Antibody Avidity in Humoral Immune Responses in Bangladeshi Children and Adults Following Administration of an Oral Killed Cholera Vaccine

Home , Avidity

Antibody Avidity in Humoral Immune Responses in Bangladeshi Children and Adults following Administration of an Oral Killed Cholera Vaccine

The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters

Citation Alam, Mohammad Murshid, Daniel T. Leung, Marjahan Akhtar, Mohammad Nazim, Sarmin Akter, Taher Uddin, Farhana Khanam, et al. 2013. “Antibody Avidity in Humoral Immune Responses in Bangladeshi Children and Adults Following Administration of an Oral Killed Cholera Vaccine.” Clinical and Vaccine Immunology 20 (10): 1541–48. https://doi.org/10.1128/CVI.00341-13.

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41542638

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Antibody Avidity in Humoral Immune Responses in Bangladeshi Children and Adults following Administration of an Oral Killed Cholera Vaccine

Mohammad Murshid Alam,a Daniel T. Leung,a,b,c Marjahan Akhtar,a Mohammad Nazim,a Sarmin Akter,a Taher Uddin,a Farhana Khanam,a Deena Al Mahbuba,a Shaikh Meshbahuddin Ahmad,a Tauﬁqur Rahman Bhuiyan,a Stephen B. Calderwood,b,c,d Edward T. Ryan,b,c,e Firdausi Qadria Centre for Vaccine Sciences, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, Bangladesha; Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts, USAb; Departments of Medicinec and Microbiology and Immunobiology,d Harvard Medical School, Boston, Massachusetts, USA; e

Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA Downloaded from

Antibody avidity for antigens following disease or vaccination increases with affinity maturation and somatic hypermutation. In this study, we followed children and adults in Bangladesh for 1 year following oral cholera vaccination and measured the avidity of antibodies to the T cell-dependent antigen cholera toxin B subunit (CTB) and the T cell-independent antigen lipopolysaccharide (LPS) in comparison with responses in other immunological measurements. Children produced CTB-specific IgG and IgA antibodies of high avidity following vaccination, which persisted for several months; the magnitudes of responses were comparable to those seen in adult vaccinees. The avidity of LPS-specific IgG and IgA antibodies in vaccinees increased significantly shortly after the second dose of vaccine but waned rapidly to baseline levels thereafter. CTB-specific memory B cells were present for only a short time following vaccination, and we did not find significant memory B cell responses to LPS in any age group. For http://cvi.asm.org/ older children, there was a significant correlation between CTB-specific memory T cell responses after the second dose of vaccine and CTB-specific IgG antibody avidity indices over the subsequent year. These findings suggest that vaccination induces a longer-lasting increase in the avidity of antibodies to a T cell-dependent antigen than is measured by a memory B cell response to that antigen and that early memory T cell responses correlate well with the subsequent development of higher-avidity antibodies.

holera is an acute dehydrating diarrheal disease caused by Antibody avidity has been used as a measure of functional matu- on October 11, 2019 by guest Ctoxinogenic strains of Vibrio cholerae serogroups O1 and ration of the humoral immune response, and increases in anti- O139 (1, 2). Cholera is endemic in over 50 countries globally; in body avidity over time have been shown after both infection and these countries, young children bear a large burden of disease vaccination (12–14). We showed previously, in adults, that the (3–5). Cholera also causes significant morbidity and death avidity of antibodies against both cholera toxin B subunit (CTB) through epidemics and outbreaks in countries in which the dis- and LPS increases following cholera infection or cholera vaccina- ease is not endemic. Along with efforts to improve access to clean tion and correlates with the levels of memory B cells against the water and sanitation, the WHO has advocated the use of oral respective antigens; the durability of high-avidity antibodies is cholera vaccines in areas with both epidemic and endemic disease longer in patients than in vaccinees (15). (6). Unfortunately, young children receiving oral killed cholera The reasons for the poor efficacy of oral cholera vaccines in vaccine achieve lower protective efficacy and a shorter duration of young children remain to be determined (16). We have shown in protection than older children and adults (7), the reasons for children receiving Dukoral (Crucell, Sweden), an oral killed which are unknown. whole-cell cholera vaccine containing recombinant CTB, that The mechanism of protection against cholera infection is still child vaccinees mount poor memory B cell responses at day 42 not fully understood. In studies of household contacts of patients after vaccination, particularly for LPS, while children with clinical with cholera, we demonstrated previously that levels of IgA anti- cholera infection develop better LPS-specific memory B cell re- bodies as well as memory B cell responses to lipopolysaccharide sponses (17). We also demonstrated that vaccinees Ͻ5 years of age (LPS), a T cell-independent antigen, on exposure in the household were unable to generate significant memory T cell responses to are associated with protection against disease (8, 9). We have also cholera antigens, in contrast to older vaccinees (18). Long-term demonstrated that adults with cholera have significant elevations in circulating cholera toxin-specific memory B cells that persist for at least 360 days after illness (10). Adult vaccinees also have chol- Received 26 May 2013 Returned for modification 13 June 2013 era toxin-specific memory B cells for up to 180 days after vaccina- Accepted 31 July 2013 tion but do not develop significant memory B cell responses to Published ahead of print 7 August 2013 LPS (11). Address correspondence to Firdausi Qadri, [email protected]. Antibody production after infection or vaccination involves S.B.C., E.T.R., and F.Q. contributed equally to this article. the process of affinity maturation and somatic hypermutation of Copyright © 2013, American Society for Microbiology. All Rights Reserved. antigen-specific B cells, most effectively in response to antigen- doi:10.1128/CVI.00341-13 specific follicular helper T cells (TFH) in germinal centers (GCs).

October 2013 Volume 20 Number 10 Clinical and Vaccine Immunology p. 1541–1548 cvi.asm.org 1541 Alam et al. immune responses following oral cholera vaccine administration these counts by ELISPOT as the percentage of antigen-specific memory B in children have yet to be reported, and the magnitude and per- cells in total IgG or IgA memory B cells (10, 19). Exclusion and inclusion sistence of antibody avidity as measures of maturation of the im- criteria for data for analyses were as described previously (17). mune response after vaccination have not been explored in chil- Vibriocidal antibody assay and detection of antigen-specific anti- dren. Therefore, in this study, we sought to characterize the V. body responses in plasma. We measured vibriocidal antibody titers in cholerae-specific antibody avidities of children who received an plasma using guinea pig complement and V. cholerae O1 Ogawa (X- oral killed whole-cell cholera vaccine, to compare these responses 25049) as the target organism, as described previously (20). The vibrio- with those of adult vaccinees, and to determine the relationship of cidal titer was defined as the reciprocal of the highest dilution resulting in Ͼ antibody avidities over time to memory T cell responses shortly 50% reduction of the optical density (OD) compared to that of control after vaccination. wells without plasma. Detection of LPS- and CTB-specific IgG and IgA antibody responses was performed with a previously described standard MATERIALS AND METHODS enzyme-linked immunosorbent assay (ELISA) procedure (20). Briefly, Study design and subject enrollment. We enrolled healthy young chil- 96-well polystyrene plates (Nunc F; Nunc, Denmark) were coated with V. dren (age, 3.5 to 5 years; n ϭ 20), older children (age, 7 to 14 years; n ϭ 20) cholerae O1 Ogawa LPS (250 ng/well) or with GM1 ganglioside (0.3 nmol/ Downloaded from (17), and adults (age, 20 to 45 years; n ϭ 33) (11), who were given two ml) followed by recombinant CTB (50 ng/well) (both gifts from A. M. doses of the oral killed whole-cell cholera vaccine Dukoral (Crucell, Swe- Svennerholm, University of Gothenburg, Gothenburg, Sweden). After in- den), with a 2-week interval. We obtained blood from children at the time cubation with the plasma samples, LPS- and CTB-specific IgG or IgA of enrollment (day 0), 3 days after administration of the first dose of antibodies were detected using horseradish peroxidase-conjugated sec- vaccine, and again on days 42 (28 days after the second dose), 90, 180, 270, ondary antibodies to human IgG or IgA (Jackson ImmunoResearch, West and 360. For adults, we collected blood at the time of enrollment (day 0), Grove, PA). We developed the plates with ortho-phenylenediamine 3 days after administration of each dose of vaccine (days 3 and 17), and (Sigma, St. Louis, MO) in 0.1 M sodium citrate buffer (pH 4.5) and 0.1% again on days 42 (28 days after the second dose), 90, 180, 270, and 360. Of hydrogen peroxide. Plates were read kinetically at 450 nm for 5 min at 14-s note, we previously reported nonavidity data for the aforementioned chil- intervals, and results were expressed as milli-absorbance units per minute. dren through 42 days of follow-up, and we now extend these observations Data were expressed as ELISA units by calculating the ratio of the optical http://cvi.asm.org/ through 360 days of follow-up and correlate avidity data with other im- density of the test sample to that of a standard of pooled convalescent- mune responses (17, 18). Similarly, we previously reported results for a phase sera prepared from previously infected cholera patients, which was subset of adults in the vaccinee cohort described in the current analysis included on each plate (10, 19, 21). (11), and we now supplement these results with data from additional Measurement of CTB- and LPS-specific antibody avidity in child adults, include avidity responses, and compare these results with those and adult vaccinees. We measured CTB- and LPS-specific IgG and IgA observed in children. In our current analysis, we include assessments of antibody avidity index (AI) values using a previously described method vibriocidal antibodies, antigen (CTB and LPS)-specific antibody re- (15, 22). Briefly, an ELISA procedure was performed to determine CTB sponses, and avidities of plasma antibodies for these antigens in both child and LPS IgG and IgA antibody responses as described above. Following and adult vaccinees. We assessed antigen-specific IgG and IgA memory B cell responses on days 0, 42, 90, 180, 270, and 360. Memory T cell re- the incubation of plasma (1:2 to 1:18,000 dilution in 0.1% bovine serum on October 11, 2019 by guest sponses were measured in children only on days 0, 21, and 42. All studies albumin-PBS-Tween) with CTB or LPS in the wells, one set of wells was were approved by the research review and ethical review committees of treated with sodium thiocyanate (NaSCN) (2 M NaSCN in PBS-0.3% the International Centre for Diarrhoeal Disease Research, Bangladesh Tween), whereas the other set of wells was treated with PBS-0.3% Tween (icddr,b) (Dhaka, Bangladesh), and the institutional review board of the alone, for 20 min at room temperature. We used horseradish peroxidase- Massachusetts General Hospital. conjugated anti-human IgG or IgA antibodies (Jackson Laboratories, Bar Isolation of peripheral blood mononuclear cells and detection of Harbor, ME) at 1:1,000 dilution to detect CTB and LPS IgG or IgA, re- memory B cell responses. We isolated peripheral blood mononuclear spectively, and plates underwent development as described above. After cells (PBMCs) and plasma from heparinized blood diluted with phos- the addition of 1 M sulfuric acid to the plates, the optical density (OD) was phate-buffered saline (PBS) by centrifugation on Ficoll-Isopaque (Phar- measured at 492 nm. We used an OD between 0.5 and 2.00 for non- macia, Piscataway, NJ). Plasma was stored at Ϫ20°C for immunological NaSCN-treated wells for each sample to calculate the avidity index (AI) as assays. We resuspended PBMCs to a concentration of 1 ϫ 107 cells/ml in the ratio of the OD of NaSCN-treated wells to the OD of the untreated RPMI complete medium (Gibco, Carlsbad, CA) supplemented with 10% wells, as described previously (15, 22). For each ELISA plate, we also used heat-inactivated fetal bovine serum (FBS) (19). PBMCs were used for the a positive-control specimen derived from pooled convalescent-phase sera memory B cell cultures and subsequent ELISPOT procedures as described from cholera patients (21). ϫ 5 previously (10). Briefly, we cultured 5 10 PBMCs/well in 24-well cell Correlation analysis with T cell responses. We used the flow cyto- culture plates (BD Biosciences, San Jose, CA) containing a mixture of metric assay of specific cell-mediated immune response in activated whole three B cell mitogens, including CpG oligonucleotide (Operon, Hunts- blood (FASCIA) method to determine lymphoblast formation in re- ville, AL), crude pokeweed mitogen extract, and Staphylococcus aureus sponse to antigenic stimulation and to measure memory T cell responses, Cowan (Sigma). The culture medium consisted of RPMI 1640 medium, as previously described (23, 24) and reported (18). We correlate our avid- 10% FBS, 200 U/ml of penicillin, 200 ␮g/ml of streptomycin, 2 mM L-glu- ity results with previously reported CTB-specific effector memory T cell tamine, and 50 ␮M ␤-mercaptoethanol (10, 19). After incubation of the (TEM) and follicular helper T cell (TFH) responses (18). plates for 5 to 6 days at 37°C in a 5% CO2 incubator, cells were harvested, washed, and plated onto 96-well nitrocellulose-bottomed plates pre- Statistical analyses. Comparisons of immune responses within and coated with either GM1 ganglioside (3 nM) followed by recombinant CTB between groups were tested for significance using the Wilcoxon (2.5 ␮g/ml), V. cholerae O1 Ogawa LPS (25 ␮g/ml), keyhole limpet he- signed-rank test and the Mann-Whitney U test, respectively. We used mocyanin (2.5 ␮g/ml, as negative control), or affinity-purified goat anti- Spearman’s correlation to assess the relationship between avidity in- human IgA or IgG (5 ␮g/ml; Jackson ImmunoResearch, West Grove, PA). dices and T cell responses. All reported P values are two sided, with P We detected IgG and IgA memory B cells using horseradish peroxidase- values of Ͻ0.05 being considered the threshold for statistical signifi- conjugated mouse anti-human IgG and IgA (Hybridoma Reagent Labo- cance. Analyses were performed using GraphPad Prism 5.0 (GraphPad ratory), followed by 3-amino-9-ethylcarbazole. We quantified the num- Software, Inc., San Diego, CA) and SigmaStat 3.1 (Systat Software, ber of antibody-secreting cells/well by stereomicroscopy and expressed Inc., San Jose, CA).

1542 cvi.asm.org Clinical and Vaccine Immunology Antibody Avidity in Children after Cholera Vaccination

TABLE 1 Demographic characteristics of study participants Characteristic Young children Older children Adults No. of participants 20 20 33 No. (%) who completed 16 (80) 16 (80) 27 (81) follow-up to day 360 Age (median [25th to 5 (4.6–5) 10 (9.2–12.7) 32 (26–39) 75th percentile]) (yr) Female (n [%]) 9 (45) 10 (50) 13 (39)

RESULTS Study population. In this study, we report results from 40 child and 33 adult vaccinees (Table 1). Among the child vaccinees, 20 Downloaded from were younger children (median age, 5 years) and 20 were older children (median age, 10 years). Eighty percent of the child vaccinees (young children, n ϭ 16; older children, n ϭ 16) completed FIG 2 Vibriocidal antibody responses in different age groups after adminis- follow-up, and 81% of the adults completed follow-up to day 360. tration of two doses of oral cholera vaccine, with a 2-week interval (day 0 and day 14). Bars, mean responses; error bars, standard errors of the mean. .statistically significant differences (P Ͻ 0.05) from the baseline (day 0) titer ,ء -The schedules for vaccination, blood collection, and immunolog ical assays for child and adult vaccinees are shown in Fig. 1. Vibriocidal and antigen-specific antibody responses in plasma. Baseline (day 0) vibriocidal antibody levels and CTB- and CTB-specific avidity indices in child and adult vaccinees. The LPS-specific IgA antibody responses were comparable among the magnitudes of avidity indices for CTB-specific IgG and IgA anti- http://cvi.asm.org/ different age groups (Fig. 2 and 3). However, baseline CTB- and bodies in adults were significantly greater than those seen in young LPS-specific IgG antibody levels in both young and older children Ͻ Ͻ children (P 0.05) at baseline (day 0) (Fig. 4). Adults also had a were significantly higher than those seen in adults (P 0.05) (Fig. higher CTB-specific IgA antibody avidity index than did older 3). Child (both young and older) and adult vaccinees showed sig- Ϯ Ͻ children. The avidity index (AI) values (mean standard error) nificant increases in vibriocidal antibody responses (P 0.05) for CTB-specific IgG antibodies in young and older children at (Fig. 2) and CTB- and LPS-specific IgG and IgA antibody re- Ϯ Ϯ Ͻ day 0 were 30% 1.7% and 36% 2.1%, respectively, which sponses within 3 days after vaccination (P 0.05) (Fig. 3), as increased significantly at day 21 to 47% Ϯ 3.8% and 58% Ϯ 4.3%, reported previously (11, 17), compared to their respective baseline respectively (Fig. 4A). The increase in avidity index over baseline on October 11, 2019 by guest values. However, these responses persisted for longer periods of persisted significantly up to day 360 for both age groups of chil- time in adult vaccinees than in child vaccinees (Fig. 2 and 3). dren. As reported previously, the adult vaccinees also showed sig- Vibriocidal and CTB-specific IgG antibody responses in adult vac- Ͻ nificant elevation in CTB-specific IgG avidity (15), although this cinees persisted up to day 270 (P 0.05), while these responses in elevation waned somewhat more quickly than in child vaccinees. child vaccinees returned to baseline levels by day 42 or 90. Anti- CTB-specific IgA antibody avidity index values in young and older CTB IgA as well as anti-LPS IgG and IgA antibody responses also Ͻ children increased significantly by day 3 and particularly by day 21 remained elevated (P 0.05) for longer periods of time in adult (AIs, 55% Ϯ 4.8% and 61% Ϯ 4.5%, respectively), compared to vaccinees than in child vaccinees. The overall magnitudes of re- baseline (AIs, 31% Ϯ 3.2% and 32% Ϯ 2.4%, respectively) (P Ͻ sponses in different age groups were comparable. 0.05) (Fig. 4B). These elevations in the avidity of CTB-specific IgA antibodies also persisted up to day 360 in both young and older children. The increased avidity index of CTB-specific IgA antibodies again waned somewhat more quickly in adult vaccinees than in child vaccinees. The magnitudes of the peak CTB-specific IgG and IgA antibody avidity indices after vaccination in different age groups were comparable. LPS-specific avidity indices in child and adult vaccinees. We detected a significantly lower magnitude of LPS-specific IgG antibody avidity indices at day 0 in young and older children than in adults (P Ͻ 0.05) (Fig. 4). However, the magnitudes of the baseline avidity indices of LPS-specific IgA antibodies in different age groups were comparable. The mean antibody avidity indices for LPS-specific IgG at day 0 (prevaccination) in healthy young and older child vaccinees were 36% Ϯ 1.8% and 37% Ϯ 1.5%, respectively. These antibody avidity index values increased by day 21 FIG 1 Schedules for vaccination, blood collection, and immunological re- (AIs, 45% Ϯ 2.9% and 44% Ϯ 2.8%, respectively) before return- sponse measurements for children and adult vaccinees. For child (younger and ing to baseline levels by day 42 (Fig. 4C). Similarly, statistically older) vaccinees, day 21 and day 42 indicate 7 and 28 days, respectively, after the second dose of vaccine at day 14. For adults, day 17 and day 42 indicate 3 significant increases in LPS-specific IgA antibody avidity indices and 28 days, respectively, after the second dose of vaccine. , vaccination; in young and older child vaccinees were detected only on day 21 Œ, study follow-up assessments. (AI ϭ 52% Ϯ 3.1% and 52% Ϯ 3.6%, respectively), compared to

October 2013 Volume 20 Number 10 cvi.asm.org 1543 Alam et al. Downloaded from http://cvi.asm.org/

FIG 3 Plasma CTB-specific (A and B) and LPS-specific (C and D) antibody responses in Bangladeshi vaccine recipients of different age groups who received two doses of oral cholera vaccine, with an interval of 2 weeks (day 0 and day 14). (A and C) IgG responses. (B and D) IgA responses. Bars, mean responses; error bars, statistically significant differences (P Ͻ 0.05) from baseline (day 0); #, statistically significant differences between age groups ,ء .standard errors of the mean (P Ͻ 0.05). on October 11, 2019 by guest baseline (AIs, 42% Ϯ 1.9% and 41% Ϯ 1.7%, respectively), and point throughout the study period (Fig. 5C and D). In children, these returned to baseline levels by day 42 (Fig. 4D). In adult we also did not detect any correlation between the antibody avid- vaccinees, the increases in LPS-specific IgG or IgA antibody avidity indices of CTB- or LPS-specific IgG or IgA antibodies at vari- ity indices were detected only at day 17 but returned to baseline ous time points after vaccination and CTB- or LPS-specific mem- levels by day 42, as reported previously (15). The magnitudes of ory B cell numbers on the corresponding days (data not shown). the peak LPS-specific IgG or IgA antibody avidity indices in dif- Correlation between early memory T cell responses and sub- ferent age groups were comparable. sequent antibody avidity in older child vaccinees. To determine Antigen-specific memory B cell responses. We analyzed the the relationship between memory T cell responses (specifically memory B cell responses to CTB and LPS by combining all child memory follicular helper T cell responses) and subsequent anti- vaccinees into one cohort (since we found no differences in mem- body avidities, we compared mutant cholera toxin-specific (18) ory B cell responses between young and older child vaccinees [data effector memory T cell (TEM) and memory follicular helper T cell not shown] and we had a paucity of matched paired data points (TFH) responses at day 21 (7 days after the second dose of vaccine) for statistical analyses), and we compared these responses with with CTB-specific IgG or IgA antibody avidity indices in the same those of adult vaccinees (a portion of which were reported previ- older child vaccinees on days 42, 90, 180, 270, and 360. We found ously) (15). For both child and adult vaccinees, significant in- that CTB-specific IgG antibody avidity indices on days 42, 90, 180, creases in CTB-specific IgG memory B cell responses were de- and 270 correlated with the memory T cell responses on day 21 tected at day 42 (P Ͻ 0.05), compared to the respective baseline (Spearman’s ␳ϭ0.45 to 0.65, P Ͻ 0.05) (Table 2); there was a responses at day 0 (Fig. 5A). These memory B cell responses to trend toward a correlation at day 360 (Spearman’s ␳ϭ0.47, P ϭ CTB persisted slightly longer in adult vaccinees than in child vac- 0.08). The correlations between memory follicular helper T cell cinees, but both responses returned to baseline levels by day 90 to responses at day 21 and CTB-specific IgG antibody avidity indices 180. The adult vaccinees also developed significantly higher CTB- on days 90, 180, and 270 were also statistically significant (Spear- specific IgA memory B cell responses at day 42, whereas the chil- man’s ␳ϭ0.45 to 0.65, P Ͻ 0.05) (Table 2). We did not find a dren demonstrated no increases in CTB-specific IgA memory B correlation between CTB-specific IgA antibody avidity indices at cell responses at any follow-up study day (Fig. 5B). We did not later time points and earlier memory T cell responses. We also did detect any significant increases in memory B cell responses to LPS not find any correlations between T cell responses and avidity for (IgG or IgA isotype) in either child or adult vaccinees at any time the younger children.

1544 cvi.asm.org Clinical and Vaccine Immunology Antibody Avidity in Children after Cholera Vaccination Downloaded from http://cvi.asm.org/

FIG 4 Avidity index (AI) values for CTB-specific (A and B) and LPS-specific (C and D) antibodies in Bangladeshi vaccine recipients of different age groups who received two doses of oral cholera vaccine, with an interval of 2 weeks (day 0 and day 14). (A and C) IgG responses. (B and D) IgA responses. Bars, mean responses; statistically significant differences (P Ͻ 0.05) from baseline (day 0) AI; #, statistically significant differences between ,ء .error bars, standard errors of the mean age groups (P Ͻ 0.05). on October 11, 2019 by guest

DISCUSSION the first study showing the development and persistence of chol- Young children are vulnerable to cholera in both endemic and era-specific antibodies with high avidity in children vaccinated epidemic settings. Despite this, current oral cholera vaccines with a cholera vaccine. achieve lower protective efficacy and shorter duration of protec- Antibody avidity represents the combined binding affinities of tion in young children than in older persons (6). Young children a variety of antibodies and their multivalent antigen (15, 32). Class generally respond poorly to polysaccharide antigens (25–27). Pre- switching, affinity maturation, and somatic hypermutation that viously, we demonstrated that memory B cell responses in young occur during B cell maturation generate high-affinity antibodies children to both the T cell-dependent antigen CTB and the T of different subclasses; this process is most efficient in the germi- cell-independent antigen LPS following vaccination were signifi- nal centers (GCs) of secondary lymph nodes (33–35). During the ϩ cantly lower than those achieved after natural infection (17). An- primary immune response, CD4 helper T cells differentiate into tibody avidity has been shown to be a correlate of immune mem- follicular helper T cells (TFH) and interact with B cells in the GC, ory and protective immunity in both infection and vaccination for inducing antigen-specific B cell proliferation and maturation. The other infectious diseases in children (28–31). In this study, we B cells that produce high-affinity antibodies during proliferation evaluated the magnitude and duration of antibody avidity in both either differentiate into plasma cells or become memory B cells children and adults who received two doses of Dukoral, a heat- (33, 36). Previously, we have shown that adult cholera patients and formalin-inactivated oral cholera vaccine. We showed that and vaccinees both develop high-avidity CTB- and LPS-specific both young and older children developed significant elevations of antibodies and that these responses correlate well with memory B antibody avidity for the T cell-dependent antigen CTB and that cell responses measured at the same time points, consistent with this increase in antibody avidity persisted for at least 360 days, a the idea that the two types of measurements reflect B cell matura- notably longer period of time than for memory B cell responses to tion in similar manners (15). However, in this study, we did not the same antigen. Increases in antibody avidity indices for LPS, the find such correlation for children after two doses of Dukoral. We T cell-independent antigen studied, however, were seen for only a also showed previously that early T cell responses to CTB correlate short time after vaccination, and we did not see memory B cells with later memory B cell responses to the T cell-dependent antigen directed to LPS at any time point following vaccination. For the T CTB in older children following vaccination but not in young cell-dependent antigen CTB, we demonstrated a significant cor- children immunized with two doses of Dukoral (18). In this study, relation between early memory T cell responses and subsequent we found a significant positive correlation between early memory increases in antibody avidity for CTB. To our knowledge, this is T cell responses (both TEM and memory TFH cell responses) to

October 2013 Volume 20 Number 10 cvi.asm.org 1545 Alam et al. Downloaded from http://cvi.asm.org/

FIG 5 CTB-specific (A and B) and LPS-specific (C and D) memory B cell responses in Bangladeshi vaccine recipients who received two doses of oral cholera vaccine, separated by 2 weeks (day 0 and day 14). (A and C) IgG responses. (B and D) IgA responses. Memory B cell responses are expressed as percent ء antigen-specific responses of total isotype-specific memory B cells. Bars, mean responses; error bars, standard errors of the mean. , statistically significant on October 11, 2019 by guest differences (P Ͻ 0.05) from baseline (day 0). cholera toxin and subsequent CTB-specific IgG antibody avidity component of protective immunity that facilitates anamnestic re- index values in older children who received vaccine but not young sponses. Both adults and children with cholera develop CTB- and children. T cell help may function to stimulate affinity maturation LPS-specific memory B cell responses within 30 days after infec- of CTB-specific B cells in older children, while similar T cell help is tion (10, 37), and we have shown that the CTB-specific responses not stimulated by vaccination in young children. in adults persist for at least 1 year following cholera infection, The precise mechanism of protective immunity against V. chol- while LPS-specific responses wane more quickly (10). In contrast, erae infection is still unknown. However, it has been shown that adults immunized with two doses of Dukoral achieve only a short CTB- and LPS-specific IgA antibodies as well as LPS-specific IgG duration of CTB-specific memory B cell responses (11), and both memory B cell responses are associated with decreased risk of adults and children who receive vaccine have no measurable subsequent infection (8, 9). The memory B cell response is a key memory B cell responses against LPS (17). These results, combined with those described above, suggest that currently available oral cholera vaccines may not induce sufficient T cell help to in- TABLE 2 Correlation between cholera toxin-specific early T cell duce longer-term memory B cell responses, particularly in responses and later antibody avidity indices in older child vaccinees younger children, in comparison with natural infection and that T cell response on this may be an explanation for the lesser degree and duration of day 21 Day of AI assessment Spearman’s ␳ P protection. Effector memory 42 0.53 0.02 Recently, we observed that adult cholera patients and vaccinees T cell 90 0.46 0.05 were capable of developing high-avidity antibodies to both CTB 180 0.58 0.01 270 0.66 0.006 and LPS; however, high-avidity antibodies to LPS did not persist 360 0.48 0.08 past 17 days after vaccination and 180 days after infection (15). Memory follicular 42 0.3 0.89 These results were consistent with LPS-specific memory B cell helper T cell 90 0.55 0.01 responses in the same groups. In this study, we have shown that 180 0.53 0.02 both young and older children are able to develop high-avidity 270 0.56 0.02 antibodies to CTB and LPS. In the case of CTB, these high-avidity 360 0.37 0.19 antibodies remained at elevated levels over baseline levels to 180 to

1546 cvi.asm.org Clinical and Vaccine Immunology Antibody Avidity in Children after Cholera Vaccination

360 days, although CTB-specific antibody responses and memory DR, Ali M, Clemens JD. 2008. The high burden of cholera in children: B cell responses waned by 42 days following vaccination. This comparison of incidence from endemic areas in Asia and Africa. PLoS suggests that measuring antibody avidity indices for T cell-depen- Negl. Trop. Dis. 2:e173. doi:10.1371/journal.pntd.0000173. 5. Glass RI, Becker S, Huq MI, Stoll BJ, Khan MU, Merson MH, Lee JV, dent antigens following vaccination may provide evidence of lon- Black RE. 1982. Endemic cholera in rural Bangladesh, 1966–1980. Am. J. ger-term immune maturation than measured by the current Epidemiol. 116:959–970. memory B cell assay, and this may explain why these vaccines 6. World Health Organization. 2010. Cholera vaccines: WHO position pa- provide protection against severe disease for 2 to 3 years following per. Wkly. Epidemiol. Rec. 85:117–128. 7. Sinclair D, Abba K, Zaman K, Qadri F, Graves PM. 2011. Oral vaccines vaccination even in the absence of memory B cell responses (7). for preventing cholera. Cochrane Database Syst. Rev. (3):CD008603. On the other hand, elevations in LPS-specific antibody avidity, 8. Patel SM, Rahman MA, Mohasin M, Riyadh MA, Leung DT, Alam MM, although detectable shortly after vaccination in both children and Chowdhury F, Khan AI, Weil AA, Aktar A, Nazim M, LaRocque RC, adults, did not persist, and memory B cells did not develop. We Ryan ET, Calderwood SB, Qadri F, Harris JB. 2012. Memory B cell hypothesize that such differences in the persistence of high-avidity responses to Vibrio cholerae O1 lipopolysaccharide are associated with protection against infection from household contacts of patients with antibodies for LPS and CTB are due to the T cell-dependent na- cholera in Bangladesh. Clin. Vaccine Immunol. 19:842–848. ture of the CTB antigen and the importance of T cell help in 9. Harris JB, LaRocque RC, Chowdhury F, Khan AI, Logvinenko T, Downloaded from affinity maturation and the induction of B cell memory. Since Faruque AS, Ryan ET, Qadri F, Calderwood SB. 2008. Susceptibility to immune responses to LPS appear to correlate with subsequent Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh. PLoS Negl. Trop. Dis. 2:e221. doi:10.1371/journal protective immunity, the failure of individuals to show evidence .pntd.0000221. of T cell-independent B cell maturation for LPS following vacci- 10. Harris AM, Bhuiyan MS, Chowdhury F, Khan AI, Hossain A, Kendall nation, measured as either increased antibody avidity indices or EA, Rahman A, LaRocque RC, Wrammert J, Ryan ET, Qadri F, Cal- the development of LPS-specific memory B cells, may explain in derwood SB, Harris JB. 2009. Antigen-specific memory B-cell responses part the shortened duration of protection following vaccination. to Vibrio cholerae O1 infection in Bangladesh. Infect. Immun. 77:3850– 3856. Animal models and human studies have suggested that affinity 11. Alam MM, Riyadh MA, Fatema K, Rahman MA, Akhtar N, Ahmed T, maturation and memory B cell development in the germinal cen- Chowdhury MI, Chowdhury F, Calderwood SB, Harris JB, Ryan ET, http://cvi.asm.org/ ter result in increases in antibody avidity (33). In studies of chil- Qadri F. 2011. Antigen-specific memory B-cell responses in Bangladeshi dren receiving polysaccharide vaccines against Haemophilus influ- adults after one- or two-dose oral killed cholera vaccination and comparison with responses in patients with naturally acquired cholera. Clin. Vac- enzae and Streptococcus pneumoniae, it has been observed that cine Immunol. 18:844–850. high-avidity antibodies are more potent than low-avidity antibod- 12. Bottiger B, Jensen IP. 1997. Maturation of rubella IgG avidity over time ies in bactericidal assays (38, 39). However, while these findings after acute rubella infection. Clin. Diagn. Virol. 8:105–111. suggest that antibody avidity may be a marker of protective im- 13. Brown SE, Howard CR, Zuckerman AJ, Steward MW. 1984. Affinity of munity for at least some diseases, the association between these antibody responses in man to hepatitis B vaccine determined with syn- thetic peptides. Lancet 2(8396):184–187. high-avidity antibodies and protection against disease has not 14. Duong YT, Qiu M, De AK, Jackson K, Dobbs T, Kim AA, Nkengasong on October 11, 2019 by guest been proven, and this association has not been investigated for JN, Parekh BS. 2012. Detection of recent HIV-1 infection using a new infections at the mucosal surface. Thus, further studies are needed limiting-antigen avidity assay: potential for HIV-1 incidence estimates to determine the role of antibody avidity for both CTB and LPS in and avidity maturation studies. PLoS One 7:e33328. doi:10.1371/journal protection against V. cholerae infection. .pone.0033328. 15. Alam MM, Arifuzzaman M, Ahmad SM, Hosen MI, Rahman MA, Rashu R, Sheikh A, Ryan ET, Calderwood SB, Qadri F. 2013. Study of ACKNOWLEDGMENTS avidity of antigen-specific antibody as a means of understanding develop- This work was supported by the icddr,b, grants from the National Insti- ment of long-term immunological memory after Vibrio cholerae O1 infection. Clin. Vaccine Immunol. 20:17–23. tutes of Health, including grants from the National Institute of Allergy 16. Leung DT, Chowdhury F, Calderwood SB, Qadri F, Ryan ET. 2012. and Infectious Diseases (AI077883 [to E.T.R. and F.Q.], AI058935 [to Immune responses to cholera in children. Expert Rev. Anti Infect. Ther. S.B.C., E.T.R., and F.Q.], and AI100923 [to D.T.L.]), and a training grant 10:435–444. in vaccine development from the Fogarty International Center 17. Leung DT, Rahman MA, Mohasin M, Patel SM, Aktar A, Khanam F, (TW005572 [to M.M.A. and F.Q.]). This study was also supported by Uddin T, Riyadh MA, Saha A, Alam MM, Chowdhury F, Khan AI, Swedish SIDA grant INT-ICDDR, B-HN-01-AV (to F.Q.), a Thrasher Charles R, LaRocque R, Harris JB, Calderwood SB, Qadri F, Ryan ET. Research Fund Early Career Award (to D.T.L.), and a postdoctoral fellow- 2012. Memory B cell and other immune responses in children receiving ship in tropical infectious diseases from the American Society for Tropical two doses of an oral killed cholera vaccine compared to responses follow- Medicine and Hygiene-Burroughs Wellcome Fund (to D.T.L.). ing natural cholera infection in Bangladesh. Clin. Vaccine Immunol. 19: 690–698. 18. Arifuzzaman M, Rashu R, Leung DT, Hosen MI, Bhuiyan TR, Bhuiyan REFERENCES MS, Rahman MA, Khanam F, Saha A, Charles RC, LaRocque RC, Weil 1. Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB. 2012. AA, Clements JD, Holmes RK, Calderwood SB, Harris JB, Ryan ET, Cholera. Lancet 379:2466–2476. Qadri F. 2012. Antigen-specific memory T cell responses after vaccination 2. Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada with an oral killed cholera vaccine in Bangladeshi children and compari- T, Takeda T, Karasawa T, Kurazano H, Pal A, Takeda Y. 1993. Emer- son to responses in patients with naturally acquired cholera. Clin. Vaccine gence of novel strain of Vibrio cholerae with epidemic potential in south- Immunol. 19:1304–1311. ern and eastern India. Lancet 341:703–704. 19. Jayasekera CR, Harris JB, Bhuiyan S, Chowdhury F, Khan AI, Faruque 3. Agtini MD, Soeharno R, Lesmana M, Punjabi NH, Simanjuntak C, AS, Larocque RC, Ryan ET, Ahmed R, Qadri F, Calderwood SB. 2008. Wangsasaputra F, Nurdin D, Pulungsih SP, Rofiq A, Santoso H, Pu- Cholera toxin-specific memory B cell responses are induced in patients jarwoto H, Sjahrurachman A, Sudarmono P, von Seidlein L, Deen JL, with dehydrating diarrhea caused by Vibrio cholerae O1. J. Infect. Dis. Ali M, Lee H, Kim DR, Han O, Park JK, Suwandono A, Ingerani Oyofo 198:1055–1061. BA, Campbell JR, Beecham HJ, Corwin AL, Clemens JD. 2005. The 20. Qadri F, Wenneras C, Albert MJ, Hossain J, Mannoor K, Begum YA, burden of diarrhoea, shigellosis, and cholera in North Jakarta, Indonesia: Mohi G, Salam MA, Sack RB, Svennerholm AM. 1997. Comparison of findings from 24 months surveillance. BMC Infect. Dis. 5:89. immune responses in patients infected with Vibrio cholerae O139 and O1. 4. Deen JL, von Seidlein L, Sur D, Agtini M, Lucas ME, Lopez AL, Kim Infect. Immun. 65:3571–3576.

October 2013 Volume 20 Number 10 cvi.asm.org 1547 Alam et al.

21. Qadri F, Ahmed F, Karim MM, Wenneras C, Begum YA, Abdus Salam immune responses after a second acellular pertussis booster vaccination in M, Albert MJ, McGhee JR. 1999. Lipopolysaccharide- and cholera toxin- children 9 years of age. Vaccine 30:51–58. speciﬁc subclass distribution of B-cell responses in cholera. Clin. Diagn. 30. Vilibic-Cavlek T, Ljubin-Sternak S, Vojnovic G, Sviben M, Mlinaric- Lab. Immunol. 6:812–818. Galinovic G. 2012. The role of IgG avidity in diagnosis of cytomegalovirus 22. Ozkul A, Demir B, Karaoglu T, Alkan F, Dincer E, Oncel T, Burgu I. infection in newborns and infants. Coll. Antropol. 36:297–300. 2008. Maturation of immunoglobulin G avidity after inactive gE deleted 31. Yam KK, Gupta J, Brewer A, Scheifele DW, Halperin S, Ward BJ. 2013. bovine herpesvirus type 1 (BHV-1) marker vaccination. Viral Immunol. Unusual patterns of IgG avidity in some young children following two 21:3–11. doses of adjuvanted pandemic H1N1 (2009) inﬂuenza vaccine. Clin. Vac- 23. Weil AA, Arifuzzaman M, Bhuiyan TR, LaRocque RC, Harris AM, cine Immunol. 20:459–467. Kendall EA, Hossain A, Tarique AA, Sheikh A, Chowdhury F, Khan AI, 32. Dimitrov JD, Lacroix-Desmazes S, Kaveri SV. 2011. Important param- Murshed F, Parker KC, Banerjee KK, Ryan ET, Harris JB, Qadri F, eters for evaluation of antibody avidity by immunosorbent assay. Anal. Calderwood SB. 2009. Memory T-cell responses to Vibrio cholerae O1 Biochem. 418:149–151. infection. Infect. Immun. 77:5090–5096. 33. Crotty S. 2011. Follicular helper CD4 T cells (TFH). Annu. Rev. Immu- 24. Ahmed T, Arifuzzaman M, Lebens M, Qadri F, Lundgren A. 2009. nol. 29:621–663. CD4ϩ T-cell responses to an oral inactivated cholera vaccine in young 34. Liu YJ, Malisan F, de Bouteiller O, Guret C, Lebecque S, Banchereau J, children in a cholera endemic country and the enhancing effect of zinc Mills FC, Max EE, Martinez-Valdez H. 1996. Within germinal centers,

supplementation. Vaccine 28:422–429. isotype switching of immunoglobulin genes occurs after the onset of so- Downloaded from 25. Clutterbuck EA, Oh S, Hamaluba M, Westcar S, Beverley PC, Pollard matic mutation. Immunity 4:241–250. AJ. 2008. Serotype-specific and age-dependent generation of pneumococ- 35. Reinhardt RL, Liang HE, Locksley RM. 2009. Cytokine-secreting follic- cal polysaccharide-specific memory B-cell and antibody responses to im- ular T cells shape the antibody repertoire. Nat. Immunol. 10:385–393. munization with a pneumococcal conjugate vaccine. Clin. Vaccine Immu- 36. Allen CD, Okada T, Cyster JG. 2007. Germinal-center organization and nol. 15:182–193. cellular dynamics. Immunity 27:190–202. 26. Huang Q, Li D, Kang A, An W, Fan B, Ma X, Ma G, Su Z, Hu T. 2013. 37. Leung DT, Rahman MA, Mohasin M, Riyadh MA, Patel SM, Alam MM, PEG as a spacer arm markedly increases the immunogenicity of meningo- Chowdhury F, Khan AI, Kalivoda EJ, Aktar A, Bhuiyan MS, LaRocque coccal group Y polysaccharide conjugate vaccine. J. Control. Release RC, Harris JB, Calderwood SB, Qadri F, Ryan ET. 2011. Comparison of [Epub ahead of print.] doi:10.1016/j.jconrel.2013.03.008. memory B cell, antibody-secreting cell, and plasma antibody responses in 27. Perrett KP, Jin C, Clutterbuck E, John TM, Winter AP, Kibwana E, Yu young children, older children, and adults with infection caused by Vibrio LM, Curtis N, Pollard AJ. 2012. B cell memory to a serogroup C menin- cholerae O1 El Tor Ogawa in Bangladesh. Clin. Vaccine Immunol. 18: http://cvi.asm.org/ gococcal conjugate vaccine in childhood and response to booster: little 1317–1325. association with serum IgG antibody. J. Immunol. 189:2673–2681. 38. Schlesinger Y, Granoff DM. 1992. Avidity and bactericidal activity of 28. Fried AJ, Altrich ML, Liu H, Halsey JF, Bonilla FA. 2013. Correlation of antibody elicited by different Haemophilus influenzae type b conjugate pneumococcal antibody concentration and avidity with patient clinical vaccines. JAMA 267:1489–1494. and immunologic characteristics. J. Clin. Immunol. 33:847–856. 39. Usinger WR, Lucas AH. 1999. Avidity as a determinant of the protective 29. Hendrikx LH, Felderhof MK, Ozturk K, de Rond LG, van Houten MA, efficacy of human antibodies to pneumococcal capsular polysaccharides. Sanders EA, Berbers GA, Buisman AM. 2011. Enhanced memory B-cell Infect. Immun. 67:2366–2370. on October 11, 2019 by guest

1548 cvi.asm.org Clinical and Vaccine Immunology