Development and Evaluation of an Antibody- Dependent Cellular Cytotoxicity (ADCC) Assay for A

A thesis submitted to the

Graduate School

of University of Cincinnati

in partial fulfillment of the

requirements for the degree of

Master of Science

in the Department of Pharmacology and System’s Physiology

of the College of Medicine

Author: Committee Chair: Dhwani Mehta Paul Spearman, MD

November 13, 2020 Abstract

Influenza is a global pathogen of major public health impact. Despite the availability of vaccines for seasonal influenza, there are still 3-5 million severe cases of influenza globally each year.

Therefore, understanding the mechanisms that the host utilizes to defend and kill the influenza virus is essential. An important host mechanism that has been associated with protection in animal models is Antibody-Dependent Cellular Cytotoxicity (ADCC). ADCC is mediated via innate immune cells that lyse the infected target cells, clearing the infection. There are several in-vitro assays that have been developed to measure influenza-specific ADCC. Most of these assays employ an endpoint that measures a surrogate marker of cell lysis. The major goal of this project was to develop and optimize an ADCC assay for (IAV) whose endpoint quantification is cellular death due to cytotoxicity, and which can also distinguish responses to the

Haemagglutinin (HA) and Neuraminidase (NA) components of 2019-2020 circulating strains.

Codon-optimized HA and NA genes were designed, synthesized, and cloned into expression systems allowing stable, inducible expression in a mammalian cell line. An additional reporter construct expressing firefly luciferase and GFP was also stably introduced into the target cells.

Robust total and cell surface expression of HA and NA along with luciferase expression was documented upon induction with doxycycline. An in-vitro assay for ADCC was then developed using an immortalized human NK cell line that is mixed with HA or NA-expressing target cells at given effector: target ratios, with lysis measured as the loss of luciferase signal compared to control cells. We next evaluated sera from recipients in this assay. H1 and H3-specific

ADCC responses were readily observed with these sera. N1-specific responses were detectable but appeared weaker in general than either H1 or H3-specific response. Evaluation with additional sera gathered from clinical trials is underway to validate the utility of this assay.

2

3 Acknowledgement

The author is grateful to NIH for funding the ADCC project. A special thanks to Cincinnati

Children’s Hospital Medical Center and University of Cincinnati for all the resources that helped to finish this project.

The researcher likes to thank her thesis advisor Dr. Paul Spearman for his valuable guidance, insights and feedback to accomplish this project. The author feels fortunate to have committee members Dr. Karnail Singh, Dr. Andrew Norman and Dr. Tongli Zhang and Department Director Katherine Hobbing for their thoughtful feedback, cooperation that helped to complete the project. A sincere and heartfelt obligation to all the lab members for their assistance, encouragement and all the help

At last, thanking all my friends and family for love and support.

4 Table of Contents

1. Introduction…………………………………………………...... 7

2. Literature Review………………………….……………………….10

a. Clinical Disease due to influenza...... ………………….10

b. Structure of Virus…………………………………………...... 10

c. Lifecycle of Virus ……………………………………………14

d. Non-Neutralizing and Neutralizing Antibody…………….….20

e. Surrogate ADCC assays………………………………………30

3. Methods and Materials …………………………………………….34

4. Results and Discussions…………………………………………....39

5. Future Direction …………………………………………………...53

6. Bibliography……………………………………………………….54

5 List of Figures

1. Structure of Influenza…………………………………………………10

2. Lifecycle of Influenza…………………………………………………14

3. Generation of Stable cell Lines and GFP expression………………….39

4. Characterization of Proteins via Western Blot of 293T and T-rex GFP-LUC

HA/NA cells……….…………………………………………………..41

5. Flow cytometry analysis of 293 T-rex GFP-LUC HA/NA cells………42

6. ADCC assay with Adult Serum Samples……………………………...44

7. ADCC assay with Baby Plasma Samples……………………………...48

6 1. Introduction

The word Influenza is derived from an Italian folk term that attributed colds, cough, and fever to the influence of the stars. Throughout the history, disease caused by influenza has been known by a variety of terms, including catarrh, grippe, the sweating sickness, the new acquaintance, the jolly rant, and knock-me-down fever. It was not until 1933 that the disease known as influenza was discovered to be caused by a virus (the influenza virus) by Drs. Christopher Andrews, Wilson

Smith, and Patrick Laidlaw(L'Vov D, Burtseva et al. 2011).

Influenza virus is a single-stranded, segmented RNA virus belonging to the family

Orthomyxoviridae. Influenza infect a variety of animal species, including birds, pigs, horses, cattle, whales, seals, and humans. They are classified into A, B, C and D viruses based on their genetic and antigenic differences. Influenza A, B, and C viruses can infect humans, while D viruses have not been found to infect humans but cause disease in pigs and cattle. The focus of this thesis proposal is influenza A viruses (IAVs), which cause significant human disease through both yearly seasonal epidemics and sporadic pandemics. While both influenza A and B viruses cause annual influenza epidemics or seasonal influenza, it is only IAVs that cause global pandemics.

Influenza viruses are also classified and named through their (H1-

H18) and Neuraminidase (N1-N11). This is based on the genetic and antigenic differences found in each , with specific combinations of Hemagglutinin and Neuraminidase responsible for seasonal epidemics along with the pandemics.

Major IAV pandemics are likely to have occurred throughout history, accounting for millions of deaths over many centuries. However, it is only since the twentieth century that we have clear records of proven influenza pandemics. The first well-documented influenza pandemic occurred in 1918. This severe pandemic, termed the “Spanish flu”, was caused by a new H1N1 virus and

7 was responsible for around 50 million deaths worldwide. The second pandemic (“Asian flu”) occurred in 1957 and was due to an H2N2 virus, resulting in 1 million casualties. The third pandemic (“Hong Kong flu”) was due to an H3N2 virus, and the most recent pandemic was in

2009 caused by swine origin H1N1 strain known as “Swine flu”(Saunders-Hastings and Krewski

2016).

Seasonal influenza virus epidemics cause 3-5 million cases of severe illness and 300000 to

500000 deaths per year globally. This high morbidity and mortality enhance the urgency of developing improved influenza vaccines. Influenza viruses may undergo major antigenic shifts through reassortment of segments in animal hosts, creating the pandemic strains discussed above.

More common and continuous is antigenic drift, caused by minor antigenic change (or ) in HA and NA or both. The gradual accumulation of overtime produces antigenically distinct viruses that host antibodies cannot neutralize, making the host susceptible to infection and disease. To counter the rapid evolution of influenza viruses, new vaccine strains are selected for inclusion in seasonal vaccines for the Northern and Southern hemisphere each year. It would be desirable to have influenza vaccines that generate a broader protective immune response and that would not require yearly administration. The development of a universal vaccine (covering all strains and extending over years) is challenging, however, because of the genetic variability and ongoing evolution of influenza viruses found in nature. One potential mechanism for broad protection is ADCC. While ADCC responses can protect against influenza in mouse models

(Jegaskanda, Reading et al. 2014) their protective capacity in humans is less certain. ADCC responses have the potential for breadth, as the antibodies mediating ADCC against HA are directed against more conserved epitopes in the stem, rather than the head of HA (Kavian, Hachim

8 et al. 2020) . This proposal seeks to develop a robust ADCC assay for both HA and NA, in order to facilitate studies of the development of immunity to influenza in humans.

9 2.Literature Review

2.a Clinical disease due to influenza

The incubation period of influenza virus is approximately 2-4 days and symptoms ranges from asymptomatic infection to sudden onset that include headache, malaise, myalgia and anorexia, cough and runny nose, fever and chills.(Moghadami 2017). Fever lasts for approximately

7 to 10 days while weakness and tiresome feeling may last for weeks. Every age group is affected, but the severity varies, in several cases children are more affected with maximum temperature, severe Gastrointestinal symptom as compared to adults while in many others cases the older aged group is affected. (Minodier, Charrel et al. 2015, Paules and Subbarao 2017). Other complications including myositis, febrile seizures, laryngotracheobronchitis are also common in severe cases.

2.b Structure of Influenza Virus

10 Influenza is an envelope virus having three surface proteins and 8 RNA segments, namely:

1. Hemagglutinin (HA) is the most variable region and is more prone to the phenomenon of

antigenic drift. HA is encoded by the 4th RNA segment. It is the major glycoprotein on the

surface of the influenza virus and plays an essential role in sialic acid binding, fusion, and

entry. HA is also the major target of the neutralizing antibody response and other functional

antibody responses.(Kosik and Yewdell 2019). The virus remains non-infectious until

HA0, precursor of the HA creates 2 subunits: N- terminal HA1, forming the head region

with the Receptor Binding Domain (RBD), and C terminal HA2, which includes the fusion

machinery required for exit from the . HA is cleaved by a host trypsin-like

proteolytic enzyme (Patterson, Swainsbury et al. 1999, Tzarum, de Vries et al. 2015). One

virion incorporates approximately 300 trimers of HA.

2. Neuraminidase (NA) is the 6th RNA segment, encoding a glycoprotein of approximately

470 amino acids (Air 2012). NA is present on the virion as a homotetrameric complex. The

NA head contains the functional catalytic site, and the stalk contains N- linked

glycosylation sites and a cysteine residue that aid in tetramer stabilization (McAuley,

Gilbertson et al. 2019). There are approximately 50 NA tetramers on the surface of the

virus. The key function of NA is cleaving sialic acids, allowing release of the progeny

virions and preventing viral aggregation (Shtyrya, Mochalova et al. 2009) Influenza virus

A Neuraminidase is classed as group 1 and group II and has about 9 subtypes, whereas

Influenza B and C have just 1 NA subtype.

11 3. M2 is a tetrameric proton channel with 97 amino acids. The essential role of this

is maintaining the pH at several phases for successful replication

of the virus and viral assembly. The residue (H37) of each protomer forms a

transportation pathway for the proton activating the ion channel by altering the protonation

state. This dissociates the M1 membrane and release of viral RNP in the cytoplasm prior

to fusion of viral and endosomal membranes (Acharya, Carnevale et al. 2010). Other

important functions of M2 are incorporation of vRNPs in the budding particles, blocking

premature conformational changes of HA by maintaining high pH in trans-Golgi network,

aid in membrane curvature or the plasma membrane, and is also involved in scission of the

bud (Wohlgemuth, Lane et al. 2018). M2 is a small component of the surface proteome,

with only 10-20 tetramers per virion, but it is essential for both escape from the ensosome

and for viral assembly (Cady, Luo et al. 2009).

4. M1 (Matrix Protein 1) - The major function of is to interact with the vRNPs

and the surface proteins. Other than HA, it’s another most abundant protein found in the

virion. M1 is a protein of 252 amino acids. The oligomeric state of M1 may differ

throughout the life cycle of the virus.(Liu, Grantham et al. 2018) (Eleonora V. Shtykova

et al, 2017). It interacts with transmembrane proteins and in this manner is recruited to the

plasma membrane as it lacks the targeting signal in sequence (Fontana and Steven 2013).

M1 is also engaged in nuclear export of vRNPs to cytoplasm. It was suggested that it also

functions as an adaptor protein between the nuclear pore complex and vRNPs. (Gómez-

Puertas et al., 2000)

12 5. Non-structural protein 2 (NS2) and the Ribonucleoprotein complex (RNP): -

Enclosed in M1 are the NS2 (Nuclear export Protein) along with the RNP complex, a

mixture of Nucleoprotein (NP), and RNA dependent RNA polymerase which includes

Polymerase Acid (PA/P3, PB1 and PB2).

6. NP- NP is a single-strand RNA (ssRNA) binding protein which is coded by segment 5 of

the RNA and it participates in translocating the RNPs via the NLS/NES signal between

cytoplasm and the nucleus and vice versa. NP is 498 amino acid long with each monomer

having a molecular mass of 56KDa. NP is particularly rich in arginine, , and glycine

residues. (Portela and Digard 2002) There are three distinct RNAs required in the viral

lifecycle: mRNA, cRNA and vRNA. The positive sense cRNA is synthesized from

negative sense vRNA, and cRNA is then coated with NP that is associated with the viral

polymerase for transcription of new vRNA.(Cianci, Gerritz et al. 2012) The other end of

the NP is attached to M1. M1-nascent RNP association promotes nuclear export as outlined

above. Influenza viruses with defects in NP cannot synthesize cRNA and vRNA and are

not viable.(Elton, Medcalf et al. 1999, Hu, Sneyd et al. 2017)

7. NS1 is encoded by RNA segment 8. NS1 has the ability to represses the innate antiviral

mechanism by inhibiting type 1 interferon production, helps in replication by hijacking the

host RNA translation mechanism in favor of translating . (Haye, Burmakina et

al. 2009). NS1 performs multiple other functions including aiding in interaction with

various components of (in cytoplasm or in the nucleus) as well as working as a virulence

factor.(de Chassey, Aublin-Gex et al. 2013)

13 8. NS2 is a structural protein and interacts with M1 protein. It is involved in nuclear export

of RNP complexes from the nucleus to the cytoplasm via the export signal. (O'Neill, Talon

et al. 1998, Paterson and Fodor 2012)

9. RNA dependent RNA polymerase- The vRNPs have major functions in transcription and

replication by modulating and using the host factors for. Transcription of mRNA is a

primed-process that requires 5’cap and 3’ poly A’s at the 3’ end. Influenza viruses cannot

generate a cap; instead they ‘cap-snatch’ the host capping mechanism.(te Velthuis and

Fodor 2016). The virus uses a PB2 cap binding domain to snatch nascent 5’ caps from the

host, while PB1 helps in elongation and PA is used to cleave the capped mRNA a little

downstream (8-15 nucleotides) of the cap structure and this is then utilized as a primer to

make mRNA. (Zhang, Hu et al. 2019)

2.c Life Cycle of the Virus

14

Entry into the host cell

Entry into the host cell is mediated by Hemagglutinin, it facilitate recognition and binding of RBS to sialic acid of human respiratory epithelial cells within the trachea, nasopharynx, bronchi and bronchioles.(Byrd-Leotis, Cummings et al. 2017). There are two major linkages found between sialic acids and the carbohydrates they are bound to: SAα(2,3)Gal and SAα(2,6)Gal.

Avian virus recognizes the SAα(2,3)Gal whereas human flu virus recognizes SAα(2,6)Gal, but swine flu viruses recognizes both generating a new strains that has a potential to cause pandemic.(Ito, Couceiro et al. 1998, Auewarakul, Suptawiwat et al. 2007).

The NA cleavage enables the movement of the virion across the mucus in the airway epithelium to the target cell. The major function of this mucus is to bind/trap the virus by presenting decoy receptors and trap the virus which is then cleared by mucociliary escalators. HA: NA balance is important for the viral infection efficiency because the HA should have a low affinity to the

Saα(2,3)Gal or there should be high activity of NA against Saα(2,3)Gal to reach the target receptor.

When the virion reached the target receptor the multivalent binding to several HA trimers results in 104 to 106 times increase in avidity, so that the binding is irreversible against various attempts by the host or the virus (NA) to block this attachment.(Kosik and Yewdell 2019)

After HA1 subunit attaching the target cell, the virus is internalized in the endosome via clatherin-mediated endocytosis involving dynamin and adaptor protein Epsin-1, various non- clatherin mediated internalization has been described as well like micropinocytosis (Nunes-

Correia, Eulalio et al. 2004). After the internalization the influenza virus also requires an active actin skeleton that transports the vesicles to microtubule to the .(Lakadamyali, Rust et al. 2003) The pH of the endosome is low (around 5 to 6), and this induces conformational changes

15 in the HA0, exposing the fusion peptide of HA2 on the N- Terminus that hooks itself leading to fusion of the endosomal and viral membrane.(White and Whittaker 2016). The acidic environment is also important for the M2 proton channel to open up which acidifies the viral core and releases the vRNP from M1 to the host cytoplasm completing the uncoating process.

Influenza replicates in the nucleus unlike other viruses, hence vRNP enter the nuclease for transcription and replication of the protein component i.e. PB1, PB2, NP, NS1, PA and NP protein that encapsulates them. All the above proteins have Nuclear Localization signal (NLS) through which they enter the nucleus using importin-α–importin-β nuclear import pathway directing the vRNP to the Nuclear pore complex finally transporting it to the nucleoplasm.(Dou, Revol et al.

2018)

Transcription

Influenza virus is a negative sense vRNA, it needs to convert to positive sense to serve as a template for the RNA replication (Samji 2009) and is achieved via RNA-dependent RNA polymerase (RdRp) (Fodor 2013). There are two steps for replication of its genome: Transcription of complementary RNA and transcription of new vmRNA (Dou, Revol et al. 2018). mRNA transcription occurs before these two steps and it requires abundant primers which increases the initiation efficiency. Normally, the mRNAs have Poly A tail and 5’cap, but the influenza vRNPs have poly A but no 5’ cap hence it was discovered that vmRNAs snatches this ability from the host. Cap-snatching mechanism is used to initiate the viral transcription, it uses

PB2 to bind to the 5’ cap of host mRNA, aided by the RNA Polymerase II C-terminal domain

(Engelhardt, Smith et al. 2005). It fixes the region of mRNA around 10-15 nucleotide downstream of 5’cap for cleavage, and PA subunit acts as endonuclease(Plotch, Bouloy et al. 1981). These

16 capped RNA fragments are further thrown back in the cytoplasm for the transcription and then imported back to start the viral transcription (Li, Rao et al. 2001). It is followed by conformation changes of PB2 subunit that repositions the mRNA cap to the PB1 subunit which has formed a base-pair with complimentary vRNA at 3’ end(Reich, Guilligay et al. 2014). Later, the polymerase acid extends the transcripts and the termination occurs by reiterative stuttering, meaning transcript is polyadenylated and the polymerase encounters poly-U sequence (about 5-7) at 5’end. But this leads to a formation of incomplete genome copies unable to serve as a template, Hence, another full length positive strand of RNA is synthesized called the cRNA(Heldt, Frensing et al. 2012).

During infection, the mRNA replication is primed, it is more efficient then the unprimed cRNA and vRNA replication

The vRNPs arises from the cRNA,(Eisfeld, Neumann et al. 2015) it is dependent on the correct complementation of free rNTPs. cRNP assembly is formed when NP molecule binds to the cRNA. Transcription is terminated when the cRNP is formed along with viral polymerase which serves as a templet for progeny vRNPs.

The negative stranded vRNPs are exported from the nucleus using NS2/NEP protein and

M1 presence via cellular CRM1 mediated pathway through nuclear pores forming a daisy-chain complex of (Crm1–RanGTP)–NEP–M1–vRNP (Boulo, Akarsu et al. 2007, Huang, Chen et al.

2013). Studies have shown that the avian viruses that can replicate in humans requires few specific mutations in the NS2 region suggesting that it’s an important replication protein(Manz, Brunotte et al. 2012). NS1 is imported and it binds to nuclear export components like TAP/NXF1, p15,

Rae1, E1B–AP5 and NP. It also has a role in inhibiting the host immune system by blocking innate and adaptive immunity via inhibiting the interferon signaling and gene expression.(Satterly, Tsai et al. 2007)

17

Maturation of the membrane proteins

The membrane proteins are synthesized in ribosomes on the rough .

Proteolytic cleavage of HA by trypsin like proteases into HA1 and HA2 occurs in a trans-Golgi network to incur efficient infectivity via fusion. M2 is critical for maintaining the pH balance to prevent premature conformation changes in HA. (Ciampor, Thompson et al. 1992)

The HA, NA and M2 are then folded in ER where HA and NA are glycosylated and transported to Golgi Complex. (Doms, Lamb et al. 1993). The membrane proteins HA, NA and

M2 are to assemble at the virus assembly site of the apical plasma membrane in polarized epithelial cell. HA and NA have 2 apical sorting signal, one in ectodomain and one in transmembrane domain

(TMD) and associates itself with the lipid raft microdomain. (Nayak, Hui et al. 2004). Along with

HA and NA, M2 is as well targeted to the apical membrane in actin dependent manner, but there is no apical sorting signal identified yet. (Hughey, Compans et al. 1992, Leser and Lamb 2005,

Pohl, Lanz et al. 2016)

Trafficking of vRNP and Viral envelop protein

Following the nuclear transport, the protein synthesis occurs and its host dependent. It occurs via cytosolic ribosomes for PB1 PB2 PA NP NS1 NS2 M1. The vRNPs are also trafficked towards the plasma membrane by Rab11A which gives a piggyback ride to vRNPs by interacting with PB2 subunit and ensures incorporation of the vRNPs into the new virions.(Amorim, Bruce et al. 2011, Eisfeld, Kawakami et al. 2011). Previously, it was known that Rab11 associate specifically with cholesterol rich recycling endosomes that uses microtubule for the trafficking towards the cell surface.(Kawaguchi, Hirohama et al. 2015) More recent studies proposed an

18 alternate approach stating that infection causes tubulation of the ER network; Rab11 binds to the vRNP that is localized to this network and transport them to the plasma membrane.(Martini,

Fournier et al. 2017) It is currently unknown how they are transported in either of the mechanism explained.

Assembly and Budding

All viruses (enveloped and non-enveloped) viral assembly needs to have a complete .

The components are bought individually or in a complex at the budding site to initiate budding, especially, in the enveloped virus, formation of capsid is a requirement. But the budding of influenza virus is complicated as not all vRNP components are required for the budding. But to bud a fully infectious viral particle, all the segments of vRNPs must be incorporated. Also, all the enveloped viral components and M1 should be transported to the bud site individually or in complex for bud initiation, bud growth and its release. (Hurst, 2011). It is known that influenza viral particles are budded off through the apical membrane and since complete virus is not found inside the cell, the assembly must happen at the plasma membrane. The assembly of the vRNPs is still unknown, there are still various proposed models like Random packaging and Specific packaging. (Nayak, Hui et al. 2004)

• In random packaging the incorporation is concentration dependent, the presence of common

structural components in RNP causing them to randomly incorporate in the newly budding

virion. Studies have showed that bud closure and bud release occur even if all 8 segments of

vRNP are not assembled or are not incorporated. This means that not all components are

required for the bud release

• In Specific Packaging, there are structural features like signals present in vRNP that is

specifically selective about incorporating in the new virion.

19 Recent studies have supported both the models the specific packaging model as well as random packaging.

The envelop proteins HA and NA play a major role for assembly and budding (Chen, Leser et al. 2007, Lai, Chan et al. 2010) But other studies also mentions that M1 is important for budding, as the major function of M1 lies in interacting and connecting the envelope proteins and the viral core by concentrating them at the bud site. M1, without other proteins, alone is sufficient to cause membrane curvature for bud initiation and release.(Gómez-Puertas, Albo et al. 2000, Dahmani,

Ludwig et al. 2019). Various mutational truncation or deletion of the M2 tail causes structural modifications of the viral particle and produce progeny virions of different shapes like elongated particles or spherical, therefore M2 is extremely important to determine the shape and size of the virus. It facilitates the release by bringing non lipid raft in a proximity as it is present at the neck of the bud. (Iwatsuki-Horimoto, Horimoto et al. 2006)

The bud closing is very efficient but can be affected by various factors. There are lot of buds that stay attached to the plasma membrane, not all buds have all 7+1 segments of vRNPs. It is energy dependent, so any metabolic inhibitors or ATP analogue can inhibit the bud closing.

Release or scission is the last step of influenza life cycle and is very inefficient as only few of them get release and the rest gets stuck on the surface. NA facilitates this release of the virus by cleaving the sialic acid from the glycans.(Palese, Tobita et al. 1974, Samji 2009)

2.d Neutralizing and Non-neutralizing antibody

The virus itself is entered via mouth or nose when infected people cough or sneeze, this releases small virus-containing droplets into the air, when breathed by healthy human it can infect the respiratory epithelial cells. After successfully invading the host, the virus spreads to all the

20 cells, immune and non-immune (Iwasaki and Pillai 2014). Beyond this point, there are different ways that the human host tries to clear the infection, one such mechanism is Antibody-Dependent-

Cellular-Cytotoxicity (ADCC). It is mediated by binding the Fab region of the antibody to the antigenic target of the infected cell and the other end is the Fc portion which serves as a link to bind to the effector cells, majorly, Natural Killer (NK) cells. Apart from the NK, neutrophils, monocytes and lymphocytes have been known to mediate Influenza ADCC (Von Holle and Moody

2019).

There are many studies which are focused on the virus neutralization Abs but little research is being conducted on Non- neutralization Abs. All envelop virus needs a receptor to bind to the host and have a mechanism to enter the cell and cellular fusion, in this case Variable HA head region does this job (Burton, Poignard et al. 2012). It is an important target for various therapeutic treatment, diagnostic tools and neutralization antibody as it is a major envelop glycoprotein. These antibodies interfere with the attachment of the HA to its Sialic acid receptor by binding themselves to the exposed area near the receptor binding domain (RBD) (Brandenburg, Koudstaal et al. 2013).

The immunity confer by them are strain specific and the protection by these antibodies after any natural infection and vaccination are limited to those homologous infections because of the high mutation state of the HA head. But antibodies that binds towards the Conserved HA stalk region exhibit much broader neutralization activity with the capacity to target entire subtype. They are traditionally being considered as the most important mechanism against influenza virus but antibodies are elicited in some individual and not everyone, irrespective they are considered as a promising agent in the development of Universal vaccine strategy (Friesen, Lee et al. 2014).

But as per the recent research, the anti-influenza function mediated by the non-neutralization antibody plays an important part as well, including different methods of quantifying them after

21 immunization via natural infection or after the vaccination. It is depended on the conserved Fc region bound to various immune system, whereas the variable Fab region binds to the infected cell. The most important antibody isotype here is IgG and IgM, with IgG3 mediating the most potent effect. (Sedova, Scherbinin et al. 2019)

Different mechanism of non-neutralizing antibodies like Antibody-dependent cellular

Phagocytosis (ADCP), Complement dependent Cytotoxicity (CDC) and Antibody-dependent

Cellular Cytotoxicity (ADCC) helps in clearing the infection. They provide a direct link between the innate and adaptive system, which eliminates the need of pattern recognition pathway that activates the adaptive immune response by facilitating antigen presentation.

Antibody-dependent cellular Phagocytosis

Various immune cells mediate ADCP e.g. Macrophages, Dendritic cells and neutrophils, but mainly it relies on macrophages to devour the infected cells. The first step is Opsonization through which phagocytes recognizes the antibody bounded antigen. The key receptors are Fcγ, they play an appreciative role as effector of cytotoxic activity, these include three activating receptors in humans FcγRIa(CD34), FcγRIIa (CD32), FcγRIIc and FcγRIIIa and one inhibitory receptor FcγRIIb all these are co-expressed in innate effector cells like Macrophages, Monocytes,

DCs, Neutrophils and many more. (Nimmerjahn, Gordan et al. 2015). It is an important defense mechanism for influenza virus. (Ana-Sosa-Batiz, Johnston et al. 2017)

Newly budded influenza virion having HA, NA and M2 proton channel on the surface, that’s where antibodies bind and opsonizes the particle. This activates the Fcγ mediated macrophages that phagocytize and clear infection. Alveolar Macrophages further activate the type

1 INF and regulate chemokine CCL2 leading to neovessel formation and tissue repair (Ridiandries,

22 Tan et al. 2018) (Huber, Lynch et al. 2001). Along with macrophages, neutrophils also participate in the ADCP as it expresses FcγRIa/b/c, FcγRIIa, FcγRIIIb and FcαRI (binds to IgA) after activation. The biding phosphorylates the ITAM via Src family that triggers the signaling cascade involving PKC and PI3 and synthesis of PIPs. Actin cytoskeleton is remodeled allowing phagocytotic advancement. The strength of the above signal lies in the amount of Fc receptor engagement.(Tay, Wiehe et al. 2019) Neutrophils and macrophages are quickly recruited at the respiratory tract, bronchoalveolar lavage and lungs to contribute to ADCP. Depletion of any of these led to reduce survival in mice.(Hashimoto, Moki et al. 2007)

Complement Dependent Lysis

Complement dependent lysis is the heat-liable component of normal plasma that clears in infection. The name given was given because it complements the action of the antibodies directed against the various pathogens and erythrocytes (Jayasekera, Moseman et al. 2007). Zymogens are distributed all throughout the body tissues and in fluid. Whenever there is an infection, these zymogens are activated which further activates a complement enzyme that cleaves the substrate that activates which further cleaves another zymogen. There is an entire cascade that follows that gives an amplified and large response (Janeway 2005)

There are three different pathways to mediate CDL, the classical, The lectin and alternative pathways.

• Classical Pathway: - This pathway begins with binding of C1(six C1q, two C1r 7, two C1s) to

the complement fixing antibodies like IgG or IgM. C1q binds the microbes or pathogen or Fc

domains of antibodies and activates the system by structurally altering the C1q, C1q then

23 causes a conformational change in the C1r, the change activates C1r leading to cleavage of

C1s proenzymes resulting in the activation of the C1s. C4 and C2 are activated because of the

C1s and the downgrade signaling leads to generation of C3 convertase that cleaves to C3a and

C3b that activates the lytic pathway

• Lectin Pathway: - A Carbohydrate pattern recognition molecule MBL, binds mannose on the

pathogens cell surface. MBL structure is similar to the C1q. This pattern recognition molecule

binds to MASP-1, MASP-2 and MASP-3. They share structural analogy with the C1s and C1r.

C2 and C4 are activated only by MASP-2 that further generate C3 convertase. MASP-1 and

MASP-3 uses an alternative pathway which is activating and cleaving the Complement Factor

D leading to enhanced activity.

• Alternative Pathway: - It takes place via the C3 thioester hydrolysis that forms a complex called

C3b(H2O). CFB, a soluble component of the pathway, binds to C3b(H2O) to generate

C3b(H2O)Bb and the cascade begins that finally uses C3 convertase to form C3bBb that

enhances C3 accumulation on the target cell that enables target cell phagocytosis and

opsonization

Antibody Dependent Cellular Cytotoxicity.

Antibody dependent cellular-cytotoxicity is mediated largely by NK cells using the receptor FcγRIIIa (CD16a) receptor. Neutrophils can also mediate some form of ADCC using

FcγRIIIb receptor but the effect is not pronounced as compared to NK cells. ADCC is initiated when the FcγR on the immune cells binds to the Fc receptor of the antibody that is bound to an infected target cell. This engagement activates the immune cell which causes degranulation releasing Perforin and Granzyme (Cytolytic granules) clearing the infection by killing the cell.

24 Release of cytokines and chemokines like IFNγ and TNF, enhances the antiviral environment limiting the spread and reproducibility of the virus. (Vanderven and Kent 2020). The cytotoxic potency of the antibodies depends largely on the affinity to the Fc receptors, the order is IgG1 and

IgG3 followed by IgG2, IgG4. IgG1 (Longer ADCC) and IgG3 (Potent ADCC) mainly binds to the CD16 receptor to mediate ADCC. IgG2 and IgG4 are poor mediators of ADCC. The binding affinity of the Fab region on the opposite side of the antibody bound to the antigen also has some effect on the mediating strong or weak cytotoxicity(Ferrari, Pollara et al. 2011, Srivastava, Yang et al. 2013). The next factor to be considered for ADCC is the post translation modification like glycosylation which is essential for the antibody structure that can determine the affinity of the Fc gamma receptors, as the expression of these is variable across different people depending on the hosts age, sex, virus infection etc.

It is known that influenza HA is a primary target for the ADCC antibody along with the contribution of other protein like NA, M2 even NP to some extent (Jegaskanda, Reading et al.

2014). HA has been more studied in its ADCC-mediating capacity than has NA. HA and NA are highly immunogenic, so antibodies are generated against both glycoproteins following natural infection or after vaccination. Anti-HA antibodies can be measured using the neutralization or hemagglutination inhibition assay (HAI)), which is often considered the standard for determining vaccine efficiency. Recent research suggests that non-neutralizating antibodies can confer protection as well. Unlike neutralizating antibody, many non-neutralizing antibodies do not bind to the Receptor Binding Site (RBS) of the HA, and typically do not inhibit hemagglutination. A subset of non-neutralizing antibodies are capable of mediating ADCC, and it is this subset that we seek to measure through the assay described in this proposal (Gao, Sheng et al. 2020).

25 NK cells

Natural Killer (NK) cells are a subset of lymphocytes and are a major component of the innate immune system. Their primary function is innate immune surveillance, and they can react quckly to virus-infected cells or tumor cells. They are important effector cells that defends against virus, bacteria and parasites. Unlike B and T cells they can kill an infected cell or a malignant cell without priming. The mature NK cells have a large reservoir of perforin and granzyme and also

IFN-y mRNA which can be stimulated when the cells are activated. The balance of signals received from the activating and inhibitory transmembrane receptor plays an important role in its function

(Pegram, Andrews et al. 2011).

• Inhibitory Receptor: - Under normal circumstances, the NK cells are in the inhibitory state.

The ligand expressed on a normal healthy cell binds to the inhibitory receptor and makes sure

that NK cells are not activated. Few inhibitory receptors are the KIR 3DL2, KIR 3DL1,

NKG2A, LIR1, TIGIT.

• Activating receptors: When the signal for the inhibitory ligand reduces or when stressed

induced ligand are activated due to an infection or malignancy that’s when the NK cells are

activated. The most dominant activating receptor on NK cell is the NKG2D, other than that

there is 2DS4, KIR 2DS1and few more. Few natural cytotoxic receptors are Nkp30, Nkp46

Hence rather than a hierarchical model the NK cell receptors combine and form a synergistic pair to tilt in favor of NK cell activation. In addition to the above Receptor family there are still numerous different receptors that can be found on the surface of the NK cell and different people have different receptor expression profile. (Long, Kim et al. 2013).

26 ADCC mediated by NK cells is regulated by FcγRIIIa receptor. It is the only receptor that has the potential to activate the NK cell on its own. Activation occurs in 3 discrete stages:

• Recognition of the target cell by the Fab region of an antibody

• Fc receptor cross-linking on the surface of the effector cell

• Cross-linking leading to Release the granules and perforin which kills the infected target

cells

FcγRIIIa Receptor downstream signaling

Also known as CD16, this is a type 1 single membrane-spanning glycoprotein that belongs to the immunoglobulin superfamily. FcγRIIIa contains immunoreceptor tyrosine based activating motifs (ITAMs) similar to those found in T cell and B cell receptor. ITAMs (YXXL) phosphorylation provides the docking site to the signaling molecules like the protein tyrosine kinase, and those molecules that have SH2 domains leading to various signaling and cytotoxic function. The important enzyme that gets activated by tyrosine phosphorylation is PLC-γ both

PLC-Y1 and PLC-Y2, it cleaves the PIP2 into IP3 and DAG that releases the intracellular calcium and protein kinase C respectively. Calcium plays an important role because when there is an increase pf Ca+ it increases the calmodulin bound calcineurin which dephosphorylates the NFAT.

NFAT translocate in the nucleus and upregulates all the ADCC promoter genes that leads to degranulation and release of antivirals eventually killing the infected target cells by forming pores in the cells, granzyme enters the cell via these pores initialize apoptosis.(Leibson 1995)

27 ADCC and influenza

Stalk Specificity of the influenza virus: - The HA stalk is immunologically subdominant to the head region, maybe because of the steric shielding caused by the head, which makes this region not so much accessible to the immune cells. However, antibodies can be detected against the HA stalk region. While the RBD is in the head of HA and is more prone to mutation, the stalk is more conserved, hence antibody against stalk can broadly recognize and bind to several strains in different groups. The role of ADCC against anti-head Abs to generate ADCC response is unclear as data shows that anti-head Abs inefficiently induce ADCC. It was also seen that the

Hemagglutinin Inhibitor (HI) positive antibody that causes neutralization could not induce ADCC.

There is a model explained by Leon and colleagues:

There are 2 synapses which are needed to induce ADCC, the 1st is binding of Fc and FcR receptor and the other is HA and Sialic acid (SA), they together confer a connection between a target cell and effector cell. There is an uncertainty with only SA binding of HA activating the

ADCC pathway, so HI positive antibody compete with the SA on the effector cell to bind with the head domain, eventually impairing one of the connections required for ADCC response (Leon, He et al. 2016).

Antibodies inducing neutralization and ADCC are not mutually exclusive. C12G6 is an antibody that was detected to induce neutralization of influenza B strain against Yamagata and

Victoria but also causes ADCC response.(Gao, Sheng et al. 2020) Therefore more research is necessary to understand the antibodies structure and function to mediate neutralization or ADCC or both. Headless HA or chimeric HA strategies can be used to validate and analyzed stalk specific antibody. Research have shown that there are anti-stalk ADCC antibodies detected after immunization against HA chimera. (Choi, Bouzya et al. 2019). Neuraminidase expression on the

28 surface of the influenza virus to HA is in the ratio of 300:40. Naturally, after vaccination low- levels of antibodies are detected against the NA. There are cases that have detected NA inducing

ADCC Abs, eg 1G01 showed blockage of release of viral particle and ADCC. Hence, mechanism of ADCC against NA is still obscure and unknown, therefore further studies are necessary.

(Stadlbauer, Zhu et al. 2019).

ADCC based vaccines against Influenza: -

Major vaccine strategy has been focused on the HA head and stalk, NA, M2e, NP isolation and characterizing for inducing broad neutralizing antibodies. (bnAbs). But, induction and effectiveness of bnAbs after natural infection is difficult after mutant virus emerges. Non- neutralizing antibody (nnAbs) induce Fc- mediated killing very efficiently and exhibit protection against the virus, making ADCC-based vaccines a strong candidate because (Gao, Sheng et al.

2020): -

• They serve against the HA and NA stalk binding antibody, so targeting more conserved

region, making them more cross-reactive than the neutralizing antibody.

• Study of severe human influenza infection survivors in Australia and china exhibited strong

ADCC activity.

• The animal model for ADCC based vaccine in mice, ferrets and Non-human primates

(NHP) was capable to inhibit influenza virus infection by stopping virus shedding.

To understand more about the ADCC, there is a need for better assay.

29 2.e Surrogate Assays

Various traditional assays are being used which may not be efficient. Here is a list of few techniques which are being used.

Lactate dehydrogenase Cytotoxicity: -

Whenever a cytotoxic compound is added on the target cell, the cell has one of the two fate, either it stops dividing and growing further or the other one is dying via one of the 2 mechanism, Necrosis and Apoptosis. When necrosis occurs, the cell loses its membrane integrity as it swells and release its intracellular content into extracellular space before shutting down. When apoptosis (programmed cell death) occurs, it follows a well-defined process of shrinkage of cytoplasm, cleavage of DNA and these pieces are phagocytosed by WBCs. Lactate dehydrogenase

(LDH) is a stable, soluble enzyme found inside all live cells. When the membrane integrity is compromised, this stable enzyme is released into the surrounding, thus this can be used as a cell death marker to quantify the amount of live/dead cell. The measurement of LDH uses a

Colorimetry or Fluorometric to quantify this cytotoxicity. LDH in the environment is experimentally measured by the enzymatic reaction that converts iodonitrotetrazolium (INT) to red color Formazan.

LDH reduces to NAD+ to NADH, and the H+ converts lactate to pyruvate. The catalyst then transfers the H to the INT that produces the red color formazan salt. Standard Spectroscopy is used to measure (At 490nm) the amount of color produce which is directly proportional to the

Damaged cell/cell death.

The similar mechanism is used for the LDH fluorometric assay, with the only difference being when NADH is produce, it further converts resazurin (A non-fluorescent compound) to a resorufin

30 (A Fluorescent) compound (Beck 2013, Vanderven, Ana-Sosa-Batiz et al. 2016, Jegaskanda,

Vanderven et al. 2019). The reading is then used to calculate %ADCC using the below formula

(LDH) test sample – (LDH) Negative control %ADCC - X 100 (LDH) Positive control – (LDH)Negative control

The major disadvantage here is that the serum and few other compounds have inherent LDH activity along with low throughput. Also, when using primary NK cell adds more variability and donor dependency.

Chromium 51 Assay

Cr51 is another type of assay which captures the direct killing of the cells in an indirect way. In this assay, the Cells are labelled with Cr51 for 2h at 37°C. Cells are washed 3 times with

PBS/ resuspended in RMPI media. Take 96 well plate and add approximately 1×105 cells/ml in triplicates. Add the antibody which is further incubated for another 1 h with subsequent addition of effector cells in 40:1, 20:1, 10:1 5:1 ratio. The entire plate is incubated for 4h. Collect the supernatant and is then quantified on the gamma counter which calculate the amount of chromium released from the lysed cell. This release directly correlates with the ADCC killing of cells.

Different controls are also added along with the experimental samples like cells are treated with water/1%NP40 for maximum release control. Target cells are incubated with effector cells for spontaneous control. No vehicle control is also added to cover all the grounds. % ADCC is then quantified using the below calculation (Mariani, Monaco et al. 1994, Neri, Mariani et al. 2001,

Karimi, Lee et al. 2014).

%ADCC- (Experimental Release - Spontaneous Release) X 100 (Maximum Release – Spontaneous Release

31 NK cell activation.

This assay is extensively used to measure the ADCC against influenza virus and HIV. In this Flow cytometry-based assay, the Target cells are coated on to the plate with a recombinant protein or virus infected cells. The antibodies or the PBMCs/isolates NK cells/NK cell lines are added on to the target cells. CD107a also known as Lysosomal associated membrane protein 1 is a degranulation cell marker that is expressed on the NK cells. This marker correlates the Activated state of NK cell Functionality using the expression level of CD107a with the cytokine secretion that damages or kills the infected target cells.

The 96 well plate were coated with the purified virus protein and kept overnight at 4 degree with 600ng/well. Plates were washed with 1xPBS and incubated with heat inactivated EDTA- anticoagulant plasma for 2 hours at 37°C. Plates were washed again with 1xPBS and then approximately 106 isolated PBMC’s were added to the plates. They were washed and resuspended and plated in RPMI medium. 5μg/ml monensin and 5μg/ml brefeldin A, were added to each well and incubated for 5h at 37°C with 5% CO2. Further Anti- human CD107a antibody, with Live/Dead dye, anti NKG2A surface antibody were added for 30 min at RT in dark room. They were further fixed with 1% paraformaldehyde and FACS permeabilization solution 2. Cells are then treated with secondary antibody like Alexa Fleur 647 or Alexa Fleur 700 for 30 mins at RT, cells are again fixed and analyzed Further (Jegaskanda, Weinfurter et al. 2013, Park, Park et al. 2013)

Hence, determining the potency of ADCC activity indirectly by analyzing the degranulation marker CD107a expression of NK cell surface.

32 ADCC Reporter Bioassay

It’s a bioluminescent reporter assay that determines the ADCC using the firefly luciferase in the modified Jurkat T-cell line. These cells are stably transfected with CD16 /FcγRIIIa receptor on the effort cell with Firefly luciferase expression which is activated with nuclear factor of activated T cells (NFAT) signal which is caused due the FcRs binding. The effector cells are simply thaw and used reducing the variability caused due to donor PBMCs as we saw in NK cell activation assay. The cells are cultured and infected with the virus. 25 microliters/well antibody dilutions are made and added in all the wells except the blank well. Add the assay buffer

(25μl/well) with RPMI media supplemented with IgG serum antibodies. Assay buffer is added in the blank wells instead of serum antibody. Effector cells were added (25 μl/well of ADCC effector cells 75,000 cells/well) to each well and the plate is kept at 37°C with 5% CO2 for 6 h. Further you added 75μl luciferase assay substrate to all the wells and luminescence readout is taken to quantify the ADCC. The NFAT activation is calculated by fold induction against the blank is further plotted on a logarithmic scale.(Tada, Ishii-Watabe et al. 2014)

All the above assay has certain disadvantages associated to them: - LDH is inherently found in serum and various other components like phenol red. Chromium 51 was discovered in 1900s and being a radioactive material, it caused lot of issues like biohazard and disposable problems along with low sensitivity issues that restricted its wide application. Flowcytometry uses the surrogate marker CD107a to resonates the ADCC, whereas the ADCC Reporter Bioassay uses

Jurkat cells which are not biologically relevant, hence we need a newer assay that irradicates all the above listed limitations. This newer assay would be helpful in evaluating the anti-IAV ADCC antibodies from the serum/plasma from various volunteers. This assay was developed using

(Singh, Marasini et al. 2018) as a reference for their Ebola ADCC assay.

33 3. Materials and Methods

Serum/Antibodies

Serum of Influenza Vaccine Recipient were used as a source of human antibodies to optimize the

ADCC assay.

Influenza H1N1 HA1 Rabbit Polyclonal (LSBio), Influenza H3N2 HA Rabbit Polyclonal Abs

(GeneTex), Influenza H1N1 NA Rabbit Polyclonal Abs (GeneTex), Influenza H3N2 N2 Rabbit

Polyclonal (ThermoFischer) were used to characterize the HA and NA protein expression.

Generation of plasmid and target cells

EGFP-IRES-Luciferase expressing reporter cassette obtained from pHAGE PGK-GFP-Luciferase

(Addgene, Cambridge, MA) This plasmid was cut by Not1 and Cla1, blunted with Klenow

Fragment and then ligated with pcDNA4/TO plasmid at EcoRV site to generating pcDNA4/TO

GFP-IRES-Luc containing Zeocine resistant gene. Another plasmid pcDNA5/TO was used to insert the HA/NA gene of interest. Here the original hygromycin resistance gene sequence pcDNA5/TO was replaced by puromycin resistance gene sequence to generate pcDNA5/TO-puro.

The deletion of hygromycin was achieved by utilizing Pml-1 digesting enzyme and then the PCR amplified puromycin gene was inserted instead and verified further by our lab members. The

Influenza A vaccine strain , A/Brisbane/02/2018 (H1N1) and A/Kansas/14/2017 (H3N2) from

2019 - 2020 was Codon optimized, synthesized, cloned by GeneScript and was placed under tetracycline-controlled cytomegalovirus promotor region of pcDNA 5/TO-puro using BamHI and

EcoRI site which generated plasmid pcDNA 5/TO-puro HA/NA GP. pcDNA6/TR plasmid with

Blasticidin resistant gene is already infused with the T-rex cells. Its function is to repress the tetracycline gene by controlling the CMV promoter and 2 tetracycline operating sites (tetO2) for

34 inducing expression. TetO2 presents 2-binding site for 2 molecules of Tet repressor. This repressor forms homodimer with the Tet sequence in the promotor region and repress the induction of gene. When tetracycline or doxycycline is added, it binds to the Tet homodimer causing conformational changes in the repressor gene such that it cannot bind to the Tet operator seq that finally allows the induction and transcription of the gene eventually producing protein of interest.

It is used to control the tetracycline system more efficiently.

To generate a stable cell line expressing the glycoprotein HA/NA, the GFP/luciferase 293 T-Rex

(Tetracycline regulating Expression) expressing cells were transfected with cells pcDNA 5/TO

HA/NA. They are cultured in DMEM (Gibco, by Life technologies) media which is supplemented with Fetal Bovine Serum (10%) (Sigma-Aldrich, St Louis, MO), Penn/Strep 100IU/ml and 100

µg/ml (Penicillin and Streptomycin, Media tech, Manassas, VA), with 1X Glutamax (Gibco),

200µg/ml Zeocine (Invivigen) and Blasticidin (Invivogen). The puromycin being the selective antibiotic was added at 1.5mg/ml. The clonal population was grown and single colony was picked up and expanded further. Doxycycline was added to one part of the clonal cells to test the GFP and protein expression. The best clone was selected and was further expanded for generating stable source of target cells for ADCC assay development against influenza virus.

Effector Cells

Immortalised NK human cell lines, CD16-176 V-NK-92 were used. The 176V high affinity variant of CD16 of NK cells were transduced in pBMN vector, to produce high amount of CD16 expression on the surface which makes it more fitting for the ADCC assay.

35 Western Blot

The protein expression from the transient and stable transfection of 239Tcells / 293 T-rex cells with HA/NA constructs were analyzed by western blot. The cells were grown in 6-well plate, they were induced with doxycycline when they reached 75% confluency. Further harvested using

500µL Versene, then incubated for 5 mins, added 3ml of PBS and transferred the cells to 15ml conical tube. Centrifuged for 5 mins at 350xg. Resuspended the pellet cells in RIPA buffer containing 1:1000 v/v protease inhibitor. Mixed 4µL of reducing agent 10X (Invitrogen) and 10µL of loading buffer 4X (Invitrogen) with 26µL cell lysate samples followed by 5 min incubation at

90°C. Protein for all three cell lines were resolved by 4-12% Bis-Tris SDA-PAGE (NuPage,

Invitrogen) using the Running buffer. The gel is further transferred to the PVDF membrane (0.45

µm, P-Immobilon, Millipore, Massachusetts) using Tris-Glycine (Bio-Rad) transfer buffer, a semidry transfer containing 10% methanol is run at 15V for 50 min. The membrane was further blocked with Intercept Blocking buffer (Li-Cor) for 1h at RT. The blocked membrane was further incubated with rabbit anti-HA1, HA3, NA1 and NA2 polyclonal antibodies at 1:1000 dilution in blocking buffer overnight at 4°C. The membrane was further washed with PBS three times containing 0.2% Tween-20 every 5 min. Next, the membrane was incubated with secondary antibody which was Goat anti-rabbit 1:10000v/v for 1 h at RT. The membrane was again washed with PBS containing 0.2% Tween-20 and it was visualized using Li-Cor Odessey CLX scanning instrument.

Flow Cytometry

The cells were grown in 10 cm2 plates. Cells were harvested by trypsinizing with Versene for 5 mins at RT then centrifuged at 350xg for 5 mins. Remove the supernatant and suspend the pellet

36 cells in 2ml of PBS and again centrifuge for 5 mins. Rewashed with 1ml of PBS. Suspended the pellet cells in PBS with 1:200 dilution of LIVE/DEAD fixable stain and incubate for 15 min at

RT. Add 2 ml of staining buffer (PBS 76.4ml, FBS 1.6ml and HEPES 2ml) to the tube and centrifuge the cells at 350xg for 5 mins. Wash again with 1 ml of staining buffer and add primary antibody in ratio 1:100v/v Influenza Rabbit anti- H1, or anti-H3, or anti-N1 and anti- N2. Keep the tubes on ice for 30 mins. Wash with 2 ml of staining buffer twice. Next, add secondary antibody in 1:10000v/v ratio with fluorochrome anti-rabbit Alexa Fluor 647 (Invitrogen) and incubate on ice for 30 mins. Wash with staining buffer, and finally add 200µL of staining buffer and keep the sample on ice until analyze on Flow Canto Machine.

Development of ADCC

Plate: 96-well plate U well

Serum: Delivered from bloods of flu vaccine recipients.

The diluted serum samples of flu vaccine recipients were added to Row C and was further serially double diluted till Row H.

Target cells: 293 Trex HA/NA cells

50,000 293 Trex HA/NA cells were add per well.

Effector cells:

Human NK cells (CD16-176 V-NK-92) were added (100,000/well) from row B to row H and the plate is incubated at 37°C, 5% CO2 for 4h. After incubation, wash the plates with PBS three times and centrifuge at 350xg for 5 mins all three times. A mixture of Britelite Plus luciferase substrate

(PerkinElmer, Waltham, MA) and DMEM growth media without Phenol red is added to all the

37 wells in ratio 1:1, Mixed well using multichannel pipette and transferred to the 96 well white plate.

The plate was further examined in Synergy HTX multi-mode reader (Bio-Tek).

Cell Transfections

JetPrime™ (Polyplus transfection, France) is used to transfect the cells, it’s a non-liposomal polymer-based transfection reagent. 293 T-rex cells were grown in 6-well plates (7 x 105/well) for

24 hours. 2μg DNA was diluted into 200μl jetPRIME® buffer (at room temperature - RT). Mixed by vortexing and incubate the mixture for 10 mins and then add 4μl jetPRIME® reagent, vortex for 10 s, spin down briefly and incubate for 20 min at RT. Add 200μl of transfection mix per well and distribute evenly by rocking the plate side-by-side. The plate was incubated at 37°C, 5% CO2 for 24 hours.

38 4.Results and Discussion

A .

Fig 3- Generation of stable target cell line. Schema of constructs used(A), Bright field images of 293 T-rex GFP-LUC HA/NA after doxycycline induction(B), Bright field images of 293 T- rex GFP-LUC HA/NA before doxycycline induction(C)

To achieve the goal of the study, there was a need to develop a reporter cell line, stably expressing influenza protein which would facilitate evaluation of Fc-mediated influenza specific functional activity of the standardized NK effector cells. Using pcDNA4/TO GFP-IRES-LUC,

Luciferase and GFP reporters were inserted in the cells to determine assay efficiency at different

39 levels. Since the target cells lines required HA/NA proteins to be expressed on the surface in an inducible way, HA/NA influenza proteins were cloned into pcDNA 5/TO puro vector to allow expression of the proteins through Doxycycline treatment. The schema for the constructs is shown in Fig 3.A. The constructs were then transfected in the 293T-Rex cells for generating stable cell lines. After 24 h of induction with Doxycycline, GFP expression was evaluated. The cells that were uninduced did not express any visible GFP and the induced cells showed robust GFP expression as observed in Fig 3.B suggesting that protein production can be tightly controlled using this inducible expression system. There is a doxycycline-dependent increase in the GFP expression which correlates to the protein production as GFP, Luciferase and Protein of interest are placed under the same CMV promotor.

40

Fig 4 Characterization of H1 H3 N1 and N2 proteins via Western Blots. Protein expression of constructs in 293 T-rex GFP-LUC HA/NA cells(A) and protein expression in Transiently

Transfected 293 T cells(B)

The next step after generating the stable 293 T-rex cell lines is to check for proper protein expression. In 293 T-rex cells stably expressing H1, H3, N1 and N2, robust protein induction was observed in western blots after doxycycline induction. The HA and NA constructs show bands at the correct molecular size as shown in Fig 4A. The H1 and H3 blot shows two bands corresponding

41 to HA0 and HA1. It is previosuly known that HA0 can be cleaved into HA1 and HA2(Bottcher-

Friebertshauser, Freuer et al. 2010). In these blots, only HA0 and HA1 are observed as the antibody used is specific to only HA0 and HA1. Protein bands of correct molecular size was observed for both N1 and N2 constructs. In the blots corresponding to the N2 construct, multiple bands of lower molecular weight was observed which could be due to some formation of multimers multiple bands or due to cleavage in the N2. To check whether the presence of other contructs used to generate the 293 Trex cells interfere with proper protein expression, these constructs were transfected into closely related 293T cells. These 293T cells showed similar protein expression patterns as seen in

293 Trex cells (Fig 4B). This suggests that the expression pattern observed for all the constructs are specfic to the construct and is unaffected by the cotransfected plasmids to generate the cell lines.

Clonal cell lines were generated with 293T-rex cells for all constructs which were stably expressing the corresponding HA/NA proteins. Multiple clones were then selected for each construct and were further expanded. Clones with robust inducible protein expression as observed through western blots were selected for further analysis (Data not shown).

Fig 5 Surface Protein confirmation via Flow Cytometry of the 293 T-rex HA/NA clones

42 For the ADCC to be mediated successfully, the HA and NA proteins should be expressed on the surface of the cells which would allow the antibodies to bind to HA/NA and also interact with the effector NK cells. Using flow cytometry without permeabilizing cells, surface expression of HA and NA proteins was validated in the generated clonal cell lines. All the 293 Trex HA/NA clones were analysed before and after induction and the ones with maximum shift in the peak after induction were selected for further expansion to be used in the ADCC Assay. Fig 3 depicts H1 H3

N1 and N2 clones showing a major peak shift. GFP expression was also analyzed in the same flow cytometry experiments to understand the amount of cells expressing HA/NA proteins. In flow cytometry experiments, robust expression of GFP was observed only after induction further confirming tight protein expression control which was observed through microscopy (Fig 3B, 3C).

The expanded cells were then used for optimization of ADCC assay.

Serum samples were collected from Influenza vaccine recipient as a source of antibody against the HA and NA. They were first tested for their reactivity with the 293 Trex GFP LUC-

HA/NA cells using Western Blot (Data not shown). The serum showing maximum expression were then chosen for ADCC development.

43 The Protocol- Influenza ADCC assay

C.

Fig 6- The 96 well-plate templete to perform the ADCC Assay(A). Schemactic diagram of the

ADCC mechanism (Singh, Marasini et al. 2018). The ADCC Assay optimization using different serum samples from Influenza Vaccine Recepients with H1, H3 and N1 cells(C)

44 Day1:-

Target Cells - Culture 293 T-Rex GFP-Luc HA/NA cells in T75 Flasks (75 cm2 tissue culture flask) in 18ml of complete DMEM media. The flask is kept horizontally in a 37 °C, 5% CO2 humidified incubator because of their semi-adherent property.

Effector cells - CD16- 176V-NK 92 were cultured in T75 Flask with 25ml of Myocult media supplemented with 200 IU/ml recombinant human IL2 (R&D Systems, Minneapolis, MN). The flask is kept vertical in a 37 °C, 5% CO2 humidified incubator.

Day 3: -

When the flask is 75% confluent, Induce the cells by adding Doxycycline (2μg/ml) to the target cell and wait for next 48h.

Day 5: -

Harvest the target cells (TC) from the plates.

Aspirate all the media out from the flask, add around 8 ml of PBS from the sides of the flask to wash the cells. Add around 1.5 ml Versene/EDTA to detach the cells from the surface. Make sure that the versene is in contact with all the cells. Keep the flask horizontally in incubator for 5 mins.

Take the flask out and add 18.5 ml of media/PBS and transfer the cells to 50 ml conical tube.

Centrifuge the cells at 350xg for 5 mins. Discard the supernatants and re-suspend the cells in 1 ml of media. Count the number of cells.

45 Harvest the effector cells from the flask.

Transfer the CD-16 NK 92 effector cells from T75 Flask to the 50ml conical tube and centrifuge the cells at 300xg for 8mins. Discard the supernatant, re-suspend the cells in 1 ml of Myocult media with recombinant IL-2. Count the number of cells.

Preparing the Influenza ADCC plate.

Take 96 U-bottom shaped well plate and add 100 μl of complete DMEM media to Row A (Target cell alone row) and add 50μl of same media to all the row except Row C

Add 50 microliters of diluted serum or antibody in DMEM media in Row C and serially dilute that till Row H in duplicates as shown in Fig 6A

4 Add the target cells 5.0 × 10 /well, incubate for 10 mins at 37°C in CO2 incubator. Next Add effector cells 1.0 × 105 /well to all the wells except Row A.

Centrifuge the plate at 300xg for 2 mins and incubate the plate at 37 °C in 5% CO2 incubator for

4 h.

After incubation, take the plates out and spin them at 300xg for 5 mins, discard the supernatant and wash the cells with PBS (200μl/well) at 300xg for 5 mins twice

Mix the DMEM growth media without phenol red (Gibco) and Britelite Firefly Luciferase

Substrate with 1:1 ratio. Re-suspend the cells in 200μl/well of the above mixture, mix well by pipetting. With the multichannel pipetted transfer 150μl/well of the reaction mix to the light protected 96 well luciferase white plate as shown in Fig 6A.

Read the plate quickly (within 5 mins) in Synergy HTX multi-mode reader (Bio-Tek) and

Calculate the %ADCC of HA/NA expressing cells using the below formula.

46

(((Luciferase units) )- ((Luciferase units) )))x100 % ADCC = no antibody control serum/antibody (Luciferase units) ) no antibody control

To mediate ADCC via this assay, the HA and NA protein on the 293 T-rex cell surfaces must interact with the antibodies in the serum. These target cell bound antibodies then interact with the effector cell (NK). The crosslinking initiates the downstream signaling that releases the cytotoxic granules from the NK cells that kills the infected target cells. The remaining luciferase cellular activity is measured and compared with luciferase expression of Row B with no-antibody control wells. This schematic representation of ADCC mechanism occurring in all the wells except

Row A is shown in fig 6 B.

Serum from influenza vaccine recipients (2019-2020) were used to evaluate the ADCC using the above-mentioned protocol. The assay recognized the ADCC mediating antibodies against HA/NA glycoproteins in the serum samples collected from the volunteers and as seen in the figure 6 C the %ADCC is highest with 1:20 dilution and decreases as the serum is being diluted to 1:640 for HAs and 1:320 for NAs concluding that there are ADCC mediating antibodies against both the proteins and the assay can distinguish the responses between HA and NA.

47

Fig 7 Specificity of ADCC assay. Chart showing % ADCC with 293 T-rex GFP-LUC cells and

293 T-rex GFP-LUC H1, N1 and H3 cells with indicated adult volunteers’ serum(A). Chart showing %ADCC in baby plasma samples with 293 T-rex GFP-LUC H1 cells(B).

It was necessary to confirm that the effect seen in Figure 6 C is due to the HA/NA ADCC antibodies, as a control, the assay was performed on the cells without HA or NA antigen, just 293

T-rex GFP-LUC cells and compared with the antigen expressing Target Cells, 293 T-rex GFP-

LUC HA/NA cells. As seen in Fig 7 A no reduction in %ADCC was seen with no antigen 293 T- rex GFP-LUC cells whereas decrease in %ADCC due to cell death with the antigen expressing

H1, H3 and N1 cells is clearly is observed.

To further validate the utility of this assay across samples from wide age groups, baby plasma samples were tested for ADCC antibodies against the H1 cells. Acute and convalescent samples were received from two babies. The acute sample is collected from infected individuals

48 and convalescent samples were collected five months after recovery. In Fig 7.B, sample 37 shows

H1-specific ADCC effect with the antibody titer at 1:320 for acute and 1:160 for convalescent sample. Whereas, sample 23 shows weak to no H1 specific ADCC in both, acute and convalescent samples. The results suggested that the assay can detect and differentiate samples showing strong or weak ADCC effect.

49 Discussion

Different ADCC assays have been reported that differ in usage of effector cells, methods of measuring cell death, and target cell/effector cell development. The oldest assay is the chromium release assay which was first described in 1958. This assay utilizes 51Cr as a measure of cell killing, requiring a scintillation counter for quantitation and generating radioactive waste. Other assays have been developed to measure target cell killing using non-radioactive compound release such as lactate dehydrogenase (LDH). Another method to quantify ADCC uses flow cytometry to quantify the degranulation marker CD107a and antiviral cytokines. This measurement does not represent the actual %ADCC as it is not measuring target cell killing, but is readily performed on

PBMCs. Each of these methods represents a potential way to measure influenza ADCC and has its own advantages and disadvantages. Our goal was to design an assay that a) utilized authentic

HA or NA on the surface of target cells; b) measured NK-mediated cell lysis; and c) could distinguish NA- and HA-specific ADCC responses in human sera.

For optimizing the assay, we obtained serum samples from flu vaccine recipient. We performed Western blotting and Flow cytometry experiments to confirm overall cellular expression and surface protein expression of target cells expressing H1, N1, H3, or N2 proteins.

Except for N2, each target cell expressed the glycoproteins well and were selected further to carry out the assay. We noted that the N2 Western blot had undesired protein bands following induction, which may be due to cleavage of various N2 monomers into different molecular size multimers.

We therefore decided to make a NA2 chimeric strain that might not exhibit this problem. We selected NA2 (A/Moscow) from the 2002-2001 Vaccine Strain and replaced its the stalk with NA2

(A/Kansas) of 2019-2020 circulating strain to understand if the effect we see is universal to all N2.

50 We reasoned that since ADCC antibodies are more stalk-specific as compare to the head, it may be important to include the stalk region of the more recent N2 strain. However, we plan to also generate the reverse chimera to evaluate this hypothesis.

Fc-dependent effector functions may play an important role in protection against influenza infection. The above data shows that HA binding Abs were able to induce strong ADCC responses, while NA responses were weaker. Murine monoclonal antibodies induce potent ADCC against influenza B NA glycoproteins, which is very well correlated in humans as well, but the NA GP is not studied with the same intensity as the HA antigen. Hence one of my research goals was to understand the NA mediating ADCC Abs and quantify them to better understand its role. Results above in Fig 6C indicated that there are NA mediating ADCC Abs present in the human serum samples. Further analysis using additional sera with in our ADCC assay will be required to better define the NA contribution towards ADCC. Perhaps because NA responses are weaker than those against HA, the ADCC measured against NA was low as compared to HA. This can be seen in figure 6C, The %ADCC killing for NA is at around 40% as compared to HA which is at 60% killing. It is important to note that the assay as developed here can detect and differentiate antibodies between HA and NA components of the virus.

In Fig 6.C we observe “Prozone effect” (also called the hook effect) in few of the serum samples.

This is an immunologic response where the efficiency of antibodies to form immune complexes is hindered when the concentrations of antibodies are very high. In this case, there is a low % ADCC in 1:20 serum dilution as compared to 1:40 serum dilution giving a hook-shaped curve. This could be due to more amount of antibody present in the well that leads to steric hindrance not allowing the effector cells (NK) to bind to the antibody-bound Target cell mediating inefficient killing. The control experiments with 293-Trex no antigen cells did not show the similar effect as seen with

51 antigen expressing cells concludes that the assay works and the %ADCC effect is specific to the

HA/NA antigen.

Currently, the efficacy of influenza vaccines is evaluated largely on neutralizing antibodies or on measurement of HAI titers. The assay outlined here will facilitate studies of the development and magnitude of ADCC in human cohorts, and will allow correlations to be derived with protection from infection or disease. This novel assay will help define the role of HA vs. NA- directed antibodies that mediate ADCC, and should help us determine if improving ADCC responses can be a means of improving future generations of influenza vaccines.

52 5.Future Directions

The assay is going to be utilize to quantify ADCC responses in infant plasma samples for the IMPRINT study. This study seeks to determine the underlying mechanism of immunologic imprinting through a birth cohort of infants and their mothers during first 3 years of life. After receiving plasma samples from initial groups of infants, we validated our ADCC assay with two samples and as seen in Figure 7 it can detect ADCC following initial exposures to influenza. This study has successfully demonstrated that the above assay is a valid option to the other conventional methods to measure in-vitro ADCC activity.

53

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