Strong SARS-CoV-2 N-Specific CD8+ T Immunity Induced by Engineered Extracellular Vesicles Associates with Protection from Lethal Infection in Mice
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Abstract
SARS-CoV-2-specific CD8+ T cell immunity is expected to counteract viral variants in both efficient and durable ways. We recently described a way to induce a potent SARS-CoV-2 CD8+ T immune response through the generation of engineered extracellular vesicles (EVs) emerging from muscle cells. This method relies on intramuscular injection of DNA vectors expressing different SARS-CoV-2 antigens fused at their N-terminus with the Nefmut protein, i.e., a very efficient EV-anchoring protein. However, quality, tissue distribution, and efficacy of these SARS-CoV-2-specific CD8+ T cells remained uninvestigated. To fill the gaps, antigen-specific CD8+ T lymphocytes induced by the immunization through the Nefmut-based method were characterized in terms of their polyfunctionality and localization at lung airways, i.e., the primary targets of SARS-CoV-2 infection. We found that injection of vectors expressing Nefmut/S1 and Nefmut/N generated polyfunctional CD8+ T lymphocytes in both spleens and bronchoalveolar lavage fluids (BALFs). When immunized mice were infected with 4.4 lethal doses of 50% of SARS-CoV-2, all S1-immunized mice succumbed, whereas those developing the highest percentages of N-specific CD8+ T lymphocytes resisted the lethal challenge. We also provide evidence that the N-specific immunization coupled with the development of antigen-specific CD8+ T-resident memory cells in lungs, supporting the idea that the Nefmut-based immunization can confer a long-lasting, lung-specific immune memory. In view of the limitations of current anti-SARS-CoV-2 vaccines in terms of antibody waning and efficiency against variants, our CD8+ T cell-based platform could be considered for a new combination prophylactic strategy.
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SciScore for 10.1101/2022.01.10.475620: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: Fourteen days after the second immunization, mice were sacrificed by either cervical dislocation or CO2 inhalation. Sex as a biological variable Animals and authorizations: Six-weeks old C57 Bl/6 and C57 Bl/6 K18-hACE-2 transgenic [19] female mice were purchased from Charles River and hosted at the Central and BSL3 Animal Facilities of the Istituto Superiore di Sanità Randomization For in vivo titration of the SARS-CoV-2/Italy INMI1#52284 isolate, age-matched K18-hACE-2 mice were randomized by body weight into groups of 4, and challenged with 5-fold serial dilutions in 1×PBS of the virus preparation containing 2.2×105, 4.4×104, 8.8×103, or 1.8×103 TCID50. Blinding not detected. Po… SciScore for 10.1101/2022.01.10.475620: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: Fourteen days after the second immunization, mice were sacrificed by either cervical dislocation or CO2 inhalation. Sex as a biological variable Animals and authorizations: Six-weeks old C57 Bl/6 and C57 Bl/6 K18-hACE-2 transgenic [19] female mice were purchased from Charles River and hosted at the Central and BSL3 Animal Facilities of the Istituto Superiore di Sanità Randomization For in vivo titration of the SARS-CoV-2/Italy INMI1#52284 isolate, age-matched K18-hACE-2 mice were randomized by body weight into groups of 4, and challenged with 5-fold serial dilutions in 1×PBS of the virus preparation containing 2.2×105, 4.4×104, 8.8×103, or 1.8×103 TCID50. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources After 16 h, cells were discarded, and the plate was incubated for 2 h at room temperature with R4-6A2 biotinylated anti-IFN-γ antibody (Mabtech) at the concentration of 100 μg/mL. anti-IFN-γsuggested: NoneNon-specific staining was minimized by pre-incubating cells with 0.5 μg of Fc blocking mAbs (i.e., anti-CD16/CD32 antibodies, Invitrogen/eBioscience Thermo Fisher) in 100 μL of 1×PBS with 2% FCS for 15 min at 4 °C. anti-CD16/CD32suggested: NoneExperimental Models: Cell Lines Sentences Resources SARS-CoV-2 preparation and in vitro titration: VERO-E6 cells were grown in DMEM (Gibco) supplemented with 2% FCS, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1× non-essential amino acids (Gibco). VERO-E6suggested: NoneTo determine SARS-CoV-2 stock TCID50 (tissue culture infectious doses 50%), 2.2 × 104 Vero E6 cells/well were added onto 96-well plates (Corning, Mediatech Inc) and, the next day, octuplicate cultures were inoculated with 10-fold serial dilutions of virus (100 μL/well). Vero E6suggested: NoneExperimental Models: Organisms/Strains Sentences Resources Animals and authorizations: Six-weeks old C57 Bl/6 and C57 Bl/6 K18-hACE-2 transgenic [19] female mice were purchased from Charles River and hosted at the Central and BSL3 Animal Facilities of the Istituto Superiore di Sanità Bl/6 K18-hACE-2suggested: NoneFor in vivo titration of the SARS-CoV-2/Italy INMI1#52284 isolate, age-matched K18-hACE-2 mice were randomized by body weight into groups of 4, and challenged with 5-fold serial dilutions in 1×PBS of the virus preparation containing 2.2×105, 4.4×104, 8.8×103, or 1.8×103 TCID50. K18-hACE-2suggested: RRID:IMSR_GPT:T037657)Recombinant DNA Sentences Resources DNA constructs: Open-reading frames coding for Nefmut fused with either S1, S2, or N SARS-CoV-2 proteins were cloned into pVAX1 plasmid (Thermo Fisher) as previously described [18]. pVAX1suggested: RRID:Addgene_137909)Software and Algorithms Sentences Resources Samples were then analyzed by a CyotFLEX LX (Beckman Coulter, Brea, CA, USA) flow cytometer and analyzed using Kaluza software (Beckman Coulter) Kaluzasuggested: (Kaluza, RRID:SCR_016182)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: We detected the following sentences addressing limitations in the study:Our study presents limitations. The use of fluorescent tetramers in ICS/flow cytometry analyses would have identified SARS-CoV-2-specific CD8+ T cell sub-populations more accurately. In addition, challenging immunized mice with sub-lethal viral doses may have revealed potentially protective effects induced by S1-specific CD8+ T cells also. Furthermore, the lack of quantification of viral loads in lungs of protected mice precluded the evaluation of the effects of N-specific immunity on viral spread within airway tissues. Emergence of variants against which anti-S neutralizing antibodies lose potency represents one of the most relevant shortcoming for current anti-SARS-CoV-2 vaccines. Notably, N-specific antiviral CD8+ T cell immunity is not expected to suffer from such a limitation. In fact, as reported in fig. 7, amino acid sequences of N protein from current variants of concern (VOCs) are well conserved, at least in part because mutations in this key viral component could have detrimental effects on optimal viral fitness. By consequence, the N-specific CD8+ T cell immune response against a single viral strain is anticipated to be effective also against other VOCs. In addition, based on the observations on patients recovered from SARS-CoV infection [11], SARS-CoV-2 N-specific CD8+ T cell immunity would wane with a kinetics much slower than that of anti-S neutralizing antibodies. We here demonstrate that adequate levels of N-specific CD8+ T cell immunity correlate with protect...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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