Influenza is an acute and highly contagious respiratory disease caused by influenza A and B viruses. Annual influenza epidemics are caused by several different seasonal influenza viruses (i.e., A/H1N1, A/H3N2, B/Yamagata, and B/Victoria). Currently, various vaccines and antiviral drugs are used to prevent and treat influenza. However, current flu vaccines may not be as effective against infection. One reason is that current seasonal influenza vaccines are produced several months before the start of the next influenza season, so the antigenicity of the vaccine strains may not exactly match the antigenicity of the dominant strains circulating the following season. Additionally, older adults often exhibit lower immune responses and vaccine effectiveness against infections.
The immunogenicity of influenza vaccines can be improved by adding adjuvants. The ideal adjuvant can increase the efficacy of the vaccine and have a good safety. Recently, FLUAD, an influenza vaccine containing the oil-in-water adjuvant MF59, was licensed in the United States and other countries for people aged 65 and older. New adjuvanted influenza vaccines may represent a future direction for future responses to influenza pandemics.
FDA Approved Influenza Vaccines
Currently, three approved seasonal influenza vaccines are available for human use: inactivated, live-attenuated, and recombinant hemagglutinin vaccines. These vaccines are made up of three or four different types of influenza viruses and are updated annually by the World Health Organization to reflect the latest circulating strains. Current influenza vaccines are typically trivalent or quadrivalent and contain two influenza A strains (i.e., A/H1N1 and A/H3N2) and one or two influenza B viruses expected to be circulating during the next season.
Inactivated Vaccines
There are three types of inactivated influenza vaccines: whole virus particle vaccine, split virus vaccine and subunit vaccine. Inactivated whole-virion vaccines, developed in the 1940s, consist of viral particles that lack infectivity but retain the shape of the viral particles, including viral genomic RNA and all viral structural proteins. Inactivated whole virus particle vaccines are effective in inducing humoral and cellular immune responses. Whole virus particle inactivated vaccines retain the ability to enter target cells without proliferating, however, due to the lipid composition of the virus particles, there are concerns about pyrogen and adverse effects.
Therefore, this vaccine was replaced by split virus vaccines, which were prepared by treating purified virus with ether or detergent to remove the viral envelope. Subunit vaccines mainly include HA and neuraminidase proteins. Split and subunit vaccines induce immunity in people previously infected with influenza virus; however, they do not induce sufficient immunity in never-infected infants. This is because split vaccines lose most of the viral single-stranded RNA and have reduced immunogenicity due to a lack of signaling to innate immune receptors.
Live Attenuated Vaccines
Live attenuated influenza vaccine (LAIV) is based on cold-adapted, temperature-sensitive and attenuated variants produced by serial passage in embryonic eggs at low temperatures. For example, FluMist® is a live attenuated influenza vaccine for healthy people ages 2 to 49 years. The virus has surface glycoproteins derived from circulating viruses, while its internal genes are derived from a cold-adapted master donor virus. Therefore, after intranasal administration of these vaccines, their replication is restricted to the upper respiratory tract, where temperatures are lower than normal body temperature. Similar to natural infection, live attenuated vaccines induce mucosal IgA responses and cross-reactive T cell responses in the upper respiratory tract. Double-stranded viral RNA produced during viral replication is recognized by the endosomal innate immune receptor TLR3 and retinoic acid-inducible gene (RIG-I), leading to the induction of interferon-mediated antiviral responses and pro-inflammatory cytokine responses.
Recombinant HA Vaccines
Recombinant HA vaccines contain purified HA produced in insect cells using a baculovirus expression system. Flublok® quadrivalent vaccine is a recombinant influenza vaccine approved for use in people 18 years of age and older in the United States and the European Union in 2017 and 2020, respectively. The vaccine has been available in the United States and some countries in the European Union since the 2020-2021 flu season. People who received the Flublok® vaccine were 30% less likely to get the flu than people who received the regular inactivated influenza vaccine, most likely because the recombinant HA vaccine contains three times the amount of HA protein. In the Flublok® production process, seed viruses and chicken embryos are not required, so recombinant vaccines can be produced faster than chicken embryo or cell-based vaccines.
Several Adjuvants at BOC Sciences
| CAS | Name | Adjuvant Classification |
| MF59 | Oil Emulsion Adjuvants | |
| AS03 | Oil Emulsion Adjuvants | |
| 7784-30-7 | aluminum phosphate | Aluminum Adjuvants |
| 21645-51-2 | aluminum hydroxide | Aluminum Adjuvants |
| 66594-14-7 | Quil A | Saponin |
| 141256-04-4 | QS-21 | Saponin |
| 24939-03-5 | Poly I:C | TLR agonists |
Adjuvants for Influenza Vaccines
One of the most effective strategies to increase the efficacy of vaccines against infection is the addition of adjuvants. In the case of influenza vaccines, adjuvants effectively enhance immunogenicity and reduce the amount of viral antigen required for protection and the number of vaccinations required. Furthermore, adjuvants can amplify reactivity to antigenic variants and are effective against antigenic mismatches between vaccine strains and circulating viruses. Many adjuvants have been approved for use in influenza vaccines, including aluminum adjuvant, MF59, AS03, AF03, and virions.
Aluminum Adjuvants
Aluminum adjuvants, including aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate, are the most common adjuvants and have been used in vaccines since the 1930s. After an aluminum-adjuvanted DPT vaccine was successfully shown to elicit protective immunity in humans, an influenza vaccine combined with an aluminum adjuvant was also developed; however, it did not demonstrate an adequate immune response. It was not until the development of MF59 that significant progress was made in influenza vaccine adjuvant research.
MF59
MF59 consists of an oil-in-water emulsion of squalene emulsified with two surfactants (polysorbate 80 and sorbitol trioleate). MF59 induces Th1 and Th2 type immune responses and the release of some specific cytokines/chemokines, such as IFN-γ, CCL2, CCL3, IL-8 and IL-5. MF59 is used in the seasonal influenza vaccine FLUAD to enhance immune responses even in the elderly. FLUAD was first approved in Italy in 1997 and subsequently in the United States and Europe in 2015 for use in people 65 and older. In addition, the FLUAD quadrivalent influenza vaccine has also been approved for this elderly population and will be available in the United States for the 2020-2021 season. The MF59-adjuvanted seasonal influenza vaccine elicited stronger immune responses in older adults and children than unadjuvanted vaccines.
AS03
AS03 is an adjuvant composed of squalene, DL-alpha-tocopherol and polysorbate 80, used as a vaccine against H1N1 pdm09 and H5N1 avian influenza viruses. AS03 triggers a transient NF-κB-dependent innate immune response, leading to the production of cytokines and chemokines, including CCL2, CCL3, IL-6, CSF3, and CXCL1, at the injection site and draining lymph nodes, thereby inducing immune cell migration. AS03 adjuvanted A (H1N1) pdm09 vaccine induces higher antibody responses than non-adjuvanted vaccine in older adults.
AF03
AF03 is an oil-in-water adjuvant used in the influenza A (H1N1) virus split vaccine Humenza, but has never been marketed. Although AF03 has been reported to enhance humoral and cellular immune responses in animal models, its mechanisms remain largely unclear.
Virions
Virions have influenza antigens on their surface, mimicking viruses, and can be considered a virus-like particle (VLP). By mimicking viruses, virions can carry antigens into antigen-presenting cells and transport them in the lymphatic system. Inflexal® V is a trivalent virion vaccine containing HA and NA from vaccine strains recommended by the World Health Organization. After being approved in Switzerland in 1997, it was introduced around the world but is no longer available. It induces strong immune responses in healthy and immunocompromised older adults, adults, and children, similar to natural viral infections.
New Developments in Influenza Vaccine Adjuvants
Saponin
Although aluminum adjuvants have been used in vaccines for a long time, this adjuvant induces humoral immunity rather than cellular immunity. Saponin-based adjuvants are already present in veterinary vaccines and induce humoral and cellular immunity. Quil A and its derivative QS-21 have been evaluated as adjuvants for human influenza vaccines, but vaccines using these adjuvants have no clear advantage in inducing immunity over vaccines that do not.
After the H7N9 avian influenza virus emerged in 2013, researchers from Novavax announced that their H7N9 vaccine clinical trial was successful. They added the saponin-based ISCOMATRIX adjuvant to the H7N9 VLP vaccine. Although H7 viruses are considered to have lower immunogenicity, the addition of saponin adjuvant induced a higher immunogenicity rate than aluminum adjuvant or no adjuvant.
Matrix M is another saponin-based adjuvant that has been evaluated as an adjuvant for virion H5N1 influenza vaccines in Phase 1 clinical trials. The Matrix M-adjuvanted quadrivalent nanoparticle vaccine for seasonal influenza is also being evaluated in a phase 3 randomized controlled trial. Compared with the licensed quadrivalent inactivated influenza vaccine, the vaccine elicited higher humoral and cellular immunity in those over 65 years of age. Matrix M requires less antigen to induce an immune response than a vaccine alone.
TLR agonists
Poly I:C is a synthetic TLR3 agonist with a dsRNA-like structure. Poly I:C12U has been tested for use as an adjuvant in nasal influenza vaccines and has been reported to display high mucosal immunogenicity. In addition, Rintatolimod can induce cross-reactive IgA against other influenza subtypes when used in combination with seasonal influenza vaccines. PIKA is also a stable dsRNA that has been shown to be effective as an adjuvant in vaccines against influenza subtypes H1, H3 and H5 in animal studies via subcutaneous injection and intranasal immunization.
Glucopyranosyl lipid A (GLA) is a TLR4 ligand and is also used as an adjuvant. In a phase 2 study, the HI antibody titers of GLA-adjuvanted H5N1 influenza virus vaccine and aluminum-adjuvanted vaccine were evaluated. The results showed that people immunized with GLA adjuvant had higher HI antibody titers than those immunized with aluminum adjuvant. degree and require less antigen.
Flagellin is the main component of bacterial flagella and a TLR5 ligand, and has been tested as an adjuvant. In a phase 1/2 trial, a recombinant protein fused to Salmonella enterica serovar Typhimurium type 2 flagellin (STF2) and the H1HA globular domain expressed from E. coli induced high HI antibody titers in healthy adults and the elderly.
Over the past decade, much research has focused on the development of a universal influenza vaccine, with an emphasis on developing vaccine strategies targeting conserved regions of influenza virus proteins. Universal influenza adjuvant vaccines that induce cross-protection against heterologous strains are also under investigation. In a recent report, the TLR7 agonist DSP-0546LP was shown to induce Th1-biased immunity in mice, such as the production of IFN-γ and IgG2c antibodies, and enhance cross-protection against other H1N1 and H3N2 subtype viruses. The mechanism mainly comes from ADCC action rather than cross-neutralizing antibodies. ADCC-induced cross-protective immunity is not only induced by TLR7 but also by TLR9. Therefore, TLR agonists as adjuvants are expected to induce high reactogenicity and cross-immune responses.