Exploring shark VNAR antibody against infectious diseases using phage display technology.

Antibody with high affinity and specificity to antigen has widely used as a tool to combat various diseases. The variable domain of immunoglobulin new antigen receptor (VNAR) naturally found in shark contains autonomous function as single-domain antibody. Due to its excellent characteristics, the small, non-complex, and highly stable have made shark VNAR can acquires the antigen-binding capability that might not be reached by conventional antibody. Phage display technology enables shark VNAR to be presented on the surface of phage, allowing the exploration of shark VNAR as an alternative antibody format to target antigens from various infectious diseases. The application of phage-displayed shark VNAR in antibody library and biopanning eventually leads to the discovery and isolation of antigen-specific VNARs with diagnostic and therapeutic potential towards infectious diseases. This review provides an overview of the shark VNAR antibody, the types of phage display technology with comparison to the other types of display system, as well as the application and case studies of phage-displayed shark VNAR antibodies against infectious diseases.

Generation of peptides using phage display technology for cancer diagnosis and molecular imaging.

Cancer is one of the leading causes of mortality worldwide; nearly 10 million people died from it in 2020. The high mortality rate results from the lack of effective screening approaches where early detection cannot be achieved, reducing the chance of early intervention to prevent cancer development. Non-invasive and deep-tissue imaging is useful in cancer diagnosis, contributing to a visual presentation of anatomy and physiology in a rapid and safe manner. Its sensitivity and specificity can be enhanced with the application of targeting ligands with the conjugation of imaging probes. Phage display is a powerful technology to identify antibody- or peptide-based ligands with effective binding specificity against their target receptor. Tumour-targeting peptides exhibit promising results in molecular imaging, but the application is limited to animals only. Modern nanotechnology facilitates the combination of peptides with various nanoparticles due to their superior characteristics, rendering novel strategies in designing more potent imaging probes for cancer diagnosis and targeting therapy. In the end, a myriad of peptide candidates that aimed for different cancers diagnosis and imaging in various forms of research were reviewed.

Bioactivities and Mode of Actions of Dibutyl Phthalates and Nocardamine from Streptomyces sp. H11809.

Dibutyl phthalate (DBP) produced by Streptomyces sp. H11809 exerted inhibitory activity against human GSK-3ß (Hs GSK-3ß) and Plasmodiumfalciparum 3D7 (Pf 3D7) malaria parasites. The current study aimed to determine DBP's plausible mode of action against Hs GSK-3ß and Pf 3D7. Molecular docking analysis indicated that DBP has a higher binding affinity to the substrate-binding site (pocket 2; -6.9 kcal/mol) than the ATP-binding site (pocket 1; -6.1 kcal/mol) of Hs GSK-3ß. It was suggested that the esters of DBP play a pivotal role in the inhibition of Hs GSK-3ß through the formation of hydrogen bonds with Arg96/Glu97 amino acid residues in pocket 2. Subsequently, an in vitro Hs GSK-3ß enzymatic assay revealed that DBP inhibits the activity of Hs GSK-3ß via mixed inhibition inhibitory mechanisms, with a moderate IC50 of 2.0 µM. Furthermore, the decrease in Km value with an increasing DBP concentration suggested that DBP favors binding on free Hs GSK-3ß over its substrate-bound state. However, the antimalarial mode of action of DBP remains unknown since the generation of a Pf 3D7 DBP-resistant clone was not successful. Thus, the molecular target of DBP might be indispensable for Pf survival. We also identified nocardamine as another active compound from Streptomyces sp. H11809 chloroform extract. It showed potent antimalarial activity with an IC50 of 1.5 µM, which is ~10-fold more potent than DBP, but with no effect on Hs GSK-3ß. The addition of ≥12.5 µM ferric ions into the Pf culture reduced nocardamine antimalarial activity by 90% under in vitro settings. Hence, the iron-chelating ability of nocardamine was shown to starve the parasites from their iron source, eventually inhibiting their growth.

Immunogenicity, antibody responses and vaccine efficacy of recombinant annexin B30 against Schistosoma mansoni.

AIMS: Schistosomes infect approximately 250 million people worldwide. To date, there is no effective vaccine available for the prevention of schistosome infection in endemic regions. There remains a need to develop means to confer long-term protection of individuals against reinfection. In this study, an annexin, namely annexin B30, which is highly expressed in the tegument of Schistosoma mansoni was selected to evaluate its immunogenicity and protective efficacy in a mouse model. METHODS AND RESULTS: Bioinformatics analysis showed that there were three potential linear B-cell epitopes and four conformational B-cell epitopes predicted from annexin B30, respectively. Full-length annexin B30 was cloned and expressed in Escherichia coli BL21(DE3). In the presence of adjuvants, the soluble recombinant protein was evaluated for its protective efficacy in two independent vaccine trials. Immunization of CBA mice with recombinant annexin B30 formulated either in alum only or alum/CpG induced a mixed Th1/Th2 cytokine profile but no significant protection against schistosome infection was detected. CONCLUSION: Recombinant annexin B30 did not confer significant protection against the parasite. The molecule may not be suitable for vaccine development. However, it could be an ideal biomarker recommended for immunodiagnostics development.

Isolation and characterization of a novel anti-salbutamol chicken scFv for human doping urinalysis.

Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant ß2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two ß2-agonists were; IC50 salbutamol = ∼0.310 µg/ml, IC50 clenbuterol = ∼0.076 µg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 µg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 µg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.

Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library.

BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability. METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken. RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13. CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.

BACKGROUND: Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs. METHODS: In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized. RESULTS: Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 µg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10(-11) M, followed by control mAb C1-13 at 1.03 × 10(-10) M and mAb D2 at 3.05 × 10(-10) M. CONCLUSIONS: Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.

Assessing the potential of NS2B/NS3 protease inhibitors biomarker in curbing dengue virus infections: In silico vs. In vitro approach.

An increase in the occurrence of viral infectious diseases is a global concern for human health. According to a WHO report, dengue virus (DENV) is one of the most common viral diseases affecting approximately 400 million people annually, with worsening symptoms in nearly 1% of cases. Both academic and industrial researchers have conducted numerous studies on viral epidemiology, virus structure and function, source and route of infection, treatment targets, vaccines, and drugs. The development of CYD-TDV or Dengvaxia® vaccine has been a major milestone in dengue treatment. However, evidence has shown that vaccines have some drawbacks and limitations. Therefore, researchers are developing dengue antivirals to curb infections. DENV NS2B/NS3 protease is a DENV enzyme essential for replication and virus assembly, making it an interesting antiviral target. For faster hit and lead recognition of DENV targets, methods to screen large number of molecules at lower costs are essential. Similarly, an integrated and multidisciplinary approach involving in silico screening and confirmation of biological activity is required. In this review, we discuss recent strategies for searching for novel DENV NS2B/NS3 protease inhibitors from the in silico and in vitro perspectives, either by applying one of the approaches or by integrating both. Therefore, we hope that our review will encourage researchers to integrate the best strategies and encourage further developments in this area.

Dengue virus infection - a review of pathogenesis, vaccines, diagnosis and therapy.

The transmission of dengue virus (DENV) from an infected Aedes mosquito to a human, causes illness ranging from mild dengue fever to fatal dengue shock syndrome. The similar conserved structure and sequence among distinct DENV serotypes or different flaviviruses has resulted in the occurrence of cross reaction followed by antibody-dependent enhancement (ADE). Thus far, the vaccine which can provide effective protection against infection by different DENV serotypes remains the biggest hurdle to overcome. Therefore, deep investigation is crucial for the potent and effective therapeutic drugs development. In addition, the cross-reactivity of flaviviruses that leads to false diagnosis in clinical settings could result to delay proper intervention management. Thus, the accurate diagnostic with high specificity and sensitivity is highly required to provide prompt diagnosis in respect to render early treatment for DENV infected individuals. In this review, the recent development of neutralizing antibodies, antiviral agents, and vaccine candidates in therapeutic platform for DENV infection will be discussed. Moreover, the discovery of antigenic cryptic epitopes, principle of molecular mimicry, and application of single-chain or single-domain antibodies towards DENV will also be presented.

Recent trends in next generation immunoinformatics harnessed for universal coronavirus vaccine design.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally devastated public health, the economies of many countries and quality of life universally. The recent emergence of immune-escaped variants and scenario of vaccinated individuals being infected has raised the global concerns about the effectiveness of the current available vaccines in transmission control and disease prevention. Given the high rate mutation of SARS-CoV-2, an efficacious vaccine targeting against multiple variants that contains virus-specific epitopes is desperately needed. An immunoinformatics approach is gaining traction in vaccine design and development due to the significant reduction in time and cost of immunogenicity studies and increasing reliability of the generated results. It can underpin the development of novel therapeutic methods and accelerate the design and production of peptide vaccines for infectious diseases. Structural proteins, particularly spike protein (S), along with other proteins have been studied intensively as promising coronavirus vaccine targets. Numbers of promising online immunological databases, tools and web servers have widely been employed for the design and development of next generation COVID-19 vaccines. This review highlights the role of immunoinformatics in identifying immunogenic peptides as potential vaccine targets, involving databases, and prediction and characterization of epitopes which can be harnessed for designing future coronavirus vaccines.

Innate and Adaptive Immunity during SARS-CoV-2 Infection: Biomolecular Cellular Markers and Mechanisms.

The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist. Historical pandemics include smallpox and influenza, with efficacious therapeutics utilized to reduce overall disease burden through effectively targeting a competent host immune system response. The immune system is composed of primary/secondary lymphoid structures with initially eight types of immune cell types, and many other subtypes, traversing cell membranes utilizing cell signaling cascades that contribute towards clearance of pathogenic proteins. Other proteins discussed include cluster of differentiation (CD) markers, major histocompatibility complexes (MHC), pleiotropic interleukins (IL), and chemokines (CXC). The historical concepts of host immunity are the innate and adaptive immune systems. The adaptive immune system is represented by T cells, B cells, and antibodies. The innate immune system is represented by macrophages, neutrophils, dendritic cells, and the complement system. Other viruses can affect and regulate cell cycle progression for example, in cancers that include human papillomavirus (HPV: cervical carcinoma), Epstein-Barr virus (EBV: lymphoma), Hepatitis B and C (HB/HC: hepatocellular carcinoma) and human T cell Leukemia Virus-1 (T cell leukemia). Bacterial infections also increase the risk of developing cancer (e.g., Helicobacter pylori). Viral and bacterial factors can cause both morbidity and mortality alongside being transmitted within clinical and community settings through affecting a host immune response. Therefore, it is appropriate to contextualize advances in single cell sequencing in conjunction with other laboratory techniques allowing insights into immune cell characterization. These developments offer improved clarity and understanding that overlap with autoimmune conditions that could be affected by innate B cells (B1+ or marginal zone cells) or adaptive T cell responses to SARS-CoV-2 infection and other pathologies. Thus, this review starts with an introduction into host respiratory infection before examining invaluable cellular messenger proteins and then individual immune cell markers.

Application of Reverse Vaccinology and Immunoinformatic Strategies for the Identification of Vaccine Candidates Against Shigella flexneri.

Reverse vaccinology (RV) was first introduced by Rappuoli for the development of an effective vaccine against serogroup B Neisseria meningitidis (MenB). With the advances in next generation sequencing technologies, the amount of genomic data has risen exponentially. Since then, the RV approach has widely been used to discover potential vaccine protein targets by screening whole genome sequences of pathogens using a combination of sophisticated computational algorithms and bioinformatic tools. In contrast to conventional vaccine development strategies, RV offers a novel method to facilitate rapid vaccine design and reduces reliance on the traditional, relatively tedious, and labor-intensive approach based on Pasteur"s principles of isolating, inactivating, and injecting the causative agent of an infectious disease. Advances in biocomputational techniques have remarkably increased the significance for the rapid identification of the proteins that are secreted or expressed on the surface of pathogens. Immunogenic proteins which are able to induce the immune response in the hosts can be predicted based on the immune epitopes present within the protein sequence. To date, RV has successfully been applied to develop vaccines against a variety of infectious pathogens. In this chapter, we apply a pipeline of bioinformatic programs for identification of Shigella flexneri potential vaccine candidates as an illustration immunoinformatic tools available for RV.

Mass spectrometry-based immunopeptidomics and computational vaccinology strategies for the identification of universal Shigella immunogenic candidates.

Shigella is a Gram-negative bacteria that cause shigellosis. Treatment with antibiotics cannot be sustained to control the bacterial infection due to the risk of antibiotic resistance. Vaccine development against the highly prevalent Shigella serotypes could provide a generous benefit in reducing the occurrence of shigellosis. The present study is aimed to identify the peptides that could be the ideal candidates for the Shigella vaccine development. THP-1 human macrophage cell lines were infected with clinical strains of Shigella flexneri 2a. The bacterial peptides bound on HLA class II molecules of infected THP-1 were analyzed and identified using the immunopeptidomics approach. Following mass spectrometry identification, a total of 14 proteins were predicted by PSORTb, CELLO, and Gneg-mPLoc as outer membrane proteins (OMPs) of Shigella. Of which, 12 OMPs were found to be conserved among Shigella species and had no significance with human proteomes. Outer membrane receptor FepA and TonB-dependent receptor were among the OMPs predicted to possess the high number of immunogenic B- and T-cell epitopes. The epitopes with high antigenicity from FepA and TonB were identified as potential peptide candidates for Shigella vaccine development. The immunoreactivity of the constructed recombinant proteins were determined using the Shigella-infected human and rabbit sera, respectively. Their protective efficacy and immune responses in controlling the Shigella infection will further be investigated in experimental animal models.

Advancements in the preparation and application of monolithic silica columns for efficient separation in liquid chromatography.

Fast and efficient separation remains a big challenge in high performance liquid chromatography (HPLC). The need for higher efficiency and resolution in separation is constantly in demand. To achieve that, columns developed are rapidly moving towards having smaller particle sizes and internal diameters (i.d.). However, these parameters will lead to high back-pressure in the system and will burden the pumps of the HPLC instrument. To address this limitation, monolithic columns, especially silica-based monolithic columns have been introduced. These columns are being widely investigated for fast and efficient separation of a wide range of molecules. The present article describes the current methods developed to enhance the column efficiency of particle packed columns and how silica monolithic columns can act as an alternative in overcoming the low permeability of particle packed columns. The fundamental processes behind the fabrication of the monolith including the starting materials and the silica sol-gel process will be discussed. Different monolith derivatization and end-capping processes will be further elaborated and followed by highlights of the performance such monolithic columns in key applications in different fields with various types of matrices.

Generation of soluble, disulfide-rich JEV NS1 protein recognizable by anti-NS1 antibodies through a simplified, in vitro refolding approach.

Co-existence of Japanese Encephalitis virus (JEV) with highly homologous antigenic epitopes results in antibody-based serodiagnosis being inaccurate at detecting and distinguishing JEV from other flaviviruses. This often causes misdiagnosis and inefficient treatments of flavivirus infection. Generation of JEV NS1 protein remains a challenge as it is notably expressed in the form of inactive aggregates known as inclusion bodies using bacterial expression systems. This study evaluated two trxB and gor E. coli strains in producing soluble JEV NS1 via a cold-shock expression system. High yield of JEV NS1 inclusion bodies was produced using cold-shocked expression system. Subsequently, a simplified yet successful approach in generating soluble, active JEV NS1 protein through solubilization, purification and in vitro refolding of JEV NS1 protein from inclusion bodies was developed. A step-wise dialysis refolding approach was used to facilitate JEV NS1 refolding. The authenticity of the refolded JEV NS1 was confirmed by specific antibody binding on indirect ELISA commercial anti-NS1 antibodies which showed that the refolded JEV NS1 was highly immunoreactive. This presented approach is cost-effective, and negates the need for mammalian or insect cell expression systems in order to synthesize this JEV NS1 protein of important diagnostic and therapeutic relevance in Japanese Encephalitis disease.

Diagnostic and therapeutic potential of shark variable new antigen receptor (VNAR) single domain antibody.

Conventional monoclonal antibodies (mAbs) have been widely used in research and diagnostic applications due to their high affinity and specificity. However, multiple limitations, such as large size, complex structure and sensitivity to extreme ambient temperature potentially weaken the performance of mAbs in certain applications. To address this problem, the exploration of new antigen binders is extensively required in relation to improve the quality of current diagnostic platforms. In recent years, a new immunoglobulin-based protein, namely variable domain of new antigen receptor (VNAR) was discovered in sharks. Unlike conventional mAbs, several advantages of VNARs, include small size, better thermostability and peculiar paratope structure have attracted interest of researchers to further explore on it. This article aims to first present an overview of the shark VNARs and outline the characteristics as an outstanding new reagent for diagnostic and therapeutic applications.

Differential expression of cytokine genes in THP-1-derived macrophages infected with mild and virulence strains of Shigella flexneri 2a.

Shigella is an intracellular bacterial pathogen that causes bacterial dysentery called shigellosis. The assessment of pro- and anti-inflammatory mediators produced by immune cells against this bacteria are vital in identifying the effectiveness of the immune reaction in protecting the host. In Malaysia, Shigella is ranked as the third most common bacteria causing diarrheal disease among children below 5 years old. In the present study, we aim to examine the differential cytokine gene expressions of macrophages in response to two types of clinical strains of Shigella flexneri 2a (S. flexneri 2a) isolated from patients admitted in Hospital Universiti Sains Malaysia, Kelantan, Malaysia. THP-1-derived macrophages, as the model of human macrophages, were infected separately with S. flexneri 2a mild (SH062) and virulence (SH057) strains for 6, 12, and 24 h, respectively. The gene expression level of inflammatory mediators was identified using real-time quantitative polymerase chain reaction (RT-qPCR). The production of nitric oxide (NO) by the macrophages was measured by using a commercialized NO assay kit. The ability of macrophages to kill the intracellular bacteria was assessed by intracellular killing assay. Induction of tumor necrosis factor-alpha (TNFα), interleukin (IL)-1ß, IL-6, IL-12, inducible NO synthase (iNOS), and NO, confirmed the pro-inflammatory reaction of the THP-1-derived macrophages in response to S. flexneri 2a, especially against the SH507 strain. The SH057 also induced a marked increase in the expression levels of the anti-inflammatory cytokine mRNAs at 12 h and 24 h post-infection. In the intracellular killing assay, both strains showed less viable, indicating the generation of pro-inflammatory cytokines in the presence of iNOS and NO was crucial in the stimulation of macrophages for the host defense against shigellosis. Transcription analysis of THP-1-derived macrophages in this study identifies differentially expressed cytokine genes that correlated with the virulence factor of S. flexneri 2a.

Designing and evaluation of an antibody-targeted chimeric recombinant vaccine encoding Shigella flexneri outer membrane antigens.

Shigellosis is one of the most common diseases found in the developing countries, especially those countries that are prone flood. The causative agent for this disease is the Shigella species. This organism is one of the third most common enteropathogens responsible for childhood diarrhea. Since Shigella can survive gastric acidity and is an intracellular pathogen, it becomes difficult to treat. Also, uncontrolled use of antibiotics has led to development of resistant strains which poses a threat to public health. Therefore, there is a need for long term control of Shigella infection which can be achieved by designing a proper and effective vaccine. In this study, emphasis was made on designing a candidate that could elicit both B-cell and T-cell immune response. Hence B- and T-cell epitopes of outer membrane channel protein (OM) and putative lipoprotein (PL) from S. flexneri 2a were computationally predicted using immunoinformatics approach and a chimeric construct (chimeric-OP) containing the immunogenic epitopes selected from OM and PL was designed, cloned and expressed in E. coli system. The immunogenicity of the recombinant chimeric-OP was assessed using Shigella antigen infected rabbit antibody. The result showed that the chimeric-OP was a synthetic peptide candidate suitable for the development of vaccine and immunodiagnostics against Shigella infection.

Reverse vaccinology approach for the identification and characterization of outer membrane proteins of Shigella flexneri as potential cellular- and antibody-dependent vaccine candidates.

PURPOSE: In the developing world, bacillary dysentery is one of the most common communicable diarrheal infections. There are approximately 169 million cases of shigellosis reported worldwide. The disease is transmitted by a group of Gram-negative intracellular enterobacteria known as Shigella flexneri, S. sonnei, S. dysenteriae, and S. boydii. Conventional treatment regimens for Shigella have been less effective due to the development of resistant strains against antibiotics. Therefore, an effective vaccine for the long term control of Shigella transmission is urgently needed. MATERIALS AND METHODS: In this study, a reverse vaccinology approach was employed to identify most conserved and immunogenic outer membrane proteins (OMPs) of S. flexneri 2a. RESULTS: Five OMPs including fepA, ompC, nlpD_1, tolC, and nlpD_2 were identified as potential vaccine candidates. Protein-protein interactions analysis using STRING software (https://string-db.org/) revealed that five of these OMPs may potentially interact with other intracellular proteins which are involved in beta-lactam resistance pathway. B- and T-cell epitopes of the selected OMPs were predicted using BCPred as well as Propred I and Propred (http://crdd.osdd.net/raghava/propred/), respectively. Each of these OMPs contains regions which are capable to induce B- and T-cell immune responses. CONCLUSION: Analysis acquired from this study showed that five selected OMPs have great potential for vaccine development against S. flexneri infection. The predicted immunogenic epitopes can also be used for development of peptide vaccines or multi-epitope vaccines against human shigellosis. Reverse vaccinology is a promising strategy for the discovery of potential vaccine candidates which can be used for future vaccine development against global persistent infections.

Molecular characterization of Schistosoma mansoni tegument annexins and comparative analysis of antibody responses following parasite infection.

Schistosomes are parasitic blood flukes that infect approximately 250 million people worldwide. The disease known as schistosomiasis, is the second most significant tropical parasitic disease after malaria. Praziquantel is the only effective drug currently licensed for schistosomiasis and there are concerns about resistance to the drug. There has been much effort to develop vaccines against schistosomiasis to produce long-term protection in endemic regions. Surface-associated proteins, and in particular, those expressed in the body wall, or tegument, have been proposed as potential vaccine targets. Of these, annexins are thought to be of integral importance for the stability of this apical membrane system. Here, we present the structural and immunobiochemical characterization of four homologous annexins namely annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni. Bioinformatics analysis showed that there was no signal peptide predicted for any annexin in this study. Further analysis showed that each of all four annexin protein possesses a primary structure consisting of a short but variable N-terminal region and a long C-terminal core containing four homologous annexin repeats (I-IV), which contain five alpha-helices. The life cycle expression profile of each annexin was assessed using quantitative PCR. The results showed that the overall transcript levels of the each of four homologous annexins were relatively low in the egg stage, but increased gradually after the transition of cercariae (the invasive schistosome larvae) to schistosomula (the post-invasive larvae). Circular dichroism (CD) demonstrated that rAnnexin B30, rAnnexin B5a and rAnnexin 7a were folded, showing a secondary structure content rich in alpha-helices. The membrane binding affinity was enhanced when rAnnexin B30, rAnnexin B5a and rAnnexin 7a was incubated in the presence of Ca2+. All annexin members evaluated in this study were immunolocalized to the tegument, with immunoreactivity also occurring in cells and in muscle of adult parasites. All four recombinant annexins were immunoreactive and they were recognized by the sera of mice infected with S. mansoni. In conclusion, the overall results present the molecular characterization of annexin B30, annexin B5a, annexin B7a and annexin B5b from S. mansoni in host-parasite interactions and strongly suggest that the molecules could be useful candidates for vaccine or diagnostic development.