Research ArticleInfectious Disease

A live vaccine rapidly protects against cholera in an infant rabbit model

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Science Translational Medicine  13 Jun 2018:
Vol. 10, Issue 445, eaap8423
DOI: 10.1126/scitranslmed.aap8423
  • Fig. 1 Major genetic alterations in HaitiV.

    (A) Deletion of the CTX prophage and adjacent sequences, including the satellite prophages TLC and RS1 and MARTX toxin genes (yellow area, deleted region). (B) Deletion of genes conferring resistance to trimethoprim (dfrA), sulfamethoxazole (sul2), streptomycin (strAB), and chloramphenicol (floR). (C) An anti-ctxA CRISPR system provides immunity to CTXΦ infection: Streptococcus pyogenes cas9 along with sequence encoding a single-guide RNA (sgRNA) targeting ctxA, integrated into the HaitiV lacZ locus; schematic showing targeting of the CTXΦ genome by the anti-ctxA Cas9-sgRNA complex; HaitiV with/without the CRISPR system (CRISPR+/−) were infected with either CTXΦ-IGKn (Target+; intergenic KanR cassette, intact ctxA) or CTX-KnΦ (Target−; ctxA replaced by KanR cassette), and the number of transductants was monitored. No detectable KanR transductants shown as “†”.

  • Fig. 2 HaitiV colonizes the infant rabbit intestine without causing cholera-like illness.

    (A) Fluid accumulation ratios (FARs) after littermates were pretreated with ranitidine-hydrochloride, to reduce stomach acidity, and inoculated with either WT (n = 11) or HaitiV (vaccine; n = 10). Plots show mean and SD derived from two litters. ****P < 0.0001, unpaired t test. (B) Successive daily weights of animals inoculated with 109 colony-forming units (CFU) of HaitiV (n = 10). (C) WT (blue circles) or HaitiV (red squares) CFU recovered from rabbit dSIs at day 1 or day 4 after inoculation (each of the three groups consists of animals from at least two litters). Lines indicate geometric means, and the open symbol indicates the limit of detection for the single animal from which no CFU were recovered. N.S. (not significant): P ≥ 0.05, Kruskall-Wallis test followed by Dunn’s multiple comparisons test. (D) Competitive indices (CIs) of dSI bacteria 1 day after inoculation with a 1:1 mixture of WT and HaitiV. The open symbol indicates limit of detection for the single animal from which no vaccine CFU were recovered; lines and bars indicate geometric means and geometric SD of CIs across two litters (n = 6). (E) WT (blue) and HaitiV (red) CFU recovered from coinoculated animals. The open symbol indicates the limit of detection the single animal from which no vaccine CFU were recovered, and lines indicate geometric means.

  • Fig. 3 HaitiV mediates colonization resistance associated with variably sized infection bottlenecks.

    (A) WT CFU (blue circles) recovered from the dSI of animals 18 hours after inoculation with WT. Littermates were pretreated with sodium bicarbonate buffer (mock, n = 8) or formalin-killed HaitiV (killed vaccine, n = 7) 24 hours before WT challenge; geometric means of each group across three litters are shown. N.S.: P ≥ 0.05, Mann-Whitney test. HaitiV and HaitiWT (B) or N16961 WT (C) CFU recovered from the dSI of animals 18 hours after challenge. Animals were pretreated with killed (n = 6) or live (n = 8) vaccine 24 hours before challenge. Open symbols indicate limit of detection for five animals in which no CFU were recovered, and lines indicate the geometric mean of each group across two litters. ***P < 0.001, *P < 0.05, Mann-Whitney test. (D) HaitiTn CFU (blue circles) and unique transposon mutants (black triangles) recovered from the dSI of individual animals (rabbits r1 to r6) 1 day after inoculation of the transposon mutant library without pretreatment. (E) HaitiTn CFU (blue circles), HaitiV CFU (red squares), and unique transposon mutants (black triangles) recovered from the dSI of individual animals (rabbits r1 to r7) 18 hours after inoculation of the transposon mutant library. Animals were pretreated with HaitiV 24 hours before challenge with the transposon mutant library. (F and G) Results of Con-ARTIST (39) analysis for single-inoculation (rabbit r4) and sequential-inoculation (rabbit r6) samples with the largest number of unique genotypes. The x axis indicates the change in relative abundance of insertion mutants per gene in vivo, and the y axis indicates the concordance of independent insertion mutants within each gene. Genes exhibiting a greater than twofold change [log2 (mean fold change) <−1 or >1] across multiple mutants (mean inverse P > 102) are considered depleted or enriched. Enriched mutants cqsS and hapR are indicated in blue. Mutations in known colonization factors, including toxin-coregulated pilus biogenesis (red circles) and the associated transcriptional regulators toxR and toxS (red asterisks), were depleted.

  • Fig. 4 HaitiV colonization protects from disease after HaitiWT challenge, and modeling demonstrates the benefit of rapid protection during a cholera outbreak.

    (A) Survival curves tracking progression to moribund disease status in animals inoculated with WT at 0 hours after pretreatment (at t = −24 hours) with killed (black, n = 8) or live vaccine (red, n = 7). ***P < 0.001, log-rank test. (B) Disease progression from the onset of diarrhea to moribund status in animals, pretreated with killed (black, n = 7) or live vaccine (red, n = 6) that developed visible diarrhea. ***P < 0.001 and **P < 0.01, log-rank test. (C) WT CFU (blue circles) recovered from the dSI of animals 41 hours after challenge [from (A)] that did not progress to moribund disease status. (D) Effect of reactive vaccination on the number of cholera infections in a simulated outbreak (R0 = 2.1) starting with a single infection in a population of 100,000 susceptible individuals where the reactive vaccination campaign (RVC) is triggered once the number of symptomatic individuals reaches 1000 (1% of the total population), indicated by the dashed line. Rollout of doses is modeled with a constant rate over 7 days until 70% of the population is vaccinated, as achieved by recent RVCs. Modeling parameters are described in fig. S3B.

  • Table 1

    Genetic alterations in HaitiV, a live attenuated cholera vaccine.

    MutationRationale
    ΔCTXΦAttenuates by removing the genes encoding
    CT and the multifunctional toxin MARTX
    (13); protects against toxigenic reversion
    by preventing chromosomal integration of
    CTXΦ (12)
    ΔflaBDEflaACAttenuates and reduces potential
    reactogenicity (15)
    ΔfloR-strAB-sul2dfrAPrevents the dispersal of antibiotic resistance
    genes
    N900_11550::Phtpg-ctxBConstitutive expression of CtxB (fig. S1)
    promotes anti-CtxB immune responses
    that may protect against diarrheal disease
    caused by V. cholerae and ETEC (16)
    ΔhupBProtects from toxigenic reversion by
    inactivating the HU complex, which is
    necessary for extrachromosomal
    replication of CTXΦ (14)
    lacZ::cas9-sgRNA_ctxAEndonuclease targeting of ctxA prevents
    toxigenic reversion
    ΔrecAPrevents homologous recombination-
    dependent gene acquisition

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/445/eaap8423/DC1

    Fig. S1. HaitiV produces only the B subunit of CT.

    Fig. S2. Results of Con-ARTIST analysis.

    Fig. S3. SEIR model and parameters.

    Fig. S4. Impact of model parameters on relative protection.

    Table S1. Strains and plasmids used in this study.

    Table S2. Oligonucleotide sequences used in this study.

    Table S3. Number of unique genotypes and the results of Con-ARTIST analysis of transposon-insertion sequencing (TIS) data from single-inoculation animals.

    Table S4. Number of unique genotypes and the results of Con-ARTIST analysis of TIS data from sequentially inoculated animals.

    Table S5. Primary data.

  • Supplementary Material for:

    A live vaccine rapidly protects against cholera in an infant rabbit model

    Troy P. Hubbard, Gabriel Billings, Tobias Dörr, Brandon Sit, Alyson R. Warr, Carole J. Kuehl, Minsik Kim, Fernanda Delgado, John J. Mekalanos, Joseph A. Lewnard, Matthew K. Waldor*

    *Corresponding author. Email: mwaldor{at}research.bwh.harvard.edu

    Published 13 June 2018, Sci. Transl. Med. 10, eaap8423 (2018)
    DOI: 10.1126/scitranslmed.aap8423

    This PDF file includes:

    • Fig. S1. HaitiV produces only the B subunit of CT.
    • Fig. S2. Results of Con-ARTIST analysis.
    • Fig. S3. SEIR model and parameters.
    • Fig. S4. Impact of model parameters on relative protection.
    • Legends for tables S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Strains and plasmids used in this study.
    • Table S2 (Microsoft Excel format). Oligonucleotide sequences used in this study.
    • Table S3 (Microsoft Excel format). Number of unique genotypes and the results of Con-ARTIST analysis of transposon-insertion sequencing (TIS) data from single-inoculation animals.
    • Table S4 (Microsoft Excel format). Number of unique genotypes and the results of Con-ARTIST analysis of TIS data from sequentially inoculated animals.
    • Table S5 (Microsoft Excel format). Primary data.

    [Download Tables S1 to S5]

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