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Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus
CLINICAL AND LABORATORY OBSERVATIONS
Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus LONE GRAFF STENSBALLE, MD, HENRIK RAVN, MSC, PHD, KIM KRISTENSEN, MD, DMSC, TIFFANY MEAKINS, PETER AABY, DMSC, ERIC A. F. SIMOES, MB, BS, DCH, MD
AND
This study used 459 prospectively sampled cord blood samples to examine the association between maternally derived respiratory syncytial virus (RSV)–neutralizing antibodies and the RSV hospitalization season in Denmark. We found a clear temporal association and suggest that RSV-neutralizing antibody level plays a role in the RSV seasonal pattern. (J Pediatr 2009;154:296-8)
igh titers of maternally derived respiratory syncytial virus (RSV)–neutralizing antibodies1 are inversely associated with the incidence of RSV acute lower respiratory tract infection (LRI) during the first 6 months of life. Incomplete transfer of maternally derived RSV-neutralizing antibodies has been implicated in the increased risk of severe RSV infection in preterm infants,2 and studies of passively administered polyclonal, or humanized monoclonal antibody against RSV have shown reduction of RSV LRI or hospitalizations among premature infants and children with chronic lung disease.3 In countries with temperate climates, RSV causes epidemics of LRI among infants and young children during late fall, winter, and early spring.4,5 In tropical or semitropical climates as well, RSV epidemics appear regularly but with different patterns of seasonality. The reason for the periodic emergence of RSV LRI epidemics is not entirely clear. Geographic and climatic factors are clearly associated with epidemics,6 but it is uncertain whether this is related to spread of the virus, behavioral factors, or cyclic changes in a population’s immunologic susceptibility. This study examined prospectively the association between maternally derived RSV-neutralizing antibodies in cord blood and the RSV seasonality in Danish newborns.
H
MATERIAL We used cord blood samples from the Danish National Birth Cohort in combination with data from the RSV database on RSV hospitalizations in the Danish infant population.
The Danish National Birth Cohort The Danish National Birth Cohort (DNBC, www.bsmb.dk)7 recruited pregnant Danish women prospectively in the period from 1997 to 2003. A cord blood sample for the DNBC bio bank was collected at birth. Among 51 701 children in the DNBC with date of birth December 3, 1998, to May 2, 2003, and a cord blood sample, we used computerized randomization to select 459 cord blood samples for the study. The RSV Database We established a database on all hospitalized Danish patients tested for RSV between January 1996 and May 20038 and calculated the weekly RSV hospitalization incidence per 100 000 Danish infants below 6 months of age during the study period. DNBC LRI
296
The Danish National Birth Cohort Lower respiratory tract infection
RSV
Respiratory syncytial virus
From the Bandim Health Project, Statens Serum Institut (L.S., H.R., P.A.), the Paediatric Clinic, Rigshospitalet (K.K.), Copenhagen, Denmark, the Bandim Health Project (L.S., H.R., P.A.), Bissau, Guinea-Bissau, and the Department of Pediatrics, University of Colorado School at Denver and Health Sciences Center and The Children’s Hospital (T.F., E.S.), Denver, CO. Conflict of interest and funding information available in the Appendix (available at www. jpeds.com). Submitted for publication Mar 18, 2008; last revision received May 20, 2008; accepted Jul 23, 2008. Reprint requests: Lone Graff Stensballe, Bandim Health Project, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. E-mail: [email protected]. 0022-3476/$ - see front matter Copyright © 2009 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2008.07.053
RSV-Neutralizing Antibody Assay We used a microneutralization assay modified from one described earlier,9 performed in 96 well microtiter plates. With each set of test plates there was a control plate that contained 3 columns of known positive and negative controls diluted 2-fold, and 2 columns for a back titer for the virus. Each of the test plates contained 4 patient sera samples (done in duplicate) diluted out 2-fold across the 96-well plate, beginning with serum dilution of 1:4. Sera were first serially diluted in 2% fetal bovine serum in minimal essential medium in the test plates followed by the addition of 15 to 25 plaqueforming units of virus per well. The serum virus mixture was allowed to incubate for 1.5 hours on a rocker, at 37.0° C. 25 000 Hep-2 cells per well in 10% fetal bovine serum minimal essential medium were then added and allowed to incubate for 6 days; plates were observed daily for visible cytopathic effects. The medium/virus/serum mixture was gently aspirated, and the cells were stained with crystal violet fixative. After 24 hours the wells were washed with water, allowed to dry, and then read under a microscope. The titer was obtained as the reciprocal of the dilution from the last row in which no cytopathic effect was seen. Statistics Titers were expressed as log base 2 and treated as continuous variables. The association between RSV season and maternally derived RSV-neutralizing antibodies was described by use of correlation analysis.
RESULTS Two serum samples had insufficient quantity for the analysis and were excluded. The geometric mean titer of maternally derived RSV-neutralizing antibody in 457 cord blood samples was 7.89 to the log base 2 (percentiles 10% 6; 25% 7; 50% 8; 75% 9; 90% 10). A temporal correlation between the incidence of RSV hospitalization and the mean RSV antibody titer in cord blood samples was observed (P ⫽ .00, correlation coefficient ⫽ ⫺0.25) (Figure). Incidence of RSV hospitalization increased when cord RSV antibody titer declined, and the RSV epidemic peaked shortly after the nadir of the mean antibody level.
DISCUSSION This study offered a unique possibility to examine the association between the maternally derived RSV-neutralizing antibody titer in consecutively sampled cord blood and RSV seasonality measured as the weekly RSV hospitalization incidence among Danish infants below 6 months of age. For antibody detection we used an assay that specifically detected neutralizing antibodies and identified titer values similar to the values of an earlier study.10 We found a clear association between the maternally derived RSV-neutralizing antibody titer and RSV seasonality in infants younger than 6 months of
Figure. The titer of maternally derived RSV-neutralizing antibody in 457 cord blood samples from Danish infants born 1998-2003 and incidence of RSV hospitalization per 100 000 Danish infants younger than 6 months of age 1998-2003. Note: The mean RSV antibody titer expressed to the log base 2 is presented by use of the cubic spline technique and based on 58 infants born in 1998, 92 infants in 1999, 112 infants in 2000, 110 infants in 2001, and 85 in 2002.
age. When the mean cord blood RSV antibody titer (to the log base 2) decreased below 7.5 (ie, below 1:181), the number of RSV hospitalizations in infants younger than 6 months of age increased steeply for 3 months, possibly until some maternally derived RSV herd immunity among the newborns was reached, decreasing the transmission rate with subsequent declining hospitalization rates. The RSV transmission in the infant population continued for another 3 months in which the maternally derived mean RSV antibody level still increased until most susceptible individuals were protected. Although the level of RSV antibodies began to decline, the infant population appeared sufficiently protected for 6 months until the mean RSV antibody titer declined below 7.5 and another cycle began. Our speculation is that infants with higher transplacental RSV-neutralizing antibody are less susceptible to hospitalization from RSV infection. As greater numbers of infants with lower cord blood titers are released into the community, RSV epidemics can occur. These findings suggest that RSV immunization of pregnant mothers might decrease the incidence of RSV hospitalizations in infants. Mothers are probably more exposed to RSV through their children than the general population. However, if maternal RSV antibody level reflects the RSV antibody level in the population, a cyclic dependency pattern between RSVneutralizing antibodies and RSV seasonality might be an important part of the explanation of RSV seasonality in temperate climates. Kenneth Agerskov is gratefully acknowledged for his valuable help in identifying the study participants and withdraw interview data from the Danish National Birth Cohort.
Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus
297
REFERENCES 1. Roca A, Abacassamo F, Loscertales MP, Quinto L, Gomez-Olive X, Fenwick F, et al. Prevalence of respiratory syncytial virus IgG antibodies in infants living in a rural area of Mozambique. J Med Virol 2002;67:616-23. 2. de Sierra TM, Kumar ML, Wasser TE, Murphy BR, Subbarao EK. Respiratory syncytial virus-specific immunoglobulins in preterm infants. J Pediatr 1993;122:787-91. 3. Connors. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. The IMpact-RSV Study Group. Pediatrics 1998;102:531-7. 4. Hall CB, Walsh EE, Schnabel KC, Long CE, McConnochie KM, Hildreth SW, et al. Occurrence of groups A and B of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children. J Infect Dis 1990;162:1283-90. 5. Eriksson M, Bennet R, Rotzen-Ostlund M, von Sydow M, Wirgart BZ. Population-based rates of severe respiratory syncytial virus infection in children with and without risk factors, and outcome in a tertiary care setting. Acta Paediatr 2002;91:593-8.
298
Stensballe et al
6. Stensballe LG, Devasundaram JK, Simoes EA. Respiratory syncytial virus epidemics: the ups and downs of a seasonal virus. Pediatr Infect Dis J 2003;22:S21-S32. 7. Olsen J, Melbye M, Olsen SF, Sorensen TI, Aaby P, Andersen AM, et al. The Danish National Birth Cohort—its background, structure and aim. Scand J Public Health 2001;29:300-7. 8. Stensballe LG, Kristensen K, Nielsen J, Aaby P. Diagnosis coding in The Danish National Patient Registry for respiratory syncytial virus infections. Scand J Infect Dis 2005;37:747-52. 9. Anderson LJ, Hierholzer JC, Bingham PG, Stone YO. Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay. J Clin Microbiol 1985;22:1050-2. 10. Piedra PA, Jewell AM, Cron SG, Atmar RL, Glezen WP. Correlates of immunity to respiratory syncytial virus (RSV) associated-hospitalization: establishment of minimum protective threshold levels of serum neutralizing antibodies. Vaccine 2003;21: 3479-82.
The Journal of Pediatrics • February 2009
APPENDIX The study received support from the Danish National Research Foundation, the Lundbeck Foundation, the Novo Nordisk Foundation, and the Augustinus Foundation. The Danish Epidemiology Science Centre was established by a grant from The Danish National Research Foundation, and the center initiated and created the Danish National Birth Cohort. The cohort is furthermore a
result of a major grant from the Danish National Research Foundation. Additional support for the Danish National Birth Cohort was obtained from the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, the Augustinus Foundation, and the Health Foundation. The University of Colorado received an unrestricted grant from Abbott Laboratories Inc. The authors declare no conflicts of interest.
Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus
298.e1
Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus LONE GRAFF STENSBALLE, MD, HENRIK RAVN, MSC, PHD, KIM KRISTENSEN, MD, DMSC, TIFFANY MEAKINS, PETER AABY, DMSC, ERIC A. F. SIMOES, MB, BS, DCH, MD
AND
This study used 459 prospectively sampled cord blood samples to examine the association between maternally derived respiratory syncytial virus (RSV)–neutralizing antibodies and the RSV hospitalization season in Denmark. We found a clear temporal association and suggest that RSV-neutralizing antibody level plays a role in the RSV seasonal pattern. (J Pediatr 2009;154:296-8)
igh titers of maternally derived respiratory syncytial virus (RSV)–neutralizing antibodies1 are inversely associated with the incidence of RSV acute lower respiratory tract infection (LRI) during the first 6 months of life. Incomplete transfer of maternally derived RSV-neutralizing antibodies has been implicated in the increased risk of severe RSV infection in preterm infants,2 and studies of passively administered polyclonal, or humanized monoclonal antibody against RSV have shown reduction of RSV LRI or hospitalizations among premature infants and children with chronic lung disease.3 In countries with temperate climates, RSV causes epidemics of LRI among infants and young children during late fall, winter, and early spring.4,5 In tropical or semitropical climates as well, RSV epidemics appear regularly but with different patterns of seasonality. The reason for the periodic emergence of RSV LRI epidemics is not entirely clear. Geographic and climatic factors are clearly associated with epidemics,6 but it is uncertain whether this is related to spread of the virus, behavioral factors, or cyclic changes in a population’s immunologic susceptibility. This study examined prospectively the association between maternally derived RSV-neutralizing antibodies in cord blood and the RSV seasonality in Danish newborns.
H
MATERIAL We used cord blood samples from the Danish National Birth Cohort in combination with data from the RSV database on RSV hospitalizations in the Danish infant population.
The Danish National Birth Cohort The Danish National Birth Cohort (DNBC, www.bsmb.dk)7 recruited pregnant Danish women prospectively in the period from 1997 to 2003. A cord blood sample for the DNBC bio bank was collected at birth. Among 51 701 children in the DNBC with date of birth December 3, 1998, to May 2, 2003, and a cord blood sample, we used computerized randomization to select 459 cord blood samples for the study. The RSV Database We established a database on all hospitalized Danish patients tested for RSV between January 1996 and May 20038 and calculated the weekly RSV hospitalization incidence per 100 000 Danish infants below 6 months of age during the study period. DNBC LRI
296
The Danish National Birth Cohort Lower respiratory tract infection
RSV
Respiratory syncytial virus
From the Bandim Health Project, Statens Serum Institut (L.S., H.R., P.A.), the Paediatric Clinic, Rigshospitalet (K.K.), Copenhagen, Denmark, the Bandim Health Project (L.S., H.R., P.A.), Bissau, Guinea-Bissau, and the Department of Pediatrics, University of Colorado School at Denver and Health Sciences Center and The Children’s Hospital (T.F., E.S.), Denver, CO. Conflict of interest and funding information available in the Appendix (available at www. jpeds.com). Submitted for publication Mar 18, 2008; last revision received May 20, 2008; accepted Jul 23, 2008. Reprint requests: Lone Graff Stensballe, Bandim Health Project, Statens Serum Institut, Artillerivej 5, 2300 Copenhagen S, Denmark. E-mail: [email protected]. 0022-3476/$ - see front matter Copyright © 2009 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2008.07.053
RSV-Neutralizing Antibody Assay We used a microneutralization assay modified from one described earlier,9 performed in 96 well microtiter plates. With each set of test plates there was a control plate that contained 3 columns of known positive and negative controls diluted 2-fold, and 2 columns for a back titer for the virus. Each of the test plates contained 4 patient sera samples (done in duplicate) diluted out 2-fold across the 96-well plate, beginning with serum dilution of 1:4. Sera were first serially diluted in 2% fetal bovine serum in minimal essential medium in the test plates followed by the addition of 15 to 25 plaqueforming units of virus per well. The serum virus mixture was allowed to incubate for 1.5 hours on a rocker, at 37.0° C. 25 000 Hep-2 cells per well in 10% fetal bovine serum minimal essential medium were then added and allowed to incubate for 6 days; plates were observed daily for visible cytopathic effects. The medium/virus/serum mixture was gently aspirated, and the cells were stained with crystal violet fixative. After 24 hours the wells were washed with water, allowed to dry, and then read under a microscope. The titer was obtained as the reciprocal of the dilution from the last row in which no cytopathic effect was seen. Statistics Titers were expressed as log base 2 and treated as continuous variables. The association between RSV season and maternally derived RSV-neutralizing antibodies was described by use of correlation analysis.
RESULTS Two serum samples had insufficient quantity for the analysis and were excluded. The geometric mean titer of maternally derived RSV-neutralizing antibody in 457 cord blood samples was 7.89 to the log base 2 (percentiles 10% 6; 25% 7; 50% 8; 75% 9; 90% 10). A temporal correlation between the incidence of RSV hospitalization and the mean RSV antibody titer in cord blood samples was observed (P ⫽ .00, correlation coefficient ⫽ ⫺0.25) (Figure). Incidence of RSV hospitalization increased when cord RSV antibody titer declined, and the RSV epidemic peaked shortly after the nadir of the mean antibody level.
DISCUSSION This study offered a unique possibility to examine the association between the maternally derived RSV-neutralizing antibody titer in consecutively sampled cord blood and RSV seasonality measured as the weekly RSV hospitalization incidence among Danish infants below 6 months of age. For antibody detection we used an assay that specifically detected neutralizing antibodies and identified titer values similar to the values of an earlier study.10 We found a clear association between the maternally derived RSV-neutralizing antibody titer and RSV seasonality in infants younger than 6 months of
Figure. The titer of maternally derived RSV-neutralizing antibody in 457 cord blood samples from Danish infants born 1998-2003 and incidence of RSV hospitalization per 100 000 Danish infants younger than 6 months of age 1998-2003. Note: The mean RSV antibody titer expressed to the log base 2 is presented by use of the cubic spline technique and based on 58 infants born in 1998, 92 infants in 1999, 112 infants in 2000, 110 infants in 2001, and 85 in 2002.
age. When the mean cord blood RSV antibody titer (to the log base 2) decreased below 7.5 (ie, below 1:181), the number of RSV hospitalizations in infants younger than 6 months of age increased steeply for 3 months, possibly until some maternally derived RSV herd immunity among the newborns was reached, decreasing the transmission rate with subsequent declining hospitalization rates. The RSV transmission in the infant population continued for another 3 months in which the maternally derived mean RSV antibody level still increased until most susceptible individuals were protected. Although the level of RSV antibodies began to decline, the infant population appeared sufficiently protected for 6 months until the mean RSV antibody titer declined below 7.5 and another cycle began. Our speculation is that infants with higher transplacental RSV-neutralizing antibody are less susceptible to hospitalization from RSV infection. As greater numbers of infants with lower cord blood titers are released into the community, RSV epidemics can occur. These findings suggest that RSV immunization of pregnant mothers might decrease the incidence of RSV hospitalizations in infants. Mothers are probably more exposed to RSV through their children than the general population. However, if maternal RSV antibody level reflects the RSV antibody level in the population, a cyclic dependency pattern between RSVneutralizing antibodies and RSV seasonality might be an important part of the explanation of RSV seasonality in temperate climates. Kenneth Agerskov is gratefully acknowledged for his valuable help in identifying the study participants and withdraw interview data from the Danish National Birth Cohort.
Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus
297
REFERENCES 1. Roca A, Abacassamo F, Loscertales MP, Quinto L, Gomez-Olive X, Fenwick F, et al. Prevalence of respiratory syncytial virus IgG antibodies in infants living in a rural area of Mozambique. J Med Virol 2002;67:616-23. 2. de Sierra TM, Kumar ML, Wasser TE, Murphy BR, Subbarao EK. Respiratory syncytial virus-specific immunoglobulins in preterm infants. J Pediatr 1993;122:787-91. 3. Connors. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. The IMpact-RSV Study Group. Pediatrics 1998;102:531-7. 4. Hall CB, Walsh EE, Schnabel KC, Long CE, McConnochie KM, Hildreth SW, et al. Occurrence of groups A and B of respiratory syncytial virus over 15 years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children. J Infect Dis 1990;162:1283-90. 5. Eriksson M, Bennet R, Rotzen-Ostlund M, von Sydow M, Wirgart BZ. Population-based rates of severe respiratory syncytial virus infection in children with and without risk factors, and outcome in a tertiary care setting. Acta Paediatr 2002;91:593-8.
298
Stensballe et al
6. Stensballe LG, Devasundaram JK, Simoes EA. Respiratory syncytial virus epidemics: the ups and downs of a seasonal virus. Pediatr Infect Dis J 2003;22:S21-S32. 7. Olsen J, Melbye M, Olsen SF, Sorensen TI, Aaby P, Andersen AM, et al. The Danish National Birth Cohort—its background, structure and aim. Scand J Public Health 2001;29:300-7. 8. Stensballe LG, Kristensen K, Nielsen J, Aaby P. Diagnosis coding in The Danish National Patient Registry for respiratory syncytial virus infections. Scand J Infect Dis 2005;37:747-52. 9. Anderson LJ, Hierholzer JC, Bingham PG, Stone YO. Microneutralization test for respiratory syncytial virus based on an enzyme immunoassay. J Clin Microbiol 1985;22:1050-2. 10. Piedra PA, Jewell AM, Cron SG, Atmar RL, Glezen WP. Correlates of immunity to respiratory syncytial virus (RSV) associated-hospitalization: establishment of minimum protective threshold levels of serum neutralizing antibodies. Vaccine 2003;21: 3479-82.
The Journal of Pediatrics • February 2009
APPENDIX The study received support from the Danish National Research Foundation, the Lundbeck Foundation, the Novo Nordisk Foundation, and the Augustinus Foundation. The Danish Epidemiology Science Centre was established by a grant from The Danish National Research Foundation, and the center initiated and created the Danish National Birth Cohort. The cohort is furthermore a
result of a major grant from the Danish National Research Foundation. Additional support for the Danish National Birth Cohort was obtained from the Pharmacy Foundation, the Egmont Foundation, the March of Dimes Birth Defects Foundation, the Augustinus Foundation, and the Health Foundation. The University of Colorado received an unrestricted grant from Abbott Laboratories Inc. The authors declare no conflicts of interest.
Seasonal Variation of Maternally Derived Respiratory Syncytial Virus Antibodies and Association with Infant Hospitalizations for Respiratory Syncytial Virus
298.e1