sexta-feira, 26 de fevereiro de 2016

Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody

  • Davide Corti1,2,*
  • John Misasi3,*
  • Sabue Mulangu3
  • Daphne A. Stanley3
  • Masaru Kanekiyo3
  • Suzanne Wollen4
  • Aurélie Ploquin3
  • Nicole A. Doria-Rose3
  • Ryan P. Staupe3
  • Michael Bailey3
  • Wei Shi3
  • Misook Choe3
  • Hadar Marcus3
  • Emily A. Thompson3
  • Alberto Cagigi3
  • Chiara Silacci1
  • Blanca Fernandez-Rodriguez1
  • Laurent Perez1
  • Federica Sallusto1
  • Fabrizia Vanzetta2
  • Gloria Agatic2
  • Elisabetta Cameroni2,
  • Neville Kisalu5,
  • Ingelise Gordon3
  • Julie E. Ledgerwood3
  • John R. Mascola3
  • Barney S. Graham3
  • Jean-Jacques Muyembe-Tamfun5
  • John C. Trefry4,
  • Antonio Lanzavecchia1,6,
  • Nancy J. Sullivan3,,§,,

  • Ebola antibodies
    Science  25 Feb 2016:

    Abstract

    Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25-90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. Here, we demonstrate that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAb) that neutralize recent and previous outbreak variants of Ebola virus, and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as five days after challenge. Treatment with a single human mAb suggests a simplified therapeutic strategy for human Ebola infection may be possible.
    Ebola virus disease (EVD) causes severe illness characterized by rapid onset of fever, vomiting, diarrhea and bleeding diathesis (12). The challenges of a large outbreak and the failure of traditional quarantine and contact tracing measures (34) to control the 2014 West Africa outbreak highlights the urgency for therapies. The success in non-human primates (NHP) of ZMapp, a cocktail of three chimeric monoclonal antibodies (mAbs) derived from immunized mice (57), illustrated the potential of mAb therapies against EVD, and it is currently being evaluated in human trials. To date, efforts in NHP to simplify the ZMapp regimen to contain fewer mAbs have not been successful (7). We sought to isolate mAbs from human EVD survivors, with the goal of identifying antibodies that confer clinical protection either as single or dual-combination agents.
    We obtained blood from two survivors of the 1995 Kikwit EVD outbreak (8) eleven years after infection. To determine if the subjects retained circulating antibodies against Ebola virus (EBOV) glycoprotein (GP), we assessed GP-specific antibodies by ELISA (Fig. 1A) (9). The reciprocal EC90 titer for subject 1 (S1) was 2,326, greater than ten-fold higher than control sera. Moreover, serum from the more severely ill subject, S1, displayed potent virus neutralizing activity (Fig. 1B), indicating that S1 maintained serologic memory against EBOV GP more than a decade following infection and suggesting the potential to clone immunoglobulins with potent neutralizing activity from S1’s memory B cells.
    Fig. 1Isolation of antigen-specific monoclonal antibodies from Ebola virus disease survivor.
    (A) Plasma obtained from two human survivors, an uninfected human donor and a non-human primate (NHP) vaccinated against EBOV GP were serially diluted and analyzed by GP ELISA, A450 (n = 1). (B) Lentivirus particles expressing luciferase and bearing EBOV GP were incubated in the presence of heat inactivated serum for 1 hour prior to addition to HEK293T. Infection was determined by measuring relative luminescence (RLU) after 3 days. Infection % = (RLU with serum / RLU without serum) X 100%, mean ± s.d. (n = 3, representative experiment shown). (C) Immortalized B cell supernatants isolated from Subject 1 were screened by EBOV GP ELISA A450 (n = 1). (D) Immortalized B cell supernatants from (C) were diluted 1:50, incubated with lentivirus particles and infection determined as in (B). Infection % = (RLU with supernatant / RLU without supernatant) X 100% (n = 1).
    Therefore, we sorted memory B-cells from S1’s peripheral blood mononuclear cells, and immortalized individual clones with Epstein-Barr virus (10). Forty clone supernatants displayed a range of GP-binding (Fig. 1C), and two, 100 and 114, expressed antibodies with markedly higher neutralizing activity than all others (Fig. 1D). A second immortalization yielded 21 clones, from which the GP-specific clones 165 and 166 were rescued (fig. S1).
    mAb100, mAb114, mAb165 and mAb166 sequences were PCR-amplified and antibodies produced by transient transfection. We assessed ELISA binding to EBOV GP and observed that mAb114, mAb165 and mAb166 displayed nearly 50% higher maximal binding than KZ52, a prototypic EBOV GP-specific human mAb (11), and 25% higher than 13C6, a component of the ZMapp cocktail (67) (Fig. 2A). The binding curve of mAb100 plateaus similarly to KZ52 (Fig. 2A). mAb100 and mAb114 achieved half maximal binding (EC50) at a concentration of 0.02 μg/mL, which is 7-19 fold lower than the other mAbs. mAb165 and mAb166 had binding profiles similar to each other, with EC50s of 0.38 μg/mL and 0.35 μg/mL respectively, while EBOV control mAbs KZ52 and 13C6 had EC50s of 0.33 μg/mL and 0.14 μg/mL (Fig. 2A).
    Fig. 2Characterization of purified EBOV GP monoclonal antibodies.
    (A) EBOV GP ELISA in the presence of purified monoclonal antibodies as indicated, A450, mean ± s.d. n = 3, representative experiment shown) (B) Pseudotyped EBOV GP lentivirus particles were incubated with increasing amounts of purified monoclonal antibodies and infection measured as in Fig. 1B. Infection % = (RLU with antibody / RLU without antibody) X 100%, mean ± s.d. (n = 3, representative experiment shown). (C) V gene usage, sequence analysis and IgG subclass of antibodies from Subject 1. (D) Schematic of mAb100 and mAb114 UCA and variants created for investigation of the binding requirements of these mAbs. Shaded areas represent sequence from unmutated common ancestor (UCA) and light regions are from the somatic, mature antibody. Wild type, somatically mutated heavy (sH) or light (sL) chains; gH or gL, germline V-gene revertants of sH or sL in which the HCDR or LCDR3 are mature; gH-FR or gL-FR, germline V-gene revertants of sH or sL in which the HCDRs or LCDRs are mature; gH-FR1-2-4, germline V-gene revertants of sH in which the HCDRs and HFR3 are mature; gH-FR3, germline V-gene revertants of sH in which the HCDRs and HFR1, HFR2 and HFR4 are mature. (E and F) Binding to EBOV GP expressed on the surface of MDCK-SIAT cells by versions of mAb100 (E) and mAb114 (F) in which all or subsets of somatic mutations were reverted to the germline sequence. Shown is the ratio between the EC50 values of the variants and the wild-type (sH/sL). Ratio values above 100 indicate a lack of detectable binding (n = 1).
    We next evaluated S1 mAbs capacity for neutralization using lentiviral particles pseudotyped with EBOV GP, a BSL-2 method that has been demonstrated to recapitulate wild type EBOV (BSL-4) results (12) (Fig. 2B and fig. S2A). mAb165 and mAb166 exhibited similar half-maximal inhibition (IC50) concentrations of 1.77 and 0.86 μg/ml, respectively. mAb100 and mAb114 were the most potent, with IC50s about one-log lower (0.06 and 0.09 μg/ml, respectively) than mAb165 and mAb166. Notably, all four of the isolated mAbs inhibited 100% of the virus unlike KZ52, which consistently displayed only 80-90% maximum inhibition, and 13C6, which neutralized < 20% at 10 μg/mL. Importantly, S1 mAbs also potently neutralized the 2014 West African Makona-variant (fig. S2B). Neutralization of wild type EBOV particles by each of the isolated antibodies was confirmed by plaque reduction assay (fig. S3).
    Sequence analysis revealed that S1 mAbs displayed between 85-95% and 89-97% germline identity for heavy and light chains, respectively (Fig. 2C). Analyses of germline gene usage and V(D)J recombination indicate that they originate from different B cell lineages. The role of somatic hypermutations for the two most potent antibodies, mAb100 and mAb114, were analyzed using variants that were partially or completely reverted to the unmutated common ancestors (UCAs) (Fig. 2D and fig. S4, A and B). The fully reverted mAb100 (UCA/UCA), as well as a variant with a single change from the UCA VL (UCA/gL, A89T), recognized cells expressing GP with 2-4 fold weaker binding compared with the fully matured antibody (Fig. 2Eand fig. S4, A and B). When three HCDR3 mutations (A96V/V103Y/Y114S) were introduced in the reverted germline antibody (gH/UCA), binding was comparable to the fully matured mAb100 (sH/sL) suggesting that three mutations are sufficient to confer the binding observed for the fully matured mAb100. In the case of the mAb114, the fully reverted version (UCA/UCA) demonstrated negligible binding to GP (Fig. 2F and fig. S4, C and D). Introduction of two mutations (A96V and Y108S) in the HCDR3 of mAb114 germline (gH/UCA) was sufficient to confer an increase in binding, although still not to the degree seen with the fully matured mAb. Since these mutations are located at the base of the HCDR3 loop, they likely do not make direct contact with GP and thus may have a stabilizing effect on the whole HCDR3. The fully matured light chain and the two HCDR3 mutations (gH/sL) were sufficient to confer binding equivalent to the fully matured mAb (sH/sL). Importantly, the fully mutated light chain gene (UCA/sL), can partially compensate for a lack of somatic mutation in the heavy chain (Fig. 2F and fig. S4, C and D). The presence of additional mutations on either VH or VL is required to achieve the level of the fully matured mAb114 binding. These results suggest a rapid pathway of mAb114 affinity maturation through one or two somatic mutations, which became redundant as further mutations accumulated, a finding that is reminiscent of what was recently observed for the generation of broadly neutralizing influenza antibodies (13).
    Since mAb100 and mAb114 were the most potently neutralizing antibodies, they were considered optimal candidates for further evaluation in NHP. In order to assess the potential for combination therapy, we wished to first rule out antagonistic binding to GP. We found that each antibody bound to GP in the presence of the other, suggesting that they recognize distinct regions on GP (Fig. 3A) and therefore could be used as combination immunotherapy to maximize efficacy (14). To define the regions targeted by mAb100 and mAb114 we employed biolayer interferometry to assess GP binding in competition with mAbs KZ52 and 13C6, which have epitopes in the GP base and glycan cap, respectively (1516). We found that mAb100 competes with KZ52 for binding at the base of GP, while mAb114 recognizes at least in part the glycan cap region, as demonstrated by competition with 13C6 (Fig. 3, B and C).
    Fig. 3Binding regions and effector function.
    (A) Inhibition of binding of biotinylated mAb114 (left) and mAb100 (right) to GP-expressing MDCK-SIAT cells by pre-incubation with increasing amounts of homologous or heterologous unlabeled antibodies. Shown is the percentage binding of biotinylated antibody (n = 1). (B and C) Biolayer interferometry competitive binding assay to soluble EBOV GP using mAb100, mAb114, KZ52, 13C6 and isotype negative control. Biosensors were preloaded with GP followed by the competitor and analyte antibodies as indicated. Analyte binding curves (B) and quantitated % inhibition (C) are reported (n = 3, representative experiment shown). (D) Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was determined at 31.6 ng/mL of mAb100, mAb114 (n = 3, representative experiment shown), control antibody or derivative antibodies with LALA mutations that abrogate Fc-mediated killing (n = 1). ADCC activity is shown as % killing, mean ± s.d.
    Since some EBOV GP antibodies have been suggested to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) (17) the ADCC activity of mAb100 and mAb114 were determined in a flow cytometric assay (Fig. 3D). We found that mAb100 and mAb114 mediated ADCC with maximal activity observed at a mAb concentration of 0.03 μg/ml. Target cell killing was mediated through Fc receptors since mAbs containing Fc LALA mutations (18) abrogated ADCC activity. Therefore, these mAbs have the potential to induce direct killing of infected cells in vivo, a key viral clearance mechanism.
    The presence of potent neutralizing and ADCC activity, and the absence of cross competition, supported testing mAb100 and mAb114 in vivo protective efficacy. We challenged four rhesus macaques with a lethal dose of EBOV. One day post-challenge, the treatment group (n = 3) received an intravenous injection with a mixture of mAb100 and mAb114 at a combined dose of 50 mg/kg, and the treatment was repeated twice at 24-hour intervals. Circulating GP-specific antibody titers in mAb recipients peaked after the second injection, and reciprocal ELISA titers remained above 105 throughout the study, suggesting minimal clearance of the mAbs (Fig. 4A). The untreated macaque succumbed to EVD on day 10 with a circulating viral load exceeding 108 ge/ml (Fig. 4, B and C). In contrast, all three mAb-treated macaques survived challenge without detectable viremia. Consistent with historic controls, the untreated animal displayed hallmark indicators of EVD including thrombocytopenia and elevations in alanine transaminase and creatinine from day 6 through the time of death (Fig. 4D and figs. S5 to S8). In contrast, the treatment group remained within normal ranges for these parameters, and free of all EVD symptoms.
    Fig. 4Passive transfer of antibody cocktail.
    Animals were challenged with a lethal dose of EBOV GP on Day 0 and given injections of antibody totaling 50 mg/kg at 24, 48 and 72 hours post-exposure (A to H) or at 120, 144 and 168 hours post-exposure (I). Surviving animals were euthanized at the conclusion of the study (Day 28). (A) to (D): Challenge data from monoclonal antibody mAb114/mAb100 mixture. mAb114 monotherapy treatment beginning 24 hours [(E) to (H)] or 120 hours (I) after exposure. Treatment animals in black, untreated controls in red. (A) and (E): Ebola GP specific ELISA titer (reciprocal EC90). (B) and (F): Viremia in blood by qRT-PCR expressed as genome equivalents (ge) per mL. (C), (G) and (I): Survival. (D) and (H): Selected hematologic and chemistry data. Platelets (PLT), alanine transaminase (ALT), creatinine (CRE).
    We next asked whether monotherapy is sufficient for protection, and focused on mAb114 since it showed higher maximal binding than mAb100. We exposed four macaques to a lethal dose of EBOV and administered 50 mg/kg of mAb114 (n = 3) to the treatment group after a one-day delay, followed by two doses at 24-hour intervals. All treated macaques survived, whereas the control animal succumbed to EVD on day 6 with a peak viral load of 1010 ge/ml (Fig. 4, E to G). In contrast to the previous experiment, transient viremia was observed in the treated animals (Fig. 4F), but remained at levels less than 0.1% of the untreated control animal. Despite transient viremia, treated animals remained free of clinical and laboratory abnormalities (Fig. 4H and figs. S9 to S12).
    Since a delay in treatment is a distinct possibility in an outbreak setting, we evaluated the therapeutic potential for mAb114 when treatment was delayed until 5 days after lethal EBOV challenge. All three animals in the treatment group survived, while the control animal succumbed to EVD on day 9. Moreover, animals in the treatment group remained symptom-free and protected against thrombocytopenia, transaminitis and renal dysfunction (Fig. 4Iand figs. S13 to S16).
    mAb114 has several characteristics that may contribute to protection as a monotherapy compared to KZ52 and 13C6, which were non-protective in NHPs (719). First, while KZ52 and mAb114 potently neutralize EBOV in vitro, only mAb114 completely neutralizes input virus. Secondly, mAb114 does not require complement for neutralizing activity (Fig. 2B) in contrast to 13C6 (6). Based on these observations, one hypothesis is that protective monotherapy may require both potent binding and complete complement-independent neutralization. In addition, mAb114’s specific mechanism of neutralization, which targets an essential step in virus entry (20), and the observed in vitro ADCC activity may contribute to mAb114’s ability to protect against lethal EVD in macaques.
    In these studies, we showed that antibodies as well as memory B cells specific to EBOV are maintained in a survivor more than a decade following infection. For the mAbs isolated from this survivor, a potential role for antibody-dependent killing is suggested by in vitro ADCC activity that in vivo may be mediated by multiple effector cells such as natural killer cells, macrophages or neutrophils. In addition, these mAbs showed potent neutralizing activity against Ebola GP variants that have evolved over a 40-year period. Together, these data demonstrate the therapeutic potential of these mAbs as dual- and/or mono-therapy and contribute to understanding the mechanisms of antibody-mediated protection against Ebola virus disease.

    Supplementary Materials

    Materials and Methods
    Figs. S1 to S16
    References (2125)

    References and Notes

    1. See supplementary materials on Science Online.

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