**1. Introduction**

262 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

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Probiotics can influence both mucosal and systemic immune responses and function as adjuvants by promoting proinflammatory cytokine production, enhancing both humoral and cellular immune responses. Adjuvant effects of several probiotic lactic acid bacteria (LAB), mostly *Lactobacillus* strains, including *L. rhamnosus* GG, *L. acidophilus* NCFM, *L. acidophilus* CRL431, *L. acidophilus* La-14, *L. fermentum* CECT5716, *L. casei* DN-114 001, and *Bifidobacterium lactis* Bl-04 have been reported in studies of influenza, polio, rotavirus and cholera vaccines and rotavirus and Salmonella typhi Ty21a infections (Boge et al., 2009; Davidson et al., 2011; Isolauri et al., 1995; Kaila et al., 1992; Mohamadzadeh et al., 2008; Olivares et al., 2007; Paineau et al., 2008; Winkler et al., 2005; Zhang et al., 2008b). The word adjuvant in the phrase "probiotic adjuvant" is not used in its traditional definition in which adjuvant implies a substance included in the vaccine formulation to aid the immune response to the vaccine antigen. Instead, probiotic adjuvants enhance the immunogenicity of vaccines when orally administered repeatedly around the time of vaccination and separately from the vaccine. By skewing the balance of pro- and anti-inflammatory innate immune responses and T helper (Th) 1 and regulatory T (Treg) cell adaptive immune responses in the context of vaccination, probiotic adjuvants act as "signal zero" to reduce Treg cell suppression and unleash effector T cell activation (Rowe et al., 2012).

Although the strain-specific effects of LAB in up- or down-regulating inflammatory immune responses have been well recognized, dose effects of probiotics on innate and adaptive immune responses are not clearly understood. The same *Lactobacillus* strain is oftentimes reported by different research groups to have opposite immune modulating effects. We hypothesized that the dose effect is at least one of the reasons for the conflicting reports. Understanding dose effects of probiotics has significant implications in their use as immunostimulatory (adjuvants) versus immunoregulatory agents.

© 2013 Yuan et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In this chapter, we discuss findings from our serial studies of gnotobiotic pigs on the dose effects of the *L. acidophilus* NCFM strain (LA) on innate and adaptive immune responses induced by an oral attenuated human rotavirus (HRV) vaccine (AttHRV). We studied the effects of low dose (total 2.11 x 106 CFU) and high dose (total 2.22 x 109 CFU) LA on the intestinal and systemic (1) rotavirus-specific IFN-γ producing CD4+ and CD8+ T cell responses; (2) CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ Treg cell responses and the regulatory cytokine TGF-β and IL-10 production; (3) rotavirus-specific antibody-secreting cell (ASC) and serum antibody responses; and (4) plasmacytoid dendritic cell (pDC) and conventional DC (cDC) frequencies, activation status, TLR expression and cytokine production profile. The protective effect of rotavirus vaccine against virus shedding and diarrhea was assessed in AttHRV-vaccinated gnotobiotic pigs fed with high, low, or no LA and challenged with the virulent HRV.

Dose Effects of LAB on Modulation of Rotavirus Vaccine Induced Immune Responses 265

AttHRV (LoLA+AttHRV), (3) AttHRV only, (4) high dose LA only (HiLA), (5) low dose LA only (LoLA), and (6) mock-inoculated control (Mock). Gnotobiotic pigs were orally inoculated at 5 days of age with the AttHRV vaccine at 5 x107 fluorescent focus-forming units (FFU) per dose. A booster dose was given 10 days later at the same dose and route. Subsets of the pigs were euthanized at post-inoculation day (PID) 28 to assess immune responses and the rest were challenged with the homotypic virulent Wa (G1,P1A[8]) strain HRV at a dose of 1 x 105 FFU to assess protection from post-challenge day (PCD) 1 to 7. The 50% infectious dose and 50% diarrhea dose of the virulent HRV in gnotobiotic pigs are approximately 1 FFU (Ward et al., 1996). The AttHRV inoculation causes virus shedding in about 6% pigs, but it does not cause any illness (Ward et al., 1996). Pigs in the high dose LA groups were fed daily with 103 to 109 CFU/dose of LA for 14 days with 10-fold incremental dose increases every other day from 3-16 days of age. The accumulative total LA dose was 2.22 x 109 CFU. Pigs in the low dose LA groups were fed with 103, 104, 105, 106, and 106 CFU/dose of LA every other day from 3-11 days of age. The accumulative total LA dose was

**2.1. Low dose LA, but not high dose LA, enhanced HRV-specific IFN-γ**

The magnitude of HRV-specific IFN-γ producing T cell responses in pigs was differentially modulated by low versus high dose LA at both prechallenge and postchallenge (PID 28 and PCD 7). AttHRV-vaccinated and low dose LA fed pigs (LoLA+AttHRV) had significantly higher frequencies of HRV-specific IFN-γ+CD8+ T cells in ileum (11- and 5-fold higher preand postchallenge, respectively), spleen (3.8- and 2.1-fold higher pre- and postchallenge, respectively) and blood (3- and 20-fold higher pre- and postchallenge, respectively) compared to the AttHRV only pigs (Table 1). The LoLA+AttHRV pigs also had significantly higher frequencies of HRV-specific IFN-γ+CD4+ T cells in blood (3-fold higher for both preand postchallenge) compared to the AttHRV only pigs. In contrast, high dose LA did not significantly alter the HRV-specific IFN-γ producing CD4+ and CD8+ T cell responses in the

> Frequencies of IFN-γ+CD8+ T cells among CD3+ cells PID 28 PCD 7

HiLA+AttHRV 0.05 0.34 0.05 0.11 0.16 0.06 LoLA+AttHRV 1.21 0.46 0.24 0.56 0.46 0.98 AttHRV only 0.11 0.12 0.08 0.11 0.22 0.05

**Table 1.** Effect of low dose vs. high dose LA on IFN-γ producing CD8+ T cell responses

Ileum Spleen Blood Ileum Spleen Blood

2.11 x 106 CFU.

**producing T cell responses** 

(Summarized from Wen et al., 2012)

HiLA+HRV pigs compared to AttHRV only pigs.

These studies clearly demonstrated differential immune modulating effects of high dose versus low dose LA on DC and T cell responses, and consequently different effects on the protection conferred by the AttHRV vaccine in gnotobiotic pigs challenged with virulent HRV. Low dose LA enhanced the protection against rotavirus diarrhea in AttHRVvaccinated pigs whereas high dose LA had negative effects on the effectiveness of the vaccine. Thus, the same probiotic strains at different doses can exert qualitatively different modulating effects on immune responses induced by rotavirus vaccines and possibly other vaccines as well.
