Review: Helicobacter: Inflammation, immunology, and vaccines.

Chronic inflammation induced by Helicobacter pylori infection is a critical factor in the development of peptic ulcer disease and gastric cancer. Central to this inflammation is the initiation of pro-inflammatory signaling cascades within epithelial cells, in particular those mediated by two sensors of bacterial cell wall components, nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and alpha-protein kinase 1 (ALPK1). H pylori is, however, also highly adept at mitigating inflammation in the host, thereby restricting tissue damage and favoring bacterial persistence. H pylori modulates host immune responses by altering cytokine signaling in epithelial and myeloid cells, which results in increased proliferation of regulatory T cells and downregulation of effector T-cell responses. H pylori vacuolating cytotoxin A (VacA) has been shown to play an important role in the dampening of immune responses and induction of immune tolerance capable of protecting against asthma. It is also possible to generate protective immune responses by immunization with various H pylori antigens or their epitopes, in combination with an adjuvant, though this for now has only been shown in mouse models. Novel non-toxic adjuvants, consisting of modified bacterial enterotoxins or nanoparticles, have recently been developed that may not only enhance vaccine efficacy, but also help translate candidate vaccines to the clinic. This review will summarize the main discoveries in the past year regarding host immune responses to H pylori infection, as well as the design of new vaccine approaches against this infection.

Comparative screening of recombinant antigen thermostability for improved leptospirosis vaccine design.

Recombinant antigens exhibit targeted protectiveproperties and offer important opportunities in the development of therapeutic technologies. Biophysical and structural methods have become important tools for the rational design and engineering of improved antigen-based vaccines. Vaccines containing Leptospira immunoglobulin-like (Lig) protein-derived antigens are currently the most promising candidates for protective immunity against the globally prevalent bacterial pathogen, Leptospira interrogans; however, vaccine trials using these domains have produced inconsistent results. Here, we compare the thermostability of domains from the main immunogenic regions from major leptospiral antigens, LigA and LigB. By measuring temperature-dependent fluorescence decay of the hydrophobic core tryptophan, 17 individual Lig protein immunoglobulin-like (Ig-like) domains were shown to display a broad range of unfolding temperatures. For a majority of the domains, stability issues begin to occur at physiologically relevant temperatures. A set of chimeric Ig-like domains was used to establish the ability of transplanted domain regions to enhance thermostability. Further insights into the determinants for domain stabilization were explored with nuclear magnetic resonance dynamics and mutational analysis. The current study has yielded a set of thermostable Ig-like domain scaffolds for use in engineering antigen-based vaccines and demonstrates the importance of incorporating thermostability screening as a design parameter.

Reverse vaccinology and subtractive genomics-based putative vaccine targets identification for Burkholderia pseudomallei Bp1651.

The Burkholderia pseudomallei is a unique bio-threat and causative agent of melioidosis. The B. pseudomallei Bp1651 strain has been isolated from a chronic cystic fibrosis patient. The genome-level DNA sequences information of this strain has recently been published. Unfortunately, there is no commercial vaccine available till date to combat B. pseudomallei infection. The genome-wide prioritization approaches are widely used for the identification of potential therapeutic candidates against pathogens. In the present study, we utilized the recently available annotated genomic information of B. pseudomallei Bp1651 through subtractive genomics and reverse-vaccinology strategies to identify its potential vaccine targets. The analyses identified more than 60 pathogen-specific, human host non-homologous proteins that may prioritize in future studies to investigate therapeutic targets for B. pseudomallei Bp1651. The potential B and T-cells antigenic determinant peptides from these pathogen-specific proteins were cataloged using antigenicity and epitope prediction tools. The analyses unveiled a promising antigenic peptide "FQWEFSLSV" from protein-export membrane protein (SecF) of Bp1651 strain, which was predicted to interact with multiple class I and class II MHC alleles with IC50 value < 100 nM. The molecular docking analysis verified favorable molecular interaction of this lead antigenic peptide with the ligand-binding pocket residues of HLA A*02:06 human host immune cell surface receptor. This peptide is predicted to be a suitable epitope capable to elicit the cell-mediated immune response against the B. pseudomallei pathogen. The putative epitopes and proteins identified in this study may be promising vaccine targets against Bp1651 as well as other pathogenic strains of B. pseudomallei.

An in silico study: Novel targets for potential drug and vaccine design against drug resistant H. pylori.

Gastric cancer risk and adverse ramifications by augmented multi-drug resistance (MDR) of Helicobacter pylori are alarming serious health concern. Combating through available drugs is a difficult task due to lack of appropriate common targets against genetically diverse strains. To improve efficacy, the effective targets should be identified and critically assessed. In the present study, we aim to predict the potential novel targets against H. pylori strains by employing computer aided approach. The genomic dataset of 53 H. pylori strains was comparatively processed and eventually predicted 826 'conserved gene products'. Further, we performed subtractive genomic approach in search of promising crucial targets through the combination of in silico analyses. Codon adaptation index (CAI) value calculation and literature surveys were also done in order to find highly expressed gene products with novelty. Consequently, four enzymes and three membrane proteins were prioritized as new therapeutic and vaccine targets respectively which found to have more interactors in network with high-confidence score, druggability, antigenicity and molecular weight <110 kDa. Therefore, our results underpin the importance of new targets may counteract with false-positive/negatives and facilitate appropriate potential targets for a new insight of reliable therapeutic development.

Genome sequencing of two Bacillus anthracis strains: a virulent strain and a vaccinal strain

ABSTRACT Bacillus anthracis strain SPV842_15 was isolated from bovine fetus, while B. anthracis strain Brazilian vaccinal was recovered from a commercial vaccine. We report here the genome sequences of both strains. The SPV842_15 genome is composed of a single circular chromosome with a length of 5,228,664 base pairs, and comprises 5911 coding sequences. In turn, the Brazilian vaccinal genome remains in 201 contigs with 5733 coding sequences. Both genomes have an overall C + G content of 35.4%, and 11 genes encoding the ribosomal RNAs (rRNAs) 5S, 16S and 23S. Only the plasmid pX01 sequence, which carries genes for toxins synthesis, was detected and completely assembled for both strains. These plasmids have a length of 181,684 base pairs and a C + G content of 32.5%. These genomic data generate insights about vaccinal B. anthracis virulence.

Helicobacter: Inflammation, immunology and vaccines.

Helicobacter pylori is usually acquired in early childhood and the infection persists lifelong without causing symptoms. In a small of cases, the infection leads to gastric or duodenal ulcer disease, or gastric cancer. Why disease occurs in these individuals remains unclear, however the host response is known to play a very important part. Understanding the mechanisms involved in maintaining control over the immune and inflammatory response is therefore extremely important. Vaccines against H. pylori have remained elusive but are desperately needed for the prevention of gastric carcinogenesis. This review focuses on research findings which may prove useful in the development of prognostic tests for gastric cancer development, therapeutic agents to control immunopathology, and effective vaccines.

Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine.

BACKGROUND: The sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis. RESULTS: A series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats. CONCLUSIONS: The established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.

Helicobacter pylori treatment: New perspectives using current experience.

Infection with Helicobacter pylori plays an essential role in the development of duodenal and gastric ulcers as well as in the pathobiology of gastric adenocarcinoma. Thus, successful elimination of the bacterium can reduce the risk of development of these diseases. Currently, most guidelines recommend standard triple therapy (amoxicillin+clarithromycin+proton pump inhibitor), although its efficacy is rapidly falling. Notably, traditional first-line therapy fails in almost 32% of H. pylori-carrying cases, suggesting the importance of choosing the best formulation for first-line therapy. Hence, due to the decreasing effectiveness of first-line treatment, we should be prepared to confront increasing H. pylori therapeutic defeat. Owing to increasing reports of antibiotic resistance worldwide, newer approaches and directions are necessary for managing this problematic infection. Developing and providing better anti-H. pylori strategies (probiotics, antibiotic therapy and non-traditional medicine) without using current clinical experience in treating the infection is impossible. Furthermore, development and examination of new preventive vaccines may also be a new therapeutic direction. Taken together, with regard to current experience, clinicians are highly recommended to consider all alternatives to eradicate H. pylori until a universal vaccine becomes practically available. This article aims to give an overview regarding the current status of H. pylori treatment, accordingly designing an actual overview to gain optimal strategies against this infection.

Inflammation, Immunity, and Vaccines for Helicobacter pylori Infection.

During the last year, a variety of studies have been published that increases our understanding of the basic mechanisms of immunity and inflammation in Helicobacter pylori infection and progression to gastric cancer. Innate immune regulation and epithelial cell response were covered by several studies that contribute with new insights in the host response to H. pylori infection. Also, the adaptive immune response to H. pylori and particularly the role of IL-22 have been addressed in some studies. These advances may improve vaccine development where new strategies have been published. Two major studies analyzed H. pylori genomes of 39 worldwide strains and looked at the protein profiles. In addition, multi-epitope vaccines for therapeutic use have been investigated. Studies on different adjuvants and delivery systems have also given us new insights. This review presents articles from the last year that reveal detailed insight into immunity and regulation of inflammation, the contribution of immune cells to the development of gastric cancer, and understanding mechanisms of vaccine-induced protection.

Novel nanoparticle vaccines for Listeriosis.

In recent years, nanomedicine has transformed many areas of traditional medicine, and enabled fresh insights into the prevention of previously difficult to treat diseases. An example of the transformative power of nanomedicine is a recent nano-vaccine against listeriosis, a serious bacterial infection affecting not only pregnant women and their neonates, but also immune-compromised patients with neoplastic or chronic autoimmune diseases. There is a major unmet need for an effective and safe vaccine against listeriosis, with the challenge that an effective vaccine needs to generate protective T cell immunity, a hitherto difficult to achieve objective. Now utilizing a gold nanoparticle antigen delivery approach together with a novel polysaccharide nanoparticulate adjuvant, an effective T-cell vaccine has been developed that provides robust protection in animal models of listeriosis, raising the hope that one day this nanovaccine technology may protect immune-compromised humans against this serious opportunistic infection.

Immunology and vaccines and nanovaccines for Helicobacter pylori infection.

Helicobacter pylori infection is very common worldwide and is an important cause of gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma. Since the eradication requires treatment with multidrug regimens, prevention of primary infection by a suitable vaccine is attractive. Developing vaccines on the spot when and where an infection is breaking out might be possible, thanks to engineered nanoparticles. In this review, the nature of the host immune response to H. pylori infection is considered. We explain recent candidate vaccines and prophylactic or therapeutic immunization strategies for use against H. pylori. We also describe identification of different types of immune responses that may be related to protection against H. pylori infection. Thus, it seems that there is still a strong need to clarify the main protective immune response against H. pylori.

Why can't we make an effective vaccine against Helicobacter pylori?

Helicobacter pylori is a major human pathogen that colonizes the stomach and is the lead etiological agent for several pathologies. An effective vaccine against these bacteria would be invaluable for protecting against gastric adenocarcinoma. However, the development of such a vaccine has stalled and the field has progressed little in the last decade. In this review, the authors provide an opinion on key problems that are preventing the development of a H. pylori vaccine. Primarily, this involves the inability to produce a completely protective immune response. The knock-on effects of this include a loss of industry investment. Overcoming these problems will likely involve defeating the immune-evasion defenses of H. pylori, in particular the mechanism(s) by which it evades antibody-mediated attack.

Immunogenicity of Bartonella henselae P26 in cats.

Cat scratch disease (CSD) has an estimated prevalence of approximately 200,000 persons in the USA, and approximately 22,000 new cases occur annually. Cats are the natural carriers of Bartonella henselae, the agent for CSD. Zoonotic transmission of B. henselae can result in CSD in immunocompetent humans and bacillary angiomatosis in immunosuppressed humans. Infection in cats often goes undetected. Development of a vaccine to prevent feline infection is warranted to reduce the prevalence of infection in the feline population and to decrease the potential for zoonotic transmission. One of the immunoreactive proteins identified from our previous study was P26. In this study, we demonstrated that B. henselae recombinant P26 (rP26) was immunogenic in cats. Four cats immunized with rP26 and four control cats were challenged with B. henselae type I and blood samples were collected for culture, PCR, and serology. Immunization with rP26 did not provide protection against B. henselae infection in cats at the doses used in this study. However, p26 PCR proved to be more sensitive for detection of infection in cats compared to gltA PCR. Furthermore, ELISA using rP26 as the substrate was more sensitive than ELISA using B. henselae type I outer membrane proteins.

Vaccine preparation by radiation processing.

A new radiation biotechnology for the acquirement of a commercial vaccine, designed for prophylaxis of ruminant infectious pododermatitis (IP), produced by gram negative bacteria Fusobacterium necrophorum (F.n.), is presented. Two different processes for preparing F.n. vaccine are used: a) the inactivation of F.n. bacteria exotoxins by microwave (MW) or/and electron beams (EB) irradiation; b) the isolation of exotoxins from F.n. cultures irradiated with MW or/and EB and the inactivation of isolated F.n. exotoxins with formalin. The EB irradiation of F.n. cultures produced simultaneously with the cells viability decrease an increasing of exotoxin quantity released in the culture supranatant as compared with classical methods. The MW irradiation is able to reduce the cells viability to zero but without an increase of exotoxin quantity in cultures supranatant. Instead of this MW irradiation, for certain conditions, is able to induce an important stimulation degree of the F.n. proliferation in cultures, from two to three log10. Two vaccine types were prepared: A1 vaccine that contains whole cell culture irradiated with MW/EB and A2 vaccine that contains cell-free culture supernatant of an MW/EB irradiated F.n. strain producing exotoxins. Also, other two vaccines are prepared: B1 and B2 that contain the same materials as A1 and A2 respectively, but without using MW/EB exposure. The vaccine efficiency is tested in ruminant farms in which IP evolves. It is expected that this new vaccine to offer a better protection, more than 60%, which is the best presently obtained result in ruminant farms.

The concept of "tailor-made", protein-based, outer membrane vesicle vaccines against meningococcal disease.

Protein-based, outer membrane vesicle (OMV) vaccines have previously proven to be efficacious against serogroup B meningococcal disease in Norway and Cuba. Currently, a public health intervention is going on in order to control a serogroup B epidemic in New Zealand. The scale-up and standardization of vaccine production required for controlling the New Zealand epidemic has allowed the establishment of large-scale GMP manufacturing for OMV vaccines. The outcome of this will be licensing of the vaccine in New Zealand and possibly other countries. The availability of licensed OMV vaccines raises the question of whether such vaccines may provide the opportunity to control other outbreaks and epidemics. For instance, such a vaccine could control a localised outbreak of group B meningococci in Normandy, France. "Tailor-made" vaccines, focusing on the sub-capsular antigens may also be considered for use in sub-Saharan Africa for the prevention of the recurrent outbreaks by serogroups A and W135 meningococci. This assumption is based on the epidemiological observation that meningococcal outbreaks in Africa are clonal and are strikingly stable regarding their phenotypic characteristics.

Sensitive and specific detection of proviral bovine leukemia virus by 5' Taq nuclease PCR using a 3' minor groove binder fluorogenic probe.

Sensitive assays are required to detect proviral bovine leukemia virus (BLV) in donor cattle used for the in vivo preparation of Australian tick fever vaccines. 5' Taq nuclease assays using 3' minor groove binder DNA probes (TaqManMGB) were developed and compared to conventional PCR assays for sensitive detection of Australian BLV. Seven beef and dairy herds were screened using DNA prepared by a variety of protocols to evaluate these tests. Comparative sensitivities of PCR tests were determined by testing log(10) dilutions of plasmids with inserted BLV sequences. Animals were also screened by the BLV standard agar-gel immunodiffusion test (AGID) and commercial enzyme linked immunosorbent assays (ELISA) for antibodies, and an ELISA for detecting viral antigens expressed (VAE) in lymphocyte cultures. The TaqMan MGB assay based on the pol region was the most sensitive and specific for the detection of BLV. This is the first report of a sensitive BLV 5' Taq nuclease assay.

Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination.

New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.