Vaccine 28 (2010) 25772579

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Short communication

Effect of probiotic supplementation in the rst 6 months of life on specic antibody responses to infant Hepatitis B vaccination
Shu E Soh a , Dave Qi Rong Ong a , Irvin Gerez a , Xiaoe Zhang b , Pavithra Chollate b , Lynette Pei-Chi Shek a , Bee Wah Lee a , Marion Aw a,
a b

Department of Paediatrics, Yong Loo Lin School of Medicine, National University Health System, 5 Lower Kent Ridge Road, Singapore 119074, Singapore Singapore Clinical Research Institute Pte Ltd., Singapore

a r t i c l e

i n f o

a b s t r a c t
Probiotics have been shown to enhance specic immune responses to vaccines. We aim to assess the effect of probiotic supplementation on specic IgG antibody responses to Hepatitis B (HepB) vaccination in infants. Compared to controls, probiotic supplementation improved HepB surface antibody responses in subjects receiving monovalent doses of HepB vaccine at 0, 1 month and a DTPaHepB combination vaccine at 6 months [placebo (n = 28): 187.97 (180.70195.24), probiotic (n = 29): 345.70 (339.41351.99) mIU/ml] (p = 0.069), but not those who received 3 monovalent doses [placebo (n = 68): 302.34 (296.31308.37), probiotic (n = 77): 302.06 (296.31307.81) mIU/ml] (p = 0.996). Probiotics may enhance specic antibody responses in infants receiving certain Hepatitis B vaccine schedules. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 23 April 2009 Received in revised form 4 January 2010 Accepted 13 January 2010 Available online 25 January 2010 Keywords: Probiotics Hepatitis B Vaccination

1. Introduction Probiotics are promising immunomodulators which enhance innate and adaptive immunity in the host [1]. Gnotobiotic animal models have shown that probiotics have a signicant immunostimulating effect on local and systemic immune responses. Furthermore, its safety record in humans has made probiotic supplementation an attractive strategy to modulate and enhance the immune system. Probiotics have been conferred GRAS (generally regarded as safe) status by the Food and Agriculture Organization (FAO)/World Health Organization expert panel [2], and are considered safe in the neonate [3,4]. Probiotic supplementation in young children has been shown to protect against gastrointestinal infections such as rotavirus gastroenteritis [5]. Probiotics have also been shown to enhance specic immune responses to vaccination in young children and adults. It increased the immunogenicity of orally administered vaccines such as that of rotavirus [6], Salmonella [7], polio [8] and cholera [9]; as well as enhanced antibody responses to parenterally administered vaccines, namely diphtheria, tetanus, and Haemophilus inuenzae type b [1014]. Probiotics therefore have an adjuvant effect by enhancing immunogenicity of vaccines. This study assessed the effect of probiotic supplementation in the rst 6 months of age on specic IgG antibody responses to Hepatitis B (HepB) vaccination. To our

knowledge, there have been no previous reports on the effect of probiotics on HepB vaccination in infants. 2. Methods 2.1. Study design This study was part of a double-blind, randomized, controlled trial (ClinicalTrials.gov Identier: NCT00318695) which evaluated the effect of supplementation of probiotics in the rst 6 months of life on eczema in at-risk infants [15]. We recruited 253 newborns with a history of allergic disease in a rst degree relative from the antenatal clinics at the National University Hospital, Singapore, between May 2004 and June 2006. Written informed consent was obtained from all families. The study was approved by the Hospitals ethics review committee (Ref Code: B/00/322). 2.2. Probiotic supplementation Subjects received at least 60 ml (9.26 g) a day of commercially available cows milk based infant formula (Nan 1 ), with either probiotic supplementation (Bidobacterium longum BL999 (ATCC: BAA-999 designation BB536, Morinaga, Japan) 1 107 colonyforming unit (cfu)/g and Lactobacillus rhamnosus LPR [CGMCC 1.3724] 2 107 cfu/g) or without, initiated within 12 h after delivery, and continued for the rst 6 months of life. The infants in the probiotic group, therefore, received at least 2.8 108 cfu of probiotic bacteria per day. Mothers were then free to decide whether

Corresponding author. Tel.: +65 67724420; fax: +65 67797486. E-mail address: paeawm@nus.edu.sg (M. Aw). 0264-410X/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.01.020

2578

S.E. Soh et al. / Vaccine 28 (2010) 25772579

Table 1 Hepatitis B surface antibody response in vaccine schedule A and B. Vaccine group Treatment group Placebo Probiotics Placebo Probiotics Sample size (n) 28 29 68 77 HBsAb IgG geometric mean titre (mIU/ml) 187.97* 345.70 302.34* 302.06 95% C.I. 180.70195.24 339.41351.99 296.31308.37 296.31307.81 p-Value Seroconversion rate (%) 96.6 93.5 93.2 97.5 p-Value

A B

0.069 0.996

1.000 0.259

Schedule A: monovalent HepB vaccines at 0, 1 month and DTPaIPVHiBHepB combination vaccine at 6 months. Schedule B: monovalent HepB vaccines at 0, 1, 6 months. * p = 0.076 (placebo group: schedule A vs. schedule B).

to make up the remainder of the baby feeds with either the trial formula, or to supplement with breast milk, or another infant formula. Both B. longum and L. rhamnosus conformed to the Food and Agriculture Organization (FAO)/World Health Organization expert panel guidelines for probiotics [16]. Computerized randomization was carried out in blocks of 6 based on a 1:1 allocation, with the lowest number allocated sequentially. Mothers (caregivers), doctors, nurses and statisticians involved in the trial were blinded to milk formula allocation. 2.3. Vaccination Depending on the attending vaccination centre, majority of the infants received either 1 of 2 schedules of HepB vaccination with intramuscular injection in the anterolateral aspect of the thigh at ages 0, 1 and 6 months, respectively, following the Singapore national immunization program. Schedule A consisted of monovalent HepB vaccination, (HBVax, MSD, Whitehouse Station, NJ, USA) at Dose 1 and 2 (2.5 g each) and a hexavalent diphtheriatetanusacellular pertussis (DTPa) combination vaccination containing a HepB component (10 g) (Infanrix HEXA, GSK Biologicals, Rixensart, Belgium) at Dose 3. Schedule B consisted of monovalent HepB (HBVax) 2.5 g/dose for all three doses. Infants born to HepB surface antigen (HBsAg)-positive mothers received the HepB vaccine with HepB immune globulin (HBIG) at birth to prevent transmission of perinatal HepB viral infection. 2.4. Antibody analysis Venous blood was collected at 12 months of age and analyzed for HepB serology with measurement of the HepB virus surface antibody (HBsAb) immunoglobulin G using ADVIA Centaur Anti-HBs (Bayer Health Care, Tarry town, NY, USA). 2.5. Statistical analysis SPSS software (version 16.0 for Windows) was used to perform t-test to compare HBsAb IgG geometric mean titres between placebo and probiotic groups. A p-value of <0.05 was considered statistically signicant for all analyses. 3. Results 3.1. Baseline characteristics and participants Families were assessed at the antenatal clinic and 253 newborns were recruited into the study. During the follow-up period, 3 subjects from the probiotic group and 6 subjects from the placebo group did not complete the study and blood samples were not collected. At the 12-month visit, 11 subjects refused blood taking. There were 20 subjects who received alternative vaccine schedules

(other than schedule A or B) involving combination vaccines and these subjects were excluded from analysis. There were 11 subjects (4.72%) including one born to a HepB carrier (HBeAg+) mother, who failed to seroconvert after 3 doses of vaccination (Table 1). Of these, 3 received vaccine schedule A (probiotic = 2) while 8 received vaccine schedule B (probiotic = 3). Seven of the 11 subjects seroconverted after an additional booster dose of vaccine (HBVax, 5 g), and the eighth subject after 2 doses. The subject born to Hepatitis B carrier mother failed to respond and was found to be HBsAg positive. The remaining 2 subjects refused further blood evaluation. There were therefore 202 evaluable subjects. Fifty-seven infants received vaccine schedule A (probiotic = 29, placebo = 28) and 145 infants received vaccine schedule B (probiotic = 77, placebo = 68). The demographic and social characteristics, gender ratio, gestational age, birth weight, number of siblings, daycare attendance, smoking at home and breastfeeding rate of the 2 vaccine groups were comparable in the probiotic and placebo group. The compliance level of consuming at least 60 ml of trial formula per day from birth to 6 months was 89% in the probiotic group and 85% in the placebo group. All subjects did not consume any other probiotic preparations or dietary products during the 6-month intervention period. As the primary clinical outcome of the study was eczema, vaccine schedule was determined by the centre the subject attended. 3.2. Effects of probiotic supplementation on Hepatitis B surface antibody response The seroconversion rates were almost similar between the 2 schedules. However, of 5 infants who were born to HepB carrier mothers, vaccine failure occurred in one infant who was randomized to the probiotic group and received schedule A vaccinations. This child was the only subject to develop HepB carrier state. Within the placebo group, the HBsAb geometric mean titres (95% CI) of subjects in schedule A [187.97 (180.70195.24) mIU/ml] were lower than subjects with schedule B [302.34 (296.31308.37) mIU/ml], although this difference did not reach statistical signicance (p = 0.076). In contrast, within the probiotic group, HBsAb geometric mean titres of those receiving schedule A [345.70 (339.41351.99) mIU/ml] and B [302.06 (296.31307.81) mIU/ml] were comparable (p = 0.575). In other words, probiotic supplementation could increase HBsAb responses in those receiving schedule A to levels more comparable with those in schedule B. Hence, for infants who received schedule A, the HBsAb geometric mean (95% CI) titres were 345.70 (339.41351.99) mIU/ml in the probiotic group and 187.97 (180.70195.24) mIU/ml in the placebo (p = 0.069). This difference was not observed for infants receiving vaccine schedule B where HBsAb geometric mean (95% CI) titres were very similar [probiotic: 302.06 (296.31307.81) mIU/ml, placebo: 302.34 (296.31308.37) mIU/ml] (p = 0.996). The data is summarized in Table 1.

S.E. Soh et al. / Vaccine 28 (2010) 25772579

2579

4. Discussion HepB vaccination is part of the WHO expanded program of immunization. Strategies that may improve immunogenicity of the vaccine are welcomed, especially in infants born to HepB carrier mothers. Probiotic supplementation has been shown to enhance the immunogenicity of various vaccines [614]. This study evaluated the effects of probiotics on HepB vaccine responses in infants vaccinated from birth. Two vaccine schedules were compared as the majority of subjects received these schedules. Our results show that the schedule with 3 monovalent doses of HepB vaccine resulted in better HBsAb responses compared to the schedule consisting of 2 monovalent doses followed by a third dose as a DTPa combination vaccine, although this difference was not statistically different (p = 0.076). These results differ from two recent studies conducted in Singapore, where HBsAb responses involving monovalent vaccine and combination vaccine were similar [17,18]. This observed difference may be related to the dose of the monovalent (2.5 g vs. 10 g in Engerix B, GSK Biologicals, Rixensart, Belgium) vaccines used, and the combination of HepB vaccines from different manufacturers in schedule A (MSD for monovalent and GSK Biologicals for combination vaccine). These schedules represent real life schedules used by vaccine centres in Singapore. There was only one documented vaccine failure in our cohort. This subject was born to Hep B (HBeAg+) mother and despite hyperimmune globulin at birth and 3 doses of HepB vaccines, developed the carrier state. This failure to respond may be genetically determined as shown in a recent report [19]. The remaining 8 subjects should not be considered non-responders as is dened by a failure to seroconvert after completion of two full 3-dose series of the HepB vaccine and for whom an acute or chronic Hepatitis B infection has been ruled out [20]. Interestingly, probiotic supplementation in the rst 6 months of life could improve HBsAb responses in subjects receiving schedule A (2 doses monovalent + 1 dose combination) but not in those receiving schedule B where higher antibody responses were observed. This difference, however, did not reach statistical significance. This is likely to have arisen from the smaller sample size of subjects in schedule A, since the vaccine schedules were not randomized for this study but were dependent on the vaccine centres attended. This study may be statistically underpowered as the number of subjects receiving vaccine schedule A were less than those in vaccine schedule B. Nonetheless, the data suggests that probiotics can potentially be used as an adjuvant for immune responses in schedules with less than optimal responses. In conclusion, our data suggests that probiotic supplementation from birth could enhance HepB antibody response in infants receiving certain vaccine schedules. These ndings however, would require larger studies to conrm these observations. Acknowledgements We sincerely appreciate the assistance of the PROMPT (PRObiotic in Milk for the Prevention of aTopy trial) team and the voluntary participation of all subjects in the study. This study

was funded by the National Medical Research Council, Singapore (NMRC/0890/2004). The study milk formula was kindly sponsored by Nestle , Vevey, Switzerland. References
[1] Isolauri E, Sutas Y, Kankaanpaa P, Arvilommi H, Salminen S. Probiotics: effects on immunity. Am J Clin Nutr 2001;73(2 Suppl.):444S50S. [2] Guidelines for the Evaluation of Probiotics in Food. FAO/WHO (Food and Agriculture Organization/World Health Organization), 2002. [3] Zhang W, Azevedo MS, Wen K, Gonzalez A, Saif LJ, Li G, et al. Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs. Vaccine 2008;26(2930):365561. [4] Zhang W, Wen K, Azevedo MS, Gonzalez A, Saif LJ, Li G, et al. Lactic acid bacterial colonization and human rotavirus infection inuence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs. Vet Immunol Immunopathol 2008;121(34):22231. [5] Guandalini S. Probiotics for children with diarrhea: an update. J Clin Gastroenterol 2008;42(Suppl 2):S537. [6] Isolauri E, Joensuu J, Suomalainen H, Luomala M, Vesikari T. Improved immunogenicity of oral D x RRV reassortant rotavirus vaccine by Lactobacillus casei GG. Vaccine 1995;13(3):3102. [7] Fang H, Elina T, Heikki A, Seppo S. Modulation of humoral immune response through probiotic intake. FEMS Immunol Med Microbiol 2000;29(1): 4752. [8] de Vrese M, Rautenberg P, Laue C, Koopmans M, Herremans T, Schrezenmeir J. Probiotic bacteria stimulate virus-specic neutralizing antibodies following a booster polio vaccination. Eur J Nutr 2005;44(7):40613. [9] Paineau D, Carcano D, Leyer G, Darquy S, Alyanakian MA, Simoneau G, et al. Effects of seven potential probiotic strains on specic immune responses in healthy adults: a double-blind, randomized, controlled trial. FEMS Immunol Med Microbiol 2008;53(1):10713. [10] Kukkonen K, Nieminen T, Poussa T, Savilahti E, Kuitunen M. Effect of probiotics on vaccine antibody responses in infancya randomized placebo-controlled double-blind trial. Pediatr Allergy Immunol 2006;17(6):41621. [11] Mullie C, Yazourh A, Thibault H, Odou MF, Singer E, Kalach N, et al. Increased poliovirus-specic intestinal antibody response coincides with promotion of Bidobacterium longum-infantis and Bidobacterium breve in infants: a randomized, double-blind, placebo-controlled trial. Pediatr Res 2004;56(5): 7915. [12] Olivares M, Diaz-Ropero MP, Sierra S, Lara-Villoslada F, Fonolla J, Navas M, et al. Oral intake of Lactobacillus fermentum CECT5716 enhances the effects of inuenza vaccination. Nutrition 2007;23(3):25460. [13] Taylor AL, Hale J, Wiltschut J, Lehmann H, Dunstan JA, Prescott SL. Effects of probiotic supplementation for the rst 6 months of life on allergen- and vaccine-specic immune responses. Clin Exp Allergy 2006;36(10):122735. [14] West CE, Gothefors L, Granstrom M, Kayhty H, Hammarstrom ML, Hernell O. Effects of feeding probiotics during weaning on infections and antibody responses to diphtheria, tetanus and Hib vaccines. Pediatr Allergy Immunol 2008;19(1):5360. [15] Soh SE, Aw M, Gerez I, Chong YS, Rauff M, Ng YP, et al. Probiotic supplementation in the rst 6 months of life in at risk Asian infantseffects on eczema and atopic sensitization at the age of 1 year. Clin Exp Allergy 2009;39:5718. [16] Joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including milk powder with live lactic acid bacteria. FAO/WHO (Food and Agriculture Organization/World Health Organization), 2001. [17] Lim FS, Han HH, Jacquet JM, Bock HL. Primary vaccination of infants against hepatitis B can be completed using a combined hexavalent diphtheriatetanus-acellular pertussis-hepatitis B-inactivated poliomyelitis-Haemophilus inuenzae type B vaccine. Ann Acad Med Singapore 2007;36(10):8016. [18] Cheng HK, Rajadurai VS, Amin Z, Sriram B, Yee MF, Han HH, et al. Immunogenicity and reactogenicity of two regimens of diphtheria-tetanus-acellular pertussis-hepatitis B-inactivated polio and Haemophilus inuenzae type b vaccines administered to infants primed at birth with hepatitis B vaccine. Southeast Asian J Trop Med Public Health 2004;35(3):68592. [19] Kamatani Y, Wattanapokayakit S, Ochi H, Kawaguchi T, Takahashi A, Hosono N, et al. A genome-wide association study identies variants in the HLA-DP locus associated with chronic hepatitis B in Asians. Nat Genet 2009. [20] Koff RS. Immunogenicity of hepatitis B vaccines: implications of immune memory. Vaccine 2002;20(3132):3695701.