Programming the lymph node immune response with Amphiphile-CpG induces potent cellular and humoral immunity following COVID-19 subunit vaccination in mice and non-human primates
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Abstract
Despite the success of currently authorized vaccines for the reduction of severe COVID-19 disease risk, rapidly emerging viral variants continue to drive pandemic waves of infection, resulting in numerous global public health challenges. Progress will depend on future advances in prophylactic vaccine activity, including advancement of candidates capable of generating more potent induction of cross-reactive T cells and durable cross-reactive antibody responses. Here we evaluated an Amphiphile (AMP) adjuvant, AMP-CpG, admixed with SARS-CoV-2 Spike receptor binding domain (RBD) immunogen, as a lymph node-targeted protein subunit vaccine (ELI-005) in mice and non-human primates (NHPs). AMP-mediated targeting of CpG DNA to draining lymph nodes resulted in comprehensive local immune activation characterized by extensive transcriptional reprogramming, inflammatory proteomic milieu, and activation of innate immune cells as key orchestrators of antigen-directed adaptive immunity. Prime-boost immunization with AMP-CpG in mice induced potent and durable T cell responses in multiple anatomical sites critical for prophylactic efficacy and prevention of severe disease. Long-lived memory responses were rapidly expanded upon re-exposure to antigen. In parallel, RBD-specific antibodies were long-lived, and exhibited cross-reactive recognition of variant RBD. AMP-CpG-adjuvanted prime-boost immunization in NHPs was safe and well tolerated, while promoting multi-cytokine-producing circulating T cell responses cross-reactive across variants of concern (VOC). Expansion of RBD-specific germinal center (GC) B cells in lymph nodes correlated to rapid seroconversion with variant-specific neutralizing antibody responses exceeding those measured in convalescent human plasma. These results demonstrate the promise of lymph-node adjuvant-targeting to coordinate innate immunity and generate robust adaptive responses critical for vaccine efficacy.
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SciScore for 10.1101/2022.05.19.492649: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animals: All animal studies were carried out under an institute-approved Institutional Animal Care and Use Committee (IACUC) protocol following federal, state, and local guidelines for the care and use of animals. Sex as a biological variable For mouse studies, female 6- to 8-week-old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Randomization For NHP studies, 8 outbred, Indian-origin, 4-5 year old female rhesus macaques (Macaca mulatta) were randomly allocated into 3 groups of 2 or 3 animals. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 106 PBMCs/well were resuspended in R10 … SciScore for 10.1101/2022.05.19.492649: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Animals: All animal studies were carried out under an institute-approved Institutional Animal Care and Use Committee (IACUC) protocol following federal, state, and local guidelines for the care and use of animals. Sex as a biological variable For mouse studies, female 6- to 8-week-old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Randomization For NHP studies, 8 outbred, Indian-origin, 4-5 year old female rhesus macaques (Macaca mulatta) were randomly allocated into 3 groups of 2 or 3 animals. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 106 PBMCs/well were resuspended in R10 media supplemented with anti-CD49d monoclonal antibody (clone: 9F10, BD) anti-CD49dsuggested: Noneanti-CD28 monoclonal antibody (clone: CD28.2, BD), and Golgi inhibitors monensin (Fisher Scientific, cat# NC0176671) and brefeldin A (Fisher Scientific, cat# 50-112-9757) and incubated at 37°C for 8 hours, then maintained at 4°C overnight. anti-CD28suggested: NoneThe next day, cells were surface-stained with antibodies against CD4 (PE-Cy5.5, clone: S3.5, Invitrogen), CD8 (AF647, clone: RPA-T8, BioLegend), CD45RA (FITC, clone: 5H9, BD), CCR7 (BV650, clone: G043H7, BioLegend), and aqua live/dead dye (Invitrogen, L34957), and subsequently fixed with BD CytoFix/CytoPerm (BD, 554714). CD4suggested: (SouthernBiotech Cat# 9522-31, RRID:AB_2796861)CD8suggested: (SouthernBiotech Cat# 9536-31, RRID:AB_2796896)CD45RAsuggested: NoneCCR7suggested: (BD Biosciences Cat# 563407, RRID:AB_2738187)Cells were further stained with antibodies against CD3 (APC-Cy7, clone: SP34-2, BD), CD69 (ECD, clone: TP1.55.3, Beckman Coulter), IFNγ (AF700, clone: B27, BioLegend), IL-2 (BV421, clone: MQ1-17H12, BioLegend), IL-4 (PE, clone: 8D4-8, BioLegend) CD69suggested: NoneIFNγsuggested: (Bio X Cell Cat# BE0245, RRID:AB_2687726)IL-2suggested: NoneBV421suggested: (BD Biosciences Cat# 562986, RRID:AB_2737933)IL-4suggested: NoneMouse antigen-specific tetramer staining of peripheral blood cells: MHC-tetramer staining on mouse samples was performed using an RBD-PE tetramer specific for sequence VNFNFNGL (NIH Tetramer Core Facility at Emory University, cat# 54971), and antibodies against CD8a (APC, clone: 53-6.7, eBioscience), CD3 (APC-Cy7, clone: 17A2, BD), CD44 (PE-Cy7, antibodies against CD8asuggested: (GeneTex Cat# GTX111860, RRID:AB_10623584)CD3suggested: (Abcam Cat# ab52305, RRID:AB_955118)CD44suggested: NonePE-Cy7suggested: NoneThe detection antibody used was horseradish peroxidase (HRP)–conjugated goat anti-human IgG (H+L) (ThermoFisher, cat# SA5-10283) at a 1:2000 dilution. anti-human IgGsuggested: (Thermo Fisher Scientific Cat# SA5-10283, RRID:AB_2868331)Experimental Models: Cell Lines Sentences Resources Briefly, a HEK293T cell line overexpressing ACE2 and TMPRSS2 was seeded at a density of 1.2×104 cells/well overnight. 3-fold serial dilutions of heat inactivated serum samples were prepared and mixed with 50 µL of pseudovirus. HEK293Tsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Gene transcript analysis by Nanostring: For mouse studies, inguinal lymph nodes were harvested from immunized C57BL/6J mice at the indicated time points, processed into single cell suspensions, and lysed with RLT buffer (Qiagen, cat# 79216) C57BL/6Jsuggested: NoneSoftware and Algorithms Sentences Resources Sample acquisition was performed on BD FACS Symphony and data were analyzed with BD FlowJo V10 software. BD FlowJosuggested: NoneThe next day, cells were surface-stained with antibodies against CD4 (PE-Cy5.5, clone: S3.5, Invitrogen), CD8 (AF647, clone: RPA-T8, BioLegend), CD45RA (FITC, clone: 5H9, BD), CCR7 (BV650, clone: G043H7, BioLegend), and aqua live/dead dye (Invitrogen, L34957), and subsequently fixed with BD CytoFix/CytoPerm (BD, 554714). BD CytoFix/CytoPermsuggested: NoneCells fixed in 1.5% formaldehyde were acquired on a BD FACS Symphony and data were analyzed with BD FlowJo V10 FlowJosuggested: (FlowJo, RRID:SCR_008520)Red blood cells were lysed in ACK lysis buffer (Quality Biological Inc., cat# no. 118156101). Quality Biologicalsuggested: NoneTranscriptional responses were assessed with nSolver software v4.0 (NanoString Technologies) and differential gene expression was carried out using ROSALIND software. nSolversuggested: NoneNanoString statistical analysis was performed using Rosalind software. Rosalindsuggested: (Rosalind, RRID:SCR_006233)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.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.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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