Fig 1.
Construction of recombinant S. mitis expressing HIV Env gp120.
(A) Recombinant S. mitis (rS. mitis) was generated by homologous recombination. The p5E3 suicide plasmid was created by inserting the S. mitis 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) S. mitis was transformed with water (control), p5E3 (Smt0163:HIVenv:ermr) or pCR2.1 containing ermr flanked by 250bp pulA 5’ and 3’ fragments (Smt0163:ermr). Growth of transformants on THB plates containing 50μg/ml erythromycin and transformation frequency are shown. (C) Integration was confirmed by PCR using S. mitis-specific primers A/B, HIV-specific primer C and ermr-specific primer D.
Fig 2.
Recombinant S. mitis expresses HIV envelope protein.
rS. mitis with the integrated HIV HXBc2 Env gp120 was designed to secrete HIV Env by ligating the HIV Env in frame with 250bp 5’ end of the pullulanase gene (pulA/Smt0163) encoding a signal peptide that allows processing and secretion of the HIV antigen. (A) The signal peptide has an amino-terminal region (N), a hydrophobic core (H), a signal peptidase cleavage site (C), and an accessory Sec transport motif (AST). Expression of HIV Env containing a C-terminal His tag was assessed by Western blotting using Penta-His-HRP from a representative recombinant clone in S. mitis lysates (B) and in culture supernatants (C) by TCA-precipitation (TCA), acetone precipitation (Acetone) and Amicon filter-concentration (Sup). HIV Env expression in lysates (B) and supernatants of control S. mitis vector (control) (C) is shown. The arrow denotes expression of the Env Ag band. 100 ng of His-tagged M. tuberculosis protein (MT0401) was used as a positive control (B and C, lane 1). (D) The expression of HIV-1 gp120 in rS. mitis containing the HIV Env gene (lane 2) in Amicon filter-concentrated supernatant was detected using human HIV patient sera. rS. mitis containing the empty plasmid was used as a negative control (lane 1).
Fig 3.
rS. mitis expressing HIV Env is stable.
To determine the stability of the integrated Env gp120 gene in rS. mitis, 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 S. mitis HIV Env clone, the number of Ermr 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).
Fig 4.
Recombinant S. mitis colonizes germ-free mice efficiently and persistently.
Germ-free Balb/c mice and conventional SPF mice were inoculated orally with 109 cfu rS. mitis expressing HIV Env gp120 (Smitis HIV Env), rS. mitis containing an integrated Ermr 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. rS.mitis 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 rS. mitis are shown.
Fig 5.
Recombinant S. mitis induces mucosal and systemic antibody responses.
Germ-free Balb/c mice were inoculated orally with 109 cfu rS. mitis expressing HIV Env gp120 (Smitis HIV Env), rS. mitis containing an integrated Ermr 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 S. mitis lysate antigens (S. mitis 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.
Fig 6.
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.