Intranasal Delivery of an IgA Monoclonal Antibody Effective against Sublethal H5N1 Influenza Virus Infection in Mice

s/p20 myeloma cells (ATCC) were cultured in modified Eagle's medium (MEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO). Madin-Darby canine kidney (MDCK) cells (ATCC) were maintained in MEM containing 5% FBS. 293-T human embryonic kidney cells (ATCC) were cultured in Opti-MEM (Gibco, Grand Island, NY) containing 5% FBS. The A/egret/Egypt/1162-NAMRU3/2006 (H5N1) (H5/egret) and A/Vietnam/1203/2004 (H5N1) (H5/Vietnam) strains were kindly provided by Ruben Donis, Centers for Disease Control and Prevention, Atlanta, GA. The attenuated avian influenza virus ΔH5N1-WF10tsHA has been previously described in detail (25). Other viruses are listed in Table 1, below. These viruses were propagated in 10-day-old embryonated specific-pathogen-free (SPF) chicken eggs at 35°C and stored at −70°C. The viruses were titrated by the Reed and Muench method to determine the 50% tissue culture infective dose (TCID₅₀) and minimal 50% mouse lethal dose (MLD₅₀) (22, 32).

Eight-week-old female BALB/c mice (n = 8; National Cancer Institute, Frederick, MD) were immunized by intraperitoneal injections with attenuated avian influenza virus ΔH5N1-WF10tsHA (25). Doses consisted of 200 μl of the allantoic fluid containing 2,048 HA units of virus. Boost immunization was given at 10, 20, and 30 days postvaccination. Animal studies using attenuated ΔH5N1-WF10tsHA recombinant viruses were conducted under animal biosafety level 2 (ABSL-2) conditions and performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland, College Park.

Mouse spleen cells collected at day 4 post-third boost with ΔH5N1-WF10tsHA virus were fused to the s/p20 myeloma cells as previously described (37). After selection of the hybridomas in hypoxanthine-aminopterin-thymidine medium (HAT; Invitrogen, Carlsbad, CA), antibody-producing cells were screened by the hemagglutination inhibition (HI) method (32) and subcloned by limiting dilution. Positive clones were checked for isotype by using the Iso-Gold Rapid mouse monoclonal isotyping test kit (BioAssay Works, Ijamsville, MD) as described in the manufacturer's protocol. The MAbs from ascites fluids were generated as previously described (37).

The culture supernatants from MAb-expressing cells were collected, and the proteins (10 μg total protein/well) were analyzed by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. After PAGE, the proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) and analyzed by Western blotting. Western blots were revealed using horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig or IgA (Santa Cruz Biotechnology, Santa Cruz, CA) and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) followed by X-ray film exposure (Thermo Scientific, Rockford, IL).

For microneutralization assays (MN) (see Table 2), serially diluted MAbs were first incubated with 100 TCID₅₀ of virus for 1 h at 37°C. The virus-MAb mixture was then adsorbed to MDCK cells for 1 h. Infected cells were washed twice with phosphate-buffered saline (PBS) and replenished with Opti-MEM (Gibco, Grand Island, NY). The infected cell supernatants were harvested at 4 days postinoculation (dpi) and were analyzed by HA assay (32). In vivo neutralization followed by in vitro evaluation were performed by incubating the MAbs (1.5 mg/ml) with 1,000 PFU of H5/Vietnam virus for 1 h at 37°C. Then the virus-MAb mixture was inoculated intranasally into 5-week-old mice (n = 5). Body weight, morbidity, and survival of mice were monitored for 14 dpi.

To find escape mutant H5N1 viruses, studies were designed as previously described (27). In brief, 100 TCID₅₀ of 2ΔH5N1-WF10 were propagated in MDCK cells in the presence of MAb DPJY01 (100 μg/ml) in a six-well plate format. After 72 h, 1 ml of the supernatant was further propagated in MDCK cells in the presence of MAb DPJY01. The supernatant from the second passage was harvested for the third virus propagation. We also performed similar studies in eggs. Briefly, MAb (100 μg/ml) was incubated with 100 TCID₅₀ of virus at room temperature for 30 min and then the mixture was inoculated into 10-day-old embryonated chicken eggs (n = 5). After 48 h, the allantoic fluid was harvested and used for two additional cycles of selection. A monoclonal antibody against NP (2B9) was used as control. Escape mutants were also evaluated using viruses found in lung, brain, and tracheal homogenates of treated mice at 48 and 72 h postinoculation (hpi) (see results shown in Fig. 5, below). The volume of tissue homogenate used corresponded to 100 TCID₅₀ of virus and it was preincubated with MAb (100 μg/ml) for 30 min at room temperature prior to infection of MDCK cells.

The ascites fluids were filtered using a 0.22-μm filter and then heat inactivated at 56°C for 30 min. Six-week-old mice (n = 5) were treated intranasally with a single dose of 50 μl PBS/mouse containing either 50 mg/kg of body weight or 10 mg/kg of MAb (the concentration corresponded to the total protein concentration, which was quantified by two independent methods, bicinchoninic acid protein assay kit [Pierce, Rockford, IL] and protein absorbance at 280 nm using the NanoDrop ND-1000 photometer [Thermo Scientific, Wilmington, DE]). The treatment was performed at different time points before or after virus inoculation as indicated in Results. Two different H5N1 strains were used for inoculation, H5/Vietnam from clade 1.0 and H5/egret from clade 2.2.1 at doses of 3 PFU of H5/Vietnam and 300 50% egg infective doses (EID₅₀) of H5/egret, respectively. The inoculation doses were lethal to between 80 and 100% (∼2 MLD₅₀) of untreated or mock-treated mice. Body weight change was monitored daily until day 21 postinoculation. Additionally, infected mice were monitored for other morbidity signs, including rough coat, inactivity, and neurological signs, following an approved scoring method. Mice showing body weight losses of ≥25% of preinoculation values were euthanized for humane reasons and counted as dead.

Virus titers in brain, lungs, and trachea were determined in 6-week-old mice that were administered intranasally a single dose of 50 μl PBS/mouse containing 50 mg/kg of DPJY01 MAb at different time points before or after inoculation with 3 PFU of H5/Vietnam. An IgA MAb 1D9 directed against the HA of 2009 H1N1 pandemic influenza viruses (H. Shao and D. R. Perez, unpublished data) was used as a control. Brain, lungs, and tracheas were collected at 6 dpi and titrated in MDCK cells by a TCID₅₀ assay with a limit of virus detection of ≤0.6999 log₁₀ TCID₅₀/g of tissue. H5N1 inoculation studies were conducted under ABSL-3 conditions approved by the U.S. Department of Agriculture and performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland, College Park.

After fusion between spleen cells from H5N1 virus-immunized mice and s/p20 myeloma cells, screening by HI assay yielded six monoclonal antibodies specific to the HA protein of the H5 virus. Subtyping showed that one of these MAbs was an IgA subtype, herein referred to as DPJY01. Because previous passive immunotherapy studies against H5 influenza virus have not made use of IgA antibodies, DPJY01 was tested as a suitable antiviral alternative. The subtype and polymeric structure of DPJY01 were confirmed by Western blotting under nonreducing conditions. The DPJY01 MAb was produced in monomeric (I) and polymeric (II) forms as revealed with an anti-IgA specific antibody or anti-mouse antibody (Fig. 1). An IgG MAb was used as a control, which as expected did not react with the anti-IgA antibody (Fig. 1). In contrast, both the IgG control and DPJY01 showed positive reactions against an anti-mouse antibody (Fig. 1).

In order to determine the specificity and the HI profile of DPJY01 for the H5 HA viruses, HI assays were performed using viruses of the H5, H1, H2, H3, H6, H7, H8, H9, H10, H11, H12, and H13 subtypes (Table 1). HI assays revealed that DPJY01 did not cross-react with H1, H2, H3, H6, H7, H8, H9, H10, H11, H12, and H13 HA subtype viruses and reacted exclusively with H5 HA viruses (Table 1). Moreover, DPJY01 showed a broad HI profile against Asian H5N1 viruses of different clades, including clades 0, 1.0, 2.1, 2.2, and 2.3. DPJY01 also showed strong HI profiles against a low-pathogenic H5 virus of the North American lineage and a low-pathogenic H5 virus from the Eurasian lineage (Table 1), suggesting that this IgA binds to a highly conserved epitope in the H5 HA protein.

To further test if the conserved epitope would be easily mutated and recognition by DPJY01 would be lost, we attempted to generate escape mutant viruses. Despite several attempts, we could not isolate a mutant virus that would not be neutralized by the DPJY01 MAb. Selection in the presence of 100 μg/ml of DPJY01 IgA in tissue culture MDCK cells or in eggs produced no escape mutant viruses. Although the use of various virus/antibody ratios could be attempted in order to select for escape mutants, this was beyond the scope of the present report.

To evaluate the neutralization activity of DPJY01 MAb, a microneutralization assay was performed in MDCK cells. As shown in Table 2, the neutralization titer of DPJY01 for H5N1 virus reached more than 10,240/50 μl, whereas there was no neutralization for H9, H7, and H1 viruses. More importantly, prior incubation of 1,000 PFU of H5/Vietnam virus with DPJY01 resulted in complete block of virus replication in mice (Fig. 2). Control mice that receive an inoculum containing the H5/Vietnam virus and MAb 2B9 (obtained against the influenza virus nucleoprotein and which cannot neutralize the virus in vitro) showed a rapid decline in body weight and did not survive (Fig. 2A and B). It is important to note that 3 PFU of H5/Vietnam represents a sublethal dose in mice, sufficient to produce between 80 and 100% mortality. Moreover, DPJY01 was ineffective against the mouse-adapted lethal strain A/WSN/33 (H1N1) and the 2009 pandemic mouse-adapted lethal strain ma-Ca/04 (data not shown). These results indicate high specificity and inhibitory activity of DPJY01 for H5 viruses in vitro and in vivo.

An acute respiratory disease characterizes H5N1 influenza virus infection in humans (1, 5, 13). The respiratory tract is the site of virus replication, and a combination of host-virus interactions modulates the severity and outcome of the disease. It is known that mucosal, virus-specific IgA antibodies play a vital role in the antiviral immune response (29, 33). To evaluate the efficacy of DPJY01 against H5N1 in vivo, the antibody was delivered to mice via intranasal droplets before H5N1 inoculation. In a prophylactic study, pretreatment with 50 mg/kg or 10 mg/kg of DPJY01 at 24 h before inoculation with H5/Vietnam provided 100% and 80% protection, respectively (Fig. 3A). Additionally, mice were fully protected against inoculation with the H5/egret virus (Fig. 3B), regardless of the dose of MAb used. Thus, DPJY101 shows efficient antiviral activity. As expected, MAb 2B9 showed no prophylactic protection against the H5N1 viruses (Fig. 3A and B). These results further support our in vitro neutralization studies with respect to the effective inhibitory activity of DPJY01 against H5 viruses of different clades. We also explored whether prophylactic protection could be achieved by providing the MAb at 48 h or at 72 h before inoculation (Fig. 3C). Here too, 100% protection against H5/Vietnam was observed with a single dose of 50 mg/kg administered at either 48 h or 72 h before inoculation (Fig. 3C), suggesting that the IgA antibody could be maintained in an active form for at least 72 h before virus inoculation and was sufficient to neutralize the virus. DPJY01 administration prevented signs of disease and body weight loss in mice that received the 50-mg/kg dose regardless of the time of administration before virus inoculation. Administration of 10 mg/kg of DPJY01 also resulted in no signs of disease and no significant body weight loss except for one mouse that died in the 24-h pretreatment group at 12 dpi (Fig. 3A). In contrast, mock-treated mice showed exacerbated signs of disease and significant body weight losses, including the few mice that ultimately recovered from the infection. These results show that DPJY01 not only increases the chances of survival but also prevents the disease associated with H5N1 infection.

In order to determine the therapeutic potential of DPJY01, the MAb was administered to mice at either 50 mg/kg or 10 mg/kg at 24 h after virus inoculation. Our results showed that the 50-mg/kg dose offered 100% protection against H5/Vietnam, whereas the 10-mg/kg dose attained 60% protection, although mortality was in general delayed 3 to 4 days compared to the mock-treated control group (Fig. 4A). As expected, the mock-treated group (treatment with 50 mg/kg of the NP MAb) showed 100% mortality within 10 dpi (Fig. 4A). Additionally, the 50-mg/kg and 10-mg/kg doses of DPJY01 also provided 100% and 80% protection against H5/egret infection (Fig. 4B). Again, mortality was delayed compared to the mock-treated group. DPJY01, at the 50-mg/kg single dose, was effective in preventing signs of disease and significant body weight loss after inoculation with either H5/Vietnam or H5/egret for the duration of the study (Fig. 4D and E). At the 10-mg/kg single dose, DPJY01 was also effective at preventing significant body weight losses for the first 14 dpi, despite the fact that some mice were euthanized due to ethical reasons (Fig. 4D). To monitor therapeutic efficacy at different times postinoculation, 50 mg/kg DPJY01 was given at 48 or at 72 after virus inoculation. As shown in Fig. 4C, DPJY01 provided 80% and 60% protection when administered at 48 and 72 hpi, respectively. Once again, mortality was delayed compared to the mock-treated group, suggesting that DPJY01 has the potential to significantly limit virus replication once the infection is well under way. Although body weight losses and signs of disease were more evident in mice that received the DPJY01 treatment at 48 and 72 h after virus inoculation (Fig. 4F), they were less severe and less significant than in the mock-treated group. We would like to emphasize that these results represent effects after a single intranasal dose administration of DPJY01 and thus suggest a strong antiviral activity by this IgA MAb.

In order to test whether DPJY01 could provide protection by decreasing virus replication, 6-week-old mice were administered 50 mg/kg of DPJY01 at 24, 48, or 72 h pre- or postinoculation with 3 PFU of H5/Vietnam (Fig. 5). Virus titers in the trachea, lung, and brain of infected mice were determined by 6 dpi in MDCK cells as described in Materials and Methods. Pretreatment with DPJY01 prior to inoculation resulted in complete inhibition of virus replication in trachea, lung, and brain of infected mice regardless of the day of DPJY01 inoculation. The exception was one mouse in the 72-h group (Fig. 5A, B, and C); however, lung virus titers in this mouse were 10,000-fold lower than in the control group mice (Fig. 5B). Treatment after inoculation resulted in complete block in virus replication in the trachea, lungs, or brain if DPJY01 was administered 24 hpi. When administrated at 48 or 72 hpi, DPJY01 could also completely block virus replication in two out of three mice in each group. The mice in which DPJY01 was ineffective showed virus titers not higher than in the control groups, with the exception of virus titers in the brain of one mouse in the 48-hpi treatment group (Fig. 5E). In order to determine whether failure of protection in these mice was the result of the emergence of H5N1 escape mutants in vivo, tissue homogenates containing the equivalent of 100 TCID₅₀ of virus were incubated in the presence of DPJY01 or 1D9 control antibody, and virus growth was analyzed in MDCK cells. No evidence of escape mutants was found in the tissue homogenates (data not shown). These results suggest that the mechanism of protective action of DPJY01 in vivo is by binding and neutralizing the virus. Failure of the antibody to control infection at 6 dpi may be due to a combination of ongoing replication and inefficient access of DPJY01 to the site of replication; however, the data presented in Fig. 5 suggest that DPJY01 can extend survival beyond the window of 6 to 10 days in which most control mice succumb to infection.