HC and ZZ produced the vaccine and involved in immunization experiments. being used in many countries. However, all the authorized vaccines are given by intramuscular injection and this may not prevent top airway illness or viral transmission. Results Here, we describe a novel, intranasally delivered COVID-19 vaccine based on a helper-dependent adenoviral (HD-Ad) vector. The vaccine (HD-Ad_RBD) generates a soluble secreted form of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and we show it induced strong mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost routine, resulted in total protection of the top respiratory tract against SARS-CoV-2 illness. Summary Our methods provide a powerful platform for constructing highly effective vaccines focusing on SARS-CoV-2 and its growing variants. Supplementary Information The online version consists of supplementary material available at 10.1186/s13578-021-00723-0. gene to increase mRNA stability (Fig.?1A). A DNA sequence encoding the 20-amino acid signal Trapidil peptide of the human being cystatin S protein was included upstream of the coding sequence of the RBD, which allows for RBD secretion. The BGH poly A tail was used to terminate transcription. Open in a separate windows Fig. 1 Building of the HD-Ad_RBD vaccine. A HD-Ad_RBD vaccine schematic. ITR, adenovirus inverted terminal repeat; Pa, adenovirus packaging signal; CBA-P, chicken beta actin gene promoter with CVM enhancer; UbC-int, the 1st intron of the human being ubiquitin C gene; SP, the human being cystatin-S transmission peptide; RBD, receptor binding website of the SARS-CoV-2 spike protein; PolyA, transcription termination transmission of the Bovine Growth Hormone gene; Stuffer DNA, noncoding human being DNA used to make the vector genome large enough to be packaged. B Western blot analysis of RBD manifestation and secretion. Epithelial cells A549 and IB3 were transfected with HD-Ad_RBD in the indicated dosages and cell lysates and tradition supernatants were prepared and subjected to Western blot analysis using anti-RBD antibodies To examine the manifestation and secretion of the RBD, epithelial cells (A549 and Trapidil IB3) were Trapidil transfected with HD-Ad_RBD at different dosages and the cell lysates and tradition supernatants were analyzed by Western blot. The results showed the RBD was indicated at high levels in both cell lines inside a dose-dependent manner and that approximately 90% of the RBD was found in the tradition supernatant (Fig.?1B). The secreted transgene product delivered by HD-Ad vectors is known to reach both airway fluid and the blood circulation system  and, as such, the secreted RBD is definitely expected to reach antigen showing cells, locally and systemically, to induce antigen-specific immune reactions. HD-Ad_RBD induces strong mucosal and systemic immunity To examine the immune reactions Trapidil induced by HD-Ad_RBD, we intranasally immunized BALB/c mice (gene specific primers, we recognized very high levels of viral RNA (108 to 109?copies/mg) in the lungs of mice vaccinated with the HD-Ad vector control. In contrast, the viral RNA levels were reduced by ?4?log10 in 16 out of the 17 mice vaccinated with HD-Ad_RBD (Fig.?3C). The very low RNA levels in the lungs of the mice vaccinated with HD-Ad_RBD may reflect an inability of the SARS-CoV-2 challenge dose to replicate in these animals. This suggestion is definitely consistent with the following two lines of reasoning. First, there was a significant increase ( ?1?log10) in both the infectious viral titer and the viral RNA copy quantity in the lungs of the control mice at day time 3 compared to day time 1, an indication of substantial viral replication in these mice (Fig.?3B, C). In contrast, no infectious computer virus was recognized in the HD-Ad_RBD vaccinated mice and the viral RNA level Trapidil remained constant at day time 1 and 3 post-infection (Fig.?3B, C). Second, the low SARS-CoV-2 RNA levels (~?104?copies/mg) observed at day time 1 and 3 in the vaccinated animals are similar to that observed at these time points in SARS-CoV-2 illness experiments in BALB/c and C57BL/6 mice (which lack Rabbit Polyclonal to ZNF329 hACE2) where replication of the input virus is not possible . Amazingly, intranasal delivery of HD-Ad_RBD offered effective protection of the top respiratory tract as judged from the absence of measurable viral RNA in the oropharyngeal swabs.
HC and ZZ produced the vaccine and involved in immunization experiments