Available online at www.sciencedirect.com

ScienceDirect

Broadly protective influenza vaccines: design and

production platforms

Husni Elbahesh, Giulietta Saletti, Thomas Gerlach and

Guus F Rimmelzwaan

Effective vaccines are the cornerstone of our defenses against and patients with chronic diseases, vaccination against

acute influenza virus that result in 500 000 annual influenza is recommended [4]. Thus, the availability of

deaths worldwide. For decades, an on-going concerted effort efficacious vaccines is important in mitigating the potential

has been to develop a universal influenza vaccine to combat impact of seasonal and future pandemic outbreaks. This

the looming threat of potentially pandemic emerging and re- was exemplified in 2009, when a novel swine origin qua-

emerging influenza viruses. To address the need for rapid druple reassortant virus of the H1N1 subtype caused a

efficacious vaccines that could mitigate the impact of seasonal pandemic outbreak.Despite huge efforts, vaccines became

and future pandemics, multiple platforms are under available after the peak of the pandemic in many countries,

development and/or investigation. What is clear is that any which is obviously an unwanted scenario.

universal vaccine must provide long-lasting cross-protective

immunity that can induce both B and T cell responses. This Although IAV of the H1N1 subtype circulated in the

review will explore some of the universal influenza vaccine human population before 2009, these viruses were anti-

platforms in the contexts of their ability to induce long-lasting genically different from the pandemic strain and conse-

and cross-protective T cell immunity. quently, the majority of individuals did not have virus

neutralizing (VN) antibodies to the H1N1pdm09 strain.

In contrast however, H1N1 viruses that circulated

Address

between 1918 and 1957 resemble the 2009 pandemic

University of Veterinary Medicine (TiHo), Research Center for Emerging

Infections and Zoonoses (RIZ), Bu¨ nteweg 17, 30559 Hannover, Germany strain antigenically. Consequently, individuals born

before 1957 had been exposed to these viruses and

Corresponding author:

developed antibodies that cross-reacted with the

Rimmelzwaan, Guus F ([email protected])

2009 pandemic strain and were spared from

to a certain extent [5–8].

Current Opinion in Virology 2019, 34:1–9

Current vaccine strategies typically aim at the induction

This review comes from a themed issue on Emerging viruses: inter-

species transmission of VN antibodies that are directed to epitopes located in

close vicinity of the receptor binding site of hemaggluti-

Edited by Adolfo Garcı´a-Sastre and Juergen A Richt

nin (HA), the receptor binding protein of influenza

For a complete overview see the Issue and the Editorial

viruses. However, update of the vaccine strains is

Available online 27th November 2018

required almost annually to match the epidemic strains

https://doi.org/10.1016/j.coviro.2018.11.005 antigenically. Furthermore, the seasonal vaccine will offer

little protection against pandemic or zoonotic influenza

1879-6257/ã 2018 Elsevier B.V. All rights reserved.

viruses with an antigenically distinct HA, often of an

alternative subtype. In contrast, IAV infections induce

a certain level of immunity against IAVs of other subtypes



(heterosubtypic immunity) (reviewed in Ref. [9 ]) and

experiments in both mouse and primate models of infec-

Introduction tion demonstrated that cross-reactive virus-specific CD8+

Influenza viruses are major cause of acute respiratory tract T-cells in particular contribute to this type of protective

infections. Both influenza A and B viruses (IAV or IBV, immunity [10–14]. In humans, it was also shown that the

respectively) cause annual epidemics in humans with sub- presence of virus-specific CD8+ T-cells induced by infec-

stantial morbidity and mortality. In addition, IAV have tion with seasonal IAV strains correlated with protection



caused pandemic outbreaks of variable severity [1]. While against pandemic and zoonotic viruses [15,16 ,17].

seasonal IAV are currently of the H1N1 and H3N2 sub- Therefore, developing vaccines that can induce potent

types, zoonotic infections with IAV of other subtypes, like T cell responses could overcome limitations of antibody-

H5N1 and H7N9, continue to be detected. It has been mediated immunity by providing heterotypic cross-pro-

demonstrated that some avian viruses only require a few tection through recognition of highly conserved internal

adaptive mutations to become transmissible between proteins. In this review, we discuss current and future

mammals and therefore pose a pandemic threat [2,3]. To universal vaccine platforms (Figure 1) with consideration

protect selected high risk groups, like children, the elderly to induction of lasting and cross-reactive T cell responses.

www.sciencedirect.com Current Opinion in Virology 2019, 34:1–9

2 Emergining virus: intraspecies transmission

Figure 1

LAIV

Broadly Protective IIV Immunity

RNA Vaccine Recombinant/VLP (mRNA/SAM-mRNA) DNA Vaccine

Influenza virus gene

Cellular correlates Vectored-Vaccine Antibody correlates of protection of protection

Influenza virus gene

Expression of Expression of internal proteins surface proteins (M1, NP) (HA, NA, M2e)

Current Opinion in Virology

Schematic of current and future ‘universal’ platforms under investigation with respective consideration of the type of lasting

immunity and correlates of protection induced.

Current vaccines Both inactivated influenza virus (IIV) and live attenu-

Design and production ated influenza virus (LAIV) vaccines make up the

The global influenza vaccination program is a major effort majority of IV vaccines being used, both of which are

with more than 400 million doses administered every largely produced in embryonated chicken eggs. Vaccine

year [18]. Because seasonal influenza viruses undergo strains used for vaccine production are typically

antigenic drift, the vaccine composition requires frequent obtained by genetic . The gene segments

updates for the vaccine to maintain efficacy. The recom- encoding HA and NA of defined antigenicity are com-

mendation of strains to be used for vaccine production bined with those obtained from an egg adapted strain

relies on a vast global network of surveillance, the and confer a high yield phenotype or for LAIV, with

WHO’s Global Influenza Surveillance and Response gene segments that confer the vaccine strains an atten-

System (GISR). Together with national centers in more uated cold-adapted and temperature-sensitive pheno-

than 100 countries, GISR is tasked with identifying type. The production and characterization of high yield

circulating strains, their global distribution patterns and reassortant strains is time-consuming. In addition, dur-

most importantly, their antigenic similarity to previous ing passaging in embryonated chicken eggs the vaccine

vaccine strains. strains may acquire adaptive changes in the HA, which

Current Opinion in Virology 2019, 34:1–9 www.sciencedirect.com

Development of universal influenza vaccines Elbahesh et al. 3

may affect its antigenicity and consequently its effec- are typically used in children. Reports of reduced vac-

tiveness [19–23]. Egg-independent vaccine production cine efficacy in children between 2 and 17 years old

using mammalian cell-culture is an attractive alterna- prompted the American Centers for Disease Control and

tive [24–27] and the production of recombinant HA in Prevention to recommend that LAIV should not be used

insect cells by a baculovirus expression system has also in this age group during the 2016/2017 and 2017/2018

received FDA approval [24–28]. Regardless of the seasons. However, the CDC did recommend the Flu-

vaccine production system, the major antigenic target Mist LIAV for the 2018/2019 season after manufacturers

of these vaccines is the viral HA; specifically the glob- showed increased efficacy by replacing the H1N1 vac-

ular head of the HA which contains at least 5 antigenic cine component with a different strain (A/Slovenia/2903/

sites that surround the receptor binding site [29]. HA- 2015) [49–51].

specific antibodies directed to these antigenic sites can

neutralize the virus by preventing binding of the virus Considering the above, there is a clear need for alterna-

to its receptor, provided that they match the virus tive influenza vaccines that can induce broadly protec-

antigenically. The acquisition of amino acid changes tive immunity. During a pandemic outbreak, the prior

at these positions can result in evasion from recognition use of these vaccines would reduce morbidity and mor-

by VN antibodies. tality in the population and would buy some time until

tailor made vaccines against the emerging pandemic

Challenges and limitations strains become available. The development of such

The emergence of viruses with novel HA and/or NA ‘universal’ influenza vaccines has been high on the

genes derived from animal influenza viruses is of great research agenda for some years now and some advance-

concern, because these reassortant viruses are well ments have been made [52].

adapted to replicate in humans and VN antibodies to

their membrane glycoproteins are virtually absent in the

human population. Consequently these viruses have the Universal vaccines

potential to cause a pandemic outbreak, which has indeed Design and limitations

occurred four times in the last century, in 1918 (H1N1), The largest efforts in designing a ‘universal’ influenza

1957 (H2N2), 1968 (H3N2) and 2009 (H1N1). In addi- vaccine have largely concentrated on HA, specifically the

tion, zoonotic human infections by avian IAV of various stalk-domain, which exhibits great conservation across

  

subtypes, including H9N2, H5N1, H7N7 and H7N9 IAV subtypes [53 ,54,55 ,56 ]. The stalk-antibodies

continue to occur [30–33]. These viruses also pose a have the ability to neutralize virus within and across

pandemic threat, because they may adapt to replicate subtypes [57,58]. In addition, stalk-antibodies can pre-

in humans by acquiring necessary mutations [2,3,34–37] vent endosomal fusion and cytosolic release of viral

or by genetic reassortment with human IAVs. genome, as well as preventing particle egress [57,59].

Recent studies also suggest that their function may

Continuous antigenic drift of seasonal influenza viruses depend on Fc Receptor (FcR), as interactions between

due to accumulation of amino acid replacements near the the stalk-antibody Fc region and the FcR were essential

receptor binding site within the HA globular head domain for protection of mice from lethal challenge [60]; in that

that is recognized by VN antibodies complicates the study however, this was dose-dependent where higher



production of efficacious vaccines [38,39 ,40]. Alterna- antibody doses did not require FcR for their effect. While

tively, posttranslational changes (i.e. glycosylation) can natural infection induces anti-stalk antibodies, they

affect recognition by VN antibodies [41,42]. Furthermore, remain at significantly lower concentrations than those

in 2009 it proved to be difficult to produce sufficient targeting the immunodominant head domain (reviewed

pandemic vaccine doses in a timely fashion and in many in Refs. [61] and [62]). To tackle this problem, several

countries the vaccination campaign started after the peak headless and/or stalk chimeric HAs have been designed

of the pandemic. and utilized for the preferential induction of stalk-specific 

antibodies [63–65,66 ,67].

Until recently, inactivated trivalent influenza vaccines

were used predominantly to target co-circulating IAV Compared to the influenza virus HA, the NA is more

H1N1 and H3N2 strains and a single IBV strain. How- conserved but less immunogenic [68]. However, a mono-

ever, difficulty in predicting whether IBV strains of the clonal antibody targeting an epitope conserved among all

B/Victoria/2/87 or B/Yamagata/16/88-lineage will be influenza A and B viruses provided heterosubtypic pro-

dominant in the next influenza season, has resulted in tection in mouse challenge experiments [69,70]. More-

production of a quadrivalent vaccine, which contain over, both human and animal studies have demonstrated

antigens of both IBV lineages which provided additional that NA-specific immunity blunts viral shedding and



protection compared to the trivalent vaccine [43 ,44]. symptom severity [71,72]. Although these studies suggest



The LAIV, that are available [45,46,47 ,48] have been that the NA of the N1 subtype can induce a potent intra-

shown to induce more broadly protective immunity and subtypic immunity, several other studies suggest that the

www.sciencedirect.com Current Opinion in Virology 2019, 34:1–9

4 Emergining virus: intraspecies transmission

response to N2 NAs is much more robust in humans ([73] on single VLPs and recent studies have explored



and reviewed in [74 ]). expression of chimeric proteins in chimeric VLPs

(reviewed in Ref. [92]). Moreover, the use of VLPs

Virus specific CD4+ and in particular CD8+ T cell containing H1, N1, M1 and NA afford cross-protection

responses are largely directed to epitopes located in against lethal challenge of mice with IAV of the H3N2

the internal viral proteins (M1 protein and nucleoprotein subtype [93]. The M2e protein has been coupled to

(NP) that are relatively conserved across subtypes of various fusion proteins or as tandem repeats and/or

influenza A viruses and are therefore highly cross-reac- adjuvants to elicit cross-protective humoral and cellular

tive [75–81]. CD4+ T cells have a variety of effector immunity [94–98].

functions and are important for establishing high affinity

class-switched memory B cells and memory CD8+ T Replication deficient or attenuated viral vectors have

cells. The main function of CD8+ cytotoxic T lympho- been investigated as candidate influenza vaccines includ-

cytes (CTL) is the recognition and elimination of virus- ing recombinant adenovirus, parainfluenza virus, modi-

infected cells, thus restricting viral replication and shed- fied vaccinia virus Ankara (MVA) and alphaviruses [99–



ding and contributing to controlling the infection. The 106,107 ,108–112]. For example, recombinant MVA

presence of pre-existing cross-reactive CTL induced by could be used for the expression of the influenza virus

infection with seasonal influenza A viruses correlated NP and induction of protective T cell responses and

with protection against infection with the pandemic heterosubtypic immunity [106,113,114]. With recombi-

H1N1 virus and zoonotic infection with avian virus of nant MVA that expressed influenza viral HA derived from



the H7N9 subtype [15,16 ,17,77]. The HLA alleles various influenza viruses, VN antibody responses were

make up of any given individual dictates which T cell induces in mice, ferrets, non-human primates and

epitopes are displayed and recognized. Differences in T humans [106,114,115].

cell repertoires due to HLA variability in and across

populations can influence the protective potential of the Vaccine production platforms based on nucleic acid pre-

virus specific CTL response and consequently suscepti- parations (DNA and RNA) have several potential advan-



bility to disease [9 ]. It is of interest that like epitopes tages over current technologies including being flexible,

recognized by antibodies, CTL epitopes are also under safe, cost effective, easy to produce and stable. In contrast

selective pressure and mutations in these epitopes at to the use of viral vectors, nucleic acid-based vaccines do

anchor residues and TCR contact residues have been not induce vector-specific immunity nor is their perfor-

identified. Additionally, amino acid residues outside mance influenced by pre-existing immunity to the vector,

epitopes have been found to affect T cell recognition which sometimes is a concern (e.g. adenoviruses).



and activation [82 ,83–90]. The latter observation should

be taken into consideration when developing vaccines DNA vaccines typically induce potent immune responses

that aim at the induction of virus specific CD8+ T cell in experimental animals, but seem less immunogenic in

responses. humans [116,117]. Boosting with a seasonal vaccine fol-

lowing vaccination with a H1 HA-recombinant DNA

Alternative vaccine production platforms vaccine resulted in the induction of stalk-specific neu-

Apart from identifying (conserved) viral targets for the tralizing antibodies [28]. Intramuscular inoculation with a

development of universal influenza vaccines, there is Vaxfectin-adjuvanted monovalent plasmid DNA vaccine

considerable interest in novel vaccine production tech- encoding H5 HA- or trivalent HA/NP/M DNA vaccines

nologies that would allow rapid production of vaccines tested in a Phase I study resulted in immune responses

able to induce protective antibodies and T cell responses. measured by HI titers and H5-specific T-cells to the

Several technologies to produce influenza vaccines have vaccine in over 60% of recipients; T cell and/or B cell

emerged in recent years that can induce either humoral or responses to at least one antigen were observed in 70%

cellular immunity or both (reviewed in Ref. [91]). of participants [118].

Virus-like particles (VLPs) are composed of viral struc- Candidate RNA vaccines developed against influenza are

tural proteins only to form non-replicative viral particles either non-replicating messenger RNA (mRNA) or self-

lacking genomes. VLPs can self-assemble in cell cul- amplifying (SAM) mRNA vaccines (reviewed in Ref.



ture systems and mimic conformational and antigenic [119 ]). Non-replicating mRNA vaccines encoding HA,

structures of native influenza viruses. Importantly, NP or NA induced protective immunity after intradermal

VLPs can activate innate immunity directly in epithe- immunization of ferrets and intravenous injection of

lial cells or by stimulation of professional antigen pre- mice; the latter study showed evidence of CD4+ T cell

senting cells (dendritic cells and macrophages), which activation after a single dose [120,121]. Interestingly,

facilitates efficient induction of virus specific B and T modified nucleoside mRNA-lipid nanoparticle vaccines

cell responses. Influenza VLP vaccines can contain HA, encoding the HA gene of H7N9 or H10N8 IAVs showed

NA, M1 or M2e proteins individually or in combination promising results in preclinical and phase I clinical trials

Current Opinion in Virology 2019, 34:1–9 www.sciencedirect.com

Development of universal influenza vaccines Elbahesh et al. 5

and induced stronger antibody responses than observed in Thus, recent developments in influenza vaccine design

response to inactivated H5 and H7 HAs split vaccines and production technology may help mitigate the impact



[122 ]. SAM-mRNA vaccines improve on non-replicating future outbreaks of influenza may have.

mRNA vaccines by enabling extended antigen expres-

sion at high levels using small immunization doses. This Conflict of interest statement

improvement is due to intracellular replication of the Nothing declared.

vaccine mRNA. Most SAM vaccines are based on antigen

expression by an alphavirus replicon lacking structural Acknowledgements

This work was funded by the Alexander von Humboldt Foundation in the

genes [123,124]. Several studies have demonstrated that

framework of the Alexander von Humboldt Professorship endowed by the

immunization with SAM-mRNA vaccines resulted in

German Federal Ministry of Education and Research.

strong HI antibody responses and afforded protection

against homologous and heterologous challenge infec-

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