The road to a more effective influenza vaccine: Up to date studies and future prospects

Vaccine xxx (2017) xxx–xxx

Contents lists available at ScienceDirect

Vaccine journal homepage: www.elsevier.com/locate/vaccine

The road to a more effective influenza vaccine: Up to date studies and future prospects Kaori Sano, Akira Ainai, Tadaki Suzuki, Hideki Hasegawa ⇑ Department of Pathology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan

a r t i c l e

i n f o

Article history: Received 18 April 2017 Received in revised form 1 August 2017 Accepted 4 August 2017 Available online xxxx Keywords: Influenza Influenza vaccine Broadly neutralizing antibody Intranasal inactivated influenza vaccine Antibody response repertoires

a b s t r a c t Influenza virus causes an acute respiratory infection in humans. Frequent point mutations in the influenza genome and occasional exchange of genetic segments between virus strains help the virus evade the pre-existing immunity, resulting in epidemics and pandemics. Although vaccination is the most effective intervention, mismatches between circulating viruses and vaccine strains reduce vaccine efficacy. Furthermore, current injectable vaccines induce IgG antibodies in serum (which limit progression of influenza symptoms) but not secretory IgA antibodies in the respiratory mucosa (which prevent virus infection efficiently). Therefore, numerous studies have attempted to improve influenza vaccines. The discovery of broadly neutralizing antibodies has progressed research into antigen design. Studies designed to improve vaccine efficacy by changing the vaccine administration route have also been conducted. A thorough understanding of the mechanisms underlying the action of various vaccines is essential if we are to develop a universal influenza vaccine. Therefore, evaluating the quality and quantity of antibodies induced by vaccines, which determine vaccine efficacy, is critical. However, at present vaccine evaluation relies on hemagglutination inhibition tests, which only measure the quantity of antibody produced. Antibody repertoires comprise a set of antibodies with specific genetic or molecular features that correspond to their functions. Genetically and functionally similar antibodies may be produced by multiple individuals exposed to an identical stimulus. Therefore, it may be possible to evaluate and compare multiple vaccine strategies in terms of the quality and quantity of an antibody response induced by a vaccine by examining antibody repertoires. Recent studies have used single cell expression and highthroughput immunoglobulin sequencing to provide a detailed picture of antibody responses. These novel methods may be critical for detailed characterization of antibody repertoires induced by various vaccination strategies. Ó 2017 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Characteristics of human antibodies specific for influenza viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rational immunogen design for universal vaccine development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Studies examining how the route of vaccine administration improves vaccine efficacy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods used to evaluate the quantity and quality of immune responses elicited by vaccination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Author disclosure statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⇑ Corresponding author at: Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan. E-mail address: [email protected] (H. Hasegawa). http://dx.doi.org/10.1016/j.vaccine.2017.08.034 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Sano K et al. The road to a more effective influenza vaccine: Up to date studies and future prospects. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.08.034

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K. Sano et al. / Vaccine xxx (2017) xxx–xxx

1. Introduction Influenza is a highly contagious acute respiratory disease that causes mild to severe respiratory symptoms in humans. The etiological agent of this disease is influenza virus, which belongs to the family Orthomyxoviridae [1]. Influenza virus contains a segmented negative-strand RNA genome, which is subject to frequent mutation, particularly point mutations within the antigenicitydetermining regions (i.e., antigenic drift) [2]. These mutations enable the virus to evade pre-existing immunity induced by past vaccination and infection, leading to large, local epidemics [3]. In addition, occasional exchange of genetic segments between genetically distinct virus strains (i.e., antigenic shift) gives rise to an unprecedented virus type [4]. Influenza virus targets epithelial cells lining the respiratory tract and causes local inflammation upon human infection [5]. The human immune system defeats viral infection chiefly via humoral immune responses; the main effectors are secretory IgA (S-IgA), which is localized at the surface of the respiratory epithelium, and IgG antibodies in the serum [6]. Thus, vaccination is currently the most effective intervention to mitigate the harm caused by this virus at both the individual and societal level. The majority of current vaccines are of the injectable inactivated type, particularly detergent-disrupted split virus vaccines. Hemagglutinin (HA) is one of the major protective antigens expressed by the influenza virus and is a potential target for anti-influenza humoral immune responses. HA is integrated into the envelope of the virus particle and plays a role in viral attachment and entry into target cells [7]. HA expressed by influenza A viruses is classified into 18 antigenically distinct subtypes (H1– H18), whereas that expressed by influenza B viruses is classified as two separate lineages (Yamagata and Victoria) [8]. Currently, human influenza vaccines contain three or four formulation strains (A/H1N1, A/H3N2, and either or both of the two influenza B lineages) that circulate among human populations. Since virus antigenicity differs greatly according to the virus strain (regardless of HA subtype), vaccine compositions are updated annually in accordance with surveillance data reported by the World Health Organization [9]. Nevertheless, antigenic mismatches between circulating and vaccine virus strains can greatly reduce vaccine efficacy [10]. In addition, current inactivated vaccines are administered via either intramuscular or subcutaneous injection. These administration routes induce virus-specific IgG antibodies in the serum; however, it is secretory (s)IgA at the site of infection (i.e., the respiratory mucosal surface) that plays the major role in preventing infection. In other words, current vaccines, which only induce serum IgG antibodies, limit the severity of symptoms after influenza virus infection but do not prevent virus infection at the primary target site [6,11]. Therefore, more effective vaccines are needed, and numerous studies have attempted to develop a novel influenza vaccine. Here, we review current progress toward a more effective influenza vaccine, along with the research methods used. We will also provide some insight into future influenza vaccine research.

2. Characteristics of human antibodies specific for influenza viruses Antibodies specific for the HA protein, induced by viral infection or vaccination, play a critical role in preventing/inhibiting viral replication, thereby limiting the severity of symptoms. HA comprises two domains: the membrane-distal globular head domain and the membrane-proximal stalk domain. Most HA-specific antibodies target the head domain [12,13]. Therefore, the head domain is constantly subject to strong evolutionary pressure, leading to

frequent point mutations that result in antigenic drift, a process that enables the virus to evade anti-viral immune responses [2]. By contrast, the stalk domain of HA is relatively well-conserved among multiple virus subtypes [14]. This fact led to the idea that an antibody clone that binds to the stalk domain of HA might be capable of neutralizing multiple strains of influenza virus; such an antibody clone would be classed as a broadly neutralizing antibody (bnAb). Indeed, in 1993 Okuno et al. succeeded in identifying C179, a murine bnAb clone that recognizes the HA stalk domain and neutralizes both the H1 and H2 subtypes [15]. In 2008, another study identified a human-derived bnAb (A06) in an H5N1 avian influenza virus infection survivor; this antibody neutralized both the H1 and H5 subtypes [16,17]. Subsequent studies have identified other bnAbs in healthy humans [18–23], seasonal influenza convalescents [24,25]/vaccinees [13,24,26–38], pandemic influenza survivors [39,40]/vaccinees [41], and H5N1 avian influenza virus infection survivors [16,17]/vaccinees [42] (Table 1.). Some exhibited surprising breadth in terms of their neutralizing potency. For example, F16v3 neutralizes virus strains belonging to antigenically distinct groups 1 (H1 and H5) and 2 (H3 and H7) [24]. The discovery of bnAbs provided a substantial amount of valuable information. Structural analysis of complexes formed between bnAbs and HA revealed conformational epitopes on the HA surface that are highly conserved among various strains and subtypes of HA. The hydrophobic groove within the HA stalk domain is an example of such a highly conserved conformational epitope. Antibodies specific for these epitopes disrupt the low pH-induced conformational rearrangement of HA, which is an essential step required for fusion of the virus with the host cell membrane [18,30,37]. Epitopes recognized by bnAbs are also present in the head domain, in particular, the receptor-binding site (RBS). Antibodies that target the RBS inhibit the binding of HA to cellular receptors, although the neutralizing breadth of these antibodies is relatively limited compared with that of stalk-targeting bnAbs [21,25,31,34]. Identification of epitopes targeted by bnAbs has led to development of vaccine antigens that induce antibodies directed to these sites: these will be discussed later. In addition, the structural and genetic characteristics of bnAbs have been studied intensively. Diversity of the heavy and light chain genes within the variable region, which binds to an epitope, is due to V(D)J recombination and somatic hypermutation (SHM). The sites within the variable region that come into direct contact with their epitopes are called complementary determining regions (CDRs). HCDR3, which is encoded by the junction site of heavy chain V, D, and J segments, is the most variable whole antibody gene in terms of genetic sequence and length [43]. Therefore, it is the main region that defines antigen binding and antibody specificity [44,45]. B cells in the germinal centers of secondary lymphoid tissues undergo SHM, and B cells harboring mutations that increase antibody affinity for specific antigens are selected and expanded. This event increases the sequence diversity of the variable region [46]. Until recently, it was believed that these events occur randomly in individuals, and that established antibody repertoires comprise antibody clones that are unique to each individual. However, bnAbs share common characteristics, such as V(D)J gene usage, HCDR3 length, and SHM rate. In addition, studies reveal preservation of genetic signatures among bnAbs that have similar functional characteristics. For example, many bnAbs identified in various individuals make use of the same VH1-69 germline heavy chain. Most heavy chains derived from the VH1-69 germline harbor exposed hydrophobic residues at the tip of the HCDR2 loop, which stabilizes binding to the highly conserved hydrophobic groove in the HA stalk domain [16,18,26,34]. Several other bnAbs utilize the VH3-30 gene; these bnAbs are characterized by a long HCDR3 loop containing hydrophobic residues that contact the hydrophobic groove [23,24,35]. Further studies may enable us to

Please cite this article in press as: Sano K et al. The road to a more effective influenza vaccine: Up to date studies and future prospects. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.08.034

Representative Ab * 1

Year

1 2

A06 CR6261

2008 2008

Kashyap et al. [16] Throsby et al. [26]

3

F10

2009

Sui et al. [18]

4

–

2010

5 6 7 8

PN-SIA49 F045-092 CR8020 FI6v3

9 10 11

Author [Reference]

Neutralizing HA subtypes

Binding site

VH gene of origin

Donor origin

Method used in study

nt*5 nt

Stalk Stalk

VH1-69 VH1-69

H5N1 avian influenza survivors Seasonal influenza vaccinees

Phage display Phage display

–

nt

Stalk

VH1-69

Non-immune human

Phage display

–

nt

Uncharacterized

Seasonal influenza vaccinees

B cell immortalization

H1, H2, H5 H1, H2, H5 – H1, H5

– H3 H3, H7 H3, H7

nt nt nt nt

Stalk Head Stalk Stalk

VH1-69 VH3-23 VH3-30 VH3-53 VH4-39 VH3-23 VH1-69 VH1-18 VH3-30

B cell immortalization Phage display B cell immortalization single plasma cell culturing *2

Whittle et al. [31]

H1

nt

nt

Head

VH1-2

Seasonal influenza vaccinee Healthy human Seasonal influenza vaccinees Seasonal influenza vaccinee/convalescents Seasonal influenza vaccinee

Krause et al. [21] Wrammert et al. [39]

H1 H1

nt nt

nt nt

Head Stalk

Head

VH4-b*01 VH1-69 VH3-30 VH3-21 VH4-30-4 VH4-31 VH4-39 VH3-9

head

VH1-18

Stalk Head Stalk Stalk

Group 1

Group 2

B

H5 H2, H5, H6, H8,

– –

H2, H5, H6, H8,

Corti et al. [13]

H1, H1, H9 H1, H9 H1,

H5, H9

2010 2011 2011 2011

Burioni et al. [28] Ohshima et al. [19] Ekiert et al. [30] Corti et al. [24]

CH65

2011

5J8 –

2011 2011

Head

12

CR8033

2012

Dreyfus et al. [34]

CR8071

*

1, 2, * 3, * 4, * 5, *

13 14

CR9114 C05 39.29

2012 2013

15

5A7

2013

Ekiert et al. [25] Nakamura et al. [35] Yasugi et al. [36]

nt

nt

nt

nt

H1 H1, H2, H9 H1, H2

H3 H3 H3

–

–

Yamagata, Victoria Yamagata, Victoria nt nt nt

Healthy human Pandemic 2009 H1N1 survivor

Unselected single cell expression B cell immortalization Unselected single cell expression

Seasonal influenza vaccinees

Phage display

VH1-69 VH3-23 VH3-30*01

Seasonal influenza convalescent Seasonal influenza vaccinee

VH3-33

Seasonal influenza vaccinees

Phage display in vivo plasmablast enrichment * 3 B cell immortalization

Yamagata, Victoria nt

Stalk

VH4-4 07

Pandemic 2009 H1N1 vaccinee

*

16

3 E1

2013

Hu et al. [41]

H1, H9

H3, H7

17

CR8043

2014

Friesen et al. [37]

–

nt

stalk

VH1-3

Seasonal influenza vaccinees

18 19 20

2014 2015 2016

Wyrzucki et al. [23] Wu et al. [40] Joyce et al. [42]

Stalk Stalk Stalk Stalk Stalk Stalk

VH3-30 VH1-18 VH6-1 VH1-18 VH1-18 VH6-1

Healthy human

B cell immortalization

22

46B8

2017

Kallewaard et al. [22] Chai et al. [38]

nt nt – – – –

Phage display ISAAC method *4 Selected single cell expression

2016

H1, H1, H1, H1, H1, H1,

Healthy human Pandemic 2009 H1N1 survivor H5N1 vaccinees

21

3.1 CT149 56.a.09 31.b.09 16.a.26 MEDI8852

H3, H7, H10 – H3, H7 H3, H7 H3, H7 H3, H7 H3, H7

Unselected single cell expression B cell immortalization

–

Yamagata, Victoria

Head

VH5-51*01

Seasonal influenza vaccinee

in vivo plasmablast enrichment * 3

–

H2, H5, H6, H2, H5, H9 H5 H5 H5 H2, H5, H6, H9

K. Sano et al. / Vaccine xxx (2017) xxx–xxx

Antibodies for which a detailed structural analysis of binding modes with hemagglutinin is available. Culture of single plasma cells with IL-6 (supports plasma cell survival), followed by culture of the supernatant. Plasmablast enrichment by intra-splenic injection of vaccinee-derived peripheral blood monomuclear cells into hSCID chimeric mice. Single cell sorting followed by screening of antigen-specific Ab-secreting cells using microwell array chips (Jin et al. 2009). nt: not tested.

3

Please cite this article in press as: Sano K et al. The road to a more effective influenza vaccine: Up to date studies and future prospects. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.08.034

Table 1 Broadly neutralizing influenza antibodies reported to date.

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K. Sano et al. / Vaccine xxx (2017) xxx–xxx

link the genetic signatures of antibody genes to their respective epitopes, meaning that the use of genetic analysis techniques to evaluate antibody specificity may become possible. Furthermore, a recent study by Joyce et al. identified the genetic signatures of three bnAb clones, which were commonly identified in multiple donors. The study indicated that the antibody gene arrangement patterns that generate bnAbs are reproduced in multiple individuals [42]. Therefore, further in-depth analysis of antibody gene repertoires may provide information about the immune status of an individual. 3. Rational immunogen design for universal vaccine development Identification and detailed characterization of conserved epitopes targeted by bnAbs prompted rational immunogen design studies to develop a ‘‘universal” vaccine that could confer broad protection against multiple influenza virus strains. Rational immunogen design is an idea based on the design and engineering of an ideal vaccine antigen based on structural biology and immunology [47–49]. In 2012, Schneemann et al. attempted to design an immunogen that can convey and display a bnAb epitope on its surface. They generated an insect virus (flock house virus)based virus-like particle (VLP) that displays the A2 helix, the epitope for bnAb CR6261. Immunization of mice with the A2 helixdisplaying VLP induced an IgG response against homologous and heterologous HA subtypes, despite insufficient induction of IgG to protect against virus challenge [50]. Though induction of antibodies against a partial region of the HA stalk domain was far from successful, several approaches have also been used to elicit an immune response against the highly conserved stalk domain. Head-stalk chimeric HA [51–54], hyperglycosylated HA [55], and headless HA [56–58] have been constructed, and their efficacy has been tested in animals. Since the rational design of an immunogen upon which to base a universal influenza vaccine is a new field of research, immune responses against engineered vaccines have only been evaluated in immunologically naïve animals. However, their efficacy must be evaluated in human populations since it is a well-known fact that immune responses in model animals cannot directly replicate those in humans. In addition, in the case of influenza virus vaccines, factors such as age, the immuno-competence of vaccinees, and the titer of pre-existing antibodies all affect the mechanisms that mediate vaccine efficacy [59]. 4. Studies examining how the route of vaccine administration improves vaccine efficacy To address unsolved issues related to currently available vaccines, studies have examined the use of intranasal vaccines as an alternative strategy. Studies focusing on human immunity against influenza virus reveal that the viruses are eliminated from the respiratory tract by innate and adaptive immune responses. S-IgA, which is secreted at the mucosal surface, bears the main responsibility for determining the quality of the adaptive immune responses triggered at the respiratory mucosa [5,6,60]. Therefore, it is expected that successful induction of influenza virus-specific S-IgA to the respiratory mucosa will contribute to efficient prevention of influenza virus infection [61,62]. In addition, individuals that have contracted influenza are less likely than uninfected individuals to be infected with a virus harboring only minor antigenic changes [63]. This phenomenon is supported by studies showing that mice exhibit cross-protection following natural infection by influenza virus [64,65]. Therefore, vaccine administration via the intranasal route, which mimics natural influenza virus infection,

may induce antibodies that elicit broad protection, as well as prevent infection of the primary target tissue. Since the 1960s, several research groups have worked on intranasally administered live attenuated virus vaccines to improve vaccine efficacy [66]. These studies came to fruition in 2002, when the Food and Drug Administration (FDA) approved a cold-adapted, live attenuated vaccine, Flumist (MedImmune Vaccines, Inc., USA), for intranasal administration [67]. This vaccine confers stronger protective immunity than the parenteral inactivated vaccines, although it is not licensed for children under the age of 2 years or adults aged over 49 years due to potential side effects. In addition, the effectiveness of this vaccine against the H1N1 pandemic strain has fallen significantly [68–70]. This may be because the virus strain used in the vaccine does not infect and replicate efficiently in the human epithelial cells of vaccinees. Therefore, an alternative vaccine that could be administered via the intranasal route and induce effective mucosal immunity in a broad range of people is needed. Since the 1970s, we have known that inactivated influenza virus induces production of S-IgA antibodies in the respiratory tract when administered intranasally, making it a promising vaccine candidate for intranasal administration [71,72]. Tamura et al. immunized mice with an inactivated split virion vaccine (derived from virus strains H1 and H3), either subcutaneously or intranasally, prior to challenge with H3 virus. As a result, intranasal vaccination induced high levels of cross-reactive S-IgA that protected mice from non-lethal local infection of the upper respiratory tract by the heterologous virus. Also, intranasal vaccination of mice induced cross-reactive IgG antibodies that protected them from lethal infection of the upper and lower respiratory tracts by the heterologous virus. By contrast, in subcutaneously vaccinated mice, cross-reactive IgG conferred protection against lethal heterologous virus infection, though cross-protection against non-lethal heterologous virus infection was not achieved [73]. Subsequent studies verified that S-IgA secreted by the nasal mucosa provides high levels of cross-reactive, as well as strain-specific, immunity [74–76]. Asahi-Ozaki et al. used a polymeric immunoglobulin receptor knockout (pIgR-KO) mouse model to confirm the role of S-IgA in the respiratory tract. pIgR is a receptor for polymeric IgA and is located at the basolateral side of mucosal epithelial cells; the receptor transports S-IgA through to the apical side of epithelial cells via transcytotic vesicular trafficking. Accordingly, knockout of pIgR reduced the amount of IgA secreted by the nasal mucosa and increased the titer of IgA in the serum. Wild-type and pIgR-KO mice were intranasally immunized and subsequently challenged with various strains of influenza B virus. The protective efficacy against homologous and heterologous virus strains was higher in wild-type mice than in pIgR-KO mice, demonstrating that the cross-reactivity against multiple virus strains induced by intranasal immunization is due to S-IgA secreted by the respiratory mucosa [77]. Antibody responses induced by intranasal inactivated vaccines have also been verified in humans. Ainai et al. examined the titers of IgG and S-IgA in serum and nasal wash samples from healthy adults who received two intranasal administrations of inactivated whole influenza virus vaccine. Induced antibody levels exceeded all criteria for vaccine efficacy evaluation used by the European Medicines Agency. In addition, cross-reactive antibody responses to a variant influenza virus strain were also identified [78]. Further analysis of the structural and functional characteristics of S-IgA in the respiratory mucosa was carried out by Suzuki et al. using IgA collected from nasal wash specimens derived from volunteers that received an intranasal inactivated vaccine. Fractionation of nasal wash specimens by size exclusion chromatography and direct observation of IgA antibodies by atomic force microscopy revealed that the antibodies on the mucosal surface exist in the form of

Please cite this article in press as: Sano K et al. The road to a more effective influenza vaccine: Up to date studies and future prospects. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.08.034

K. Sano et al. / Vaccine xxx (2017) xxx–xxx

monomers, dimers, timers, and tetramers. Additionally, polymerized S-IgA showed the greatest ability to neutralize homogenous and heterologous virus [79]. This indicates that the crossneutralization potency observed in intranasally vaccinated individuals is due, at least in part, to polymerization of IgA. Accumulating data has proved the effectiveness of intranasally administered inactivated virus vaccines. However, if we are to develop a universal influenza vaccine, we must acquire a deep understanding of the mechanisms underlying various vaccine strategies. The factors that define the magnitude of a vaccineelicited immune response are the quality and quantity of the antibodies induced. Therefore, evaluating the antibody repertoires induced by vaccinees is an important aspect of vaccine research, which may be useful as a means of comparing different vaccine strategies. For decades, evaluation of antibody responses against influenza vaccination has relied on hemagglutination inhibition (HAI) tests, which use serum from vaccinated individuals. Approval of influenza vaccines is also based on the results of HA tests; the FDA defines seroconversion as the percentage of subjects with either a pre-vaccination HAI titer of <1:10 and a post-vaccination HAI titer of 1:40, or a pre-vaccination HAI titer of 1:10 and a minimum 4-fold increase in the post-vaccination HAI titer [80]. HAI tests are easy to conduct and convenient for measuring the levels of virus-specific antibodies induced by vaccination. However, the method cannot evaluate the quality of the vaccineelicited antibody response. There are three main reasons for this: first, HAI tests only measure IgG in serum, not S-IgA in the mucosa (which is induced by intranasally administered vaccines); second, HAI tests cannot detect bnAbs, especially HA stalk-targeting antibodies in serum. A large number of bnAbs do not possess HAI activity. These antibodies contribute to protection via effector functions such as antibody-dependent cellular cytotoxicity, antibodydependent phagocytosis (ADPC), and complement activation

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rather than by inhibiting virus binding to the cells [81,82]. Recent studies emphasize the benefits of bnAbs induced by vaccines; therefore, the bnAb population within serum should not be neglected. The third reason is that HAI tests cannot characterize individual antibody clones that comprise the antibody repertoire. As noted above, there are certain patterns of antibody gene arrangement that induce bnAbs, and these patterns are likely to affect the antibody repertoire in an individual. Nakaya et al. used a systems biology approach to compare immune responses elicited by an intramuscularly administered inactivated virus vaccine and an intranasally administered live attenuated vaccine. The results indicated that cell populations activated by immunization differ according to the vaccine strategy used, resulting in different adaptive and innate immune responses [83]. Thus, there is a high chance that the antibody repertoire induced may vary according to the route of vaccine administration, leading to differences in the quality and quantity of the antibody response, although this cannot be measured by bulk serological tests. Novel approaches to evaluating and comparing the quality and quantity of antibodies induced by multiple vaccination strategies, including intranasally administered inactivated virus vaccines, need to be identified. 5. Methods used to evaluate the quantity and quality of immune responses elicited by vaccination To date, various methods have been developed to study antibody populations induced by vaccination or infection (Fig. 1). These methods can be classified into three groups: lowthroughput immunoglobulin (Ig) sequencing, high-throughput Ig sequencing, and shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS). Low-throughput Ig sequencing is a method in which antibody sequences with the desired specificity and functional characteris-

Fig. 1. Research methods used by antibody-focused studies. Cells and antibodies in the peripheral blood used in antibody focused vaccine studies are depicted on the right. Research methods exploited by using PBMC components and antibodies, along with their respective research target of interest are listed on the left. Qualitative and quantitative antibody data can be obtained by classical Ig sequencing/cloning and serological analysis respectively, whereas antibody data with both qualitative and quantitative aspects can be obtained by use of novel research tools such as high-throughput sequencing and shotgun liquid chromatography-tandem mass spectrometry. Plasmablasts are present at high numbers in the circulation following infection by pathogens or vaccination. By contrast, memory B cells are present at low numbers and encode antibody genes generated by past infections. PBMC: Peripheral blood mononuclear cells, NGS: Next generation high throughput sequencing, LC-MS/MS: Liquid chromatography-tandem mass spectrometry, NT test: Virus neutralizing test, HI test: Hemagglutination inhibition test, Ig-seq: Immunoglobulin gene sequencing.

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tics (encoded by genes expressed by antibody-producing cells) are identified using classical sequencing methods. This method can be further sub-classified as phage display technology, B cell immortalization, and single cell expression, according to the procedure that precedes direct sequencing of the antibody genes [84]. Phage display starts with the collection of memory B cells from peripheral blood followed by RT-PCR of the VH (variable region of the heavy chain) and VL (variable region of the light chain) genes. PCR products are cloned, and phage display libraries expressing scFvs (single-chain variable antibody fragments) are constructed. The scFvs that bind to the antigen of interest are selected and sequenced. Since scFvs result from a random pairing of VH and VL genes, the phage display library does not necessarily reflect the native antibody repertoire. Therefore, this method is used only to mine antibody clones with the highest possible pathogen neutralizing potency, not for evaluating antibody repertories [16,18,19,23,25,26,34]. B cell immortalization is used to expand memory B cells that only exist in small numbers in the peripheral blood. B cells are then subjected to single cell antibody expressing and screening protocols [13,21,22,28,30,36,37]. The target cells of single cell expression technology are plasmablasts, which exist in high numbers in the peripheral blood after a certain period of time post-vaccination or post-infection. Single plasmablasts are sorted from blood by fluorescence activated cell sorting, followed by cloning and sequencing of VH and VL genes. Sequences obtained by these methods enable in vitro expression of the antibodies of interest [31,39,41,42]. These methods have led to the identification and functional characterization of various bnAb clones. The introduction of high-throughput sequencing (nextgeneration sequencing; NGS) into the field of antibody research field has allowed us to obtain genetic information about a larger subset of antibody-encoding B cells [85]. NGS can characterize the antibody repertoire in detail, including the frequency of V(D)J germline gene usage and SHM rates. Jiang et al. determined the lineage (a cluster of descendant antibody clones derived from an unmutated common ancestor) structures within antibody repertoires of influenza vaccinees by analyzing a VH sequence database constructed by NGS. They found that the pre- and post-vaccination repertoires of elderly vaccinees harbored fewer antibody lineages and showed higher mutation rates than those of younger vaccinees [86]. Jackson et al. identified a specific pattern of clonal expansion exhibited by specific antibody clones by monitoring timedependent changes in the antibody repertoire postadministration of a vaccine against pandemic H1N1 2009 [87]. As noted above, detailed characterization of bnAbs identified common genetic signatures. Schmidt et al. examined these genetic signatures in combination with NGS. By searching an antibody database constructed from plasmablasts derived from seasonal influenza vaccinees, they identified numerous bnAb CH65-like antibodies originating from 11 different VH genes [88]. Thus, NGS data can be held in a database, which can then be searched to identify antibodies with specific genetic characteristics. Until recently, VH and VL sequence databases were constructed separately; however, technological advances have enabled us to construct paired VH and VL repertoires, which reflect naïve VH and VL pairings [89– 91]. This can provide more detailed and accurate information about the antibody repertoire. In addition, in-depth characterization of antibodies identified by NGS is possible, since the native pairing of the VH and VL genes is known. Various studies are making use of NGS to reveal the dynamic antibody repertoire induced by vaccination [92–95]. Although most research on the antibody repertoire has been carried out by sequencing BCR repertoires, a proteomics approach called LC-MS/MS can directly characterize the monoclonal composition of polyclonal antibodies present in body fluids. The approach enables identification of the amino acid sequences that comprise

the HCDR3 region of trypsin-digested antibody fragments. Antibodies can be grouped into clonotypes based on their HCDR3 sequence [96–99]. Lee et al. conducted proteomics-based characterization of vaccine-specific IgG antibodies collected from seasonal influenza virus vaccinees. They found that the majority of cross-reactive antibody clonotypes were already present in prevaccination serum. This suggests that, upon vaccination, preexisting cross-reactive antibodies bind to the conserved region of an antigen; thus the response elicited by vaccination becomes focused on the nonconserved regions of the antigen [100]. It is said that LC-MS/MS can capture antibody fractions that are produced by B cells present in very low numbers and that cannot be detected by previously used Ig gene sequencing techniques. LC-MS/MS analysis of s-IgA purified from nasal wash specimens in combination with high- throughput sequencing of IgA genes may be used to characterize the IgA repertoire and evaluate the quality of antibody response induced by natural influenza virus infection or intranasal influenza vaccines. Therefore, LC-MS/MS can provide a more detailed and accurate picture of the antibody repertoire in bodily fluids. Studies of antibody responses began at the monoclonal level but evolved rapidly to such a level that they now examine detailed antibody repertoires. The new high-throughput Ig analysis methods may well allow us to define the detailed characteristics of the antibody repertoire induced by various vaccination strategies.

6. Conclusions Numerous studies have attempted to improve the efficacy of current influenza vaccines. Most studies adopted two main approaches: designing immunogens to induce antibodies with more protective potency, or using a vaccine administration route that mimics the natural mode of infection. In contrast to the knowledge and strategies developed from this research, classical serological methods used to evaluate antibody responses to vaccines are rather primitive. These methods can only evaluate the quantity of bulk antibody responses and cannot provide detailed characterization of antibody repertoires, which are the best indicator of the quality of an antibody response. Antibody repertoires are now recognized as comprising a set of antibodies that harbor specific genetic or molecular features corresponding to their functions. In addition, antibodies that share similar genetic and functional characteristics may be generated in multiple individuals exposed to an identical stimulus. Therefore, there is a possibility that we could evaluate and compare multiple vaccine strategies in terms of the quality and quantity of the antibody responses they induce by examining antibody repertoires. The research methods used to analyze and evaluate antibodies have become highly sophisticated. By applying novel methods such as highthroughput Ig analysis to evaluate various vaccination strategies, we expect to increase our understanding of antibody repertoires that may be induced by vaccination, and of the underlying immune system. The results of future studies will contribute to the development of more efficient influenza vaccines. Funding This work was supported by grants from the Research Program on Emerging and Re-emerging Infectious Diseases, of the Japan Agency for Medical Research and Development (AMED).

Author disclosure statement The authors have no competing interests to declare.

Please cite this article in press as: Sano K et al. The road to a more effective influenza vaccine: Up to date studies and future prospects. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.08.034

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