Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and β-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli

Demographic and clinico-epidemiological features of dengue fever in Faisalabad, Pakistan.

This cross-sectional study was carried out to explore the epidemiological and clinical features of dengue fever in Faisalabad, Pakistan during 2011 and 2012. During the study period, anti-dengue IgM positive cases were reported in the post-monsoon period during the months of August-December. Certain hotspots for the dengue infection were identified in the city that coincide with the clusters of densely populated urban regions of the city. Out of total 299 IgM positive patients (male 218 and female 81); there were 239 dengue fever (DF) and 60 dengue hemorrhagic fever (DHF) patients. There was decrease in the median age of dengue patients from 31 years in 2011 to 21.5 years in 2012 (p<0.001). Abdominal pain was seen in 35% DHF patients followed by nausea in 28.3%, epistaxis in 25% and rash in 20% patients (p<0.05). Patients reported to be suffering from high-grade fever for an average of 8.83 days in DHF as compared to 5.82 days in DF before being hospitalized. Co-morbidities were found to be risk factor for the development of DHF in dengue patients. Clinical and laboratory features of dengue cases studied could be used for the early identification of patients at risk of severe dengue fever

Molecular model of fusion proteins and their CPORT analyses.

<p>(A-C) Molecular models of HSPx-tnPstS1 (A), HSPX-PE35 (B) and HSPX-FbpC1 (C). HSPX in each of the three fusion protein is shown as pink, whereas tnPstS1, PE35 and FbpC1 are shown in cyan, green and yellow color respectively. CPORT analyses of these fusion molecules (D-F) show the region marked as red, green and blue that are active, supporting and non-supporting, respectively in antibody binding. The epitope region in HSPX-tnPstS1 (D) and HSPX-PE35 (F) are shown mostly in red or green. However, the epitope regions of HSPX-FbpC1 Shown in black (C) appear blue in F.</p

Expression and purification of native antigens of <i>M</i>. <i>tuberculosis</i> and their fusion proteins.

<p>Expression and purification of native antigens of <i>M</i>. <i>tuberculosis</i> and their fusion proteins.</p

Primers used in PCR with the restriction sites shown as underlined.

<p>Primers used in PCR with the restriction sites shown as underlined.</p

Circular dichroism spectra of individual and fusion proteins.

<p>CD spectrum of HSPX, PE35 and HSPX-PE35 (A); HSPX, tnPstS1 and HSPX-tnPstS1 (B) and HSPX, FbpC1 and HSPX-FbpC1 (C) over the range of 190nm to 280nm.</p

Scheme for the construction of the fusion molecules HSPX-PE35, HSPX-tnPstS1 and HSPX-FbpC1.

<p>Scheme for the construction of the fusion molecules HSPX-PE35, HSPX-tnPstS1 and HSPX-FbpC1.</p

Antibody response of individual and fusion proteins with polyclonal antisera.

<p>(A) Western blot analyses of HSPX-PE35, HSPX-tnPstS1 and HSPX-FbpC1 with anti-HSPX (lanes 2, 4 & 6); anti-PE35 (lane 3); anti-tnPstS1 (lane 5) and anti-FbpC1 (lane 7). (B) Bar graph of OD_450/630 obtained from ELISA of individual and fusion antigens against anti-HSPX, anti-PE35, anti-PstS1 and anti-FbpC1.</p