RESUMEN
Diarrhoeagenic Escherichia coli (DEC) pathotypes are one of the major causative agents of diarrhoea induced childhood morbidity and mortality in developing countries. Licensed vaccines providing broad spectrum protection against DEC mediated infections are not available. Outer membrane vesicles (OMVs) are microvesicles released by gram-negative bacteria during the growth phase and contain multiple immunogenic proteins. Based on prevalence of infections, we have formulated a pentavalent outer-membrane vesicles (POMVs) based immunogen targeting five main pathotypes of DEC responsible for diarrhoeal diseases. Following isolation, OMVs from five DEC pathotypes were mixed in equal proportions to formulate POMVs and 10 µg of the immunogen was intraperitoneally administered to adult BALB/c mice. Three doses of POMVs induced significant humoral immune response against whole cell lysates (WCLs), outer membrane proteins (OMPs) and lipopolysaccharides (LPS) isolated from DEC pathotypes along with significant induction of cellular immune response in adult mice. Passive transfer of POMVs immunized adult mice sera protected neonatal mice significantly against DEC infections. Overall, this study finds POMVs to be immunogenic in conferring broad-spectrum passive protection to neonatal mice against five main DEC pathotypes. Altogether, these findings suggest that POMVs can be used as a potent vaccine candidate to ameliorate the DEC-mediated health burden.
Asunto(s)
Diarrea , Lipopolisacáridos , Humanos , Adulto , Animales , Ratones , Niño , Animales Recién Nacidos , Inmunidad Humoral , Escherichia coliRESUMEN
In today's world and mostly in low and middle income countries, Shigella flexneri and Shigella sonnei remains the major causative agent of clinical bacillary dysentery. Based on contemporary epidemiology, a tetravalent Outer Membrane Vesicle (OMVs) based immunogen was formulated using the most commonly circulating Shigella strains, namely, S. flexneri 2a, S. flexneri 3a, S. flexneri 6 and S. sonnei I, in a 1:1:1:1 ratio. Adult BALB/c mice were orally immunized in a prime-boost-boost manner. Tetravalent Shigella OMVs immunogen induced significant and persistent serum and mucosal antibodies against OMVs, Outer Membrane Proteins (OMPs) and lipopolysaccharides (LPS). Tetravalent OMVs also primed cell mediated immune response effectively. Protective efficacy against six heterologous Shigella strains was checked in an intra-peritoneal mouse model. Immunized mice survived lethal infection better than the non-immunized mice cohort with fewer replicating bacteria isolated from their gut. This study establishes the possibilities of tetravalent OMVs immunogen to become a potent vaccine candidate against human shigellosis, overcoming the limitations of sero-specific cross-protection of Shigella species.
Asunto(s)
Disentería Bacilar , Vacunas contra la Shigella , Shigella , Vacunas , Animales , Adulto , Humanos , Ratones , Serogrupo , Disentería Bacilar/prevención & control , Disentería Bacilar/microbiología , Shigella flexneri , Anticuerpos AntibacterianosRESUMEN
Shigellosis has been a menace to society for ages. The absence of an effective vaccine against Shigella, improper sanitation, and unhygienic use of food and water allow the disease to flourish. Shigella can also be transmitted via natural water bodies. In the absence of a good animal model, the actual nature of pathogenesis and transmission remains unclear. Zebrafish larvae have previously been described as a model for Shigella pathogenesis. However, larval fish lack a mature intestinal microbiota and immune system. Here, the adult zebrafish was assessed as a potential model for Shigella pathogenesis. Their well-developed innate and adaptive immune responses mimic the mammalian immune system. Shigella showed a clear dose-, time-, and temperature-dependent colonization of the adult zebrafish gut. Efficacy of a three-dose immunization regime was tested using bath immunization with heat-killed trivalent Shigella immunogen. The present study demonstrates the efficacy of an adult zebrafish model for pathogenesis, transmission, and vaccine efficacy studies. IMPORTANCE Shigellosis is a diarrheal disease that is prevalent in developing countries and especially dangerous in young children. Currently, animal models for shigellosis are unable to model some aspects of the infectious cycle. Here, we describe a new shigellosis model in adult zebrafish, an increasingly common model organism for studying bacterial pathogens. The zebrafish model can be used to study Shigella colonization, transmission, and immune responses, as well as test vaccine efficacy.
Asunto(s)
Disentería Bacilar , Shigella , Animales , Modelos Animales de Enfermedad , Mamíferos , Eficacia de las Vacunas , Agua , Pez Cebra/microbiologíaRESUMEN
With the acquirement of antibiotic resistance, Shigella has resulted in multiple epidemics of shigellosis, an infectious diarrheal disease, causing thousands of deaths per year. Unfortunately, there are no licensed vaccines, primarily due to low or serotype-specific immunogenicity. Thus, conserved subunit vaccines utilizing recombinant invasion plasmid antigens (Ipa) have been explored as cross-protective vaccine candidates. However, achieving cross-protection against Shigella dysenteriae 1, which caused multiple pandemics/epidemics in the recent past, has been difficult. Therefore, a rational approach to improve cross-protection in the preparation for a possible pandemic should involve conserved proteins from S. dysenteriae 1 (Sd1). IpaC is one such conserved immunogenic protein that is less explored as an independent vaccine due to its instability/aggregation. Therefore, to improve cross-protection and potential immunogenicity and to be prepared for a future epidemic/pandemic, herein, we stabilized recombinant Sd1 IpaC, expressed without its chaperone, using a previously reported stabilizing detergent (LDAO) in a modified protocol and assessed its vaccine potential without an adjuvant. The protein assembled into heterogeneous complex spherical structures in the presence of LDAO and showed improved stability at storage temperatures of -80, -20, 4, 25, and 37 °C while providing enhanced yield and concentration. The protein could also be stably lyophilized and reconstituted, increasing the convenience of transportation and storage. Upon intranasal administration in BALB/c mice, the stabilized-IpaC-immunized groups generated significant antibody response and were not only protected against a high intraperitoneal dose of homologous S. dysenteriae 1 but also showed 100% survival against heterologous Shigella flexneri 2a without an adjuvant, while the control animals showed visible diarrhea (bloody-Sd1 challenge), lethargy, and weight loss with 0% survival. Overall, this work demonstrates that stabilized IpaC can be explored as a minimalist, self-adjuvanting, cross-protective, intranasal, single-antigen Shigella vaccine.
Asunto(s)
Disentería Bacilar , Vacunas contra la Shigella , Shigella , Administración Intranasal , Animales , Disentería Bacilar/prevención & control , Ratones , Shigella/genética , Vacunas contra la Shigella/genética , Vacunas Sintéticas/genéticaRESUMEN
Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.
Asunto(s)
Gastroenteritis , Fiebre Tifoidea , Vacunas Tifoides-Paratifoides , Animales , Anticuerpos Antibacterianos , Gastroenteritis/prevención & control , Ratones , Ratones Endogámicos BALB C , Salmonella typhimuriumRESUMEN
Multiple diarrheagenic enteric bacterial infections cause global morbidity and mortality. A combination vaccine is needed to combat different diarrhea-causing organisms. In our present work, we formulated a combination of antigens from three different diarrheagenic Escherichia coli strains and three different Vibrio cholerae strains. We demonstrated that our newly formulated combination immunogen was able to raise species-specific immunogenicity. This formulation also gave protection against different diarrheagenic E. coli strains in the removable intestinal tie-adult rabbit diarrhea model. However, protective efficacy was not found against the V. cholerae El Tor Ogawa Haitian variant, but challenged with V. cholerae El Tor Inaba or O139 showed protection in rabbits. This is the first report of a single formulated nonliving heat-killed combination immunogen from different diarrheagenic E. coli and V. cholerae that could bestow protection against different bacteria in an animal model.
Asunto(s)
Vacunas contra el Cólera/inmunología , Cólera/prevención & control , Diarrea/prevención & control , Infecciones por Escherichia coli/prevención & control , Vacunas contra Escherichia coli/inmunología , Escherichia coli/inmunología , Vibrio cholerae/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/inmunología , Vacunas contra el Cólera/administración & dosificación , Protección Cruzada/inmunología , Diarrea/microbiología , Modelos Animales de Enfermedad , Escherichia coli/genética , Vacunas contra Escherichia coli/administración & dosificación , Inmunización , Inmunoglobulinas/sangre , Conejos , Vacunas Combinadas/inmunología , Vacunas de Productos Inactivados/inmunología , Vibrio cholerae/genéticaRESUMEN
Salmonella Typhi and Salmonella Paratyphi A are the leading causative agents of enteric fever which cause morbidity and mortality worldwide. Currently, there is no combination vaccine which could protect infection from both the strains. In this paper, we are focusing on the development of a novel bivalent typhoidal Outer Membrane Vesicles (OMVs) based immunogen against enteric fever. We have isolated Salmonella Typhi and Paratyphi A OMVs and also characterized OMVs associated antigens. Then we immunized adult mice with three doses of our newly formulated bivalent immunogen orally (25 µg/200 µl). After three doses of oral immunization, we found our immunogen could significantly induce humoral response. We have also found serum IgG against LPS, Vi-polysaccharide etc. OMV immunization induces CD4, CD8 and CD19 population in immunized mice spleen. It also induces Th1 and Th17-cell mediated immunity. We also found bivalent OMVs immunization can prevent more than lethal dose of heterologous Salmonella strains mediated systemic infection in adult mice model. We determined that, the protective immune responses depend on the humoral and cell-mediated immune response. Furthermore, we have evaluated the mode of protective immune response carried out by anti-OMVs antibody by significantly inhibiting bacterial motility and mucin penetration ability. Taken together, these findings suggest that our bivalent immunogen could be used as a novel candidate vaccine against enteric fever.
Asunto(s)
Salmonella typhi/inmunología , Fiebre Tifoidea/inmunología , Vacunas Tifoides-Paratifoides/química , Animales , Anticuerpos Antibacterianos/inmunología , Femenino , Ratones , Ratones Endogámicos BALB C , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fiebre Tifoidea/prevención & controlRESUMEN
Enteric fever has been one of the leading causes of severe illness and deaths worldwide. S. Typhi and S. Paratyphi A, B and C are important enteric fever-causing organisms globally. This infection causes about 21 million cases among which 222,000 typhoid related deaths occurred in 2015. These estimates do not reflect the ultimate and real status of the disease due to the lack of unified diagnostic and proper reporting system from typhoid endemic and other regions. Current control strategies have become increasingly ineffective due to the emergence of multi-drug resistance among the strains. This situation worsens the disease-burden in developing as well as in developed countries. Moreover the emergence of S. Paratyphi A as a major enteric fever-causing organism in several Asian countries, demands a prophylactic measure at this hour. Other than two licensed vaccines of S. Typhi, there are no exsisting vaccines for S. Paratyphi A. Moreover, travelers returning from endemic regions are becoming more susceptible to have these infections. In this situation, a need for bivalent approach is required where a single immunogen (consisting from each organism) will be effective against the disease. In this review, we have focused on the general information about typhoidal fever, its spread and epidemiology in brief and the present status of typhoidal vaccines and its future. This review highlights existing gaps in the typhoidal salmonellae research with a special emphasis on the status of present typhoidal salmonellae vaccine research.
Asunto(s)
Descubrimiento de Drogas/tendencias , Salmonella paratyphi A/inmunología , Salmonella typhi/inmunología , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides/inmunología , Vacunas Tifoides-Paratifoides/aislamiento & purificación , Salud Global , Humanos , Fiebre Tifoidea/epidemiologíaRESUMEN
Salmonella-induced gastroenteritis causes massive morbidity and mortality in both adults and children of developing countries. However, it is difficult to study the mode of infection and vaccine efficacy due to inadequacies of current animal models. For this reason, we have explored using zebrafish as an improved model for non-typhoidal Salmonella (NTS) infection, including Salmonella enterica Typhimurium, Salmonella enterica Enteritidis and Salmonella enterica Weltevreden. In this study, we found that after infection of zebrafish with NTS, severe diarrhea like symptoms were observed and NTS significantly colonized the zebrafish intestine without any manipulation of the normal intestinal microbiota of the fish. Furthermore, these strains can colonize for longer than 72h and induce severe inflammation in the intestine, which may induce fish death. We also found that infected fish can transmit the pathogen into naïve fish. Moreover, we have established that zebrafish is an excellent model for vaccine study. Successive triple bath vaccination with heat-killed single serotype S. Typhimurium and S. Enteritidis immunogen induced protective efficacy against a high dose (10(8)CFU/ml) of infection with these pathogens. This study provides a natural infection model for the study of NTS infection, transmission and vaccine efficacy.