Emerging Concepts in Cholera Vaccine Design.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae and constitutes a significant public health threat in many areas of the world. V. cholerae infection elicits potent and long-lasting immunity, and efforts to develop cholera vaccines have been ongoing for more than a century. Currently available inactivated two-dose oral cholera vaccines are increasingly deployed to both prevent and actively curb cholera outbreaks, and they are key components of the global effort to eradicate cholera. However, these killed whole-cell vaccines have several limitations, and a variety of new oral and nonoral cholera vaccine platforms have recently been developed. Here, we review emerging concepts in cholera vaccine design and implementation that have been driven by insights from human and animal studies. As a prototypical vaccine-preventable disease, cholera continues to be an excellent target for the development and application of cutting-edge technologies and platforms that may transform vaccinology.

Dissecting serotype-specific contributions to live oral cholera vaccine efficacy.

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into the Ogawa and Inaba serotypes. The O-antigen is V. cholerae's immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogeneous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germfree mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single- and mixed-strain infection assays, we found that all three HaitiV variants-InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa)-were immunogenic and protective. None of the vaccine serotypes were superior across both of these vaccine metrics, suggesting that the impact of O1-serotype variation in OCV design, although detectable, is subtle. However, all three live vaccines significantly outperformed formalin-killed HikoV, supporting the idea that live OCV usage will bolster current cholera control practices. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

Proteomic analysis of the host-pathogen interface in experimental cholera.

The microbial cell surface is a site of critical microbe-host interactions that often control infection outcomes. Defining the set of host proteins present at this interface has been challenging. Here we used a surface-biotinylation approach coupled to quantitative mass spectrometry to identify and quantify both bacterial and host proteins present on the surface of diarrheal fluid-derived Vibrio cholerae in an infant rabbit model of cholera. The V. cholerae surface was coated with numerous host proteins, whose abundance were driven by the presence of cholera toxin, including the C-type lectin SP-D. Mice lacking SP-D had enhanced V. cholerae intestinal colonization, and SP-D production shaped both host and pathogen transcriptomes. Additional host proteins (AnxA1, LPO and ZAG) that bound V. cholerae were also found to recognize distinct taxa of the murine intestinal microbiota, suggesting that these host factors may play roles in intestinal homeostasis in addition to host defense.

Genomic and Phenotypic Insights for Toxigenic Clinical Vibrio cholerae O141.

Vibrio cholerae remains a major public health threat worldwide, causing millions of cholera cases each year. Although much is known about the evolution and pathogenicity of the O1/O139 serogroups of V. cholerae, information is lacking on the molecular epidemiology of non‒O1/O139 strains isolated from patients who have diarrheal illnesses. We performed whole-genome sequence analysis and in vivo infections to investigate characteristics of V. cholerae O141 isolated from sporadic diarrheal cases in 4 countries. The strains formed a distinct phylogenetic clade distinguishable from other serogroups and a unique multilocus sequence type 42, but interstrain variation suggests that O141 isolates are not clonal. These isolates encode virulence factors including cholera toxin and the toxin-coregulated pilus, as well as a type 3 secretion system. They had widely variable capacities for intestinal colonization in the infant mouse model. We propose that O141 isolates comprise a distinct clade of V. cholerae non‒O1/O139, and their continued surveillance is warranted.

Transient Intestinal Colonization by a Live-Attenuated Oral Cholera Vaccine Induces Protective Immune Responses in Streptomycin-Treated Mice.

Current mouse models for evaluating the efficacy of live oral cholera vaccines (OCVs) have important limitations. Conventionally raised adult mice are resistant to intestinal colonization by Vibrio cholerae, but germfree mice can be colonized and have been used to study OCV immunogenicity. However, germfree animals have impaired immune systems and intestinal physiology; also, live OCVs colonize germfree mice for many months, which does not mimic the clearance kinetics of live OCVs in humans. In this study, we leveraged antibiotic-treated, conventionally raised adult mice to study the effects of transient intestinal colonization by a live OCV V. cholerae strain. In a single-dose vaccination regimen, we found that HaitiV, a live-attenuated OCV candidate, was cleared by streptomycin-treated adult mice within 2 weeks after oral inoculation. This transient colonization elicited far stronger adaptive immune correlates of protection against cholera than did inactivated whole-cell HaitiV. Infant mice from HaitiV-vaccinated dams were also significantly more protected from choleric disease than pups from inactivated-HaitiV-vaccinated dams. Our findings establish the benefits of antibiotic-treated mice for live-OCV studies as well as their limitations and underscore the immunogenicity of HaitiV.IMPORTANCE Oral cholera vaccines (OCVs) are being deployed to combat cholera, but current killed OCVs require multiple doses and show little efficacy in young children. Live OCVs have the potential to overcome these limitations, but small-animal models for testing OCVs have shortcomings. We used an antibiotic treatment protocol for conventional adult mice to study the effects of short-term colonization by a single dose of HaitiV, a live-OCV candidate. Vaccinated mice developed vibriocidal antibodies against V. cholerae and delivered pups that were resistant to cholera, whereas mice vaccinated with inactivated HaitiV did not. These findings demonstrate HaitiV's immunogenicity and suggest that this antibiotic treatment protocol will be useful for evaluating the efficacy of live OCVs.

Animal models for dissecting Vibrio cholerae intestinal pathogenesis and immunity.

The human diarrheal disease cholera is caused by the bacterium Vibrio cholerae. Efforts to develop animal models that closely mimic cholera to study the pathogenesis of this disease began >125 years ago. Here, we review currently used non-surgical, oral inoculation-based animal models for investigation of V. cholerae intestinal colonization and disease and highlight recent discoveries that have illuminated mechanisms of cholera pathogenesis and immunity, particularly in the area of how V. cholerae interacts with the gut microbiome to influence infection. The emergence of high-throughput tools for studies of pathogen-host interactions, along with continued advances in host genetic engineering and manipulation in animal models of V. cholerae will deepen understanding of cholera pathogenesis, uncovering knowledge important for control of this globally important bacterial pathogen.

Nontoxigenic Vibrio cholerae Challenge Strains for Evaluating Vaccine Efficacy and Inferring Mechanisms of Protection.

Human challenge studies are instrumental for testing cholera vaccines, but these studies use outdated strains and require inpatient facilities. Here, we created next-generation isogenic Ogawa and Inaba O1 V. cholerae challenge strains (ZChol strains) derived from a contemporary Zambian clinical isolate representative of current dominant pandemic V. cholerae. Since the primary mechanism of immune protection against cholera is thought to be antibody responses that limit V. cholerae colonization and not the diarrheagenic actions of cholera toxin, these strains were rendered nontoxigenic. In infant mice, the ZChol strains did not cause diarrhea and proved to accurately gauge reduction in intestinal colonization mediated by effective vaccination. ZChol strains were also valuable as targets for measuring vibriocidal antibody responses. Using barcoded ZChol strains, we discovered that vaccination and passive immunity in the infant mouse model tightens the infection bottleneck without restricting pathogen expansion during intestinal infection. Collectively, our findings suggest that ZChol strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera. IMPORTANCE Human challenge studies are a valuable method for testing the efficacy of cholera vaccines. However, challenge studies cannot be performed in countries of cholera endemicity due to safety concerns; also, contemporary pandemic Vibrio cholerae strains are not used in current challenge studies. To facilitate cholera research, we derived nontoxigenic challenge strains of both V. cholerae serotypes from a 2016 clinical isolate from Zambia and demonstrated how they can be used to gauge cholera immunity accurately and safely. These strains were also genetically barcoded, adding the potential for analyses of V. cholerae population dynamics to challenge studies. Preclinical analyses presented here suggest that these strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera.

Oral immunization with a probiotic cholera vaccine induces broad protective immunity against Vibrio cholerae colonization and disease in mice.

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiV's immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera.