Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Infection and Immunity
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About IAI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Microbial Immunity and Vaccines

A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB, Encoding a Putative Capsular Biosynthesis Protein, Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge

Qingmei Jia, Bai-Yu Lee, Richard Bowen, Barbara Jane Dillon, Susan M. Som, Marcus A. Horwitz
Qingmei Jia
1Division of Infectious Diseases, Department of Medicine, 37-121 Center for Health Sciences, School of Medicine, University of California—Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1688
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Bai-Yu Lee
1Division of Infectious Diseases, Department of Medicine, 37-121 Center for Health Sciences, School of Medicine, University of California—Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1688
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Richard Bowen
2Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Barbara Jane Dillon
1Division of Infectious Diseases, Department of Medicine, 37-121 Center for Health Sciences, School of Medicine, University of California—Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1688
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Susan M. Som
1Division of Infectious Diseases, Department of Medicine, 37-121 Center for Health Sciences, School of Medicine, University of California—Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1688
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marcus A. Horwitz
1Division of Infectious Diseases, Department of Medicine, 37-121 Center for Health Sciences, School of Medicine, University of California—Los Angeles, 10833 Le Conte Avenue, Los Angeles, California 90095-1688
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: mhorwitz@mednet.ucla.edu
DOI: 10.1128/IAI.00192-10
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • FIG. 1.
    • Open in new tab
    • Download powerpoint
    FIG. 1.

    Construction and characterization of LVS ΔcapB. (A) Construction of LVS ΔcapB. LVS ΔcapB was constructed by allelic exchange between LVS (top) and a plasmid carrying an exchange cassette containing truncated capB (FTL_1416/FTT0805) and its flanking nucleotide sequences, including the upstream region of FTL_1417 (FTL_1417′) and the downstream region of capC and capA (capCA′) (middle). The resultant LVS ΔcapB strain (bottom) retains 6 amino acids from the N terminus and 4 amino acids from the C terminus of CapB and contains no antibiotic resistance marker (unmarked). (B) Schematic representations of the structure of the capB locus and its upstream and downstream regions in the genomes of LVS (top) and LVS ΔcapB (bottom), the position of a probe spanning the EcoRV restriction site located in FTL_1417 and the EcoRV site located in capA, and the predicted EcoRV, HindIII, and AvaII restriction fragments (in kilobases) detected by the probe by Southern blot analysis. A, restriction enzyme AvaII; E, EcoRV; H, HindIII. (C) Southern blot analysis of LVS ΔcapB (Δ) and the parental LVS genomic DNA. The genomic DNAs of LVS (lanes 1, 3, and 5, indicated below the panels) and LVS ΔcapB (lanes 2, 4, and 6) were isolated from bacteria cultured on chocolate agar for 2 days. The DNA was digested with EcoRV, HindIII, or AvaII, as indicated at the top of each panel; electrophoresed with a DNA ladder (L); blotted; and hybridized with the biotin-labeled probe as shown in B and the biotin-labeled DNA ladder. The left of each panel shows a 1-kb Plus DNA ladder, and to the right of each panel are the fragments resulting from restriction enzyme digestion of the bacterial DNA detected by the probe. (D and E) Quantitative RT-PCR analysis of capB and its neighboring genes. Total RNA was isolated from LVS, LVS ΔcapB, and the capB-complemented strain LVS ΔcapB/CapB; reverse transcribed in the presence (+) or absence (−) of reverse transcriptase; and amplified by using primer pairs specific to capB, its upstream region of FTL_1417, and downstream regions of capC and capA. 16S rRNA (16S) was used as an internal control. Transcript levels are presented as the mean fold changes versus LVS ± SE. ***, P < 0.001 by two-way ANOVA. qRT-PCR products were analyzed on a 2% agarose gel. The predicted sizes of the qRT-PCR product for capA, capB, capC, FTL_1417, and 16S rRNA are 231, 231, 212, 209, and 156 bp, respectively. The sizes of the DNA in the DNA ladder are shown at the left of each panel in E. F. tularensis strains from which the RNA was isolated are shown at the right of each panel in E.

  • FIG. 2.
    • Open in new tab
    • Download powerpoint
    FIG. 2.

    In vitro characterization of LVS ΔcapB. (A) Analysis of LVS and LVS ΔcapB LPS and proteins. (Left) LPS was analyzed by immunoblotting using a monoclonal antibody to LPS (note the characteristic LPS ladder pattern). (Right) Proteins were evaluated by Coomassie blue staining to provide a loading control. (B) Complement sensitivity assay. Each bacterial strain was incubated with either fresh human AB serum (ABS) or heat-inactivated AB serum (HI-ABS) for 10 min and then assayed for CFU on chocolate agar. This experiment was performed three times, with similar results. (C) Competition for growth in human macrophage-like THP-1 cells. THP-1 cells were coinfected with LVS ΔcapB carrying a hygromycin resistance gene (ΔcapB-h) and with LVS carrying a kanamycin resistance gene (LVS-k) (solid circles, left vertical axis) or with the same two strains carrying the opposite resistance markers (solid squares, right vertical axis). At 0, 7, and 22 h postinfection, the cell monolayer was lysed, serial dilutions of the lysate were plated onto chocolate agar supplemented with either kanamycin or hygromycin, CFU were enumerated, and the ratio between the numbers of LVS ΔcapB and LVS CFU was calculated. The ratios of LVS ΔcapB to LVS CFU on the left and right vertical axes differ because of the variability among the four strains in the numbers of CFU recovered from THP-1 monolayers at the start of the experiment. For the competition data shown on the left axis, the initial number of LVS ΔcapB organisms was greater than the initial number of LVS organisms (initial ratio of ∼10:1), and for the competition data shown on the right axis, the initial number of LVS ΔcapB organisms was fewer than the initial number of LVS organisms (initial ratio of ∼0.4:1). In both cases, LVS ΔcapB was outcompeted by LVS, and therefore, the ratio of LVS ΔcapB CFU to LVS CFU declined with time in culture. This experiment was performed twice, with similar results.

  • FIG. 3.
    • Open in new tab
    • Download powerpoint
    FIG. 3.

    LVS ΔcapB is more attenuated than parental LVS and complemented LVS ΔcapB/CapB in mice. Groups of four BALB/c mice were nonimmunized, immunized with PBS (sham), or immunized i.n. or i.d. with LVS, LVS ΔcapB, or LVS ΔcapB/CapB at the indicated doses (CFU) and monitored for survival, weight change, local lesions (after i.d. immunization), and other signs of illness for 2 or 3 weeks. (A1 and A2) Mice were weighed daily from days 2 to 9 postimmunization and monitored for survival for 3 weeks. (B1 to B3) Mice were weighed daily from day 0 (the day of immunization) to day 7 postimmunization and monitored for survival for 2 weeks. Values are means ± SE. RF, ruffled fur; HB, hunched back; SE, swollen eyes.

  • FIG. 4.
    • Open in new tab
    • Download powerpoint
    FIG. 4.

    Dissemination and clearance of LVS ΔcapB in mice. Groups of four BALB/c mice were immunized i.n. with 112 CFU LVS or 1 × 105 CFU LVS ΔcapB or i.d. with 1 × 105 CFU LVS or 1 × 106 CFU LVS ΔcapB. At the indicated times postimmunization, mice were euthanized, and numbers of CFU in the spleen, liver, lung, and skin at the site of injection (after i.d. immunization) were assayed. LVS ΔcapB is cleared faster than LVS in spleen, liver, and lung after i.n. immunization and at the site of injection in the skin after i.d. immunization. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by ANOVA).

  • FIG. 5.
    • Open in new tab
    • Download powerpoint
    FIG. 5.

    Immunization and challenge protocols. Groups of BALB/c mice were immunized i.n. or i.d. with LVS ΔcapB or LVS. Mice immunized with PBS (sham) or LVS served as controls. At the indicated times postimmunization, mice were either euthanized for immunology studies (A), challenged with approximately 4,000 CFU LVS (>5× the LD50) by the i.n. route (B and C), or challenged with 10× the LD50 of type A F. tularensis strain SchuS4 by the aerosol route (D). For assessments of organ bacterial burden, four or eight mice per group challenged with LVS i.n. were euthanized at 5 days postchallenge, and the organs were homogenized and assayed for CFU of LVS (B). For assessments of survival, eight mice per group challenged with LVS i.n. or SchuS4 by aerosol were monitored for signs of illness and death for 3 weeks after challenge (C and D). LPA, lymphocyte proliferation assay.

  • FIG. 6.
    • Open in new tab
    • Download powerpoint
    FIG. 6.

    Immunization with LVS ΔcapB induces cell-mediated immune responses comparable to those induced by LVS. Groups of four BALB/c mice were immunized i.n. with 112 CFU LVS or 1 × 105 CFU LVS ΔcapB or i.d. with 1 × 105 CFU LVS or 1 × 106 CFU LVS ΔcapB. At 4 weeks postimmunization, mice were euthanized, and single-cell suspensions of splenocytes were prepared. A total of 1 × 106 splenocytes were incubated with 2 × 105 or 2 × 106 CFU heat-inactivated (HI) LVS as indicated, and splenic lymphocyte proliferation was assayed. The lines above the bars indicate a statistical comparison between the bar beneath the left end of the line and the bar beneath the right end of the line. Only comparisons where differences are statistically significant are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (by two-way ANOVA).

  • FIG. 7.
    • Open in new tab
    • Download powerpoint
    FIG. 7.

    Immunization with LVS ΔcapB induces potent antibody responses. Groups of four BALB/c mice were immunized i.n. with 112 CFU LVS or 1 × 105 CFU LVS ΔcapB or i.d. with 1 × 105 CFU LVS or 1 × 106 CFU LVS ΔcapB. At the indicated times postimmunization, mice were euthanized, and serum was collected and analyzed for IgG, IgA, IgM, IgG1, and IgG2 antibodies specific to heat-inactivated LVS. The antibody level was calculated as the log10 of the reciprocal of the endpoint dilution of the test serum. Data represent means ± SE. The lines above the bars indicate a statistical comparison between the bar beneath the left end of the line and the bar beneath the right end of the line. Only comparisons where differences were statistically significant between LVS and LVS ΔcapB by the same route are shown. *, P < 0.05; ***, P < 0.001 (by two-way ANOVA).

  • FIG. 8.
    • Open in new tab
    • Download powerpoint
    FIG. 8.

    Immunization with LVS ΔcapB induces protective immunity against F. tularensis LVS i.n. challenge in a dose-dependent manner. BALB/c mice (four mice per group) were immunized i.n. (left) or i.d. (right) with LVS ΔcapB at the doses indicated on the horizontal axes. Mice immunized with PBS (sham) (eight per group) or LVS (four per group) served as controls. Four weeks later, the mice were challenged i.n. with 4,000 CFU LVS. At 5 days postchallenge, the spleen (A and B), liver (C and D), and lung (E and F) were removed and assayed for bacterial burden. Symbols represent each animal in a group. CFU data were compared by one-way ANOVA (Prism 5.01 software) with Bonferroni's posttest. Dashed lines indicate the limit of detection. Differences in CFU between the sham group and each of the groups vaccinated with either LVS or LVS ΔcapB were statistically significant (P < 0.001). The lines above the bars indicate a statistical comparison between the bar beneath the left end of the line and the bar beneath the right end of the line. Only comparisons between the LVS-immunized group and the LVS ΔcapB-immunized group where differences were statistically significant are shown.

  • FIG. 9.
    • Open in new tab
    • Download powerpoint
    FIG. 9.

    Immunization with LVS ΔcapB induces protective immunity against i.n. F. tularensis LVS challenge comparable to that of the parental LVS. Groups of 12 mice were sham immunized, immunized i.d. with 1 × 105 CFU LVS or 1 × 106 CFU LVS ΔcapB, or immunized i.n. with 200 CFU LVS or 1 × 105 CFU LVS ΔcapB. Four weeks (A and C) or 8 weeks (B and D) later, mice were challenged i.n. with 4,000 CFU LVS. (A and B) At 5 days postchallenge, four mice per group were euthanized, and the spleen, liver, and lung were assayed for bacterial burden. CFU values are shown as means ± SE. The dashed line indicates the limit of detection. The mean CFU in each organ among different groups was analyzed by grouped ANOVA with Bonferroni's posttest (Prism 5.01 software). The lines above the bars indicate a statistical comparison between the bar beneath the left end of the line and the bar beneath the right end of the line. Only comparisons where differences were statistically significant are shown. **, P < 0.01; ***, P < 0.001. (C and D) The remaining mice in each group were monitored for survival and signs of illness for 3 weeks. The differences in survival between the mice in the vaccinated groups and the mice in the sham-vaccinated group were evaluated by using a log-rank (Mantel-Cox) test (Prism 5.01). ***, P < 0.0001 versus Sham. a, 3 of 12 mice (25%) died after i.n. immunization with LVS; of the remaining 9 mice, 4 were studied for the data shown in A, and 5 were studied for the data shown in C.

  • FIG. 10.
    • Open in new tab
    • Download powerpoint
    FIG. 10.

    Immunization with LVS ΔcapB induces protective immunity against F. tularensis SchuS4 aerosol challenge. Groups of eight mice were sham immunized, immunized i.d. (A and C) with 1 × 105 CFU LVS or 1 × 106 CFU LVS ΔcapB, or immunized i.n. (B and D) with 200 CFU LVS or 1 × 105 CFU LVS ΔcapB. Six weeks later, all mice were challenged by the aerosol route with 10× the LD50 of F. tularensis strain SchuS4. Mice were monitored for weight change (A and B) and survival (C and D) for 3 weeks. Weights at each time point are shown as means ± SE. Mean survival time was calculated by dividing the sum of the survival times of all mice in a group by the total number of mice challenged, with animals surviving until the end of the experiment given a survival time of 21 days, when the experiment was terminated. The differences in survival between the mice in the vaccinated groups and mice in the sham-vaccinated group were evaluated by using a log-rank (Mantel-Cox) test (Prism 5.01 software).

PreviousNext
Back to top
Download PDF
Citation Tools
A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB, Encoding a Putative Capsular Biosynthesis Protein, Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge
Qingmei Jia, Bai-Yu Lee, Richard Bowen, Barbara Jane Dillon, Susan M. Som, Marcus A. Horwitz
Infection and Immunity Sep 2010, 78 (10) 4341-4355; DOI: 10.1128/IAI.00192-10

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Infection and Immunity article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB, Encoding a Putative Capsular Biosynthesis Protein, Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge
(Your Name) has forwarded a page to you from Infection and Immunity
(Your Name) thought you would be interested in this article in Infection and Immunity.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
A Francisella tularensis Live Vaccine Strain (LVS) Mutant with a Deletion in capB, Encoding a Putative Capsular Biosynthesis Protein, Is Significantly More Attenuated than LVS yet Induces Potent Protective Immunity in Mice against F. tularensis Challenge
Qingmei Jia, Bai-Yu Lee, Richard Bowen, Barbara Jane Dillon, Susan M. Som, Marcus A. Horwitz
Infection and Immunity Sep 2010, 78 (10) 4341-4355; DOI: 10.1128/IAI.00192-10
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Bacterial Proteins
Bacterial Vaccines
Francisella tularensis
tularemia

Related Articles

Cited By...

About

  • About IAI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #IAIjournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0019-9567; Online ISSN: 1098-5522