Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mouse and non-human primates

  1. Nerea Zabaleta
  2. Wenlong Dai
  3. Urja Bhatt
  4. Jessica A Chichester
  5. Julio Sanmiguel
  6. Reynette Estelien
  7. Kristofer T Michalson
  8. Cheikh Diop
  9. Dawid Maciorowski
  10. Wenbin Qi
  11. Elissa Hudspeth
  12. Allison Cucalon
  13. Cecilia D Dyer
  14. M. Betina Pampena
  15. James J. Knox
  16. Regina C LaRocque
  17. Richelle C Charles
  18. Dan Li
  19. Maya Kim
  20. Abigail Sheridan
  21. Nadia Storm
  22. Rebecca I Johnson
  23. Jared Feldman
  24. Blake M Hauser
  25. Eric Zinn
  26. Aisling Ryan
  27. Dione T Kobayashi
  28. Ruchi Chauhan
  29. Marion McGlynn
  30. Edward T Ryan
  31. Aaron G Schmidt
  32. Brian Price
  33. Anna Honko
  34. Anthony Griffiths
  35. Sam Yaghmour
  36. Robert Hodge
  37. Michael R. Betts
  38. Mason W Freeman
  39. James M Wilson
  40. Luk H Vandenberghe

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Abstract

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

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  1. Version published to 10.1101/2021.01.05.422952v3 on bioRxiv
  2. Version published to 10.1101/2021.01.05.422952v2 on bioRxiv
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    SciScore for 10.1101/2021.01.05.422952: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Mouse studies: All the mouse studies were performed in compliance with the Schepens Eye Research Institute IACUC.
    IRB: Human subject investigation was approved by the institutional Review Board of the Massachusetts General Hospital.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableFour 3 to 7 year-old Rhesus macaques (Macaca mulatta) were treated with the clinical candidates (n=2/vector, 1 female and 1 male) intramuscularly at a dose of 1012 gc/animal.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membranes were probed with an anti-SARS-CoV-2 RBD rabbit polyclonal antibody (Sino Biological Inc., 40592-T62) followed by a goat anti-rabbit HRP-conjugated secondary antibody (Thermo Fisher Scientific, Cat# A16110
    anti-SARS-CoV-2 RBD
    suggested: None
    , RRID AB_2534782).
    detected: (Thermo Fisher Scientific Cat# A16110, RRID:AB_2534782)
    An anti-GAPDH antibody (Cell Signaling Technology Cat# 2118, RRID:AB_561053) was used as loading control.
    anti-GAPDH
    detected: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)
    SARS-CoV-2 Spike-binding antibody detection ELISA: Nunc MaxiSorpTM high protein-binding capacity 96 well plates (Thermo Fisher Scientific, Cat# 44-2404-21) were coated overnight at 4 °C with 1µg/ml SARS-CoV-2 RBD, SARS-CoV-2 ectodomain (LakePharma, Cat# 46328) or SARS-CoV-1 RBD diluted in phosphate-buffered saline (PBS).
    SARS-CoV-2 Spike-binding
    suggested: None
    After an hour of incubation, the plates were washed and 100 µl of secondary Peroxidase AffiniPure Rabbit Anti-Mouse IgG (Jackson ImmunoResearch, Cat# 315-035-045, RRID: AB_2340066) antibody diluted 1:1000 in blocking solution or rabbit Anti-Monkey IgG (whole molecule)-Peroxidase antibody (Sigma-Aldrich Cat# A2054, RRID:AB_257967) were added to each well.
    Anti-Mouse IgG
    detected: (Jackson ImmunoResearch Labs Cat# 315-035-045, RRID:AB_2340066)
    Anti-Monkey IgG ( whole molecule)-Peroxidase
    suggested: None
    Anti-Monkey IgG whole molecule)-Peroxidase
    detected: (Sigma-Aldrich Cat# A2054, RRID:AB_257967)
    For mouse serum SARS-CoV-2 RBD-specific antibody isotyping, the same ELISA was performed but using the secondary antibodies from SBA Clonotyping System-HRP kit (SouthernBiotech, 5300-05, RRID:AB_2796080) diluted accordingly to manufacturer’s instructions.
    SBA Clonotyping System-HRP kit
    detected: (SouthernBiotech Cat# 5300-05, RRID:AB_2796080)
    Then, horseradish peroxidase (HRP)-conjugated secondary antibody against rhesus IgG1 (NIH Nonhuman Primate Reagent Resource supported by AI126683 and OD 010976 Cat# PR-7110, RRID:AB_2819310) and IgG4 (NIH Nonhuman Primate Reagent Resource supported by AI126683 and OD 010976 Cat# PR-7180, RRID:AB_2819322) for 1 h.
    rhesus IgG1
    detected: (NIH Nonhuman Primate Reagent Resource Cat# PR-7110, RRID:AB_2819310)
    IgG4
    detected: (NIH Nonhuman Primate Reagent Resource Cat# PR-7180, RRID:AB_2819322)
    Neutralizing antibody titers or 50% inhibitory concentration in the serum sample (EC50 or ID50) were calculated as the reciprocal of the highest dilution showing less RLU signal than half of the average RLU (maximum infectivity) of Virus Control group (cells + virus, without serum).
    EC50
    suggested: None
    Briefly, 96-well PVDF plates (Millipore) were pre-coated with 10 μg/ml anti-mouse IFN-γ ELISPOT capture antibody (BD Biosciences Cat# 551881, RRID:AB_2868948) or 4 μg/ml anti-mouse IL-4 ELISPOT capture antibody (BD Biosciences Cat# 551878, RRID:AB_2336921) at 4°C overnight, and then blocked with complete RPMI-1640 medium for 3 hours at 37°C.
    anti-mouse IFN-γ ELISPOT
    suggested: None
    ELISPOT
    detected: (BD Biosciences Cat# 551881, RRID:AB_2868948)
    anti-mouse IL-4
    detected: (BD Biosciences Cat# 551878, RRID:AB_2336921)
    Co-stimulation was added with peptides: 1μg/mL anti-CD49d (Clone 9F10, BioLegend Cat# 304301, RRID:AB_314427) and CD28-ECD (Clone CD28.2
    anti-CD49d
    detected: (BioLegend Cat# 304301, RRID:AB_314427)
    , Beckman Coulter Cat# 6607111, RRID:AB_1575955) at the start of stimulation.
    detected: (Beckman Coulter Cat# 6607111, RRID:AB_1575955)
    CD107a BV650 (clone H4A3, BioLegend Cat# 328643, RRID:AB_2565967) was added at the start of stimulation.
    CD107a
    detected: (BioLegend Cat# 328643, RRID:AB_2565967)
    The following antibodies were used: PD1 BV421 (clone EH12.2H7, BioLegend Cat# 329919, RRID:AB_10900818), CD14 BV510 (clone M5E2, BioLegend Cat# 301842, RRID:AB_2561946) and APC-Cy7 (clone M5E2, BioLegend Cat# 301819, RRID:AB_493694), CD16 BV510 (clone 3G8, BioLegend Cat# 302048, RRID:AB_2562085) and APC-Cy7 (clone 3G8, BioLegend Cat# 302017, RRID:AB_314217), CD20 BV510 (clone 2H7, BioLegend Cat# 302339, RRID:AB_2561721) and BV650 (clone 2H7, BioLegend Cat# 302335, RRID:AB_11218609), CD69 BV605 (clone FN50, BioLegend Cat# 310937, RRID:AB_2562306), CD21 PECy7 (clone Bu32, BioLegend Cat# 354911, RRID:AB_2561576), CD4 BUV661 (clone SK3, BD Biosciences Cat# 612962, RRID:AB_2870238), CD95 BUV737 (clone DX2, BD Biosciences Cat# 612790, RRID:AB_2870117), CD8 BUV563 (clone RPA-T8, BD Biosciences Cat# 612914, RRID:AB_2870199), KI67 BV786 (clone B56, BD Biosciences Cat# 563756, RRID:AB_2732007)
    The PD1 BV421
    detected: (BioLegend Cat# 329919, RRID:AB_10900818)
    CD14 BV510
    detected: (BioLegend Cat# 301842, RRID:AB_2561946)
    APC-Cy7
    detected: (BioLegend Cat# 354911, RRID:AB_2561576)
    APC-Cy7
    detected: (BioLegend Cat# 301819, RRID:AB_493694)
    detected: (BioLegend Cat# 302048, RRID:AB_2562085)
    APC-Cy7
    suggested: (BD Biosciences Cat# 557758, RRID:AB_396864)
    APC-Cy7
    detected: (BioLegend Cat# 302017, RRID:AB_314217)
    CD20 BV510
    detected: (BioLegend Cat# 302339, RRID:AB_2561721)
    BV650 ( clone 2H7
    detected: (BioLegend Cat# 302335, RRID:AB_11218609)
    CD69
    detected: (BioLegend Cat# 310937, RRID:AB_2562306)
    CD4 BUV661
    detected: (BD Biosciences Cat# 612962, RRID:AB_2870238)
    BUV737
    detected: (BD Biosciences Cat# 612790, RRID:AB_2870117)
    BUV563
    detected: (BD Biosciences Cat# 612914, RRID:AB_2870199)
    KI67
    suggested: (Fluidigm Cat# 3172024B, RRID:AB_2858243)
    detected: (BD Biosciences Cat# 563756, RRID:AB_2732007)
    , IL2 PE (clone MQ1-17H12, BD Biosciences Cat# 554566, RRID:AB_395483), IFNγ BV750 (clone B27, BD Biosciences Cat# 566357, RRID:AB_2739707), CD3 BUV805 (clone SP34-2, BD Biosciences Cat# 742053, RRID:AB_2871342)
    IL2 PE
    detected: (BD Biosciences Cat# 554566, RRID:AB_395483)
    IFNγ
    detected: (BD Biosciences Cat# 566357, RRID:AB_2739707)
    CD3 BUV805
    detected: (BD Biosciences Cat# 742053, RRID:AB_2871342)
    Granzyme B AF700 (clone GB11, BD Biosciences Cat# 560213, RRID:AB_1645453)
    Granzyme B
    detected: (BD Biosciences Cat# 560213, RRID:AB_1645453)
    , CD3 APC-Cy7 (clone SP34-2, BD Biosciences Cat# 557757, RRID:AB_396863), IgM PECy5 (clone G20-127, BD Biosciences Cat# 551079, RRID:AB_394036), CD27 BV421 (clone M-T271, BD Biosciences Cat# 562513, RRID:AB_11153497), HLA-DR BV605 (clone G46-6, BD Biosciences Cat# 562844, RRID:AB_2744478), CD80 BV786 (clone L307.4, BD Biosciences Cat# 564159, RRID:AB_2738631), CXCR3 AF488
    CD3 APC-Cy7
    detected: (BD Biosciences Cat# 557757, RRID:AB_396863)
    IgM PECy5
    detected: (BD Biosciences Cat# 551079, RRID:AB_394036)
    CD27
    detected: (BD Biosciences Cat# 562513, RRID:AB_11153497)
    HLA-DR
    detected: (BD Biosciences Cat# 562844, RRID:AB_2744478)
    CD80
    detected: (BD Biosciences Cat# 564159, RRID:AB_2738631)
    (clone 1C6, BD Biosciences Cat# 558047, RRID:AB_397008), CXCR5 SB702 (clone MU5BEE, Thermo Fisher Scientific Cat# 67-9185-42, RRID:AB_2717183), Tbet PerCP-Cy5.5 (clone 4B10, Thermo Fisher Scientific Cat# 45-5825-82, RRID:AB_953657), CD11c PECy5.5 (clone 3.9, Thermo Fisher Scientific Cat# 35-0116-42, RRID:AB_11218511)
    ( clone 1C6
    detected: (BD Biosciences Cat# 558047, RRID:AB_397008)
    SB702
    detected: (Thermo Fisher Scientific Cat# 67-9185-42, RRID:AB_2717183)
    Tbet
    detected: (Thermo Fisher Scientific Cat# 45-5825-82, RRID:AB_953657)
    CD11c PECy5.5
    detected: (Thermo Fisher Scientific Cat# 35-0116-42, RRID:AB_11218511)
    TNFα PE-Cy7 (clone Mab11, Thermo Fisher Scientific Cat# 25-7349-41, RRID:AB_1257208), and polyclonal anti-IgD PE Tx Red (SouthernBiotech Cat# 2030-09, RRID:AB_2795630).
    TNFα PE-Cy7 ( clone Mab11
    detected: (Thermo Fisher Scientific Cat# 25-7349-41, RRID:AB_1257208)
    anti-IgD PE
    detected: (SouthernBiotech Cat# 2030-09, RRID:AB_2795630)
    AAV Neutralizing Antibody (NAb) Assay: NAb responses against AAV1, AAV2, AAV5, AAV8, AAV9 and AAVrh32.33 capsids were measured in serum using an in vitro HEK293 cell-based assay and LacZ expressing vectors (Vector Core Laboratory, University of Pennsylvania, Philadelphia, PA) as previously described (Calcedo et al., 2018).
    AAV1
    suggested: (ARP American Research Products Cat# 03-651150, RRID:AB_1540399)
    AAV2
    suggested: None
    AAV5
    suggested: None
    AAV8
    suggested: (Fitzgerald Industries International Cat# 10R-3126, RRID:AB_11202215)
    Experimental Models: Cell Lines
    SentencesResources
    In vitro expression studies: 105 HEK293 cell/well were seeded in 12-well plates (Corning, MA, USA) plates and incubated at 37°C overnight.
    HEK293
    suggested: None
    In addition, 5×104 HuH7 cell/well were seeded in 12-well plates and incubated overnight at 37°C.
    HuH7
    suggested: None
    Pseudovirus Neutralization Assay: HEK293T cells expressing ACE2 were seeded at 1.5×104 cells/well in poly-L-Lysine (0.01%) coated 96-well black plates.
    HEK293T
    suggested: None
    Two hundred microliters of each dilution or control were added to confluent monolayers of NR-596 Vero E6 cells in triplicate and incubated for 1 hour at 37°C and 5% CO2.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    BALB/c, C57BL/6 or C57BL/6 diet-induced obese (DIO) animals were intramuscularly (right gastrocnemius muscle) treated at 1010 gc/mouse or 1011 gc/mouse.
    BALB/c
    suggested: None
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    The final concentration and Tm’s of primers were determined using the DINAMelt application of the UNAfold software package (Markham and Zuker, 2005, 2008) and set to hybridize the target with a Tm of just under 60°C (59.0-59.9°C) for high specificity.
    UNAfold
    suggested: (UNAFold, RRID:SCR_001360)
    The resulting 67 bp amplicon was inspected for specificity via NCBI BLAST® using the somewhat similar algorithm in the suite against human, NHP, mouse, ferret, and betacoronavirus databases and determined to be highly specific for our vaccine candidates.
    NCBI BLAST®
    suggested: (NCBI BLAST, RRID:SCR_004870)
    Data were analyzed using FlowJo software (versions 9.9.6 and 10.6.2, Tree Star).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Phylogenetic Analysis: First, fourteen representative AAV capsid sequences were aligned by Clustal Omega (Sievers and Higgins, 2018)
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    A maximum-likelihood approach was then used to infer the evolutionary relationships among the included sequences using MEGA X (Kumar et al., 2018) and the resultant phylogeny rooted along the midpoint of the branch between AAV4 and AAV5 for purposes of visualization.
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    Graphs and statistical analysis: GraphPad Prism 8 was used for graph preparation and statistical analysis.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    We hypothesized that this methodology can address several limitations of first generation COVID vaccines. Indeed, both AAVCOVID vaccine candidates were shown in mice and NHPs to elicit robust neutralizing antibody responses following a single dose administration. This contrasts with front-runner vaccines, which require 2 injections spaced by 3 or more weeks (Folegatti et al., 2020b; Jackson et al., 2020; Logunov et al., 2020; Walsh et al., 2020). Compared to a single dose vaccine, a multiple dose regimen complicates a vaccination campaign by increasing cost, reducing compliance, and multiplying the manufacturing needs. Similarly, durable vaccine responses prolong or prevent the need for boost injections over time, resulting in similar benefits just described for single prime injection vaccines. AAVCOVID candidates have been shown in NHPs to retain peak immunogenicity for at least 5 months following a single dose injection. The durability of other vaccine candidates remains to be determined, although a recent report on the Moderna follow up from a Phase 1 study reported the maintenance of a robust, albeit modestly declining, antibody response in 34 participants across age groups (Widge et al., 2020). While it remains difficult to project the efficacy of the AAVCOVID vaccines based on immunological readouts alone, current data suggest an important role of neutralizing antibody responses in the prevention or mitigation of disease caused by SARS-CoV-2 (Chandrashekar et al., 2020;...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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  4. Version published to 10.1101/2021.01.05.422952v1 on bioRxiv