Administration of a probiotic associated with nasal vaccination with inactivated Lactococcus lactis-PppA induces effective protection against pneumoccocal infection in young mice.

Streptococcus pneumoniae is a serious public health problem, especially in developing countries, where available vaccines are not part of the vaccination calendar. We evaluated different respiratory mucosa immunization protocols that included the nasal administration of Lactococcus lactis-pneumococcal protective protein A (PppA) live, inactivated, and in association with a probiotic (Lc) to young mice. The animals that received Lc by the oral and nasal route presented the highest levels of immunoglobulin (Ig)A and IgG anti-PppA antibodies in bronchoalveolar lavages (BAL) and IgG in serum, which no doubt contributed to the protection against infection. However, only the groups that received the live and inactivated vaccine associated with the oral administration of the probiotic were able to prevent lung colonization by S. pneumoniae serotypes 3 and 14 in a respiratory infection model. This would be related to a preferential stimulation of the T helper type 1 (Th1) cells at local and systemic levels and with a moderate Th2 and Th17 response, shown by the cytokine profile induced in BAL and by the results of the IgG1/IgG2a ratio at local and systemic levels. Nasal immunization with the inactivated recombinant strain associated with oral Lc administration was able to stimulate the specific cellular and humoral immune response and afford protection against the challenge with the two S. pneumoniae serotypes. The results obtained show the probiotic-inactivated vaccine association as a valuable alternative for application to human health, especially in at-risk populations, and are the first report of a safe and effective immunization strategy using an inactivated recombinant strain.

Intestinal pathway of internalisation of lactic acid bacteria and gut mucosal immunostimulation.

The induction of the gut mucosal immune response is dependent on the antigen interacting with the M cells of Peyer's patches and with the immune cells associated with this lymphoid organ. In previous studies we showed that the mucosal immunostimulation by LAB varied depending upon the strain being studied. Some of them increased the inflammatory immune response and others enhanced the level of secretory antibody (S-IgA). Our aim was to determine the pathway of the internalisation of LAB strains in the intestine, to provide a basis for understing the different behaviour exhibited by them. The presence of LAB on Peyer's patches or in the immune cells associated with the villi of small intestine was determined using fluorescein-labelled bacteria and by transmission electron microscopy. Mice were dosed orally by intubation, with 101 cells of labelled single strains of Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. For electron microscopy studies unlabelled bacteria were used. Histological preparations of the small and large intestine were performed 1 hour after administration of the bacteria. Fluorescent bacteria of L. casei and S. thermophilus were found only in Peyer's patches while L. delbrueckii ssp. bulgaricus and L. acidophilus were observed in Peyer's patches and in the small intestine. L. acidophilus was also found in the large intestine. We confirmed these findings by electron microscopy. We determined that for L. casei the pathway of internalisation was via the M and FAE cells of Peyer's patches, while for S. thermophilus, L. acidophilus and L. delbrueckii ssp. bulgaricus the interaction with the immune cells of Peyer's patches was through the follicle associated epithelium (FAE). L. delbrueckii ssp. bulgaricus and L. acidophilus also interacted with the epithelial cells of the small intestine and L. acidophilus with epithelial cells of the large intestine. These results suggest that the different effects of LAB on the mucosal immunostimulation, are related to the different pathways of gut internalisation used to take contact with the immune cells associated with the lamina propria intestinal.

Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria.

The induction of a mucosal immune response is not easy due to the development of oral tolerance, but under some conditions, bacteria can activate this immune system. Antigens administered orally can interact with M cells of Peyer's patches or bind to the epithelial cells. We have demonstrated that certain lactic acid bacteria are able to induce specific secretory immunity, and others will enhance the gut inflammatory immune response. The aim of this work was to establish the reason for these different behaviors and to define possible mechanisms involved in the interaction of lactic acid bacteria at the intestinal level. We studied IgA+ and IgM+ B cells comparatively in bronchus and intestine and CD4+ T cells and IgA anti-lactic acid bacteria antibodies in the intestinal fluid, induced by oral administration of Lactobacillus casei, Lb. delbrueckii ssp. bulgaricus, Lb. acidophilus, Lb. plantarum, Lb. rhamnosus, Lactococcus lactis, and Streptococcus salivarius ssp. thermophilus. The increase in the IgA+ B cells in the bronchus means that these lactic acid bacteria were able to induce the IgA cycle by interaction with M cells from Peyer's patches or intestinal epithelial cells. The IgM+ cells increased when the stimulus did not induce the switch from IgM+ to IgA+. The increase in the CD4+ cells suggests interaction of Peyer's patches and enhancement of the B- and T-cell migration. The anti-lactic acid bacteria antibody is related to the processing and presentation of the microorganisms to the immune cells. We demonstrated that Lb. casei and Lb. plantarum were able to interact with Peyer's patch cells and showed an increase in IgA-, CD4+ cells, and antibodies specific for the stimulating strain. Lactobacillus acidophilus induced gut mucosal activation by interaction with the epithelial cells without increase in the immune cells associated with the bronchus. Although Lb. rhamnosus and Strep. salivarius ssp. thermophilus interact with epithelial cells, they also induced an immune response against their epitopes. Lactococcus lactis and Lb. delbrueckii ssp. bulgaricus induced an increase of IgA+ cells entering the IgA cycle but not CD4+ cells; thus, these bacteria would have been bound to epithelial cells that activated B lymphocytes without processing and presenting of their epitopes. We did not determine specific antibodies against Lc. lactis or Lb. bulgaricus.

Influence of the oral administration of lactic acid bacteria on iga producing cells associated to bronchus.

Intestinal, respiratory and genitourinary mucosal surfaces are the most important routes of entry for microbial pathogens. The stimulus of the mucosal immunity is not easy because the trigger keys for the activation do not follow the ones of the systemic immune response. In previous works we have demonstrated that some Lactic Acid Bacteria (LAB), when orally administered, can induce an enhance of the gut immune response. Taking into account the concept of a common mucosal response, we studied the effect of orally administered mice with Lactobacillus casei, L. acidophilus, L. rhamnosus, L. delbrueckii subsp. bulgaricus, Streptococcus salivarius subsp. thermophilus and Lactococcus lactis on the IgA secreting cells associated to bronchus. As shown before, oral immunostimulation with LAB induced an increase of the IgA* cells at intestinal level by a dose depending effect. In this study it is also showed that the LAB assayed, with exception of L. acidophilus, were able to enhance IgA+ cells at bronchial level, being also this effect dose dependent. The increment induced by some LAB in the number of IgA+ cells on the mucosa surface of the lower respiratory tract may be very important to prevent bronchus diseases.