WO2007136789A2

WO2007136789A2 - Methods of preventing, treating or ameliorating enhanced respiratory disease via innate immune mechanisms

Info

Publication number: WO2007136789A2
Application number: PCT/US2007/011972
Authority: WO
Inventors: Jennifer Lynne Reed
Original assignee: Medimmune, Inc.
Priority date: 2006-05-19
Filing date: 2007-05-18
Publication date: 2007-11-29
Also published as: WO2007136789A3

Abstract

The present invention encompasses methods of preventing, treating or ameliorating bronchiolitis, ARDS, or asthma or a symptom thereof, the method comprising administering to a patient an effective amount of an anti-inflammatory, wherein the anti-inflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, an isolated anti-CD 19 monoclonal antibody or fragment thereof, an isolated anti-CD20 monoclonal antibody or fragment thereof, an isolated anti-CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti-BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist that may further be in combination with an anti-viral, wherein said anti-viral is one or more antibodies or fragments thereof that immunospecifically bind to one or more RSV antigens or is a synthetic anti-viral compound.

Description

Methods of Preventing, Treating or Ameliorating Enhanced Respiratory Disease via

Innate Immune Mechanisms

Background of the Invention Innate Immune System

(0001] Host defenses to microbial invasion include the rapidly developing antigen-independent or innate immunity and the much more slowly developing specific or adaptive immunity. Innate immune responses are triggered by bacteria, viruses, protozoa, and fungi, as non-self, and involve nonspecific activation of neutrophils, monocytes and macrophages, dendritic cells (DCs), natural killer (NK) cells, and complement. Innate recognition of pathogens is the first step in inducing adaptive immunity.

|0002] Phagocytosis as a mechanism of innate immune defense has served as the classical model for studying host-parasite interactions. An important humoral component of innate immunity is the activation of the complement system, which can lead to antibody-independent opsonization and opsonophagocytosis.

10003] Recently, Toll-like receptors (TLRs) have emerged as central points of innate immunity. TLRs represent a conserved family of immune, receptor sensing molecules on a wide variety of pathogens. These receptors recognize pathogen- associated molecular patterns, which results in activation of NF-κB and other transcription factors including interferon (IFN) regulatory factors. TLRs are expressed on the surface of monocytes, macrophages, DCs, and epithelial cells or in the cytoplasm of cells from different tissues. Ligand binding to innate receptors generates intracellular signals, initiates gene activation, and enhances the release of cytokines and chemokines at the site of immune activation. Chemokines recruit innate immune effector cells such as granulocytes, monocytes, macrophages, and NK cells.

|0004] Human neonates and young infants are more vulnerable to infectious agents than older children and adults and are especially susceptible to infections with intracellular pathogens. Some of the pathogens causing infections in utero, intrapartum, and postpartum evoke fetal and neonatal innate immune responses. Innate immunity against pathogens represents the critical first-line barrier of host defenses, as newborns have a naϊve adaptive immune system. Tt is suggested that neonatal innate responses may not be fully developed, allowing early dissemination of infections which can be responsible for significant morbidity and mortality in newborns. A better understanding of molecular mechanisms that underlie neonatal immune functions may improve the ability to prevent and treat neonatal infections. Bronchiolitis [0005] Bronchiolitis is a common illness of the respiratory tract caused by a respiratory infection that affects the small airways, called the bronchioles, that lead to the alveoli, where gas exchange occurs. As bronchioles become inflamed, they swell and become occluded, making it difficult for a child to breathe.

|0006] Why this illness affects infants and young children most often is incompletely understood, but is most likely linked to the smaller size of airways which can become blocked more easily than those of older children or adults. Bronchiolitis typically occurs during the first 2 years of life, with the peak occurrence at about 3 to 6 months of age. It's more common in males, children who have not been breastfed, and children who live in crowded conditions. Day-care attendance and exposure to cigarette smoke can also increase the likelihood that an infant will develop bronchiolitis.

|0007j _* Although it's often a mild illness, some infants are at risk for a more severe disease that requires hospitalization. Conditions that increase the risk of severe infection include prematurity, prior chronic heart or lung disease, and a weakened immune system due to illness or medications. Children who have had bronchiolitis may be more likely to develop asthma later in life, but it's unclear whether bronchiolitis causes or triggers asthma, or whether children who eventually go on to develop asthma were simply more prone to developing bronchiolitis as infants.

[0008] Bronchiolitis frequently results from a mild upper respiratory infection that, over a period of 2 to 3 days, can develop into increasing respiratory distress with wheezing and a "tight," wheezy cough. As the effort of breathing increases (tachypnea), parents may see the nostrils flaring with each breath and the muscles between the ribs retracting (intercostal retractions) as the child tries to inhale air. This can be exhausting for the child, and very young infants may become so fatigued that breathing becomes difficult to maintain. As a result, the infant may become irritable or anxious-looking. Tf the disease is severe enough, the infant may turn bluish (cyanotic), an indication of a critical emergency. 2007/011972

|00091 Bronchiolitis is an inflammation of the bronchioles (small passages in the lungs) usually caused by a viral infection, most commonly respiratory syncytial virus (RSV). RSV infections are responsible for more than half of all cases of the illness and are most widespread in the winter and early spring. Other viruses that can cause bronchiolitis include: parainfluenza (PIV), particularly PIV-3, metapneumovirus (MPV), influenza and adenovirus (i.e., non-RSV associated bronchiolitis).

|0010] RSV has a direct cytopathic effect on cells in the lung epithelium, leading to loss of specialized functions such as cillial motility and sometimes to epithelial destruction. In addition, a peribronchiolar mononuclear cell infiltrate forms and is accompanied by airway obstruction with patchy atelectasis and areas of compensatory emphysema. Syncytium formation is not often seen in vivo and varies considerably from one RSV strain to another in vitro. In explanted human epithelial cultures, RSV infects the apical surface of ciliated columnar cells, is shed exclusively from the luminal surface, and spreads to neighboring cells by cillial motion.

Acute Respiratory Distress Syndrome (ARDS)

10011] Acute respiratory distress syndrome (ARDS) is a syndrome of severe respiratory failure associated with the presence of pulmonary edema in the absence of volume overload or depressed left ventricular function. This is distinguished from acute lung injury, which is similar to ARDS with the exception of a PaO 2 /FIO 2 ratio of less than 300 mm Hg.

[0012] ARDS occurs in children as well as adults. The condition originates from a number of insults involving damage to the alveolocapillary membrane with subsequent fluid accumulation within the airspaces of the lung. Histologically, these changes have been termed diffuse alveolar damage.

[0013] The development of ARDS starts with damage to the alveolar epithelium and vascular endothelium resulting in increased permeability to plasma and inflammatory cells into the interstitium and alveolar space. Damage to the surfactant- producing type II cells and the presence of protein-rich fluid in the alveolar space disrupts the production and function of pulmonary surfactant leading to microatelectasis and impaired gas exchange. 11972

|0014] Some patients have an uncomplicated course and rapid resolution, whereas others may progress to fibrosing alveolitis, which involves the deposition of collagen in alveolar, vascular, and interstitial spaces leading to poor lung compliance. Fibrosing alveolitis has been reported histologically as early as 5-7 days. |0015] The mortality rate is approximately 30-40%. Deaths usually result from multisystem organ failure rather than lung failure alone.

[0016] There are a number of clinical conditions are associated with development of ARJDS, however, the most common predisposing factors are sepsis and the systemic inflammatory response syndrome (SIRS). These conditions may result from the indirect toxic effects of neutrophil-derived inflammatory mediators in the lungs.

Asthma

[0017] About 12 million people in the U.S. have asthma and it is the leading cause of hospitalization for children. The Merck Manual of Diagnosis and Therapy (17th ed., 1999).

|0018] Asthma is an inflammatory disease of the lung that is characterized by airway hyperresponsiveness ("AHR"), bronchoconstriction (i.e., wheezing), eosinophilic inflammation, mucus hypersecretion, subepithelial fibrosis, and elevated IgE levels. Asthmatic attacks can be triggered by environmental triggers (e.g., acarids, insects, animals (e.g., cats, dogs, rabbits, mice, rats, hamsters, guinea pigs, mice, rats, and birds), fungi, air pollutants (e.g., tobacco smoke), irritant gases, fumes, vapors, aerosols, chemicals, or pollen), exercise, or cold air. The cause(s) of asthma is not fully elucidated. It has been speculated that family history of asthma (London et al., 2001 , Epidemiology 12(5):577-83), early exposure to allergens, such as dust mites, tobacco smoke, and cockroaches (Melen et al., 2001, 56(7):646-52), and respiratory infections (Wenzel et al., 2002, Am J Med, 1 12(8):672-33 and Lin et al., 2001 , J Microbiol Immuno Infect, 34(4):259-64), such as RSV₅ may increase the risk of developing asthma. A review of asthma, including risk factors, animal models, and inflammatory markers can be found in O'Byrne and Postma (1999), Am. J. Crit. Care. Med. 159:S41 -S66, which is incorporated herein by reference in its entirety.

|0019] Current therapies are mainly aimed at managing asthma and include the administration of β-adrenergic drugs (e.g., epinephrine and isoproterenol), US2007/011972

theophylline, anticholinergic drugs (e.g., atropine and ipratorpium bromide), corticosteroids, and leukotriene inhibitors. These therapies are associated with side effects such as drug interactions, dry mouth, blurred vision, growth suppression in children, and osteoporosis in menopausal women. Cromolyn and nedocromil are administered prophylatically to inhibit mediator release from inflammatory cells, reduce airway hyperresponsiveness, and block responses to allergens. However, there are no current therapies available that prevent the development of asthma in subjects at increased risk of developing asthma. Thus, new therapies with fewer side effects and better prophylactic and/or therapeutic efficacy are needed for asthma.

Wheezing

(00201 Wheezing (also known as sibilant rhonchi) is generally characterized by a noise made by air flowing through narrowed breathing tubes, especially the smaller, tight airways located deep within the lung. It is a common symptom of RSV infection, and secondary RSV conditions such as asthma and bronchiolitis. The clinical importance of wheezing is that it is an indicator of airway narrowing, and it may indicate difficulty breathing.

[00211 Wheezing is most obvious when exhaling (breathing out), but may be present during either inspiration (breathing in) or exhalation. Wheezing most often comes from the small bronchial tubes (breathing tubes deep in the chest), but it may originate if larger airways are obstructed.

Bronchiolitis obliterans (OB)

10022] Bronchiolitis obliterans (OB) describes an intraluminal polypoid plug of granulation tissue found within the terminal and respiratory bronchioles. OB is initiated by damage or chronic scarring process to the epithelium of the small conducting airways and progresses to inflammation of the airways, frequently to the adjacent alveolar tissue as well. The clinical -consequence of this injury and inflammation is a progressive obliteration of the small airways with resultant obstructive lung disease or irreversible airway obstruction. Histologic examination can show bronchiolitis obliterans with marked intraluminial proliferation of fibrous tissue. OB is seen in most infectious pneumonias, diffuse alveolar damage, aspiration, usual interstitial pneumonia, cryptogenic organizing pneumonia, among other conditions, and therefore is often thought of as a symptom of bronchiolitis itself.

(0023) The most common symptoms of OB include cough, dyspnea and fever. Exposure to very high levels of oxides of nitrogen may cause acute respiratory failure. Following recovery, symptoms may reoccur in 3 to 6 weeks.

Respiratory Syncytial Virus (RSV)

[0024] Virus families containing enveloped single-stranded RNA of the negative-sense genome are classified into groups having non-segmented genomes (Paramyxoviridae, Rhabdoviridae) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). Paramyxoviridae have been classified into three genera: paramyxovirus (sendai virus, parainfluenza viruses types 1 -4, mumps, newcastle disease virus); morbillivirus (measles virus, canine distemper virus and rinderpest virus); and pneumovirus (respiratory syncytial virus and bovine respiratory syncytial virus).

[0025] RSV possesses a single-stranded nonsegmented negative-sense

RNA genome of 15,221 nucleotides (Collins, 1991 , In The Paramyxoviruses pp. 103-162, D.W. Kingsbury (ed.) Plenum Press, New York). The genome of RSV encodes 10 mRNAs (Collins et al., 1984, J. Virol. 49: 572-578). The genome contains a 44 nucleotide leader sequence at the 3' termini followed by the NS1-NS2-N-P-M-SH-G-F- M2-L and a 155 nucleotide trailer sequence at the 5' termini (Collins. 1991 , supra). Each gene transcription unit contains a short stretch of conserved gene start (GS) sequence and a gene end (GE) sequences. Two antigenically diverse RSV subgroups A and B are present in human populations. [0026J The viral genomic RNA is not infectious as naked RNA. The RNA genome of RSV is tightly encapsidated with the major nucleocapsid (N) protein and is associated with the phosphoprotein (P) and the large (L) polymerase subunit. These proteins form the nucleoprotein core, which is recognized as the minimum unit of infectivity (Brown et al., 1967, J. Virol. 1 : 368-373). The RSV N, P, and L proteins form the viral RNA dependent RNA transcriptase for transcription and replication of the RSV genome (Yu et al., 1995, J. Virol. 69:2412-2419; Grosfeld et al., 1995, J. Virol. 69:5677- 7 011972

86). Recent studies indicate that the M2 gene products (M2-1 and M2-2) are involved and are required for transcription (Collins et al., 1996, Proc. Natl. Acad. Sci. 93 :81-5).

[0027] The M protein is expressed as a peripheral membrane protein, whereas the F and G proteins are expressed as integral membrane proteins and are involved in virus attachment and viral entry into cells. The G and F proteins are the major antigens that elicit neutralizing antibodies in vivo (as reviewed in Mclntosh and Chanock, 1990 "Respiratory Syncytial Virus" 2nd ed. Virology (D. M. Knipe et al., Ed.) Raven Press, Ltd., N.Y.). Antigenic dimorphism between the subgroups of RSV A and B is mainly linked to the G glycoprotein, whereas the F glycoprotein is more closely related between the subgroups.

[0028] Respiratory syncytial virus (RSV) is one of the leading causes of respiratory disease worldwide. In the United States, it is responsible for tens of thousands of hospitalizations and thousands of deaths per year (see Black, CP. , Resp. Care 2003 48(3):209-31 for a recent review of the biology and management of RSV). Infants and children are most at risk for serious RSV infections which migrate to the lower respiratory system, resulting in pneumonia or bronchiolitis. In fact, 80% of childhood bronchiolitis cases and 50% of infant pneumonias are attributable to RSV. The virus is so ubiquitous and highly contagious that almost all children have been infected by two years of age. Although infection does not produce lasting immunity, re-infections tend to be less severe so that in older children and healthy adults RSV manifests itself as a cold or flu-like illness affecting the upper and/or lower respiratory system, without progressing to serious lower respiratory tract involvement. However, RSV infections can become serious in elderly or immunocompromised adults. (Evans, A.S., eds., 1989, Viral Infections of Humans. Epidemiology and Control, 3rd ed., Plenum Medical Book, New York at pages 525-544; Falsey, A. R., 1991 , Infect. Control Hosp. Epidemiol. 12:602-608; and Garvie et al., 1980, Br. Med. J. 281 :1253-1254; Hertz et al., 1989, Medicine 68:269- 281 ).

[0029| The goal of RSV immunoprophylaxis is to induce sufficient resistance to prevent the serious disease which may be associated with RSV infection. The current strategies for developing RSV vaccines principally revolve around the administration of purified viral antigen or the development of live attenuated RSV for intranasal administration. However, to date there have been no approved vaccines or highly effective synthetic antiviral compound therapy for RSV. Recent clinical data has failed to support the early promise of the antiviral compound ribavirin, which is approved for the treatment of RSV infection (Black, C.P., Resp. Care 2003 48(3):209-31). Consequently, the American Academy of Pediatrics issued new guidelines suggesting that use of ribavirin be restricted to only the most severe cases (Committee on Infectious Disease, American Academy of Pediatrics. 1996. Pediatrics 97:137-140; Randolph, A. G., and E.E. Wang., 1996, Arch. Pediatr. Adolesc. Med. 150:942-947).

|0030| A formalin-inactivated virus vaccine failed to provide protection against RSV infection and exacerbated symptoms during subsequent infection by the wild-type virus in infants (Kapikian et ah, 1969, Am. J. Epidemiol. 89:405-21 ; Chin et al., 1969, Am. J. Epidemiol. 89:449-63) Efforts since have focused on developing live attenuated temperature-sensitive mutants by chemical mutagenesis or cold passage of the wild-type RSV (Gharpure et al., 1969, J. Virol. 3: 414-21 ; Crowe et al., 1994, Vaccine 12: 691 -9). However, earlier trials yielded discouraging results with these live attenuated temperature sensitive mutants. Virus candidates were either underattenuated or overattenuated (Kim et al., 1973, Pediatrics 52:56-63; Wright et al., 1976, J. Pediatrics 88:931-6) and some of the vaccine candidates were genetically unstable which resulted in the loss of the attenuated phenotype (Hodes et al., 1974, Proc. Soc. Exp. Biol. Med. 145:1158-64). 10031] Attempts have also been made to engineer recombinant vaccinia vectors which express RSV F or G envelope glycoproteins. However, the use of these vectors as vaccines to protect against RSV infection in animal studies has shown inconsistent results (Olmsted et al. 1986, Proc. Natl. Acad. Sci. 83:7462-7466; Collins et al., 1990, Vaccine 8: 164-168). |00321 While a commercially available vaccine is not yet available, success has been achieved in the area of prevention for infants at high risk of serious lower respiratory tract disease caused by RSV, as well as a reduction of LRl. In particular, there are two immunoglobulin-based therapies approved to protect high-risk infants from serious LRl: RSV-IGIV (RSV-immunoglobulin intravenous, also known as RespiGam™) and palivizumab (SYN AGIS®). However, neither RSV-IGIV nor palivizumab has been approved for use other than as a prophylactic agent for serious lower respiratory tract (LRT) acute RSV disease. Influenza Virus

[0033] Influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein. Influenza A and B viruses each contain eight segments of single stranded RNA with negative polarity. The influenza A genome encodes at least eleven polypeptides. Segments 1-3 encode the three polypeptides, making up the viral RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2. The remaining polymerase proteins PBl and PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza A strains encodes a small protein, PBl -F2, produced from an alternative reading frame within the PBl coding region. Segment 4 encodes the hemagglutinin (HA) surface glycoprotein involved in cell attachment and entry during infection. Segment 5 encodes the nucleocapsid nucleoprotein (NP) polypeptide, the major structural component associated with viral RNA. Segment 6 encodes a neuraminidase (NA) envelope glycoprotein. Segment 7 encodes two matrix proteins, designated Ml and M2, which are translated from differentially spliced mRNAs. Segment 8 encodes NSl and NS2 (NEP), two nonstructural proteins, which are translated from alternatively spliced mRNA variants.

|0034] The eight genome segments of influenza B encode 1 1 proteins. The three largest genes code for components of the RNA polymerase, PBl, PB2 and PA. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both proteins, NB and NA, are translated from overlapping reading frames of a biscistronic mRNA. Segment 7 of influenza B also encodes two proteins: M l and BM2. The smallest segment encodes two products: NSl is translated from the full length RNA, while NS2 is translated from a spliced mRNA variant.

10035] Vaccines capable of producing a protective immune response specific for influenza viruses have been produced for over 50 years. Vaccines can be characterized as whole virus vaccines, split virus vaccines, surface antigen vaccines and live attenuated virus vaccines. While appropriate formulations of any of these vaccine types is able to produce a systemic immune response, live attenuated virus vaccines are also able to stimulate local mucosal immunity in the respiratory tract. 2007/011972

[0036] FluMist™ is a live, attenuated vaccine that protects children and adults from influenza illness (Belshe et al. (1998). The efficacy of live attenuated, cold- adapted, trivalent, intranasal influenza virus vaccine in children, N Engl J Med 338:1405-12; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282:137- 44). FluMist™ vaccine strains contain HA and NA gene segments derived from the currently circulating wild-type strains along with six gene segments, PBl, PB2, PA, NP, M and NS, from a common master donor virus (MDV). The MDV for influenza A strains of FluMist (MDV-A), was created by serial passage of the wt A/Ann Arbor/6/60 (A/AA/6/60) strain in primary chicken kidney tissue culture at successively lower temperatures (Maassab (1967) Adaptation and growth characteristics of influenza virus at 25"C Nature 213:612-4). MDV-A replicates efficiently at 25 "C (ca, cold adapted), but its growth is restricted at 38 and 39 "C (ts, temperature sensitive). Additionally, this virus does not replicate in the lungs of infected ferrets (att, attenuation). The ts phenotype is believed to contribute to the attenuation of the vaccine in humans by restricting its replication in all but the coolest regions of the respiratory tract. The stability of this property has been demonstrated in animal models and clinical studies. In contrast to the ts phenotype of influenza strains created by chemical mutagenesis, the ts property of MDV-A did not revert following passage through infected hamsters or in shed isolates from children (for a recent review, see Murphy & Coelingh (2002) Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines Viral Immunol 15:295-323).

|00371 Clinical studies in over 20,000 adults and children involving 12 separate 6:2 reassortant strains have shown that these vaccines are attenuated, safe and efficacious (Belshe et al. (1998) The efficacy of live attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine in children N Engl J Med 338:1405-12; Boyce et al. (2000) Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines administered intranasally to healthy adults (Vaccine 19:217-26; Edwards et al. (1994) A randomized controlled trial of cold adapted and inactivated vaccines for the prevention of influenza A disease J Infect Dis 169:68-76 ; Nichol et al. (1999) Effectiveness of live, attenuated intranasal influenza virus vaccine in healthy, working adults: a randomized controlled trial JAMA 282:137-44). Reassortants carrying the six internal genes of MDV-A and the two HA and NA gene segments of the wt virus (6:2 reassortant) consistently maintain ca, ts and att phenotypes (Maassab et al. (1982) Evaluation of a cold-recombinant influenza virus vaccine in ferrets J Infect Dis 146:780- 900).

Human Metapneumovirus (hMPV)

[0038] Recently, a new member of the Paramyxoviridae family has been isolated from 28 children with clinical symptoms reminiscent of those caused by human respiratory syncytial virus ("hRSV") infection, ranging from mild upper respiratory tract disease to severe bronchiolitis and pneumonia (Van Den Hoogen et al., 2001 , Nature Medicine 7:71 9-724). The new virus was named human metapneumovirus (hMPV) based on sequence homology and gene constellation. The study further showed that by the age of five years virtually all children in the Netherlands have been exposed to hMPV and that the virus has been circulating in humans for at least half a century. Additionally, the seasonality of the infection is similar to RSV, peaking in the winter months (Robinson, 2005, J. Med. Virol. 76:98-105; Williams, 2004, New Engl. J. Med. 350:443- 450). However, unlike RSV, hMPV can be isolated year-round, albeit at a lower rate (Robinson, 2005, J. Med. Virol. 76:98-105; Williams, 2004, New Engl. J. Med. 350:443- 450). Risk factors for hMPV infection are also similar to those found for RSV. Highest incidence of infection with human metapneumovirus has been found in young children, in the elderly and immunocompromised humans. Infection with human metapneumovirus is a significant burden of disease in at-risk premature infants, chronic lung disease of prematurity, congestive heart disease, and immunodeficiency (Robinson, 2005, J. Med. Virol. 76:98-105; Williams, 2004, New Engl. J. Med. 350:443-450). [0039] The genomic organization of human metapneumovirus is described in van den Hoogen et al., 2002, Virology 295:1 19-132. Human metapneumovirus has recently been isolated from patients in North America (Peret et al., 2002, J. Infect. Diseases 185: 1660-1663).

[0040J hMPV shares a similar genetic structure to RSV but lacks the non- structural genes found in RSV (van den Hoogen, 2002, Virology. 295:1 19-132). Both viruses code for similar surface proteins that are defined as the surface glycoprotein (G) protein and the fusion (F) protein. Based upon differences between the amino acid sequences of the G and F proteins, both RSV and hMP V have been subdivided into A and B groups. However, in hMPV there is a further bifurcation of A and B subgroups into Al, A2, Bl , and B2 groupings (Boivin, 2004, Emerg. Infect. Dis.l 0:1 154-1157, 25). For both RSV and hMPV viruses, the sequences of the G proteins display a wide variance between subgroups; with hMPV the G protein has only 30% identity between A and B subgroups. For both RSV and hMPV the F protein is more conserved; across the known hMPV isolates the F protein amino acid sequence is 95% conserved (Biacchesi, 2003, Virology 315:1 -9; Boivin, 2004, Emerg. Infect. Dis.lO:l 154-1 157; van den Hoogen, 2004, Emerg. Infect. Dis. 10:658-666) . Despite the similarities in structure of the viruses, the F proteins of hMPV and RSV share only a 33% amino acid sequence identity and antisera generated against either RSV or hMPV do not neutralize across the pneumoviridae group (Wyde, 2003, Antiviral Research. 60:51 -59). With RSV a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection^'. Similarly, because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies.

Inflammatory Responses in Respiratory Disease

|0041] It is increasingly appreciated that symptoms and signs of many viral diseases are caused less by viral cytopathic effects than by the host's response to infection. The peak of viral infection often precedes the period of maximal illness, which coincides with cellular infiltration of infected tissues and the release of inflammatory mediators. In particular, RSV bronchiolitis is thought to arise in part from excessive immune responses to RSV virus infection, resulting in lung tissue destruction. |0042] Inflammation is often induced by proinflammatory cytokines, such as type 1 interferons, tumor necrosis factor (TNF), interleukin (IL)-I a, IL-I β, IL-6, platelet- activating factor (PAF), macrophage migration inhibitory factor (MIF), and other compounds. These proinflammatory cytokines are produced by several different cell types, most importantly immune cells (for example, monocytes, macrophages and neutrophils), but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, and neurons. These proinflammatory cytokines contribute to various disorders during the early stages of an inflammatory cytokine cascade. I0043J Inflammatory cytokine cascades contribute to deleterious characteristics, including inflammation and apoptosis, of numerous disorders. Included are chronic and acute disorders characterized by both localized and systemic reactions, including, without limitation, diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, COPD, acute respiratory distress syndrome, pneumoultramicroscopicsilico-volcanoconiosis, alvealitis, bronchiolitis, non-RSV associated bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as, for example, influenza, respiratory syncytial virus, parainfluenza virus (PIV), metapneumovirus (MPV), HIV, hepatitis B virus, hepatitis C virus and herpes).

[0044] Cysteinyl leukotrienes (CysLTs) are bioactive lipids that have been shown to contribute to allergic and inflammatory diseases. Eosinophils and mast cells have the capacity to produce large amounts of CysLTs after allergic or non-allergic stimulation. Molecular identification of both the synthetic and signalling proteins in the CysLT pathway allows the investigation of expression of the CysLT enzymes and receptors in active allergic rhinitis. CysLTs are potent chemoattractants for eosinophils and have direct actions on smooth muscle and other cell type which contribute to airways hyperreactivity.

Summary of the Invention

[0045] The present invention contemplates the following embodiments:

[0046] A method of preventing, treating or ameliorating bronchiolitis or a symptom thereof, the method comprising administering to a patient an effective amount of an anti-inflammatory. ]0047] A composition comprising an effective amount of an antiinflammatory, wherein said composition is sterile and used for preventing, treating or ameliorating bronchiolitis or a symptom thereof, bronchiolitis obliterans or a symptom thereof, or the development of asthma or a symptom thereof.

[0048] The composition of the above, wherein said anti -inflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment US2007/011972

thereof, an isolated anti-CD19 monoclonal antibody or fragment thereof, an isolated anti- CD20 monoclonal antibody or fragment thereof, an isolated anti-CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti-BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist.

[0049| A composition comprising an effective amount of an antiinflammatory, wherein said anti-inflammatory is selected from the group consisting of the above, and an effective amount of an anti-viral, wherein said anti-viral is either an antibody or a synthetic anti-viral, wherein said composition is sterile and used for preventing, treating or ameliorating bronchiolitis or a symptom thereof, bronchiolitis obliterans or a symptom thereof, or the development of asthma or a symptom thereof.

[0050] The composition of the above, wherein said anti-viral is selected from the group consisting of palivizumab, motavizumab, ribavirin, Amantadine, Oseltamivir, Peramivir, Rimantadine, Zanamivir, Fomivirsen, Enfuvirtide, Imiquimod, Interferon, or Viramidine.

[0051] (1) A method of preventing, treating or ameliorating bronchiolitis or a symptom thereof, the method comprising administering to a patient an effective amount of an anti-inflammatory, wherein the anti-inflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, an isolated anti-CD19 monoclonal antibody or fragment thereof, an isolated anti-CD20 monoclonal antibody or fragment thereof, an isolated anti-CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti- BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist.

[0052] (2) The method of embodiment 1 , wherein the bronchiolitis or a symptom thereof is acute and caused by a respiratory syncytial virus infection.

[0053] (3) The method of embodiment 1, wherein the bronchiolitis or a symptom thereof is non-RSV associated bronchiolitis.

[0054] (4) The method of embodiment 1 , wherein the symptom is bronchiolitis obliterans or wheezing. |0055] (5) The method of preventing, treating or ameliorating acute respiratory distress syndrome (ARDS) or a symptom thereof caused by systemic or pulmonary infection, the method comprising administering to a patient an effective amount of an anti-inflammatory, wherein the anti-inflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, an isolated anti-interleukin 9 monoclonal antibody or fragment thereof, an isolated anti-CD19 monoclonal antibody or fragment thereof, an isolated anti-CD20 monoclonal antibody or fragment thereof, an isolated anti-CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti-BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist.

[0056] (6) The method of embodiment 1, wherein said method further prevents, treats or ameliorates the development of asthma.

[0057] (7) The method of embodiments 1 through 6, wherein the isolated anti-human HMGBl monoclonal antibody or fragment thereof includes, but is not limited to, particular antibodies (and fragments thereof) that specifically bind HMGI with high affinity which comprise the following: [0058] S6 clone comprising the VH domain SEQ ID NO:9 and VL domain

SEQ ID NO: 10 or an antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRI (SEQ ID NO: 1 1), CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13) and the light chain CDRs as follows: CDRl (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 16); or [0059| S 16 clone comprising the VH domain SEQ ID NO: 17 and VL domain SEQ ID NO:18 or an antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ ID NO: 19), CDR2 (SEQ ID NO: 20) and CDR3 (SEQ ID NO: 21 ) and the light chain CDRs as follows: CDRl (SEQ ID NO: 22), CDR2 (SEQ TD NO: 23) and CDR3 (SEQ ID NO: 24); or [00601 El l clone comprising the VH domain SEQ ID NO:25 and VL domain SEQ ID NO:26 or an antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ ID NO: 27), CDR2 (SEQ ID NO: 28) and CDR3 (SEQ ID NO: 29) and the light chain CDRs as follows: CDRl (SEQ ID NO: 30), CDR2 (SEQ ID

NO: 31) and CDR3 (SEQ ID NO: 32); or

[0061J G4 clone comprising the VH domain SEQ ID NO: 1 and VL domain SEQ ID NO:2 or an antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ ID NO: 3), CDR2 (SEQ ID NO: 4) and CDR3 (SEQ ID

NO: 5) and the light chain CDRs as follows: CDRl (SEQ ID NO: 6), CDR2 (SEQ ID

NO: 7) and CDR3 (SEQ ID NO: 8); or

[0062] which have been deposited with the American Type Culture

Collection (10801 University Boulevard, Manassas, Va. 201 10-2209) and assigned ATCC Deposit Nos. PTA-6143 (Deposited August 4, 2004), PTA-6259 (Deposited

October 19, 2004) and PTA-6258 (Deposited October 19, 2004) (also referred to herein as "S6", "S 16", and "G4", respectively) as described in U.S. Patent Publication No. 2006-

0099207 A l filed October 21 , 2005, which is incorporated herein by reference in its entirety. [0063J (8) The method of embodiments 1 through 6, wherein the isolated anti- interferon alpha monoclonal antibody or fragment thereof includes, but is not limited to an antibody or fragment thereof which comprises: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

33, 34 and 35; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37 and 38; wherein the antibody inhibits biological activity of at least one interferon alpha subtype, as described in U.S. serial no. 1 1/009,410 filed December 10, 2004, which is incorporated herein by reference in its entirety.

|0064] (9) The method of embodiments 1 through 6, wherein the isolated anti-interferon receptor monoclonal antibody or fragment thereof comprises the amino acid sequence (SEQ ID NO: 28) of the heavy chain variable region and (SEQ ID NO: 32) of the light chain variable region, both of the 9D4 human monoclonal antibody.

Alternatively, the isolated anti-interferon receptor monoclonal antibody or fragment thereof comprises the heavy chain CDRs as follows: CDRl (SEQ ID NO: 39), CDR2 (SEQ ID NO: 40) and CDR3 (SEQ ID NO: 41 ) and the light chain CDRs as follows:

CDRl (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43) and CDR3 (SEQ ID NO: 44), as described in U.S. Patent Publication No. 2006-0029601 Al , filed June 20, 2005, which is incorporated herein by reference in its entirety.

[0065] (1 1 ) The method of embodiments 1 through 6, wherein the mast cell product antagonist is a cysteinyl leukotriene antagonist selected from the group consisting of montelukast sodium (Singulair©) or zileutin (ZYFLO®).

|0066] (12) The method of embodiments 1 through 6, wherein the method further comprises administering an effective amount of an anti-viral.

[0067] (13) The method of embodiment 12, wherein said anti-viral is one or more antibodies or fragments thereof that immunospecifically bind to one or more RSV antigens.

[0068] (14) The method of embodiment 13, wherein the one or more RSV antigens is the RSV F protein.

[0069] (15) The method of embodiment 14, wherein the one or more antibodies or fragments thereof that immunospecifically binds the RSV F protein is palivizumab or motavizumab.

10070] (16) The method of embodiment 12, wherein said anti-viral is a synthetic anti-viral compound.

[0071] (17) The method of embodiment 16, wherein the synthetic antiviral compound is selected from the group consisting of ribavirin, Amantadine, Oseltamivir, Peramivir, Rimantadine, Zanamivir, Fomivirsen, Enfuvirtide, Imiquimod, Interferon,-or Viramidine.

[0072J (18) The method of embodiment 1 -6, wherein said patient is a human infant.

[0073] (19) The method of embodiment 1-6, wherein said patient is a human infant born prematurely or is at risk of hospitalization for an RSV infection.

[0074] (20) The method of embodiment 1-6, wherein said patient is an elderly human patient.

|0075] (21) The method of embodiment 1-6, wherein said patient lives in a nursing home or assisted living facility. [0076] (22) The method of embodiment 1 -6, wherein the antiinflammatory is administered after the onset of bronchiolitis in order to mitigate the predisposition of asthma development. |00771 (23) The method of embodiment 1 -6, wherein the antiinflammatory is administered during the convalescent phase of bronchiolitis in order to mitigate the predisposition of asthma development.

|0078| (24) The method of embodiment 1 -6, wherein the anti- inflammatory antibody therapeutics are administered one or twice a month.

|0079] (25) The method of embodiment 1-6, wherein the mast cell product antagonist is administered once, twice, three time or four times daily.

[0080] (26) The method of embodiment 13, wherein the anti-viral antibodies or fragments thereof that immunospecifically bind to one or more RSV antigens is administered once or twice a month.

[0081] (27)The method of embodiment 16, wherein the synthetic anti-viral compound is administered once or twice a day.

|0082] (28) A method of screening anti-virals for RSV, the method comprising: a. infecting an epithelial cell line with a labeled RSV in the presence of candidate anti-virals for RSV;

b. challenging the infected epithelium with MHC matched respiratory macrophages; and

c. measuring the uptake of infected epithelium cells by the macrophages based on detecting the labeled RSV.

|0083[ (29) The method of embodiment 28, wherein the candidate anti- virals for RSV are targeted against the NS or M proteins of RSV.

Brief Description of the Drawings |0084] Figure IA-D: A histological assessment of human pediatric bronchiolitis necropsy lung tissue. Hematoxylin and eosin (HE) staining was performed on formalin fixed, paraffin embedded lung tissue. Panel A is normal pediatric lung tissue. Panels B-D are pediatric lung tissue affected by RSV bronchiolitis demonstrating airways occlusion. The occlusion is typified by a dense cellular infiltrate with areas of consolidation. The occlusion is also characterized as Periodic acid-Schiff (PAS)-negative material or devoid of any mucus in most bronchioles and alveolar spaces.

[0085] Figure 2A-B: Panel A: A histological characterization of viral burden and cellular infiltrate in human lung tissue with RSV bronchiolitis by IHC. Panel B: Detection of viral proteins in RSV bronchiolitis human lung tissues by IHC.

[0086] -Figure 3A-B: Panel A: Inflammatory infiltrate in human lung tissue with RSV bronchiolitis showing a strong CDl 6 detection by IHC indicating a predominant neutrophil and macrophage presence in immune cell infiltrate in RSV lower respiratory tract infection (LRTI). In contrast, CDl 6 antigen is not observed in normal infant lung tissue (data not shown). Panel B: Inflammatory infiltrate in human lung tissue with RSV bronchiolitis showing a strong CDl 4 detection by IHC.

|0087[ Figure 4: Human lymphocyte positive controls for tissue staining of CD8 in tonsil tissue, CD4 in tonsil tissue and CD56 in lung tumor tissue with NK cell infiltrate, all by IHC. |0088| Figure 5A-C: Panel A: Inflammatory infiltrate in human lung tissue with RSV bronchiolitis showing a minimal CD4 detection by IHC indicating a sparse T lymphocyte presence in immune cell infiltrate in RSV lower respiratory tract infection (LRTI). Compare to Fig. 4 above. Panel B: Inflammatory infiltrate in human lung tissue with RSV bronchiolitis showing a minimal CD8 detection by IHC indicating a sparse T lymphocyte presence in immune cell infiltrate in RSV lower respiratory tract infection (LRTI). Compare to Fig.4 above. Panel C: Inflammatory infiltrate in human lung tissue with RSV bronchiolitis showing a minimal CD56 detection by IHC indicating a sparse NK lymphocyte presence in immune cell infiltrate in RSV lower respiratory tract infection (LRTI) and therefore a diminished NK cell activity. Compare to Fig. 4 above. CD56 antigen is also not typically observed in normal infant lung tissue (data not shown).

[0089] Figure 6A-F: Histopathologic assessment of RSV infected lung tissue in BALB/c and NZB mice. Five to six week old BALB/c and NZB mice were inoculated via intranasal route with 10^A7 pfu RSV A2. Lung- tissue was collected on day 6 post infection. Hematoxylin and eosin staining was performed on formalin fixed, paraffin embedded lung tissue sections. Panel A shows naϊve BALB/c lung tissue as control. Panel B shows BALB/c on day 6 post RSV A2 infection. Panels C-F show NZB mice on day 6 post RSV A2 infection. The NZB mice show occluded bronchioles, demonstrating enhanced RSV disease.

[0090] Figure 7A-B: Panel A shows normal human lung tissue as control.

Panel B shows inflammatory infiltrate in human lung tissue with RSV bronchiolitis showing strong CD20 detection by IHC, indicating a strong B lymphocyte presence.

[0091] Figure 8: MxA protein, induced by type I interferons, is strongly detected in Balb/c mice lung macrophages early, 24 hours post RSV infection. However, in NAB mice, macrophage expression of antiviral type I interferon induced proteins is essentially absent at the same time point. NZB and BALB/c mice were inoculated via intranasal route with 10^Λ7 pfu RSV A2. HE staining and histopathology were performed as described.

[0092] Figure 9: RSV recovery from lung tissue. NZB and BALB/c mice

(n-5 per group) were inoculated via intranasal route with 10^A7 pfu RSV A2. On day 5 post infection, lung tissue was collected. Lung tissue homogenates were used to infect Hep2 epithelial monolayers. Plaque formation was assessed on day 10. p=0.05. Increased RSV viral load was recovered from NZB mice as compared to BALB/c mice.

[0093] Figure 10A-B: Panel A shows lung-associated lymphocyte and pDC populations. BALB/c and NZB mice were inoculated with 10^Λ7 pfu RSV A2, or with vehicle only (sham group). On days 2 and 4 post-infection, animals were euthanized and lung tissue was lavaged with saline. Recovered cells were analyzed by flow cytometry. Panel A shows recovered NK cells (CD49b+CD3-) and pDC(CDl 1 c+Grl .1+) populations. Panel B shows CD4 (CD3+CD8-CD49b-) and CD8 (CD3+CD8+CD49b-) populations. P=O.05. This demonstrates an increased plasmacytoid dendritic cell (pDC) and natural killer cell (NK) accumulation in NZB mice post RSV infection. [0094] Figure 1 1 A-F: Analysis of cytokine and arachadonic acid metabolites. BALB/c and NZB mice were inoculated with 10^Λ7 pfu RSV A2. On days 4 and 6 post-infection, animals were euthanized and lung tissue was lavaged with saline (n=4 per time point). Panels A and B and C show IL- 12 and IL-5 and interferon gamma levels, respectively, were assessed by Luminex multiplex ELISA. Panels D-F show arachadonic acid metabolites PGD2, PGE2 and CysTL as measured by competition ELISA. Stippled bars indicate BALB/c mice. Solid bars indicate NZB mice. p=0.05. These results show a differential cytokine and lipid mediator profile for each mouse post RSV infection.

|0095J Figure 12: Cytolytic activity assay. BALB/c and NZB mice (n=5 per group) were inoculated via intranasal route with 10^A7 pfu RSV A2. On day 6 post- infection, animals were euthanized and lung tissue lavaged with saline. Lymphocytes recovered from bronchoalveolar lavage were quantitated, and used as effector cells in a FACS-based cytotoxic activity assay. Briefly, NZBK (H2d) epithelial cells were double labeled with fluorescent indicator dye and Granzyme B substrate, which yields fluorescence with cleaved (Oncalmmunin, Gaithersburg, MD). Effector cells and target cells were coincubated for 2 hours at the indicated ratio. Cytolytic activity was determined by percentage of double-positive cells. This study demonstrates that RSV- directed cytolytic activity is detectable in NZB mice.

[0096] Figure 13: Prophylactic RSV-F and IFNaR-directed monoclonal antibodies reduce RSV disease in NZB mice. NZB mice received irrelevant control antibodies, humanized anti-RSVF, anti-IFNaR, or both anti-RSVF and anti-lFNaR via intraperitoneal route 12 hours prior to challenge and again at the midpoint of RSV infection on day 3. A separate, group received vehicle alone without virus (sham) as a negative control. A) Lung tissue was harvested on day 6 and histopathology was assessed in formalin-fixed tissue by HE staining (n=5-7 per group). Overall inflammation (gray bars; mean +/- s.e.m.) and bronchiolar occlusion (open bars; mean +/- s.e.m.) were separately assessed. Representative experiment of two separate studies is shown. B) Body weight was assessed on day 6 post RSV infection and expressed as a percentage of starting weight. Combined results of two separate studies, total n=15-20 per group. Mean +/- s.e.m. is reported. C) Lung tissue was harvested on day 6 and lavaged with saline. Total and differential cell recovery is reported; results from two. separate studies are combined, total n=10-15 per group. Mean recovery +/- s.e.m. is reported. Asterisks denote P values <0.05. Double asterisks denote P values <0.01.

[0097] Figure 14A-D: Combinations of innate factors like BAFF interferon and toll ligands appear adequate to promote antibody production fast, in the absence of T lymphocyte help. B lymphocytes were purified from human donors, and cultured for 7 days in the presence of B cell activating factor (BAFF (BlyS/TALL- l/zTNF4), a TNF-related ligand that promotes B cell survival and binds to three receptors (BCMA, TACI₃ and the recently described BAFF-R), type 1 interferon, and TLR7 ligand plus anti-B-cell receptor (BCR is a multiprotein structure that provides important signalling cues for the development, and activation or inactivation, of B cells), to mimic the conditions observed in acute RSV bronchiolitis. Under those conditions, B lymphocytes produced IgG, IgM, IgA, and IL-6 all in the absence of T lymphocyte help.

J0098] Figure 15: NPS from subjects recruited for cytokine analysis were tested using the Bio-Plex Human Cytokine 17-Plex panel (Bio-Rad Laboratories, Hercules, CA). The panel includes IL (interleukin)-lbeta, IL2, IL4, IL5, IL6, IL7, IL8, ILl O, IL12 (p70), ILl 3, ILl 7, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, interferon gamma (IFNg), monocyte chemoattractant protein (MCP)-I (referred to as CCL2), macrophage inflammatory protein (MIP)-I beta (referred to as CCL4), and tumor necrosis factor alpha (TNFa). The lower limit of detection of mediators was 1 pg/ml. See Figure 15. Therefore, the data demonstrates that neonates demonstrate robust B lymphocyte responses during primary RSV infection while T lymphocyte responses are weak. Such an antibody response during primary RSV infection appears to be independent of cognate T lymphocyte assistance.

Detailed Description of the Invention [0099] There are many who postulate that severe RSV disease is the result of overly aggressive T lymphocyte responses (see Openshaw and Tregoning, Clin. Microbiol. Rev. 2005; 18: 541-5). Both CD4 and CD8 cells have been proposed as sources of immunopathology. fOOlOO] For instance, CD4-mediated processes, there is lung deposition of non-neutralizing antibodies, a recruitment of granulocytes, activation of complement. All of this was observed in the formalin-inactivated RSV vaccine trial (see Kim et al., 1969 AmJ. Epidemiol. 89:422). It was hypothesized that similar processes occurs in natural infection. This was observed in animal models of RSV disease (see Graham et al., 1991 J.Virol. 65: Connors et al. 1994 J. Virol. 68:5321 ; Polack et al. J. Ex. Med. 2002 196: 859). Neutralizing and non-neutralizing antibodies were observed after challenge with virus, immunization with F or G protein skews lymphocyte response toward ThI or Th2. [00101 j For CD8-mediated processes, the hypothesis that CD8+ T cells engage in 'bystander' killing. Cytokine, cell-mediated responses injure non-infected neighboring cells resulting in lung damage.

[001021 CD8+ T lymphocyte responses predominate in RSV animal models:

[00103] IFNγ production associated with both weight loss and viral clearance in mouse model of primary infection (Ostler et al. 2002 Eur. J. Immunol 32: 21 17; van Schaik et al. 2002 J. Med Virol 62: 257).

[00104] Antagonism of the adaptive response with steroids was proposed as a therapeutic approach to bronchiolitis in late 1960s. Anecdotal evidence that steroidal treatment helpful in serious RSV LRTI/bronchiolitis. Systematic clinical studies are less convincing (Springer et al. 1990 Pediatr. Pulmonol. 9: 181 observed no benefit; Roosevelt et al. 1996 Lancet 348: 292 observed no benefit; Buckingham et al. 2002 J. Infect. Dis.

185: 1222 found .increased viral recovery; van Woensel et al. 1997 Thorax 52:634 and van Woensel et al., 2003 Thorax 58:383. Overall, steroidal therapy shortened hospital stay only in the most severe LRT/bronchiolitis cases (a small subgroup).

[00105] There are problems with the Enhanced Disease Hypothesis. Infants are only capable of adaptive immune responses under the right circumstances. Dampened

T lymphocyte responses in infants are more typical. It seems incongruous that an aggressive T lymphocyte response to primary infection is a critical factor in most cases of

RSV disease in infants. The understanding of RSV pathogenesis has been based largely on experimental models of primary infection in adult rodents, which generate a robust, T lymphocyte- dominant immune response to RSV. However, the most serious consequences of RSV infection occur primarily in neonates and in the elderly, and immunological immaturity or deterioration of the immune response to infection have been implicated in disease exacerbation.

[00106] To date, there have been no careful studies of the immune cellular response to primary RSV infection in infants for a number of reasons: (a) a conservative approach to sample collection from infants; (b) excellent supportive care for bronchiolitis in the industrialized world; and (c) therefore few lung biopsy tissues available for study.

|00107] Therefore, in order to develop effective therapies to combat RSV bronchiolitis and similar respiratory diseases, it is imperative that good animal models exist for the advancement of experimental strategies. Thus far, there is no animal model that completely replicates the entire spectrum of RSV induced respiratory disease as seen in humans. A review by Domachowske et al., Ped.Inf.Dis. J, vol. 23(11 ):S228-S234 (2004) examines the various animal models known and used to model RSV infection and disease. In this paper, Domachowske suggests the pneumonia virus of mice (PVM) as a potential alternative model to study the proinflammatory responses during acute infection. However, the disease observed in PVM does not appear to mirror that of RSV bronchiolitis in that the inflammatory cells are observed in the alveolar space and in the bronchiolar lumen. No bronchial occlusion, a hallmark of severe human RSV disease, is observed.

|OOI08] Durbin presents the idea that innate responses such as type 1 IFN and the elicitation of chemokines are critical for limited viral spread and in recruiting the adaptive, cytolytic T cell response (see, Durbin J and Durbin K, Viral Immunol., Vol. 17(3):370-380 (2004)). However, all the animal data cited within were performed in animal models with good to excellent macrophage function. In this context, innate responses like type I IFN become uncontrolled because of the inefficiency or failure of viral clearance, and therefore contribute heavily to pathogenesis. Further, there is no suggestion or teaching of how an innate response might contribute to asthma.

|00109] In standard animal models of primary viral infection, immunologically normal rodents are inoculated with supersphysiologic doses of RSV. Because of host tropism differences, RSV replication is limited in rodents. Strong T lymphocyte responses are rapidly elicited, which promote viral clearance. However, strong adaptive immune responses have been linked with enhanced disease by prolonging lung inflammation and increasing airways responsiveness ( see Connors et al. 1992; J. Virol. 66: 7444-7451). In immunologically normal rodents, ablation of CD4 and CD8 T cells is associated with greater virus recovery but greatly attenuated disease (see Graham et al. 1991; J. Clin. Invest. 88: 1026-33). The relevance of these observations to primary RSV disease in humans is unclear, as CD4 and CD8 responses to RSV appear minimal in human bronchiolitis (see Figures 6-8 herein). lOOllOJ Finally, a review by Smit, JJ., et al., J. Exp. Med. 203, 1 153-9

(2006), suggests that pDCs, involved in the innate response and a major producer of interferon alpha, are protective in RSV disease. In this study, Smit and his colleagues use a standard mouse strain, BALB/c. Accumulation of pDCs were observed after RSV infection (see Figure 1 in Smit). PDCs were depleted with a commercially-available reagent and see some enhancement of AHR, histopathology and marginally increased viral load (see Figure 2 in Smit). An increase in cytokines detected post-RSV infection was observed, which was associated with pDC depletion (Fig 3 and 4 in Smit). Thus, Smit et al conclude that pDCs functions include a reduction of viral load and down- modulation of lymphocyte responses, i.e. limiting immunopathology. While the idea that interferon-alpha should down modulate T lymphocyte responses is valid, data shown in the present specification demonstrate that T lymphocytes do not contribute to RSV disease, therefore, the 'protective' function of pDCs in the mouse model as suggested by Smit et al, does not translate to the human setting. Further, one of type 1 IFN's primary "antiviral" roles is to help induce apoptosis in infected cells. If macrophages are unable to phagocytose the apoptotic cells promptly, this antiviral action severely damages lung tissue, an outcome Smit et al would not be able to be observed in the BALB/c mouse model.

100111] As a result, it was decided to investigate RSV bronchiolitis pathology during natural infection with a particular focus on cellular infiltrate by immunohistochemistry (see Example 1 herein). Cytokine profiles in aspirates from intubated infants were examined with an eye to any link with clinical outcomes (see Example 3 herein). The contribution of adaptive immunity to protection and pathology was determined as was the relationship of viral burden to protection and pathology.

[00112] Animal models clearly show that a range of immune responses can enhance disease severity, particularly after vaccination with formalin-inactivated RSV. Prior immune sensitization leads to exuberant chemokine production, an excessive cellular influx, and an overabundance of cytokines during RSV challenge. Under different circumstances, specific mediators and T-cell subsets and antibody-antigen immune complex deposition are incriminated as major factors in disease. Animal models of immune enhancement permit a deep understanding of the role of specific immune responses in respiratory diseases, assist in viral vaccine design, and indicate which immunomodulatory therapy might be beneficial to patients afflicted with respiratory disease, such as those describe above. [00113] Spontaneously autoimmune New Zealand Black (NZB) mice develop lupus-like disease at approximately 6-12 months of age. It was hypothesized that RSV infection might accelerate the symptoms of lupus in these animals. NZB mice display constitutive deficiencies in macrophage function including poor recognition and uptake of apoptotic cells. Briefly, NZB animals were inoculated via intranasal route with approximately 10^A7 pfu of RSV A2 strain, and examined for lung inflammatory endpoints and virus recovery up to six days post infection. It was observed that pre- autoimmune NZB mice, and the related strains NZW and BWFl , are highly susceptible to RSV infection at 5 to 6 weeks of age, well prior to any appearance of lupus symptoms. RSV infection at 10^A7 pfu was lethal in 25-30% of NZB mice but not in control mice such as BALB/c and C57B1/6, which are the standard mouse strains for RSV infection modeling.

[00114] In the present .study disclosed herein, NZB mice was most extensively studied, and were compared to the MHC matched strain BALB/c. RSV disease in NZB mice is characterized as follows: a) increased viral recovery (approximately 1 log increase) compared with BALB/c; b) extensive occlusion of airway lumens, both bronchiolar and alveolar spaces, with protein and fluid derived from serum and interstitial spaces; this airways occlusion is not observed in BALB/c nor in any other rodent model of RSV infection; c) lower IFNg and no IL-12 post infection in lung post infection compared with standard strain; d) recovery of cysteinyl leukotrienes from lung post infection, never reported in standard strain; e) recovery of eosinophils from lung post infection. These features of RSV infection in NZB mice closely match features of human RSV bronchiolitis. For this reason it was proposed that the NZB model of RSV infection offers significant advantages over other in vivo models currently used. [00115] Autoimmune prone animals including NZB mice have well known deficiencies in macrophage function (see Licht, R., et al., J. Autoirnmuno. 22, 139-45 (2004) and Potter, P. et al., J. Immunol. 170, 3223-3232 (2003)). It was proposed that NZB mice are highly susceptible to RSV because of: 1) decreased ability of macrophages to detect and induce apoptosis in infected cells; 2) inability of macrophages to phagocytize apoptotic infected epithelium and apoptotic neutrophils; 3) reduced and/or inappropriate presentation of RSV antigens to T lymphocytes. These macrophage deficiencies result in a higher viral burden and contribute to persistent inflammation. [00116] Degeneration of bronchiolar epithelium in NZB mice post infection was observed, even though these cells are not infected by RSV as determined by a lack of viral antigen staining by THC. Bursts of cysteinyl leukotriene production was also observed that coincides with decline in lung function and airways occlusion. It is proposed that bronchiolar epithelium is driven to apoptosis by type I IFN expressed during RSV infection. Apoptotic bronchiolar epithelial cells are not efficiently cleared by NZB and therefore are able to contribute chemokines and other factors that prolong inflammation. Failure to clear virus occurs despite increased expression of type I IFN- induced genes, and may be related to deficient early responses of macrophages to viral insult. It is further proposed that either type I interferon itself or chemokines such as TNFα derived from degenerating bronchiolar epithelium, or perhaps both, may be responsible for triggering mast cell production of cysteinyl leukotriene in NZB animals. Alternatively or in addition, toll ligands such as single stranded RNA or double stranded RNA intermediates may directly act in a previously unrecognized manner on respiratory mast cells, to produce activation and cysteinyl leukotriene release. Previous studies implicate cysteinyl leukotrienes in altered endothelial permeability in blood vessels. Cysteinyl leukotrienes in the lung may contribute to vascular leak and protein transudate into alveolal and bronchiolar lumens.

[00117] NZB macrophages demonstrated poor uptake of infected or apoptotic epithelial targets, and very limited cytokine release to a variety of stimuli including Toll ligands, compared with controls. In particular, RANTES production, which has been identified as a macrophage survival factor during respiratory infection, was produced at very low levels by NZB macrophages (data not shown).

[00118] While recent studies do document that mast cells bear TLRs including TLR3, the mast cells utilized in these studies were either derived from the peritoneal cavity, from bone marrow mast cells, or from PBMC progenitors. TLR signaling in non-respiratory mast cells was associated with LTB4 and IFNα production and was hypothesized to stimulate the adaptive CD8 response. The data disclosed herein are suggestive that respiratory mast cells may respond differently to TLR stimulation and/or type I interferon, compared with mast cells derived from other sources. Elaboration of cysteinyl leukotrienes in the proposed model is a novel observation and would be expected not to promote protective CD8 responses, but instead to heighten asthma-promoting events such as the recruitment of eosinophils and promotion of airways hyper responsiveness (AHR) through direct effects on airway smooth muscle cells. Additional respiratory mast cell products released by TLR3 or interferon-alpha stimulation may include pleiotropic factors such as VEGF, histamine, neurokinins, neurotrophins, and serotonin. Respiratory mast cell products released as a result of TLR signaling and/or type I interferon have a great potential to condition lung tissue through, for example, recruitment of mast cell or eosinophil progenitors, modulation of respiratory innervation, and remodeling of vascular tissue and smooth muscle. All of these activities can predispose for asthma responses. In particular, recruitment of mast cell and eosinophil precursors may be linked to TGFβ production in tissue, airways remodeling and the promotion of Th2 lymphocyte responses to respiratory allergens.

[00119] It is envisioned that stressed airway smooth muscle (due to increased work of breathing) and hypoxia (due to occlusion of airway lumens) which occur as a result of lung vascular leak, are likely triggers for the accumulation of mast cells and possibly eosinophil progenitors. Accumulation of mast and/or eosinophil precursors is believed to predispose for an asthmatic phenotype, by conditioning lung tissue and shaping responses to respiratory allergens as described above.

[00120] A dampening of the adaptive T lymphocyte response to RSV in

NZB mice compared with BALB/c was observed, which may in part explain the higher viral recovery in NZB. Similarly, in recent studies of bronchiolitis necropsy tissue in human infants, a high viral burden was found with no evidence of a strong T lymphocyte response to infection. Indeed, there appears to be no evidence of T lymphocyte activation in the respiratory secretions of infants hospitalized for RSV LRTl. It was shown that classical T lymphocyte cytokines IL-2, IL-4, IFN-g, and IL-17 were nearly undetectable in such respiratory secretions (see Welliver, T., et. al., JID 2007:195(15 April). In NZB mice, increases in innate immune responder cells including natural killer (NK) cells and plasmacytoid dendritic cells (pDCs) in NZB lung tissue post infection was observed. There is thus far, no evidence of NK or pDC accumulation in necropsy tissue yet in RSV bronchiolitis; however, one recent report does document pDC accumulation in aspirates from intubated infants with flu or RSV bronchiolitis (Gill et al 2006). In that study pDC accumulation coincided with viral burden but not with clinical outcomes. The present data are suggestive that the innate responder cells pDCs and NKs are not sufficient to control RSV infection, and in fact may promote pathogenesis and an asthma predisposition possibly through a type 1 IFN-mediated pathway as discussed above. Accumulation of pDCs in lung tissue may be useful as a diagnostic marker, for individuals in whom the adaptive response is insufficiently controlling respiratory virus infection.

[00121] Despite the lack of T lymphocyte response, there is a robust B lymphocyte response as measured by the presence of CD20 staining (see Fig. 7). Further, these B cells are mature, i.e., RSV bronchiolitis human infant tissue stained positive for the presence of IgD (data not shown). 100122] Studies of innate B lymphocyte responses to RSV infection was performed. It is proposed that the same factors that promote T independent autoantibody production in lupus are present in bronchiolitis, and can activate naive, mature B lymphocytes in infants to produce antibodies and cytokines without T lymphocyte help. To this end, B lymphocytes were purified from human donors, and cultured for 7 days in the presence of B cell activating^' factor (BAFF (BlyS/T ALL-I /zTNF4), a TNF-related ligand that promotes B cell survival and binds to three receptors (BCMA, TACI, and the recently described BAFF-R), type I interferon, and TLR7 ligand plus anti-B-cell receptor (BCR is a multiprotein structure that provides important signalling cues for the development, and activation or inactivation, of B cells), to mimic the conditions observed in acute RSV bronchiolitis. Under those conditions, B lymphocytes produced IgG, IgM, IgA, IL-6 and TL-I O - all in the absence of T lymphocyte help. See Figure 14. Combinations of innate factors like BAFF interferon and toll ligands appear adequate to promote antibody production fast, in the absence of T lymphocyte help.

100123] Next, nasopharyngeal aspirates were acquired from infants who were admitted to the hospital with acute RSV infection in order to analyze their antibody responses. Briefly, twenty-five (25) inpatients and outpatients less than 12 months of age with RSV infection were seen at Women and Children's Hospital of Buffalo. These nasopharyngeal aspirates were analyzed for BAFF, IFN, cytokines, and chemokines using a multiplex ELISA detection assay. BAFF is detectable in most infant aspirates and correlates both with inflammatory cytokine production (i.e., IL-6 and IL-I O) and with total antibody production. It would appear that the antibody response generated during RSV infection appears independent of cognate T cell help. A second group of infants had fatal LRTI. Post-mortem lung tissue was obtained from infants with fatal RSV (n = 9) LRTI who were autopsied at Hospital Roberto del Rio, Santiago, Chile. Two pathologists (L.V. and L.M.) independently judged that the cause of death in all cases was severe LRTI, with typical sloughing of bronchiolar epithelium, plugging of the terminal bronchioles, and infiltration of the airway wall and the alveoli macrophages and neutrophils. There was no histological evidence of bacterial infection of the lung. Dying infants had not been subjected to prolonged mechanical ventilation or to the use of antiinflammatory agents or to antivirals.

|00124| NPS from subjects recruited for cytokine analysis were tested using the Bio-Plex Human Cytokine 17-Plex panel (Bio-Rad Laboratories, Hercules, CA). The panel includes IL (interleukin)-lbeta, IL2, IL4, IL5, IL6, IL7, IL8, ILl O, ILl 2 (p70), IL13, ILl 7, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, interferon gamma (IFNg), monocyte chemoattractant protein (MCP)-I (referred to as CC L2), macrophage inflammatory protein (MIP)-I beta (referred to as CCL4), and tumor necrosis factor alpha (TNFa). The lower limit of detection of mediators was 1 pg/ml. See Figure 15. Therefore, the data demonstrates that neonates demonstrate robust B lymphocyte responses during primary RSV infection while T lymphocyte responses are weak. Such an antibody response during primary RSV infection appears to be independent of cognate T lymphocyte assistance. |00125J Therefore, one aspect of the present invention are therapeutics directed against plasmacytoid DCs, type I IFN alpha and its receptor, TLRs particularly TLR3, mast cells and their products, particularly cysteinyl leukotrienes, for the prevention, treatment or amelioration of bronchiolitis and symptoms thereof associated with RSV, influenza, hMPV, or other respiratory viruses, the method comprising administering to a patient an effective amount of an anti-inflammatory, wherein the antiinflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist. [00126] Another aspect of the present invention is the therapeutic targeting of type I interferon in cases of RSV bronchiolitis. This may be accomplished by administering to a patient in need thereof, an effective amount of an anti-interferon antibody, such as, for example, an anti-interferon alpha antibody or fragment thereof, an anti-interferon alpha receptor antibody or fragment thereof.

|00127] A further aspect of the present invention is the therapeutic targeting of primary B lymphocyte response through CD 19, CD20, CD21 in cases of RSV bronchiolitis. This may be accomplished by administering to a patient in need thereof, an effective amount of an anti-CD19 or anti-CD20 or anti-CD21 antibody or fragment thereof.

[00128] A further aspect of the present invention is the therapeutic targeting of primary B lymphocyte response in cases of RSV bronchiolitis through an anti-BAFF antibody or fragment thereof or anti-BAFF receptor antibody or fragment thereof.

[00129] It is also another aspect of the present invention that continued antagonism of TLR signaling and antagonism of mast cell survival, proliferation, and activation (for example, by IL-9 or IL-9R blockade) may be beneficial both during acute bronchiolitis and during the convalescent phase weeks to months after infection, in order to block tissue conditioning that may lead to asthma predisposition.

J00130J It is also another aspect of the present invention that antagonism of mast cell products such as cysteinyl leukotrienes, VEGF, neurotransmitters, neurotrophins, neurokinins, and histamine, may be beneficial in the treatment of bronchiolitis and in the convalescent phase weeks to months after infection. |00131] It is proposed that these therapeutics may be effective alone or in the context of antiviral therapy such as RSV-F protein antibodies and synthetic anti-viral compounds, or other antiviral approaches. Contemplated RSV-F protein antibodies include, but are not limited to, palivizumab (Synagis® as described in Johnson et al JID vol. 176:1215-1224 (1997) and in U.S. Patent No. 5,824,307) or motavizumab (Numax™). Other contemplated RSV F protein antibodies may be found in Wu et al., JMB vol. 350:126-144 (2005), and in U.S. Serial No. 11/263,230 filed October 31 , 2005, both of which are incorporated herein by reference in their entirety. Contemplated synthetic anti-viral compounds include, but are not limited to, ribavirin, Amantadine, Oseltamivir, Peramivir, Rimantadine, Zanamivir, Fomivirsen, Enfuvirtide, Imiquimod, Interferon,-or Viramidine.

|00132] It is proposed that antiviral approaches may be useful therapeutically, after the onset of bronchiolitis and during the convalescent stage, to antagonize the production of TLR ligands (particularly TLR3), reduce the elaboration of type I interferon, enhance macrophage recognition and uptake of infected cells (see below), dampen the subsequent engagement of mast cells, and block tissue reconditioning that may predispose for asthma. |00133] Therefore, it is contemplated that the method of the invention administer an anti-inflammatory antibody therapeutic once or twice a month.

[00134] It is also contemplated that the method of the invention administer a mast cell product antagonist once, twice, three times or four times daily.

[001351 It is also contemplated that the method of the invention administer an anti-viral antibody or fragments thereof that immunospecifϊcally bind to one or more RSV antigens once or twice a month.

[00136] It is also contemplated that the method of the invention administer a synthetic anti-viral compound once or twice a day.

[00137] The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred earner when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[00138] The amount of the therapeutic or compound used in the method of the invention is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[00139 J The amount of the therapeutic or compound used in the method of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, tri ethyl amine, 2-ethylamino ethanol, histidine, procaine, etc.

[00140] The amount of the therapeutic or compound used in the method of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[00141] For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[00142] Various delivery systems are known and can be used to administer a therapeutic in the method of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 ( 1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The therapeutics or compounds described herein may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. [00143] In yet another embodiment, the therapeutic or compound can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201 ; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61 ; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351 ; Howard et al.,1989, J. Neurosurg. 71: 105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). [00144] Accordingly, therapeutic compounds used in the method of the invention, designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.

|00145] Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth or gelatin. Examples of excipients include starch or lactose. Some examples of disintegrating agents include alginic acid, corn starch and the like. Examples of lubricants include magnesium stearate or potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used. |00146| Another embodiment of present invention are antibodies used in the method of the invention and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10^"5 M, or of less than 10^"6 M, or of less than 10^"7 M, or of less than 10^"8 M, or of less than 10^'9 M, or of less than 10^"'° M₃ or of less than 10^'" M, or of less than 10^"12 M, or of less than 10^'13 M, or of less than 5x 10^'13M, or of less than 10^" ¹⁴M, less than 5x l O^"14M, or of less than 10^"15M, or of less than 5xlO^{"l 5}M. In still another embodiment, antibodies used in the method of the invention and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between about 10^"7M and about 10^" ⁸M, between about 10^"8M and about 10^"9M, between about 10^"9M and about 10^"10M, between about 10^"10 M and about 10^{"1 1}M, between about 10^{"1 1}M and about 10^"12M, between about 10^"12M and about 10^'13M, between about 10^"13M and about 10^"14M. In still another embodiment, antibodies used in the method of the invention and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between 10^"7M and 10^" ⁸M, between 10^"8M and 10^"9M, between 10^"9M and 10^"10M₅ between 10^"'⁰M and 10^{"1 1}M, between 10^{"1 1}M and 10^"12M, between 10^"12M and 10^"13M, between 10^"13M and 10^"14M.

[00147) Another embodiment of present invention are antibodies used in the method of the invention and antigenic fragments thereof with a dissociation constant or Kd (koff/kon) of less than 10^"5 M, or of less than 10^"6 M, or of less than 10^"7 M, or of less than 10^"8 M, or of less than 10^'9 M, or of less than 10^"' ° M, or of less than 10^"" M, or of less than 10^"12 M, or of less than 10^"13 M₅ or of less than 5x10^"13M, or of less than 10^" ¹⁴M, less than 5x 10^"14M, or of less than 10^"15M, or of less than 5x10^"15M. In still another embodiment, antibodies used in the method of the invention and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between about 10^"7M and about 10^" ⁸M, between about 10^"8M and about 10^"9M, between about 10^'9M and about 10^"10M, between about 10^"10 M and about 10^{"1 1}M, between about 10^{"1 1}M and about 10^"12M₅ between about 10^"12M and about 10^"13M, between about 10^"13M and about 10^{" 14}M. In still another embodiment, antibodies used in the method of the invention and antigenic fragments thereof has a dissociation constant or Kd (koff/kon) of between 10^"7M and 10^" ⁸M, between 10^"8M and 10^"9M₅ between 10^"9M and 10^"10M₅ between 10^'10M and 10^{"1 1}M₅ between 10^{"1 1}M and 10^"12M, between 10^"12M and 10^"13M, between 10^"13M and 10^"14M.

[00148] It is well known in the art that the equilibrium dissociation constant

(Kd) is defined as koff/kon. It is generally understood that a binding molecule (e.g., and antibody) with a low Kd (i.e., high affinity) is preferable to a binding molecule (e.g., and antibody) with a high Kd (i.e., low affinity). However, in some instances the value of the kon or koff may be more relevant than the value of the Kd. One skilled in the art can determine which kinetic parameter is most important for a given antibody application. In certain embodiments, the antibodies used in the method of the invention have a lower Kd for one antigen than for others.

[00149] In another embodiment, the antibodies used in the method of the invention and antigenic fragments thereof with a koff of less than 1x10-3 s-1 , or of less than 3x10-3 s-1 . In other embodiments, the antibodies used in the method of the invention and antigenic fragments thereof with a koff of less than 10^"V, less than 5x10^" ³S^{" 1}, less than 10^"4S^"1, less than 5xl 0^"V, less than 10^"5S^"1, less than 5x10^"V, less than 10^" V, less than 5x l 0^"V, less than 10^"V, less than 5x10^"V, less than 10^"8S^'1, less than 5x10^"8s^"'₃ less than 10^"V, less than 5x10^'V, or less than 10^"1V.

[00150] In another embodiment, the antibodies used in the method of the invention and antigenic fragments thereof with a koff of less than 1x10^"V, or of less than 3x 10^"3s^"'. In other embodiments, the antibodies and antigenic fragments thereof with a Koff of less than 10^"V, less than 5xl0^"V, less than 10^"4S^"1, less than 5xl0^"V, less than 10^"5s^"', less than 5xl 0^"V, less than 10^'V, less than 5xl O^"6s^"', less than 10^"7S^'1, less than 5xlO^"V, less than 10^"V, less than 5x10^" V, less than 10^"V, less than 5x10^'V, or less than 10^"1V .

[00151] In another embodiment, the antibodies used in the method of the invention and/or antigenic fragments thereof with an association rate constant or kon rate of at least 10⁵ M^"V, at least 5xlO⁵ M^"'s^"', at least 10⁶ M^-1S^"1, at least 5 x 10⁶ IvT¹S^"1, at least 10⁷ M^"'s^"', at least 5 x 10⁷ M^-1S^-1Or at least 10^s M^'V¹, or at least 10⁹ M^'V. [00152] In another embodiment, the antibodies used in the method of the invention and/or antigenic fragments thereof binds to its target with an association rate constant or kon rate of at least 10⁵ M^"V, at least 5xl O⁵ M^'V, at least 10⁶ M^"'s^"', at least 5 x 10⁶ M^"'s^"', at least 10⁷ IvT¹S^'1, at least 5 x 10⁷ M^'Vor at least 10⁸ NT's^"1, or at least 10⁹ M-¹S^"1. [00153] Antibodies like all polypeptides have an Isoelectric Point (pi), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein. As used herein the pi value is defined as the pi of the predominant charge form. The pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et al., 1993, Electrophoresis 14:1023). In addition, the thermal melting temperatures (Tm) of the Fab domain of an antibody, can be a good indicator of the thermal stability of an antibody and may further provide an indication of the shelf-life. A lower Tm indicates more aggregation/less stability, whereas a higher Tm indicates less aggregation/ more stability. Thus, in certain embodiments antibodies having higher Tm are preferable. Tm of a protein domain (e.g., a Fab domain) can be measured using any standard method known in the art, for example, by differential scanning calorimetry (see, e.g., Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000, Biophys. J. 79: 2150-2154).

100154] Accordingly, an additional nonexclusive embodiment of the present invention includes high affinity antibodies of the invention that have certain preferred biochemical characteristics such as a particular isoelectric point (pi) or melting temperature (Tm).

[00155] More specifically, in one embodiment, the high affinity antibodies of the present invention have a pi ranging from 5.5 to 9.5. In still another specific embodiment, the high affinity antibodies of the present invention have a pi that ranges from about 5.5 to about 6.0, or about 6.0 to about 6.5, or about 6.5 to about 7.0, or about 7.0 to about 7.5, or about 7.5 to aboutδ.O, or about 8.0 to about 8.5, or about 8.5 to about 9.0, or about 9.0 to about 9.5. In other specific embodiments, the high affinity antibodies of the present invention have a pi that ranges from 5.5-6.0, or 6.0 to 6.5, or 6.5 to 7.0, or 7.0-7.5, or 7.5-8.0, or 8.0-8.5, or 8.5-9.0, or 9.0-9.5. Even more specifically, the high affinity antibodies of the present invention have a pi of at least 5.5, or at least 6.0, or at least 6.3, or at least 6.5, or at least 6.7, or at least 6.9, or at least 7.1, or at least 7.3, or at least 7.5, or at least 7.7, or at least 7.9, or at least 8.1, or at least 8.3, or at least 8.5, or at least 8.7, or at least 8.9, or at least 9.1 , or at least 9.3, or at least 9.5. In other specific embodiments, the high affinity antibodies of the present invention have a pi of at least about 5.5, or at least about 6.0, or at least about 6.3, or at least about 6.5, or at least about 6.7, or at least about 6.9, or at least about 7.1, or at least about 7.3, or at least about 7.5, or at least about 7.7, or at least about 7.9, or at least about 8.1 , or at least about 8.3, or at least about 8.5, or at least about 8.7, or at least about 8.9, or at least about 9.1 , or at least about 9.3, or at least about 9.5.

[00156J It is possible to optimize solubility by altering the number and location of ionizable residues in the antibody to adjust the pi. For example the pi of a polypeptide can be manipulated by making the appropriate amino acid substitutions (e.g., by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine). Without wishing to be bound by any particular theory, amino acid substitutions of an antibody that result in changes of the pi of said antibody may improve solubility and/or the stability of the antibody. One skilled in the art would understand which amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pT. In one embodiment, a substitution is generated in an antibody of the invention to alter the pi. It is specifically contemplated that the substitution(s) of the Fc region that result in altered binding to FcgR may also result in a change in the pi. In another embodiment, substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in FcgR binding and any desired change in pi.

[00157| In one embodiment, the high affinity antibodies of the present invention have a Tm ranging from 65°C to 120⁰C. In specific embodiments, the high affinity antibodies of the present invention have a Tm ranging from about 75°C to about 120⁰C, or about 75°C to about 85°C, or about 85°C to about 95°C, or about 95°C to about 105°C, or about 105⁰C to about 1 15°C, or about 1 15°C to about 120⁰C. In other specific embodiments, the high affinity antibodies of the present invention have a Tm ranging from 75°C to 120⁰C₃ or 75°C to 85°C, or 85°C to 95°C, or 95°C to 105⁰C, or 105⁰C to 1 15°C, or 1 15⁰C to 120⁰C. In still other specific embodiments, the high affinity antibodies of the present invention have a Tm of at least about 65°C, or at least about 70⁰C, or at least about 75°C, or at least about 80⁰C, or at least about 85°C, or at least about 90⁰C, or at least about 95°C, or at least about 100⁰C, or at least about 105⁰C, or at least about 1 10⁰C, or at least about 1 15°C, or at least about 120⁰C. In yet other specific embodiments, the high affinity antibodies of the present invention have a Tm of at least 65°C, or at least 70⁰C, or at least 75°C, or at least 80⁰C₃ or at least 85°C, or at least 90⁰C, or at least 95°C, or at least 100⁰C, or at least 105⁰C, or at least 1 10⁰C, or at least 1 1 5°C, or at least 120⁰C. (00158| A new strategy for selection of antivirals for RSV is proposed.

Because macrophages represent the first line of defense against RSV, and their responses appear deficient in infants, a selection strategy that enhances macrophage recognition of or ingestion of infected epithelium seems promising. NS protein is known to antagonize type 1 interferon signaling. In the infected cell, the type I interferon pathway drives the infected cell to apoptosis and to the production of a stress protein profile that is recognized by macrophages. By antagonizing type I interferon in this way, RSV may escape the notice of respiratory macrophages and prolong infection. A small molecule anti-NSl approach might be effective in helping respiratory macrophages recognize and phagocytize RSV infected epithelium, limiting infection and making a protective cytolytic response more likely. Similarly, it has been previously observed that M protein antagonists result in smaller plaques in vitro. In vivo, smaller syncytia would be easier for macrophages to take up, process, and present to ThI T lymphocytes. In vitro, one skilled in the art could infect an epithelial line such as NZBK with a labeled RSV such as RSV-luciferase, in the presence of potential small molecule inhibitors. At given times after infection, one could challenge the infected epithelium with MHC matched macrophages (in this example, one could use BALB/c macrophages), and measure the uptake of cells by the respiratory macrophages based on luciferase expression in macrophages using a plate-based or cytometric assay. [00159| Similarly it is proposed that live attenuated vaccines for respiratory viruses are successful at least in part because they prime respiratory macrophages for responses to wild type infection. In designing respiratory viral vaccines, one skilled in the art might use a similar in vitro approach as described above, and select for virus attenuations that best promote recognition/uptake by respiratory macrophages. Based on these observations, it is predicted a major biomarker of a protective vaccine will be the production of IL-12 by macrophages. In an in vivo model or in people, it is proposed that a successful attenuated vaccine will be characterized by local IL-12 production, and will elicit minimal to no plasmacytoid dendritic cell recruitment.

|00160| Acute Respiratory Distress Syndrome

|001611 Rapid and catastrophic serum fluid and protein transudation in

NZB lung post RSV infection has been observed. The tissue changes are reminiscent of acute respiratory distress syndrome (ARDS). It is proposed that in at least some cases of ARDS, particularly cases associated with sepsis or lung infection, innate immune responses may strongly contribute to pathogenesis. It is proposed that type I interferon and toll ligands may directly activate mast cell responses resulting in the release of cysteinyl leukotrienes and other factors that promote permeability changes in lung endothelium. Alternatively or at the same time type I interferon and toll ligands may act on epithelium, resulting in the release of epithelial-derived factors (e.g. TNFa) that activates mast cells. Alternatively or additively, type I interferon may engage lung epithelium, driving apoptotic responses that reduce the integrity of alveolar and bronchiolar spaces, contributing to serum and/or interstitial fluid leak. The NZB model of RSV infection represents a novel in vivo model for ARDS.

|00162] Several new therapeutic approaches for treating ARDS are proposed, particularly ARDS with an infectious etiology (e.g. sepsis or respiratory infection), which will be tested in the NZB mouse model: a) blockade of mast cells or mast cell-derived vasoactive factors such as cysteinyl leukotrienes; b) blockade of type I interferon receptors in the lung; c) blockade of TLR signaling in the respiratory tract; d) blockade of HMGB-I in the respiratory tract to antagonize presentation of TLR ligands and further dampen TLR signaling; e) increasing activity of alveolar macrophages, to promote clearance of infected epithelium and resolve inflammation by timely removal of apoptotic immune cells.

Examples

|00163] The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example 1. Study of Bronchiolitis Pathology in RSV-Infected Infants

100164] The purpose was to investigate RSV bronchiolitis pathology during natural infection with a particular focus on cellular infiltrates by immunohistochemistry and by an analysis of viral burden in tracheal aspirates by qPCR. In this way, both the contribution of adaptive immunity and the relationship of viral burden to protection and pathology could be studied. 100165] For the immunohistochemistry of lung tissue, bronchiolitis necropsy tissue samples were obtained from Dr. Luis Avendano at the University of Chile and processed through formalin-fixation and paraffin embedding. After sectioning samples, slides were deparaffinized using consecutive xylene washes, and were rehydrated using an ethanol gradient. Following rehydration, heat induced epitope retrieval was performed, treating samples in a pH 6.0 HIER Buffer (pH 8.0 for CD 16) (DakoCytomation) for 20 minutes. Endogenous Peroxidase and nonspecific protein blocking- using 10% Hydrogen Peroxide/Methanol and 5% BSA (Sigma- Aldrich)/TBST solutions, respectively- were employed to eliminate nonspecific background. Antibodies were diluted to appropriate concentrations in 1% BSA/TBST, and then applied to samples for two hours at room temperature; Anti-Flu A and Anti-RSV (both from Chemicon International) and Anti-CD56 (Lab Vision Corporation) were used at 1 mg/mL; Anti-CD4 (Chemicon International), Anti-CD8 (Zymed Laboratories), Anti-CD14, and Anti-CD16 (both from Lab Vision Corporation) were all prediluted antibodies. Following a stringent wash in TBST, the samples underwent a double treatment of appropriate secondary antibody followed by Streptavidin: Horseradish Peroxidase conjugate (All from DakoCytomation), with TBST washes intermittent between treatments; the secondary reagents were all used at 1 mg/mL for 30 minutes for each treatment. Samples were developed through application of Peroxidase-based DAB substrate kit (Sigma-Aldrich)- developing approximately 4 minutes- before being washed. Samples were counterstained using Mayer's Hematoxylin (approximately two minutes) stain, and Scott's Substitute Tap Water bluing agent (Surgipath). Samples were washed and processed through an ethanol-based dehydration gradient. Samples were briefly cleared using xylene, and mounted using coverslips and DPX mounting agent (Sigma-Aldrich). |00166] Histologic analysis of RSV bronchiolitis necropsy tissue sections revealed extensive damage epithelial cells in alveoli, respiratory bronchioles (Figure 1). Immune infiltrates were strongly present and consisted of macrophages and neutrophils, with few lymphocytes. Airway spaces were found to be occluded with immune cells and apoptotic debris, mixed with fluid and protein transudated from serum (Figure I ). Based on widespread detection of RSV-associated antigens viral burden appeared to be high in these necropsy tissues. Although positive controls yielded strong detection of CD4, CD8 and CD56 antigens, minimal detection of these lymphocyte associated antigens in RSV necropsy tissue was observed (see Figure 5 A-C). Conversely, CD 16 (associated with activated macrophages and granulocytes) and CD 14 (associated with monocytes) positive cells were found throughout the tissue. (Figure 3A-B). These results suggest that innate immune responders such as macrophages and neutrophils contribute more to RSV bronchiolitis pathology than adaptive immune responders (e.g., T lymphocytes). This is in contrast to what has been observed in RSV disease that is 'enhanced' by vaccination, and also contrasts with standard rodent models of RSV disease in which a vigorous adaptive response is associated with both virus clearance and (mild) immunopathology.

J00167] For the qRT-PCR, viral RNA was isolated from tracheal aspirate samples obtained from nasopharyngeal secretions from intubated infants with lower respiratory tract RSV infection or bronchiolitis from Dr. Robert Welliver at Children's Hospital in Buffalo, NY, using QlAamp Viral RNA Mini Kit (Qiagen, Inc.) according to kit instructions. Genomic DNA was digested using RNase-Free Dnase Set (Qiagen). Viral RNA was translated into cDNA using Sprint Powescript Preprimed Plate (Clontech), according to instructions; all samples were normalized so that 1 μg of RNA for each sample was used to make cDNA. cDNA was subsequently diluted 1 : 15 in RNase free water. These diluted samples were then processed by qPCR in separate reactions for Flu A and RSV. The following primers (All from Applied Biosystems) were used: Flu A forward primer, TGGCCAGCACTACAGCTAAGG; Flu A reverse primer, CCATGGCCTCTGCTGCTT; RSV forward primer,

AGATCAACTTCTGTCATCCAGCAA; RSV reverse primer,

TTCTGCACATCATAATTAGGAGTATCAAT. In each reaction, the samples were processed through 40 cycles. The results were collected and analyzed using software.

|00168] RSV viral load in tracheal aspirates was found to not correlate with oxygen saturation nor with length of hospital stay, but may inversely correlate with age of infant (r=-0.365; p=0.08). Thus absolute viral load alone does not appear to predict disease severity in this population.

Luminex Assay 100169] The tracheal aspirates were processed for cytokine detection using a Luminex cytokine kit and beadmates (Upstate Cell Signaling Solutions) according to included protocol. [00170] Detection of lymphocyte-derived cytokines such as 1L-2, IL-4, IL-

5, IL-9 and IL-13 was low to absent in tracheal aspirates derived from infants with RSV bronchiolitis. In contrast, cytokines and chemokines derived from epithelium, macrophages, and granulocytes such as 1L-6, IL-8, MCP-I and IL-10 were more easily detected. Infants with influenza bronchiolitis, which is generally milder than RSV bronchiolitis, demonstrated higher detection of cytokines and chemokines across the board, with I L- 12 as a particularly strong discriminator between influenza and RSV.

These results suggest severe RSV bronchiolitis is not characterized by a strong lymphocyte response, and that IL- 12 production by macrophages may be a useful diagnostic marker.

Example 2: Spontaneously autoimmune New Zealand Black (NZB) mouse model studies

[00171 ] Spontaneously autoimmune New Zealand Black (NZB) mice develop lupus-like disease at approximately 6-12 months of age. It was hypothesized that RSV infection might accelerate the symptoms of lupus in these animals.

[00172] Briefly, NZB animals were inoculated via intranasal route with approximately 10^A7 pfu of RSV A2 strain, and examined lung inflammatory endpoints and virus recovery up to six days post infection. [00173| Experimental Animals: Spontaneously lupus-prone mouse strains

NZB, NZW, and BWFl were obtained from Harlan Labs. Control mouse strains BALB/c and C57B1/6 were obtained from Jackson Laboratories.

[00174] RSV Infection Procedure: Animals were lightly anesthetized with isofluorane. A 100 microliter inoculum of RSVA2 (10^A7 pfu) was placed on the nares using a pipettor. Mice were permitted to inhale the inoculum, then were placed in microisolator cages to recover from anesthesia. Weights and signs of illness were monitored over the course of 6 days.

|00175] Endpoints were measured at the termination of the experiment on day 6. |00176] Broncho-alveolar Lavage: On the day of harvest, animals were euthanized using CO2. For broncho-alveolar lavage (BAL) collection, lung tissue was lavaged with 0.5 ml of saline. BAL-associated cells were recovered by low-speed centrifugation.

100177] Endpoints: For some studies, collected BAL cells were analyzed by flow cytometry for the presence of surface markers. All antibodies for flow cytometry (GrI , CD3, CD4, CD8, CDl Ib₃ CDl Ic, Ly49, CD69) were obtained from Becton Dickinson Biosciences. Additionally, the presence of cytotoxic cells was tested in BAL fluid acquired cells. Briefly, an RSV-permissive kidney cell line (NZBK, provided by Jay Levy, UCSF) was double-labeled using a proprietary fluorescent dye and a granzyme B substrate which yields a fluorescent product when cleaved (both purchased from Oncolmmunin, Gaithersburg, MD). Labeled cells were then infected with RSVA2 at an MOI of 10. Twenty-four hours after infection, BAL acquired cells were quantitated and were added to the culture at effector: target ratio of 5: 1 to 20:1. After two hours conversion of the granzyme B substrate to its fluorescent product was assessed (see Figure 12). [00178] Cytokine and lipid mediators were measured in BAL fluid using commercial kits (Linco for Luminex multiplex cytokine detection; Cayman Chemical for leukotriene and prostaglandin analysis; R+D Systems for standard ELISA of IFNg and IL-10 and Mig). For quantitation of virus, whole lung tissue was collected and homogenized in 1 ml saline buffer. From this sample, 100 microliters was retained for RNA extraction (RNEasy kit, QIAgen) and quantitation of viral nucleic acid by qPCR. The remaining sample was serially diluted and added to monolayers of RSV-permissive Hep2 cells. Cultures were maintained for 10 days to allow plaques to develop; then, cell monolayers were fixed and stained with crystal violet to aid in plaque identification. Virus recovery was calculated in plaque forming units per milliliter. For histopathology assessment, entire lungs were inflated with buffered formalin, and fixed tissue was embedded in paraffin blocks for processing.

|00179| It was observed that pre-autoimmune NZB mice, and the related strains NZW and BWFl , are highly susceptible to RSV infection at 5 to 6 weeks of age, well prior to any appearance of lupus symptoms. RSV infection at 10^Λ7 pfu was lethal in 25-30% of New Zealand mice but not in control mice such as BALB/c and C57B1/6, which are the standard mouse strains for RSV infection modeling. 100180] RSV disease in NZB mice is characterized as follows: a) increased viral recovery (approximately 1 log increase) compared with BALB/c; b) extensive occlusion of airway lumens, both bronchiolar and alveolar spaces, with protein and fluid derived from serum and interstitial spaces; this airways occlusion is not observed in BALB/c nor in any other rodent model of RSV infection; c) lower IFNg and no IL- 12 post infection in lung post infection compared with standard strain; d) recovery of cysteinyl leukotrienes from lung post infection, never reported in standard strain; e) recovery of eosinophils from lung post infection. These features of RSV infection in NZB mice closely match features of human RSV bronchiolitis. For this reason it is proposed the NZB model of RSV infection to offer significant advantages over other in vivo models currently used.

Example 3: NZB mouse model challenge

|00181] To determine whether modulation of innate immunity could determine the outcome of RSV infection, an TFNaR -neutralizing monoclonal antibody was administered to NZB mice on the day prior to virus infection. Another group of NZB mice received a neutralizing RSV F protein-directed humanized monoclonal antibody on the same schedule; other groups received anti-RSVF and anti-IFNaR antibodies together, or irrelevant isotype-matched antibodies. [00182 j Antibodies: Antibodies were administered via intraperitoneal route at 40 mg/kg in a total volume of 0.5 milliliters saline buffer. A monoclonal antibody directed against the extracellular domain of the interferon-alpha receptor (IFNAR; antibody provided by Dr. R. Schreiber), was used to block type I interferon signaling. RSV-F protein blockade was accomplished using an affinity-matured human monoclonal antibody motavizumab (Numax™, Medlmmune Inc., Gaithersburg, MD). Control mAbs of irrelevant specificity used in these studies were 1A7 (mulgGl) generated against E. coli FimH protein, and humanized anti-huCD2 which fails to cross-react with murine CD2, both obtained from Medlmmune, Inc. Antibodies were administered 12 hours prior to virus and on day 3 post infection. |00183] RSV Infection Procedure: Animals were lightly anesthetized with isofluorane. A 100 microliter inoculum of RSVA2 (10^Λ7 pfu) was placed on the nares using a pipettor. Mice were permitted to inhale the inoculum, then were placed in microisolator cages to recover from anesthesia. Weights and signs of illness were monitored over the course of 6 days. Endpoints were measured at the termination of the experiment on day 6.

[00184] Results: Blockade of type I interferon signaling via IFNaR blockade by an anti-IFNaR monoclonal antibody greatly reduced inflammatory infiltrate and therefore ameliorates RSV-associated inflammation in NZB animals as measured, 6 days post-infection, by (a) histopathologic evaluation, (b) loss of lung function as evaluated by bronchoalveolar lavage (BAL) cell differential analysis-; and (c) weight loss, as measured from starting weight. See Figure 13. |00185] The histopathology was performed as follows: animals were euthanized and the lungs removed, inflated with formalin, embedded in paraffin, then sectioned. HE staining was performed to assess histopathology. A histopathologist was blinded to the study groups before analyzing the HE stained lung tissue. A score was assigned on a scale from 0 = normal tissue to 3 = highly inflamed and occluded tissue. [00186] The BAL cell differential analysis was performed as follows: on the day of harvest, animals were euthanized using CO₂. The airway was cannulated and lavaged with 0.5 ml of saline. Cytokine and lipid mediators were measured in BAL fluid using commercial kits (Linco for Luminex. multiplex cytokine detection; R+D Systems for standard ELlSA of IFNg, IL-10, and Mig). BAL-associated cells were recovered by low speed centrifugation, and were analyzed for surface markers by flow cytometry. Antibodies recognizing GrI, Ly49, CDl I c, CDl Ib, CD3, CD4, and CD8 were obtained from Becton Dickinson Biosciences.

[00187] Even though IFNaR neutralization increased lung detection of

RSV, this therapeutic regimen did not exacerbate lung histopathology, weight loss or BAL cellularity after RSV infection. Instead, lung macrophage recovery was reduced in IFNaR antibody recipients, and total immune cell recovery from BAL trended lower although the difference did not reach statistical significance. In contrast RSV-F recipients demonstrated less bronchiolar occlusion without changes in overall inflammation. RSV- F blockade did not reduce immune cell recovery or weight loss compared with control Ig recipients. Combined RSV-F blockade and IFNaR neutralization showed significant reductions in lung inflammation, airways occlusion, and weight loss observed after RSV infection. Example 4: Phase I Clinical Trials in Healthy Adults

|00188] Antibodies of the invention or fragments thereof tested in in vitro assays and animal models, as described above, may be further evaluated for safety, tolerance and pharmacokinetics in groups of normal healthy adult volunteers. The volunteers are administered intramuscularly, intravenously or by a pulmonary delivery system a single dose of 1 mg/kg, 3 mg/kg, 5 mg/kg, 10 mg/kg or 15 mg/kg of an anti- inflammatory antibody or fragment thereof as previously described. Each volunteer is monitored at least 24 hours prior to receiving the single dose of the antibody or fragment thereof and each volunteer will be monitored for at least 48 hours after receiving the dose at a clinical site. Then volunteers are monitored as outpatients on days 3, 7, 14, 21 , 28, 35, 42, 49, and 56 post-dose.

[00189] Blood samples are collected via an indwelling catheter or direct venipuncture using 10 ml red-top Vacutainer tubes at the following intervals: (1 ) prior to administering the dose of the antibody or antibody fragment; (2) during the administration of the dose of the antibody or antibody fragment; (3) 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours after administering the dose of the antibody or antibody fragment; and (4) 3 days, 7 days 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administering the dose of the antibody or antibody fragment. Samples are allowed to clot at room temperature and serum will be collected after centrifugation.

[00190| The antibody or antibody fragment is partially purified from the serum samples and the amount of antibody or antibody fragment in the samples will be quantitated by ELlSA. Briefly, the ELlSA consists of coating microtiter plates overnight at 4°C. with an antibody that recognizes the antibody or antibody fragment administered to the volunteer. The plates are then blocked for approximately 30 minutes at room temperate with PBS-Tween-0.5% BSA. Standard curves are constructed using purifϊed- antibody or antibody fragment, not administered to a volunteer. Samples are diluted in PBS-Tween-BSA. The samples and standards are incubated for approximately 1 hour at room temperature. Next, the bound antibody is treated with a labeled antibody (e.g., horseradish peroxidase conjugated goat-anti-human IgG) for approximately 1 hour at room temperature. Binding of the labeled antibody is detected, e.g., by a spectrophotometer.

[001911 The concentration of antibody or antibody fragment levels in the serum of volunteers are corrected by subtracting the predose serum level (background level) from the serum levels at each collection interval after administration of the dose. For each volunteer the pharmacokinetic parameters are computed according to the model- independent approach (Gibaldi et al., 1982, Pharmacokinetics, 2nd edition, Marcel Dekker, New York) from the corrected serum antibody or antibody fragment concentrations.

Example 5 — Clinical Trials — Treatment of Pediatric Patients [00192] A randomized (2 treatment to 1 control), double-blind, placebo- controlled trial can be conducted to evaluate the safety and effectiveness of an administration of an an ti -inflammatory therapy (such as, for example, an isolated anti- human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, an isolated anti-interleukin 9 monoclonal antibody or fragment thereof, an isolated anti-CD19 monoclonal antibody or fragment thereof, an isolated anti-CD20 monoclonal antibody or fragment thereof, an isolated anti- CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti-BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist), which may or may not be combined with an anti-viral (such as, for example, an isolated anti-RSV F protein monoclonal antibody or fragment thereof, or a synthetic anti-viral compound, such as ribavirin), for the prophylaxis for serious bronchiolitis in high-risk children. The studies will be performed during an RSV season.

[00193| In choosing the test group, children can be eligible if they are either: 1 ) 35 weeks gestation or less and 6 months of age or younger; or 2) 24 months old or younger and have a clinical diagnosis of bronchopulmonary dysplasia (BPD) requiring ongoing medical treatment (ie, supplemental oxygen, steroids, bronchodilators, or diuretics within the past 6 months). Children will be excluded if they have any of the following: hospitalization at the time of entry that lasts more than 30 days; mechanical ventilation at the time of entry; life expectancy less than 6 months; active or recent RSV infection; known hepatic or renal dysfunction, seizure disorder, immunodeficiency, allergy to IgG products; receipt of RSV immune globulin within the past 3 months; or previous receipt of palivizumab, other monoclonal antibodies, RSV vaccines, or other investigational agents. Children with congenital heart disease will also be excluded.

[00194| Once the test group of children is appropriately screened and approved, they can be randomized to receive administrations of either (a) an anti- inflammatory therapy and/or an anti-viral therapy or (2) a placebo (for example, the same formulation, except without the therapeutic) in coded vials that does not identify the contents.

[00195] The primary endpoint can-be hospitalization with confirmed RSV infection. Children can be followed for 150 days (30 days from the last injection). Those with hospitalization as a result of RSV infection can be evaluated for total number of days in the hospital, total days with increased supplemental oxygen, total days with moderate or severe lower respiratory tract illness, and incidence and total days of intensive care and mechanical ventilation. The incidence of hospitalization for respiratory illness not caused by RSV and the incidence of otitis media can also evaluated. The placebo and test groups can be balanced at entry for demographics and RSV risk factors.

[00196] Patients will be followed by the clinical investigator for 150 days from randomization (30 days after the last scheduled injection), regardless of the amount of study drug they receive. At each visit and on each hospital day, children will be evaluated using the Lower Respiratory Tract Illness/Infection (LRI) Score as follows: 0 = no respiratory illness/infection; 1 = upper respiratory tract illness/infection; 2 = mild LRI; 3 = moderate LRI; 4 = severe LRI; 5 = mechanical ventilation. [00197] To capture all primary endpoints, all hospitalizations are identified and children with respiratory hospitalizations are tested for RSV antigen in respiratory secretions using commercially available tests. Children are considered to have reached the primary endpoint if: 1) they are hospitalized for a respiratory illness and the RSV antigen test of respiratory secretions is positive; or 2) if children already hospitalized for reasons other than RSV illness have a positive RSV test, and a minimum LRl score of 3 and at least 1 point higher compared with their last pre-illness visit. 100198] All hospitalized children are monitored to determine the total days of hospitalization. Children with RSV hospitalization are also monitored for the total days with an increased supplemental oxygen requirement, total days with a moderate or severe respiratory illness (based on the LRl score), and frequency and total days of ICU and mechanical ventilation. The incidence of clinically diagnosed otitis media will be recorded for all randomized patients.

(00199] Adverse events will be reported throughout the study period and each will be assessed by the investigators with regard to severity (using a standard toxicity table modified from the Pediatric AIDS Clinical Trials Group) and potential relationship to the study drug.

[00200] All randomized patients will be included in the safety and efficacy analyses. Statistical comparison of groups can be performed using Fisher's exact test for categorical variables and Wilcoxon rank sum test for continuous variables. The proportion of children with RSV hospitalization at 150 days can be estimated by the K.aplan-Meier method as an alternative analysis of the primary endpoint. Logistic regression can also be performed on the primary endpoint to evaluate predefined co van at es (gender, age, weight, BPD vs premature, without BPD).

EQUIVALENTS |00201| Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

J00202] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of preventing, treating or ameliorating bronchiolitis or a symptom thereof, the method comprising administering to a patient an effective amount of an antiinflammatory, wherein the anti-inflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti- interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, an isolated anti-CD 19 monoclonal antibody or fragment thereof, an isolated anti-CD20 monoclonal antibody or fragment thereof, an isolated anti-CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti-BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist.

2. The method of claim 1 , wherein the bronchiolitis or a symptom thereof is acute and caused by a respiratory syncytial virus infection.

3. The method of claim 1 , wherein the bronchiolitis or a symptom thereof is non-RSV associated bronchiolitis.

4. The method of claim 1 , wherein the symptom is bronchiolitis obliterans or wheezing.

5. A method of preventing, treating or ameliorating acute respiratory distress syndrome (ARDS) or a symptom thereof caused by systemic or pulmonary infection, the method comprising administering to a patient an effective amount of an anti-inflammatory, wherein the anti-inflammatory is selected from the group consisting of an isolated anti-human HMGBl monoclonal antibody or fragment thereof, an isolated anti-interferon alpha monoclonal antibody or fragment thereof, an isolated anti-interferon receptor monoclonal antibody or fragment thereof, an isolated anti-interleukin 9 monoclonal antibody or fragment thereof, an isolated anti-CD 19 monoclonal antibody or fragment thereof, an isolated anti- CD20 monoclonal antibody or fragment thereof, an isolated anti-CD21 monoclonal antibody or fragment thereof, an isolated anti-BAFF monoclonal antibody or fragment thereof, an isolated anti-BAFF receptor monoclonal antibody or fragment thereof, or an isolated mast cell product antagonist.

6. The method of claim 1 , wherein said method further prevents, treats or ameliorates the development of asthma.

7. The method of claim 1 -6, wherein the isolated anti-human HMGBl monoclonal antibody or fragment thereof is selected from the group consisting of: the S6 antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ ID NO: 1 1), CDR2 (SEQ ID NO: 12) and CDR3 (SEQ ID NO: 13) and the light chain CDRs as follows: CDRl (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 16); the Sl 6 antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ ID NO: 19), CDR2 (SEQ ID NO: 20) and CDR3 (SEQ ID NO: 21) and the light chain CDRs as follows: CDRl (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 24); the E l 1 antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ ID NO: 27), CDR2 (SEQ ID NO: 28) and CDR3 (SEQ ID NO: 29) and the light chain CDRs as follows: CDRl (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31 ) and CDR3 (SEQ ID NO: 32); or the G4 antibody or fragment thereof comprising the heavy chain CDRs as follows: CDRl (SEQ.ID NO: 3), CDR2 (SEQ ID NO: 4) and CDR3 (SEQ ID NO: 5) and the light chain CDRs as follows: CDRl (SEQ ID NO: 6), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 8).

8. The method of claim 1 -6, wherein the isolated anti- interferon alpha monoclonal antibody or fragment thereof comprises:

(a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 33, 34 and 35;

(b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 37 and 38;

wherein the antibody inhibits biological activity of at least one interferon alpha subtype.

9. The method of claim 1-6, wherein the isolated anti-interferon receptor monoclonal antibody or fragment thereof comprises the 9D4 heavy chain CDRs as follows: CDRl (SEQ ID NO: 39), CDR2 (SEQ ID NO: 40) and CDR3 (SEQ ID NO: 41) and the light chain CDRs as follows: CDRl (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43) and CDR3 (SEQ ID NO: 44).

10. The method of claim 6, wherein the mast cell product antagonist is a cysteinyl leukotriene antagonist selected from the group consisting of montelukast sodium (Singulair®) or zileutin (ZYFLO©).

1 1. The method of claim 1-6, wherein the method further comprises administering an effective amount of an anti -viral.

12. The method of claim 1 1 , wherein said anti-viral is one or more antibodies or fragments thereof that immunospecifically bind to one or more RSV antigens.

13. The method of claim 12, wherein the one or more RSV antigens is the RSV F protein.

14. The method of claim 13, wherein the one or more antibodies or fragments thereof that immunospecifically binds the RSV F protein is palivizumab or motavizumab.

15. The method of claim 1 1 , wherein said anti-viral is a synthetic anti-viral compound.

16. The method of claim 15, wherein the synthetic anti-viral compound is selected from the group consisting of ribavirin, Amantadine, Oseltamivir, Peramivir, Rimantadine, Zanamivir, Fomivirsen, Enfuvirtide, Imiquimod, Interferon,-or Viramidine.

17. The method of claim 1 -6, wherein said patient is a human infant.

18. The method of claim 1-6, wherein said patient is a human infant born prematurely or is at risk of hospitalization for an RSV infection.

19. The method of claim 1-6, wherein said patient is an elderly human patient.

20. The method of claim 1-6, wherein said patient lives in a nursing home or assisted living facility.

21. The method of claim 6, wherein the anti-inflammatory is administered after the onset of bronchiolitis in order to mitigate the predisposition of asthma development.

22. The method of claim 6, wherein the anti-inflammatory is administered during the convalescent phase of bronchiolitis in order to mitigate the predisposition of asthma development.

23. The method of claim 1-6, wherein the anti-inflammatory antibody therapeutics are administered one or twice a month.

24. The method of claim 1-6, wherein the mast cell product antagonist is administered once, twice, three time or four times daily.

25. The method of claim 12, wherein the anti-viral antibodies or fragments thereof that immunospecifically bind to one or more RSV antigens is administered once or twice a month.

26. The method of claim 15, wherein the synthetic anti-viral compound is administered once or twice a day.

27. A method of screening anti-virals for RSV, the method comprising:

a. infecting an epithelial cell line with a labeled RSV in the presence of candidate anti-virals for RSV;

28. The method of claim 27, wherein the candidate anti-virals for RSV are targeted against the NS or M proteins of RSV.