Abstract
Despite the widespread use of seasonal influenza vaccines, there is urgent need for a universal influenza vaccine to provide broad, long-term protection. A number of factors underpin this urgency, including threats posed by zoonotic and pandemic influenza A viruses, suboptimal effectiveness of seasonal influenza vaccines, and concerns surrounding the effects of annual vaccination. In this article, we discuss approaches that are being investigated to increase influenza vaccine breadth, which are near-term, readily achievable approaches to increase the range of strains recognized within a subtype, or longer-term more challenging approaches to produce a truly universal influenza vaccine. Adjuvanted and neuraminidase-optimized vaccines are emerging as the most feasible and promising approaches to extend protection to cover a broader range of strains within a subtype. The goal of developing a universal vaccine has also been advanced with the design of immunogenic influenza HA-stem constructs that induce broadly neutralizing antibodies. However, these constructs are not yet sufficiently immunogenic to induce lasting universal immunity in humans. Advances in understanding how T cells mediate protection, and how viruses are packaged, have facilitated the rationale design and delivery of replication-incompetent virus vaccines that induce broad protection mediated by lung-resident memory T cells. While the lack of clear mechanistic correlates of protection, other than haemagglutination-inhibiting antibodies, remains an impediment to further advancing novel influenza vaccines, the pressing need for such a vaccine is supporting development of highly innovative and effective strategies.
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Current influenza vaccines provide only moderate protection that is largely strain-specific. |
The development of influenza vaccines with increased breath and efficacy is an achievable short-term goal that should be pursued. |
Universal influenza vaccine development represents a considerable challenge that relies on inducing rare B cells that make fully cross-protective antibodies, and/or T cells that can be retained in sufficient numbers in the mucosa. |
1 Introduction
Influenza viruses comprise four types, A, B, C and D, of which types A, B and C infect humans and types A and B cause influenza epidemics. Influenza A viruses are subtyped based on the antigenicity of the envelope haemagglutinin (HA) and neuraminidase (NA) proteins into 18 HA and 11 NA subtypes, which occur in a wide range of animal species, including waterfowl and shorebirds, pigs, horses and dogs. The HA is a trimer, with a globular head that bears the receptor-binding pocket and neutralizing antibody-binding sites, and a stem (or stalk) that includes a peptide that mediates fusion of virus and host membranes during virus entry [1]. The 18 influenza A virus HAs are classified into groups 1 and 2 based on phylogenetic similarity of the stem region. Influenza viruses that currently circulate in humans include two influenza A subtypes, H1N1 (group 1) and H3N2 (group 2), and two influenza B lineages, Yamagata and Victoria. Influenza viruses mutate readily because the RNA-dependent RNA polymerase lacks proof-reading function, and selection pressure leads to the rapid generation of viruses containing mutations in antibody binding sites. This process, termed antigenic drift, is ongoing, difficult to predict and necessitates frequent updates of the influenza vaccine. The establishment of an influenza virus with a novel HA subtype to which the majority of the population lacks immunity is referred to as antigenic shift. Other avian influenza A subtypes, including H5N1, H7N9, H6N1, and H7N7, have infected humans but have not transmitted efficiently between humans, whereas H2N2 virus caused a pandemic in 1957 and caused epidemic influenza in humans until 1968.
Seasonal trivalent or quadrivalent influenza vaccines, including inactivated influenza vaccine (IIV), live attenuated influenza vaccine (LAIV), and recombinant HA vaccines, include antigens from each of the type A and B viruses that cause epidemic influenza. They induce strain-specific antibody-mediated immunity to the HA, with limited effectiveness against antigenic drift variants within the same subtype and virtually none against other influenza A virus subtypes. Ongoing antigenic drift in seasonal influenza A and B viruses, suboptimal effectiveness of currently licensed influenza vaccines [2] that can be of very limited duration in the elderly [3], and the sporadic threats of zoonotic and pandemic influenza A viruses are driving efforts to develop broadly protective influenza vaccines. This goal encompasses a spectrum that extends from a near-term achievable goal of a vaccine with enhanced efficacy against antigenic drift variants within the same subtype, to a long-term, more distant goal of a truly universal influenza vaccine that protects against all influenza A and B viruses (Fig. 1). Different strategies along this spectrum will provide increasing breadth of coverage against antigenic drift variants, zoonotic strains and pandemic influenza viruses.
Comparison of strategies to increase the breadth of influenza vaccines in terms of the anticipated duration of the immune responses invoked and timescales for product development. IIV inactivated influenza vaccine, HA hemagglutinin, NA neuraminidase
2 Strategies to Induce a More Broadly-Reactive Immune Response
2.1 New Approaches for Vaccine Strain Selection
The WHO Global Influenza Surveillance and Response System (GISRS) has operated since the 1950s to select seasonal influenza vaccine strains that closely match circulating strains, genetically and antigenically. GISRS also characterizes zoonotic influenza viruses to update candidate vaccine viruses for pandemic preparedness (http://www.who.int/influenza/vaccines/virus/201602_zoonotic_vaccinevirusupdate.pdf). It is not known which animal influenza virus may emerge to cause a pandemic. Therefore, the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH)/MedImmune collaborative programme undertook identification of animal influenza viruses that would generate antibodies with broad cross-reactivity against viruses within the same subtype. Ferret anti-sera were generated against 10–20 viruses each for H2 [4], avian and equine H3 [5], H5 [6], H6 [7, 8], H7 [9] and H10 [10] subtypes, including viruses that covered a wide geographic and temporal distribution and that represented different phylogenetic clades. Virus(es) that induced the greatest breadth of cross-reactivity in haemagglutination-inhibiting (HI) and neutralization assays were used to generate candidate pandemic LAIVs, which were evaluated in animal models and in phase I clinical trials [4, 11,12,13,14,15,16]. An alternative approach has been to design a computationally optimized broadly reactive antigen (COBRA) based on multiple rounds of generating HA consensus sequences. COBRA-HA virus-like particle (VLP) vaccines incorporating H5-HA or H1-HA induce cross-clade antibodies in mice [17, 18].
Several groups have explored forecasting of influenza A(H3N2) and A(H1N1) virus antigenic evolution by incubating viruses with human and/or ferret convalescent sera and driving positive selection [19, 20]. This approach suggests that it may be possible to make vaccines containing future antigenic escape variants and pre-empt antigenic drift.
2.2 Adjuvants
Adjuvants augment the adaptive immune response to vaccines, either by triggering innate immunity through pathogen recognition receptors (PRRs) with pathogen- or damage-associated molecular patterns (PAMPs and DAMPs) or by facilitating antigen delivery. Humoral immunity following influenza virus infection is facilitated by a broad array of PRRs, TLR7, TLR3, RIG-I/MAVS and the NLRP3 inflammasome (reviewed by Iwasaki and Pillai [21]). In contrast, the standard subunit and split virion IIV are unadjuvanted, and immunogenicity is more reliant on activation of TLR7, which recognizes single-stranded viral RNA [22].
The modest immunogenicity of IIV for high-risk groups has led to the clinical development of adjuvants. MF59, a squalene-based oil-in-water emulsion, was approved by the US FDA in 2015 for use in the elderly. Compared with IIV, adjuvanting with MF59 has been shown to increase antibody titre and cross-protection in older individuals [23, 24] and children [25]. For example, among elderly vaccinees, MF59-adjuvanted vaccine generates protective levels of antibody in 98%, whereas unadjuvanted vaccine generates this in 70–80% [23]. AS03, developed by GlaxoSmithKline, is another oil-in-water emulsion that uses squalene and dl-α-tocopherol. AS03 has been approved for several different pandemic influenza vaccine formulations, including Pandemrix™ used in Europe during the 2009 pandemic, Adjupanrix™ for A/H5N1 in Europe, and Q-Pan for A/H5N1 in the US. In general, AS03 induces robust antibody responses, with observations of 100% seroconversion against homologous strains and 69% seroconversion against heterologous strains in clinical trials [26, 27]. A head-to-head comparison showed that H7N9 vaccine titres were far higher when adjuvanted with AS03 compared with MF59 [28]. ISCOMATRIX, a formulation comprised of saponin, phospholipid and cholesterol, also increases seroconversion rates against an H7N9 VLP vaccine [29, 30]. Antibodies induced by unadjuvanted and ISCOMATRIX-adjuvanted VLPs were compared for binding to an HA gene fragment phage display library [30]. Antibodies induced by unadjuvanted VLPs predominantly bound epitopes in the C-terminus of the HA1 domain, whereas antibodies induced by ISCOMATRIX-adjuvanted VLPs also bound more accessible, conformational epitopes spanning the receptor binding domain, and are therefore more likely to mediate HI and protection [30].
AS03 and MF59 augment humoral immunity by increasing naive B-cell activation, B-cell receptor (BCR) adaptation by recalled memory B cells [31], and antibody persistence in ferrets [32] and humans [33]. It is assumed that they facilitate antigen uptake, but the actual mechanisms of adjuvancy are not known. Neither include PAMPs, so there remains scope to combine PAMPs, DAMPs and other antigen delivery modalities to promote robust adaptive immunity. Indeed, several experimental adjuvants using PAMPs and DAMPs show promise. TLR5 sensing is critical for optimal antibody responses to IIV, even though IIV does not directly engage TLR5 [34]. It is thought that flagellin, produced by gut microbiota, triggers TLR5-mediated priming of innate mechanisms that promote plasma cell differentiation [34]. Fusion proteins of influenza antigens and flagellin are being clinically tested [35, 36]. S-[2,3-bis(palmitoyloxy)propyl]-cysteine or Pam2Cys and R4Pam2Cys are TLR2 agonists that increase the immunogenicity of influenza-derived peptides and detergent-split IIV, respectively, leading to increased protection against homologous and heterologous challenge in mice [37,38,39]. R4Pam2Cys associates electrostatically with oppositely charged regions on protein antigens, forming antigen complexes that can be directed to TLR2 on dendritic cells, and induce both CD8+ T-cell and antibody responses [38, 40].
The inclusion of adjuvants can lead to higher rates of vaccine-associated side effects. These are generally very mild, such as inflammation at the injection site, fever and headache, but very rarely they can be more pronounced and serious. For example, small but significant increases in the rates of narcolepsy were observed in recipients of the Pandemrix™ vaccine in a number of countries [41, 42]. While there was initial concern that the AS03 adjuvant triggered narcolepsy, the adjuvant has since been used in a number of other vaccine formulations without similar observations. The precise cause of narcolepsy with Pandemrix™ remains unknown but is being extensively examined.
As a possible alternative to adjuvants, antibodies have been used to form antigen immune complexes that can engage Fcγ receptors on B cells [43]. Stimulation of Fcγ receptors, such as CD23, is dependent on the level of immunoglobulin (Ig)G-Fc glycosylation [44]. Interestingly, levels of Fc-glycosylated anti-HA IgG peak approximately 1 week after vaccination with the trivalent IIV, and complexing with Fc-glycosylated anti-HA IgG induces more potent and broadly reactive antibodies [45]. Fc-glycosylated IgG facilitates affinity selection by engaging CD23 on antigen-specific B cells; this induces FcγRIIB and inhibits the maturation of low but not high affinity B cells [45]. IIV complexed with a broadly HA-reactive mAb that engages CD23 protects mice against influenza H5N1 virus challenge [46].
As outlined above, there is both scope for, and a need for, improved adjuvants in influenza vaccines, and this will continue to drive development and refinement of formulations into the future.
2.3 Neuraminidase-Inhibiting Antibodies
The viral NA cleaves sialic acids from the surface of infected cells, releasing progeny virions [47, 48]. Therefore, although NA-inhibiting (NI) antibodies do not block virus entry or neutralize infectivity [47], they reduce virus release and spread, and in turn reduce illness severity in animal models [49, 50]. The evidence for a protective role of NI antibodies in humans is strong. When H3N2 viruses emerged in the 1968 pandemic, replacing previously circulating H2N2 viruses, H3-specific HI antibodies were lacking but partial protection associated with N2-reactive NI antibodies was demonstrated [51,52,53]. Clinical trials conducted in the 1970s and 1980s also demonstrated that NA-only vaccines protected children [54] and adults [55] against influenza illness, while being permissive to infection. Since then, more reliable, higher throughput NI assays such as the enzyme-linked lectin assay (ELLA) have been developed [56, 57] and several studies have correlated NI titres with protection against illness [58,59,60,61]. Antigenic drift of the HA and NA are not coordinated, therefore NI antibodies may provide protection against HA drift variants [62]. More importantly, NI antibodies provide cross-strain protection in animal models [63,64,65], consistent with the presence of conserved epitopes in N1 and N2 that are recognized by NI antibodies from mice [66] and humans [67]. A striking observation has been that while antibodies with broad NI activity are induced in humans following influenza infection, they are not induced following vaccination, suggesting that the NA content or structural integrity of NA in IIV is suboptimal [67]. A potential ‘universal NA’-inhibiting antibody epitope has also been identified [68].
While the combined evidence supports the development of NA-optimized vaccines, the NA concentration is not standardized in current vaccines; it varies widely and tends to be low [69]. To optimize NA concentration, it is important to first define the optimal protective NI titre, using assays such as ELLA. It may also be necessary to use NA-only vaccines to define protective NI titres and overcome the immunodominance of the HA head [70,71,72], with the potential benefit that NA-only vaccines should prevent symptoms but permit natural infection that will boost a broader range of immune responses than conventional IIV [73, 74].
2.4 Haemagglutinin Stem-Based Vaccines
HA head-directed neutralizing antibodies rarely recognize conserved regions such as the receptor binding site (reviewed by Neu et al. [75]). The highly conserved HA stem elicits protective antibodies that neutralize virus infectivity without inhibiting haemagglutination, and is a more attractive target for the development of a universal influenza vaccine. The HA stem also induces non-neutralizing antibodies that inhibit virus infection by mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of both neutralizing and non-neutralizing HA stem antibodies to protect humans against influenza has not yet been documented. Moreover, the two categories of HA stem antibodies may not be equivalent in their protective efficacy. Four epitopes have been identified in the stem region of group 1 and 2 influenza A and B viruses (reviewed by Neu et al. [75]). Although escape mutations arise readily in the HA head, the stem epitope is not normally under immune pressure, and attempts to generate escape mutants in vitro suggest that they will not arise easily [76].
The biggest technical challenge in designing an HA stem vaccine is to direct the antibody response towards the stem and away from the immunodominant HA head. The first approach that was attempted was a ‘headless’ construct [77], but a portion of the stem epitope that is part of the HA1 domain was missing from this construct. Subsequently, a strategy was developed for serial immunization with chimeric HA molecules that were engineered with different head domains atop an identical stem (reviewed by Neu et al. [75]). The underlying principle is that the antibody response to each new head domain will be a primary response, while the response to the stem will be boosted because it is identical in all the chimeric constructs [78]. The plan is to administer prime-boost vaccinations with chimeric HAs as LAIV and IIV, respectively. This approach has shown great promise in animal models and is now in a phase 1 clinical trial.
Polypeptides mimicking the native, pre-fusion HA stem epitope of group 1 influenza A viruses have also shown very promising results in animal models [79,80,81,82]. Yassine et al. used iterative cycles of a structure-based design to develop an H1 HA stabilized-stem immunogen lacking the immunodominant head domain, presented on a ferritin nanoparticle [83]. This immunogen conferred complete heterosubtypic protection against lethal H5N1 challenge in mice and partial protection in ferrets (4/6 survived) by eliciting broadly cross-reactive antibodies. However, the development of a group 2 HA stem vaccine has been more challenging, likely due to structural and/or biochemical differences between group 1 and group 2 HAs [84]. In collaboration with scientists from the Indian Institute of Science, we evaluated a bacterially expressed group 2 stem immunogen that was confirmed by biophysical characterization to form folded trimeric proteins. Vaccination induced stem-directed antibodies that protected mice from lethal homologous and intrasubtypic challenge and provided moderate protection against lethal heterologous virus challenge. However, in ferrets, vaccination induced relatively low levels of HA stem-directed antibodies that did not significantly reduce weight loss or nasal wash titres following robust H7N9 virus challenge. Epitope mapping revealed that ferrets developed lower titres of antibodies that bound a narrower range of HA stem epitopes than mice. We infer that this likely explains the lower efficacy in ferrets [84]. Our findings indicate that while group 2 stem immunogen showed promise in inbred mice, their immunogenicity and efficacy in larger outbred animals was not yet optimal and needs to be enhanced.
Once the technical challenge of inducing a robust stem-specific immune response is solved, downstream challenges include demonstrating efficacy against novel viruses in humans, or identifying correlate(s) of protection, characterizing the longevity of the response and developing appropriate assays to quantify them.
2.5 Vaccines to Induce Influenza-Reactive T Cells
T cells recognize internal proteins of the virus that are conserved and are not accessible to antibodies, have the capacity to kill virus-infected cells in vitro, and contribute to influenza virus clearance and control of infection in mouse models [85, 86]. Therefore, vaccines that induce robust T-cell responses have the potential to provide protection against influenza viruses bearing novel envelope proteins. IIVs induce little, if any, T-cell response. LAIV induces influenza-reactive T cells in animal models [87,88,89] and humans [90,91,92,93], and may induce greater heterologous immunity than IIV, at least in young children [94,95,96], but induces little or no heterosubtypic immunity [97]. The US Advisory Committee on Immunization Practices recommended against the use of LAIV for two recent seasons due to low effectiveness against the A(H1N1)pdm09 virus, which was postulated to reflect decreased replicative fitness [98]. A fitter A(H1N1)pdm09 strain has been included in 2018–2019 LAIV, and the recommendation against LAIV has been removed [98].
Depletion or functional impairment of individual CD4+ or CD8+ T-cell subsets does not uniformly exacerbate disease, indicating that multiple mechanisms contribute to heterosubtypic immunity [99, 100]. Some studies indicate that the contribution of CD4+ T cells is limited in the absence of CD8+ T and B cells [101], and that CD8+ T cells are the effectors of virus clearance [102, 103]. The requirement for more control mechanisms as the virus dose increases [104] could account for discrepant findings regarding the need for CD4+ [105] versus CD8+ T cells [106, 107] for protection against influenza illness in humans.
CD4+ T cells play a central role in the generation of high-affinity, isotype-switched B cells and antibodies that provide long-term protection against re-infection. Affinity selection occurs within germinal centres and is mediated by CD4+ T follicular helper (Tfh) cells that characteristically express the chemokine receptor CXCR5 to facilitate entry into lymphoid follicles [108, 109]. Whether changes in circulating Tfh [108, 110] can inform vaccine development [111] is an area of active investigation. Influenza vaccination causes transient expansion of peripheral CXCR3+, ICOS+, CD38+ Tfh cells, which include influenza-specific cells [111, 112]. Limited studies in mice indicate that optimal HI antibody responses require HA-specific Tfh [110]. This contrasts with reports that healthy human CD4+ T-cell responses focus on the internal viral nucleoprotein (NP) and matrix (M) proteins [113]. Vaccine-induced Tfh expansion is positively associated with antibody production [111] and benefits of high-dose vaccine in the elderly [114]. Therefore, CD4+ T-cell specificity or Tfh magnitude should be considered for the development of more effective vaccines.
Accumulating evidence indicates that influenza-reactive T cells must reside in the respiratory mucosa for timely control of viral replication and ensuing illness since it takes 4–5 days for memory T cells to track from lymph nodes to the respiratory tract, although they produce interferon (IFN)-γ within 6 h of infection [86]. Additionally, delayed T-cell responses, combined with large amounts of virus in lungs, could contribute to immunopathology [115]. It has been established that tissue resident memory (Trm) T cells represent a distinct subset [116,117,118,119,120,121] that co-express CD69 and CD103, unlike circulating T cells [120, 122,123,124]. The induction of Trm following pulmonary, but not systemic, immunization coincides with reduced viral replication and lung damage [121], and increased protection of mice against lethal influenza virus challenge [125]. Similarly, CD4+ and influenza-reactive CD8+ Trm are induced in mice by LAIV, but not IIV, and mediate heterosubtypic protection independent of circulating T cells and antibody [126].
The susceptibility of humans to repeated influenza infections indicates that T-cell-mediated heterosubtypic immunity may be weak or poorly maintained. A few studies have examined influenza-reactive T-cell frequencies over time in small sample sizes. The frequency of influenza-reactive cells with lytic capacity declines rapidly after infection, with an approximate half-life of 2–3 years [127], but tetramer staining indicates that CD8+ memory T-cell frequencies remain stable in the absence of documented re-infection [128]. Studies to determine whether vaccines induce and maintain Trm will not be possible in humans, but the evidence from preclinical models suggests that T-cell-based vaccines should be delivered mucosally.
Plasmid-based reverse genetics is being used to rationally design influenza vaccines that can infect airway cells to induce Trm, but not replicate because a crucial gene has been rendered defective [129]. For example S-FLU can only undergo a single cycle of replication because the HA packaging signal sequence has been inactivated [129]. HA is provided in trans during pseudotype particle production in cell lines, therefore S-FLU virus can infect cells and express the NA and conserved internal viral proteins in the cytosol, but cannot replicate or donate its HA to other influenza strains. Other single-cycle vaccines have also been produced, most of which lack fully functional HA [130,131,132] or M2 alone [133, 134], whereas Si et al. target multiple gene segments by introducing premature stop codons that can only be translated using a packaging cell line that expresses the appropriate non-host tRNA–tRNA synthetase pair [135]. Similarly, codon-pair deoptimization of specific gene segments yields influenza virus that is highly attenuated for growth, but not immunogenicity, in mice [136], although the optimal balance between attenuation and immunogenicity for other species requires fine tuning [137]. Single replication cycle influenza viruses represent a safer alternative to LAIV that can be delivered intranasally to high-risk and immunocompromised patients [135, 138], with potential to stimulate immune responses in the lower airways. S-FLU vaccines induce a strong cross-reactive T-cell response in the lungs, a specific antibody response to the expressed NA, but a minimal antibody response to the HA pseudotyping the particle [88, 139]. H1N1 or H5N1 S-FLU vaccines induce robust protection against the homologous and heterologous H1N1, H6N1 (group 1), H3N2 and H7N9 (group 2) viruses in mice [88, 139]. Importantly, ferrets immunized with one dose of H1 S-FLU then challenged with the homologous H1N1pdm09 virus did not transmit challenge virus to naive ferrets by the airborne route [88]. In pigs, immunization reduced the viral load in nasal swabs and lungs following challenge with a swine H1N1pdm09 isolate [139]. The ability of aerosol-administered H3N2 S-FLU vaccine to protect against heterosubtypic H1N1pdm09 challenge has been evaluated in pigs and ferrets [140]. H3N2 S-FLU reduced heterosubtypic challenge viral replication and aerosol transmission in ferrets, and induced lung Trm cells and reduced lung pathology, but not the viral load in the upper respiratory tract samples collected daily or bronchoalveolar lavage collected on day 5 at necropsy in pigs. Taken together, S-FLU vaccines showed protective efficacy in pigs and ferrets, demonstrating that in the absence of antibody, lung T-cell immunity can reduce disease severity and lung pathology, even without reducing challenge viral replication in the upper respiratory tract.
Vaccines based on peptide, DNA or viral vectors may induce more robust T-cell responses (discussed below), but further development of T-cell-based vaccines is hindered by a lack of a correlate of protection, and of strategies to induce and maintain influenza-reactive T cells in the respiratory tract. The minimal magnitude, breadth and functional capacity of T cells required for protection need to be defined [105,106,107].
2.5.1 Peptide-Based T-Cell Vaccines
Synthetic peptides are relatively safe and can be readily modified in the event that mutations are identified. However, the peptide vaccine approach requires peptides that are recognized by a diverse spectrum of HLA A and B types in the population. The majority of influenza-specific CD8+ T cells recognize NP and M proteins [141,142,143,144]. Importantly, NP and M contain epitopes presented by common HLAs such as A2, and responses are boosted by infection with different strains [143, 145, 146], reflecting the presence of epitopes that are highly conserved across the majority of influenza A viruses characterized [147, 148]. However, the focus on common HLA types means that immunogenic peptides identified to date do not cover the entire population. For example, the coverage of immunogenic NP peptides is approximately 16–57%, with an absence of epitopes for HLA-A*0101, A*6801, B*1501 and A*2402 [149].
Two influenza peptide vaccines have been assessed in phase I and II clinical trials, and both induce T-cell responses. Flu-v contains 21–33 amino acid stretches of M1, M2 and NP [150, 151], and includes five known CD8+ T-cell epitopes [149]. M-001 contains five T-cell and four B-cell linear peptide epitopes from NP, M1 and HA. Participants immunized twice with M-001 followed by trivalent IIV had significantly elevated T-cell responses [152], seroconversion rates, and antibody titres against drifted strains [153] compared with those who received trivalent IIV alone. While it is assumed that a combination of immune responses may be desirable for optimal protection, validation is required. Clinical trials that examined adjuvant effects [150] indicate the need to target peptides to professional antigen-presenting cells for optimal T-cell activation [154].
2.5.2 Vectored Vaccines
Replication-deficient viral vectors such as modified Vaccinia Ankara (MVA) can be engineered to express multiple protein antigens intracellularly, and thereby induce CD8+ T-cell responses without the need to identify epitopes for particular HLA types, or to use adjuvants [155]. The development of immunity to the vector can limit the use of the same viral vector for repeated (prime and boost) vaccine doses [155]. Therefore, DNA or distinct viral vectors are often used for priming. MVA expressing influenza NP + M1 induces T-cell responses and reduces clinical illness in human challenge studies [156], and can be coadministered with IIV without compromising antibody responses [157]. Similarly, viral vectors incorporating HA, NP and M1 induce robust T-cell responses, long-lived antibody secreting cells and HA-reactive neutralizing antibodies in mice [158]. However, in macaques, two doses of MVA-HA induced potent serum antibody responses against viruses with homologous HAs, but did not stimulate strong T-cell responses prior to challenge [159]. Although post-challenge CD4+ and CD8+ T-cell boosting was observed in animals that received either MVA-HA or MVA-NP, only MVA-HA reduced challenge virus replication and provided protection [159].
2.6 Combining Vaccine Platforms
The combination of different vaccine platforms has been demonstrated to increase the magnitude and breadth of the immune response. In phase I clinical trials evaluating H5N1 vaccine candidates, priming with LAIV [160, 161], or DNA expressing the influenza H5 HA, or adenovirus-H5 HA followed by IIV boosting was more immunogenic than priming and boosting with IIV [160, 162]. Similar results have been obtained from studies combining H7N7 and H7N9 vaccine candidate formulations [16, 163], and, in all studies, immunogenicity was increased by extending the interval between prime and boost [162, 163]. The mechanism underlying the superior priming effects of LAIV over IIV were explored in a non-human primate model [164]. Intranasal H5N1 LAIV elicits a highly localized germinal centre B-cell response in the mediastinal lymph node, which is rapidly recalled following IIV boost, eliciting germinal centre reactions at numerous distant immune sites [164]. These data provide mechanistic insights for the generation of robust humoral responses via prime-boost vaccination.
3 Conclusions
A number of avenues are being pursued to develop broadly protective influenza vaccines against seasonal influenza and, importantly, viruses with pandemic potential. Key drivers are the high rate of enzootic H5Nx and H7N9 viruses in poultry, with hundreds of spillover infections in humans, and the challenges in implementing a monovalent H1N1pdm09 vaccine following the 2009 H1N1 pandemic, in a timely fashion. Clearly, it is possible to increase the breadth of protection conferred by conventional vaccines through a number of approaches, such as the use of adjuvants or viral vectors, the addition of NA, vaccines containing computationally optimized or antigenically advanced HAs, and the selection of strains for inclusion in the vaccine that elicit a more broadly reactive immune response than conventional vaccines, including naturally occurring viruses. New vaccines that target conserved epitopes on the HA stem, or induce cellular immunity against internal viral proteins, induce greater breadth of reactivity and protection than conventional influenza vaccines and show great promise in animal models. Hopefully one or more of these avenues will prove to be safe and effective in humans.
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A. Fox, K. Quinn and K. Subbarao declare that they have no conflicts of interest.
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Fox, A., Quinn, K.M. & Subbarao, K. Extending the Breadth of Influenza Vaccines: Status and Prospects for a Universal Vaccine. Drugs 78, 1297–1308 (2018). https://doi.org/10.1007/s40265-018-0958-7
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DOI: https://doi.org/10.1007/s40265-018-0958-7