A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC
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Abstract
Most approved vaccines against COVID-19 have to be administered in a prime/boost regimen. We engineered a novel vaccine based on a chimeric human adenovirus 5 (hAdV5) vector. The vaccine (named CoroVaxG.3) is based on three pillars: (i) high expression of Spike to enhance its immunodominance by using a potent promoter and an mRNA stabilizer; (ii) enhanced infection of muscle and dendritic cells by replacing the fiber knob domain of hAdV5 by hAdV3; (iii) use of Spike stabilized in a prefusion conformation. The transduction with CoroVaxG.3-expressing Spike (D614G) dramatically enhanced the Spike expression in human muscle cells, monocytes and dendritic cells compared to CoroVaxG.5 that expressed the native fiber knob domain. A single dose of CoroVaxG.3 induced a potent humoral immunity with a balanced Th1/Th2 ratio and potent T-cell immunity, both lasting for at least 5 months. Sera from CoroVaxG.3-vaccinated mice was able to neutralize pseudoviruses expressing B.1 (wild type D614G), B.1.117 (alpha), P.1 (gamma) and B.1.617.2 (delta) Spikes, as well as an authentic P.1 SARS-CoV-2 isolate. Neutralizing antibodies did not wane even after 5 months, making this kind of vaccine a likely candidate to enter clinical trials.
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SciScore for 10.1101/2021.08.11.455942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Mice were maintained under specific pathogen-free conditions at the Institute Leloir animal facility and all experiments were conducted in accordance with animal use guidelines and protocols approved by the Institutional Animal Care and Use Committee (IACUC protocol 69). Sex as a biological variable Mice immunization: Six- to eight-week-old male BALB/c mice (obtained from the animal facility of the Veterinary School, University of La Plata, Argentina) were immunized with 109 or 1010 viral particles (vp) of Ad.C (empty vector), CoroVaxG.5 or CoroVaxG. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies S… SciScore for 10.1101/2021.08.11.455942: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Mice were maintained under specific pathogen-free conditions at the Institute Leloir animal facility and all experiments were conducted in accordance with animal use guidelines and protocols approved by the Institutional Animal Care and Use Committee (IACUC protocol 69). Sex as a biological variable Mice immunization: Six- to eight-week-old male BALB/c mice (obtained from the animal facility of the Veterinary School, University of La Plata, Argentina) were immunized with 109 or 1010 viral particles (vp) of Ad.C (empty vector), CoroVaxG.5 or CoroVaxG. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources ELISA: Sera from all mice were collected at different time points after immunization and evaluated for SARS-CoV-2-S-specific IgG antibodies using ELISA. SARS-CoV-2-S-specific IgGsuggested: NoneSera collected at week 4 after vaccination were also tested for SARS-CoV-2-S-specific IgG1 and IgG2a antibodies using ELISA. SARS-CoV-2-S-specific IgG1suggested: NoneIgG2asuggested: NonePlates were washed and bound specific IgG was detected with a HRP-conjugated goat anti-mouse IgG H&L antibody (ab6789, Abcam) diluted 1: 10,000 in blocking buffer. anti-mouse IgGsuggested: (Abcam Cat# ab6789, RRID:AB_955439)Neutralization of authentic SARS-CoV-2 virus: Neutralizing antibody (nAb) titers against SARS-CoV2 were defined according to the following protocol. SARS-CoV2suggested: NoneExperimental Models: Cell Lines Sentences Resources CRL-3216), Vero cells (CCL-81), Hs 729T (HTB-153) and THP-1 (TIB-202) cells were obtained from the ATCC (Manassas, VA, USA) Verosuggested: NoneTHP-1suggested: NoneHEK293 cells were purchased from Microbix Biosystems Inc (Mississauga, Canada) and 911 cells were already described63. HEK293suggested: NoneHEK293T-hACE2 cells were already described64. HEK293T-hACE2suggested: RRID:CVCL_A7UK)For the selection of the most appropriate promoter, HEK293T cells grown in 24-well plates were co-transfected with 1 μg of the different plasmids and 100 ng of pS-CMV-Renilla, using Lipofectamine 2000 (Thermo Fisher Scientific, CA, USA). HEK293Tsuggested: NoneThe viruses were propagated in HEK-293 cells in CellSTACK® cell culture chambers (Corning, Arizona, USA), purified by double CsCl density gradient centrifugation and stored in 10% glycerol in single-use aliquots at –80°C. HEK-293suggested: NoneHEK-293T cells growing in Optimem media (Gibco, MD, USA) with 2% of FBS were transduced with G*ΔG-VSV at a multiplicity of infection of four. HEK-293Tsuggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)Next, 100 μl of 5×105/mL HEK293T-ACE2 cells were added to the pseudovirus-serum mixture and incubated at 37 °C, 5% CO2 for 20-24 h. HEK293T-ACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources For RBD inoculations eight-week-old male BALB/c mice were immunized with 7.5 μg of the receptor binding domain of Spike protein (kindly gifted by Andrea Gamarnik, Argentina) in 75 μL Complete Freund’s Adjuvant (CFA, Sigma, St. Louis, MO) via subcutaneous injection and boosted 2 weeks later with 5 μg of RBD in 100 μL Incomplete Freund’s Adjuvant (IFA, Sigma). BALB/csuggested: NoneRecombinant DNA Sentences Resources Promoter and fiber selection: Promoters were synthesized by Genscript (NJ, USA) with NotI / (XhoI-StuI) flanking restriction sites and cloned in the NotI / StuI sites of the vector pShuttle-I-XP-Luc63 to obtain pShuttle-Pr1-Luc and pShuttle-Pr2-Luc. pShuttle-I-XP-Luc63suggested: NonepShuttle-Pr1-Lucsuggested: NoneThe vectors pAd-SV40-Luc, and pS-CMV-Renilla were previously described63. pAd-SV40-Lucsuggested: NonepS-CMV-Renillasuggested: NoneThe synthesized 4,650 bp fragment was cloned into the pShuttle-Pr2-Luc vector digested with StuI / SalI to exchange the luciferase ORF by the designed Spike cassette, downstream of Pr2. pShuttle-Pr2-Lucsuggested: NoneThe sequence of the resulting plasmid pS-Spike(D614G)-PP was confirmed by sequencing (Macrogen, Seul, Korea). pS-Spike(D614G)-PPsuggested: NoneTo construct the non-replicating adenoviruses, the plasmid pS-Spike(D614G)-PP was linearized with PmeI and co-transformed with E1/E3 (pCoroVaxG.5) or E1 (pCoroVaxG.3) deleted adenoviral backbone vectors in electrocompetent BJ5183 bacteria. pCoroVaxG.5suggested: NonepCoroVaxG.3suggested: NoneBriefly, the full length cDNA of Spike-D614G, and the Spike variants B.1.1.7 (alpha, first identified in the UK) and P.1 (gamma, first identified in Manaos, Brazil), were cloned into the eukaryotic expression vector pcDNA3.1, using the EcoRV restriction site and blunt-end ligation strategy, to generate the recombinant plasmids pcDNA-3.1-Spike-D614G, pcDNA-3.1-Spike-B,1,1,7 and pcDNA-3.1-Spike-P.1.. pcDNA3.1suggested: RRID:Addgene_79663)pcDNA-3.1-Spike-D614Gsuggested: NonepcDNA-3.1-Spike-B,1,1,7suggested: NonepcDNA-3.1-Spike-P.1suggested: NoneSoftware and Algorithms Sentences Resources Protein extracts were separated by SDS-PAGE with a 10% gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories). Bio-Rad Laboratoriessuggested: (Bio-Rad Laboratories, RRID:SCR_008426)Semi-quantifications of WB assays were performed by densitometry using the ImageJ software 1.53 (Wayne Rasband, NIH, USA), normalizing by β-actin expression. ImageJsuggested: (ImageJ, RRID:SCR_003070)For each IgG subclass reference, a standard curve was plotted using GraphPad Prism 8.0 generating a four-parameter logistical (4PL) fit of the OD 450 nm at each serial antibody dilution. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Cells were acquired on a FACSAria Fussion cytometer and analysis was performed using Flow Jo version 10.7.1. Flow Josuggested: (FlowJo, RRID:SCR_008520)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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