Systemic and local immune responses to Salmonella enterica serovar Typhi by prime boost strategy with different preparations of Salmonella antigen(s)

Abstract

Typhoid fever, caused by Salmonella enterica serovar Typhi (Salmonella Typhi), is an important medical and public health problem in South East Asia including Bangladesh and may be prevented and controlled by vaccines. Two existing licensed vaccine namely parenteral Vi CPS and oral Ty21a for typhoid are modestly effective having 3 year cumulative efficacy of 51-55%. Though immune response to the Vi capsular polysaccharide (Vi CPS) vaccine confers protection against Salmonella Typhi, but it is non immunogenic in children below 2 years of age, provides protection only for 3-5 year and unable to induce booster effect. In view of the above, the present study investigated the effect of prime boost method using Vi CPS in combination with different salmonella antigens on the anti-Vi antibody response in mice model. Also, the immunogenic antigens (s) as well as humoral immune responses to Vi CPS and to other structural components of Salmonella Typhi were evaluated in human. Six groups of BALB/c mice were immunized with Vi CPS in combination with Vi CPS, Vi-TT, killed whole cell (KWC) antigens through different routes. Sera from a total of 70 culture confirmed typhoid and 25 healthy/diseased cases were included in the study. Six adult human volunteers were enrolled and immunized with single dose of Vi-CPS vaccine. Anti-Vi IgM, IgG and IgA antibody secreting cell (ASC) in spleen and Peyer‟s patch (PP) of mice were estimated by ELISPOT assay and in serum by ELISA on 28 th day of immunization. Production of functional antibodies in mice and human typhoid cases was measured by colorimetric serum salmonellacidal assay. S. Typhi surface, envelop and whole cell proteins were extracted by water extraction method (WEM), Tris-Sucrose-EDTA (TSE) buffer and sonication method respectively and analyzed by SDS-PAGE and Western blot (WB) methods. Antibodies to surface, envelop and whole cell extract (WCE) proteins in human sera from typhoid and control cases were measured by ELISA method. In mice, anti-Vi IgM, IgG and IgA producing ASCs were detected in spleen following prime boosting with KWC-Vi and ViTT-ViTT antigens whereas no anti-Vi IgG ASC response was observed following prime-boosting with Vi antigen alone. Anti-Vi IgM, IgG and IgA producing ASCs were found significant (p<0.05) in PPs in groups primed with KWC followed by boosting with Vi in comparison to other groups. Serum Ant-Vi IgG antibody titer response was found significantly high (p<0.05) in groups immunized with ViTT-ViTT and KWC-Vi antigen combinations. No significant (p>0.05) difference of anti-Vi ASC response was detected in spleen (except IgM), PPs and in serum when heterologous antigen (KWC-Vi) was given in similar (i.p-i.p) or alternative (oral-i.p) routes. Salmonellacidal antibody (bactericidal titer) response was observed in low titer (15±3.3) in sera of BALB/c mice immunized with KWC- Vi (i.p-i.p) and ViTT-ViTT (i.p-i.p) combinations. Similarly, very low salmonellacidal titers (2.5±1.5 and 2.3±1.5) were also detected 14 and 21 days after single dose of Vi CPS vaccine among the human volunteers. However, sera from typhoid and paratyphoid A patient showed significant (p<0.05) levels of salmonellacidal antibody titer (549.9±108.5 and 528.7±187.3) compared to control population (0.133±0.1). Moreover, salmonellacidal titer increased significantly (p<0.05) in samples collected between 7 to 10 days and between 10 to 25 days of fever (titer 535.7± 119.2 and 794.6± 235.6) compared to samples collected from cases having fever for less than 7 days (Mean titer 136.4± 52.7). The mean titer significantly (p<0.05) decreased to 5.5± 2.1 after 6-8 weeks onset of illness. Mean titer of anti-Vi IgG antibody of post vaccinated sera (titer 1050 ± 530.5) was 3.4 fold high from pre vaccinated sera (titer 312.5± 129.2) though it was not statistically significant (p>0.05). Anti-Vi IgG antibody titer response of pre and post vaccinated human sera exhibited some extent of correlation with bactericidal titer in our study (spearman‟s correlation coefficient test r=0.6, p<0.05). Anti-Vi IgG antibody response (>cut off OD 0.41 at 1:100 serum dilution) was positive in 67.5% (46/70) and 77.8% (7/9) of acute and convalescent typhoid cases, respectively, compared to 52% (13/25) in control cases. IgG antibody against S.Typhi surface, envelope and whole cell proteins was positive in all (100%) acute and convalescent typhoid cases compared to 30.8% to 55.6% in control participants. Mean IgG antibody levels against S.Typhi surface, envelope and whole cell proteins were significantly raised (p<0.05) in acute stage of typhoid fever and persisted during convalescent period (after 6-8 wks) compared to individuals in healthy and diseased groups. Water extracted surface protein of S.Typhi showed similar SDS-PAGE pattern to those of envelope protein extracted by Tris-sucrose-EDTA buffer. A total of ten (10) protein components, viz, 180, 100, 82, 66, 58, 50, 41, 37, 33, 25 kDa were common in surface and envelope proteins and three additional proteins (126 kDa, 35 kDa, 21 kDa) were also identified in whole cell extract of S.Typhi. Seven (7) out of 10 surface protein of S. Typhi (i.e. 180, 100, 82, 66, 58, 50, 37 kDa) were immunoreactive by Western blot analysis using sera from typhoid cases. Also, the 58 kDa and 37 kDa surface proteins of S.Typhi showed most frequent (92% and 80%) immunoreactivity. The results of the present study showed that prime boost strategy could be applied to get enhanced systemic and mucosal anti-Vi antibody responses using Vi CPS antigen in combination with KWC. The method was able to stimulate secondary booster effect of T independent plain Vi polysaccharide antigen. High salmonellacidal antibody detected in early period of typhoid fever could be a promising serological marker for S. Typhi acute infection. Highly immunoreactive surface or envelope protein of S. Typhi could be used as potential vaccine antigen or diagnostic marker.