Inactivated recombinant influenza vaccine: the promising direction for the next generation of influenza vaccine

ABSTRACT

Introduction

Vaccination is the most effective method to control the prevalence of seasonal influenza and the most widely used influenza vaccine is the inactivated influenza vaccine (IIV). Each season, the influenza vaccine must be updated to be most effective against current circulating variants. Therefore, developing a universal influenza vaccine (UIV) that can elicit both broad and durable protection is of the utmost importance.

Area covered

This review summarizes and compares the available influenza vaccines in the market and inactivation methods used for manufacturing IIVs. Then, we discuss the latest progress of the UIV development in the IIV format and the challenges to address for moving these vaccine candidates to clinical trials and commercialization. The literature search was based on the Centers for Disease Control and Prevention (CDC) and the PubMed databases.

Expert opinion

The unmet need for UIV is the primary aim of developing the next generation of influenza vaccines. The IIV has high antigenicity and a refined manufacturing process compared to most other formats. Developing the UIV in IIV format is a promising direction with advanced biomolecular technologies and next-generation adjuvant. It also inspires the development of universal vaccines for other infectious diseases.

KEYWORDS:

• Beta-propiolactone
• formaldehyde
• inactivated influenza vaccine
• hemagglutinin
• universal influenza vaccine
• reverse transgenic rescued influenza virus
• adjuvant

1. Introduction

Every year, the seasonal influenza viruses are responsible for ~ 3–5 million cases of severe illness and result in 290,000 to 650,000 global deaths [1]. Influenza viruses belong to a family of enveloped single-strand negative-sense segmented RNA viruses. There are four types of influenza virus: A, B, C, and D, with only types A and B causing human infection by person-to-person transmission [2]. Influenza A virus (IAV) can cause very severe pandemic outbreaks. For example, the 1918 pandemic caused by influenza A H1N1 resulted in over 40 million deaths worldwide [3]. However, influenza B virus (IBV) has only caused seasonal epidemics, and symptoms are usually less severe than those caused by IAV [4]. Currently, the circulating strains in human populations are influenza A H1N1 and H3N2, and influenza B B/Victoria/2/1987-like and B/Yamagata/16/1988-like lineages [4].

The U.S. Centers for Disease Control and Prevention (CDC) recommends prophylactic vaccination as the primary method to prevent outbreaks of seasonal influenza illness [5,6]. Based on the information on the CDC website, different types of influenza vaccines are available now, including inactivated influenza vaccine (IIV), live attenuated influenza vaccines (LAIV), and recombinant HA (RIV). To broaden the protection, current flu shots consist of multiple inactivated influenza strains, including two IAVs (one H1N1 and one H3N2 strain) and two IBVs [7]. Those vaccine strains are selected ~9 months prior to each annual influenza season (one selection for the North Hemisphere and one for the South Hemisphere) from isolated strains reported by the Global Influenza Surveillance and Response System (GISRS) [8]. The influenza vaccine must be updated annually due to the high viral mutation rate. The entire process of surveillance and vaccine production is expensive and labor-intensive [9]. However, the virus can evolve, or new variants arise over the production period, and the manufactured vaccines may not be a good match with the viruses circulating in the next influenza season. For instance, the CDC reported that the selected H3N2 vaccine strain was mismatched with the circulating H3N2 influenza viruses during the 2021–2022 season, leading to an increased number of cases due to H3N2 viral infections in the U.S [10].

2. Inactivated influenza vaccine: the most common flu shot

The first influenza virus was isolated in 1933, and the whole inactivated virus (WIV) was one of the first developed influenza vaccines in the early 1940s [11,12]. WIV vaccines were produced by inactivating live influenza viruses produced from embryonic chicken eggs [13]. However, it was reported to induce reactogenicity; therefore, the split inactivated influenza vaccine (SIV) with improved safety replaced the WIV later [14]. SIV is produced based on WIV by adding detergent, primarily ester or Triton-X100, to disrupt the viral membrane and split the WIV [15]. However, SIV vaccines elicit lower immunogenicity, particularly in the elderly, compared to WIV vaccines [16]. Even though there are other commercial influenza vaccine formats in the U.S., such as live attenuated influenza vaccine (LAIV) or recombinant HA vaccine (RIV), currently, SIV vaccines are the most commonly produced influenza vaccine due to its safety and efficacy [17,18]. It has been reported that IIV is suitable for more age groups and has fewer side effects than LAIV and RIV [16]. Formaldehyde (FA) is an effective inactivation agent for manufacturing IIV. However, beta-propiolactone (BPL), an inactivation agent used initially for rabies vaccine, has more advantages than FA [19]. The Afluria Quadrivalent (Seqirus) influenza vaccine is the only vaccine formulation inactivated by BPL amongst current influenza vaccines (2023–2024). With improvements in inactivation and purification techniques, the reactogenicity caused by WIV could be ameliorated, and it could be brought back to the market for better protection. All types of available influenza vaccines in the U.S. are listed on the CDC website, and there is access to detailed information on each product including the manufacturing process on the Food and Drug Administration (FDA) website. Based on it, I compared all available influenza vaccines (2023–2024) and listed them in Table 1.

Table 1. 2023–2024 available influenza vaccines in the U.S. aIIV: adjuvanted IIV; IM: intramuscular; IN: intranasal.

Vaccine type	Dose	Brand name (manufacture)	Administration route	Suitable age group
IIV4 (egg-based)	Standard dose	Afluria Quadrivalent (Seqirus), Fluarix Quadrivalent (GlaxoSmithKline), FluLaval Quadrivalent (GlaxoSmithKline), Fluzone Quadrivalent (Sanofi Pasteur)	IM	≥6 months
	High dose	Fluzone High-Dose Quadrivalent (Sanofi Pasteur)	IM	≥65 years old
IIV4 (cell culture-based)	Standard dose	Flucelvax Quadrivalent (Seqirus)	IM	≥4 years old
aIIV4 (egg-based)	Standard dose	Fluad Quadrivalent (Seqirus)	IM	≥65 years old
RIV4 (recombinant HA)	Standard dose	Flublok Quadrivalent (Sanofi Pasteur)	IM	≥18 years old
LAIV4 (egg-based)	Standard dose	FluMist Quadrivalent (AstraZeneca)	IN	2–49 years old

Herein, we compare different activation and splitting methods and the effectiveness of WIV and SIV.

2.1. Inactivation reagents: formaldehyde or β-propiolactone

Formaldehyde, commonly known as formalin, can cross-link protein, RNA, or DNA via the alkylation of proteins and purine bases, consequently abolishing the infectivity [20,21]. The mechanism of inactivation of formaldehyde is shown in Figure 1. Formalin was first used as an inactivation reagent for producing a vaccine for the Russian Autumnal Encephalitis virus in the 1930s, and it continues to be used in the manufacturing of influenza virus vaccines [11]. The formalin used during inactivation is diluted during manufacturing but can be detected in some vaccines at very low concentrations that do not cause harm to people [7].

Figure 1. Reaction mechanism of formaldehyde with influenza viral RNA and proteins.

Reaction with (a) viral RNA (uracil) and (b) amino acids of proteins.

BPL is another commonly used inactivating chemical in manufacturing inactivated viral vaccines. BPL alkylates or acylates nucleic acid bases, thereby inhibiting the viral genome replication [22]. The interaction between RNA and BPL is shown in Figure 2. Initially, PBL was used to manufacture acrylic acid in the 1950s and to sterilize various biological materials obtained from people, such as blood plasma, tissue grafts, and enzymes [23]. Due to its excellent inactivation ability, BPL has been used to manufacture inactivated vaccines, including the recent flu shots [24]. Even though BPL is labeled a human carcinogen following exposure, fortunately, BPL has a short half-life in aqueous media (~3 hours at 25°C), and it dissipates even faster in higher temperatures [25]. Therefore, it is easy to detoxify BPL after the inactivation is finished.

Figure 2. Reaction mechanism of β-propiolactone with influenza viral RNA.

Nu: nucleophilic molecule.

Inactivation by BPL blocks viral replication while retaining the protein antigenicity better than formalin because, mechanically, it only alkylates nucleic acids, whereas formalin affects both proteins and nucleic acids. However, BPL can cross-link viral proteins and form the cross-links between the viral genome and viral proteins, link intra-and inter-strand DNA, and link viral RNA loops, which affect the HA structure and, therefore, the antigenicity and immunogenicity of the molecules [26]. The effectiveness of formalin and BPL was compared for inactivating different influenza viral subtypes (H1N1, H3N2, H5N1, and H7N9) and the use of these inactivated proteins as vaccine immunogens [27]. There was a range of inactivation using formalin against strains in each viral subtype [27]. These differences may be due to the different formalin sensitivities of each viral protein. In contrast, BPL completely inactivated all the strains from each of the four subtypes. BPL inactivation generated slightly more hydrophobic HA proteins, but this change did not affect the HA immunogenicity [28]. Moreover, in vivo, the BPL-inactivated H5N1 strain provided better protection against the heterosubtypic H1N1 viral infections following viral challenge than the formalin-inactivated vaccines [29]. BPL also showed superiority in inactivating SARS-CoV-2 over formaldehyde by retaining more antigenicity [30]. IIV can stimulate innate immune responses using internal proteins or genomes without the use of adjuvants. The viral RNA genome can activate the Toll/like receptor 7 (TLR7) pathway, which is a single-strand RNA sensor that triggers the innate immune response. In contrast, formalin-inactivated virus does not efficiently activate the TLR7 pathway [27].

Formalin and BPL inactivation of influenza viruses can be affected by various reaction conditions, such as inactivation time, temperature, and inactivation agent concentration [20]. Depending on the viral families, formalin inactivation varies vastly based on concentration (from 0.08 to 0.009% w/v), time of inactivation (from days to months), and temperature (4 or 37°C) [20]. The higher concentrations and temperatures require shorter inactivation time but may cause alterations in the antigenicity of the protein and/or the elicited immune responses with higher residual formalin concentrations in the final vaccine products. Typically, the final concentration of formalin for inactivating influenza viruses is 0.02% (v/v) at 2–7°C for 24 h. BPL usually takes 35–170 min to completely inactivate the influenza viruses at the final concentration between 0.011–0.055 M at room temperature [31]. At lower temperatures (i.e. 4°C), the procedure takes 3–14 days to inactivate influenza viruses completely [32].

Currently, formalin is more widely used to manufacture influenza virus vaccines than BPL, most likely due to government regulations for licensing new manufacturing methods [33]. However, BPL can inactivate multiple influenza viruses more completely and faster than formalin, which ensures the safety and antigenicity of the vaccine. Recently, the FDA approved two BPL-inactivated influenza vaccines for human use, Afluria® quadrivalent and Fluvirin®. Overall, for developing the next generation of influenza vaccines, the BPL-inactivation bioprocess with lower temperatures but longer inactivation time may be an effective and economic strategy.

2.2. WIV or SIV

WIV vaccines have been associated with adverse effects, such as soreness, swelling at the administration site, headache, fever, nausea, muscle aches, and fatigue. SIV replaced the WIV in 1968 when it was first approved in the U.S. market [34] because they produce fewer side effects and have higher safety profiles for all age groups, especially in children and the elderly who have weaker immune systems [35]. Split-inactivation technology was introduced in the 1960s by using diethyl-ether to disrupt the structure of viral particles [36]. However, the toxicity of ether and HA alternation limited its use for commercial vaccines [37]. Alternatively, splitting the virion with Triton X-100 improved HA structure and reduced toxicity following administration [38]. Using BPL inactivation, the Triton X-100 SIV had only slightly higher HA concentration than ether split SIV [39].

The SIV is safe in all age groups in full-dose and half-dose shots [40]. Children under three years old were vaccinated with either a full-dose of quadrivalent SIV or a half-dose and the results showed only limited side effects. However, local and systemic adverse effects, such as rashness and fever at the administration site, are common in young kids [40]. In the long-term clinical trials testing commercial SIV, only a few mild systemic reactions were reported in the elderly [41]. However, with improved safety, SIV elicits reduced immune responses, particularly for individuals with weaker immune systems [35]. The WIV is similar to the infectious viral particles that activate the pattern recognition receptor (PRRs) and initiate innate immune responses that bridge to the adaptive immune response [42]. However, the mechanism of action by SIV is not clear and results in a weaker stimulation of the immune system. In mouse models, the WIV can induce higher HAI titers and virus-neutralizing antibody titers than the virosome (VS) and subunit vaccine (SV) [43]. WIV can stimulate the T help (Th) type 1 (Th1) response by producing type I interferon and other pro-inflammatory cytokines, but the SIV or subunit vaccine cannot induce a comparable response [16,43]. SIV can activate several immune cells and signal pathways, but these responses differ from the common TLR activation by WIV immunization or live viral infection [44]. SIV only elicits a protective immune response in an IgG-dependent manner via the rapid formation of the immune complexes, which provides potent protection only for previously exposed individuals [44]. This may explain why SIV provides poor protection in immunologically naïve children. Moreover, WIV could elicit cross-protection against heterologous influenza strains by stimulating anti-neuraminidase antibodies [45]. WIV still maintains the fusion ability and ‘infects’ the host cells, which later is presented via the major histocompatibility complex (MHC) I pathway and activates the cytotoxic T lymphocytes (CTL). Overall, formalin- and BPL-inactivated WIVs retain HA fusion activity and the elicitation of cross-protective immune responses, resulting in protection against heterologous infection [29,46].

3. New influenza vaccine: inactivated recombinant influenza vaccine

In general, it takes ~6 months to manufacture the seasonal influenza vaccine each season. To improve the yield of influenza vaccines during manufacturing, recombinant influenza viruses are produced using the backbone viral gene segments of the A/Puerto Rico/8/1934 (PR8) due to its fast growth in embryonic eggs [33]. The eggs are co-infected with PR8 and target strains, and then all amplified viruses are screened to select the correct reassorted strains by incubating them in the presence of antibodies. The selected strains are cloned and sequenced for confirmation. Typically, influenza A viruses are reassorted, but influenza B viruses are directly used to produce the IIV. Still, every year, the huge demand for influenza virus vaccines is a burden for the conventional manufacturing process. Moreover, the vaccine itself still has limitations and deficiencies in its poor efficacy for high-risk populations and mismatch between the vaccine strains and circulating strains. Therefore, the new influenza vaccines with better protection and improved manufacturing processes are under research. Some of them have been used in commercial strains, and some are still under development.

In addition to growing influenza viruses in conventional egg-based culture, these viruses can also be amplified in cell-based culture. Cell culture-produced influenza viruses for vaccine production have some advantages over egg-based ones. It can reduce the risks, including possible contamination by avian pathogens or microbes during the supply of eggs and allergic response to eggs [47]. Moreover, the cell-cultured viruses have fewer alternations to the HA glycans [47]. Currently, three mammalian cell lines, Vero (monkey kidney cells), Madin-Darby canine kidney (MDCK) cells, and human-derived PER.C6 cells, have been used successfully to amplify influenza viruses for over 20 years [48]. Recently, the Flucelvax amplified in MDCK was approved by the U.S. FDA and reported to be safe and effective in eliciting protection against influenza virus infection by stimulating sustained and balanced cellular and humoral immune responses [49,50].

To improve the yield of cell-based culture, reverse genetics technology is employed for influenza subtypes that grow slowly. This technology was developed in the 1990s to generate influenza viruses from cDNA [51]. The genes encoding each viral protein are inserted into the plasmid, and then plasmids can transfect host cells. Plasmids can use the cellular machinery to express viral proteins and RNA and then automatically assemble to generate the target influenza viruses [52]. The 8-plasmid system that each plasmid can generate one negative-sense viral RNA is the most commonly used [53]. The diagram of the 8-plasmid system for generating the recombinant influenza viruses is shown in Figure 3 [53]. This technology shortens the amplification time by eliminating the selection process and, moreover, allows for the introduction of precise mutations. However, the current influenza vaccines still poorly prevent infection during flu seasons because of the rapid evolutionary rate of influenza. With the development of this technology, influenza viruses can be generated to express antigens that can elicit broadly reactive protective immunity in the inactivated IIV format.

Figure 3. Eight-plasmid system for generating the reassortant influenza a virus. the A/PR/8/34 (H1N1) serves as the master strain to generate six plasmids representing its six different proteins. The plasmids containing HA or NA of interest were generated. 6 + 2 plasmids co-transfected 293T-MDCK cells and within 2–3 days after transfection, the recombinant influenza a virus was successfully generated. This figure is cited from ‘A DNA transfection system for generation of influenza a virus from eight plasmids.’ by Hoffmann, E., et al., 2000. Proceedings of the National Academy of Sciences. 97(11): p. 6108–6113 [53].

3.1. HA stalk-based strategy

Hemagglutinin (HA) is a primary target of the immune system and the most studied of the viral antigens. It has a globular head domain and a stem region, and antibodies against both sites were identified [54]. The HA stem region has a significantly lower evolutionary rate than the HA globular head domain, which means the stem region naturally elicits broader protective immunity than the globular head domain [55]. Many stem-specific monoclonal antibodies have neutralizing activity for both heterologous and heterosubtypic influenza strains, many of which are currently being tested in clinical trials as a potential therapeutic strategy against influenza virus infection and disease [56]. To elicit the stem-specific antibody, the immunodominant effect of the globular head domain needs to be inhibited or blocked [57]. One approach is the hyperglycosylation of the HA head region to shield immunodominant antigenic domains by introducing multiple N-linked glycosylation sites over immunodominant sites [58]. Animal studies proved that the hyperglycosylated HA could direct the antibody responses toward the HA stalk region and then protect vaccinated animals from the cross-clade viral challenge via the antibody-dependent cellular cytotoxicity (ADCC) [58,59]. The headless HA construct is another strategy that shields the head domain. Starting from 1983, researchers have been trying various methods to produce stable, native conformational stalk regions. Later, the mini HA structure expressed on the virus-like-particle (VLP) was administrated to experimental mice and increased the survival rate from 0 to around 100% against heterologous and heterosubtypic strains [60]. More recently, researchers delivered ferritin nanoparticles expressing headless HA to non-human primates and detected around 2 Log more neutralizing antibodies than the HA head-only vaccine. Moreover, the neutralizing antibodies are specific against H1N1 strains circulating from 1934 to 2015, and H3N2 strains circulating from 1968 to 2016 [61]. This strategy fully focuses on eliciting HA stalk-binding antibodies. However, the instability and low antigenicity of headless HA remain as a problem, therefore, the chimeric HA (cHA) was developed as an alternative method that designs the full-length HA with the same stalk region, but different head domains [62] In the mouse study, the cHA recombinant proteins successfully increased the survival rate from 0 to 100% after heterologous and heterosubtypic challenges [62]. Mosaic HA (mHA) is similar to the cHA strategy, but it only replaces the most antigenic epitopes on the HA-head domain with exotic sequences [63]. Both strategies elicited robust HA stalk-binding antibodies with strong neutralizing activities, but also HA head-binding antibodies even though the HI activities were highly specific [63]. Moreover, sequential vaccinations are required to strengthen the stem-specific antibody response for both cHA and mHA strategies, which is inconvenient and costly for the market.

Most of these strategies used a RIV or VLP format to determine vaccine efficacy [63–66]. Many of these same immunogens have been tested in the SIV-based and WIV-based vaccines. Both SIV and WIV platforms elicited antibodies with broadly-reactive HAI activity, resulting in protection following the challenge [67,68]. The bivalent recombinant SIV expressing cHA was approved for a clinical phase-I trial and the results showed strong stem-binding antibodies with multifunction, such as stimulating the antibody-dependent cellular cytotoxicity & phagocytosis (ADCC and ADCP), and those antibodies maintained 2-fold higher than the placebo group 420 days post the vaccination [69].

3.2. HA head-based strategy

Even though the stem region is more conserved, the antibody binding affinities to the stem are lower than the head-specific antibodies [70]. Consensus strategies using HA head regions with common epitopes among various drifted strains are designed to overcome the globular head domain hypervariability. Firstly, researchers computationally designed the full-length HA molecules containing the common structures from various strains [71]. However, the popular strains dominated the subtypes of IAVs, which made this strategy less broad. Therefore, the centralized HA strategy that picks the virus strains that can best represent the diversity in each branch of the phylogenetic tree was designed to overcome the bias of sampling and provide a broader protection [72,73]. The computationally optimized broadly reactive antigen (COBRA) is similar to the centralized HA strategy but used multiple rounds of layered consensus building to generate the HA structures [74,75].

All those strategies were reported to generate expected broad protection in vivo, and they were mostly formulated in rHA [73–75]. However, owing to the reduced antigenicity of rHA, multiple doses (as many as three vaccinations), and the addition of adjuvant are necessary for the expected effectiveness. To improve the antigenicity, researchers expressed the COBRA HA antigens in PR8 viruses and then split-inactivated it to generate the COBRA-SIV. This vaccine was proved that it effectively elicited cross-reactive antibodies against multiple drifted H1N1 and H3N2 strains and protected ferrets against viral challenge [15]. In a more recent study, researchers designed a swine H3 antigen, rescued as a recombinant influenza A/WSN/1933 (H1N1) virus, and then inactivated it to generate the WIV [76]. Pigs vaccinated with this WIV vaccine had 8-fold more broadly reactive antibodies against various swine and human influenza H3 strains than the commercial swine WIV vaccine.

With those promising pre-clinical and clinical data, the IIV has advantages for the development of next-generation influenza vaccines. Moreover, there is more space for designing the recombinant viruses, for example, by replacing both HA and Neuraminidase (NA) to produce the 6 + 2 recombinant viruses. NA, as the secondary target for universal influenza vaccine design, is more conserved than HA and naturally elicits cross-protective antibodies. The NA-specific antibodies can block the enzymic activities of NA, which inhibits the release of newly generated virions from the infected cells [77].

4. Next-generation adjuvant

To enhance the VE, an adjuvant is usually used in both research and clinic, for example, the quadrivalent inactivated influenza vaccine manufactured by Seqirus was formulated with MF59, an oil-in-water emulsion of squalene oil [78]. The addition of MF59 to the trivalent influenza vaccine was reported to reduce 58.5% of hospitalizations after influenza infection in the elderly [79]. Therefore, besides the antigens, the next-generation adjuvant is also developed for the goal of UIV.

Agonists for the pathogen recognition patterns (PPPs) are a promising direction of new adjuvant development to stimulate innate immunity which later bridges the adaptive immunity [80]. The TLR activators were designed and proved to significantly enhance the humoral response, Th1/2 activation, and protection against homologous, heterologous, and heterosubtypic viral challenges [81–83]. Besides, the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway which is activated during the viral infection is another popular target [84]. The 2′3′-cyclic GMP-AMP (cGAMP), effectively activating the cGAS-STING, showed promising effectiveness in stimulating the antiviral status [85]. More importantly, the cGAMP-adjuvanted vaccines could increase the survival rate 4–5 times higher than the unadjuvanted ones in immunosenescent mice without increasing the antigen dose, which showed huge advantages for the elderly populations [86].

Typically, adjuvants aim to enhance the antibody responses, and this has been well established in the adjuvants summarized above. The adjuvants stimulating T cells, especially CTL, deserve more attention since the CTL is majorly responsible for eliminating intracellular pathogens [87]. Inspiringly, a recent study formulated the cHA protein vaccines with C34, a novel glycolipid adjuvant, and this formulation elicited 2-fold more neutralizing antibodies and CD4 and CD8 T cells specific for multiple strains than with the Alum that primarily induces the humoral immunity [66]. Moreover, the T cell reactivity is naturally more diverse than antibodies by recognizing internal viral proteins [88]. To better promote T-cell immunity, scientists developed the R-DOTAP, a cationic nanoparticle that can deliver protein or peptide antigens into cells, which can stimulate the cross-presentation and activate the CTL [89]. The COBRA rHA adjuvanted with R-DOTAP has been reported to elicit 6 Log2 higher broadly reactive antibody levels than unadjuvanted rHA and increase the survival rate from 30% (unadjuvanted rHA) to 100% [90].

5. Conclusion

Seasonal influenza vaccines have been used for over 80 years to prevent seasonal epidemics and pandemic influenza outbreaks [11]. Regardless of the long history of formaldehyde usage, BPL was recently approved for influenza virus vaccine manufacturing and has improved efficacy and safety compared to formalin. To overcome the high mutation rate of influenza viruses, optimizing the bioprocess of manufacturing and developing a universal influenza virus vaccine may be a promising approach to meet the urgent need for the next generation of influenza vaccines. Various strategies aiming to design the antigen that can elicit broad protection against multiple influenza strains have been under development and generated exciting results. The IIV which has been well-regulated for a long time in the U.S. is the primary format to apply those designed antigens on. Moreover, the addition of next-generation adjuvant could further expand the cross-reactivities and optimize the VE. Conclusively, the adjuvanted BPL-inactivated recombinant influenza virus carrying modified antigen structures will be a promising candidate for the UIV.

6. Expert opinion

Each year, the healthcare and pharmaceutical industries spend millions of dollars designing and manufacturing annual influenza vaccines. However, due to the unreliable vaccine effectiveness and high-risk populations, hospitalizations and even death are caused by influenza virus infection each season. The next generation of influenza vaccines may elicit protective immunity to prevent severe disease, and even transmission, in all human populations against multiple drifted strains for a longer period of time. The use of recombinant virus-based IIV vaccines is a promising candidate to protect people from drifted influenza virus strains in the next ten or twenty years. This will result in considerable savings of money and time. More importantly, it can save many lives by protecting high-risk populations who respond poorly to vaccination, especially with the current progress in developing next-generation adjuvants. The broader protection and suitableness for more age groups will also provide better herd immunity in the community. This will protect the populations who cannot get vaccinated, such as patients with compromised immunity.

However, influenza virus infection may always be challenging because of the antigenic evolution and zoonotic reservoir of influenza viruses. Even though various strategies are designed to overcome the hypervariation of HA/NA, once it is applied to influenza vaccines, it may become a part of the selection pressure that affects the direction of evolution. Eventually, the influenza viruses will evolve into new strains that are distinct from the vaccine strains and escape from the preexisting immunity. This could also be a potential issue because most people will be immunologically naïve to the evolved new strain, which could cause a worldwide burden. Indeed, with current seasonal influenza vaccines, selection pressure also exists, and mutated influenza strain is always a threat to public health. Strict surveillance of the global isolates will still be a powerful tool to monitor the emergence of new mutations and prevent them from becoming widespread. Therefore, the next generation of broadly-reactive, universal influenza virus vaccines will need to be updated within time. Still, it will be less frequent than current annual seasonal influenza vaccines.

Another concern for the recombinant influenza virus is the safety issue during the research. The rescued artificial viruses in labs did provide advantages to design the next-generation influenza vaccines. However, their pathogenesis has not been studied yet. The recombination of different antigens in one virus could change the pathogenesis and/or infectivity of the backbone strain, which may lead to different biosafety requirements for the experiments. Even though they are not supposed to be more pathogenic since no new antigen was introduced into the recombinant virus, with more recombinant viruses rescued, their pathogenesis needs to be evaluated in the future.

The battle between host and influenza viruses has been lasting for centuries with the earliest evidence of influenza infection in 412BC [91]. With current influenza vaccines, most vaccinated people only have mild symptoms. Nevertheless, the spreading of influenza is still prevalent, and severe compliances are still reported. The next-generation influenza vaccines, if applied to most populations, would be able to control the prevalence of influenza and ameliorate the disease severity by inducing long-lasting antibodies and more diverse immune memories.

The technology of computationally designed antigens and the development of new adjuvants will also facilitate the vaccine design for other pathogens, especially those with a rapid mutation rate. With the advanced reverse transgenic virus technology and the optimized bioprocess of inactivation, the inactivated recombinant virus vaccine provides a potential solution for all infectious diseases.

Article highlights

• There are inactivated influenza vaccine (IIV), live attenuated influenza vaccine (LAIV), and recombinant influenza vaccine (RIV) available in the U.S. market, but IIV has the longest history of human use.

• Beta-propiolactone (BPL) inactivates multiple influenza subtypes more completely and retains antigenicity better than formaldehyde (FA).

• Reverse transgenic technology is used to enhance the yield of IIV by reassorting the selected strain with high-speed growth strain.

• Various strategies are developed to elicit cross-protection against multiple drifted strains. Designing the inactivated recombinant influenza virus carrying modified antigens provides a promising direction for developing a UIV with a simple manufacturing process.

Declaration of interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author contributions

HS and TMR conceptualized and designed this work. HS drafted this manuscript. TMR reviewed and revised this manuscript. All authors contributed to, revised, and improved the final manuscript.

Acknowledgments

TM Ross is also supported in part as an Eminent Scholar by the Georgia Research Alliance, GRA-001. Benjamin Chadwick helped proofread this work for language editing.

Additional information

Funding

This manuscript was funded by the National Institute of Allergy and Infectious Diseases, a component of the NIH, Department of Health and Human Services, under the Collaborative Influenza Vaccine Innovation Center (CIVIC) Contract [75N93019C00052].