WHO consultation on ETEC and Shigella burden of disease, Geneva, 6-7th April 2017: Meeting report.

According to the 2015 Global Burden of Disease Study, diarrhea ranked ninth among causes of death for all ages, and fourth among children under 5 years old, accounting for an estimated 499,000 deaths in this young age group. It was also the second most common cause of years lived with disability (2.39 billion YLDs). The goal of the WHO/UNICEF Integrated Global Action Plan for the Prevention and Control of Pneumonia and Diarrhea (GAPPD) is to reduce deaths from diarrhea in children under 5 years of age to less than 1 per 1000 live births, by 2025. Development of new and improved vaccines against diarrheal infections is a fundamental element of the strategy towards achieving this goal. Enterotoxigenic Escherichia coli (ETEC) and Shigella are enteropathogens that cause significant global mortality and morbidity, particularly in low- and middle-income countries. In 2016, WHO's Product Development for Vaccines Advisory Committee (PDVAC) recommended that the WHO's Initiative for Vaccine Research (IVR) engage in this area, based on PDVAC's criteria of prioritizing the development of vaccines against pathogens that will address a major unmet public health need, and for which clinical candidates with a good probability of technical success are in the pipeline. As a first step, WHO's IVR convened global subject matter experts to discuss the current global ETEC and Shigella disease burden estimates, including the current understanding of the long-term indirect effects of ETEC and Shigella infection, and how these data may affect future decision making on vaccine development for both pathogens. The available global burden estimates for ETEC and Shigella differ with respect to the relative importance of these two pathogens. The mortality estimates vary between iterations published by the same group, as well as between estimates of different groups, although the uncertainty intervals are broad and overlapping. These variances are attributable to differences in the data available and incorporated in the models; the methods used to detect the pathogens; the modelling methodologies; and, to actual changes in the total number of diarrheal deaths over time. The changes in the most recently reported mortality estimates for these pathogens, as compared to previous iterations, has led to debate as to whether investment in development of stand-alone vaccines, rather than combined vaccines, is warranted from cost-effectiveness and vaccine impact perspectives. Further work will be needed to understand better the variances and uncertainties in the reported mortality estimates to support investment decision making, and ultimately policy recommendations for vaccine use. In addition, a comprehensive assessment of the value proposition for vaccines against these pathogens is needed and will be strengthened if the long-term health consequences associated with diarrhea and dysentery due to these pathogens are better defined.

Improving chances for successful clinical outcomes with better preclinical models.

In order to avoid expensive clinical failures, better and more predictive animal models of vaccine efficacy are needed to screen Shigella and ETEC vaccine candidates for protective efficacy. The 2016 Vaccines Against Shigella and ETEC (VASE) Conference included a workshop focused on the strengths and weaknesses of current models, particularly in terms of the correlation to vaccine efficacy in human clinical trials. Workshop presenters shared information on existing preclinical animal models for assessing the immunogenicity and protective efficacy of Shigella and ETEC vaccines. The presentations were followed by a discussion about how to best utilize these models, how the models can be improved, and best practices for Shigella and ETEC vaccine developers. The workshop concluded with three major recommendations for the field: (1) develop better and more consistent reagents for animal studies and make them widely available, (2) prioritize harmonization of animal models and immunology assays, and (3) develop preclinical correlates of protection, which will be key in selecting the best vaccine candidates for further clinical development.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part II: Vaccines for Shigella, Salmonella, enterotoxigenic E. coli (ETEC) enterohemorragic E. coli (EHEC) and Campylobacter jejuni.

In Part II we discuss the following bacterial pathogens: Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic) and Campylobacter jejuni. In contrast to the enteric viruses and Vibrio cholerae discussed in Part I of this series, for the bacterial pathogens described here there is only one licensed vaccine, developed primarily for Vibrio cholerae and which provides moderate protection against enterotoxigenic E. coli (ETEC) (Dukoral(®)), as well as a few additional candidates in advanced stages of development for ETEC and one candidate for Shigella spp. Numerous vaccine candidates in earlier stages of development are discussed.

Detection of secretory immunoglobulin A in human colostrum as mucosal immune response against proteins of the type III secretion system of Salmonella, Shigella and enteropathogenic Escherichia coli.

BACKGROUND: Some enteropathogens use the type III secretion system to secrete proteins that allows them to interact with enterocytes and promote bacterial attachment or intracellular survival. These proteins are Salmonella invasion proteins (Sip), invasion plasmid antigens (Ipa) of Shigella and Escherichia coli secreted proteins (Esp) of enteropathogenic E. coli. There are no previous studies defining the presence of colostral sIgA against all these 3 major enteric pathogens. OBJECTIVE: To evaluate the presence of sIgA in colostrum against proteins of the type III secretion system of Salmonella, Shigella and enteropathogenic E. coli. METHODS: We collected 76 colostrum samples from puerperal women in Lima, Peru. These samples were reacted with type III secretion system proteins extracted from bacterial culture supernatants and evaluated by Western Blot. RESULTS: Antibodies were detected against Salmonella antigens SipA in 75 samples (99%), SipC in 62 (82%) and SipB in 31 (41%); against Shigella antigens IpaC in 70 (92%), IpaB in 68 (89%), IpaA in 66 (87%) and IpaD in 41 (54%); and against enteropathogenic E. coli EspC in 70 (92%), EspB-D in 65 (86%) and EspA in 41 (54%). Ten percent of samples had antibodies against all proteins evaluated and 42% against all except 1 protein. There was no sample negative to all these proteins. CONCLUSIONS: The extraordinarily high frequency of antibodies in colostrum of puerperal women detected in this study against these multiple enteric pathogens shows evidence of immunological memory and prior exposure to these pathogens, in addition to its possible protective role against infection.

Specific IgG activity against diarrheagenic bacteria in bovine immune milk and effect of pH on its antigen-binding activity upon heating.

Bovine colostrum and milk antibodies of calving and lactating cows immunized with a multivalent vaccine consisting of whole cells of three different species of pathogenic bacteria including four strains of enterotoxigenic Escherischia coli, five strains of enteropathogenic Esch. coli, three strains of enteroinvasive Esch. coli, two strains of Samonella typhi, and one strain each of Shigellia dysenteriae, Sh. sonnei and Sh. flexneri were generated, respectively. A significantly elevated activity and titre of specific IgG from bovine immune colostrum were seen for only 5 days after calving of immunized cows, however, the levels of specific IgG could be obtained continuously from the milk of immunized lactating cows until the 11th week of the entire experiment period. Subsequently, we observed that the high specific IgG activity in immune milk was relatively stable under pH 5.0-7.0 at 37 degrees C. Of importance, we identified that the specific IgG preserved its biological function for high antigen-binding activity at pH 5.5-6.5 for 30 min of heat treatment at 70 degrees C and for 350 s at 72 degrees C. Our findings suggest that the specific IgG from milk antibodies of immunized lactating cows may be used as an abundant source of hyper-immune products for prevention of multibacteria-induced diarrhea, however, the effect of pH on its antigen-binding activity upon heating should be carefully considered and designed.

A standard immunoglobulin preparation produced from bovine colostra shows antibody reactivity and neutralization activity against Shiga-like toxins and EHEC-hemolysin of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) causes a variety of clinical conditions, the most important being hemorrhagic colitis and hemolytic uremic syndrome. A curative therapy of EHEC diseases is not yet feasible. This study investigates the antibody reactivity of Lactobin, a standardized immunoglobulin (Ig) preparation, obtained from the colostra of non-immunized cows. Three different batches of Lactobin exhibited equally high titers of specific antibodies against Shiga-like toxins (SLTs, verocytotoxins) and EHEC hemolysin (EHEC-Hly) produced by E. coli O157. In addition, Lactobin blocked the cytotoxic effect of SLT-I and SLT-II on Vero cell monolayers and inhibited the cytolytic effects of EHEC-Hly on human erythrocytes. Since Lactobin contains high levels of antibodies and neutralizing activity against important virulence factors of EHEC O157, this drug has potential use in the treatment of diarrhea and the prevention of EHEC-associated hemolytic uremic syndrome.

Titres of class-specific antibodies against Shigella and Salmonella lipopolysaccharide antigens in colostrum and breast milk of Costa Rican, Swedish and Vietnamese mothers.

Enzyme immunoassays (EIA) were used to estimate titres of class-specific antibodies against purified and chemically defined phenol-water-extracted lipopolysaccharide (LPS) antigens of Salmonella serogroup B (BO), Shigella dysenteriae type I, Plesiomonas shigelloides (the same O-antigen as Shigella sonnei) and Shigella flexneri Y. Titres in colostrum and breast milk of Swedish, Vietnamese and Costa Rican mothers from various socioeconomic conditions were compared. The antibodies were mainly of the IgA isotype. IgM antibodies were also present, but only very low concentrations of IgG were found. In Costa Rican mothers, the IgA antibody titres were significantly higher (P less than 0.05) in women of low and middle socioeconomical conditions than were those in mothers of high socioeconomical level. The low titres in the last group were comparable to those found in Swedish mothers. The IgA antibody titres found in Vietnamese mothers were similar to those of Costa Rican mothers from the low and middle socioeconomic conditions, being highest against S. flexneri Y LPS. The IgM antibody titres were also highest in Vietnamese mothers, immediately followed by the Costa Rican mothers of low socioeconomic conditions. The low IgM titres in the Costa Rican women of high socioeconomic level were comparable to those seen in Swedish mothers. The results suggest that, in Costa Rica and Vietnam, S. flexneri is the most prevalent Shigella sp. causing infection and that Salmonella serogroup B infections are rare in all three countries. The results also show that the antibody repertoire in colostrum and breast milk varies. Furthermore, in addition to the prevalence of a specific micro-organism in a determined geographical area, such differences may be associated mainly with exposure to certain pathogens in particular socioeconomic conditions.

Antibodies against invasion plasmid coded antigens of shigellae in human colostrum and milk.

Colostral and milk samples of Swedish, Vietnamese and Costa Rican mothers living under various socioeconomic conditions were tested for the presence of shigella invasion plasmid coded antigen (Ipa) specific antibodies. IgA antibodies of this specificity were found in significantly higher titres in samples of Vietnamese (600 +/- 338) than in samples of Swedish or high income Costa Rican mothers (190 +/- 224 and 290 +/- 241, respectively; p < 0.05). Specific IgA titres in the low income group of Costa Rican mothers (470 +/- 338) did not differ significantly from the values obtained in Vietnam. While no Ipa specific IgM could be detected in any of the samples tested, specific IgG was found in 90% of the Vietnamese colostrum. These data indicate that antibodies which could be responsible for the dysentery-preventing effect of breast feeding are indeed present in human colostrum and milk in areas where shigellosis occurs with relatively high incidence.

Human milk secretory immunoglobulin A to Shigella virulence plasmid-coded antigens.

Although antibodies to the lipopolysaccharide antigens of Shigella have been demonstrated in human milk, such antibodies do not explain the putative protective effect of breast-feeding against symptomatic Shigella infection. Shigella species do not share related lipopolysaccharides, but they do possess closely related virulence plasmids that code for the proteins essential for cell invasion. We therefore sought to determine the frequency, amount, and duration of excretion of human milk antibodies to these shared virulence plasmid-associated antigens in populations of different rates of Shigella infection frequency (Mexico City, high; Houston, low). Such antibodies were present in the milk of virtually all the Mexican women but also were present in a large proportion of milk samples from the women living in Houston. The amounts of these antibodies were highest in colostrum but after 2 weeks of lactation fell to stable levels. The frequency and persistence of these antibodies in the milk of the women from Houston suggest that the memory and drive for secretion of these antibodies is extremely long lived.