Nanoparticles in influenza subunit vaccine development: Immunogenicity enhancement

Abstract The threat of novel influenza infections has sparked research efforts to develop subunit vaccines that can induce a more broadly protective immunity by targeting selected regions of the virus. In general, subunit vaccines are safer but may be less immunogenic than whole cell inactivated or live attenuated vaccines. Hence, novel adjuvants that boost immunogenicity are increasingly needed as we move toward the era of modern vaccines. In addition, targeting, delivery, and display of the selected antigens on the surface of professional antigen‐presenting cells are also important in vaccine design and development. The use of nanosized particles can be one of the strategies to enhance immunogenicity as they can be efficiently recognized by antigen‐presenting cells. They can act as both immunopotentiators and delivery system for the selected antigens. This review will discuss on the applications, advantages, limitations, and types of nanoparticles (NPs) used in the preparation of influenza subunit vaccine candidates to enhance humoral and cellular immune responses.


| INTRODUC TI ON
Influenza virus infection is a global public health problem, causing a huge morbidity and mortality burden due to annual epidemics and pandemics. Worldwide, annual epidemics cause 3 to 5 million cases of severe illness, and about 290,000 to 650,000 deaths. 1 Vaccination is the most effective method to prevent influenza infection. Current influenza vaccines mainly rely on hemagglutinin (HA) proteins as antigens to induce neutralizing antibodies that can inhibit virus infection and replication in humans. These antibodies are mostly targeting the immunodominant epitopes of the influenza virus that are highly variable between different virus strains. Each year, new variants of influenza virus may emerge due to antigenic drift, which necessitates the reformulation of influenza vaccines on a yearly basis. 2 Previously, mismatches between predicted and actual circulating strain have resulted in reduced vaccine protection and increased clinical cases. 3 A "universal" vaccine that targets the conserved regions of influenza viruses and induces a broadly protective immunity may dramatically improve protection against seasonal and pandemic influenza viruses.
Antibodies that target conserved sites in the HA stalk have been isolated from humans and shown to confer protection in animals challenged with various influenza virus strains and subtypes. 4 However, it is noteworthy that antibodies specifically targeting the conserved HA2 region can also increase disease severity by enhancing viral fusion to target cells, hence should be given sufficient consideration during universal vaccine design and evaluation. 5 Licensed influenza vaccines are currently available as inactivated (whole inactivated virus vaccine, split virus vaccine or subunit vaccine), live attenuated, and recombinant vaccines (Table 1). These vaccines are produced in eggs or cell cultures and mainly induce antibodies against strain-specific HA. 6 Today, research is more focused on the development of subunit vaccines, as they are safer and easier to produce. With recombinant technology, the production of epitopes of interest, such as the conserved stalk domain of HA, can be done.
In addition to vaccine antigens, adjuvants are also sometimes added to vaccines to boost immunogenicity. Adjuvants are particularly important in the development of influenza vaccines for the elderly population who has decreased immune capacity and during pandemics, where a rapid antibody response is required. 7 In addition, adjuvants are also required in the development of novel peptide-based influenza vaccines, which are known to have low immunogenicity. There are currently six types of adjuvants that have been included in licensed influenza vaccines; alum, AS03, AF03, MF59, heat labile enterotoxin, and virosome, which is a nanoparticle (NP).
Recently, the potential use of NPs as adjuvants in vaccines has been gaining interest. The inclusion of NPs in vaccine formulations has been reported to enhance antigen stability, 8-10 promote targeted antigen delivery, 11,12 and assist slow release of antigens 13,14 to eliminate the need for booster shots. 15,16 Different NPs have been evaluated for their ability to deliver antigens and increase immune responses against influenza antigens in vaccines. The current review focuses on the latest scientific advancement in the application of different NPs in influenza subunit vaccine development to enhance immunogenicity (Table 2).

| NANOPARTICLE S IN INFLUENZ A VACCINE DE VELOPMENT
NPs are comparable to pathogens in terms of their size (1-1000 nm), and thus, they can be efficiently recognized by immune cells and can therefore act as carriers to induce desirable immune-activating effects. NPs can facilitate the delivery of loaded antigens to the primary antigenpresenting cells (APCs). 17,18 For efficient protection against influenza, influenza vaccines are required to induce specific antibody responses, such as antibodies belonging primarily to the immunoglobulin G (IgG) subclass that can block the function of HA, either via blocking host receptor binding or preventing fusion. 19,20 To promote vaccine immunogenicity, various types of NPs have been employed in the design of influenza subunit vaccines such as bacterial spores, virus-like particles (VLPs), bacteriophages, polysaccharides, liposomes, virosomes, immune-stimulating complexes (ISCOM), and inorganic NPs, which are reviewed here under "Natural" and "Synthetic" nanoparticle categories.

| Bacterial spore
Spores are quiescent cells that can be produced by certain bacterial species such as the Gram-positive Bacilli and Clostridia. 21 Spore formation is a survival strategy that enables the bacteria to survive in harsh environmental conditions. Typically, mature spores are 800-1200 nm in size and have either a spherical or ellipsoidal shape. 22 Interestingly, a spore can self-assemble into its functional structure and when used as a vaccine carrier, it can protect the antigens on its surface from degradation and stimulate an immune response. 23,24 The spores of B subtilis have high stability, low production cost, facile construction, and a good safety profile which earns them the Generally Recognized as Safe (GRAS) status. 25 Moreover, they can be administered via the oral pathway, where they can protect the antigens from degradation by stomach acid prior to reaching immune cells within the small intestine. 26 In vaccine design, these bacterial spores are usually conjugated to vaccine antigens through recombinant technology. However, for them to work efficiently, the vaccine antigens need to be of certain size and complexity to effectively activate antigen presentation. 27 Apart from that, the possibilities of transferring selectable marker genes, and release of live recombinant bacterial spores, are major concerns. Recently, antigen co-administration and antigen adsorption to non-recombinant spores were reported as safer alternatives. 28,29 Bacillus subtilis spores have been used in the design of an oral influenza vaccine, where the spore coat protein of B subtilis PY79 (CotB) was fused with three copies of conserved matrix protein M2eH-A-S-H was reported to elicit specific antibody production in the absence of any other adjuvant. However, the levels of antibody titers were relatively low, suggesting that the induced immunity was inadequate for protection and some modifications in vaccine preparation may be required to increase immunogenicity. 30 Live and heat-inactivated spores of B subtilis can also be directly used in vaccine production due to their ability in binding to influenza virions. In a previous study, mice that were intranasally immunized with killed spores adsorbed to H5N1 virions (NIBRG-14) were fully protected even after being challenged with a lethal dose of the virus (˃20 times LD 50 ). Particularly interesting was the observation that in the absence of influenza antigens, the killed spores alone were able to confer about 60% partial protection in the animals, suggesting that the spores themselves are immunogenic in nature. 31 This type of protection however was short-lived and has been attributed to the recruitment of natural killer cells into lungs in response to the killed spores.

| Virus-like particles
Virus-like particles (VLPs) are self-assembling and non-replicating particles that are devoid of infectious genetic material. 32 VLPs can be produced from different host cells, which include bacteria, yeast, insect, and animal cell lines. They can be used as both particulate carriers and immunopotentiators in vaccine development due to their immunogenic characteristics such as having similar size to original pathogen, repetitive surface geometry, and ability to induce innate and adaptive immune responses. 33 The main advantage of VLP-based vaccines is that the immune system of the host can recognize VLPs in a similar way to the original virus to promote a robust immune response. 34 They have been primarily designed to promote B-cell activation and induce potent antibody responses following activation of T helper cells. 35 104 There are several licensed human prophylactic VLP-based vaccines such as Cervarix®, Gardasil®, and Gardasil9® against human papillomavirus (HPV) and the third generation Sci-B-Vac™ vaccine against hepatitis B virus (HBV). VLP-based approaches are also explored as a promising approach for the development of a universal influenza vaccine. 37 To design a successful VLP-based vaccine, the most applicable VLP construct has to be selected and antigens need to be incorporated without destabilizing the VLPs. To achieve this, each biological virus-derived particle needs to be studied in detail for their properties and possible side effects before use in human. to be safe and immunogenic at low doses (0.3-1.0 μg). 51 However, at higher doses (3 and 10 μg), the vaccine was associated with severe symptoms due to increased levels of C-reactive protein.

| Bacteriophage VLPs
Phage VLPs are not pathogenic, and there is no pre-existing immunity against them in humans; consequently, they are safer than other VLPs. 52 Barfoot

Liposomes
Liposomes are formed through self-assembly upon dispersion of certain amphiphilic lipids in aqueous buffer. 71 These structures can be modified accordingly to achieve desirable features that suit their application purpose, such as achieving particular size and charge to enable entrapment of antigens to be used in vaccines. Liposomes can provide controlled release of antigen, while their plasticity and versatility enable them to overcome biological barriers, such as mucosa and skin. They can gain access to APCs via IM or SC injection routes and be used as both delivery vehicle and immunopotentiator. 72 Advanced methods to produce liposomal vaccines, including lyophilization, cryoprotection, and sterilization, can enhance chemical stability of the lipids and widen their applicability in vaccine development. 73 Hong and colleagues reported higher virus-specific antibodies with long-lasting protective immunity and 100% survival rate against lethal viral challenges for a cationic liposome-DNA complex (CLDC)adjuvanted influenza vaccine candidate compared to un-adjuvanted formulation. 74

Virosomes
Virosomes are lipid vesicles that incorporate virus-derived protein and are devoid of viral genome and internal proteins. 80 The membrane proteins can either be produced via recombinant technology or purified from the corresponding viruses. During surface protein purification, virus membrane is normally solubilized and reconstructed using mild detergents without causing denaturation. After solubilization, nucleocapsid and other viral components will be removed via ultracentrifugation. 81 Virosomes are biodegradable, non-toxic and do not induce antiphospholipid antibody responses. 82

Immune-stimulating complexes
Immune-stimulating complexes (ISCOMs) are particulate adjuvant systems composed of antigen, cholesterol, phospholipid, and saponin. 88 They are hollow, cage-like particles of around 40 nm in diameter. 89 ISCOMs combine the advantages of a particulate carrier system with the presence of an in-built immunopotentiator (Quil A) and consequently have been found to be more immunogenic than liposomes. 90 They also required substantially less antigen and other adjuvant to induce immunity in the host than vaccination with simple mixtures of free antigen and saponins. 91 The use of ISCOM in vaccine formulations needs standardized procedures to produce highquality finished vaccines with assured batch-to-batch consistency.
Heterogeneous mixture of ISCOM components can be separated and purified by reversed phase HPLC to eliminate potential toxic fractions in the vaccine preparation. 92,93 The use of ISCOM containing influenza viral proteins has been reported to enhance the CD8+ immune responses in mice and humans. 94

| Inorganic NPs
There is now increasing interest in the use of inorganic NPs as adju- AuNP-HA/AuNP-FliC also stimulated antigen-specific IFN-γ secreting CD4+ cell proliferation. 104

| Polymer NPs
Synthetic polymers have unique characteristics such as biocompatibility and versatility due to their chemical structure. 105 They can be modified in terms of size, surface properties, and composition, which results in a controlled release and protection of drugs. PLGA (poly D,L-lactide-co-glycolide) is a FDA-approved, biodegradable synthetic polymer used for drug delivery in humans. 106 110,111 Molecules that can target mucosal APCs can be covalently attached to PLGA NPs for the induction of long-lasting and potent immune responses. 11

| CON CLUS ION
There is great potential in the use of NPs in influenza vaccine development as they can be used to deliver antigens to target cells, as cytokine production and complement activation should also be characterized in detail, as these responses can be protective but pathological when in excess. In addition, it should be explored if different routes of immunization can impact on the generation of longterm immunity induced by these NP-based vaccines. Future studies can also investigate the potential use of several types of NPs in one vaccine formulation to enhance immunogenicity. By enhancing our understanding on these issues, a safer, highly immunogenic and affordable influenza vaccine can be expected in the near future.

ACK N OWLED G EM ENTS
This work was supported by Taylor Award.

AUTH O R CO NTR I B UTI O N S
AK-H involved in writing, reviewing, and editing processes. CLL, PS, and KW involved in supervision, editing, and review process.