Universal Flu Vaccine—Multivalent mRNA Vaccine for All Known Seasonal Influenza Viruses

Introduction

According to information from the World Health Organization (WHO), seasonal influenza (or flu) is an acute respiratory infection caused by influenza A and B viruses, which circulate in all parts of the world. Illnesses range from mild to severe and can be deadly; hospitalization and death occur mainly among high-risk groups, such as young children, pregnant women, and the elderly. Worldwide, annual flu epidemics are estimated to result in ~3 to 5 million cases of severe illness, and ~290,000 to 650,000 respiratory deaths.

The Centers for Disease Control and Prevention (CDC) provides details for classification and naming of flu viruses. In brief, influenza A viruses are divided into subtypes based on two kinds of proteins on the surface of the virus, namely, hemagglutinin (H) and neuraminidase (N) (Figure 1). There are 18 different H subtypes (H1-H18) and 11 different N subtypes (N1-N11). Current subtypes of influenza A viruses that routinely circulate in people include A/H1N1 and A/H3N2. Influenza B viruses are not divided into subtypes, but instead are further classified into two lineages, namely, B/Yamagata and B/Victoria. 

The National Institutes of Health (NIH) notes that each year, before the flu season begins, scientific experts must predict which influenza strains are likely to be most common during the upcoming months and then select three or four of these strains to include in the next seasonal flu vaccine. Traditional vaccine manufacturers then need time to produce and distribute the vaccine, during which the dominant strains of the virus can change in unexpected ways, potentially decreasing the efficacy of the vaccine. 

Overall, these factors have led to an average flu vaccine efficacy of only ~40% for the years 2005-2022, based on CDC data. An effective universal flu vaccine could eliminate these problems by protecting its recipients against a wide variety of strains.

A Multivalent Nucleoside-Modified mRNA Universal Flu Vaccine

Flu vaccines based on modern mRNA technology provide significant advantages over traditional flu vaccines that are produced using an egg-based manufacturing process that has been employed for more than 70 years. Growing flu viruses in eggs can introduce changes that can cause differences between the viruses in the vaccine and the ones that are circulating. These egg-adapted changes could result in less effective antibodies leading to less effective protection against the flu viruses in circulation. This problem is obviated by encoding each flu antigen in mRNA that can be individually manufactured for each known flu variant and pooled into a multivalent mRNA universal flu vaccine.

A report on this universal mRNA strategy by Arevalo et al. in Science in November, 2022, has been currently downloaded more than 22,000 times, indicating a very high-level of interest. The following sections provide highlights of this first-of-kind study, which used TriLink products to prepare CleanCap® nucleoside-modified mRNAs to obtain the multivalent vaccine.

Design of Flu Immunogens

As noted above, there are 18 different H-antigen influenza A virus subtypes and 2 distinct H-antigen lineages of influenza B that circulate seasonally in the human population. Because it is not possible to accurately predict which of these influenza viruses will cause the next outbreak of flu, the approach used by Arevalo et al. for inducing universal immunity was to design a multivalent mRNA vaccine that encodes all 20 H-antigens from all of these known influenza strains. 

Briefly, they used publicly available databases to download the available full-length H-antigen genes for influenza A types H1-H18 and influenza B/Victoria and B/Yamagata lineages. Phylogenetic trees were built using the Nextstrain pipeline in order to select a representative mRNA sequence encoding the H-antigen for each of the 20 different flu strains, as detailed in the Supplementary Materials and Fig. S1 therein. 

mRNA/Lipid Nanoparticle (LNP) Production

All of the 20 mRNAs were co-transcriptionally synthesized using N1-methylpseudouridine-5’-triphosphate (TriLink), instead of uridine-5’-triphosphate, and the trinucleotide Cap1 analog, CleanCap® reagent (TriLink), as previously described (Freyn et al. 2021). One-step cellulose purification (Baiersdörfer et al. 2019) was followed by gel analysis of each mRNA and frozen storage at -20 oC. 

The nucleoside-modified mRNAs were encapsulated in LNPs using a self-assembly process (Pardi et al. 2015) wherein an ethanolic lipid mixture of ionizable cationic lipid, phosphatidylcholine, cholesterol, and polyethylene glycol-lipid was rapidly mixed with an aqueous solution containing mRNA at acidic pH. The mRNA-loaded particles were characterized and subsequently stored at -80oC at a concentration of 1 mg/mL. 

Robust and Durable Universal Immunity in Mice

Groups of mice were injected intramuscularly with a low dose (3 µg) of each individual mRNA-LNP vaccine to first verify that each mRNA vaccine component was immunogenic. Each individual mRNA vaccine elicited antibodies that reacted more efficiently to the encoded H-antigen compared with the 19 other H-antigens that were tested, indicating a low-level or no detectable cross-reactivity among antibodies elicited by single H-antigen mRNA vaccinations.

Arevalo et al. then vaccinated mice with the 20 H-antigen mRNA-LNPs simultaneously using a combined dose of 50 µg of H-antigen mRNA, i.e., 2.5 µg of each individual H-antigen mRNA-LNP. As controls, mice were vaccinated with a 50-µg dose of mRNA-LNPs

encoding single H-antigens from either influenza A/H1N1 or A/H3N2, which commonly circulate during flu season, or phosphate buffered saline (PBS). 

Mice vaccinated with the 20 H-antigen mRNA-LNPs produced antibodies that strongly reacted (P < 0.05) to all 20 encoded H-antigens, whereas mice vaccinated with either single influenza A/H1N1 or A/H3N2 mRNA-LNPs or PBS did not. Antibody levels in mice immunized with the 20 H-antigen mRNA-LNP vaccine remained largely unchanged 4 months post-vaccination, indicating durable immunization.

Prime-Boost Universal Immunity in Ferrets

Finally, Arevalo et al. carried out a prime-boost vaccination experiment with the 20 H-antigen mRNA-LNPs in ferrets (a non-rodent larger than mice) to mimic the dosing schedule initially used for evaluating SARS-CoV-2 mRNA vaccines for COVID-19 (Wang et al. 2021). Each ferret produced antibodies reactive to all 20 H-antigens after a single vaccination, and antibody levels increased after a booster vaccination delivered 28 days later. 

Fully vaccinated and unvaccinated ferrets were then challenged with an avian H1N1 virus that has a H1 antigen distinct (81.8% amino acid homology) from the H1 antigen included in the 20 H-antigen mRNA-LNP vaccine to mimic a hypothetical flu outbreak featuring an unknown viral strain. Unvaccinated animals lost >16% of their initial weight by day-5 post-infection and 50% of the animals died, whereas fully vaccinated ferrets lost only ~8.5% of their initial weight by day-5 post-infection and 100% of the animals survived. 

In addition, unvaccinated ferrets displayed more clinical signs of disease (Brown et al. 2022) relative to fully vaccinated animals after infection. Viral titers in nasal washes were similar in unvaccinated and fully vaccinated animals at day-1 to day-4 after infection, but the virus was cleared more efficiently in fully vaccinated animals at day-5 and day-6 after infection. Thus, the 20 H-antigen mRNA-LNP vaccine protected ferrets against an antigenically mismatched avian H1N1 virus.

Concluding Comments

Arevalo et al. suggest that significant advantages of this 20-plex multivalency approach include the following factors.

• - Flu strain-match may be more accurate because there will be no need to grow the virus in eggs. This can also improve manufacturing, because making mRNA is less cumbersome compared to recombinant technology, facilitating vaccine approval and distribution.  - Vaccine immunity may be better or broader because the viral proteins will be expressed at high fidelity by human cells likely preserving the natural structure. - In vitro transcribed mRNA makes it easier to incorporate a larger number of antigens, which may stimulate cellular immunity or expand protection beyond flu virus H-antigens to N, nucleoprotein, and matrix-2 ion-channel antigens, discussed elsewhere (Pecetta et al. 2022). It was further suggested that this multivalent mRNA-LNP approach will likely be useful for other infectious diseases caused by genetically variable pathogens. Especially those infectious pathogens prone to mutation-driven diversification, such as coronaviruses and rhinoviruses. 

Arevalo et al. note that future studies will be required to determine the maximum number of antigens that can be simultaneously delivered through mRNA-LNP vaccines and the underlying immunological mechanisms that allow for the induction of responses against multiple antigens. In the meantime, a news report about this work by Arevalo et al. states that “initial clinical trials are being designed.” While no further information is available at this time., the ClinicalTrials.Gov database provides the status for Phase 1 trials of quadrivalent influenza mRNA vaccines independently developed by Sanofi (NCT05553301) and Moderna (NCT05606965).