Full Paper

Genes and Immunity (2004) 5, 122–129. doi:10.1038/sj.gene.6364051 Published online 22 January 2004

Genetic regulation of immune responses to vaccines in early life

This study was supported by the Medical Research Council (UK), the WHO Global Program for Vaccines and Immunization, the Wellcome Trust and the Royal Society.

M J Newport1,4, T Goetghebuer2,4, H A Weiss3, The MRC Gambia Twin Study Group7H Whittle4, C-A Siegrist5 and A Marchant4,6

  1. 1Department of Medicine, University of Cambridge, UK
  2. 2University Department of Paediatrics, John Radcliffe Hospital, Oxford, UK
  3. 3MRC Tropical Epidemiology Unit, London School of Hygiene and Tropical Medicine, UK
  4. 4Medical Research Council Laboratories, PO Box 273, Banjul, The Gambia
  5. 5WHO Collaborative Centre for Neonatal Vaccinology, University of Geneva, Switzerland
  6. 6MRC Human Immunology Unit, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Oxford, UK

Correspondence: Dr MJ Newport, Department of Medicine, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK. E-mail: melanie.newport@cimr.cam.ac.uk

7Members listed at the end of the paper

Financial disclosures: A Marchant received research support from Glaxo SmithKline Biologicals. C-A Siegrist has served as consultant and received research support from Glaxo SmithKline Biologicals, Aventis and Wyeth.

Received 15 July 2003; Revised 6 October 2003; Accepted 6 October 2003; Published online 22 January 2004.

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Abstract

Infant immunization is the most cost-effective strategy to prevent infectious diseases in childhood, but is limited by immaturity of the immune system. To define strategies to improve vaccine immunogenicity in early life, the role of genetic and environmental factors in the control of vaccine responses in infant twins was studied. Immune responses to BCG, polio, hepatitis B, diphtheria, pertussis and tetanus vaccines were measured at 5 months of age in 207 Gambian twin pairs recruited at birth. Intrapair correlations for monozygous and dizygous pairs were compared to estimate the environmental and genetic components of variation in responses. High heritability was observed for antibody (Ab) responses to hepatitis B (77%), oral polio (60%), tetanus (44%) and diphtheria (49%) vaccines. Significant heritability was also observed for interferon-gamma and interleukin-13 responses to tetanus, pertussis and some BCG vaccine antigens (39–65%). Non-HLA genes played a dominant role in responses to Ab-inducing vaccines, whereas responses to BCG were predominantly controlled by genes within the HLA class II locus. Genetic factors, particularly non-HLA genes, significantly modulate immune responses to infant vaccination. The identification of the specific genes involved will provide new targets for the development of vaccines and adjuvants for young infants that work independently of HLA.

Keywords:

Gambia, vaccines, twins, heritability, HLA

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Introduction

Infectious diseases are the main cause of mortality in young children, causing 4 million infant deaths each year.1,2 The vast majority of these deaths occur in developing countries. Early immunization is required to impact on this mortality, but the development of vaccines that are effective at birth is impeded by the immaturity of the immune system.3,4,5 The antibody (Ab) responses to T-cell-independent and many T-cell-dependent vaccine antigens are lower in young infants when compared to older children or adults.6,7,8,9,10 Qualitative differences in Ab responses in infants include reduced Ab avidity and repertoire, and different immunoglobulin G (IgG) subclass distribution.11,12,13,14 Furthermore, the ability of young infants to develop interferon-gamma (IFN-gamma)-producing CD4+ T lymphocytes in response to a number of vaccines also matures during the first year of life.15,16

The induction of protective immunity in infants generally requires the administration of multiple doses of vaccines. In many parts of the world, especially in developing countries, acceptable coverage with multiple vaccine doses has been difficult to achieve.17 Another major limitation to immunization of young infants is the presence of maternal antibodies that are transferred across the placenta in the last months of pregnancy and decrease the Ab response to vaccines given during the first months of life.18 To avoid this inhibition, the administration of many currently licensed vaccines has to be delayed until several weeks or months after birth. The development of more immunogenic vaccines that can be administered earlier during life requires a better understanding of the factors controlling immune responses in infants.

Studies in gene-disrupted animal models and rare human Mendelian immunodeficiency disorders19 prove an essential role for specific genes in the regulation of immune responses. However, with the exception of HLA, none have been shown definitively to have a role in the regulation of variation in polygenic immune responses at the population level.20,21 Although studies in adults indicate a potential role for the genetic regulation of immune responses,22 the genetic contribution to the regulation of immune responses in early human life has never been quantified. Since the immune system in early life is in a state of maturation, the relative contribution of host genes is likely to differ significantly from that observed in adults. In addition to host genes, a number of environmental factors, including maternal antibodies and nutritional factors, particularly vitamin A status, can influence immune responses in early life.23

Thus, immune responses are inherited as complex quantitative traits with variation resulting from both genetic and environmental factors. Twin studies are a powerful method with which to dissect the relative roles of genetic and environmental factors in the etiology of disease or other phenotype. Genetic effects are revealed if the concordance for a trait is higher within monozygous (MZ) twin pairs, who are genetically identical, than in dizygous (DZ) twin pairs, who share on average 50% of their genes. The administration of vaccines to infants represents a controlled situation in which a primary immune response is triggered by a well-characterized antigen. All vaccinees are the same age at the time of vaccination and responses can be measured at the same interval postvaccination, while the young age of vaccine administration minimizes the exposure to environmental agents that might influence the immune response. Thus the study of vaccine responses in an infant twin cohort provides a novel paradigm in which to dissect the relative contribution of genetic and environmental factors to variation in immune responses in early life. If genetic variation is an important determinant of immune responses in this age group, it is possible to then use genetic methods to identify specific genes and pathways that regulate immune responses. New vaccines or adjuvants could then be developed that target these pathways and lead to enhanced vaccine immunogenicity. Furthermore, although HLA has been associated with variation in immune responses in adults, non-HLA genes also influence susceptibility to pathogens.24,25 The unique twin study presented here also provided an opportunity to quantify for the first time the relative contribution of HLA to the genetic control of immune responses in infancy.

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Subjects and methods

Study design and population

This study was conducted in The Gambia and had the approval of The Gambia Government/Medical Research Council (MRC) Ethics Committee. Twin pairs were enrolled at birth at the Royal Victoria Hospital (the referral hospital in the capital Banjul) or at one of the two health centres (Serrekunda and Fajikunda) in the same district. The exclusion criteria were death of one or both twins at or shortly after birth, residence outside study area and BCG vaccine given in hospital before our first contact. Informed consent was obtained and demographic data including ethnicity, family history and maternal health during pregnancy were collected by parental interview. Enrolled twins were examined within 3 days of birth, and birth weight, length and gestational age, using the Dubowitz method,26 recorded. Twins were reviewed monthly until 5 months of age and vaccinated following the Expanded Programme on Immunization schedule. Blood samples were collected at birth (umbilical cord blood), 2 and 5 months of age. Hepatitis B serology is not performed antenatally in The Gambia, nor is passive immunization given to infants of mothers known to be infected.

Vaccines

For each vaccine, a single batch was used throughout the study and vaccines were given at the same time to each twin of a pair. BCG (0.05 ml, Statens Serum Institut, Copenhagen, Denmark) was given intradermally at birth (or at 1 month of age if either twin weighed <2.5 kg). Oral polio vaccine (OPV, Sabin, Glaxo SmithKline Biologicals, Rixensart, Belgium) was given at birth, 1, 2, 3 and 9 months. Hepatitis B vaccine (HBV, Korea Green Cross, Seoul, South Korea) was given intramuscularly (i.m.) at birth, 2 and 4 months, and whole-cell diphtheria, tetanus and pertussis (DTP, Aventis Pasteur) in which Haemophilus influenzae type b (Hib) vaccine (ActHIB, Aventis Pasteur) was diluted, were given i.m. at 2, 3 and 4 months.

Zygosity determination

The zygosity of same-sex pairs was determined genetically by typing 10 microsatellite markers, as described elsewhere.27 Twin pairs with identical genotypes for all markers were classified as MZ.

Antibody assays

Neutralizing Abs to poliovirus type 1 and Ab concentrations to tetanus toxoid (TT), diphtheria toxin (DT) and hepatitis B surface antigen were measured as described previously.28 Concentration of total serum IgG was determined by ELISA on goat anti-human IgG- (Zymed Laboratories San Francisco, CA, USA) coated plates. The incubation of serum samples was followed by successive addition of peroxidase-coupled goat anti-human IgG (Cappell-ICN, Costa Mesa, CA, USA) and ABTS substrate. Ab concentrations were calculated with the Softmax PRO software (Molecular Devices, Sunnydale, CA, USA) in comparison with standard curves using purified human IgG (Chemicon, Temecula, CA, USA). Values below the assay cutoff were arbitrarily given a value of half the cutoff value for the determination of geometric mean titers.

In vitro cytokine responses to vaccine antigens

In vitro cytokine responses to vaccine antigens were measured as described previously.28 Briefly, peripheral blood mononuclear cells were incubated with mycobacterial antigens, including purified protein derivative (PPD, RT49, 10 mug/ml; Statens Serum Institut), short-term culture filtrate (STCF, 10 mug/ml; a gift from Dr Peter Andersen, Statens Serum Institut), killed Mycobacterium tuberculosis and Ag85 (both at 10 mug/ml, and given by Dr Kris Huygen, Institut Pasteur du Brabant, Belgium) and Hsp 65 (10 mug/ml, GBF, Braunschweig, Germany), and to other vaccine antigens including TT (2 mug/ml; Chiron Behring, Marburg, Germany), Bordetella pertussis antigens (pertactin (PER), 5 mug/ml, pertussis toxin (PT), 5 mug/ml and filamentous haemagglutinin (FHA), 5 mug/ml, kindly provided by Glaxo SmithKline Biologicals) and phytohaemagglutinin (PHA, 5 mug/ml; Sigma Chemicals, Poole, Dorset, UK) or medium alone. IFN-gamma and interleukin-13 (IL-13) concentrations were measured in supernatants collected on day 2 (PHA) or day 6 (antigens and medium) using commercially available reagents (Biosource Europe, Fleurus, Belgium).

HLA typing

Dynal RELI™ SSO kits (Dynal Biotech ASA, Oslo, Norway) were used to determine the HLA DRB1 type for DZ twins. This method utilizes a generic PCR amplification with biotinylated primers flanking the HLA DRB1 locus, followed by hybridization of the resulting amplicon to an array of Sequence Specific Oligonucleotide probes immobilized as lines on a nylon membrane. DZ twin pairs were genotyped according to their pattern of hybridization, using the Dynal RELI™ SSO Pattern Matching Program, and classified as HLA identical or nonidentical.

Statistical analysis

Differences in sociodemographic characteristics and logarithmic Ab responses between MZ and DZ twin pairs were analysed in Stata 7.0. (Stata Corporation, TX, USA) using linear regression, adjusting for nonindependence of twin pairs. Standardized cytokine responses were calculated by subtracting the background production of cytokines measured in control wells from that measured in antigen-simulated wells. Individuals for whom the response was equal to or lower than the control response were termed 'nonresponders'. Antigens with a substantial proportion of nonresponders (>16%) were analysed using categorical methods, using three categories: nonresponders, responses below the median positive response and responses above the median positive response. Median values are presented for these antigens (Table 3). The analysis of continuous data was based on the natural logarithm of the standardized response and geometric means presented.


Intratwin pair correlations were calculated separately for MZ and DZ twins using Pearson's correlation coefficient for continuous responses and Spearman's rank correlation for categorical responses. Heritability (the genetic contribution to the total phenotypic variation in the population) was estimated using Mx path analysis.29 Briefly, the total population variance observed for a given phenotype results from the sum of additive genetic variance (A) caused by the sum of the average effects of all the genes that influence the phenotype, dominant genetic variance (D) associated with dominant gene action, common environmental variance (C) caused by the effects of environmental factors shared within families (and therefore affecting each twin in a pair similarly regardless of zygosity) and random environmental variance (E) specific to each individual. Mx path analysis fits models to the raw data and determines the relative contribution of each of the above factors. We initially fitted a model that allowed for additive genetic, common environment and unique environment contributions to variation (ACE). The fit of the data was then compared under the ACE, AE and CE models, respectively. While genetic factors (A) increase correlations within MZ pairs, common environmental factors will increase intrapair correlations for both MZ and DZ pairs (C) and unique environmental factors will decrease intrapair correlations for both MZ and DZ pairs (E). Results show heritability under the ACE model, unless the AE model provided a significantly better fit to the data than ACE (P<0.1), generally reflected in lower DZ correlations, and there was no evidence that the CE model fitted better than the ACE model (P>0.1). In this case, heritability under the AE model is shown. Point estimates and 95% confidence intervals for the heritability under the final model are presented. The assessment of the contribution of HLA genes and non-HLA genes to the observed variation was estimated by comparing the correlation of responses in HLA-identical DZ twins with HLA-nonidentical twins and MZ twins, respectively, using the Mx path analysis.30

Standard multiple comparison adjustments (such as the Bonferroni) may be overly conservative when data are correlated and/or not normally distributed. We have therefore not applied any corrections to the statistical analysis reported here, and P-values should be consequently be interpreted with caution.

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Results

In all, 560 twin pairs were identified between March 1998 and May 2000 (398 from Banjul, 124 from Serrekunda and 38 from Fajikunda), and of these, 345 (62%) were eligible for the study. The reasons for ineligibility were as follows: death of one or both twins at or shortly after birth (92), residence outside study area (101) and BCG vaccine given by hospital staff before enrolment (22). Of the 345 eligible pairs, 297 (86%) were enrolled. Reasons for nonenrolment were refusal (22) and no traceable address (26). All twin pairs lived together for the duration of the study. Of the 297 twin pairs enrolled in the study, immune responses were measured at 5 months and zygosity determined in 207 pairs (48 MZ and 159 DZ pairs). There were no significant differences in sex, ethnic group, gestational age, birth weight or birth centre between MZ and DZ twins. Parity was higher in DZ than in MZ twin pairs (4.5 vs 3.6, P=0.02).

Antibody responses

The geometric mean and range for Ab levels for each antigen tested and total IgG are shown in Table 1. The wide range of Ab responses confirm high interindividual variability in these responses. There were no significant differences in geometric mean Ab responses between male and female twins (and adjustment for gender did not influence results) or in mean Ab responses between MZ and DZ twins (Table 1). Intrapair correlations for MZ and DZ twins and heritabilities for Ab responses at 5 months are shown in Table 2. Higher correlations were observed within the MZ twin pairs for all specific Ab responses as well as for total serum IgG levels. All Ab responses were found to be significantly heritable (Table 2), with heritabilities ranging from 44 to 77% for antigen-specific responses and 78% for total IgG levels. The highest heritabilities (with correspondingly narrow 95% confidence intervals) were observed for HBV and OPV responses, and the best model fitting the data included additive genetic and unique environment factors. No significant correlation was found between HBV and OPV responses in the total study population (r=0.02, P=0.76). In contrast, TT and DT Ab responses were significantly correlated (r=0.62, P<0.001). The contribution of maternal antibodies to intrapair correlations and heritabilities of Ab levels was estimated by measuring anti-TT Ab levels before immunization, at 2 months of age. Extremely high correlations were observed in both MZ (r=0.99) and DZ pairs (r=0.95), resulting in very low heritability (7%, 95% CI 4–11%, data not shown). Maternal HBV serology was not studied.



Cytokine responses

Cytokine responses to vaccine antigens were also highly variable in the study population (Table 3). There was no difference between male and female responses within the total population (and adjustment for gender did not influence the results). Cytokine responses were similar in MZ and DZ twins, except IFN-gamma responses to PER and PT, and IL-13 responses to PHA, HSP65 and FHA that were higher in DZ compared to MZ twins. Intrapair correlations and heritabilities for MZ and DZ twin cytokine responses at 5 months are shown in Table 4. Genetic factors had an important role in responses to mycobacterial antigens (BCG). Significant heritability was observed for IFN-gamma, and IL-13 responses to PPD, IFN-gamma response to KMTB and IL-13 response to Hsp65. No significant heritability was detected for Ag85 or STCF. Cytokine response to TT and pertussis antigens were also genetically controlled, with significant heritability detected for IFN-gamma response to PER and FHA, and IL-13 response to PT and TT. In contrast, cytokine responses to PHA showed no evidence of genetic regulation. As both Ab and cytokine responses to TT were studied, we evaluated whether they were correlated. TT-specific Ab responses were significantly correlated with IL-13 (r=0.18, P=0.0002) but not with IFN-gamma (r=0.09, P=0.09) production.


Contribution of HLA to the overall genetic variation

The contribution of HLA class II genes was estimated using path analysis by comparing correlations within HLA-identical and HLA-different DZ pairs, whereas the contribution of non-HLA genes was estimated by comparing correlation within HLA-identical DZ and MZ pairs. Of the 159 DZ pairs studied at 5 months, DNA was available for 145 twin pairs, and HLA typing technically successful in both twins, for 125 pairs. Genes outside the HLA DRB1 locus significantly influenced Ab responses to all vaccines as well as total IgG levels, whereas no significant effect was observed for HLA class II genes (Table 5). Similar results were obtained for cytokine responses to TT and pertussis antigens. In contrast, IFN-gamma responses to BCG antigens were predominantly influenced by HLA class II genes.


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Discussion

We report here the findings of a twin cohort study that has quantified the relative contribution of environmental and genetic factors to variation in immune responses to vaccines in early life. This study is the first to document the large genetic component for both Ab and cytokine immune responses to vaccines given in early life. We estimated that between 44 and 78% of the variation observed in Ab responses to vaccines is genetic in origin. This high heritability reflects the response to vaccines and not the passive transfer of maternal antibodies, as Ab levels measured before immunization were highly correlated within both MZ and DZ pairs and therefore showed no significant heritability. A high heritability of total IgG levels was observed at 5 months of age, suggesting that genetic factors also play an important role in the control of Ab responses to nonvaccine antigens in early life. The highest heritabilities were observed for HBV and OPV for which the first dose was given at birth. Significant but lower heritabilities were detected for DT and TT, for which the first dose is given at 2 months. These differences suggest an increasing role for environmental factors in the variability of Ab responses during the first months of life. However, infant Ab responses to multiple vaccine doses depend on the age at which the last vaccine dose is given,2 which was 3–4 months of age for all vaccines studied here. Alternatively, the lower heritability for both DT and TT responses, which were also highly correlated, may have resulted from the influence of environmental factors on the response to the shared adjuvant (aluminium hydroxide). We may also speculate that differences in heritability reflect differences in the nature of the vaccines. This would be in accordance with previous twin studies providing evidence for the genetic regulation of Ab responses to pneumococcal polysaccharide and HBV in older children and adults.31,32 With the exception of TT and DT, which are both protein antigens given in a combined vaccine, Ab responses to the different vaccines were not correlated in our study population, suggesting that different genes may be involved. The response of B-lymphocytes to T cell-dependent antigens is a complex phenomenon involving a large number of molecules, including cytokines.33

The HLA system has evolved to provide diversity in host responses to multiple, varied and frequently changing pathogen antigens. However, this polymorphism presents a major challenge to the development of effective vaccines against globally important diseases such as malaria, HIV and tuberculosis.34 The HLA class II locus has been shown to play an important role in the control of Ab response to HBV and measles vaccines in older individuals.32,35 Indeed, HLA-associated vaccine failure has been reported for several vaccines.36,37,38 In contrast to such reports, we found that non-HLA genes played a dominant role in Ab responses including total IgG in Gambian infants. These observations suggest that non-HLA genes exert a strong control on B-cell responses during the postnatal immune maturation process, whereas data from other studies in older population samples suggest HLA genes influence Ab responses triggered in immunologically mature individuals.32

Genetic factors exerted distinct influences on antigen-specific cytokine responses. As observed for Ab responses, IL-13 responses to Ab-inducing vaccines (tetanus and pertussis) were controlled by non-HLA genes. The correlation we observed between Ab and IL-13 responses to TT suggests that similar genes may be involved in the control of B and CD4+ T-lymphocyte responses to this vaccine. Cytokine responses to some BCG antigens were also found to be highly heritable.

In contrast to Ab-inducing vaccines, where a non-HLA effect was observed for IL-13, HLA class II genes were found to influence IFN-gamma responses to BCG. Thus the relative role of HLA in the genetic regulation of a given cytokine response may also depend on the nature of the immune response induced by different antigens: mycobacteria classically induce CD4 T-helper lymphocyte type 1 (Th1) responses, while tetanus and pertussis induce balanced Th1/Th2 responses. It could be hypothesized that there is less genetic variation in the downstream Th1 cytokine pathways than in the type 2 pathways accentuating the relative HLA contribution to Th1 cytokines. Studies in rare families in which BCG vaccination has led to disseminated infection have identified mutations in five genes within IFN-gamma/IL-12 axis, none of which are encoded within the HLA locus.39 Our data suggest that while the IFN-gamma pathway genes are crucial for immunity to mycobacteria, they are not responsible for variation at the population level in IFN-gamma responses to BCG. Alternative interpretations of the differential influence of HLA on responses according to antigen and cytokine considered are that HLA class II alleles vary in their capacity to stimulate cytokine production by CD4 T cells,40 or that non-HLA genes may play a more important role when antigen presentation to CD4+ T cells involves B-lymphocytes.

No significant heritability was detected for the cytokine responses to the Ag85 and the STCF that contains high concentrations of Ag85. This negative result may be related to the promiscuity of immunodominant Ag85 peptides that can be presented by multiple HLA class II molecules.41 Antigen 85 is one of the major candidates for new antituberculosis vaccines.42 Our results suggest that environmental factors may play a more important role than genetic factors in the variability of the responses to these subunit vaccines.

We conclude that genetic factors play a central role in the regulation of immune responses to vaccines in early life, essentially through non-HLA-mediated genetic pathways. The identification of the non-HLA genes influencing Ab responses in infants should help in the development of novel and improved vaccines and adjuvants for young infants that act independent of HLA restrictions. The high additive genetic heritability that we estimate suggests that, if adequately powered, whole genome approaches towards the identification of the genes that regulate immune responses will be feasible. In addition to the impact on vaccine development, quantitative trait loci controlling immune responses are likely to be involved in the regulation of discrete phenotypes, such as infectious and autoimmune diseases, which are major causes of morbidity and mortality worldwide.

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Notes

The MRC Twin Study Group

A Allen, W Banya, D Jackson Sillah, KPWJ McAdam, M Mendy, M Ota, J Vekemans (The Medical Research Council Laboratories, The Gambia); K Jobe (The Gambian Expanded Programme on Immunisation, Department of State for Health, Banjul, The Gambia); S Bennett (the MRC Tropical Epidemiology Unit, London School of Hygiene and Tropical Medicine, UK); P Aaby (the Danish Epidemiology Science Centre, Statens Serum Institut, Copenhagen, Denmark); JC Stockton (Department of Medicine, University of Cambridge, UK); G Cadau, P-H Lambert, S Schlegel-Hauter, P Valenti (the WHO Collaborative Centre for Neonatal Vaccinology, University of Geneva, Switzerland).

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Acknowledgements

We thank the staff of the three enrolment centres and all the twin families for their participation: to Omar Badjie, Dawda Baldeh, Isatou Drammeh, Malick John, Bunja Kebbeh, Suleyman Manjang, Mamadi Sidibeh and Samba Sowe for expert assistance with field work; to Dr Tumani Corrah for help with clinical care of infants; to Dr Mariama Jallow (RVH, Banjul) and Mr JS Saidykhan (Divisional Health Team, Western Kanifing, The Gambia) for guidance during the planning of this project; to Momodou Jobe and Momodou Cham for sample processing, Albert Magnusen, Mariamma Sanneh and Dr Michael Kidd for assistance with antibody assays; to Helen Rance and Sarah Nutland for assistance with HLA typing; to Drs Kris Huygen and Peter Andersen, and to Glaxo SmithKline Biologicals for generously providing antigens; and to Drs Karen Butterworth and Giorgio Sirugo for their contribution to this and ongoing studies in this cohort.

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