Figure 2.5: Vaccination schedule used on BALB/c mice showing bleeds and inoculations. Balb/c mice were acclimatized from days -7 to 0, and pre-bled before intramuscular inoculation with 3 x 105 pfu on days 0, 21 and 42.
Bleeds were taken on days 14, 35 and 56.
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The immunogenicity of MVA-SARS-2-SΔTM was assessed in BALB/c mice. A group of five mice were inoculated intramuscularly with 3 x 105 pfu of the recombinant MVA-SARS-2-SΔTM, according to an experimental schedule (Table 2.4) that was approved by the University of Cape Town Animal Ethics Committee (reference UCTAEC020_024). Prior to the inoculations mice were anaesthetised with a mixture of ketamine/xylazine which was done with a peritoneal injection. Three inoculations were done at three week intervals with 50 µl MVA-SARS-2-SΔTM resuspended in 100 µl 1 X PBS (Gibco, South Africa). The vaccine was prepared by pipetting up and down to mix before being vortexed gently for 30 seconds. Mice were injected with 50 µl by needle in the right tibialis muscle.
2.7.1.1 Binding antibody Enzyme-Linked Immunosorbent Assay (ELISA)
The serum binding antibody ELISA was used to quantify SARS-CoV-2 spike glycoprotein antibody titres as previously described by Margolin et al., [228].
A 96-well Maxisorb™ microtitre plate (NunC) was coated overnight at 4 ˚C with 10 ng/well of recombinant SARS-CoV-2 Spike Protein (S-ECD) (aa14-1213) His Tag Recombinant Protein (Invitrogen, USA).
The plate was washed three times with 1 X PBS (Gibco, South Africa) to remove any unbound antigen.
The plate was blocked with 5% blocking buffer (Appendix A) for 1 hour at room temperature to decrease non-specific binding. Five PBST (PBS containing 0.1% Tween (Roche, USA) washes followed, with tapping on paper towel to remove any residual buffer.
Pooled mice sera were diluted 1:10 and thereafter serially diluted 1:4 before plating in duplicate and incubation overnight. The plate was washed with PBST five times.
A goat polyconal anti- mouse IgG HRP conjugate antibody (1:10 000) (ab97023) (Abcam, USA), was added and incubated at room temperature for 1 hour before repeating the five PBST washes.
The plate was developed by adding 100 µl/well of TMB ELISA Substrate (Abcam, USA) and incubating in the dark for 10 minutes to allow the signal to develop. The reaction was stopped by
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adding 100 µl/well of 1N H2SO4. Readings were done on the SoftMax Pro software at 450nm and the data were calculated as previously described [229] before being plotted using GraphPad Prism.
2. 7.1.2 IFNγ- Enzyme-Linked Immune Absorbent Spot (ELISpot)
The ELISpot assay was used to measure the frequency of splenocytes secreting IFNγ as a measure of the induction of cellular immunity by the vaccine. A PVDF membrane 96-well plate was coated with mouse IFNγ SARS-CoV-2 capture antibody (1:200) and incubated at 4 °C overnight in a fridge.
The capture antibody was discarded, and wells were washed with 200 µl 1 x R10 ([1 ml Penicillin- Streptomycin antibiotic (Gibco, South Africa); 200mM L-Glutamine (Gibco, South Africa); 10 ml CTL-Test Medium (ImmunoSpot, Germany); 50mM 2-Mercapto-ethanol (Sigma-Aldrich, USA)].
Aliquots of 200 µl R10 were dispensed into each well and the plate was incubated for 2 hours at room temperature.
Spleens from the five vaccinated mice were harvested on day 56 and pooled. A cell suspension of splenocytes was prepared and centrifuged at 2500 rpm for 10 minutes on the Jouan CR422 Centrifuge (Thermo Scientific, USA). The supernatant was decanted, and the pellet was resuspended in 50 ml 1 X Rosewell Park Memorial Institute Medium 1640 with Glutamax (Gibco, South Africa) The solution was centrifuged again at 2500 rpm for 10 minutes, before resuspension in 1 X RPMI and removal of fibrin clots. After an additional round of centrifugation, the pellet was resuspended with 1 ml home-made Red Blood Cell Lysis buffer (Appendix A) to lyse red blood cells. The solution was mixed by inverting 5-6 times and then the samples was incubated for 1 -2 minutes at room temperature before being topped up with 1 X RPMI (Sigma-Aldrich, USA) to a final volume of 40 ml. The lysed cells were centrifuged at 1350 rpm for 7 minutes. The pellet was resuspended in 2 ml R10 growth media (Gibco, South Africa) and inverted several times for adequate mixing. The cells were counted as described in Section 2.2.4 and adjusted to a concentration of 5 x 106 / ml.
Peptides corresponding to the SARS-CoV-2 spike protein were synthesized (GenScript Biotech, USA), diluted in R10 medium to 0.2 µg/well, and used to stimulate splenocytes. The peptide pool included 316 peptides for the Severe Acute Respiratory Syndrome-related Coronavirus Virus 2
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spike glycoprotein (cat#RP30020) (158 peptides for subunit A and 158 for subunit B) (Appendix C). A stimulant, ConA (Sigma-Aldrich, USA), was added at a concentration of 0.1 µg/well as a positive control to confirm the assay’s functionality. An irrelevant peptide (0.2 µg/well) was included as a negative control. Peptides or stimulants were added in triplicate for each mouse.
Additional controls of blank wells with R10 medium and no cells were added to determine background responses.
The blocking solution was discarded from each well and 100 µl peptides or stimulants were added, with ConA being added last. Splenocytes were added and plates were incubated at 37 ℃, 5% CO2
for 22-24 hours.
Cells were removed and wells were washed 3 times with 200 µl H20 before being washed three times with PBST wash buffer (Appendix A). Excess buffer was removed by blotting the plate on a paper towel. Biotinylated detection antibody was added (1:250), and the plate was incubated for 2 hours at room temperature in the dark. The detection antibody was discarded, and wells were washed 3 times with PBST. The enzyme Streptavidin-HRP (0.5 mg/ml) (BD Biosciences, South Africa) was added prior to a 1-hour incubation at room temperature in the dark.
The Streptavidin solution was discarded, and plates were washed 3 times with PBST followed by three subsequent washes with 1 X PBS. The NovaRED Substrate (Vector Laboratories, USA) was added as per manufacturer’s instructions. The reaction was allowed to continue for 10 minutes and thereafter was stopped by washing of the plate 5-6 times in running cold water. The plate was stored in the dark at room temperature overnight to facilitate before being viewed and spots counted with an automatic ELISPOT reader (CTL Technologies, Cleveland, Ohio) and the Immunospot Version 3.2 software. Spot numbers were averaged for the triplicate reactions per mouse.
The number of spots was averaged for each reaction from triplicate wells. The standard deviation and mean were calculated, adjusted and expressed as 1 x 106 spots forming units (sfu)/ml. The mean number of spots and standard deviation of the irrelevant peptide group were used as background responses. Positive responses to the SARS-CoV-2 spike A and B peptides were those greater than the mean plus two standard deviations. Background responses were subtracted from the positive SARS-CoV-2 spike A and B responses, and these were expressed as net sfu/106 splenocytes.
71 2. 7.1.3 Neutralization assays
Neutralizing antibody titres were assessed using a SARS-CoV-2 pseudovirus as previously described [228] for which I fully acknowledge Dr. Georgia Schafer (Full member, Institute of Infectious Diseases and Molecular Medicine) who conducted the assay. Briefly, the pseudoviruses were generated by co-transfection of HEK-293TT cells with an HIV plasmid pNL4-3.Luc.R-.E- (aidsreagent #3418) and pcDNA3.3-SARS-CoV-2-spike Δ18 prepared by Rogers et al., [135].
Supernatants were harvested at 3 days post transfection and incubated with serially diluted sera (five-fold) heat-inactivated mouse plasma for 60 minutes at 37˚ C. HEK-293T cells expressing the ACE2 receptor were then transfected with the psuedovirus mixture and lysed at 3 days post infection using the Cell Culture Lysis Reagent (Promega). Luciferase activity was assessed using the GloMax® Explorer Multimode Microplate Reader (Promega) [230] and the Luciferase assay system (Promega). The half-maximal inhibitory dilution (ID50) was calculated and plotted using a non-linear regression on GraphPad Prism [231].
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