Status of Vaccine Research and Development of Universal Vaccine Prepared for WHO PD-VAC

I. About the Disease and Pathogen Basic information on pathogen, including transmission, estimated global disease burden for those at risk, for morbidity and for mortality, including uncertainties/data gaps, geographical distribution, economic burden if available, age groups affected and target groups for vaccination. Existing preventive, diagnostic and treatment measures and their limitations.

Influenza viruses are one of the major infectious disease threats to the human population owing to both the health impact of annual influenza and the tremendous global consequences of influenza pandemics. Seasonal influenza alone causes 250,000 to 500,000 deaths and three to five million cases of severe illness worldwide each year [1]. A public health concern is that a highly virulent influenza strain could lead to millions of deaths in today’s highly interconnected world. The recent 2009 influenza A/H1N1 pandemic illustrated the unpredictability of the influenza virus and the challenges to mounting a global response against a newly recognized virus strain.

Human influenza viruses are members of the family and are classified into three types, A, B, and C, based on internal proteins which are highly conserved across all sub-types and strains of a particular influenza type; matrix protein 1(M1), nucleoprotein (NP) and membrane matrix protein M2. Type A influenza viruses are further divided into subtypes based on the antigenicity of the hemagglutinin (H or HA) and neuraminidase (N or NA) surface glycoproteins. Currently, 16 HA and 9 NA subtypes are known, all of which exist in aquatic birds that are their natural reservoirs. Influenza B viruses infect only humans but two antigenically and phylogenetically distinct lineages co-circulate. Major outbreaks of human influenza are associated with influenza A or B. The virus depends on sialic acid receptors on the host tissue, which allow attachment of viral HA proteins and cell entry. NA acts as a cleaving enzyme, ensuring release of the virus from the cell, and the development of the . The presence and anatomical location of different types of sialic acid receptors vary between humans, birds and other animals, accounting for the tropism of influenza A [2].

The virus circulates worldwide, with a seasonal nature that is often hemisphere-specific and mostly observed in temperate climates. Existing methods of vaccine selection, production and distribution are best suited for seasonal nature of influenza and not well suited for preventing influenza in tropical countries where the virus circulates year-round. Therefore low-resource countries are mostly unprotected against seasonal influenza and are particularly vulnerable to future pandemics because individuals may be more susceptible to severe outcomes from influenza due to underlying nutritional deficiencies and concomitant illness, poor access to healthcare, and the lack of widespread vaccine use against Streptococcus pneumoniae and Haemophilus influenzae Type b. Additionally, many low-resource countries do not have a full picture of their influenza disease burden due to limited surveillance, and further information is becoming available only recently [3].

Influenza affects all age groups but the highest risk of complications occur among young children (under 2 years), older adults (65 years and over), pregnant women, and those with certain medical conditions, such as chronic heart, lung, kidney, liver, blood or metabolic diseases (such as diabetes), or weakened immune systems. The economic cost can also be very high as a severe epidemic may affect a significant part of the working population, affecting productivity and overwhelming public health systems. Anti-viral drugs – specific ones such as neuraminidase inhibitors and , or M2 inhibitors such as and non-specific ones such as arbidol (used in only few countries)- appear to have a limited benefit as they need to be administered early in the infection and tend to provide only some reduction in the duration and perhaps severity of symptoms of influenza. In addition, some strains of

1 influenza have developed anti-drug resistance. Vaccination remains the primary approach for controlling influenza in persons and populations. In reality the challenge of immunizing entire populations on an annual basis is currently beyond the capacities of most countries, driving the need to develop improved influenza vaccines that would offer both broad-spectrum and long-lasting protection to the populations most vulnerable to the consequences of influenza.

II. Overview of Current Efforts

A. EITHER Vaccines currently available and their limitations OR Biological feasibility for vaccine development

Include perceived limitations with available vaccines for low and middle income country markets (LMIC). These could include safety, effectiveness, serotype/strain coverage, supply, affordability, financing, number of WHO prequalified vaccines, WHO policy recommendations for available vaccines, perceived lack of priority from endemic country authorities. Where there are no vaccines available, this section should focus on the evidence that vaccine development is biologically feasible including from development of naturally acquired immunity, from vaccine development for related pathogens, from animal models or in vitro data

Several types of vaccines against influenza have been licensed and in long-time use around the world. They range from inactivated vaccines made of killed virus and administered parenterally to live attenuated (LAIVs) made from weakened virus and administered intranasally. Inactivated vaccines may be produced in eggs or in cultured eukaryotic cells while LAIV vaccines are produced in embryonated hen eggs. Both types of vaccines share the common goal of inducing immunity primarily to the hemagglutinin (HA) protein and possibly to the neuraminidase (NA) protein of the virus [4].

The HA-specific neutralizing antibody is a recognized correlate for immunity induced by inactivated, seasonal influenza vaccines [4]. Although the precise roles of other immune responses induced by vaccination in disease protection remain largely unknown, and no other correlates have been yet defined, LAIVs are known to induce both humoral and cellular immunity—mimicking the responses derived after natural infection. Other influenza vaccines have entered the market more recently. The first recombinant vaccine based exclusively on hemagglutinin was licensed in 2013 and the first produced in mammalian cell culture also achieved licensure. Most existing influenza vaccines have an excellent safety record but their efficacy in various age groups varies significantly. For example, LAIV efficacy in young children was reported as 83% [5] but it was much less effective in the elderly [6]. Furthermore, a wide discrepancy between vaccine efficacy and effectiveness is often reported; most recent systematic vaccine effectiveness studies indicate that overall, influenza vaccines provide a moderate level of protection; between 50 and 60% [7].

While some countries (such as USA since 2010) recommends universal influenza immunization (in this context, vaccination of almost entire populations) for all individuals 6 month and older, other countries either have more limited recommendations for specific age or high-risk groups, or no recommendations at all. Current WHO SAGE recommendations include the following risk groups for influenza vaccination: pregnant women (being the highest priority), healthcare workers, children 6-59 month of age, the elderly and those with high-risk conditions. Given the epidemiology and complexity of influenza and varied efficacy afforded by different types of vaccines, greater public health benefit could be achieved by separating the use of particular vaccines to particular age groups. Even though it may not be in best commercial interest of all companies supplying the vaccines, current evidence indicates that policy-wise, one size does not fit all. Recent evidence-based Advisory Committee on Immunization Practices (ACIP) recommendation for preferential use of nasally administered LAIV in children 2-8 years old in the USA and a similar one in Germany offer an example of such an approach that may be effective in for controlling influenza in LMIC. Some experts in the field have long suggested that focusing influenza

2 immunization on children could provide the greatest health impact given increased susceptibility of children to be infected with and transmit the disease. Recently implemented immunization policy in the UK with LAIV vaccine made available free of charge to school-aged children may soon provide data to support that notion.

At least four companies offer WHO prequalified seasonal inactivated influenza vaccine (GSK, Green Cross, Novartis, Sanofi Pasteur). Few other companies (CSL, MedImmune, and Serum Institute of India) prequalified their pandemic influenza vaccine only for 2009 H1N1 virus. With most seasonal vaccines produced by developed countries and despite sufficient influenza vaccine production capacity for annual needs, influenza vaccination coverage among high-risk groups even in industrialized countries is below the targets set by national governments and recommended by the WHO. In low and medium resource countries (LMIC), different health priorities and constrains on health budgets limit influenza vaccine use primarily to private markets, perhaps with the exception of PAHO countries. With low, but growing local capacity to manufacture influenza vaccines in LMIC, vaccines currently available on the market in these countries are often either imported by a multinational pharmaceutical company or supplied in bulk to a local manufacturer (usually through a joint-venture with a multinational company) for local packaging and distribution.

It can be assumed that in the absence of data from cost-benefit studies, expansion of influenza vaccine coverage to public markets in LMIC is unlikely to happen. Local production is expected to reduce the cost and increase timely availability of vaccines in some LMIC but the lack of local recommendations and government funding for use remain as limiting factors. While the vaccine cost is another factor, use of LAIV vaccines may contribute positively to coverage rates in children if they cost less than inactivated vaccines to produce and administer, and if the low age indication for current LAIV (currently 2 years) can be safely extended to a younger population. There are ongoing and planned age de-escalation clinical studies in LMIC to determine if seasonal LAIV is safe and efficacious in children as young as 6 months old. While the elderly also suffer from increased morbidity and mortality from influenza, all currently existing vaccines demonstrate very modest effectiveness in this age group. It can be partly explained by immune senescence and usually higher levels of co-morbidities in the elderly, but more efforts are required to develop more efficacious vaccines for the elderly. Use of adjuvants or higher dose of antigen have improved immunogenicity in recently approved influenza vaccines for those 65 years and older but the impact of these vaccines on decreasing the burden of disease in this age group needs to be evaluated further [8].

Although the current portfolio of subtype specific seasonal influenza vaccines do provide important medical benefit, their limitations have driven the field towards the “universal influenza” vaccine concept that can overcome the virological phenomenon of antigenic drift and antigenic shift. Antigenic drift refers to the frequent emergence of strains with different antigenicity, due to the ability of the virus to escape pre-existing immunity by point mutations in genes encoding HA and neuraminidase (NA). This necessitates frequent, sometimes annual updates to one or more component of trivalent influenza vaccine (TIV), which comprise strains representing H1, H3 and type B virus. The current system of influenza evolution monitoring, coupled with consensus-based bi-annual strain selection and subsequent recommendations to vaccine manufacturing is unique. However, accurately predicting which strains will circulate in the upcoming year remains difficult, and antigenic mismatch does occur [9]. In particular, influenza B viruses are often poorly predicted. As such, a quadrivalent influenza vaccine (QIV) vaccine that includes HA from viruses from the two different phylogenetic influenza B lineages has been suggested as a way to improve protection and has recently been approved in some markets. However, QIV while reducing the risk of mismatch may cost more than TIV.

Antigenic shift involves the introduction of novel strains with HA genes (from zoonotic reservoir) that the human population has not previously experienced. The lack of pre-existing immunity in

3 the population can enable the novel virus, if it is easily transmissible, to spread to a large percentage of the human population, increasing pandemic risk, such as in the shift from H1 hemagglutinin subtype to H2 in 1957 and from H2 to H3 in 1968 [10]. Currently H1 and H3 sub-types co-circulate. An additional threat is presented by direct transmission of highly lethal avian strains to humans (such as influenza A/H5N1 and, more recently, influenza A/H7N9), but such strains have not demonstrated the capacity for efficient transmission in humans as yet. However, future adaptation of these viruses for efficient human- to-human transmission cannot be discounted and should be anticipated. Thus the need for broadly- reactive, long-lasting and cross-protective “universal” influenza vaccine is clear.

No single definition of a universal influenza vaccine currently exists. It is assumed that a universal vaccine would induce heterosubtypic immunity (HSI), which is defined as immunity generated by a given IAV subtype or its antigens that protects against challenge with other IAV subtypes (e.g., immunity to influenza A/H1N1 protecting against an infection with influenza A/H3N2 or A/H2N2) [11, 12]. However, it is unlikely that a single vaccine product could protect and prevent infection with any type of influenza in light of the virus constant evolution and individuals varied immunological experiences with influenza (or previous vaccinations), their age, degree of immune system maturity, genetic background, and underlying conditions (such as pregnancy). Thus one can define “universal vaccines” in this context as those which protect against influenza viruses of two or more subtypes most commonly found in humans. Ideally, the universal vaccine would provide protection against multiple hemagglutinin subtypes and would target both influenza A viruses (IAV) and influenza B viruses (IBV) by having a separate antigen component for each. However, a reasonable first step could be a vaccine that is more “broadly reactive” within a single given subtype than the currently licensed influenza vaccines, and that consequently would not need to be administered or updated annually.

While studies in animals and human data from most recent 2009 pandemic support the notion that broadly-reactive immune responses against influenza naturally exist, which strengthens the case for “universal” influenza vaccine development, only further studies in humans can advance the field. Studies in animal models are limited as they do not completely and faithfully mimic the complex and layered nature of immune responses to natural and vaccinations in human populations. As mentioned, a highly desirable element of the universal influenza vaccine profile would be to eliminate the need for annual vaccination while maintaining protection. The main question is how to elicit responses leading to long-lasting production of broadly-neutralizing antibodies. In summary, to increase the likelihood of success, vaccine development efforts will need to address the following major issues: • Selecting a target (or multiple targets) that ensures breadth of protection against strains and subtypes • Achieving sufficient durability of the elicited response • Establishing a correlate of protection

B. General approaches to vaccine development for this disease for low and middle income country markets

What are the scientific approaches and indications and target/age/geographic groups being pursued? What public health needs will these vaccines meet if successfully developed? Where there are several different possible indications/target groups, how much consensus is there as to prioritization between these for vaccine development in LMIC.

While the influenza field has seen substantial investment over the last ten years, the majority of US- centered funding has focused on ensuring the sustainability of domestic manufacturing and broadening manufacturing options that are not dependent on egg-based production. Comparatively little has been invested in research designed to provide a rational basis for developing universal or broadly reactive vaccine against influenza.

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There is no consensus on what primary clinical benefit “universal” influenza vaccine should try to accomplish – prevent any influenza infection or merely reduce severity of acquired infection. The field also seems to be somewhat artificially divided between those who see either humoral or cell-mediated response as the best way to address the goal of universal influenza vaccine development. Very few approaches currently consider combination of approaches to elicit multifaceted and complementing immune responses that involves antibodies, T cells and mucosal immunity.

Common approaches for both pandemic and seasonal broadly reactive influenza vaccine development exist. The two main protein antigen targets from the influenza virus generally considered to be promising for antibody-driven, broadly reactive vaccine development are HA and the ectodomain of Matrix 2 protein (M2e) [13]. The immunodominant regions of HA located with protein “head” are highly variable, but HA also contains conserved epitopes in its “stalk” region [14]. Several broadly-neutralizing antibodies recently isolated from humans target those conserved sites in the HA stalk. M2e is well conserved among influenza type A viruses and is considered as an attractive target [15]. Other well-conserved influenza proteins, such as the NP and matrix (M) protein are mainly targeted for developing strong cross-protective T cell responses. Some research programs attempt to combine antigen targets in a single vaccine to engage both arms of immune response and to utilize various delivery systems for their optimal presentation. Among these approaches are 1) live attenuated influenza vaccines (LAIVs), and 2) vaccines created by more targeted and rational modifications to the influenza genome. Another such approach is a whole virion vaccine strategy that utilizes novel methods of virus inactivation with claims of broader protection [16].

III. Technical and Regulatory Assessment Highlight perceived positive/negative aspects in clinical/regulatory pathways e.g. well established product development and regulatory pathway to licensure, accepted immune correlates and/or functional assays, accepted surrogate efficacy endpoints, existence of well accepted animal or challenge models, agreed trial designs and endpoints. Possibilities to develop case for correlates/surrogates should be included.

Lacking a recognized correlate of protective immunity such as the HA-specific neutralizing antibody for inactivated seasonal influenza vaccines, universal influenza vaccine development will most likely follow a similar regulatory pathway that was established by LAIV (FluMist® by MedImmune), where efficacy against a clinically meaningful end-point will have to be demonstrated. Lack of correlates of efficacy does not prevent the development of novel influenza vaccines, but could increase the risk, cost and duration of clinical development. Using systems biology approaches should be encouraged to identify surrogate markers which can predict protection afforded by combining the best characteristics of M2e-based and T cell vaccines.

The issue of vaccine strain “match” with circulating strains to qualify vaccine efficacy would be expected to become unimportant if vaccine candidates inducing cross-strains protection advance to licensure but the path forward to establish the claim of protection against multiple strains and, significantly, over more than one influenza season, would mean large and long Phase 3 clinical trials. The question of whether a novel “universal” vaccine would need to demonstrate superiority over the existing vaccine(s) or simple efficacy against placebo would need to be resolved relatively early on. Should any of the current universal vaccine candidates under development reach licensure, an issue that may need to be considered will be the possibility that it might not keep up with the evolving nature of influenza few years down the road.

The existing paradigm in currently licensed influenza vaccines is to define protection as the vaccine’s ability to prevent infection (sterilizing protection), with protection understood as mediated by antibody- dependent prevention of cell infection driven by HA. This is distinct from vaccine’s ability to reduce disease severity, which may be an important component of vaccine-mediated protection, in particular,

5 broadly reactive vaccines that could mediate protection from various medically and public health relevant outcomes through different mechanisms [17]. Reducing disease severity may allow for reduced virus shedding and only mild or short-lived infection with reduced morbidity. This could potentially translate in lower economic and public health burdens but while a similar approach is accepted for other infections (e.g. rotavirus) no influenza vaccine has been licensed based on reduction of severe disease. Demonstrating vaccine efficacy with a generally accepted measure of infection is simpler than demonstrating reduced disease severity, and the latter will require significant clinical trials and may not be feasible in all high-risk population groups. Much discussion has occurred of late about the appropriateness of influenza vaccine trials using outcomes other than laboratory-confirmed infection—a subject that will have to be addressed if a vaccine is expected to elicit non-sterilizing protection.

Addressing a reduction in disease severity can be a worthwhile strategy in the development of broadly reactive vaccines particularly for countries that do not have an established system of annual vaccination against seasonal influenza, but a solid business case and countries’ acceptance of the new concept need to be built to support it. Additionally, the success of a broadly reactive vaccine is likely going to be constrained in the very young and the elderly, who experience a particularly acute burden of influenza due to their respective immature or aging immune systems.

IV. Status of vaccine R&D activities Summarize status of vaccine design, pre-clinical and clinical trial activity, including platforms, vectors, and adjuvants. Note academic, government, biotech and industry entities engaged. Summarize antigenic targets (if subunit approaches). Section on major advances in last 3-5 years, including key opportunities highlighted by recent science developments in the area.

Table 1 summarizes viral targets for the development of universal influenza vaccine, and Table 2 the current development pipeline. The development landscape can be divided into two general areas – those seeking to elicit antibodies, mainly to structurally conserved elements of HA, and to smaller extent to M2e, and those targeting cross-protective T-cell response against conserved internal influenza proteins, NP and M1. Multiple platforms are being used to deliver target antigens – recombinant proteins, synthetic peptides, virus-like particles (VLP), DNA vector, mRNA, viral vectors (MVA, Ad), and attenuated or whole virion influenza backbone.

Oil-in-water adjuvants such as MF-59 (from Novartis) and ASO3 (from GSK) have been demonstrated to increase the number of epitopes within influenza antigenic proteins (HA) and corresponding increase in in vitro cross-recognition of non-matched strains. This was mainly conducted for pandemic H5N1 vaccines where normal cross-protection against various clades of this influenza sub-type is poor. Use of MF-59 adjuvant in seasonal vaccine increased efficacy of vaccine in children [18] but it is not known whether it was due to enhanced cross-recognition of poorly-matched strain. Use of AS03 adjuvanted pandemic 2009 vaccine led to some concerns about its association with narcolepsy [19].

Major advances in the field over the last 3-5 years include identification of several broadly-neutralizing antibodies (bnAbs) from human samples which target conserved epitopes either on HA stalk or around its receptor-binding site [13]. Borrowing from similar advances in the HIV vaccine field, efforts are afoot to develop antigens and identify delivery platforms capable of eliciting these bnAbs. One of the most promising approaches appear to be a design of a series of chimeric HA which possess the same stalk from currently circulating HA (H1, H3 or B type) fused to the “head” regions derived from exotic strains of influenza (those not circulating in humans) [20]. The idea is that sequential vaccination with those constructs is going to divert immune response from dominant but variable “head” of hemagglutinin to more conserved “stalk” thereby eliciting bnAbs. Animal data support this hypothesis and planning of human studies is underway.

6 Table 1. Viral targets for broadly reactive influenza vaccines.

Protein antigen Targeted function Proposed mechanism of protection Receptor binding and Hemagglutinin (HA) Inhibition of fusion, maturation of HA, ADCC membrane fusion Complement-mediated lysis, ADCC, antibody- Ectodomain of M2 (M2e) Ion channel dependent NK cell activity Cleaves sialic acid Neuraminidase (NA) releasing virus from the Inhibition of viral spread surface of infected cells Matrix 1 (M1) Cell lysis by CD8+ cytotoxic T lymphocytes T cell stimulation (CTLs), CD4+ T lymphocyte-mediated cytolysis Nucleoprotein (NP) and B cell stimulation

Table 2. Development Status of Current Vaccine Candidates (POC = Proof of concept trial)

Organization Pre Phase Phase Phase Approach, Target, Adjuvant POC Identifier clinical 1 2 3 Use of MF-59 adjuvant to achieve broadly X cross-reactive antibody response. (and

Phase Novartis (USA) 4) Rational antigen design (HA) based on preferential presentation of conserved epitopes X for antibody response. Fusion protein between influenza M2e and VaxInnate bacterial flagellin (TLR5 ligand). Self- X (USA) adjuvanted. Proposed to be used with conventional TIV. Medicago Recombinant HA expressed as virus-like X (Canada) particle in tobacco plants. Requires adjuvant. Six long peptides from four core influenza Immune proteins conjugated to fluorocarbon chain, Targeting X elicits strong T cell response, proposed to be Systems (UK) used with conventional TIV. Proposed as “universal primer” to be followed by conventional TIV boost to potentiate HAI BiondVax responses. Consists of a mixture of peptides Pharmaceutical X comprising nine B and T cell conserved linear s (Israel) epitopes derived from three influenza proteins HA, M1, and NP. Mixture of 4 chemically synthesized peptides SEEK (formerly targeting conserved T cell epitopes present in X PepTcell, U.K.) M1, NPA, NPB and M2. Proposed to be used with oil-in-water adjuvant Flanders Fusion between M2e and hepatitis B virus core Institute protein for virus-like particle expression and X (Belgium) antibody-directed response. DNA plasmids encoding consensus sequences of HA, NA, and NP delivered by intradermal Inovio (USA) X electroporation for eliciting antibody and T cell responses. Fusion protein comprised of two highly Dynavax (USA) X conserved influenza antigens, NP, and M2e,

7 Organization Pre Phase Phase Phase Approach, Target, Adjuvant POC Identifier clinical 1 2 3 covalently linked to proprietary immunostimulatory sequence. Envisioned to be used with conventional TIV. Synthetic peptides derived from conserved B cell epitopes from HA modified with MHC Antigen Express Class 2 for facilitated T activity. Envisioned to X (USA) h be combined with traditional seasonal vaccine for improved response. Adenovirus encoding HA to prime followed by National inactivated vaccine (TIV) boost. X Institute of Allergy and Fusion protein between self-assembling ferritin Infectious protein and HA for nanoparticle presentation of X Diseases (USA) HA. Replication-deficient modified vaccinia Ankara (MVA) virus expressing both NP and M1. X Designed for strong cross-reactive T cell response. Self-adjuvanted. Jenner Institute, University of Replication-deficient simian adenovirus Oxford (UK) expressing both NP and M1. Designed for X strong cross-reactive T cell response. MVA expressing NP, M1 and conserved X portion of HA. Wistar Institute Fusion protein between M2e and NP, expressed X (USA) in chimpanzee adenovirus vector. Whole virion gamma-irradiated virus for Gamma intranasal application. Elicits B and T cell Vaccines X responses which are cross-protective. Self- (Australia) adjuvanted. Florida Vaccine Computer optimized consensus HA sequence. and Gene Elicits broad antibody response. Alum X Therapy adjuvanted. Institute (USA) Single-replication influenza virus which is not attenuated but unable to shed and designed to FluGen (USA) X elicit humoral, mucosal, and cell mediated immunity. University of Rearranged genome of influenza virus Maryland, permitting expression of two HA on the same X College Park virus, while also attenuating it. (USA) Various approaches to target conserved broadly reactive epitopes on HA stalk, such as Icahn School of “headless” HA or functional chimeric HA Medicine at (comprised of non-matched “head” and “stalk”) X Mount Sinai expressed either in the context of whole virus (USA) or as a rHA. Use of recombinant cHA protein requires adjuvant. Synthetic mRNA encoding HA and NP. CureVac Temperature-stable product, elicits both B and X (Germany) T cell response, self-adjuvanted. University of Adenovirus expressing broadly-neutralizing X

8 Organization Pre Phase Phase Phase Approach, Target, Adjuvant POC Identifier clinical 1 2 3 Pennsylvania monoclonal antibody against HA delivered by (USA) intranasal administration. Multiple, including support for Flanders Sanofi Pasteur Institute and the Vaccine and Gene Therapy X (USA) Institute. Internal work attempts to develop sequence-optimized HA. Georgia State M2e expressed in a virus-like particle (VLP). University X (USA) M2e-based vaccine comprised of peptide Merck (USA) X fusion to KLH carrier protein. Bionor Peptide-based approach targeting conserved X (Norway) epitopes. Unique technology using a mixture of 8 to 32 VBI (formerly peptides which represent hypervariable Variation X epitopes of HA to elicit polyclonal immune Biotechnologies) response University of “Headless” HA expressed together with NA Wisconsin and M1 in Drosophila S2 cell line for induction X (USA) of anti-stalk antibodies

9 References

1. WHO Fact sheet No. 211; March 2014. http://www.who.int/mediacentre/factsheets/fs211/en/ 2. Palese P, Shaw ML. Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors.Fields Virology.5 Edition. Philadelphia: Lippincott, Williams & Wilkins; 2007. pp. 1647–90 3. Radin JM, Katz MA, Tempia S, et al. Influenza Surveillance in 15 Countries in Africa, 2006– 2010. JID. 2012; 206 (Suppl 1), S14–21 4. Li CK, Rappuoli R, Xu X. Correlates of protection against influenza infection in humans--on the path to a universal vaccine? Curr Opin Immunol.2013;25:470–6. 5. Ambrose CS(1), Wu X, Knuf M, Wutzler P. The efficacy of intranasal live attenuated influenza vaccine in children 2 through 17 years of age: a meta-analysis of 8 randomized controlled studies. Vaccine. 2012; 30(5):886-92 6. Forrest BD(1), Steele AD, Hiemstra L, Rappaport R, Ambrose CS, Gruber WC. A prospective, randomized, open-label trial comparing the safety and efficacy of trivalent live attenuated and inactivated influenza vaccines in adults 60 years of age and older. Vaccine. 2011; 29(20):3633-9 7. Osterholm MT, Kelley NS, Sommer A, Belongia EA (2012) Efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis. Lancet Infect Dis 12: 36–44 8. Puig-Barbera J, Natividad-Sancho A, Calabuig-Perez J, Lluch-Rodrigo JA, Pastor-Villalba E, et al. MF59-adjuvanted and virosomal influenza vaccines for preventing influenza hospitalization in older people: comparative effectiveness using the Valencia health care information system.Vaccine 2013; 31: 3995–4002 9. Beyer, WE, Palache AM, , de Jong JC, et al. Cold-adapted live influenza vaccine versus inactivated vaccine: systemic vaccine reactions, local and systemic antibody response, and vaccine efficacy. A meta-analysis. Vaccine 2002; 20:1340–1353. 10. Taubenberger JK, Kash JC. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe.2010;7:440–51 11. Subbarao K, Matsuoka Y. The prospects and challenges of universal vaccines for influenza Trends Microbiol. 2013 July; 21(7): 350–358 12. Lee Y, Kim K, Ko E, Lee Y, Kim M, Kwon Y, Tang Y, Cho M, Lee Y, Kang S. New vaccines against influenza virus. Clin Exp Vaccine Res. 2014;3:12–28 13. Corti D, Lanzavecchia A. Broadly neutralizing antiviral antibodies. Annu Rev Immunol. 2013; 31:705–42. 14. Krammer F, Palese P (2013) Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr Opin Virol 3(5)521–30 15. Fiers W, De Filette M, Birkett A, Neirynck S, Min Jou W. A "universal" human influenza A vaccine. Virus Res. 2004;103:173–176 16. Alsharifi M, Furuya Y, Bowden T R, Lobigs M, Koskinen A, Regner M, Trinidad L, Boyle D B and Müllbacher A Intranasal protective against seasonal and H5N1 Infections. PLoS One (2009), 4 (4) e 5336 17. Wilkinson TM, Li CK, Chui CS, Huang AK, Perkins M, Liebner JC, Lambkin-Williams R, Gilbert A, Oxford J, Nicholas B. et al. Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans. Nat Med. 2012;10:274–280. doi: 10.1038/nm.2612. 18. Vesikari T(1), Knuf M, Wutzler P, Karvonen A, Kieninger-Baum D, Schmitt HJ, Baehner F, Borkowski A, Tsai TF, Clemens R Oil-in-water emulsion adjuvant with influenza vaccine in young children. N Engl J Med. 2011; 365(15):1406-16. 19. Nohynek H, Jokinen J, Partinen M, et al: AS03 adjuvanted AH1N1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in Finland. PloS one 2012, 7:e33536. 20. Krammer F, Pica N, Hai R, Margine I, Palese P Chimeric hemagglutinin influenza virus vaccine constructs elicit broadly protective stalk-specific antibodies. J Virol, 2013, 87: 6542–6550

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