Generation of a high yield vaccine backbone for influenza b virus in eggs

Influenza B virus strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens' eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for influenza B viruses, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 influenza B virus strains from 1940 to present day, including as much temporal and genetic variability as possible. This panel contains strains from the ancestral lineage, the B/Victoria/2/87-like lineage, and B/Yamagata/16/88-like lineage to provide a diverse set that would help to generate a suitable backbone that can be used in combination with hemagglutinin and neuraminidase glycoproteins from any influenza B strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 influenza B virus strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to generate reassortant viruses. After serial passaging, we performed two rounds of plaque purification to isolate monoclonal viruses. We screened 95 plaques by RNA-seq and identified 31 monoclonal plaques. We selected 10 clonal isolates based on hemagglutinin units and plaque-forming units to test their growth profiles. Then we selected three clones that performed the best and generated recombinant versions. Two of the selected backbones were used to generate different reassortant viruses with high and low yield HA/NA combinations. When the growth profiles of the recombinant reassortant viruses were tested the low yield HA/NA viruses with the two backbones yielded higher titers then the wild type viruses. Introduction Influenza B viruses (IBV) belong to the Orthomyxoviridae family and primarily infect humans. Influenza B viruses are segmented, single stranded, negative sense RNA viruses (1). Influenza B viruses are comprised of eight segments: hemagglutinin (HA), neuraminidase (NA), polymerase basic 1 (PB1), polymerase basic 2 (PB2), polymerase acid (PA), nucleoprotein (NP), matrix (M) and non-structural (NS) proteins. The influenza B virus was first isolated in 1940 and it is primarily a human virus virus, and although it has been found in seals, there are no other known animal reservoirs. The influenza B has been known to cause epidemics, but its restriction to humans has reduced the chances of a pandemic, since there are no animal reservoirs where the strain can reassort. Around 1980s, two different strains of influenza B virus were isolated, indicating co-circulation of two different strains. Since then, the influenza B virus is divided into two different lineages based on the sequences of the hemagglutinin (HA) segment of the virus. All the strains from 1940 to 1980s, before the split into the two different lineages, are referred to as the ancestral strain. Antigenic drift caused by variation in strains, due to immune selection of variant strains, has resulted in two different lineages formed, which started to co-circulate over time. The two lineages are referred to as B/Victoria-like and B/Yamagata-like and their assignment is based on the HA sequence. The initial identification of the co-circulation was also based on the results of hemagglutination-inhibition and neutralization tests used to monitor antigenic changes (2). Every year one strain is more dominant than the other, but in some years they also co-circulate (2). Influenza viruses are names by genus type, species isolated, location of isolate, isolate number, and year of isolation, for example, B/Texas/06/2011. Influenza B viruses are enveloped viruses with the HA and NA glycoproteins on the surface of the virion, and the matrix protein is the transmembrane protein. Eight single stranded RNA segments are located inside the virus particle. Each segment of influenza B virus has a specific role in the replication cycle of the virus. PB2, PB1, and PA are part of the RNA polymerase. PB2 is involved in cap recognition, PB1 is involved in elongation and PA is involved in endonuclease activity (3). HA is a surface glycoprotein, and it is involved in receptor binding and fusion activity. HA is one of the major antigens on the surface of the virus and the current target for most influenza vaccines. NP is involved in RNA binding and RNA synthesis. NA is a glycoprotein present at the surface, and it plays a role in the release of the new virions. M is a matrix protein and interacts with vRNPs and surface glycoproteins. M protein is made up of M1 and BM2; BM2 is a membrane protein, and it works as an ion channel and plays a role in assembly. NS protein is consisted of NS1 and NEP; NS1 is a multifunctional protein (4) and a viral IFN (interferon) antagonist and NEP is a nuclear export protein and plays a role in the export of vRNPs. Virus life cycle begins with the binding of virus to sialic acids on the cell surface to start infection and replication. The influenza B virus preferentially binds to N-acetylneuraminic acid, which is attached to the galactose sugar by an α2,6 linkage (3). Influenza virus internalization is dependent on pH, low pH is required to initiate fusion of the HA protein to the endosomal membrane (3). Other mechanisms of entry that have been discovered are clathrin mediated endocytosis (3). The fusion activity is induced by structural change in HA protein, where HA0 is cleaved into two subunits HA1 and HA2. The conformational change of HA exposes the fusion peptide, allowing it to interact with the membrane of the endosome (3). vRNPs are released into the endosome and nuclear export occurs. In the nucleus, transcription of mRNA and replication through a positive-sense complimentary ribonucleoprotein intermediate occurs. Viral mRNA is translated into viral proteins in the cytoplasm and these proteins are used to assemble new virions with the newly synthesized vRNPs (5). Over the years, influenza B viruses has caused many deaths during the influenza season. According to weekly flu reports by Centers for Disease Control and Prevention (CDC), pediatric death reports for 2019-2020 season has been mainly because of influenza B virus (6). And roughly half of the deaths in the US during the 2019-2020 season was due to influenza B virus (7). According to world health organization (WHO) the annual influenza epidemic results in about 1 billion infections, 3-5 million cases of severe illness and about 300,000-500,000 deaths (5). The influenza B virus infection can be treated by administering NA inhibitors, such as oseltamivir and zanamivir, which target the enzymatic activity of the viral NA protein by mimicking the binding of sialic acid in the active site of NA on influenza B viruses (5). However, there has been an emergence of antiviral resistant strains of influenza B viruses, leading to limited efficacy of the drugs, and most of the antivirals are only efficient when given within the first 24-48 hours of infection (5). The primary focus of targeting against influenza virus is yearly vaccination. The first influenza virus vaccine was a live-attenuated vaccine containing an influenza A virus strain. In 1942, a bivalent vaccine was produced that included an influenza A and influenza B strain. The first form of vaccine was made with the virus being serial passaged several times to reduce the virulence and the pathogenicity of the virus. The first virus used in USSR in 1936 was passaged in eggs thirty times before being used as a live influenza vaccine and it was given to factory workers (8). Due to antigenic drift and shift, there were many epidemics because of mismatch in the vaccine strain. In 2012, the first quadrivalent vaccine was approved where two strains of influenza A virus and two strains of influenza B virus were recommended in the seasonal vaccine. The first approved quadrivalent vaccine was Fluarix®, a split vaccine (9). The vaccine strain selection process is based on the determination of the prevalence of recent human circulating strains by multiple laboratories that are involved in the influenza surveillance program by the WHO. These laboratories sequence and test strains that are collected throughout the world, and, around February and March, a committee meets to decide the vaccine strains for the upcoming influenza season. Since the northern and southern hemisphere have influenza season in opposite times in the year, the committee decides for the northern hemisphere, based on influenza activity in the southern hemisphere and vice versa (5). The isolated viruses are also tested for their antigenicity, to confirm if the newly isolated viruses are antigenically similar to the previous strains, or the slow change in amino acids has generated an antigenically different virus. Antigenicity is normally tested by hemagglutinin inhibition (HAI) assay, where serum samples are incubated with the virus followed by blood, to see if hemagglutination occurs. If the antibodies in the serum cause inhibition, then the virus is not antigenically different from the previous viruses. There has been great progress in the generation of the seasonal influenza vaccines. There are several platforms that are currently being used, such as cell based, protein based, and egg based. Seasonal vaccines utilize the internal segment of a high yield laboratory strain, in combination with HA and NA of the recommended strain for the season. The laboratory strain for influenza A virus is A/PR/8/34, while there is no specific laboratory strain for influenza B virus. Historically, various strains have been used as the high yield backbone strains for influenza B viruses, such as, B/Lee/1940, and many times the wild type strain has been used to generate the vaccine strain. There have been several attempts made to generate a high yield seed virus