J. Matthews, A. Langey, M. E. Tanner, M. E. Martin, R. M. Scearce, J. E. Kim, J. A. Berzofsky, D. P. Bolognesi, and B. Haynes. 1989. Polyvalent human immunodeficiency virus synthetic immunogens composed of envelope gp120 T helper cell sites and B cell neutralization epitopes. J. Immunol. 142:3610–3619. 19. Partidos, C. D., and M. W. Steward. 1990. Prediction and identification of a T cell epitope in the fusion protein of measles virus immunodominant in mice and humans. J. Gen. Virol. 71:2099–2105. 20. Portner, A., R. A. Scroggs, and C. W. Naeve. 1987. The fusion glycoprotein of Sendai virus: sequence analysis of an epitope involved in fusion and virus neutralization. Virology 57:556–559.
7245
21. Rath, S., C. M. Stanley, and M. W. Steward. 1988. An inhibition enzyme immunoassay for the estimation of relative antibody affinity and affinity heterogeneity. J. Immunol. Methods 106:245–249. 22. Richardson, C. D., D. Hull, P. Greer, K. Hasel, A. Berkovich, G. Englund, W. J. Bellini, B. Rima, and R. A. Lazzarini. 1986. The nucleotide sequence of the mRNA encoding the fusion protein of measles virus (Edmonston strain); a comparison of fusion proteins from several different paramyxoviruses. Virology 155:508–523. 23. Schneider-Schaulies, J., L. M. Dunster, R. Schwartz-Albiez, G. Krohne, and V. ter Meulen. 1995. Physical association of moesin and CD46 as a receptor complex for measles virus. J. Virol. 69:2248–2256. 24. Steward, M. W., C. M. Stanley, and O. E. Obeid. 1995. A mimotope from a solid-phase peptide library induces a measles virus-neutralizing and protective antibody response. J. Virol. 69:7668–7673. 25. Steward, M. W., and R. E. Petty. 1972. The use of ammonium sulphate globulin precipitation for determination of affinity of antiprotein antibodies in mouse serum. Immunology 22:747–756. 26. Toyoda, T., B. Gotoh, T. Sakaguchi, H. Kida, and Y. Nagai. 1988. Identification of amino acids relevant to three antigenic determinants on the fusion protein of Newcastle disease virus that are involved in fusion inhibition and neutralization. J. Virol. 62:4427–4430. 27. Whittle, H. C., P. Hanlon, K. O’Neill, L. Hanlon, V. Marsh, E. Jupp, and P. Aaby. 1988. Trial of high-dose Edmonston-Zagreb measles vaccine in the Gambia: antibody responses and side-effects. Lancet i:811–814. 28. Wiesmuller, K. H., G. Spahn, D. Handtmann, F. Schneider, G. Jung, and C. P. Muller. 1992. Heterogeneity of linear B cell epitopes of the measles virus fusion protein reacting with late convalescent sera. J. Gen. Virol. 73:2211–2216. 29. Wild, T. F., and R. Buckland. 1997. Inhibition of measles virus interaction and fusion with peptide corresponding to the leucine zipper region of the fusion protein. J. Gen. Virol. 78:107–111. 30. Wild, T. F., J. Fayolle, P. Beaunverger, and R. Buckland. 1994. Measles virus fusion: role of the cysteine-rich domain of the fusion glycoprotein. J. Virol. 68:7546–7548. 31. World Health Report. 1996. Fighting disease, fostering development. World Health Organization, Geneva, Switzerland. 32. Yilma, T., D. Hsu, L. Jones, S. Owens, M. Grubman, C. Mebus, M. Yamanaka, and B. Dale. 1988. Protection of cattle against rinderpest with vaccinia virus recombinants expressing the HA or F gene. Science 242:1058– 1061.
