In a novel proof of concept study, published on the bioRxiv* preprint server, U.S. researchers used attenuated influenza viral particles that express severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain to induce neutralizing antibodies in mice – unveiling another viable vaccine candidate for preventing coronavirus disease (COVID-19).
The unprecedented spread and seriousness of the disease caused by the SARS-CoV-2 virus put the need for effective control measures in the spotlight. An effective, safe, and scalable vaccine is the most promising option to limit the dire public-health impact of COVID-19.
The influenza virus represents a promising and already studied platform for vaccine development. More specifically, not only attenuated influenza viruses are already used as vaccines against influenza itself, but well-established reverse genetics systems enable the incorporation of foreign genes into the influenza genome.
Furthermore, intranasal infection with influenza induces mucosal immune responses not only within the respiratory tract but also in other mucosal surfaces, which may prove crucial for SARS-CoV-2. Finally, the infrastructure for large-scale production of influenza virions for vaccine usage already exists.
In this study, the researchers from Fred Hutchinson Cancer Research Center set out to incorporate a membrane-anchored form of SARS-CoV-2 receptor-binding domain (RBD) into the influenza genome.
Developing influenza-virus-based vaccine platform
The aforementioned RBD of the SARS-CoV-2 spike glycoprotein represents a key antigen candidate for SARS-CoV-2 vaccines, as it is small in size, demonstrates autonomous folding, and induces the production of potent neutralizing antibodies against SARS-CoV-2.
The researchers engineered the neuraminidase segment of the A/WSN/33 influenza virus in order to replace the neuraminidase coding sequence with a sequence coding for the membrane-anchored RBD. The derived construct was named ΔNA(RBD).
Then, the reverse-genetics approach was utilized to generate influenza virions encoding the ΔNA(RBD) segment, which was named ΔNA(RBD)-Flu. Alongside the ΔNA(RBD) segment, these viruses harbored the hemagglutinin (H.A.) segment from the A/Aichi/2/1968 (H3N2) influenza strain.
"We next set out to determine if cells transfected with the membrane-anchored RBD gene or infected with ΔNA(RBD)-Flu expressed RBD on their surface," further explain study authors in their bioRxiv paper.
"To do this, we transfected cells with a mammalian protein expression plasmid encoding the membrane-anchored RBD or the SARS-CoV-2 spike glycoprotein – lacking the last 21 amino acids of the cytoplasmic tail", they add.
To determine whether the ΔNA(RBD)-Flu virus successfully induces an anti-RBD antibody response in vivo, the researchers intranasally infected four groups of four mice. Finally, they have tested if the mice had also mounted an immune response to the influenza virus by performing viral neutralization assays.
A 'double whammy' approach
This study has shown that ΔNA(RBD)-Flu virus can stably maintain the gene encoding the RBD over multiple passages, while the cells infected with this constructed virus express high levels of RBD on their surface.
Furthermore, the intranasal inoculation of mice with a single dose of ΔNA(RBD)-Flu elicited neutralizing antibodies against SARS-CoV-2 in mice, with titers akin to those observed in humans after natural infection or administration of two doses of an mRNA-based vaccine in recent clinical trials.
Remarkably, the ΔNA(RBD)-Flu elicited these neutralizing antibody titers after only a single intranasal inoculation, and also at a viral dose that is much lower than those typically used for live-attenuated influenza vaccines.
"It, therefore, seems possible that neutralizing antibody titers against SARS-CoV-2 could be further enhanced by using a higher viral dose in a single inoculation, and/or by boosting either with recombinant RBD or a second administration of a ΔNA(RBD)-Flu variant containing a different H.A. protein", say study authors.
Implications and advantages
Vaccines based on ΔNA(RBD)-Flu have a myriad of possible advantages. As already mentioned, the vaccine successfully induces neutralizing antibodies after using just one dose, while mucosal immune responses may be pivotal for protective immunity against SARS-CoV-2.
Moreover, it might be feasible to leverage additional known influenza-virus engineering approaches to generate virions that express N.A. as well as RBD, serving in turn as dual influenza and SARS-CoV-2 vaccines.
Finally, there is already existing infrastructure for large scale production of influenza-virus based vaccines. And since one of the significant hurdles in front of a COVID-19 vaccine is scaling and global distribution, this could indeed be a viable option to produce live-virus vaccines similar to ΔNA(RBD)-Flu at scale.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
- Loes, A.N. et al. (2020). Attenuated influenza virions expressing the SARS- CoV-2 receptor-binding domain induce neutralizing antibodies in mice. bioRxiv. https://doi.org/10.1101/2020.08.12.248823.