Laboratory procedures
Blood sample collection and processing
After signing the consent form, with aseptic precautions 4 ml venous blood samples were collected at each time in Plain vacutainer tubes (1ml) and sodium heparin vacutainer tubes(3ml). The blood collected in plain vacutainer tubes were allowed to clot by leaving it undisturbed at room temperature for 30 minutes, followed by centrifugation at 1,000 x g for 10 minutes in a refrigerated centrifuged (cells in pellet), the supernatant was collected and transferred into a clean micro centrifuge tube, aliquoted and stored at –20°C after proper labelling. Sodium heparin containing vacutainer tubes were processed for the collection of PBMCs by density gradient centrifugation and immediately used in ELISpot assay. An extra 1ml Blood was collected in EDTA vacutainer tubes from the participants before 1st dose of vaccination, were labelled properly and sent to Department of Biochemistry, BSMMU for HbA1c determination by an automated analyzer (Sebia capillary).
Detection of IgG antibodies specific to SARS-CoV-2
Before 1st dose and 4 weeks after 1st dose of BNT162b2 mRNA (Pfizer-BioNTech) vaccination, using processed blood serum, IgG antibody specific to SARS-CoV-2 infection were detected by Automated LIAISON® XL Analyzer using FDA approved SARS-CoV-2 IgG chemiluminescence enzyme immunoassays kit (LIAISON® SARS-CoV-2 TrimericS IgG assay, REF 311510, Diasorin, Italy) following manufacturer instructions to assess the seroconversion rate. Detection range of assay was 4.80 to 2080 BAU/mL (Binding Antibody Unit/mL) and test results were reported as positive (\(\ge\) 33.8 BAU/mL) or negative (<33.8 BAU/mL).
Ex-vivo IFN-\(\gamma\) and IL-2 ELISpot assay
On each time point for each participant, sample collected in the sodium heparin vacutainer tubes was processed for the collection of PBMCs by density gradient centrifugation using Histopaque®-1077 (SigmaAldrich, Germany) by previous method\cite{Nilsson_2008} and ex-vivo ELISpot assays for IFN-\(\gamma\) and IL-2 were done using ELISpot assay-kit (AutoImmuneDiagnostika GmbH, Germany) according to manufacturer instructions. The pre-coated multi-test-plate (MTP) was adjusted to room temperature and was taken out of the bag under sterile conditions in Biosafety cabinet class II type A2. A 100 \(\mu\)L of RPMI-1640 medium was added in negative control well, a 100 \(\mu\)L of mix 1(80 \(\mu\)L medium + 20\(\mu\)L Pokeweed mitogen (PWM) (Thermo Fisher Scientific Inc., USA) (final conc. of Mix 1 working solution was 2\(\mu\)g/mL) was added in positive control\cite{Ra_kov__2005} and a 100 \(\mu\)L of mix 2 (80 \(\mu\)L medium + 20 \(\mu\)L of SARS-CoV-2 Spike RBD (RBM) Peptide Pool (SinoBiological Inc, China) stock solution (final conc. of Mix 2 working solution was 2\(\mu\)g/mL)\cite{King_2021} was added in test well. Finally, 100 \(\mu\)L of freshly isolated PBMC cell suspension adjusted at about 2x106 cells/ml was added in each well. After 24 hours incubation, plate was removed from the incubator. Wells were emptied and washed for 6 times with 200 \(\mu\)L of washing buffer in each well. A 10 \(\mu\)L of diluted AP-conjugated secondary antibody was added per well and incubated for 2 h at room temperature in a humid chamber. A 100 \(\mu\)L of diluted streptavidin was added per well and incubated for 1 h at room temperature in a humid chamber. Wells were emptied and washed for 6 times with 200 \(\mu\)L of washing buffer / well. A 100 \(\mu\)L of Substrate was added per well and incubated for 5-20 minutes at room temperature until spots were clearly visible. To stop the reaction, washing was done for 3 times with distilled water. The plate was allowed to dry thoroughly overnight by putting upside down over tissue paper. After drying the plate was evaluated with AID ELISpot Reader 7.0 System and the number of spots in the sample well was calculated at a prefixed count setting which indicates the number of PBMCs secreting IFN-\(\gamma\) or IL-2 accordingly.
Statistical analysis
After collection of all the required data, these were checked, verified for consistency and tabulated. Collected data processed using Microsoft Excel® 2019. The statistical analysis was conducted and the 95% confidence intervals for each category were calculated at 10% acceptable error level using using IBM® SPSS Statistics 25.0, jamovi 2.2.5 (based on R software), and DATAtab online statistical calculator. Dataset was tested for normality (Shapiro-Wilk and Kolmogorov-Smirnov) and for non-normal data non-parametric test as Mann-Whitney U-Test (two-tailed), Friedman Test etc. was used. A p value of <0.05 were considered significant.
Results
Out of 35 participants, 19 (54.3%) were diagnosed cases of type 2 diabetes mellitus (Group 1) and 16 (45.7%) were non-diabetic healthy control (Group 2). The mean age of type 2 diabetic group and healthy control group was 53.2±9.12 (SD) and 50.4±11.4 (SD) years respectively. Among type 2 diabetic participants (n=19), male and female ratio was 1.11:1, while it was 1:1.29 in case of healthy control group (n=16). Majority of the participants, were from urban areas (n=20, 57.1%) and belonged to middle-income families (n=23, 65.7%). Mean duration of sample collection after 2nd dose was 24.2±3.39 days. Among all participants, 16 (45.7%) were hypertensive and 19 (54.3%) were normotensive. Subdividing based on body mass index (BMI), 12 (34.29%) had a BMI \(\ge\)25 kg/m2 and 23 (65.71%) had a BMI <25 kg/m2 (Table 1).