**2. Dose effects of LA on T cell responses**

Probiotics have been reported to exert adjuvant properties by inducing pro-Th1 cytokines and promote Th1 type immune responses. For example, *L. lactis* and *L. plantarum* induced production of IL-12 and IFN-γ by splenocytes when the LAB and an allergen were coadministered intranasally to mice (Repa et al., 2003). *L. fermentum* strain CECT5716 enhanced the Th1 responses induced by an influenza vaccine in addition to enhancing virus neutralizing antibody responses (Olivares et al., 2007). Eleven different probiotic strains were tested for cytokine production in human peripheral blood mononuclear cells (MNC) and each tested bacterium was shown to induce production of TNF-α and some strains also induced production of IL-12 and IFN-γ (Kekkonen et al., 2008). Previous studies of gnotobiotic pigs showed that a mixture of LA NCFM strain and *L. reuteri* strain enhanced both Th1 (IL-12, IFN-γ) and Th2 (IL-4 and IL-10) cytokine responses to virulent HRV infection (Azevedo et al., 2012)*.* LA NCFM strain enhanced the HRV-specific IFN-γ producing CD8+ T cell response to a rotavirus vaccine in gnotobiotic pigs, indicating adjuvanticity of the LA strain (Zhang et al., 2008b).

Dose effects of probiotics on modulating T cell immune responses have not been well studied. To address this question, we examined the dose effects of LA NCFM (NCK56) strain on IFN-γ producing CD4+ and CD8+ T cell immune responses induced by an oral rotavirus vaccine in gnotobiotic pigs (Wen et al., 2012). The animal treatment groups included (1) high dose LA plus AttHRV vaccine (HiLA+AttHRV), (2) low dose LA plus 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 2.11 x 106 CFU.

## **2.1. Low dose LA, but not high dose LA, enhanced HRV-specific IFN-γ producing T cell responses**

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 HiLA+HRV pigs compared to AttHRV only pigs.


(Summarized from Wen et al., 2012)

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

and challenged with the virulent HRV.

**2. Dose effects of LA on T cell responses** 

adjuvanticity of the LA strain (Zhang et al., 2008b).

vaccines as well.

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

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

Probiotics have been reported to exert adjuvant properties by inducing pro-Th1 cytokines and promote Th1 type immune responses. For example, *L. lactis* and *L. plantarum* induced production of IL-12 and IFN-γ by splenocytes when the LAB and an allergen were coadministered intranasally to mice (Repa et al., 2003). *L. fermentum* strain CECT5716 enhanced the Th1 responses induced by an influenza vaccine in addition to enhancing virus neutralizing antibody responses (Olivares et al., 2007). Eleven different probiotic strains were tested for cytokine production in human peripheral blood mononuclear cells (MNC) and each tested bacterium was shown to induce production of TNF-α and some strains also induced production of IL-12 and IFN-γ (Kekkonen et al., 2008). Previous studies of gnotobiotic pigs showed that a mixture of LA NCFM strain and *L. reuteri* strain enhanced both Th1 (IL-12, IFN-γ) and Th2 (IL-4 and IL-10) cytokine responses to virulent HRV infection (Azevedo et al., 2012)*.* LA NCFM strain enhanced the HRV-specific IFN-γ producing CD8+ T cell response to a rotavirus vaccine in gnotobiotic pigs, indicating

Dose effects of probiotics on modulating T cell immune responses have not been well studied. To address this question, we examined the dose effects of LA NCFM (NCK56) strain on IFN-γ producing CD4+ and CD8+ T cell immune responses induced by an oral rotavirus vaccine in gnotobiotic pigs (Wen et al., 2012). The animal treatment groups included (1) high dose LA plus AttHRV vaccine (HiLA+AttHRV), (2) low dose LA plus

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