Journal of Medical Virology 27:215-223 (1989)

Immunoglobulin-Class-Specific Immune Response to Respiratory Syncytial Virus Structural Proteins in Infants, Children, and Adults Therese Popow-Kraupp, Evelyne Lakits, Gabriele Kellner, and Chr is t ian Kunz Institute of Virology, University of Vienna. Austria

The protein specificities of IgG, IgM, and IgA an- tibodies induced during respiratory syncytial vi- rus (RSV) infection in 74 patients (4 weeks to 81 years of age) were investigated using the tech- nique of immunoblotting. Although the pattern of antibody reactivity varied among patients, most of the humoral immune response in all age groups was directed against the 48, 42, 35, and 27 K proteins. An infant’s own antibody re- sponse was discernible in 55 of the 57 children below 1 year of age, despite the presence of ma- ternally derived antibodies. Antibody against the 90 K surface glycoprotein was not detectable in those less than 1 year of age. Primary RSV in- fection induced antibodies only against a subset of RSV proteins. Although a broadening of the antibody response occurred with increasing age and in the course of reinfection, an immune re- sponse to all the viral structural proteins was observed rarely.

K E Y WORDS: immunoblotting, pattern of an- tibody reactivity, age groups

INTRODUCTION

Respiratory syncytial virus (RSV) is one of the most important respiratory pathogens of infants and young children causing considerable morbidity and which of- ten requires hospitalization IChanock and Parrott, 1965; Kim et al., 1973; Belshe et al., 19831. Severe disease due to RSV is most common during the first 6 months of life, although maternally transmitted serum antibodies are almost universal. The severity of RSV- induced disease, however, seems to depend on the level of passively acquired maternal viral neutralizing anti- bodies at the time of birth [Glezen e t al., 1981 1. Follow- ing natural infection, 60-704 of infants less than 6 months of age fail to develop a detectable complement fixing and neutralizing antibody response IParott et al., 1961,19731. Repeated infections with RSV arecom- mon, and an appreciable reduction in the severity of illness associated with reinfection is usually not ob-

c 1989 ALAN R. LISS, INC.

served until the third infection [Henderson e t al., 19791. All these observations indicate that natural in- fection induces only a state of partial immunity to re- infection. The precise role of the humoral immune re- sponse in immunity and pathogenesis of RSV infections is not fully understood, and information on the antibody response to the different structural com- ponents of the virus is scarce I Ward e t al., 1983; Vain- ionpaa e t al., 1985; Gimenez et al., 19871.

RSV is composed of ten different proteins and at least seven of these serve structural functions [ Venkatesan e t al., 1983; Collins and Werzt, 19851. Two glycopro- teins are expressed at the virus surface, the large gly- coprotein G (molecular weight [MWl = 90 K ) and the fusionprotein F (MW = 70 K), which in infectious virus particles consists of disulfide-linked fragments of 48 K (F,) and 20 K (F2) IPeeples and Levine, 1979; Gruber and Levine, 19831. The 90 K glycoprotein is involved in virus adsorption to the cell surface I Walsh et al., 1984bl, and the F protein mediates both viral penetra- tion and cell-cell spread via membrane fusion [Fernie and Gerin, 1982; Walsh and Hruska, 1983; Walsh e t al., 19851. Both envelope glycoproteins have been iden- tified as the major viral antigens responsible for induc- ing protective antibodies IOlmsted e t al., 1986; Johnson e t al., 1987; Walsh e t al., 1987; Routledge et al., 19881.

Three proteins are present in the nucleocapsid: the nucleoprotein N (MW = 42 K), the phosphoprotein P (MW = 35 K), and the large protein L (MW approxi- mately 200 K). The matrix protein M tMW = 27 K ) is part of the virion envelope, and the function of a second putative matrixprotein (MW = 23 K ) is not clear IStott and Taylor, 1985 I.

In order to obtain more information on the relevance of the immune response to the different viral structural proteins, we attempted to define the protein specifici-

Accepted for publication October 21, 1988. Address reprint requests to Therese Popow-Kraupp, Institute

of Virology. University of Vienna, Kinderspitalgasse 15. A-1095 Vienna, Austria.

2 16

ties of maternally derived antibodies in RSV-infected and -uninfected infants using the technique of immu- noblotting. We further investigated the immunoglobu- lin (Ig)-class-specific immune response of infected in- fants and adults to the main structural proteins of RSV and examined the persistence of these antibodies for 1 year after RSV infection.

MATERIALS AND METHODS Patients and Specimens

The study group consisted of 74 hospitalized patients with confirmed RSV infections and 24 RSV-negative children under 1 year of age. Patients infected with RSV ranged in age from 4 weeks to 81 years, with 77% of them under 1 year of age (median: 6 months), and the majority (95%) suffered from an infection of the lower respiratory tract. Of these, 31 were diagnosed as having tracheobronchitis, 16 having obstructive bron- chitis, and 23 having pneumonia. Of the 24-RSV neg- ative infants, 21 were also hospitalized with respira- tory symptoms (rhinopharyngitis: seven, bronchitis: seven, obstructive bronchitis: three, pneumonia: four). Using the techniques of virus antigen detection by en- zyme-linked immunosorbent assay (ELISA) and virus isolation, two were found to have infections due to para- influenza virus type 3, one had an enterovirus-caused disease, and one patient was adenovirus positive. In the remaining 17 and in those three who were hospi- talized for nonrespiratory ailments (hepatitis), viral agent was not found.

Acute-phase sera (A) were collected on the day of admission, which was usually within the first week after the onset of symptoms (median: 3 days; range: 1-25 days). Convalescent-phase sera (B) were usually drawn in the second week of illness (median: 12 days; range: 7-110 days). Follow-up serum samples (one to three per patient) were available from 13 of our RSV- infected patients, covering an observation period of 2 months up to 1 year after the onset of symptoms.

Documentation of RSV Infection RSV infection was confirmed by detection of virus-

specific antigens in nasopharyngeal secretions (NPS) by ELISA, as described previously IPopow-Kraupp et al., 19861 and/or by recovery of virus in tissue culture using HeLa cells, strain “Ohio” (kindly provided by Dr. D.A.J. Tyrrell, Clinical Research Centre, Common Cold Research Unit, Salisbury, Wiltshire, England). Isolates were identified as RSV by indirect immunof- luorescence using calf immune serum against RSV and fluorescein-isothiocyanate ( FITCbconjugated anti-bo- vine immunoglobulin from Wellcome Reagents, Ltd. (Temple Hill, Dartford, England). Since an RSV-spe- cific complement-fixing antibody response is uncom- mon in those under 1 year of age IParrott et al., 1961, 19731 and, in addition, only small amounts of serum were available, only those above this age were screened for RSV-specific complement-fixing antibodies. The complement-fixation (CF) test was carried out accord-

Popow-Kraupp et al.

ing to standard procedures, using 2 U of antigen IHawkes, 19791.

IMMUNOBLOT ANALYSIS OF SERUM SAMPLES

Preparation of Viral Antigens and Control Antigens

Hep-2 cells (Flow Laboratories, Rockville, MD) grown in Roux-bottles and infected with the Long strain of RSV (100 TCID5,,/ml, 10 ml per Rouxbottlej were maintained in minimal essential medium (Earle’s MEM) supplemented with 2 7 heat-inactivated fetal calf serum (FCS), 2 mM glutamine, penicillin (200 U/ml), streptomycin (200 Fgirnl), neomycin (100 pg/ml), and fungizon (5 ~ g i m l ) a t 35°C. Supernatants of infected cultures were harvested and pooled on day 5 postinfection and contained infectious virus a t a titre of lo7. After freezing at -80°C and thawing, the super- natant was clarified by centrifugation at 180 g for 15 min a t 4°C. Subsequently, virions and viral antigens were pelleted by high-speed centrifugation a t 130,OOOg for 2.5 hr. The pellet was resuspended in phosphate- buffered saline (PBS), pH 7.2, and aliquots were stored a t -80°C until use. Uninfected cells were treated in the same way and served as control antigen.

SDS-PAGE Proteins were separated on polyacrylamide gels us-

ing the discontinuous buffer system of Laernmli 119701. Gels contained 12% acrylamide cross-linked with N , N ‘ - methylenbisacrylamide. Protein samples were solubi- lized by addition of 3 vol of sample buffer (0.125 R i s - HCI, pH 6.8, containing 2% SDS, 10% glycerol, 5 1 mercaptoethanol, and 0.0025% bromophenol blue) and heated to 100°C for 5 min. For electrophoresis, the pro- tein content was adjusted to 100 p g per 10-mm lane. Gels were run at 100 V for 2 hr at 4°C.

Western Blot Analysis Polypeptides resolved by SDS-PAGE were electro-

phoretically transferred to nitrocellulose membranes (Trans-Blot Transfer Medium, Bio-Rad Laboratories, Richmond, CA) with blotting buffer (25 mM Tris-buff- ered saline, pH 8.3, containing 192 mM glycine and 20% methanol) at 4°C and 50 V for 15 min and then at 100 V for 1 hr. Nitrocellulose membranes were then treated overnight with PBS containing 5% bovine se- rum albumin to prevent nonspecific binding. Thereaf- ter, nitrocellulose filters were cut into strips and stored at -80°C until use.

Testing of Sera Tests were carried out simultaneously on strips with

virus-specific proteins ( AG) and on those prepared from mock-infected Hep-2 cells (CoAG). All incubation steps were carried out at room temperature. Prior to the screening for virus-specific IgM antibodies, sera were absorbed with anti-human IgG (RF-Absorbent, Be- hringwerke AG, Marburg, FRG) to avoid false-positive results due to the presence of rheumatoid factor. Nitro- cellulose strips were incubated overnight with the pa-

Immune Response to RSV Structural Proteins 217

Fig. 1 . Immunoglobulin-class-specific antibody response to RSV structural proteins of three patients. ST - biotinylated standard pro- teins of known molecular weights; MAB monoclonal antibody di- rected against the 35 K protein; AG antigen prepared from virus-

infected cells; CoAG = antigen prepared from mock-infected cells, GP:PS = guinea pig preimmunization serum; IS = guinea pig RSV immune serum; A = acute-phase serum sample; B = convalescent- phase serum sample.

tient sera diluted 1:25 in blotting buffer containing 5% heat-inactivated goat serum and 5% nonfat milk pow- der followed by extensive washing with washing buffer (Tris-buffered saline containing 2% Tween-20). There- after, strips were incubated with biotinylated goat anti-human IgG (DuPont de Nemours GmbH, Bad Nauheim, FRG) and IgM and IgA (Amersham In- ternational plc, Amersham, UK). For the IgG assay, the conjugate dilution was 1:1,000, and for the IgM and IgA assay, 1:100, in blotting buffer. After another washing procedure, the avidin-horseradish peroxidase conjugate (DuPont de Nemours) diluted 1:1,200 was added and incubated for 1 hr, followed by a final wash- ing procedure. For visualization of membrane-bound peroxidaseactivity,substratesolutioncontaining4-chlo- ro-1-naphthol and hydrogenperoxid 1:l (DuPont de Ne- mours) was added. The reaction was allowed to proceed for 20 min and was stopped by washing with distilled water.

For control, a guinea pig preimmunization and RSV immune serum (CF antibody titre 1:64) and a RSV- specific monoclonal antibody directed against the 35 K protein (kindly provided by Prof. C. Orvell, Depart- ment of Virology, Karolinska Institute, School of Med- icine, Stockholm, Sweden) were included in the assay. Protein molecular weights were determined by co-elec- trophoresis with the following biotinylated standard proteins of known molecular weights: rabbit muscle phosphorylase b (97,400), bovine serum albumin (66,200), hen egg white ovalbumin (42,699), bovine car- bonic anhydrase B (31,000), soybean trypsin inhibitor (21,500), and hen egg white lysozyme (14,400) (Bio-Rad Laboratories ).

Statistical Methods The Fisher exact probability test was used to test

significant differences.

RESULTS Immunoblot analysis of sera from RSV patients re-

vealed great differences with respect to the protein

specificities in all three Ig classes tested. Typical ex- amples are shown in Figure 1. Blots of patient 1 present the immune response against RSV proteins of an infant aged 3 months suffering from RSV tracheo- bronchitis. IgG antibodies in the acute-phase serum (A) reacted with the 90, 48, and 42 K proteins and most probably represent maternally transmitted antibodies. In addition, IgG and IgM antibodies binding to the 35 K protein were found in the convalescent-phase serum (B) sample (IGG, B; IGM, B). The IgA blot revealed antibodies reactive with the 27 K protein in the first (IGA, A) and with the 35 and 27 K proteins in the second serum samples (IGA, B).

A different reactivity pattern was observed in pa- tient 2, a 6-month-old child with RSV-induced obstruc- tive bronchitis. In the first serum sample, the IgG blot revealed a pattern of maternally derived antibodies quite similar to that of the patient described above. In addition to the antibodies reactive with the 90,48, and 42 K proteins, antibodies binding to the 35 K protein were also present (IGG, A). These remained detectable in the second serum, drawn 67 days after onset of symptoms, whereas the others initially present dropped below detectable levels (IGG, B). IgM antibod- ies were directed against the 20 K protein, and IgA antibodies bound to the proteins of 35, 27, and 20 K (IGA, B). When sera containing antibodies against the 48 and/or 20 K proteins were tested using virus pro- teins resolved under nonreducing conditions as anti- gen, only a 70 K reactivity was observed. This is con- sistent with the 20 K protein identified in our blots being a part of the fusion protein, either F2 or a frag- ment of F1 INorrby et al., 19861.

Blots of patient 3 demonstrate the immune response to RSV proteins of an 8.5-year-old child suffering from RSV pneumonia. In contrast to the two previously de- scribed patients, a significant rise of RSV-specific com- plement-fixing antibodies between the two sera was observed. In the first serum sample, IgG antibodies strongly reacted with the proteins of 90, 48, and 42 K (IGG, A) and IgA antibodies binding to the 90 K pro-

2 18 Popow-Kraupp et al.

TABLE I. IgG Antibodies to RSV Structural Proteins of Infants Without Evidence of RSV Infection

Age groups (months) FtSV structural proteins 1 - 3 ( N = 11) 4-6" = 6) 7-12(N = 7 )

Ig class (MW x 10 A = B A = B A = B InG 90 4

70 48 42 35 30 27 20

5 15 Total IgG positive 9 6 6 A 7 B: Identical antibody reactivity pattern of acute (A ) - and convalescent (81- phase serum sample. N: number of patients

tein were detectable. A marked broadening of the IgG and IgA antibody reactivity was observed between the collection of the two sera. Newly formed IgG antibodies were directed against the 35 K protein, a 30 K protein, which was not detectable on strips of mock-infected cells, and against the 27 K protein (IGG, B). IgA anti- bodies in the second serum sample bound to the pro- teins of 90,48,42,35, and 27 K (IGA, B). IgM antibod- ies formed in the course of the disease showed weak reactivity with the proteins of 35 and 27 K (IGM, B).

In order to assess whether such differences reflect age-dependant variations in antibody formation possi- bly influenced by the protein specificities of transpla- centally derived antibodies in those under 1 year of age, we first examined 24 infants with no evidence or history of RSV infection in order to obtain information on the pattern of maternally derived antibodies. We then analysed the protein specificities of the IgG, IgM, and IgA immune reponse of infected patients with re- spect to their age.

Immunoglobulin (1g)-Class-Specific Antibodies to RSV Structural Proteins in Different

Age Groups

Antibody patterns of RSV-negative infants. The RSV-specific IgG antibody patterns of the 24 children under 1 year of age whose nasopharyngeal secretions were negative for RSV by ELISA and virus isolation are shown in Table I. Consistent with the results ob- tained by antigen detection and virus isolation, in none of the RSV-uninfected infants were virus-specific IgM andlor IgA antibodies against any of the structural pro- teins detected and no child showed a change in the IgG antibody spectrum between the collection of the acute ( = A), and the convalescent-phase ( = B) serum sample ( A = B). Since IgA antibodies are known to persist for a long period of time I Welliver et al., 1980; also our results in this study], the IgG pattern of the RSV-neg- ative infants most probably reflects the reactivity of maternally transmitted antibodies, which were prima-

rily directed against the proteins of 48, 42, 35, and 20 K.

Antibody patterns of RSV-infected infants, chil- dren, and adults. Results on the Ig-class-specific antibody response in relation to the age of the patients are summarized in Table 11. In age group 1-3 months, all infants had virus-specific IgG antibodies in their acute-phase serum sample and their protein specifici- ties were identical to those of the RSV-negative infants of the same age, reflecting the pattern of maternal antibodies. However, in addition to these, IgM and IgA antibodies revealing the infants own antibody response were already detectable. The ongoing immune re- sponse was also reflected by a broadening of the IgG antibody pattern in the convalescent serum in eight of the 21 infants. Newly formed IgG antibodies were di- rected primarily against one or more of the following proteins: 48, 35, and 27 K.

Results of the IgM and IgA blots provided further evidence that the infants' own immune responses were primarily directed against one or more of the 48,42,35, and 27 K proteins. A broadening of the IgM antibody response between the first and second serum sample was not observed, whereas a change in the IgA anti- body spectrum between the collection of the two sera was noticed in eight infants.

IgG blots of acute-phase sera in the age groups 4-6 and 7-12 months revealed a narrower antibody pattern as compared with those aged 1-3 months. The protein specificities of the antibodies were quite similar to those observed in the RSV-negative infaiits of corre- sponding age, consistent with the presence of tranpla- centally derived antibodies. It was, however, of interest that a significantly higher proportion of RSV-negative infants aged 4-6 months possessed IgG antibodies di- rected against the 27 and 20 K proteins ( P < . O l ) and that significantly more ( P < .0015) of the RSV-nega- tive children aged 7-12 months had antibodies binding to the 20 K protein.

Also in these two age groups, the majority of the immune responses was directed against one or more of

Immune Response to RSV Structural Proteins 2 19

TABLE 11. Ig-Class-Specific Antibody Response to RSV Structural Proteins in Different Age Groups

R S V structural Age groups (months) Droteins 1-3(N = 21) 4-6" = 19) 7-12" = 17) ' 12 ( N = 17)

Ig class ( M W x 10 A B A B A B A

kc 90 1 1 1 1 2 2 2 2 9 70 5 48 17 42 21 35 19 30 1 27 5 20 4

5 15 Total IgC positive 21

20 15 15 21 13 16 20 14 16

1 1 8 4 5 3 3

1 1 21 18 19

8 1 1 12 1 1 12 13 4 1 1 1 1

4 4 6 9

1 2 2

15 16 14

B 1 1 6

13 14 15 7

12 5 2

15

-

90 1 70 48 42

1 2 3 2 3 3 5 2 4 8 3 9 3 6 7 8

35 4 13 6 10 1 4 3 9 30 1 27 1 4 1 4 6 20 1 1 1 1 1

5 15 3 2 Total IgM positive

90 70 48 42 35 30 27 20

c_ 15

IRA

5 15 9 13 5 10 1 1 5

1 1 10 9 14 2 3 10 10 4

12 15 8 12 10 13 6 14 16 14 15 8 9 6

2 12 13 12 17 1 1 10 3

3 2 3 2 2 1 2

15 7 9 8

10 9 3 8

1 Total IgA positive 16 19 16 19 16 17 14 16

A acute-phase serum sample. B convalescent-phase serum sample, N :. number of patients.

the 48,42,35, and 27 K proteins. In the age group 4-6 months, a change in the polypeptide profiles between acute- and convalescent-phase sera was observed in five children in the IgG and IgM classes and in six children in the IgA class. An IgA immune response against a 70 K protein, most probably the uncleaved precursor of the fusion protein, was detectable in one infant of this age group. In this patient, no antibodies directed against the 48 K protein were found in any of the three Ig classes. In those aged 7-12 months, a broadening of antibody reactivity between the first and second serum samples was observed in five children in the IgG blot and in four in the IgA blot. An immune response against the 90 K surface glycoprotein was not detectable in any of the patients under 1 year of age.

It was apparent that a broadening of the antibody response occurred with increasing age. In those over 1 year of age, the 90 K protein and the 70 K fusion pre- cursor protein induced an immune response with the same frequency as the 48, 42, 35, and 27 K proteins, and this was the most striking difference when com- pared with those under this age. In some patients, an- tibodies against a protein of 30 K, which was not de- tectable on strips of mock-infected Hep-2 cells, could also be observed.

Patients' sera that reacted with the 70 K protein in the IgG blot were also reactive with the 48 K protein, and two of them also possessed antibodies binding to the 20 K protein. IgM antibodies binding to the 70 K protein were found in the acute-phase serum sample of a single patient who had IgG antibodies but no addi- tional IgM antibodies against the 48 K protein. In the IgA blot, four of the 10 patients with antibodies bind- ing to the 70 K protein in the first serum sample and five of the nine with a 70 K reactivity in the second serum additionally had antibodies directed against the 48 K protein.

A broadening of the immune response between the collection of the two sera was observed in the IgG and IgA blots in 10 patients and in the IgM blot in three.

From the 17 patients over 1 year of age, 11 showed a significant rise of RSV-specific complement-fixing t CF) antibodies (acute-phase sera: CF titre < 1:2; convales- cent phase sera: CF titres 1:32-1:128). The median age of those with a rise of CF antibody titres was 12 years (range: 1.5-81 years), and of those without, 2 years (range: 1.5-3 years). The IgG blots of the six patients without an antibody response detectable by the CF test revealed no IgG antibodies in the acute-phase sera of three, indicating primary infection and a narrow band

220 Popow-Kraupp et al.

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1 1 5 30 35 n weeks after onset f r 0 1 lira1 RSV inlection

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pattern in the remaining three, corresponding also to primary or secondary infection. In contrast, all the pa- tients with a significant rise of CF antibody titres had detectable IgG antibodies in the acute-phase sera and the pattern of IgG antibody reactivity was broader with an intensive staining of the individual proteins (Fig. 1, patient 3, IGG, A), a finding consistent with repeated infections. This observation, combined with the higher age of the CF-antibody-positive patients, indicates that repeated infections are required to in- duce antibody levels traceable by the CF test and is in agreement with the observation that CF antibodies are generally not detectable in infants and young children in contrast to older children and adults (Parrott et al., 1961, 19731.

Further evidence that a single RSV infection induced only an immune response to a subset of viral proteins, especially in infants and children, was provided by the follow-up of one of our patients 2 years of age, experi- encing two laboratory-confirmed RSV infections dur- ing an observation period of 33 weeks (Fig. 2). At the onset of the first infection, this patient had no detect- able RSV-specific IgG antibodies, and IgM antibodies formed were directed against the 42 K protein (Fig. 2, serum 1). Twenty-six weeks after the first recovery of RSV from the nasopharyngeal secretion (NPS) of this patient, the IgG antibodies detectable were directed against the 42, 35, 30, 27, and 20 K proteins and IgA antibodies against the 42 K protein (Fig. 2, serum 2). The third serum sample was collected simultaneously with the NPS, when the patient was again admitted to hospital, this time suffering from obstructive bronchi- tis. RSV was detected for the second time in the NPS and the pattern of antibody reactivity of this serum was identical to that of the second serum sample, drawn 2 weeks earlier. A broadening of IgG antibody reactivity including an immune response to the surface glycoprotein of 90 K was observed 6 weeks after the

onset of symptoms of the second RSV infection. IgM antibodies formed during the second RSV episode were directed against the 27 K protein.

Detectability of Immunoglobulin (1g)-Class-Specific Antibodies to RSV Structural Proteins Within 1 Year After Onset of Symptoms

We determined the persistence of protein-specific an- tibodies in the three Ig classes in 11 patients (six over 1 year of age) after a single RSV infection (without evidence of reinfection). Follow-up serum samples (one to three per patient) covered an observation period of 2 months to 1 year after the onset of symptoms. IgG an- tibodies directed against the surface glycoproteins could never be detected in all follow-up sera of the 11 patients. The pattern of IgG, IgM, and IgA antibody reactivity of those four patients followed for more than half a year after onset of symptoms are presented as characteristic examples in Figure 3. In two of our six patients over 1 year of age, IgG antibodies binding to the 90 K surface glycoprotein dropped below detectable levels, in one of the two by the 23rd week and in the second ( = patient 2, Fig. 3) by the 27th week after onset of symptoms.

In those patients, in whom IgM antibodies were de- tectable, formation of IgM antibodies took place within 3 weeks after onset of symptoms, but irrespective of the time following RSV disease, not all infected patients revealed a detectable IgM antibody response. Never- theless, in three follow-up serum samples from four patients obtained between 25 and 52 weeks (Fig. 3) , IgM antibodies were still detectable although their spectrum of reactivity had considerably narrowed. IgM antibodies found at that time were directed only against the 35 or the 27 K protein. An RSV specific IgA-antibody response was observed in all the patients by the fourth week and remained detectable in those

Immune Response to RSV Structural Proteins 221

K protein. The prolonged detectability of antibodies re- active with both fragments of the fusion protein in the uninfected group indicates that these antibodies were initially present in a high concentration. In addition, there are studies reporting that a polyclonal immune serum and a monoclonal antibody that recognize epitopes on the 48 and 20 K fragments most efficiently neutralize the virus and inhibit the fusion of virus- infected cells [ Walsh et al., 1985; Samson et al., 19861. The higher proportion of uninfected infants possessing antibodies directed against the 20 K fragment in addi- tion to antibodies reactive with the 48 K protein may indicate a more efficient immune response against the biologically important epitopes on the fusion protein, thus providing a protective effect for the infants.

The development of an infant’s own immune re- sponse was discernible in 19 of the RSV-infected chil- dren aged 1-3 months despite the presence of mater- nally derived antibodies. A differentiation between passively acquired and actively formed antibodies was not only made possible by the determination of RSV protein specific IgM and IgA antibodies, but also by a broadening of the IgG antibody response between the collection of the acute and convalescent sera in eight of the 21 infants. This differs from the results obtained by Ward et al. I 1983 I using radioimmunoprecipitation analysis, which indicate that an infant’s own immune response is discernible only above the age of 3 months. In all age groups of RSV-infected patients, the bulk of the humoral immune response was directed against the 48, 42, 35, and 27 K proteins. The 90 K surface glyco- protein seemed to be less immunogenic, and an anti- body response against this protein was not detectable in our study in those under 1 year of age. Although antibodies binding to conformation-dependent epitopes are not traceable by immunoblotting, a significantly lower immune response to both surface glycoproteins in those aged 1-8 months compared with those above this age was also observed in an ELISA using undena- tured glycoproteins as antigen IMurphy et al., 19861.

A broadening of the antibody response, however, oc- curred with increasing age, indicating that more than one RSV infection is necessary to induce detectable an- tibody levels against the remaining structural pro- teins. This broadening of antibody reactivity with in- creasing age also included an immune response to the 70 K uncleaved fusion precursor protein. Antibodies reactive with this uncleaved protein, but not with the cleavage products of 48 and 20 K were found in the IgM and IgA Ig classes. This indicates that different epitopes, present on the uncleaved and the cleaved fu- sion protein, are recognized in the course of infection.

The immune response to RSV structural proteins varied not only according to the age of the patients, but also within the age groups. Consistent with the sug- gestion of Gimenez et al. I1987 1, our results clearly demonstrate 1 ) that during primary RSV infections an- tibodies only against a subset of RSV proteins are found and 2 ) also, that in the course of reinfection an

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four patients with follow-up sera a t 25-52 weeks (Fig. 3 ).

DISCUSSION The data available on the protection of infants

against KSV by maternally derived antibodies are con- flicting, but there are several studies indicating that passively acquired antibodies, especially those directed against the surface glycoproteins, may protect against RSV-induced lower respiratory tract infection and that their concentration is important for a protective effect IGlezen et al., 1981; Ogilvie et al., 1981; Walsh et al., 1984a; Prince et al., 19851. Using immunoblot analysis, antibodies could be detected up to 1 year of age. These are most probably of maternal origin, which is in agreement to the data presented by Ward e t al. 119831. Transplacentally derived antibodies were predomi- nantly directed against the 48, 42, and 35 K proteins and their protein specificities in infected and uninfect- ed children under 1 year of age were quite similar. The only observable difference between the two groups was that up to the age of 1 year a higher proportion of uninfected infants revealed antibodies binding to the 20 K protein, in addition to those reactive with the 48

222

immune response against all the proteins is only rarely observed. It may be that the induction of an incomplete immune response by one RSV infection and the decay of antibodies within a relatively short period following RSV disease leads to inadequate immunity, thus pro- viding a precondition for reinfection.

As far as laboratory diagnosis is concerned, the tech- nique o f imniunoblotting permitted a sensitive differ- entiation between virus-specific antibodies already present (transplacentally derived or resulting from previous infections) and the ongoing immune response, thus providing a tool for the diagnosis of RSV infection in those cases where viral antigen detection has been neglected. However, i t has to be considered that IgM antibodies may remain detectable for a period of at least 1 year following RSV disease and that their pres- ence as a marker for a recent RSV infection has to be interpreted with caution.

ACKNOWLEDGMENTS We are greatful to Prof. C. Orvell (Department of

Virology, Karolinska Institute, Stockholm, Sweden) for providing us with monoclonal antibodies. We further wish to thank Mrs. R. Lang and Mrs. I. Hartnett for their excellent technical assistance and Miss S. Pfauser for typing the manuscript.

Popow-Kraupp et al.

Chanock HH. Parrott RH 119731: Epidemiology of respiratory syn- cytial virus infection in Washington. DC: 1. Importance of the vi- rus in different respiratory tract disease syndromes and temporal distribution of infection. American Journal of Epidemiology 98216-222.

Laemmli UK (19701: Cleavage ofstructural protein during the iiss(*m- bly to the head of bacteriophage T4. Nature 227:680-685.

Murphy BR, Alling DW. Snyder MH. Walsh EE, Prince GA. Chanock RM. Hemming VG. Rodriguez WJ, Kim HW. Graham BS. Wright P F (19861: Effect of age and preexisting antibody on serum ant i - body response of infants and children to the F and G glycoproteins during respiratory syncytial vlrus infection Journal of Clinical Microbiology 24:894 -898.

Norrby E, Mufson MA, Sheshberadaran H 1 1986l: Structural d1ffi.r- ences between subtype A and B s t ra ins of respiratory syncytial virus. Journal of General Virology 67.2721-2729.

Ogilvie MM. Vathenen S, Kadford M. Codd J. Key S I 1981 I: Maternal antibody and respiratory syncytial virus infection in infkncy. Journal of Medical Virology 7963-27 1.

Olmsted RA, Elango N, Prince GA. Murphy BR, Johnson 1%. Moss 8. Chanock RM. Collins PL I 19861: Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: Comparison of the individual contributions of the P and G glyco- proteins to host immunity. Proceedings of the National Academy of Sciences USA 83:7462-7466.

Parrott RH. Vargosko AJ. Kim HW. Cumming C. Turner H, Huebner R J . Chanock RM (1961): Respiratory syncytial virus. 11. Serologic studies over a 34-month period of children with bronchiolitis. pneumonia. and minor respiratory disease .Journal of the Amer- ican Medical Association 176:653-657.

Parrott RH. Kim HW. Arrobio JO, H d e s DS, Murphy HR. Rrandt CD. Camargo E. Chanock RM ( 19731: Epidemiology of resptratory syn- cytial virus infection in Washington, DC. 11. Infection and diseases with respect to age, immunologic s ta tus , race and sex. American Journal of Epidemiology 98289-300.

Peeples M, Levine S ( 19791: Respiratory syncytial virus polypeptides. Their location in the virion. Virology 95:137-145.

Popow-Kraupp Th, Kern G. Binder C, Tuma W. Kundi M. Kunz C: 119861: Detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay. indirect im- munofluorescence, and virus isolation: A comparative study. .Jour- nal of Medical Virology 19:123-134.

Prince GA, Horswood RL. Chanock MR I 19851: Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats. Journal of Virology 55:517-520.

Routledge EG, Willcocks MM, Samson ACR. Morgan 1,. Scott H. Anderson JJ. Toms GL 1 19881: The purification of four respiratory syncytial virus proteins and their evaluation a s protective agents against experimental infection in BALBic mice. Journal of (;en- era I Virology 69293 -303.

Samson ACR, Willcocks MM, Routledge EG, Morgan LA, Toms GI, ( 19861: A neutralizing monoclonal antibody to respiratory syncy- tial virus which binds to both Fi and F2 components of the fusion protein. Journal of General Virology 67:1479-1483

Stott EJ. Taylor G 119851: Respiratory syncytial virus. Brief review Archives of Virology 84:l-52.

VainionpAB R. Meurmann 0, Sarkkinen H (19851: Antibody response to respiratory syncytial virus structural proteins in children with acute respiratory syncytial virus infection. Journal of Virology 53:976-979.

Venkatesan S, Elango N. Satake M. Camargo E. Chanock RM (19831- Organization and expression of respiratory syncytial virus ge- nome. In Lerner R, Chanock HM cedsl: “Modern Approaches to Vaccines.” Cold Spring Harbor. New York: Cold Spring Harbor Laboratory. pp 37-44.

Walsh EE, Hruska J (1983): Monoclonal antibodies to respiratory syncytial virus proteins: Identification of the fusion protein. Jour - nal of Virology 47:171-177.

Walsh EE. Schlesinger JJ. Brandriss MW 11984al. Protection from respiratory syncytial virus infection in cotton rots by passive transfer of monoclonal antibodies. Infection and Immunity

Walsh EE, Schlesinger JJ, Brirqdriss MW (1984bi: Purification and char- acterization of G P 90, one of the envelope glycoproteins of respi- ratory syncytial virus. Journal of General Virologv 65:761-767.

43:756-758.

REFERENCES Belshe HB. VanVoris LP. Mufson MA 11983): Impact of viral respira-

tory diseases on infants and young children in a rural and urban area of southern West Virginia. American Journal of Epidemiol- ogy 117:467-474.

Chanock RM, Parrott RH I 19651: Acute respiratory dise and childhood: Present understanding and prospec tion. Pediatrics 3621-39.

Collins PI,. Wertz GM 11985): Gene products and genome organization of human respiratory syncytial lRSl virus. In Lerner RA. Chanock RM, Brown F leds,: “Modern Approaches to Vaccines.” Cold Spring Harbor. New York: Cold Spring Harbor Laboratory. pp 297-3 10

Fernie HF. Gerin J L ~198‘21: Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion. Infection and Immunity 37243-249.

Gimenez HB. Keir HM. Cash P (19871: Immunoblot analysis of the human antibody response to respiratory syncytial virus infection. Journal of General Virology 68:1267-1275.

Glezen WP. Paredes A, Allison JE. Taber LH. Prank AI, (1981 I: Risk of respiratory syncytial virus infection for infants from low-income families in relationsip to age. sex. ethnic group and maternal an- tibody level. Journal of Pediatrics 98:708-715.

Gruber C. Levine S ( 1983,: Respiratory syncytial virus polypeptides. I l l The envelope-associated proteins. Journal of General Virology 64:825-832.

Hawkes RA ( 19791. General principks underlying laboratory diagno- sis of viral infections. In Lennette EH. Schmidt NJ (eds): “Diag- nostic Procedures for Viral, Rickettsia1 and Chlamydia1 Infec- tions.” Sth Ed. Washington. DC- American Public Health Association Inc., pp 3-48.

Henderson FW. Collier AM. Clyde WA. Denny FW (19791: Respira- tory syncytial virus infections. reinfections and immunity. A pro- spective, longitudinal study in young children. New England Journal of Medicine 300:.530-534.

Johnson PH, Olmsted RA. Prince GA. Murphy RR, Alling DW. Walsh EE. Collins PL 119871: Antigenic relatedness between glycopro- teins of human respiratory syncytial virus subgroups A and B: Evaluation of the contributions of F and G glycoproteins to immu- nitv. Journal of Virology 61:3163-3166.

Kim HW. Arrobio JO. Brandt CD. Jeffries BC, Pyles G. Reid J L ,

Immune Response to RSV Structural Proteins

Walsh EL.:. Hrandriss MW. Schlesinger JJ 11985,: Purification and characterization of t h e respiratory syncytial virus fusion protein. Jou rna l of General Virology 66:409-415.

Walsh EE. Hall CB. Briselli M. Brandriss MW. Schlesinger JJ 11987): Immunization with glycoprotein subun i t s of respiratory syncytial virus to protect cotton r a t s against viral infection. J o u r n a l of In- fectious Diseases 155:1198-1204.

223

Ward KA. Lambden PK. Ogilvie MM, Wat t PJ (1983). Antibodies t o respiratory syncytial virus polypeptides and their significance in h u m a n infection. Jou rna l of General Virology 64: 1867-1876.

Welliver KC. Kaul TN, P u t n a m TI, S u n M, Riddlesherger K , Ogra P (1980): T h e antibody response to pr imary and secondary infection with respiratory syncytial virus: Kinetics of class-specific re- sponses. T h e Journa l of Pediatrics 96:808-81:3.