Passive IgA Monoclonal Antibody Is No More Effective Than IgG at Protecting Mice from Mucosal Challenge with Respiratory Syncytial Virus

Abstract

Respiratory syncytial virus (RSV) is a mucosally restricted pathogen that can cause severe respiratory disease. Although parenteral administration of sufficient RSV-specific IgG can reduce severity of lower respiratory tract infection in high-risk infants, delivery of antibody by direct airway administration is an attractive alternative. Topical and parenteral administration of an IgA monoclonal antibody (MAb) specific for the RSV F glycoprotein was compared with an IgG MAb, specific for the same antigenic site, for ability to protect mice against RSV infection. Administration of RSV-specific IgG was more effective in reducing RSV titers in lung (4.6 log₁₀ pfu/g) than IgA MAb (3.6 log₁₀ pfu/g) when given intranasally immediately prior to infection (P = .005). RSV titers in the nose were reduced only by prophylactic administration of IgG parenterally. Therefore, topical administration of IgA is no more effective than topically administered IgG and is less effective than systemically administered IgG for protecting against RSV infection.

Respiratory syncytial virus (RSV) is the most important viral respiratory pathogen of infancy and childhood. After inoculation into the upper respiratory tract, RSV replicates for 1–3 days before producing lower respiratory tract symptoms affecting almost 60% of infants and up to 25% of toddlers and preschoolers [1]. Current treatment approaches for severe RSV-induced disease are ineffective. Therefore, prevention of disease is a high priority.

Active immunization has been an elusive ideal. Formalin-inactivated, alum-precipitated virus vaccine failed to protect and caused enhanced disease in vaccinees on exposure to wild-type virus [2]. Trials using live attenuated virus vaccines have not yet found the proper balance between attenuation and im-munogenicity [3, 4].

In contrast, passive immunoprophylaxis has shown promise in both animal [5] and human [6] trials. RSV-specific immunoglobulin, administered parenterally, reduces hospitalizations when given prophylactically [6] and positively affects some aspects of disease when given as treatment [7].

IgA is the most abundant immunoglobulin in mammals. Unlike other antibody isotypes, IgA is targeted to mucosal tissues [8], and virus-specific IgA in mucosal secretions has been shown to protect from reinfection [9, 10]. IgA, unlike IgG, is able to bind and neutralize viral proteins intracellularly at the site of initial replication in epithelial cells [11]. Mucosal IgA may be of particular importance in immunity against RSV, which is a mucosally restricted pathogen. Whereas RSV infection in humans begins with replication in the upper respiratory tract, it is possible that direct prophylaxis of the nasal mucosa may prevent subsequent progression to lower respiratory tract disease.

A purified murine monoclonal IgA directed at a specific epitope of the F protein of RSV has been tested in a mouse model of RSV infection and has been shown to reduce RSV titers in lung tissue if given immediately prior to challenge [12]. Comparison with an isotype control has not been reported, nor has its effect on the subsequent native antibody response to primary RSV infection. We report the outcome of primary RSV infection treated with IgA or IgG monoclonal antibody (MAb) directed against the same antigenic site on the RSV F glycoprotein compared with treatment with isotype control antibodies.

Methods

Mice

Pathogen-free female BALB/c mice (8 weeks old; Charles River, Raleigh, NC) were cared for as described elsewhere [13].

Antibody

Purified murine RSV F-specific IgA MAb (HNK-20) was provided by Oravax (Cambridge, MA). A dose of 5 mg/kg (∼100 μg) was administered in 20 or 100 μL volumes intran-asally. This dose had been shown to be effective at preventing primary infection in a prior study [12]. Isotype control antibody was a similarly prepared IgA MAb (2D6) directed against a control antigen (cholera toxin). It was administered at the same doses as the RSV-specific IgA MAb. The IgG MAb (133–1H) is reportedly specific for the same antigenic site on F by competitive binding assays [14] and was generously provided by Dr. Larry Anderson (CDC, Atlanta, GA). It was purified by precipitation with caprylic acid. Purity was evaluated by Coomassie-stained SDS-PAGE gels, and IgG concentration was measured by ELISA. An isotype control IgG MAb (HB151) specific for human HLA-Dr5 was prepared in a similar fashion. The IgG MAbs were used in the same concentrations and doses as the IgA MAbs. The 60% plaque reduction neutralization end point [13] for HNK-20 was 416 ng/mL and for 133-1H was 169 ng/mL

Cells and virus

HEp-2 cells were maintained in Eagle MEM supplemented with glutamine, amphotericin, gentamicin, penicillin G, and 10% fetal bovine serum. The A2 strain of RSV was kindly provided by Dr. Robert Chanock (National Institutes of Health). Working stocks of the virus were prepared as described elsewhere [13].

Mouse infection

Anesthetized mice were infected nasally with 100 μL of RSV stock. An illness grading scale was used to assign numbers to a set of clinical features detected in mice with different degrees of illness: 0, healthy; 1, barely ruffled fur; 2, ruffled fur, but active; 3, ruffled fur and inactive; 4, ruffled fur, inactive, hunched, and gaunt; 5, dead. Illness scores were assigned by a blinded observer. Illness was also assessed by weighing the mice daily.

Plaque assays and neutralization tests

Two-day-old HEp-2 monolayers, 80% confluent in 12-well plates (Costar, Cambridge, MA) were used for plaque assay and neutralization tests. The assays were done as described elsewhere [13].

Nose washes (NWs) and bronchoalveolar lavages (BALs)

After mice were euthanized by CO₂ inhalation, the trachea was exposed by blunt dissection. A 25-gauge needle attached to a syringe containing 1 mL of sterile PBS was used to enter the trachea. The needle was immobilized by using 2–0 nylon suture material, and the PBS was gently flushed into the lungs, aspirated, and then reinfused. Lungs were flushed 3 times, and then the sample was aspirated and placed into a cryovial for freezing. NWs were performed by retrograde flushing of 1.5-mL sterile PBS from the back of the nasopharynx through the nares and collecting the sample in a cryovial. Samples were quick-frozen in dry ice and ethanol.

ELISAs for antibodies in mucosal samples

ELISAs were performed on BAL and NW specimens by using 96-well microtiter plates (Costar) coated with BCH4 cells, which are persistently RSV-infected BALB/c fibroblasts (provided by Dr. Bruce Fernie). Samples were placed into the wells and serially diluted. Alkaline phosphatase-conjugated sheep anti-mouse IgG and IgA (Sigma, St. Louis), were used to quantify amounts of RSV-specific antibody of each isotype in the samples. The colorimetric reaction was done with Sigma 104 phosphatase tablets dissolved in diethanolamine buffer. Samples were read at a wavelength of 405 nm by a Titertek Multiscan (Flow Laboratories, McLean, VA) at 30 and 60 min of incubation.

Study design and statistical analysis

Thirteen mice in each group were given 20 μL of either the RSV F-specific murine IgA (HNK-20) or murine IgG (133-1H) intranasally 1 h prior to infection with 10⁷ pfu of RSV. Two other groups were given the same amount of the RSV F-specific IgA and the IgG product, but diluted in a 10-fold larger volume, and administered intraperitoneally, 24 h prior to infection with the same RSV challenge dose. Controls included 1 group of 13 mice that was given control IgA intranasally and 1 group given control IgG intraperitoneally. Five mice from each group were killed on day 4 to assess peak virus replication in both lung and nose. Daily weights and illness scores were performed on another 5 mice in each group. Lungs from 3 mice in each group were submitted for histopathologic examination on day 8. Primary antibody response was assessed on serum collected from 5 mice in each group on day 42. Bronchoalveolar lavages and nasal washes were performed in 5 mice from each group to evaluate mucosal antibody production on day 8.

One-way analysis of variance and 2-tailed t tests were used to determine statistically significant differences between groups, defined as P < .05. For calculating geometric mean titers, a sample with no detectable virus was assigned a value of 50% the lower limit of detection.

Results

Virus replication

Prophylactic RSV-specific IgA MAb given intranasally reduced virus replication in the lungs by 3.6 Iog₁₀/g lung, as reported elsewhere [12]. However, it had no effect on virus replication in the nose, which differs from the prior report [12]. RSV-specific IgA MAb given intraperitoneally had no effect on RSV titer in either lung or nose. In contrast, RSV-specific IgG MAb given intraperitoneally decreased replication in lung by 4.5 Iog₁₀/g lung. Intraperitoneal administration of IgG had no effect upon virus replication in the nose, as noted in previous experiments. The same RSV-specific IgG MAb administered intranasally eliminated replication in the nose almost completely and also had a greater effect on lung virus replication (reduction of 4.6 Iog₁₀/g) than did the RSV-specific IgA MAb (P = .005; figure 1).

Figure 1

Virus replication and illness patterns in mice given prophylactic antibodies. A, geometric mean titer ± SD of virus recovered per gram of lung (solid box) tissue or per nose (diagonal striped) from BALB/c mice given respiratory syncytial virus F-specific IgA or IgG intranasally (in) or intraperitoneally (ip), control IgA in, or control IgG ip Virus replication is suppressed to the greatest degree by in IgG. B, weight loss and illness were most pronounced in mice who received IgA ip (▲) or groups receiving the control antibodies (dashed lines) in (*) or ip (●). Other curves represent groups treated with HNK-20 in (■), HNK-20 ip (▲), 133-1H in (▼), and 133-1H ip (◆). Data are from 1 representative experiment of 2. Each value represents data from 5 mice.

Illness and weight loss

Illness was not severe in any of the groups but was worst in the groups treated with RSV-specific or control IgA MAb intraperitoneally. Weight loss curves reflected illness score data, with peak values on days 6 and 7 (figure 1).

Neutralizing antibody production

Primary serum antibody response was measured in 5 mice from each group at day 42. Mean geometric log₂ 60% neutralization titers were somewhat suppressed in groups receiving either the RSV F-specific IgA MAb intranasally or the IgG MAb by any route. Although the differences did not reach statistical significance, HNK-20 (IgA)-treated mice had slightly higher neutralizing activity than 133-1H (IgG)-treated mice. Titers in the group that received RSV F-specific IgA MAb intraperitoneally were not distinguishable from those of control groups. Neutralizing antibody response was most suppressed in the group that received RSV-specific IgG intranasally (figure 2).

Figure 2

ELISA and neutralizing antibody responses. IgG in sera (■), bronchoalveolar lavage (BAL) fluid (left diagonal stripes), and nasal wash (NW; right diagonal stripes), from mice given prophylactic antibodies intranasally (in) or intraperitoneally (ip) is shown in comparison with serum neutralizing (NT) activity (□). No IgA was detected in any of the samples. Results represent the geometric mean log₂ titer ± SD for each measurement. Suppression of native antibody response is noted to be highest in the group given in IgG and is inversely correlated with virus replication (shown in figure 1A). Each point represents data pooled from 5 mice. RSV, respiratory syncytial virus.

Systemic and mucosal antibody production

RSV-specific IgA was not detected in serum, BAL, or NW from any group. Levels of RSV-specific IgG in mucosal samples paralleled those seen in serum and were most suppressed in groups that received the RSV-specific IgG MAb product intranasally or intraperitoneally. Interestingly, the RSV-specific IgG response in HNK-20 (IgA)-treated mice was significantly higher than in 133-1H (IgG)-treated mice, despite similar suppression of RSV titer in lung (figure 2).

Histopathology

Inflammatory changes were minimal in animals that received IgG or IgA intranasally or IgG intraperitoneally. Moderate periarteriolar, peribronchial, and interstitial lymphocytic infiltration was present in the control groups and in mice given RSV-specific IgA MAb intraperitoneally, typical of primary RSV infection. No infiltration of neutrophils or eosinophils was noted (data not shown).

Discussion

The present study examines the role of IgA in passive immunization against infection with RSV. Like previously studied monoclonal IgG antibodies, this monoclonal IgA antibody is able to protect against RSV replication in lungs if given immediately prior to challenge [5] but is unable to protect the nasal mucosa. In contrast to our findings, a prior study evaluating the RSV-specific IgA intranasally given 1 h prior to RSV challenge reported a decrease in RSV titers in nose [12]. The major difference was that in our study, the volume used for RSV challenge was 4-fold higher, and the viral titer was 50-fold higher, perhaps diluting and overcoming the neutralizing capacity of the product.

These data suggest that prevention of infection may be more dependent on the neutralization titer of the passive antibody administered than on its isotype. The RSV-specific IgG antibody, which had ∼3-fold higher in vitro neutralization titer than the RSV-specific IgA antibody, was more effective at preventing virus replication on primary challenge than was the IgA antibody directed at the same antigenic site. Furthermore, the RSV-specific IgG was effective whether given intranasally or intra-peritoneally, whereas the RSV-specific IgA antibody was only effective intranasally. This is probably because the half-life of IgA in serum is extremely short, especially in mice, in which hepatocytes express polymeric immunoglobulin receptor, and actively remove IgA from the circulation [15].

Suppression of RSV-specific antibody response by passive antibody was affected by the isotype of the treatment antibody. IgA prophylaxis had slightly less effect on the IgG response to RSV than IgG prophylaxis (figure 2), suggesting the possibility that RSV-IgA immune complexes may have different antigenic properties than RSV-IgG immune complexes. An alternative explanation is that the IgA treatment caused less reduction in virus titer, thereby providing a higher antigen load to stimulate the primary antibody response.

The potent antiviral activity of naturally produced and secreted mucosal IgA is related to its production in the lamina propria, active transport across epithelial cells, and secretion at their apical surfaces. Topically administered antibody delivered by currently available approaches may be unable to achieve sufficient concentrations between the mucociliary blanket and the apical cell membrane to prevent infection of respiratory epithelium. In summary, we have shown that the properties of topically or parenterally administered IgA are not as effective as those of IgG for preventing or aborting RSV infection and that passively administered antibody is more effective for lower than upper respiratory tract RSV infection.

Acknowledgments

We thank Frances Robinson and Rauf Kuli-Zade for technical assistance.

All animals used in these experiments were cared for according to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and according to the guidelines established by Vanderbilt University.

No author of this paper has a commercial or other association that might pose a conflict of interest in objective evaluation of this data.

Grant support: Oravax, Inc.