Title: Influenza Vaccines: Challenges and Solutions
Abstract: Vaccination is the best method for the prevention and control of influenza. Vaccination can reduce illness and lessen severity of infection. This review focuses on how currently licensed influenza vaccines are generated in the U.S., why the biology of influenza poses vaccine challenges, and vaccine approaches on the horizon that address these challenges. Vaccination is the best method for the prevention and control of influenza. Vaccination can reduce illness and lessen severity of infection. This review focuses on how currently licensed influenza vaccines are generated in the U.S., why the biology of influenza poses vaccine challenges, and vaccine approaches on the horizon that address these challenges. Influenza viruses are members of the Orthomyxoviridae family and are comprised of segmented negative-sense single-stranded RNA genomes. Infection with an influenza virus can result in a sudden onset of fever, cough, rhinitis, malaise, headache, and sore throat following an incubation period of 1 to 3 days. There are three genera of influenza viruses (A, B, and C) that are divided based on antigenic differences in the viral nucleoprotein (NP) and matrix protein (M). Both influenza A and B viruses result in annual epidemics, with an attack rate of 5%–10% and 20%–30% in adults and in children each year, respectively (WHO, 2014WHOInfluenza. Seasonal, 2014Google Scholar). This results in 3 to 5 million infections annually and 250,000 to 500,000 excess deaths worldwide (WHO, 2014WHOInfluenza. Seasonal, 2014Google Scholar). Influenza A viruses from zoonotic sources can also result in occasional pandemics; four have occurred within the past 100 years (Taubenberger and Kash, 2010Taubenberger J.K. Kash J.C. Influenza virus evolution, host adaptation, and pandemic formation.Cell Host Microbe. 2010; 7: 440-451Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). The influenza A and B virus genomes each contain eight gene segments. Influenza A viruses are further divided based on the antigenic properties of their surface glycoproteins into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. While only a subset of these have been known to result in human infections, all have been isolated from their natural hosts—waterfowl and shorebirds (Yoon et al., 2014Yoon S.W. Webby R.J. Webster R.G. Evolution and ecology of influenza A viruses.Curr. Top. Microbiol. Immunol. 2014; 385: 359-375PubMed Google Scholar). The genomes of two additional subtypes of HA and NA have recently been sequenced from bats, but these viruses have not yet been isolated (Tong et al., 2012Tong S. Li Y. Rivailler P. Conrardy C. Castillo D.A. Chen L.M. Recuenco S. Ellison J.A. Davis C.T. York I.A. et al.A distinct lineage of influenza A virus from bats.Proc. Natl. Acad. Sci. USA. 2012; 109: 4269-4274Crossref PubMed Scopus (778) Google Scholar, Tong et al., 2013Tong S. Zhu X. Li Y. Shi M. Zhang J. Bourgeois M. Yang H. Chen X. Recuenco S. Gomez J. et al.New world bats harbor diverse influenza A viruses.PLoS Pathog. 2013; 9: e1003657Crossref PubMed Scopus (913) Google Scholar). Two influenza A subtypes (H1N1 and H3N2) and two antigenically distinct lineages of influenza B viruses currently co-circulate in humans (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). The best method for the prevention and control of influenza is vaccination (WHO, 2014WHOInfluenza. Seasonal, 2014Google Scholar). Vaccination can reduce illness and lessen severity of infection, particularly in groups at risk for complications of influenza, including young children and the elderly. This review is focused on how currently licensed influenza vaccines are generated in the US, why the biology of influenza poses vaccine challenges, and vaccine approaches on the horizon that address these challenges. Currently licensed influenza vaccines focus on the production of antibodies against the viral HA protein, which binds host receptors to mediate viral entry. Strain-specific antibodies produced against the HA neutralize the virus and prevent infection (Figure 1). The current seasonal vaccines require annual evaluation and reformulation to keep pace with the antigenic drift of circulating strains. This process is completed twice a year, once each for the northern and southern hemispheres (WHO, 2014WHOInfluenza. Seasonal, 2014Google Scholar). Antigenic drift results from mutations that occur because the error-prone viral RNA-dependent RNA polymerase lacks proofreading function, resulting in mutations in the HA and other viral proteins. Additionally, the HA is under positive selection for antigenic escape from neutralization by pre-existing antibodies. Selection of the vaccine composition for the upcoming season’s vaccine must take place 7 to 8 months in advance of “flu season” to accommodate the steps of vaccine production (WHO, 2014WHOInfluenza. Seasonal, 2014Google Scholar). There are three classes of licensed seasonal vaccines including inactivated, live attenuated, and recombinant HA vaccines (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). All three vaccines are multivalent, with components representing influenza A and B viruses anticipated to circulate in the next influenza season. The inactivated influenza vaccine (IIV) is a split virion or subunit vaccine that contains 15 μg of each purified HA protein administered intramuscularly or 9 μg of each purified HA protein administered intradermally (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). There is also a higher dose of antigen available for the elderly population aged 65 years and older, in which 60 μg of each HA is administered in order to increase the immunogenicity of the vaccine. The trivalent inactivated vaccine (TIV) contains H1N1 and H3N2 subtypes of influenza A along with the predicted dominant lineage of influenza B. A recently licensed quadrivalent influenza vaccine (QIV) includes two lineages of influenza B along with the H1N1 and H3N2 subtypes of influenza A. The IIVs induce a strain-specific serum IgG antibody response and are licensed for individuals aged 6 months and older. The second licensed vaccine product is the live attenuated influenza vaccine (LAIV). This vaccine also contains a mixture of the same four influenza strains as the QIV but is administered intranasally as a spray. The LAIV contains live viruses with temperature-sensitive and attenuating mutations (Coelingh et al., 2014Coelingh K.L. Luke C.J. Jin H. Talaat K.R. Development of live attenuated influenza vaccines against pandemic influenza strains.Expert Rev. Vaccines. 2014; 13: 855-871Crossref PubMed Scopus (37) Google Scholar). As a result of these mutations, the vaccine virus is restricted in replication at the temperature of the lower respiratory tract but can replicate at the cooler temperature of the nasal cavity. Vaccination with LAIV results in the production of strain-specific serum IgG as well as mucosal IgA and T cell responses (Coelingh et al., 2014Coelingh K.L. Luke C.J. Jin H. Talaat K.R. Development of live attenuated influenza vaccines against pandemic influenza strains.Expert Rev. Vaccines. 2014; 13: 855-871Crossref PubMed Scopus (37) Google Scholar). LAIV is also effective against some antigenically drifted strains of influenza (Coelingh et al., 2014Coelingh K.L. Luke C.J. Jin H. Talaat K.R. Development of live attenuated influenza vaccines against pandemic influenza strains.Expert Rev. Vaccines. 2014; 13: 855-871Crossref PubMed Scopus (37) Google Scholar). The LAIV is licensed for healthy individuals between the ages of 2 and 49 years (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). The third licensed product is FluBlok, which is a recombinant HA vaccine with HA proteins that are expressed in insect cells from baculovirus vectors. FluBlok is currently licensed for adults aged 18 to 49 years and can be used in individuals who are allergic to eggs (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). The manufacturing process for this vaccine has a shorter timeframe, which would be valuable during a pandemic response. The safety of seasonal influenza vaccines is well accepted. The most common adverse events reported for IIV involve reactions at the site of injection, including pain, redness, and swelling (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). For the LAIV, the most common events involve a runny nose and nasal congestion, although fever and sore throat have also been reported in specific age groups (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). Current recommendations in the US are for annual vaccination in individuals 6 months and older, with an emphasis on children; persons over 65 years of age; pregnant women; individuals with chronic health conditions; and healthcare workers (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar, WHO, 2014WHOInfluenza. Seasonal, 2014Google Scholar). Although the currently licensed influenza vaccines are effective in healthy young adults, Table 1 summarizes several challenges that remain. They include the dependence on embryonated eggs for vaccine production, the lengthy timeline for vaccine production, the need for annual vaccination, the emergence of antigenically novel viruses, the need for improved immunogenicity in the elderly, and the need for an improved correlate of protection. Several approaches have been developed to overcome these challenges and improve the immunogenicity and efficacy of influenza vaccines.Table 1Summary of Current Vaccine Approaches against Influenza VirusesVaccine FormatViral TargetsMode of ActionAdvantagesSolution to Vaccine ChallengeCurrently LicensedIIVHANeutralizing serum antibodiesInactivated vaccine; Low reactivityLAIVHA; NASerum antibodies; mucosal antibodies; CTL activityMucosal administration; Higher response in childrenMore cross-reactiveEmerging ApproachesRecombinant DNAVarious viral epitopesAntibodies; CTL activityNon-replicating; No egg requirementDependence on eggs for production; Lengthy production timeCOBRAHANeutralizing antibodiesIncreases cross-reactivityNeed for annual vaccination; Broader immune responseStem HA AntibodiesHA stemBlocks viral fusion; blocks HA maturation; increases ADCCBroad protection; Works at multiple steps in life cycleNeed for annual vaccination; Broader immune responseViral VectorsVarious viral epitopesIncreases ADCC; CTL activityNon-replicating; multiple delivery methodsLengthy production timeM2 AntibodiesM2Increases ADCCBroad protection; “Universal vaccine”Need for annual vaccination; broader immune response; emergence of novel virusesAdjuvantsN/AInnate immune signaling; broadens immune responseIncreases immunogenicity; dose sparingBroader immune response Open table in a new tab One disadvantage that is shared by IIV and LAIV is the need for embryonated eggs for production. A pandemic will likely result in a higher demand for vaccine, and embryonated eggs may be in short supply if the pandemic virus is pathogenic for poultry (Hannoun, 2013Hannoun C. The evolving history of influenza viruses and influenza vaccines.Expert Rev. Vaccines. 2013; 12: 1085-1094Crossref PubMed Scopus (141) Google Scholar). Several new influenza vaccines have been licensed within recent years that do not rely on production in eggs. Flucelvax is a newly licensed vaccine that is produced in a mammalian cell line and subsequent manufacturing steps are similar to egg-based IIV (Grohskopf et al., 2014Grohskopf L.A. Olsen S.J. Sokolow L.Z. Bresee J.S. Cox N.J. Broder K.R. Karron R.A. Walter E.B. Centers for Disease Control and PreventionPrevention and control of seasonal influenza with vaccines: recommendations of the Advisory Committee on Immunization Practices (ACIP) — United States, 2014-15 influenza season.MMWR Morb. Mortal. Wkly. Rep. 2014; 63: 691-697PubMed Google Scholar). As mentioned previously, the recently licensed recombinant HA vaccine FluBlok is expressed in insect cells. Also, DNA vaccines and virus-like particles (VLPs) are vaccine strategies that are in clinical development and are not manufactured in eggs. The selection of strains to include in annual influenza vaccines is based on global surveillance of circulating influenza viruses. Predictions are made months ahead of the arrival of “flu season” in order to accommodate all the steps of vaccine production, including the generation of three or four vaccine seed viruses, amplification, inactivation, purification, and dispensing into vials for IIV and blending and filling of sprayers for LAIV. Antigenic characterization of circulating viruses is the most critical criterion for the selection of vaccine strains. The antigenic relationship between circulating viruses is determined by hemagglutination inhibition (HAI) assays, in which their reactivity is tested against a panel of ferret antisera generated against reference strains including the previous year’s vaccine virus. Antigenic change among influenza viruses can be visualized by antigenic cartography (Smith et al., 2004Smith D.J. Lapedes A.S. de Jong J.C. Bestebroer T.M. Rimmelzwaan G.F. Osterhaus A.D. Fouchier R.A. Mapping the antigenic and genetic evolution of influenza virus.Science. 2004; 305: 371-376Crossref PubMed Scopus (1202) Google Scholar), which is a computational tool for the analysis of HAI assay data that provides a mathematical foundation for quantitative analysis of antigenic data (University of Cambridge, 2014University of Cambridge (2014). W.C.C. “Mapping the Evolution of Pathogens.” Retrieved Nov 1, 2014, from http://www.whocc.infectiousdisease.cam.ac.uk/antigenic-cartography.Google Scholar). Antigenic cartography is now applied to the selection of strains for influenza vaccines. The 2009 pandemic revealed the difficulty in producing and distributing a vaccine against a newly emerged virus within a short timeframe (Lee et al., 2014Lee Y.T. Kim K.H. Ko E.J. Lee Y.N. Kim M.C. Kwon Y.M. Tang Y. Cho M.K. Lee Y.J. Kang S.M. New vaccines against influenza virus.Clin. Exp. Vaccine Res. 2014; 3: 12-28Crossref PubMed Google Scholar). The 2009 H1N1pdm IIV was not available in time to prevent the second wave of the pandemic (National Center for Immunization and Respiratory Diseases, CDC, 2009National Center for Immunization and Respiratory Diseases, CDCCenters for Disease Control and Prevention (CDC)Use of influenza A (H1N1) 2009 monovalent vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP), 2009.MMWR Recomm. Rep. 2009; 58: 1-8Google Scholar). One approach to avoid this predicament in the future would be to stockpile vaccine seed viruses against different subtypes that have pandemic potential. This process involves the selection of representative viruses from each subtype prioritized based on epidemiological data and testing of the candidate vaccines in preclinical studies and clinical trials (Coelingh et al., 2014Coelingh K.L. Luke C.J. Jin H. Talaat K.R. Development of live attenuated influenza vaccines against pandemic influenza strains.Expert Rev. Vaccines. 2014; 13: 855-871Crossref PubMed Scopus (37) Google Scholar, Subbarao and Joseph, 2007Subbarao K. Joseph T. Scientific barriers to developing vaccines against avian influenza viruses.Nat. Rev. Immunol. 2007; 7: 267-278Crossref PubMed Scopus (201) Google Scholar). The decline in vaccine-specific antibodies and the antigenic drift of influenza viruses over time necessitates annual revaccination. Several strategies are being explored to increase the breadth of protection, or cross-reactivity, of influenza vaccines to avoid the need for annual revaccination. These include the use of a computationally designed HA sequence, induction of antibodies directed at the conserved HA stem, immunization with conserved influenza proteins that target T cell responses, incorporation of an adjuvant, and strategies that combine different vaccine platforms in “prime-boost” formats. One approach aimed to increase the breadth of the antibody response against the HA protein involves a computationally optimized broadly reactive antigen, or COBRA HA presented in a VLP vaccine (Giles and Ross, 2011Giles B.M. Ross T.M. A computationally optimized broadly reactive antigen (COBRA) based H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets.Vaccine. 2011; 29: 3043-3054Crossref PubMed Scopus (129) Google Scholar). The sequence of the COBRA HA represents a consensus sequence from a vast collection of influenza viruses that incorporates the most common amino acid at each position. This retention of conserved regions within the HA results in the generation of cross-reactive antibodies (Giles et al., 2012aGiles B.M. Bissel S.J. Dealmeida D.R. Wiley C.A. Ross T.M. Antibody breadth and protective efficacy are increased by vaccination with computationally optimized hemagglutinin but not with polyvalent hemagglutinin-based H5N1 virus-like particle vaccines.Clin. Vaccine Immunol. 2012; 19: 128-139Crossref PubMed Scopus (66) Google Scholar). An H5N1 COBRA vaccine has been shown to induce broadly reactive antibodies against multiple clades of H5N1 viruses and results in less pathology following challenge than a non-consensus VLP vaccine in nonhuman primates (Giles et al., 2012bGiles B.M. Crevar C.J. Carter D.M. Bissel S.J. Schultz-Cherry S. Wiley C.A. Ross T.M. A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection.J. Infect. Dis. 2012; 205: 1562-1570Crossref PubMed Scopus (90) Google Scholar). Another approach to enhance the breadth of the antibody response is to elicit antibodies directed at the conserved stalk or stem of the HA. A majority of antibodies elicited during infection target the immunodominant HA head domain that contains the receptor binding site and several well-defined antigenic sites that accumulate mutations as the virus drifts under immune pressure. The HA stem domain is much more conserved and antibodies targeting this region are more broadly reactive. Based on phylogenetic analysis HA proteins fall into two groups; group 1 includes subtypes H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 while group 2 includes H3, H4, H7, H10, H14, and H15 HAs (reviewed in Krammer and Palese, 2013Krammer F. Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr. Opin. Virol. 2013; 3: 521-530Crossref PubMed Scopus (254) Google Scholar). HA stem antibodies recognize subtypes within the same group, and a few demonstrate binding across both groups (Krammer and Palese, 2013Krammer F. Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr. Opin. Virol. 2013; 3: 521-530Crossref PubMed Scopus (254) Google Scholar). These stem antibodies do not block receptor binding and therefore are not detected by HAI assays (Corti and Lanzavecchia, 2013Corti D. Lanzavecchia A. Broadly neutralizing antiviral antibodies.Annu. Rev. Immunol. 2013; 31: 705-742Crossref PubMed Scopus (348) Google Scholar). Stem antibodies are believed to function at several steps of the viral lifecycle. They can inhibit fusion of the viral membrane through steric hindrance, inhibit maturation of the virus if the antibody binds to the uncleaved HA protein, and clear virally infected cells through antibody-dependent cell-mediated cytotoxicity (ADCC) (Krammer and Palese, 2013Krammer F. Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr. Opin. Virol. 2013; 3: 521-530Crossref PubMed Scopus (254) Google Scholar). Stem antibodies are not easily detected after vaccination with IIV but are found after natural infection in low amounts (Krammer and Palese, 2013Krammer F. Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr. Opin. Virol. 2013; 3: 521-530Crossref PubMed Scopus (254) Google Scholar). They are more readily produced following infection with an antigenically distinct virus and were found in higher amounts following infection with the 2009 pandemic virus (Krammer and Palese, 2013Krammer F. Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr. Opin. Virol. 2013; 3: 521-530Crossref PubMed Scopus (254) Google Scholar, Li et al., 2012Li G.M. Chiu C. Wrammert J. McCausland M. Andrews S.F. Zheng N.Y. Lee J.H. Huang M. Qu X. Edupuganti S. et al.Pandemic H1N1 influenza vaccine induces a recall response in humans that favors broadly cross-reactive memory B cells.Proc. Natl. Acad. Sci. USA. 2012; 109: 9047-9052Crossref PubMed Scopus (323) Google Scholar, Sangster et al., 2013Sangster M.Y. Baer J. Santiago F.W. Fitzgerald T. Ilyushina N.A. Sundararajan A. Henn A.D. Krammer F. Yang H. Luke C.J. et al.B cell response and hemagglutinin stalk-reactive antibody production in different age cohorts following 2009 H1N1 influenza virus vaccination.Clin. Vaccine Immunol. 2013; 20: 867-876Crossref PubMed Scopus (50) Google Scholar). Several vaccine approaches have been designed to elicit stem antibodies. Such techniques include “headless HAs” that lack the immunodominant head portion of the protein, sequential immunization with chimeric HAs bearing different head domains on a constant stalk domain to boost stem antibody responses, and vaccines expressing specific sections of the HA, including the conserved alpha-helix of the stem or the fusion peptide (Krammer and Palese, 2013Krammer F. Palese P. Influenza virus hemagglutinin stalk-based antibodies and vaccines.Curr. Opin. Virol. 2013; 3: 521-530Crossref PubMed Scopus (254) Google Scholar). An alternative method of vaccination has focused on expressing additional influenza antigens using viral vectors. Viral vectors are replication-defective viruses that can express high, sustained levels of antigens (Tripp and Tompkins, 2014Tripp R.A. Tompkins S.M. Virus-vectored influenza virus vaccines.Viruses. 2014; 6: 3055-3079Crossref PubMed Scopus (38) Google Scholar). Viral vectors can target specific cell types and allow delivery through multiple routes, and the vectors themselves can act as adjuvants to improve the immune response. An example of a viral vectored influenza vaccine is a modified vaccinia virus Ankara (MVA) expressing a fusion protein of influenza NP and matrix 1 protein (M1) that induce T cell responses but not neutralizing antibodies. In phase I clinical trials, the MVA-NP+M1 vaccine was immunogenic in healthy older adults aged 50–85 years, both alone and combined with seasonal TIV (Antrobus et al., 2014Antrobus R.D. Berthoud T.K. Mullarkey C.E. Hoschler K. Coughlan L. Zambon M. Hill A.V. Gilbert S.C. Coadministration of seasonal influenza vaccine and MVA-NP+M1 simultaneously achieves potent humoral and cell-mediated responses.Mol. Ther. 2014; 22: 233-238Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, Antrobus et al., 2012Antrobus R.D. Lillie P.J. Berthoud T.K. Spencer A.J. McLaren J.E. Ladell K. Lambe T. Milicic A. Price D.A. Hill A.V. Gilbert S.C. A T cell-inducing influenza vaccine for the elderly: safety and immunogenicity of MVA-NP+M1 in adults aged over 50 years.PLoS ONE. 2012; 7: e48322Crossref PubMed Scopus (97) Google Scholar). Also, a phase 1/2a trial evaluating a MVA-hemagglutinin-based H5N1 vaccine was safe and immunogenic in young adults, with a booster immunization a year later resulting in a substantial boost in antibody titers in all recipients (Kreijtz et al., 2014Kreijtz J.H. Goeijenbier M. Moesker F.M. van den Dries L. Goeijenbier S. De Gruyter H.L. Lehmann M.H. Mutsert Gd. van de Vijver D.A. Volz A. et al.Safety and immunogenicity of a modified-vaccinia-virus-Ankara-based influenza A H5N1 vaccine: a randomised, double-blind phase 1/2a clinical trial.Lancet Infect. Dis. 2014; 14: 1196-1207Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Adenoviral vectors have also been utilized to express the influenza matrix 2 protein (M2) and NP proteins that induced strong IgA and T cell responses after a mucosal administration and provided heterosubtypic immunity in mice and ferrets (Price et al., 2010Price G.E. Soboleski M.R. Lo C.Y. Misplon J.A. Quirion M.R. Houser K.V. Pearce M.B. Pappas C. Tumpey T.M. Epstein S.L. Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses.PLoS ONE. 2010; 5: e13162Crossref PubMed Scopus (108) Google Scholar). Another technique to broaden the cross-reactivity of influenza vaccines is to target T cell responses. The T cell response to influenza is targeted primarily against the internal proteins of the virus, including the NP and M1 proteins (Lee et al., 2014Lee Y.T. Kim K.H. Ko E.J. Lee Y.N. Kim M.C. Kwon Y.M. Tang Y. Cho M.K. Lee Y.J. Kang S.M. New vaccines against influenza virus.Clin. Exp. Vaccine Res. 2014; 3: 12-28Crossref PubMed Google Scholar). These viral proteins are highly conserved; therefore, they induce cytotoxic T lymphocyte (CTL) responses that are more cross-reactive than antibody responses directed at the HA. T cell immunity does not prevent infection but can reduce the severity and duration of infection (Lee et al., 2014Lee Y.T. Kim K.H. Ko E.J. Lee Y.N. Kim M.C. Kwon Y.M. Tang Y. Cho M.K. Lee Y.J. Kang S.M. New vaccines against influenza virus.Clin. Exp. Vaccine Res. 2014; 3: 12-28Crossref PubMed Google Scholar). The role of T cell immunity in ameliorating the severity of disease was demonstrated during the 2009 H1N1 pandemic, where the magnitude of the pre-existing CTL response inversely correlated with disease severity in individuals without detectable neutralizing antibody (Sridhar et al., 2013Sridhar S. Begom S. Bermingham A. Hoschler K. Adamson W. Carman W. Bean T. Barclay W. Deeks J.J. Lalvani A. Cellular immune correlates of protection against symptomatic pandemic influenza.Nat. Med. 2013; 19: 1305-1312Crossref PubMed Scopus (588) Google Scholar). Of the already licensed vaccines, LAIV results in higher CTL responses than IIV (He et al., 2006He X.S. Holmes T.H. Zhang C. Mahmood K. Kemble G.W. Lewis D.B. Dekker C.L. Greenberg H.B. Arvin A.M. Cellular immune responses in children and adults receiving inactivated or live attenuated influenza vaccines.J. Virol. 2006; 80: 11756-11766Crossref PubMed Scopus (271) Google Scholar, Lee et al., 2014Lee Y.T. Kim K.H. Ko E.J. Lee Y.N. Kim M.C. Kwon Y.M. Tang Y. Cho M.K. Lee Y.J. Kang S.M. New vaccines against influenza virus.Clin. Exp. Vaccine Res. 2014; 3: 12-28Crossref PubMed Google Scholar). Including adjuvants to boost and broaden the immune response is an additional approach to broaden the immune response of vaccines (Lee et al., 2014Lee Y.T. Kim K.H. Ko E.J. Lee Y.N. Kim M.C. Kwon Y.M. Tang Y. Cho M.K. Lee Y.J. Kang S.M. New vaccines against influenza virus.Clin. Exp. Vaccine Res. 2014; 3: 12-28Crossref PubMed Google Scholar). Adjuvants can also result in dose sparing of antigen. Several adjuvants are approved for use in human vaccines in other countries, but adjuvanted influenza vaccines are not yet approved in the U.S. (Even-Or et al., 2013Even-Or O. Samira S. E