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antibodies that recognized H5 HA by ELISA, but did not neutralize live virus, were also identified from each donor (unpublished data).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 (). Significantly, more distant HPAI H5N1 strains, including the Clade II H5N1 virus A/Indonesia/5/2005, were neutralized by FLA3.14 and FLD20.19 (). In contrast, none of these clones neutralized an H3N2 influenza virus, A/California/7/2004 (). IgG1,κ mAbs of irrelevant specificity (diphtheria toxin and anthrax protective antigen) were used as negative controls and did not neutralize any influenza virus ( and ). 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.
Neutralizing Titers of mAbs against Influenza A H5N1 Influenza Viruses Isolated from Viet Nam
Neutralizing Titers of mAbs against Influenza A H3N2 and a Range of H5N1 Influenza Reference Viruses
BALB/c mice are highly susceptible to infection with the HPAI H5N1 viruses isolated in Asia in 1997 and since 2003. Following i.n. administration, these H5N1 viruses replicate to high titer in the lungs of the mice and some isolates disseminate to extrapulmonary sites and are lethal for mice [22
]. To explore the efficacy of mAbs FLA3.14 and FLA5.10 for pre-exposure prophylaxis, BALB/c mice were passively immunized by i.p. administration of graded doses of mAbs and then challenged i.n. with A/Vietnam/1203/04 (H5N1) 24 h later. All preparations of mAb FLA5.10 conferred 100% protection from lethality by A/Vietnam/1203/04 (p
= 0.001) (). mAb FLA3.14 also conferred some protection from lethal A/Vietnam/1203/04 (H5N1) infection, but with lower efficacy and in a dose-dependent manner (). Mice receiving the highest dose of FLA3.14 were afforded almost complete protection (p
= 0.001), whilst mice receiving the lowest dose of FLA3.14 were as susceptible as mice that received a human mAb of irrelevant specificity, though time to death was delayed (p
= 0.02). Mice that received hyperimmune anti-H5 polyclonal sheep antiserum were afforded complete protection. These data, demonstrating the relatively greater in vivo activity of FLA5.10 over FLA3.14 against A/Vietnam/1203/04, are consistent with the in vitro neutralization titers presented in .
Passive Immunization and Survival after Challenge with A/Vietnam/1203/04 (H5N1)
To better understand the mechanism by which mAbs FLA3.14 and FLA5.10 conferred protection from lethality, the kinetics of viral infection in passively immunized mice was defined. Mice that were passively immunized with FLA3.14, FLA5.10, or a human mAb of irrelevant specificity (D2.2) were challenged with A/Vietnam/1203/04 (H5N1) 24 h later, and the level of virus replication in different organs was determined 2 and 4 d later. Mice that received the control mAb, D2.2, had high titers of virus in the lungs (A), with evidence of extrapulmonary dissemination indicated by viral replication in the brain (B) and spleen (C). In contrast, mice passively immunized with FLA3.14 or FLA5.10 had significantly (10- to 100-fold) lower titers of virus in the lungs (A) (p = 0.01, FLA3.14 versus D2.2; p = 0.001, FLA5.10 versus D2.2), undetectable viral burdens in the brain (B) and a low titer of virus detected in the spleens of mice that received FLA5.10 (C). Alongside the reduction in lung viral titers, mice that received prophylaxis with FLA5.10 had less dramatic pathological changes in the pulmonary airways and parenchymal tissue (). Thus, the percentage of abnormal bronchioles with necrosis and viral antigen in lung sections from mice (n = 2 per group) that received FLA5.10 prophylaxis was less (13%) than in control mice (80%). Similarly, there were fewer inflammatory interstitial lesions in which H5 antigen was detected by immunohistochemical staining in the lung sections of mice given FLA5.10 relative to the control antibody, D2.2 (1 versus >10) (). To a slightly lesser extent, FLA3.14 prophylaxis also limited bronchiolitis (31% versus 80%) and H5-associated interstitial pathology (2 versus >10) when compared with control mice (n = 2). These data suggest that prophylaxis with FLA3.14 or FLA5.10 probably confers protection from lethal challenge through a combination of limiting viral replication in the lung, attenuating virus-induced lung pathology, and blocking extrapulmonary dissemination of virus to distant organs.
Pulmonary Virus Titers and Extrapulmonary Dissemination of A/Vienam/1203/04 (H5N1) Following Passive Immunization
Histopathology in Pulmonary Tissue of Passively Immunized and Challenged Mice
Attenuation of established infection represents a clinically relevant endpoint for antiviral therapy against H5N1 infection. To this end, the efficacy of treatment with FLA3.14, FLA5.10, FLD20.19, and FLD21.140 was determined in BALB/c mice i.n. infected 24, 48, or 72 h previously with 5 LD50 of A/Vietnam/1203/04 (H5N1). FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality in A/Vietnam/1203/04 (H5N1) infected mice at all time points, whilst an irrelevant control mAb, D2.2, gave no protection (p = 0.003) (). These promising therapeutic results against a Clade I virus from Viet Nam led us to examine the therapeutic efficacy of mAbs FLA3.14, FLA5.10, FLD20.19, and FLD21.140 against A/Indonesia/5/2005, an antigenically divergent H5N1 virus from Clade II. The efficacy of treatment with FLA3.14, FLA5.10, FLD20.19, and FLD21.140 was determined in BALB/c mice infected i.n. 24 h previously with 5 LD50 of A/Indonesia/5/2005. Consistent with the in vitro neutralization data (), mice treated with FLA3.14 and FLD20.19, but not FLA5.10 or the control mAb D2,2, were significantly protected from A/Indonesia/5/2005 lethal infection (p = 0.003) (). Although FLD21.140 did not demonstrate neutralizing activity in vitro against A/Indonesia/5/2005 (), treatment with this mAb significantly protected the mice (p = 0.003) from A/Indonesia/5/2005 lethal infection. This observation of neutralization in vivo suggests that a factor found in vivo enhances the neutralizing activity of this mAb, accounting for its efficacy in vivo in preventing mortality associated with infection. These data provide proof of concept that mAb therapy for at least 72 h postinfection in the mouse model can markedly improve survival from highly virulent H5N1 infection. Importantly, these data also imply it is possible to obtain significant cross-protection against a Clade II H5N1 virus using a mAb elicited by a Clade I virus.
mAb Therapy and Survival in Mice with Established A/Vietnam/1203/04 (H5N1) Infection
mAb Therapy and Survival in Mice with Established A/Indonesia/5/2005 (H5N1) Infection