Secretion and anchoring of proteins in Lactobacillus plantarum: Studies of a dendritic cell-targeted Mycobacterium tuberculosis antigen

The work described in this thesis is part of a larger project where the goal is to develop oral vaccines based on lactic acid bacteria (LAB). LAB have been used in food for centuries, are a natural inhabitant of the gastrointestinal (GI) tract of humans and are generally recognized as safe (GRAS). These characteristics make LAB attractive candidates for use as delivery vectors of therapeutic proteins to mucosal sites. The ability of Lactobacillus plantarum to persist in the GI tract of humans for up to a week together with its resistance to bile and low pH makes it well suited as an oral delivery vector. Dendritic cells (DC) are major contributors to the initiation of an immune response and it has previously been shown that targeting oral vaccines to DCs may enhance the subsequent immune response. This thesis describes studies on the use of L. plantarum as a live delivery vector for a Mycobacterium tuberculosis antigen fused with a DC binding peptide (DC-pep). Vectors for secretion, cell wall anchoring and membrane anchoring of Ag85B-ESAT6 fused with a DC-pep were constructed. Secretion and surface display of this antigen in L. plantarum was successfully accomplished with the efficiency seemingly unaffected by the DC-pep fusion. However, correct anchoring of the antigen could not be determined with certainty since the secreted version of antigen was also detected on the surface of the cells. Experiments with DCs showed that their internalization of L. plantarum was higher for strains producing antigen with DC-pep than for the corresponding strains without the DC-pep. Further experiments showed that all antigen producing L. plantarum strains as well as the strain harbouring the empty vector (pEV) were able to induce expression of the maturation marker CD83. Mice were immunized with L. plantarum strains producing DC-pep fused Ag85B-ESAT6 and further challenged with BCG. Upon stimulation with antigen, peripheral blood mononuclear cells (PBMCs) isolated from these mice showed elevated production of the cytokines IFN-γ and IL-17A, both of which are pro-inflammatory cytokines; an up-regulation indicates initiation of a correct immune response. In mice which were immunized with strains producing Ag85B-ESAT6 without DC-pep, the PBMCs showed no elevation in the production of these cytokines. This experiment strongly indicates that the DC-pep stimulates the immune response. In conclusion, the work described in this thesis shows that L. plantarum is able to secrete and possibly anchor DC-pep fused Ag85B-ESAT6. The presence of the DC-pep enhanced the immune response indicating that further development of the constructs described in this study is a promising strategy for developing novel vaccines against M. tuberculosis

Comparison of eight Lactobacillus species for delivery of surface-displayed mycobacterial antigen

Lactobacillus spp. comprise a large group of Gram-positive lactic acid bacteria with varying physiological, ecological and immunomodulatory properties that are widely exploited by mankind, primarily in food production and as health-promoting probiotics. Recent years have shown increased interest in using lactobacilli for delivery of vaccines, mainly due to their ability to skew the immune system towards pro-inflammatory responses. We have compared the potential of eight Lactobacillus species, L. plantarum, L. brevis, L. curvatus, L. rhamnosus, L. sakei, L. gasseri, L. acidophilus and L. reuteri, as immunogenic carriers of the Ag85B-ESAT-6 antigen from Mycobacterium tuberculosis. Surface-display of the antigen was achieved in L. plantarum, L. brevis, L. gasseri and L. reuteri and these strains were further analyzed in terms of their in vitro and in vivo immunogenicity. All strains activated human dendritic cells in vitro. Immunization of mice using a homologous prime-boost regimen comprising a primary subcutaneous immunization followed by three intranasal boosters, led to slightly elevated IgG levels in serum in most strains, and, importantly, to significantly increased levels of antigen-specific mucosal IgA. Cellular immunity was assessed by studying antigen-specific T cell responses in splenocytes, which did not reveal proliferation as assessed by the expression of Ki67, but which showed clear antigen-specific IFN-γ and IL-17 responses for some of the groups. Taken together, the present results indicate that L. plantarum and L. brevis are the most promising carriers of TB vaccines.publishedVersio

Lactobacillus plantarum producing a Chlamydia trachomatis antigen induces a specific IgA response after mucosal booster immunization

publishedVersio

Kostholdets betydning for fysisk og psykisk helse - ny kunnskap. Svar på oppdrag fra Helse- og omsorgsdepartementet til Folkehelseinstituttet

Som del av forberedelsene til Helse- og omsorgsdepartementets "Handlingsplan for bedre kosthold" ble Folkehelseinstituttet bedt om å oppdatere kunnskapsgrunnlaget for de norske kostholdsanbefalingene fra 2011. Dette er én av to rapporter

Kostholdets betydning for fysisk og psykisk helse, ny kunnskap Svar på oppdrag fra Helse- og omsorgsdepartementet til Folkehelseinstituttet

Som del av forberedelsene til Helse- og omsorgsdepartementets "Handlingsplan for bedre kosthold" ble Folkehelseinstituttet bedt om å oppdatere kunnskapsgrunnlaget for de norske kostholdsanbefalingene fra 2011. Dette er én av to rapporter

Comparison of eight Lactobacillus species for delivery of surface-displayed mycobacterial antigen

Lactobacillus spp. comprise a large group of Gram-positive lactic acid bacteria with varying physiological, ecological and immunomodulatory properties that are widely exploited by mankind, primarily in food production and as health-promoting probiotics. Recent years have shown increased interest in using lactobacilli for delivery of vaccines, mainly due to their ability to skew the immune system towards pro-inflammatory responses. We have compared the potential of eight Lactobacillus species, L. plantarum, L. brevis, L. curvatus, L. rhamnosus, L. sakei, L. gasseri, L. acidophilus and L. reuteri, as immunogenic carriers of the Ag85B-ESAT-6 antigen from Mycobacterium tuberculosis. Surface-display of the antigen was achieved in L. plantarum, L. brevis, L. gasseri and L. reuteri and these strains were further analyzed in terms of their in vitro and in vivo immunogenicity. All strains activated human dendritic cells in vitro. Immunization of mice using a homologous prime-boost regimen comprising a primary subcutaneous immunization followed by three intranasal boosters, led to slightly elevated IgG levels in serum in most strains, and, importantly, to significantly increased levels of antigen-specific mucosal IgA. Cellular immunity was assessed by studying antigen-specific T cell responses in splenocytes, which did not reveal proliferation as assessed by the expression of Ki67, but which showed clear antigen-specific IFN-γ and IL-17 responses for some of the groups. Taken together, the present results indicate that L. plantarum and L. brevis are the most promising carriers of TB vaccines

<i>Lactobacillus plantarum</i> producing a <i>Chlamydia trachomatis</i> antigen induces a specific IgA response after mucosal booster immunization

<div><p>Mucosal immunity is important for the protection against a wide variety of pathogens. Traditional vaccines administered via parenteral routes induce strong systemic immunity, but they often fail to generate mucosal IgA. In contrast, bacteria-based vaccines comprise an appealing strategy for antigen delivery to mucosal sites. Vaginal infection with <i>Chlamydia trachomatis</i> can develop into upper genital tract infections that can lead to infertility. Therefore, the development of an effective vaccine against Chlamydia is a high priority. In the present study, we have explored the use of a common lactic acid bacterium, <i>Lactobacillus plantarum</i>, as a vector for delivery of a <i>C</i>. <i>trachomatis</i> antigen to mucosal sites. The antigen, referred as Hirep2 (H2), was anchored to the surface of <i>L</i>. <i>plantarum</i> cells using an N-terminal lipoprotein anchor. After characterization, the constructed strain was used as an immunogenic agent in mice. We explored a heterologous prime-boost strategy, consisting of subcutaneous priming with soluble H2 antigen co-administered with CAF01 adjuvant, followed by an intranasal boost with H2-displaying <i>L</i>. <i>plantarum</i>. The results show that, when used as a booster, the recombinant <i>L</i>. <i>plantarum</i> strain was able to evoke cellular responses. Most importantly, booster immunization with the <i>Lactobacillus</i>-based vaccine induced generation of antigen-specific IgA in the vaginal cavity.</p></div

Plasmids and strains used in this study.

<p>Plasmids and strains used in this study.</p

Estimation of the total amount of antigen by semi-quantitative Western blotting.

<p>Serial dilutions of bacterial cell lysate from 1 x 10⁹ CFU of induced <i>Lp_</i>H2 and dilutions of purified Hirep1 with known concentration were subjected to Western blotting and proteins were detected using the anti-H2 antibody (which also binds to Hirep1 protein). Panel A shows a typical Western blot: wells 1–5, standard protein (20 ng, 10 ng, 7.5 ng, 5 ng and 2.5 ng, respectively); wells 6–9, lysate of <i>Lp</i>_H2, diluted 20-fold, 30-fold, 40-fold and 50-fold, respectively. Signal intensities of different dilutions of the standard protein were used to make a standard curve that is presented in panel B. The marked points on the standard curve correspond with the amount of standard protein used: (1) - 20 ng, (2) - 10 ng, (3)– 7.5 ng, (4) - 5 ng, (5) - 2.5 ng.</p

Immune responses induced by the booster <i>Lp_</i>H2 vaccine.

<p>Panel A presents the cellular response measured by H2-specific IFN-γ production in splenocytes. Cells were purified from individual mice and stimulated with H2 for 72 h. Produced IFN-γ was measured in harvested supernatants, by ELISA. Each point represents an individual mouse and the overall results per group are presented as a mean ± SEM (<i>n</i> = 8 for naive, H2 and H2/Lp_H2 groups, <i>n</i> = 7 for Lp_H2). Statistical significance was determined using one-way ANOVA with Tukey's post hoc test and is indicated as follows: **, p < 0.01. Panels B—E show antibody responses: H2-specific IgG in plasma (B) and H2-specific IgA in plasma (C), vaginal washes (D) and lung fluids (E). The samples from individual mice were serially diluted and added to H2-coated plates and specific IgG and IgA levels were measured by ELISA. The individual points represent the average OD₄₅₀ values per group ± SEM (<i>n</i> = 8 for naive, H2 and H2/Lp_H2 groups, <i>n</i> = 7 for Lp_H2) at each dilution. Panels F and G show H2-specific IgA in 10-fold diluted plasma samples (F) and 5-fold diluted vaginal samples (G). Each point represents an individual mouse and the overall results per group are presented as a mean ± SEM (<i>n</i> = 8 for naive, H2 and H2/Lp_H2 groups, <i>n</i> = 7 for Lp_H2). Statistical significance was determined using one-way ANOVA with post hoc Dunnett's test and is shown as follows: *, p < 0.05; **, p < 0.01.</p