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Adaptive immune determinants of viral clearance and protection in mouse models of SARS-CoV-2

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DISCUSSION

Here we describe our use of mouse models of SARS-CoV-2 infection to identify components of adaptive immunity responsible for both viral clearance during primary infection and protection from reinfection. We found that, similar to COVID-19 patients (23) (24) (34) (35), mice require an adaptive immune response to clear SARS-CoV-2. This indicates that in this model, innate immunity is insufficient to clear infection, which may be due to antagonism of interferon pathway by the virus (36). We found that either cellular or humoral arms of the adaptive immune system are sufficient to promote viral clearance, as either B cell deficient mice or mice treated with αCD8 or αCD4 antibodies alone can clear SARS-CoV-2 infection, albeit slower than in the setting of a fully competent adaptive immune system. However, depletion of both CD4+ and CD8+ T cells in WT mice or depletion of CD8+ T cells in μΜΤ mice led to the inability to clear the virus during primary infection. We also found that the major contribution of CD4+ T cells to viral clearance during acute infection is likely the promotion of antibody production, as treatment of μΜΤ mice, devoid of antibodies, with αCD4 only led to a small increase in viral RNA. These results indicate that CD4+ cytotoxic T cells do not play a major role in viral clearance during primary infection. Consistent with these results, antigen specific CD4+ T cell profiling of acute and convalescent COVID-19 patients has shown that circulating T follicular helper cells are associated with reduced disease severity (37), indicating the importance of antibody promoting CD4+ T cells clearance of acute infection. COVID-19 patient studies have shown that antigen specific CD4+ T cells can be detected as early as 2-4 days from symptom onset, and their early detection was found to be associated with improved outcomes and viral clearance (37, 38). These data clearly show that both humoral and cellular adaptive immunity contribute to clearance of SARS-CoV-2 during primary infection, and are consistent with recent patient studies showing a correlation between clinical outcome and a robust coordinated adaptive response requiring CD4+ T cells, CD8+ T cells, and antibodies (37, 39).
Adaptive immune memory has been detected for as long as 8 months after primary infection with SARS-CoV-2 consisting of memory CD4+ T cells, CD8+ T cells, B cells and antibodies (1, 4, 40, 41). Epidemiologic evidence also indicates that immune memory is sufficient to protect against reinfection in patients who have seroconverted, and mRNA-based vaccines have also show >90% efficacy in preventing COVID-19 in both phase III clinical trials and in real world settings (3, 1315). As both vaccines and natural infection induce multiple types of immune memory and because reinfections in humans have been so rare, identifying the distinct components of adaptive immune memory that confer protection remain unknown. While protective immunity after primary infection or vaccine has been shown in multiple animal models, few have identified correlates of protection (512). Many of these studies have also shown that adoptive transfer of serum from convalescent or vaccinated animals or from humans confers protection. However, the role that T cells play in conferring protection either from vaccination or natural infection has received less attention than antibodies until recently, due to emerging immune evasive variants. To begin to assess the individual components of adaptive immunity that confer protection, McMahan et al. (6) recently showed that macaques treated with CD8-depleting antibody had higher RNA viral loads in nasal swab at 1 DPI than mock treated animals. However, this difference normalized by 2 DPI, and no difference at any time post infection was noted in BAL, suggesting that CD8 TRM may only confer added protection in the upper respiratory tract of macaques. In mouse models, nucleocapsid (N) based vaccines, which rely mostly on T cell-based protection, have shown differing results. Matchett et al. found that IV administration of Adenovirus 5 vector expressing N protein conferred protection (42), while Dinnon et al. showed no protection via foot pad injection of Venezuelan equine encephalitis virus replicon particle expressing N (43). While it is possible that the different vectors led to differential protection, it seems more likely that IV administration led to antigen specific TRM in the lungs, which may not have developed in the setting of footpad inoculation.
To expand upon these studies in identifying the individual roles of humoral and cellular immunity in protection, we performed adoptive transfer experiments in mice deficient in adaptive immunity (RAG−/−) and found that both T cells and convalescent serum from previously infected animals could reduce viral load. However, only serum was able to achieve viral clearance. To identify the level of antibodies required for protection we performed variable mRNA vaccine dosing and showed that dose dependent protection highly correlated with antibody levels and identified IC50 of ~1:30 as providing 50% protection in K18 mice. Consistent with McMahan et al. (6), we showed that memory CD8+ T cells, either TRM or circulating memory cells, are not required to confer significant protective immunity in the lower respiratory tract in either convalescent or vaccinated animals. In these experiments, it is likely that the CD8+ T cell contribution is limited in the setting of overwhelming humoral response, given that prime/boost vaccine or convalescent mice maintained IC50 >1:1000 to B.1.351. These data support antibody responses as the key determinant of protection from mRNA vaccines or natural infection against SARS-CoV-2 reinfection by current circulating VOCs. One caveat to this conclusion is that we did not specifically address whether TRM cell mediated immunity, in the absence of humoral responses is sufficient for protection as was shown by Matchett et al. (42). Additionally, we did not specifically address the role of CD4+ T cells in protective immunity, these have been shown to be important in SARS-CoV-1 protection in the absence of antibody mediated immunity (44).
SARS-CoV-2 variants that significantly evade humoral immunity have recently been identified. The variant which has consistently shown the strongest ability to evade humoral responses in in vitro neutralization assays is B.1.351 (17, 33, 45). We found that while there was some loss of protection against infection in either vaccinated or convalescent mice, humoral immunity was sufficient to completely protect from disease in mice infected by B.1.351. These data build upon a recent case control study that reported increased rates of vaccine breakthrough with B.1.351 in fully vaccinated subjects, though most cases were asymptomatic (16). However, the degree to which mRNA vaccinated people are susceptible to clinical disease by B.1.351 remains unclear. While we identified antibodies as a correlate of protection and a IC50 titer of ~1:30 as proving 50% protection in K18 mice, extreme caution should be used with application of this number beyond this model. Further studies that build on the work of Khoury et al. (46) will be required to assess the neutralizing antibody titers required to prevent COVID-19 in humans.

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

Study design

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.

Safety

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

As reported in previous manuscripts (20), Vero E6 kidney epithelial cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% sodium pyruvate (NEAA) and 5% fetal bovine serum (FBS) at 37°C and 5% CO2. Huh7.5 and 293T cells were cultured in DMEM supplemented with 1% sodium pyruvate and 10% fetal bovine serum (FBS) at 37°C and 5% CO2. The cell line was obtained from the ATCC and has been tested negative for contamination with mycoplasma. SARS-CoV-2 isolate hCOV-19/USA-WA1/2020 (NR-52281) and SARS-CoV-2 Isolate hCoV-19/South Africa/KRISP-K005325/2020 (NR-54009) was obtained from BEI Resources and was amplified in either VeroE6 (ATCC CRL-1586) cells or VeroE6 cells overexpressing hACE2 and TMPRSS2 (kindly provided by Barney Graham NIH-VRC). Cells were infected at a MOI 0.01 for two-three days to generate a working stock and after incubation the supernatant was clarified by centrifugation (500 g × 5min) and filtered through a 0.45-micron filter. To concentrate virus, filtered supernatants were applied to Amicon Ultra- 15 centrifugal filter (Ultracel 100k) and spun at 2000 rpm for 15 min. The supernatant was then aliquoted for storage at −80°C. Viral titers were measured by standard plaque assay using Vero E6 cells described below.

Mice

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.

AAV infection

As previously described (20), adeno-associated virus 9 encoding hACE2 was purchased from Vector biolabs (AAV-CMV-hACE2). Animals were anaesthetized using a mixture of ketamine (50 mg kg−1) and xylazine (5 mg kg−1), injected intraperitoneally. The rostral neck was shaved and disinfected. A 5mm incision was made and the salivary glands were retracted, and trachea was visualized. Using a 32 g insulin syringe a 50μL bolus injection of 1011 genomic copies of AAV-CMV-hACE2 was injected into the trachea. The incision was closed with 4-0 Vicryl suture. Following intramuscular administration of analgesic (Meloxicam and buprenorphine, 1 mg kg−1), animals were placed in a heated cage until full recovery. Mice were used for SARS-CoV-2 infection at 14-21 days post AAV administration.

SARS-CoV-2 infection

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.

Vaccination

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.

Viral titer

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

ELISAs were performed as previously described (20, 47) and reproduced here for convenience. Briefly, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5mg/ml respectively and incubated at room temperature (RT) for 30 min before use to reduce risk from any potential virus in serum. 96-well MaxiSorp plates (Thermo Scientific #442404) were coated with 50 μl/well of recombinant SARS CoV-2 S1 protein (ACROBiosystems S1N-C52H3) and RBD (ACROBiosystems SPD-C52H3) at a concentration of 2 μg/ml in PBS and were incubated overnight at 4°C. The coating buffer was removed, and plates were incubated for 1h at RT with 250 μl of blocking solution (PBS with 0.1% Tween-20, 3% milk powder). Serum was diluted in dilution solution (PBS with 0.1% Tween-20, 1% milk powder) and 100 μl of diluted serum was added for two hours at RT. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20) and 50 μl of HRP anti-mouse IgG (Cell Signaling Technology #7076, 1:3,000) diluted in dilution solution added to each well. After 1 hour of incubation at RT, plates were washed three times with PBS-T. Plates were developed with 100 μl of TMB Substrate Reagent Set (BD Biosciences #555214) and the reaction was stopped after 15 min by the addition of 2 N sulfuric acid. Plates were then read at a wavelength of 450 nm and 570nm.

Pseudovirus production

Vesicular stomatitis virus (VSV)-based pseudotyped viruses were produced as previously described (4850). Vector pCAGGS containing the SARS-CoV-2 Wuhan-Hu-1 spike glycoprotein gene was produced under HHSN272201400008C and obtained through BEI Resources (NR-52310). The sequence of the Wuhan-Hu-1 isolate spike glycoprotein is identical to that of the USA-WA1/2020 isolate. The spike sequence of the B.1.351 variant of concern was generated by introducing the following mutations: L18F, D80A, D215G, R246I, K417N, E484K, N501Y, and A701V. 293T cells were transfected with either spike plasmid, followed by inoculation with replication deficient VSV expressing Renilla luciferase for 1 hour at 37°C (9). The virus inoculum was then removed, and cells were washed three times with warmed PBS. Supernatant containing pseudovirus was collected 24 and 48 hours post inoculation, clarified by centrifugation, concentrated with Amicon Ultra Centrifugal Filter Units (100 kDa), and stored in aliquots at -80°C. Pseudoviruses were titrated in Huh7.5 cells to achieve a relative light unit (RLU) signal of ~600 times the cell-only control background.

Pseudovirus neutralization assay

3×104 Huh7.5 cells were plated in each well of a 96-well plate the day before infection. On the day of infection sera for neutralization assay were heat-inactivated for 30 min at 56°C. Sera were tested at a starting dilution of 1:10 (Fig. 4) and 1:50 (Fig. 5) for USA-WA1/2020 pseudovirus and 1:40 for B.1.351 pseudovirus, with 8 or 12 two-fold serial dilutions. Serial dilutions of sera were incubated with pseudovirus for 1 hour at 37°C. Growth media was then aspirated from the cells and replaced with 100 μl of serum/virus mixture. Luciferase activity was measured at 24 hours post infection using the Renilla-Glo Luciferase Assay System (Promega). Each well of cells was lysed with 20μl Passive Lysis Buffer, freeze thawed 1x, and then mixed with 20 μl Luciferase Assay reagent. Luminescence was measured on a microplate reader (SpectraMax i3, Molecular Devices). Half maximal inhibitory concentration (IC50) was calculated as using Prism 9 (GraphPad Software) non-linear regression.

Intravascular labeling, cell isolation, and flow cytometry

To discriminate intravascular from extravascular cells, mice were anesthetized with 30% Isoflurane and injected i.v. with APC/Fire 750 CD45 Ab (30-F11, AB_2572116 (BioLegend, #103154) and after 3 min recovery, mice were euthanized. Tissues were harvested and analyzed as previously described (20). In short, lungs were minced with scissors and incubated in a digestion cocktail containing 1 mg/ml collagenase A (Roche) and 30 μg/ml DNase I (Sigma-Aldrich) in RPMI at 37°C for 45 min. Tissue was then filtered through a 70-μm filter. Cells were treated with ammonium-chloride-potassium buffer and resuspended in PBS with 1% BSA. Single cell suspensions were incubated at 4°C with Fc block and Aqua cell viability dye for 20 min. After washing with PBS, cells were stained with (anti-CD103(BV42, 2E7, AB_2562901, BioLegend #121422)), (anti-CD3(BV605, 17A2, AB_2562039, BioLegend #100237)), (anti-CD44(BV711, IM7, AB_2564214, BioLegend #103057)), (anti-CD62L(FITC, MEL-14, AB_313093, BioLegend # 104406)), (anti-CD8a(PerCP/Cy5.5, 16-10A1, AB_2566491, BioLegend #305232)), (anti-CD69(PE/Cy7, H1.2F3, AB_493564, BioLegend #104512)), (anti-CD183(CXCR3)(APC, CXCR3-173, AB_1088993, BioLegend #126512)), (anti-CD4(AF700, GK 1.5, AB_493699, BioLegend #100430)), and (PE-SARS-CoV-2 S 539-546 MHC class I tetramer (H-2K(b)) for 30 min at 4°C. After washing with PBS, cells were fixed using 4% paraformaldehyde. Cell population data were acquired on an Attune NxT Flow Cytometer and analyzed using FlowJo Software (10.5.3; Tree Star). See (Fig. S5) for gating strategy.

Statistical analysis

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.

Graphics

Created with BioRender.com

Acknowledgments

We thank H. Dong and M. Linehan for technical and logistical assistance. We thank Patrick Roberts and Bryan Cretella from the Yale Health Pharmacy for providing residual vaccine used in this study. We thank Craig Wilen, Jin Wei, and Jennifer Chen for technical assistance and providing reagents. We thank Carolina Lucas for technical expertise and for critical feedback on the manuscript. We thank Sidi Chen and Lei Peng for kindly providing the plasmid to express SARS-CoV-2 B.1.351 Spike. We thank Barney Graham (NIH-VRC) for kindly providing VeroE6 cells overexpressing ACE2 and TMPRSS2. We thank the NIH Tetramer Core Facility for providing PE labeled SARS-CoV-2 S 539-546 tetramer (H-2K(b)). We also give special recognition to Ben Fontes and the Yale EH&S Department for their on-going assistance in safely conducting biosafety level 3 research. Funding: This work was supported by NIH grants T32AI007517 and K08AI163493 to B.I., T32AI007019 to T.M., T32GM007205 and F30CA239444 to E.S., and R01AI157488 to A.I. Support for this work was also provided by Fast Grant from Emergent Ventures at Mercatus Center, the Ludwig Family Foundation, and the G. Harold and Leila Y. Mathers Foundation to A.I. A.I. is an investigator of the Howard Hughes Medical Institute. Author contributions: B.I., S.B.O., and A.I. conceived of and designed the study. B.I, T.M., J.K., and E.S., performed investigation, B.M. generated critical reagents. B.I. and T.M. performed data analysis and visualization. Manuscript written by B.I. and A.I. All authors discussed the result and reviewed and commented on the manuscript. Competing interests: AI served as a consultant for Spring Discovery and Adaptive Biotechnologies. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. Further information and requests for resources and reagents should be directed to corresponding author A.I. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

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