key: cord-0266589-jb7f58g5
authors: Greer, Christopher D.; Kasden, Coral M.; Morales, Leon; Lundgreen, Kendall A.; Hicks, Philip D.; Carpenter, Lilia J.; Salas-Quinchucua, Cristhian; Tichy, Elisia D.; Maguire, Albert M.; Hoffman, Jennifer G.; Bailey, Robin J.; Zhou, Shangzhen; Luo, Angela; Chomistek, Steven J.; Kozyak, Benjamin W.; Bridges, Charles R.; Gordon, Gilad S.; Tabin, Geoffrey C.; Bates, Paul F.; Osorio, Jorge E.; Bennett, Jean; Stedman, Hansell H.
title: T Cell Predominant Response to AAV-Spike Protects hACE2 Mice from SARS-CoV-2 Pneumonia
date: 2021-08-17
journal: bioRxiv
DOI: 10.1101/2021.08.16.456441
sha: 990d4c0ed391f34adebace678c8ac96503a1a196
doc_id: 266589
cord_uid: jb7f58g5

Prevention of COVID-19 is widely believed to depend on neutralization of SARS-CoV-2 by vaccine-induced humoral immunity1,2, raising concern that emerging escape variants may perpetuate the pandemic3–6. Here we show that a single intramuscular injection of Adeno-Associated Virus-6 (AAV6) or AAV9 encoding a modified, N-terminal domain deleted (ΔNTD) spike protein induces robust cellular immunity and provides long-term protection in k18-hACE2 transgenic mice from lethal SARS-CoV-2 challenge, associated weight loss and pneumonia independent of vaccine-induced neutralizing humoral immunity. In both mice and macaques, vaccine-induced cellular immunity results in the clearance of transduced muscle fibers coincident with macrophage and CD8+ cytotoxic T cell infiltration at the site of immunization. Additionally, mice demonstrate a strong Type-1 polarized cellular immunophenotype and equivalent ex vivo T cell reactivity to peptides of wt and alpha (B.1.1.7) variant spike. These studies demonstrate not only that AAV6 and AAV9 can function as effective vaccine platforms, but also that vaccines can provide long-term efficacy primarily through the induction of cellular immunity. The findings may provide an alternative approach to containment of the evolving COVID-19 pandemic and have broader implications for the development of variant-agnostic universal vaccines against a wider range of pathogens.

Spike protein is a class I homotrimeric fusion glycoprotein that initiates viral entry of host cells via high-affinity S1 and S2 fragments of spike protein of the alpha variant and there was no change in the number of IFNg secreting 138 splenocytes (Fig. 2d) . Together, these experiments indicate that the cellular memory response against the spike is 139 strongly Type 1-polarized, and this response is not diminished by mutations present in the alpha variant spike.

To assess vaccine efficacy against lethal SARS-CoV-2 challenge and associated viral pneumonia, 6-8-week-141 old k18-hACE2 mice were administered a single dose IM injection of either 1E12 vg AAV9-M8, AAV9-M8B, AAV6-142 M8B, or AAV buffer as a mock vaccinated control group before intranasal inoculation with a lethal dose of 143 2.5x10^4 PFU of SARS-CoV-2 at 25 dpa (Extended Data Fig. 3a) . In order to compare tissues and viral RNA levels 144 between groups, we decided to terminate the experiment at 7 days post infection (dpi) with SARS-CoV-2, by which 145 time the majority of k18-hACE2 mice will have died or met objective criteria for euthanasia by the masked/blinded 

While the experimental design (termination at 7 dpi) did not allow for a longer assessment of differential 152 survival, it did permit assessment of other objective surrogates of vaccine-induced protection. Mock-vaccinated mice began progressively losing weight starting at 4 dpi whereas animals in the vaccinated groups maintained minutes according to a predetermined ramping protocol. Whereas 80% of the mock vaccinated mice performed revealed signs of severe interstitial pneumonia characterized by collapse of the alveolar spaces (Fig. 3e) . We 

Interestingly, our analysis of serum obtained at necropsy revealed that this robust protection occurred 169 despite the absence of vaccine-induced neutralizing humoral immunity (Fig. 3g) . This finding was confirmed in 170 mice by giving either AAV9-M8 or AAV9-M8B followed by a booster 5 weeks later. Three weeks post booster there 171 was still an absence of detectable neutralizing antibody (Extended Data Fig. 4) . However, to support the claim infection, we show that neutralizing antibodies in the challenged AAV6-M8B and AAV9-M8B groups, but not the 174 challenged mock vaccinated group, are significantly elevated relative to the unchallenged AAV6-M8B vaccinated 175 group at the completion of the experiment (Fig 3g) . To further support the claim of accelerated post-vaccination, 176 post-infection humoral immunity kinetics, we assayed for IgG titers against recombinant spike RBD. Some of the 177 unchallenged AAV6-M8B group did have detectable, albeit low levels, of anti-RBD IgG whereas all the challenged 178 mock vaccinated animals were below the limit of detection (Fig. 3h) . However, the challenged AAV6-M8B and 179 AAV9-M8B vaccinated mice were elevated 6.6-fold and 14.3-fold, respectively, relative to the unchallenged AAV6-180 M8B group and the challenged AAV9-M8B groups RBD IgG titer was significantly greater than that of the 181 challenged mock vaccinated group (Fig. 3h ). Together, these serological studies reveal that AAV-M8/M8B 182 vaccination alone produces a very mild humoral immune response with no detectable neutralizing antibody 183 component; however, upon infection vaccinated mice respond with an accelerated humoral response.

We next assessed long-term therapeutic efficacy as well as vaccine stability in real-world storage 185 conditions, specifically following long-term refrigeration. A cryofrozen vial of AAV6-M8B was thawed and used to 186 vaccinate k18-hACE2 mice (Cohort 1). The AAV6-M8B vial was then stored at 4 o C for 4 months before being used In aggregate, these challenge experiments demonstrate that vaccination with AAV-M8/M8B provides a 194 highly significant survival advantage to k18-hACE2 mice upon subsequent lethal challenge with SARS-CoV-2 ( Fig.   195 3). 

To preliminarily assess capsid efficacy and dose-response in non-human primates, four cynomolgus macaques 219 were vaccinated IM with either AAV6-M8B or AAV9-M8B at a dose of either 1E11 vg or 1E12 vg. At 29 dpa there 220 is significant mononuclear cell infiltration and the presence of cytotoxic CD8 T cells and CD11b+ macrophages 221 surrounding M8B expressing muscle fibers (Fig. 4a, b) . IFNg ELISpots performed on peripheral blood mononuclear 222 cells (PBMCs) isolated at 29 dpa demonstrated the presence of spike antigen-specific T cells in both macaques 223 that received the higher dose of 1E12 vg, but neither animal that received the lower dose of 1E11 vg (Fig. 4c ).

Finally, as observed in mice, there was a small increase in the anti-Spike-ECD IgG titers of both macaques that 225 received the 1E12 vg dose, but neither animal that received the dose of 1E11 vg and no vaccine-induced 226 neutralizing antibodies were detectable at 29 dpa at any dose (Fig. 4d, e) . These data strongly support our data 227 generated from mice and suggest a similar immune response to AAV-M8B in primates. 

Cells were then transfected with 0.4 ug plasmid DNA using lipofectamine 3000 in growth media for 5 hours at 37 o C 296 before being switched to C2C12 differentiation medium (DMDM high glucose, 5% horse serum, 1x anti-anti, 1x 297 NEAA, 1x glutamax) for 72 hours. Cells were then fixed with 10% NBF for 10 min at room temperature and then 298 washed for 5 minutes with PBS before being permeabilized in 0.5% Triton X-100 solution 10 minutes at room 299 temp. Cells were washed in PBS for 5 min before being blocked with 10% normal donkey serum (Abcam, ab7475, 300 Lot GR3234297-32) diluted in PBS for 20 minutes at room temperature, followed by incubation with rabbit Immediately prior to IM injection, the AAV was removed from the -80 o C freezer and thawed at room temperature.

The AAV vector was then diluted in formulation buffer (PBS + 0.001% Pluronic F-68) such that 50 ul of diluted 333 vector contained the desired dose of viral genomes (either 6.4E10 or 1E12 vg), this controlled for volume injected 334 across either dose administered. Mixed gender 2-5-month-old C57BL/10 mice or 6-9-week-old k18-hACE2 mice 335 were randomly assigned to groups and anesthetized with isoflurane before receiving an intra gastrocnemius 

Following another 3x10 min washes in PBS, slides were mounted in VECTASHIELD mounting media containing 364 DAPI (Vector Laboratories, H-1500). Images were taken using a Zeiss Observer 7 widefield microscope (Zeiss). 

50% pseudovirus-neutralization titer as measured by focus reduction neutralization test (FRNT50)).

Extended Data Figure 4 : Boosting fails to elicit a neutralizing humoral following booster vaccination. a, Mixed gender, 2-5-month-old C57BL/10 mice received 6.4E10 vg IM injection of either AAV9-M8 (n=5) or AAV9-M8B (n=3) and then received an identical booster vaccination 5 weeks later. Three weeks after the booster dose animals were evaluated for the presence of SARS-CoV-2 neutralizing antibodies. Data is reported as log10(inverse SARS-CoV-2 50% pseudovirus-neutralization titer as measured by focus reduction neutralization test (FRNT50)). a

An Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing 533 antibody and T cell responses in mice and nonhuman primates

A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate. antigen

The cytoplasmic tail of the severe acute respiratory syndrome 570 coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds COPI and 571 promotes interaction with membrane protein

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-573 reactivity with SARS-CoV

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and 577 Neutralizing Effects on Infection in Non-human Primates

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

SARS-CoV-2 spike D614G change enhances replication and transmission

COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice

SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung 588 inflammation and impaired function

Mutation in Mpzl3, a novel [corrected] gene encoding a predicted [corrected] adhesion harboring the following elements in sequence AAV_ITR-CMV-UTR5'-M8B-3'UTR-pA-AAV_ITR and then grown on 600 chamber slides. Later cells were fixed and stained for spike protein and nuclei labeled with DAPI

Extended Data Figure 1: In vitro characterization. a, c2c12 cells were lipofectamine transfected with plasmid harboring the following elements in sequence AAV_ITR-CMV-UTR5'-M8B-3'UTR-pA-AAV_ITR and then grown on chamber slides. Later cells were fixed and stained for spike protein and nuclei labeled with DAPI

Both cohorts were challenged days 630 apart from each other with IN inoculation of 2.5E4 PFU SARS-CoV-2

All animals were tracked until a terminal concluding date. b, e, Kaplan Meier survival curve post SARS-CoV-2 infection. Survival was compared by the log-ranked Mantel-Cox test. c, f, Change in body weight post SARS-CoV-2 infection. Weight was compared by the nonparametric Mann-Whitney U-test. The average mock vaccinated body weight in (f) switches from