Influenza virus causes millions of illnesses and up to 650,000 deaths worldwide every year [1
]. Approved seasonal influenza vaccines protect from well-matched circulating strains but are not effective against drifted seasonal and pandemic viruses; thus, there is an urgent need for more broadly protective vaccines. Recent publications have discussed the challenges that need to be resolved by a broadly protective or universal influenza virus vaccine, such as: lack of protection from antigenically drifted or shifted viral strains, suboptimal epitope-specific antibody responses caused by pre-existing host immunity (original antigenic sin), short-lived protective immunity after vaccine administration, potential side effects of live virus vaccines, or inhibitory effects of maternal antibodies on vaccine immunogenicity in infants [2
]. The data available to date suggest that RNA-based influenza vaccines have the potential to induce broadly protective immune responses and may address some of the above-mentioned challenges.
Vaccination with in vitro transcribed (IVT) messenger RNA (mRNA) was not widely considered as a viable approach until the early 2000s; however, several early studies using various types of mRNA vaccines demonstrated promising preclinical results [7
]. Seminal publications from the past years made it clear that mRNA vaccines—targeting cancer and infectious diseases—have significant therapeutic potential and might represent the next generation of prophylactic and therapeutic vaccines (reviewed in [10
Early studies using naked (uncomplexed) unmodified RNA for immunization via the intramuscular route demonstrated some efficacy against various infectious pathogens [11
] but substantial improvements were necessary to make mRNA into a potent vaccine. The major goals were to increase the in vivo half-life of mRNA and achieve high in vivo translatability to produce large amounts of immunogen for extended periods of time after vaccine administration. Apart from intranodally administered naked mRNA vaccines (discussed in [10
]), most potent iterations of directly injectable mRNA vaccines have two major components: (1) a fairly stable, highly translatable, optimized mRNA and (2) a carrier molecule that encapsulates mRNA. (1) mRNA is transcribed by a bacteriophage RNA polymerase from a linear DNA template (linearized plasmid or PCR product) containing a T3, T7 or Sp6 phage promoter [12
]. Introduction of naturally occurring modified nucleosides, optimization of the codon composition (replacing rare codons with frequently used synonymous codons) and purification of the in vitro transcribed mRNA can yield increases of many orders of magnitude in protein translation from mRNA via the silencing of various innate immune sensing pathways [13
]. Further optimization steps, such as the incorporation of 5′ and 3′ UTR elements that increase mRNA stability and translatability, optimization of the length of poly(A) tail and addition of a 5′ cap structure also proved to be critical in achieving therapeutic potency [19
]. (2) The vast majority of directly injectable mRNA vaccines have a component that protects mRNA from extracellular RNases, facilitates cellular uptake, and often serves as an adjuvant to improve immune responses. Lipid nanoparticles (LNPs) are probably the most frequently used mRNA carriers, but several natural and synthetic polymers have also proved to be efficacious mRNA delivery tools (reviewed in [23
Various formats of RNA vaccines against influenza virus have demonstrated potent immunogenicity in preclinical models (Table 1
and discussed in Section 2
). Moreover, RNA-based influenza vaccines offer critical advantages over other approaches (detailed in Table 2
) such as a favorable safety profile (RNA is a non-infectious, non-integrating molecule degraded by normal cellular processes), highly controllable immunogen production, the absence of anti-vector immunity that enables repeated administration, and, importantly, rapid, scalable production without the use of eggs or complex cell culture systems. The latter is particularly important because some viruses do not grow well in eggs or they can develop egg-adaptive mutations that can alter the antigenicity of the viral surface proteins [25
]. Additionally, generation of FDA-approved conventional influenza virus vaccines can take several months, which can be too long to influence the outcome of an influenza virus pandemic [28
]. In contrast, once the genetic sequences of the circulating influenza virus strains are known, RNA vaccines can be easily updated giving an adequate response to viral antigenic drift.
3. Industrial Development of Influenza Virus RNA Vaccines—Global Players in the Field
RNA vaccines have recently received significant attention and attracted massive academic and industrial investment. A limited number of medium-size biotechnology companies have successfully raised capital to develop innovative RNA vaccines (Table 4
and discussed in [10
]). Additionally, multiple Phase I and Phase II clinical trials with various RNA vaccines have been conducted to target cancer and infectious pathogens [10
]. The first directly injectable infectious disease RNA vaccines that entered clinical trials were against rabies, Zika and influenza viruses [34
]. Several other RNA vaccines are in preclinical development against a wide spectrum of pathogens, including HIV-1, RSV, HCMV and Coxiella burnetii
]. Initiation of new clinical trials—including trials targeting influenza virus—is underway.
Currently, there is one ongoing influenza virus RNA vaccine trial reported on clinicaltrials.gov (NCT03076385) conducted by Moderna Therapeutics (Cambridge, MA, USA). Early results from this Phase I trial using nucleoside-modified mRNA-LNPs are discussed in Section 2.2
. CureVac AG (Tübingen, Germany) has plans to initiate its first human seasonal influenza virus RNA vaccine trial, using unmodified mRNA-LNPs, in 2018. Moreover, they announced that an RNActive prophylactic vaccine for seasonal influenza virus will also be evaluated in a Phase I clinical trial in 2018 (www.curevac.com
). Both CureVac and Moderna have invested in internal production capabilities. Expected in 2018, CureVac’s “GMP III” facility will have the capacity to produce 10 million doses of GMP mRNA per year, while the commercial scale “GMP IV” facility is currently under construction and will be able to produce up to 30 million mRNA doses per year (www.curevac.com
). The company envisions a “GMP V” facility that will be able to increase the capacity to 400 million doses per year (presented at the 5th mRNA Health Conference, Berlin, Germany). Other companies have also announced investments in developing RNA-based influenza virus vaccines; however, little is currently known about their research strategies (Table 4
4. Considerations for Developing a Highly Effective Influenza Virus RNA Vaccine
Companies focused on developing RNA-based influenza vaccines aim to create a product that is superior to the currently available split (inactivated) or LAIV vaccines. Requirements for innovative, broadly protective influenza virus vaccines have been extensively discussed in the influenza vaccine scientific community [57
] and the World Health Organization (WHO) has summarized the guidelines in a recent publication, titled Preferred Product Characteristics (PPCs) for Next-Generation Influenza Vaccine [58
]. PPCs describe WHO’s preferences for vaccine parameters, including the indications, target groups, immunization strategies, and clinical data for assessment of safety and efficacy. PPCs provide early guidance for the improvement of current influenza virus vaccines and the development of new vaccines, with five and ten-year time horizons (Table 5
). The Target Product Profile (TPP) for each RNA vaccine should reflect these PPCs so that the corresponding development plan will meaningfully demonstrate the vaccine candidate’s superior value. The PPC advisory group defined two strategic goals and preferred vaccine characteristics based on WHO’s evaluation of the possible impact on public health. The first goal includes incremental vaccine improvements, such as greater protection against vaccine-matched or drifted influenza virus strains. The second goal aims at greater research and development advances towards vaccines providing broader protection against influenza virus disease (protection across groups or universal influenza virus vaccines) and increased duration of protection (at least five years).
Currently used seasonal influenza vaccines show some level of protection against matched viruses but they offer little protection against drifted seasonal or pandemic viruses. A critical requirement for a new class of vaccine is to greatly improve efficacy and breadth of protection. Prophylactic RNA vaccines demonstrated heterologous protection in preclinical models (Table 1
), but translation of this benefit to humans is uncertain and needs to be demonstrated with clinical data. Additionally, individual variations in immune responses to RNA vaccines need to be carefully investigated. The only candidate that has entered clinical development induced protective levels of HAI titers against the homologous virus strain [34
], but evaluation of broad protection will require multi-year clinical-endpoint trials. A potential requirement for licensure of a novel RNA influenza vaccine could be the ability to prevent influenza virus disease over several years regardless of antigenic drift in a large-scale field trial. To demonstrate superiority over the conventional vaccine platforms, RNA vaccine candidates will need to be tested in head to head trials, and may be considered to be superior if they protect for longer durations and/or show protection against mismatched strains.
Large-scale efficacy trials are expensive, and thus, an early indication of the level of protection and cross-protection against drifted strains could be provided by Controlled Human Influenza Virus Infection Models (CHIVIM). The utility of such a challenge system for predicting vaccine effectiveness depends on the nature of the challenge virus, its infectious dose, and route of administration. Currently available challenge viruses (influenza H1N1 [59
] and H3N2 [61
]) were isolated four to eight years ago; therefore, finding highly susceptible adult subjects is difficult because of prior exposure. Moreover, the challenge virus dose is large (e.g., 105–7
PFU or TCID50
) and it is in a liquid suspension or large droplet spray that induce only an upper respiratory tract infection after administration into the nares [59
]. This does not model the typical natural infection, in which an inhaled virus inoculum reaches the upper and lower respiratory tracts. These attributes of the current CHIVIM limit the generalizability of challenge trial outcomes, and restrict its suitability for testing broadly protective influenza countermeasures that are now entering early phase clinical development. Improved CHIVIM are needed to support the development of broadly protective influenza vaccines, including potent RNA vaccines. Ideal CHIVIM would use influenza virus strains to which young adults are susceptible and a suitable but low challenge dose to better mimic natural exposure.
Immune correlates of protection are invaluable in vaccine development as they minimize complexity, size and cost of clinical trials. While HAI titers are used as a correlate of protection against laboratory-confirmed influenza viruses, no correlates are available for severe disease outcome (or even for lower respiratory tract infection). Non-HA based vaccines or LAIV do not have a known correlate and need to rely on large-scale field efficacy trials. The most potent RNA vaccines induce very high HAI titers in small and large animals and HAI titers in the protective range in humans (discussed in Section 2
). Some RNA vaccines have been shown to increase survival in a lethal challenge preclinical model, even in the absence of protective antibodies [34
], which suggests that both the cellular and humoral immune responses contribute to RNA vaccine protection. CHIVIM that induce lower respiratory tract disease could be used to explore the correlation of pre-challenge immune measures with reduced risk of illness. This could be an approach to reduce the risk of large trials, and perhaps a pathway for accelerated approval of improved influenza vaccines.
Durability of protective immunity is also a critical consideration for influenza vaccines. The results of non-human primate studies [34
] raise the possibility that RNA-based influenza vaccines may elicit more durable immune responses in humans compared to split virus or recombinant protein vaccines. Clinical studies have demonstrated that when naïve subjects are immunized with a vaccine that induces potent cellular immune responses such as LAIV, antibody titers could be boosted with an inactivated virus vaccine up to five years after the original LAIV prime [62
]. Since some RNA vaccines have been shown to confer protection before antibodies reached protective titers [34
], it would be tempting to test if prime-boost regimens using RNA as a prime would give similar results in preclinical models and humans.
The ultimate goal of an improved and broadly protective influenza virus vaccine is the prevention of influenza virus disease, especially among infants who are at increased risk for severe disease because their respiratory system is immature and they have little to no anti-influenza virus immune memory. This implies that improved vaccines should be indicated for children soon after birth, or not later than three to four months of age if maternal immunization is used to provide passive protection for the first three to four months of life. The lack of an indication for LAIV below two years of age due to increased risk of hospitalization and wheezing is a major limitation of LAIV [63
]. RNA vaccines could potentially be evaluated in this patient population, particularly if they are shown to be well-tolerated with an acceptable safety profile in children age two and older. Of note, if RNA vaccines proved to be superior to currently licensed vaccines, a benefit-risk balance could be favorable despite some level of adverse events.
A high level of safety is a critical requirement for vaccines, particularly for those that are administered prophylactically to healthy individuals. RNA vaccine production does not require toxic materials or cell cultures that could be contaminated with viruses; thus, it circumvents the major risk factors associated with manufacturing of live virus, inactivated virus, protein subunit or viral vector-based platforms. Additionally, RNA does not have the ability to integrate into the host cell DNA, thus avoiding the risk of insertional mutagenesis. Potential adverse events such as fever can arise from the potent induction of type I interferons and proinflammatory cytokines by some RNA vaccines [64
]. Moreover, the presence of extracellular RNA following vaccine administration could potentially raise safety concerns by contributing to formation of pathological thrombus or oedema [66
]. Although various mRNA vaccine formats have proved to be safe and well tolerated in clinical trials (reviewed in [10
]), continuous evaluation of safety of this new platform is critically important.
Due to the limitations of egg-based influenza vaccine production, new manufacturing technologies that do not use eggs to grow viruses have been actively investigated [68
]. Vaccines that are produced in cell lines have several advantages, such as a lower risk of adverse events after vaccine administration to people with egg allergies, a sterile manufacturing process that eliminates the use of antibiotics, the absence of egg-adapted mutations, and most importantly, a potentially higher effectiveness [68
]. Egg-adapted mutations in the sequence of HA have been associated with low vaccine effectiveness during the 2016–17 influenza season in the United States [26
]. Zost and colleagues demonstrated that the loss of a glycosylation site by a mutation in the HA of the egg-adapted H3N2 vaccine strain resulted in poor neutralization of the circulating H3N2 viruses in vaccinated humans and ferrets [26
]. On the contrary, H3 antigens expressed in the baculovirus-insect cell system did not contain the mutation, and, therefore humans vaccinated with these antigens generated potent neutralizing antibodies against the circulating H3N2 virus [26
]. While recombinant protein production by the baculovirus-insect cell system has several critical advantages over egg-based vaccine production, improper glycosylation of HA antigens made by insect cells can be a potential limitation of this approach as reported in a recent study [69
]. Vaccine production in mammalian cell lines such as Madin-Darby Canine Kidney (MDCK) cells or Vero cells resolves potential glycosylation issues and allows the production of large amounts of influenza vaccines under carefully controlled conditions, but they require complex and expensive infrastructure [68
]. As a comparison, RNA vaccines are produced without the use of eggs or cell culture systems and properly folded and glycosylated mRNA-encoded proteins (vaccine antigens) are made by the host cells after vaccine administration, thus avoiding the risk of the production of incorrect antigens.
Finally, additional characteristics for a potent RNA vaccine would include considerations on WHO programmatic suitability. Optimal presentation, packaging, thermostability, formulation and disposal are some of the parameters that need to be achieved. WHO has published several documents on programmatic suitability [70
] and these requirements should be considered early on during development. In principle, there are no barriers for an RNA influenza vaccine to meet these criteria, and could even be superior to existing vaccines, if, for example, needle-free administration and vaccine stability at ambient temperature could be achieved.
5. Conclusions and Future Directions
The past six to eight years brought a clear breakthrough in the fields of cancer and infectious disease RNA vaccines, demonstrating proof-of-concept in both preclinical and clinical settings [10
]. Influenza virus RNA vaccines comprise the best-studied RNA vaccines to date. As discussed above, multiple vaccine formats have elicited potent influenza-virus specific, protective immune responses in various preclinical models (Table 1
One of the biggest uncertainties of the field is the translatability of the promising animal data to humans. Encouraging early results from the first influenza virus RNA vaccine trial have been published [34
]. Long term safety and immunogenicity data from this and other future trials are required to confidently judge the impact of RNA vaccines on the influenza virus vaccine field; thus the coming years will be critical for this new vaccine approach. The authors are optimistic and believe that one or more influenza virus RNA vaccine platform(s) will enter the clinic; however, it is possible that further significant optimization of the current vaccines will be required to decrease the cost of production and increase potency and safety.
There are multiple ways to improve the current influenza virus RNA vaccines. Here, we consider some of the possibilities. As discussed above, most influenza virus RNA vaccine studies used a single full-length HA as an immunogen. Immunization with antibody-accessible conserved influenza virus proteins such as the stalk domain of HA, various domains of NA, and the ectodomain of matrix protein 2 (M2e) has been shown to correlate well with protection in preclinical [71
] and clinical settings [72
]; thus it would be intriguing to include these antigens encoded as RNAs in multivalent RNA vaccines. Similarly to the conventional tri- and quadrivalent vaccines, RNA-encoded HAs from various antigenically distant influenza virus strains could be included in a single vaccine regimen to increase neutralization breadth (similarly to the recent study by Vogel and colleagues [31
]). In fact, multivalency of RNA vaccines could be easily increased due to the simple manufacturing process and the same supply chain for each coding sequence. The use of optimized RNA-encoded influenza virus immunogens or more efficient immunization schemes (prime-boost) that can elicit favorable immune responses could also lead to more protective vaccines. Finally, addition of various adjuvants (traditional small molecules or RNAs encoding immune modulatory proteins) to RNA vaccines could also increase efficacy.
As discussed in several recent publications [4
], an optimal broadly protective/universal influenza vaccine would confer durable protection from various antigenically distant (for example group 1 and group 2 influenza A and influenza B) viruses without causing severe adverse events after vaccine administration. Some RNA vaccines elicit durable influenza virus-specific immune responses in preclinical models, including non-human primates [34
], and induce protection from heterologous influenza viruses [29
] but protective efficacy across groups has not yet been reported. The production time of broadly protective/universal influenza vaccines should be relatively short (several weeks) which would allow for the protection of the majority of the population from disease caused by a newly emerging seasonal or pandemic virus. The flexibility of RNA vaccine immunogen design and short production time (without the use of eggs and cell culture) are critical advantages over currently approved influenza vaccines. Additionally, storage of all licensed influenza vaccines requires cold chain, while RNActive vaccines have been reported to be active after lyophilization and storage at 5–25 °C for 3 years and at 40 °C for six months [55
] and development of mRNA-LNP vaccines that are stable at ambient temperature is underway (Arbutus Biopharma, personal communications).
Collectively, RNA-based vaccines represent a new vaccine class that can be used to effectively combat influenza virus. As noted, more data from clinical trials, including improved controlled human influenza virus infection models, and large-scale field efficacy trials will be critical to demonstrate the viability of this vaccine technology.