Probiotic Lactobacillus rhamnosus GG enhanced Th1 cellular immunity but did not affect antibody responses in a human gut microbiota transplanted neonatal gnotobiotic pig model.

Ke Wen,Lijuan Yuan,Ernawati Giri-Rachman,Xingdong Yang,Guohua Li,Sherrie Clark-Deener,Jacob Kocher,Tammy Bui,Christine Tin,Haifeng Wang,Shan Lu

doi:10.1371/journal.pone.0094504

Abstract

This study aims to establish a human gut microbiota (HGM) transplanted gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and diarrhea, and to verify the dose-effects of probiotics on HRV vaccine-induced immune responses. Our previous studies using the Gn pig model found that probiotics dose-dependently regulated both T cell and B cell immune responses induced by rotavirus vaccines. We generated the HGM transplanted neonatal Gn pigs through daily feeding of neonatal human fecal suspension to germ-free pigs for 3 days starting at 12 hours after birth. We found that attenuated HRV (AttHRV) vaccination conferred similar overall protection against rotavirus diarrhea and virus shedding in Gn pigs and HGM transplanted Gn pigs. HGM promoted the development of the neonatal immune system, as evidenced by the significantly enhanced IFN-γ producing T cell responses and reduction of regulatory T cells and their cytokine production in the AttHRV-vaccinated pigs. The higher dose Lactobacillus rhamnosus GG (LGG) feeding (14 doses, up to 109 colony-forming-unit [CFU]/dose) effectively increased the LGG counts in the HGM Gn pig intestinal contents and significantly enhanced HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine. Lower dose LGG (9 doses, up to 106 CFU/dose) was ineffective. Neither doses of LGG significantly improved the protection rate, HRV-specific IgA and IgG antibody titers in serum, or IgA antibody titers in intestinal contents compared to the AttHRV vaccine alone, suggesting that an even higher dose of LGG is needed to overcome the influence of the microbiota to achieve the immunostimulatory effect in the HGM pigs. This study demonstrated that HGM Gn pig is an applicable animal model for studying immune responses to rotavirus vaccines and can be used for studying interventions (i.e., probiotics and prebiotics) that may enhance the immunogenicity and protective efficacy of vaccines through improving the gut microbiota.

Highlights

Gnotobiotic (Gn) pig models have been extensively used in studies of human rotavirus (HRV) because of the similar susceptibility to the Wa strain (G1P1A[8]) HRV infection and clinical manifestations in neonatal Gn pigs as in human infants [14]
Our previous studies demonstrated that probiotic Lactobacillus acidophilus NCFM (LA) at the appropriate dose was effective in reducing rotavirus diarrhea as well as enhancing immunogenicity of oral rotavirus vaccines in Gn pigs [5]
High dose Lactobacillus rhamnosus GG (LGG) feeding significantly enhanced the fecal and intestinal LGG counts in human gut microbiota (HGM) transplanted Gn pigs

Summary

Introduction

Gnotobiotic (Gn) pig models have been extensively used in studies of human rotavirus (HRV) because of the similar susceptibility to the Wa strain (G1P1A[8]) HRV infection and clinical manifestations in neonatal Gn pigs as in human infants [14]. Our previous studies demonstrated that probiotic Lactobacillus acidophilus NCFM (LA) at the appropriate dose was effective in reducing rotavirus diarrhea as well as enhancing immunogenicity of oral rotavirus vaccines in Gn pigs [5]. LA regulated rotavirus vaccine-induced immune responses in a dose-dependent manner in Gn pigs [5,6]. Intermediate dose LA (9 doses, up to 106 CFU/dose) significantly enhanced rotavirus-specific antibody secreting cell (ASC) and memory B cell responses induced by rotavirus vaccines in Gn pigs [5]. LA dose-dependently regulated innate immune responses of cytokine producing dendritic cells and Toll-like receptor expressing antigen presenting cells [7]. The probiotic Lactobacillus rhamnosus (LGG) strain enhanced the immunogenicity of rotavirus vaccines in Gn pigs at the appropriate dose in our previous study (Manuscript in preparation). LGG, but not LA has been evaluated in several human clinical trials as a vaccine adjuvant [8], including rotavirus vaccine [9] and influenza vaccine [10], we chose LGG for the present study

Objectives

Methods

Results

Conclusion