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Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

Xie E, Kotha A, Biaco T, Sedani N, Zou J, Stashenko P, Duncan MJ, Campos-Neto A, Cayabyab MJ - PLoS ONE (2015)

Bottom Line: Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses.Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance.Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV.

View Article: PubMed Central - PubMed

Affiliation: Global Infectious Disease Research Center and the Department of Immunology and Infectious Diseases, The Forsyth Institute, 245 First Street, Cambridge, Massachusetts, United States of America.

ABSTRACT
The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis) that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

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rS. mitis induces systemic T cell tolerance.(A) Germ-free mice were vaccinated orally with 109 cfu rS. mitis expressing HIV Env gp120 (Smitis HIV Env) or rS. mitis containing an integrated Ermr gene without Env gp120 (Smitis). Ten weeks later, peripheral blood monuclear cells were isolated from vaccinated mice and stimulated with media alone (media), S. mitis lysate antigens (S. mitis Ag), the HIV Env gp120 protein (HIV Ag), or PMA and Ionomycin (PMA/IONO). Production of IFNγ, TNFα, and IL-2 by the stimulated cells measured by % IFNγ+, TNFα+, and IL-2+ CD4 or CD8 T cells from each group (3 mice/group) is shown. (B) To assess tolerance induction, germ-free mice were inoculated orally with 109 cfu to allow colonization and three months later, these mice were injected intraperitoneally (IP) with 2x108 cfu rS. mitis HIV Env (Balb/c (colonized) IP group). Non-colonized mice were either non-immunized (Non-immunized group) or inoculated IP with 2x108 cfu rS. mitis HIV Env (Balb/c IP group). Two weeks after intraperitoneal immunization of the colonized and non-colonized mice, the spleen and mesenteric lymph nodes (MLN) were harvested and stimulated in vitro with media, 10μg/ml purified HIV Env gp120 protein (HIV Ag), 10μg/ml of S. mitis antigens (Smitis Ag), Concanavalin A (ConA), or PMA/IONO. T cell proliferative responses were measured by using 3H-Thymidine incorporation assay. Mean CPM from 3 mice/group is shown. The production of IL-2, IL-4, IL-6, IL-10, IL-13, IL-17A, TGFβ, TNFα and IFNγ by cells stimulated with media, HIV antigen, or S. mitis antigens was measured using multiplex luminex assay (C). The assay was performed in duplicates and the mean concentration (pg/ml) for each cytokine from pooled supernatants samples (3 mice/group) is shown.
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pone.0143422.g006: rS. mitis induces systemic T cell tolerance.(A) Germ-free mice were vaccinated orally with 109 cfu rS. mitis expressing HIV Env gp120 (Smitis HIV Env) or rS. mitis containing an integrated Ermr gene without Env gp120 (Smitis). Ten weeks later, peripheral blood monuclear cells were isolated from vaccinated mice and stimulated with media alone (media), S. mitis lysate antigens (S. mitis Ag), the HIV Env gp120 protein (HIV Ag), or PMA and Ionomycin (PMA/IONO). Production of IFNγ, TNFα, and IL-2 by the stimulated cells measured by % IFNγ+, TNFα+, and IL-2+ CD4 or CD8 T cells from each group (3 mice/group) is shown. (B) To assess tolerance induction, germ-free mice were inoculated orally with 109 cfu to allow colonization and three months later, these mice were injected intraperitoneally (IP) with 2x108 cfu rS. mitis HIV Env (Balb/c (colonized) IP group). Non-colonized mice were either non-immunized (Non-immunized group) or inoculated IP with 2x108 cfu rS. mitis HIV Env (Balb/c IP group). Two weeks after intraperitoneal immunization of the colonized and non-colonized mice, the spleen and mesenteric lymph nodes (MLN) were harvested and stimulated in vitro with media, 10μg/ml purified HIV Env gp120 protein (HIV Ag), 10μg/ml of S. mitis antigens (Smitis Ag), Concanavalin A (ConA), or PMA/IONO. T cell proliferative responses were measured by using 3H-Thymidine incorporation assay. Mean CPM from 3 mice/group is shown. The production of IL-2, IL-4, IL-6, IL-10, IL-13, IL-17A, TGFβ, TNFα and IFNγ by cells stimulated with media, HIV antigen, or S. mitis antigens was measured using multiplex luminex assay (C). The assay was performed in duplicates and the mean concentration (pg/ml) for each cytokine from pooled supernatants samples (3 mice/group) is shown.

Mentions: The nature of the T cell response to S. mitis in humans is not clearly known. We investigated whether rS. mitis induced T cell responses in the vaccinated germ-free mice. Vaccinated mice that harbored rS. mitis persistently for three months developed no vaccine-specific T cell-responses systemically. Intracellular cytokine staining analysis revealed that peripheral blood T cells isolated from vaccinated mice stimulated with either HIV or S. mitis antigens did not produce IFNγ, TNFα and IL-2 (Fig 6A). In contrast, T cells expressed large amounts of IFNγ, TNFα and IL-2 in response to PMA and Ionomycin stimulation (Fig 6A).


Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

Xie E, Kotha A, Biaco T, Sedani N, Zou J, Stashenko P, Duncan MJ, Campos-Neto A, Cayabyab MJ - PLoS ONE (2015)

rS. mitis induces systemic T cell tolerance.(A) Germ-free mice were vaccinated orally with 109 cfu rS. mitis expressing HIV Env gp120 (Smitis HIV Env) or rS. mitis containing an integrated Ermr gene without Env gp120 (Smitis). Ten weeks later, peripheral blood monuclear cells were isolated from vaccinated mice and stimulated with media alone (media), S. mitis lysate antigens (S. mitis Ag), the HIV Env gp120 protein (HIV Ag), or PMA and Ionomycin (PMA/IONO). Production of IFNγ, TNFα, and IL-2 by the stimulated cells measured by % IFNγ+, TNFα+, and IL-2+ CD4 or CD8 T cells from each group (3 mice/group) is shown. (B) To assess tolerance induction, germ-free mice were inoculated orally with 109 cfu to allow colonization and three months later, these mice were injected intraperitoneally (IP) with 2x108 cfu rS. mitis HIV Env (Balb/c (colonized) IP group). Non-colonized mice were either non-immunized (Non-immunized group) or inoculated IP with 2x108 cfu rS. mitis HIV Env (Balb/c IP group). Two weeks after intraperitoneal immunization of the colonized and non-colonized mice, the spleen and mesenteric lymph nodes (MLN) were harvested and stimulated in vitro with media, 10μg/ml purified HIV Env gp120 protein (HIV Ag), 10μg/ml of S. mitis antigens (Smitis Ag), Concanavalin A (ConA), or PMA/IONO. T cell proliferative responses were measured by using 3H-Thymidine incorporation assay. Mean CPM from 3 mice/group is shown. The production of IL-2, IL-4, IL-6, IL-10, IL-13, IL-17A, TGFβ, TNFα and IFNγ by cells stimulated with media, HIV antigen, or S. mitis antigens was measured using multiplex luminex assay (C). The assay was performed in duplicates and the mean concentration (pg/ml) for each cytokine from pooled supernatants samples (3 mice/group) is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664415&req=5

pone.0143422.g006: rS. mitis induces systemic T cell tolerance.(A) Germ-free mice were vaccinated orally with 109 cfu rS. mitis expressing HIV Env gp120 (Smitis HIV Env) or rS. mitis containing an integrated Ermr gene without Env gp120 (Smitis). Ten weeks later, peripheral blood monuclear cells were isolated from vaccinated mice and stimulated with media alone (media), S. mitis lysate antigens (S. mitis Ag), the HIV Env gp120 protein (HIV Ag), or PMA and Ionomycin (PMA/IONO). Production of IFNγ, TNFα, and IL-2 by the stimulated cells measured by % IFNγ+, TNFα+, and IL-2+ CD4 or CD8 T cells from each group (3 mice/group) is shown. (B) To assess tolerance induction, germ-free mice were inoculated orally with 109 cfu to allow colonization and three months later, these mice were injected intraperitoneally (IP) with 2x108 cfu rS. mitis HIV Env (Balb/c (colonized) IP group). Non-colonized mice were either non-immunized (Non-immunized group) or inoculated IP with 2x108 cfu rS. mitis HIV Env (Balb/c IP group). Two weeks after intraperitoneal immunization of the colonized and non-colonized mice, the spleen and mesenteric lymph nodes (MLN) were harvested and stimulated in vitro with media, 10μg/ml purified HIV Env gp120 protein (HIV Ag), 10μg/ml of S. mitis antigens (Smitis Ag), Concanavalin A (ConA), or PMA/IONO. T cell proliferative responses were measured by using 3H-Thymidine incorporation assay. Mean CPM from 3 mice/group is shown. The production of IL-2, IL-4, IL-6, IL-10, IL-13, IL-17A, TGFβ, TNFα and IFNγ by cells stimulated with media, HIV antigen, or S. mitis antigens was measured using multiplex luminex assay (C). The assay was performed in duplicates and the mean concentration (pg/ml) for each cytokine from pooled supernatants samples (3 mice/group) is shown.
Mentions: The nature of the T cell response to S. mitis in humans is not clearly known. We investigated whether rS. mitis induced T cell responses in the vaccinated germ-free mice. Vaccinated mice that harbored rS. mitis persistently for three months developed no vaccine-specific T cell-responses systemically. Intracellular cytokine staining analysis revealed that peripheral blood T cells isolated from vaccinated mice stimulated with either HIV or S. mitis antigens did not produce IFNγ, TNFα and IL-2 (Fig 6A). In contrast, T cells expressed large amounts of IFNγ, TNFα and IL-2 in response to PMA and Ionomycin stimulation (Fig 6A).

Bottom Line: Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses.Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance.Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV.

View Article: PubMed Central - PubMed

Affiliation: Global Infectious Disease Research Center and the Department of Immunology and Infectious Diseases, The Forsyth Institute, 245 First Street, Cambridge, Massachusetts, United States of America.

ABSTRACT
The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis) that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

Show MeSH
Related in: MedlinePlus