After preliminary characterization of the protein by SDS-PAGE, the immunogenicity of the fractionated protein was analyzed by dot EIA. In order to perform comparative characterization on the feasibility of the differentially extracted protein fractions derived from S.Typhi and S.spp, the antigens were probed with pooled sera of typhoid patients (PTS), pooled sera of healthy subjects (PHS), pooled sera of typhoid vaccinated subjects (PVS), pooled sera of subjects with other disease (POS) and invasive non-typhoidal patient serum (iNTS). In this study, the immunological characterization by dot EIA revealed the dynamic profile of humoral immune response by PTS sera, PHS sera, PVS sera, POS sera and iNTS serum against the arrays of differentially extracted protein fractions derived from S.Typhi and S.spp.
The dot EIA result of WCP derived from S.Typhi in fraction 1 to fraction 3 revealed positive reaction against PTS sera, PHS sera, PVS sera and POS sera at various degree of intensity. Similar reaction was observed in WCP of S.spp fractions whereby fraction 1 to fraction 7 showed positive reaction against PTS sera, PHS sera and POS sera. The sdWCP fractions derived from S.spp also revealed similar pattern of antigenicity on the first 7 fractions probed with PTS sera, PHS sera, PVS sera and POS sera. Parallel response was observed in CSP of S.spp fractions, with fraction 1 showing high antigenicity to PTS, PHS, PVS sera and POS sera. Despite the variation in term of antigenic intensity, these conserved antigenic protein fractions identified in both differentially extracted Salmonella strains serve as a tool in initiating humoral immune response without potentially developing the disease. Therefore, these arrays of antigenic fractions can be a platform for the development of vaccine for typhoid fever.
Variation in term of the antigenic intensity of the protein fractions is also important is determining the diagnostic cut off in differentiating typhoid patient, patient with other diseases, vaccinated subjects and healthy subjects. From the results, all the differentially extracted fractions of both strains probed with PTS sera had the strongest antigenic intensity compared to other pooled categories of sera. Many patients with mild or lower dose of infections are diagnosed with Covid-19 after their infectious phase has ended, resulting in unneeded quarantine and unnecessary contact tracing.[14]–[16] Similar cases was observed in typhoid fever whereby high false positive result due to overdiagnosis led to inappropriate antibiotic treatment and the emergence of antibiotic resistance.[17]–[19] Therefore, the selective use of fractionated protein highlighted in this study could be harnessed to set up parameters as a proxy for differential serodiagnosis of typhoid fever and increase the sensitivity of diagnosis in order to reduce false positive results.
On the other hand, the dot EIA result observed in CSP of fractions derived from S.Typhi showed highest degree of sensitivity and positive predictive value (PPV) whereby only PTS elicited reactivity in F1 to F7 with the strongest degree of intensity observed in IgA antibody isotype. The strong distinction between PTS and PHS, PVS, and POS sera conclusively proved that CSP probed against IgA antibody isotype is important and useful as seromarker for acute infection. The role of IgA antibody isotype in recognizing early and acute infection which lead to improved serodiagnosis of infectious disease caused by Helicobacter pylori, Toxoplasma gondii, dengue virus, Mycobacterium tuberculosis, Epstein-Barr virus and coronavirus has been elegantly documented in literature.[20]–[26] Therefore, greater accuracy in serodiagnosis can be achieved by detecting IgA in addition to IgG and IgM antibody isotypes.
In addition, the results observed in Fig. 3 showed that all the 20 sdWCP fractions probed with iNTS serum against all three antibody isotypes expressed enhanced antigenic reaction compared to WCP fractions. This study reveals a previously unreported effect of depleting highly abundant proteins in enhancing the antigenicity of target proteins, in addition to improving the detection of low abundance proteins. To the best of our knowledge, this is the first report of such an effect, which highlights the importance of considering the impact of sample preparation on downstream analyses. Findings from this comparative analysis of the role of depleted protein in the diagnosis of typhoid fever clearly demonstrated that the detection using combination of differential extracts were the most suitable for the serodiagnosis of typhoid fever.
Further experiment was performed to confirm the separation of protein upon fractionation and identify the location of O and dH antigens. The O antigens was observed in F1 to F3 of the WCP and sdWCP derived from S.Typhi. Strong reactivity was observed in F4 to F6 of the CSP derived from S.Typhi when probed with dH polyclonal agglutinating sera. As a result, the differentially extracted protein fractions probed with anti-Salmonella 9-O and dH polyclonal agglutinating sera against IgG antibody clearly indicated the location of flagellar and somatic antigens. This separation technique performed after differential extraction procedure, highlighted the powerful, discriminatory capacity of modified 2D preparative IEF fractionation.
Further comparative analysis revealed that the dH antigens identified in F4 to F6 as mentioned above was not recognize by typhoid patients. Overall, typhoid patients and iNTS patient recognized antigens beyond the location O9 and dH antigen. This discovery suggested that this could represent a novel class of antigens and therefore highlighted the importance of moving beyond serotypes.
A detailed and comprehensive comparison was carried out between the differently extracted fraction derived from S.Typhi and S.spp probed with PTS sera and iNTS sera. Based on the dot EIA of the fractions shown in Table 1, 14 out of the 120 protein fractions were successfully identified as potential biomarker for diagnosis and vaccine development. Eight of the 14 fractions identified were specifically reactive to iNTS patients, three were specifically reactive to typhoid patients, and the remaining three antigens were reactive in both typhoid patients and iNTS patients. This novel finding from the spectrum of fractions can be used to develop a highly sensitive and accurate diagnostic test, that incorporate the selectively reactive antigens. On the other hand, the cross-reactive antigens can be utilized for the development of a vaccine with universal protection against diseases caused by different Salmonella serovars.
The 14 discriminative antigens derived from S.Typhi and S.spp were selected for further characterization by WB assay. Herein, comparative analysis was performed between PTS sera and iNTS serum that were conjugated with IgM, IgG and IgA antibody isotypes. The WB results based on Fig. 5 demonstrated a wide range of immunological diversities and notable heterogenicity of antibody responses in typhoid patients and iNTS patient.
Numerous studies have reported on the role of IgM in detecting acute infection of various diseases including acute hepatitis, acute HIV1 and HIV2, parvovirus and dengue virus.[27], [28] This study demonstrated the antigenic and serologic relationship between the highly specific and reactive antigens against IgM antibody isotypes. In Fig. 5, the IgM antibody response of PTS sera was directed primarily towards NTb3 protein fraction with smearing bands at the location between 25 to 60 kDa. Both PTS sera and iNTS serum elicited reaction in the USb2 protein fraction at a markedly different location. Additionally, low molecular wight protein band at the location of 18kDa was observed in PTS whereas high molecular weight protein bands at the location of 55 and 60kDa was observed in the iNTS serum blot. In the dot EIA results above, protein fractions designated as USB are categorized as antigens that were recognized by both typhoid and iNTS patient. Additionally, the WB result revealed that the antigenic proteins in typhoid and iNTS patient were expressed at different location. In contrast with PTS sera, smearing bands with high degree of intensity was observed in USb3 and NTb6 protein fraction in the iNTS serum blot against IgM antibody isotype. Overall, stronger reaction in terms of antigenic intensity and number of bands from protein fractions derived from S.spp was observed in iNTS patient compared to typhoid patient.
Paradoxically, the opposite response was observed in IgG antibody isotypes from PTS blots whereby stronger reaction from USb3 and NTb6 protein fractions derived from S.spp at the location between 34 kDa to 200 KDa was detected at a low degree of intensity. The WB result for iNTS blot against IgG antibody isotype showed diffuse and smearing high molecular weight bands at the region between 50 to 200 kDa for USb3 and NTb6 protein fraction. Overall, the antigenicity in both PTS sera and iNTS serum were observed at the same protein fraction with typhoid patient recognizing additional low molecular weight protein bands. The IgG antibody isotype is not the preferred primary choice in serologic tests due to its longevity in the body for up to 2 years after infection and its difficulty in differentiating between acute and convalescent infections.[29] Contrastingly, in this result the faint bands observed in IgG antibody isotype blots compared to the respond against IgM and IgA isotypes indicate the potential of IgG antibody isotype as an indicator in differentiating between acute and convalescent infection.
IgA antibody isotype response in iNTS serum blot was observed in the USb3 and NTb6 protein fractions as well. Overall, the NTb6 protein fraction was highly reactive in the WB result of iNTS serum against all three antibody isotypes. Based on the dot EIA results, NTb6 protein fraction was identified as potential diagnostic biomarker for iNTS disease. Therefore, results obtained from the WB assay validates the potential role of NTb6 protein fraction for definitive diagnosis of iNTS disease. On the other hand, PTS sera blot against IgA antibody isotype in Fig. 5 revealed identification of well-defined bands with the molecular weights of 28 kDa, 32 kDa, and 34 kDa in all lanes with the exception of TSb1 and TSb2 protein fractions. Previous report has delineated the role of 34 kDa antigenic protein as a potential biomarker due to its natural ability in eliciting humoral immune response in typhoid patient.[30] However, the antigenic nature of 34 kDa protein in iNTS disease have not been reported before. Based on the results, these highly conservative proteins derived from both S.Typhi and S.spp serve as a potential vaccine candidate for typhoid fever and iNTS disease.
The highly ubiquitous Salmonella strains are known to cause different diseases within the same species and therefore complicates diagnosis and early health intervention.[31] Herein, the extensive characterization of proteins in this study answers the dilemma in the need of a well-defined serologic assays to monitor typhoid fever and iNTS disease in natural communities and to optimise vaccine development for early medical intervention in an epidemic setting.