In conclusion, we provide insights into both the immunologic determinants of viral clearance and protection. While T cells were important in the clearance of primary infection, they were not required for protection against reinfection or vaccine-mediated protection, likely due to sufficient antibody-mediated immunity. These results are reassuring as they indicate that a robust humoral immune response is sufficient even in the setting of decreased neutralizing capacity. These results also have important public health and vaccine development implications, as they suggest that antibody mediated immunity may be a sufficient correlate of protection.
MATERIALS AND METHODS
The objective of this study was to identify the adaptive immune determinants of SARS-CoV-2 viral clearance and protection. To address the determinants of SARS-CoV-2 clearance, we chose to employ our recently developed mouse model of SARS-CoV-2 infection as it allowed us to utilize an array of genetic models combined with well-established cellular depletion techniques. To investigate the determinants of protection mediated by either prior infection or mRNA vaccine currently being used in humans, we utilized a commercially available human ACE2 transgenic mouse that has been well established as a model of SARS-CoV-2 lethality and combined this with cellular depletion techniques and infection by immune evasive SAR-CoV-2 variants.
All procedures were performed in a BSL-3 facility (for SARS-CoV-2–infected mice) with approval from the Yale Institutional Animal Care and Use Committee and Yale Environmental Health and Safety.
Cell lines and viruses
Six to twelve-week-old mixed sex C57Bl/6J (WT), B6.129S7-Rag1tm1Mom/J (Rag1−/−), B6.129S2-Ighmtm1Cgn/J (μMT), and B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18) were purchased from Jackson laboratories, and were subsequently bred and housed at Yale University. All procedures used in this study (sex-matched, age-matched) complied with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee.
Mice were anesthetized using 30% v/v Isoflurane diluted in propylene glycol. Using a pipette, 50μL of SARS-CoV-2 was delivered intranasally.
CD4+ and CD8+ T cell depletion
Indicated mice were injected intraperitoneally with PBS or 200ug in 200ul diluted in PBS of either anti-mouse CD4 (BioXcell InVivoMab Clone GK 1.5), anti-mouse CD8 (BioXcell InVivoMab Clone 2.43) or both at indicated time points. For local pulmonary CD8 depletion,100ug of anti-mouse CD8 Ab was given intranasally in 50ul of volume diluted in PBS.
Adoptive transfer experiments
WT AAV-hACE2 mice were infected as indicated above. At 14 DPI animals were euthanized and blood and mediastinal lymph nodes collected. Blood was allowed to coagulate at room temperature for 30minutes and then was centrifuged at 3900rpm for 20 min at 4c. Serum was collected, and anesthetized mice (30% v/v Isoflurane diluted in propylene glycol) were injected with 200ul serum with a 32 g 8mm syringe via retro orbital route. Mediastinal lymph nodes were mechanically dissociated in 500ul of cold PBS and passed through a 40um filter. Cells were counted in duplicate on the Countess II (Invitrogen) and total T-cell isolation was performed via negative selection using the EasySepTM mouse T cell isolation kit (Stemcell). Isolated cells were counted and 2×106 total T cells were diluted in 200ul PBS and retro-orbitally injected into anesthetized mice.
Used vials of Pfizer/BioNTec BNT162b2 mRNA vaccine were acquired from Yale Health pharmacy within 6 hours of opening. No vaccines were diverted for the purposes of this study and the vaccine residual volumes described in this study were obtained only after usage by Yale Health and prior to discarding. All vials contained residual vaccine volumes (less than 1 full dose per vial, diluted to 100ug/ml per manufacturer’s instructions) and remaining liquid was removed with spinal syringe and pooled. Vaccine was not stored or refrozen prior to use, but was directedly used. Mice were anaesthetized using a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1), and 10ul (1mg) of undiluted vaccine was injected into left quadriceps muscle with a 32 g syringe.
Viral RNA analysis
At indicated time points mice were euthanized in 100% Isoflurane. ~50% of total lung was placed in a bead homogenizer tube with 1ml of PBS+2%FBS +2% antibiotic/antimycotic (Gibco). After homogenization 250ul of this mixture was placed in 750ul Trizol LS (Invitrogen), and RNA was extracted with RNeasy mini kit (Qiagen) per manufacturer protocol. To quantify SARS-CoV-2 RNA levels, we used the Luna Universal Probe Onestep RT-qPCR kit (New England Biolabs) with 1 ug of RNA, using the US CDC real-time RT-PCR primer/probe sets for 2019-nCoV_N1.
Lung homogenates were cleared of debris by centrifugation (3900rpm for 10 min). Infectious titers of SARS-CoV-2 were determined by plaque assay in Vero E6 cells in DMEM supplemented NaHCO3, 2% FBS 0.6% Avicel RC-581. Plaques were resolved at 48hrs post infection by fixing in 10% Neutral buffered formalin for 1 hour followed by staining for 1 hour in 0.5% crystal violet in 20% ethanol for 30 min. Plates were rinsed in water to visualize plaques.
SARS-CoV-2 specific-antibody measurements
Pseudovirus neutralization assay
Intravascular labeling, cell isolation, and flow cytometry
Prism 9 (GraphPad) was used for all analysis. Statistical significance was determined using one-way ANOVA or two-way Anova with Tukey’s multiple comparison test or Student’s two-tailed, unpaired t test where indicated in the figure ledged. P < 0.05 was considered statistically significant.
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