key: cord-1049379-r72zt6fp
authors: Shilovskiy, Igor P.; Yumashev, Kirill V.; Nikolsky, Alexandr A.; Vishnyakova, Liudmila I.; Khaitov, Musa R.
title: Molecular and Cellular Mechanisms of Respiratory Syncytial Viral Infection: Using Murine Models to Understand Human Pathology
date: 2021-03-15
journal: Biochemistry (Mosc)
DOI: 10.1134/s0006297921030068
sha: 2ec137c34a82b6dbb74636d1d9152e0d07e8e306
doc_id: 1049379
cord_uid: r72zt6fp

Respiratory syncytial virus (RSV) causes severe pathology of the lower respiratory tract in infants, immunocompromised people, and elderly. Despite decades of research, there is no licensed vaccine against RSV, and many therapeutic drugs are still under development. Detailed understanding of molecular and cellular mechanisms of the RSV infection pathology can accelerate the development of efficacious treatment. Current studies on the RSV pathogenesis are based on the analysis of biopsies from the infected patients; however deeper understanding of molecular and cellular mechanisms of the RSV pathology could be achieved using animal models. Mice are the most often used model for RSV infection because they exhibit manifestations similar to those observed in humans (bronchial obstruction, mucous hypersecretion, and pulmonary inflammation mediated by lymphocytes, macrophages, and neutrophils). Additionally, the use of mice is economically feasible, and many molecular tools are available for studying RSV infection pathogenesis at the molecular and cellular levels. This review summarizes new data on the pathogenesis of RSV infection obtained in mouse models, which demonstrated the role of T cells in both the antiviral defense and the development of lung immunopathology. T cells not only eliminate the infected cells, but also produce significant amounts of the proinflammatory cytokines TNFα and IFNγ. Recently, a new subset of tissue-resident memory T cells (T(RM)) was identified that provide a strong antiviral defense without induction of lung immunopathology. These cells accumulate in the lungs after local rather than systemic administration of RSV antigens, which suggests new approaches to vaccination. The studies in mouse models have revealed a minor role of interferons in the anti-RSV protection, as RSV possesses mechanisms to escape the antiviral action of type I and III interferons, which may explain the low efficacy of interferon-containing drugs. Using knockout mice, a significant breakthrough has been achieved in understanding the role of many pro-inflammatory cytokines in lung immunopathology. It was found that in addition to TNFα and IFNγ, the cytokines IL-4, IL-5, IL-13, IL-17A, IL-33, and TSLP mediate the major manifestations of the RSV pathogenesis, such as bronchial obstruction, mucus hyperproduction, and lung infiltration by pro-inflammatory cells, while IL-6, IL-10, and IL-27 exhibit the anti-inflammatory effect. Despite significant differences between the mouse and human immune systems, mouse models have made a significant contribution to the understanding of molecular and cellular mechanisms of the pathology of human RSV infection.

Respiratory syncytial virus (RSV; order Mononega virales, family Pneumoviridae, genus Orthopneumovirus) is one of the most common pathogens [1] that cause severe upper and lower respiratory tract infection in children [2] , elderly [3] , and patients with immunodeficiencies [4] . Individuals with bronchial asthma and chronic obstructive pulmonary disease also often suffer from this infection [5] . As many as 3 million RSV infected children BIOCHEMISTRY (Moscow) Vol. 86 No. 3 2021 are hospitalized annually, resulting in 66,000 199,000 deaths [6] . The economic impact caused by the RSV infection in the USA alone was estimated to be 1.15 bil lion US dollars [7] .

No efficacious RSV vaccine has been developed so far [8] . Although preparations based on specific mono clonal antibodies (mAbs) are used for immunopreven tion, but their wider application is limited by the high cost [9] . New vaccines are being developed; at present, 16 candidate vaccines undergo various stages of clinical tri als, six of which have reached phase II [10] . It may be expected that a preventive RSV vaccine will be registered for the market in the next few years.

Anti RSV therapeutics are also being developed, such as virus inhibitors based on small interfering RNAs (siRNAs) [11] , nanoparticles [12] , peptide compounds [13] , and other small molecules [14] . One of the promis ing approaches is the use of siRNAs which specifically suppress important genes of the virus, thereby blocking viral replication. Among them is ALN RSV01, which contains siRNA molecules specifically recognizing the viral nucleocapsid encoding n gene. In clinical studies, the intranasal administration of ALN RSV01 for 5 days significantly (by 38%) decreased the number of patients with the verified RSV infection [15] . Despite these posi tive results, Alnylam Pharmaceuticals discontinued the trial for ALN RSV01, making it difficult to predict when this preparation will be registered.

Understanding molecular and cellular mechanisms of the RSV infection pathogenesis is essential necessary for creating efficacious and safe therapeutics and preven tive agents, the development of which is impossible with out the use of experimental animal models. Many models of RSV infection have been proposed by now are devel oped in animals such as mice, rats, ferrets, calves, sheep, chimpanzees, etc. [16, 17] . Although chimpanzees are the only species naturally susceptible to human RSV, mice are commonly used to model this infection [16, 17] due to the ease of use and low cost of veterinary care, as well as the availability of diverse scientific tools (mAbs, probes, specialized reagents, and equipment) for reveal ing detailed mechanisms of the pathogenesis. Here, we summarized new data on the RSV infection pathogenesis obtained in mouse models.

RSV genome is a single stranded non segmented negative sense RNA molecule that contains 10 genes encoding 11 proteins: NS1, NS2, N, P, M, SH, G, F, M2 1, M2 2, and L (the m gene encodes two proteins -M2 1 and M2 2). Genomic RNA is encapsulated in the nucleocapsid consisting of the N protein, RNA poly merase (L protein), its cofactor (P protein), and M2 1 protein. The M protein surrounds the nucleocapsid and interacts with the lipid bilayer of the virion envelope and the cytoplasmic domain of the F protein. Several RSV glycoproteins are embedded into the envelope, such as the fusion protein F, protein G, and small hydrophobic protein SH. The M2 2 protein and two nonstructural proteins (NS1 and NS2) are lacking in the virion struc ture [18] .

The RSV life cycle begins after virion attachment to the target cell followed by the fusion between the viral and the host cell membranes. The crucial role in this event plays glycoproteins F and G. The G protein exists in two forms. The membrane bound form enables virion attach ment to the cells via binding to the cognate receptor or cell surface attachment factors. Of note, in the recent studies, cell surface proteins that bind the G protein are designated as attachment factors, whereas cell proteins initiating the fusion process between the virus and the cell are designated as cell receptors [13, 18] . The soluble form of the G protein (sG) functions as an antigenic trap for binding the anti G protein antibodies, which is necessary for evading the host immune system [19] .

Some of the most studied attachment factors are heparan sulfates (HSGAGs) belonging to glycosamino glycans (GAGs). They are disaccharide polymers bound to the transmembrane proteins on the surface of many cell types. The binding between the G protein and HSGAGs occurs via electrostatic interactions of positive ly charged heparin binding domain of the G protein to the negatively charged HSGAGs [20] . Other attachment factors have also been identified, e.g., the chemokine CX3CR1 receptor that binds to the CX3C motif in the G protein [21] . The F protein can also be involved in the virion attachment by binding to the factors such as HSGAG [22] , ICAM1 [23] , EGFR [24] , and nucleolin [25] (the latter functions as both the attachment factor and the receptor) [25] .

Although the F protein participates in the attach ment, its major function is to ensure the fusion between the viral and cell membranes. This glycoprotein is synthe sized by the infected host cells as a precursor molecule (F0) to be further proteolytically cleaved into the F1 and F2 subunits covalently bound into a heterodimer. The functional F protein on the virion surface is a trimer com posed of F1/F2 heterodimers in the prefusion conforma tion. The fusion peptide (FP) with the adjacent α helical HR N motif is located at the N terminus of the F1 sub unit, whereas the C terminus contains a transmembrane domain (TM) with the α helical HR C motif that ensures protein anchoring in the viral envelope. During the fusion, FP interacts with the membrane of the target cell, resulting in the F protein rearrangement from the prefusion to the postfusion conformation. During such rearrangement, the HR C and HR N motifs approach each other and form a hairpin structure that brings the membranes of the host cell and the virion into close con tact followed by their final fusion. Recent studies have BIOCHEMISTRY (Moscow) Vol. 86 No. 3 2021 shown that the virion cell fusion is a two step process: macropinocytosis occurs first followed by the immediate membrane fusion inside the endosome [13, 18] . The SH glycoprotein does not play any role in the attachment and fusion events [13, 18] .

After the fusion, the viral genome enters the cytosol, where it is transcribed into mRNAs that are translated into the viral proteins. There are three viral proteins involved in the viral genome replication: N, P, and L. The P and L proteins act as subunits of the RNA dependent RNA polymerase, whereas the N protein binds to the genomic RNA and protects it from cell nucleases. Viral mRNAs are transcribed by the same enzyme involved in the replication of viral genomic RNA [26] .

The virion assembly involves cell actin cytoskeleton, which transports viral glycoproteins (F, G, and SH) to the apical surface of the cell plasma membrane. The N pro tein binds to the de novo synthesized viral genomic RNA together with the RNA dependent RNA polymerase, thus forming the ribonucleoprotein complex that addi tionally associates with the M protein, which is also trans ported to the apical surface of the cell plasma membrane (i.e., to the location of viral glycoproteins). These events result in the formation of viral particles that bud off from the infected cells as mature infectious virions [13, 18] .

In humans, RSV targets the upper and lower respira tory tract. The infection occurs via direct contact between the viral particles and the respiratory epithelium. After the incubation period (4 5 days), the viral replication proceeds in the nasopharyngeal epithelium, followed by the virus spread into the lower respiratory tract. The severity of the disease varies from minor symptoms of common cold to the airway obstruction, hypoxia, and pneumonia [27] .

RSV is mainly replicated in the respiratory ciliated epithelium, as well as in the type II and I alveolar pneu mocytes [28] . Histological data on the changes in the res piratory tract have been obtained by the analysis of post mortem lung samples from patients who died from severe RSV infection. Such changes include perivascular and peribronchial infiltration by mononuclear and T cells, pneumonia symptoms, necrosis of bronchial epithelium, obstruction of bronchiole lumen by cell conglomerates (desquamated epithelial cells, macrophages, and neu trophils), and mucus hypersecretion [28] . On the con trary, CD4 + and CD8 + T cells have been rarely found in the respiratory tract [29] . Analysis of bronchoalveolar lavage (BAL) samples from children with the RSV induced bronchiolitis revealed a significantly elevated content of neutrophils [30] . In addition, the BAL samples were also enriched in the pro inflammatory cytokines, such as TNFα, IL 6, IL 1a, IL 8, MIP 1a, MCP 1, RANTES, IFNγ, IL 4, IL 5, IL 10, IL 9, and IL 17 [31, 32] , which impact the pathogenesis of the RSV infec tion.

Both clinical data from patients and results of animal studies (mice, rats, ferrets, calves, sheep, etc.) [16, 17] are used to reveal the pathogenesis of RSV infection. The biggest limitation of RSV studies in animal models is that all used species are only partially sensitive to RSV. The virus poorly replicates in the animal respiratory tract, resulting in mild symptoms. Chimpanzees are the only animal species naturally susceptible to human RSV [16, 17] . However, the use of chimpanzees for modeling the RSV infection is limited due to the high cost of animal care; hence, most of such experiments are conducted in rodents, particularly, mice. A great body of evidence on the molecular and cellular mechanisms of the RSV infec tion pathogenesis has been obtained in the mouse models. BALB/c mice are commonly used, in which RSV repli cates (although in a limited manner) in the respiratory tract (table) . It was also found that the viral load is higher and the pathological symptoms are more pronounced in the aged mice [33] . In most studies, mice are infected intranasally at a dose of 10 4 10 7 pfu (plaque forming units) per animal. Generally, the dose of 10 6 pfu is suffi cient to induce visible symptoms (mucus hypersecretion, lung infiltration with pro inflammatory cells, etc.). The three laboratory RSV strains commonly used for model ing the infection are A2, line 19, and long; however, some studies used clinical isolates of RSV received from patients (table) [34] .

The first studies on the mouse models of RSV infec tion were published in the late 1970s early 1980s [35 37 ]. The very first study by Prince et al. [35] investigated the ability of RSV to replicate in the mouse respiratory tract. The authors infected 20 mouse strains with RSV (strain long) at a dose of ~10 4 pfu. CBA/CaHN mice was found to be most resistant and DBA/2N turned out to be most susceptible, whereas BALB/c mice displayed inter mediate susceptibility to this RSV strain. However, after the ability of RSV to replicate in the mouse respiratory tract had been documented, no studies on the pathologi cal changes occurred during the infection (table) [35] .

Several years later, two research groups [36, 37] inde pendently confirmed that RSV (strain A2) was able to infect the upper and lower respiratory tract in BALB/c mice, with the viral replication peaking on days 4 6 post infection. It was also shown that the viral replication occurred mainly in the alveolar (but not bronchial) epithelium and resulted in the respiratory tract patholog ical changes, such as lung infiltration with macrophages and neutrophils, thickening of bronchial wall, and desquamation of the respiratory epithelium (table) [36, 37] . Later [38] , RSV replication was intravitally visu alized in the respiratory tract of live mouse using recom binant RSV rHRSV Cherry and rHRSV Luc strains carry ing reporter genes. It was confirmed that RSV reproduc tion in the lower respiratory tract peaked on days 4 5 post infection, and the virus targeted both lungs equally (table) [38] .

The impact of RSV infection on the bronchial hyperreactivity (BHR) has been speculated for a long time. Jafri et al. [39] demonstrated that mice, infected with high dose RSV (~10 7 pfu) developed BHR that peaked on day 5 post infection which lasted for more than 40 days. It was assumed that BHR was induced due to the mucus hypersecretion by the bronchial epithelium, as well as to the lung infiltration with pro inflammatory cells (lymphocytes, macrophages, and neutrophils) detected for over 150 days post infection. However, no correlation between the BHR and viral load in the lungs was found (table) [39] . One year later, Bitko et al. con firmed that RSV at a dose of 10 7 pfu triggered BHR in mice, which was believed to be due to the elevated pro duction of leukotrienes interacting with the cognate receptors on the bronchial smooth muscle cells and elic iting bronchoconstriction. Moreover, leukotrienes pro voked mucus hypersecretion in the respiratory tract epithelium, which also contributed to the BHR develop ment (table) [40] .

However, not all RSV strains trigger BHR and mucus hypersecretion. It was demonstrated that the RSV line 19 strain was more mucogenic and induced prominent BHR even at a dose of 10 4 pfu, as well as upregulated IL 13 pro duction responsible for the BHR development and mucus hypersecretion (this effect was abrogated in the IL 13 knockout animals) (table) [41] . These data were con firmed in another study that used chimeric RSV rA2 line19F strain (modified strain A2 with the F protein derived from the line 19 strain). Compared to the strain A2, the chimeric virus was able to activate IL 13 produc tion, thus inducing mucus hypersecretion and BHR (table) [42] . Hence, surface glycoprotein F might account for the pathogenicity of RSV strains. This hypothesis was further confirmed by Stokes et al. [34] , who investigated the possibility to induce the respiratory tract pathology not only by the laboratory RSV strains (A2, line 19, and long), but also by clinical isolates. Some clinical isolates induced no pathological changes in mice; however, 2 out of 6 examined isolates triggered pathological changes manifested as lung infiltration by the pro inflammatory cells, as well as IL 13 mediated BHR and mucus produc tion (table) [34] . These data demonstrated different severity of pathological changes observed in clinical prac tice during epidemiological seasons. Taking into consid eration that IL 13 and IFNγ are mainly produced by T cells and act as mutual antagonists, it was proposed that RSV induced IL 13 expression in alternatively activated macrophages rather than in Th2 cells, because the IL 13 gene knockout did not result in the increased pulmonary IFNγ levels after RSV infection in mice [34] .

Later, it was shown that the RSV strain A2 triggered BHR and mucus hypersecretion in the bronchial epithe lium, but only when applied at a high dose (5 × 10 6 TCID 50 /mouse). No elevated IL 13 expression was observed during the infection, so that the aforementioned pathological manifestations have probably developed via the TNFα mediated pathway (table) [43] . These data indicated that the development of the same pathological changes might occur via different molecular mechanisms.

The impact of T cells. The use of mouse models allowed to demonstrate the role of T cells in the antiviral defense against the RSV infection. Mice lacking the thy mus and, therefore, T cells (nu/nu BABLB/c) or exposed to gamma irradiation were more susceptible to the RSV infection and developed more severe pathology compared to the animals with functionally active T cells [44] . The adoptive transfer of the virus primed T cells to these mice accelerated RSV clearance from the respiratory tract. Primed T cells exhibited the antiviral activity due to their cytotoxic effects independently of the humoral immunity, because the virus clearance in the lungs occurred in the absence of virus specific antibodies [44] . However, the presence of virus specific antibodies accelerated RSV clearance from the lungs, indicating an important role not only of the cell mediated immunity, but also of the humoral component of the antiviral defense [44] . Similar observations were made in clinical practice. It was found that an increased number of CD8 T cells in BAL samples correlated with lower viral load in human respiratory tract during RSV infection [45] .

Later, the role of T cells in the RSV infection patho genesis has been repeatedly confirmed (see reviews else where [46 48] ). According to the obtained data, during the primary RSV infection, dendritic cells (DCs) engulf and present virus derived antigens to activate virus spe cific CD8 T cells in the local draining lymph node. CD8 T cells then migrate to the respiratory tract and provide protection. Moreover, the number of activated T cells in the bronchial aspirate from the RSV infection patients peaked on day 10 after the onset of disease symptoms; the virus specific CD8 T cells accumulated mainly in the lung parenchyma rather than circulated in the blood stream. During the infection, activated T cells produce pro inflammatory cytokines IFNγ and TNFα. After res olution of infection, CD8 T cells are maintained for sev eral months as memory cells and provide defense against subsequent infections. Upon the reinfection, T cells accumulate in the lungs at a much faster rate primarily due to the migration rather than cell proliferation (Fig. 1a) [38] [43] Models used in studying RSV infection pathogenesis Results RSV was for the first time shown to reproduce in the mouse respiratory tract: CBA/CaHN and DBA/2N mouse strains showed the highest resistance and the highest susceptibility, respectively, to the viral replication RSV induced pathological changes in the respiratory tract were described: viral reproduction peaked on days 3 4 in the upper respiratory tract and on days 4 6 post infection in the lower respiratory tract; replication took place in the alveo lar rather than bronchial epithelium; the loss of the host activity and body weight (up to 20%) was found to be a crucial manifestation of the virus induced patho logy; lung infiltration with lymphocytes and macrophages occurred along with the thickening of bronchial wall and respiratory epithelium desquamation; multi nuclear giant cells were observed \in the bronchial epithelium; anti RSV anti bodies developed 2 weeks post infection RSV infection induces bronchial hyperreactivity: viral replication in the respira tory tract peaked on days 3 5 post infection, but was almost absent on day 7; the maximum bronchial hyperreactivity and mucus hypersecretion were observed on day 5 post infection and were sustained for 42 days; bronchial hyperreactivity (BHR) significantly correlated with the mucus hypersecretion and intensity of lung infiltration with pro inflammatory cells, but not with the viral load; patho logical changes in the lung tissue were most prominent on days 4 5 post infec tion and were sustained for 154 days; RSV infection was associated with the upregulated expression of pro inflammatory cytokines (TNFα, IL 6, IFNγ, IL 4, IL 10, KC, MIG, RANTES, MIP 1a, and eotaxin) RSV caused pulmonary inflammation and BHR, which were manifested on days 2 10 days post infection; RSV induced leukotriene production that mediated mucus hypersecretion and BHR chimeric RSV A2 line 19 strain encoding F protein was created that replicated more actively in the respiratory tract compared to the long and A2 strains; the virus induced the IL 13 dependent mucus hypersecretion and BHR, as well as IFNα production (to a lesser extent) different RSV strains caused varying severity of pulmonary pathology: strain A2 was mainly observed in the alveolar regions, whereas virus clinical isolates were found in the bronchial epithelium; clinical isolates A2001/2 20 and A2001/3 12 elicited IL 13 mediated BHR and mucus hypersecretion, as well as more pro nounced lung pathology compared to the laboratory strains; the knockout of the IL 13 gene markedly lowered mucus hypersecretion in the mouse bronchial epithelium infected with the viral isolates; RSV induced IL 13 expression in alternatively activated macrophages (and not in Th2 cells), as the IL 13 knock out caused no increase in the IFNγ level in the lungs RSV reproduction was visualized in the respiratory tract of live mice: viral repro duction peaked on day 3 and days 4 5 post infection in the upper and lower res piratory tract, respectively; both lungs were equally affected; RSV at the used dose caused no prominent pathological manifestations RSV induced BHR developed via the IL 13 independent pathway: RSV infec tion caused body weight loss, BHR, lung infiltration with macrophages and lym phocytes (but not with eosinophils and neutrophils), and hyperplasia of mucus producing goblet cells; RSV infection upregulated expression of IFNγ and TNFα, but not of IL Results for the first time were shown that T cells provide RSV clearance in the lungs for the first time were shown that T cells play a role in of RSV induced lung immunopathology: introduction of T cells into infected mice resulted in lower viral clearance, but exacerbated lung pathology depletion of CD4 and CD8 T cells resulted in the elevated viral reproduction accompanied by the decrease in the pathology manifestation the role of tissue resident memory CD8 T cells (T RM ) in the antiviral defense in lungs was shown for the first time: local (intranasal) immunization with the viral antigen resulted in a higher number of mouse lung T RM cells that caused no prominent lung immunopathology compared to other subsets of memory T cells; intranasal administration of CD8 T RM cells induced a marked antiviral effect, whereas administration of CD4 T RM cells suppressed expression of pro inflam matory TNFα and ameliorated pathological changes, but did not affect viral replication IFNγ and TNFα produced by the memory CD8 T cells did not affect viral repli cation, but caused pulmonary immunopathology: in the absence of CD4 T cells and antibodies, memory CD8 T cells exhibited protective antiviral response, but induced lung immunopathology after RSV infection; lung immunopathology developed due to IFNγ and TNFα produced by CD8 T cells, as their neutraliza tion with specific mAbs abrogated lung immunopathology, but did not affect the viral load; T RM cells exhibited the antiviral activity without inducing lung immunopathology anti IgG mAbs lowered the viral load and the severity of pulmonary inflamma tion Higher virus neutralizing potential was identified for mAb targeting the antigenic site Ø in the F protein vs. mAb against site II (experimental analog of palivi zumab) the role of STAT1 (transcription factor required for type I and type II IFN sig naling) in the anti RSV defense was demonstrated: the STAT1 gene knockout resulted in the elevated viral load in the lungs and promoted BHR, mucus hyper secretion, and infiltration of pro inflammatory cells into the lungs; production of Muc5ac (major component of bronchial epithelial secretion), as well as of cytokines IL 5, IL 13, IFNγ, and IL 17A was increased in the infected STAT1 deficient mice RSV was shown to interact with the TLR receptors and to activate innate immu nity: the knockout of the TLR2 and TLR6 genes resulted in lower neutrophil infiltration into the lungs and increase in the viral load; RSV interacted with TLR2 and TLR6 and activated NF κB dependent production of cytokines and chemokines, but not of IFN I; RSV induced IFN I production via the macrophage TLR3 signaling cascade Results aberrant interferon response to RSV infection was observed in the neonatal mice, that promoted Th2 mediated lung immunopathology: expression of pulmonary IFNα and IFNβ was 2 4 times higher in adult vs. neonatal mice; the number of IFN producing plasmacytoid dendritic cells (DCs) was 10 times higher in the lungs of adult vs. neonatal mice; IFNα administration to the neonatal mice prior to the infection resulted in the lower BHR and less pronounced lung immunopathology, as well as caused a decline in the number of Th2 cells and in the amount of cytokines released by these cells (IL 4 and IL 13), while no effect on the level of Th1 cytokines (IFNγ and IL 12) was observed a dual role for IFNα as an antiviral agent and an activator of adaptive immunity was demonstrated: IFNα administered intranasally to the neonatal mice result ed in a lower viral load after primary RSV infection and in the reduced inflam mation in the lungs after reinfection; compared to adult mice, neonatal animals produced no mucosal IgA antibodies in the respiratory tract after the RSV infec tion, while administration of exogenous IFNα activated production of protec tive IgA intranasally administrated IFNγ lowered the viral load in the lungs, but promot ed production of pro inflammatory cytokine IL 6 and chemokine CXCL1 IFNγ exerted a protective effect during RSV infection: the IFNγ gene knockout resulted in the increased RSV replication after both primary and secondary infec tions the beneficial role of IL 10 in the RSV induced lung inflammation was demon strated: RSV infection elicited production of IL 10 by the airway CD4 + and CD8 + T cells; the IL 10 gene knockout augmented lung immunopathology, but did not affect viral replication IL 13 participate in the RSV induced BHR: both RSV strains (line 19 and A2) induced inflammation in the lungs; RSV line 19, but not A2, induced IL 13 expression, BHR, and mucus hypersecretion; IL 13 gene knockout profoundly reduced BHR and mucus hypersecretion the role for thymic stromal lymphopoietin (TSLP) in RSV induced BHR and mucus production was uncovered: RSV infection resulted in the elevated IL 13 expression and increased number of type 2 innate lymphoid (ILC2) cells pro ducing this cytokine; mice deficient by the TSLP receptor (TSLPR) demonstrat ed upregulated IL 13 production, as well as augmented BHR and mucus hyper production; the TSLPR gene knockout did not affect the RSV replication IL 17A was shown to be involved in the pathogenesis of RSV induced inflam mation: RSV upregulated expression of the IL 17A gene, but not of the IL 17F gene, in CD4 + T cells; neutralization of the IL 17A protein with mAbs or the IL 17A gene knockout did not affect BHR, but lowered hypersecretion and intensi ty of viral replication in the airways; the IL 17A gene knockout led to the decreased neutrophil infiltration into the lungs IL 6 and IL 27 were found to suppress RSV induced inflammation: expression of IL 6 and IL 27 in the respiratory tract was elevated during RSV infection; inactivation of IL 6 and IL 27 by mAbs resulted in the increased count of CD8 T cells producing the pro inflammatory cytokines TNFα and IFNγ that aggra vated pathological events in the lungs; administration of IL 27 promoted Treg maturation and ameliorated inflammatory process directly destroying them with granzymes and perforins (Fig. 1b) . Moreover, production of IFNγ and TNFα ini tiates the death of the infected cells [46, 47] .

Several memory T cell subsets have been identified, including T CM -central memory T cells located in the secondary lymphoid organs, and T EM -effector memory T cells migrating to the respiratory tract and realize effec tor functions. Both T CM and T EM subsets are simultane ously found in the circulation and the respiratory tract. In addition, another T cell type was found solely in the lungs -T RM (tissue resident memory CD8 T cells) serv ing as the first line defense against repeated infections [45 47, 49] (Fig. 1a) .

The role of T cells in the RSV induced pulmonary pathology. Despite their antiviral activity, T cells can elic it pulmonary immunopathology [50] . It was shown that depletion of CD8 T cells resulted in the elevated viral replication in the lungs, accompanied by the amelioration of pathological changes in the respiratory tract [51] . On the contrary, an increased number of CD8 T cells aggra vated pathological events in the lungs, but lowered the viral load [52] . According to the recent data, this type of immunopathology involves cytokines TNFα and IFNγ produced by the virus specific T cells. TNFα neutraliza tion with specific mAbs prior to the infection resulted in the alleviation of pathological changes in the lungs [52] . Similar mAb mediated neutralization of IFNγ or the knockout of the IFNγ gene decreased the severity of lung immunopathology after RSV infection (table; Fig. 1a ) [52, 53] .

It is remarkable that such T cell mediated immunopathology is typical for the RSV infection, but not for all respiratory viruses. For instance, memory CD8 T cells provide protection against influenza and coron avirus (strain MA15), causing no severe lung immuno pathology [54, 55] .

The data on the role of T cells in the immuno pathol ogy obtained in mouse models correlate with the clinical observations. It was found that the elevated number CD8 T cells in BAL led to a more severe pathology of the respi ratory tract of patients with the RSV infection [56] . The increase in the count of CD8 T cells after the bone mar row transplantation correlated with a lower RSV titer in the nasal washings, while the appearance of CD8 T cells negatively affected the patients' respiratory function, being a marker of pathological changes in the lungs [57] .

Regulatory T cells (Treg) play a role in negating the pathology mediated by CD8 T cells. In particular, they alleviate the severity of lung pathology. Treg depletion during RSV infection resulted in the increased number of CD8 T cells, elevated production of TNFα and IFNγ, and more pronounced lung pathology [58] . On the con trary, increasing Treg number led to the opposite effect [59] due to the upregulated production of the anti inflammatory cytokine IL 10. Neutralization of IL 10 with mAbs or the knockout of the corresponding gene restore lung pathology (table; Fig. 1a ) [60, 61] .

Recent studies have shown that T RM cells are able to provide the antiviral defense without inducing lung immunopathology. Kinnear et al. [62] intranasally administered T RM cells isolated from infected mouse lungs to na ve mice prior to the RSV infection, which substantially lowered the viral load in the lungs and alle viated lung pathology. In this case, CD8 T RM cells exhib ited the antiviral defense, whereas CD4 T RM cells did not affect RSV reproduction, but exhibited the anti inflam Refe rences [104] [107] Results the role of IL 33 in the RSV infection was demonstrated: expression of IL 13 and IL 33 in the lungs of neonatal mice was substantially higher than in adult ani mals; the content of pulmonary ILC2 cells in the neonatal mice was elevated 3 4 fold compared to adult animals; neutralization of IL 33 with specific mAbs in the neonatal mice resulted in the decreased number of ILC2 cells and lower expression of IL 13 and ameliorated lung pathology, but did not affect the viral load; administration of recombinant IL 33 during RSV infection aggravated pathological events in the respiratory tract; the knockout of ST2 gene (receptor for IL 33) fully abrogated the Th2 mediated lung immunopathology during RSV infection the gene encoding IRG1 factor and ROS were shown to contribute to the RSV induced lung inflammation and tissue damage: RSV infection activated IRG1 gene controlling ROS formation in the lungs; IRG1 knockdown with siRNA low ered ROS production in the lungs and abrogates inflammation [62] . It should be noted that formation of T RM cells in the lungs occurred after local (rather than systemic) immunization with viral antigens [49] . Most likely, local (e.g., aerosol) vaccination route will hold promise for preventing RSV infection.

It should be mentioned that the pro inflammatory cytokine TNFα is produced not only by CD8 T cells as a result of viral infection, but by macrophages as well. Macrophages isolated from TLR2 deficient mice pro duced much less TNFα after the virus dependent stimu lation compared to the macrophages isolated from the wild type mice [63] . Respiratory epithelial cells also release TNFα after virus stimulation, as it was shown in the in vitro study using anti TNFα mAbs [64] . Thus, like CD8 T cells, other cell types (macrophages and epithelial cells) are involved in the RSV induced lung immuno pathology.

The impact of humoral immunity. The role of adaptive humoral immunity in the RSV infection pathogenesis has not been examined in animal models due to the profound differences in the structure and functions of human and mouse antibodies. Numerous data have been obtained in the human RSV infection model (volunteers infected with the purified virus). It was found that class IgA anti bodies isolated from the nasal washings and serum IgG antibodies demonstrated the anti viral protection [65] , IgA antibodies exhibiting more prominent and long last ing anti RSV effect than IgG antibodies [66] .

The neutralizing antibodies are mainly produced against the F protein, which, according to the immuno logical mapping, has seven major antigenic sites: Ø, I, II, III, IV, V, and VIII. The sites Ø, V, and VIII exist solely in the prefusion conformation of the F protein [67, 68] . It was demonstrated that the majority of virus neutralizing antibodies (>60%) isolated from the B cells of patients recovered from the RSV infection were directed against the Ø, V [69] and VIII sites [68] . At the same time, anti bodies specific to the sites II, III, and IV possess much lower (100 to 1000 fold) virus neutralizing potential.

It may be due to the fact that the antigenic sites Ø, V and VIII are found in the prefusion rather than postfusion conformation of the F protein. Antibody binding to such sites stabilizes F protein, hindering its rearrangement into the postfusion conformation and preventing the virion cell fusion [67, 68] .

Palivizumab contains antibodies against the anti genic site II found in the postfusion F protein isoform. mAbs against the Ø, V, and VIII sites had a much higher (100 1000 fold) virus neutralizing potential compared with palivizumab (table) [68, 70] . New data on the anti genic structure of F protein will allow not only to create more efficient palivizumab analogues, but also to design efficacious RSV vaccines.

The neutralizing antibodies have been also developed against another surface viral glycoprotein -G protein, that target its central conserved domain [71] ; however, the soluble form of this protein may negate the antiviral effects of these antibodies (table) [72] .

IgE antibodies are also induced during the RSV infection, but they provide no antiviral effect, and, con versely, play an unfavorable role in the infection patho genesis. RSV specific IgE antibodies initiate histamine and leukotriene release from the mast cells, thereby elic iting inflammatory reactions [73] .

The role of interferons. Three types of interferon have been identified in humans: IFN I, IFN II, and IFN III, each of them exerting biological activity via binding to the cognate receptor. IFN I and IFN III are directly involved in the host antiviral defense [74, 75] . Interferon production is induced after interaction of RSV glycopro teins with the TLR molecules (TLR2, TLR6, TLR3, TLR4, and TLR7) found on the surface of leukocytes, plasmacytoid DCs, and alveolar macrophages [76] . The F protein is recognized by TLR4 that elicits IFN I produc tion [77] . The knockout of the TLR4 gene lowered mouse potential to eliminate RSV [76] . TLR2 and TLR6 recep tors are also involved in the recognition of RSV ligands. The knockout of the corresponding genes resulted in the elevated viral load (table; Fig. 2 ) [63] . Endosomal TLR3 and TLR7 receptors, as well as the cytosolic NLR family member Nod2, recognize viral RNA and trigger IFN I production [78 80] .

The role of interferons in the RSV infection patho genesis has been demonstrated in mouse models. Inactivation of the gene for the transcription factor STAT1 (involved in IFN I/II mediated signaling) result ed in the elevated viral load in the lungs, more severe BHR and mucus hypersecretion in the bronchial epithe lium, and increased lung infiltration by the pro inflam matory cells [81] (table; Fig. 2 ). Analysis of patients' bio logical samples showed that IFN I and IFN III become upregulated within the two days post infection [82] . It should be mention that type III interferons (IFN III) include four members: IFN λ1, IFN λ2, IFN λ3 (also known as IL 29, IL 28A, and IL 28B), and IFN λ4. Selvaggi et al. [82] demonstrated that children hospital ized with the verified RSV infection had a markedly upregulated expression of the IFN λ1-IFN λ3 genes in the nasal washings, while expression levels of IFN λ1 directly correlated with the disease severity and impaired lung function. At the same time, the impact of the RSV infection on the IFN λ4 expression in the bronchial epithelium has not been studied yet.

Compared to other respiratory viruses, RSV induces mild interferon response, especially in children [83] . The data obtained in the RSV infection model in the neonatal mice confirmed it: infection of mice at the age of 5 days vs. adult mice (6 8 weeks) induced no IFNα and IFNβ expression in the lungs, while administration of exoge nous IFNα abrogated pathological changes (table) [84] . Another study showed that IFNα administered BIOCHEMISTRY (Moscow) Vol. 86 No. 3 2021 intranasally in the neonatal mice before the infection resulted in a markedly decreased viral load in the lungs via elevated production of protective IgA antibodies in the respiratory tract mucosa (table) [85] .

On the one hand, aberrant IFN response in early age may be accounted for by a low number of plasmacytoid DCs and macrophages, which are the major source of IFN I in the respiratory tract [84, 86] . The data obtained in mouse models corroborate the results of the large scale INFANT clinical study showing that in children, the RSV infection does not induce any prominent IFN response in the respiratory tract mucosa [87] . The aforementioned studies demonstrate the similarity between the human and mouse immune responses to RSV, thereby justifying the use of mouse models to study the properties of innate immunity in humans. IFNγ not only participates in the pathology development, but also accounts for the host antiviral defense. RSV induces necrosis in the respiratory epithelium, resulting in the release of cytokines IL 33 and thymic stromal lym phopoietin (TSLP) that activate ILC2 cells producing their own cytokines (IL 5 and IL 13). IL 5 and IL 13 are involved in the development of pathological manifestations by causing pulmonary eosinophilic inflammation, mucus hypersecretion, and BHR. Polarization of Th2 and Th17 cells occurs during RSV infection due to specific cytokine microenvironment. Th2 cells produce cytokines IL 4, IL 5, and IL 13, which trigger BHR, mucus hypersecretion, and pulmonary eosinophilic inflammation, whereas Th17 cells secrete IL 17A that induces pulmonary neutrophilic inflammation and mucus hypersecretion. Immunoregulatory functions are executed by the cytokine IL 10 released by regulato ry T cells (Treg). IL 10 suppresses the pro inflammatory activity of CD8 T cells. Similar activity was described for IL 6 and IL 27, which are able to directly suppress CD8 T cells and activate Treg. RSV is recognized by surface TLR proteins on the macrophages (MPhs) and plasma cytoid DCs (pDCs) producing limited amounts of type I interferons (IFN I) with the antiviral activity.

Despite a large body of accumulated data on the antiviral activity of IFN I and IFN III, their efficacy for the anti RSV therapy remains controversial due to the ability of RSV to evade the IFN response [48] . In partic ular, two viral nonstructural proteins (NS1 and NS2) bind to the cytosolic RIG I receptor inside the infected cells and suppress the downstream signaling pathways. Moreover, it was found that the viral G protein is also involved in the inhibition of the IFN related antiviral effects via suppressor proteins SOCS1 and SOCS3, which are activated during RSV infection through the TLR dependent pathway and block IFN production [88] . Therefore, RSV deploys several mechanisms not only to block the antiviral signaling from the IFN receptors, but also to suppress production of endogenous IFNs, thereby limiting their medical application for treating the RSV infection. In some cases, IFN may even promote more severe course of RSV infection [82] .

The impact of cytokines. RSV infection leads to the upregulated production of some pro inflammatory cytokines (IFNγ, TNFα, IL 4, IL 6, IL 8, IL 17A, etc.) both during the natural infection in humans [89] and in mouse models [39] . The majority of pro inflammatory cytokines are secreted by the virus specific CD8 T cells, as well as by macrophages, epithelial cells and ILC2 cells [39, 45, 52] .

Patients with the RSV infection have elevated IFNγ levels in nasal washings and blood serum [89] . Moreover, severe infection course is associated with low IFNγ levels [90] . Similar data were obtained in mice. Thus, the intranasal delivery of IFNγ in mice promoted virus clear ance in the lungs [91] , and conversely, ablation of the IFNγ gene resulted in more pronounced RSV replication (table; Fig. 2 ) [92] . Taken together, these data suggest the protective antiviral effect of IFNγ. However, this cytokine may also play an unfavorable pathogenetic role, as its administration during RSV infection aggravated inflam mation and caused bronchial obstruction in mice (table; Fig. 2 ) [91] , whereas IFNγ neutralization with specific mAbs resulted in the reduced lung immunopathology [52] . Hence, IFNγ exerts protective effects during RSV infection, but its role in the development of lung immunopathology remains controversial.

TNFα also affects lung immunopathology during the RSV infection. In particular, its neutralization with mAbs does not affect RSV replication, but reduces lung inflammation (Fig. 2) [52] .

The production of both pro inflammatory and regu latory (e.g., IL 10) cytokines is increased during the RSV infection. Thus, upregulated IL 10 expression was observed in the mouse models [61] and clinical samples [89] . IL 10 is mainly secreted by Treg cells [61] and exhibit the anti inflammatory effects that may partially ameliorate the virus induced inflammation in the respi ratory tract. Mice deficient by the IL10 gene experience more severe pulmonary inflammation during the RSV infection, because IL 10 suppresses production of the pro inflammatory cytokines IFNγ and TNFα by CD8 T cells (table; Fig. 2 ) [61] . Similar observations were obtained in clinical practice, indicating that downregula tion of the IL10 gene and genes involved in the IL 10 sig naling pathway correlates with the disease severity [93] .

It was also found in mouse models that the IL 4 pro duction was associated with the RSV infection severity [94] . Similar data were obtained by assessing nasal wash ings from patients with the RSV infection [95] . In con trast, neutralizing IL 4 with mAbs abrogated infection related inflammation (Fig. 2) [94] , because this cytokine suppresses the antiviral cytotolytic activity of CD8 T cells [96] . Another Th2 cytokine -IL 13 -is involved in the development of mucus hypersecretion and BHR; knock ing out the IL 13 gene abolished these infection symp toms (table; Fig. 2 ) [41] . Apparently, IL 13 production by ILC2 cells is regulated by TSLP, because its neutraliza tion downregulates IL 13 expression and reduces mucus hypersecretion (table; Fig. 2 ) [97] .

The cytokine IL 17A, which is produced mainly by Th17 cells, induces pulmonary neutrophil inflammation and mucus hypersecretion in mice infected with RSV (table; Fig. 2 ) [98] . Secretion by the epithelial cells and macrophages of another pro inflammatory cytokine, IL 6 (necessary for Th17 skewing), is also elevated during the RSV infection. Despite the pro inflammatory properties of IL 6, its neutralization by mAbs prior to the infection resulted in the aggravated pathological process. This might be due to the fact that IL 6 neutralization is asso ciated with the increase in the number of virus specific CD8 + T cells in the lungs and, consequently, with the increased production of the pro inflammatory cytokines TNFα and IFNγ, which is accompanied by the decreased secretion of regulatory cytokines IL 10 and IL 27, as well as significant reduction in the IL 17A concentration (table; Fig. 2 ) [99] . Based on these results, no clinical tri als assessing the efficacy of anti IL 6 and anti IL 17 therapy in RSV infection have been conducted. Likewise, the IL 27 level is upregulated during murine RSV infec tion, but its inactivation with specific mAbs results in the aggravated lung pathology due to the increased number of CD8 + T cells producing IFNγ and TNFα. Hence, IL 6 and IL 27 play a similar role in the RSV infection immunopathogenesis by decreasing the activity of CD8 + T cells and activating Tregs (table; Fig. 2 ) [99] .

Despite the lack of studies on the clinical efficacy of anti IL 6 therapy in patients with the RSV infection, a number of papers have been published recently on the use of anti IL 6 mAbs (Tocilizumab, Sarilumab, and Siltuximab) for treating COVID 19 patients; however, none of the 10 clinical trials demonstrated prominent favorable effects of such therapy [100, 101] . The use of anti IL 17 mAbs in the COVID 19 therapy has been also discussed [102] , although no related publications are available so far. Anti IL 17 mAbs were applied to treat BIOCHEMISTRY (Moscow) Vol. 86 No. 3 2021 pulmonary neutrophil inflammation in moderate to severe bronchial asthma, but no clinical efficacy was demonstrated for Brodalumab (mAbs targeting the IL 17RA subunit of the receptor for IL 17A, IL 17F, and IL 25). Examination of another anti IL 17 preparation (anti IL 17A mAb CJM112) is currently underway [103] .

Recently, the role of epithelium produced cytokines (IL 33 and TSLP) in the RSV infection pathogenesis has been revealed. It was found that neonatal mice demon strated an elevated expression of the pro inflammatory cytokine IL 33 in the lung tissue [104] . IL 33 activates ILC2 cells that produce large amounts of IL 13, which contributes to the development of type 2 immune response against the RSV infection. IL 33 neutralization or knockout of the gene for its cognate receptor resulted in the alleviated lung pathology, but did not impact the viral replication (table; Fig. 2 ) [104] . Similar data were obtained in our studies: IL 33 silencing by siRNA result ed in the reduced lung pathology, without affecting the viral load [105] . TSLP as another bronchial epithelial cytokine is also involved in the RSV induced pulmonary pathology, and its inactivation lowers the number of ILC2 cells in the respiratory tract [97] .

Reactive oxygen species. A convincing body of evi dence shows that ROS serve as crucial cues in lung inflammation and tissue damage (mainly by affecting bronchial epithelium) in the RSV infection. In 2004, it was demonstrated that RSV triggers production of ROS that induce the inflammatory cascade via activating STAT transcription factors in the epithelial cells [106] . The IRG1 gene is involved in the ROS regulation. RSV infec tion of the bronchial cells in vitro resulted in the IRG1 activation and elevated ROS production. These data were corroborated in the in vivo experiments in RSV infected mice. Thus, the IRG1 gene knockdown with specific siRNA lowered ROS production, which reduced lung inflammation (table) [107] .

Despite obvious similarities in the features of RSV infection in humans and mice, there are substantial dif ferences. Mouse models are often criticized for the fact that RSV is not a natural mouse pathogen, which accounts for its poor replication in the mouse respiratory tract. Hence, pathological changes are induced only after infecting mice with a high dose of the virus. Moreover, human RSV infection is often accompanied by a marked neutrophilic inflammation of the respiratory tract, so that the percentage of neutrophils in the BAL samples reach es up to 80%, whereas in mice, these leukocytes do not dominate and comprise no more than 20%. Natural human RSV infection induces both Th1 and Th2 dependent immune responses, which are accompanied by the eosinophilic infiltration of the respiratory tract, whereas in mice, it triggers solely Th1 immune response, so that the lungs virtually lack eosinophils [48] .

Despite the aforementioned limitations, modeling the RSV infection in mice allows to reproduce main clin ically relevant pathological symptoms, such as BHR, mucus hypersecretion, and infiltration of pro inflamma tory cells (mainly lymphocytes and macrophages) into the lungs. The studies conducted using such in vivo mod els contribute to the understanding molecular and cellu lar mechanisms of human RSV pathology.

Funding. The study was supported by the Russian Science Foundation (project no. 18 74 10002) .

Ethics declarations. The authors declare no conflict of interests. This article does not contain description of studies with the involvement of humans or animal sub jects.

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