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Authors:Rice et al.   

Journal/ Pre-Print:Biorxiv 

Tags: Immunology/Immunity, Vaccines, Structural Biology 

Research Highlights

  1. In contrast to most vaccines now in trials based on the S protein, here Rice et al. generate a bivalent hAd5 SARS-CoV-2 vaccine containing S but also N protein, both modified for optimal expression, antigen processing and presentation, mimicking on the natural infection and most described T cell responses 

  1. The vaccine platform hAd5 has some deletions that allow potent immune responses in individuals with Ad5 pre-existing immunity, expanded gene carrying capacity and not to suppress innate immune signalling, providing an improved platform on terms of efficacy and safety. 

  1. In mice, this vaccine elicits both humoral and cell-mediated responses skewed towards a Th1 response. CD4 response was found to be mostly against N protein, which highlights the importance of multiple antigen vaccines.  


Rice et al. develop next generation bivalent hAd5 SARS-CoV-2 vaccine and test its immunogenicity in mice. The vaccine platform used (Ad5) is modified to elicit potent immune responses in individuals with Ad5 pre-existing immunity, expanded gene carrying capacity and not to suppress innate immune signalling, providing an improved platform on terms of efficacy and safety. The vaccine carries two modified SARS-CoV-2 proteins; S-Fusion, that the authors show has an optimised cell-surface expression and physiologically-relevant folding compared to S-WT, and N-ETSD (Enhanced T-cell Stimulation Domain) that is found to localise to endosomal/lysosomal subcellular compartment for increased antigen presentation. The immunisation of mice with this vaccine elicited both humoral and cell-mediated responses skewed towards a Th1-specific response. Notably, while CD8 responses were found against the two antigens CD4 responses preferentially targeted the N protein. The IgG responses were high in some immunized mice, with a potent neutralising activity. Combined antigen vaccines may be more successful in eliciting a potent and durable T cell response, and this vaccine could be a promising candidate for further evaluation.  

Impact for SARS-CoV2/COVID19 research efforts  

Develop a vaccine for SARS-CoV2/COVID19 

Study Type  

  • In vitro study 

  • In vivo study (CD-1 mouse)

Strengths and limitations of the paper 

Novelty: New bivalent hAd5 vaccine containing optimised N and S proteins with high capacity to elicit Ab and T cell responses. Also, next generation hAd5 with some deleted genes allows vaccination to individuals with a pre-existing immunity to Ad5.  

Standing in the field:The vaccine is designed in line with results found from natural infection for SARS-CoV-2.   

Appropriate statistics: Yes, but in some graphs Student t-test assumes a normal distribution and by the number of samples would be better not to assume a normal distribution. Mann-Whitney, without the assumption of a normal distribution, is applied in other graphs.  

Viral model used: 

Constructs used:  

  • hAd5 [E1-, E2b-, E3-] vector was used (Fig. 1c) to create viral vaccine candidate constructs. hAd5 [E1-, E2b-, E3-] backbones 

  • S-WT: S protein comprising 1273 amino acids and all S domains: extracellular (1-1213), transmembrane (1214-1234), and cytoplasmic (1235-1273) (Unitprot P0DTC2) 

  •  S RBD-ETSD: S Receptor Binding Domain (S RBD) with an ETSD 

  • N-ETSD: Nucleocapsid (N) with ETSD 

  • S-WT + N-ETSD: S-WT with an Enhanced T-cell Stimulation Domain (ETSD) 


  • S Fusion: S optimized to enhance surface expression and display of RBD 

  • Bivalent S-Fusion + N-ETSD; 

Translatability:This study designs a promising vaccine that may be better than solo antigen vaccines. Further animal studies are to be performed before clinical trials. The authors state that this vaccine is their candidate to be further evaluated and explored for oral, intranasal, and sublingual formulations to induce mucosal immunity in addition to cell-mediated and humoral immunity. 

Main limitations:  

  • The authors claim a synergistic effect of their vaccine by carrying both N and S proteins, but never show the effect of the vaccine carrying only one of these same optimised proteins.   

  • All the studies on cellular localisation and expression are performed by transfection to HEK293, to mimic the vaccine it would have been more accurate to infect the cells.  

  • The authors claim that their antigens are optimised for a better antigen presentation, and while they show an optimised expression in the cell surface or in the endosomal/lysosomal compartments; there is no evidence of a better antigenic presentation.  

  • The samples for antibody and T cell responses are only evaluated one week after last immunisation, but the authors claim this response may be indicative of long-lasting immunity.