The Journal of Infectious Diseases MAJOR ARTICLE
Modeling of EBV Infection and Antibody Responses in Kenyan Infants With Different Levels of Malaria Exposure Shows Maternal Antibody Decay is a Major Determinant of Early EBV Infection Arnold Reynaldi,1 Timothy E. Schlub,2 Erwan Piriou,3 Sidney Ogolla,4 Odada P. Sumba,4 Ann M. Moormann,5 Rosemary Rochford,6 and Miles P. Davenport1 1 Infection Analytics Program, Kirby Institute for Infection and Immunity, UNSW Australia, Sydney, and 2Sydney School of Public Health, Sydney University, New South Wales, Australia; 3Médecins Sans Frontières, Amsterdam, The Netherlands; 4Centre for Global Health Research, Kenya Medical Research Institute, Kisumu; 5Program in Molecular Medicine, University of Massachusetts Medical School, Worcester; and 6Department of Immunology and Microbiology, University of Colorado Denver, Aurora
The combination of Epstein-Barr virus (EBV) infection and high malaria exposure are risk factors for endemic Burkitt lymphoma, and evidence suggests that infants in regions of high malaria exposure have earlier EBV infection and increased EBV reactivation. In this study we analyzed the longitudinal antibody response to EBV in Kenyan infants with different levels of malaria exposure. We found that high malaria exposure was associated with a faster decline of maternally derived immunoglobulin G antibody to both the EBV viral capsid antigen and EBV nuclear antigen, followed by a more rapid rise in antibody response to EBV antigens in children from the high-malaria-transmission region. We also observed the long-term persistence of anti–viral capsid antigen immunoglobulin M responses in children from the high-malaria region. More rapid decay of maternal antibodies was a major predictor of EBV infection outcome, because decay predicted time to EBV DNA detection, independent of high or low malaria exposure. Keywords. Epstein-Barr virus; P. falciparum malaria; Burkitt Lymphoma; antibody; immunity.
Epstein-Barr virus (EBV) is a gammaherpesvirus causing infectious mononucleosis and also associated with a variety of cancers in humans [1]. In Africa, EBV is associated with endemic Burkitt lymphoma in the presence of Plasmodium falciparum malaria infection [2]. Previous studies in Africa have shown that most children seroconvert to EBV between 6 and 18 months of age [3–5] and that living in an area of high malaria transmission is associated with higher EBV load [4] and earlier time of the first EBV infection [5]. Moreover, the presence of P. falciparum infection is correlated with increased EBV load over time and with more frequent detectable EBV in children [5, 6] and pregnant women [7]. High levels of antibody against the EBV viral capsid antigen (VCA) are associated with a higher risk of Burkitt lymphoma in both Ugandan [8, 9] and Kenyan [10] children. High malaria exposure was also associated with impaired transfer of maternal VCA and EBV nuclear antigen 1 (EBNA1)–specific immunoglobulin (Ig) G from mother to infant [11], and later in life, higher level of EBV-specific IgG antibodies [12]. The measurement of antibodies specific for EBV VCA and EBNA1 have been used in numerous seroepidemiology studies to assess primary and persistent EBV infections (reviewed in
[13]). Typically, anti-VCA IgG appears early after infection, with a delay to detection of anti-EBNA1 IgG. Both are then found at relatively stable levels throughout the life of the host. Anti-EBV early antigen diffuse complex (EAd) IgG has been used as a serological marker for evidence of viral reactivation, because its levels are generally much lower than levels of antiVCA IgG, they decrease after primary infection (in contrast to VCA IgG), and are elevated during viral reactivation. The EBV Z-transactivation antigen (Zta) is an EBV immediate early protein. It has not been used in clinical settings to detect EBV reactivation to our knowledge, but we have found that the serology of Zta is similar to that of EAd-IgG and that Zta serology can also be used as a marker of viral reactivation [12]. In this study, we used a modeling approach to investigate how malaria exposure might affect the serological profiles of EBVinfected children, and EBV infection kinetics. This study demonstrated rapid decay of maternal antibodies in the region of high malaria exposure, followed by early EBV DNA detection and higher expansion rate of EBV-specific antibodies. This expansion of EBV antibodies can be observed from 1–2 months of age, suggesting very early EBV exposure. MATERIALS AND METHODS
Received 7 June 2016; accepted 17 August 2016; published online 28 August 2016. Correspondence: M. P. Davenport, Kirby Institute for Infection and Immunity, UNSW Australia, Sydney, New South Wales 2052, Australia ([email protected]). The Journal of Infectious Diseases® 2016;214:1390–8 © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail [email protected]. DOI: 10.1093/infdis/jiw396
1390
•
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Reynaldi et al
Patients
Detailed methods of the cohort and analysis of viral loads from birth to 24 months have been described elsewhere [5]. Briefly, 2 cohorts of infants enrolled within 1 month of birth were monitored in Kisumu (77 children; high-malaria-transmission region)
and Nandi (86 children; low-malaria-transmission region) in Kenya. The first data collection was performed when the children were about 1 month of age (range, 1–4 months), and then roughly every month until 24 months of age. Luminex bead assays were used to capture 4 different types of EBV-specific IgG to VCA, EBNA1, z-transactivation antigen (Zta), and EAd [12]. Plasma was diluted either 1:100 or 1:6400. We expressed the results as median fluorescence intensity (MFI) level of ≥75 beads. Detection of anti-VCA IgM is used clinically to evaluate whether primary EBV infection has occurred [13]. We performed the enzyme-linked immunosorbent assays [14] to detect anti-VCA IgM, and we had limited sample volume to perform more extensive serological analysis. For all serological assays, pooled serum from EBV negative donors was used as negative controls. The EBV seronegative plasma was used to create baseline values for the MFI, these values were then averaged and the mean (±3 standard deviations) was subtracted from the MFI or optical density. Quantitative polymerase chain reaction was used to detect the presence of EBV DNA in the blood, with a limit of detection of 2 copies/µg of DNA [4, 15]. Modeling the Kinetics of EBV-Specific Antibodies
Throughout this study, we modeled the kinetics of EBV-specific IgG antibodies as having potentially 2 phases. The first phase was a decline in antibody present at birth (representing the presence and loss of maternal antibodies), and the second was the subsequent increase in EBV-specific antibodies (representing the production of endogenous EBV-specific antibodies). Note that in some cases, we did not observe an initial loss of maternal antibodies. Maternal antibodies (M [t]) transferred in utero will decay at a rate δ (exponentially), while, at the same time, endogenous EBV-specific antibodies (E[t]) reflective of infection will be formed (expanding at rate g) and will later reach a constant level K. This can be written as follows: MðtÞ ¼ Aedt EðtÞ ¼
KE0 egt K þ E0 ðegt 1Þ
ð1Þ
MFI(tÞ ¼MðtÞþEðtÞ where MFI (t) represents MFI level at time t; A, initial maternal EBV-specific antibody at birth; δ, decay of maternal antibody; g, intrinsic expansion rate of EBV antibody level; and K, stable level of EBV-specific antibodies. Note that in the cases of Ztaand EAd-specific IgG, we did not observe an initial presence or loss of maternal antibodies (ie, A = 0). For the kinetics of VCA-specific IgM, we used a piecewise log-linear model on the level of VCA IgM titer against age. The level of VCA IgM increases exponentially until a certain time (Tlast), after which it decreases exponentially. This was chosen based on the overall trend in the dynamics of VCA IgM. Maternal IgM does not cross the placenta, and detection
of VCA-specific IgM in the infants is an indicator of primary infection. Consistent with the inability of IgM to cross the placenta, we did not observe the initial presence or loss of maternal antibodies (Figure 1B). Fitting Procedures and Statistical Analysis
We used a nonlinear mixed-effect model with fixed treatment effect to capture the difference in antibody expansion and decay parameters between the 2 regions. Significance was determined based on the value of this covariate, which can be calculated using the Wald test from the standard errors calculated in package nlme in R software (version 3.1.2). The models were fitted with the nonlinear mixed-effect model R function nlme in library nlme (version 3.1-113). The fit was weighted using varPower, and we assumed a diagonal variance-covariance matrix for the random effect, using the pdDiag option in R. To find the 95% confidence interval (CI), we used the R function intervals in library nlme. We used Cox proportional hazard regression to determine if there was an association between the predicted parameters in the IgG responses and the time to be DNA positive. The analysis was done using the coxph function in R software from the library survival (version 2.38-1). RESULTS General Description
Examples of antibody levels of various EBV-specific antibodies are shown in Figure 1 (individual data in Supplementary Figure 1). For VCA-and EBNA1-specific IgG antibodies, we observed an initial high level of antibodies, which dropped during about the first 6 months of life (assumed to reflect the initial presence and loss of the maternally transmitted antibodies). After this, we observed an increase in VCA- and EBNA1specifc IgG, assumed to be from endogenous production by infants. Anti-Zta and anti-EAd antibodies are typically used as indicators of EBV reactivation and are transiently detected. In a separate study we have shown that maternal antibodies to Zta and EAd are not efficiently transferred to infants as assessed in cord blood [11]. Consistent with this, we did not observe the initial presence or subsequent loss of maternal antibody to these antigens. Faster Loss of Maternal IgG Antibody to VCA and EBNA1 for Children in High-Malaria Region
We first analyzed the kinetics of VCA- and EBNA1-specific IgG over time from 1 to 24 months of age, fitting a model with loss of maternal antibodies, and increase in endogenously produced antibodies (Eq 1) [3, 16]. We found that the estimated initial amount of VCA-specific IgG at birth was similar between the 2 cohorts (P = .77), consistent with the fact that the levels at enrollment 1 month later were also not significantly different (P = .84). However, the subsequent decay rate of maternal antibody to VCA was significantly faster in the Kinetics of EBV-Specific Antibodies
•
JID 2016:214 (1 November)
•
1391
A
EBNA1 IgG
VCA IgG 100 000
EBNA1 MFI
VCA MFI
100 000
10 000
Low malaria High malaria
10 000
1000
Low malaria High malaria
1000
100 0
10
20
30
10
20
Age, mo
Zta IgG
EAd IgG
30
10 000
EAd MFI
10 000
Zta MFI
0
Age, mo
1000
1000
Low malaria High malaria
Low malaria High malaria
100
100 0
10
20
30
0
10
Age, mo
20
30
Age, mo
B
VCA IgM
VCA IgM Titer
1000
100
10
Low malaria High malaria 1 0
10
20
30
Age, mo Figure 1. Serological profiles in children over time. Kinetics of Epstein-Barr virus (EBV)–specific immunoglobulin (Ig) G antibodies to 4 EBV antigens: viral capsid antigen (VCA), EBV nuclear antigen 1 (EBNA1), Z-transactivation antigen (Zta), and early antigen diffuse complex (EAd). Each graph shows the model prediction (solid line) with the 95% confidence band of the model fit (dashed lines); error bars represent standard error of the mean in the data. A, Initial drop during the first few months is assumed to reflect the presence and loss of maternal antibodies ( particularly antibodies to VCA and EBNA1). The later increase in antibody levels is assumed to indicate the production of endogenous EBV-specific antibodies. A complete time course of the data for individual subjects (with the model fitting) can be seen in Supplementary Figure 1. MFI, median fluorescence intensity. B, Different kinetics of VCA-specific IgM in the low- and high-malaria regions with age. In the low-malaria region, we see a later drop in the level of VCA IgM; in the high-malaria region, the level of VCA IgM is maintained.
high-malaria region (P < .001), with a rate of 0.23 month−1 (high-malaria region) and 0.15 month−1 (low-malaria region). The kinetics of maternal EBNA1-specific IgG showed the same trends. We found no difference in the measured level of EBNA1 IgG at first sampling (P = .75) or the level estimated to have been present at birth (P = .23). However, we found that similar to the decay of anti-VCA antibody, those living in a high-malaria region had faster decay of EBNA1 IgG 1392
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Reynaldi et al
than that in the low-malaria region (rates, 0.97 and 0.24 month−1, respectively; P < .001). Faster Rate of Increase of VCA and EBNA1 IgG for Children in High-Malaria Region
Using the same model (Eq 1), we found that the rate of increase of VCA-specific IgG was higher for children in the high-malaria than for those in the low-malaria region (0.93 month−1 vs 0.76
month−1; P = .046). However, the plateau level of VCA-specific IgG achieved at a later age did not differ significantly between the 2 cohorts (P = .52). This similar plateau level could arise owing to the limitations of the assay, in which maximal binding of antibodies is reached. However, when we repeated the same analysis using data obtained when plasma was diluted 1:6400, we still observed similar levels of saturation in the 2 groups (P = .55). The same trend can also observed in the kinetics of EBNA1specific IgG, with faster expansion in EBNA1-specific in the high-malaria region (0.55 month−1) than in the low-malaria region (0.32 month−1; P = .06). The stable levels reached by EBNA1-specific IgG did not differ significantly between those 2 cohorts (P = .48). For a complete summary, see Table 1. Steady Increase in Levels of Antibodies to Zta and EAd From a Very Early age
In the majority of study participants, we could not see evidence of maternal antibodies to Zta and EAd IgG (Figure 1A and Supplementary Figure 1), as the level of antibody rose continuously
Table 1.
Dynamics of VCA- and EBNA1-Specific IgGa
from the first sampling at age 1–4 months (mean of differences for Zta, 250 MFI [95% CI, 55.42–443.7; P = .01 ( paired t test)]; mean of differences for EAd, 181 MFI [29.50–308 ; P = .03]). The rise in both Zta and EAd IgG from the time of enrollment at 1 month of age suggests ongoing antigenic stimulation from very early in life. The estimated initial levels of Zta-specific IgG at birth did not differ significantly between infants in high- versus low-malaria regions (P = .76), nor did the levels at the time of first sample (P = .78). The stable levels of Zta-specific IgG were also not significantly different (P = .19). However, we found that the expansion rate of Zta-specific IgG was significantly faster for children in the high-malaria region than for those in the low-malaria region (0.4 vs 0.28 month−1; P = .006). Next, we used the same approach to analyze the kinetics of EAd-specific antibodies, where again we saw no evidence of maternal antibody decay. We found that neither the initial amounts of EAd-specific IgG nor the plateau levels differed significantly between groups (P = .87 and P = .80, respectively). Although there was a trend to faster expansion of antibodies in the high-malaria region (0.4 month−1) compared with the low-malaria region (0.36 month−1), this difference was not significant (P = .38). The summary for both Zta and EAd IgG can be seen in Table 2.
Value (95% CI) Parameter
Low-Malaria Region
High-Malaria Region
P Valueb
VCA-specific IgG Initial maternal antibody, MFI
11 560 (9113–14 007)
12 086 (6037–18 136)
.77
Decay rate, mo−1
0.15 (.13–.17)
0.23 (.18–.27)
Initial EBV antibody, MFI
14.9 (2–27.75)
68.5 (6.4–130.6)
.03
Expansion rate, mo−1
0.76 (.65–.87)
0.93 (.65–1.22)
.046
Threshold, MFI
21 722 (20 141–23 303)
19 932 (16 232–23 614)
Modeling of EBV Infection and Antibody Responses in Kenyan Infants With Different Levels of Malaria Exposure Shows Maternal Antibody Decay is a Major Determinant of Early EBV Infection Arnold Reynaldi,1 Timothy E. Schlub,2 Erwan Piriou,3 Sidney Ogolla,4 Odada P. Sumba,4 Ann M. Moormann,5 Rosemary Rochford,6 and Miles P. Davenport1 1 Infection Analytics Program, Kirby Institute for Infection and Immunity, UNSW Australia, Sydney, and 2Sydney School of Public Health, Sydney University, New South Wales, Australia; 3Médecins Sans Frontières, Amsterdam, The Netherlands; 4Centre for Global Health Research, Kenya Medical Research Institute, Kisumu; 5Program in Molecular Medicine, University of Massachusetts Medical School, Worcester; and 6Department of Immunology and Microbiology, University of Colorado Denver, Aurora
The combination of Epstein-Barr virus (EBV) infection and high malaria exposure are risk factors for endemic Burkitt lymphoma, and evidence suggests that infants in regions of high malaria exposure have earlier EBV infection and increased EBV reactivation. In this study we analyzed the longitudinal antibody response to EBV in Kenyan infants with different levels of malaria exposure. We found that high malaria exposure was associated with a faster decline of maternally derived immunoglobulin G antibody to both the EBV viral capsid antigen and EBV nuclear antigen, followed by a more rapid rise in antibody response to EBV antigens in children from the high-malaria-transmission region. We also observed the long-term persistence of anti–viral capsid antigen immunoglobulin M responses in children from the high-malaria region. More rapid decay of maternal antibodies was a major predictor of EBV infection outcome, because decay predicted time to EBV DNA detection, independent of high or low malaria exposure. Keywords. Epstein-Barr virus; P. falciparum malaria; Burkitt Lymphoma; antibody; immunity.
Epstein-Barr virus (EBV) is a gammaherpesvirus causing infectious mononucleosis and also associated with a variety of cancers in humans [1]. In Africa, EBV is associated with endemic Burkitt lymphoma in the presence of Plasmodium falciparum malaria infection [2]. Previous studies in Africa have shown that most children seroconvert to EBV between 6 and 18 months of age [3–5] and that living in an area of high malaria transmission is associated with higher EBV load [4] and earlier time of the first EBV infection [5]. Moreover, the presence of P. falciparum infection is correlated with increased EBV load over time and with more frequent detectable EBV in children [5, 6] and pregnant women [7]. High levels of antibody against the EBV viral capsid antigen (VCA) are associated with a higher risk of Burkitt lymphoma in both Ugandan [8, 9] and Kenyan [10] children. High malaria exposure was also associated with impaired transfer of maternal VCA and EBV nuclear antigen 1 (EBNA1)–specific immunoglobulin (Ig) G from mother to infant [11], and later in life, higher level of EBV-specific IgG antibodies [12]. The measurement of antibodies specific for EBV VCA and EBNA1 have been used in numerous seroepidemiology studies to assess primary and persistent EBV infections (reviewed in
[13]). Typically, anti-VCA IgG appears early after infection, with a delay to detection of anti-EBNA1 IgG. Both are then found at relatively stable levels throughout the life of the host. Anti-EBV early antigen diffuse complex (EAd) IgG has been used as a serological marker for evidence of viral reactivation, because its levels are generally much lower than levels of antiVCA IgG, they decrease after primary infection (in contrast to VCA IgG), and are elevated during viral reactivation. The EBV Z-transactivation antigen (Zta) is an EBV immediate early protein. It has not been used in clinical settings to detect EBV reactivation to our knowledge, but we have found that the serology of Zta is similar to that of EAd-IgG and that Zta serology can also be used as a marker of viral reactivation [12]. In this study, we used a modeling approach to investigate how malaria exposure might affect the serological profiles of EBVinfected children, and EBV infection kinetics. This study demonstrated rapid decay of maternal antibodies in the region of high malaria exposure, followed by early EBV DNA detection and higher expansion rate of EBV-specific antibodies. This expansion of EBV antibodies can be observed from 1–2 months of age, suggesting very early EBV exposure. MATERIALS AND METHODS
Received 7 June 2016; accepted 17 August 2016; published online 28 August 2016. Correspondence: M. P. Davenport, Kirby Institute for Infection and Immunity, UNSW Australia, Sydney, New South Wales 2052, Australia ([email protected]). The Journal of Infectious Diseases® 2016;214:1390–8 © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail [email protected]. DOI: 10.1093/infdis/jiw396
1390
•
JID 2016:214 (1 November)
•
Reynaldi et al
Patients
Detailed methods of the cohort and analysis of viral loads from birth to 24 months have been described elsewhere [5]. Briefly, 2 cohorts of infants enrolled within 1 month of birth were monitored in Kisumu (77 children; high-malaria-transmission region)
and Nandi (86 children; low-malaria-transmission region) in Kenya. The first data collection was performed when the children were about 1 month of age (range, 1–4 months), and then roughly every month until 24 months of age. Luminex bead assays were used to capture 4 different types of EBV-specific IgG to VCA, EBNA1, z-transactivation antigen (Zta), and EAd [12]. Plasma was diluted either 1:100 or 1:6400. We expressed the results as median fluorescence intensity (MFI) level of ≥75 beads. Detection of anti-VCA IgM is used clinically to evaluate whether primary EBV infection has occurred [13]. We performed the enzyme-linked immunosorbent assays [14] to detect anti-VCA IgM, and we had limited sample volume to perform more extensive serological analysis. For all serological assays, pooled serum from EBV negative donors was used as negative controls. The EBV seronegative plasma was used to create baseline values for the MFI, these values were then averaged and the mean (±3 standard deviations) was subtracted from the MFI or optical density. Quantitative polymerase chain reaction was used to detect the presence of EBV DNA in the blood, with a limit of detection of 2 copies/µg of DNA [4, 15]. Modeling the Kinetics of EBV-Specific Antibodies
Throughout this study, we modeled the kinetics of EBV-specific IgG antibodies as having potentially 2 phases. The first phase was a decline in antibody present at birth (representing the presence and loss of maternal antibodies), and the second was the subsequent increase in EBV-specific antibodies (representing the production of endogenous EBV-specific antibodies). Note that in some cases, we did not observe an initial loss of maternal antibodies. Maternal antibodies (M [t]) transferred in utero will decay at a rate δ (exponentially), while, at the same time, endogenous EBV-specific antibodies (E[t]) reflective of infection will be formed (expanding at rate g) and will later reach a constant level K. This can be written as follows: MðtÞ ¼ Aedt EðtÞ ¼
KE0 egt K þ E0 ðegt 1Þ
ð1Þ
MFI(tÞ ¼MðtÞþEðtÞ where MFI (t) represents MFI level at time t; A, initial maternal EBV-specific antibody at birth; δ, decay of maternal antibody; g, intrinsic expansion rate of EBV antibody level; and K, stable level of EBV-specific antibodies. Note that in the cases of Ztaand EAd-specific IgG, we did not observe an initial presence or loss of maternal antibodies (ie, A = 0). For the kinetics of VCA-specific IgM, we used a piecewise log-linear model on the level of VCA IgM titer against age. The level of VCA IgM increases exponentially until a certain time (Tlast), after which it decreases exponentially. This was chosen based on the overall trend in the dynamics of VCA IgM. Maternal IgM does not cross the placenta, and detection
of VCA-specific IgM in the infants is an indicator of primary infection. Consistent with the inability of IgM to cross the placenta, we did not observe the initial presence or loss of maternal antibodies (Figure 1B). Fitting Procedures and Statistical Analysis
We used a nonlinear mixed-effect model with fixed treatment effect to capture the difference in antibody expansion and decay parameters between the 2 regions. Significance was determined based on the value of this covariate, which can be calculated using the Wald test from the standard errors calculated in package nlme in R software (version 3.1.2). The models were fitted with the nonlinear mixed-effect model R function nlme in library nlme (version 3.1-113). The fit was weighted using varPower, and we assumed a diagonal variance-covariance matrix for the random effect, using the pdDiag option in R. To find the 95% confidence interval (CI), we used the R function intervals in library nlme. We used Cox proportional hazard regression to determine if there was an association between the predicted parameters in the IgG responses and the time to be DNA positive. The analysis was done using the coxph function in R software from the library survival (version 2.38-1). RESULTS General Description
Examples of antibody levels of various EBV-specific antibodies are shown in Figure 1 (individual data in Supplementary Figure 1). For VCA-and EBNA1-specific IgG antibodies, we observed an initial high level of antibodies, which dropped during about the first 6 months of life (assumed to reflect the initial presence and loss of the maternally transmitted antibodies). After this, we observed an increase in VCA- and EBNA1specifc IgG, assumed to be from endogenous production by infants. Anti-Zta and anti-EAd antibodies are typically used as indicators of EBV reactivation and are transiently detected. In a separate study we have shown that maternal antibodies to Zta and EAd are not efficiently transferred to infants as assessed in cord blood [11]. Consistent with this, we did not observe the initial presence or subsequent loss of maternal antibody to these antigens. Faster Loss of Maternal IgG Antibody to VCA and EBNA1 for Children in High-Malaria Region
We first analyzed the kinetics of VCA- and EBNA1-specific IgG over time from 1 to 24 months of age, fitting a model with loss of maternal antibodies, and increase in endogenously produced antibodies (Eq 1) [3, 16]. We found that the estimated initial amount of VCA-specific IgG at birth was similar between the 2 cohorts (P = .77), consistent with the fact that the levels at enrollment 1 month later were also not significantly different (P = .84). However, the subsequent decay rate of maternal antibody to VCA was significantly faster in the Kinetics of EBV-Specific Antibodies
•
JID 2016:214 (1 November)
•
1391
A
EBNA1 IgG
VCA IgG 100 000
EBNA1 MFI
VCA MFI
100 000
10 000
Low malaria High malaria
10 000
1000
Low malaria High malaria
1000
100 0
10
20
30
10
20
Age, mo
Zta IgG
EAd IgG
30
10 000
EAd MFI
10 000
Zta MFI
0
Age, mo
1000
1000
Low malaria High malaria
Low malaria High malaria
100
100 0
10
20
30
0
10
Age, mo
20
30
Age, mo
B
VCA IgM
VCA IgM Titer
1000
100
10
Low malaria High malaria 1 0
10
20
30
Age, mo Figure 1. Serological profiles in children over time. Kinetics of Epstein-Barr virus (EBV)–specific immunoglobulin (Ig) G antibodies to 4 EBV antigens: viral capsid antigen (VCA), EBV nuclear antigen 1 (EBNA1), Z-transactivation antigen (Zta), and early antigen diffuse complex (EAd). Each graph shows the model prediction (solid line) with the 95% confidence band of the model fit (dashed lines); error bars represent standard error of the mean in the data. A, Initial drop during the first few months is assumed to reflect the presence and loss of maternal antibodies ( particularly antibodies to VCA and EBNA1). The later increase in antibody levels is assumed to indicate the production of endogenous EBV-specific antibodies. A complete time course of the data for individual subjects (with the model fitting) can be seen in Supplementary Figure 1. MFI, median fluorescence intensity. B, Different kinetics of VCA-specific IgM in the low- and high-malaria regions with age. In the low-malaria region, we see a later drop in the level of VCA IgM; in the high-malaria region, the level of VCA IgM is maintained.
high-malaria region (P < .001), with a rate of 0.23 month−1 (high-malaria region) and 0.15 month−1 (low-malaria region). The kinetics of maternal EBNA1-specific IgG showed the same trends. We found no difference in the measured level of EBNA1 IgG at first sampling (P = .75) or the level estimated to have been present at birth (P = .23). However, we found that similar to the decay of anti-VCA antibody, those living in a high-malaria region had faster decay of EBNA1 IgG 1392
•
JID 2016:214 (1 November)
•
Reynaldi et al
than that in the low-malaria region (rates, 0.97 and 0.24 month−1, respectively; P < .001). Faster Rate of Increase of VCA and EBNA1 IgG for Children in High-Malaria Region
Using the same model (Eq 1), we found that the rate of increase of VCA-specific IgG was higher for children in the high-malaria than for those in the low-malaria region (0.93 month−1 vs 0.76
month−1; P = .046). However, the plateau level of VCA-specific IgG achieved at a later age did not differ significantly between the 2 cohorts (P = .52). This similar plateau level could arise owing to the limitations of the assay, in which maximal binding of antibodies is reached. However, when we repeated the same analysis using data obtained when plasma was diluted 1:6400, we still observed similar levels of saturation in the 2 groups (P = .55). The same trend can also observed in the kinetics of EBNA1specific IgG, with faster expansion in EBNA1-specific in the high-malaria region (0.55 month−1) than in the low-malaria region (0.32 month−1; P = .06). The stable levels reached by EBNA1-specific IgG did not differ significantly between those 2 cohorts (P = .48). For a complete summary, see Table 1. Steady Increase in Levels of Antibodies to Zta and EAd From a Very Early age
In the majority of study participants, we could not see evidence of maternal antibodies to Zta and EAd IgG (Figure 1A and Supplementary Figure 1), as the level of antibody rose continuously
Table 1.
Dynamics of VCA- and EBNA1-Specific IgGa
from the first sampling at age 1–4 months (mean of differences for Zta, 250 MFI [95% CI, 55.42–443.7; P = .01 ( paired t test)]; mean of differences for EAd, 181 MFI [29.50–308 ; P = .03]). The rise in both Zta and EAd IgG from the time of enrollment at 1 month of age suggests ongoing antigenic stimulation from very early in life. The estimated initial levels of Zta-specific IgG at birth did not differ significantly between infants in high- versus low-malaria regions (P = .76), nor did the levels at the time of first sample (P = .78). The stable levels of Zta-specific IgG were also not significantly different (P = .19). However, we found that the expansion rate of Zta-specific IgG was significantly faster for children in the high-malaria region than for those in the low-malaria region (0.4 vs 0.28 month−1; P = .006). Next, we used the same approach to analyze the kinetics of EAd-specific antibodies, where again we saw no evidence of maternal antibody decay. We found that neither the initial amounts of EAd-specific IgG nor the plateau levels differed significantly between groups (P = .87 and P = .80, respectively). Although there was a trend to faster expansion of antibodies in the high-malaria region (0.4 month−1) compared with the low-malaria region (0.36 month−1), this difference was not significant (P = .38). The summary for both Zta and EAd IgG can be seen in Table 2.
Value (95% CI) Parameter
Low-Malaria Region
High-Malaria Region
P Valueb
VCA-specific IgG Initial maternal antibody, MFI
11 560 (9113–14 007)
12 086 (6037–18 136)
.77
Decay rate, mo−1
0.15 (.13–.17)
0.23 (.18–.27)
Initial EBV antibody, MFI
14.9 (2–27.75)
68.5 (6.4–130.6)
.03
Expansion rate, mo−1
0.76 (.65–.87)
0.93 (.65–1.22)
.046
Threshold, MFI
21 722 (20 141–23 303)
19 932 (16 232–23 614)
