Expression and purification of TolC as a recombinant protein vaccine against Shigella flexneri and evaluation of immunogenic response in mice.

BACKGROUND: Shigella is one of the major causes of dysenteric diarrhea, which is known shigelosis. Shigelosis causes 160,000 deaths annually of diarrheal disease in the global scale especially children less than 5 years old. No licensed vaccine is available against shigelosis, therefore, efforts for develop an effective and safe vaccine against Shigella as before needed. The reverse vaccinology (RV) is a novel strategy that evaluate genome or proteome of the organism to find a new promising vaccine candidate. In this study, immunogenicity of a designed-recombinant antigen is evaluated through the in silico studies and animal experiments to predict a new immunogenic candidate against Shigella. METHODS: In the first step, proteome of Shigella flexneri was obtained from UniProtKB and then the outer membrane and extracellular proteins were predicted. In this study TolC as an outer membrane protein was selected and confirmed among candidates. In next steps, pre-selected protein was evaluated for transmembrane domains, homology, conservation, antigenicity, solubility, and B- and T-cell prediction by different online servers. RESULT: TolC as a conserved outer membrane protein, using different immune-informatics tools had acceptable scores and was selected as the immunogenic antigen for animal experiment studies. Recombinant TolC protein after expression and purification, was administered to BALB/c mice over three intraperitoneal routes. The sera of mice was used to evaluate the IgG1 production assay by indirect-ELISA. The immunized mice depicted effective protection against 2LD50 of Shigella. Flexneri ATCC12022 (challenge study). CONCLUSION: Therefore, the reverse vaccinology approach and experimental test results demonstrated that TolC as a novel effective and immunogenic antigen is capable for protection against shigellosis.

Facile synthesis of multi-faceted, biomimetic and cross-protective nanoparticle-based vaccines for drug-resistant Shigella: a flexible platform technology.

BACKGROUND: No commercial vaccines are available against drug-resistant Shigella due to serotype-specific/narrow-range of protection. Nanoparticle-based biomimetic vaccines involving stable, conserved, immunogenic proteins fabricated using facile chemistries can help formulate a translatable cross-protective Shigella vaccine. Such systems can also negate cold-chain transportation/storage thus overcoming challenges prevalent in various settings. METHODS: We explored facile development of biomimetic poly (lactide-co-glycolide)/PLGA 50:50 based nanovaccines (NVs), encapsulating conserved stabilized antigen(s)/immunostimulant of S. dysenteriae 1 origin surface-modified using simple chemistries. All encapsulants (IpaC/IpaB/LPS) and nanoparticles (NPs)-bare and modified (NV), were thoroughly characterized. Effect of IpaC on cellular uptake of NPs was assessed in-vitro. Immunogenicity of the NVs was assessed in-vivo in BALB/c mice by intranasal immunization. Cross-protective efficacy was assessed by intraperitoneally challenging the immunized groups with a high dose of heterologous S. flexneri 2a and observing for visible diarrhea, weight loss and survival. Passive-protective ability of the simplest NV was assessed in the 5-day old progeny of vaccinated mice. RESULTS: All the antigens and immunostimulant to be encapsulated were successfully purified and found to be stable both before and after encapsulation into NPs. The ~ 300 nm sized NPs with a zeta potential of ~ - 25 mV released ~ 60% antigen by 14th day suggesting an appropriate delivery kinetics. The NPs could be successfully surface-modified with IpaC and/or CpG DNA. In vitro experiments revealed that the presence of IpaC can significantly increase cellular uptake of NPs. All NVs were found to be cytocompatible and highly immunogenic. Antibodies in sera of NV-immunized mice could recognize heterologous Shigella. Immunized sera also showed high antibody and cytokine response. The immunized groups were protected from diarrhea and weight loss with ~ 70-80% survival upon heterologous Shigella challenge. The simplest NV showed ~ 88% survival in neonates. CONCLUSIONS: Facile formulation of biomimetic NVs can result in significant cross-protection. Further, passive protection in neonates suggest that parental immunization could protect infants, the most vulnerable group in context of Shigella infection. Non-invasive route of vaccination can also lead to greater patient compliance making it amenable for mass-immunization. Overall, our work contributes towards a yet to be reported platform technology for facile development of cross-protective Shigella vaccines.

Development of a Self-Adjuvanting, Cross-Protective, Stable Intranasal Recombinant Vaccine for Shigellosis.

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.

Rationalizing the design of a broad coverage Shigella vaccine based on evaluation of immunological cross-reactivity among S. flexneri serotypes.

No vaccine to protect against an estimated 238,000 shigellosis deaths per year is widely available. S. sonnei is the most prevalent Shigella, and multiple serotypes of S. flexneri, which change regionally and globally, also cause significant disease. The leading Shigella vaccine strategies are based on the delivery of serotype specific O-antigens. A strategy to minimize the complexity of a broadly-protective Shigella vaccine is to combine components from S. sonnei with S. flexneri serotypes that induce antibodies with maximum cross-reactivity between different serotypes. We used the GMMA-technology to immunize animal models and generate antisera against 14 S. flexneri subtypes from 8 different serotypes that were tested for binding to and bactericidal activity against a panel of 11 S. flexneri bacteria lines. Some immunogens induced broadly cross-reactive antibodies that interacted with most of the S. flexneri in the panel, while others induced antibodies with narrower specificity. Most cross-reactivity could not be assigned to modifications of the O-antigen, by glucose, acetate or phosphoethanolamine, common to several of the S. flexneri serotypes. This allowed us to revisit the current dogma of cross-reactivity among S. flexneri serotypes suggesting that a broadly protective vaccine is feasible with limited number of appropriately selected components. Thus, we rationally designed a 4-component vaccine selecting GMMA from S. sonnei and S. flexneri 1b, 2a and 3a. The resulting formulation was broadly cross-reactive in mice and rabbits, inducing antibodies that killed all S. flexneri serotypes tested. This study provides the framework for a broadly-protective Shigella vaccine which needs to be verified in human trials.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study.

BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.

Live attenuated Salmonella Typhimurium with monophosphoryl lipid A retains ability to induce T-cell and humoral immune responses against heterologous polysaccharide of Shigella flexneri 2a.

Shigella flexneri 2a (Sf2a) is one of the most frequently isolated Shigella strains that causes the endemic shigellosis in developing countries. In this study, we used recombinant attenuated Salmonella vaccine (RASV) strains to deliver Sf2a O-antigen and characterized the immune responses induced by the vectored O-antigen. First, we identified genes sufficient for biosynthesis of Sf2a O-antigen. A plasmid containing the identified genes was then introduced into the RASV strains, which were manipulated to produce only the heterologous O-antigen and modified lipid A. After oral immunization of mice, we demonstrated that RASV strains could induce potent humoral immune responses as well as robust CD4+ T-cell responses against Sf2a Lipopolysaccharide (LPS) and protect mice against virulent Sf2a challenge. The induced serum antibodies mediated high levels of Shigella-specific serum bactericidal activity and C3 deposition. Moreover, the IgG+ B220low/int BM cell and T follicular helper (Tfh) cell responses could also be triggered effectively. The live attenuated Salmonella with the modified lipid A delivering Sf2a O-antigen polysaccharide showed the same ability to induce immune responses against Sf2a LPS as the strain with the original lipid A. These findings underscore the potential of RASV delivered Sf2a O-antigen for induction of robust CD4+ T-cell and IgG responses and warrant further studies toward the development of Shigella vaccine candidates with RASV strains.

Functional Annotation and Curation of Hypothetical Proteins Present in A Newly Emerged Serotype 1c of Shigella flexneri: Emphasis on Selecting Targets for Virulence and Vaccine Design Studies.

Shigella flexneri is the principal cause of bacillary dysentery, contributing significantly to the global burden of diarrheal disease. The appearance and increase in the multi-drug resistance among Shigella strains, necessitates further genetic studies and development of improved/new drugs against the pathogen. The presence of an abundance of hypothetical proteins in the genome and how little is known about them, make them interesting genetic targets. The present study aims to carry out characterization of the hypothetical proteins present in the genome of a newly emerged serotype of S. flexneri (strain Y394), toward their novel regulatory functions using various bioinformatics databases/tools. Analysis of the genome sequence rendered 4170 proteins, out of which 721 proteins were annotated as hypothetical proteins (HPs) with no known function. The amino acid sequences of these HPs were evaluated using a combination of latest bioinformatics tools based on homology search against functionally identified proteins. Functional domains were considered as the basis to infer the biological functions of HPs in this case and the annotation helped in assigning various classes to the proteins such as signal transducers, lipoproteins, enzymes, membrane proteins, transporters, virulence, and binding proteins. This study contributes to a better understanding of growth, survival, and disease mechanism at molecular level and provides potential new targets for designing drugs against Shigella infection.

Towards a subunit vaccine from a Shigella flexneri ΔtolR mutant.

Disruption of one or more components of the Tol-Pal system, involved in maintaining the integrity of the outer membrane of Gram-negative bacteria, has been proposed as a method to increase the yield obtained from natural production of outer membrane vesicles (OMV). We present a new OMV-based product, obtained from genetically modified Shigella flexneri 2a with a non-polar deletion in tolR and heat-inactivated (HT-ΔtolR). The S. flexneri ΔtolR strain lead to a higher release of vesicles, more than 8-times when compared to the yield obtained from chemically inactivated wild type strain. S. flexneri mutant strain appeared to be more sensitive to different chemical compounds, including antibiotics, bile salts or human complement and it was also less virulent in both in vitro and in vivo assays. The mutation produced some changes in the LPS O-chain and protein expression. S. flexneri ΔtolR was enriched in long and very long LPS O-chain and expressed a different pattern of surface proteins or lipoproteins. In vitro toxicity and activation properties were determined in Raw 267.4 macrophage cell line. HT-ΔtolR antigenic complex was non-cytotoxic and activation markers, such as MHC-II or CD40, were highly expressed during incubation with this product. Finally, preliminary studies on the antibody response elicited by HT-ΔtolR demonstrated a robust and diverse response in mice. Considering these promising results, HT-ΔtolR antigenic extract appears as a new potential vaccine candidate to face shigellosis.

Cyclic OmpC peptidic epitope conjugated to tetanus toxoid as a potential vaccine candidate against shigellosis.

In earlier works we have described that mice immunized with outer membrane protein OmpC survive the challenge with live Shigella flexnerii 3a. We have also identified conformational epitope of this protein, that was recognized by mice antibodies. The aim of current work was to investigate whether synthetic OmpC epitope homologs can elicit immunological response sufficient in protecting mice against shigellosis. Several linear peptides containing RYDERY motif were synthesized and conjugated to poly-lysine. These conjugates appeared to be poor immunogens and to boost the immunological response an addition of the adjuvant (MPL) was required. Unfortunately, the MPL alone caused a very high immunological reaction that was masking response to peptidic epitope. Under those circumstances we used tetanus toxoid (TT) as the carrier protein for the peptides and the agent stimulating immunological response. Series of cyclic peptides, homologs of the OmpC main epitope were synthesized and conjugated to TT. The loop size in cyclic peptides varied by number of glycine residues, i.e., 1-3 residues added to the GLNRYDERYIGK motif. The linear GLNRYDERYIGC-TT was also prepared as the control. The latter conjugate gave the highest immunological response, followed by the cyclic-GGLNRYDERYIGC-TT and cyclic-GLNRYDERYIGC-TT. The third peptide, cyclic-GGGLNRYDERYIGC-TT, gave a very low response, although it was the most resistant to proteolysis. However, antibodies obtained against cyclic-GGLNRYDERYIGC-TT were more potent to recognize both OmpC and Shigella flexnerii 3a cells than the antibodies against linear GLNRYDERYIGC-TT. Furthermore, the monoclonal antibodies raised against linear GLNRYDERYIGC-TT showed 20-fold lower dissociation constant (KD) than the naturally occurring polyclonal antibodies from umbilical cord sera. Monoclonal antibodies also gave a weaker signal in electron microscope than mice and human polyclonal antibodies. In overall, our results point to cyclic peptides as better candidates for a vaccine development, since they are eliciting production of the higher affinity antibodies against Shigella cells and OmpC.

In silico design of a novel chimeric shigella IpaB fused to C terminal of clostridium perfringens enterotoxin as a vaccine candidate.

This study aimed to design a novel chimeric protein in silico to serve as a serotype-independent vaccine candidate against Shigella. The chimera contains amino acid residues 240-460 of Shigella invasion plasmid antigen B (IpaB) and the C-terminus of Clostridium perfringens enterotoxin (C-CPE). Amino acid sequences of 537 peptide linkers were obtained from two protein linker databases. 3D structures of IpaB-CPE290-319, IpaB-CPE184-319, IpaB-CPE194-319 and 537 newly designed IpaB-linker-CPE290-319 constructs with varying linker regions were predicted. These predicted 3D structures were merged with the 3D structures of native IpaB240-460, CPE194-319, CPE184-319 and CPE290-319 to select the structure most similar to native IpaB and C-CPE. Several in silico tools were used to determine the suitability of the selected IpaB-C-CPE structure as a vaccine candidate. None of the 537 linkers was capable of preserving the native structure of CPE290-319 within the IpaB-linker-CPE290-319 structure. In silico analysis determined that the IpaB-CPE194-319 3D structure was the most similar to the 3D structure of the respective native CPE domain and that it was a stable chimeric protein exposing multiple B-cell epitopes. IpaB-CPE194-319 was designed for its capability to bind to human intestinal epithelial and M cells and to accumulate on these cells. The predicted B-cell epitopes are likely to be capable of inducing a mucosal antibody response in the human intestine against Shigella IpaB. This study also showed that the higher binding affinities of CPE184-319 and CPE194-319 to claudin molecules than those of CPE290-319 is the result of preserving the 3D structures of CPE184-319 and CPE194-319 when they are linked to the C-termini of other proteins.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a.

We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

Development of a Live Attenuated Bivalent Oral Vaccine Against Shigella sonnei Shigellosis and Typhoid Fever.

Shigella sonnei and Salmonella Typhi cause significant morbidity and mortality. We exploited the safety record of the oral, attenuated S. Typhi vaccine (Ty21a) by using it as a vector to develop a bivalent oral vaccine to protect against S. sonnei shigellosis and typhoid fever. We recombineered the S. sonnei form I O-antigen gene cluster into the Ty21a chromosome to create Ty21a-Ss, which stably expresses S. sonnei form I O antigen. To enhance survivability in the acid environment of the stomach, we created an acid-resistant strain, Ty21a-AR-Ss, by inserting Shigella glutaminase-glutamate decarboxylase systems coexpressed with S. sonnei form I O-antigen gene. Mice immunized intranasally with Ty21a-AR-Ss produced antibodies against S. sonnei and S. Typhi, and survived lethal intranasal S. sonnei challenge. This paves the way for proposed good manufacturing practices manufacture and clinical trials intended to test the clinical effectiveness of Ty21a-AR-Ss in protecting against S. sonnei shigellosis and typhoid fever, as compared with the current Ty21a vaccine.

Development of recombinant vaccine candidate molecule against Shigella infection.

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella's IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90-95% of mice whereas 80-85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60-70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe, efficacious and cost-effective recombinant vaccine against Shigella serotypes.

An Approach to Identify and Characterize a Subunit Candidate Shigella Vaccine Antigen.

Shigellosis remains a serious issue throughout the developing countries, particularly in children under the age of 5. Numerous strategies have been tested to develop vaccines targeting shigellosis; unfortunately despite several years of extensive research, no safe, effective, and inexpensive vaccine against shigellosis is available so far. Here, we illustrate in detail an approach to identify and establish immunogenic outer membrane proteins from Shigella flexneri 2a as subunit vaccine candidates.

In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation.

BACKGROUND: Glycoconjugated vaccines composed of polysaccharide antigens covalently linked to immunogenic carrier proteins have proved to belong to the most effective and safest vaccines for combating bacterial pathogens. The functional transfer of the N-glycosylation machinery from Campylobacter jejuni to the standard prokaryotic host Escherichia coli established a novel bioconjugation methodology termed bacterial glycoengineering. RESULTS: In this study, we report on the production of a new recombinant glycoconjugate vaccine against Shigella flexneri 2a representing the major serotype for global outbreaks of shigellosis. We demonstrate that S. flexneri 2a O-polysaccharides can be transferred to a detoxified variant of Pseudomonas aeruginosa carrier protein exotoxin A (EPA) by the C. jejuni oligosaccharyltransferase PglB, resulting in glycosylated EPA-2a. Moreover, we optimized the in vivo production of this novel vaccine by identification and quantitative analysis of critical process parameters for glycoprotein synthesis. It was found that sequential induction of oligosaccharyltransferase PglB and carrier protein EPA increased the specific productivity of EPA-2a by a factor of 1.6. Furthermore, by the addition of 10 g/L of the monosaccharide N-acetylglucosamine during induction, glycoconjugate vaccine yield was boosted up to 3.1-fold. The optimum concentration of Mg2+ ions for N-glycan transfer was determined to be 10 mM. Finally, optimized parameters were transferred to high cell density cultures with a 46-fold increase of overall yield of glycoconjugate compared to the one in initial shake flask production. CONCLUSION: The present study is the first attempt to identify stimulating parameters for improved productivity of S. flexneri 2a bioconjugates. Optimization of glycosylation efficiency will ultimately foster the transfer of lab-scale expression to a cost-effective in vivo production process for a glycoconjugate vaccine against S. flexneri 2a in E. coli. This study is an important step towards this goal and provides a starting point for further optimization studies.

Fusion of HPV L1 into Shigella surface IcsA: a new approach in developing live attenuated Shigella-HPV vaccine.

Despite the success of L1 virus-like particles (VLPs) vaccines in prevention of high-risk human papillomavirus (HPV) infection and cervical cancer, extraordinary high cost for the complete vaccination has impeded widespread use of the vaccine in resource-poor countries, where cervical cancers impose greater challenge. Presentation of HPV L1 protein by attenuated pathogenic bacteria through natural infection provides a promising low-cost and convenient alternative. Here, we describe the construction and characterization of attenuated L1-expressing Shigella vaccine candidate, by fusion of L1 into the autotransporter of Shigella sonnei, IcsA, an essential virulence factor responsible for actin-based motility. The functional α domain of IcsA was replaced by codon-optimized L1 gene with independent open reading frames (ORFs) facilitated by suicide vector pJCB12. The L1 gene was stabilized in the genome of recombinant S. sonnei with protein expression and assembly of VLPs in the bacterial cytoplasm. Through conjunctival route vaccination in guinea pigs, L1-containing S. sonnei was able to elicit specific immune response to HPV16 L1 VLP as well as bacterial antigens. The results demonstrated the feasibility of the novel stratagem to develop prophylactic Shigella-HPV vaccines.

Immunogenicity of a new recombinant IpaC from Shigella dysenteriae type I in guinea pig as a vaccine candidate.

BACKGROUND: Recombinant vaccine technology is one of the most developed means in controlling infectious diseases. However, an effective vaccine against Shigella is still missing. OBJECTIVE: To evaluate recombinant IpaC protein of Shigella as a vaccine candidate. METHODS: In this study we cloned IpaC gene into an expression vector in prokaryotic system. The protein expression was evaluated by SDS-PAGE and Western-Blotting analysis. The recombinant protein was purified using Ni-NTA affinity chromatography. Guinea pigs were immunized with the recombinant protein and the level of immunogenicity was examined by ELISA and Western blotting of IpaC. Challenge test was done through the intraoculary injection of Shigella dysenteriae (6×108 CFU/eye) and after 48 hours was scored for keratoconjunctivitis. RESULTS: The results showed a remarkable level of immunogenicity in terms of antibody response and protection against keratoconjunctivitis in tested animals. The recombinant IpaC protein provided a protective system against Shigella dysenteriae type I during the challenge test. CONCLUSION: The results showed the potential of using recombinant IpaC in preparation of vaccine in perspective studies.

Cross-protection provided by live Shigella mutants lacking major antigens.

The immune response elicited by Shigella infections is dominated by serotype-specific antibodies recognizing the LPS O-antigens. Although a marked antibody response to invasion plasmid antigens (Ipa-s) shared by all virulent strains is also induced, the varying level of immunity elicited by natural infections is serotype-restricted. Previous vaccines have tried to mimic and achieve this serotype-specific, infection-induced immunity. As, however, the four Shigella species can express 50 different types of O-antigens, current approaches with the aim to induce a broad coverage use a mixture of the most common O-antigens combined in single vaccines. In the current study we present data on an alternative approach to generate immunity protective against multiple serotypes. Mutants lacking both major immune-determinant structures (i.e. the Ipa and O-antigens) were not only highly attenuated, but, unlike their avirulent counterparts still expressing these antigens, elicited a protective immune response to heterologous serotypes in a murine model. Evidence is provided that protection was mediated by the enhanced immunogenic potential of minor conserved antigens. Furthermore, the rough, non-invasive double mutants triggered an immune response different from that induced by the smooth, invasive strains regarding the isotype of antibodies generated. These non-invasive, rough mutants may represent promising candidates for further development into live vaccines for the prophylaxis of bacillary dysentery in areas with multiple endemic serotypes.

Two live attenuated Shigella flexneri 2a strains WRSf2G12 and WRSf2G15: a new combination of gene deletions for 2nd generation live attenuated vaccine candidates.

Shigella infections are a major cause of inflammatory diarrhea and dysentery worldwide. First-generation virG-based live attenuated Shigella strains have been successfully tested in phase I and II clinical trials and are a leading approach for Shigella vaccine development. Additional gene deletions in senA, senB and msbB2 have been engineered into second-generation virG-based Shigella flexneri 2a strains producing WRSf2G12 and WRSf2G15. Both strains harbor a unique combination of gene deletions designed to increase the safety of live Shigella vaccines. WRSf2G12 and WRSf2G15 are genetically stable and highly attenuated in both cell culture and animal models of infection. Ocular immunization of guinea pigs with either strain induces robust systemic and mucosal immune responses that protect against homologous challenge with wild-type Shigella. The data support further evaluation of the second-generation strains in a phase I clinical trial.