We evaluated virus-specific B and T cell immune responses induced by the two-dose oral AttHRV vaccine with or without LA feeding in Gn pigs. We demonstrated that LA significantly enhanced the immunogenicity of the AttHRV vaccine as indicated by the significantly higher magnitude of HRV-specific IFN-γ producing CD8+ T cell responses in ileum and spleen, IgA and IgG ASC responses in ileum, and serum IgM, IgA, IgG and VN antibody responses in the AttHRV-vaccinated and LA-fed pigs compared to the AttHRV vaccinated pigs without LA. These findings indicate that L. acidophilus
NCFM has a strong potentiating effect on both cellular and humoral immunity induced by the oral rotavirus vaccine. We also evaluated the effect of LA, AttHRV and LA plus AttHRV on the development of total B cell compartment by comparing the total IgSC responses in intestinal and systemic lymphoid tissues and the Ig titers in serum among the 4 groups. Total Ig responses were induced by either LA or AttHRV, but LA induced significantly lower IgA and IgG SC in ileum, IgM and IgG SC in spleen and IgM and IgA SC in blood compared to AttHRV, indicating that AttHRV was more effective in promoting development of the neonatal immune system than LA colonization. However, LA enhanced development of the intestinal IgA and IgG SC responses in the LA+AttHRV+ pigs, which were in parallel with the enhanced virus-specific intestinal IgA and IgG ASC responses. Challenge studies are needed to assess the effect of LA on improving the protective efficacy of the AttHRV vaccine in Gn pigs. The significantly elevated virus-specific immune responses after AttHRV vaccination in LA-fed pigs are likely to improve the protective efficacy of the vaccine, because previous studies of rotavirus protective immunity in neonatal Gn pigs have shown that the magnitude of virus-specific serum IgA antibody, intestinal IgA ASC and IFN-γ producing T cell responses are positively correlated with protection rates against rotavirus diarrhea upon challenge the Gn pigs with the virulent Wa HRV [15
]. Serum antibody titers were also correlated with protection against reinfection in humans [24
A recent study by Olivares et al. showed that oral intake of L. fermentum
CECT5716 significantly enhanced serum Th1 type cytokine and influenza-specific IgA antibody responses to an intramuscular influenza vaccine in adults [6
]. Hori et al. reported that mice infected with influenza virus and fed L. casei
had significantly higher IFN-γ production by nasal lymphocytes than non-fed mice [27
]. These and our results collectively demonstrated that probiotic lactobacilli can increase both mucosal and systemic T and B cell responses to viral infection or vaccines. Therefore, these particular Lactobacillus
strains may be used as oral adjuvants to improve the immunogenicity and protective efficacy of oral or parental vaccines, such as rotavirus and influenza vaccines.
Oral intake of lactobacilli during a period around vaccination is a safe and easy way for enhancing the effectiveness of vaccines. Because neonatal Gn pigs are totally devoid of maternal antibodies, they are likely more susceptible than human infants to potential adverse effects of the lactobacilli or vaccines. We confirmed that the LA feeding regimen did not induce any adverse effect in neonatal Gn pigs, yet was highly effective in enhancing the immunogenicity of the AttHRV vaccine. The AttHRV vaccine caused transient diarrhea in 13% (2/15) Gn pigs after the first dose. Although LA feeding did not prevent the diarrhea, there was a delay in the onset and a reduction in the diarrheic days of the pig, and the mean cumulative fecal consistency scores in the LA+AttHRV+ group were significantly lower than the LA−AttHRV group. LA mixed with L. reuteri
and Bifidobacterium infantis
BBI significantly decreased the duration and incidence of diarrhea in children of 12–32 mo of age [11
]. The effect of LA alone in reducing rotavirus diarrhea has not been reported [11
]. Further studies with a larger number of pigs in each group may be needed to confirm if LA alone has an effect on reducing the severity of rotavirus diarrhea after rotavirus infection, and in this case, reducing the side effects of AttHRV vaccine.
After the first dose of AttHRV inoculation, none of the LA+AttHRV+ pigs shed virus nasally compared to 50% of LA−AttHRV+ pigs, whereas both groups shed virus fecally (29% versus 25%). These results are consistent with a previous study that AttHRV induced a higher rate of nasal shedding than of fecal shedding (79% versus 17%) in neonatal Gn pigs [28
]. The potential mechanism by which LA inhibited the AttHRV nasal shedding might be that LA inhibited the virus adhesion to the upper respiratory tract epithelial surfaces. It has been suggested that Lactobacillus
may bind to receptors on the cell surface so that the receptors were not accessible or not recognized by the pathogen [29
]. We found that after LA feeding, LA colonized effectively not only in the gut, but also in the oral/nasal cavity and on the tonsils (Zhang and Yuan, unpublished data). However, LA did not affect the AttHRV fecal shedding, except delayed the onset. It is likely that the differences in the biological environment (temperature, density of virus receptors, density of colonizing LA, proteolytic enzymes, etc.) between intestinal and upper respiratory tract have led to the different replication capability of the AttHRV and the observed differential effects of LA. Further studies are needed to identify the mechanism. Nonetheless, our results showed that LA intestinal colonization did not reduce vaccine replication rate in the gut.
In our previous studies, the protective efficacy of the Wa two-dose AttHRV vaccine (G1P1A) against rotavirus diarrhea in Gn pigs was substantially lower (33%) than that of the two-dose RIX4414 AttHRV vaccine (Rotarix™, G1P1A) in human infants (63.5% efficacy against all case diarrhea) [3
]. This difference may be partially explained by the lack of immunostimulation of the neonatal immune system by commensal microflora in Gn pigs. However, the Wa three-dose AttHRV vaccine conferred a protection rate against rotavirus diarrhea of 67% in Gn pigs [32
], similar to the efficacy of RIX4414 vaccine in humans. Because human infants are colonized with lactobacilli hours after birth, the effect of LA observed in Gn pigs may not be extrapolated directly to humans. Further challenge studies in Gn pigs and “conventionalized” Gn pigs (Gn pigs seeded with the gut microflora from a suckling conventional pig to mimic formula-fed human infants) are needed to confirm LA’s effect on improving the protective efficacy of oral live HRV vaccines before clinical trials in humans.
In conclusion, we demonstrated that L. acidophilus NCFM has a significant immunostimulating effect on the intestinal and systemic HRV-specific T and B cell immune responses induced by the AttHRV vaccine and is safe in neonates; therefore it may have the potential to be used as an adjuvant for rotavirus vaccines.