In a recent study published in Nature Communications, researchers developed a novel intranasal vaccine candidate, DelNS1-RBD4N-DAF, for coronavirus disease 2019 (COVID-19).

This live attenuated influenza virus (LAIV) vector-based vaccine lacked the non-structural protein 1 (NS1) gene but expressed the receptor-binding-domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein.

Study: An intranasal influenza virus-vectored vaccine prevents SARS-CoV-2 replication in respiratory tissues of mice and hamsters. Image Credit: OrpheusFX/Shutterstock.com

Background

All current COVID-19 vaccines, including messenger ribonucleic acid (mRNA) and adenoviral vector vaccines, prevent progression to severe disease and death post-infection. However, the extent to which they induce immunity in the upper respiratory tract (URT) is less clear, where SARS-CoV-2 infection begins.

Many people, including vaccinees, frequently contract breakthrough infections, raising the need for alternative vaccine approaches that could enhance mucosal immunity in the upper respiratory tract (URT).

Since SARS-CoV-2 will continue to co-circulate with seasonal respiratory viruses, e.g., influenza virus, a dual-function vaccine for both might be more suitable for people worldwide.

It will be cost-effective and work as an effective post-pandemic mitigation strategy to reduce SARS-CoV-2 circulation and possibly cease annual epidemics. Thus, in the next phase of COVID-19 vaccine development, the enhancement of URT immunity would be a key focus.

The influenza virus was not used as a vaccine vector, partly due to its segmented & compact genome. However, after discovering adaptative mutations in NS1-deleted (DelNS1) influenza viruses, it became feasible to replicate them in embryonated chicken eggs and Madin-Darby canine kidney (MDCK) mammalian cell lines. It opened doors to develop novel live attenuated influenza viruses (LAIV) combating respiratory viruses.

Intranasal administration of DelNS1-RBD4N-DAF LAIV mimicked natural influenza virus infection and delivered the SARS-CoV-2 RBD (antigen) to the URT.

Phase I/II clinical trials showed that the NS1-deleted mutant virus (DelNS1) was a great immunogen for triggering the host's immunity and was immensely safe for human use. Moreover, it induced high levels of interferon β in infected cells, thus, could also serve as an adjuvant to enhance the immune response against SARS-CoV-2.

About the study

In the present study, researchers first tested if including the decay accelerating factor (DAF), a gene for a membrane-anchored protein, enhanced immune responses to the receptor-binding domain (RBD) in mice primed by DelNS1-RBD-DAF LAIVs.

So, they measured total anti-RBD immunoglobulins six weeks following the primary vaccination and their neutralization potential against a pseudovirus expressing the SARS-CoV-2 S protein.

Next, they constructed an RBD with the substitution of four residues, viz., A372T, G413N, D428N, and P521N, outside the receptor-binding motif (RBM) to target them for N-glycosylation (4N).

They hypothesized that shielding of non-angiotensin-converting enzyme 2 (ACE2) competing epitopes nested outside the RBM might help better present RBM epitopes.

This helped the researchers evaluate immune responses to RBD and RBD4N in mice triggered by the DelNS1-RBD-DAF LAIV vaccine system. They vaccinated the test animals intranasally with DelNS1-RBD4N-DAF or DelNS1-RBD-DAF LAIVs, DelNS1 vector, or their Beta or Delta versions.

Next, they measured the total antibody response to the RBD of wild-type (WT) and lineage A virus. Further, they used enzyme-linked immunosorbent assay (ELISA) and pseudovirus assays to measure neutralizing activity, respectively.

Finally, the researchers compared the immunogenicity of Beta4N-, Delta4N-, and Omi4N-DAFs with the mRNA vaccine in animals. To this end, they measured anti-RBD immunoglobulin A (IgA) in bronchoalveolar lavage (BAL) fluid taken from vaccinated mice.

In addition, they measured acute phase T cell responses in the lungs and spleens of mice ten days following boost vaccination. They vaccinated these animals with two doses of DelNS1 vector or DelNS1-RBD4N-DAF containing Delta or Omicron RBDs at an interval of four weeks.

Briefly, the team analyzed immune responses and protection against the live SARS-CoV-2 challenge following intranasal administration of DelNS1-RBD4N-DAF vaccines in mice.

Since hamsters are more susceptible to SARS-CoV-2 infection, they are a better animal model to simulate the clinical and pathological manifestations of COVID-19 in humans.

Thus, the team compared the findings made in the mouse model with antibody induction in hamsters following prime-boost immunization with intramuscular BNT162b2 mRNA or intranasal DelNS1-RBD4N-DAF vaccines.

Results

In animal models, the LAIV vector system expressing SARS-CoV-2 RBD from the site of NS1 deletion (DelNS1-RBD) effectively boosted systemic and mucosal immune responses.

Fusing SARS-CoV-2 RBD to a short peptide spanning DAF's transmembrane and cytoplasmic domains ensured it was processed and presented on the cell surface DelNS1-RBD-DAF LAIV infected cells.

Its inclusion for cell surface presentation and selective glycosylation of non-ACE2 competing epitopes in the new DelNS1-RBD4N-DAF LAIV vector further enhanced its immunogenicity.

Western blotting confirmed an increase in the RBD's molecular weights where the researchers replaced residues to promote N-glycosylation. Intriguingly, N-glycosylation also increased RBD expression in mammalian cell lines, e.g., MDCK cells.

So, while DAF optimized cell surface RBD expression, introducing four N-glycosylation sites shielded epitopes outside the RBM and encouraged the generation of nAbs-specific for ACE2 competing epitopes.

The SARS-CoV-2 Beta and Delta RBDs termed Beta4N and Delta4N induced higher nAb levels than RBDs with no 4N modifications. Neutralization assays showed enhanced nAb responses against pseudoviruses expressing Beta, Delta, and Omicron BA.1 S proteins.

As expected, intranasal delivery of the DelNS1-RBD4N-DAF vaccine triggered a greater T cell response in the lungs than in the spleens of vaccinated mice, reinforcing the specificity of intranasal vaccination to URT.

During live SARS-CoV-2 challenge, immunization with Delta or Omicron RBDs-based DelNS1-RBD4N-DAF LAIVs prevented body weight loss in mice and protected their lung tissues, with the virus being undetectable between days two and four after inoculation with a mouse-adapted (MA) strain of Omicron BA.1 subvariant.

In hamsters, all vaccination schemes triggered similar levels of anti-RBD antibodies, especially that of the ancestral SARS-CoV-2 strain belonging to lineage A. Conversely, two doses of the Omi4N-DAF vaccine did not induce marked levels of neutralizing antibodies (nAbs) against Omicron BA.1 & BA.2 subvariants.

Notably, the DelNS1-RBD4N-DAF LAIV vaccine, but not BNT162b2, prevented replication of SARS-CoV-2 variants, including Delta and Omicron BA.2, in the respiratory tissues, and provided almost sterilizing immunity against SARS-CoV-2 infection in hamsters.

Conclusions

The newly developed DelNS1-RBD4N-DAF LAIV vaccine candidate remarkably induced immunity in URT to prevent disease and viral replication, an attribute deemed necessary in the next-generation COVID-19 vaccines.

In the future, it could serve as a bi-functional vaccine to combat influenza and SARS-CoV-2 annual epidemics. However, this vaccine system warrants further evaluation in clinical trials before use as annual vaccination in humans.

Written by

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.