A Systems Biology Approach Identifies B Cell Maturation Antigen (BCMA) as a Biomarker Reflecting Oral Vaccine Induced IgA Antibody Responses in Humans.

Vaccines against enteric diseases could improve global health. Despite this, only a few oral vaccines are currently available for human use. One way to facilitate such vaccine development could be to identify a practical and relatively low cost biomarker assay to assess oral vaccine induced primary and memory IgA immune responses in humans. Such an IgA biomarker assay could complement antigen-specific immune response measurements, enabling more oral vaccine candidates to be tested, whilst also reducing the work and costs associated with early oral vaccine development. With this in mind, we take a holistic systems biology approach to compare the transcriptional signatures of peripheral blood mononuclear cells isolated from volunteers, who following two oral priming doses with the oral cholera vaccine Dukoral®, had either strong or no vaccine specific IgA responses. Using this bioinformatical method, we identify TNFRSF17, a gene encoding the B cell maturation antigen (BCMA), as a candidate biomarker of oral vaccine induced IgA immune responses. We then assess the ability of BCMA to reflect oral vaccine induced primary and memory IgA responses using an ELISA BCMA assay on a larger number of samples collected in clinical trials with Dukoral® and the oral enterotoxigenic Escherichia coli vaccine candidate ETVAX. We find significant correlations between levels of BCMA and vaccine antigen-specific IgA in antibodies in lymphocyte secretion (ALS) specimens, as well as with proportions of circulating plasmablasts detected by flow cytometry. Importantly, our results suggest that levels of BCMA detected early after primary mucosal vaccination may be a biomarker for induction of long-lived vaccine specific memory B cell responses, which are otherwise difficult to measure in clinical vaccine trials. In addition, we find that ALS-BCMA responses in individuals vaccinated with ETVAX plus the adjuvant double mutant heat-labile toxin (dmLT) are significantly higher than in subjects given ETVAX only. We therefore propose that as ALS-BCMA responses may reflect the total vaccine induced IgA responses to oral vaccination, this BCMA ELISA assay could also be used to estimate the total adjuvant effect on vaccine induced-antibody responses, independently of antigen specificity, further supporting the usefulness of the assay.

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.

Retinoic acid elicits a coordinated expression of gut homing markers on T lymphocytes of Zambian men receiving oral Vivotif, but not Rotarix, Dukoral or OPVERO vaccines.

All-trans retinoic acid (ATRA) up-regulates, in laboratory animals, the expression of the gut homing markers α4ß7 integrin and CCR9 on lymphocytes, increasing their gut tropism. Here, we show that, in healthy adult volunteers, ATRA induced an increase of these gut homing markers on T cells in vivo in a time dependent manner. The coordinated increase of α4ß7 and CCR9 by ATRA was seen in 57% (12/21) of volunteers and only when given together with an oral Vivotif vaccine. When this coordinated response to ATRA and Vivotif vaccine was present, it was strongly correlated with the gut immunoglobulin A (IgA) specific response to vaccine LPS (ρ = 0.82; P = 0.02). Using RNA-Seq analysis of whole blood transcription, patients receiving ATRA and Vivotif in conjunction showed transcriptomic changes in immune-related pathways, particularly including interferon α/ß signaling pathway, membrane-ECM interactions and immune hubs. These results suggest that exogenous ATRA can be used to manipulate responses to a subclass of oral vaccines, so far limited to a live attenuated Vivotif vaccine.

Retinoic acid pre-treatment down regulates V. cholerae outer membrane vesicles induced acute inflammation and enhances mucosal immunity.

Bacterial outer membrane vesicles have been extensively investigated and considered as a next generation vaccine. Recently, we have demonstrated that the cholera pentavalent outer membrane vesicles (CPMVs) immunogen induced adaptive immunity and had a strong protective efficacy against the circulating V. cholerae strains in a mouse model. In this present study, we are mainly focusing on reducing outer membrane vesicle (OMV) -mediated toxicity without altering its antigenic property. Therefore, we have selected All-trans Retinoic Acid (ATRA), active metabolites of vitamin A, which have both anti-inflammatory and mucosal adjuvant properties. Pre-treatment of ATRA significantly reduced CPMVs induced TLR2 mediated pro-inflammatory responses in vitro and in vivo. Furthermore, we also found ATRA pre-treatment significantly induced mucosal immune response and protective efficacy after two doses of oral immunization with CPMVs (75µg). This study can help to reduce OMV based vaccine toxicity and induce better protective immunity where children and men suffered from malnutrition mainly in developing countries.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part I: Overview, vaccines for enteric viruses and Vibrio cholerae.

Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided into 2 articles, of nearly 50 vaccine candidates against the most relevant viral and bacterial pathogens that cause acute gastroenteritis. In order to facilitate reading, sections for each pathogen are organized as follows: i) a discussion of the main epidemiological and pathogenic features; and ii) a discussion of vaccines based on their stage of development, moving from current licensed vaccines to vaccines in advanced stage of development (in phase IIb or III trials) to vaccines in early stages of clinical development (in phase I/II) or preclinical development in animal models. In this first article we discuss rotavirus, norovirus and Vibrio cholerae. In the following article we will discuss Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic), and Campylobacter jejuni.

CD4+ T-cell responses to an oral inactivated cholera vaccine in young children in a cholera endemic country and the enhancing effect of zinc supplementation.

Immunization of young children with the oral inactivated whole cell cholera vaccine Dukoral((R)) containing recombinant cholera toxin B subunit (CTB) induces antibody responses which can be further enhanced by zinc supplementation. We have investigated if immunization with the cholera vaccine induces specific T-cell responses in young children and also whether zinc supplementation influences these responses. Bangladeshi children (10-18 months old) received vaccine alone, vaccine together with zinc supplementation or only zinc. T-cell blast formation indicating a proliferative response was analyzed by the flow cytometric assay of cell-mediated immune response in activated whole blood (FASCIA) and cytokines were measured by ELISA. Stronger T-cell responses were detected if a modified CTB molecule (mCTB) with reduced binding to GM1 ganglioside was used for cell stimulation compared to normal CTB. After vaccination, CD4+ T cells responded to mCTB with significantly increased blast formation (P<0.01) and IFN-gamma production (P<0.05) compared to before vaccination. No responses to mCTB were detected in children receiving zinc alone (P>0.05). The IFN-gamma production was significantly higher (P<0.01) but the blast formation comparable (P>0.05) in children receiving zinc plus vaccine compared to in children receiving vaccine alone. The vibriocidal antibody responses induced by the vaccine were also significantly higher in children receiving zinc supplementation (P<0.001). Our results thus show that oral cholera vaccination induces a Th1 T-cell response in young children, and that the IFN-gamma as well as the vibriocidal antibody responses can be enhanced by zinc supplementation.

Enhanced immunogenicity of an oral inactivated cholera vaccine in infants in Bangladesh obtained by zinc supplementation and by temporary withholding breast-feeding.

The killed oral cholera vaccine Dukoral is recommended for adults and only children over 2 years of age, although cholera is seen frequently in younger children and there is an urgent need for a vaccine for them. Since decreased immunogenicity of oral vaccines in children in developing countries is a critical problem, we tested interventions to enhance responses to Dukoral. We evaluated the effect on the immune responses by temporarily withholding breast-feeding or by giving zinc supplementation. Two doses of Dukoral consisting of killed cholera vibrios and cholera B subunit were given to 6-18 months old Bangladeshi children (n=340) and safety and immunogenicity studied. Our results showed that two doses of the vaccine were safe and induced antibacterial (vibriocidal) antibody responses in 57% and antitoxin responses in 85% of the children. Immune responses were comparable after intake of one and two doses. Temporary withholding breast-feeding for 3 h before immunization or supplementation with 20 mg of zinc per day for 42 days resulted in increased magnitude of vibriocidal antibodies (77% and 79% responders, respectively). Administration of vaccines without buffer or in water did not result in reduction of vibriocidal responses. This study demonstrates that the vaccine is safe and immunogenic in children under 2 years of age and that simple interventions can enhance immune responses in young children.

Suppressive effect of zinc on antibody response to cholera toxin in children given the killed, B subunit-whole cell, oral cholera vaccine.

In a previous study, children aged 2-5 years old in Bangladesh were supplemented orally with a single dose of Vitamin A (200,000 IU) and a placebo for zinc (zinc equivalent to 20 mg of elemental zinc) everyday for 42 days (group A), zinc and a placebo for Vitamin A (group Z), zinc and Vitamin A (group AZ) or both placebos (group P). All children were orally immunised with two doses of the killed cholera vaccine containing whole cells and a recombinant B subunit of cholera toxin (CT). The number of children who responded with > or = 4-fold vibriocidal antibody (a proxy indicator of protection against cholera) was significantly greater among the zinc-supplemented groups than among the non-zinc-supplemented groups, while Vitamin A supplementation did not appear to have any effect. The sera from these children were assayed for antibody to CT. Antibody to CT is known to exert a synergistic protective effect against cholera in animal studies, and offer significantly higher short-term protection against cholera and significant short-term protection against enterotoxigenic Escherichia coli diarrhoea in humans on oral immunisation with the cholera vaccine. Children who received zinc had significantly reduced levels of serum antibodies to CT than children who received placebos only. Factorial analysis showed a trend for zinc showing a reduction in the number of children responding with CT-antibody, while Vitamin A did not appear to have any effect. Thus, zinc enhanced vibriocidal antibody response, but suppressed CT-antibody response, suggesting that zinc supplementation has different modulating effects on vibriocidal antibody response and CT-antibody response.

Intestinal and systemic immune responses to an oral cholera toxoid B subunit whole-cell vaccine administered during zinc supplementation.

Zinc plays a critical role in the normal functioning of the immune system. We investigated whether zinc sulfate administered orally to adult zinc-replete volunteers modulates systemic and intestinal immune responses to an oral killed cholera toxoid B subunit (CTB) whole-cell cholera vaccine. The 30 participants were immunized twice, with a 17-day interval. The vaccinees in the intervention group ingested 45 mg of elemental zinc thrice daily for 9 days starting 2 days before each vaccine dose. The median serum anti-CTB immunoglobulin A (IgA) and IgG responses from day 0 to day 30, i.e. after two vaccine doses, were 13-fold lower (P value for identical distribution, <0.005) in the zinc-supplemented compared to the nonsupplemented vaccinees. The median serum vibriocidal responses from baseline to after one (day 0 to day 17) and two (day 0 to day 30) vaccine doses were at least sixfold (P = 0.033) and fourfold (P = 0.091) higher, while the median fecal anti-CTB IgA response after two doses was estimated to be fourfold higher (P = 0.084) in the zinc-supplemented vaccinees. These observations show that zinc reduces the antitoxin and may enhance the antibacterial responses in serum. Zinc may also improve the intestinal antitoxin immune response. Oral zinc administration has the potential to modify critical immune responses to antigens applied to mucosal surfaces.

Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues.

We tested the notion that the mucosal adjuvant cholera toxin (CT) could target, in addition to nasal-associated lymphoreticular tissues, the olfactory nerves/epithelium (ON/E) and olfactory bulbs (OBs) when given intranasally. Radiolabeled CT ((125)I-CT) or CT-B subunit ((125)I-CT-B), when given intranasally to mice, entered the ON/E and OB and persisted for 6 days; however, neither molecule was present in nasal-associated lymphoreticular tissues beyond 24 h. This uptake into olfactory regions was monosialoganglioside (GM1) dependent. Intranasal vaccination with (125)I-tetanus toxoid together with unlabeled CT as adjuvant resulted in uptake into the ON/E but not the OB, whereas (125)I-tetanus toxoid alone did not penetrate into the CNS. We conclude that GM1-binding molecules like CT target the ON/E and are retrograde transported to the OB and may promote uptake of vaccine proteins into olfactory neurons. This raises concerns about the role of GM1-binding molecules that target neuronal tissues in mucosal immunity.

Estudio del patrón de excresión y la capacidad protectora en conejos inmunizados de forma oral con cepas atenuadas de Vibrio cholerae O1 biotipo El Tor

Con el fin de estudiar los patrones de excreción, colonización y la capacidad protectora de cepas vivas atenuadas de Vibrio cholerae O1El Tor, se inmunizaron conejos Nueva Zelandia con estas cepas y su correspondiente parental, con 2 dosis por el modelo de inoculación oral en conejos adultos. Fueron retados 2 semanas después de la segunda dosis por el modelo de intestino ligado, con cepas altamente virulentas de V. choleraeO1 serotipos Ogawa e Inaba y serogrupo O139. Se comprobó que las cepas manupuladas de forma genética no afectan los patrones de excreción, cuando se compara con su parental. Se observó en el reto una disminución en los niveles de colonización de las cepas virulentas de ambos serotipos; tanto en los conejos inmunizados con las cepas atenuadas como con la parental en comparación con animales controles inmunizados con la cepa Escherichia coli K-12, lo que indica que hubo cierto grado de protección. En el caso de los animales retados con la cepa 0139 se demostró que la protección es específica para cada serogrupo pues en este caso no se observó disminución de la colonización.

In order to study the excretion patterns, colonization and protective capacity of live sttenuated strains of Vibrio cholerae O1. E1 Tor, rabbits were immunized in New Zealand with these strains and their corresponding parental strains. 2 doses were administered by the model of oral inoculation in adult rabbits. Rabbits were rotated 2 weeks after the second dose by the model of ligated intestine with highly virulent strains of V. cholerae O1 Ogawa and Inaba serotypes and O139 serogroup. It was proved that the genetically manipulated strains do not effect the excretion patterns when they are compared with their parental strains. It was observed in the challenge a decrease in the levels of colonization of virulent strains of both serotypes, not only among the rabbits immunized with the attenuated strains, but also among those immunizedwith the parental strains in comparison with control animals immunized with the strain of Escherichia coli K-12, which means that there was certain degree of protection. In the case of the animals challenged with the O139 strain it was demonstrated that the protection is specific for each serogroup, since in this case there was no reduction of the colonization.

[The excretion pattern and protective capacity in rabbits orally immunized with attenuated strains of Vibrio cholerae O1 biotype El Tor]. / Estudio del patrón de excreción y la capacidad protectora en conejos inmunizados de forma oral con cepas atenuadas de Vibrio cholerae O1 biotipo El Tor.

In order to study the excretion patterns, colonization and protective capacity of live attenuated strains of Vibrio cholerae O1. El Tor, rabbits were immunized in New Zealand with these strains and their corresponding parental strains. 2 doses were administered by the model of oral inoculation in adult rabbits. Rabbits were rotated 2 weeks after the second dose by the model of ligated intestine with highly virulent strains of V. cholerae O1 Ogawa and Inaba serotypes and O139 serogroup. It was proved that the genetically manipulated strains do not effect the excretion patterns when they are compared with their parental strains. It was observed in the challenge a decrease in the levels of colonization of virulent strains of both serotypes, not only among the rabbits immunized with the attenuated strains, but also among those immunized with the parental strains in comparison with control animals immunized with the strain of Escherichia coli K-12, which means that there was certain degree of protection. In the case of the animals challenged with the O139 strain it was demonstrated that the protection is specific for each serogroup, since in this case there was no reduction of the colonization.

[The outer membranes of Vibrio cholerae as a potential component in a chemical vaccine]. / Naruzhnye membrany kholernogo vibriona kak potentsial'nyi komponent khimicheskoi vaktsiny.

The results of the study of the preparation of V. cholerae eltor membrane, obtained by the lysis and inactivation of microbial cells with urea and the subsequent differential centrifugation and nuclease treatment. As revealed in this study, the outer membrane preparation, when introduced parenterally and orally to mice, induced pronounced immunity to experimental cholera infection and the production of vibriocidal antibodies in high titers. The treatment of V. cholerae eltor membranes with trypsin led to further increase of the immunogenic potency of the preparation. The protective action of V. cholerae eltor outer membranes considerably exceeded the protective effect of currently used whole-cell eltor vaccine. This opens prospects for using the above-mentioned preparation for the improvement of chemical vaccine as a component ensuring the formation of antibacterial immunity.

[Combined (associated) vaccination against quarantinable infections]. / Kompleksnaia (assotsiirovannaia) vaktsinatsiia protiv karantinnykh infektsii.

The presence of several active foci of infection of different etiology is an indication for complex (combined) immunization against these diseases. The scheme of complex (combined) immunization against plague, cholera and yellow fever has been experimentally substantiated and successfully tested on volunteers.

Impaired mucosal antibody response to cholera toxin in vitamin A-deficient rats immunized with oral cholera vaccine.

To investigate the importance of vitamin A in the ability to respond to oral antigen administration, rats were fed a vitamin A-free diet. The animals were immunized perorally three times with a mixture of cholera toxin (CT) and a commercial cholera vaccine. The total immunoglobulin A (IgA) concentration as well as the specific IgA anti-CT antibody levels in serum and bile was significantly lower in the vitamin A-deficient animals than in the paired fed controls (animals that were fed a normal commercial diet in an amount equal to the amount the deficient animals consumed), while the levels of total and specific anti-CT IgG were not affected to the same extent by the vitamin A deficiency. The number of IgA anti-CT antibody-producing cells in the mesenteric lymph nodes after immunization was also significantly lower in the vitamin A-deficient rats than in the control rats. Supplementation of the diet with retinyl palmitate restored the ability to mount an IgA antibody response to the antigen, since the level of specific IgA anti-CT antibodies in relation to the total IgA concentration was as high in the vitamin A-supplemented group as in the paired fed control group. Restricted diet intake by itself did not affect the ability to respond adequately to the antigen since there was no difference in IgA anti-CT antibody level between paired fed rats and those being fed ad libitum. Assessment of transforming growth factor beta in cell cultures revealed no difference between vitamin A-deficient and paired fed animals. In summary, vitamin A deficiency resulted in a decreased number of IgA-producing cells, decreased IgA production, and a reduced ability to respond with IgA antibodies to the oral cholera vaccine.

[An oral chemical vaccine from the hypertoxigenic strains of the causative agent of cholera KM-76 Inaba and KM-68 Ogawa]. / Oral'naia khimicheskaia vaktsina iz gipertoksigennykh shtammov KM-76 Inaba i KM-68 Ogava vozbuditel'a kholery.

The material on the development of chemical vaccine, prepared from two newly formed strains (KM-76 Inaba and KM-68 Ogawa) and intended for oral administration, is presented. The conditions for the submerged cultivation of these strains have been established, which makes it possible to increase the production of choleragen 8- to 10-fold and O-antigen 3- to 4-fold in comparison with V. cholerae natural strain 569B. The maximum accumulation of neuraminidase, protease, phospholipase, along with choleragen, has been registered in the logarithmic phase and that of O-antigen, in the stationary phase of growth. The use of strains KM-76 and KM-68 has led to the fourfold increase of the specific activity of the main immunogens, thus permitting the respective increase of the yield of the oral vaccine without changes in its high capacity for the formation of specific antibodies and its low residual toxigenicity.