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A novel influenza (H1N1) virus caused an influenza pandemic in 2009, while highly pathogenic H5N1 avian influenza viruses have continued to infect humans since 1997. Influenza, therefore, remains a serious health threat. Currently, neuraminidase (NA) inhibitors are the mainstay for influenza therapy; however, drug-resistant mutants of seasonal H1N1 and H5N1 viruses have emerged highlighting the need for alternative therapeutic approaches. One such approach is antibody immunotherapy. Here, we show that the monoclonal antibody C179, which recognizes a neutralizing epitope common among H1, H2, H5, and H6 hemagglutinins (HAs), protected mice from a lethal challenge with various H5N1 and pandemic (H1N1) 2009 viruses when administered either intraperitoneally or intranasally. The protective efficacy of intranasally inoculated C179 was comparable to that of intraperitoneal administration. Our results suggest that direct administration of this anti-influenza antibody to viral replication sites is an effective strategy for prophylaxis and therapy.
The first fatal case of highly pathogenic avian H5N1 influenza virus infection of a human was reported in Hong Kong in 1997 (Claas et al., 1998; Subbarao et al., 1998). Since then, over 470 people have been infected with H5N1 viruses, with a mortality rate of about 60% (http://www.who.int/csr/disease/avian_influenza/en/). As outbreaks of H5N1 virus infection of poultry have spread worldwide, the virus' glycoprotein, hemagglutinin (HA), has evolved into multiple phylogenetically distinct clades and subclades (Chen et al., 2006; Webster and Govorkova, 2006; WHO, 2008). Meanwhile, in 2009, a novel influenza virus, pandemic (H1N1) 2009 virus, caused a pandemic with serious public health issues (Khan et al., 2009).
Vaccines offer effective prophylaxis for influenza, whereas, for therapy, two classes of drugs are available: M2 ion channel blockers (amantadine and rimantadine) (Dolin et al., 1982; Hay et al., 1985) and neuraminidase (NA) inhibitors (oseltamivir and zanamivir) (Zambon and Hayden, 2001). However, viruses resistant to M2 ion channel blockers have emerged (Belshe et al., 1989; Bright et al., 2006; He et al., 2008; Shiraishi et al., 2003) and spread worldwide. In fact, currently circulating seasonal H1N1 and H3N2 viruses, as well as some H5N1 viruses, are resistant to M2 ion channel blockers (Bright et al., 2006; He et al., 2008). M2 ion channel blockers, therefore, are no longer the first choice for influenza therapy. The NA inhibitors are effective against seasonal influenza viruses, including those resistant to M2 ion channel blockers; however, oseltamivir-resistant viruses have appeared sporadically (Kiso et al., 2004). Indeed, during the 2007–2008 season, oseltamivir-resistant seasonal H1N1 viruses emerged and spread worldwide (Cheng et al., 2010; Meijer et al., 2009). Moreover, oseltamivir-resistant pandemic (H1N1) 2009 and H5N1 viruses have also been reported (Le et al., 2005; Wang et al., 2010), raising concerns regarding the management of influenza virus infections. Taken together, these resistance patterns underscore the need for novel antiviral therapeutic options.
Antibody immunotherapy is an effective therapeutic strategy with high potency. In fact, human monoclonal antibody (mAb)-based immunotherapy has been used to treat numerous human diseases, including respiratory tract infections (Desjardin and Snydman, 1998; Frogel et al., 2010; Groothuis and Simoes, 1993). Monoclonal antibody-based immunotherapy may, therefore, be of value in the management of influenza outbreaks, including those caused by highly pathogenic avian H5N1 and pandemic (H1N1) 2009 viruses. Influenza A viruses are sub-classified according to the antigenicity of their two major surface proteins: HA (H1–H16), which mediates cell entry and membrane fusion, and NA (N1–N9), which facilitates virion release. However, antigenic drift occurs in human influenza viruses, necessitating modification and re-administration of the vaccine to ensure protection. Therefore, the ideal antibodies for influenza immunotherapy would be cross-reactive among the HAs of different subtypes and their drift variants. Recently, several monoclonal antibodies cross-reactive with influenza A viruses of different HA subtypes, including H5, have been shown to be highly protective in mouse and ferret models (Chen et al., 2010; Chen et al., 2009; Friesen et al., 2010; Khurana et al., 2009; Sui et al., 2009; Throsby et al., 2008; Yoshida et al., 2009). C179, which was raised against H2 HA more than 15 years ago, was the first anti-HA monoclonal antibody shown to be subtype cross-reactive, neutralizing viruses of the H1, H2, and H5 subtypes (Okuno et al., 1993). C179 recognizes a common epitope among H1, H2, H5, and H6 (Smirnov Iu et al., 1999), and neutralizes virus by inhibiting the fusion process (Okuno et al., 1993). It has been shown to protect mice from a mouse-adapted seasonal H1N1 virus strain (Okuno et al., 1994), but its efficacy against highly pathogenic H5N1 avian and pandemic (H1N1) 2009 influenza viruses has not been demonstrated in animal models.
Although the studies to date with anti-HA monoclonal antibodies have shown these antibodies to be highly protective in animal models, these studies all used intraperitoneal or intravenous inoculation. Other inoculation routes (e.g., intranasal inoculation) have not been evaluated (Friesen et al., 2010; Throsby et al., 2008; Yoshida et al., 2009). Here, to bridge this gap, we evaluated the prophylactic and therapeutic efficacy of C179 against various H5N1 viruses and a pandemic (H1N1) 2009 virus, comparing its efficacy via intraperitoneal and intranasal administration in a mouse model.
The mouse monoclonal antibody (mAb) H3-32.1 recognizes only H3 HA and was used as a control.
The mAbs C179 and H3-32.1 were purified from acsites by affinity chromatography using an IgG purification kit (Pierce) or HiTrap Protein G Columns (GE Healthcare) before use.
The following virus strains were used in this study: A/Vietnam/UT3040/04 (H5N1, clade1; VN3040), A/Vietnam/UT31312III/07 (H5N1, clade2.3.4; VN31312III), A/swine/Indonesia/UT2071/05 (H5N1, clade2.1.1; SwIDN2071), A/Indonesia/UT3006/05 (H5N1, clade2.1.3; IDN3006), A/chicken/Miyazaki/K11/07 (H5N1, clade2.2; CkMiyazaki), A/California/04/09 (pandemic (H1N1) 2009; CA04), mouse-adapted A/California/04/09 (pandemic (H1N1) 2009; MA-CA04), and A/WSN/33 (H1N1; WSN). These viruses were propagated in Madin-Darby canine kidney (MDCK) cells, which were maintained in Eagle’s minimal essential medium (MEM) supplemented with 5% newborn calf serum (SIGMA) and cultured at 37°C in 5% CO2.
Viruses were serially diluted by ten-fold in MEM and mixed with C179 (100µg) or phosphate-buffered saline (PBS). The virus-C179 or -PBS mixture was then added to MDCK cells in 12-well tissue culture plates. After a 72 h inoculation, cytopathic effects (CPEs) were assessed. The ratio of the 50% tissue culture infectious dose (TCID50) of the virus-PBS mixture and virus-C179 mixture was determined and used as the neutralizing activity. TCID50 was calculated by the methods of Reed and Muench (1938).
We used 4- to 5-week-old female BALB/c mice (Japan SLC) for all animal experiments. For the prophylactic efficacy study, C179 or the control antibody H3-32.1 was inoculated into four mice per group via the intraperitoneal (15 mg/kg) or intranasal (2.5 mg/kg) route in 0.5 ml or 50 µl, respectively. One or 24 h after administration, mice were infected with H5N1 viruses, WSN, CA04, or MA-CA04 via the intranasal route in 50 µl volumes per mouse with 50 MLD50 (dose required to kill 50% of mice), 50 MLD50, 105 plaque-forming units (PFU), or 10 MLD50, respectively. Mice were monitored daily for mortality (for H5N1 viruses, WSN, and MA-CA04) or body weight changes (for CA04) for 14 days after virus challenge.
For the therapeutic efficacy study, the experimental design duplicated that of the prophylaxis study, with the following exceptions. We first inoculated mice intranasally with 50 MLD50, 50 MLD50, 105 PFU, or 10 MLD50 of H5N1 viruses, WSN, CA04, or MA-CA04, respectively. At 24, 48, or 72 h post-challenge, the virus-infected mice received C179 or the control mAb H3-32.1 via the intraperitoneal or intranasal route.
Three mice per group challenged with CA04 were euthanized on day 3 or 6 post-challenge. Their lungs were collected in a 9-fold volume of MEM supplemented with 0.3% bovine serum albumin (BSA: SIGMA). The amount of virus in the lung homogenates was then determined by plaque assays in MDCK cells.
Statistically significant differences in survival between mouse groups treated with C179 and the untreated group were determined by the log-rank and generalized Wilcoxon (Gehan) tests by use of R software ver. 2.11.1.
Dunnett’s multiple comparison method was used to evaluate statistically significant differences between virus titers in the lungs of mice treated with C179 and the untreated groups.
The epitope of C179 is believed to consist of two regions, the A region (amino acids 318–322 in HA1) and the B region (amino acids 47–58 in HA2) (H2 numbering) (Okuno et al., 1993). An amino acid change at position 318 in HA1 or position 52 in HA2 abolishes C179 recognition (Okuno et al., 1993). Comparison of the amino acid sequence of the A region showed complete conservation of the sequence among A/Okuda/57 (H2N2; Okuda57), A/WSN/33 (H1N1; WSN), all H5N1 viruses examined, and A/California/04/09 (H1N1; CA04). For the B region, the H5N1 and CA04 virus sequences differed from those of Okuda57 or WSN at two or one positions, respectively (Table 1). In addition, the amino acid at position 55 in HA2 differed among Okuda57, WSN, H5N1, and CA04 viruses. However, Val or Ile at this position did not appear to affect C179 binding to the viruses since the antibody efficiently neutralized WSN (see below).
To examine the efficacy of C179 against H5N1 and pandemic (H1N1) 2009 viruses in vitro, the neutralizing activity of the antibody was measured. Neutralization assays, in which the mAb concentration that neutralizes a given infectious dose of virus is determined, are commonly used to evaluate the neutralizing activity of antibodies against influenza viruses. However, complete neutralization of H5N1 viruses was observed only at the highest concentration of the C179 antibody tested (i.e., 100ug/ul) and virus growth was observed at the next dilution (i.e., 50ug/ul). We, therefore, evaluated the neutralizing activity of C179 by comparing virus titers in the absence (PBS served as a control) or presence of this antibody (Table 2). C179 neutralized WSN most efficiently, followed by pandemic (H1N1) 2009 virus, and to a much lesser extent, H5N1 viruses.
To examine the prophylactic and therapeutic efficacy of C179 against H5N1 viruses in vivo, mice were inoculated intraperitoneally with a single dose of C179 (15 mg/kg) at the following time points (prophylaxis, 24 h or 1 h pre-virus challenge; and therapy, 24 h, 48 h, or 72 h post-virus challenge). Mice were then challenged by intranasal inoculation of 50 MLD50 (dose required to kill 50% of mice) of various H5N1 viruses (A/Vietnam/UT3040/04 (clade 1; VN3040), A/Vietnam/UT31312III/07 (clade 2.3.4; VN31312III), A/swine/Indonesia/UT2071/05 (clade 2.1.1; SwIDN2071), A/Indonesia/UT3006/05 (clade 2.1.3; IDN3006), A/chicken/Miyazaki/K11/07 (clade 2.2; CkMiyazaki)) or WSN virus. Survival of infected mice was monitored daily for 14 days (Fig. 1). While all of the control mice, who received a control mAb H3-32.1 instead of C179 (see Supplementary Fig. S1), and the untreated control mice died by day 9 post-challenge, most (74%) of the C179-inoculated mice survived during the monitoring period. Furthermore, the groups with 100% survival did not show any clinical signs. The prophylactic and therapeutic efficacies of C179 were high for VN3040 and CkMiyasaki, as well as for WSN, and relatively high for SwIDN2071; they were, however, low for IDN3006 and VN31312III (Fig. 1). These results indicate that C179 can protect mice from lethal challenge of various clades and subclades of H5N1 viruses, albeit to differing degrees.
Next, we examined the efficacy of C179 against CA04, a representative pandemic (H1N1) 2009 virus. Because a high dose of CA04 is required for lethality in mice (MLD50: 6.3 × 105 PFU) (Itoh et al., 2009; Maines et al., 2009), we evaluated changes in body weight of mice intranasally challenged with 105 PFU of CA04 (Fig. 2A). Mice inoculated with C179 before virus challenge exhibited little to no weight loss, whereas all of the control mice that received H3-32.1 instead of C179 lost a substantial amount of body weight. For therapy, recovery of body weight loss was improved by C179 when administrated 24 h.
To further evaluate the efficacy of C179 against CA04, three mice per group were euthanized on days 3 and 6 post-challenge and virus titers in the lungs were determined by plaque assays in MDCK cells (Fig. 2B). On day 3 post-challenge, the virus titers in the lungs of the C179-inoculated mice were significantly lower than those of the untreated control mice when C179 was administered 24 h, but not 1 h, before challenge or after challenge. However, on day 6 post-challenge, a significant reduction in lung virus titer as a result of C179 inoculation was more prominent in most treatment regimens, with the exception of the animals treated with the antibody 1 h after challenge.
We also examined the efficacy of C179 against a mouse-adapted CA04, MA-CA04 (MLD50: 3.7×104 PFU) (Sakabe et al., unpublished data) in a murine lethal infection model (Fig. 3). When mice were inoculated with C179 prior to challenge with this virus, C179 effectively protected them (≥75%) from a lethal dose (10 MLD50) of MA-CA04, which killed all of H3-32.1-treated control mice (see Supplementary Fig. S2) and the untreated mice by seven days post-challenge. The prophylactic efficacy of C179 against MA-CA04 (Fig. 3) was significant. By contrast, therapeutic treatment with C179 afforded limited protection against lethal challenge with MA-CA04. These results indicate that C179 protects mice from pandemic (H1N1) 2009 virus infection prophylactically, but not therapeutically when administered intraperitoneally.
For intraperitoneal treatment, we used 15 mg/kg of C179. To examine whether we could reduce the amount of antibody for treatment, we next examined the protective efficacy of intranasal treatment with C179 against H5N1 and pandemic (H1N1) 2009 viruses. Mice were inoculated with C179 (2.5 mg/kg) intranasally 24 or 1 h before or 24, 48 or 72 h after virus challenge. Mice were challenged with WSN, SwIDN2071, IDN3006 (50 MLD50 respectively), or MA-CA04 (10 MLD50; we could not use a higher dose because of the lower titer of the virus stock compared with that of the other test viruses) and monitored daily for survival for 14 days (Fig. 4). While all of H3-32.1-treated control mice (see Supplementary Fig. S3) and untreated control mice died by day 12 post-challenge. Some (68%) of C179-inoculated mice survived during the monitoring period. Mice in groups with 100% survival did not show any clinical signs. The prophylactic and therapeutic efficacies of intranasally inoculated C179 were significantly high for WSN, relatively high for SwIDN2071, but low for IDN3006 and MA-CA04 (Fig. 4). Interestingly, the protective efficacy of intranasally inoculated C179 at a dose of 2.5 mg/kg was comparable to that achieved via the intraperitoneal route with a 15 mg/kg dose for all of the viruses tested, suggesting that direct administration of this anti-influenza antibody to the viral replication site may reduce the amount of antibody needed for prophylaxis and therapy.
Here, we show that the subtype cross-reactive monoclonal antibody C179 protects mice from H5N1 and pandemic (H1N1) 2009 viruses, despite its low neutralizing activity in vitro. Furthermore, we found that C179 provides protective efficacy via two different routes of administration. In particular, a substantially lower dose of intranasally administered antibody was as effective as intraperitoneal administration. Recently, multiple cross-reactive anti-HA antibodies with high potency have been reported (Chen et al., 2010; Chen et al., 2009; Friesen et al., 2010; Kashyap et al., 2008; Sui et al., 2009; Yoshida et al., 2009). Our results suggest that intranasal administration of anti-influenza virus antibodies should be considered as a route of inoculation for these antibodies.
The epitope recognized by C179 is highly conserved among H1N1, H5N1, and H2N2 viruses (Table 2). However, the in vitro neutralizing activity of C179 varied among these viruses, suggesting that regions other than the proposed C179 epitope (Okuno et al., 1993) affect C179 recognition.
C179 protected mice from H5N1 and pandemic (H1N1) 2009 viruses effectively, although it showed low neutralizing activity in vitro. It may be that this antibody exhibits its protective efficacy by not only inhibiting HA fusion (Okuno et al., 1993), but also by inducing other mechanisms, such as opsonization. Further studies are needed to gain more insights into the protective mechanism of this antibody.
Prophylactic administration of C179 to mice was effective for all of the virus strains examined in this study. In addition, C179 therapeutic use was also effective for some H5N1 viruses. Although the therapeutic efficacy for pandemic (H1N1) 2009 virus was limited, C179 did extend the lives of the test mice (Figs. 3 and and4).4). These results suggest that daily administration of C179 after challenge may be more effective for pandemic (H1N1) 2009 virus.
In this study, we found that intranasal administration required a lower dose of antibody for protection against influenza virus infection compared to intraperitoneal administration. Thus, the intranasal administration of antibody for influenza treatment may offer cost-effective benefits.
It is difficult to predict both when an influenza pandemic will occur and what kind of virus will cause a pandemic, as exemplified by the pandemic of 2009. Due to the limitations with existing vaccines and currently licensed anti-influenza drugs, it is prudent to develop new prophylactic and therapeutic measures. Intranasal administration of an antibody as immunotherapy may be a promising option for controlling future pandemics and epidemics.
We thank Susan Watson for editing the manuscript. This work was supported by a Grant-in-Aid for Specially Promoted Research, by a contract research fund for the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science, and Technology, by grants-in-aid from the Ministry of Health and by ERATO (Japan Science and Technology Agency), by National Institute of Allergy and Infectious Diseases Public Health Service research grants, and by an NIAID-funded Center for Research on Influenza Pathogenesis (CRIP, HHSN266200700010C). Saori Sakabe is supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists.
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