Journal of Functional Foods 33 (2017) 1–10

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Journal of Functional Foods

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Drinking fermented milk containing Lactobacillus paracasei 431(IMULUSTM) improves immune response against H1N1 and cross-reactiveH3N2 viruses after influenza vaccination: A pilot randomized triple-blinded placebo controlled trial

http://dx.doi.org/10.1016/j.jff.2017.03.0161756-4646/� 2017 Elsevier Ltd. All rights reserved.

Abbreviations: HAI, Hemagglutination Inhibition Assay; ELISA, Enzyme-LinkedImmunosorbent Assay; IMULUSTM, Lactobacillus paracasei 431.⇑ Corresponding authors at: Institute of Nutrition, Mahidol University, 999

Phutthamonthon Sai 4 Road, Salaya, Nakhon Pathom 73170, Thailand.E-mail addresses: dunyaporn.tra@mahidol.ac.th (D. Trachootham), chalat.san@

mahidol.ac.th (C. Santivarangkna).

Dunyaporn Trachootham a,⇑, Chaowanee Chupeerach a, Siriporn Tuntipopipat a, Lilly Pathomyok b,Kobporn Boonnak c,d, Kemika Praengam a, Chadamas Promkam a, Chalat Santivarangkna a,⇑a Institute of Nutrition, Mahidol University, Nakhon Pathom 73170, ThailandbDepartment of Medicine, Golden Jubilee Medical Center, Mahidol University, Nakhon Pathom 73170, ThailandcDepartment of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, ThailanddVaccine Trial Centre, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand

a r t i c l e i n f o

Article history:Received 17 November 2016Received in revised form 25 January 2017Accepted 6 March 2017

Keywords:Immune responseInfluenzaMilkProbioticVaccineLactobacillus casei

a b s t r a c t

A randomized triple-blinded placebo controlled trial was conducted in healthy adults to evaluate theeffects of daily consumption of fermented probiotic milk on immune response towards influenza virustype H1N1, H3N2 and Flu-B. Study and placebo groups (n = 30 each) consumed fermented milk contain-ing 109 Lactobacillus casei 431 and acidified milk respectively, and the vaccination was administered twoweeks thereafter. The consumption continued for another four weeks. Anti-viral response was tested byHemagglutination Inhibition (HAI) and ELISA assays. Results showed that study group had higher sero-conversion rates for H1N1 and cross-reactive H3N2; higher HAI response (�2-fold) rates for cross-reactive H3N2 virus; and increased ratios of geometric mean (study/placebo) for HAI titers and IgM ofH1N1 and H3N2 viruses following the vaccination. These pilot results indicated that drinking fermentedmilk may be an approach to raise immune response against influenza A, especially for participants withlow pre-existing immune.Clinical Trial Registry: ClinicalTrials.gov NCT02909842

� 2017 Elsevier Ltd. All rights reserved.

1. Introduction ple, and most cases were found in people aged between 15 and

Upper respiratory tract infections such as influenza are theworld’s most frequent reason for seeking medical care and spend-ing medical expenditure by patients (Footitt & Johnston, 2009).According to World Health Organization (WHO), influenza occursglobally with an annual attack rate of 5–10% in adults and 20–30% in children. Globally, these annual epidemics result in approx-imately 3–5 million cases of severe illness and 250,000–500,000deaths (WHO Fact sheet N�211, 2014). Consistently, data from Cen-ter of Disease Control, Ministry of Health of Thailand reported thatthe incidence of influenza in Thai people was 34.9 per 100,000 peo-

44 years old (Center of Disease Control, Ministry of Health ofThailand, 2016). Therefore, influenza is considered as an importantmedical burden of many parts of the world including Thailand.Since there are no effective anti-viral therapies for such infections,preventive strategies such as immune protection by vaccinationare the most important approach to reduce the risk and severityof the diseases (Footitt & Johnston, 2009). Furthermore, vaccinechallenge model has been considered one of the best tools forintervention studies involving immune response (Albers et al.,2005; Burleson & Burleson, 2007). Thus, the guideline of EuropeanFood Safety Authority (EFSA) recommends the model for clinicaltrial of food product with immunomodulating effects (Guidanceon the scientific requirements for health claims related to gutand immune function-EFSA, 2011).

Probiotics are living micro-organisms that provide health bene-fits if given at an appropriate dose (FAO/WHO Guidelines for theevaluation of probiotics in food, 2002). Many probiotics have been

2 D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10

shown to promote proper functions of gastrointestinal andimmune systems. The efficacy of probiotics mostly depends onthe type of micro-organisms (Rijkers et al., 2010). Lactobacillusparacasei subsp. paracasei 431 or L. casei 431 is a probiotic withgood in vitro and in vivo evidences of immunomodulatory activity(Haro, Villena, Zelaya, Alvarez, & Aguero, 2009; Marranzino,Villena, Salva, & Alvarez, 2012). Furthermore, clinical trials usingvaccine models also demonstrated that the probiotic L. casei 431raised antibody titer (the most diluted concentration of antibodiesexhibiting immune response) against polio and influenza viruses(de Vrese et al., 2005; Rizzardini et al., 2012). A randomizeddouble-blind, placebo-controlled trial conducted in Italy in 211healthy volunteers aged between 20 and 60 years reported thatcontinuous intake of probiotic fermented milk with L. casei 431for 2 weeks prior to influenza vaccination against H1N1, H3N2and B-Florida influenza virus, and continued for additional 4 weeksafter vaccination resulted in an elevation of vaccine-specific anti-bodies (vaccine-specific plasma IgG, IgG1 and IgG3), compared toplacebo control group. This trial used Enzyme-Linked Immunosor-bent Assay (ELISA) to measure plasma level of antibodies(Rizzardini et al., 2012). In contrast, another study conducted inDenmark in 1104 healthy volunteers aged between 18 and60 years found an opposite result in which probiotic fermentedmilk with L. casei 431 had no effects on antibody titers measuredby Hemagglutination Inhibition Assay (HAI or HI) (Jespersen,Tarnow, & Eskesen, 2015). Nevertheless, these two trials consis-tently demonstrated that daily intake of probiotic fermented milkwith L. casei 431 at 1 � 109 colony-forming units/dose was safe. Nosignificant differences in safety parameters between the probioticand control groups were found (Jespersen et al., 2015; Rizzardiniet al., 2012). Correspondingly, a clinical study also found that babyfood containing L. casei 431 were well-tolerated and safe for infantage 0–6 months (Vlieger, Robroch, & Buuren, 2009).

Even though the efficacy of probiotic L. casei 431 had been stud-ied in few countries, its effect on immune response was inconclu-sive. Furthermore, there were no studies regarding theimmunomodulating effect of L. casei 431 or any probiotics againstPhuket B influenza virus, a recently epidemic virus. In fact, WHOrecommended that the influenza vaccine used in the northernhemisphere influenza season (2015–2016) and in the southernhemisphere influenza season (2015) raised immune responseagainst H1N1, H3N2 and Phuket-B viruses (WHO CollaboratingCentre for Reference and Research on Influenza, 2014, 2015).

Drinking fermented milk containing 1 � 109 colony-formingunits/dose of IMULUSTM (Lactobacillus paracasei 431) was registeredby Thai FDA. This essential fermented milk was commerciallymade available throughout Thailand and many other countriesglobally. It passed food safety test according to Thai Food and DrugAdministration (FDA). However, the immunomodulating effect ofthe product had never been tested. The goal of this study was toassess whether daily drinking of fermented milk containing 109

live cells of the probiotic L. casei 431 could increase the immuneresponse against influenza virus type H1N1, H3N2 and Flu-B (Phu-ket strain) in a vaccine challenge model. A randomized triple-blinded placebo controlled trial was conducted in healthy Thaiadults receiving 2015/2016 influenza vaccine. Four weeks afterthe vaccination, improved immune responses were determinedby an increase in antibody titers through HAI and ELISA assays.

2. Methods

2.1. Materials

Drinking fermented milk products containing IMULUSTM andplacebo acidified milk were obtained from Dutchmill, Co, Inc.

(Thailand). Viral antigens of A/California/07/09 (H1N1), A/Songkhla/308/13 (H3N2) (A/Victoria/361/2011 (H3N2)-like strain)and B/Phuket/287/13 (B/Massachusetts/02/2012-like strain) wereobtained from Vaccine Trial Centre, Faculty of Tropical Medicine,Mahidol University, Thailand. The commercial ELISA kit for IgMand IgG against Influenza A (H1N1 and H3N2) was fromImmuno-Biological Laboratories, Inc. (Minnesota, USA). Standarddose of 2015–2016 trivalent seasonal influenza vaccine wasobtained from Agrippal�2015: Novartis, USA. The vaccine pro-duced from inactivated surface antigens of an A/California/07/09(H1N1) pdm09-like virus, an A/Switzerland/9715293/2013(H3N2)-like virus and a B/Phuket/3073/2013-like virus.

2.2. Ethical approval

This study protocol no. MU-IRB 2015/145.2019 was approvedby the Mahidol University Central Institutional Review Board(MU-CIRB), with COA no. 2015/150.0112.

2.3. Design, blinding and random allocation

This study is a triple-blinded placebo randomized controlledtrial. Participants were randomly allocated into two groups, i.e.study and placebo groups. For this pilot trial, minimization accord-ing to age and sex was used in randomization. Subjects, physician,clinical data collector, laboratory data analyzer and statistical ana-lyzer were blinded. The product manufacturer and randomassigner did not involve in data collection and statistical analysis.

2.4. Sample size and power

The main objective of this work was to compare immuneresponse rate between two groups. Therefore, the sample sizewas calculated using non-centrality parameter of chi-square test.Since there were no similar studies conducted in Thai people, wecalculated the sample size for this pilot study by using hypotheticallarge effect size for chi-square test which is 0.5. Level of signifi-cance was set as 0.05, power of 0.95 and degree of freedom of 1.The calculated total sample size was 52. To account for possible15% drop-out, a total of 60 participants (30 each group) wasrequired. To ensure that this pilot study had sufficient sample sizefor the observed effect, we performed a post hoc power analysis.

2.5. Participants

Participants were recruited to the outpatient clinic, departmentof medicine, Golden Jubilee Medical Center, Mahidol University,Thailand. Prior to the recruitment, all participants were screenedbased on the following criteria. Inclusion criteria: being 18–45 yearsold; normal vital signs; having no history of uncontrolled systemicdiseases or autoimmune diseases; having no signs of respiratoryinfections; normal range of blood chemistry (result of less than1 month): complete blood count (CBC), fasting blood sugar (FBS),lipid profiles (Cholesterol, HDL, LDL, Triglycerides), liver functions(SGOT, SGPT), kidney functions (BUN, Creatinine); never haveinfluenza vaccine during the past 6 months; able to continue tak-ing the fermented milk product daily for 6 weeks; able to refrainfrom eating fermented dairy products or products containing pro-biotics from screening until the end of the study; agreed to receiveinfluenza vaccination; able to refrain from receiving other vaccina-tion during the study; able to refrain from traveling to high riskarea of influenza infections during the study; able to communicateand understand Thai language and self-decide on written consent.Exclusion criteria: under pregnancy; lactose/milk protein intoler-ance; acute/terminal disease; chronic alcoholism; frequent gas-trointestinal disorders, gastrointestinal surgery; long-term

D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10 3

treatment with any other drug known to affect the immuneresponse (e.g. glucocorticoids, chemotherapy, antibodies, cyclos-porin, hormone, opioids).

All participants signed their written informed consent prior todata collection. Their identities have been protected, followingICH-GCP.

2.6. Intervention

Participants in study group were instructed to daily take a bot-tle of 100 mL drinking fermented milk products containing IMU-LUSTM. Participants in placebo group were instructed to daily takeone bottle of 100 mL placebo acidified milk products. Both groupsreceived an intramuscular injection with 0.5 mL of influenza vac-cine (Agrippal�2015: Novartis, USA) 2 weeks after the start of theintervention. The vaccine used was the seasonal influenza vaccinefor the 2015–2016 season and contained antigens from anA/California/7/2009 (H1N1) pdm09-like virus, an A/Switzer-land/9715293/2013 (H3N2)-like virus and a B/Phuket/3073/2013-like virus.

2.7. Study procedures

The study was performed according to the Declaration of Hel-sinki and ICH-GCP, as indicated in Appendix 1. First, a physicianscreened volunteers to identify eligible participants based oninclusion/exclusion criteria. A 15 mL of blood was drawn for bloodchemistry and baseline immune measure. Second, a researcherdescribed the project to the participants. Upon agreement, partic-ipants signed their written informed consent. Third, baseline mea-surements were performed for consented participants includinganti-viral titer against H1N1, H3N2 and FluB, measured by HAIand ELISA assays. Forth, randomization was carried and partici-pants were divided by a researcher into study or placebo controlgroups, using minimization based on equal distribution of sexand age. The participants and the physician were blinded (onlyknown as group 1 or 2). Then, the participants were given IMU-LUSTM drink or placebo product, accordingly for 2 weeks. Subjectdiaries were provided and returned to researchers at everyfollow-up visit. For each follow-up visits, a volume of 5 mL bloodwas collected for measurement of antibody titer and then partici-pants returned their subject diaries. When the participantsreturned for the first follow-up (2 weeks after baseline), the2015/16 influenza vaccine was given to each subject in bothgroups. The, IMULUSTM drink or placebo products were providedfor additional 4 weeks and subjects returned for the secondfollow-up thereafter (6 weeks after baseline). Then, IMULUSTM drinkor placebo products were terminated for 2 weeks, and participantsreturned for the final follow-up (8 weeks after baseline).

2.8. Monitoring

Adherence to intervention was monitored by subject diaries.During home use of the products, the subjects daily recorded theirintake of product, influenza-like symptoms and other adversesymptoms relevant to product intake. They also did their 3-daydietary record weekly. Any participant with intake less than5 weeks (out of 6 weeks) would be excluded from the study. Ineach follow-up visits, the physician provided medical history tak-ing and physical exam for their influenza-like symptoms or otheradverse events. If any occurs, it would be managed by the physi-cian. Any subjects who exhibit serious adverse events (SAE) wouldbe withdrawn from the project and reported to the MU-CIRB andthe sponsor as soon as possible.

2.9. Outcome

2.9.1. Blood specimen processingAt each point of sample collection, 5 mL of blood was drawn

from each participant. Plasma was obtained after centrifugationat 1500 rpm for 10 min. Then, the sample was divided into twoparts for HAI and ELISA assays. All the samples were kept at�80 �C until all collection was completed. Then, HAI or ELISAassays were performed for all samples together.

2.9.2. Anti-viral response using Hemagglutination Inhibition (HAI)assay

HAI or HI assay was used to determine the serum titer which ismost diluted concentration of serum that can inhibit bindingbetween virus and red blood cells. Comparison of HAI titer beforeand after vaccination can predict efficacy of influenza vaccine(Luytjes et al., 2012). Serum samples were tested by hemagglutina-tion inhibition (HAI) assay against A/California/07/09 (H1N1), A/Songkha/308/13 (H3N2) (A/Victoria/361/2011 (H3N2)-liked strain)and B/Phuket/287/13 (B/Massachusetts/02/2012-likedstrain).Serum samples were treated with receptor destroying enzyme(RDE, SEIKEN, Campbell, CA), and 2-fold serially diluted in 96-well V-bottom plates starting at a dilution of 1:10. Then, fourhemagglutination (HA) units of virus were added to each sample.Control wells received phosphate buffer saline (PBS) alone or PBSwith virus in the absence of antibody. Virus and sera were incu-bated together for 30 min at room temperature. Subsequently,50 lL of a 0.5% (vol/vol) suspension of goose erythrocytes wasadded for detection of antibodies against H1N1 and Flu B, while50 mL of a 0.75% (vol/vol) suspension of guinea pig erythrocyteswas added for detection of H3N2. The results were recorded afterincubating for 45–60 min at room temperature. The HAI titer wasrecorded as the reciprocal of the highest dilution of serum thatcompletely inhibited agglutination of goose or guinea pig red bloodcells.

2.9.3. Antibody response using ELISA assay for IgM and IgG againstinfluenza A

Specific immunoglobulin (antibody) against virus (de Vreseet al., 2005) was measured by ELISA according to the manufac-turer’s instructions (IBL America, Minneapolis, MN, USA). Dupli-cated serum samples were tested by ELISA against influenza Aantigen. An influenza A antigen (common epitope recognized bothBeijing265/95 (H1N1) and Sydney 5/97 (H3N2) strains) was coatedon the surface of microtiter strips. A volume of 100 mL of dilutedserum (1:100 for IgM and 1:200 for IgG) or various standardswas pipetted into the wells of the microtiter plate coated withthe antigen and incubated at room temperature for 1 h. Thereafter,each well was washed with diluted washing solution (phosphatebuffer saline (PBS) containing Tween�-20) three times to removethe unbound material. Then, 100 mL of ready-to-use anti-human-IgM peroxidase conjugate or anti-human-IgG peroxidase conjugatewas added in each well and incubated for 30 min. After furtherwashing three times consecutively, 100 mL of substrate (TMB) solu-tion was added and incubated for 5–10 min in the dark. The reac-tion was terminated by adding 100 mL/well of the stop solution(0.5 M sulfuric acid). The absorbance of the product was measuredat 450 nm. The concentrations of the IgM and IgG antibodies wereproportionated to the color intensity. For a quantitative evaluation,the absorptions of the standards [expressed in arbitrary units (U/mL)] are graphically drawn point-to-point against their concentra-tions. Antibody concentration (U/mL) in each sample was calcu-lated from a standard curve plotted between absorbance anddifferent concentrations of the ready-to-use standards of the influ-enza A IgM and IgG antibodies.

4 D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10

2.10. Statistical analyses

All analyses were done by a researcher who was blind to ran-domization. Graphing and statistical analyses were performedusing GraphPad Prism 6.0. Power analysis and sample size calcula-tion were performed using G-power 3.1. A significance level of 5%(p < 0.05) was used for all analyses. Seroresponse rate (percentageof responder showing an increase in HAI titer by a factor of >4),seroconversion rate (percentage of responder going from a pre-vaccination titer of <40 to a post-vaccination titer �40), and sero-protection rate (percentage of responder with a post-vaccinationtiter �40) were calculated. Furthermore, immune response ratesfor �2-fold rising in HAI titers and �4-fold rising in IgM or IgGlevels were also calculated. Comparison of all response ratesbetween groups was analyzed by Fisher’s exact test. Geometricmeans of HAI titers, IgG or IgM were calculated as previously sug-gested (Reverberi, 2008). Ratios between geometric mean of studyand placebo groups were determined in similar fashion as a previ-ous study (Xie et al., 2015). Comparison of antibody titer over timebetween probiotic and placebo groups was analyzed usingrepeated measured two-way ANOVA. Mann-Whitney test was alsoused to compare the difference between groups at each time point.

3. Results

3.1. Participants

As shown in Fig. 1, sixty-six volunteers were screened by aphysician. Sixty-one passed the criteria and were randomlyassigned into study and placebo groups. All subjects had takenthe products completely and returned to follow-up visits. Finaldata analysis was obtained from sixty subjects (thirty each). Asshown in Table 1, all characteristics of participants in study andplacebo groups were matched (p > 0.05). No significant differencesin baseline immune response detected by HAI and ELISA assayswere observed between groups (p > 0.05).

3.2. Compliance of participants to study protocol

As shown in Table 2, data from subject diaries confirmed that allparticipants adhered to the intervention protocol. Most of themdrank the assigned milk every day. One participant each from bothgroups forgot to take the milk for a couple of days. Furthermore,one participant from each group had common cold after vaccina-tion and was prescribed with antibiotics by the physician. Also,one participant from each group accidentally drank a 100 mL bot-tle of other branded fermented milk containing Lactobacillus caseiShirota strain for 1 day. Since these events happened equally inboth study and control groups, the results of both groups can stillbe compared.

3.3. Comparison of immune response rate for H1N1, H3N2 and Flu-Bvirus detected by HAI assay

As shown in Fig. 2, at 4 weeks after vaccination, study group hadsignificantly higher seroconversion rate than that of placebo group(72% vs 55%, p < 0.05 for H1N1; 100% vs 67%, p < 0.0001 for cross-reactive H3N2). However, as shown in Appendices 2 and 3, therewere no differences noted in seroresponse and seroprotection rateof all the viruses between groups. Interestingly, Fig. 3B showedhigher immune response rate (�2-fold rising) to H3N2 than thatof placebo group (97% vs 56%, p < 0.001, Fisher’s exact test). In con-trast, Fig. 3A and C showed no differences in immune response ratefor H1N1 and Flu-B virus between groups (p > 0.05).

3.4. Geometric mean titers for H1N1, H3N2 and Flu-B viruses, IgM andIgG levels

Geometric mean titers in study and placebo group at pre andpost vaccination were compared using ratio. As shown in Table 3,the ratio of geometric mean titers (study group/placebo group)for H1N1 and H3N2 but not Flu-B increased after vaccination. Fur-thermore, the ratio of geometric mean for IgM but not IgGincreased slightly after vaccination.

3.5. Comparison of HAI titers for H1N1, H3N2 and Flu-B virus

As shown in Fig. 4A–C and Appendix 4, at 4 weeks after vaccina-tion both study and placebo groups showed response to vaccina-tion by increasing immune titer. Although study group seems tohave higher increase in immune titer for all viruses, the differenceswere not statistically significant in both analyses (two-way ANOVAand Mann-Whitney test for each time point).

3.6. Comparison of IgM and IgG antibodies for influenza A (H1N1 andH3N2) virus detected by ELISA assay

There were no differences in IgM or IgG immune response(�2-fold rising) rate between groups. As shown inFig. 5A and B, 4 weeks after vaccination both study and placebogroups showed response to vaccination by increasing serum levelsof IgM and IgG, respectively. Although subjects in study group seemto have higher increase in IgM (Fig. 5A), the difference was notstatistically significant. Furthermore, there were no differences inIgG for influenza A between both groups (Fig. 5B). It is worth notingthat the baseline IgG levels in both groups were relatively high(above 200 U/mL).

4. Discussion

Seasonal influenza vaccination is recommended by WorldHealth Organization as a preventive strategy for influenza (WHOCollaborating Centre for Reference & Research on Influenza, 2014,2015). However, the vaccine effectiveness in recent years (2012–2013 and 2014–2015) was low due to poor response againstH3N2 virus (Brendan et al., 2015; Centers for Disease Control &Prevention, 2013; Pebody et al., 2015). Several attempts are beingproposed to improve the response including selecting a better viralantigen or providing an adjuvant (Center for Disease Control andPrevention (CDC), USA, 2016; Song, Chen, Sakwiwatkula, Lia, &Hua, 2010). Owing to the importance of H3N2 infection burdenand poor response of seasonal flu vaccination, the adjuvant effectof IMULUSTM observed in this pilot trial shed the light of using pro-biotics to enhance response rate of vaccine against H3N2. Post-hocpower analysis for the data of seroconversion rate for H3N2yielded power of 0.98, which was more than the required powerof 0.8. This confirms the adequacy of sample size for the observedeffect. However, this finding warrants further confirmation in alarge-scale study.

Results from previous studies on the efficacy of drinking fer-mented milk containing L. casei 431 were inconclusive. A clinicaltrial in Italy reported its positive effect in raising vaccine-specificIgG antibodies for influenza A when detected by ELISA assay(Rizzardini et al., 2012). In contrast, another study conducted inGermany and Denmark found no effects of the probiotic producton antibody titers after vaccination when detected by HAI assay(Jespersen et al., 2015). In the present study conducted in Thai peo-ple, immune response against viral vaccine was measured by bothHAI and ELISA assays. The result showed a positive effect in sero-conversion rate and geometric mean ratio (study/placebo) of HAI

Assessed for eligibility (n=66)

Excluded (n= 5) ¨ Not meeting inclusion criteria (n= 4) ¨ Declined to participate (n= 1)

Analysed (n=30) Excluded from analysis (n=0)

Allocated to study group (n= 30 ) ¨ Received allocated intervention (n= 30) ¨ Did not receive allocated intervention (n=0 )

Allocated to placebo group (n= 31) ¨ Received allocated intervention (n=31) ¨ Did not receive allocated intervention (n= 0 )

Analysed (n=30) Excluded from analysis (very high titer prior to vaccination) (n=1)

Allocation

Analysis

Follow-Up

Randomized (n= 61)

Enrollment

Follow-up (n = 30) Lost to follow-up (n= 0) Discontinued intervention (n=0)

Follow-up (n=31) Lost to follow-up (n= 0) Discontinued intervention (n= 0)

Fig. 1. Participant flow diagram. The diagram depicts number of recruited volunteer and actual number of participants included in data analysis.

Table 1Characteristics of participants in study and placebo groups.

Characteristics Study Group (N = 30) Placebo group (N = 30) P value*

Sex Male 4 (13.3%) 4 (13.3%) 1.00Female 26 (86.7%) 26 (86.7%)

Education Undergraduate 13 (43.3%) 13 (43.3%) 1.00Graduate 17 (56.7%) 17 (56.7%)

Age (years) Mean ± S.D. 29.1 ± 6.6 28.75 ± 6.5 0.847Height (cm) Mean ± S.D. 160.20 ± 5.60 161.57 ± 8.53 0.625Weight (kg) Mean ± S.D. 58.49 ± 12.64 59.72 ± 11.57 0.530BMI (kg/m2) Mean ± S.D. 22.68 ± 4.07 22.82 ± 3.86 0.859Fat percentage Mean ± S.D. 28.88 ± 7.52 28.88 ± 7.19 0.994Muscle mass Mean ± S.D. 38.68 ± 6.69 39.74 ± 7.39 0.503Systolic blood pressure (mmHg) Mean ± S.D. 114.9 ± 10.6 117.9 ± 12.8 0.273Diastolic blood pressure (mmHg) Mean ± S.D. 78.8 ± 8.3 78.1 ± 6.4 0.762Pulse (pulse/min) Mean ± S.D. 76 ± 11 74 ± 6 0.271White blood cell count (K/lL) Mean ± S.D. 6.0 ± 1.3 6.2 ± 1.3 0.912Lymphocyte (%) Mean ± S.D. 35.0 ± 6.6 33.1 ± 8.39 0.210Monocyte (%) Mean ± S.D. 6.1 ± 1.9 5.5 ± 1.7 0.151Eosinophil (%) Mean ± S.D. 2.4 ± 1.4 2.6 ± 2.0 0.890Basophil (%) Mean ± S.D. 0.5 ± 0.5 0.4 ± 0.5 0.436Baseline HAI response H1N1 Median ± IQR 0 ± 0 0 ± 0 0.494H3N2 Median ± IQR 40 ± 60 40 ± 60 0.678PhuB Median ± IQR 40 ± 160 40 ± 80 0.343Baseline IgM response Median ± IQR 14.25 ± 12.69 10.75 ± 17.88 0.086Baseline IgG response Median ± IQR 233.5 ± 81.4 245.8 ± 93.2 0.763

S.D. = standard deviation; IQR = interquartile range.* p-values for sex and education were from Fisher’s exact, while others were from Mann-Whitney test.

D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10 5

titer and IgM against H1N1 and H3N2 viral antigens. The variationin results from different studies may be due to individual pre-

vaccinated immune status. Study group seems to have greaterincrement in HAI titer, IgG and IgM against all influenza viruses

Table 2Compliance of participants to study protocol.

Factors Study group(n = 30)

Placebo(n = 30)

Intervention� Complete (6 weeks) 29 29� Forget to take it for 1–2 days 1 1

Medications during study� No 29 29� Antibiotics for treatment of commoncold

1 1

Intake of other probiotic products� No 29 29� Drink a 100 mL bottle of other brandedfermented milkcontaining Lactobacillus casei Shirotastrain for 1 day

1 1

Fig. 2. The effect of drinking fermented milk containing IMMULUSTM on seroconversionbetween study and placebo groups: each stacked bar represents the percentage of partvaccination titer of <40 to a post-vaccination titer �40, at 4 weeks after vaccination com

6 D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10

than placebo group. However, the results were not significant dueto large standard deviation within group by individual variation.Furthermore, our study demonstrated that the seroconversionrates of both H1N1 and H3N2 in study group were significantlygreater than that of placebo group. However, the seroresponseand seroprotection rates were statistically insignificantly different.Seroconversion rate is defined as the percentage of participantswhose HAI titer increases from pre-vaccinated titer of <40 topost-vaccinated titer of �40 (Xie et al., 2015). This suggested thatprobiotic milk might enhance immune responses effectively in par-ticipants with low pre-vaccinated immune titer (<40). Similarly,the relatively high pre-vaccinated level of IgG in both groups(>200 U/mL) may explain the lack of probiotic effect on IgG in thisstudy. Future research should screen for pre-vaccinated immunetiter prior to recruitment and only include participants with lowimmune titer (<40).

rate after vaccination detected by HAI assay. Comparisons of seroconversion rateicipants who had (black) or did not have (white) increase in HAI titer from a pre-pared to baseline, for H1N1 (a), H3N2 (b) or Flu-B (c) viruses.

Fig. 3. The effect of drinking fermented milk containing IMMULUSTM on immune response rate after vaccination detected by HAI assay. Comparisons of immune response ratebetween study and placebo groups. Each stacked bar represents the percentage of participants who had (black) or did not have (white) �2-fold increase in HAI titer, i.e.response (black) or unchanged of HAI titer, i.e. no response (white), at 4 weeks after vaccination compared to baseline, for H1N1 (a), H3N2 (b) or Flu-B (c) viruses.

D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10 7

Our study showed that the ratios of study/placebo geometricmean titers for H1N1 and H3N2 but not Flu-B increased after vac-cination. These data suggested that study group has better HAIresponse to influenza A after vaccination. Since all kinds ofresponse rate for Flu-B (Phuket strain) were relatively high (80–90%) even in placebo group, this may explain why no positiveeffect of probiotic against Phuket strain was observed in the pre-sent study. Therefore, this study still does not exclude the possibleeffect of probiotic on Influenza type B. Further studies should uti-lize quadrivalent vaccine, which includes two influenza A andtwo Influenza B viruses.

Generally, assays for immune response should be performedusing identical viral strains as vaccinated. Unfortunately, theH3N2 viral strains in seasonal influenza vaccination program in

Thailand keep changing every year. Thus, the availability of identi-cal viral antigen and antibodies for such strains (A/Switzer-land/9715293/2013 (H3N2)-like virus) in Thailand was limited.Therefore, we needed to perform the assay using another viralstrain (A/Victoria/361/2011 (H3N2)-like strain), which wasincluded in the influenza vaccination program of the previous year(2012–2013). Interestingly, some evidences suggest that immuneresponses to the same type of virus (H3N2) can cross-react amongdifferent viral strains and recent vaccination may generate suffi-cient immune protection against early strains of influenza(Levandowski et al., 1991). For example, significant cross reactiveantibodies detected by HI assay were found in two differentH3N2 influenza strains used in 2002 and 2007 vaccines(Mandelboim et al., 2014). In contrast, a recent study found no

0

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100

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HA

I tite

r, fo

ld o

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p=0.0897

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HA

I tite

r, fo

ld o

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p=0.1716

Baseline 2 weekswith drink

4 weeksafter vaccine

2 weeksafter stop

Baseline 2 weekswith drink

4 weeksafter vaccine

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Baseline 2 weekswith drink

4 weeksafter vaccine

2 weeksafter stop

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I tite

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a

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Fig. 4. The effect of drinking fermented milk containing IMMULUSTM on immune titer (fold of baseline) after vaccination detected by HAI assay. Bar graph showed fold of HAItiter (the highest dilution of serum having with immunoreactivity with specific virus) at each time point compared to baseline. Each bar represents mean ± 95% CI of HAI titerfor H1N1 (a), H3N2 (b) or Flu-B (c) viruses in study group (black) or placebo group (white).

Table 3Geometric mean of HAI titer, IgM and IgG at various time points.

Virus Baseline 2 Weeks with drinks 4 Weeks after vaccinated 2 Weeks after drinks stopped

HAI titer for H1N1 Study group 1.53 1.53 50.48 48.20Placebo group 1.94 2.14 41.65 40.70Ratio (study/placebo) 0.79 0.71 1.21 1.18

HAI titer for H3N2 Study group 40.03 35.66 327.48 320.00Placebo group 34.05 31.77 191.06 178.26Ratio (study/placebo) 1.18 1.12 1.71 1.80

HAI titer for Flu-B Study group 22.06 23.14 225.50 238.37Placebo group 15.11 16.16 177.64 185.77Ratio (study/placebo) 1.46 1.43 1.27 1.28

IgM (U/ml) Study group 8.72 8.53 12.20 12.10Placebo group 4.75 4.27 5.81 6.30Ratio (study/placebo) 1.84 2.00 2.10 1.92

IgG (U/ml) Study group 239.35 229.74 260.08 264.05Placebo group 230.74 225.41 257.36 260.57Ratio (study/placebo) 1.04 1.02 1.01 1.01

8 D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10

A

B

-5

0

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Compare IgM change from baseline

Cha

nge

of Ig

M fr

om b

asel

ine

(

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p=0.0847

-20

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Cha

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ine

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p=0.4156

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IgM

(U/m

l)

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Baseline 2 weekswith drink

4 weeksafter vaccine

2 weeksafter stop

Baseline 2 weekswith drink

4 weeksafter vaccine

2 weeksafter stop

Baseline 2 weekswith drink

4 weeksafter vaccine

2 weeksafter stop

Baseline 2 weekswith drink

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2 weeksafter stop

p=0.1705

0

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compare IgG

IgG

(U/m

l)

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p=0.7956

Fig. 5. The effect of drinking fermented milk containing IMMULUSTM on IgM and IgG levels after vaccination detected by ELISA assay. Bar graph shows levels (left panel) orchanges from baseline (right panel) of serum IgM (a) or IgG (b) for influenza A (H1N1 and H3N2) at each time point. Each bar represents geometric mean ± 95% CI (left panel)of the levels or mean ± 95% CI (right panel) of the changes in study group (black) or placebo group (white).

D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10 9

cross-reactions between 2014–2015 vaccine (A/Texas/50/2012(H3N2)-like virus strains) and 2009–2010 vaccine A/Bris-bane/10/2007 (H3N2)-like virus or 2009–2010 vaccine (A/Perth/16/2009-like (H3N2)-like virus) (Xie et al., 2015). However,cross-reactions between H3N2 virus used in 2015–2016 vaccine(Switzerland/2013) and other viruses have never been reportedyet. Based on the present study, after vaccination with 2015–2016 vaccine raised against A/Switzerland/9715293/2013(H3N2)-like virus), the HAI responses against A/Victoria/361/2011(H3N2)-like strain) were detected in both placebo and study group.Since the Victoria strain was used for 2012–2013 vaccination, ourstudy suggested a potential cross-reaction between H3N2 virusesused in 2015–2016 vaccine and that of 2012–2013 vaccine. In thisstudy, we observed the probiotic effect on cross-reactive H3N2.Nevertheless, we still cannot conclude the probiotic effect onimmune response against H3N2 strain Switzerland/2013. Futurestudies using the identical strains of viral vaccine are stillwarranted.

The mechanisms behind the probiotic effects of bacteria are notentirely understood (Butel, 2014). Compelling evidences suggestthat mechanism of a probiotic on immune response could be localto gut community or systemic (Butel, 2014). A recent study in micesuggested that the immunomodulating effect of Lactobacilli sp.against influenza virus involves crosstalk between adaptive andinnate immunity (Jung et al., 2015). Furthermore, effect of Lacto-bacilli on adaptive immune response could be from both humoraland cell-mediated immunity (Wells, 2011). The present study onlymeasured humoral response with HAI and ELISA assays. The resultshowed that drinking fermented milk containing Lactobacilli para-casei 431 (IMULUSTM) improved the seroconversion rate of totalantibodies (by HAI assay) against H1N1 and H3N2 viral antigens.These data suggested significant effect of Lactobacilli paracasei431 on antibody response. Nevertheless, previous in vitro andin vivo studies showed that some Lactobacilli species can activate

regulatory T cells and differentiation of T helper lymphocytes(Th) inducing the production of pro- or anti-inflammatory cytoki-nes (Butel, 2014; Jung et al., 2015; Wells, 2011). Further studiesare warranted to investigate whether the immunomodulatingmechanism of Lactobacilli paracasei 431 on influenza virus alsoinvolves regulatory T cells in human.

In conclusion, the present study showed for the first time that aprobiotic product may enhance response rate to influenza vaccine.Since drinking fermented milk is already marketed and the sensoryproperties are acceptable by general consumers, it could be a prac-tical approach to raise our immune response after influenza vacci-nation, especially for people with low pre-existing immune titer.Additional studies in large scale are warranted to confirm this find-ing and support future recommendation to drink IMULUSTM fer-mented milk prior to and after influenza vaccination.

Potential conflict of interest statement

Dr. Trachootham received a research grant and obtained milkproducts from Dutch Mill Co., Ltd., Thailand. Other conflicts ofinterest: none. No staffs of the company had any role in the designand conduct of the study; the collection, analysis, and interpreta-tion of the data; or the preparation, review, or approval of themanuscript.

Acknowledgement

The study was funded by a research grant from Dutch Mill Co.,Ltd., Thailand to Mahidol University and registered in ClinicalTri-als.gov with identifier of NCT02909842, and approved by MahidolUniversity Central Institutional Review Board (MU-CIRB), with COAno. 2015/150.0112. C.S., D.T. and S.T. conceptualized and designedthe trial and obtained the grant. DT submitted protocol for ethicapproval and clinical trial registry. CC conducted random alloca-

10 D. Trachootham et al. / Journal of Functional Foods 33 (2017) 1–10

tion; LP and CP blindly collected clinical data and biological sam-ples. ST, KB and KP conducted laboratory analysis for HAI and ELISAassays; DT performed statistical analysis of the data; DT and CSwrote the paper and had primary responsibility for final content.All authors read and approved the final manuscript. The authorsthank Ms. Kanoknun Vitayakasemson, Ms. Tassanee Petthai andMs. Kanoknad Kaengamkam for technical assistances. We aregrateful for the statistical advice from Asst. Prof. Nipa Rojroong-wasinkul and professional editing from Dr. Mehraj Ahmad.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jff.2017.03.016.

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