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Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines a
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Nguyen V Trang , Tessa Braeckman , Tinne Lernout , Vu T B Hau , Le T K Anh , Le T Luan , b
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Pierre Van Damme & Dang D Anh a
National Institute of Hygiene and Epidemiology; Hanoi, Vietnam
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Center for the Evaluation of Vaccination; VAXINFECTIO; Faculty of Medicine and Health Sciences; University of Antwerp; Antwerpen, Belgium c
Center for Research and Production of Vaccines and Biologicals; Hanoi, Vietnam Published online: 10 Feb 2015.
Click for updates To cite this article: Nguyen V Trang, Tessa Braeckman, Tinne Lernout, Vu T B Hau, Le T K Anh, Le T Luan, Pierre Van Damme & Dang D Anh (2014) Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines, Human Vaccines & Immunotherapeutics, 10:12, 3681-3687, DOI: 10.4161/21645515.2014.980204 To link to this article: http://dx.doi.org/10.4161/21645515.2014.980204
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RESEARCH PAPER Human Vaccines & Immunotherapeutics 10:12, 3681--3687; December 2014; © 2014 Taylor & Francis Group, LLC
Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines Nguyen V Trang1, Tessa Braeckman2, Tinne Lernout2, Vu T B Hau1, Le T K Anh1, Le T Luan3, Pierre Van Damme2, and Dang D Anh1,* 1
National Institute of Hygiene and Epidemiology; Hanoi, Vietnam; 2Center for the Evaluation of Vaccination; VAXINFECTIO; Faculty of Medicine and Health Sciences; University of Antwerp; Antwerpen, Belgium; 3Center for Research and Production of Vaccines and Biologicals; Hanoi, Vietnam
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Keywords: breast milk, IgA, neutralizing antibody, rotavirus, Vietnam Abbreviations: DTP, diphtheria–tetanus–pertussis; ELISA, Enzyme linked immunosorbent assay; G1-VN, neutralizing antibodies against G1P[8]; G4-VN, neutralizing antibodies against G4P[6]; HCl, Hydrogen chloride; HRP, Horseradish peroxidase; IgA, Immunoglobulin A; IgG, Immunoglobulin G; MEM, Minimal essential medium; NIHE, National Institute of Hygiene and Epidemiology; OD, Optical density; PBS, Phosphate-buffered saline; PFU, Plaque-forming unit; POLYVAC, Center for Production of Vaccines and Biologicals; P-RV, Rotavirus strain P; RRV, Rhesus rotavirus; RV, Rotavirus; SE, Standard error; TMB, 3, 30 , 5, 50 -Tetramethylbenzidine.
Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n D 145) and mothers living in an urban area (n D 147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45– 48%), whereas the majority of the remaining samples had low antibody titers (2–16). Despite these low titers, the breast milk still reduced vaccine strain titers (2 £ 106 plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (P < 0.05) in rural infants over time suggests a continuous exposure to circulating RV. These results contribute to the understanding of the potential interference of breast milk with RV vaccine efficacy and immunogenicity in Vietnamese infants.
Introduction Rotavirus (RV) remains the most common cause of vaccinepreventable severe diarrhea in children under the age of 5 y and is associated with 28% of fatal cases.1 The majority of these deaths occur in developing countries.2 Nearly every child will have experienced at least one episode of RV gastroenteritis by the age of 5 y3 Therefore, the World Health Organization recommends that RV vaccines should be included in all national immunization programs, with the first dose of RV vaccine administered as soon as possible after 6 weeks of age, together with the diphtheria–tetanus–pertussis (DTP) vaccine, to ensure protection prior to natural RV infection.4 The Vietnamese government’s proposal to introduce the RV vaccine into the national immunization program was supported by numerous national surveillance data collected from 1998 to present, which enabled the quantification of RV gastroenteritis disease burden and the evaluation of the cost-effectiveness of the
RV vaccine.5-9 Up to 60% of children hospitalized for acute diarrhea are infected with RV. RV infection seasonality differs between regions of Vietnam: RV peaks in the winter in northern Vietnam but is high year-round in central and southern Vietnam. Surveillance data also identified RV serotypes G1, G2, G3, and G4 as the most prevalent serotypes circulating in Vietnam.6,10-12 Currently, Vietnam is considering the use of the 2 licensed, oral, live attenuated vaccines recommended in infants: RotarixÒ (GlaxoSmithKline, Rixensart, Belgium), which is based on the human RV G1P[8] strain, and RotaTeqÒ (Merck and Company, Whitehouse Station, New Jersey, USA), consisting of 5 humanbovine recombinant strains. In addition, 3 RV vaccine candidates derived from human strains (G1P[8], G1P[4] and G4P[6]) have been developed by the Center for Production of Vaccines and Biologicals (POLYVAC) in Vietnam.13 The Rotavin-M1 vaccine, which is based on the G1P[8] strain, is similar to the monovalent Rotarix vaccine, but the viral strain of the Rotavin-M1 vaccine is different from the Rotarix strain in both viral sequence
*Correspondence to: Dang D Anh; Email: [email protected] Submitted: 02/11/2014; Revised: 08/26/2014; Accepted: 09/08/2014 http://dx.doi.org/10.4161/21645515.2014.980204
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and replication ability compared to the viral strain used in Rotarix. Rotavin-M1 is currently available on the Vietnamese private market. The G4P[6] strain is also of interest as a inactivated vaccine due to its high production efficiency (Le Luan, unpublished data). Moon et al. hypothesized that the neutralizing effect of breast milk could play an important role in reducing the antigen dose, when breast milk is consumed immediately prior to vaccination.14 These authors demonstrated a neutralizing effect of breast milk in vitro against Rotarix, RotaTeq-G1, and the Indian vaccine strain 115E. Breast milk from Indian mothers had the highest RV-neutralizing titers compared to breast milk from Vietnamese, South Korean, and American women. The neutralizing antibody activity against Rotarix and RotaTeq-G1 was also analyzed in a small set of breast milk samples from Vietnam, and a striking similarity with the antibody profile of American women was demonstrated.14 The aim of the current study is to determine whether there is a rural/urban residency effect on the impact of breastfeeding on the performance of the RV vaccine. In particular, we examine whether there is a difference in antibody levels between mothers from urban and rural areas (level of total IgA, RV-specific IgA, and RV-neutralizing antibodies). In addition, the in vitro effect of breast milk with different antibody titers on the Vietnamese RV vaccine strain (Rotavin-M1) was investigated to assess whether and how antibody levels affect the vaccine response.
Results Subjects A total of 292 breast milk samples were collected from mothers aged between 18–45 y (145 from rural Thanh Son, Phu Tho, and 147 from urban Thai Binh city, both in northern Vietnam). Data from the 2010 national survey of household living standard showed that the poverty rates in Phu Tho and Thai Binh were 10.7% and 19.2%, respectively (the poverty cut-off level set by the Vietnamese government was 20 USD for rural and 25 USD for urban areas).15 The living conditions were quite different between the 2 study sites; 14.9% of people in Phu Tho lived in temporary housing, whereas no one in Thai Binh reportedly lived in temporary housing. The illiteracy rate of women between 15– 39 y of age in Phu Tho and Thai Binh was 1.3% and 0.34%, respectively. Mothers living in the urban region (mean age, 30.0 years) were significantly older (P < 0.05) than those living in the rural region (mean age, 25.0 years). At the time of sampling, the age of the infants born to mothers living in the rural region was between 1.1–5.9 months (mean age, 3.5 months), which was comparable to those born to mothers living in the urban region (age range, 1.5–5.9 months, mean age, 3.6 months). Total IgA and anti-RV IgA antibodies in breast milk The total IgA antibody concentration in breast milk samples ranged from 29–1,395 mg/dL (median, 165 mg/dL) in samples from mothers living in the rural area, compared to a range of
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27–432 mg/dL (median, 72 mg/dL) in samples from mothers living in the urban area (Fig. 1A). The total IgA concentration was significantly higher in milk from rural mothers than in milk from urban mothers (P < 0.001). Conversely, samples from urban mothers had significantly higher RV-specific IgA antibody titers (median, 80 mg/dL) than did samples from rural mothers (median, 40) (P < 0.001) (Fig. 1B). This difference was present in breast milk samples for infants aged 2 to less than 4 months and from 4 to less than 6 months (Fig. 2). A high percentage (>90 %) of samples contained anti-RV IgA antibodies (titers 20). The majority of samples (117 samples or 81%) from rural mothers had RV-specific IgA titers in the range of 20–160, whereas the RV-specific IgA titers of samples from urban mothers were in the range of 40–320. A significantly higher proportion of samples from urban mothers (37 samples or 25.2%) had high titers of RV-specific IgA (320 or above) than did samples from rural mothers (14 samples or 9.7%; p 0.05), suggesting a higher degree of exposure to circulating RV in the urban region. There was a significant negative correlation (Spearman’s rho r D ¡0.29; P < 0.001) between the age of the mother and the concentration of total IgA found in breast milk over the total population (rural and urban samples) but neither for rural and urban samples separately nor for RV-specific IgA. Additionally, there was no correlation between the concentration of RV-specific IgA and the moment of lactation (infant age), neither in the rural (r D 0.02; p D 0.84) nor in the urban (r D 0.08; p D 0.34) region.
Neutralizing antibodies in breast milk We also investigated the level of breast milk-neutralizing activity that interfered with RV vaccine strains. Figure 3 shows the distribution of neutralizing antibody titers against RV G1P[8] and G4P[6] vaccine strains (G1-VN and G4-VN, respectively), stratified by region. There was no significant difference between titers in milk samples from the rural and urban areas for either of the strains. Close to one-half of milk samples (65 samples or 45% of rural samples; 71 samples or 48% of urban samples) had no detectable G1-VN antibodies, and the majority of the remaining samples had low G1-VN antibody titers (2–16). In breast milk samples from rural mothers, G1-VN antibodies significantly increased as time since delivery increased (p D 0.03), whereas antibody levels remained unchanged in samples from urban mothers. G4-VN antibodies followed a trend similar to that observed for G1-VN, although G4-VN titers were significantly higher than were G1-VN titers for the same samples in both regions (Z D ¡2.4 and P < 0.05 for rural; Z D ¡3.8 and P < 0.001 for urban). The percentage of samples with undetectable G4-VN levels were comparable for rural (37%, n D 54) and urban samples (46%, n D 68) (p D 0.92). As with G1-VN, lower G4-VN titers (2–16) were more common in both regions, whereas higher G4VN titers (>16) were only found in nine rural samples (6%) and in seven urban samples (5%).
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Neutralization activity of breast milk on Rotavin-M1 Breast milk’s neutralizing activity on the Rotavin-M1 viral strain correlated with RV-specific IgA (r2 D 0.81; P < 0.0001) and G1VN antibody titers (r2 D 0.88; P < 0.0001), but less strongly with G4-VN antibody titers (r2 D 0.64; P D 0.0001) (Fig. 4). At G1-VN titers of only 2 and equal parts breast milk to virus, a 20% to 90% reduction in the vaccine strain occurred in all of the tested samples (n D 6). With G1-VN titers from 2 Figure 1. Cumulative frequency profile of (1A) total IgA antibodies (mg/dL) or (1B) RV-specific IgA titers in to 16 and equal parts breast milk to breast milk of mothers living in a rural (dotted line) or urban (solid line) Vietnamese region. virus, virus titers were reduced more than 90% (n D 7). Virus titers were reduced at least 80%, even with a milk-to-virus ratio of exposure can differ regionally, regional differences in maternal 1:8. With high G1-VN titers of 32 or more, there was a greater antibody levels are expected. In our study, higher titers of total than 95% reduction in the virus titers, even when the milk-to- IgA were observed in the breast milk of rural mothers than in the breast milk of urban mothers, suggesting that there may be virus ratio was 1:32 (n D 7; data not shown). higher exposure to pathogens in rural areas, where living standards and sanitation tend to be poorer. This difference was not reflected in RV-specific IgA antibody titers, as breast milk from Discussion rural mothers had lower RV IgA titers than did that of urban In this study, we explore for the first time in Vietnam the pos- mothers. This finding corroborates the results of a RV vaccine sible regional and socioeconomic affects on total and RV-specific clinical trial in Vietnam, in which a higher proportion of urban antibody levels in breast milk, focusing on the level of neutraliz- infants receiving a placebo vaccine was seroconverted into RV19 ing activity against the Rotavin-M1 vaccine strain. The study specific IgA than did rural infants receiving a placebo vaccine. results, therefore, directly impact the practice of RV vaccination Breast milk from Venezuelan mothers of newborns or infants 4– titers to RRV (G3) and P than and continuous assessment of vaccine efficacy in Vietnam, espe- 10 months old had higher VN 20 mothers in the United States, and a study by Moon et al. demcially since the RV vaccine is scheduled to be implemented onstrated significantly higher titers of RV-specific IgA in breast nationwide in 2015. For instance, breast feeding could be hold milk from mothers in India than in that from American off at the time of vaccination and resume 1h after vaccine delivery 14 women. On the other hand, as in our study, Ray et al. observed to enhance the immune responses in children. Furthermore, this study has added to the scarce literature available on the effects of breast milk on the efficacy of RV vaccines used in different socioeconomic settings. Similar to findings by Moon et al. on milk samples from Vietnam, the milk samples of Vietnamese women in this study mirrored those from industrialized countries.14 The range of total IgA and RV-specific IgA titers found here are similar to those from previous studies in Australia, the United States, and Spain.16-18 Because antibody concentraFigure 2. RV-specific IgA titer distribution in breast milk of mothers living in (2A) rural or (2B) urban Vietnamtions in serum and milk indicate ese region, categorized according to infant age group. Six outliners (titers > 1200) have been removed, one the level of exposure to different in each age group of both rural and urban mothers. pathogens, and because RV
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The present study also found undetectable G1-VN (titers < 2) in 45–48% of samples, with low titers in the remaining samples, corroborating the findings of Moon et al. for a collection of milk samples from different regions in Vietnam.14 However, even with these low antibody titers, the presence of breast milk could still neutralized and reduced vaccine virus by up to 80% or more, even when the milk to virus ratio Figure 3. Distribution profile of neutralizing titers against (3A) the G1P[8] strain of the Rotavin-M1 vaccine was 1:8. These results confirm the and (3B) G4P[6] strain in breast milk of mothers living in a rural or urban Vietnamese region. Four and 5 outfindings of Moon et al.14 and sugliners (titers > 60) were removed for the G1P[8] Rotavin-M1 and G4P[6] groups, respectively. gest that breast milk antibodies have a substantial neutralizing activity against the RV vaccine lower RV-specific IgG antibody titers in cord blood and sera of strains tested. Thus, the common practice breastfeeding at the mothers from a low socioeconomic group than in mothers from clinic just before or after vaccination could easily interfere with a high socioeconomic group, with nutrition as a correlating vari- the immune response and vaccine efficacy. Interestingly, anti-G4P[6] antibody (G4-VN) titers were as able.21 Thus these results again demonstrate differences between socioeconomic regions in breast milk’s antibody level specific to high as anti-G1P[8] antibody (G1-VN) titers in breast milk from RV. In our study, both socioeconomic status and nutrition mothers living in both rural and urban settings. Our findings cor(based on the household survey data in the regions) could play roborate the observations made by Shin et al., in which serum roles: Women living in a rural area (Thanh Son-Phu Tho) might samples of children between 6–12 weeks of age have similar G1have a lower socioeconomic level than do women living in an VN and G4-VN titers, whereas G2-VN and G3-VN titers were urban area (Thai Binh city); additionally, women living in a rural lower.22 The presence of these antibodies in maternal serum or area might have less access to food than women in an urban area breast milk transferred to the baby via the placenta or through due to low income. breastfeeding could potentially interfere with oral RV vaccines comprised of these genotypes. Therefore, despite differences in the amount of total IgA and RVspecific IgA between mothers living in rural and urban areas, there are no differences in the levels of neutralizing antibodies. There are limitations of this study that must be considered. Because we did not collect demographic information directly from the participants, we used data from the national survey of household living standards to reflect the socioeconomic conditions of mothers living in the 2 study sites. The observed age difference between the mothers can be explained by the fact that mothers who live in an urban setting tend to have children at a later age.23 Nevertheless, this difference should be taken into account as a possible confounding Figure 4. Correlation between neutralizing activity of breast milk to the Rotavin-M1 vaccine strain (G1P[8]) variable when interpreting the difA B C and the RV-IgA (4 ), G1 (4 ), and G4-VN (4 ) antibody titers measured in the samples. ferences in total IgA antibody
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concentrations between rural and urban mothers. Furthermore, we could not control for factors related to sample collection, such as the time interval between last feeding and sampling, and method of milk expression. Due to the cross-sectional study design, milk samples from the same mothers were not collected longitudinally, which would have facilitated the study of antibody kinetics. Also, more detailed analysis of the relationship between total IgA or RV-specific IgA and lactation status (colostrum, transitional milk, or mature milk) was not possible due to the small number (n D 19) of children drinking transitional milk (age 0–2 months). Finally, sample collection occured during RV peak season in northern Vietnam, which could cause elevated RV-specific IgA levels in breast milk. However, because both study sites (rural and urban) are located in northern Vietnam, this is likely not a confounding factor for the comparisons performed in this study. Future studies should collect data on a wide range of potentially confounding factors, including nutrional status and socioeconomic information. In conclusion, our study confirmed the neutralizing activity of breast milk antibodies against the G1-based RV vaccine strain in vitro, even at low neutralizing antibody titers and independent of RV-specific IgA antibody titers. Other breast milk factors, such as lactadherin or lactoferrin, could contribute to the reduction in vaccine virus titers, as shown previously.24-27 Strategies to avoid the immediate neutralization of the vaccine antigen by antibodies in breast milk should be considered to improve the immunogenicity and efficacy of live oral RV vaccines for children living in lowand middle-income countries. The existing regional differences in RV exposure between countries and within the same country should be taken into consideration when testing an RV vaccine.
Materials and Methods Study participants and sample collection In January 2010, a cross-sectional study was conducted among 292 breastfeeding women. The women lived in a rural mountainous area in Vietnam (Thanh Son district, Phu Tho province, n D 145) and in urban Thai Binh city, Thai Binh province (n D 147). The study objectives, and the benefits and risks involved were explained to the mothers. If they agreed to participate, written informed consent forms were signed. This study was approved by the Human Ethical Committee of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. The mothers were asked if they were aware of any acute illness, and only those who appeared healthy with no signs of fever were included in the study. Information regarding the ages of the mothers and children was also collected; infants had to be at least 1.5 months of age and less than 6 months of age, according to the age range for which RV immunization is recommended. To ensure that any observed differences between milk samples were related to the region of residence, both groups of mothers were compared, taking age into consideration. Through power analysis, we determined that a sample size of at least 17 for each infants’ age group was necessary to observe a difference in antibody titers of at least 0.5 logs, with a power of 80% and a
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probability of type I error 0.05 (www.statstodo.com). Milk samples (15 to 40 mL) were collected from the mothers at a single time point and were stored at 4 C before and during transport to the National Institute of Hygiene and Epidemiology (NIHE), where the samples were divided into aliquots and stored at ¡20 C before analysis. Virus and cell cultures The G1P[8] and G4P[6] RV strains were isolated from infected children, cloned, and serially propagated through cell cultures as described by Luan et al.13 and were used in this study to produce virus stocks for virus neutralization assays.13 The RRV (donated by Dr. Carl Kirkwood, Melbourne Children Hospital) were used to produce viral antigen stocks for enzyme-linked immunosorbent assay (ELISA). Viral strains were propagated in confluent MA104 cells maintained in Minimum Essential Medium (MEM) (Gibco Invitrogen Corp., catalog number 41500–034) without serum and supplemented with trypsin (concentration of 10–20 mg/mL, depending on the virus strain). Virus titers were determined using a plaque assay, as previously described.14 Treatment of samples Milk samples were centrifuged at 3,000 rpm for 30 minutes at 4 C to remove fat and cell debris, followed by, and heat-treatment at 56 C for 30 minutes to inactivate complements. Total IgA antibody determination The total amount of IgA antibodies in breast milk was measured by ELISA. Microplate wells were coated with 100 mL goat anti-human IgA antibodies (Invitrogen, catalog number 627400) at 5 mg/mL, using an in-house test and IgA standards (Sigma Immunochemicals, catalog number I1010). The samples were diluted to 1:10,000 and 1:40,000 in phosphate-buffered saline (PBS) in duplicates. The concentration of each sample was calculated from a standard curve of purified human IgA, ranging from 4 ng/mL to 1.05 mg/mL. After a 1-hour incubation at 37 C, the samples were washed 5 times with PBS containing 0.1% Tween20, and 100 mL of peroxidase-conjugated anti-human IgA goat antibodies (Kirkegaard & Perry Laboratories, catalog number 14–10–01) were added to each well. After five washes, the reactions were developed using TMB and 0.2% H2O2 in citrate buffer (pH 5.5) and stopped with 1 M hydrogen chloride (HCl). The optical density (OD) at 450 nm was determined using Benchmark Plus (Biorad). The adjusted average concentrations from duplicate samples were reported. ELISA for the detection of RV-specific IgA RV-specific IgA titers in milk were measured using an endpoint ELISA.14 Briefly, 96-well microtiter plates (NUNC, catalog number 456537) were coated with rabbit-anti RRV hyperimmune serum (obtained from Dr. Baoming Jiang, US-CDC). The virus (RRV) and mock-infected supernatant were added to the plates in alternate wells. Despite the circulation of many different RV serotypes, RRV was used as the antigen to perform RV-specific IgA ELISA, because this strain shares the major VP6 capsid
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protein with all group-A RV. Furthermore, an initial comparison using either RRV (genotype G3) or Wa (genotype G1) as the ELISA antigen was performed and resulted in no difference in the RV-IgA antibody titer (data not shown). Milk samples at 2-fold serial dilutions starting at 1:10 were added to the virus/ mock wells. Biotinylated anti-human IgA (Kirkegaard and Perry Laboratory, catalog number 16–10–01) and peroxidase-labeled extravidin (Sigma-Aldrich, Inc., catalog number E2886) were added for the detection of RV-specific IgA antibodies. Positive and negative control sera were tested in the same manner. The antibody titers in the milk samples were calculated as the reciprocal of the highest dilution that gave a mean OD greater than the cut-off value (mean C3 standard deviations of the negative control and mock wells). An RV-specific IgA titer of 20 or higher was considered positive. Neutralizing antibody assays Microneutralizing assays for the detection of neutralizing antibodies to G1P[8] and G4P[6] (G1-VN and G4-VN, respectively) were carried out with the Rotavin-M1 vaccine strain and the G4P[6] cell culture adapted strain developed in Vietnam,13 following published procedures with some modifications.14 Briefly, milk samples were incubated with 4,000 pfu virus for 1 hour, followed by adsorption of the MA104 cell monolayer for 2 hours at 37 C. After incubation, the residual virus-milk mixture was removed, and the cells were washed with medium and incubated for 48–60 hours in MEM with 5 mg/mL trypsin. The cells were fixed with formaldehyde (4% final concentration) at 4 C for 30 minutes, and the plates were dried. The plates were developed using rabbit anti-RV antibodies, HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG (Invitrogen, catalog number 627420), and TMB/H2O2 substrate. Virus-neutralizing antibody titers were defined as the highest dilutions that produced a signal reduction of 80% or more compared to the virus control. Samples were analyzed in duplicate, and the geometric mean was used for further analysis. Breast milk-mediated neutralization activity of Rotavin-M1 Breast milk-mediated neutralization of RV vaccine strain Rotavin-M1 by different antibody titers was performed using the plaque reduction assay.14 Milk samples were serially diluted 2– 4–fold, and 50 mL Rotavin-M1 (100 pfu) were added to 50 mL diluted milk samples and incubated for 1 hour. The virus-sample mixtures were then added to the cell monolayer and incubated with occasional rocking for 1 hour at 37 C. At the end of the incubation period, residual virus/samples were washed and the cell monolayers were overlaid with 0.3% agarose in MEM containing 5 mg/mL trypsin and incubated at 37 C in an References 1. Walker CL, Rudan I, Liu L, Nair H, Theodoratou E, Bhutta ZA, O’Brien KL, Campbell H, Black RE. Global burden of childhood pneumonia and diarrhoea. Lancet 2013; 381:1405-16; PMID:23582727; http:// dx.doi.org/10.1016/S0140-6736(13)60222-6 2. Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, Parashar UD. 2008 estimate of worldwide rotavirus-associated mortality in children younger than
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atmosphere of 5% CO2. RV plaques were stained after 3 d with neutral red (66 mg/L) and counted in duplicate wells for 2 d Reduction in virus titers (compared to the virus-only controls) was recorded. Statistical analysis The results were analyzed using SPSS 20.0 software (IBM). Mothers were categorized into 3 different groups based on the age of the infant, when appropriate (0–2 months, 2–4 months, 4–6 months) and were further analyzed as categorical variables. A Kruskal-Wallis test was used to compare the total IgA and neutralizing antibody titers between groups, which were stratified according to geographical region and categorized according to the infant age group. The distribution of RV-specific IgA across the different regions and different age categories was analyzed using a Mann-Whitney U-test. The comparison of the neutralizing activity of breast milk against G1P[8] and G4P[6] was analyzed using the related samples Wilcoxon signed ranks test. Correlation analyses were performed using the Spearman correlation coefficient. A p value less than 0.05 was considered significant.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We thank Dr. Baoming Jiang for providing technical advice and protocols for the ELISA and neutralization assays. We thank the mothers from Thanh Son district, Phu Tho province, and Thai Binh city, Thai Binh province for participating in our study. We appreciate the support from the medical staff at the Center of Preventive Medicine in Thanh Son, Phu Tho, and Thai Binh city for the recruitment of participants and collection of the samples. We thank Deborah Hong (International Vaccine Institute) and Dr. Lay Mint Yoshida (Nagasaki University) for valuable comments on the manuscript. This study was approved by the Human Ethical Committee of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. Written consent was obtained from the human subjects. Funding
This work was funded by the National Foundation for Science and Technology, grant number, 106.99.182.09, PI Prof. Dr. Dang D. Anh.
5 years before the introduction of universal rotavirus vaccination programmes: a systematic review and metaanalysis. Lancet Infect Dis 2012; 12:136-41; PMID:22030330; http://dx.doi.org/10.1016/S14733099(11)70253-5 3. Parashar UD, Hummelman EG, Bresee JS, Miller MA, Glass RI. Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 2003; 9:565-72;
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PMID:12737740; http://dx.doi.org/10.3201/eid0905. 020562 4. Rotavirus vaccines WHO position paper: January 2013 -recommendations. Vaccine 2013; 31:6170-1; PMID:23746456; http://dx.doi.org/10.1016/j.vaccine. 2013.05.037 5. Anh DD, Thiem VD, Fischer TK, Canh DG, Minh TT, Tho le H, Van Man N, Luan le T, Kilgore P, von Seidlein L, et al. The burden of rotavirus diarrhea in
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Khanh Hoa Province, Vietnam: baseline assessment for a rotavirus vaccine trial. Pediatr Infect Dis J 2006; 25:37-40; PMID:16395100; http://dx.doi.org/ 10.1097/01.inf.0000195635.05186.52 Ngo TC, Nguyen BM, Dang DA, Nguyen HT, Nguyen TT, Tran VN, et al. Molecular epidemiology of rotavirus diarrhoea among children in Haiphong, Vietnam: the emergence of G3 rotavirus. Vaccine 2009; 27(Suppl 5):F75-80; PMID:19931725 Nguyen VM, Nguyen VT, Huynh PL, Dang DT, Nguyen TH, Phan VT, Nguyen TL, Le TL, Ivanoff B, Gentsch JR, et al. The epidemiology and disease burden of rotavirus in Vietnam: sentinel surveillance at 6 hospitals. J Infect Dis 2001; 183:1707-12; PMID:11372022; http://dx.doi.org/10.1086/318084 Fischer TK, Anh DD, Antil L, Cat ND, Kilgore PE, Thiem VD, Rheingans R, Tho le H, Glass RI, Bresee JS. Health care costs of diarrheal disease and estimates of the cost-effectiveness of rotavirus vaccination in Vietnam. J Infect Dis 2005; 192:1720-6; PMID:16235169; http://dx.doi.org/10.1086/497339 Kim SY, Goldie SJ, Salomon JA. Cost-effectiveness of Rotavirus vaccination in Vietnam. BMC Public Health 2009; 9:29; PMID:19159483; http://dx.doi.org/ 10.1186/1471-2458-9-29 Van Man N, Luan le T, Trach DD, Thanh NT, Van Tu P, Long NT, Anh DD, Fischer TK, Ivanoff B, Gentsch JR, et al. Epidemiological profile and burden of rotavirus diarrhea in Vietnam: 5 years of sentinel hospital surveillance, 1998-2003. J Infect Dis 2005; 192 Suppl 1:S127-32; PMID:16088796; http://dx.doi. org/10.1086/431501 Nguyen TA, Yagyu F, Okame M, Phan TG, Trinh QD, Yan H, Hoang KT, Cao AT, Le Hoang P, Okitsu S, et al. Diversity of viruses associated with acute gastroenteritis in children hospitalized with diarrhea in Ho Chi Minh City, Vietnam. J Med Virol 2007; 79:58290; PMID:17385670; http://dx.doi.org/10.1002/ jmv.20857 Trang NV, Vu HT, Le NT, Huang P, Jiang X, Anh DD. Association between Norovirus and Rotavirus Infection and Histo-Blood Group Antigen Types in Vietnamese Children. J Clin Microbiol 2014; PMID:24523471
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13. Luan le T, Trang NV, Phuong NM, Nguyen HT, Ngo HT, Tran HB, Tran HB, Dang HN, Dang AD, Gentsch JR, et al. Development and characterization of candidate rotavirus vaccine strains derived from children with diarrhoea in Vietnam. Vaccine 2009; 27 Suppl 5:F130-8; PMID:19931712 14. Moon SS, Wang Y, Shane AL, Nguyen T, Ray P, Dennehy P, Baek LJ, Parashar U, Glass RI, Jiang B. Inhibitory effect of breast milk on infectivity of live oral rotavirus vaccines. Pediatr Infect Dis J 2010; 29:91923; PMID:20442687; http://dx.doi.org/10.1097/ INF.0b013e3181e232ea 15. GeneralStatisticsOffice. Result of the Vietnam household living standards survey 2010. 2011 16. Bishop RF, Bugg HC, Masendycz PJ, Lund JS, Gorrell RJ, Barnes GL. Serum, fecal, and breast milk rotavirus antibodies as indices of infection in mother-infant pairs. J Infect Dis 1996; 174 Suppl 1:S22-9; PMID:8752287; http://dx.doi.org/10.1093/infdis/ 174.Supplement_1.S22 17. Pickering LK, Morrow AL, Herrera I, O’Ryan M, Estes MK, Guilliams SE, Jackson L, Carter-Campbell S, Matson DO. Effect of maternal rotavirus immunization on milk and serum antibody titers. J Infect Dis 1995; 172:723-8; PMID:7658064; http://dx.doi.org/ 10.1093/infdis/172.3.723 18. Asensi MT, Martinez-Costa C, Buesa J. Anti-rotavirus antibodies in human milk: quantification and neutralizing activity. J Pediatr Gastroenterol Nutr 2006; 42:560-7; PMID:16707981; http://dx.doi.org/ 10.1097/01.mpg.0000221892.59371.b3 19. Trang NV, Anh DD. National development and licensure of a human monovalent rotavirus vaccine (Rotavin-M1) in Vietnam. Proceedings to the Tenth International Rotavirus symposium- Bangkok -Thailand 2012:2 20. Rimer HC, Wasserman SS, Flores J, Pichichero ME, Losonsky GA. Rotavirus-specific breast milk antibody in two populations and possible correlates of interference with rhesus rotavirus vaccine seroconversion. J Infect Dis 1992; 165:826-30; PMID:1373753; http:// dx.doi.org/10.1093/infdis/165.5.826 21. Ray PG, Kelkar SD, Walimbe AM, Biniwale V, Mehendale S. Rotavirus immunoglobulin levels
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among Indian mothers of two socio-economic groups and occurrence of rotavirus infections among their infants up to six months. J Med Virol 2007; 79:341-9; PMID:17245723; http://dx.doi. org/10.1002/jmv.20804 Shin S, Anh DD, Zaman K, Yunus M, Mai le TP, Thiem VD, Azim T, Victor JC, Dallas MJ, Steele AD, et al. Immunogenicity of the pentavalent rotavirus vaccine among infants in two developing countries in Asia, Bangladesh and Vietnam. Vaccine 2012; 30 Suppl 1: A106-13; PMID:22520119; http://dx.doi.org/ 10.1016/j.vaccine.2011.11.091 Tran TK, Nguyen CT, Nguyen HD, Eriksson B, Bondjers G, Gottvall K, Ascher H, Petzold M. Urban rural disparities in antenatal care utilization: a study of two cohorts of pregnant women in Vietnam. BMC Health Serv Res 2011; 11:120; PMID:21605446; http://dx.doi.org/10.1186/1472-6963-11-120 Kvistgaard AS, Pallesen LT, Arias CF, Lopez S, Petersen TE, Heegaard CW, Rasmussen JT. Inhibitory effects of human and bovine milk constituents on rotavirus infections. J Dairy Sci 2004; 87:4088-96; PMID:15545370; http://dx.doi.org/10.3168/jds. S0022-0302(04)73551-1 Newburg DS, Peterson JA, Ruiz-Palacios GM, Matson DO, Morrow AL, Shults J, Guerrero ML, Chaturvedi P, Newburg SO, Scallan CD, et al. Role of humanmilk lactadherin in protection against symptomatic rotavirus infection. Lancet 1998; 351:1160-4; PMID:9643686; http://dx.doi.org/10.1016/S01406736(97)10322-1 Newburg DS. Human milk glycoconjugates that inhibit pathogens. Curr Med Chem 1999; 6:117-27; PMID:9927761 Moon SS, Tate JE, Ray P, Dennehy PH, Archary D, Coutsoudis A, Bland R, Newell ML, Glass RI, Parashar U, et al. Differential Profiles and Inhibitory Effect on Rotavirus Vaccines of Non-Antibody Components in Breast Milk from Mothers in Developing and Developed Countries. Pediatr Infect Dis J 2013; PMID:23584581
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Human Vaccines & Immunotherapeutics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/khvi20
Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines a
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b
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Nguyen V Trang , Tessa Braeckman , Tinne Lernout , Vu T B Hau , Le T K Anh , Le T Luan , b
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Pierre Van Damme & Dang D Anh a
National Institute of Hygiene and Epidemiology; Hanoi, Vietnam
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Center for the Evaluation of Vaccination; VAXINFECTIO; Faculty of Medicine and Health Sciences; University of Antwerp; Antwerpen, Belgium c
Center for Research and Production of Vaccines and Biologicals; Hanoi, Vietnam Published online: 10 Feb 2015.
Click for updates To cite this article: Nguyen V Trang, Tessa Braeckman, Tinne Lernout, Vu T B Hau, Le T K Anh, Le T Luan, Pierre Van Damme & Dang D Anh (2014) Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines, Human Vaccines & Immunotherapeutics, 10:12, 3681-3687, DOI: 10.4161/21645515.2014.980204 To link to this article: http://dx.doi.org/10.4161/21645515.2014.980204
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RESEARCH PAPER Human Vaccines & Immunotherapeutics 10:12, 3681--3687; December 2014; © 2014 Taylor & Francis Group, LLC
Prevalence of rotavirus antibodies in breast milk and inhibitory effects to rotavirus vaccines Nguyen V Trang1, Tessa Braeckman2, Tinne Lernout2, Vu T B Hau1, Le T K Anh1, Le T Luan3, Pierre Van Damme2, and Dang D Anh1,* 1
National Institute of Hygiene and Epidemiology; Hanoi, Vietnam; 2Center for the Evaluation of Vaccination; VAXINFECTIO; Faculty of Medicine and Health Sciences; University of Antwerp; Antwerpen, Belgium; 3Center for Research and Production of Vaccines and Biologicals; Hanoi, Vietnam
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Keywords: breast milk, IgA, neutralizing antibody, rotavirus, Vietnam Abbreviations: DTP, diphtheria–tetanus–pertussis; ELISA, Enzyme linked immunosorbent assay; G1-VN, neutralizing antibodies against G1P[8]; G4-VN, neutralizing antibodies against G4P[6]; HCl, Hydrogen chloride; HRP, Horseradish peroxidase; IgA, Immunoglobulin A; IgG, Immunoglobulin G; MEM, Minimal essential medium; NIHE, National Institute of Hygiene and Epidemiology; OD, Optical density; PBS, Phosphate-buffered saline; PFU, Plaque-forming unit; POLYVAC, Center for Production of Vaccines and Biologicals; P-RV, Rotavirus strain P; RRV, Rhesus rotavirus; RV, Rotavirus; SE, Standard error; TMB, 3, 30 , 5, 50 -Tetramethylbenzidine.
Rotavirus (RV) is the most common cause of childhood diarrhea worldwide, and several vaccines have been successfully developed to reduce the burden of disease. However, lower vaccine immunogenicity and efficacy in developing countries might be related to the virus-neutralizing activity of breast milk. We examined possible differences in breast milk antibody levels (total IgA antibody, RV-specific antibodies, and RV-neutralizing antibodies) between healthy mothers living in a rural area (n D 145) and mothers living in an urban area (n D 147) of Vietnam. Total IgA concentration was significantly higher in samples from mothers in the rural region than in samples from mothers in the urban region, whereas urban mothers had significantly higher RV-specific IgA antibody titers than did rural mothers. Neutralizing antibodies against RV strain G1P[8] were undetected in nearly one-half of the breast milk samples (45– 48%), whereas the majority of the remaining samples had low antibody titers (2–16). Despite these low titers, the breast milk still reduced vaccine strain titers (2 £ 106 plaque forming units/mL) up to 80% or more, even at a milk-to-virus ratio of 1:8. An increase in neutralizing anti-G1P[8] antibody titers (P < 0.05) in rural infants over time suggests a continuous exposure to circulating RV. These results contribute to the understanding of the potential interference of breast milk with RV vaccine efficacy and immunogenicity in Vietnamese infants.
Introduction Rotavirus (RV) remains the most common cause of vaccinepreventable severe diarrhea in children under the age of 5 y and is associated with 28% of fatal cases.1 The majority of these deaths occur in developing countries.2 Nearly every child will have experienced at least one episode of RV gastroenteritis by the age of 5 y3 Therefore, the World Health Organization recommends that RV vaccines should be included in all national immunization programs, with the first dose of RV vaccine administered as soon as possible after 6 weeks of age, together with the diphtheria–tetanus–pertussis (DTP) vaccine, to ensure protection prior to natural RV infection.4 The Vietnamese government’s proposal to introduce the RV vaccine into the national immunization program was supported by numerous national surveillance data collected from 1998 to present, which enabled the quantification of RV gastroenteritis disease burden and the evaluation of the cost-effectiveness of the
RV vaccine.5-9 Up to 60% of children hospitalized for acute diarrhea are infected with RV. RV infection seasonality differs between regions of Vietnam: RV peaks in the winter in northern Vietnam but is high year-round in central and southern Vietnam. Surveillance data also identified RV serotypes G1, G2, G3, and G4 as the most prevalent serotypes circulating in Vietnam.6,10-12 Currently, Vietnam is considering the use of the 2 licensed, oral, live attenuated vaccines recommended in infants: RotarixÒ (GlaxoSmithKline, Rixensart, Belgium), which is based on the human RV G1P[8] strain, and RotaTeqÒ (Merck and Company, Whitehouse Station, New Jersey, USA), consisting of 5 humanbovine recombinant strains. In addition, 3 RV vaccine candidates derived from human strains (G1P[8], G1P[4] and G4P[6]) have been developed by the Center for Production of Vaccines and Biologicals (POLYVAC) in Vietnam.13 The Rotavin-M1 vaccine, which is based on the G1P[8] strain, is similar to the monovalent Rotarix vaccine, but the viral strain of the Rotavin-M1 vaccine is different from the Rotarix strain in both viral sequence
*Correspondence to: Dang D Anh; Email: [email protected] Submitted: 02/11/2014; Revised: 08/26/2014; Accepted: 09/08/2014 http://dx.doi.org/10.4161/21645515.2014.980204
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and replication ability compared to the viral strain used in Rotarix. Rotavin-M1 is currently available on the Vietnamese private market. The G4P[6] strain is also of interest as a inactivated vaccine due to its high production efficiency (Le Luan, unpublished data). Moon et al. hypothesized that the neutralizing effect of breast milk could play an important role in reducing the antigen dose, when breast milk is consumed immediately prior to vaccination.14 These authors demonstrated a neutralizing effect of breast milk in vitro against Rotarix, RotaTeq-G1, and the Indian vaccine strain 115E. Breast milk from Indian mothers had the highest RV-neutralizing titers compared to breast milk from Vietnamese, South Korean, and American women. The neutralizing antibody activity against Rotarix and RotaTeq-G1 was also analyzed in a small set of breast milk samples from Vietnam, and a striking similarity with the antibody profile of American women was demonstrated.14 The aim of the current study is to determine whether there is a rural/urban residency effect on the impact of breastfeeding on the performance of the RV vaccine. In particular, we examine whether there is a difference in antibody levels between mothers from urban and rural areas (level of total IgA, RV-specific IgA, and RV-neutralizing antibodies). In addition, the in vitro effect of breast milk with different antibody titers on the Vietnamese RV vaccine strain (Rotavin-M1) was investigated to assess whether and how antibody levels affect the vaccine response.
Results Subjects A total of 292 breast milk samples were collected from mothers aged between 18–45 y (145 from rural Thanh Son, Phu Tho, and 147 from urban Thai Binh city, both in northern Vietnam). Data from the 2010 national survey of household living standard showed that the poverty rates in Phu Tho and Thai Binh were 10.7% and 19.2%, respectively (the poverty cut-off level set by the Vietnamese government was 20 USD for rural and 25 USD for urban areas).15 The living conditions were quite different between the 2 study sites; 14.9% of people in Phu Tho lived in temporary housing, whereas no one in Thai Binh reportedly lived in temporary housing. The illiteracy rate of women between 15– 39 y of age in Phu Tho and Thai Binh was 1.3% and 0.34%, respectively. Mothers living in the urban region (mean age, 30.0 years) were significantly older (P < 0.05) than those living in the rural region (mean age, 25.0 years). At the time of sampling, the age of the infants born to mothers living in the rural region was between 1.1–5.9 months (mean age, 3.5 months), which was comparable to those born to mothers living in the urban region (age range, 1.5–5.9 months, mean age, 3.6 months). Total IgA and anti-RV IgA antibodies in breast milk The total IgA antibody concentration in breast milk samples ranged from 29–1,395 mg/dL (median, 165 mg/dL) in samples from mothers living in the rural area, compared to a range of
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27–432 mg/dL (median, 72 mg/dL) in samples from mothers living in the urban area (Fig. 1A). The total IgA concentration was significantly higher in milk from rural mothers than in milk from urban mothers (P < 0.001). Conversely, samples from urban mothers had significantly higher RV-specific IgA antibody titers (median, 80 mg/dL) than did samples from rural mothers (median, 40) (P < 0.001) (Fig. 1B). This difference was present in breast milk samples for infants aged 2 to less than 4 months and from 4 to less than 6 months (Fig. 2). A high percentage (>90 %) of samples contained anti-RV IgA antibodies (titers 20). The majority of samples (117 samples or 81%) from rural mothers had RV-specific IgA titers in the range of 20–160, whereas the RV-specific IgA titers of samples from urban mothers were in the range of 40–320. A significantly higher proportion of samples from urban mothers (37 samples or 25.2%) had high titers of RV-specific IgA (320 or above) than did samples from rural mothers (14 samples or 9.7%; p 0.05), suggesting a higher degree of exposure to circulating RV in the urban region. There was a significant negative correlation (Spearman’s rho r D ¡0.29; P < 0.001) between the age of the mother and the concentration of total IgA found in breast milk over the total population (rural and urban samples) but neither for rural and urban samples separately nor for RV-specific IgA. Additionally, there was no correlation between the concentration of RV-specific IgA and the moment of lactation (infant age), neither in the rural (r D 0.02; p D 0.84) nor in the urban (r D 0.08; p D 0.34) region.
Neutralizing antibodies in breast milk We also investigated the level of breast milk-neutralizing activity that interfered with RV vaccine strains. Figure 3 shows the distribution of neutralizing antibody titers against RV G1P[8] and G4P[6] vaccine strains (G1-VN and G4-VN, respectively), stratified by region. There was no significant difference between titers in milk samples from the rural and urban areas for either of the strains. Close to one-half of milk samples (65 samples or 45% of rural samples; 71 samples or 48% of urban samples) had no detectable G1-VN antibodies, and the majority of the remaining samples had low G1-VN antibody titers (2–16). In breast milk samples from rural mothers, G1-VN antibodies significantly increased as time since delivery increased (p D 0.03), whereas antibody levels remained unchanged in samples from urban mothers. G4-VN antibodies followed a trend similar to that observed for G1-VN, although G4-VN titers were significantly higher than were G1-VN titers for the same samples in both regions (Z D ¡2.4 and P < 0.05 for rural; Z D ¡3.8 and P < 0.001 for urban). The percentage of samples with undetectable G4-VN levels were comparable for rural (37%, n D 54) and urban samples (46%, n D 68) (p D 0.92). As with G1-VN, lower G4-VN titers (2–16) were more common in both regions, whereas higher G4VN titers (>16) were only found in nine rural samples (6%) and in seven urban samples (5%).
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Neutralization activity of breast milk on Rotavin-M1 Breast milk’s neutralizing activity on the Rotavin-M1 viral strain correlated with RV-specific IgA (r2 D 0.81; P < 0.0001) and G1VN antibody titers (r2 D 0.88; P < 0.0001), but less strongly with G4-VN antibody titers (r2 D 0.64; P D 0.0001) (Fig. 4). At G1-VN titers of only 2 and equal parts breast milk to virus, a 20% to 90% reduction in the vaccine strain occurred in all of the tested samples (n D 6). With G1-VN titers from 2 Figure 1. Cumulative frequency profile of (1A) total IgA antibodies (mg/dL) or (1B) RV-specific IgA titers in to 16 and equal parts breast milk to breast milk of mothers living in a rural (dotted line) or urban (solid line) Vietnamese region. virus, virus titers were reduced more than 90% (n D 7). Virus titers were reduced at least 80%, even with a milk-to-virus ratio of exposure can differ regionally, regional differences in maternal 1:8. With high G1-VN titers of 32 or more, there was a greater antibody levels are expected. In our study, higher titers of total than 95% reduction in the virus titers, even when the milk-to- IgA were observed in the breast milk of rural mothers than in the breast milk of urban mothers, suggesting that there may be virus ratio was 1:32 (n D 7; data not shown). higher exposure to pathogens in rural areas, where living standards and sanitation tend to be poorer. This difference was not reflected in RV-specific IgA antibody titers, as breast milk from Discussion rural mothers had lower RV IgA titers than did that of urban In this study, we explore for the first time in Vietnam the pos- mothers. This finding corroborates the results of a RV vaccine sible regional and socioeconomic affects on total and RV-specific clinical trial in Vietnam, in which a higher proportion of urban antibody levels in breast milk, focusing on the level of neutraliz- infants receiving a placebo vaccine was seroconverted into RV19 ing activity against the Rotavin-M1 vaccine strain. The study specific IgA than did rural infants receiving a placebo vaccine. results, therefore, directly impact the practice of RV vaccination Breast milk from Venezuelan mothers of newborns or infants 4– titers to RRV (G3) and P than and continuous assessment of vaccine efficacy in Vietnam, espe- 10 months old had higher VN 20 mothers in the United States, and a study by Moon et al. demcially since the RV vaccine is scheduled to be implemented onstrated significantly higher titers of RV-specific IgA in breast nationwide in 2015. For instance, breast feeding could be hold milk from mothers in India than in that from American off at the time of vaccination and resume 1h after vaccine delivery 14 women. On the other hand, as in our study, Ray et al. observed to enhance the immune responses in children. Furthermore, this study has added to the scarce literature available on the effects of breast milk on the efficacy of RV vaccines used in different socioeconomic settings. Similar to findings by Moon et al. on milk samples from Vietnam, the milk samples of Vietnamese women in this study mirrored those from industrialized countries.14 The range of total IgA and RV-specific IgA titers found here are similar to those from previous studies in Australia, the United States, and Spain.16-18 Because antibody concentraFigure 2. RV-specific IgA titer distribution in breast milk of mothers living in (2A) rural or (2B) urban Vietnamtions in serum and milk indicate ese region, categorized according to infant age group. Six outliners (titers > 1200) have been removed, one the level of exposure to different in each age group of both rural and urban mothers. pathogens, and because RV
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The present study also found undetectable G1-VN (titers < 2) in 45–48% of samples, with low titers in the remaining samples, corroborating the findings of Moon et al. for a collection of milk samples from different regions in Vietnam.14 However, even with these low antibody titers, the presence of breast milk could still neutralized and reduced vaccine virus by up to 80% or more, even when the milk to virus ratio Figure 3. Distribution profile of neutralizing titers against (3A) the G1P[8] strain of the Rotavin-M1 vaccine was 1:8. These results confirm the and (3B) G4P[6] strain in breast milk of mothers living in a rural or urban Vietnamese region. Four and 5 outfindings of Moon et al.14 and sugliners (titers > 60) were removed for the G1P[8] Rotavin-M1 and G4P[6] groups, respectively. gest that breast milk antibodies have a substantial neutralizing activity against the RV vaccine lower RV-specific IgG antibody titers in cord blood and sera of strains tested. Thus, the common practice breastfeeding at the mothers from a low socioeconomic group than in mothers from clinic just before or after vaccination could easily interfere with a high socioeconomic group, with nutrition as a correlating vari- the immune response and vaccine efficacy. Interestingly, anti-G4P[6] antibody (G4-VN) titers were as able.21 Thus these results again demonstrate differences between socioeconomic regions in breast milk’s antibody level specific to high as anti-G1P[8] antibody (G1-VN) titers in breast milk from RV. In our study, both socioeconomic status and nutrition mothers living in both rural and urban settings. Our findings cor(based on the household survey data in the regions) could play roborate the observations made by Shin et al., in which serum roles: Women living in a rural area (Thanh Son-Phu Tho) might samples of children between 6–12 weeks of age have similar G1have a lower socioeconomic level than do women living in an VN and G4-VN titers, whereas G2-VN and G3-VN titers were urban area (Thai Binh city); additionally, women living in a rural lower.22 The presence of these antibodies in maternal serum or area might have less access to food than women in an urban area breast milk transferred to the baby via the placenta or through due to low income. breastfeeding could potentially interfere with oral RV vaccines comprised of these genotypes. Therefore, despite differences in the amount of total IgA and RVspecific IgA between mothers living in rural and urban areas, there are no differences in the levels of neutralizing antibodies. There are limitations of this study that must be considered. Because we did not collect demographic information directly from the participants, we used data from the national survey of household living standards to reflect the socioeconomic conditions of mothers living in the 2 study sites. The observed age difference between the mothers can be explained by the fact that mothers who live in an urban setting tend to have children at a later age.23 Nevertheless, this difference should be taken into account as a possible confounding Figure 4. Correlation between neutralizing activity of breast milk to the Rotavin-M1 vaccine strain (G1P[8]) variable when interpreting the difA B C and the RV-IgA (4 ), G1 (4 ), and G4-VN (4 ) antibody titers measured in the samples. ferences in total IgA antibody
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concentrations between rural and urban mothers. Furthermore, we could not control for factors related to sample collection, such as the time interval between last feeding and sampling, and method of milk expression. Due to the cross-sectional study design, milk samples from the same mothers were not collected longitudinally, which would have facilitated the study of antibody kinetics. Also, more detailed analysis of the relationship between total IgA or RV-specific IgA and lactation status (colostrum, transitional milk, or mature milk) was not possible due to the small number (n D 19) of children drinking transitional milk (age 0–2 months). Finally, sample collection occured during RV peak season in northern Vietnam, which could cause elevated RV-specific IgA levels in breast milk. However, because both study sites (rural and urban) are located in northern Vietnam, this is likely not a confounding factor for the comparisons performed in this study. Future studies should collect data on a wide range of potentially confounding factors, including nutrional status and socioeconomic information. In conclusion, our study confirmed the neutralizing activity of breast milk antibodies against the G1-based RV vaccine strain in vitro, even at low neutralizing antibody titers and independent of RV-specific IgA antibody titers. Other breast milk factors, such as lactadherin or lactoferrin, could contribute to the reduction in vaccine virus titers, as shown previously.24-27 Strategies to avoid the immediate neutralization of the vaccine antigen by antibodies in breast milk should be considered to improve the immunogenicity and efficacy of live oral RV vaccines for children living in lowand middle-income countries. The existing regional differences in RV exposure between countries and within the same country should be taken into consideration when testing an RV vaccine.
Materials and Methods Study participants and sample collection In January 2010, a cross-sectional study was conducted among 292 breastfeeding women. The women lived in a rural mountainous area in Vietnam (Thanh Son district, Phu Tho province, n D 145) and in urban Thai Binh city, Thai Binh province (n D 147). The study objectives, and the benefits and risks involved were explained to the mothers. If they agreed to participate, written informed consent forms were signed. This study was approved by the Human Ethical Committee of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. The mothers were asked if they were aware of any acute illness, and only those who appeared healthy with no signs of fever were included in the study. Information regarding the ages of the mothers and children was also collected; infants had to be at least 1.5 months of age and less than 6 months of age, according to the age range for which RV immunization is recommended. To ensure that any observed differences between milk samples were related to the region of residence, both groups of mothers were compared, taking age into consideration. Through power analysis, we determined that a sample size of at least 17 for each infants’ age group was necessary to observe a difference in antibody titers of at least 0.5 logs, with a power of 80% and a
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probability of type I error 0.05 (www.statstodo.com). Milk samples (15 to 40 mL) were collected from the mothers at a single time point and were stored at 4 C before and during transport to the National Institute of Hygiene and Epidemiology (NIHE), where the samples were divided into aliquots and stored at ¡20 C before analysis. Virus and cell cultures The G1P[8] and G4P[6] RV strains were isolated from infected children, cloned, and serially propagated through cell cultures as described by Luan et al.13 and were used in this study to produce virus stocks for virus neutralization assays.13 The RRV (donated by Dr. Carl Kirkwood, Melbourne Children Hospital) were used to produce viral antigen stocks for enzyme-linked immunosorbent assay (ELISA). Viral strains were propagated in confluent MA104 cells maintained in Minimum Essential Medium (MEM) (Gibco Invitrogen Corp., catalog number 41500–034) without serum and supplemented with trypsin (concentration of 10–20 mg/mL, depending on the virus strain). Virus titers were determined using a plaque assay, as previously described.14 Treatment of samples Milk samples were centrifuged at 3,000 rpm for 30 minutes at 4 C to remove fat and cell debris, followed by, and heat-treatment at 56 C for 30 minutes to inactivate complements. Total IgA antibody determination The total amount of IgA antibodies in breast milk was measured by ELISA. Microplate wells were coated with 100 mL goat anti-human IgA antibodies (Invitrogen, catalog number 627400) at 5 mg/mL, using an in-house test and IgA standards (Sigma Immunochemicals, catalog number I1010). The samples were diluted to 1:10,000 and 1:40,000 in phosphate-buffered saline (PBS) in duplicates. The concentration of each sample was calculated from a standard curve of purified human IgA, ranging from 4 ng/mL to 1.05 mg/mL. After a 1-hour incubation at 37 C, the samples were washed 5 times with PBS containing 0.1% Tween20, and 100 mL of peroxidase-conjugated anti-human IgA goat antibodies (Kirkegaard & Perry Laboratories, catalog number 14–10–01) were added to each well. After five washes, the reactions were developed using TMB and 0.2% H2O2 in citrate buffer (pH 5.5) and stopped with 1 M hydrogen chloride (HCl). The optical density (OD) at 450 nm was determined using Benchmark Plus (Biorad). The adjusted average concentrations from duplicate samples were reported. ELISA for the detection of RV-specific IgA RV-specific IgA titers in milk were measured using an endpoint ELISA.14 Briefly, 96-well microtiter plates (NUNC, catalog number 456537) were coated with rabbit-anti RRV hyperimmune serum (obtained from Dr. Baoming Jiang, US-CDC). The virus (RRV) and mock-infected supernatant were added to the plates in alternate wells. Despite the circulation of many different RV serotypes, RRV was used as the antigen to perform RV-specific IgA ELISA, because this strain shares the major VP6 capsid
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protein with all group-A RV. Furthermore, an initial comparison using either RRV (genotype G3) or Wa (genotype G1) as the ELISA antigen was performed and resulted in no difference in the RV-IgA antibody titer (data not shown). Milk samples at 2-fold serial dilutions starting at 1:10 were added to the virus/ mock wells. Biotinylated anti-human IgA (Kirkegaard and Perry Laboratory, catalog number 16–10–01) and peroxidase-labeled extravidin (Sigma-Aldrich, Inc., catalog number E2886) were added for the detection of RV-specific IgA antibodies. Positive and negative control sera were tested in the same manner. The antibody titers in the milk samples were calculated as the reciprocal of the highest dilution that gave a mean OD greater than the cut-off value (mean C3 standard deviations of the negative control and mock wells). An RV-specific IgA titer of 20 or higher was considered positive. Neutralizing antibody assays Microneutralizing assays for the detection of neutralizing antibodies to G1P[8] and G4P[6] (G1-VN and G4-VN, respectively) were carried out with the Rotavin-M1 vaccine strain and the G4P[6] cell culture adapted strain developed in Vietnam,13 following published procedures with some modifications.14 Briefly, milk samples were incubated with 4,000 pfu virus for 1 hour, followed by adsorption of the MA104 cell monolayer for 2 hours at 37 C. After incubation, the residual virus-milk mixture was removed, and the cells were washed with medium and incubated for 48–60 hours in MEM with 5 mg/mL trypsin. The cells were fixed with formaldehyde (4% final concentration) at 4 C for 30 minutes, and the plates were dried. The plates were developed using rabbit anti-RV antibodies, HRP (horseradish peroxidase)-labeled goat anti-rabbit IgG (Invitrogen, catalog number 627420), and TMB/H2O2 substrate. Virus-neutralizing antibody titers were defined as the highest dilutions that produced a signal reduction of 80% or more compared to the virus control. Samples were analyzed in duplicate, and the geometric mean was used for further analysis. Breast milk-mediated neutralization activity of Rotavin-M1 Breast milk-mediated neutralization of RV vaccine strain Rotavin-M1 by different antibody titers was performed using the plaque reduction assay.14 Milk samples were serially diluted 2– 4–fold, and 50 mL Rotavin-M1 (100 pfu) were added to 50 mL diluted milk samples and incubated for 1 hour. The virus-sample mixtures were then added to the cell monolayer and incubated with occasional rocking for 1 hour at 37 C. At the end of the incubation period, residual virus/samples were washed and the cell monolayers were overlaid with 0.3% agarose in MEM containing 5 mg/mL trypsin and incubated at 37 C in an References 1. Walker CL, Rudan I, Liu L, Nair H, Theodoratou E, Bhutta ZA, O’Brien KL, Campbell H, Black RE. Global burden of childhood pneumonia and diarrhoea. Lancet 2013; 381:1405-16; PMID:23582727; http:// dx.doi.org/10.1016/S0140-6736(13)60222-6 2. Tate JE, Burton AH, Boschi-Pinto C, Steele AD, Duque J, Parashar UD. 2008 estimate of worldwide rotavirus-associated mortality in children younger than
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atmosphere of 5% CO2. RV plaques were stained after 3 d with neutral red (66 mg/L) and counted in duplicate wells for 2 d Reduction in virus titers (compared to the virus-only controls) was recorded. Statistical analysis The results were analyzed using SPSS 20.0 software (IBM). Mothers were categorized into 3 different groups based on the age of the infant, when appropriate (0–2 months, 2–4 months, 4–6 months) and were further analyzed as categorical variables. A Kruskal-Wallis test was used to compare the total IgA and neutralizing antibody titers between groups, which were stratified according to geographical region and categorized according to the infant age group. The distribution of RV-specific IgA across the different regions and different age categories was analyzed using a Mann-Whitney U-test. The comparison of the neutralizing activity of breast milk against G1P[8] and G4P[6] was analyzed using the related samples Wilcoxon signed ranks test. Correlation analyses were performed using the Spearman correlation coefficient. A p value less than 0.05 was considered significant.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed. Acknowledgments
We thank Dr. Baoming Jiang for providing technical advice and protocols for the ELISA and neutralization assays. We thank the mothers from Thanh Son district, Phu Tho province, and Thai Binh city, Thai Binh province for participating in our study. We appreciate the support from the medical staff at the Center of Preventive Medicine in Thanh Son, Phu Tho, and Thai Binh city for the recruitment of participants and collection of the samples. We thank Deborah Hong (International Vaccine Institute) and Dr. Lay Mint Yoshida (Nagasaki University) for valuable comments on the manuscript. This study was approved by the Human Ethical Committee of the National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. Written consent was obtained from the human subjects. Funding
This work was funded by the National Foundation for Science and Technology, grant number, 106.99.182.09, PI Prof. Dr. Dang D. Anh.
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