Identification and analysis of immunoreactive proteins of Shigella flexneri in human sera and stool specimens.

Background: The method currently available to diagnose shigellosis is insensitive and has many limitations. Thus, this study was designed to identify specific antigenic protein(s) among the cell surface associated proteins (SAPs) of Shigella that would be valuable in the development of an alternative diagnostic assay for shigellosis, particularly one that could be run using a stool sample rather than serum. Methods: The SAPs of clinical isolates of S. dysenteriae, S. boydii, Shigella flexneri, and S. sonnei were extracted from an overnight culture grown at 37 °C using acidified-glycine extraction methods. Protein profiles were observed by SDS-PAGE. To determine if antibodies specific to certain Shigella SAPs were present in both sera and stool suspensions, Western blot analysis was used to detect the presence of IgA, IgG, and IgM. Results: Immunoblot analysis revealed that sera from patients infected with S. flexneri recognized 31 proteins. These SAP antigens are recognized by the host humoral response during Shigella infection. Specific antibodies against these antigens were also observed in intestinal secretions of shigellosis patients. Of these 31 S. flexneri proteins, the 35 kDa protein specifically reacted against IgA present in patients' stool suspensions. Further study illustrated the immunoreactivity of this protein in S. dysenteriae, S. boydii, and S. sonnei. This is the first report that demonstrates the presence of immunoreactive Shigella SAPs in stool suspensions. The SAPSs could be very useful in developing a simple and rapid serodiagnostic assay for shigellosis directly from stool specimens.

Deletion of Salmonella enterica serovar Typhi tolC reduces bacterial adhesion and invasion toward host cells.

Background: S. Typhi is a Gram-negative bacterium that causes typhoid fever in humans. Its virulence depends on the TolC outer membrane pump, which expels toxic compounds and antibiotics. However, the role of TolC in the host cell adhesion and invasion by S. Typhi is unclear. Objective: We aimed to investigate how deleting the tolC affects the adhesion and invasion of HT-29 epithelial and THP-1 macrophage cells by S. Typhi in vitro. Methods: We compared the adhesion and invasion rates of the wild-type and the tolC mutant strains of S. Typhi using in vitro adhesion and invasion assays. We also measured the expression levels of SPI-1 genes (invF, sipA, sipC, and sipD) using quantitative PCR. Results: We found that the tolC mutant showed a significant reduction in adhesion and invasion compared to the wild-type strain in both cell types. We also observed that the expression of SPI-1 genes was downregulated in the tolC mutant. Discussion: Our results suggest that TolC modulates the expression of SPI-1 genes and facilitates the adhesion and invasion of host cells by S. Typhi. Our study provides new insights into the molecular mechanisms of S. Typhi pathogenesis and antibiotic resistance. However, our study is limited by the use of in vitro models and does not reflect the complex interactions between S. Typhi and host cells in vivo.

Perceived risk of infection and death from COVID-19 among community members of low- and middle-income countries: A cross-sectional study.

Background: Risk perceptions of coronavirus disease 2019 (COVID-19) are considered important as they impact community health behaviors. The aim of this study was to determine the perceived risk of infection and death due to COVID-19 and to assess the factors associated with such risk perceptions among community members in low- and middle-income countries (LMICs) in Africa, Asia, and South America. Methods: An online cross-sectional study was conducted in 10 LMICs in Africa, Asia, and South America from February to May 2021. A questionnaire was utilized to assess the perceived risk of infection and death from COVID-19 and its plausible determinants. A logistic regression model was used to identify the factors associated with such risk perceptions. Results: A total of 1,646 responses were included in the analysis of the perceived risk of becoming infected and dying from COVID-19. Our data suggested that 36.4% of participants had a high perceived risk of COVID-19 infection, while only 22.4% had a perceived risk of dying from COVID-19. Being a woman, working in healthcare-related sectors, contracting pulmonary disease, knowing people in the immediate social environment who are or have been infected with COVID-19, as well as seeing or reading about individuals infected with COVID-19 on social media or TV were all associated with a higher perceived risk of becoming infected with COVID-19. In addition, being a woman, elderly, having heart disease and pulmonary disease, knowing people in the immediate social environment who are or have been infected with COVID-19, and seeing or reading about individuals infected with COVID-19 on social media or TV had a higher perceived risk of dying from COVID-19. Conclusions: The perceived risk of infection and death due to COVID-19 are relatively low among respondents; this suggests the need to conduct health campaigns to disseminate knowledge and information on the ongoing pandemic.

Vaccine hesitancy among communities in ten countries in Asia, Africa, and South America during the COVID-19 pandemic.

Vaccine hesitancy is considered one of the greatest threats to the ongoing coronavirus disease 2019 (COVID-19) vaccination programs. Lack of trust in vaccine benefits, along with concerns about side effects of the newly developed COVID-19 vaccine, might significantly contribute to COVID-19 vaccine hesitancy. The objective of this study was to determine the level of vaccine hesitancy among communities in particular their belief in vaccination benefits and perceived risks of new vaccines. An online cross-sectional study was conducted in 10 countries in Asia, Africa, and South America from February to May 2021. Seven items from the WHO SAGE Vaccine Hesitancy Scale were used to measure a construct of belief in vaccination benefit, and one item measured perceived riskiness of new vaccines. A logistic regression was used to determine which sociodemographic factors were associated with both vaccine hesitancy constructs. A total of 1,832 respondents were included in the final analysis of which 36.2% (range 5.6-52.2%) and 77.6% (range 38.3-91.2%) of them were classified as vaccine hesitant in terms of beliefs in vaccination benefits and concerns about new vaccines, respectively. Respondents from Pakistan had the highest vaccine hesitancy while those from Chile had the lowest. Being females, Muslim, having a non-healthcare-related job and not receiving a flu vaccination during the past 12 months were associated with poor beliefs of vaccination benefits. Those who were living in rural areas, Muslim, and those who did not received a flu vaccination during the past 12 months had relatively higher beliefs that new vaccines are riskier. High prevalence of vaccine hesitancy in some countries during the COVID-19 pandemic might hamper COVID-19 vaccination programs worldwide. Programs should be developed to promote vaccination in those sociodemographic groups with relatively high vaccine hesitancy.

Willingness-to-pay for COVID-19 vaccine in ten low-middle-income countries in Asia, Africa and South America: A cross-sectional study.

Vaccine hesitancy is considered as one of the greatest challenges to control the ongoing coronavirus disease 2019 (COVID-19) pandemic. A related challenge is the unwillingness of the general public to pay for vaccination. The objective of this study was to determine willingness-to-pay (WTP) for COVID-19 vaccine among individuals from ten low- middle-income countries (LMICs) in Asia, Africa, and South America. Data were collected using an online questionnaire distributed during February - May 2021 in ten LMICs (Bangladesh, Brazil, Chile, Egypt, India, Iran, Nigeria, Pakistan, Sudan, and Tunisia). The major response variable of in this study was WTP for a COVID-19 vaccine. The assessment of COVID-19 vaccine hesitancy was based on items adopted from the World Health Organization (WHO) Strategic Advisory Group of Experts (SAGE) vaccine hesitancy scale constructs. In this study, 1337 respondents included in the final analysis where the highest number of respondents was from India, while the lowest number was from Egypt. A total of 88.9% (1188/1337) respondents were willing to pay for the COVID-19 vaccination, and 11.1% (149/1337) were not. The average WTP for COVID-19 vaccination was 87.9 US dollars ($), (range: $5-$200). The multivariate model analysis showed that the country, monthly household income, having a history of respiratory disease, the agreement that routine vaccines recommended by health workers are beneficial and having received the flu vaccination within the previous 12 months were strongly associated with the WTP. Based on the country of origin, the highest mean WTP for COVID-19 vaccine was reported in Chile, while the lowest mean WTP for the vaccine was seen among the respondents from Sudan. The availability of free COVID-19 vaccination services appears as a top priority in the LMICs for successful control of the ongoing pandemic. This is particularly important for individuals of a lower socio- economic status. The effects of complacency regarding COVID-19 extends beyond vaccine hesitancy to involve less willingness to pay for COVID-19 vaccine and a lower value of WTP for the vaccine.

Performance of Immunodiagnostic Tests for Typhoid Fever: A Systematic Review and Meta-Analysis.

Typhoid fever, also known as typhoid, is a life-threatening bacterial infection that remains a global health concern. The infection is associated with a significant morbidity and mortality rate, resulting in an urgent need for specific and rapid detection tests to aid prevention and management of the disease. The present review aims to assess the specificity and sensitivity of the available literature on the immunodiagnostics of typhoid fever. A literature search was conducted using three databases (PubMed, ProQuest and Scopus) and manual searches through the references of identified full texts to retrieve relevant literature published between 1 January 2011 and 31 December 2020. Of the 577 studies identified in our search, 12 were included in further analysis. Lipopolysaccharides (LPS) and hemolysin E (HlyE) were the most frequently studied antigens. The specimens examined in these studies included serum and saliva. Using blood culture as the gold standard, anti-LPS IgA gave the highest sensitivity of 96% (95% CI: 93-99) and specificity of 96% (95% CI: 93-99) for distinguishing between typhoid cases and healthy controls, whereas the combination of anti-LPS and anti-flagellin total IgGAM gave the highest sensitivity of 93% (95% CI: 86-99) and specificity of 95% (95% CI: 89-100) for distinguishing typhoid cases and other febrile infections. A comparably high sensitivity of 92% (95% CI: 86-98) and specificity of 89% (95% CI: 78-100) were shown in testing based on detection of the combination of anti-LPS (IgA and IgM) and anti-HlyE IgG as well as a slightly lower sensitivity of 91% (95% CI: 74-100) in the case of anti-50kDa IgA. Anti-50kDa IgM had the lowest sensitivity of 36% (95% CI: 6-65) against both healthy and febrile controls. The development of a rapid diagnostic test targeting antibodies against lipopolysaccharides combined with flagellin appeared to be a suitable approach for the rapid detection test of typhoid fever. Saliva is added benefit for rapid typhoid diagnosis since it is less invasive. As a result, further studies could be done to develop additional approaches for adopting such samples.

Nucleic Acid-Based Lateral Flow Biosensor for Salmonella Typhi and Salmonella Paratyphi: A Detection in Stool Samples of Suspected Carriers.

A multiplex rapid detection system, based on a PCR-lateral flow biosensor (mPCR-LFB) was developed to identify Salmonella Typhi and Salmonella Paratyphi A from suspected carriers. The lower detection limit for S. Typhi and S. Paratyphi A was 0.16 and 0.08 ng DNA equivalent to 10 and 102 CFU/mL, respectively. Lateral flow biosensor was used for visual detection of mPCR amplicons (stgA, SPAint, ompC, internal amplification control) by labeling forward primers with fluorescein-isothiocyanate (FITC), Texas Red, dinitrophenol (DNP) and digoxigenin (DIG) and reverse primers with biotin. Binding of streptavidin-colloidal gold conjugate with the amplicons resulted in formation of a red color dots on the strip after 15-20 min of sample exposure. The nucleic acid lateral flow analysis of the mPCR-LFB was better in sensitivity and more rapid than the conventional agarose gel electrophoresis. Moreover, the mPCR-LFB showed 100% sensitivity and specificity when evaluated with stools spiked with 100 isolates of Salmonella genus and other bacteria. A prospective cohort study on stool samples of 1176 food handlers in outbreak areas (suspected carriers) resulted in 23 (2%) positive for S. Typhi. The developed assay has potential to be used for rapid detection of typhoid carriers in surveillance program.

Acceptance of COVID-19 vaccination at different hypothetical efficacy and safety levels in ten countries in Asia, Africa, and South America.

Vaccine hesitancy, defined as the reluctance or rejection in receiving a vaccine despite its availability, represents a major challenge to global health efforts aiming to control the ongoing COVID-19 pandemic. Understanding the possible factors correlated with COVID-19 vaccine hesitancy using a refined well-informed approach can be helpful to address the phenomenon. The current study aimed to evaluate COVID-19 vaccine acceptance rates using four hypothetical scenarios of varying levels of vaccine efficacy and safety profiles in ten Asian, African and South American countries. These scenarios included: 95% efficacy and 20% side effects (Vaccine A), 75% efficacy and 5% side effects (Vaccine B); 75% efficacy and 20% side effects (Vaccine C) and 50% efficacy and 5% side effects (Vaccine D). This study used a self-administered online survey that was distributed during February-May 2021. The total number of study respondents was 1337 with countries of residence as follows: India (21.1%), Pakistan (12.9%), Sudan (11.2%), Nigeria (9.3%), Iran (8.2%), Bangladesh and Brazil (7.9%), Chile (7.7%), Tunisia (7.6%), and Egypt (6.2%). The overall acceptance rates for COVID-19 vaccination were variable based on varying degrees of safety and efficacy as follows: 55.6% for Vaccine C, 58.3% for Vaccine D, 74.0% for Vaccine A and 80.1% for Vaccine B. The highest levels of COVID-19 vaccine acceptance were observed in Brazil followed by Chile across the four different safety and efficacy scenarios. The lowest COVID-19 vaccine acceptance rates were reported in Egypt and Tunisia for the low safety scenarios (20% side effects), and the low efficacy scenario (50% efficacy). The study revealed the potential effect of vaccine safety and efficacy on the intention to get COVID-19 vaccination. At the same efficacy level, higher possibility of side effects caused a large drop in COVID-19 vaccine acceptance rate. This indicates the importance of accurate communication regarding vaccine safety and efficacy on attitude towards the vaccine and intentions to get vaccinated. Regional differences in COVID-19 vaccine acceptance were observed with the Middle East/North African countries showing the lowest rates and the South American countries displaying the highest vaccine acceptance rates.

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein.

With the development of de novo binders for protein targets from non-related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single-chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking "disembodied" amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein-antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen-based detection agents for typhoid diagnostics.

Salivary Anti-50 kDa Antibodies as a Useful Biomarker for Diagnosis of Typhoid Fever.

INTRODUCTION: Typhoid fever remains a scourge of humanity, especially in developing and under-developed countries due to poor sanitation and food hygiene. Diagnostic methods available for detection of this disease are not satisfactory due to a lack of sensitive, specific, rapid and convenient diagnostic test kits available in the market. AIM: To evaluate the feasibility of a Dot-EIA method for Ig-class specific salivary antibody detection for diagnosis of typhoid fever. MATERIALS AND METHODS: Paired saliva and serum samples were collected in the year 2010 from patients and normal volunteers in Hospital Universiti Sains Malaysia, Kelantan, Malaysia, which is endemic for typhoid fever. A total of 11 culture-confirmed typhoid fever patients, 43 non-typhoid fever patients and 53 normal human control subjects were evaluated for antibodies against a 50 kDa antigen specific for Salmonella Typhi using Dot-EIA. RESULTS: Ig class-specific screening of the test samples showed a higher sensitivity for IgA (90.9%) compared to either IgG (72.7%) or IgM (72.7%) antibodies in saliva, but for serum, IgG (90.9%) had a higher degree of sensitivity compared to IgA (36.4%) and IgM (63.6%). Combining all isotypes (IgA, IgG or IgM), serum showed a higher sensitivity (100.0%) compared to saliva (90.9%). Also, the specificity for serum (100.0%) was much higher than saliva (85.4%). CONCLUSION: Salivary IgA anti-50kDa antibody was found to be more suitable biomarker for routine screening, whereas serum IgG was more suitable for confirmatory test as it has higher specificity. Nevertheless, salivary IgA Dot-EIA is a convenient method for rapid testing, such as for Point-of-Care Diagnostics (POCD) and field epidemiological studies, due to its non-invasive nature and ease of use.

Outer membrane proteins analysis of Shigella sonnei and evaluation of their antigenicity in Shigella infected individuals.

Bacillary dysentery caused by infection with Shigella spp. remains as serious and common health problem throughout the world. It is a highly multi drug resistant organism and rarely identified from the patient at the early stage of infection. S. sonnei is the most frequently isolated species causing shigellosis in industrialized countries. The antigenicity of outer membrane protein of this pathogen expressed during human infection has not been identified to date. We have studied the antigenic outer membrane proteins expressed by S. sonnei, with the aim of identifying presence of specific IgA and IgG in human serum against the candidate protein biomarkers. Three antigenic OMPs sized 33.3, 43.8 and 100.3 kDa were uniquely recognized by IgA and IgG from patients with S. sonnei infection, and did not cross-react with sera from patients with other types of infection. The antigenic proteome data generated in this study are a first for OMPs of S. sonnei, and they provide important insights of human immune responses. Furthermore, numerous prime candidate proteins were identified which will aid the development of new diagnostic tools for the detection of S. sonnei.

Delineation of B-cell Epitopes of Salmonella enterica serovar Typhi Hemolysin E: Potential antibody therapeutic target.

Hemolysin E (HlyE) is an immunogenic novel pore-forming toxin involved in the pathogenesis of typhoid fever. Thus, mapping of B-cell epitopes of Salmonella enterica serovar Typhi (S. Typhi) is critical to identify key immunogenic regions of HlyE. A random 20-mer peptide library was used for biopanning with enriched anti-HlyE polyclonal antibodies from typhoid patient sera. Bioinformatic tools were used to refine, analyze and map the enriched peptide sequences against the protein to identify the epitopes. The analysis identified both linear and conformational epitopes on the HlyE protein. The predicted linear GAAAGIVAG and conformational epitope PYSQESVLSADSQNQK were further validated against the pooled sera. The identified epitopes were then used to isolate epitope specific monoclonal antibodies by antibody phage display. Monoclonal scFv antibodies were enriched for both linear and conformational epitopes. Molecular docking was performed to elucidate the antigen-antibody interaction of the monoclonal antibodies against the epitopes on the HlyE monomer and oligomer structure. An in-depth view of the mechanistic and positional characteristics of the antibodies and epitope for HlyE was successfully accomplished by a combination of phage display and bioinformatic analysis. The predicted function and structure of the antibodies highlights the possibility of utilizing the antibodies as neutralizing agents for typhoid fever.

Generation of a naïve human single chain variable fragment (scFv) library for the identification of monoclonal scFv against Salmonella Typhi Hemolysin E antigen.

Antibody phage display is a useful tool for the isolation and identification of monoclonal antibodies. Naive antibody libraries are able to overcome the limitations associated with the traditional hybridoma method for monoclonal antibody generation. Antibody phage display is also a preferred method for antibody generation against toxins as it does not suffer from toxicity mediated complications. Here, we describe a naïve multi ethnic scFv antibody library generated via two-step cloning with an estimated diversity of 2 × 10(9). The antibody library was used to screen for monoclonal antibodies against Hemolysin E antigen, a pore forming toxin produced by Salmonella enterica serovar Typhi. A soluble monoclonal scFv antibody against the HlyE toxin (IgM scFv D7 anti-hlyE) was isolated from the library. This shows the value of the naïve library to generate antibodies against toxin targets in addition to the potential use of the library to isolate antibodies against other immunogenic targets.

Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning.

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.

Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism.

ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a ß-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-ß-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.

Multi-isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen.

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

Identification of carriers among individuals recruited in the typhoid registry in Malaysia using stool culture, polymerase chain reaction, and dot enzyme immunoassay as detection tools.

Chronic carriers of Salmonella Typhi act as reservoirs for the organism and become the agents of typhoid outbreaks in a community. In this study, chronic carriers in Kelantan, Malaysia were first identified using the culture and polymerase chain reaction method. Then, a novel serological tool, designated Typhidot-C, was evaluated in retrospect using the detected individuals as control positives. Chronic carriage positive by the culture and polymerase chain reaction method was recorded at 3.6% (4 out of 110) among individuals who previously had acute typhoid fever and a 9.4% (10 out of 106) carriage rate was observed among food handlers screened during outbreaks. The Typhidot-C assay was able to detect all these positive carriers showing its potential as a viable carrier screening tool and can be used for efficient detection of typhoid carriers in an endemic area. These findings were used to establish the first carrier registry for S Typhi carriers in Malaysia.

Principles and application of antibody libraries for infectious diseases.

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

Overexpression, purification and validation of antigenic Salmonella enterica serovar Typhi proteins identified from LC-MS/MS.

In our earlier study, an immunoblot analysis using sera from febrile patients revealed that a 50-kDa band from an outer membrane protein fraction of Salmonella enterica serovar Typhi was specifically recognized only by typhoid sera and not sera from other febrile illnesses. Here, we investigated the identities of the proteins contained in the immunogenic 50-kDa band to pinpoint antigens responsible for its immunogenicity. We first used LC-MS/MS for protein identification, then used the online tool ANTIGENpro for antigenicity prediction and produced recombinant proteins of the lead antigens for validation in an enzyme-linked immunosorbent assay (ELISA). We found that proteins TolC, GlpK and SucB were specific to typhoid sera but react to antibodies differently under native and denatured conditions. This difference suggests the presence of linear and conformational epitopes on these proteins.