Inhaled Countermeasures for Respiratory Tract Viruses
Ralph A. Tripp and Jarod M. Hanson
Aerosol biology (aerobiology) as it relates to respiratory viral diseases is an understudied area considering the vast microbiome in the nasopharynx and related upper respiratory tract (URT) airways [1-3]. Despite the abundance of microbes that coexist at mucosal surfaces, comparatively few of these microorganisms are pathogenic with most occupying commensal or opportunistic niches in the URT microbiome. However, several pathogens have evolved airborne transmission as a primary route of infection in part to gain direct access to the respiratory system in order to facilitate host-to-host transmission. One example is influenza virus, which can be considered an obligate airborne pathogen. Influenza infection induces host generation of large quantities of virus-laden aerosols via coughs and sneezes that typically serve as a means for virus transmission via the respiratory system. Infectious aerosols produced by the affected host typically range in size from 0.1 to 20 pm . Aerosol generation induced by a pathogen is one way to efficiently enable multiple passages through several hosts, an effect that may ultimately lead to a general increase in relative virulence via virus modifications induced by passaging in successive hosts. Aerosols generated during infection vary in both droplet size and virus content; that is, those larger than 5 pm in diameter are classified as droplets (and have a relatively low virus content), while those smaller than 5 pm are classified as particles and typically have increased virus content [4,5].
Experimentally, intranasal inoculation is a surrogate method used to emulate aerosol infection and involves putting an inoculum directly into the nasal passage(s). It should be noted that the aerosol dispersion patterns and droplet sizes in the respiratory tract change when surrogate methods are used compared to natural aerosol inhalation of respiratory droplets. Typically, intratracheal instillation is used in animal models to provide better control of the inoculation process, although this surrogate method often does not adequately reproduce the full spectrum of clinical disease typically seen following natural infection via inhalation using the same virus concentration . Importantly, the aerosolized particulates include variable numbers of viral particles that can also affect infection and disease outcome . Others have shown direct links between aerosol particle size and the quantity of influenza A virus (IAV) carried in the particle during natural infection with fine particles (<5 pm) containing significantly more viral copies than coarse particles (>5 pm), which indicates potential particle size effects on the spatial dynamics of IAV infection in the respiratory tract .
The respiratory tract consists of a large mucosal surface and is second only to the digestive system in terms of mucosal surface area . Thorough knowledge of mucosal immunity is necessary not only for the conceptualization and actualization of the next-generation vaccines but also for the advancement of other prevention and treatment strategies targeting infectious respiratory diseases, particularly those involving viruses [6,7]. Mucosal immunity influences aerobiology in the large airways found in the respiratory tract as well as in the microenvironments found within the small airways and alveoli .
The mammalian respiratory tract is often referred to as the upper (trachea) and lower (lung) respiratory tracts based in part on anatomical differences between the regions . The URT can be further subdivided into the nasopharynx (from nose to trachea) and oropharynx (from oral cavity to trachea). Historically, in healthy individuals, the lower respiratory tract was presumed to be largely sterile; therefore, once infection occurred in this area, subsequent host inflammatory responses then led to the development of pneumonia . However, that paradigm has begun to shift with the identification of various airway microbiota in the lower respiratory tract identified via PCR-based methods, many of which are implicated as having significant roles in disease prevention especially in those with preexisting airway complications such as smokers and asthmatics . The URT is colonized not only by resident microbes but also by opportunistic and pathogenic microorganisms, for example, influenza virus and various bacteria, and other foreign substances such as particulates including pollen, smoke, and dust . Interestingly, there is evidence that aerobiology and susceptibility to respiratory infection can be programmed by signals from the gut microbiome , which is not unexpected given the multisystemic nature of mucosal immunity . Several studies have elucidated the enhanced susceptibility of germ- free mice to respiratory pathogens including influenza virus, Coxsackievirus, and Friend leukemia virus [1,13,14]. However, resistance to infection is generally attributed to facets of the innate immune response mediated by cells lining the respiratory tract, including the respiratory epithelium’s role as a physical barrier as well as its associated mucociliary clearance mechanisms [3,15-17].
The respiratory epithelium serves as the initial, and highly critical, line of defense against viral infections of the respiratory tract . The physical barrier function of the epithelia is augmented by innate immune cell-like macrophages that traverse the respiratory tract in part to destroy pathogenic organisms including viruses and bacteria [3,18]. There are adaptive immune cells that also affect aerobiology via contributions made to respiratory tract immunity as well as epithelia cells contributing further to innate defense via mucin production, mucociliary clearance, etc. Type I epithelial cells have been shown to internalize particles in the respiratory tract directly, while type II cells are unable to do so, implicating cell targeting as a crucial mechanism for drug or particle-based vaccine uptake, with at least one study indicating pulmonary surfactants are also involved in uptake by epithelial cells . Additionally, the relative size of a particle determines its ability to successfully penetrate the respiratory epithelium as ~50 nm particles can enter epithelial cell cytoplasm via passive diffusion, while ~100 nm particles rely on clathrin- or caveolin-mediated endocytosis, and yet neither particle can cross cellular tight junctions, which demonstrates the added importance of particle size in drug and/or particle-based vaccine uptake . Also of note is the potential for particle size to directly influence drug uptake as was shown with ciprofloxacin delivered to the respiratory tract via successively increasing diameter liposomes with direct implications for drug concentrations relative to the diameter of the delivery particle . The size and cellular specificity afforded by the respiratory epithelium likely includes additional screening interactions facilitated by transmembrane mucins, pattern recognition receptors (PRRs), PAMPs, etc. that directly affects the uptake of a given particle based on a multitude of host and particle factors.
Mucins can have somewhat contradictory roles during viral infection in the airways in that they can not only potentially induce innate immunity and act as a physical barrier to virus entry to host cells but may also enable viral penetration to host cells depending on specific viral properties such as the neuraminidase (NA) associated with influenza. Mucins can be split into two groups based on which layer they partition to in vivo: the transporting mucous layer (TML) on the apical cell surface and the periciliary layer (PCL) located above the TML and spanning the distance between the TML and the apical portion of the cilia. Two polymeric mucins, MUC5AC and MUC5B, are enriched in the TML (as well as many additional globular proteins), while the PCL is rich in keratin sulfate as well as the tethered mucins MUC1, MUC4, and MUC16, but it has relatively few proteoglycans compared to the TML . While these mucins may be enriched in their respective layers, they are not solely found there.
With respect to innate immunity and viral infections, MUC1 in particular has been implicated in T cell responses as well as being a modulator of the airway during inflammation and infection . This would imply a potential immunomodulatory effect of mucins themselves (in the absence of any viral or inflammatory initiator). Additionally, the mucins mentioned earlier have associated alpha-2,6-linked sialic acids (but not alpha-2,3-) with them, which has been postulated to help neutralize influenza virus. However, this effect is probably transient at best in that NA has been shown to aid mucous penetration via the cleavage of sialic acid molecules although at least one study showed greater than 80% neutralization of influenza virus in vitro by cellular subunit preparations containing MUC1.
If penetration of the initial mucin barrier is achieved, actual infection of respiratory epithelial cells by pathogenic viruses and live-attenuated vaccines results in the production of antiviral cytokines and other defensive compounds such as type I and III interferons (INFs) and nitric oxides . This stimulates the release of various chemokines involved in the recruitment of inflammatory and immune cells that influence adaptive immunity . The respiratory tract’s adaptive immune response includes B and T cells, immunoglobulins, and antigen-presenting cells that are typically diffusely associated with the epithelium. Alternately, they can be found within the lungs, airways, and nasal passages in specific locations defined as mucosal-associated lymphoid tissues (MALTs)  consisting of aggregated lymphoid tissues and immune cells . Additionally, bronchial-associated lymphoid tissues (BALTs) and nasal-associated lymphoid tissues (NALTs) have been described in mice [25-27] to have a similar functional role as MALTs in humans. The MALT, NALT, and BALT appear to be sites of antibody production within the respiratory tract .
T cells subsist in MALTs and are broadly classified by their receptors. The alpha- beta T cells found in the respiratory tract may be either effector or effector memory T cells, while those located in associated lymphatic tissues, such as lymph nodes, may be of the effector or central memory phenotypes [3,28-32]. CD8 T cells are primarily cytotoxic T lymphocytes involved in the destruction of host cells infected with viruses, while CD4 T cells are primarily of the Th1 variety and are involved in the initiation of cell-mediated immunity [3,23,33,34]. Gamma-delta T cells can be found in various mucosal tissues, including those lining the respiratory tract [3,35]. This T cell type is predominately an innate immune cell type, but pulmonary gamma-delta T lymphocytes can produce IL-17, which functions in host adaptive immune-mediated responses [3,36]. B cells produce immunoglobulins that can directly, or with the addition of complement, bind to and destroy pathogens such as viruses [3,37,38]. Plasma cells capable of secreting antibodies make up the majority of B cells, or memory B lymphocytes (these are present in germinal centers [lymph nodes] and follicle-associated epithelium) [3,39]. Plasma cells lining the respiratory tract can produce immunoglobulin, which is expressed as IgG and IgA [40-42]. IgA is the predominant antibody type present in mucous secretions. It is also present in the alveolar spaces although IgG is the primary immunoglobulin isotype in this locale [3,43].
Mucosally induced S-IgA antibodies are an essential arm of adaptive immunity for protecting the host from infection, but take several days to generate at the site(s) of infection and replication. During that generation time, the innate immune response, which is antigen nonspecific, can respond and confront the pathogen by producing inflammatory cytokines, IFNs, and IFN-stimulated cytokines .
The proinflammatory cytokines include IL-6, IL-12, and TNFa  that are needed for the induction of antigen-specific T and B cell responses to pathogens; thus, the innate immune response is needed to induce, activate, and expand adaptive immune responses .
Influenza virus infection serves as a useful example of a prominent respiratory tract pathogen as the virus affects aerosol biology when it first attaches to respiratory mucosal epithelial cells and then invades the cell cytoplasm by endocytosis . This event signals PRRs and Toll-like receptors (TLRs), particularly when the viral single-strand RNA (ssRNA) is released into the cytoplasmic region of the epithelial cell . ssRNA viruses like influenza virus are recognized by TLR7 in humans located on the inside of the endosome . Also, retinoic-acid inducible gene I (RIG-I) is a prominent PRR during the innate immune response [48,49].
RIG-I, a cytoplasmic RNA helicase, is especially important because it is capable of binding virus-specific RNA structures . RIG-I recognizes genomic RNA released into the cytoplasmic region of infected cells . TLR7 and RIG-I activated by ssRNA trigger intracellular signal transductions, hence leading to the expression and production of IFNs, IFN-stimulated genes, and proinflammatory cytokines. A better understanding of host aerobiology features as they relate to respiratory tract pathogens is important for developing infection countermeasures utilizing aerosol or pulmonary methods of delivery for vaccines or therapeutics.