THE JOURNAL OF INFECTIOUS DISEASES • VOL. 134, NO.3· © 1976 by the University of Chicago. All rights reserved.
SEPTEMBER 1976
MAJOR ARTICLES Role of Maternal Antibody in Pneumonia and Bronchiolitis Due to Respiratory Syncytial Vims Catherine L. Lamprecht, * Helen E. Krause, and Maurice A. Mufson
From the Departments of Medicine and of Preventive Medicine and Community Health, Abraham Lincoln School of Medicine, University of Illinois; and West Side Veterans Administration Hospital, Chicago, Illinois
pneumonia and bronchiolitis caused by RSV. The data suggested that the level of preexisting antibody was inversely related to the severity of illness in pneumonia but not in bronchiolitis, and that the presence of preexisting antibody did not worsen either disease.
Epidemiologic studies among several different populations have established respiratory syncytial virus (RSV) as a major cause of serious disease of the lower respiratory tract in infants and children [1-3]. In infants less than six months of age, infection with RSV is especially severe, and often the patient requires hospitalization. Until the sixth or seventh month of life infants frequently possess preexisting, circulating, homologous neutralizing antibody of maternal origin, but this antibody does not prevent infection or severe respiratory illness caused by RSV [4, 5]. Since RSV causes serious respiratory tract illnesses in the youngest infants, even in those with high levels of neutralizing antibody to RSV, we investigated the effect of circulating homologous antibody on the severity and course of illness in infants with
Materials and Methods
Population. Infants less than nine months of age who were admitted to Cook County Hospital with acute disease of the lower respiratory tract were studied for evidence of RSV infection by isolation of virus and serologic techniques. Only infants from whose oropharyngeal secretions RSV was recovered were included in the study group. The epidemiology of disease caused by RSV in this population has been described [2]. Infants were diagnosed as having pneumonia on the basis of radiographic evidence of pulmonary infiltrates. Infants with bronchiolitis had respiratory distress, expiratory wheezing, and substernal or subcostal retractions, without pneumonia. Diagnoses were recorded without knowledge of data from viral isolation and serologic studies. Isolation of virus. Oropharyngeal swabs for isolation of virus were obtained within 24 hr of
Received for publication June 30, 1975, and in revised form March 8, 1976. We thank Carmen Castellanos and Mattie Boyd who provided technical assistance. Please address requests for reprints to Dr. Maurice A. Mufson, Department of Medicine, Marshall University, School of Medicine, Huntington, West Virginia 25701. * Present address: Department of Pediatrics, University of Michigan Hospitals, Ann Arbor, Michigan.
211
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
Fifteen infants with pneumonia caused by respiratory syncytial virus (RSV) and 19 infants with bronchiolitis caused by RSV were studied for the influence of homologous, circulating neutralizing antibody on the severity of their illness. All infants were under nine months of age. Although maternal neutralizing antibody did not prevent infection with RSV and illness, the severity of pneumonia caused by RSV was inversely related to the level of neutralizing antibody. The severity of bronchiolitis caused by RSV was unrelated to maternal antibody levels. Chest roentgenograms showed pneumonia to be slightly more severe than bronchiolitis. Neither the severity of illness nor the presence of maternal neutralizing antibody was related to the development of complement-fixing antibody.
212
defects in the confluent cell monolayer. Microscopically the plaques consisted of attached and unattached RSV-infected syncytial cells. Sera were inactivated at 56 C for 30 min, diluted, and mixed with an equal volume of virus that had been diluted so that it contained approximately 20-50 pfujO.2 ml. All dilutions were made in Eagle's minimal essential medium. The mixtures were incubated for 2 hr at 24 C before inoculation onto duplicate cell culture plates. Control cultures included virus alone and diluent alone. The titer of neutralizing antibody was expressed as the reciprocal of the serum dilution that reduced the plaque count to 50% of the count in the virus control cultures. Clinical score. A standard data sheet was used for assessment of symptoms and signs at the time of admission, and the severity of each infant's illness was quantified by means of a weighted scoring system. The severity of various physical findings, the extent of pulmonary involvement by chest roentgenogram, the necessity for administration of oxygen, and the duration of illness were the parameters of the scoring system (table 1). The duration of illness was measured from the onset as reported by the parents until discharge from the hospital. Scores increased with severity of illness. By this scoring system, one-third of the patients studied were scored 3-9, one-third were scored 10-13, and one-third were scored 13-22. Results
Characteristics of study group. The study group was composed of 15· infants with pneumonia caused by RSV and 19 infants with bronchiolitis caused by RSV (table 2). The mean age of infants with pneumonia was 4.9 months, and the mean age of infants with bronchiolitis was 4.2 months. Males outnumbered females in both groups and accounted for two-thirds of the study group. Age and neutralizing antibody to RSV. The mean titer of neutralizing antibody to RSV decreased from 41 in infants one to two months of age to < 8 at five to six months of age, a decrease reflecting the loss of passively acquired maternal antibody (table 3). All eight infants aged one to two months possessed antibody.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
the patient's admission to the hospital. Swab specimens were treated with penicillin, streptomycin, and amphotericin B for 1 hr at 4 C before inoculation into duplicate roller-tube cultures of HEp-2, rhesus monkey kidney, and fetal human diploid fibroblast cells (WI-38) [2]. All cultures were incubated at 33 C on a rotating drum for at least 18 days and often as long as 28 days; subpassages of negative cultures were not done. Details of these procedures have been reported [2]. Inoculated HEp-2 and WI-38 cell cultures were examined at intervals of three to five days for evidence of CPE, and the culture media were changed at that time. Rhesus monkey kidney cell cultures were examined by the hemadsorption test at intervals of five to seven days [2]. Determinations of antibody. Blood samples were obtained soon after patients were admitted to the hospital, and convalescent blood samples were collected 14-36 days later. Paired sera were tested for CF antibody by microtiter techniques with use of overnight fixation at 4 C with 1.8-2.0 units of complement and 8 units of RSV antigen. Preexisting neutralizing antibody was determined in the acute-phase sera by plaque-reduction techniques [6]. HEp-2 cell cultures were grown in 32-oz plastic bottles. When the monolayers were confluent, the cells were treated with 0.25 % trypsin for 15-20 min, and the cells were dispersed in 5 ml of Eagle's minimal essential medium supplemented with 10% calf serum and antibiotics. The suspended cells were distributed to plastic 60-mm tissue culture petri dishes and incubated in 5% CO 2 at 37 C. Within two to four days, the cell sheets were confluent; they were washed twice with Hanks' balanced salt solution and inoculated with 0.2 ml of a virus-serum mixture and 0.2 ml of diluent (which was added to prevent drying during adsorption of virus). The plates were rotated every 15 min during the 2-hr period of adsorption so that the inoculum was equally distributed. After one rinsing with Hanks' balanced salt solution, the cultures were overlaid with 10 ml of a fresh 2% methylcellulose solution in Eagle's minimal essential medium and incubated for six to seven days in 5% CO 2 at 37 C. The overlay was removed, 10% formalin was added for preservation of the cells, and cultures were rinsed in tap water and dried. RSV plaques approximately 1-2 mm in diameter appeared as
Lamprecht, Krause, and Mujson
213
Maternal Antibody in RSV Disease
Table 1.
Clinical scoring system. Score assigned to severity of attribute
1
Attribute
3
1-7
8-14
Unilateral Unilateral
Bilateral Bilateral Unilateral Unilateral Moderate ~37.8 C Yes Yes
Mild ~37.7
C
Unilateral
5
4
15-21
22-28
29-35
Bilateral Bilateral
Bilateral
3
2 Yes
Only two of four infants aged three to four months and three of 15 infants aged five to six months had measurable antibody. All seven infants aged seven to eight months lacked detectable neutralizing antibody in serum. Neutralizing antibody to RSV and severity of illness. Among the 15 infants with pneumonia caused by RSV, there was a significant inverse relationship between the severity of clinical illness and the level of preexisting neutralizing antibody (r == -0.62; P < 0.02). The linear regression of clinical illness score on level of neutralizing antibody for pneumonia and bronchiolitis caused by RSV is shown in figure 1. Thus neutralizing antibody appeared to lessen the severity of pneumonia caused by RSV, but it did not prevent the development of disease. However, no correlation could be demonstrated between the severity of illness with bronchiolitis and the level of preexisting antibody (r == 0.20; P, not significant). The mean illness score for infants with pneumonia or bronchiolitis caused by RSV could also be correlated with the presence or absence of preexisting neutralizing antibody (table 4). The infants with pneumonia who had preexisting neu-
tralizing antibody had significantly lower scores for clinical illness than did those who lacked antibody (P == 0.004). In contrast, among infants with bronchiolitis, mean scores for clinical illness of the children with neutralizing antibody did not differ from those for children free of antibody. In either case, the presence of preexisting neutralizing antibody did not predispose infants with either illness to more severe disease of the respiratory tract. CF antibody responses. No relationship was found between the presence of preexisting neutralizing antibody to RSV and a fourfold or greater rise in CF antibody among infants with pneumonia (X2 == 5.0; df == 3; P == 0.17) or bronchiolitis (X2 == 5.48; df == 3; P == 0.14) (table 5). Three of the nine infants with pneumonia who had rises in titer of CF antibody and two of the six infants who did not have such rises had preexisting neutralizing antibody. Eight of the infants with bronchiolitis caused by RSV developed fourfold rises in titer of CF antibody, but only one of these infants had preexisting neutralizing antibody. Of the 11 children who lacked preexisting neutralizing antibody, seven had CF antibody responses.
Table 2. Distribution by age and sex of infants with disease of the lower respiratory tract caused by respiratory syncytial virus. Age (months) Sex Illness (no.) Pneumonia (15) Bronchiolitis (19) Total (34)
Male
Female
9 13
6 6
22
12
3-4
5-6
4
0 4
7 8
7-8 4 3
8
4
15
7
1-2 4
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
Duration of illness (days) Physical findings Harsh breath sounds Rhonchi Wheezing Rales Retractions Fever Respiratory distress Palpable liver/spleen Chest roentgenogram Air-trapping Extent of pneumonic infiltrate (no. lobes) Requirement for oxygen
2
Lamprecht, Krause, and Mujson
214
Table 3. Relation of preexisting reciprocal titer of neutralizing antibody to respiratory syncytial virus (RSV) in serum and age of infants with respiratory tract disease caused by RSV. Titers of neutralizing antibody to RSV Age (months)
Pneumonia
Bronchiolitis
1-2 3-4
14,48,66,103
5-6
SEPTEMBER 1976
MAJOR ARTICLES Role of Maternal Antibody in Pneumonia and Bronchiolitis Due to Respiratory Syncytial Vims Catherine L. Lamprecht, * Helen E. Krause, and Maurice A. Mufson
From the Departments of Medicine and of Preventive Medicine and Community Health, Abraham Lincoln School of Medicine, University of Illinois; and West Side Veterans Administration Hospital, Chicago, Illinois
pneumonia and bronchiolitis caused by RSV. The data suggested that the level of preexisting antibody was inversely related to the severity of illness in pneumonia but not in bronchiolitis, and that the presence of preexisting antibody did not worsen either disease.
Epidemiologic studies among several different populations have established respiratory syncytial virus (RSV) as a major cause of serious disease of the lower respiratory tract in infants and children [1-3]. In infants less than six months of age, infection with RSV is especially severe, and often the patient requires hospitalization. Until the sixth or seventh month of life infants frequently possess preexisting, circulating, homologous neutralizing antibody of maternal origin, but this antibody does not prevent infection or severe respiratory illness caused by RSV [4, 5]. Since RSV causes serious respiratory tract illnesses in the youngest infants, even in those with high levels of neutralizing antibody to RSV, we investigated the effect of circulating homologous antibody on the severity and course of illness in infants with
Materials and Methods
Population. Infants less than nine months of age who were admitted to Cook County Hospital with acute disease of the lower respiratory tract were studied for evidence of RSV infection by isolation of virus and serologic techniques. Only infants from whose oropharyngeal secretions RSV was recovered were included in the study group. The epidemiology of disease caused by RSV in this population has been described [2]. Infants were diagnosed as having pneumonia on the basis of radiographic evidence of pulmonary infiltrates. Infants with bronchiolitis had respiratory distress, expiratory wheezing, and substernal or subcostal retractions, without pneumonia. Diagnoses were recorded without knowledge of data from viral isolation and serologic studies. Isolation of virus. Oropharyngeal swabs for isolation of virus were obtained within 24 hr of
Received for publication June 30, 1975, and in revised form March 8, 1976. We thank Carmen Castellanos and Mattie Boyd who provided technical assistance. Please address requests for reprints to Dr. Maurice A. Mufson, Department of Medicine, Marshall University, School of Medicine, Huntington, West Virginia 25701. * Present address: Department of Pediatrics, University of Michigan Hospitals, Ann Arbor, Michigan.
211
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
Fifteen infants with pneumonia caused by respiratory syncytial virus (RSV) and 19 infants with bronchiolitis caused by RSV were studied for the influence of homologous, circulating neutralizing antibody on the severity of their illness. All infants were under nine months of age. Although maternal neutralizing antibody did not prevent infection with RSV and illness, the severity of pneumonia caused by RSV was inversely related to the level of neutralizing antibody. The severity of bronchiolitis caused by RSV was unrelated to maternal antibody levels. Chest roentgenograms showed pneumonia to be slightly more severe than bronchiolitis. Neither the severity of illness nor the presence of maternal neutralizing antibody was related to the development of complement-fixing antibody.
212
defects in the confluent cell monolayer. Microscopically the plaques consisted of attached and unattached RSV-infected syncytial cells. Sera were inactivated at 56 C for 30 min, diluted, and mixed with an equal volume of virus that had been diluted so that it contained approximately 20-50 pfujO.2 ml. All dilutions were made in Eagle's minimal essential medium. The mixtures were incubated for 2 hr at 24 C before inoculation onto duplicate cell culture plates. Control cultures included virus alone and diluent alone. The titer of neutralizing antibody was expressed as the reciprocal of the serum dilution that reduced the plaque count to 50% of the count in the virus control cultures. Clinical score. A standard data sheet was used for assessment of symptoms and signs at the time of admission, and the severity of each infant's illness was quantified by means of a weighted scoring system. The severity of various physical findings, the extent of pulmonary involvement by chest roentgenogram, the necessity for administration of oxygen, and the duration of illness were the parameters of the scoring system (table 1). The duration of illness was measured from the onset as reported by the parents until discharge from the hospital. Scores increased with severity of illness. By this scoring system, one-third of the patients studied were scored 3-9, one-third were scored 10-13, and one-third were scored 13-22. Results
Characteristics of study group. The study group was composed of 15· infants with pneumonia caused by RSV and 19 infants with bronchiolitis caused by RSV (table 2). The mean age of infants with pneumonia was 4.9 months, and the mean age of infants with bronchiolitis was 4.2 months. Males outnumbered females in both groups and accounted for two-thirds of the study group. Age and neutralizing antibody to RSV. The mean titer of neutralizing antibody to RSV decreased from 41 in infants one to two months of age to < 8 at five to six months of age, a decrease reflecting the loss of passively acquired maternal antibody (table 3). All eight infants aged one to two months possessed antibody.
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
the patient's admission to the hospital. Swab specimens were treated with penicillin, streptomycin, and amphotericin B for 1 hr at 4 C before inoculation into duplicate roller-tube cultures of HEp-2, rhesus monkey kidney, and fetal human diploid fibroblast cells (WI-38) [2]. All cultures were incubated at 33 C on a rotating drum for at least 18 days and often as long as 28 days; subpassages of negative cultures were not done. Details of these procedures have been reported [2]. Inoculated HEp-2 and WI-38 cell cultures were examined at intervals of three to five days for evidence of CPE, and the culture media were changed at that time. Rhesus monkey kidney cell cultures were examined by the hemadsorption test at intervals of five to seven days [2]. Determinations of antibody. Blood samples were obtained soon after patients were admitted to the hospital, and convalescent blood samples were collected 14-36 days later. Paired sera were tested for CF antibody by microtiter techniques with use of overnight fixation at 4 C with 1.8-2.0 units of complement and 8 units of RSV antigen. Preexisting neutralizing antibody was determined in the acute-phase sera by plaque-reduction techniques [6]. HEp-2 cell cultures were grown in 32-oz plastic bottles. When the monolayers were confluent, the cells were treated with 0.25 % trypsin for 15-20 min, and the cells were dispersed in 5 ml of Eagle's minimal essential medium supplemented with 10% calf serum and antibiotics. The suspended cells were distributed to plastic 60-mm tissue culture petri dishes and incubated in 5% CO 2 at 37 C. Within two to four days, the cell sheets were confluent; they were washed twice with Hanks' balanced salt solution and inoculated with 0.2 ml of a virus-serum mixture and 0.2 ml of diluent (which was added to prevent drying during adsorption of virus). The plates were rotated every 15 min during the 2-hr period of adsorption so that the inoculum was equally distributed. After one rinsing with Hanks' balanced salt solution, the cultures were overlaid with 10 ml of a fresh 2% methylcellulose solution in Eagle's minimal essential medium and incubated for six to seven days in 5% CO 2 at 37 C. The overlay was removed, 10% formalin was added for preservation of the cells, and cultures were rinsed in tap water and dried. RSV plaques approximately 1-2 mm in diameter appeared as
Lamprecht, Krause, and Mujson
213
Maternal Antibody in RSV Disease
Table 1.
Clinical scoring system. Score assigned to severity of attribute
1
Attribute
3
1-7
8-14
Unilateral Unilateral
Bilateral Bilateral Unilateral Unilateral Moderate ~37.8 C Yes Yes
Mild ~37.7
C
Unilateral
5
4
15-21
22-28
29-35
Bilateral Bilateral
Bilateral
3
2 Yes
Only two of four infants aged three to four months and three of 15 infants aged five to six months had measurable antibody. All seven infants aged seven to eight months lacked detectable neutralizing antibody in serum. Neutralizing antibody to RSV and severity of illness. Among the 15 infants with pneumonia caused by RSV, there was a significant inverse relationship between the severity of clinical illness and the level of preexisting neutralizing antibody (r == -0.62; P < 0.02). The linear regression of clinical illness score on level of neutralizing antibody for pneumonia and bronchiolitis caused by RSV is shown in figure 1. Thus neutralizing antibody appeared to lessen the severity of pneumonia caused by RSV, but it did not prevent the development of disease. However, no correlation could be demonstrated between the severity of illness with bronchiolitis and the level of preexisting antibody (r == 0.20; P, not significant). The mean illness score for infants with pneumonia or bronchiolitis caused by RSV could also be correlated with the presence or absence of preexisting neutralizing antibody (table 4). The infants with pneumonia who had preexisting neu-
tralizing antibody had significantly lower scores for clinical illness than did those who lacked antibody (P == 0.004). In contrast, among infants with bronchiolitis, mean scores for clinical illness of the children with neutralizing antibody did not differ from those for children free of antibody. In either case, the presence of preexisting neutralizing antibody did not predispose infants with either illness to more severe disease of the respiratory tract. CF antibody responses. No relationship was found between the presence of preexisting neutralizing antibody to RSV and a fourfold or greater rise in CF antibody among infants with pneumonia (X2 == 5.0; df == 3; P == 0.17) or bronchiolitis (X2 == 5.48; df == 3; P == 0.14) (table 5). Three of the nine infants with pneumonia who had rises in titer of CF antibody and two of the six infants who did not have such rises had preexisting neutralizing antibody. Eight of the infants with bronchiolitis caused by RSV developed fourfold rises in titer of CF antibody, but only one of these infants had preexisting neutralizing antibody. Of the 11 children who lacked preexisting neutralizing antibody, seven had CF antibody responses.
Table 2. Distribution by age and sex of infants with disease of the lower respiratory tract caused by respiratory syncytial virus. Age (months) Sex Illness (no.) Pneumonia (15) Bronchiolitis (19) Total (34)
Male
Female
9 13
6 6
22
12
3-4
5-6
4
0 4
7 8
7-8 4 3
8
4
15
7
1-2 4
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 25, 2015
Duration of illness (days) Physical findings Harsh breath sounds Rhonchi Wheezing Rales Retractions Fever Respiratory distress Palpable liver/spleen Chest roentgenogram Air-trapping Extent of pneumonic infiltrate (no. lobes) Requirement for oxygen
2
Lamprecht, Krause, and Mujson
214
Table 3. Relation of preexisting reciprocal titer of neutralizing antibody to respiratory syncytial virus (RSV) in serum and age of infants with respiratory tract disease caused by RSV. Titers of neutralizing antibody to RSV Age (months)
Pneumonia
Bronchiolitis
1-2 3-4
14,48,66,103
5-6
