Bringing influenza vaccines into the 21st century

Abstract

The recent H7N9 influenza outbreak in China highlights the need for influenza vaccine production systems that are robust and can quickly generate substantial quantities of vaccines that target new strains for pandemic and seasonal immunization. Although the influenza vaccine system, a public-private partnership, has been effective in providing vaccines, there are areas for improvement. Technological advances such as mammalian cell culture production and synthetic vaccine seeds provide a means to increase the speed and accuracy of targeting new influenza strains with mass-produced vaccines by dispensing with the need for egg isolation, adaptation, and reassortment of vaccine viruses. New influenza potency assays that no longer require the time-consuming step of generating sheep antisera could further speed vaccine release. Adjuvants that increase the breadth of the elicited immune response and allow dose sparing provide an additional means to increase the number of available vaccine doses. Together these technologies can improve the influenza vaccination system in the near term. In the longer term, disruptive technologies, such as RNA-based flu vaccines and ‘universal’ flu vaccines, offer a promise of a dramatically improved influenza vaccine system.

Keywords: :

• influenza
• vaccine
• cell culture
• synthetic seed
• potency assay
• RNA-based

Introduction

Influenza virus infections are responsible for significant morbidity and mortality in a broad age range, with particularly severe outcomes in the elderly and young children. Seasonal epidemics are responsible for about 250 000 to 500 000 deaths and between three and five million cases worldwide. Concern that a new pandemic could arise like the 1918 pandemic that claimed ~50 million lives highlights the need for both constant surveillance and improved preparedness.¹

Vaccines against influenza are unique because they require nearly annual reformulation due to continuous viral evolution through antigenic drift (changes in hemagglutinin [HA] surface residues), antigenic shift (new viruses resulting from genome segment swaps), and zoonotic transmission (introduction of non-human animal influenza viruses into the human population).² The solution to this important problem has been the implementation of a worldwide influenza vaccine production system that includes both public and private components and carries out four major activities: influenza surveillance, vaccine virus generation, vaccine manufacturing, and strain-specific release assay development (Fig. 1). The system must be sufficiently robust to reliably produce seasonal vaccines and bring on line surge capacity for pandemic vaccine production when pandemics arise.

Figure 1. Broad overview of the (A) current influenza vaccine global system from clinical isolate to final commercial vaccine and (B) anticipated system. Advancements at every step streamline the process, generating a new global system in which information on genetic sequences, antigenicity testing, and release assays rather than materials are the primary items shared, resulting in accelerated and increased vaccine supply. Images courtesy of Debora Cartagena and Brian Judd/James Gathany.

Currently, most licensed influenza vaccines (inactivated whole virus, detergent or solvent split vaccines, live attenuated vaccines, and some subunit vaccines) require a viral vaccine seed to produce vaccine, and the virus or viral genes for this seed typically are provided by a WHO Collaborating Center. Generating an influenza vaccine seed begins when an influenza virus is isolated from an infected individual’s respiratory secretions at a WHO-linked National Influenza Center. The virus is then adapted for growth in eggs, generally resulting in egg-specific adaptations that change the virus binding specificity from the receptors dominant in human respiratory epithelium (α2,6-linked sialosides) to those dominant in the egg allantoic cavity (α2,3-linked sialosides).³ Egg adaptation can generate mutations in the important neutralizing epitopes that surround the sialoside binding site.⁴

The HA generated from this process can be a genetic mismatch to the clinical isolate and is sometimes an antigenic mismatch.⁵ Historically, vaccine antigenic mismatch has been particularly prominent during seasons in which there has been high influenza-like illness leading to particular stresses on the worldwide vaccine production system (Fig. 2). Human immunization with vaccines produced from the egg adapted vaccine seed viruses for both A/H3N2 in the 2010–11 season and influenza B in the 2009–10 and 2010–11 seasons elicited low neutralizing and hemagglutination inhibition (HI) titers to circulating viruses and to the isolates of these viruses on mammalian (Madin-Darby canine kidney [MDCK]) cells, despite high titers to the egg-adapted vaccine strain.⁶ As recently as the 2012–13 season, egg-adaptations in the A/H3N2 vaccine virus may have contributed to lower vaccine effectiveness.⁷A WHO report from after the 2012-13 vaccination campaign began showed that the egg-adapted reference strain appeared to be a mismatch from the circulating strains.⁸ An equivalent strain isolated in MDCK cells was a match to circulating strains, but even vaccine manufacturers that can produce vaccine in mammalian cells were required to produce the season’s influenza vaccine using the mismatched egg-adapted vaccine seed virus.

Figure 2. Percentage of patient visits for influenza-like illness in the US for selected seasons and years of mismatch. Chart on patient visits are from the US. Outpatient Influenza-like Illness Surveillance Network, Centers for Disease Control and Prevention (http://www.cdc.gov/flu/weekly/). Mismatch data are from the World Health Organization vaccine formulation recommendation from the relevant years.^8,35–37

Two newer approaches can address the limitations of egg isolation: MDCK cells as an alternate substrate for virus isolation and the use of synthetic seed viruses.^9,10 Data on virus isolation rates in 2009 demonstrated that for the H3N2 subtype, a much higher proportion of circulating viruses could be isolated in MDCK cells than in chicken eggs.¹¹ The large difference in isolation rates and declining egg isolation rates over time raised the concern that, if trends continued, a suitable virus match to circulating H3N2 strains would not be available. MDCK cells have both α2,3 and α2,6 sialosides on their surfaces, decreasing the degree of adaptation needed for growth in the laboratory and resulting in higher virus isolation rates.^12,13 A collaboration was formed between the World Health Organization (WHO) and International Federation of Pharmaceutical Manufacturers and Associations (IFPMA) to further explore the use of a mammalian cell lines in vaccine virus isolation. For the 2013–14 influenza season, for the first time, the WHO has recommended that the viruses used to produce influenza vaccines may be antigenically “like” a MDCK-isolated H3N2 reference virus that is well matched to circulating influenza strains.⁸ In the past, only vaccine viruses that match egg-adapted reference strains were allowed. Once regulatory hurdles to the use of purely MDCK-cultured seasonal vaccine viruses are overcome, manufacturers who produce vaccine antigens on mammalian cells will be able to produce better matched vaccines, no longer constrained to match viruses with egg-adaptive mutations. While these efforts are ongoing, a recently published approach shows how the generation of vaccine viruses using synthetic DNA also alleviates the issues of egg-adaptation while providing a number of additional advantages.

Synthetic Seed and Cell Culture Technology

Synthetic vaccine viruses are influenza viruses that have been generated using reverse genetics techniques with synthetic genes for the purpose of preparing vaccine seeds. These viruses are rescued (generated from nucleic acids) in a vaccine-approved MDCK cell line, and the process has robustly rescued viruses (>33) from multiple relevant subtypes while maintaining the original viral HA and neuraminidase (NA) sequences.¹⁰ From the time HA and NA sequences are obtained, synthetic vaccine viruses with high growth backbones can be rescued in <5 d, eliminating the additional time required in traditional processes to adapt viruses to growth in eggs and “mate” them with donor strains to improve their growth and antigen productivity for vaccine manufacturing.¹⁴ The process decreases the risk of adventitious agent contamination because the key strain-specific starting material is an electronically transmitted gene sequence, not a sick patient’s respiratory secretions. When this becomes the method for routine preparation of vaccine viruses, the WHO’s global influenza surveillance system will likely retain its primarily role in information gathering and sharing and in making recommendations, but the production of strain-specific starting materials used in vaccine manufacturing is likely to shift to organizations with established track records for timely, well-controlled, and highly compliant production of pharmaceutical products.

The benefits of improved vaccine seed virus production can be increased through the use of newer cell-culture based production technologies. Cell culture production systems, such as those using MDCK cells or Vero cells, allow for faster scale up in case of pandemics and enable greater safety of supply, because exhaustively tested cell banks are far better characterized than the 100s of millions of chicken eggs used for influenza vaccine manufacturing each year. Unlike the eggs laid by large commercial bird flocks, the mammalian cells are propagated in carefully controlled environments, using well-defined media and have been tested for a number of qualities, including absence of adventitious agents, purity, and genetic stability, to ensure homogeneity and safety. Overall, completely egg-free, mammalian cell culture-based vaccine manufacturing can be expected to introduce fewer mutations away from circulating influenza antigens, increase a timely vaccine supply, and meet modern standards of pharmaceutical production.

Alternative Potency Assays

Although the most rapid vaccine response in history was executed during the 2009 H1N1 pandemic, substantial vaccine quantities were available only after the second pandemic wave had peaked.¹⁵ The time required to generate calibrated reagents for the antiquated potency assay (the assay used to measure the amount of vaccine antigen put in each dose), serial radial immunodiffusion (SRID), was rate limiting.¹⁶ Reagent generation for this assay requires the time-consuming steps of immunizing sheep with antigen from the new strain, waiting for the sheep to seroconvert, and then calibrating the strain-specific sera and antigen standards. The timing of SRID reagent availability determines the speed of vaccine release because blending, filling, finishing, and distributing the vaccine only occur after vaccine potency is determined. Public health agencies and vaccine manufacturers worldwide have recognized that replacing the current influenza potency assay, which involves a complicated international system to generate and calibrate reagents, with alternative approaches can benefit public health by accelerating vaccine availability.

Alternative influenza vaccine potency assay approaches can be placed in two main categories: antibody-independent and antibody-dependent. To date, antibody-independent assays, such as reversed phase high pressure liquid chromatography (RP-HPLC),^17,18 isotope dilution mass spectrometry¹⁹ and SDS-PAGE,²⁰ can determine the amount of HA present but denature the HA before measurement. Therefore, they are non-conformational and do not distinguish HA that can elicit protective antibodies (immunologically active HA) from misfolded or denatured HA that is unlikely to elicit potently protective immune responses. Antibody-dependent assays, such as ELISA^21,22 and surface plasmon resonance,²³ can distinguish immunologically active HA if conformationally specific antibodies to certain regions are used. However, when a strain change occurs, new antibodies would often need to be generated and characterized, slowing vaccine release as occurs today.

Thus, although these alternative approaches for measuring potency have promising aspects, none robustly and accurately measure immunologically active HA quickly. An optimal influenza vaccine antigen potency assay must combine conformational selectivity with speed and use only non-strain-specific reagents. This should be possible. For example, if a biochemical pre-step can separate immunologically active HA from damaged or misfolded HA and then an accurate, reproducible, and rapid but denaturing measurement technique measures only the HA in the immunologically active fraction, then speed, conformational selectivity, and accuracy can be combined. For use in testing seasonal vaccine stability during storage, it would be desirable for the separation and measurement procedure to determine accurately and simultaneously the amount of immunologically active HA of each strain present in a trivalent or quadrivalent mixture. This is an area of active exploration. As other manufacturing advances, such as the use of synthetic seeds, speed vaccine production it will become increasingly important to have such an alternative potency assay to speed vaccine release.

Adjuvants

Adjuvants have been an exciting area of exploration for vaccines and have been reviewed extensively elsewhere.²⁴ Only the oil-in-water emulsion adjuvants MF59 and ASO3 are used in contemporary influenza vaccines. Influenza vaccines using these adjuvants elicit increased quantities of neutralizing antibodies, increase the breadth of the immune response for coverage of mismatched and drifted influenza strains, and enable antigen dose sparing.²⁵ A particularly striking example of how adjuvants can improve vaccine efficacy in the pediatric population has been shown in a Phase III clinical trial for Fluad^® (MF59-adjuvanted trivalent inactivated vaccine [TIV], Novartis) compared with un-adjuvanted TIV and a control non-influenza vaccine. Over 4500 vaccine-naïve 6–72 mo-old children were immunized with the respective vaccines and were monitored for PCR-confirmed influenza-like illness over two influenza seasons.²⁶ Fluad^® had an 86% absolute efficacy against all influenza strains, and TIV had a 43% absolute efficacy. These data demonstrate the ability of adjuvants to improve the efficacy of influenza vaccination.

Disruptive Technologies

Two disruptive technologies recently described in the literature could bring about more dramatic changes in the current system: RNA-based influenza vaccines and ‘universal’ influenza vaccines. RNA technologies promise quick, strain-independent production with high yields from a small manufacturing footprint. Recently, Hekele et al. (2013) demonstrated that an H7N9 SAM^® influenza vaccine candidate based on self-amplifying mRNA delivered by synthetic lipid nanoparticles could be produced within 8 d of the posting of the first H7N9 HA sequence on a web-based data sharing platform (GISAID) by the Chinese CDC. This vaccine candidate was immunogenic in mice, raising an HI geometric mean titer (GMT) of 238 three wk after the second immunization when there was a three week interval between the first and second immunization.²⁷ If the interval between immunizations was 8 wk, an HI GMT of 807 was elicited. If this vaccine is similarly immunogenic, safe and well-tolerated in humans, rapidly generated SAM pandemic influenza vaccines could be new tools to slow an influenza pandemic.

Universal influenza vaccine technologies seek to generate robust, durable memory responses that are broadly protective against current and future influenza viruses. Different groups are developing vaccines that target T cell- and/or B cell-mediated immunity.²⁸ T cell-based vaccines rely on the generation of a response to conserved T cell epitopes on both internal and surface exposed proteins. T cell responses against conserved buried epitopes generally have greater breadth than antibody responses against more variable surface-exposed epitopes. However, the protective efficacy of vaccine-elicited T cell responses upon natural challenge is uncertain, and demonstrating their value in human subjects may require expensive efficacy trials because, unlike for antibody-mediated protection, there is no established laboratory test of cell-mediated immunity that is accepted as a correlate of protection for the purpose of vaccine licensure.

In 2009, efforts to generate a B cell-mediated universal influenza vaccine were bolstered by the structural elucidation of a broadly neutralizing antibody, CR6261, bound to the HA stem region.²⁹ The structure provided evidence that, despite HA antigenic variability, a broadly neutralizing epitope exists, and it provided a target epitope for structure-guided antigen design efforts. Initial efforts to generate a stem-only modified HA antigen have proven challenging with limited in vivo success.³⁰

An alternative approach for directing the immune response was supported by data from the 2009 H1N1 pandemic, during which the B cell antibody repertoire of patients exposed to the 2009 H1N1 pandemic virus contained a higher proportion of cells expressing broadly neutralizing antibodies than similar samples from subjects who had not yet been exposed to the pandemic virus.³¹ This observation led to the hypothesis that a vaccination strategy of sequential immunization with immunogens that only share broadly neutralizing epitopes could have the effect of boosting a pre-existing B cell response to these conserved regions.³² Recently, this approach has been tested through the generation of HA immunogens that contain a stem region from a single virus strain but head regions from exotic HA subtypes.³³ Mice immunized in succession with these antigens have shown both an increase in anti-stem antibody titers and expanded breadth of protection in challenge models. On the other hand, an efficacy study in a pig model of influenza infection provides a cautionary example.³⁴ In pigs, immunization with an inactivated influenza virus that shared HA stem epitopes but not HA head epitopes or NA epitopes with a live influenza challenge strain elicited cross-reactive HA stem antibodies but enhanced disease upon challenge.

Conclusions

The current influenza system provides a valuable public health service through the large-scale production of influenza vaccines. Recent technological advances can modernize several aspects of vaccine virus generation and vaccine production and have the potential to accelerate the speed of vaccine production and release. Adjuvants increase the neutralizing immune response, increase vaccine breadth, and allow dose sparing. While disruptive technologies show promise for how the system could evolve in the future, there is a real opportunity to improve the influenza vaccine system to address vaccine needs using technologies that are ready today.

Disclosure of Potential Conflicts of Interest

The authors are Novartis stockholders and employees of Novartis Vaccines and Diagnostics.

10.4161/hv.27600