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

Abstract

Outbreaks of cholera, a rapidly fatal diarrheal disease, often spread explosively. The efficacy of reactive vaccination campaigns-deploying Vibrio cholerae vaccines during epidemics-is partially limited by the time required for vaccine recipients to develop adaptive immunity. We created HaitiV, a live attenuated cholera vaccine candidate, by deleting diarrheagenic factors from a recent clinical isolate of V. cholerae and incorporating safeguards against vaccine reversion. We demonstrate that administration of HaitiV 24 hours before lethal challenge with wild-type V. cholerae reduced intestinal colonization by the wild-type strain, slowed disease progression, and reduced mortality in an infant rabbit model of cholera. HaitiV-mediated protection required viable vaccine, and rapid protection kinetics are not consistent with development of adaptive immunity. These features suggest that HaitiV mediates probiotic-like protection from cholera, a mechanism that is not known to be elicited by traditional vaccines. Mathematical modeling indicates that an intervention that works at the speed of HaitiV-mediated protection could improve the public health impact of reactive vaccination.

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: S. 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 Kan^R cassette, intact ctxA) or CTX-KnΦ (Target-; ctxA replaced by Kan^R cassette), and the number of transductants was monitored. No detectable Kan^R transductants shown as (†).

Fig. 2:

HaitiV colonizes the infant rabbit intestine without causing cholera-like illness. A) Fluid accumulation ratios after littermates were pre-treated with ranitidine-hydrochloride, to reduce stomach acidity, and inoculated with either WT (n=11) or HaitiV (vaccine; n=10). Plots show mean and standard deviation derived from 2 litters. ****P<0.001, unpaired t-test. B) Successive daily weights of animals inoculated with 10⁹ CFU HaitiV (n=10). C) WT (blue circles) or HaitiV (red squares) CFU recovered from rabbit distal small intestines (dSI) at Day 1 or 4 post-inoculation (each of the 3 groups consists of animals from at least 2 litters). Lines indicate geometric means, and the open symbol indicates limit of detection for the single animal from which no CFU were recovered. NS: P≥0.05, Kruskall-Wallis test followed by Dunn’s multiple comparisons test. D) Competitive indices (CI) of dSI bacteria 1 day post-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 standard deviation of CIs across 2 litters, (n=6). E) WT (blue) and HaitiV (red) CFU recovered from co-inoculated animals. The open symbol indicates limit of detection the single animal, from which no vaccine CFU were recovered; lines indicate geometric means.

Fig. 3:

HaitiV mediates colonization resistance associated with variably-sized infection bottlenecks. A) WT CFU (blue circles) recovered from 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 prior to WT challenge; geometric means of each group across 3 litters are shown. NS: P>0.05, Mann-Whitney test. HaitiV and HaitiWT (B) or N16961 WT (C) CFU recovered from the dSI of animals 18 hours post-challenge. Animals were pretreated with killed (n=6) or live (n=8) vaccine 24 hours prior to challenge. Open symbols indicate limit of detection for 5 animals in which no CFU were recovered, and lines indicate the geometric mean of each group across 2 litters. ***P<0.001, *P<0.05, Mann-Whitney test. D) WT CFU (blue circles), and unique transposon mutants (black triangles) recovered from the dSI of individual animals (rabbits r1 to r6) one 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) 18hrs after inoculation of the transposon mutant library. Animals were pretreated with HaitiV 24 hours prior to 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 2-fold change (Log₂(mean fold change) <−1 or > 1) across multiple mutants (mean inverse P-value > 10²) are considered depleted/enriched. Enriched mutants cqsS and hapR are indicated in blue. Mutations in known colonization factors, including toxin co-regulated pilus biogenesis (red circles), and the associated transcriptional regulators toxR and toxS (red asterisks), were depleted.

Fig. 4:

HaitiV colonization protects from disease following 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, Log-rank test. C) WT CFU (blue circles) recovered from dSI of animals 41 h post-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 are modeled with a constant rate over 7 days until 70% of the population is vaccinated, as achieved by recent reactive vaccination campaigns. Modeling parameters are described in Fig. S3B.