Rational design of polymer‐based particulate vaccine adjuvants

Vaccination is considered one of the major milestones in modern medicine, facilitating the control and eradication of life‐threatening infectious diseases. Vaccine adjuvants are a key component of many vaccines, serving to steer antigen‐specific immune responses and increase their magnitude. Despite major advances in the field of adjuvant research over recent decades, our understanding of their mechanism of action remains incomplete. This hinders our capacity to further improve these adjuvant technologies, so addressing how adjuvants induce and control the induction of innate and adaptive immunity is a priority. Investigating how adjuvant physicochemical properties, such as size and charge, exert immunomodulatory effects can provide valuable insights and serve as the foundation for the rational design of vaccine adjuvants. Most clinically applied adjuvants are particulate in nature and polymeric particulate adjuvants present advantages due to stability, biocompatibility profiles, and flexibility in terms of formulation. These properties can impact on antigen release kinetics and biodistribution, cellular uptake and targeting, and drainage to the lymphatics, consequently dictating the induction of innate, cellular, and humoral adaptive immunity. A current focus is to apply rational design principles to the development of adjuvants capable of eliciting robust cellular immune responses including CD8+ cytotoxic T‐cell and Th1‐biased CD4+ T‐cell responses, which are required for vaccines against intracellular pathogens and cancer. This review highlights recent advances in our understanding of how particulate adjuvants, especially polymer‐based particulates, modulate immune responses and how this can be used as a guide for improved adjuvant design.


Introduction
Whilst live attenuated and inactivated vaccines, developed based on the principles of Pasteur, continue to be safely and effectively used (e.g.most of the current flu vaccines are based on inactivated viruses [1]), vaccine development over the recent decades has shifted from whole to subunit antigens.These vaccines, incor-Vaccine adjuvants are key components of many modern vaccines that aim to initiate, propagate, and steer vaccine-induced immune responses.Successful vaccine adjuvants can modulate the magnitude and type of adaptive immunity, facilitate dose sparing, and improve vaccine responses in challenging cohorts including the elderly and neonates [3].Following decades of research, today there are 10 vaccine adjuvants incorporated in vaccines approved for use in humans -aluminum salts, MF59, adjuvant system (AS)01, AS03, AS04, matrix M, lipid nanoparticles, CpG 1018, granulocyte-macrophage colony-stimulating factor, and Imiquimod [4][5][6].This is a significant advance and adjuvant research is now a dynamic research field.However, with the exception of the Toll-like receptors (TLR) agonist adjuvants, monophosphoryl lipid A (MPLA), CpG 1018, and Imiquimod, and the cytokine/growth factor adjuvant granulocyte-macrophage colony-stimulating factor, we still have an incomplete understanding of the mechanism(s) by which these adjuvants promote protective immunity [7].This arises in part from the empirical manner by which these adjuvants were discovered and developed.Ultimately, a shift toward a more rational vaccine adjuvant design, based on defined physicochemical properties and characterized mechanisms of action may facilitate the development of next-generation tunable vaccine adjuvants, eliciting specific types of immune responses.This would allow for tailoring of adjuvants for different vaccination approaches, informed through established pathogen/disease-specific correlates of protection [8].In this vein, polymeric particulates are an attractive class of vaccine adjuvant in terms of stability and biocompatibility and are extensively customizable which facilitates rational design to elicit distinct immune responses [9].This review addresses the potential of these systems, highlights their limited progress to date, and proposes focus areas for future research that should contribute to more effective adjuvant development.

Polymeric particulate adjuvants
The first polymers used in the design of polymeric particulate adjuvants were nonbiodegradable, such as polystyrene or poly(methyl methacrylate), due to cost-effectiveness, stability, and uniformity in size.Concerns over toxicity and accumulation in cells after repeated administration led to a shift toward biodegradable polymers [10].These included natural polymers, such as chitosan, albumin, gelatine, and sodium alginate; or synthetic polymers, such as polylactide (PLA), poly(lactide co-glycolic acid) (PLGA), and polyethylene glycol (PEG).Nanoparticles provide favorable drainage properties, high surface area for the presentation of antigens adhered on the particle surface, and enhanced cell-particle interactions [2,3].In addition, the presentation of vaccine antigens on the nanoparticle surface creates a pathogenmimetic structure that can enhance immune responses, particularly B-cell responses [11].For example, ferritin-based vaccines have recently emerged as vaccine candidates for pathogens such as Influenza, Epstein-Barr, and SARS-CoV-2 viruses due to the ability to self-assemble into a virus-like particle with comparable size and structure to these pathogens, with the added advantage of surface antigen display [12].Although it is considered unlikely that synthetic polymeric nanoparticles directly engage specific pathogen or damage-sensing receptors, these adjuvants may promote immune responses via the induction of cell stress or immunogenic cell death [13].The capacity of particles to promote (or suppress) immune responses is associated with specific tunable physicochemical properties of the adjuvant, including diameter, charge, and surface properties.These properties can additionally modulate processes such as antigen-release kinetics and biodistribution [14] while enhancing cellular uptake, targeting, and drainage to the lymphatics.Polymeric nanoparticles have commonly been employed as delivery vehicles to improve tissue-specific targeting of therapeutics [15], including anticancer drugs, antivirals, gene therapy, and ophthalmic treatments [9].

Why have no polymeric particle vaccine adjuvants been approved?
Polymeric nanoparticles currently offer untapped potential as immunostimulatory adjuvants.Indeed, there are ongoing clinical trials using these nanomaterials, particularly in the context of anti-cancer therapy such as (1) cationic ethylcellulose polymeric nanoparticles loaded with Cetuximab and decorated with somatostatin analog (octreotide) to selectively target colorectal cells as oral anticancer therapy in phase 1; (2) a phase 1 trial with a docetaxel-containing polymeric nanoparticle to evaluate the maximum tolerated dose, safety, and pharmacokinetics in patients with advanced solid malignancies; (3) a phase 2 clinical trial for prostate cancer using CRLX101, a nanoparticle drug conjugate composed of 20(S)-camptothecin (a potent selective topoisomerase I inhibitor with anti-HIF-1α properties) conjugated to a linear cyclodextrin-polyethylene glycol-based polymer.
However, there are currently no clinically approved vaccines containing polymeric-based particulate adjuvants.The empirical approach to adjuvant development yields limited insight into adjuvant mechanism(s) of action, leading to a lack of consensus on how particle physicochemical properties impact immune responses [14].A lack of standardized protocols to characterize physicochemical parameters of particles as well as issues such as polydispersity and variation in formulation processes has led to conflicting preclinical results [16,17].There is a growing urgency to identify safe, efficient, and accessible adjuvants for vaccines against existing and emerging pathogens and cancer, in particular adjuvants that promote robust cell-mediated and mucosal immunity.For optimal rational adjuvant design and development, the impact that physicochemical properties (such as surface charge, shape, and size) exert on adjuvant-induced immune responses should be extensively characterized (Fig. 1).

Figure 1.
The basis for rational adjuvant design.A major focus is to understand how adjuvants enhance vaccine efficacy providing a basis for rational design of vaccines.For this, we must begin by describing in detail the optimal physicochemical properties of adjuvants, such as composition, size, charge, and route of administration (A).It is then crucial to establish the impact of these adjuvant properties on specific outputs, such as enhancing the kinetics of antigen delivery to lymph nodes, increasing the efficiency of targeting dendritic cell populations and the capacity to activate and modulate cell-mediated and humoral immunity.At a mechanistic level, understanding how specific adjuvant properties influence innate signaling pathways that promote antigen-specific immunity can provide a strong basis for further adjuvant optimization (B).This can then allow tailoring of adjuvants with specific properties to promote protective immune responses for specific pathogens and diseases across different cohorts (C).

Changing views on adjuvanticity and the limitations of empirical adjuvant discovery
Historically, there was a major focus on how vaccine adjuvants could facilitate controlled and sustained release of antigens with the aim of inducing robust neutralizing antibody responses [18].Aluminum salts were the first adjuvants to be widely used in human vaccines and have remained among the leading vaccine adjuvants for over 80 years owing to established efficacy and safety.As such, they have been widely incorporated into vaccines including those against diphtheria, tetanus, pertussis, rabies, and anthrax [19].
Until relatively recently, the efficacy of aluminum salts as adjuvants was attributed to the "depot effect" at the injection site [20].Alum was hypothesized to facilitate the slow and sustained release of antigen by acting as a platform for antigen adsorption and improving its localization and persistence, as well as inducing infiltration of innate immune cells to the injection site [21,22].Depot formation has also been proposed to contribute to the efficacy of other adjuvants including oil-in-water emulsions which were identified as effective adjuvants in the 1900's.The oil-in-water emulsion MF59 was found to be a safe and biodegradable alternative, superior to alum at enhancing antigen-presenting cells (APC) recruitment and activation, cytokine secretion, and antigen drainage to the lymph nodes [23].These properties led to enhanced antibody titers and promoted a more balanced Thelper (Th)1/Th2 responses [24,25].As a result, oil-in-water emulsions were included in seasonal influenza vaccines with evidence for superior immunogenicity compared with unadjuvanted vaccines including in older age cohorts.However, although capable of inducing robust humoral responses, the precise mechanism of action is not fully established, and it is thought that these traditional adjuvants have limited potency in promoting the type of cell-mediated immunity (CMI) necessary for many viral and cancer vaccines [24].
The empirical approach demonstrated in the discovery of alum and oil-in-water emulsions was effective in generating adjuvants that drive strong and sustained antigen-specific antibody responses, but these systems are not optimal for enhancing CMI.Instead, an approach is required that leverages the mechanisms by which adjuvants direct and drive the specific innate immune responses and dendritic cell (DC) activation profiles needed for induction of robust CMI.
The discovery that live-attenuated vaccines such as those against yellow fever and smallpox could induce potent antigenspecific effector and memory CD8 + T cells in humans through the activation of several TLRs and MyD88 signaling suggested the potential for combination adjuvant systems that engage multiple innate immune receptors.The generation of adjuvant systems combining immunostimulatory components, such as TLR agonists with traditional adjuvants, demonstrated a capacity to promote T-cell responses by enhancing DC maturation, with a favorable Th1 bias, leading to their incorporation into several effective vaccines [5].Specifically, the TLR4 agonist MPLA was included in the liposome-based adjuvant AS01, in combination with the saponin fraction Quillaja saponaria (QS)-21, resulting in enhanced CMI and humoral responses in humans, regardless of the antigen used and the age or challenging immune statuses of the individuals involved [26].Recently, 77nm hydrodynamic polymeric particles (PLA mixed with poly(ethylene glycol)-b-poly(lactic-coglycolic acid)) containing a TLR7 agonist (gardiquimod), were shown to induce broad antibody protection against Influenza and SARS-CoV-2 while enhancing CD8 + T-cell responses [27,28] .
Synthetic biodegradable polymeric particles have been a focus of vaccine adjuvant research for a number of decades, initially due to the success of biodegradable and biocompatible polymers for drug delivery.The tunable size and shape of polymeric particles offer the advantage of microbe-mimetics which can activate innate immune responses and increase antigen internalization by APC populations [2,3].Furthermore, they offer potential to tai-lor other physicochemical properties, such as the antigen localization, rate of degradation and charge, to modulate the kinetics of antigen delivery to draining lymph nodes, the efficiency of targeting DC populations, and the capacity to activate and direct cellmediated and humoral immunity [14].Through greater understanding and conceptualization of how physicochemical properties of polymeric particles affect the ensuing innate and adaptive immune response, it is proposed that they could hold exciting prospects as a next-generation adjuvant.

Novel mechanistic insights into the induction of cellular immune responses by polymeric particulate adjuvants
Conventional type 1 DCs (cDC1s) are specialized at crosspresenting exogenous antigens on MHC class I molecules and consequently activating antigen-specific CD8 + T cells [29].Therefore, targeting the activation of these cells utilizing adjuvants is a promising approach to trigger cellular immune responses by vaccine adjuvants.
Upon injection, adjuvants can travel to different regions of the draining lymph nodes resulting in the activation of different DC populations and subsequently triggering different T-cell responses [30].This targeting may be achieved either by incorporating specific molecules such as antibodies directed to cell surface molecules, for example, cDC1s can be targeted through CLEC9A antibody presentation on the surface of nanoparticles [31] or through rational design of the physicochemical properties directing adjuvants to specific tissues.Conventional type 2 dendritic cells are normally located in close contact with the cortical and medullary sinuses, where they principally activate CD4 + T cells, whereas cDC1s are usually in close contact with the paracortical area (T-cell area) in the center of the lymph nodes, where they activate CD8 + T cells by cross-presentation of exogenous antigens [32].Coating mannan onto polylactic acidpolyethyleneimine nanoparticles with adsorbed CpG and ovalbumin (OVA) has proven to target deep paracortical regions and trigger the activation and maturation of resident cDC1s (CD8α + ) through mannan receptors and synergistic activation of TLR9 and TLR4 signaling, enhancing cross-presentation of exogenous antigens to CD8 + T cells, and reducing tumor progression [33].All these processes can be modulated with attention to the specific physicochemical properties of a polymeric particulate adjuvant.
Targeting of cDC1 populations must then be followed by activation of specific pathways leading to cross-presentation of exogenous antigens on MHC class I molecules, which are key in modulating CD8 + T-cell responses.In this regard, the size of particulate adjuvants is pivotal in modulating antigen interactions with receptors and their internalization and presentation by APCs.Smaller cylindrical and spherical nanoparticles of an optimal radius of 15-30 nm can overcome membrane resistive forces more efficiently than micron-sized particles, achieving higher thermodynamic forces and leading to more efficient internalization [34] through scavenger receptor recognition and caveolae/clathrin-mediated endocytosis, mimicking the entry mechanism proposed for several viruses [35].In fact, this high antigen uptake has been demonstrated to be directly related to the rupture of overloaded phagocytic compartments and the release of their content into the cytosol [36,37] facilitating the processing of the antigens and crosspresentation of exogenous peptides on MHC class I molecules and increasing the activation of antigen-specific CD8 + T cells and type 1 immunity [13,38,39].On the other hand, larger particles (>250 nm in diameter) enter DCs preferentially by micropinocytosis, a clathrin-independent pathway involving the extension of membrane ruffles, leading to the classical presentation of peptides by MHC class II molecules to CD4 + T cells [40,41].Fifis et al. [39] demonstrated a size-dependent IFNy-producing CD4 + and CD8 + T-cell responses alongside IgG production following intradermal vaccination with polystyrene beads ranging from 20 to 2000 nm in size conjugated with OVA, where the 40 nm beads were most efficient.In addition, in a different study, 70-100 nm silica particles correlated with increased rupture of endosomes and antigen cross-presentation compared with 300 and 1000 nm size particles [42].
However, there are conflicting reports within the literature regarding the correlation of particle size and induced cellular and humoral immune responses [43].Some of these contradictory results likely stem from different methods of antigen association with particles (ultimately affecting particle size), different vaccination routes utilized, and different metrics used to measure the induction of CMI (e.g. the use of IgG1/IgG2a ratios to provide an indication of Th1/Th2 responses, instead of addressing CD4 + and CD8 + T-cell responses directly).
Particulate adjuvants can be formulated either with the antigen adsorbed on their surface (protein corona) or encapsulated within.Encapsulation of antigens within the polymer matrix may prove ineffective, particularly in the induction of humoral responses.This is a result of limited B cell and antigen interaction, as well as the risk of antigen degradation within the acidic micro-environment generated during the degradation of some polymeric nanoparticles [44], compromising the generation of antigen-specific antibodies.Second, although strongly influenced by the size of the formulation, T-cell-mediated responses have also been shown to be altered depending on the location of the antigen in the formulation.Polymeric nanoparticles composed of PLGA [45] or PEG-poly(propylene sulfide) [32,46] with encapsulated antigens were shown to be transported from endosomes to lysosomes (Rab7 + or LAMP-1 + ) in DCs, where the antigens are cleaved and presented on MHC class II molecules through the classical pathway, activating CD4 + T cells.On the contrary, by adsorbing antigen to the nanoparticle surface, it is possible to mimic the shape and structure of pathogens, a property shown to be associated with optimal antibody responses and CD8 + Tcell activation [11].This is attributed to the retention of particulate adjuvants with adsorbed antigens in stable early endosomes (Rab5 + and EEA1 + ) upon internalization by DCs, which due to their reducing conditions, promote the escape of their content to the cytosol.This allows the processing and presentation of exoge-nous antigens as if these were self-proteins, ending up loaded on MHC class I molecules and facilitating presentation to CD8 + T cells [32,46].Furthermore, increased B-cell responses have been associated with the high density of repetitive epitopes on the surface of pathogens.This density-dependent antibody response was corroborated by Jegerlehner et al. [47] using virus-like particles with covalently attached epitopes at various densities.
When adsorbing antigens to the surface of nanoparticles, the nanoparticle surface charge can inadvertently become modified [6].Surface charge has been shown to significantly impact antigen trafficking to lymph nodes, as well as antigen internalization, processing, and presentation by APCs and consequently, Tcell responses.Highly charged particles offer the advantage of increased stability because of the electrostatic repulsion between them and enhanced protein adsorption to the surface based on their own charge, improving their transport and delivery [48,49].Cationic biodegradable polymeric nanoparticles were better internalized by DCs than anionic nanoparticles due to the electrostatic attraction to the negative charge of cell membranes.For instance, cationic chitosan nanoparticles showed increased internalization by APCs and more efficient trafficking to lymph nodes compared to neutral and anionic nanoparticles [50].However, negatively charged nanoparticles were shown to be more effective at inducing an antigen depot at the injection site, prolonging delivery and immune responses over time [50].Pulmonary vaccination through primary and secondary orotracheal instillation of OVAconjugated cationic nanoparticles enhanced protective immunity at mucosal tissues in mice by inducing CD4 + T cells and humoral responses in lungs and draining lymph nodes to a greater degree than vaccination with antigen and anionic nanoparticles [51].In addition, positively charged polymeric nanoparticles can present high buffering capacity, leading to the induction of a "protonsponge effect" upon endocytosis by DCs.This is due to the cotransport of protons and chloride ions into the endosome or lysosome to regain the neutral charge of the compartment, causing osmotic swelling of the polymer or the vesicle membrane, leading to physical rupture, antigen escape, and finally increased crosspresentation of exogenous peptides and activation of antigenspecific CD8 + T cells [52].In fact, this was described following subcutaneous administration of 220 nm lipid-polymer hybrid nanoparticles (DSPE-PEG-mannose and DOTAP) with encapsulated TLR7 agonist (Imiquimod) and adsorbed TLR4 agonist (MPLA) and OVA [53].
Thus, to enhance CMI, it is pivotal to target the cDC1 population and enhance the cross-presentation of antigens in these cells.This can be achieved by modulating the physicochemical properties of adjuvants, such as the particle size, antigen localization in the formulation, and charge (Fig. 2).

Immunogenic cell death is a key mediator of polymer particle adjuvanticity
In addition to the previously established mechanisms by which polymeric particles can activate DCs to enhance cellular immunity, the induction of cell stress or death by polymeric particles can also trigger this type of response through the release of damageassociated molecular patterns and activation of danger receptors [13].There have been a wide range of regulated cell death modalities described, the most prominent forms being apoptosis, necroptosis, and pyroptosis, these regulated cell death forms may also fall into the bracket of immunogenic cell death (ICD) if certain criteria are fulfilled, providing antigenicity and adjuvanticity, resulting in the capacity to mount adaptive immune responses to antigens expressed by the dying cell.Therefore, vaccine adjuvants that can be modified to induce ICD may prove an effective method to induce antigen-specific immune responses.One such example of ICD is pyroptosis, involving the activation of caspase-1 and/or caspase-11 (caspase 4/5 in humans) which mediate the activation of the pore-forming effector protein gasdermin D (GSDMD).These GSDMD pores facilitate the secretion of cleaved IL-1 family cytokines (IL-1α, IL-1β, and IL-18), which can function as damage-associated molecular patterns enhancing APC maturation, naïve T-cell priming, and T-cell effector functions [54].Polymeric nanoparticles were recently demonstrated to activate the noncanonical inflammasome (caspase 11 and GSDMD formation) leading to enhanced CD8 + T cell and Th1-biased responses.Furthermore, this response was restricted to particles of 50-60 nm size in diameter [55].The enhancement of this type of immune response is crucial for prophylactic vaccines but also for therapeu-tic vaccines against cancer.Therapeutic vaccination has advantages over chemotherapy and radiotherapy, as it is more specific and reduces undesirable side effects.Therefore, this work provides a rationale not only for the use of polymeric small nanoparticulate adjuvants (50-60 nm in size) but also highlights the potential for enhancing the targeting and activation of this inflammasome pathway to improve the magnitude of cellular immune responses.

Conclusion and perspectives: The future of polymeric particulate adjuvant design and development
As previously mentioned, despite decades of research there are no approved polymeric nanoparticulate adjuvants for use in humans.This may be attributed to the empirical way by which these adjuvants were developed and a lack of consensus in terms of characterizing the basic physicochemical properties that govern their adjuvanticity.Ultimately these adjuvants are highly amenable to customization, and as such, a shift to more rational design of this adjuvant class should prove more fruitful, as is the case with the design of vaccines as a whole [18].The core principle underlying rational adjuvant design relies on deciphering an adjuvant mechanism of action [7], facilitating the goal of developing a comprehensive toolset of polymeric adjuvants with physicochemical properties that can be tailored for specific vaccines according to the required correlates of protection.
For instance, an optimal polymeric particulate adjuvant for the induction of CMI should target cDC1 populations and pathways leading to efficient cross-presentation of exogenous antigens, whereas optimal adjuvants for induction of humoral responses should effectively target follicular DCs (fDC) regions [56].Smaller nanoparticles have been shown to have improved localization and preferentially target these cDC1s (CD8α + DCs) in the lymph nodes [57], whereas positively charged nanoparticles [52,53] and those with the antigen located on their surface enhance cross-presentation of exogenous antigens in these cells [32,46].Furthermore, smaller nanoparticles can activate the noncanonical inflammasome, highlighting the potential for enhancing the magnitude of cellular immune responses induced, which could be further improved perhaps by utilizing co-administration of exogenous or endogenous ligands of pathogen recognition receptors.Similarly, effective targeting of fDCs, inhibition of extracellular protease activity, and promotion of immune complex formation may improve the induction of antibody responses [56].For example, the MBL/complement pathway has been demonstrated to target ferritin-based trimer nanoparticles to fDCs as opposed to soluble trimer formulations [56].These are key considerations for vaccines aiming to elicit potent neutralizing antibody titers.The future development of this adjuvant class will rely on further unveiling of the key cellular and molecular mediators underlying the induction of diverse immune responses and the development of effective targeting techniques through physicochemical modifications.These principles align with an overhaul of the framework for adjuvant development suggested by Pulendran et al. [5] whereby small-scale clinical phase 0/I trials are conducted with systems immunology approaches utilized to analyze the induction of immune responses and vaccine safety.These data are then used to feedback into further preclinical testing and optimization before larger scale trials are conducted.This not only would allow for further optimizations of the parameters highlighted in this review but provide insights into the mechanism of action and crucially, into mouse-human translatability early in adjuvant/vaccine design.
This strategy would additionally allow for the optimization of formulations for delivery via alternative vaccination routes, such as mucosal administration.Intramuscular vaccines against respiratory viruses, such as Influenza are generally ineffective at generating mucosal immunity which is critical in protection against infection [58].Mucosal vaccines on their own or combined with systemic vaccines could be a promising strategy to induce longterm protection at mucosal surfaces by inducing cellular and local immune responses mediated by resident memory T cells.This type of response has been shown to be critical following mucosal viral and bacterial infection [59].The choice of vaccine adjuvant is also crucial for this application [60] and the highly tunable properties of polymeric adjuvants may prove advantageous mucosally, where properties such as charge, shape, and size can influence targeting and uptake at the mucosal interface.For example, chitosan is a cationic polymer with bioadhesive properties [61] that allow it to remain in contact with mucosal surfaces and therefore sustain antigen release and innate immune activation.This was shown by Sawaengsak et al. [58] who demonstrated protection against Influenza challenge in mice that were vaccinated intranasally with hemagglutinin-split influenza antigen encapsulated in chitosan-based nanoparticles.In theory, adjuvants optimized for parenteral use and those optimized for mucosal routes could be combined in systemic prime and mucosal boost push-pull vaccination strategies, with the goal of eliciting effective systemic immunity sustained over time alongside sterilizing immunity at the mucosal sites.Lapuente et al. [62] achieved mucosal and systemic immunity against SARS-CoV-2 variants of concern after systemic prime of mice with plasmid DNA or mRNA vaccine followed by intranasal boost with adenovirus 5 and 19a vectored vaccines.
Another emerging consideration regarding the influence of adjuvant properties on both resident and circulating APCs is the capacity to mediate "innate immune memory" which could offer a compelling route to generating sustained specific immune response.Netea et al. [63] described trained immunity in innate immune cells such as macrophages, DCs, monocytes, and neutrophils which retain a form of memory after exposure to a pathogen, promoting a significantly upregulated response to a secondary unrelated challenge despite having returned to an unactivated state.For instance, epidemiological studies into the non-specific effects of Bacillus Calmette-Guérin [64], smallpox [65], oral polio [66], and measles, mumps, and rubella [67] vaccine suggested enhanced protection against unrelated infections attributed to a trained innate immune phenotype [68].Emerging evidence suggests that nanoparticulate polymeric adjuvants might be capable of mediating innate immune training by inducing epigenetic and/or metabolic modifications, as previously described with other nanoparticulate adjuvants such as aluminum salts [69], gold [70], titanium dioxide [71], carbon nanotubes [72], and silica [73].An understanding of how nanomaterials regulate innate immune training is still in its infancy and therefore warrants increased attention for how they could influence the longterm phenotype of innate immune cells systemically and within resident populations.With this novel insight, the innate immune system could be harnessed as a much more influential accessory to vaccine-mediated immunity and a possible advantage in terms of extending and heightening the local inflammatory microenvironment during adaptive cell priming.
The use of nonbiodegradable nanoparticles such as polystyrene provides useful tools to investigate polymeric adjuvants and optimize physicochemical properties, nevertheless, they ultimately are unlikely to be approved for use clinically, stemming from concerns surrounding potential toxicities and accumulation [74].Therefore, a key area of focus should be the translation of knowledge gained from their use preclinically to biodegradable polymers such as PLA and PLGA, which have a proven track record of biocompatibility and safety.However, the production of these adjuvants must also be amenable to scalability.This constituted a major hurdle until recent advances in highly scalable microfluidic technology, facilitating the reliable production of small nanoparticulate adjuvants (<100 nm in diameter) and the improvement in the consistency between batches of adjuvants produced.Technologies including NanoAssemblr may overcome this challenge, allowing the manufacturing of larger quantities in shorter time scales and enhancing the scalability of vaccine adjuvants [75].In addition, the cost price of vaccines and the storage conditions required for transport are key determinants for real-world practicality as demonstrated by the recent widespread rollout of COVID-19 vaccines.As such a key focus should be the development of effective adjuvants which can be efficiently freeze-dried at source, negating the need for costly cold-chain transport.
To improve the likelihood of clinical success and real-world practicality of this class of adjuvant, rational design principles must also be applied to develop cost-effective adjuvants that possess favorable stability profiles, with production processes amenable to scalability.First, the ease of sterilization must be considered and constitutes an often-overlooked area of adjuvant research.Smaller adjuvants amenable to filter sterilization are therefore attractive candidates; however, it must be considered that endotoxins, such as lipopolysaccharides, cannot be removed through common filer-sterilizing techniques utilizing 0.22 μm filters, and this can be further compounded through direct binding of endotoxins to positively charged components in the adjuvant formulation.This necessitates the use of endotoxin-free reagents during adjuvant manufacture.In addition, the nature of formulation must be carefully considered as traditional sterilization methods (high-pressure steam, sterile filtration, or γ-irradiation) are not compatible with formulations involving encapsulated antigens, leading to an increase in manufacturing costs, as expensive aseptic conditions are required [76].Therefore, the physicochemical properties of an adjuvant and precise formulation can dictate the method and cost of suitable sterilization techniques, with knock-on effects on the feasibility of scalability.

Figure 2 .
Figure 2. Tailoring polymer-based particulate adjuvants to enhance CMI.The modulation of size, antigen adsorption to polymeric-based particulate adjuvants, and charge can direct the trafficking of antigens to draining lymph nodes, targeting of the cDC1 population, rupture of phagocytic compartments, and the cross-presentation of exogenous antigens, leading to an enhanced CMI crucial for vaccines against existing and emerging viruses and cancer.