Profiling of Humoral Immune Response in Typhoid Patients against Differentially Extracted Whole Cell Bacterial Protein Derived from S. typhi and S. spp

Typhoid fever is a multiorgan infectious disease caused by Salmonella typhi. It is transmitted through fecal oral route and can be fatal without proper treatment. Therefore, early diagnosis of typhoid fever is crucial. In the previous study, we have developed TYPHOIDYNE EIA, which showed excellent synergy between the genus conserved and species-specific antigens in the serodiagnosis of typhoid fever. TYPHOIDYNE EIA can effectively detect and differentiate typhoid patients, typhoid vaccinated subjects, healthy subjects, and subjects with other febrile illnesses. Following the successful development of TYPHOIDYNE EIA, in this report, we further characterize the antigenic components of differentially extracted S. typhi and S. spp recognized by IgM, IgG, and IgA antibody isotypes in typhoid patients and possible typhoid carrier by the western blot (WB) assay. The WB characterization revealed a dynamic pattern of recognition, with significant variations in the number of antigenic bands observed between the differentially extracted arrays of antigens. The reactivity of patient's sera was divided into 3 regions, with region 1 (≥55 kDa) showing the strongest reactivity followed by region 2 (54 kDa–34 kDa) and region 3 (<34 kDa). Overall, the good synergy expressed in these bands suggests the potential role of these proteins in differentiating typhoid patients with possible typhoid carrier. The antigenic bands highlighted in this study are also identified as prospective biomarkers for diagnostic use and vaccine development.


Introduction
Typhoid fever is an infectious disease that is transmitted through the fecal oral route.It is caused by the bacterial agent, Salmonella typhi (S. typhi).Tis typhoid agent can elicit a spectrum of infection outcomes including chronic typhoid, mild infection, relapse after recovery, and passive or acute carrier of typhoid [1].Salmonella carriers are frequently asymptomatic, and they regularly shed the bacteria which cause the spread of the disease [2].On the other hand, drug resistance against S. typhi is increasingly becoming a major cumbersome issue in managing this disease.With the emergence of extensively drug resistance (XDR) strains, there is potential that typhoid fever will become untreatable owing to a lack of antibiotic options [3].
Over the years, numerous studies have been conducted to investigate and discover specifc antigenic proteins from various preparations derived from S. typhi for the serodiagnosis of typhoid fever.Tis included characterization of heat shock protein (HSP), capsular-polysaccharide (Vi) antigen, somatic (O) antigen, and fagellar (H) antigen.In this regard, WB analysis of the HSP revealed immunodominant antigenic proteins with molecular weights of 58, 68, and 88 kDa [4].HSP has been known to play a major role in activating a high degree of humoral immune response in infected individuals [5].HSP has also been identifed as the target of immune response in a wide range of infections [6].
On the other hand, the Vi antigen found in S. typhi, S. Paratyphi C, and S. Dublin has been shown to aid in escaping innate immune detection and elevate systemic virulence of infection.In 1994, the Vi capsular-polysaccharide vaccine was licensed and has been commercially available throughout the years.However, this vaccine has been shown to elicit paucity of immune response to young children [2,7].Terefore, a few other vaccines derived from the Vi antigen which includes Vi-rEPA vaccine and Typbar-TCV have been introduced to elicit a high degree of immune response for better protection from typhoid fever in a variety of age groups, from children to the elderly [8,9].However, several limitations such as the short-term efect of these vaccines due to absence of immune memory and the inability of protection against iNTS and other salmonellosis impede the performance of the vaccines [10,11].
Te O and H antigens have been used as a serological diagnostic biomarker in the classical Widal test that was invented in 1896.Te Widal test measures the antibody titre against O and H antigens by the agglutination method.Despite the long-term deployment of this method in primary and secondary healthcare facilities in rural settings, the high rate of false positive and false negative cases were observed in endemic areas [12].Patients that have been previously infected with typhoid or other corelated febrile disease such as coliforms, Shigella, or other salmonellae stimulate production of homogenous antibodies that resulted in a high rate of false positive cases.In addition, vaccinated individuals possessed antigens that stimulated antibody production, resulting in inaccurate diagnosis [13].Tese drawbacks in the application of O and H antigens in the Widal test have resulted in a disparate level of its acceptability around the world.
Tese fndings demonstrated a heterogenous antibody response following typhoid infection against diferent antigens as well as a signifcant number of drawbacks in the use of single antigen for the diagnosis and prevention of typhoid fever.Terefore, the combination of more than one antigen would be essential to detect the humoral immune response in typhoid patients and contribute to both diagnostic value and vaccine development against typhoid fever.Te generation of multiantigens -ased immunoassay may assist in the development of serological assay with improved sensitivity and specifcity.
Previously, we have successfully developed TYPHOI-DYNE EIA that have successfully detected and diferentiated typhoid cases, typhoid vaccinated individuals, typhoid carriers, and healthy individuals.TYPHOIDYNE EIA has efectively highlighted the synergistic efect of the genus conserved and species specifc antigens in the serodiagnosis of typhoid fever.
Terefore, in this present study, we aim to further characterize the WCP, CSP, and sdWCP derived from S. typhi and S. spp using WB analysis and highlight the signifcance of multiple antigens inclusion in the development of serodiagnosis immunoassay.Te WB analysis was carried out to assess the presence of the specifc IgM, IgG, and IgA antibody isotypes response in typhoid patients against the diferentially extracted antigens.Subsequently, we performed a comparative analysis to evaluate the degree of immune response and diferentiate the reactive bands elicited in the WB profles of typhoid patients compared to the possible carrier from healthy subjects based on TYPHOIDYNE EIA evaluation in our previous report [14].

Serum Sample Collection.
Te overall profle of the humoral immune response against antigens derived from S. typhi and S. spp were analyzed by WB assay using representative patient's sera from culture and serology positive typhoid cases.Healthy subjects that were categorized as carrier in the previous report were included in this study [14].To perform the WB assay, sera from typhoid patients and healthy subjects were analyzed as pooled sera, respectively.Tere were two categories of pooled sera used in WB assay to determine recognizable and representative antigenic bands present in diferentially extracted protein of S. typhi and S. spp and to analyze the diversity in humoral immune response between typhoid patients and the possible typhoid carrier.Pool sera designated as PTS was made up of 4 positive IgM, IgG, and IgA.Pooled sera designated as PHPS was made up from 4 sera taken from possible carrier subjects.Te categorized pooled sera are tabulated and summarized in Table 1.

Protein Extraction.
Te diferential protein extraction of WCP, CSP, and sdWCP of S. typhi and S. spp was performed as described previously [14].For WCP, each bacterial strain was isolated as a single colony from sheep blood agar, inoculated into 10 ml Luria broth, and cultured overnight at 37 °C.Te broth was centrifuged (10000 rpm, 4 °C, and 10 minutes) after three successive overnight subcultures, and the pellet was resuspended in 1X SPB (pH 6.8).After boiling for 5 minutes, the suspension was centrifuged (10000 rpm, 4 °C, and 10 minutes).Te suspension was precipitated with ethanol and incubated at −20 °C overnight.Te suspension was then centrifuged (10000 rpm, 4 °C, and 10 minutes).Te pellet was dissolved in Tris-PMSF and kept at −20 °C until further use.For CSP extraction, the bacterial strain was cultured in the same manner as for WCP extraction.Te pellet was resuspended in glycine-HCL (pH 2.2) and incubated for 15 minutes at room temperature before being centrifuged (10000 rpm, 4 °C, 10 minutes).Te pH of the supernatant was lowered to 7.4 before proceeding with ice cold ethanol precipitation.Te sdWCP was extracted using the same method as WCP from the pellet left after the CSP extraction.

Western Blot Analysis.
Te antigens employed for immunoblotting were WCP, CSP, and sdWCP derived from the two strains.Te diferentially extracted proteins were separated by SDS-PAGE following the method described by Leammli [15].After the separation of protein by SDS-PAGE, the gel was incubated in Towbin transfer bufer for 15 minutes.Te separated components on the gel were electroblotted to 0.45 µm pore size nitrocellulose(NC) membrane.Te Bio-Radsemi-dry transblot apparatus was used to perform the electroblotting.Te electroblotting condition was set to 20 V for 30 minutes according to 2 Interdisciplinary Perspectives on Infectious Diseases manufacturer's instructions.After the transfer to NC membrane was done, the NC membrane was incubated in Ponceau S staining solution for 15 minutes.Te reference lane with the protein marker was then cut of before proceeding with western blot analysis.Next, blocking was done with 5% skimmed milk in 1X TBS for one hour.Te NC membrane was then incubated with primary antibody (serum diluted 1 : 100 in 1XTBS) for 2 hours followed by washing three times, for 5 minutes each.Te NC membrane was then incubated with the secondary antibody (IgM, IgG, and IgA) for another 2 hours before washing with skimmed milk three times, for 5 minutes each.Te membrane strips were than incubated for 10 minutes with AP conjugate for optimum colour development.Te reaction was stopped by washing with distilled water for 10 minutes.

Characterization of O9 (Somatic Antigens) and dH (Flagellar Antigens
) against WCP, CSP, and sdWCP Derived from S. typhi and S. spp.WB assay was performed to identify the location of somatic and fagellar antigens recognized by goat anti-rabbit IgG antibody isotypes in diferentially extracted proteins derived from S. typhi and S. spp.As shown in Figure 1, the reactivity of the WB profle probed with Salmonella O9 serum were largely confned in cluster 1 and cluster 2. Te zone of smearing bands with a high degree of intensity was observed in cluster 1. Te O9 serum also recognized low molecular weight protein bands at the region of 13 kDa and 15 kDa in cluster 3.
On the other hand, well separated and distinct bands were observed in the WB profle in Figure 2. Te reactivity against diferentially extracted antigens derived from S. typhi and S. spp probed with dH serum did not show any presence of reactive protein bands in cluster 3. Te diferentially extracted proteins derived from both S. typhi and S. spp recognized protein bands at cluster 1 and cluster 2 with two protein bands at the location of 34 kDa and 55 kDa recognized across the six lanes of diferentially extracted antigens derived from S. typhi and S. spp.

Characterization of Antibody Isotypes Response in PTS
against WCP, CSP, and sdWCP Derived from S. typhi and S. spp.WB analysis was performed to determine the three main antibody isotypes responses in the panel of pooled sera from typhoid patients designated as PTS against the differentially extracted antigens derived from S. typhi and S. spp.As shown in Figures 3 and 4, the IgM WB profle of reactive bands recognized by diferentially extracted S. typhi antigens was closely similar to the WB profle of reactive bands detected by diferentially extracted S. spp antigens.Te IgM WB profles against S. typhi and S. spp antigens showed the recognition of reactive bands in cluster 1, cluster 2, and cluster 3 at diferent degrees of intensity.
On the other hand, the IgG and IgA WB profles against diferentially extracted S. typhi and S. spp antigens did not show any presence of protein bands in cluster 3. Te WB reactivity studied on the PTS serum pool against IgG and IgA antibody isotypes are shown in Figures 5-8.Te WB profle of IgG and IgA antibody isotypes against diferentially extracted S. typhi antigens was relatively similar to diferentially extracted S. spp antigens.However, the degree of intensity in the IgG and IgA WB profles elicited by diferentially extracted S. spp antigens was lower than differentially extracted S. typhi antigens.

Characterization of Antibody Isotypes Responses in PHPS
against WCP, CSP, and sdWCP Derived from S. typhi and S. spp.WB analysis was also performed to analyze the reactivity of the panel of pooled sera from TYPHOIDYNE EIA test positive healthy subjects designated as PHPS against the diferentially extracted antigens derived from S. typhi and S.     profles of the diferentially extracted antigens derived from S. typhi and S. spp against IgM, IgG, and IgA antibody isotypes in the PTS sera showed stronger reactivity in terms of the degree of intensity and number of protein bands compared to PHPS sera.Te summary of the overall WB reactivity profles produced by the pooled sera from typhoid patients and possible carriers is summarized in Tables 2-4.
Based on Tables 3 and 4, reactive low molecular weight bands were not detected against IgG and IgA antibody isotypes in the WB assay for both PTS and PHPS serum pool.

Discussion
Te diferentially extracted antigens derived from S. typhi and S. spp were also classifed according to the somatic (O9) and fagellar (dH) antigens by WB assay.Te pattern of recognition in WB profles probed with O9 and dH serum was analyzed to further understand the signifcance of these antigens in the serodiagnosis of typhoid fever.Based on Figures 1 and 2, the somatic and fagellar antigens observed in the WB assay were heterogenous and generally recognized by diferentially extracted proteins derived from S. typhi and S. spp in PTS-and PHPS-pooled serum against all three antibody isotypes.Based on this fnding, it is interesting to note that the diferentially extracted crude proteins provided a moderate sense of specifcity in the WB assay, particularly when variants are present.Due to the complexity of differentially extracted crude protein, a better separation technique such as liquid phase preparative isoelectric focusing (IEF) can be used to further identify the location of somatic and fagellar antigens.
To further analyze the host humoral immune response in typhoid patients against the multiantigen consisting of WCP, CSP, and sdWCP derived from S. typhi and S. spp, WB analysis was performed with typhoid sera and possible carrier sera.Te sera from typhoid patients which tested positive in IgM, IgG, and IgA antibody isotypes in TYPHOIDYNE EIA were selected and tested by the WB assay in pooled samples.Healthy sera that were tested positive and categorized as possible carrier in TYPHOI-DYNE EIA from our previous report were selected and pooled for the WB assay evaluation [14].Comparative analysis of the multiantigen profle recognized by IgM, IgG, and IgA antibody isotypes in the WB assay probed with PTS and PHPS was performed.Te use of pool sera is to minimize the variation of the reactivity in IgM, IgG, and IgA antibody isotypes against the multiantigens derived from S. typhi and S. spp as antigen recognition by individual sera which are varied [16,17].Tese two pools of sera were selected for WB assay in order to discriminate and defne the profles of reactive antigenic bands between diferentially extracted antigens produced by each antibody isotypes in 8 Interdisciplinary Perspectives on Infectious Diseases typhoid patients compared to healthy subjects with a possible history of exposure to typhoid fever.Based on the results, we identifed up to 16 distinct antigenic bands with molecular weights ranging from 100 kDa to 20 kDa in the WB assay probed with PTS and PHPS against three antibody isotypes with various degree of intensity and frequency at a given concentration.Tese antigenic bands were categorized into three main clusters of antigens consisting of broadly difused antigenic bands (RI), regularly spaced antigenic bands (R2) and low molecular weight antigenic bands (R3).Based on the WB assay results for PTS, the IgM, IgG and IgA antibody isotypes elicited strong reactivity to all three diferentially extracted antigens derived from both S. typhi and S. spp majorly in cluster 1 and 2. Tese results indicated that the highly reactive antigens in cluster 1 and 2 were responsible in triggering strong immune response from all three antibody isotypes in typhoid infection.Tis fnding also demonstrated that closely related Salmonella species share number of antigens with S. typhi, which may indicate the cross-protective roles of these antigens.
On the other hand, WB assay probed with PHPS against IgM, IgG, and IgA antibody isotypes expressed similar antigenic reactivity as PTS with the reactive bands from S. typhi and S. spp largely confned in cluster 1 and 2. As the protein in cluster 1 and 2 in both serovars were recognized by PHPS in this study, it is highly likely that these proteins are conserved protein among Salmonella and are able to stimulate the immune response among both typhoid patients and healthy subjects suggesting that they could be good vaccine candidates.However, the degree of antigenic intensity between PTS and PHPS can clearly be diferentiated as PTS elicited a stronger degree of antigenic intensity compared to PHPS.Overall, the diverse degree of antigenic intensity of the bands between PTS and PHPS highlighted the heterogenicity in the humoral immune response in typhoid patients and healthy subjects with a possible history of exposure to typhoid fever.
In the subsequent review of the WB results, we compared the reactivity of multiantigens against IgM, IgG, and IgA antibody isotypes to obtain a distinct fngerprint pattern in the WB assay.We observed that PTS patients and PHPS subjects both produced markedly heterogenous reactivity against the IgM, IgG, and IgA antibody isotypes response with respect to the intensity, number, and location of the bands.
IgM, which is the initial antibody isotype produced during an immune response, serves as the body's main Interdisciplinary Perspectives on Infectious Diseases defense against infections [18].In the previous study, the role of IgM as an indicator for acute infection of M. pneumonia has been well documented [19].Tese fndings is in line with the results we have achieved in our study whereby the PTS serum pool produced profles of difuse bands with visible background reaction in clusters 1 and 2 based on the overall view of the humoral immune response in IgM antibody isotype of PTS and PHPS against the genus conserved and species-specifc antigens derived from S. typhi and S. spp in Table 2.In addition, most of the low molecular weight antigenic bands derived from both S. typhi and S. spp were recognized by IgM antibody isotypes in PTS.Surprisingly, WB assay performed with the PHPS serum pool revealed no visible IgM antibody isotypes reactive bands in cluster 3. Previous data analysis report on the Typhidot diagnostic test has revealed the high avidity of IgM antibody in the detection of acute typhoid infection [20].Several authors have suggested that IgM antibody isotypes are mostly generated during the early stages of infection [21][22][23].IgM antibodies have been shown to identify a variety of microbial components, including viral antigens and bacterial toxins [24].As mentioned in the previous study, IgM may remain for a very long time; hence, this antibody isotype can still be detected in carriers but at a lower degree of intensity.
Tere have been frequent reports on the persistence of IgG antibodies for more than two years following infection.
In addition, IgG antibodies that were generated later than IgM antibodies frequently persist for a lifetime [25][26][27].Our fndings extent these previous evaluations signifcantly as we observed a relatively similar pattern of humoral immune response in IgG antibody isotype in both PTS and PHPS.Both categories of the subjects elicited well-separated antigenic bands at the location above 34 kDa.Although the location of the bands observed in the WB assay for IgG antibody isotypes was analogous, a clear diference in terms of the degree of antigenic intensity was demonstrated in the WB assay whereby PTS demonstrated a higher intensity of reaction compared to PHPS.In addition, a number of reports in the literature have delineated the presence and reactivity of Salmonella fagellin antigen with the expected molecular weight of 55 kDa [28][29][30].In this study, the 55 kDa antigen was also detected in PTS-and PHPS-pooled serum against the IgG antibody isotype.Te presence of reactive 55 kDa antigenic band in the WB profles of both typhoid and possible carrier subjects suggests the role of this fagellin antigen as a marker for protective immunity.
Based on the WB assay result for the IgA antibody isotype, we remarked a certain degree of homogenous antigenic protein pattern with two major bands recognized in both PTS and PHPS at the location of 34 and 55 kDa.In the previous study, the antigenic protein with the molecular weight of 34 kDa has been identifed, purifed, and characterized as cytolysin, a cytotoxic protein from the outer  10 Interdisciplinary Perspectives on Infectious Diseases membrane of S. typhi.Te utility of 34 kDa cytotoxin protein against the IgA antibody has been well demonstrated as a diagnostic biomarker for typhoid fever [31,32].On the other hand, the WB profle of PHPS recognized the IgA antibody isotype antigenic band at the region of 32 kDa which was not seen in other antibody isotypes.In addition, no visible background reaction was detected in IgA antibody isotypes, which indicated a high specifcity for IgA antibody isotype reaction against the multiantigens characterized by the WB assay.Emerging data also highlighted the role of the IgA antibody isotype as a good biomarker of acute stage typhoid [33,34].
Based on the WB assay, additional reactive bands located at diferent location in IgM, IgG, and IgA antibody isotypes were spotted and are described in Tables 2-4.Generally, we observed a clear distinguishment of the reactive multiantigen band profles for IgM, IgG, and IgA antibody isotypes probed with PTS and PHPS.High intensities of reaction were observed in IgM followed by IgA and IgG antibody isotypes of PTS.On the other hand, the degree of intensity of the reactive bands produced by PHPS serum pool in WB assay were lower and can be distinguished from the reactive bands found in the PTS serum pool.Based on the results in our study, we found out that the pattern of the humoral immune response in all three antibody isotypes were diversifed and served as a potential fngerprint profle that could be conclusive for the serodiagnosis of typhoid fever.
We also noted the discrepancies between the reactivity of antigens exhibited between closely related Salmonella species which include S. typhi and S. spp in the WB assay.PTS pool sera recognized a greater number of antigenic bands derived from S. typhi compared to S. spp.Subsequently, PHPS pool sera recognized several reactive multiantigens derived from S. typhi and S. spp that were not present in PTS pool sera.
Te production of several antigens at various stages of infection in malaria is also linked to the diversity of immune responses by Plasmodium spp.[35].On the other hand, M. Tuberculosis is associated with the expression of antibodies against a range of antigens in most active TB patients [36].Our fndings extent these reports whereby the WB assay displayed a dynamic pattern of recognition with clear discrepancies in the number of antigenic bands present between three diferentially extracted arrays of antigens derived from S. typhi and its closely related species.Overall, the arrays of antigens derived from S. typhi and S. spp successfully diferentiated typhoid patients from healthy subjects with possible prior exposure to typhoid fever.

Figure 1 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :
Figure 1: Te WB analysis of goat anti-rabbit IgG antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP derived from S. typhi and S. spp.Te antigens were probed with Salmonella O9 serum.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers stained with Ponceau S are indicated to the most left.

Figure 7 :
Figure 7: Te WB analysis of IgA antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP of S. typhi against PTS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the most left.

Figure 8 :Figure 9 :
Figure 8: Te WB analysis of IgA antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP of S. spp against PTS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the most left.

Figure 10 :
Figure 10: Te WB analysis of IgM antibody isotype reactivity against SDS-PAGE separated antigens from the WCP, CSP and sdWCP of S. spp against PHPS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the left most.

Figure 11 :
Figure 11: Te WB analysis of IgG antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP of S. typhi against PHPS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the left most.

Figure 12 :
Figure 12: Te WB analysis of IgG antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP of S. spp against PHPS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the left most.

Figure 13 :
Figure 13: Te WB analysis of IgA antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP of S. typhi against PHPS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the left most.

Figure 14 :
Figure 14: Te WB analysis of IgA antibody isotype reactivity against SDS-PAGE-separated antigens from the WCP, CSP, and sdWCP of S. spp against PHPS.Te immunoreactive bands identifed in the WB reaction are classifed according to the region (top right) and indicated by coloured arrows on the right.Te reference lane molecular weight markers along with the protein profle stained with Coomassie are indicated to the left most.

Table 1 :
Description of the pooled sera and the designated codes for characterization by WB assay.

Table 2 :
Summary of the overall view of the humoral immune response in the IgM antibody isotype of PTS and PHPS against the antigens in the WCP, CSP, and sdWCP of S. typhi and S. spp analyzed by WB assay.