This article was downloaded by: [University of Southern Queensland] On: 13 March 2015, At: 02:18 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Polio Virus Neutralizing Antibody Dynamics Among Children in a NorthCentral and South-Western Nigeria State ab
a
Adekunle Johnson Adeniji , Anyebe Bernard Onoja & Moses Olubusuyi Adewumi
a
a
Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria b
Click for updates
World Health Organization National Reference Polio Laboratory, Ibadan, Nigeria Accepted author version posted online: 25 Feb 2014.Published online: 22 Aug 2014.
To cite this article: Adekunle Johnson Adeniji, Anyebe Bernard Onoja & Moses Olubusuyi Adewumi (2015) Polio Virus Neutralizing Antibody Dynamics Among Children in a North-Central and South-Western Nigeria State, Journal of Immunoassay and Immunochemistry, 36:1, 45-53, DOI: 10.1080/15321819.2014.893889 To link to this article: http://dx.doi.org/10.1080/15321819.2014.893889
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Immunoassay and Immunochemistry, 36:45–53, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.893889
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
POLIO VIRUS NEUTRALIZING ANTIBODY DYNAMICS AMONG CHILDREN IN A NORTH-CENTRAL AND SOUTH-WESTERN NIGERIA STATE
Adekunle Johnson Adeniji,1,2 Anyebe Bernard Onoja,1 and Moses Olubusuyi Adewumi1 1 Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria 2 World Health Organization National Reference Polio Laboratory, Ibadan, Nigeria
Northern Nigeria accounts for the highest number of confirmed wild polio viruses globally. Transmission to neighboring countries is worrisome after the country failed to meet several deadlines for polio eradication. Most studies on neutralizing antibody have focused on the Northeastern and Northwestern regions. This study measured polio virus neutralizing antibody levels among children in North-central and South-western Nigeria. Children between the ages of 10 months and 13 yr were randomly selected from Abanishe-lolu Hospital Ilorin (North-central) and Oni Memorial and Adeoyo Hospitals in Ibadan (South-west) Nigeria. The alpha neutralization method was employed. Herd immunity was 1.4% in Ilorin, 36.6% in Oni Memorial Hospital, and 49.5% in Adeoyo Hospital. Out of 299 children studied, 49 (16.4%) children had no protection against all poliovirus serotypes while 100 (33.4%) were fully protected against all three serotypes. Five (7.9%) children in Ilorin and 5 (3.4%) children in Oni Memorial Hospital Ibadan had no detectable neutralizing antibody. A significant difference was observed (p=0.000) when the GMT against poliovirus 1, 2, and 3 was compared. A significant proportion of children were not fully protected. Sero-surveillance is recommended for effective monitoring of vaccination efforts to guide health policy formulators. Keywords Polio virus, vaccination, children, Nigeria
INTRODUCTION The global polio eradication drive started in 1988 with the specific goal of eradicating wild polio viruses (WPV) by the year 2000.[1] This target was not met; instead, it was set again for 2005 and that date too was still unmet.[2] Polio virus (PV) transmission was interrupted in the Americas, Europe, Address correspondence to Anyebe Bernard Onoja, Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria. E-mail: [email protected] Color versions of one or more of the figures in this article can be found online at www.tandfonline. com/ljii.
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
46
A. J. Adeniji et al.
and Western Pacific regions, and by the end of 2002 more than 180 countries were polio-free. The number of polio-infected countries decreased to seven with PV eliminated from three continents. Eradication of polio and eventual cessation of polio immunization will save the world US$1.5 billion per year.[3] The average number of outbreaks experienced by countries in Africa declined with physical distance from Nigeria. Cameroon, Chad, and Niger, all bordering Nigeria, reported 9 outbreaks each from 2004–2010, and Niger reported 33 outbreaks of which 21 were single cases. Benin, also bordering Nigeria, reported seven outbreaks during this period.[4] In 2002, three states in northern Nigeria boycotted vaccination because of the belief that the vaccine was laced with anti-fertility, cancerous agents and HIV by Western countries. This led to a resurgence of poliomyelitis to about 1,300–1,500 cases per year worldwide,[5] and by 2004 there was a 30% increase in polio cases in Nigeria.[6] The epidemic resulted in reinfection of 13 neighboring countries and importation by 21 countries such that in 2006 Nigeria ranked highest with 675 cases out of the 1,749 confirmed cases of PV infection globally.[7] Multiple chains of transmission have been shown to occur in religious communities which actively oppose or resist immunization.[8] In 2012, Nigeria contributed 95% of the polio burden in the WHO-AFRO-region with 121 WPVs and 8 circulating vaccine derived polio viruses (cVDPVs).[9] Nigeria relies on a combination of routine immunization services using trivalent OPV (tOPV, types 1, 2, and 3) and Supplementary Immunization Activities (SIAs) to immunize children against polio.[10] In some states in Nigeria, Immunization Plus Days (IPDs) are conducted monthly at state level while Supplementary Immunization Activities (SIAs) are less than once a month for high risk local government areas and mop-ups are usually carried out at ward level after a confirmed case. In some parts of the country, these activities are not conducted on a monthly basis but depend upon the epidemiology of poliovirus and the recommendations of the Nigeria Expert Review Committee for Polio Eradication.[11] In addition, during the routine immunization sessions children receive trivalent oral polio vaccine (tOPV) types 1, 2, and 3 which is usually administered at birth and at ages 6, 10, and 14 weeks.[12] Also, monovalent oral polio vaccine types 1 and 3, bivalent oral polio vaccine types 1 and 3, or tOPV types 1, 2, and 3 are used during Supplementary Immunization Activities.[12] In Nigeria, research activities on the PV neutralizing antibody have concentrated on Northeast and Northwestern Nigeria,[11,13] however scant information exists on the North-central States. Also, low PV neutralizing antibody level was demonstrated many years ago in Ibadan, South-western Nigeria.[14] In this study, we investigate PV neutralizing antibody levels in children in selected locations in North-central and South-western Nigeria in order to assess their immune status.
Polio virus neutralizing antibody in Nigeria
47
MATERIALS AND METHODS
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
Study Design Children aged between 10 months and 13 years whose parents consented were enrolled for the study, irrespective of their vaccination status. They visited physicians for various ailments in the General Out-Patient Clinics of the various hospitals and belonged to different socioeconomic groups. In Ilorin–Kwara State (North-central Nigeria), Abanishe-lolu Children Hospital (63 children: Male = 34; Female = 29) was the location for this study while in Ibadan–Oyo State (South-western Nigeria) it was carried out in Oni Memorial Children’s Hospital (145 children: Male = 82; Female = 63) and Adeoyo Maternity Hospital (91 children: Male = 55; Female = 36). The few number of samples from Ilorin was due to logistical problems and religious apathy towards participation in the study. Sample Collection and Processing About 3 mL of blood was collected from each child into appropriately labeled anti-coagulant-free tubes by venipuncture. The blood was allowed to clot and then centrifuged for 15 min at 2,000 × g. The serum was carefully removed with fine pipette to avoid extracting red blood cells, then transferred to a sterile cryovial and stored away at -20◦ C until ready for analysis. Cell Culture L20B cell line was propagated in T75cm2 flask for virus isolation and neutralization procedures. The cell line was maintained with Gibco® Dulbecco’s Modified Eagle Medium (Massy Cedex, France). Cell count was done using Neuber counting chamber (Hatfield, PA, USA). Virus Preparation Sabin strain of PV subtypes 1, 2, and 3 were propagated in L20B cells in modified Eagles Minimum Essential Medium supplemented with 10% fetal calf serum until full cytopathic effects developed. Culture supernatant was clarified and freed of aggregates by cold centrifugation for 15 min at 2,000 × g. The supernatant was extracted with 10% arklone at +4◦ C for 45 min with constant shaking, followed by centrifugation for 30 min at 2,000 × g. The supernatant was concentrated by direct ultrafiltration with membranes.[15] Tissue culture infectious dose 50 (TCID50 ) was calculated using Spearman- Karber method[16,17] and found to be 7.0 for PV1, 6.75 for PV2, and 7.0 for PV3. The working infectious dose was 100TCID50 per
48
A. J. Adeniji et al.
inoculum, a 1:10 dilution was made from the stock polioviruses and built up to 10−5 for PV1, 10−4.75 for PV2, and 10−5 for PV3 to work with.
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
Neutralization Test (NT) The alpha (α) method of virus neutralization was employed using constant virus varying serum.[18] Sera were heat inactivated in water bath at 56◦ C for 30 min before use to destroy complements and other labile non specific neutralizing or potentiating factors. 50 µL of 0% medium was initially dispensed serially into all wells except well 11 and 12. A 1:8 dilution of each sera was then added to well 1 and 2, a serial 8-fold dilution was then carried out from well 2–10 and 50 µL of the serum/medium mixture was discarded from well 10 to maintain uniform volume of 50 µL sera throughout. This was repeated in triplicate microtiter plates for serotypes PV1, PV2, and PV3, then 50 µL of PV1, PV2, and PV3 was separately added from well 1–10 leaving wells 11 and 12, this was done for microtiter plate 1, 2, and 3. It was kept in the incubator for 1 hr so that the Ag and Ab will react to form an Ag-Ab complex. A confluent flask of L20B was then trypsinized at this point and medium containing 5% calf serum was used to stop the action of trypsin. Each well then received 50 µL of cell suspension including wells 11 and 12 (cell control), sealer was placed over it to avoid escape of CO2 . The sides of each microtiter plate were sealed with sterile paper tape to prevent contamination. Cultures were incubated for 7 days at 37◦ C and observed daily for cytopathic effects (rounding up of cells). The minimum antibody titer was 8 and protective titer ≥32 while full protection was based on protection against all 3 serotypes.[18] Statistical Analysis Data analysis was done using SPSS 20 software (IBM Corp. released 2011, IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY, USA). Chi square test was used to determine association between neutralizing antibody against polio serotypes and immunization status in a univariate analysis. P value
Journal of Immunoassay and Immunochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ljii20
Polio Virus Neutralizing Antibody Dynamics Among Children in a NorthCentral and South-Western Nigeria State ab
a
Adekunle Johnson Adeniji , Anyebe Bernard Onoja & Moses Olubusuyi Adewumi
a
a
Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria b
Click for updates
World Health Organization National Reference Polio Laboratory, Ibadan, Nigeria Accepted author version posted online: 25 Feb 2014.Published online: 22 Aug 2014.
To cite this article: Adekunle Johnson Adeniji, Anyebe Bernard Onoja & Moses Olubusuyi Adewumi (2015) Polio Virus Neutralizing Antibody Dynamics Among Children in a North-Central and South-Western Nigeria State, Journal of Immunoassay and Immunochemistry, 36:1, 45-53, DOI: 10.1080/15321819.2014.893889 To link to this article: http://dx.doi.org/10.1080/15321819.2014.893889
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Immunoassay and Immunochemistry, 36:45–53, 2015 Copyright © Taylor & Francis Group, LLC ISSN: 1532-1819 print/1532-4230 online DOI: 10.1080/15321819.2014.893889
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
POLIO VIRUS NEUTRALIZING ANTIBODY DYNAMICS AMONG CHILDREN IN A NORTH-CENTRAL AND SOUTH-WESTERN NIGERIA STATE
Adekunle Johnson Adeniji,1,2 Anyebe Bernard Onoja,1 and Moses Olubusuyi Adewumi1 1 Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria 2 World Health Organization National Reference Polio Laboratory, Ibadan, Nigeria
Northern Nigeria accounts for the highest number of confirmed wild polio viruses globally. Transmission to neighboring countries is worrisome after the country failed to meet several deadlines for polio eradication. Most studies on neutralizing antibody have focused on the Northeastern and Northwestern regions. This study measured polio virus neutralizing antibody levels among children in North-central and South-western Nigeria. Children between the ages of 10 months and 13 yr were randomly selected from Abanishe-lolu Hospital Ilorin (North-central) and Oni Memorial and Adeoyo Hospitals in Ibadan (South-west) Nigeria. The alpha neutralization method was employed. Herd immunity was 1.4% in Ilorin, 36.6% in Oni Memorial Hospital, and 49.5% in Adeoyo Hospital. Out of 299 children studied, 49 (16.4%) children had no protection against all poliovirus serotypes while 100 (33.4%) were fully protected against all three serotypes. Five (7.9%) children in Ilorin and 5 (3.4%) children in Oni Memorial Hospital Ibadan had no detectable neutralizing antibody. A significant difference was observed (p=0.000) when the GMT against poliovirus 1, 2, and 3 was compared. A significant proportion of children were not fully protected. Sero-surveillance is recommended for effective monitoring of vaccination efforts to guide health policy formulators. Keywords Polio virus, vaccination, children, Nigeria
INTRODUCTION The global polio eradication drive started in 1988 with the specific goal of eradicating wild polio viruses (WPV) by the year 2000.[1] This target was not met; instead, it was set again for 2005 and that date too was still unmet.[2] Polio virus (PV) transmission was interrupted in the Americas, Europe, Address correspondence to Anyebe Bernard Onoja, Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria. E-mail: [email protected] Color versions of one or more of the figures in this article can be found online at www.tandfonline. com/ljii.
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
46
A. J. Adeniji et al.
and Western Pacific regions, and by the end of 2002 more than 180 countries were polio-free. The number of polio-infected countries decreased to seven with PV eliminated from three continents. Eradication of polio and eventual cessation of polio immunization will save the world US$1.5 billion per year.[3] The average number of outbreaks experienced by countries in Africa declined with physical distance from Nigeria. Cameroon, Chad, and Niger, all bordering Nigeria, reported 9 outbreaks each from 2004–2010, and Niger reported 33 outbreaks of which 21 were single cases. Benin, also bordering Nigeria, reported seven outbreaks during this period.[4] In 2002, three states in northern Nigeria boycotted vaccination because of the belief that the vaccine was laced with anti-fertility, cancerous agents and HIV by Western countries. This led to a resurgence of poliomyelitis to about 1,300–1,500 cases per year worldwide,[5] and by 2004 there was a 30% increase in polio cases in Nigeria.[6] The epidemic resulted in reinfection of 13 neighboring countries and importation by 21 countries such that in 2006 Nigeria ranked highest with 675 cases out of the 1,749 confirmed cases of PV infection globally.[7] Multiple chains of transmission have been shown to occur in religious communities which actively oppose or resist immunization.[8] In 2012, Nigeria contributed 95% of the polio burden in the WHO-AFRO-region with 121 WPVs and 8 circulating vaccine derived polio viruses (cVDPVs).[9] Nigeria relies on a combination of routine immunization services using trivalent OPV (tOPV, types 1, 2, and 3) and Supplementary Immunization Activities (SIAs) to immunize children against polio.[10] In some states in Nigeria, Immunization Plus Days (IPDs) are conducted monthly at state level while Supplementary Immunization Activities (SIAs) are less than once a month for high risk local government areas and mop-ups are usually carried out at ward level after a confirmed case. In some parts of the country, these activities are not conducted on a monthly basis but depend upon the epidemiology of poliovirus and the recommendations of the Nigeria Expert Review Committee for Polio Eradication.[11] In addition, during the routine immunization sessions children receive trivalent oral polio vaccine (tOPV) types 1, 2, and 3 which is usually administered at birth and at ages 6, 10, and 14 weeks.[12] Also, monovalent oral polio vaccine types 1 and 3, bivalent oral polio vaccine types 1 and 3, or tOPV types 1, 2, and 3 are used during Supplementary Immunization Activities.[12] In Nigeria, research activities on the PV neutralizing antibody have concentrated on Northeast and Northwestern Nigeria,[11,13] however scant information exists on the North-central States. Also, low PV neutralizing antibody level was demonstrated many years ago in Ibadan, South-western Nigeria.[14] In this study, we investigate PV neutralizing antibody levels in children in selected locations in North-central and South-western Nigeria in order to assess their immune status.
Polio virus neutralizing antibody in Nigeria
47
MATERIALS AND METHODS
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
Study Design Children aged between 10 months and 13 years whose parents consented were enrolled for the study, irrespective of their vaccination status. They visited physicians for various ailments in the General Out-Patient Clinics of the various hospitals and belonged to different socioeconomic groups. In Ilorin–Kwara State (North-central Nigeria), Abanishe-lolu Children Hospital (63 children: Male = 34; Female = 29) was the location for this study while in Ibadan–Oyo State (South-western Nigeria) it was carried out in Oni Memorial Children’s Hospital (145 children: Male = 82; Female = 63) and Adeoyo Maternity Hospital (91 children: Male = 55; Female = 36). The few number of samples from Ilorin was due to logistical problems and religious apathy towards participation in the study. Sample Collection and Processing About 3 mL of blood was collected from each child into appropriately labeled anti-coagulant-free tubes by venipuncture. The blood was allowed to clot and then centrifuged for 15 min at 2,000 × g. The serum was carefully removed with fine pipette to avoid extracting red blood cells, then transferred to a sterile cryovial and stored away at -20◦ C until ready for analysis. Cell Culture L20B cell line was propagated in T75cm2 flask for virus isolation and neutralization procedures. The cell line was maintained with Gibco® Dulbecco’s Modified Eagle Medium (Massy Cedex, France). Cell count was done using Neuber counting chamber (Hatfield, PA, USA). Virus Preparation Sabin strain of PV subtypes 1, 2, and 3 were propagated in L20B cells in modified Eagles Minimum Essential Medium supplemented with 10% fetal calf serum until full cytopathic effects developed. Culture supernatant was clarified and freed of aggregates by cold centrifugation for 15 min at 2,000 × g. The supernatant was extracted with 10% arklone at +4◦ C for 45 min with constant shaking, followed by centrifugation for 30 min at 2,000 × g. The supernatant was concentrated by direct ultrafiltration with membranes.[15] Tissue culture infectious dose 50 (TCID50 ) was calculated using Spearman- Karber method[16,17] and found to be 7.0 for PV1, 6.75 for PV2, and 7.0 for PV3. The working infectious dose was 100TCID50 per
48
A. J. Adeniji et al.
inoculum, a 1:10 dilution was made from the stock polioviruses and built up to 10−5 for PV1, 10−4.75 for PV2, and 10−5 for PV3 to work with.
Downloaded by [University of Southern Queensland] at 02:18 13 March 2015
Neutralization Test (NT) The alpha (α) method of virus neutralization was employed using constant virus varying serum.[18] Sera were heat inactivated in water bath at 56◦ C for 30 min before use to destroy complements and other labile non specific neutralizing or potentiating factors. 50 µL of 0% medium was initially dispensed serially into all wells except well 11 and 12. A 1:8 dilution of each sera was then added to well 1 and 2, a serial 8-fold dilution was then carried out from well 2–10 and 50 µL of the serum/medium mixture was discarded from well 10 to maintain uniform volume of 50 µL sera throughout. This was repeated in triplicate microtiter plates for serotypes PV1, PV2, and PV3, then 50 µL of PV1, PV2, and PV3 was separately added from well 1–10 leaving wells 11 and 12, this was done for microtiter plate 1, 2, and 3. It was kept in the incubator for 1 hr so that the Ag and Ab will react to form an Ag-Ab complex. A confluent flask of L20B was then trypsinized at this point and medium containing 5% calf serum was used to stop the action of trypsin. Each well then received 50 µL of cell suspension including wells 11 and 12 (cell control), sealer was placed over it to avoid escape of CO2 . The sides of each microtiter plate were sealed with sterile paper tape to prevent contamination. Cultures were incubated for 7 days at 37◦ C and observed daily for cytopathic effects (rounding up of cells). The minimum antibody titer was 8 and protective titer ≥32 while full protection was based on protection against all 3 serotypes.[18] Statistical Analysis Data analysis was done using SPSS 20 software (IBM Corp. released 2011, IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY, USA). Chi square test was used to determine association between neutralizing antibody against polio serotypes and immunization status in a univariate analysis. P value
