Biomaterials-Based Opportunities to Engineer the Pulmonary Host Immune Response in COVID-19

SARS-CoV-2 etiology and host cell invasion

Coronaviruses are viral pathogens that belong to the Coronaviridae family which are enveloped, single-stranded, positive-sense RNA viruses with a genome length of ~30 kb.^{7, 8} Structurally, coronaviruses have four main proteins including spike (S) glycoproteins, small envelope (E) glycoproteins, membrane (M) glycoproteins, and nucleocapsid (N) proteins, along with several other proteins.⁹ The spike glycoprotein is a transmembrane, trimeric glycoprotein that facilitates binding and invasion into the host cell.¹⁰ Techniques like electron microscopy have identified SARS-CoV-2 to be a round or oval shaped virus with an approximate diameter of 60–140 nm and a crown-shaped appearance.¹¹ SARS-CoV-2 is closely related to two other coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV), which have the potential to replicate in the lower respiratory tract and result in fatal pneumonia.¹² SARS-CoV-2 shares 79% genome sequence identity with SARS-CoV-1, another human coronavirus, and has approximately 98% genomic similarity to the bat coronavirus, RaTG13.^{13, 14}

SARS-CoV-2 is primarily transmitted via exhaled aerosol particulates containing virus that cause infection following inhalation.¹⁵ Upon infection with SARS-CoV-2, the median incubation period is around 4–5 days^{16, 17} followed by symptom onset within 11.5 days.¹⁷ Invasion of SARS-CoV-2 into the host cell initiates with viral binding to angiotensin-converting enzyme 2 (ACE2) receptor (Figure 1A).^{10, 18} Previous studies with SARS-CoV-1 have identified increased ACE2 receptor expression in pulmonary type II epithelial cells, making them likely targets for invasion.^19–21 ACE2 expression decreases from higher amounts in the upper airways to lower expression in the lower respiratory tract, with high SARS-CoV-2 infectivity in the nose compared to the alveolar region.²² The spike (S) protein on the viral surface is comprised of two functional subunits: S1 and S2. S1 subunits facilitate binding with the host cell receptor and comprises an amino-terminal domain and a receptor-binding domain (RBD).²³ The RBD binds to the ACE2 receptor followed by endocytosis of viral components into the host cell.^{24, 25} Following the binding of SARS-CoV-2 to ACE2 and endocytosis, the spike protein undergoes protease cleavage, exposing the S2 site, which results in membrane fusion and release of viral contents into the cell cytoplasm.²⁶ Studies have identified increased affinity of ACE2 receptor with RBD of SARS-CoV-2 relative to SAR-CoV-1, which might explain the greater relative infectivity of SARS-CoV-2.^{24, 27} The role of the cellular serine protease TMPRSS2 was also identified in promoting host cell entry of the virus in SARS-CoV-2 infections.²⁸ In addition to TMPRSS2, recent literature has implicated other proteases such as TMPRSS4, Mpro, PLpro and RdRp, which might play a role in host cell invasion.^{29, 30}

Host response to SARS-CoV-2

Both innate and adaptive immune responses are engaged to neutralize the SARS-CoV-2 infection; however, an impaired immune response can result in severe lung pathology and infection manifestations in multiple organs,⁴ including coagulation abnormalities and thromboembolic events that are common in COVID-19 deaths.^{31, 32} The severity of initial infection and viral load, combined with the timing and extent of the host immune response, can result in symptoms ranging from asymptomatic or mild to severe pneumonia and acute respiratory distress syndrome (ARDS) with multiple organ failure, as discussed in the following sections. It is critical to increase our understanding of the kinetics, progression, and timing of both healthy and improper host immune involvement prior to developing immune-modifying therapeutics.

Role of biological factors, pre-existing conditions, and lifestyle in COVID-19 host response

The severity of COVID-19 is primarily dependent on the host immune system.⁷⁸ Many factors play a role into the background immune state of the host including genetics, pre-existing conditions, environmental factors, and even circadian rhythms.^79–81 We summarize the current state of understanding for each of these factors as they relate to COVID-19 to identify design criteria for therapeutic intervention.

Age quickly emerged as a major biological risk factor linked to increased susceptibility to COVID-19 infections. Large-sample studies in China, along with other countries, have observed a higher rate of infection in the elderly population (65 or older) along with higher mortality rates.^82–85 One potential reason for the increased mortality might be age-related impairment of the innate and adaptive immune systems, termed immunosenescence.^{86, 87} Type I IFN production in response to viral infection is reduced with age, making elderly patients more susceptible to severe COVID-19 infection.^{48, 88} Furthermore, increased inflammation with age or “inflammaging” may be attributed to a heightened innate inflammatory baseline in older patients, resulting in worse clinical outcomes and increased severity relative to the younger population.^89–92 However, reports of COVID-19 manifestations in children and adolescents have included multisystem inflammatory syndrome and Kawasaki-like symptoms lasting a considerable period after infection, but mechanistic understandings of these phenomena remain unclear.^{93, 94} Sex disparities have also been found in COVID-19, with increased incidence and mortality in males compared to females.^{84, 85, 95–97} Broadly, females and males have been observed to exhibit differences in immunological responses to foreign and self antigens, where differences in both genes and hormones are thought to play a role.⁹⁸ Increased susceptibility of COVID-19 in the male population could be because of the presence of the ACE2 gene on the X-chromosome and higher circulating ACE2 levels in men relative to women.⁹⁹ Males have also been found to demonstrate increased production of pro-inflammatory cytokines such as IL-8 and IL-18 in addition to worse T cell responses in males,¹⁰⁰ indicating the potential need for different treatment options based on patients’ sex.

Along with biological factors, certain pre-existing health conditions increase infection risk and fatality rates. As the lungs are the primary site of infection for COVID-19, respiratory patients with pre-existing health conditions such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, and pulmonary fibrosis are also at an increased risk.¹⁰¹ COPD, a chronic inflammation disease associated with limitation in airflow, was identified to increase COVID-19 infection risk by a factor of over 4 times and correlated with increased disease severity.^{32, 102–104} Pulmonary fibrosis, a chronic fibrotic disorder resulting in scarring of the lungs, shares similar risk factors associated with COVID-19.¹⁰⁵ ARDS, which is observed in close to 40% of COVID-19 cases, is often linked as a precursor to pulmonary fibrosis.¹⁰⁶ Conversely, pre-existing dysregulated wound healing in idiopathic pulmonary fibrosis can increase the risk of contracting COVID-19 infection.¹⁰⁵ Currently, the functional relationship between chronic pulmonary conditions and COVID-19 remains speculative, and further studies would be required to formally investigate pulmonary fibrosis as a COVID-19 risk factor. Other health conditions including hypertension, chronic respiratory disease, diabetes, and cardiovascular diseases have also been identified as potential risk factors.^{32, 106, 107}

Furthermore, recent evidence suggests that approximately 10% of people diagnosed with COVID-19 experience symptoms months after testing negative for the virus.^{108, 109} These people, termed “COVID-19 long haulers,” experience a wide range of symptoms which, while inconsistent between individuals, include coughing, fatigue, body and joint pain, headache, and brain fog among many others. The existence of these long haulers potentially implicates permanent changes in the immune response and lung microenvironment, indicating incomplete recovery from the virus.^{109, 110} One study from Italy found that approximately 87% of recovered COVID-19 patients aged 19–84 reported at least one persistent symptom, particularly fatigue or dyspnea.¹¹¹ Another study from a Paris hospital reported a high daily average number of COVID-19 long hauler patients with an average patient age around 40 years and a 4:1 ratio of women to men. Their study suggested that the long term impacts of COVID-19 may be experienced by people as a function of age, sex, and/or disease severity.^{112, 113} The reasons for the long-term health impacts of COVID-19 are still not clear; however, permanent organ damage from the cytokine storm,¹¹⁴ persistent inflammation,²³ and inadequate/variable memory antibody response¹¹⁵ might contribute to long-term impairment of innate and adaptive immune response in COVID-19 long haulers. A recent study showed higher impairment of natural killer T cells (NKT cells) in individuals recovering from COVID-19 resulting in reduced immune response¹¹⁶. Further, similar phenomena were observed with recovered SARS-CoV-1 patients, who experienced fatigue months or even years after recovery, which was hypothesized to result from persistent low-level inflammation caused by the infection.^117–119 Further studies would be critical in understanding the relationship between COVID-19 and other chronic diseases along with various biological factors, which will help in guiding therapeutic advances toward the COVID-19 disease both in the short and long terms.

Corticosteroids

Corticosteroids have been widely used for inflammatory and autoimmune diseases in addition to treating pneumonia symptoms and other respiratory conditions, such as asthma and COPD, and can be administered through oral, intravenous, or inhalable routes.^126–128 Corticosteroids exert anti-inflammatory effects through glucocorticoid receptor binding and interference with inflammatory gene transcription factors (e.g. NF-κB).¹²⁹ Because of the hyperinflammatory state caused by the SARS-CoV-2 infection and other viruses causing pneumonia and ARDS, corticosteroids have been proposed to mitigate pulmonary and systemic inflammation caused by cytokine storm. However, the delivery of corticosteroids has had mixed results, with studies indicating that the dosage, timing, and type of corticosteroid used may be important to achieving positive outcomes.^130–132 Corticosteroids have previously been evaluated in SARS-CoV-1 and MERS-CoV; treatment showed limited success with some possible harmful effects in SARS-CoV-1 patients,¹³³ while some improvement was observed with methylprednisolone treatment in patients with ARDS.¹³⁴ There is a strong case against the use of certain corticosteroids for COVID-19 complications based on clinical data from MERS-CoV and SARS-CoV-1, with patients receiving corticosteroids becoming more likely to be placed on mechanical ventilation and showing delayed recovery relative to patients that had not received corticosteroid treatment.¹³⁵ Several studies investigating methylprednisolone, including a randomized, double blind, placebo-controlled trial, found no significant prevention of deaths or improvement of clinical outcomes following a short-course treatment,^{131, 136, 137} while others showed modest improvement.¹³⁸ Budesonide has recently been shown to reduce the number of deaths in patients with severe COVID-19 as compared to untreated patients.^{139, 140} A recent study with a relatively greater number of participants has shown efficacy with dexamethasone with a six mg daily oral or intravenous dose reducing the number of deaths and requirement of invasive mechanical ventilation in hospitalized COVID-19 patients.¹⁴¹ While these results indicate the potential for some corticosteroids improving outcomes for COVID-19 patients, more studies are required to elucidate the mechanism of dexamethasone compared to other corticosteroids in preventing clinical deterioration in COVID-19. Because of the widespread availability of corticosteroids and the potential utility by physicians for COVID-19 patients, caution must be exercised to avoid undesirable and potentially harmful side effects. The main concern with systemically administered corticosteroids is the immunosuppressive effects that could hinder the host’s innate antiviral responses. In some studies, 50% of non-surviving COVID-19 patients had secondary infections,³² indicating that broad immunosuppressive strategies may be detrimental to the host’s ability to fight infections. While evaluation of corticosteroids in SARS-CoV-1, MERS-CoV, and, in many cases, SARS-CoV-2 has shown disappointing results partly because of the lack of randomized and properly controlled trials, the recent, promising results with dexamethasone treatment emerging from a thorough design and a large number of participants have sparked cautious optimism and pushed dexamethasone into becoming a new standard of care for treating COVID-19. More studies are required to confirm these preliminary results and investigate critical factors such as dosage, frequency, route of administration, and timing of treatment.

Pro-inflammatory cytokine blockade and pathway inhibition

Given the overabundance of pro-inflammatory signaling molecules, approaches to dampen or regulate the immune response and prevent cytokine storm have been attempted in various clinical trials. Unlike corticosteroids, blocking specific cytokine targets and pathways does not risk broad immunosuppression and will likely mitigate individual pro-inflammatory responses without impairing the host’s ability to clear the SARS-CoV-2 virus. It is important to point out that most of these treatments are still under investigation for potential COVID-19 complications, and further trials are required before approval.

IL-6 has been identified as a primary pro-inflammatory marker in COVID-19 and represents a key therapeutic target. Tocilizumab is a recombinant monoclonal antibody (mAb) formulation designed to block both membrane-bound and soluble forms of the IL-6 receptor (IL-6R). It has previously been used for rheumatoid arthritis¹⁴² and is approved in the United States for treatment of cytokine storms that occur as a result of CAR-T cell therapy.¹⁴³ In studies of patients with severe COVID-19, intravenous or subcutaneous administration of Tocilizumab resulted in some improvements in the context of reduction in the instances of invasive mechanical ventilation or death,¹⁴⁴ in addition to normalization of body temperature, lymphocyte counts, and C-reactive protein (CRP) levels, indicating the potential for IL-6R blockade to improve clinical symptoms in severe COVID-19.¹⁴⁵ However, because there are no clinical guidelines or thresholds for cytokine storm or IL-6 levels, more studies are needed to determine proper dosage and examine the effectiveness of treatment for different levels of disease severity. Preliminary studies with sarilumab, an anti-IL-6Rα mAb, had mixed results, with some studies showing clinical improvements,^{146, 147} while others did not observe significantly improved outcomes compared to standard of care.¹⁴⁶ These studies highlight the importance of dosage and timing of administration, two factors that must be investigated to thoroughly evaluate the efficacy of IL-6R blockade for COVID-19. In addition to IL-6R blockade, siltuximab, a mAb against IL-6 itself has been preliminarily investigated in COVID-19 patients with respiratory failure. A reduction in serum CRP levels and likelihood of death were observed following one or two treatments of siltuximab.¹⁴⁸ Results showing lower than expected efficacy could potentially signal the need for localized treatment to the lung, as opposed to systemic IL-6 or IL-6R blockade, in an effort to relieve local hyperinflammatory regions at the primary SARS-CoV-2 infection site. More randomized, placebo-controlled studies are required to validate these findings and further elucidate the role of IL-6 neutralization in mitigating the effects of cytokine storm and ARDS. Furthermore, with IL-6 serving a role in fighting infections, total hindrance or blockage may harm the host’s ability to clear the SARS-CoV-2 infection, indicating the need for more studies to optimize dosage, route of administration, and timing of treating with IL-6 and IL-6R blocking strategies.¹⁴⁹

GM-CSF is a cytokine responsible for maintaining homeostasis in the lungs through regulation of immune cell proliferation and differentiation of immune cells, especially macrophages and neutrophils.¹⁵⁰ GM-CSF targeting has emerged as a possible therapeutic strategy because of the high numbers of infiltrating inflammatory cells in the lungs and elevated blood levels of GM-CSF in severe COVID-19.³⁹ Aberrant GM-CSF levels may enhance the activation of inflammatory phagocytes and exacerbate cytokine storm by stimulating the secretion of other pro-inflammatory cytokines.¹⁵¹ A study investigating mavrilimumab, a mAb against the GM-CSF receptor (GM-CSFRα), has shown promising results in patients with severe COVID-19. Some of these improvements include faster recovery of respiratory function and clinical improvement as well as reduced CRP levels after a single intravenous dose. Furthermore, administration of three doses of lenzilumab, a mAb targeting GM-CSF itself, has shown notable improvements including lower CRP and pro-inflammatory cytokine levels and higher absolute lymphocyte counts, indicating potential recovery.¹⁵²

Inflammasome activation as a result of the SARS-CoV-2 infection results in increased secretion of IL-1β, which has been observed in severe COVID-19.⁷⁶ Anakinra is a mAb with specificity to the IL-1 receptor (IL-1R) and blocks the binding of IL-1α and IL-1β, thereby preventing the propagation of pro-inflammatory cascades, including the expression of IL-6 as a result of IL-1β secretion.¹⁵³ High-dose intravenous administration of anakinra, in combination with antivirals, promoted higher survival and lower risk for invasive mechanical ventilation in addition to improvement in CRP levels and respiratory function.¹⁵⁴ However, in the same study, a subcutaneously-administered, lower dose was not associated with any indicators of clinical improvement and, as a result, the subcutaneous administration study was ended prior to completion. Another study investigating daily intravenous anakinra treatment without the use of antivirals found a rapid response characterized by sharp reduction in fever and CRP within 48 hours of administration.¹⁵⁵

In addition to IL-1β, TNF-α is present in blood and lungs of patients in severe cases of COVID-19 and is a common inflammatory agent in cytokine storm. Anti-TNF-α mAbs have been widely used to alleviate inflammation in many autoimmune and inflammatory diseases.¹⁵⁶ Infliximab, an anti-TNF-α mAb was used to treat a Crohn’s disease pediatric patient suffering from Kawasaki-like symptoms as a result of severe COVID-19.¹⁵⁷

The JAK-STAT pathway is responsible for the transcription of multiple pro-inflammatory cascades¹⁵⁸ and is hypothesized to be activated as a result of the SARS-CoV-2 infection.¹⁵⁹ JAK inhibitors such as ruxolitinib have been used to treat hemophagocytic lymphohistiocytosis,¹⁶⁰ an inflammatory disease caused by the cytokine-mediated hyperactivation of immune cells, and is similar to the inflammatory profile in severe COVID-19.¹⁶¹ Therefore, the use of JAK inhibitors has been proposed to control the runaway inflammation in severe COVID-19. Baricitinib, a potent and specific inhibitor of JAK 1 and 2, has shown promising signs of clinical improvement in a preliminary, non-controlled, retrospective study with a resolution of fever and a reduction of CRP levels,¹⁶² indicating potential for expansive investigation of baricitinib to treat COVID-19 complications. In addition to disrupting transcription of pro-inflammatory cytokines, baricitinib is hypothesized to block the cellular entry of SARS-CoV-2, although robust clinical evidence is required to support this hypothesis.¹⁶³ Another small, non-controlled study supported similar conclusions, with baricitinib treatment shown to promote improvements in respiratory function and reduction of CRP levels within one to two weeks of treatment.¹⁶⁴ Ruxolitinib, another potent JAK1 and JAK2 inhibitor, has also been investigated for mitigating the effects of cytokine storm in severe COVID-19 patients. In a small study without a randomized control group, researchers investigated compassionate use of ruxolitinib in older and high risk COVID-19 patients. Treatment showed notable improvement in respiratory function and led to eventual discharge of a large majority of patients.¹⁶⁵ Furthermore, higher lymphocyte counts and lower expression of serum inflammatory cytokines was observed 14 days after treatment, and a majority of patients tested negative for SARS-CoV-2 at the end of the study. These results were supported by another small study that reported a sharp decrease in CRP and IL-6 levels as well as a general recovery in lymphocyte counts after treatment with ruxolitinib.¹⁶⁶ A single-blind, randomized controlled study with a 5 mg daily, oral dose of ruxolitinib showed significant recovery from lymphopenia and reduction in levels of pro-inflammatory cytokines including IL-6,¹⁶⁷ which supports the conclusion from other studies discussed here. Albeit small in sample size, the study is the first randomized controlled trial evaluating ruxolitinib for severe COVID-19.

Overall, preliminary results of various cytokine blockades are promising, and more studies, including placebo-controlled studies, are warranted to confirm the observed trends and investigate potential systemic and pulmonary immunomodulatory effects of blocking these molecules. A major concern regarding the use of inhibitors is the broad suppression of the expression of other pro-inflammatory cytokines including interferons;¹⁶⁸ specific cytokine targets must be thoroughly investigated to avoid these undesirable side effects. Administration at incorrect timing could also disrupt the host antiviral response and impair the ability to clear the SARS-CoV-2 infection, while increasing susceptibility to secondary infections.¹⁶⁹ Current evaluations are often limited with small sample sizes. Additionally, general impact on antiviral IFNs and host safety must be evaluated in larger cohorts to determine whether there should be wider use of these inhibitors for severe COVID-19 treatment.

Exogenous Interferons

Because of the impaired type I IFN response in severe COVID-19, IFN administration has been proposed as a possible therapeutic strategy to overcome viral immune evasion and promote immune-mediated antiviral pathways. In a prospective non-controlled trial with a small cohort, severe COVID-19 patients that received subcutaneous injections of a type I IFN (IFN-β−1a) showed some improvements in fever, viral clearance, and reduction in airway pathology in chest X-rays and CT scans.¹⁷⁰ However, another study found no significant improvement in survival in severe COVID-19 patients receiving subcutaneous IFN-β−1b with or without hydroxychloroquine or other protease inhibitors relative to the control group.¹⁷¹ In a third randomized, open-label trial without placebo groups, subcutaneous administration of IFN-β−1b in combination with protease inhibitors yielded significantly faster viral clearance and suppression of IL-6 levels compared to the control group receiving protease inhibitors only. Relative to the aforementioned studies, this trial showed encouraging results at earlier stages of the disease, indicating the importance of early intervention with antiviral therapies and IFNs. These conflicting results warrant the investigation of factors that affect treatment, especially disease severity and timing of treatment, because the strength of the early IFN response may determine the severity of the disease and clinical outcome.¹⁷² Inhalable IFN-α−2b has preliminarily demonstrated potential in enhancing viral clearance and reduction of IL-6 and CRP levels in moderate COVID-19 patients,¹⁷³ indicating that the route of administration may be important in the efficacy of immunomodulatory treatments.

Immunoglobulins and Convalescent Plasma

Intravenous immunoglobulins (IVIGs) are used to intercept cytokines, antibodies, and inflammatory factors especially in autoimmune diseases and other inflammatory dysfunctions through non-specific interactions.¹⁷⁴ IVIGs are also used to block Fcγ receptors on macrophages and other phagocytic cells to prevent involvement of inflammatory cells.¹⁷⁵ Some hypotheses are suggesting that a possible route for immune cell infection with SARS-CoV-2 is through Fcγ receptor-mediated internalization of active SARS-CoV-2 bound to antibodies or immune complexes, which could ultimately result in infection and activation of macrophages and inflammatory cells.¹⁷⁶ Therefore, there is potential for using IVIGs to block Fcγ receptors on infiltrating inflammatory cells in the lung, while simultaneously depleting some aberrant cytokines that contribute to cytokine storm. High dose IVIG in three patients with severe COVID-19 resulted in a rapid drop in fever and recovery of respiratory function.¹⁷⁷ However, a retrospective trial did not find significant improvement in survival or inflammatory response as a result of IVIG treatment,¹⁷⁸ although it is unclear whether the lack of effectiveness was a result of the treatment or more severe disease in the IVIG cohort. A study investigating timing of IVIG administration showed evidence suggesting that early treatment (within 48 hours of admission) resulted in better survival than delayed treatment in severe or critical COVID-19 patients.¹⁷⁹ Altogether, the conflicting studies from IVIG treatment warrant more robust and larger controlled studies to elucidate conclusions for treatment effectiveness and safety in COVID-19.

Unlike IVIG, which provides non-specific protection, convalescent plasma is derived from plasma of patients with high titers of antibodies specific to SARS-CoV-2 and has recently emerged as a possible therapeutic avenue for COVID-19.¹⁸⁰ In an open-label randomized trial of severe and critical COVID-19 patients, convalescent plasma treatment did not result in significant clinical improvement or survival within 28 days, possibly because treatment was initiated at least 14 days after onset of symptoms, indicating the potential importance of early clinical intervention for more effective results.¹⁸¹ A larger, multicenter open-label study demonstrated strong evidence for improved outcomes for patients receiving convalescent plasma treatment within three days or a delayed administration after four days of COVID-19 diagnosis, with a significant improvement in survival in earlier administration.¹⁸² Furthermore, the study evaluated the effect of antibody titers and showed higher survival in patients receiving plasma with high antibody titers compared to those receiving plasma with lower titers, indicating a concentration-dependent effect on recovery and showing no evidence of worsening because of antibody-dependent enhancement (ADE). These promising results must be validated in randomized, placebo-controlled trials to further assess its effectiveness as well as quantify secondary clinical outcomes including systemic inflammation and respiratory function.

Prophylactic vaccine strategies

No discussion of immunotherapies for COVID-19 could be complete without acknowledging the heroic effort behind the highly accelerated SARS-CoV-2 vaccine development. An impressive number of potential SARS-CoV-2 vaccine candidates are currently being investigated in accelerated clinical trials.^183–185 Several SARS-CoV-2 vaccine formats are currently in development including whole virus, subunit, and nucleic acid vaccines.¹⁸⁶ Formats involving whole viruses under investigation include live-attenuated virus vaccine,¹⁸⁷ inactivated virus vaccine,^{188, 189} and adenovirus-based vaccines.^{190, 191} Subunit vaccines have also had promising results with some progressing to ongoing clinical studies.¹⁹² Nucleic acid-based vaccines have recently come into the spotlight with Moderna’s mRNA vaccine showing promising results¹⁹³ and progressing rapidly in the development pipeline. Other nucleic acid-based (DNA) candidates are also currently under investigation.¹⁹⁴ Although there is much optimism surrounding the rapid vaccine development and early positive results, caution is warranted to avoid taking shortcuts. Robust clinical trials must be performed to ensure that vaccines promote protective and proper immune responses in healthy individuals from all ages, genders, races, and genetic backgrounds and avoid potential ADE from non-neutralizing antibodies that exacerbates cell activation and inflammatory response.^{195, 196}

Localizing Therapeutics to the Lung

Orchestrating the host response in the lung requires consideration of the unique pulmonary microenvironment as well as physiological barriers that arise from both the air and vascular sides of the lung. Delivery of agents which need to act in the lung can be accomplished by systemic delivery (primarily oral or intravenous)^{208, 209} and diffusion into the tissue. This delivery is non-specific and delivery to the lungs likely should be enhanced through the use of active targeting ligands to aid in crossing the endothelium into lung tissue.²¹⁰ Efforts to target nanoparticle drug carriers to the inflamed pulmonary vasculature intravenously can be achieved with ligand functionalization that targets upregulated receptors in the leukocyte adhesion cascade occurring during pulmonary infiltration and by tuning carrier modulus.^{211, 212} Alternatively, high drug concentrations can be achieved through oral inhalation via aerosols, reducing off-target effects associated with systemic delivery routes while increasing the effective dose to affected lung tissue.^{6, 213} Here, particle size dominates in terms of effect on deposition location and therapeutic efficacy.²⁰³ Particles with aerodynamic diameters significantly smaller than 1 μm or larger than 5 μm will have deposition fractions below 0.5 in the alveolar region of the lung, our primary therapeutic target region for treatment and/or prevention of COVID-19.^{203, 214} Accordingly, it is critical to achieve aerodynamic particle sizes within the 1 to 5 μm range, along with low dispersity, for effective, consistent dosage.²⁰³ In the case of aerosol delivery, the delivery device also can be impactful on delivery because of the formulated particle size and patient compliance. For the case of COVID-19 treatment, nebulizers have the advantages of delivering large dosages without particular breathing techniques or coordination by patients that may have difficulty breathing and also allow for ease of formulation chemistry, as the nebulizer will generate aerosols in the desired size range.^{6, 215} Other delivery devices such as dry powder inhalers and metered dose inhalers have the advantages of higher delivery efficiency and lower cost, but have drawbacks of potential aggregation, which could cause reduced deposition in the alveoli, and requirement of coordinated inspiration by the patient.²¹⁵ Regardless of the delivery method, pulmonary delivery of biomaterials can offer great advantages in increasing dosage to the lungs and avoiding off-target, systemic effects, while simultaneously targeting immune cells in the lung through size and charge considerations or avoid clearance through the same parameters;^{216, 217} smaller (50 to a few hundred nm) nanoparticles will be preferentially internalized by alveolar macrophages, while larger (greater than 10 μm) particles will be more likely to avoid internalization. However, to induce immunomodulatory effects and achieve high delivery efficiency to diseased areas and immune cells, pulmonary delivery of therapeutic particles must overcome mucosal and epithelial barrierss.²¹⁸ Hydrophilic and neutral/positively charged moieties may be incorporated in the particle design to avoid entrapment by mucus and promote localization to APCs.²¹⁹ Because of the tailorability of biomaterial-based particles, these parameters can be factored into their design and increase the effectiveness of potential therapeutic treatments of SARS-CoV-2.

Biomaterials in vaccine development

Nanoparticles and microparticles have distinct advantages in terms of delivery of vaccines, as they can be passively targeted to immune cells because of their size and can either co-deliver adjuvants or act as their own adjuvants.^{220, 221} Furthermore, the modular nature of many nanoparticles, whether liposomes, polymer nanoparticles, or others, allows for the design of smart materials to actively target immune cells²²² and be biodegradable.²⁰⁵ Some nanovaccines, termed as virus-like particles, utilize components or structures of viruses to achieve effective vaccination owing to their size and ability to enter cells effectively to deliver vaccine components.²²³ Several virus-like particle vaccines for COVID-19 are rapidly progressing through the pipeline.²²⁴ Nanovaccine formulations can have controlled degradation based on nanoparticle properties such as swellability and modulus,²²⁵ and adjusted immunogenicity and localization as a function of particle properties such as charge and density of antigen for efficient dendritic cell association and lymph node trafficking (Figure 3A).^226–228 Nanoparticles, such as those based on gold,²²⁹ lipids (liposomes),²³⁰ PLGA,²²² and HPMA/NIPAM²³¹ among many others, have been used previously for vaccine approaches. These approaches have been described in depth in previous reviews regarding the use of biomaterials for vaccine applications.^{218, 222, 232–237} Additionally, we point readers to excellent works reviewing other considerations in design of particle-based vaccines.^238–240

Particle-based formulations are rising to the forefront of vaccine approaches to combat COVID-19, with Moderna’s lipid nanoparticle-formulated mRNA vaccine currently in Phase III clinical trials, having shown significant promise in non-human primates.²⁴¹ The vaccine delivers mRNA in a four-lipid nanoparticle which encodes for the SARS-CoV-2 spike glycoprotein in its prefusion conformation.^{193, 242} In addition, other leading vaccine candidates including the Pfizer and BioNTech formulations also utilize mRNA technology with lipid nanoparticle designs.²⁴³ Respiratory infections like SARS-CoV-2 could benefit from inhalable vaccine strategies to achieve high mucosal immunity in the lungs, the primary target for SARS-CoV-2. Therefore, particle-based, inhalable formulations could be a significant, untapped opportunity that could improve current vaccines in development.

Engineered viral decoys

Engineered particles offer opportunities as novel antivirals by blocking SARS-CoV-2 binding to host ACE2 receptors through host-cell mimicry.¹⁴⁰ Delivery of free ACE2 is currently being pursued in clinical trials,²⁴⁴ which would occupy RBD to limit viral binding to host cells. This decoy approach would benefit from nanoparticle-mediated delivery, particularly to the lung, to prevent or abate infection. Nanoparticles can be decorated with ACE2-mimicing moieties that will bind to SARS-CoV-2 RBD and limit viral binding to host cells (Figure 3B). Engineered biomaterials are an ideal class of materials for this purpose, as they can be delivered to the pulmonary space, and can be designed to display high densities of ACE2.^{245, 246} For example, PLGA polymer nanoparticles could be functionalized and decorated with the aforementioned ACE2 receptor proteins. Recently, Zhang, et al. developed a method to distract SARS-CoV-2 and reduce infectivity using PLGA nanoparticles coated with membranes from alveolar epithelial cells and macrophages, respectively.²⁴⁷ Both of the membrane-coated nanoparticles were able to neutralize infectivity of SARS-CoV-2 in a dose dependent manner up to ~80% at treatments of 5 mg/mL of the nanoparticles in an infection model using Vero E6 cells using authentic SARS-CoV-2 viruses.⁴⁴ Other promising attempts include using extracellular vesicles containing ACE2 as decoys to block infection by a lentivirus containing SARS-CoV-2 spike protein²⁴⁸ or gene delivery approaches that package ACE2 mRNA inside lipid nanoparticles.²⁴⁹ Collectively, these nanoparticle platforms perform critical host-mimicry to distract the SARS-CoV-2 virus from host binding.

Drug delivery approaches to cytokine sequestration and cytokine storm abatement

As mentioned previously, a number of cytokines and factors have been identified as contributors to the inflammation and severe symptoms of COVID-19 including IL-1β, IL-2, IL-6, IL-18, and granulocyte colony-stimulating factor (G-CSF), among others.^{23, 140} Treatments have been proposed for the sequestration of IL-6, IL-18, and IL-1β through the use of systemically-delivered antibodies.^{140, 250, 251} Possible downsides of this approach may be the lack of specificity to the lung space, leading to body or system-wide immune suppression, reducing the recruitment of antiviral immune cells in other instances in which it is desired or necessary. Accordingly, localized delivery to the lung and efficient sequestration of inflammatory cytokines is a potentially untapped opportunity for pulmonary delivery of biomaterials and therapeutic particles. Similar to some of the prophylactic treatments in which ACE2 can be conjugated to the surface of a biomaterial-based nanoparticle, antibodies against one or multiple inflammatory molecules involved in the cytokine storm can be conjugated to the surface of the biomaterial-based nanoparticles to allow for efficient and effective removal of the markers to alleviate the burden caused by the cytokine storm (Figure 3C). Some previously employed strategies have included chitosan and hyaluronic acid-based, PLGA-based, and mesoporous silica nanoparticles to sequester IL-6,^252–254 all of which can be adapted for delivery to the lung. The mesoporous silica nanoparticles in particular were designed for aerosol delivery for an asthma model and may have capacity to be translated to cytokine storm abatement during SARS-CoV-2 infection.²⁵⁴ The incorporation strategies utilized, including carbodiimide conjugation of antibodies and formulation of nanoparticles with loaded antibodies, can be utilized in addition to other synthetic strategies, such as thiol-Michael click chemistry to incorporate antibodies into nanoparticles for delivery.^252–254 Simultaneous delivery of multiple cues is a secondary advantage through conjugation of multiple antibodies to sequester cytokines, inclusion of active-targeting moieties, and co-delivery of anti-inflammatory agents to limit activated macrophage polarization.^{222, 255–258} All of these strategies could improve COVID-19 treatment through more efficient, localized delivery, reduction in off-target effects, and simultaneous delivery of anti-inflammatory agents alongside particles sequestering inflammatory molecules.

Delivery of anti-inflammatory agents for repolarization of immune cells

Another approach of promise for the treatment of the COVID-19 pathology is the repolarization of inflammatory macrophages to prevent or abate their cytokine contributions. Macrophages fall along a spectrum of activation, traditionally described as skewing toward either classically activated pro-inflammatory (M1) or alternatively activated anti-inflammatory (M2) phenotypes,²⁵⁹ although the reality of macrophage phenotype is much more fluid. M1 macrophages are abundant in the SARS-CoV-2-infection following monocyte infiltration and cell differentiation in the lung. When present, they cause the further release of inflammatory cytokines including IL-6 and exacerbate existing inflammation.²⁶⁰ Accordingly, therapeutic strategies that aid in the repolarization of macrophages from the M1-like phenotype toward the M2 phenotype (Figure 3D) could be useful in the treatment of COVID-19 symptoms by reducing the cycle of inflammation caused by release of the aforementioned cytokines and further recruitment of inflammatory leukocytes.²⁶¹ M2 macrophages not only reduce secretion of inflammatory cytokines such as IL-6, TNF-α, and IL-1β,²⁶² but promote secretion of anti-inflammatory factors such as IL-10 that may restore the microenvironment homeostasis.²⁵⁹ When introduced at the right time during COVID-19 host response, macrophage repolarization at the air-liquid interface using inhaled, engineered particles may allow for local restoration of host immune response and mitigation of subsequent inflammatory airway pathologies.

Biomaterials are particularly suited for this purpose because they often do not persist in the body, can deliver cytokines and other materials to aid in the polarization, can be targeted to immune cells like macrophages, and can, themselves, repolarize macrophages.²⁶² A plethora of strategies have been developed to influence inflammatory macrophage response. Some examples include an alginate-based nanoparticle system was developed to deliver IL-10 plasmid DNA to repolarize M1 macrophages to M2 macrophages,²⁶³ polyethyleneimine nanoparticles were used to deliver the gene for CD163, an M2 macrophage marker,²⁶⁴ and PLGA nanoparticles have been used to mimic apoptotic bodies via a phospholipid membrane coating supplemented with phosphatidylserine to trigger the memory of uptake of apoptotic bodies, inducing an M2 polarization.²⁶⁵ All of these strategies led to decreases in the release of inflammatory cytokines including IL-6, IL-1β, IFN-γ, TNF-α, and MCP-1 and led to the repolarization of macrophages toward the M2 phenotype. These materials and methods were used in vitro, but their resultant reduction in the release of inflammatory cytokines and macrophage repolarization indicates their potential to be used to reduce or eliminate the inflammation induced by macrophages during SARS-CoV-2 infection.

Another way in which biomaterials could be used for the therapeutic treatment of COVID-19 is the delivery of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13. IL-4 has previously been administered both in vitro and in vivo via conjugation to gold nanoparticles²⁶⁶ and within mesoporous silica nanoparticles.²⁶⁷ Both treatments led to an increase in the population of M2 macrophages alongside a corresponding reduction in the population of M1 macrophages. The silica nanoparticles would also have the benefit of larger aerodynamic diameters than their 180 nm geometric diameter, making them good candidates for pulmonary delivery of IL-4 for the treatment of COVID-19, which, as noted previously, would aid in minimizing off-target effects and increasing efficacy of treatment.^{213, 267, 268} Other studies have also indicated that mesoporous silica nanoparticles can themselves stimulate the immune system, leading to the production of IFN-γ, IL-2, IL-4, and IL-10, and are preferentially internalized by M1 macrophages.^{269, 270} This may lead to greater efficiency of treatment relative to other materials, as the M1 macrophages, primary drivers of inflammation and thus key therapeutic targets, will internalize the silica nanoparticles more than other cells, which are less desirable targets. Similar treatments with IL-10 have been developed using nanoparticles including the previously mentioned alginate nanoparticles that deliver genes encoding for IL-10²⁶³ and nanoparticulate polyester (PLGA and PEG) polymers nanoprecipitated with IL-10.²⁷¹ Many of these studies have demonstrated efficacy in vivo, giving more credence to their potential for combatting COVID-19. Many of the nanoparticles have potential for pulmonary delivery as well, which would make them even better candidates for combatting the inflammation associated with COVID-19.

Delivery of corticosteroids can also impact macrophage polarization and thus are another category of therapeutics that may be of use for COVID-19 treatment. A primary concern of corticosteroid use is systemic immunosuppression, which could lead to impaired antiviral response.¹⁴⁰ In this regard, pulmonary drug delivery could be advantageous, as particle platforms have already been developed for corticosteroid delivery and can likely be adapted to the use of particular steroids proven to be effective, such as budesonide and dexamethasone.²⁷² For example, budesonide has been loaded into PLGA nanoparticles with 90% encapsulation efficiency and high drug loading of relevant sizes for cellular internalization.²⁷³ Similarly, metal-organic framework (MOF) nanoparticles were loaded with dexamethasone and evaluated for pulmonary delivery.²⁷⁴ Some other methods that have been developed to deliver corticosteroids include delivery of solid lipid nanoparticles loaded with budesonide for pulmonary drug delivery²⁷⁵ and dexamethasone for potential lung targeting,²⁷⁶ itraconazole nanoparticle-based dry powders for aerosol delivery of budesonide,²⁷⁷ crosslinked copolymers of methyl methacrylate, N,N-dimethylaminoethyl methacrylate, and butyl methacrylate monomers designed to load hydrophobic corticosteroids.²⁷⁸ Notably, dexamethasone-loaded PLGA microparticles were utilized to polarize macrophages towards an anti-inflammatory phenotype and resulted in a significant suppression of pro-inflammatory genes and TNF-α secretion.^{279, 280} All of these methods may be viable options to deliver these anti-inflammatory agents, especially if they can be formulated and delivered directly to the lungs to reduce or preclude systemic immunosuppression that may result in worse outcomes for patients treated with corticosteroids.

In addition to the delivery of anti-inflammatory agents, biomaterials, themselves, can be used to either induce or reduce inflammation. Their properties of immune skewing, particularly of macrophages, depend on the size, surface chemistry, and hydrophobicity of the material, in addition to other properties.²⁶² In particular, larger materials (greater than 10 μm in at least one dimension), particles in particular, tend to induce an M1 phenotype in macrophages because of frustrated phagocytosis that occurs when macrophages are unable to effectively engulf the large particles.²⁸¹ Accordingly, smaller particles would be desired for treatment of COVID-19 to reduce inflammation, as was the case for folate-conjugated silicate and calcium-based nanoparticles²⁸² and RGD-conjugated gold nanorods,²⁸³ which both utilized sub-250 nm sized nanoparticles and showed anti-inflammatory properties of the respective nanoparticles. In both of these cases, surface modifications were necessary to aid in M2 polarization; however, there are instances demonstrating non-functionalized materials aiding in the polarization toward M2 phenotypes, such as non-functionalized PLGA nanoparticles and microparticles that reduce the expression of M1 markers and the secretion of pro-inflammatory cytokines, including IL-6 in vitro.^{284, 285} It has also been shown that titanium implants with calcium and strontium surface modifications and a particular nanotopography can induce an M2 immune response, as were some titania nanoparticles,²⁸⁶ which may be another material type that could be explored to reduce inflammation in the form of titanium or titania nanoparticles containing these ions.²⁸⁷ Any of the aforementioned systems could be used to aid in inducing anti-inflammatory phenotypes in macrophages as part of COVID-19 treatment, though they lack distinct advantages over polymeric biomaterials capable of delivering anti-inflammatory agents and ensuring intracellular degradation. In both cases, active targeting ligands for macrophages may be beneficial to ensure uptake or to increase their anti-inflammatory effects. For example, clustering mannose receptors on macrophages through carbohydrate inclusion can aid in their M2 polarization,²⁸⁸ while conjugation of dextran ligands can target M1 macrophages for delivery of cargo and re-education toward an M2 phenotype.²⁸⁹ Functionalization with molecular “markers of self”, such as CD47 or phosphatidylserine can also promote macrophage polarization for localized immune modulation.^{290, 291}

Phagocyte Distraction

Neutrophils, in addition to macrophages, serve as part of the innate immune response to SARS-CoV-2 infection, contributing significantly to the aforementioned cycle of inflammation and cytokine storm.²³ A potential approach to abating the cycle of inflammation in COVID-19 is to direct inflammatory phagocytic cells away from the lung and reduce the number of recruited leukocytes from vasculature or surrounding tissue (Figure 3E). Vascular drug delivery systems have previously been used to distract neutrophils from sites of inflammation, including previous work which demonstrated that intravenous injections of 2 and 0.5 μm carboxylated polystyrene nanoparticles can distract neutrophils from infiltrating into the lung as part of an acute lung injury model.²²⁸ Instead, the neutrophils are diverted to the liver, reducing the pulmonary inflammation associated with their response. In addition, negatively charged 500 nm polystyrene and PLGA particles were used to limit trafficking of inflammatory monocytes to inflammation loci in several murine inflammation models including West Nile virus encephalitis.²⁹² Similar methods have been employed for the distraction of monocytes and neutrophils using intravenous injections of PLGA nanorods and nanoparticles.^{293, 294} PLGA-based nanorods have been shown to be preferentially internalized by neutrophils, which can be used to distract said neutrophils during inflammation to improve pathogenesis, while PLGA nanoparticles have been used to reduce the recruitment of both monocytes and neutrophils to the site of injury using a spinal cord injury model.^{293, 294} In the latter case, the recruitment of all innate immune cells at the site of injury was reduced by 75%, allowing for much more rapid recovery from injury. Recent work has shown that asymmetric lysozyme-dextran nanogels and crosslinked albumin nanoparticles with agglutinated proteins in amorphous nanostructures particularly target neutrophils during lung injury as demonstrated both in vivo in mice and also using human lungs, which could be critically valuable for selective treatment and/or distraction of neutrophils during COVID-19.²⁹⁵ These examples demonstrate that inflammation can be reduced and innate immune cells can be redirected from the sites of injury, which could be very valuable in the therapeutic treatment of COVID-19, especially in light of increased neutrophil-lymphocyte ratios found in 80% of patients infected with SARS-CoV-2.³² These distraction methods can also potentially be used in tandem with previously discussed drug delivery approaches, leveraging vascular targeting.

Opportunities for biomaterials-based model system development for addressing long-term effects of COVID-19

As noted above, COVID-19 infection can have long-term health implications in addition to short-term difficulties. These effects are attributed to permanent changes in the lung microenvironment and altered innate and adaptive immunity. These long-term issues can be further exacerbated by age, pre-existing health conditions, and COVID-19 infection severity.¹¹³ While there is a push to find vaccine candidates and clinical treatments for COVID-19 in the short term, there is also an emerging need to better understand the long-term ailments associated with COVID-19 and develop therapeutics for persistent health conditions for millions of recovering patients worldwide. Screening and developing such new therapeutics will require advances in mechanistic understanding of the resultant multicellular crosstalk involved in COVID-19 resolution as well as novel testing platforms able to recapitulate these effects. Traditional in vitro culture models (i.e., tissue culture plastic systems)²⁹⁶ and in vivo models (e.g., murine animal models)²⁹⁷ may be useful for initial hypothesis testing and translation of winnowed drug candidates; however, such systems alone may limit the development of effective therapeutics for COVID-19 long haulers because of anatomical inaccuracies, lack of complexity, and/or species-related differences. In that vein, biomaterials-based model systems that integrate human cells, including those from each sex, allow for an interesting opportunity to recapitulate the permanent changes in the lung microenvironment such as increased tissue stiffness, altered extracellular matrix composition, and a modified cellular microenvironment. These model systems would enable hypothesis testing in more physiologically relevant conditions that could help in understanding aspects of how the immune response is altered in the long term and consequently allow for the development and testing of therapeutics. Work from several groups, including ours, is utilizing such biomimetic model systems to reproduce key aspects of pulmonary fibrosis, which shares similar pathophysiology to COVID-19, which both cause organ damage and dysregulated wound healing.^298–303 Additionally, there exist underlying opportunities in utilizing organ-on-a-chip model systems to study the effects on other organs systems in recovered COVID-19 patients.^304–306 Incorporating these model systems along with primary cells derived from recovered patients would facilitate a hypothesis-based approach to understanding post-COVID-19 symptoms and recovery in a physiological manner and could allow for pre-screening of therapeutics in a microenvironment resembling that of COVID-19 long haulers.