Vaccines against human diarrheal pathogens: current status and perspectives

Abstract

Worldwide, nearly 1.7 billion people per year contract diarrheal infectious diseases (DID) and almost 760 000 of infections are fatal. DID are a major problem in developing countries where poor sanitation prevails and food and water may become contaminated by fecal shedding. Diarrhea is caused by pathogens such as bacteria, protozoans and viruses. Important diarrheal pathogens are Vibrio cholerae, Shigella spp. and rotavirus, which can be prevented with vaccines for several years. The focus of this review is on currently available vaccines against these three pathogens, and on development of new vaccines. Currently, various types of vaccines based on traditional (killed, live attenuated, toxoid or conjugate vaccines) and reverse vaccinology (DNA/mRNA, vector, recombinant subunit, plant vaccines) are in development or already available. Development of new vaccines demands high levels of knowledge, experience, budget, and time, yet promising new vaccines often fail in preclinical and clinical studies. Efficacy of vaccination also depends on the route of delivery, and mucosal immunization in particular is of special interest for preventing DID. Furthermore, adjuvants, delivery systems and other vaccine components are essential for an adequate immune response. These aspects will be discussed in relation to the improvement of existing and development of new vaccines against DID.

Keywords: Shigellaspp. Campylobacterspp.; Vibrio cholerae; diarrheal diseases; human pathogen; oral vaccine; recombinant vaccine; rotavirus.

Figure 1. The gastrointestinal tract, interactions between pathogens and host and approaches to vaccine design. (A) Vibrio cholerae secrets an enterotoxin, which is an AB Toxin and comprises a single catalytic A subunit and a pentameric B subunit for specific binding to host cells. The receptor for CT is the glycolipid ganglioside GM1. Following internalization by receptor-mediated endocytosis, transport to the Golgi and the ER, the A1 subunit is finally transferred to the cytoplasm. The A1 fragment is a NAD-dependent ADP ribosyltransferase and activates the G protein G_sα (GTP-bound), thereby continually stimulating adenylate cyclase (AC) produce cAMP. The high cAMP level enables the protein kinase A which induces a dramatic electrolyte transport, which is typically for diarrhea. Possible vaccine approaches (indicated by syringes) are the cholera toxin (CT), different virulence genes and use of inactivated/attenuated strains.(B) Salmonella spp. translocates effector proteins by a T3SS, encoded by Salmonella Pathogenicity Island 1, inducing pro-inflammatory responses and uptake of the pathogen via macropinocytosis. Uptake also occurs via M-cells or phagocytes. After phagocytosis by macrophages apoptosis is triggered, thereby triggering inflammation reactions with recruitment of neutrophils. Internalized Salmonella survive and replicate within the ‘Salmonella-containing vacuole’. Salmonella enterica Serovar Typhi Ty21 represents a promising live vector for presentation of foreign antigens from unrelated bacterial, viral and parasitic pathogens. (C) Shigella spp. infects the epithelium from the intestinal lumen of the terminal small intestine and colon through M-cells. After phagocytosis, the bacteria are able to escape from the macrophage by triggering apoptosis. By remodeling the host cell actin cytoskeleton and forming large membrane protrusions, invasion occurs similar to S. enterica. Within host cells, Shigella is motile in the cytoplasm by a mechanism involving the formation of actin tails, also leading to infection into neighboring host cells. Spreading from cell to cell within intestinal tissue is accompanied by emission of bloody mucopurulent stools. Strains deficient in intracellular motility, in enterotoxin and further virulence genes present good vaccine candidates. Furthermore, cross-linked O-antigen polysaccharides of the relevant Shigella serotypes to a carrier protein is also a vaccine design approach.

Figure 3. Mucosal immune responses. The gastrointestinal tract (GI) is covered by a mucus layer covering a monolayer of epithelial cells and M-cells that are connected by tight junctions (not shown). Inside the GI pathogens come in contact with the mucus, natural barrier not only against pathogens, but also against the gastric acid in the stomach. Mucus also protects against drying of the nasal mucosa and serves as an adhesion surface for the intestinal flora. Components of the mucus are mucin glycoprotein chains, antimicrobial peptides such as lysozyme, histatine and cystatine, defensins and specific secretory IgA. The mucus is produced from subjacent monolayer of enterocytes; ultimately they form the protective barrier. A further layer is the lamina propria with its gut-associated lymphoid tissues (GALT) such as Peyer’s patches and isolated lymphoid follicles. It also contains a high amount of immune cells such as CD4⁺- and CD8⁺-T-cells, B-lymphocytes and plasma cells, dendritic cells and macrophages. For long-term immunization IgA producing plasma cells are very important. M-cells transport antigens via transcytosis to antigen presenting cells such as dendritic cells. These and macrophages interact with various types of T-cells (such as CD4⁺- and CD8⁺-cells) in the Peyer’s patches, lamina propria and other lymphatic tissues through their receptors and various signal molecules. After T-cell activation, they also interact with B-cells, which then move to the target side and change into IgA producing plasma cells. IgA is secreted as mono- or dimer and binds to pathogens (e.g., viruses, bacteria and parasites) and antigens. Thus, the adaptive immune response does not proceed in an uncontrolled manner there are also regulatory T-cells. However, there are other defense mechanisms for instance antimicrobial peptides, defensins, digestive enzymes, the complement system and the mucin glycoprotein chains (not shown). Modified according to Macdonald and Monteleone et al. 2005.

Figure 2. Traditional vaccine production and reverse genetics strategies. (A) Simplified representation of the individual steps of the traditional vaccine production by isolation of the pathogen over the cultivation, processing, up to the final vaccine. With this approach, different vaccines such as inactivated and attenuated live vaccines or subunit vaccines and special types like conjugate and toxoid vaccines can be produced and combined. Dashed line means destroyed DNA or RNA. (B) Simplified scheme of steps for reverse vaccine manufacturing. Different from traditional methods, reverse vaccinology starts with the analysis of the pathogen genome and epitope libraries searching for possible antigens used for immunization. After cloning/synthesis of the candidate sequence, it is transformed into vectors, antigen-expressing microorganisms or conjugated to gold particles. Vectors then can be used as vaccine or for plant modification. Antigen-expressing plants may be used as edible vaccines or are further processed to plant vaccines. Recombinant subunit vaccines are derived from antigen-expressing microorganisms. DNA- or mRNA-conjugated gold particles are also used as DNA or mRNA vaccines.