**5. Dose effects of LA on protection conferred by the oral AttHRV vaccine against virulent HRV challenge**

To examine the effects of low and high dose LA on improving the protection conferred by the AttHRV vaccine, subsets of gnotobiotic pigs from each treatment group were challenged with the virulent HRV Wa strain at PID 28. Clinical signs and virus shedding were monitored for 7 days postchallenge (Table 4).

After challenge, although the proportion of pigs that developed virus shedding and diarrhea did not differ significantly among the three AttHRV vaccinated pig groups, the LoLA+AttHRV group had the shortest mean durations of fecal virus shedding and diarrhea and the lowest mean cumulative fecal consistency score among all the treatment groups. The durations of diarrhea in the LoLA+AttHRV pigs were significantly shorter compared to the AttHRV only and the mock-vaccinated control pigs. The durations of virus shedding in the LoLA+AttHRV pigs were significantly shorter compared to the HiLA+AttHRV and the mock control pigs. The mean cumulative fecal consistency scores in all the pigs in the LoLA+AttHRV and AttHRV only groups (8.4 and 9.0, respectively) were significantly lower than the control group, indicating significant protection against the severity of diarrhea. Thus, low dose LA slightly, but clearly improved the protection conferred by the AttHRV vaccine against rotavirus diarrhea. In contrast, high dose LA reduced the protection conferred by the AttHRV vaccine as indicated by the significantly longer mean duration of virus shedding (3.8 versus 1.3 days) and higher mean cumulative fecal scores compared to the AttHRV only pigs.


<sup>∆</sup> The data was partially presented previously (Wen et al., 2012). a 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.

b Standard error of the mean.

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

molecular mechanisms of the dose responses of different MAPM.

p < 0.05), while shared letters indicate no significant difference.

**against virulent HRV challenge** 

monitored for 7 days postchallenge (Table 4).

on DCs and consequently on adaptive immune responses induced by rotavirus vaccines. It has been reported that the effect of low dose microbe-associated pattern molecular (MAPM), such as lipopolysaccharide, was strikingly different as compared to that of high dose on macrophage cell functions: low dose lipopolysaccharide induced a strong inflammatory response in macrophages (Maitra et al., 2011). It is plausible that a similar interaction occurs between the MAPM from LA and DCs in the gut. Future studies are needed to identify the

**Figure 5.** Cytokine production profiles of pDC and cDC in intestinal and systemic lymphoid tissues of Gn pigs vaccinated with AttHRV vaccine with high dose, low dose or no LA at PID 28. MNC were stained freshly without *in vitro* stimulation before flow cytometry analyses. Data are presented as mean frequency ± standard error of the mean (n = 3-8). Different letters on top of bars indicate signicant differences in frequencies among groups for the same cytokine in the same tissue (Kruskal–Wallis test,

**5. Dose effects of LA on protection conferred by the oral AttHRV vaccine** 

To examine the effects of low and high dose LA on improving the protection conferred by the AttHRV vaccine, subsets of gnotobiotic pigs from each treatment group were challenged with the virulent HRV Wa strain at PID 28. Clinical signs and virus shedding were

After challenge, although the proportion of pigs that developed virus shedding and diarrhea did not differ significantly among the three AttHRV vaccinated pig groups, the LoLA+AttHRV group had the shortest mean durations of fecal virus shedding and diarrhea c Mean cumulative score calculation included all the pigs in each group.

d FFU, fluorescent focus forming units. Geometric mean peak titers were calculated among pigs that shed virus.

\* Proportions in the same column with different superscript letters (A, B) differ significantly (Fisher's exact test,

<sup>p</sup>≤0.05). \*\* Means in the same column with different superscript letters (A, B, C) differ significantly (Kruskal Wallis Test, p≤0.05).

**Table 4.** Clinical signs and rotavirus fecal shedding in Gn pigs after virulent HRV challenge<sup>∆</sup>

We reported previously that protection rates against rotavirus diarrhea are correlated with virus-specific intestinal IgA ASC and IFN-γ producing T cell responses at PID 28 in Gn pigs (Yuan et al., 1996; Yuan et al., 2008). A balanced Th1 and Th2 type response is needed for the optimal protective immunity against rotavirus. Although low dose LA further reduced the duration of diarrhea in the AttHRV-vaccinated pigs postchallenge, neither low nor high dose LA significantly altered protection rate against rotavirus challenge (proportions of pigs that were infected and developed diarrhea after challenge). Because virus-specific intestinal IgA ASC responses probably play a more important role in rotavirus protective immunity than the IFN-γ producing CD8+ T cell responses (Yuan et al., 1996; Yuan et al., 2008), the effect of LA on virus-specific ASC responses also need to be taken into consideration regarding the differences in the protection conferred by the AttHRV vaccine with high or low dose LA. Although the low dose LA enhanced IFN-γ producing CD8+ T cell responses, it had negative effects on the serum antibody and ASC responses induced by the AttHRV vaccine. To improve the AttHRV vaccine efficacy, a different dose of LA (possible an intermediate dose) or a different probiotic strain (i.e. LGG) may be optimal to promote a balanced Th1 and Th2 response without increasing Treg cell responses.

Dose Effects of LAB on Modulation of Rotavirus Vaccine Induced Immune Responses 277

Studies of probiotics' immune modulating effects on the rotavirus vaccine in gnotobiotic pigs were supported by a grant (R01AT004789) from the National Center of Complementary

Armah, G.E., Sow, S.O., Breiman, R.F., Dallas, M.J., Tapia, M.D., Feikin, D.R., Binka, F.N., Steele, A.D., Laserson, K.F., Ansah, N.A., Levine, M.M., Lewis, K., Coia, M.L., Attah-Poku, M., Ojwando, J., Rivers, S.B., Victor, J.C., Nyambane, G., Hodgson, A., Schodel, F., Ciarlet, M. & Neuzil, K.M. (2010). Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa: a randomised,

Azevedo, M.S., Zhang, W., Wen, K., Gonzalez, A.M., Saif, L.J., Yousef, A.E. & Yuan, L. (2012). Lactobacillus acidophilus and Lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus. *Benef Microbes*. 3, 1, (Mar 1, 2012), 33-42. Boge, T., Remigy, M., Vaudaine, S., Tanguy, J., Bourdet-Sicard, R. & van der Werf, S. (2009). A probiotic fermented dairy drink improves antibody response to influenza vaccination in the

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**Author details** 

**Acknowledgement** 

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Lijuan Yuan, Ke Wen, Fangning Liu and Guohua Li *Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, USA* 

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## **6. Conclusion**

Differential modulating effects on innate and adaptive immune responses by low dose versus high dose of the same LA NCFM strain were clearly demonstrated in gnotobiotic pigs. Low dose LA significantly enhanced the Th1 type effector T cell responses and decreased Treg cell functions in AttHRV-vaccinated pigs. Meanwhile, low dose LA resulted in a suppressed Th2 response, as evidenced by significantly reduced virus-specific ASC responses and serum antibody titers compared to the AttHRV only group. The dose effects of LA on IFN-γ producing T cell and CD4+CD25-FoxP3+ Treg cell immune responses were similar between the intestinal and systemic lymphoid tissues. Thus the same probiotic strain used in different doses can either increase or reduce mucosal and systemic immune responses induced by vaccines. These findings have significant implications in the use of probiotic lactobacilli as immunostimulatory versus immunoregulatory agents. Probiotic products are increasingly used to improve health, alleviate disease symptoms, and enhance vaccine efficacy. Our findings suggest that probiotics can be ineffective or even detrimental if not used at the optimal dosage for the appropriate purposes, highlighting the importance of not only strain but also dose selection in probiotic studies.

The gnotobiotic pig model is a valuable animal model for study of probiotic-virus-host interaction because of the many similarities between human and porcine intestinal physiology and mucosal immune system (Meurens et al., 2012). The gnotobiotic status prevents confounding factors from commensal microflora that are present in conventionally reared animals or in humans. Unlike gnotobiotic mice, gnotobiotic pigs are devoid of maternal antibodies, thus providing an immunologically naïve background that allows clear identification of the immune responses to a single vaccine in hosts colonized with a qualitatively and quantitatively defined probiotic bacterial strain (Butler, 2009; Yuan & Saif, 2002). Although data from studies of gnotobiotic animal models may not be generalized directly to normal animals or humans, gnotobiotic animals provide a medium in which investigating the complex interrelationships of the host and its associated microbes become possible (Coates, 1975). Our findings provide a good starting point for identification of the optimal dosage of a probiotic strain. But nonetheless, the optimal dosage needs to be confirmed in conventionalized gnotobiotic pigs and in human clinical trials in order to achieve the appropriate adjuvant effect for rotavirus and other vaccines.
