Immune response against Pythium Insidiosum in Thai patients with immunotherapy

Abstract

The highest incidence of human pythiosis, the life threatening disease, has been found in Thailand. In the present, immunotherapy has been commonly combined with surgery and antifungal drug for treatment. It has been reported that after vaccination, the immune response switches from TH2 to TH1, however, no clear evidence of the isotypes were demonstrated. Thus, here, the immune responses, IgG, IgG1 (represent of TH1 type) and IgG4 (represent of TH2 type) isotype approach, were analyzed. Three groups of 13 patients were divided, base on the clinical, manifestation and the period of treatment. There were long term response (LRg, ≥14-34 mo.; n = 4), short term response (SRg, <14 mo.; n = 6), and non response (NRg, n = 3) group. First, the IgGs level from pre-vaccination serum against crude protein, prepared from each of P. insidiosum clade, ATH (MTPI19), BTH (PMS), and CTH (PC), were determined using ELISA. The result showed that total IgG antibody level against C-PC ( =283.2, SD=302.2) were significantly higher than those against other two antigens C-MTPI19 ( =31.15, SD=19.81) and C-PMS ( =46.85, SD=25.50) (P<0.001). For the specific IgG1 antibody level recognized C-PC ( =458.0, SD=735.7) was significantly higher (P<0.05) than C-MTPI19 ( =54.23, SD=43.48) but not C-PMS ( =119.0, SD=162.6). In terms of specific IgG4 antibody, the levels against C-PC ( =37.38, SD=50.0) and C-PMS ( =27.23, SD=27.87) were not significantly higher (P>0.05) than C-MTPI19 ( =18.15, SD=28.03). After that, all the IgGs levels in PIA-treated patient sera against C-MTPI19 (prepared from the PIA strain) were compared with pre-vaccination sera in each individual. Two times difference in level was the criteria as the level changing. The IgG1 level increasing was found in only 2/4 case (PY22, PY51) in LRg whereas very slightly change was found in the other 2 cases (PY5, PY30). This might implied that these patients were recovered. In addition, no typical change was observed in both SRg and NRg. Finally, all the sera were exposed to C-MTPI19 antigen to study their antigenic profiles by western blot. Four bands, ~120, ~55 and ~40-~34 kDa, only one band, ~120 kDa, and three bands, ~32-~28 and ~24 kDa, were commonly found against total IgG, IgG1 and IgG4 antibody, in order, in all 13 pre-vaccination sera. In contrast, the variation of profile against IgG (11 profiles), IgG1 (9 profiles) and IgG4 (10 profiles), antibody in post-vaccination sera in both number and the density were revealed. These were found mostly in the low molecular weight antigen, ~55, ~40-~34 and~28 kDa. Since the limitation of the case number and their different clinical status, it was hardly to conclude the switching TH2 to TH1 response. More study of their cytokine is required. However, this study demonstrated the optimum antigen for the ELISA technique (C-PC) and western blot analysis (C-MTPI19).