Date Published: May 29, 2007
Publisher: Public Library of Science
Author(s): Cameron P Simmons, Nadia L Bernasconi, Amorsolo L Suguitan, Kimberly Mills, Jerrold M Ward, Nguyen Van Vinh Chau, Tran Tinh Hien, Federica Sallusto, Do Quang Ha, Jeremy Farrar, Menno D de Jong, Antonio Lanzavecchia, Kanta Subbarao, Peter Palese
Abstract: BackgroundNew prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection.Methods and FindingsUsing Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1).ConclusionsThese studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.
Partial Text: The continued circulation of highly pathogenic avian influenza (HPAI) strains of subtype H5N1, and occasional coincident cases of human infection (274 patients as of 19 February 2007, with 167 fatalities), has triggered international public health concern. On the basis of haemagglutinin (HA) sequences, these circulating HPAI H5N1 viruses fall into different lineages, termed clades; viruses isolated in Viet Nam and Indonesia in 2004 and 2005, respectively, were designated as reference strains for Clades I and II . The HA sequences of Clade I and Clade II viruses differ by 4% to 5% at the amino acid level and the viruses of the two clades are antigenically distinguishable. The H5N1 viruses are not efficiently transmitted from person to person. Potentially, a virus capable of efficient human-to-human transmission could result from either adaptation of the HPAI H5N1 viruses and/or reassortment of the H5N1 virus genome with that of a circulating human influenza virus. Widespread dissemination of such a virus could cause significant morbidity and mortality, since humans are generally immunologically naïve to H5 influenza subtypes.
Blood samples from four Vietnamese adults (CL26, CL36, CL114, and CL115) who had recovered from HPAI H5N1 infection were collected 3–15 mo postinfection. IgG+ memory B cells recovered from frozen PBMC were immortalized with EBV. Cultures secreting neutralizing antibodies were identified by a microneutralization assay against the prototype Clade I virus, A/Vietnam/1203/04 (H5N1), and cloned by limiting dilution. Supernatants from approximately 11,000 wells were screened to identify 15 independent clones secreting a neutralizing antibody. Of these clones, three were isolated from donor CL26, one from donor CL114, and eleven from donor CL115. The number of clones isolated from each donor did not correlate with the plasma titer of neutralizing antibody, though this was not surprising given the small sample size. Clones producing antibodies that recognized H5 HA by ELISA, but did not neutralize live virus, were also identified from each donor (unpublished data). Clones FLA3.14 and FLA5.10, isolated from donor CL26, were the first obtained and were studied more extensively. Subsequently, clones FLD20.19 and FLD21.140, isolated from donor CL115, became available and were studied in parallel with FLA3.14 and FLA5.10. Clones FLA3.14, FLA5.10, FLD20.19, and FLD21.140 secreted IgG1,κ antibodies with neutralizing activity against the autologous virus A/Vietnam/CL26/2004 and other more recent Clade I viruses circulating in Viet Nam during 2005 and 2006 (Table 1). Significantly, more distant HPAI H5N1 strains, including the Clade II H5N1 virus A/Indonesia/5/2005, were neutralized by FLA3.14 and FLD20.19 (Table 2). In contrast, none of these clones neutralized an H3N2 influenza virus, A/California/7/2004 (Table 2). IgG1,κ mAbs of irrelevant specificity (diphtheria toxin and anthrax protective antigen) were used as negative controls and did not neutralize any influenza virus (Tables 1 and 2). Thus, the mAbs selected for further study demonstrated broad in vitro neutralizing activity against H5N1 viruses isolated from 1997 to 2005, albeit with some variation in potency.
The risk of a devastating human influenza pandemic caused by an H5N1 influenza strain remains difficult to quantify. What is clear is that zoonotic infections with HPAI H5N1 viruses continue to occur in Southeast Asia with a mortality in 2006 of 67% and for which there are few specific interventions [20,24]. Here we report on the generation of four fully human mAbs with a spectrum of neutralizing activity against multiple strains of HPAI H5N1 viruses in vitro and in vivo. These mAbs could have potential in the adjunctive treatment of human pandemic or zoonotic H5N1 cases.