Fig 1.
Quality-controlled, heterologous expression of MU-Ag85A by BCG and M. smegmatis.
A. The plasmid map for pSL401 is shown. Constitutive expression of MU-Ag85A (purple) fused to an HA epitope tag (yellow) is driven by the promoter region from hsp60 (Phsp60, black). Plasmid replication is regulated in E. coli using oriE (blue) and in mycobacteria using oriM (red). Plasmid retention is selected by the presence of a kanamycin resistance cassette (hashed) B. Three vaccine accession lot vials of BCG or M. smegmatis (Msmeg) transformed with pSL401 were chosen at random for Western analysis of MU-Ag85A expression using anti-HA antibody. The expected band size was 35 kDa. Negative controls are lysates from BCG or Msmeg transformed with empty vector (BCG pHA, Msmeg pHA). Loading controls were performed by detection of Hsp65. Plasmid DNA was isolated from BCG (C) and Msmeg (D) vaccine lots and transformed into chemically competent E. coli. 10 transformants were plasmid prepped and restriction digested (DraI/NheI) to assess presence of MU-Ag85A insert and correct band size (1.4 kb). Undigested and digested pSL401 plasmids were run as negative and positive controls, respectively.
Fig 2.
BCG MU-Ag85A vaccination induces proliferation of antigen-specific CD4+ T cells and immunological memory.
A. C57BL/6 mice were left unprimed (dotted black) or were intravenously primed using 107 BCG transformed with empty vector (BCG pHA, gray) or with pSL401 (BCG MU-Ag85A, black). At various time points, peripheral submandibular blood was collected. T lymphocytes were isolated by centrifugation and flow cytometric analysis was performed to quantify levels of CD4+ T cells bound to MHCII-Ag85 tetramer. At 8 weeks post-prime (black arrow), mice were intravenously boosted with 107 Msmeg expressing MU-Ag85A. Blood was similarly collected and analyzed for percent MU-Ag85A-specific CD4+ T cells. Asterisks indicate statistical analysis by the student’s t-test (n = 5 for each group) comparing the BCG pHA to BCG MU-Ag85A groups. Error bars represent standard deviation. *p<0.05, **p<0.005 B-D. At 3 weeks post-prime, mice were bled retro-orbitally and peripheral CD4+ T cells were stained for memory markers. Percentages of total CD62LloCD44lo naïve (B) and CD4+ T cells (C) were quantified, as were effector memory T cells (CD62LloCD44hi) and central memory T cells (CD62LhiCD44hi) which specifically bound Ag85-MHCII-tetramer (D). Asterisks indicate statistical analysis by the student’s t-test (n = 4 for each group). Error bars represent standard deviation. **p<0.005, ***p<0.001
Fig 3.
Expression of MU-Ag85A increases responsiveness of Th1 cells to M. ulcerans antigens.
C57BL/6 mice were left unprimed (white) or were intravenously primed with 107 BCG transformed with empty vector (BCG pHA, gray), Msmeg pHA (checkered), BCG MU-Ag85A (black), or Msmeg MU-Ag85A (hashed). At 8 weeks post-prime, mice were intravenously boosted with 107 Msmeg expressing MU-Ag85A. Two weeks following the boost, mice were euthanized and splenocytes were isolated for stimulation with MU-Ag85A peptide or heat killed M. ulcerans 1615 (HKMU). ELISPOT spot forming units (SFU) were used to determine number of IFNγ-producing splenocytes after 24 hours of stimulation. Asterisks indicate statistical analysis by the student’s t-test (n = 5 for each group). Error bars represent standard deviation. *p<0.05, **p<0.005
Fig 4.
Footpad swelling and tissue pathology are reduced upon vaccination with BCG MU-Ag85A.
C57BL/6 mice were left unprimed or were subcutaneously primed with 107 BCG transformed with empty vector (BCG pHA), or BCG MU-Ag85A. At 8 weeks post-prime, mice were intravenously boosted with 107 Msmeg expressing MU-Ag85A. Two weeks following the boost, mice were challenged with 105 MU1615 intradermally via the left hind leg footpad. Shown are representative images of infected footpads at 3, 6, and 15 weeks post-challenge. Mice were euthanized if vertical swelling surpassed 4.5 mm, as was the case for unprimed mice before week 15. B. Representative H&E stained tissue sections from 12 week MU-infected mouse footpads are shown. Paws of unprimed mice (UN) consistently showed ulceration (loss of epidermis) with abundant necrosis (N) and infiltrates of inflammatory cells (*). Edema (E) remained prominent in BCG pHA-primed (B) mice but was rarely associated with BCG MU-Ag85A-primed (BA) mouse footpads. Scale bar, 100 μm. C. Representative Ziehl-Neelson (ZN, top row) and H&E (bottom row)-stained tissue sections from 12 week MU-infected mouse footpads are shown. Granulomatous lesions (G) and large masses of acid fast bacilli (AFB,*) are apparent in unprimed footpads (UN), while groups of extracellular AFB were detected in BCG-primed mice (B). AFB generally presented as single scattered extracellular bacteria in footpad tissues from BCG MU-Ag85A-primed mice (BA) and could not be detected in the footpad sections available for ZN staining. Scale bars indicate 100 μm.
Fig 5.
Superior protective effects of subcutaneous vaccination with BCG MU-Ag85A.
A. Naïve C57BL/6 mice (dotted black) or mice subcutaneously primed with empty-vector BCG pHA (gray) or BCG MU-Ag85A (black) were challenged with 105 MU1615 via the footpad ten weeks post-vaccination. Area of footpad swelling was measured at various time points post-challenge. Mice were euthanized if vertical footpad swelling surpassed 4.5 mm and survival represents time to euthanasia. Asterisk indicates statistical analysis of BCG pHA versus BCG MU-Ag85A by the Mantel-Cox test (n = 10 for each group). B. Survival was assessed in MU1615-challenged mice which had been subcutaneously primed with BCG pHA (gray) or BCG MU-Ag85A (black) for eight weeks, followed by a two week boost with Msmeg MU-Ag85A. Asterisk indicates statistical analysis of BCG pHA versus BCG MU-Ag85A by the Mantel-Cox test (n = 10 for each group). *p<0.03, **p<0.002
Fig 6.
Vaccination with BCG MU-Ag85A significantly reduces footpad bacterial load at weeks 5 and 12 over BCG.
A. C57BL/6 mice were subcutaneously vaccinated as previously described (unprimed; white, empty-vector BCG pHA; gray, BCG MU-Ag85A; black) and challenged with 105 MU1615. At 5 and 12 weeks post-challenge, mice were euthanized and footpad homogenates were smeared on glass slides for auramine-rhodamine staining. Acid-fast bacilli (AFB) were quantified under 1000x magnification. Asterisks indicate statistical analysis by the student’s t-test (n = 16 images per group). Error bars represent standard deviation. ***p<0.001, *p<0.02 B. Shown are representative images from unprimed, BCG pHA, and BCG MU-Ag85A primed footpad smears at 5 and 12 weeks post-challenge.