Research develops a new intranasal influenza virus vector vaccine for SARS-CoV-2.

In a recent study published in Nature Communications, Researchers have developed a new intranasal vaccine candidate, DelNS1-RBD4N-DAF, against coronavirus disease 2019 (COVID-19).

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

study: An intranasal influenza virus vector vaccine prevents replication of SARS-CoV-2 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 post-infection death. However, the extent to which SARS-CoV-2 infection initiates immunity in the upper respiratory tract (URT) is less clear.

Many people, including vaccinated people, suffer frequent breakthrough infections, raising the need for alternative vaccine approaches that can enhance mucosal immunity of the upper respiratory tract (URT).

SARS-CoV-2 continues to coexist with seasonal respiratory viruses such as the influenza virus, so dual-function vaccines against both may be more suitable for people worldwide.

It could serve as a cost-effective and effective post-pandemic mitigation strategy, reducing SARS-CoV-2 circulation and possibly stopping its annual epidemic. Enhancing URT immunity will therefore be an important focus in the next phase of COVID-19 vaccine development.

Influenza virus was not used as a vaccine vector due to its segmented and compact genome. It is now possible to replicate them in the Darby canine kidney (MDCK) mammalian cell line. This has opened the door to the development of new live attenuated influenza viruses (LAIV) to combat respiratory viruses.

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

Phase I/II clinical trials have shown that the NS1-deleted mutant virus (DelNS1) is an excellent immunogen for inducing host immunity and is very safe for human use. I was. In addition, it induced high levels of interferon-β in infected cells and thus may serve as an adjuvant to enhance the immune response against SARS-CoV-2.

About research

In the present study, researchers first investigated the gene for the membrane-anchored protein, attenuation accelerating factor (DAF ) is included.

So they measured total anti-RBD immunoglobulin and its neutralizing capacity 6 weeks after primary vaccination. pseudovirus It expresses the SARS-CoV-2 S protein.

We then substituted four residues outside the receptor binding motif (RBM), namely A372T, G413N, D428N, and P521N, to construct RBDs and target them for N-glycosylation (4N). .

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

This helped researchers to assess immune responses to RBD and RBD4N in mice induced by the DelNS1-RBD-DAF LAIV vaccine system. They intranasally vaccinated test animals with DelNS1-RBD4N-DAF or DelNS1-RBD-DAF LAIV, DelNS1 vectors, or their beta or delta versions.

Next, they measured total antibody responses against RBD of wild-type (WT) and lineage A viruses. In addition, they used an enzyme-linked immunosorbent assay (ELISA) and a pseudovirus assay, respectively, to measure neutralizing activity.

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

In addition, they measured acute-phase T-cell responses in the lungs and spleens of mice 10 days after boost vaccination. They inoculated these animals twice with the DelNS1 vector or with the DelNS1-RBD4N-DAF containing the Delta or Omicron RBD, 4 weeks apart.

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

Hamsters are a better animal model for simulating the clinical and pathological manifestations of COVID-19 in humans because they are susceptible to SARS-CoV-2 infection.

Therefore, the team compared antibody induction in hamsters after prime-boost immunization with intramuscular BNT162b2 mRNA or intranasal DelNS1-RBD4N-DAF vaccines with findings obtained in mouse models.

result

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

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

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

Western blotting confirmed the increase in molecular weight of RBD in which the researchers substituted residues to promote N-glycosylation. Interestingly, N-glycosylation also increased her RBD expression in mammalian cell lines such as MDCK cells.

Therefore, DAF optimized cell surface RBD expression, protected epitopes outside the RBM by introducing four N-glycosylation sites, and facilitated the generation of nAbs specific for ACE2-competing epitopes.

SARS-CoV-2 Beta and Delta RBDs, termed Beta4N and Delta4N, induced higher nAb levels than RBDs without the 4N modification. Neutralization assays showed enhanced nAb responses to pseudoviruses expressing beta, delta, and omicron BA.1 S proteins.

As expected, intranasal delivery of the DelNS1-RBD4N-DAF vaccine elicited greater T-cell responses in the lung than in the spleen of vaccinated mice, enhancing the specificity of intranasal vaccination against URT.

During live SARS-CoV-2 challenge, immunization with Delta or Omicron RBD-based DelNS1-RBD4N-DAF LAIV prevented weight loss in mice and protected lung tissue. Virus was undetectable from day 2 to day 4 after inoculation of mice. Omicron BA.1 subvariant adaptation (MA) strain.

In hamsters, all vaccination schemes elicited similar levels of anti-RBD antibodies, particularly those of ancestral SARS-CoV-2 strains belonging to lineage A. Neutralizing antibody (nAbs) against Omicron BA.1 & BA.2 subvariants.

Notably, the DelNS1-RBD4N-DAF LAIV vaccine prevented replication of SARS-CoV-2 variants including delta and omicron BA.2, but not BNT162b2, in respiratory tissues, making them nearly sterile against SARS-CoV-2 infection. provided effective immunity. with a hamster.

Conclusion

The newly developed DelNS1-RBD4N-DAF LAIV vaccine candidate significantly induced immunity in the URT to prevent disease and viral replication, properties thought to be required for the next generation of COVID-19 vaccines.

In the future, it may serve as a bifunctional vaccine to combat annual influenza and SARS-CoV-2 epidemics. to warrant further evaluation in clinical trials.