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. Author manuscript; available in PMC: 2026 Mar 9.
Published in final edited form as: J Pediatr Gastroenterol Nutr. 2015 Jun;60(6):834–843. doi: 10.1097/MPG.0000000000000694

Lactobacillus rhamnosus GG Dosage Affects the Adjuvanticity and Protection Against Rotavirus Diarrhea in Gnotobiotic Pigs

Ke Wen 1, Fangning Liu 1, Guohua Li 1, Muqun Bai 1, Jacob Kocher 1, Xingdong Yang 1, Haifeng Wang 1, Sherrie Clark-Deener 1, Lijuan Yuan 1
PMCID: PMC12967290  NIHMSID: NIHMS2147352  PMID: 25564808

Abstract

Objectives:

The use of immunostimulatory strains of probiotics as adjuvants has been increasingly recognized as a promising approach in enhancing vaccine immunogenicity; however, dose effects of probiotic adjuvants are not well defined. In the present study, we examined dose effects of a commonly used probiotic strain, Lactobacillus rhamnosus GG (LGG), on immunomodulation with 2 different dosages.

Methods:

Neonatal gnotobiotic pigs were inoculated with 2 oral doses of attenuated human rotavirus (AttHRV) vaccines and fed with 5 doses (LGG5X; total 2.1 × 106 colony-forming units) or 9 doses (LGG9X; total 3.2 × 106 colony-forming units) of LGG, starting at 3 days of age.

Results:

Both LGG feeding regimens enhanced the protection rate of AttHRV vaccine against diarrhea on virulent human rotavirus challenge. LGG5X, but not LGG9X, significantly enhanced rotavirus-specific intestinal memory B-cell responses to AttHRV; LGG5X also significantly enhanced virus-specific intestinal immunoglobulin A (IgA) antibody-secreting cell responses. Both regimens significantly enhanced rotavirus-specific serum IgA antibody responses to AttHRV. They also enhanced rotavirus-specific interferon-γ–producing effector/memory T-cell responses to AttHRV vaccine, with LGG9X being more effective than LGG5X, and both regimens downregulated CD4+CD25–FoxP3+ regulatory T (Treg) cell responses in most lymphoid tissues examined prechallenge and postchallenge and maintained the CD4+CD25+FoxP3+ Treg population in the ileum and intraepithelial lymphocyte postchallenge. LGG9X, however, did not significantly reduce total CD4+CD25–FoxP3+ Treg frequencies in the intestine and transforming growth factor-β–producing and interleukin (IL)-10–producing Treg frequencies in the blood.

Conclusions:

These results indicate that LGG at both dosages functioned as effective probiotic adjuvant for AttHRV vaccine, but different dosages differentially modulated immune responses to favor either the mucosal IgA response (LGG5X) or the T-cell response (LGG9X).

Keywords: B cells, dosage, gnotobiotic pigs, Lactobacillus rhamnosus GG, probiotics, rotavirus

Strain-specific effects of probiotics on enhancing proinflammatory immune responses (1) or inducing regulatory T (Treg)-cell responses (2) have been well recognized. Some Lactobacillus strains were known to have immunoregulatory functions on controlling inflammatory and autoimmune diseases and allergies (3–8); some other strains were found to enhance cellular and/or humoral immune responses (9–16). Dose effects of probiotics on differential immune modulation are also beginning to be recognized. For example, administration of Lactobacillus casei at high doses suppressed proinflammatory cytokine expression by CD4+ T cells and upregulated the levels of immunoregulatory cytokine interleukin (IL)-10 and transforming growth factor (TGF)-β (5,6), whereas another study found that lower doses of L casei were pure TH1 inducers (17). Being immunostimulatory without being proinflammatory or affecting the intestinal regulatory cell homeostasis is the ideal characteristic for probiotics as enteric vaccine adjuvants. Our goal is to identify the optimal strain and dosage of the probiotic adjuvant regimen, especially for rotavirus vaccines.

In our previous studies, we found that the probiotic strain Lactobacillus acidophilus NCFM (LA) regulated antibody-secreting cell (ASC), memory B-cell, effector T-cell, and Treg-cell responses to the attenuated human rotavirus (AttHRV) vaccine in a dose-dependent manner in gnotobiotic (Gn) pigs (18,19). At the appropriate dosage, LA alone also reduced rotavirus diarrhea (18). Another probiotic strain Lactobacillus rhamnosus GG (LGG) has been tested more extensively than the LA strain in randomized controlled human clinical trials for reducing the severity of diarrhea (20–24). LGG had a modest efficacy in preventing or shortening the duration of diarrhea in 9 of 10 trials. Clinical studies also demonstrated that LGG promoted antibody production (9,25,26) and induced proinflammatory cytokine responses in human mononuclear cells (MNCs) (27). LGG enhanced rotavirus-specific immunoglobulin A (IgA) ASC responses in adults (9) and rotavirus-specific immunoglobulin MASC responses and IgA seroconversion in 2- to 5-month-old infants (15). Interestingly, however, a study in healthy human volunteers and patients with Crohn disease showed that oral supplementation with 5 × 1010 colony-forming units (CFU) LGG for 2 weeks induced hyporesponsiveness in T cells, including TH subset 1 and 2 responses, without upregulation of immunoregulatory cytokines (28). Here, we hypothesize that the dosage of LGG plays an important role in its divergent immunomodulatory effects. The present study investigated the adjuvant effects of LGG at 2 different dosing regimens on the B- and T-cell immune responses induced by the AttHRV vaccine and protection against rotavirus diarrhea in Gn pigs. We found that different dosages of LGG preferentially promoted AttHRV vaccine–induced virus-specific adaptive immune responses in terms of lymphoid tissue distribution (intestinal vs systemic), B-cell versus T-cell responses, and differences in protective efficacy against rotavirus diarrhea.

METHODS

Virus

The cell culture–adapted human rotavirus Wa strain (G1P1A[8]), derived from the 35th passage in African green monkey kidney cells (MA104), was used as the AttHRV vaccine for inoculation of Gn pigs at a dose of 5 × 107 fluorescent focus-forming units (FFU) (29). AttHRV was also used as the detector antigen in the enzyme-linked immunosorbent assay (ELISA) (18) and as the stimulating antigen in the intracellular interferon (IFN)-γ staining assay as described previously (30).

The virulent human rotavirus (VirHRV) Wa strain was passed through Gn pigs, and the pooled intestinal contents from the 27th passage were used to challenge Gn pigs at a dose of 105 FFU. The median infectious dose and median diarrhea dose of the VirHRV in Gn pigs were determined as approximately 1 FFU (31).

Probiotic Bacteria

LGG strain (ATCC 53103) was used in the present study and was propagated in lactobacilli MRS broth (Weber, Hamilton, NJ). LGG inoculum was prepared and titrated as previously described (32). The enumeration of LGG in Gn pig fecal samples was performed weekly throughout the experiment to confirm colonization as previously described (32).

Treatment Groups and Inoculation of Gn Pigs

Near-term pigs were derived by hysterectomy and maintained in germ-free isolator units as described (33). All of the pigs were confirmed seronegative for anti-rotavirus antibodies and germ-free before AttHRV and LGG exposure. Gn pigs (both males and females) were randomly assigned to 6 treatment groups. Pigs in AttHRV groups were orally inoculated twice with 5 × 107 FFU/dose of AttHRV in 5 mL of a diluent (Minimum Essential Medium, Invitrogen, Grand Island, NY) at 5 and 15 days of age (postinoculation days [PIDs] 0 and 10, respectively). Pigs not inoculated with AttHRV were given an equal volume of the diluent. Pigs in the LGG5X group were orally dosed with 103, 104, 105, 106, and 106 CFU/dose of LGG in 3 mL of 0.1% peptone water (BD Biosciences, Sparks, MD) at 3, 5, 7, 9, and 11 days of age, respectively, and pigs in the LGG9X groups were orally dosed daily with 103, 103, 104, 104, 105, 105, 106, 106, and 106 CFU/dose of LGG at 3, 4, 5, 6, 7, 8, 9, 10, and 11 days of age, respectively, as described (18). Pigs not fed LGG were given an equal volume of 0.1% peptone water. At PID 28, subsets of pigs from controls (n = 4), AttHRV (n = 6), AttHRV + LGG5X (n = 4), and AttHRV + LGG9X (n = 5) groups were euthanized for examining rotavirus-specific immune responses (Fig. 1A). Other subsets of pigs from controls (n = 9), AttHRV (n = 12), AttHRV + LGG5X (n = 4), and AttHRV + LGG9X (n = 5) groups and all of the pigs from LGG5X (n = 4) and LGG9X (n = 4) groups were orally challenged with 105 FFU VirHRVat PID 28 and euthanized on postchallenge day (PCD) 7 as previously described (18,19) (Fig. 1A). Pigs were given 4 mL of 200 mM sodium bicarbonate to reduce gastric acidity 20 minutes before human rotavirus (HRV) inoculation/infection. Postchallenge, pigs were examined daily for clinical signs, and fecal swabs were collected daily as previously described (29). MNCs were isolated from the ileum, spleen, and peripheral blood of pigs euthanized on PID 28 or PCD 7 (19,29). All of the animal experimental procedures were conducted in accordance with protocols approved by Institutional Animal Care and Use Committees of Virginia Polytechnic Institute and State University.

FIGURE 1.

FIGURE 1.

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Numbers of pigs in each group (A), LGG feeding regimen (B), and LGG counts in fecal samples of Gn pigs fed with different doses of LGG or inoculated with/without AttHRV (C). Fecal samples were collected on PIDs 0, 5, 10, 21, and 28 for counting LGG numbers by plating the fecal samples on MSR (de Man, Rogosa, and Sharpe) selective medium. The LGG counts were recorded as CFU/mL. Data in “C” are presented as geometric mean counts standard error of the mean (n = 8–10). The symbol “*” indicates significant difference in LGG numbers between groups at the same time points (Kruskal–Wallis test, P < 0.05). AttHRV =attenuated human rotavirus; CFU = colony-forming units; Gn = gno-gnotobiotic; LGG = Lactobacillus rhamnosus GG; LGG5X = 5 doses of Lactobacillus rhamnosus GG; LGG9X = 9 doses of Lactobacillus rhamnosus GG; PCD = postchallenge day; PID = postinoculation day; PPD = postpartum day.

Detection of LGG Counts and Rotavirus Shedding and Assessment of Rotavirus-Caused Diarrhea

Fecal swabs were collected at PIDs 0, 5, 10, 21, and 28 for enumeration of LGG counts as previously described (18). They were also collected for 7 days after VirHRV challenge to assess rotavirus diarrhea and shedding. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid. Pigs with daily fecal scores of ≥2 were considered diarrheic. The mean cumulative score was calculated as the sum of daily fecal scores from PCDs 1 to 7 in each group divided by the numbers of pigs. Virus shedding was detected by ELISA and cell culture immunofluorescence assay in processed rectal swab fluids as described previously (34,35). A sample was positive for rotavirus shedding if the mean absorbance of replicates was greater than the mean absorbance of the negative controls plus 3 times standard deviations. Fecal samples from mock-infected Gn pigs were used as negative controls. Protection rate against diarrhea or shedding on challenge is calculated as (1 – [percentage of AttHRV-inoculated pigs with diarrhea or shedding/percentage of mock-inoculated control pigs with diarrhea or shedding]) × 100.

Assessment of Rotavirus-Specific IgA and IgG Antibody Responses in Serum and ASC and Memory B-Cell Responses in Intestinal and Systemic Lymphoid Tissues

Serum rotavirus-specific IgA and immunoglobulin G (IgG) antibody titers were measured using indirect isotype-specific antibody ELISAs as previously described (36,37). The MNCs from the ileum, spleen, and peripheral blood were stimulated in vitro with semipurified AttHRV antigen and then were subjected to enzyme-linked ImmunoSpot assays for detection of virus-specific IgA and IgG ASC (29) and IgA and IgG memory B cells (38) as previously described. The enzyme-linked ImmunoSpot plates were scanned with a CTL-ImmunoSpot S5 Core Analyzer (CTL Analyzers LLC, Shaker Heights, OH). Numbers of rotavirus-specific ASC or memory B cells were determined by counting blue spots in the wells and were reported as numbers per 5 × 105 of MNCs.

Flow Cytometry Analysis of Frequency of IFN-γ–Producing CD4+ and CD8+ T Cells, CD4+CD25–FoxP3+ Treg Cells, and TGF-β–Producing and IL-10–Producing CD4+CD25–FoxP3+ Treg Cells

The frequencies of HRV-specific IFN-γ–producing CD4+/CD8+ T cells among CD3+ cells, the frequencies of CD4+CD25–FoxP3+ Treg cells among MNCs, and the frequencies of TGF-γ–producing and IL-10–producing Treg cells in the ileum, intraepithelial lymphocytes (IEL), spleen, and blood of Gn pigs were determined by using intracellular cytokine staining and flow cytometry as previously described (19,30). MNCs were stimulated with semipurified AttHRV antigen in vitro for 17 hours before staining for detecting IFN-γ–producing T cells, and they were stained freshly without in vitro stimulation for detecting Treg cells. Only CD4+CD25–FoxP3+ Treg cells were included in the present study because our previous study (19) found that CD25– Treg cells, but not CD25+ Treg cells, were the predominant responding Treg cells after HRV infection or vaccination in Gn pigs.

Statistical Analysis

Nonparametric Kruskal-Wallis rank sum test was performed to compare LGG counts, frequencies of cytokine-producing T-cell subsets, frequencies of Treg cells, titers of serum IgA and IgG antibodies, and numbers of ASC and memory B cells among treatment groups at each time point. When differences among the groups were detected, the same test was used in a pairwise fashion to identify the nature of the differences. One-way analysis of variance–general linear model, followed by Duncan multiple range test, was used to compare mean onset days and mean duration days of virus shedding and diarrhea, mean cumulative fecal consistence scores, and mean peak fecal titers among treatment groups. Fisher exact test was used to compare percentage of pigs with diarrhea or virus shedding among treatment groups. Correlations between protection rates against diarrhea and virus shedding and mean numbers of ASC and memory B cells of the treatment groups were analyzed using Spearman rank correlation coefficient. The statistical significance was assessed at P < 0.05 and indicated with capital letters or asterisks. Different letters (A, B, C, D) indicate significant differences, whereas any shared letters (A, AB, and ABC) indicate no significant difference among groups (Table 1; Fig. 4). The asterisks above 2 bars connected by bracket or different numbers of asterisks on the top of bars indicate significant differences in Figures 1C, 2, 3A, 5, and 6. All of the statistical analyses were performed using SAS program 9.2 (SAS Institute, Cary, NC).

TABLE 1.

Clinical signs and rotavirus fecal shedding in Gn pigs after VirHRV challenge

Treatments n Clinical signs Fecal virus shedding (by CCIF and/or ELISA)
Percentage with diarrhea *,† Mean days to onset‡ Mean duration, days‡,§ Mean cumulative fecal score‡,|| Percentage of shedding virus* Mean days to onset‡ Mean duration, days‡ Mean peak titer, FFU/mL‡,
Controls 9 100A 1.4 (0.2#)C 5.6 (0.3)A 14.4 (1.0)A 100A 2.0 (0.3)B 4.7 (0.7)A 4558.1 (18473.7)A
LGG5X 4 100AB 4.0 (0.0)A 2.8 (0.3)BC 9.1 (1.3)BC 100AB 1.8 (0.5)B 6.3 (0.5)A 2111.4 (1974.0)A
LGG9X 4 100AB 1.8 (0.8)BC 3.8 (0.9)AB 11.9 (0.8)AB 100AB 2.0 (0.0)B 5.3 (0.5)A 7601.3 (2070.0)A
AttHRV 12 67AB 4.4 (0.8)AB 3.1 (0.7)BC 9.8 (1.4)BC 50B 6.0 (0.7)A 0.7 (0.2)B 4.9 (116.0)B
AttHRV+LGG5X 4 25B 6.5 (1.5)A 0.3 (0.3)D 6.3 (0.1)C 25B 6.3 (1.8)A 1.0 (1.0)B 6.3 (49.5)B
AttHRV+LGG9X 5 40B 5.6 (1.2)A 1.0 (0.4)CD 8.5 (0.5)BC 60AB 5.2 (1.2)A 0.8 (0.4)B 5.0 (39.6)B
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AttHRV = attenuated human rotavirus; CCIF = cell culture immunofluorescence; ELISA = enzyme-linked immunosorbent assay; FFU = focus-forming units; Gn = gnotobiotic; LGG5X = 5 doses of Lactobacillus rhamnosus GG; LGG9X = 9 doses of Lactobacillus rhamnosus GG; PCD = postchallenge day; VirHRV = virulent human rotavirus.

*

Fisher exact test followed by Duncan multiple range test was used for comparisons. Different letters indicate significant differences among treatment groups (P < 0.05), whereas shared letters indicate no significant difference.

†

Pigs with daily fecal scores of ≥2 were considered diarrheic. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid.

‡

One-way analysis of variance–general linear model followed by Duncan multiple range test was used for comparisons. Different letters indicate significant differences among treatment groups (P < 0.05), whereas shared letters indicate no significant difference.

§

For durations of diarrhea and virus shedding, if no diarrhea or virus shedding up to the euthanasia day (PCD 7), the duration (days) was recorded as 0 and the onset (days) was recorded as 8 for statistical analysis.

||

Mean cumulative fecal score calculation included all of the pigs in each group.

Geometric mean peak titers were calculated among pigs that shed virus.

#

Standard error of the mean.

FIGURE 4.

FIGURE 4.

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Rotavirus-specific IgA (A) and IgG (B) antibody responses in serum of Gn pigs vaccinated with AttHRV and fed with 2 different doses of LGG. Rotavirus-specific antibody titers were measured by an indirect isotype-specific antibody ELISA and presented as geometric mean titers + standard error of the mean (n = 4–12). Samples negative at a dilution of 1:4 were assigned a titer of 1:2 for the calculation of geometric mean antibody titers. Different letters (A, B, or C) indicate significant differences in antibody titers compared among different groups at the same time points (Kruskal-Wallis test, P < 0.05), whereas shared letters indicate no significant difference. AttHRV =attenuated human rotavirus; ELISA = enzyme-linked immunosorbent assay; Gn = gnotobiotic; IgA = immunoglobulin A; IgG = immunoglobulin G; LGG = Lactobacillus rhamnosus GG; LGG5X = 5 doses of Lactobacillus rhamnosus GG; LGG9X = 9 doses of Lactobacillus rhamnosus GG; PID = postinoculation day.

FIGURE 2.

FIGURE 2.

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Rotavirus-specific IgA and IgG memory B-cell responses in Gn pigs vaccinated with AttHRV and fed with 2 different doses of LGG at PID 28. The MNCs isolated on PID 28 (PCD 0) were stimulated with semipurified AttHRV antigen for 96 hours. The numbers of rotavirus-specific IgA and IgG memory B cells were enumerated by using the ELISPOT assay. Data are presented as geometric mean numbers ± standard error of the mean (n = 4–12). The symbol “*” indicates significant difference between groups for the same isotype (Kruskal-Wallis test, P < 0.05). AttHRV =attenuated human rotavirus; Gn = gnotobiotic; IgA = immunoglobulin A; IgG = immunoglobulin G; LGG = Lactobacillus rhamnosus GG; LGG5X = 5 doses of Lactobacillus rhamnosus GG; LGG9X = 9 doses of Lactobacillus rhamnosus GG; MNC = mononuclear cell; PCD = postchallenge day; PID = postinoculation day.

FIGURE 3.

FIGURE 3.

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Rotavirus-specific IgA and IgG ASC responses in Gn pigs vaccinated with AttHRV and fed with 2 different doses of LGG at PID 28 (A) and correlations of mean IgA ASC and memory B-cell numbers at challenge (PID 28) with protection rates against diarrhea (B). The numbers of rotavirus-specific IgA and IgG ASCs were enumerated by using the ELISPOT assay. Data in “A” are presented as geometric mean numbers ±- standard error of the mean (n = 4–12). See legend of Figure 2 for statistical analysis. Correlations between protection rates against diarrhea and mean numbers of ASC and memory B cells of the treatment groups were analyzed using Spearman rank correlation coefficient and are shown in “B.” ASC = antibody-secreting cell; AttHRV = attenuated human rotavirus; Gn = gnotobiotic; IgA = immunoglobulin A; IgG = immunoglobulin G; LGG = Lactobacillus rhamnosus GG; LGG5X = 5 doses of Lactobacillus rhamnosus GG; LGG9X = 9 doses of Lactobacillus rhamnosus GG; MNC = mononuclear cell; PID = postinoculation day.

FIGURE 5.

FIGURE 5.

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Rotavirus-specific IFN-γ–producing T-cell responses in Gn pigs vaccinated with AttHRV and fed with 2 different doses of LGG. MNCs were stimulated with semipurified AttHRV antigen in vitro for 17 hours. Brefeldin A was added for the last 5 hours to block secretion of cytokines produced by T cells. IFN-γ production was detected by intracellular staining and flow cytometry. Data are presented as mean frequency ± standard error of the mean (n = 4–12). The top 2 figures show the frequencies of IFN-γ + CD4+ T cells among CD3+ cells and the bottom 2 show those of IFN-γ + CD8+ T cells among CD3+ cells. Figures in the left panel show the prechallenge data, and those in the right panel show the postchallenge data. The symbol “*” or the different numbers of symbol “*” indicate significant difference between groups (Kruskal–Wallis test, P < 0.05). AttHRV = attenuated human rotavirus; Gn = gnotobiotic; IEL = intraepithelial lymphocyte; IFN = interferon; LGG = Lactobacillus rhamnosus GG; LGG5X = 5 doses of Lactobacillus rhamnosus GG; LGG9X = 9 doses of Lactobacillus rhamnosus GG; MNC = mononuclear cell; PCD = postchallenge day; PID = postinoculation day.

FIGURE 6.

FIGURE 6.

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Treg cell responses in Gn pigs vaccinated with AttHRV and fed with 2 different doses of LGG. MNCs were stained freshly without in vitro stimulation. Data are presented as mean frequency standard error of the mean (n = 4–9). The 2 figures in the first row show the frequencies of CD4+CD25+FoxP3+ Tregs among MNCs and those in the second row show the frequencies of CD4+CD25–FoxP3+ Tregs among MNCs, and the 2 figures in the third row and the bottom 2 show the frequencies of TGF-γ+ cells and those of IL-10+ cells among the CD4+CD25–FoxP3+ Tregs, respectively. See Figure 5 for additional panel description and statistical analysis. AttHRV =attenuated human rotavirus; Gn = gnotobiotic; IEL = intraepithelial lymphocyte; IL = interleukin; LGG = Lactobacillus rhamnosus GG; MNC = mononuclear cell; PCD = postchallenge day; PID = postinoculation day; Treg = regulatory T; TGF = transforming growth factor.

RESULTS

Fecal LGG Counts in AttHRV + LGG9X Pigs Were Significantly Higher Than the AttHRV + LGG5X Pigs at PID 10 But Were the Same at PID 28

LGG colonization in all of the LGG-fed Gn pigs was confirmed by LGG enumeration in rectal swab samples collected on PIDs 0, 5, 10, 21, and 28, and the fecal shedding data from AttHRV-vaccinated and LGG-fed pigs are presented in Fig. 1C. Exposure to AttHRV did not significantly influence LGG CFU in the LGG5X pigs, but significantly increased LGG CFU in LGG9X pigs on PIDs 5 and 10. The similar effect of VirHRV on LGG shedding was discussed in detail in our previously published study (39). AttHRV + LGG9X pigs shed higher counts of LGG than AttHRV + LGG5X pigs at PID 5 (LGG feeding period is between postpartum days 3 and 11) and significantly higher counts at PID 10 (4 days after the LGG feeding period ends), reflecting the difference in LGG feeding doses. By PIDs 21 to 28, LGG shedding counts in the 2 vaccine groups become similar and maintained between 1.9 × 107 and 2.9 × 107 CFU/mL.

Protection of Both LGG Feeding Regimens Enhanced AttHRV Vaccine Against Rotavirus Diarrhea With LGG5X Being Slightly More Effective

Data of clinical signs and fecal virus shedding of the Gn pigs after VirHRV challenge are summarized in Table 1. All of the AttHRV-vaccinated pigs had significantly shorter duration of diarrhea and significantly reduced cumulative fecal scores compared with the controls. Compared with AttHRV-alone pigs, vaccinated and LGG-fed pigs (AttHRV + LGG5X and AttHRV + LGG9X groups) had further reduced incidence (%), duration, and cumulative fecal score of rotavirus diarrhea and delayed onset of diarrhea, but these differences were not statistically significant (except the duration of diarrhea between AttHRV + LGG5X and AttHRV groups). Further comparing the 2 vaccine groups, pigs in the AttHRV + LGG5X group had a lower percentage of diarrhea and virus shedding, shorter duration of diarrhea, and lower cumulative fecal scores than those in the AttHRV + LGG9X group, but these differences were not statistically significant. Interestingly, LGG5X, but not LGG9X, feeding alone significantly delayed onset of diarrhea, shortened duration of diarrhea, and lowered cumulative fecal scores compared with the controls.

LGG5X Significantly Enhanced Intestinal Memory B-Cell Responses to AttHRV

Virus-specific intestinal ASC and memory B-cell responses at challenge have been most closely correlated with protective immunity against rotavirus diarrhea in Gn pigs (40). For memory B-cell responses in the present study, AttHRV + LGG5X pigs had the highest numbers of IgA and IgG memory B cells in the ileum among the 4 groups at PID 28 (Fig. 2). AttHRV + LGG5X pigs had significantly higher numbers of IgA and IgG memory B cells than AttHRV + LGG9X pigs and significantly higher numbers of IgG memory B cells than AttHRV-alone pigs in the ileum. Both groups had significantly higher IgA memory B cells than AttHRV-alone pigs in the blood. AttHRV + LGG5X pigs also had significantly higher IgG memory B cells than AttHRV + LGG9X pigs in the blood. Conversely, AttHRV + LGG9X pigs had significantly higher numbers of IgA and IgG memory B cells than the other groups in the spleen (Fig. 2). Overall memory B-cell responses induced by AttHRV + LGG5X occurred primarily in the ileum and blood (no memory B cells were detected in the spleen), whereas memory B cells induced by AttHRV + LGG9X reside primarily in the spleen.

LGG5X Significantly Enhanced Virus-Specific Intestinal IgA ASC Responses to AttHRV

AttHRV + LGG5X pigs had significantly higher numbers of virus-specific IgA ASC in the ileum compared with AttHRV-alone pigs at PID 28 (Fig. 3A). AttHRV + LGG9X pigs had significantly higher numbers of IgG ASC in the spleen compared with AttHRV + LGG5X and AttHRV-alone pigs, although the numbers are much lower than those in the ileum (note the different Y scale) (Fig. 3A). Thus, LGG5X was slightly more effective than LGG9X in enhancing virus-specific ASC responses to the AttHRV vaccine and the 2 regimens had a differential effect on the tissue distribution (ileum vs spleen) of the ASC and memory B-cell responses. There were no significant differences in virus-specific ASC responses in the blood among AttHRV, AttHRV + LGG5X, and AttHRV + LGG9X pig groups. The mean numbers of virus-specific IgA ASC in the ileum and blood and IgA memory B cells in the blood at PID 28 among the 4 treatment groups were positively correlated with protection rates against HRV diarrhea (Fig. 3B). The IgG ASC and memory B cells in all of the tissues, IgA memory B cells in the ileum and spleen, and IgA ASC in the spleen did not significantly correlate with protection rates (Fig. 3B; only ileum IgA memory B cells are shown as an example).

Responses of Both LGG Feeding Regimens Enhanced Rotavirus-Specific IgA Serum Antibody to AttHRV

Both LGG5X and LGG9X feedings significantly increased virus-specific serum IgA antibody titers with the same magnitude in AttHRV-vaccinated pigs from PID 21 to PCD 7 compared with AttHRV-alone pigs (Fig. 4). At PCD 7, both LGG feeding regimens significantly enhanced virus-specific serum IgG antibody titers compared with AttHRV-alone pigs. AttHRV + LGG9X pigs had significantly higher IgG antibody titers at PID 28 and PCD 2 than AttHRV + LGG5X and AttHRV-alone pigs, and significantly higher IgG antibody titers at PID 14 and PCD 4 compared with AttHRV-alone pigs. Notably, AttHRV + LGG9X pigs had significantly lower IgA antibody titers at PID 10 compared with AttHRV-alone pigs, and also significantly lower IgA titers at both PIDs 10 and 14 compared with AttHRV + LGG5X pigs. Corroborating with the trends in ASC and memory B-cell responses, LGG9X promoted stronger systemic IgG antibody responses than LGG5X (Fig. 4).

Both LGG Feeding Regimens Were Effective in Enhancing Rotavirus-Specific IFN-γ–Producing T-Cell Responses to AttHRV Vaccine, With LGG9X Being More Effective

Rotavirus-specific IFN-γ–producing T cells also play an important role in protective immunity against rotavirus diarrhea (30). LGG9X feedings overall enhanced virus-specific IFN-γ–producing T-cell responses in all of the tissues of AttHRV-vaccinated pigs (Fig. 5). AttHRV + LGG9X pigs had significantly higher frequencies of IFN-γ + CD4+ T cells in the spleen and blood and IFN-γ + CD8+ T cells in all of the tissues at PID 28 than AttHRV-alone pigs. Compared with AttHRV + LGG5X pigs, AttHRV + LGG9X pigs had significantly higher frequencies of IFN-γ + CD4+ T cells in the spleen and blood and IFN-γ + CD8+ + T cells in the ileum, IEL, and spleen at PID 28. Postchallenge, AttHRV + LGG9X pigs again had significantly higher frequencies of IFN-γ + CD8+ T cells in the ileum and blood than AttHRV-alone pigs and significantly higher frequencies of IFN-γ + CD4+ and IFN-g + CD8+ T cells in the blood compared with AttHRV + LGG5X pigs (Fig. 5). Notably, compared with AttHRV-alone pigs, AttHRV + LGG5X pigs also had significantly higher frequencies of virus-specific IFN-γ + CD4+ T cells in IEL at PID 28 and IFN-γ + CD8+ T cells in the ileum and blood at both PID 28 and PCD 7 but significantly lower frequencies of IFN-γ + CD4+ T cells in the spleen at PID 28 (Fig. 5).

Both LGG Feeding Regimens Maintained the CD4+CD25+ Treg Population in Ileum and IEL at PCD 7

Compared with AttHRV-alone pigs, AttHRV + LGG5X pigs had significantly lower frequencies of CD4+CD25+ Tregs in the spleen and blood at PID 28 but significantly higher frequencies of CD4+CD25+ Tregs in the ileum and IEL at PCD 7 (Fig. 6). AttHRV + LGG9X pigs also had significantly lower frequencies of CD4+CD25+Tregs in the blood at PID 28 but significantly higher frequencies of CD4+CD25+ Tregs in the ileum and IEL at PCD 7 (Fig. 6).

LGG Feeding Downregulated CD4+CD25– Tregs in Most Tissues of AttHRV-Vaccinated Pigs, but LGG9X Maintained the CD4+CD25–Treg Population in Ileum and IEL, As Well As TGF-β–Producing and IL-10–Producing CD4+CD25– Tregs in Blood

AttHRV + LGG5X pigs had significantly lower frequencies of CD4+CD25– Tregs in the spleen and blood at PID 28 and in all of the tissues at PCD 7 than AttHRV-alone pigs (Fig. 6). LGG5X feedings also significantly reduced frequencies of TGF-β + CD4+CD25– Tregs in the ileum, IEL, and blood and of IL-10 + CD4+CD25– Tregs in the ileum, spleen, and blood at PID 28, as well as of TGF-β + CD4+CD25– Tregs in the ileum and spleen and of IL-10 + CD4+CD25– Tregs in the ileum at PCD 7 (Fig. 6).

Compared with AttHRV-alone pigs, AttHRV + LGG9X pigs also had significantly lower frequencies of CD4+CD25– Tregs in the blood at PID 28 and in the spleen and blood at PCD 7, significantly lower frequencies of TGF-β + CD4+CD25– Tregs in the ileum, IEL, and spleen at PID 28 and in the ileum at PCD 7, and significantly lower frequencies of IL-10 + CD4+CD25– Tregs in the ileum and spleen at PID 28 and in the ileum at PCD 7 (Fig. 6). There were no significant differences in the frequencies of CD4+CD25– Tregs in the ileum and IEL or TGF-β–producing and IL-10–producing Tregs in the blood between AttHRV + LGG9X and AttHRV-alone groups at both PID 28 and PCD 7.

DISCUSSION

Our previous studies of probiotics have suggested that the strain, the timing of initial colonization, and the dosage of probiotics are critical factors for establishing the optimal immune-enhancing effects of probiotics for vaccines against VirHRV infection and for their direct effect on reducing rotavirus diarrhea (18,41). With 2 different LGG feeding regimens in the present study, we demonstrated for the first time that different probiotic dosages preferentially promoted intestinal and systemic IgA and IgG memory B-cell responses and IFN-γ–producing T-cell responses. Preferential immunoregulatory effects of probiotics also resulted in different outcomes in protection against rotavirus diarrhea. The most intriguing findings from the present study include the following: LGG5X and LGG9X dosages differentially enhanced rotavirus-specific memory B-cell responses in different lymphoid tissues (LGG5X in the ileum and LGG9X in the spleen), but only LGG5X significantly enhanced virus-specific IgA ASC responses in the ileum at challenge (PID 28); both dosages enhanced rotavirus-specific serum IgA responses, but only LGG9X enhanced virus-specific serum IgG responses at challenge and soon after (PCD 2); both dosages enhanced virus-specific IFN-γ–producing CD8+ T-cell responses in the ileum and blood, but LGG9X was more effective and only LGG9X enhanced IFN-γ–producing CD4+ T-cell responses in the spleen and blood at PID 28; both dosages downregulated Treg cell responses in most tissues, but LGG9X maintained normal levels of CD4+CD25– Treg cells in the ileum and IEL and TGF-β–producing and IL-10–producing CD4+CD25– Treg cells in the blood of the AttHRV + LGG9X pigs; and both LGG doses enhanced the AttHRV vaccine’s protection rate against rotavirus diarrhea, with LGG5X being slightly more effective; however, only LGG5X alone significantly reduced the mean duration of diarrhea.

In the present study, there were significant correlations between the numbers of virus-specific IgA ASC in the ileum and blood and IgA memory B cells in the blood at challenge (PID 28) and protection rates against rotavirus diarrhea among groups. AttHRV + LGG5X significantly enhanced the IgA ASC responses in the ileum and blood, corresponding to the highest protection rate. In comparison, although AttHRV + LGG9X significantly enhanced the virus-specific IgG ASC response and IgA and IgG memory B-cell responses in the spleen, the regimen was less effective than AttHRV + LGG5X in conferring protection. These results are consistent with our previous findings (29,40) and support the notion that the magnitude of virus-specific intestinal IgA ASC responses induced by rotavirus vaccines are a reliable indicator of protective efficacy and circulating IgA B-cell responses may be used as a window to monitor intestinal immunity against rotavirus diarrhea.

The mechanism for the observed dose effects is unknown. Although pigs between the 2 dosage groups showed no significant difference in fecal LGG shedding at PID 28, they received LGG in different frequencies. Pigs in LGG5X group received 1 dose of LGG every other day and pigs in LGG9X group received 1 dose daily between 3 and 11 days of age. The difference in LGG dosing frequencies may have regulated intestinal antigen-presenting cells differently, leading to different adaptive immune responses. In order to examine this hypothesis, the immunomodulating effects on antigen-presenting cell responses of different LGG dosing regimens will need to be investigated during and shortly after LGG feedings in future studies.

LGG alone has been extensively used to prevent or treat rotavirus diarrhea, showing a modest effect in reducing the diarrhea duration (20,22–24,39,42–44). Our study concurred with those reports in that LGG5X alone, compared with mock controls, delayed the onset of diarrhea, shortened the duration of diarrhea, and lowered the cumulative fecal scores. We did not measure the total intestinal immunoglobulin levels in the pigs. The stronger mucosal antibody responses induced by the AttHRV + LGG5X regimen, the higher virus-specific intestinal IgA responses, suggest that the LGG5X alone may have also stimulated production of more nonspecific intestinal IgA antibody than LGG9X, which may explain the increased protection against the severity of rotavirus diarrhea.

The present study was intended to expand our previous findings on the dose effects of LA on modulating T- and B-cell immune responses and protection against rotavirus diarrhea (18,19). The LA dose-response studies demonstrated that low-dose LA (5 doses) significantly enhanced effector T-cell responses to the AttHRV vaccine and reduced Treg responses, but high-dose LA (14 doses) significantly downregulated effector T-cell responses and upregulated Treg responses (19). Intermediate-dose LA (9 doses) did not have significant effects on T-cell responses (unpublished data), but significantly enhanced ASC and memory B-cell responses and serum rotavirus-specific antibody titers induced by the AttHRV vaccine. LA enhanced the protection rate against rotavirus diarrhea on VirHRV challenge only at the intermediate dose (18). It also dose-dependently regulated innate immune responses of cytokine-producing dendritic cells and Toll-like receptor– expressing antigen-presenting cells (45). Our studies revealed that the dose-dependent immunomodulating characteristics and the optimal dose for LA and LGG as vaccine adjuvant are different. The optimal dose for LA is the intermediate 9 doses, whereas for LGG it depends on the desired type of the protective immune response, that is, LGG5X promotes better mucosal IgA response and LGG9X stronger systemic IgG and IFN-γ–producing T-cell responses.

This is the first time that divergent dosage-dependent immunomodulating effects of probiotics are comprehensively described in an in vivo study. Specific probiotics at different doses differentially modulated immune responses to an oral vaccine to favor either mucosal IgA or systemic IgG and TH1-type responses. Both humoral and cellular immune responses can be enhanced to increase the vaccine-induced protective immunity against rotavirus diarrhea; enhancing intestinal and circulating IgA B-cell responses is likely to be the most effective approach. Properly selecting the specific strain of probiotics and the dosage based on the understanding of their immunomodulating characteristics is critical for establishing the optimal immune-enhancing effects of probiotic vaccine adjuvants.

What Is Known

  • Specific strains of lactobacilli have adjuvanticity.

  • Differential immunomodulatory dose effects of probiotics have started to be recognized.

  • In our previous study, 5 doses of Lactobacillus acidophilus NCFM enhanced interferon (IFN)-γ+ T-cell responses, whereas 9 doses enhanced B-cell and serum antibody responses to the attenuated human rotavirus vaccine in neonatal gnotobiotic pigs.

What Is New

  • The present study demonstrated that Lactobacillus rhamnosus GG (LGG) at both doses enhanced IFN-γ+ T-cell and serum immunoglobulin A antibody responses to attenuated human rotavirus.

  • LGG5X, however, promotes better mucosal immunoglobulin A responses, whereas LGG9X promotes stronger systemic immunoglobulin G and IFN-γ+ T-cell responses.

  • Probiotic strain and dose selection is critical for archiving optimal adjuvant effects.

Acknowledgments:

We thank Pete Jobst, Andrea Pulliam, Mariah Weiss, Kimberly Allen, and Shannon Viers for animal care, and Dr Kevin Pelzer, Dr Marlice Vonck, Dr Nicole Lindstrom, and Betsy Midkiff for veterinary service.

The work was supported by a grant (R01AT004789) from the National Center of Complementary and Alternative Medicine (NCCAM), National Institutes of Health, Bethesda, MD.

Footnotes

The authors report no conflicts of interest.

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