Host immunity in the protective response to nasal immunization with a pneumococcal antigen associated to live and heat-killed Lactobacillus casei

Abstract

BackgroundAt present, available pneumococcal vaccines have failed to eradicate infections caused by S. pneumoniae. Search for effective vaccine continues and some serotype independent pneumococcal proteins are considered as candidates for the design of new vaccines, especially a mucosal vaccine, since pneumococci enter the body through mucosal surfaces. Selection of the appropriate adjuvant is important for mucosal vaccines, and lactic acid bacteria (LAB) with immunostimulant properties are promissory candidates. In this work, we assessed the adjuvant effect of a probiotic strain, Lactobacillus casei (L. casei), when nasally administered with a pneumococcal antigen (pneumococcal protective protein A: PppA) for the prevention of pneumococcal infection. Adjuvanticity of both live (LcV) and heat-killed (LcM) was evaluated and humoral and cellular antigen-specific immune response was assessed in mucosal and systemic compartments. The potential mechanisms induced by nasal immunization were discussed.ResultsNasal immunization of young mice with PppA+LcV and PppA+LcM induced anti-PppA IgA and IgG antibodies in mucosal and systemic compartments and levels of these specific antibodies remained high even at day 45 after the 3rd Immunization (3rd I). These results were correlated with IL-4 induction by the mixture of antigen plus LcV and LcM. Also, PppA+Lc (V and M) induced stimulation of Th1 and Th17 cells involved in the defence against pneumococci. The protection against pneumococcal respiratory challenge at day 30 after the 3rd I showed that PppA+LcV and PppA+LcM immunizations significantly reduced pathogen counts in nasal lavages while prventing their passage into lung and blood. Survival of mice immunized with the co-application of PppA plus LcV and LcM was significantly higher than in mice immunized with PppA alone and control mice when intraperitoneal challenge was performed. No significant differences between the treatments involving LcV and LcM were found.ConclusionsLive and heat-killed L. casei enhanced the antigen-specific immune response when administered nasally with a pneumococcal antigen. Considering the potential risk associated with live bacteria, the design of a nasal vaccine based on pneumococcal antigens and heat-killed L. casei emerges as a safe and effective strategy for the prevention of pneumococcal infections and opens new possibilities of application of dead LAB as adjuvants in vaccine formulations against other pathogens.

Specific Immune Response Induced by a Lactobacillus Associated with a Pneumococcal Antigen in an “in vitro” Human Cells Model

“…Lactic acid bacteria are considered GRAS (Generally Recognized as Safe) microorganisms and their capacity to stimulate the immune system is amply documented, so that they are good candidates to be used as mucosal adjuvants. In previous reports, the immunostimulant properties of an inactivated recombinant lactococcus expressing PppA [18] and live/heat-killed Lactobacillus casei associated with PppA were demonstrated in mice [15]. The animal model allowed us to obtain crucial information about the mucosal adjuvant effect of dead and live Lactobacillus casei.…”

“…rPppA concentration was determined by Bradford's method and 10 ug of this protein was used in immunization protocols. Th s same concentration of PppA was employed in a previous report in an animal model [15].…”

“…Lactobacillus casei CRL 431 (L. casei) [15,18], obtained from the CERELA culture collection, was cultured overnight at 37°C (fi al log phase) in Man-Rogosa-Sharpe broth (Oxoid), harvested and washed twice with sterile 0.01 M phosphate buffer saline (PBS), pH 7.2, and resuspended in PBS containing 20% glycerol. Aliquots of these suspensions were stored at -80°C until use.…”

“…In the development of vaccines, LAB were used mainly as antigen vehicles [13,14] while there are few studies concerning the use of non-recombinant strains as adjuvants in mucosal vaccine formulations. In this sense, it was demonstrated that nasal administration of live and heat-killed L. casei CRL 431 associated with pneumococcal protective A protein (PppA) effectively prevented lung colonization in a pneumococcal respiratory infection and increased the survival of mice intraperitoneally challenged with the pathogens [15]. Animal models are very useful tools to study the effect of vaccines on the protection against human pathogens but the results obtained with them do not guarantee their effectiveness in human health.…”

“…The aim of this work was to evaluate the immune response induced by experimental vaccines containing live or heat-killed lactobacilli as adjuvants associated with a pneumococcal antigen on cytokine production and cellular immunity in a human cells model (peripheral blood mononuclear cells: PBMCs) and to analyze the potential mechanism/s involved in this effect. On the other hand, in a previous work [15], the same vaccines administered nasally were evaluated in an animal model. Thus, correlation of results obtained "in vitro" and "in vivo" will be discussed in order to analyze the usefulness of PBMCs as an alternative to assess the effect of a mucosal vaccine on humans.…”