Immunocytochemical colocalization of specific immunoglobulin A with sendai virus protein in infected polarized epithelium.

Immunoglobulin (Ig)A provides the initial immune barrier to viruses at mucosal surfaces. Specific IgA interrupts viral replication in polarized epithelium during receptor-mediated transport, probably by binding to newly synthesized viral proteins. Here, we demonstrate by immunoelectron microscopy that specific IgA monoclonal antibodies (mAbs) accumulate within Sendai virus-infected polarized cell monolayers and colocalize with the hemagglutinin- neuraminidase (HN) viral protein in a novel intracellular structure. Neither IgG specific for HN nor irrelevant IgA mAbs colocalize with viral protein. Treatment of cultures with viral-specific IgA but not with viral-specific IgG or irrelevant IgA decreases viral titers. These observations provide definitive ultrastructural evidence of a subcellular compartment in which specific IgA and viral envelope proteins interact, further strengthening our hypothesis of intracellular neutralization of virus by specific IgA antibodies. Our results have important implications for intracellular protein trafficking, viral replication, and viral vaccine development.

Comparison of IgA versus IgG monoclonal antibodies for passive immunization of the murine respiratory tract.

The protective efficacy of anti-Sendai virus IgA was compared to that of IgG after topical application of monoclonal antibodies (MAb) to the respiratory tract of mice. BALB/c mice were passively intranasally immunized with 50 microliters ascites containing equivalent ELISA titers of MAb 1 h before and 4 and 24 h after intranasal challenge with Sendai virus. Lung viral titers were determined by plaque assay 3 days following challenge. In most instances IgA MAb afforded equivalent protection to IgG MAb in that there was no significant difference in virus recovery from the lungs of animals treated with either IgA or IgG MAb, including subclasses of IgG. When IgA MAb was fractionated into monomers and oligomers, there was no inherent advantage to the oligomeric form with respect to passive protection against viral challenge. The data indicate that IgA and IgG antibodies are equally efficacious in protecting the airways from viral infection. The experiments suggest that the advantage of IgA for protecting mucosal surfaces, such as the respiratory tract, relates to the presence of a specialized mechanism for transporting oligomeric IgA across epithelial surfaces. The results also support the rationale for active mucosal immunization protocols designed to generate an IgA response.

The contribution of respiratory viruses to severe exacerbations of asthma in adults.

Viral infections are known to be associated with severe exacerbations of asthma in children. In contrast, there is limited data that viral infections evoke acute episodes of asthma that require emergency care in adults. To determine the role of viral infections in exacerbations of asthma in adults, we examined 33 patients who presented to the emergency room with 35 exacerbations of asthma between September 1990 and March 1991 for the presence of a viral infection. A nasal swab was obtained for virus isolation by culture and rapid antigen detection by fluorescent staining. In 16 patients, serum was collected at initial presentation and 3 to 4 weeks later for acute and convalescent viral antibody titers. All patients had acute episodes of asthma ascertained by medical history and physical examination. About 56% of the patients with asthma exacerbations had symptoms suggestive of viral illness. Rapid antigen detection and viral cultures for influenza A and B, parainfluenza-1, 2, 3, respiratory syncytial virus, adenovirus, and rhinovirus were negative on all patients. Likewise, in all 16 patients tested, acute and convalescent serologic studies did not show a significant rise in titer by complement fixation test. Thus, despite symptoms consistent with viral infection, viral pathogens could not be shown by current virologic techniques. This study suggests that viral infection may not be as prevalent a precipitate of asthma in adults requiring emergency room treatment as is generally thought.

IgA and mucosal defense.

The traditional role of IgA antibodies in mucosal defense has been considered as providing an immune barrier to keep exogenous substances, including microbial pathogens, from penetrating the mucosa. In this way infections can be prevented. More recently, studies in vitro and in vivo are providing evidence to suggest that IgA may have additional roles in mucosal defense. For example, during their passage through the lining epithelial cells of mucous membranes en route to the secretions, IgA antibodies may have an opportunity to neutralize intracellular pathogens like viruses. Also, IgA antibodies in the mucosal lamina propria have opportunities to complex with antigens and excrete them through the adjacent mucosal epithelium, again by the same route to the secretions that is taken by free IgA. These latter functions could aid in recovery from infection.

Immunoglobulin A monoclonal antibodies protect against Sendai virus.

Immunoglobulin A anti-Sendai virus HN protein monoclonal antibodies, generated via a mucosal immunization protocol, were shown to neutralize virus in vitro and, when passively administered to the mouse respiratory tract, to protect against Sendai virus in vivo. Thus, immunoglobulin A antibodies by themselves can protect against respiratory virus infection.

Intracellular neutralization of influenza virus by immunoglobulin A anti-hemagglutinin monoclonal antibodies.

Traditionally, immunoglobulin A (IgA) was thought to neutralize virus by forming complexes with viral attachment proteins, blocking attachment of virions to host epithelial cells. Recently we have proposed an intracellular action for dimeric IgA, which is actively transported through epithelial cells by the polymeric immunoglobulin receptor (pIgR), in that it may be able to bind to newly synthesized viral proteins within the cell, preventing viral assembly. To this effect, we have previously demonstrated that IgA monoclonal antibodies against Sendai virus, a parainfluenza virus, colocalize with the viral hemagglutinin-neuraminidase protein within infected epithelial cells and reduce intracellular viral titers. Here we determine whether IgA can interact with influenza virus hemagglutinin (HA) protein within epithelial cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the pIgR were infected on their apical surfaces with influenza virus A/Puerto Rico/8-Mount Sinai. Polymeric IgA anti-HA, but not IgG anti-HA, delivered to the basolateral surface colocalized with HA protein within the cell by immunofluorescence. Compared with those of controls, viral titers were reduced in the supernatants and cell lysates from monolayers treated with anti-HA IgA but not with anti-HA IgG. Furthermore, the addition of anti-IgA antibodies to supernatants did not interfere with the neutralizing activity of IgA placed in the basal chamber, indicating that IgA was acting within the cell and not in the extracellular medium to interrupt viral replication. Thus, these studies provide additional support for the concept that IgA can inhibit replication of microbial pathogens intracellularly.

Transport of serum IgA into murine respiratory secretions and its implications for immunization strategies.

Both nonlabeled and radiolabeled IgA mAb with specificity toward Sendai virus, a respiratory pathogen, were used to investigate the transport of serum polymeric and monomeric IgA into murine upper and lower respiratory secretions as well as into the gut. After purification by affinity chromatography, IgA mAb were fractionated into monomers and polymers by gel filtration and radiolabeled with 125I. Mice were injected i.v. with either 125I-monomer and 131I-albumin or 125I-polymer and 161I-albumin. At various times after injection, serum and gut, nasal and bronchoalveolar lavage samples were collected. The TCA precipitable radioactivities were determined and the selective transport indices calculated. The results indicated selective transport of polymeric IgA but not monomeric IgA from serum into upper respiratory and intestinal secretions. The degree of TCA precipitability in nasal lavage and to a lesser extent gut secretions suggested significant degradation of the antibody during or after transport. To investigate further the integrity of the IgA in mucosal secretions, ELISA viral binding activity of nonradiolabeled IgA was determined for both IgA incubated with nasal secretions in vitro and polymeric IgA recovered by nasal lavage 4 h after i.v. injection. Although reconstitution experiments indicated no significant loss of antibody binding activity after incubation of antibody with lavage fluid in vitro, only negligible ELISA binding activity was detected in nasal washes after i.v. injection of antibody. The data overall suggest that although there is a quantitatively small, but selective transport of polymeric IgA into the upper respiratory tract, this transport results in minimal functional antibody activity. Implications of these and other findings for strategies of oral immunization in prophylaxis against respiratory infections are discussed.

Location of amino acid residues important for the structure and biological function of the haemagglutinin-neuraminidase glycoprotein of Sendai virus by analysis of escape mutants.

To locate sites important for the structure and function of the haemagglutinin-neuraminidase glycoprotein (HN) of Sendai virus, the biological characteristics of antibody-selected escape mutants were correlated with mutations in the primary HN amino acid sequence. An escape mutant virus deficient only in neuraminidase function but with an HN content equal to that of the wild-type virus had an amino acid change at residue 184, implying that this position may be important for maintaining a functionally active enzymic site. In contrast, other escape mutant viruses with reductions in haemagglutination (eightfold) and neuraminidase activities (70 to 80%) had a sharply diminished HN content and substitutions either at residue 375, or double mutations at residues 279 and 461. The loss of biological activity with the concomitant loss of HN content suggests that these sites may be important for the processing and transport of HN, or in maintaining a structure resistant to proteolytic degradation; residue 451 was shown to have an undefined role in fusion activity. The monoclonal antibodies (MAbs) used to isolate the mutant viruses included those of the IgA and IgG classes and were divided into four operational groups based on their haemagglutination-inhibition pattern against the selected mutants. MAbs of the IgA class recognized epitopes overlapping with (group A) as well as epitopes distinct from (groups C and D) those recognized by the IgG class; group B included only IgG antibodies. The epitopes recognized by IgA antibodies may identify residues important for the secretory immune response to the HN molecule.

Intracellular neutralization of virus by immunoglobulin A antibodies.

IgA is thought to neutralize viruses at the epithelial surface of mucous membranes by preventing their attachment. Since IgA, a polymeric immunoglobulin, is transported through the lining of epithelial cells by the polymeric-immunoglobulin receptor and since viruses are obligate intracellular parasites, we hypothesized that IgA antibodies may also interfere with viral replication by binding to newly synthesized viral proteins within infected cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the polymeric-immunoglobulin receptor were infected on the apical surface with Sendai virus. Anti-Sendai virus IgA monoclonal antibody delivered from the basolateral surface colocalized with viral protein within the cell, as documented by immunofluorescence. More importantly, anti-viral IgA reduced virus titers greater than 1000-fold (P less than 0.0001) in apical supernatants and greater than 10-fold (P less than 0.0001) in cell lysates from monolayers treated with anti-viral IgA compared with those treated with either anti-viral IgG or an irrelevant IgA monoclonal antibody. We believe that the differences in viral titers between cell layers treated with specific IgA, which enters the epithelial cell by binding to the polymeric-immunoglobulin receptor, and those treated with specific IgG, which does not enter the cells, or irrelevant IgA indicate that specific intracellular IgA antibodies can inhibit viral replication. Thus, in addition to the classical role of humoral antibodies in extracellular defense, IgA antibody may be able to neutralize microbial pathogens intracellularly, giving IgA a role in host defense that has traditionally been reserved for cell-mediated immunity.

A three-tiered view of the role of IgA in mucosal defense.

Mucosal IgA has generally been viewed as an immune barrier to prevent the adherence and absorption of antigens. Recent studies employing polarized epithelial monolayers have suggested two additional functions for mucosal IgA. One is to neutralize intracellular microbial pathogens, such as viruses, directly within epithelial cells. The second is to bind antigens in the mucosal lamina propria and excrete them through the adjacent epithelium into the lumen, thereby ridding the body of locally formed immune complexes and decreasing their access to the systemic circulation.

Hepatic pathology resulting from mouse hepatitis virus S infection in severe combined immunodeficiency mice.

Mouse hepatitis virus (MHV) is a pervasive pathogen that causes morbidity and mortality in mouse colonies worldwide. Although it is not a major cause of mortality in immunocompetent mice, infections from MHV strains of lower virulence can be fatal to athymic nude mice. The histopathologic features and alterations of serum biochemical parameters resulting from infection with a low-virulence MHV strain in severe combined immunodeficiency (scid) mice has not been well described. Thus we recently studied the disease caused by MHV-S in scid mice after intranasal inoculation. Mouse hepatitis virus infection in scid mice, which have severe defects of B and T cells, may be highly lethal, resulting in immediate mortality. However, our results indicate that scid mice survived for an average of 12 to 14 days after infection with doses of MHV up to 10(7) PFU/mouse. The virus caused a significant increase in serum enzyme activities and bilirubin concentration associated with histologically demonstrable hepatocellular injury at postinoculation days 3, 4, and 8. Furthermore, virus was detected in mouse liver homogenates and nasal and bronchial lavage specimens. These results provide valuable information regarding the histopathologic and biochemical consequences of MHV-S infection in scid mice.