GENERATION OF A HIGH YIELD VACCINE BACKBONE FOR INFLUENZA B IN EGGS

by

Sadaf Aslam

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Arts in Biology

Queens College The City University of New York

July 2021

Alicia Meléndez, Ph.D. Approved by______Committee Chair

______Signature

July 19, 2021 Date:______

Table of Content

i. Acknowledgment

1. Abstract

2. Introduction

3. Materials and Methods

4. Results

5. Conclusion/Discussion

6. Reference

Acknowledgment

This work was done in Dr. Adolfo García-Sastre’s laboratory at Icahn School of Medicine at

Mount Sinai. The work was done under the supervision of Dr. Juan Ayllón and Dr. Adolfo

García-Sastre. Madhusudan Rajendran helped performed plaque assays for samples from figure

1. Dr. Juan Ayllón and Dr. Alicia Meléndez helped edit this manuscript.

Abstract

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 , 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 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 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. 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 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 for influenza B virus, through , and by introducing several growth enhancing mutations into the six internal segments of a selected strain (10). However, the disadvantage of this method was that instead of

one seed virus, two separate seed viruses were generated for the two different lineages of influenza

B viruses, instead of one seed virus that can be used for all influenza B viruses (10). Recombinant influenza vaccines were made by co-infecting the selected strain with the laboratory strain, in the presence of antibodies against the HA and NA of the laboratory strains, to generate recombinant viruses that had the internal segments of the laboratory strains and HA and NA of the desired strains. However, due to the advancement in reverse genetic techniques, influenza viruses were generated by transfecting plasmids that contained internal segments of the laboratory strains, and plasmids that expressed HA and NA of the desired circulating strain. This allowed a faster method of generating recombinant viruses that can be used for vaccine production (11-13). The current influenza manufacturing process takes about 6 months, from the day strains are selected, to the availability of enough vaccine doses (14, 15). It is important to have a high yield backbone strain, because you can produce more vaccines with a strain that grows to high titers versus one that grows at low titers. High yield strains are also very efficient at generating reassortant viruses, to help expediate the vaccine production process.

In this study, we tried to generate a high yield vaccine backbone for influenza B virus.

We wanted to generate a backbone that can be used to generate high titers for any influenza B virus strain, regardless of the lineage of the wild type strain. We also wanted to generate a backbone that produced HA levels similar or higher than the wild type strains.

Material and Methods

Cell culture

Madin-Darby canine kidney (MDCK) epithelial cells and human embryonic kidney 293T cells were cultured in Dulbecco’s modified Eagle ’s medium (Gibco) supplemented with 10%

fetal bovine serum (HyClone) and 1% penicillin-streptomycin (Gibco) and grown at 37 °C and

5% CO2.

Virus growth in eggs, plaque assay, and hemagglutination assay

Influenza B virus strains were grown in 8-10 day old embryonated chicken eggs (Charles

River) for various time points and temperatures. Allantoic fluid was harvested and spun at 1200 rpm for 5 minutes at 4°C and supernatant was aliquoted and frozen at -80°C. Viral titer was determined using standard plaque assay on MDCK cells and immunostained using pool of monoclonal mouse antibodies against various strains of influenza B virus. Hemagglutination assay was done as described before (16), briefly, virus supernatant is serially diluted (1:2) with phosphate buffered saline (GIBCO) in a V-bottom 96-well plate (fisher) and 50 µl of 0.5% chicken red blood cells are added and incubated at 4°C for 30 minutes. Hemagglutination units are determined by the reciprocal of the last dilution which showed complete hemagglutination.

Cloning influenza B virus segments into rescue plasmids

Viral RNA was extracted from the allantoic supernatant using E. Z. N. A. Viral RNA kit

(Omega Bio-Tek) according to the manufacturer guidelines. Viral segments were amplified by

RT-PCR using Superscript III one-step RT-PCR kit (Invitrogen) and segment specific primers.

RT-PCR products were gel purified (QIAGEN). pDZ plasmid was digested with Sap I restriction enzyme (New England biolabs) and treated with alkaline phosphatase (New England biolabs) and gel purified. Purified RT-PCR products were cloned into digested pDZ plasmid using In-

Fusion kit (Takara bio). Plasmid sequences were confirmed by Sanger sequencing (Psomogen).

Generation of recombinant influenza B viruses

Recombinant influenza B viruses were generated by transfection of 293T with plasmids containing viral segments as described previously (13). Briefly, 8 plasmids encoding viral

segments are co-transfected using TransIT LT1 (Mirus) and optimem (Gibco) according to manufacturer guidelines. 293T cells (2x105 cells/ml) are mixed with DNA/TransIT LT1 mixture and plated in a 6-well plate. 24 hours later media is changed to infectious media (1XMEM

(GIBCO), 1% penicillin/streptomycin, 0.2% BSA (MP biomedicals), 1mg/ml of N-tosyl-L- phenylalanine chloromethyl ketone (TPCK) trypsin) and the cells are incubated for 2 days at

33°C. The supernatant is harvested and injected into 8-10 day old embryonated chicken eggs and incubated for 3 days at 33°C. Allantoic fluid is harvested and hemagglutination assay is performed to check for presence of virus. Viruses were plaque purified and stocks were aliquoted and stored at -80°C.

Deep sequencing and data analysis

In order to get consensus sequences and monitor polymorphisms during passage, viruses were deep sequenced. Viral RNA was extracted and prepared for RNA-seq as described previously (19). Briefly, viral RNA was extracted using E. Z. N. A. viral RNA kit (Omega Bio-

Tek) and the samples were prepared to run on illumine. Raw data was aligned using NGEN

DNASTAR lasergene and Integrative genomics viewer (IGV) (20).

Virus purification/concentration

Viruses were grown in 10-day old eggs and allantoic fluid was harvested and clarified by centrifugation at 1200 rpm using a benchtop centrifuge (Eppendorf) at 4 ℃ for 5 minutes. The clarified supernatant was concentrated using a 30% sucrose cushion in NTE buffer (100 mM

NaCl, 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 8) by centrifugation in a Beckman L7-65 ultracentrifuge at 22,000 rpm for 2 hours at 4℃ using a

Beckman SW28 rotor. Supernatant was aspirated and pellets were resuspended in PBS and stored at -80 ℃ in small aliquots.

Western blot and protein quantification

Purified viruses were quantified using pierce BCA protein assay kit (thermo fisher) and

20ng of protein was mixed with pierce lane marker reducing sample buffer (thermo fisher).

Purified viruses were run on 10% reducing denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad) and the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) using turbo transfer system (Bio-Rad).

The membrane was blocked with 5% (w/v) non-fat milk in PBS containing 0.1% (v/v) Tween 20

(PBST) for 1 h at room temperature on a shaker. The membrane was incubated with KL-BHA-

4C10 (1:2000) diluted in 5% non-fat milk in PBST overnight at 4℃ on a shaker. The membrane was washed with PBST three times (5 minutes incubation each time) on a shaker at room temperature. The membrane was incubated with anti-mouse-HRP IgG diluted in 5% milk in

PBST (1:2000) for 1 h at room temperature on a shaker. The membrane was washed three times with PBST and developed using Brightstar HCL (ASI). The HRP was killed by incubating the membrane with 1% sodium azide for 15 minutes on a shaker at room temperature. The membrane is washed overnight with PBST (changed several times) on a shaker. The membrane was incubated with polyclonal antibody against NP (Invitrogen, PA5-81758) diluted in 5%

Milk/PBST (1:2000) overnight at 4℃ on a shaker. The membrane was washed three times and incubated with anti-rabbit-HRP diluted in 5% milk/PBST (1:2000) and incubated for 1hr at room temperature on a shaker. The membrane was washed three times and developed with Brightstar

HCL.

Results

Optimal conditions and testing a panel of influenza B virus strains

Initially, we wanted to determine the best possible growth conditions for influenza B virus strains, so we could use them later for the selection of the optimized backbones. We started testing for optimal temperature, and time post infection, using three model strains representative of B/Phuket/3073 (B/Yamagata-like), B/Malaysia/2506/2004 (B/Victoria-like), and B/Hong

Kong/8/1973 (pre-lineage). We tested two different temperatures and several time post infections. The three representative strains grew to the highest infectious titers and HA titers at

33 ℃ and 48 hours post infection (Figure 1A), and they seemed to be the same along the model strains, independently of their lineage. Thus, from there onward we continued using just one of them as an internal control and as a reference strain. Then we tested for the optimal inoculum by testing three different inoculums (250, 500, and 1000 pfu) and two different egg ages (8 and 10 days old) at 33°C and 48 hours post infection. The B/Malaysia/2506/2004 had the highest HA and plaque titers in 10-day old eggs, with the 250 pfu inoculum (Figure 1B).

Once the optimal conditions were established as 33℃, 48hr post infection, 10 day old eggs, and 250 pfu inoculum, we proceeded to use them to assess the peak growth titers of a broad collection of influenza B virus strains (Figure 2), which has been described before (16).

This allowed us to show a continuous distribution of peak titers, and to discriminate those strains with potentially high-yield genetic constitutions (from which the optimized backbone would be selected) and those low-yield strains which would be used later for assessing the yield increase provided by said backbones. Phylogenetic analysis was done on the HA and NP segments

(Figure 3) of all the viruses used in this collection to show the broad distribution and temporal diversity of this panel.

Co-infection and reassortant generation

Next, we selected 15 strains that had high infectious titers, from the panel of 71 strains. We

selected five strains with the highest titers from each group pre-lineage, B/Yamagata/16/1988-

like, and B/Victoria/2/1987-like for co-infection. The selected strains are highlighted in red in

the two phylogenetic trees. We pooled all the strains at a constant titer of 40 pfu/strain and

infected three 10-day old eggs labelled Mix 1, Mix 2, and Mix 3, respectively. These samples

were serially passaged in eggs for 10 passages. Between each passage the virus was diluted to

10-5 and 200µl was injected into the eggs (Figure 4). We monitored hemagglutination and plaque

titers during the passages. Throughout the passages viral titers were relatively high. After

passage 10, RNA-seq was done on all three samples and we saw a polyclonal population in every segment of each of the samples.

Clonal Isolates

Since viruses isolated after passage 10 had many polymorphisms, we picked multiple clones from each of the three samples (total of 19 clones) and amplified them in eggs. All 19 clones were deep sequenced and analyzed. Since the plaque purified viruses still had many polymorphisms in every segment, we selected 5 clones per initial plaque and amplified them in eggs for a total of 95 clonal isolates. After analyzing the data, we found 31 clonal isolates out of the 95 that were monoclonal in every segment. The identities of each segment were identified by aligning the fastq files from RNA-seq with the consensus sequences of each of the fifteen strains used for co-infection. The strain that aligned closest to the given segment was assigned that strain name. There was a lot of diversity in each segment. Most of the sequences matched 100%

with the original sequence but there were also some segments that had a few nucleotide

mutations.

Growth kinetics and selection of backbone

Next, we determined the hemagglutinin and plaque titers of all 31 clonal isolates to select

the top 10 clonal isolates to move to further test for determining the best backbone. After

comparing hemagglutination and plaque titers (Table 1), we selected 10 clonal isolates (Table 2)

to perform growth kinetics under the optimal conditions that we had defined earlier.

Based on the growth kinetics we identified three clonal isolates that grew to high titers at 48 hours post infection (Figure 5). The three clonal isolates selected to establish reverse genetics were M1P21, M3P84 and M3P67.

Recombinant virusses

We generated recombinant viruses using the three selected backbones. We cloned all 8 segments of M1P21, M3P84 and M3P67 into pDZ vector (17) for generating recombinant viruses

(13) (Figure 6). These 8 plasmids were transfected into 293T cells and recombinant viruses were generated. Next, we selected a high titer and low titer strain from pre-lineage,

B/Yamagata/16/1988-like, and B/Victoria/2/1987-like groups and cloned out their hemagglutinin and neuraminidase into pDZ vector. We used B/Hong Kong/1972 (Pre-lineage), B/Hawaii/2001

(B/Victoria/2/1987-like) and B/Utah/2014 (B/Yamagata/16/1988-like) as the low titer yielding viruses.

We also compared the high titer yielding viruses to see if we can maintain or increase

their titer even further with our backbone constructs. We used B/Great Lakes/1954 (Pre-lineage),

B/Bangladesh/5278/2006 (B/Victoria/2/1987-like) and B/Texas/06/2011 (B/Yamagata/16/1988-

like) as the high titer yielding viruses (Figure 7). We generated recombinant M1P21 and M3P67

(considered WT) viruses and in combination with the different hemagglutinin and neuraminidase

with each of the backbones. We dropped M3P84 backbone because the viruses did not rescue

very efficiently with the different combinations. We were able to rescue different combinations

of viruses with M1P21 and M3P67 backbones in the first attempt.

Growth kinetics using recombinant viruses

To validate that the high yield backbone truly increases the titer of the virus we

performed growth kinetics using the 14 recombinant viruses that we generated. We tested

different combinations of hemagglutinin and neuraminidases with the reassortant viruses. We

used the optimal conditions to perform the growth kinetics. We saw that the titers for the

recombinant viruses with the low yield HA/NA increased by 100-fold in comparison to the wild

type viruses in our original screen (Figure 8). And the titers for recombinant viruses with high

yield HA/NA were still high and similar to the original titers. This indicates that these backbones are efficient at generating reassortant viruses and can increase the titer for low yielding viruses regardless of the lineage of the virus.

HA level comparison

Finally, we wanted to determine if the HA levels of the recombinant viruses were similar to the wild type viruses. We purified the recombinant viruses and wild type viruses using 30% sucrose gradient and ran 10% reducing denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting by probing with KL-BHA-4C10 (18). We compared HA2 levels across all the viruses and saw that the HA2 levels were comparable across the recombinant and wild type viruses (Figure 9). A polyclonal antibody against NP was used as a loading control and NP levels were similar across the viruses as well. This result indicates that the recombinant viruses express similar levels of HA2 to the wild type indicating that the

backbone can yield high titers and express comparable levels of HA protein, the protein used for standardizing the vaccine batches.

Discussion/ Conclusion

In this study, we generated two backbones that can be used as vaccine backbones for producing influenza B strains for vaccines. We tested these backbones in combination with different HA and NA from different lineages of influenza B viruses and saw that the low yield viruses grew to higher titers when in combination with the two backbones. We also tested HA2 levels of the wild type strains and with the reassortant viruses and saw that the HA2 levels were relatively similar in both viruses. These backbones can be used in the future to generate vaccine strains that grow to high titers and can yield several doses of the vaccine in the egg based system, the most used system currently available. In future these backbones can be tested in other vaccine platforms to see if they still yield high titers and can be used across platforms.

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