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Vaccines

Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

R. L. Brocato, M. J. Josleyn, V. Wahl-Jensen, C. S. Schmaljohn, J. W. Hooper
R. L. Brocato
aVirology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
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M. J. Josleyn
aVirology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
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V. Wahl-Jensen
aVirology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
bIntegrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Frederick, MD, USA
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C. S. Schmaljohn
cScience and Technology, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
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J. W. Hooper
aVirology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA
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DOI: 10.1128/CVI.00546-12
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Figures

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  • Fig 1
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    Fig 1

    PUUV DNA vaccine testing in nonhuman primates. (A) Neutralization of PUUV strain K27. Groups of three rhesus macaques were vaccinated (indicated by arrows) with either pWRG/PUU-M(s1), pWRG/PUU-M(s2), or pWRG/PUU-M(x22). Sera collected on the indicated weeks were tested for the capacity to neutralize PUUV strain K27. Each symbol represents the GMT for 2 or 3 PRNT50 assays. (B) Cross-neutralization of PUUV strain Sotkamo. Sera collected after 3 (week 5) or 4 (week 19) vaccinations were tested by a PRNT using PUUV strain Sotkamo. The PRNT limit of detection was a titer of 20 (dashed line).

  • Fig 2
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    Fig 2

    In vivo stability testing of HTNV and PUUV DNA vaccines delivered using a handheld single-use gene gun (ND10). (A) Lot 1 of each vaccine was tested for immunogenicity over 1 year. (B) Lot 2 of each vaccine was tested for immunogenicity over 2 years. Symbols represent the neutralizing antibody PRNT50 titers for individual hamsters vaccinated with either pWRG/HTN-M(x) or pWRG/PUU-M(s2). Hamsters vaccinated with the HTNV DNA vaccine were evaluated for HTNV neutralizing antibodies, and hamsters vaccinated with the PUUV DNA vaccine were evaluated for PUUV strain K27 neutralizing antibodies. The PRNT limit of detection was a titer of 20 (dashed line).

  • Fig 3
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    Fig 3

    Evaluation of protection (PUUV) and cross-protection (DOBV) in hamster infection models. Groups of 6 to 8 hamsters were vaccinated (week 0, 2, or 3) with PUUV DNA vaccine, HTNV DNA vaccine, or no vaccine and then challenged with PUUV strain K27 or DOBV. (A) Hamsters were challenged either 4 weeks or 12 weeks after the last vaccination, using an intramuscular challenge of 2,000 PFU PUUV. (B) Hamsters were challenged 11 weeks after the last vaccination, using an intranasal challenge of 2,000 PFU DOBV. Sera were collected at 35 days postchallenge, and N-ELISA log10 titers were plotted. The N-ELISA limit of detection was a titer of 2 log10 (dashed line).

  • Fig 4
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    Fig 4

    PUUV DNA vaccine protects against lethal hantavirus disease. Groups of 8 to 10 hamsters were vaccinated with the indicated vaccine or not vaccinated. Sixteen weeks (A) or 20 weeks (B) elapsed before an intranasal challenge of 4,000 PFU ANDV. (C) Groups of 9 or 10 hamsters were vaccinated with the indicated vaccine or not vaccinated before an intramuscular challenge of 2,000 PFU ANDV. Survival of all animals was monitored for 35 days postchallenge. ns, not significant.

Tables

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  • Table 1

    Comparison of differences in deduced M segment open reading frame amino acids for four strains of PUUV and three DNA vaccine constructsf

    Amino acid positionaPUUV M segment amino acidDNA vaccine amino acid
    DTK/Ufa-97bK27cP360dHallnas B1ePUU-M(x22)PUU-M(s1)PUU-M(s2)
    10YYCYYYY
    38VIIIIII
    187QQQDQQQ
    207HHHHYYH
    312AAAVAAA
    416LSLLLLL
    448MMMMTTM
    492WWWWRRW
    501FLFFFFF
    604TTTRTTT
    649LLLHLLL
    650EEEHEEE
    675GGGRGGG
    690PPPQPPP
    972QQQQRRQ
    1077GGGPGGG
    1097SSSSLLS
    • ↵a M gene segment open reading frame amino acid position.

    • ↵b DTK/Ufa-97, PUUV strain DTK/Ufa (GenBank accession no. BAF49040).

    • ↵c K27, PUUV strain K27 (GenBank accession no. P41265).

    • ↵d P360, PUUV strain P360 (GenBank accession no. P41266).

    • ↵e Hallnas B1, PUUV strain Hallnas B1 (GenBank accession no. P21400).

    • ↵f Amino acids shown in bold are differences with respect to the PUUM(s2) sequence.

  • Table 2

    PUUV neutralizing antibodies and protection against PUUV infection

    VaccineaHamster IDPrechallenge PUUV PRNT50 titerdPostchallenge N-ELISA titer (log10)b,dProtectionc
    pWRG/PUU-M(s2)2695,1203N
    2622,5601Y
    261, 2631,2801Y
    2436401Y
    2483202N
    2663201Y
    245, 264, 2671601Y
    242, 244, 249, 270801Y
    241401Y
    246, 250, 268201Y
    26512N
    None298403N
    281, 284203N
    282202N
    28312N
    285, 28613N
    28714N
    288, 290, 978, 98013N
    29112N
    292, 293–295, 29713N
    pWRG/HTN-M(x)251, 252, 254, 258–260, 232, 236, 239, 24013N
    253, 256, 231, 23714N
    • ↵a Hamsters were vaccinated on weeks 0, 2, and 3 by use of ND10 delivery devices.

    • ↵b Log10 endpoint titers represent GMTs for two N-ELISAs.

    • ↵c A postchallenge N-ELISA titer of <2 indicates that the hamster was protected. N, not protected; Y, protected.

    • ↵d A value of 1 indicates that the titer was below the limit of detection. The limit of detection for the PRNT was 20, and the limit of detection for the ELISA was 2 (log10).

  • Table 3

    Calculation of estimated probabilities of protective immunity against PUUV infection based on the protection data from Table 2

    Estimated probability of protective immunityaPrechallenge PUUV PRNT50 titerLower 95% fiducial limitUpper 95% fiducial limit
    0.4024877
    0.503713148
    0.605922305
    0.709735713
    0.80177582,122
    0.9043811911,700
    • ↵a Determined by logistic regression by Probit. Protection status by titer was used to calculate estimated probabilities of protective immunity.

Additional Files

  • Figures
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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Fig. S1. Presynthetic PUUV full-length M gene-based DNA vaccine.

      PDF, 309K

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Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine
R. L. Brocato, M. J. Josleyn, V. Wahl-Jensen, C. S. Schmaljohn, J. W. Hooper
Clinical and Vaccine Immunology Jan 2013, 20 (2) 218-226; DOI: 10.1128/CVI.00546-12

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Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine
R. L. Brocato, M. J. Josleyn, V. Wahl-Jensen, C. S. Schmaljohn, J. W. Hooper
Clinical and Vaccine Immunology Jan 2013, 20 (2) 218-226; DOI: 10.1128/CVI.00546-12
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