Long-Term Immunogenicity of Live Oral Cholera Vaccine CVD 103-HgR in Adolescents Aged 12-17 Years in the United States.

As part of a phase 4, randomized, double-blind, placebo-controlled trial to assess the immunogenicity and safety of PXVX0200 in children and adolescents aged 2-17 years, a subset of 73 adolescent subjects aged 12-17 years was followed for 2 years after vaccination and had blood collected for antibody assays on days 1, 11, 29, 91, 181, 365, 547, and 730. Endpoints included serum vibriocidal antibody (SVA) seroconversion, defined as a 4-fold or greater rise in antibody titer over baseline; geometric mean titers (GMTs); and geometric mean fold increase (GMFI) over baseline. Serum vibriocidal antibody seroconversion persisted in most subjects, with a rate of 64.5% noted at day 730. Geometric mean titers and GMFI both peaked at day 11 and remained greater than baseline at all time points, including day 730. Vaccination with PXVX0200 produces an immune response which persists for at least 2 years in adolescents aged 12-17 years.

Protective effects of egg yolk immunoglobulins (IgYs) developed against recombinant immunogens CtxB, OmpW and TcpA on infant mice infected with Vibrio cholerae.

Vibrio cholerae causes cholera and other infections, especially in children under five years of age. Cholera toxin (CT), toxin-coregulated pilus (TCP) and outer membrane protein W (OmpW) are three major virulence factors of this bacterium. The emergence of antimicrobial-resistant (AMR) strains and the absence of a comprehensive and flawless vaccine, has prompted other treatments. There are several advantages of egg yolk antibodies (IgY) over other immunotherapy agents, such as economic feasibility, high yield simple production, and better immune responsiveness to mammalian antigens due to phylogenetic distance. Accordingly, in the present study, IgYs against recombinant proteins CtxB (responsible for the CT binding to eukaryotic cells), TcpA (enhances bacterial attachment to enterocytes) and OmpW were produced, in single, coupled or combined forms, to evaluate and compare their protectivity potency. Immunoreactivity of IgYs were examined through protein and whole cell ELISA, their specificity was confirmed by western blotting, and their neutralizing effects on CT was evaluated in Y1 cell culture. Produced IgYs were gavage administered to different groups of infant mice infected with V. cholerae. The results indicated that IgYs produced against CtxB had the highest titers, and were able to neutralize cytotoxicity effects in Y1 cell culture, while the highest protection in the mice challenge was obtained by IgY-TcpA. No considerable increase was observed in immunoreactivity or protectivity of antibodies produced against combined antigens. The produced IgYs showed a good antigen-specificity and protectivity which can be used in passive immunotherapy against cholera.

Non-O1/non-O139 Vibrio cholerae bacteraemia in mainland China from 2005 to 2019: clinical, epidemiological and genetic characteristics.

In mainland China, the clinical, epidemiological and genetic features of non-O1/non-O139 Vibrio cholerae (NOVC) bacteraemia have been scarcely investigated. Herein, we describe a patient with NOVC bacteraemia diagnosed in our hospital and present a retrospective analysis of literature reports of 32 other cases in China, detailing the clinical epidemiology, antibiotic resistance and molecular characteristics of isolates. Most patients were male (84.8%; median age, 53 years) and had predisposing factors, such as cirrhosis, malignant tumours, blood diseases and diabetes. In addition to fever, gastroenteritis was the most frequent presenting symptom. The mortality rate during hospitalisation was 12.1%. NOVC bacteraemia cases were more common in June-August, with the majority in coastal provinces and the Yangtze River basin. Only 42.4% of cases were attributed to consumption of marine (aquatic) products. Tetracycline, third-generation cephalosporins, and fluoroquinolones were the most effective antimicrobial agents, and the highest frequencies of resistance were recorded for ampicillin/sulbactam (37.5%), amoxicillin/clavulanic acid (33.3%), ampicillin (29.2%) and sulfamethoxazole (20%). Multi-drug resistant isolates were not detected. Limited data indicate that ctxAB and tcpA genes were absent in all NOVC isolates but other putative virulence genes (hlyA, toxR, hap and rtxA) were common. Ten multilocus sequence types were identified with marked genetic heterogeneity between different isolates. As clinical manifestations of NOVC bacteraemia may vary widely, and isolates exhibit genetic diversity, clinicians and public health experts should be alerted to the possibility of infection with this pathogen because of the high prevalence of liver disease in China.

Selenium Nanoparticles Induce Potent Protective Immune Responses against Vibrio cholerae WC Vaccine in a Mouse Model.

The aim of this study was to evaluate the efficacy of selenium nanoparticle (an immune booster) and naloxone (an opioid receptor antagonist) as a new adjuvant in increasing immune responses against killed whole-cell Vibrio cholerae in a mouse cholera model. The Se NPs were synthesized and characterized by UV-visible, DLS, and zeta potential analysis. The SEM image confirmed the uniformity of spherical morphology of nanoparticle shape with 34 nm in size. The concentration of the Se NPs was calculated as 0.654 µg/ml in the ICP method. The cytotoxic activity of Se NPs on Caco-2 cells was assessed by the MTT assay and revealed 82.05% viability of cells after 24 h exposure with 100 µg/ml of Se NPs. Female BALB/C mice were orally immunized three times on days 0, 14, and 28, and challenge experiments were performed on immunized neonates with toxigenic V. cholerae. Administration of Se NP diet led to significant increase in V. cholerae-specific IgG and IgA responses in serum and saliva and caused protective immunity and 83.3% survival in challenge experiment against 1 LD50 V. cholerae in a group receiving diet of Se NPs compared with other groups including Dukoral vaccine. The IL-4 and IL-5 were significantly increased in response to WC+daily diet of Se NPs with or without naloxone. Naloxone proved no effect on IL-4 and IL-5 increase and is proposed as null in the cytokine and antibody production process. These results reveal that daily diet of Se NPs could efficiently induce immune cell effectors in both humoral and mucosal levels.

The roles of mesoporous silica and carbon nanoparticles in antigen stability and intensity of immune response against recombinant subunit B of cholera toxin in a rabbit animal model.

Vaccines are the front line in the fight against diseases. However, setbacks with existing cholera vaccines have ignited a considerable effort to develop more suitable vaccine formulations. In this study, we aim to investigate the effect of antigen stability and controlled release in inducing an immune response. Therefore, two types of silica and carbon mesoporous nanoparticles of the same size and shape but different pore architectures were synthesized and loaded with recombinant cholera toxin subunit B to serve as a model for antigen stability and controlled release of antigenic CTB. In order to evaluate immune response efficacy for these model formulations, IgG and IgA responses and fluid accumulation (FA) index were measured in immunized rabbits, which were challenged with wild-type Vibrio cholerae. Our result suggests that mesoporous silica nanoparticles have greater efficacy in inducing mucosal immune responses, and it proved more proficiency in overall immune responses in challenge experiments and FA index (p < 0.05). These findings indicate that mesoporous nanoparticles and, in particular, mesoporous silica nanoparticles, could be used in oral vaccine formulation against cholera.

Estimating cholera incidence with cross-sectional serology.

The development of new approaches to cholera control relies on an accurate understanding of cholera epidemiology. However, most information on cholera incidence lacks laboratory confirmation and instead relies on surveillance systems reporting medically attended acute watery diarrhea. If recent infections could be identified using serological markers, cross-sectional serosurveys would offer an alternative approach to measuring incidence. Here, we used 1569 serologic samples from a cohort of cholera cases and their uninfected contacts in Bangladesh to train machine learning models to identify recent Vibrio cholerae O1 infections. We found that an individual's antibody profile contains information on the timing of V. cholerae O1 infections in the previous year. Our models using six serological markers accurately identified individuals in the Bangladesh cohort infected within the last year [cross-validated area under the curve (AUC), 93.4%; 95% confidence interval (CI), 92.1 to 94.7%], with a marginal performance decrease using models based on two markers (cross-validated AUC, 91.0%; 95% CI, 89.2 to 92.7%). We validated the performance of the two-marker model on data from a cohort of North American volunteers challenged with V. cholerae O1 (AUC range, 88.4 to 98.4%). In simulated serosurveys, our models accurately estimated annual incidence in both endemic and epidemic settings, even with sample sizes as small as 500 and annual incidence as low as two infections per 1000 individuals. Cross-sectional serosurveys may be a viable approach to estimating cholera incidence.

High-Resolution Crystal Structures Elucidate the Molecular Basis of Cholera Blood Group Dependence.

Cholera is the prime example of blood-group-dependent diseases, with individuals of blood group O experiencing the most severe symptoms. The cholera toxin is the main suspect to cause this relationship. We report the high-resolution crystal structures (1.1-1.6 Å) of the native cholera toxin B-pentamer for both classical and El Tor biotypes, in complexes with relevant blood group determinants and a fragment of its primary receptor, the GM1 ganglioside. The blood group A determinant binds in the opposite orientation compared to previously published structures of the cholera toxin, whereas the blood group H determinant, characteristic of blood group O, binds in both orientations. H-determinants bind with higher affinity than A-determinants, as shown by surface plasmon resonance. Together, these findings suggest why blood group O is a risk factor for severe cholera.

Plasma Leptin Levels in Children Hospitalized with Cholera in Bangladesh.

Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infected humans. Leptin is a hormone that plays a role in both metabolism and mediating immune responses. We characterized leptin levels in 11 children with cholera in Bangladesh, assessing leptin levels on days 2, 7, 30, and 180 following cholera. We found that patients at the acute stage of cholera had significantly lower plasma leptin levels than matched controls, and compared with levels in late convalescence. We then assessed immune responses to V. cholerae antigens in 74 children with cholera, correlating these responses to plasma leptin levels on day 2 of illness. In multivariate analysis, we found an association between day 2 leptin levels and development of later anti-cholera toxin B subunit (CtxB) responses. This finding appeared to be limited to children with better nutritional status. Interestingly, we found no association between leptin levels and antibody responses to V. cholerae lipopolysaccharide, a T cell-independent antigen. Our results suggest that leptin levels may be associated with cholera, including the development of immune responses to T cell-dependent antigens.

[The development of biochip to detect anti-cholera antibodies in human blood serum].

The full-scaled agglutinating immunoassay is commonly applied to detect content of antibodies to cholera agent Vibrio cholerae human in blood serum under application of serological diagnostic. The time of analysis implementation amounts to 18 hours. To shorten time of detection of antibodies a biological microchip (biochip) was developed. The biochip represents an activated slide with immobilized corpuscle and soluble antigen cholera agent (O-antigens, cholera toxin). The experimental work resulted in development of scheme of biochip and selection of optimal conditions of sorption and implementation of immunologic analysis using biochip. The application of biochip facilitated to detect specific antibodies to antigens of cholera agent in commercial experimental animal serums and blood serums of ill patients. The time of analysis implementation amounted to 2-3 hours. The results are substantiated by bacteriological and serological methods.

[Serologic study of experimental cholera polymer antigen diagnosticums].

AIM: Evaluation of quality indicators of constructed cholera antigen polymer diagnosticums by using a complex of specific anti-cholera sera. MATERIALS AND METHODS. Cell lysates of cholera vibrio strains Vibrio cholerae cholerae 1395, V. eltor Ogawa 2044, V. eltor Inaba 13020, V. cholerae O139 16064 were sensitins for experimental preparations. 3 sera from cholera patients, normal human sera, cholera O1 (Ogawa, Inaba) commercial horse, cholera O139 commercial rabbit and heterologic sera against shigella, salmonella, escherichia and yersinia as well as experimental cholera rabbit sera against O1 and O139 were used as control. RESULTS: The study established that diagnosticums based on V. cholerae cholerae 1395 and V. cholerae O139 16064 strain sensitins by quality indicators may be used in the future for construction of these diagnosticums. CONCLUSION: Antibody containing preparations--commercial horse O1 sera, rabbit experimental and commercial sera and MCA O139 demonstrating titers not lower than 1/5120-1/10240 may serve as a control of experimental diagnosticums in the absence of human sera from cholera patients.

Molecular characterization of the circulating strains of Vibrio cholerae during 2010 cholera outbreak in Nigeria.

This study aimed at characterizing the phenotypic and toxigenic status of circulating strains of cholera during outbreaks in Nigeria, employing molecular typing techniques. Two hundred and one samples of rectal swabs, stool, vomitus, water (from the well, borehole, sachet, stream, and tap) and disinfectants (sodium hypochlorite) were collected from three states in the country. The samples were inoculated on thiosulphate-citrate bile salt-sucrose (TCBS), Cary-Blair transport medium and smeared on glass slides for direct examination. The Vibrio cholerae isolates were serotyped, biotyped, and characterized using PCR of the cytotoxin gene A (ctxA), wbeO1, and wbfO139 gene primer. Of the 201 samples screened, 96 were positive for V cholerae O1 (48%), with 69 (72%) positive for ctxA gene. The results from this study showed that the circulating strains of cholera in Nigeria were of Ogawa serotype, also observed in other outbreaks in Nigeria (1991, 1992, and 1996). However, the strains were of the Classical biotype and were mainly (72%) ctxA gene-positive. This current investigation has confirmed the production of cholera toxin by the circulating strains, and this could be harnessed for possible cholera vaccine production in Nigeria.

[The application of monoclonal peroxidase conjugates to identify comma bacillus of serum groups 01 and 0139 in the reaction of dot-immune analysis].

The source of monoclonal antibodies was chosen the cultural fluid of hybridoma-producers deposited in the specialized collection of cell cultures of vertebrates (St. Petersburg) with numbers RKKK(P) 386D and RKKK(P) 674D. The specific immunoglobulin (Ig) from cultural fluid was concentrated by precipitation with saturated solution of ammonium sulfate. The scheme of obtaining monoclonal antibodies included activation of peroxidase, conjugation of activated peroxidase with Ig, removal of unbounded proteins, storage and control. The preservation of activity of conjugates was supported with BSA (10%) or glycerin (50%). The last on is preferable to be applied for this purpose. The test of monoclonal antibody-01 and monoclonal antibody-0139 of peroxidase conjugates with kit of strains of comma bacillus 01 and 0139 demonstrated their strict specificity because they interacted only with corresponding serum groups under absence of crossed reactions with representatives of geterologic microorganisms. The direct dot-immune analysis is carried out during 1.5 hour and its sensitivity is within the limits 105-106. The application of diagnostic monoclonal peroxidase conjugates 01, 0139 in laboratory practice can promote the increase of specificity of serologic analysis of cholera and saving time-frame of its application.

Evaluation of TcpF-A2-CTB chimera and evidence of additive protective efficacy of immunizing with TcpF and CTB in the suckling mouse model of cholera.

The secreted colonization factor, TcpF, which is produced by Vibrio cholerae 01 and 0139, has generated interest as a potential protective antigen in the development of a subunit vaccine against cholera. This study evaluated immunogenicity/protective efficacy of a TcpF holotoxin-like chimera (TcpF-A2-CTB) following intraperitoneal immunization compared to TcpF alone, a TcpF+CTB mixture, or CTB alone. Immunization with the TcpF-A2-CTB chimera elicited significantly greater amounts of anti-TcpF IgG than immunization with the other antigens (P<0.05). Protective efficacy was measured using 6-day-old pups reared from immunized dams and orogastrically challenged with a lethal dose of El Tor V. cholerae 01 Inaba strain N16961. Protection from death, and weight loss analysis at 24 and 48 hours post-infection demonstrated that immunization with TcpF alone was poorly protective. However, immunization with TcpF+CTB was highly protective and showed a trend toward greater protection than immunization with CTB alone (82% vs 64% survival). Immunization with the TcpF-A2-CTB chimera demonstrated less protection (50% survival) than immunization with the TcpF+CTB mixture. The TcpF-A2-CTB chimera used for this study contained the heterologous classical CTB variant whereas the El Tor CTB variant (expressed by the challenge strain) was used in the other immunization groups. For all immunization groups that received CTB, quantitative ELISA data demonstrated that the amounts of serum IgG directed against the homologous immunizing CTB antigen was statistically greater than the amount to the heterologous CTB antigen (P≤0.003). This finding provides a likely explanation for the poorer protection observed following immunization with the TcpF-A2-CTB chimera and the relatively high level of protection seen after immunization with homologous CTB alone. Though immunization with TcpF alone provided no protection, the additive protective effect when TcpF was combined with CTB demonstrates its possible value as a component of a multivalent subunit vaccine against Vibrio cholerae 01 and 0139.

Molecular basis of cholera blood-group dependence and implications for a world characterized by climate change.

Climate change has the potential to increase the threat of water-borne diseases, through rises in temperature and sea-level, and precipitation variability. Cholera poses a particular threat, and the need to develop better intervention tools is imminent. Cholera infections are particularly severe for blood group O individuals, who are less protected by the current vaccines. Here we derive a hypothesis as to the molecular origins of blood-group dependence of this disease, based on relevant epidemiological, clinical and molecular data, and give suggestions on how to plan prevention strategies, and develop novel and improved pharmaceuticals.

Memory T-cell responses to Vibrio cholerae O1 infection.

Vibrio cholerae O1 can cause diarrheal disease that may be life-threatening without treatment. Natural infection results in long-lasting protective immunity, but the role of T cells in this immune response has not been well characterized. In contrast, robust B-cell responses to V. cholerae infection have been observed. In particular, memory B-cell responses to T-cell-dependent antigens persist for at least 1 year, whereas responses to lipopolysaccharide, a T-cell-independent antigen, wane more rapidly after infection. We hypothesize that protective immunity is mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue, and T-cell responses may be required to generate and maintain durable memory B-cell responses. In this study, we examined B- and T-cell responses in patients with severe V. cholerae infection. Using the flow cytometric assay of the specific cell-mediated immune response in activated whole blood, we measured antigen-specific T-cell responses using V. cholerae antigens, including the toxin-coregulated pilus (TcpA), a V. cholerae membrane preparation, and the V. cholerae cytolysin/hemolysin (VCC) protein. Our results show that memory T-cell responses develop by day 7 after infection, a time prior to and concurrent with the development of B-cell responses. This suggests that T-cell responses to V. cholerae antigens may be important for the generation and stability of memory B-cell responses. The T-cell proliferative response to VCC was of a higher magnitude than responses observed to other V. cholerae antigens.

Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh.

BACKGROUND: Despite recent progress in understanding the molecular basis of Vibrio cholerae pathogenesis, there is relatively little knowledge of the factors that determine the variability in human susceptibility to V. cholerae infection. METHODS AND FINDINGS: We performed an observational study of a cohort of household contacts of cholera patients in Bangladesh, and compared the baseline characteristics of household members who went on to develop culture-positive V. cholerae infection with individuals who did not develop infection. Although the vibriocidal antibody is the only previously described immunologic marker associated with protection from V. cholerae infection, we found that levels of serum IgA specific to three V. cholerae antigens-the B subunit of cholera toxin, lipopolysaccharide, and TcpA, the major component of the toxin-co-regulated pilus-also predicted protection in household contacts of patients infected with V. cholerae O1, the current predominant cause of cholera. Circulating IgA antibodies to TcpA were also associated with protection from V. cholerae O139 infection. In contrast, there was no association between serum IgG antibodies specific to these three antigens and protection from infection with either serogroup. We also found evidence that host genetic characteristics and serum retinol levels modify susceptibility to V. cholerae infection. CONCLUSIONS: Our observation that levels of serum IgA (but not serum IgG) directed at certain V. cholerae antigens are associated with protection from infection underscores the need to better understand anti-V. cholerae immunity at the mucosal surface. Furthermore, our data suggest that susceptibility to V. cholerae infection is determined by a combination of immunologic, nutritional, and genetic characteristics; additional factors that influence susceptibility to cholera remain unidentified.

Blood group, immunity, and risk of infection with Vibrio cholerae in an area of endemicity.

Individuals with blood group O are more susceptible than other individuals to severe cholera, although the mechanism underlying this association is unknown. To assess the respective roles of both intrinsic host factors and adaptive immune responses that might influence susceptibility to infection with Vibrio cholerae, we prospectively followed a cohort of household contacts of patients with cholera in Bangladesh. In this study, we made the novel observation that persons with blood group O were less likely than those with other blood groups to become infected with V. cholerae O1 (odds ratio [OR], 0.67; 95% confidence interval [CI], 0.53 to 0.85; P = 0.008). Consistent with prior studies, however, household contacts with blood group O were more likely to develop severe illness if infected with V. cholerae O1 (OR, 2.3; 95% CI, 0.98 to 5.59; P = 0.05). While blood group O protected significantly against infection with V. cholerae O1, there was no evidence of protection against V. cholerae O139. A multivariate analysis demonstrated that the association between blood group O and protection from infection with V. cholerae O1 was independent of age, gender, and baseline anti-cholera toxin and vibriocidal antibody titers. Based on this epidemiologic evidence, we propose a hypothesis for understanding the association between blood group O and the risk of infection with V. cholerae O1 and O139 as well as the risk of developing severe symptoms once infected.

Enfermedades emergentes y reemergentes: un reto de nunca acabar: segunda parte / Emergent and re-emergent diseases: a never-ending challenge

El presente artículo muestra cómo la humanidad se enfrenta a nuevos agentes patógenos (emergentes) y a otros, ya conocidos, pero que en los últimos años han aumentado su mortalidad (reemergentes). Hasta dónde la “mano del hombre” es responsable de éste fenómeno?La segunda parte pretende analizar los diferentes factores, sociales, políticos, morales y económicos, que incidieron para que el S.I.D.A apareciera, no como epidemia, sino como pandemia y cómo los intereses económicos primaron sobre las políticas de salud, al precio de millones de vidas.

[Rapid laboratory detection of antigens of infective agents of infections and technical means for their realization]. / Uskorennaia laboratornaia indikatsiia antigenov vozbuditelei osobo opasnykh infektsii i tekhnicheskie ustroistva dlia ee obespecheniia.

A system of new accelerated and rapid methods for the detection of the antigens of the infective agents of plague, cholera, tularemia and brucellosis were developed on the basis of solid phase immunosuspension tests: the passive hemagglutination (PHA) test and the latex agglutination (LA) test. The immunological and physico-chemical properties of suspensions in the PHA and LA tests made it possible to use extraneous sources of energy (centrifugal acceleration and the electric field) to accelerate these tests. The results of the PHA and LA tests were registered with the use of a densitometer, model Ultrascan 2202, and a tester, model C 34014.2. To apply centrifugal acceleration and the electric field, a laboratory centrifuge and an electrophoretic microchamber were designed. Densitometry was carried out on modified plates and conductometry, with the use of modified electrodes. The time of obtaining the results of the PHA and LA tests was 15-30 minutes with the use of centrifugation and 2-5 minutes in the electric field, which made it possible to regard these tests as rapid.