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

<div><p>The pioneer human oral commensal bacterium <i>Streptococcus mitis</i> has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. <i>S</i>. <i>mitis</i> 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 <i>S</i>. <i>mitis</i> (<i>rS</i>. <i>mitis</i>) 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 <i>rS</i>. <i>mitis</i> 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 <i>rS</i>. <i>mitis</i> having the ability to elicit T cell tolerance suggest the potential use of <i>rS</i>. <i>mitis</i> as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.</p></div

<i>rS</i>. <i>mitis</i> induces systemic T cell tolerance.

<p>(A) Germ-free mice were vaccinated orally with 10⁹ cfu <i>rS</i>. <i>mitis</i> expressing HIV Env gp120 (Smitis HIV Env) or <i>rS</i>. <i>mitis</i> containing an integrated Erm^r gene without Env gp120 (Smitis). Ten weeks later, peripheral blood monuclear cells were isolated from vaccinated mice and stimulated with media alone (media), <i>S</i>. <i>mitis</i> lysate antigens (<i>S</i>. <i>mitis</i> 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 r<i>S</i>. <i>mitis</i> HIV Env (Balb/c (colonized) IP group). Non-colonized mice were either non-immunized (Non-immunized group) or inoculated IP with 2x10⁸ cfu r<i>S</i>. <i>mitis</i> 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 <i>S</i>. <i>mitis</i> 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 <i>S</i>. <i>mitis</i> 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.</p

<i>rS</i>. <i>mitis</i> expressing HIV Env is stable.

<p>To determine the stability of the integrated Env gp120 gene in <i>rS</i>. <i>mitis</i>, four clones (HIVEnvA, B, C, and D) were picked at random and grown anaerobically for 24 hours which represents approximately three generations (7.1 hours per generation) in THB media without erythromycin. Cultures were grown for approximately 30 generations without erythromycin. From each generation 100 and 150 colonies were picked at random and streaked to THB plates with and without erythromycin. For each <i>S</i>. <i>mitis</i> HIV Env clone, the number of Erm^r colonies/total number of colonies streaked after 6, 12, 18, 24, and 30 generations was determined (Fig 3A). Expression of Env in the same daughter clones was analyzed by Western blot analysis. Env production in a representative daughter clone (after 30 generations) and the original HIVgp120A clone is shown (Fig 3B).</p

Construction of recombinant <i>S</i>. <i>mitis</i> expressing HIV Env gp120.

<p>(A) Recombinant <i>S</i>. <i>mitis</i> (<i>rS</i>. <i>mitis</i>) was generated by homologous recombination. The p5E3 suicide plasmid was created by inserting the <i>S</i>. <i>mitis</i> codon-optimized HIV-1 HXBc2 Env gp120-His-tag (HIV Env) in-frame with the 250bp 5’ end of the pullulanase gene (pulA/Smt0163) followed by ermr gene and the 250bp 3’ end of the pulA gene into pCR2.1 for transformation and integration into the Smt0163 locus. (B) <i>S</i>. <i>mitis</i> was transformed with water (control), p5E3 (<i>Smt0163</i>:<i>HIVenv</i>:<i>erm</i>^<i>r</i>) or pCR2.1 containing ermr flanked by 250bp pulA 5’ and 3’ fragments (<i>Smt0163</i>:<i>erm</i>^<i>r</i>). Growth of transformants on THB plates containing 50μg/ml erythromycin and transformation frequency are shown. (C) Integration was confirmed by PCR using <i>S</i>. <i>mitis</i>-specific primers A/B, HIV-specific primer C and ermr-specific primer D.</p

Recombinant <i>S</i>. <i>mitis</i> colonizes germ-free mice efficiently and persistently.

<p>Germ-free Balb/c mice and conventional SPF mice were inoculated orally with 10⁹ cfu <i>rS</i>. <i>mitis</i> expressing HIV Env gp120 (Smitis HIV Env), <i>rS</i>. <i>mitis</i> containing an integrated Erm^r gene without Env (Smitis) or PBS (Non-Immunized). Following inoculation the upper right buccal cheek was swabbed and two pellets of feces were collected from each mouse at various time points. <i>rS</i>.<i>mitis</i> colonization was assessed by growth on THB plates containing 50 μg/ml erythromycin. The mean colony-forming-units, cfu (±SEM) from 3 mice/group at various timepoints, present in the mouth (A) and feces (B) of germ-free mice and in the mouth (C) and feces (D) of conventional mice inoculated with <i>rS</i>. <i>mitis</i> are shown.</p

Recombinant <i>S</i>. <i>mitis</i> induces mucosal and systemic antibody responses.

<p>Germ-free Balb/c mice were inoculated orally with 10⁹ cfu <i>rS</i>. <i>mitis</i> expressing HIV Env gp120 (Smitis HIV Env), <i>rS</i>. <i>mitis</i> containing an integrated Erm^r gene without Env gp120 (Smitis), or PBS (Non-Immunized). Following immunization, the presence of IgA, IgG1, and IgG2a antibodies specific to the HIV Env gp120 protein (HIV Ag) or <i>S</i>. <i>mitis</i> lysate antigens (<i>S</i>. <i>mitis</i> Ag) was measured by ELISA in the saliva (A) and serum (B) of mice. The mean optical density (OD) (±SEM) values of the undiluted samples from 3 mice/group at various timepoints post-immunization are shown. The saliva and serum antibody dilution factor was 1:3 or indicated otherwise.</p