Primary Vaccination during InfancyCorrelates with Humoral Immune Response following
Induced Cell-Mediated Immunity−Hepatitis B Virus Vaccine
Laura Gelinas, Bahaa Abu-Raya, Candice Ruck, Bing Cai and Tobias R. Kollmann
http://www.immunohorizons.org/content/1/4/42https://doi.org/10.4049/immunohorizons.1700015doi:
2017, 1 (4) 42-52ImmunoHorizons
This information is current as of November 8, 2018.
Referenceshttp://www.immunohorizons.org/content/1/4/42.full#ref-list-1
, 5 of which you can access for free at: cites 48 articlesThis article
Email Alertshttp://www.immunohorizons.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:
ISSN 2573-7732.All rights reserved.1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,
is an open access journal published byImmunoHorizons
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
by guest on N
ovember 8, 2018
http://ww
w.im
munohorizons.org/
Dow
nloaded from
Hepatitis B Virus Vaccine–Induced Cell-Mediated ImmunityCorrelates with Humoral Immune Response following PrimaryVaccination during Infancy
Laura Gelinas,*,1 Bahaa Abu-Raya,†,‡,1 Candice Ruck,* Bing Cai,‡ and Tobias R. Kollmann†,‡
*Department of Experimental Medicine, University of British Columbia, Vancouver, British Columbia V5Z 1M9, Canada; †Vaccine Evaluation Center,
University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada; and ‡Department of Pediatrics, University of British Columbia,
Vancouver, British Columbia V6H 3V4, Canada
ABSTRACT
Data on hepatitis B virus (HBV) vaccine–induced cell-mediated immunity (CMI) and humoral immune response during infancy is
scarce. We assessed HBV vaccine–induced CMI among infants stratified as nonresponders (Ab against HBV surface Ag [anti-HBs]
levels ,10 IU/l), low-responders (anti-HBs levels 10–100 IU/l), and high-responders (anti-HBs levels $100 IU/l) following their
primary vaccination against HBV. Moreover, we assessed the association between HBV vaccine–induced CMI and anti-HBs levels.
Infants were immunized with HBV vaccine at ages 2, 4, and 6 mo. Hepatitis B surface Ag (HBsAg)-specific proliferation, intracellular
cytokine production, and bulk cytokine secretion were assessed on PBMCs collected at 1 y and anti-HBs levels were measured at 1
and 2 y of age. Infants classified at 2 y of age as low-responders (n = 28) had lower median levels of secreted IFN-g than high-
responders (n = 29), 17.15 pg/ml versus 33.16 pg/ml, respectively, p = 0.009. Infants classified at 2 y of age as nonresponders (n = 15)
had lower median levels of secreted TNF-a than high-responders (n = 29), 116.11 pg/ml versus 162.27 pg/ml, respectively, p = 0.032.
There was a positive correlation between HBsAg-specific secreted IFN-g levels at 1 y and anti-HBs levels at 1 and 2 y of age, rho =
0.269 and 0.302, respectively, (p = 0.019 and p = 0.01, respectively). There was a positive correlation between anti-HBs levels at age 1
y and the levels of secreted IL-10, rho = 0.297, p = 0.009. HBsAg-specific IFN-g, IL-10, and TNF-a secretion correlated with HBV
vaccine–induced humoral immune response. Assessment of CMI is a useful adjunct in demonstrating the persistence of HBV
vaccine–induced memory immune response. ImmunoHorizons, 2017, 1: 42–52.
INTRODUCTION
Infection with hepatitis B virus (HBV) is a global public healthconcern, with a worldwide estimate of more than two billionindividuals infected at somepoint in their lives (1). In some regionsof the world, including sub-Saharan Africa, HBV is hyperendemic
($8% hepatitis B surface Ag [HBsAg] prevalence) (2). The burdenofHBV infection is chieflydrivenby infections acquired in thefirstyears of life (3). In southern and eastern sub-Saharan Africa, anincrease in chronic hepatitis B infection occurred among youngchildren from 1990 to 2005, reaching a prevalence of 8–9% inchildren 0–9 y old (3). InNorth America, although the prevalence
Received for publication May 2, 2017. Accepted for publication May 25, 2017.
Address correspondence and reprint requests to: Dr. Bahaa Abu-Raya, Division of Infectious Diseases, Department of Pediatrics, University of British Columbia, BCChildren’s Hospital, Ambulatory Care Building, K4-165 - 4480 Oak Street, Vancouver, BC V6H 3V4, Canada. E-mail address: [email protected]
ORCID: 0000-0003-3713-4275 (L.G.).1L.G. and B.A. are joint first authors.
This work was supported in part by National Institute of Allergy and Infectious Diseases, National Institutes of Health Grant N01 AI50023. T.R.K. is supported in part by aCareer Award from the Michael Smith Foundation for Health Research.
Abbreviations used in this article: anti-HBs, Ab against HBV surface Ag; CMI, cell-mediated immunity; HBsAg, hepatitis B surface Ag; HBV, hepatitis B virus; IQR,interquartile range; rcf, relative centrifugal force; RT, room temperature.
The online version of this article contains supplemental material.
This article is distributed under the terms of the CC BY 4.0 Unported license.
Copyright © 2017 The Authors
42 https://doi.org/.4049/immunohorizons.1700015
RESEARCH ARTICLE
Adaptive Immunity
ImmunoHorizons is published by The American Association of Immunologists, Inc.
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
ofHBV infection has declined in the postvaccination era, children0–4 y of age still haveHBsAg positivity rates of nearly 2% in 2005,representing the highest age-specific positivity rate (3). Infectionsacquired during thefirst 5 y of life carry a high risk (nearly 30%) ofprogression to chronic disease (3, 4). Among immigrants to NorthAmerica, the prevalence of chronicHBV infection is ~5% and 40%of them develop advanced liver disease (5). In Canada, nearly300,000 individuals who were born abroad have chronic HBVinfection. The socioeconomic burden of this condition among thispopulation is significant (5).
Although a decline in the prevalence of HBV infectionfollowing expanded HBV vaccination has been established(3), breakthrough infections have been reported after primaryvaccination against HBV (6–9).
Measurement of the humoral immune response (Ab againstHBV surface Ag [anti-HBs] Ab levels) is currently the mostcommonly employed immune marker that correlates with pro-tection fromHBV infection. It is generally accepted that anti-HBslevels$10 IU/l are considered protective against HBV infection(10–12). In the vaccinated population, up to 10% of individualsonly achieve anti-HBs levels of,10 IU/l despite full vaccination(termed “nonresponders”) (1). However, some data suggest thatmany individuals whose levels are ,10 IU/l are still protectedagainst HBV infection (13, 14). On the other hand, there is noabsolute protection against HBV infection at any anti-HBs levels(11).CasesofHBV infectionoccur inhealthy individualswithanti-HBs levels between 10 and 100 IU/l (termed “low-responders”)(11, 15, 16) and those with anti-HBs levels $100 IU/l (termed“high-responders”) (11).
The finding that protection from HBV infection in subjectswith anti-HBs levels ,10 IU/l (13, 14) and the potential forinfectionwithHBV inpersonswhose anti-HBs levels are.10 IU/l(11, 15, 16) indicates that the full mechanisms of vaccine-inducedprotection are yet to be determined.
Cell-mediated immunity (CMI) may correlate with humoralimmune response to HBV vaccination. To date, studies that haveattempted to elucidate the biological mechanisms driving thehumoral response to theHBVvaccinehavebeenconductedmainlyin adult populations and have thus far yielded inconsistent results.Sufficient data on these questions in infant cohorts is currentlylacking.
Adult studies, inherently limited by the necessity of including aheterogeneous population of postprimary and postbooster vacci-nated subjects, reported a correlation betweenanti-HBs levels andT cell proliferation (17–19). Other studies reported a lack of T cellproliferative responses to HBsAg among nonresponders to HBVvaccine booster dose (19–21). Although one study reported thatinfant nonresponders to primary vaccination secreted less IFN-gthan responders (22), the correlation between anti-HBs levels andIFN-g production remained inconclusive, as this finding wasconfirmed in some (23) but refuted in other studies (14, 24, 25). Inone study in infants, nonresponders to primary vaccinationagainst HBV secreted less IL-10 than responders (22).Similarresults have been observed in adult populations (23, 26, 27). Last,TNF-a was found to be produced at significantly lower levels in
adult nonresponders as compared with responders following invitro stimulation with HBsAg (23).
To the best of our knowledge, systematic dissection of CMImeasurements and the relationship between CMI and humoralimmunity in infants after primary vaccination against HBV islacking. Thus, the purpose of the current study was to assess HBVvaccine–induced CMI (HBsAg-specific proliferation, intracellularcytokine production, and bulk cytokine secretion) amonghealthy infants stratified as nonresponders, low-responders,and high-responders based on anti-HBs Ab levels achievedfollowing their primary HBV vaccination. Moreover, we soughtto ascertain if there is an association between the levels ofdifferent functionalmeasures ofHBVvaccine–inducedCMI andthemagnitude ofHBVvaccine–inducedhumoral response (anti-HBs Ab levels).
MATERIALS AND METHODS
Prospective birth cohort study designStudy settings, eligibility, and follow-up. Healthy full-termnewborns born at the British Columbia Women’s Hospital wereassessed for eligibility during theyears 2005–2008.Thepurpose ofthe cohortwas to study the ontogeny of the immune systemduringthe first 2 y of life (28). As such, study participants were recruitedat birth andprospectively followedandenrolled further at 1 and2yof age.
Inclusion criteria were full-term healthy newborns born afterelective caesarean sectionswithout labor. Itwas previously shownthat the mode of delivery (unlabored, elective caesarean sectionversus labored normal spontaneous vaginal) has significantimpact on the immune status (29). Thus, the current studyincluded only infants born after elective caesarean sections asdetailed in the original study (28). All the participants in thestudy were considered healthy based on history-driven healthassessments. Exclusion criteria have been described in aprevious publication (28).
Vaccination. Participants were vaccinated with a pediatricformulation of the Recombivax HB vaccine (Merck) i.m. at 2, 4,and 6 mo of age. Each 0.5 ml dose contained 5 mg of recombinantHBsAg (adw subtype) with amorphous aluminum hydroxyphos-phate sulfate (alum) adjuvant. Concomitantly with RecombivaxHB vaccine, infants also received diphtheria, tetanus, acellularpertussis-inactivated polio-Haemophilus influenzae type b(DTaP-IPV-Hib) vaccine at 2, 4, and 6 mo of age, seven-valentpneumococcal conjugate vaccine at 2 and 4 mo of age, andmeningococcal C conjugate at 2 mo of age, in accordance withthe British Columbia childhood immunization schedule. Theimmunization history of each subject was recorded andupdated at each visit.
Timing of blood collection. Peripheral venous blood was drawnprior to any vaccinations received on the same day at 1 and 2 yof age.
https://doi.org/.4049/immunohorizons.1700015
ImmunoHorizons HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY 43
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
Definition of response to primary HBV vaccination. The standardclassification defines nonresponders, low-responders, and higherresponders based on anti-HBs levels measured 1 or 2 mo afterprimary immunization. In this study, participants at year 1 and 2with anti-HBs levels,10, 10–100 and$100 IU/l were consideredas nonresponders, low-responders, and high-responders, respec-tively (10–12, 30–33).Consent obtainment and ethics statement.During initial recruitment,parentswere contactedprior to giving birth at theBritishColumbiaWomen’s Hospital and written informed consent was obtainedfromboth parents. Prior to postnatal blood draws at year 1 and year2, written informed consent was reobtained from the parent orlegal guardian. The study was approved by the Institutional EthicsReview Board at the University of British Columbia (Certificate #H06-03339) and the University of Washington.
Blood sample processingSeven to ten milliliter of venous peripheral blood was drawn intosodium–heparin tubes (Becton Dickinson, Franklin Lakes, NJ).Tubes were spun at 300 relative centrifugal force (rcf ) for 10 min.Plasma was collected and frozen at 280°C until further analysis.The remaining blood products (WBCs, RBCs, and platelets) werediluted with Dulbecco’s PBS (Life Technologies, Burlington, ON)at a 1:1 ratio and layered on Ficoll-Paque Plus (GEHealthcare, Baied’Urfe, QC). Cells were spun at 900 rcf for 25 min with no brake.PBMCs were isolated as per the manufacturer’s protocol. Cellswere cryopreserved as previously described (29). PBMCs werestored in a liquid nitrogen storage unit (vapor phase) until furtheranalysis.
Determination of HBsAg-specific AbsHBsAg-specific IgG (anti-HBs IgG) was measured in plasma bycommercial enzyme immunoassay (DiaSorin, Brussels, Belgium)as per themanufacturer’s instructions. The assay’s standard curverange is 1.25–1000 IU/l.All sampleswere testedat a 1:2dilution.Allsamples with anti-HBs levels exceeding the upper limit of thestandard curve were retested at a 1:10 dilution.
Cell culture and proliferation assayPBMCs were thawed and washed twice with prewarmed R10medium,RPMI 1640medium, 10%FCS (ThermoFisher Scientific,Logan,UT), penicillin (100U/ml) (LifeTechnologies), streptomycin(100 mg/ml) (Life Technologies), and 50 mM b-mercaptoethanol(Sigma-Aldrich, Oakville, ON). Cells were then treated withDNase I (60 mg/ml) (Sigma-Aldrich) for 5 min at 37°C with 5%CO2. Following this, cells were then washed with RPMI 1640medium, resuspended in 5 ml of R10 medium and transferred toa six-well polystyrene culture plate (Becton Dickinson) and in-cubated overnight at 37°C with 5% CO2.
After 24 h, EDTA (Sigma-Aldrich) was added to the cells ata final concentration of 2 mM and incubated for 10 min at 37°Cwith 5%CO2. Cells were then harvested, washed, and counted.Viability was assessed by trypan blue dye exclusion and foundto be above 90% for all samples. Cells were resuspended in500 ml of staining buffer (Dulbecco’s PBS with 5% FCS) and
passed through a 70-mm cell strainer (Becton Dickinson).CellTrace Oregon Green 488 carboxylic acid diacetate,succinimidyl ester (OG488) (Life Technologies), a derivativeof CFSE, was added to the cell suspension at a final concentrationof 5 mM.
For unstained controls, an equal volume of staining buffer wasadded tocells and treated identically as stainedsamples.Cellswereincubated with OG488 for 5 min, after which an equal volume ofFCS was added to quench any residual OG488. To remove excessOG488, cells were washed three times with staining buffer. Cellswere resuspended in R10 at a final concentration of 4 3 106
cells/ml andplatedat0.53 106 cellsperwell in a96-wellU-bottompolystyrene tissue culture plate (Becton Dickinson). The finalvolume of cultures in the 96-well plates is 200 ml. Samples ofunstimulated controls were incubated with anti-CD28 (1 mg/ml)and anti-CD49d (1 mg/ml) monoclonal Abs (eBioscience, SanDiego, CA) in R10 medium. Ag-specific samples were incubatedwith recombinant HBsAg (5 mg/ml) (Aldevron, Fargo, ND), anti-CD28 (1 mg/ml) and anti-CD49d (1 mg/ml) monoclonal Abs(eBioscience) in R10 medium. Positive controls were stimulatedwith anti-CD3 (30 ng/ml) and anti-CD28 (1 mg/ml) monoclonalAbs (eBioscience) in R10 medium.
Plateswere incubated for 7 d at 37°Cwith 5%CO2. Plateswerethen spun at 300 rcf for 10 min to pellet cells and 50 ml per well ofcell-free supernatantwas transferred to a clean plate and frozen at280°C until further analysis. Cells were then resuspended andrestimulated with a mixture containing PMA (10 ng/ml) (Sigma-Aldrich), ionomycin (1 mM) (Sigma-Aldrich), and brefeldin A(2.5mg/ml) (Sigma-Aldrich) for 6 h at 37°C and 5%CO2. After 6 h,EDTA (2 mM) was added to each well and incubated for anadditional 10min at 37°Cwith 5%CO2. The plates were then spunand supernatants were removed. Cells were fixed in a 13 FACSlyse solution (BectonDickinson) and frozen at280°Cuntil furtheranalysis.
Determination of bulk cytokine levelsThe cell-free supernatant was thawed at room temperature (RT),and the levels of IL-10, IL-13, IL-17A, IL-21, IFN-g, and TNF-awere measured by Luminex assay (Millipore, Billerica, MA) asper the manufacturer’s recommendations. Assays were readusing the Luminex 200 Total System (Luminex, Austin, TX),and data were acquired and analyzed using MasterPlexsoftware (MiraiBio Group, San Francisco, CA). The standardcurves for the Luminex assay were as follows: 10–40,000 pg/mlfor IFN-g, 1–5000 pg/ml for IL-10, 7–30,000 pg/ml for IL-13,12–50,000 pg/ml for IL-17A, 5–20,000 pg/ml for IL-21, and2.5–10,000 pg/ml for TNF-a.
Immunophenotypic analyses by flow cytometryPreviously frozen plates containing cells fixed in FACS lysesolution were thawed in 37°C incubator for 10 min. Cells werewashed once in PBSAN solution (PBS [Lonza, Basel, Switzerland],0.5%BSA [Sigma-Aldrich], 0.1% sodiumazide [Teknova,Hollister,CA]), resuspended in 13 FACS permeabilization solution (BectonDickinson), and incubated for 10 min at RT.
https://doi.org/.4049/immunohorizons.1700015
44 HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY ImmunoHorizons
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
Following one wash with PBSAN, cells were stained in afinal volume of 100 ml per well of PBSAN for 30 min at RT.The following Abs were used: anti–CD3-APCeFluor780 (cloneUCHT1; eBioscience), anti–CD4-eFluor450NC (clone RPA-T4;eBioscience), anti–CD8-PE-Cy7 (clone SK1; eBioscience), anti–IFN-g-PE-CF594 (clone B27; Becton Dickinson), anti–IL-2-PerCPeFluor710 (clone MA1-17H12; eBioscience), anti–IL-13-APC(clone JES10-5A2; Becton Dickinson), anti–IL-21-PE (cloneeBio3A3-N2;eBioscience), andanti–TNF-a-AlexaFluor700(cloneMAb11; eBioscience). All Abs were used at a 1:100 dilution. OG488was detected using the same filter as FITC. Following twowasheswith PBSAN, cells were resuspended in 200ml perwell of PBSANand immediately analyzed using an LSRII Flow Cytometer(Becton Dickinson). FACSDiva (Version 9) (Becton Dickinson)was used as acquisition software. Compensation beads (BectonDickinson)were used as single stain controls for all purchasedAbs.For OG488, an equal number of unstained and stained PBMCswere combined and used as single stain control for compensationpurposes.
Prior to each acquisition, Cytometer Setup and Trackingbeads (Becton Dickinson) were run on the LSRII and previouslystandardized and saved cytometer settingswere applied. Cells andbeads were acquired uncompensated using a high-throughputsampler. The maximum number of cells were acquired from eachwellandrangedfrom144,000(116,000–179,000),median(interquartilerange [IQR]), for unstimulated and HBsAg-stimulated samples.Compensationmatrices were calculated and applied using FlowJo(Version 9.4) (TreeStar, Ashland, OR). All samples were analyzedcompensated.Biological controlswereused to set appropriate gatesfor analyses.
Determination of cellular proliferationProliferation data were analyzed using the proliferation platformon FlowJo. Positive controls were used to set the gates identify-ing successive rounds of cell division (generation gates)and sub-sequently copied to unstimulated and HBsAg-stimulated samples on a per subject basis. Number of cells ineach generationgate were determined and used to calculate thepercentage of the starting population that divided at least once.The gating strategy employed for the flow cytometric data arerepresented (Supplemental Fig. 1).
StatisticsAll demographicdatawerecomparedbetween studygroups by theuse of the Pearson Chi-Square test.
Anti-HBs Ab levels below the lower limit of detection wereassigned the lower limitof detectionof the assay.Ab levelswere logtransformed prior to analysis. Geometric mean concentration and95% confidence interval for infants’ sera at the age of 1 and 2 ywere calculated. The Mann–Whitney U test was used to assessdifferences in log transformed Ab levels between low-responders(anti-HBs levels 10–100 IU/l) and high-responders (anti-HBslevels$100 IU/l) at year 1 and theKruskal–Wallis testwas usedto test for differences in log transformed Ab levels between
nonresponders (anti-HBs levels ,10 IU/l), low-responders,and high-responders.
For CMI parameters, technical replicates were averaged andunstimulated values were subtracted from stimulated values.Wilcoxon matched pairs signed rank test was used to test fordifferences between unstimulated and stimulated values fromthe same subject.
Nonparametrical tests were used to compare the proliferation,bulk cytokine production, and secretion between study groupsstratified according to anti-HBs levels. Cytokines expressed belowthe lower limit of detection were assigned the value of 0. TheMann–Whitney U test was used to test for differences in CMIresponses (proliferation, bulk cytokine production, and secretion)between low-responders and high-responders. The Kruskal–Wallis test was used to test for differences in CMI responses(proliferation, bulk cytokine production, and secretion) betweennonresponders, low-responders, andhigh-responders.Correlationsbetween anti-HBs levels andCMI responseswere studied by use ofSpearman rank analysis.
Data was analyzed using SPSS software version 24.0 (SPSS,Chicago, IL). Significance was set at p, 0.05.
RESULTS
Baseline characteristics and humoral immune responseBlood for anti-HBs Abs and CMI measurement was avail-able from 76 and 72 participants at age 1 and 2 y, respectively(Fig. 1).
The demographic, clinical characteristics and anti-HBs levelsof the study population are presented (Table I). At 1 y of age, therewereno statistical differences between the low-responders (n = 16)and high-responders (n = 57) in sex or ethnicity. Immunizationrecords confirmed that by 1 y of age, all participants received threedoses of HBV vaccine. At 2 y of age, there were no statistical dif-ferences between the nonresponders, low-responders, and high-responders in sex or ethnicity (Table I). A correlation analysisbetween anti-HBs levels measured at the age of 1 and 2 yshowed a significant positive correlation between anti-HBslevels measured at the age of 1 and 2 y (Spearman correlationcoefficient [rho] 0.558, p = 0.000). This indicates that thosewith higher anti-HBs levels at 1 y of age were likely to havehigher anti-HBs levels at 2 y of age.
HBsAg-specific cellular proliferationThe median percentage of proliferating CD4 T cells was higher ascompared with the percentage of proliferating CD8 T cells,0.575 (n = 76, IQR = 0.635) versus 0.168 (n = 76, IQR = 0.309),respectively (p = 0.000, Wilcoxon signed rank test). There wasa significant positive correlation between the percentage ofproliferating CD4 and proliferating CD8 T cells in response toHBsAg (Spearman rho 0.346, p = 0.002 [two-tailed]) indicatingthat those with a greater percentage of proliferating CD4T cells were likely to have a greater percentage of proliferatingCD8 T cells as well.
https://doi.org/.4049/immunohorizons.1700015
ImmunoHorizons HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY 45
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
Age 1 y: comparing HBsAg-specific CMI and humoralimmune responseAge 1 y: HBsAg-specific CMI of low-responders versus high-responders. At 1 yof age, three infantshadanti-HBs levels,10 IU/l.This small number of subjects precluded the analysis of a non-responder group at the 1-y time point. Thus, the comparisonbetween groups at 1 y reported below is between low-respondersand high-responders.HBsAg-specific cellular proliferation and HBsAg-specificintracellular cytokine production. The median percentage ofstartingCD4Tcells, startingCD8Tcells, IFN-g+CD4Tcells, IL-13+
CD4 T cells, and IL-21+ CD4 T cells that proliferated were notsignificantly different among infants classified as low-respondersversus high-responders (Fig. 2).HBsAg-specific bulk cytokine secretion. Several differenceswere observed in the bulk cytokine levels when comparingdifferent responder groups (Table II). The median levels ofIL-10 were lower in low-responders as compared with high-responders (p = 0.05). Lower median levels of TNF-a wereobserved in low-responders as compared with high-responders(p = 0.06).
Age 1 y: correlation between HBsAg-specific CMI and anti-HBslevels. To determine if there is a relationship between anti-HBslevels and HBsAg-specific CMI, a correlation analysis was per-formed at the 1-y time point. Therewas no significant correlationbetween anti-HBs levels at age 1 y andHBsAg-specificproliferationor intracellular cytokineproduction (Table III). Interestingly, therewasa significantpositivecorrelationbetweenanti-HBs levels at age1 y and the levels of secreted IFN-g and IL-10with rho of 0.269 and0.297, respectively, (p = 0.019 and 0.009, respectively) (Table III).
Age 2 y: comparing HBsAg-specific CMI and humoralimmune responseAge 2 y: HBsAg-specific CMI of nonresponders, low-responders,and high-responders. HBsAg-specific cellular proliferation andHBsAg-specific intracellular cytokine production. The medianpercentage of startingCD4Tcells andCD8Tcells that proliferatedwere not significantly different among infants classified as nonre-sponders, low-responders, and high-responders (Fig. 3).
The median percentage of IFN-g+ CD4 T cells that prolifer-atedwas 8.02 (n = 12, IQR = 10.47), 12.25 (n = 25, IQR = 16.67), and
FIGURE 1. Flow algorithm of study participants.
(A) Flow algorithm of study participants at year 1. (B) Flow algorithm of study participants at year 2.
https://doi.org/.4049/immunohorizons.1700015
46 HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY ImmunoHorizons
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
21.05 (n = 23, IQR = 18.60) among infants classified as nonre-sponders, low-responders, and high-responders, respectively,p = 0.013 (Fig. 3C). Moreover, the median percentage of IFN-g+
CD4 T cells was significantly lower in infants classified asnonresponders as comparedwith high-responders, (p = 0.006);and infants classified as low-responders as compared withhigh-responders (p = 0.033).
Themedian percentage of IL-13+ CD4 T cells that proliferatedwas 7.88 (n = 12, IQR=8.11), 14.21 (n = 25, IQR=8.24), and 17.12 (n =23, IQR = 16.39) among infants classified as nonresponders, low-responders, and high-responders, respectively, p = 0.089 (Fig. 3D).Moreover, the median percentage of IL-13+ CD4 T cells wassignificantly lower in infants classified as nonresponders ascompared with high-responders (p = 0.032) and a trend wasnoted in infants classified as nonresponders when comparedwith low-responders (p = 0.065).
Themedian percentage of IL-21+ CD4 T cells that proliferatedwas 7.18 (n = 12, IQR = 10.23), 13.09 (n = 25, IQR = 14.93), and 19.12(n = 23, IQR = 18.21) among infants classified as nonresponders,low-responders, and high-responders, respectively, p = 0.075 (Fig.3E). In addition, the median percentage of IL-21+ CD4 T cells wassignificantly lower in infants classified as nonresponders versushigh-responders (p = 0.034). It should be noted that despite beinglower than responder groups in several parameters, nonre-sponders do demonstrate evidence ofHBsAg-specificCMI as theyhad measurable HBsAg-specific CD4 and C8 T cell proliferation.HBsAg-specific bulk cytokine secretion. Several differenceswereobserved in the bulk cytokine levels when comparing differentresponder groups (Table II). Specifically, nonresponders hadlower median levels of IL-10 than low-responders (p = 0.05). Aswell, the median levels of TNF-a were significantly lower innonresponders than high-responders (p = 0.032). The medianlevels of IFN-gwere significantly different in infants stratifiedby humoral immune response (p = 0.025). Specifically, low-responders had significantly lower median levels of IFN-g
secretion thanhigh-responders (p = 0.009).Moreover, it should benoted that nonresponders demonstrated evidence of HBsAg-specific CMI as they had measurable levels of secreted cytokines.
Age 1 y: correlation between HBsAg-specific CMI. There was apositive correlation between anti-HBs levels at 2 y of age andthe percentage of IFN-g+ CD4 T cells, rho = 0.350, p = 0.006(Table III). In addition, there was a significant positive corre-lation between anti-HBs levels at 2 y of age and the levels ofsecreted IFN-g, rho = 0.3, p = 0.01 (Table III).
DISCUSSION
In the current study, we interrogated the HBsAg-specific CMIafterprimaryHBVvaccinationduring infancyasmeasuredbyCD4and CD8 T cell proliferation, CD4 T cell intracellular cytokineproduction, and bulk cytokine secretion at the age of 1 y ascompared with humoral immune response at 1 and 2 y of age.
Our study provides evidence on a significant associationbetween humoral immune response at the age of 1 and 2 y andIFN-g secretion. IFN-g is produced by CD4 and CD8 T cells,supports the differentiation of CD4 T cells to Th1 cells, andsuppresses the development of Th2 cells (34). It also supportsT cell proliferation (34). Moreover, IFN-g enhances HBV-specifichumoral immune response in vitro as the addition of IFN-gresulted in a dose-dependent increase of anti-HBsAg secretingB cells (35). IFN-g produced by HBV-specific CD8 T cells isnecessary for controlling HBV infection (36–38). Our resultscorrelatewithprevious literature onadult cohorts,which showedthat low- or nonresponder adults had low or undetectable ex-pression or secretion of IFN-g (18, 20, 21, 23, 39). However, thecohort included in these studies was a mixed adult population ofthose having their primary and booster vaccination against HBV.In the pediatric population, only one study reported that infant
TABLE I. Clinical characteristics and anti-HBs levels of subjects stratified by humoral immune response to primary series of HBV vaccine at age1 and 2 y
ClinicalCharacteristics
High-Responders
at 1 y (n = 57)Low- Responders at 1
y (n = 16)p
Value
High-Responders
at 2 y (n = 29)Low-Responders at 2
y (n = 28)Nonresponders at 2
y (n = 15)p
Value
Sex, n (%) 0.614a 0.330a
Male 29 (50.8) 7 (43.8) 14 (48.3) 16 (57.1) 5 (33.3)Female 28 (49.2) 9 (56.2) 15 (51.7) 12 (42.9) 10 (66.4)
Ethnicity, n (%)b 0.649a 0.376a
Caucasian 23 (40.3) 9 (56.2) 11 (40) 16 (57.1) 8 (53.3)Asian 6 (10.5) 3 (18.7) 5 (17.2) 1 (3.4) 3 (20)South Asian 8 (14) 1 (6.2) 4 (13.8) 4 (14.3) 2 (13.3)Asian-Caucasian 5 (8.8) 1 (16.6) 4 (13.8) 2 (7.1) 0 (0)
Anti-HBs IgG (IU/l)
(GMC, 95% CI)
340.24
(275.42–407.38)
57.37
(44.66–72.44)
0.000c 231.64
(186.20–281.83)
36.03
(27.52–45.70)
1.73 (1.17–2.51) 0.000d
Nonresponders: anti-HBs levels ,10 IU/l; low-responders: anti-HBs levels 10–100 IU/l; high-responders: anti-HBs levels $100 IU/l. Categorical data are presented asa number (%).aPearson Chi-Square test.b17 cases with missing data.cMann–Whitney U test.dKruskal–Wallis test.CI, confidence interval; GMC, geometric mean concentration.
https://doi.org/.4049/immunohorizons.1700015
ImmunoHorizons HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY 47
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
nonresponders to primary vaccination against HBV secreted lessIFN-g than responders (22). The correlation between anti-HBslevels and IFN-g production is controversial, as some studiesshowed a correlation (23), whereas others have not (14, 24, 25).
Our data showed that there was a significant positive cor-relation between anti-HBs Ab levels at age 1 y and the levels ofsecreted IL-10. IL-10 is a potent growth and differentiationfactor for activated B cells inducing secretion of Igs (40). Our
findings are in agreement with evidence from adult studies,which included a heterogeneous population following primaryand booster vaccination and showed that individuals who didnot mount a protective humoral immune response followingvaccination against HBV secreted significantly lower levels ofIL-10 in response to HBsAg stimulation (23, 26, 27).
We also found that the median levels of TNF-a weresignificantly lower in nonresponders than high-responders
FIGURE 2. HBsAg-specific proliferation and intracellular cytokine production stratified by humoral immune response to primary immunization
against HBV at age 1 y.
(A) Percentage of starting CD4 T cells that proliferated. (B) Percentage of starting CD8 T cells that proliferated. (C) Percentage of IFN-g+ proliferating
CD4 T cells. (D) Percentage of IL-13+ proliferating CD4 T cells. (E) Percentage of IL-21+ proliferating CD4 T cells. Data are presented in boxplots as
median (lines) and IQR (boxes). Small circles indicate outliers and stars indicate extreme values.
TABLE II. HBsAg-specific bulk cytokine secretion stratified by humoral immune response to primary immunization against HBV at age 1 and 2 y
CMI Parameter at 1 y
Low-Responders
at 1 y (n = 16)
High-Responders
at 1 y (n = 57)p
ValueNonrespondersat 2 y (n = 15)
Low- Respondersat 2 y (n = 28)
High-Responders
at 2 y (n = 29) p Value
IFN-g secretion (pg/ml),
median (IQR)
18.64 (32.18) 24.23 (30.98) 0.119 22 (37.84) 17.15 (34.26) 33.16 (44.87) 0.025 (0.009a)
IL-10 secretion (pg/ml),
median (IQR)
32.11 (87.17) 82.32 (163.16) 0.05 35.09 (114.69) 79.87 (162.62) 79.06 (119.45) 0.112 (0.05b,0.069c)
IL-13 secretion (pg/ml),
median (IQR)
529.61 (1219.63) 978.30 (957.53) 0.125 796.25 (1104.05) 1157.21 (1313.74) 860.19 (988.54) 0.262
TNF-a secretion (pg/ml),
median (IQR)
103.50 (131.24) 171.24 (209.45) 0.06 116.11 (161) 178.65 (231.64) 162.27 (175.13) 0.115 (0.032c)
Nonresponders: anti-HBs levels ,10 IU/l; low-responders: anti-HBs levels 10–100 IU/l; high-responders: anti-HBs levels $100 IU/l. Categorical data are presented asa number (%). The bold text indicates statistical significance.aLow-responders versus high-responders.bNonresponders versus low-responders.cNonresponders versus high-responders.
https://doi.org/.4049/immunohorizons.1700015
48 HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY ImmunoHorizons
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
at the age of 2 y. TNF-a is a proinflammatory cytokine (41)and studies have shown that TNF-a produced by HBV-specific CD8 T cells is necessary for controlling HBVinfection (36–38). In our study, the source of TNF-a wasnot demonstrated. Despite the importance of TNF-a in
controlling HBV infection, there is limited data on its role inthe response to vaccination against HBV, particularly ininfants. In line with our results, adult nonrespondersproduced significantly less TNF-a than strong respondersfollowing stimulation with HBsAg (23).
FIGURE 3. HBsAg-specific proliferation and intracellular cytokine production stratified by humoral immune response to primary immunization
against HBV at age 2 y.
(A) Percentage of starting CD4 T cells that proliferated. (B) Percentage of starting CD8 T cells that proliferated. (C) Percentage of IFN-g+ proliferating
CD4 T cells. (D) Percentage of IL-13+ proliferating CD4 T cells. (E) Percentage of IL-21+ proliferating CD4 T cells. Data are presented in boxplots as
median (lines) and IQR (boxes). Small circles indicate outliers and stars indicate extreme values. p , 0.05 indicates significance. 1nonresponders
versus low-responders versus high-responders, 2nonresponders versus high-responders, 3low-responders versus high-responders.
TABLE III. Correlations between anti-HBs levels at 1 and 2 y of age and HBsAg-specific CMI at 1 y of age
CMI Parameter at age 1 yCorrelation Coefficienta (Rho)to Anti-HBs Levels at 1 y (n)
p Value(Two-Tailed)
Correlation Coefficienta (Rho)to Anti-HBs Levels at 2 y (n)
p Value(Two-Tailed)
Percentage of starting CD4 T cells
that proliferated
0.115 (n = 76) 0.321 20.001 (n = 72) 0.994
Percentage of starting CD8 T cells
that proliferated
0.176 (n = 76) 0.129 0.179 (n = 72) 0.132
Percentage of IFN-g+ proliferating
CD4 T cells
0.170 (n = 63) 0.182 0.350 (n = 60) 0.006
Percentage of IL-13+ proliferating
CD4 T cells
0.097 (n = 63) 0.447 0.106 (n = 60) 0.420
Percentage of IL-21+ proliferating
CD4 T cells
0.068 (n = 63) 0.598 0.180 (n = 60) 0.169
IFN-g secretion (pg/ml) 0.269 (n = 76) 0.019 0.302 (n = 72) 0.01IL-10 secretion (pg/ml) 0.297 (n = 76) 0.009 0.088 (n = 72) 0.461IL-13 secretion (pg/ml) 0.114 (n = 76) 0.325 20.029 (n = 72) 0.812TNF-a secretion (pg/ml) 0.091 (n = 76) 0.433 0.122 (n = 72) 0.308
The bold text indicates statistical significance.aSpearman rank analysis.
https://doi.org/.4049/immunohorizons.1700015
ImmunoHorizons HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY 49
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
Inour study,we foundthat themedianpercentageof IL-13+CD4Tcellswas significantly lower in infants classified as nonrespondersas compared with high-responders. Limited knowledge is availableon the role of IL-13 in the response to vaccination against HBV.Following primary or booster vaccination of adults, nonrespondershad a lower percentage of IL-13+ CD4 T cells than responders (20).Although immunization of naive adults againstHBVwas associatedwith increased levels of secreted IL-13, infants showednodifferencein IL-13 following full vaccination series against HBV (24). Thecorrelation between IL-13 genotype and response to immunizationagainst HBV is controversial. Certain genotypes in the IL-13 genewere associatedwith nonresponse to vaccination againstHBV in anadult population (42). However, no associations between anti-HBslevels and genetic variation in IL-13 genes was reported in a cohortof patients undergoing hemodialysis (43).
We found that the median percentage of IL-21+ CD4 T cellswas significantly lower in infants classified at the age of 2 y asnonresponders when compared with high-responders. IL-21 is aneffector cytokine of follicular helper T cells, a subset of T helpercells that are critical in the formation andmaintenance of germinalcenters as well the differentiation of germinal center B cells intoplasma and memory B cells (44, 45). The role of IL-21 in theimmune response to HBV infection remains relatively undeter-mined and there is limited data in humans for the role of IL-21 inthe immune response to vaccination against HBV.
In our study, we did not find a correlation between the pro-liferative response of CD4 and CD8 T cells in infants categorizedaccording to their humoral immune response to immunizationagainst HBV. Previous literature on this topic is controversial.Although some adult studies reported on a correlation betweenanti-HBs levels and T cell proliferation (17–19), others showed alack of T cell proliferative response to HBsAg among nonre-sponders to HBV vaccine booster (19–21).
It should be noted that we demonstrated that infants whowere defined as nonresponders have evidence of HBsAg-specific CMI as they had some degree of HBsAg-specific CD4and CD8 T cell proliferation and measurable levels of secretedcytokines following stimulation with HBsAg. Our findings andthe evidence that there is clinical protection from HBVinfection in subjects with anti-HBs levels ,10 IU/l (13, 14),clearly indicates that an alternative or additional immunologicalmechanism might also correlate with HBV vaccine–induced pro-tection. Further support for the role of CMI generated aftervaccination in the prevention of clinical infection was reported forinfluenza. CMI to influenza correlatedwith protection from clinicalinfection, whereas serum Ab response did not (46).
Our study has several limitations. First, we did not assess peakanti-HBsAg levels (1 mo following primary vaccination againstHBV), thus limitingourability toclassify thecohort into thestandardgroupings of nonresponders, low-responders, and high-responders.It should be noted that there is increased anti-HBs response to thethird dose of primary immunization against HBV as comparedwiththe first two doses (47), with the vaccine-induced anti-HBs levelsachieving a peak usually 4 wk after immunization (48). This is thenfollowed by a rapid decline during the next few years after
immunization (especially the first year) and more slowly thereafter(48, 49). Second, the small sample size of our cohort limited ourcomparison at age 1 y to low-responders and high-responders,which might have limited our ability to detect significantcorrelation between CMI and humoral immune response. Itshould be emphasized that the vast majority of studies on HBVvaccine responses have concentrated on older children, adoles-cents, or adults. Thus, the strength of our study is that we addsignificant data on CMI at age 1 y and following primaryvaccination against HBV.
In conclusion, in the current studywe demonstrateHBsAg-specific CMI in infants following primary immunizationagainst HBV. We found that HBsAg-specific IFN-g and IL-10secretion correlated with HBV vaccine–induced humoralimmune response indicating that there is a balanced Th1/Th2 cytokine profile in response to vaccination against HBV.This is consistent with previous literature (18, 24). Althoughour results were less robust with TNF-a, this cytokine shouldalso be targeted by future research as another CMI candidatethat correlates with an HBsAg-specific humoral immuneresponse.
Furthermore, the demonstration in our study of evidence ofHBsAg CMI response among nonresponders and the fact thatindividuals whose levels are ,10 IU/l are protected againstHBV infection clearly argue that an alternative or additionalimmunological mechanism might also correlate with vaccine-induced protection.
Assessment ofT cell responses could be a useful adjunct indem-onstrating the persistence of HBsAg-specific immune response.
DISCLOSURES
The authors have no financial conflicts of interest.
REFERENCES
1. Shepard, C. W., E. P. Simard, L. Finelli, A. E. Fiore, and B. P. Bell.2006. Hepatitis B virus infection: epidemiology and vaccination.Epidemiol. Rev. 28: 112–125.
2. Amponsah-Dacosta, E., R. L. Lebelo, J. N. Rakgole, R. J. Burnett, S. G.Selabe, and M. J. Mphahlele. 2014. Evidence for a change in the epide-miology of hepatitis B virus infection after nearly two decades of uni-versal hepatitis B vaccination in South Africa. J. Med. Virol. 86: 918–924.
3. Ott, J. J., G. A. Stevens, J. Groeger, and S. T. Wiersma. 2012. Globalepidemiology of hepatitis B virus infection: new estimates of age-specificHBsAg seroprevalence and endemicity. Vaccine 30: 2212–2219.
4. Hyams, K. C. 1995. Risks of chronicity following acute hepatitis Bvirus infection: a review. Clin. Infect. Dis. 20: 992–1000.
5. Wong, W. W., G. Woo, E. J. Heathcote, and M. Krahn. 2013. Diseaseburden of chronic hepatitis B among immigrants in Canada. Can. J.Gastroenterol. 27: 137–147.
6. Dentinger, C. M., B. J. McMahon, J. C. Butler, C. E. Dunaway, C. L.Zanis, L. R. Bulkow, D. L. Bruden, O. V. Nainan, M. L. Khristova, T. W.Hennessy, and A. J. Parkinson. 2005. Persistence of antibody tohepatitis B and protection from disease among Alaska natives im-munized at birth. Pediatr. Infect. Dis. J. 24: 786–792.
https://doi.org/.4049/immunohorizons.1700015
50 HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY ImmunoHorizons
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
7. Teoharov, P., A. Kevorkyan, N. Petrova, I. Baltadzhiev, and P. VanDamme. 2013. Immune memory and immune response in childrenfrom Bulgaria 5-15 years after primary hepatitis B vaccination.Pediatr. Infect. Dis. J. 32: 51–53.
8. Chaves, S. S., G. Fischer, J. Groeger, P. R. Patel, N. D. Thompson, E. H.Teshale, K. Stevenson, V. M. Yano, G. L. Armstrong, T. Samandari,et al. 2012. Persistence of long-term immunity to hepatitis B amongadolescents immunized at birth. Vaccine 30: 1644–1649.
9. van der Sande, M. A., M. Mendy, P. Waight, C. Doherty, S. J.McConkey, A. J. Hall, and H. C. Whittle. 2007. Similar long-termvaccine efficacy of two versus three doses of HBV vaccine in earlylife. Vaccine 25: 1509–1512.
10. Plotkin, S. A. 2010. Correlates of protection induced by vaccination.Clin. Vaccine Immunol. 17: 1055–1065.
11. Jack, A. D., A. J. Hall, N. Maine, M. Mendy, and H. C. Whittle. 1999.What level of hepatitis B antibody is protective? J. Infect. Dis. 179:489–492.
12. Andre, F. E. 1990. Overview of a 5-year clinical experience with ayeast-derived hepatitis B vaccine. Vaccine 8(Suppl.): S74–S78; dis-cussion S79–80.
13. Mendy, M., I. Peterson, S. Hossin, T. Peto, M. L. Jobarteh, A. Jeng-Barry, M. Sidibeh, A. Jatta, S. E. Moore, A. J. Hall, and H. Whittle.2013. Observational study of vaccine efficacy 24 years after the startof hepatitis B vaccination in two Gambian villages: no need for abooster dose. PLoS One 8: e58029.
14. Zhu, C. L., P. Liu, T. Chen, Z. Ni, L. L. Lu, F. Huang, J. Lu, Z. Sun,and C. Qu. 2011. Presence of immune memory and immunity tohepatitis B virus in adults after neonatal hepatitis B vaccination.Vaccine 29: 7835–7841.
15. Szmuness, W., C. E. Stevens, E. J. Harley, E. A. Zang, H. J. Alter, P. E.Taylor, A. DeVera, G. T. Chen, and A. Kellner. 1982. Hepatitis Bvaccine in medical staff of hemodialysis units: efficacy and subtypecross-protection. N. Engl. J. Med. 307: 1481–1486.
16. Whittle, H. C., H. Inskip, A. J. Hall, M. Mendy, R. Downes, and S.Hoare. 1991. Vaccination against hepatitis B and protection againstchronic viral carriage in the Gambia. Lancet 337: 747–750.
17. Leroux-Roels, G., E. Van Hecke, W. Michielsen, P. Voet, P.Hauser, and J. Petre. 1994. Correlation between in vivo humoraland in vitro cellular immune responses following immunizationwith hepatitis B surface antigen (HBsAg) vaccines. Vaccine 12:812–818.
18. Larsen, C. E., J. Xu, S. Lee, D. P. Dubey, G. Uko, E. J. Yunis, and C. A.Alper. 2000. Complex cytokine responses to hepatitis B surface an-tigen and tetanus toxoid in responders, nonresponders and subjectsnaive to hepatitis B surface antigen. Vaccine 18: 3021–3030.
19. Chedid, M. G., H. Deulofeut, D. E. Yunis, M. L. Lara-Marquez, M.Salazar, R. Deulofeut, Z. Awdeh, C. A. Alper, and E. J. Yunis. 1997.Defect in Th1-like cells of nonresponders to hepatitis B vaccine.Hum. Immunol. 58: 42–51.
20. Goncalves, L., B. Albarran, S. Salmen, L. Borges, H. Fields, H. Montes,A. Soyano, Y. Diaz, and L. Berrueta. 2004. The nonresponse to hep-atitis B vaccination is associated with impaired lymphocyte activation.Virology 326: 20–28.
21. Vingerhoets, J., G. Vanham, L. Kestens, G. Penne, G. Leroux-Roels,and P. Gigase. 1994. Deficient T-cell responses in non-responders tohepatitis B vaccination: absence of TH1 cytokine production.Immunol. Lett. 39: 163–168.
22. Jafarzadeh, A., and F. Shokri. 2003. The antibody response to HBsantigen is regulated by coordinated Th1 and Th2 cytokine productionin healthy neonates. Clin. Exp. Immunol. 131: 451–456.
23. Velu, V., S. Saravanan, S. Nandakumar, E. M. Shankar, A. Vengatesan,S. S. Jadhav, P. S. Kulkarni, and S. P. Thyagarajan. 2008. Relationshipbetween T-lymphocyte cytokine levels and sero-response to hepatitisB vaccines. World J. Gastroenterol. 14: 3534–3540.
24. Ota, M. O., J. Vekemans, S. E. Schlegel-Haueter, K. Fielding, H.Whittle, P. H. Lambert, K. P. McAdam, C. A. Siegrist, and A.Marchant. 2004. Hepatitis B immunisation induces higher antibodyand memory Th2 responses in new-borns than in adults. Vaccine22: 511–519.
25. Carollo, M., R. Palazzo, M. Bianco, E. Pandolfi, P. Chionne, G.Fedele, A. E. Tozzi, R. Carsetti, L. Romano, and C. M. Ausiello. 2013.Hepatitis B specific T cell immunity induced by primary vaccinationpersists independently of the protective serum antibody level. Vac-cine 31: 506–513.
26. Jarrosson, L., M. N. Kolopp-Sarda, P. Aguilar, M. C. Bene, M. L.Lepori, M. C. Vignaud, G. C. Faure, and C. Kohler. 2004. Most hu-moral non-responders to hepatitis B vaccines develop HBV-specificcellular immune responses. Vaccine 22: 3789–3796.
27. Kardar, G. A., M. Jeddi-Tehrani, and F. Shokri. 2002. Diminished Th1and Th2 cytokine production in healthy adult nonresponders torecombinant hepatitis B vaccine. Scand. J. Immunol. 55: 311–314.
28. Corbett, N. P., D. Blimkie, K. C. Ho, B. Cai, D. P. Sutherland, A. Kallos,J. Crabtree, A. Rein-Weston, P. M. Lavoie, S. E. Turvey, et al. 2010.Ontogeny of Toll-like receptor mediated cytokine responses of humanblood mononuclear cells. PLoS One 5: e15041.
29. Blimkie, D., E. S. Fortuno III, H. Yan, P. Cho, K. Ho, S. E. Turvey,A. Marchant, S. Goriely, and T. R. Kollmann. 2011. Variables tobe controlled in the assessment of blood innate immune re-sponses to Toll-like receptor stimulation. J. Immunol. Methods366: 89–99.
30. Dienstag, J. L., B. G. Werner, B. F. Polk, D. R. Snydman, D. E. Craven, R.Platt, C. S. Crumpacker, R. Ouellet-Hellstrom, and G. F. Grady. 1984.Hepatitis B vaccine in health care personnel: safety, immunogenicity,and indicators of efficacy. Ann. Intern. Med. 101: 34–40.
31. Zuckerman, J. N., C. Sabin, F. M. Craig, A. Williams, and A. J.Zuckerman. 1997. Immune response to a new hepatitis B vaccinein healthcare workers who had not responded to standard vac-cine: randomised double blind dose-response study. BMJ 314:329–333.
32. Wiedmann, M., U. G. Liebert, U. Oesen, H. Porst, M. Wiese, S.Schroeder, U. Halm, J. Mossner, and F. Berr. 2000. Decreased im-munogenicity of recombinant hepatitis B vaccine in chronic hepatitisC. Hepatology 31: 230–234.
33. Dervisoglu, E., M. Simsek, and A. Yilmaz. 2011. Antibody responsefollowing hepatitis B vaccination in peritoneal dialysis patients:does normalized urea clearance matter? Clinics (Sao Paulo) 66:1559–1562.
34. Young, H. A., and K. J. Hardy. 1995. Role of interferon-gamma inimmune cell regulation. J. Leukoc. Biol. 58: 373–381.
35. Bocher, W. O., S. Herzog-Hauff, J. Schlaak, K. H. Meyer zumBuschenfeld, and H. F. Lohr. 1999. Kinetics of hepatitis B surfaceantigen-specific immune responses in acute and chronic hepatitis B orafter HBs vaccination: stimulation of the in vitro antibody response byinterferon gamma. Hepatology 29: 238–244.
36. Phillips, S., S. Chokshi, A. Riva, A. Evans, R. Williams, and N. V.Naoumov. 2010. CD8(+) T cell control of hepatitis B virus replica-tion: direct comparison between cytolytic and noncytolytic func-tions. J. Immunol. 184: 287–295.
37. Guidotti, L. G., T. Ishikawa, M. V. Hobbs, B. Matzke, R. Schreiber, andF. V. Chisari. 1996. Intracellular inactivation of the hepatitis B virus bycytotoxic T lymphocytes. Immunity 4: 25–36.
38. Carey, I., and D. Vergani. 2007. The role of viral-specific immuneresponses on the outcome of chronic hepatitis B (HBV) infection inchildren: new insights into immunopathology. J. Pediatr. Gastro-enterol. Nutr. 45: 15–18.
39. Weihrauch, M. R., M. von Bergwelt-Baildon, M. Kandic, M. Weskott,W. Klamp, J. Rosler, and J. L. Schultze. 2008. T cell responses tohepatitis B surface antigen are detectable in non-vaccinated individ-uals. World J. Gastroenterol. 14: 2529–2533.
https://doi.org/.4049/immunohorizons.1700015
ImmunoHorizons HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY 51
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
40. Rousset, F., E. Garcia, T. Defrance, C. Peronne, N. Vezzio, D. H.Hsu, R. Kastelein, K. W. Moore, and J. Banchereau. 1992. Inter-leukin 10 is a potent growth and differentiation factor for acti-vated human B lymphocytes. Proc. Natl. Acad. Sci. USA 89:1890–1893.
41. Vassalli, P. 1992. The pathophysiology of tumor necrosis factors.Annu. Rev. Immunol. 10: 411–452.
42. Chen, J., Z. Liang, F. Lu, X. Fang, S. Liu, Y. Zeng, F. Zhu, X. Chen, T.Shen, J. Li, and H. Zhuang. 2011. Toll-like receptors and cytokines/cytokine receptors polymorphisms associate with non-response tohepatitis B vaccine. Vaccine 29: 706–711.
43. Grzegorzewska, A. E., D. Pajzderski, A. Sowinska, A. Mostowska, and P. P.Jagodzinski. 2013. IL4R and IL13 polymorphic variants and developmentof antibodies to surface antigen of hepatitis B virus in hemodialysis pa-tients in response to HBV vaccination or infection. Vaccine 31: 1766–1770.
44. Crotty, S. 2011. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol.29: 621–663.
45. Kurosaki, T., K. Kometani, and W. Ise. 2015. Memory B cells. Nat. Rev.Immunol. 15: 149–159.
46. McElhaney, J. E., D. Xie, W. D. Hager, M. B. Barry, Y. Wang, A.Kleppinger, C. Ewen, K. P. Kane, and R. C. Bleackley. 2006. T cellresponses are better correlates of vaccine protection in the elderly.J. Immunol. 176: 6333–6339.
47. European Consensus Group on Hepatitis B Immunity. 2000. Arebooster immunisations needed for lifelong hepatitis B immunity?Lancet 355: 561–565.
48. Zuckerman, J. N. 1996. Nonresponse to hepatitis B vaccines and thekinetics of anti-HBs production. J. Med. Virol. 50: 283–288.
49. Jilg, W., M. Schmidt, and F. Deinhardt. 1989. Four-year experiencewith a recombinant hepatitis B vaccine. Infection 17: 70–76.
https://doi.org/.4049/immunohorizons.1700015
52 HEPATITIS B VIRUS VACCINE–INDUCED CELL-MEDIATED IMMUNITY ImmunoHorizons
by guest on Novem
ber 8, 2018http://w
ww
.imm
unohorizons.org/D
ownloaded from
