Simultaneous CD8+ T-Cell Immune Response against SARS-Cov-2 S, M, and N Induced by Endogenously Engineered Extracellular Vesicles in Both Spleen and Lungs
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Most advanced vaccines against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 are designed to induce antibodies against spike (S) protein. Differently, we developed an original strategy to induce CD8+ T cytotoxic lymphocyte (CTL) immunity based on in vivo engineering of extracellular vesicles (EVs). This is a new vaccination approach based on intramuscular injection of DNA expression vectors coding for a biologically inactive HIV-1 Nef protein (Nefmut) with an unusually high efficiency of incorporation into EVs, even when foreign polypeptides are fused to its C-terminus. Nanovesicles containing Nefmut-fused antigens released by muscle cells can freely circulate into the body and are internalized by antigen-presenting cells. Therefore, EV-associated antigens can be cross-presented to prime antigen-specific CD8+ T-cells. To apply this technology to a strategy of anti-SARS-CoV-2 vaccine, we designed DNA vectors expressing the products of fusion between Nefmut and different viral antigens, namely N- and C-terminal moieties of S (referred to as S1 and S2), M, and N. We provided evidence that all fusion products are efficiently uploaded in EVs. When the respective DNA vectors were injected in mice, a strong antigen-specific CD8+ T cell immunity became detectable in spleens and, most important, in lung airways. Co-injection of DNA vectors expressing the diverse SARS-CoV-2 antigens resulted in additive immune responses in both spleen and lungs. Hence, DNA vectors expressing Nefmut-based fusion proteins can be proposed for new anti-SARS-CoV-2 vaccine strategies.
Article activity feed
-
-
SciScore for 10.1101/2020.12.18.423420: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Mice immunization: Both 6-weeks old C57 Bl/6 and, for S2 immunization (in view of the lack of already characterized H2b immunodominant S2 epitopes), Balb/c female mice were obtained from Charles River. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources To minimize nonspecific staining, cells were pre-incubated with 0.5 μg of Fc blocking mAbs (i.e., anti-CD16/CD32 antibodies, Invitrogen/eBioscience) in 100 μL of PBS with 2% FCS for 15 minutes at 4 °C. anti-CD16/CD32suggested: NoneExperimental Models: Cell Lines Sentences Re… SciScore for 10.1101/2020.12.18.423420: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Mice immunization: Both 6-weeks old C57 Bl/6 and, for S2 immunization (in view of the lack of already characterized H2b immunodominant S2 epitopes), Balb/c female mice were obtained from Charles River. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources To minimize nonspecific staining, cells were pre-incubated with 0.5 μg of Fc blocking mAbs (i.e., anti-CD16/CD32 antibodies, Invitrogen/eBioscience) in 100 μL of PBS with 2% FCS for 15 minutes at 4 °C. anti-CD16/CD32suggested: NoneExperimental Models: Cell Lines Sentences Resources Cell cultures and transfection: Human embryonic kidney (HEK) 293T cells (ATCC, CRL-11268) were grown in DMEM (Gibco) plus 10% heat-inactivated fetal calf serum (FCS, HEKsuggested: None293Tsuggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)Human immature dendritic cells (iDCs) were obtained after 5 to7 days of culture of monocytes in the presence of both IL-4 and GM-CSF. Immature DCs were challenged by engineered EVs uploading either Nefmut/N or Nefmut alone isolated from supernatants of HEK293T transfected cells. HEK293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Briefly, 2×105 PBLs recovered from cross-primed cultures were co-cultivated for 5 hours with equal number of HLA-matched target cells, i.e., MCF-7 previously treated with 1 μg/mL of either SARS-CoV-2 N-specific or unrelated HLA-A. MCF-7suggested: NCI-DTP Cat# MCF7, RRID:CVCL_0031)Experimental Models: Organisms/Strains Sentences Resources Mice immunization: Both 6-weeks old C57 Bl/6 and, for S2 immunization (in view of the lack of already characterized H2b immunodominant S2 epitopes), Balb/c female mice were obtained from Charles River. Balb/csuggested: RRID:IMSR_ORNL:BALB/cRl)Software and Algorithms Sentences Resources Samples were then assessed by a Gallios 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: 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.
- 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.
-
