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Vaccines | Spotlight

A Cation-Binding Surface Protein as a Vaccine Antigen To Prevent Moraxella catarrhalis Otitis Media and Infections in Chronic Obstructive Pulmonary Disease

Timothy F. Murphy, Aimee L. Brauer, Antoinette Johnson, Gregory E. Wilding, Mary Koszelak-Rosenblum, Michael G. Malkowski
Marcela F. Pasetti, Editor
Timothy F. Murphy
aDivision of Infectious Diseases, Department of Medicine, University at Buffalo, the State University of New York, Buffalo, New York, USA
bClinical and Translational Research Center, University at Buffalo, the State University of New York, Buffalo, New York, USA
cDepartment of Microbiology and Immunology, University at Buffalo, the State University of New York, Buffalo, New York, USA
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Aimee L. Brauer
aDivision of Infectious Diseases, Department of Medicine, University at Buffalo, the State University of New York, Buffalo, New York, USA
bClinical and Translational Research Center, University at Buffalo, the State University of New York, Buffalo, New York, USA
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Antoinette Johnson
aDivision of Infectious Diseases, Department of Medicine, University at Buffalo, the State University of New York, Buffalo, New York, USA
bClinical and Translational Research Center, University at Buffalo, the State University of New York, Buffalo, New York, USA
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Gregory E. Wilding
dDepartment of Biostatistics, University at Buffalo, the State University of New York, Buffalo, New York, USA
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Mary Koszelak-Rosenblum
eDepartment of Structural Biology, University at Buffalo, the State University of New York, Buffalo, New York, USA
fHauptman Woodward Medical Research Institute, Buffalo, New York, USA
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Michael G. Malkowski
eDepartment of Structural Biology, University at Buffalo, the State University of New York, Buffalo, New York, USA
fHauptman Woodward Medical Research Institute, Buffalo, New York, USA
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Marcela F. Pasetti
University of Maryland School of Medicine
Roles: Editor
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DOI: 10.1128/CVI.00130-17
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  • FIG 1
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    FIG 1

    (A) Schematic illustration of the afe gene cluster in the M. catarrhalis genome. Arrows indicate direction of transcription, and numbers indicate size of genes in bp. (B) Results of reverse transcriptase PCR (RT-PCR) with RNA extracted from M. catarrhalis O35E to detect afe gene cluster transcript in the regions that span genes as noted at the bottom. a lanes, PCR product from genomic DNA template; b lanes, RT-PCR in the absence of reverse transcriptase; c lanes, RT-PCR product. DNA standards are noted in kilobases. Bands in c lanes indicate that the gene cluster expresses a single transcript.

  • FIG 2
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    FIG 2

    Ethidium bromide-stained agarose gel showing amplicons of the afeA gene amplified from genomic DNA of 20 clinical isolates of M. catarrhalis. Lane a, O35E; lanes b to j, sputum isolates from adults experiencing exacerbations of COPD (lane b, 6P29B1; lane c, 10P66B1; lane d, 14P30B1; lane e, 39P33B1; lane f, 47P31B1; lane g, M2; lane h, M3; lane i, M4; lane j, M5); lanes k to t, middle-ear fluid isolates obtained by tympanocentesis from children experiencing acute otitis media (lane k, 2015; lane l, 5193; lane m, 6955; lane n, 7169; lane o, 9483; lane p, 0701057V1L; lane q, 0701064V3L; lane r, 0702076SV4R; lane s, 0701062V1L; lane t, 0701067V3L). Molecular size markers are on the left in kilobases.

  • FIG 3
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    FIG 3

    Left, lane a, purified AfeA in Coomassie blue-stained sodium dodecyl (SDS) gel; lane b, purified AfeA in silver-stained SDS gel. Center, immunoblot assay with rabbit antiserum to recombinant purified AfeA (1:106 dilution). Right, immunoblot assay with rabbit antiserum to recombinant purified BCAA SBP1 (branched-chain amino acid substrate binding protein 1). c lanes, whole-cell lysate of wild-type strain O35E; d lanes, whole-cell lysate of afe knockout mutant; e lanes, whole-cell lysate of afe complemented mutant. Molecular weight markers are shown in kilodaltons. Arrows denote AfeA.

  • FIG 4
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    FIG 4

    Immunoblot assay of whole-cell lysates of 9 clinical isolates of M. catarrhalis probed with rabbit antiserum to recombinant purified AfeA (1:106 dilution). Strains are sputum isolates in lanes as follows: lane a, 6P29B1; lane b, 10P66B1; lane c, 14P30B1; lane d, 39P33B1; lane e, 47P31B1; lane f, M2; lane g, M3; lane h, M4; lane i, M5. Molecular size markers are shown in kilodaltons on the left.

  • FIG 5
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    FIG 5

    Results of whole-cell ELISA with M. catarrhalis strain O35E, afe knockout mutant, and complemented afe mutant used to coat wells and assayed with antisera as noted. x axes are serum dilutions, and y axes are optical density at 450 nm. Results are shown with preimmune and immune antisera. (A) AfeA antiserum with WT and afe knockout mutant. (B) AfeA antiserum with complemented afe mutant. (C) OppA antiserum with WT and afe knockout mutant (positive control; OppA is a surface protein). (D) OppA antiserum with complemented afe mutant. (E) BCAA antiserum with WT and afe knockout mutant (negative control; BCAA is a nonsurface protein). (F) BCAA antiserum with complemented afe mutant. Error bars indicate the standard deviations of the results from three independent experiments.

  • FIG 6
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    FIG 6

    Results of flow cytometry with M. catarrhalis wild-type (WT) O35E, afe knockout mutant, and complemented afe mutant. x axes are fluorescence, and y axes are cell counts. (A) WT strain O35E, afe knockout mutant, and complemented afe mutant assayed with AfeA antiserum (1:100) and preimmune serum (1:100). (B) WT strain O35E and afeA knockout mutant assayed with OppA antiserum (1:100) and preimmune serum (1:100) (positive control; OppA is a surface protein). (C) WT strain O35E and afeA knockout mutant assayed with BCAA antiserum (1:100) and preimmune serum (1:100) (negative control; BCAA is a nonsurface protein).

  • FIG 7
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    FIG 7

    (A) Immunoblot assays with sera (1:2,000) pooled from mice immunized with PBS (negative control), purified recombinant AfeA (25-μg and 50-μg schedules as noted), and whole cells of M. catarrhalis O35E. a lanes, whole bacterial cell lysate of wild-type strain O35E; b lanes, whole-cell lystate of afe knockout mutant. Arrows denote AfeA. Molecular size markers are shown in kilodaltons on the left. (B) Results of pulmonary clearance 3 h after aerosol challenge with M. catarrhalis O35E following immunization of groups of mice with PBS (negative control), recombinant AfeA, and whole cells of M. catarrhalis strain O35E (positive control). y axis is colony count (in CFU per milliliter) in lung homogenates. Error bars represent the standard deviations (n = 6). Statistically significant overall group differences were observed with a P value of 0.0003. Results of pairwise comparisons with the PBS group (negative control) and associated P values are shown.

  • FIG 8
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    FIG 8

    Results of ELISA to purified AfeA with 19 pairs of preexacerbation and postexacerbation serum samples (1:4,000) from adults with COPD followed longitudinally. x axis shows results from individual patients. y axis shows % change in optical density from preexacerbation to postexacerbation values. Dotted line represents the cutoff for a significant change based on assays with control serum pairs (see the text).

Tables

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  • TABLE 1

    Oligonucleotide primers used in the study

    Name of primerExptaSequenceb
    Afe frag1 F1Mutant fragment 1 upstream of afeATTTAAATAAAAAGCCATACG
    Afe frag1 R1Mutant fragment 1 upstream of afeATAGTTAGTCAAAATTAACCTAATTGCTTGA
    Afe frag2 F1Mutant fragment 2 kanamycin cassetteAGGTTAATTTTGACTAACTAGGAGGAATAA
    Afe frag2 R1Mutant fragment 2 kanamycin cassetteAAAAATATAACATTATTCCCTCCAGGTACT
    Afe frag3 F1Mutant fragment 3 downstream of afeAGGGAATAATGTTATATTTTTAATATATTTT
    Afe frag3 R1Mutant fragment 3 downstream of afeACCTGCTGGTGTCATGTATCA
    Afe lipoprotein F1Clone AfeA geneGTACCCATGGAGGTCAGCAGACCAAAGAAGA
    Afe lipoprotein R1Clone AfeA geneGATCGGATCCCTTTTCAAAACCGCTGGCGA
    AfeA 5RT-PCR afeA-afeBGCACTCATTAAGCAAGACCC
    AfeB 3RT-PCR afeA-afeBGAGCCAAAGCCCTTGCCA
    AfeB 5RT-PCR afeB-afeCGGCAAGGGCTTTGGCTCAAG
    AfeC 3RT-PCR afeB-afeCCAGTCACTTGATACATATC
    AfeC 5RT-PCR afeC-afeDGATATGTATCAAGTGACTG
    AfeD 3RT-PCR afeC-afeDCAATCGTCGCACCTGTCGC
    AfeA F1Amplify afeA to assess conservation among strainsATGAAATCAATCAAAACTTT
    AfeA R1Amplify afeA to assess conservation among strainsTCACTTTTCAAAACCGCTGG
    Afe comp F1Complement afe mutationGATCGGATCCCAATTCATGATTAAGTGGTG
    Afe comp R1Complement afe mutationGATCGAGCTCGGTCTTGAACGGTGTTTGTT
    Afe PET F1Clone nonlipidated AfeA for thermal shift assaysCACCTGCGGTCAGCAGACCAAAGA
    Afe PET 2Clone nonlipidated AfeA for thermal shift assaysTCACTTTTCAAAACCGCTGG
    • ↵a Reverse transcriptase PCR.

    • ↵b Underlined sequences indicate restriction enzyme sites.

  • TABLE 2

    Melting temperatures and results of thermal shift assays with purified nonlipidated recombinant AfeA

    SampleCationTm (°C)aΔTm (°C)b
    Nonlipidated AfeA alone 53.1, 76.7
    +1 mM MgCl2Mg2+53.6, 76.90.5
    +1 mM MnCl2Mn2+76.423.3
    +1 mM ZnCl2Zn2+77.124.0
    +1 mM Fe(NO3)3Fe3+76.823.7
    +1 mM FeCl3Fe3+76.723.6
    +1 mM FeCl2Fe2+76.723.6
    • ↵a Tm, melting temperature. Two values indicate peaks at two temperatures due to trace cations in buffers.

    • ↵b ΔTm, thermal shift.

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A Cation-Binding Surface Protein as a Vaccine Antigen To Prevent Moraxella catarrhalis Otitis Media and Infections in Chronic Obstructive Pulmonary Disease
Timothy F. Murphy, Aimee L. Brauer, Antoinette Johnson, Gregory E. Wilding, Mary Koszelak-Rosenblum, Michael G. Malkowski
Clinical and Vaccine Immunology Sep 2017, 24 (9) e00130-17; DOI: 10.1128/CVI.00130-17

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A Cation-Binding Surface Protein as a Vaccine Antigen To Prevent Moraxella catarrhalis Otitis Media and Infections in Chronic Obstructive Pulmonary Disease
Timothy F. Murphy, Aimee L. Brauer, Antoinette Johnson, Gregory E. Wilding, Mary Koszelak-Rosenblum, Michael G. Malkowski
Clinical and Vaccine Immunology Sep 2017, 24 (9) e00130-17; DOI: 10.1128/CVI.00130-17
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    • ABSTRACT
    • INTRODUCTION
    • RESULTS
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KEYWORDS

ABC transporters
Moraxella catarrhalis
immunization
otitis media
pulmonary infection
surface antigens
vaccines

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