time data were evaluated using noncompartmental modeling with winnonlin professional edition ( version 5.2 ; pharsight ) . the area under the curve from zero to infinity ( auc0 ) values were calculated using the linear trapezoidal method . we used a validated lethal ebola zaire mouse model developed at the us army medical research institute of infectious diseases ( usamriid ) , with a double plaque - purified , mouse - adapted , ebola isolate , ez'76 mp3 vp2 mp9 gh . the virus was inoculated intraperitoneally ( i.p . ) at 100 pfu ( 3000  ld50 ) producing uniformly lethal disease in c57b6 mice using biosafety level-4 facilities . research was conducted in compliance with the animal welfare act and federal regulations in a fully accredited facility . to assess the effect of rhmbl on virus lethality i.p . with either 4.3 mg / kg or 20 mg / kg of rhmbl twice daily 12 hours apart for 10 days . on the day of virus exposure , mice were treated and exposed to 100 pfu of mouse - adapted ebola zaire either 12 hours before or 1 hour after the first dose of rhmbl as indicated in figure 1 . survival and laboratory indices of filovirus - infected mice treated with recombinant human mannose - binding lectin ( rhmbl ) . sham - treated wild - type mice were compared with wild - type mice receiving 350 g of rhmbl ( referred to as rx ) administered intraperitoneally ( i.p . ) every 12 hours starting 1 hour before mouse - adapted ebola virus ( ebo ) challenge ( 100 pfu ) . shown is a kaplan - meier probability curve for mouse survival at the indicated times ( * log - rank mantel - cox test , p  =  .0075 ) . sham - treated mice were compared with mice receiving 350 g of rhmbl administered i.p . every 12 hours starting 12 hours after mouse - adapted ebola virus challenge ( 100 pfu ) . both wild - type ( wt ) and knockout mice lacking complement component 3 ( c3 ko ) were compared . shown is a kaplan - meier probability curve for mouse survival at the indicated times ( * log - rank analyses ; wt : sham - treated vs rhmbl - treated , p  =  .0013 ; rhmbl - treated : wt versus c3 ko , p  =  .0003 ) . rhmbl - treated mice had significantly lower platelet counts on day 5 after ebola virus inoculation than sham - treated mice ( * p  =  .014 ) . rhmbl - treated mice tended to have lower viral titers than sham - treated mice but the differences were not statistically significant . rhmbl - treated mice had lower proinflammatory and t helper cell type 2 ( th2 ) cytokine titers in liver homogenates on day 5 after inoculation ( p values as shown ) . fifteen wild - type mice received a 10-day course of rhmbl administered every 12 hours that was started 1 hour before inoculation with 100 pfu of mouse - adapted ebola virus as indicated . antibody ( ab ) titers were obtained on day 21 and again on day 56 . ebola virus antibody titers in 5 mice successfully treated with rhmbl are shown on the indicated days after initial and repeat challenges with ebola virus . viremia was assessed by reverse transcription - polymerase chain reaction ( rt - pcr ) and plaque assays as described elsewhere , and anti ebola virus antibodies were measured using standard enzyme - linked immunosorbent assays ( elisas ) . standard blood counts were evaluated with a coulter at diff ( beckman coulter ) . for analysis with flow cytometry after incubation with fc block ( bd ) , cells were washed and incubated with antibody ( cd3 fitc bd no . 45 - 0141 , cd4 pe ebio no . 12 - 0041 - 82 , cd11b apc bd no . 553312 , and cd19 pe - cy7 bd no . data were immediately acquired with a bd facscantoii and analyzed with flowjo ( version 7 ) . the bio - plex mouse cytokine 23-plex panel assay ( bio - rad 171-f11241 ) was used to measure multiple cytokines , chemokines , and growth factors in serum and tissue supernatants according to the manufacturer 's instructions . mice that survived the initial infection were tested for ebola - specific serological response on day 21 and rechallenged with the same virus dose without further treatment , and antibody titers were retested 28 days later . time data were evaluated using noncompartmental modeling with winnonlin professional edition ( version 5.2 ; pharsight ) . the area under the curve from zero to infinity ( auc0 ) values were calculated using the linear trapezoidal method . we used a validated lethal ebola zaire mouse model developed at the us army medical research institute of infectious diseases ( usamriid ) , with a double plaque - purified , mouse - adapted , ebola isolate , ez'76 mp3 vp2 mp9 gh . the virus was inoculated intraperitoneally ( i.p . ) at 100 pfu ( 3000  ld50 ) producing uniformly lethal disease in c57b6 mice using biosafety level-4 facilities . research was conducted in compliance with the animal welfare act and federal regulations in a fully accredited facility . to assess the effect of rhmbl on virus lethality i.p . with either 4.3 mg / kg or 20 mg / kg of rhmbl twice daily 12 hours apart for 10 days . on the day of virus exposure , mice were treated and exposed to 100 pfu of mouse - adapted ebola zaire either 12 hours before or 1 hour after the first dose of rhmbl as indicated in figure 1 . survival and laboratory indices of filovirus - infected mice treated with recombinant human mannose - binding lectin ( rhmbl ) . sham - treated wild - type mice were compared with wild - type mice receiving 350 g of rhmbl ( referred to as rx ) administered intraperitoneally ( i.p . ) every 12 hours starting 1 hour before mouse - adapted ebola virus ( ebo ) challenge ( 100 pfu ) . shown is a kaplan - meier probability curve for mouse survival at the indicated times ( * log - rank mantel - cox test , p  =  .0075 ) . sham - treated mice were compared with mice receiving 350 g of rhmbl administered i.p . every 12 hours starting 12 hours after mouse - adapted ebola virus challenge ( 100 pfu ) . both wild - type ( wt ) and knockout mice lacking complement component 3 ( c3 ko ) were compared . shown is a kaplan - meier probability curve for mouse survival at the indicated times ( * log - rank analyses ; wt : sham - treated vs rhmbl - treated , p  =  .0013 ; rhmbl - treated : wt versus c3 ko , p  =  .0003 ) . rhmbl - treated mice had significantly lower platelet counts on day 5 after ebola virus inoculation than sham - treated mice ( * p  =  .014 ) . rhmbl - treated mice tended to have lower viral titers than sham - treated mice but the differences were not statistically significant . rhmbl - treated mice had lower proinflammatory and t helper cell type 2 ( th2 ) cytokine titers in liver homogenates on day 5 after inoculation ( p values as shown ) . fifteen wild - type mice received a 10-day course of rhmbl administered every 12 hours that was started 1 hour before inoculation with 100 pfu of mouse - adapted ebola virus as indicated . antibody ( ab ) titers were obtained on day 21 and again on day 56 . ebola virus antibody titers in 5 mice successfully treated with rhmbl are shown on the indicated days after initial and repeat challenges with ebola virus viremia was assessed by reverse transcription - polymerase chain reaction ( rt - pcr ) and plaque assays as described elsewhere , and anti ebola virus antibodies were measured using standard enzyme - linked immunosorbent assays ( elisas ) . standard blood counts were evaluated with a coulter at diff ( beckman coulter ) . for analysis with flow cytometry after incubation with fc block ( bd ) , cells were washed and incubated with antibody ( cd3 fitc bd no . 45 - 0141 , cd4 pe ebio no . 12 - 0041 - 82 , cd11b apc bd no . 553312 , and cd19 pe - cy7 bd no . 557655 ) . cells were washed with pbs and fixed in bd cytofix . data were immediately acquired with a bd facscantoii and analyzed with flowjo ( version 7 ) . the bio - plex mouse cytokine 23-plex panel assay ( bio - rad 171-f11241 ) was used to measure multiple cytokines , chemokines , and growth factors in serum and tissue supernatants according to the manufacturer 's instructions . mice that survived the initial infection were tested for ebola - specific serological response on day 21 and rechallenged with the same virus dose without further treatment , and antibody titers were retested 28 days later . we previously found that rhmbl bound ebola ( zaire ) and marburg ( musoke ) envelope gps . rhmbl effectively blocked ebola gp interactions with dc - sign , and hiv particles lacking gp120/gp41 pseudotyped with ebola or marburg gps were neutralized by the lectin complement pathway . to develop an in vivo test of rhmbl effectiveness , we determined that 100 ng / ml of rhmbl was the minimum concentration needed to inhibit 90% infectivity of hepg2 cells using ebola gp pseudotyped lentiviral particles and to inhibit 90% infectivity of vero e6 cells using recombinant ebola zaire virus ( mayinga strain)-egfp ( data not shown ) . we had previously found that a single intraperitoneal dose of 75 g of rhmbl reconstituted the lectin complement pathway in mbl - knockout mice . we compared the pharmacokinetic parameters ( table 1 ) of that single reconstitution dose ( 4.3 mg / kg ) with a higher single intraperitoneal dose of 350 g ( 20 mg / kg ) to identify a potentially supraphysiological dose to test in model infections . the average maximum serum concentration ( cmax ) of both doses exceeded the minimum concentration of mbl that inhibited infection in vitro by at least 55-fold . the average ratio of maximum to baseline complement component 4 cleavage activity was 1.7 for the 75-g rhmbl dose and 5.4 for the 350-g dose . pharmacokinetic parameters of low- vs high - dose recombinant human mannose - binding lectin ( rhmbl ) therapy in uninfected mice note . statistical differences were analyzed with the student t - test ( 2-tailed ) . a value of p  <  .05 was considered to indicate a statistically significant difference . intraperitoneal administration of 100 pfu of native ebola zaire virus ( 3000  ld50 ) is uniformly fatal in mice . treatment with 75 g of rhmbl per dose every 12 hours failed to protect mice from that virus inoculum . therefore , we increased rhmbl to 350 g administered every 12 hours for 10 days starting either 1 hour before or 12 hours after ebola virus challenge ( figure 1a and 1b ) . when treatment was started 1 hour before virus infection , the supraphysiological dose increased survival to > 40% of mice in several trials ( figure 1a ) . we compared survival in wild - type and complement component 3 ( c3)deficient mice as the inhibitory effects of mbl on ebola virus are mediated by complement in cell culture . once again we saw an increase in survival from 0% to > 40% in rhmbl - treated mice , and survival was dependent on an intact complement pathway , since c3-deficient mice did not survive ( figure 1b ) . all inoculated mice showed signs of infection according to standardized observation scores and weight loss , and surviving mice had detectable ebola virus  specific antibodies 28 days after infection ( data not shown ) . we monitored the effect of treatment started 12 hours after infection on a variety of laboratory indices . mean white blood cell counts were 9100 cells / ml in mbl - treated mice ( n  =  5 ) compared with 4525 cells / ml on day 7 after infection in the surviving sham - treated mice ( n  =  4 ) . average lymphocyte counts were also higher in mbl - treated mice compared with controls ( 5500 cells / ml vs 2800 cells / ml , respectively ) . a similar trend was seen for platelet counts , which averaged 726,000 cells / ml in the treatment group and 239,000 cells / ml in the controls . these differences were statistically significant for platelet counts on day 5 ( 672,000 cells / ml vs 322,000 cells / ml , p  =  .014 ; figure 1c ) . in a separate experiment , spleens were harvested on day 5 after infection ( 4 sham - treated and 4 mbl - treated mice ) . numbers of splenic cd3cd19 cells ( b lymphocytes ) and cd11b granulocytes were higher in mbl - treated mice ( 89.2% vs 85.1% , p  =  .019 ; 17.6% vs 12.8% , p  =  .04 , respectively ) . the rna viral loads as determined by rt - pcr in blood , liver , and spleen 5 days after infection were similar in sham- and rhmbl - treated mice ( p  >  .05 ) . virus titers in blood were generally lower on days 1 and 3 in rhmbl - treated mice as determined by plaque assays ( p  >  .05 ; figure 1d ) . of 23 cytokines and chemokines tested in serum , liver , and spleen on day 5 after inoculation , lower values ( fluorescence intensity units ) for interleukin ( il)-1b ( 170 vs 253 , p  =  .07 ) , il-5 ( 89 vs 112 , p  =  .03 ) , il-10 ( 379 vs 518 , p  =  .004 ) , il-13 ( 264 vs 384 , p  =  .008 ) , and il-17 ( 120 vs 174 , p  =  .028 ) were found in liver homogenates from rhmbl - treated mice ( figure 1e ) . we tested protective immunity in 5 seropositive mice that survived initial infection by rechallenging them with native ebola virus 28 days after initial infection . it is noteworthy that all mbl - treated survivors also survived the second viral challenge . similar or higher immunoglobulin g , a , and m antibody titers were seen 28 days after the second challenge with the virus ( figure 1f ) . in the past 3 decades , approved antivirals have increased from a few nucleoside analogues to well over 40 drugs . the human immunodeficiency virus ( hiv ) and hepatitis c virus ( hcv ) epidemics particularly drove antiviral discovery toward rationally designed drugs targeting specific viral enzymes . although this approach was remarkably effective , the advent of newly emerging or drug - resistant viruses that threaten humans calls for the development of more broadly active agents targeting viral components shared among viruses . n - glycosylation of viral envelopes is an important such target shared between influenza , hiv , hcv , west nile virus , sars - cov , hendra virus , nipah virus , and filoviruses ( ebola and marburg viruses ) . to assess one possible strategy against n - glycosylated viruses , we tested a stringent ebola virus infection model ( 3000  ld50 ) in mice . filovirus infections are characterized by marked lymphopenia , severe degeneration of lymphoid tissues , dysregulated dendritic cell function , and cytokine storms  all hallmarks of pathogens that subvert both innate and adaptive immune responses . therefore , we hypothesized that administration of a recombinant innate immune molecule that targets glycosylated viruses might bridge an infected individual to recovery . here we show for the first time that rhmbl can be used as a therapeutic agent to achieve serum concentrations in mice that correspond to levels in humans that are 724-fold higher than average human concentrations and complement cleaving activity that is > 5-fold higher than baseline values in mice . this result confirms our previous in vitro data showing that mbl possesses complement - dependent intrinsic antimicrobial activity . biological responses of the infected mice to rhmbl treatment further indicated that our strategy targeted the main pathogenic effects of ebola viruses . mbl - treated mice had higher b lymphocyte and cd11b granulocyte counts and demonstrated down - regulation of intrahepatic proinflammatory ( il-1b and il-17 ) and th2 cytokines ( il-5 , il-10 , and il-13 ) early in the course of infection ( figure 1e ) , suggesting that rhmbl may mitigate the detrimental effects of the characteristic cytokine storm . mbl - treated mice tended to have greater inhibition of viral replication on days 1 and 3 after infection ( p  >  .05 ; figure 1d ) . most important , rhmbl treatment bridged surviving mice to development of an effective adaptive immune response ( figure 1f ) . future experiments will be needed to scale high - dose rhmbl therapy for use in larger animal models and to test rhmbl in combination with other promising experimental therapies such as small molecule inhibitors , coagulation modulators , antisense technologies , therapeutic antibodies and cytokines , and postexposure vaccination . in summary , we report that supraphysiologic rhmbl therapy may be an effective immunotherapeutic strategy against ebola virus , and since ebola glycoproteins potentially model other glycosylated viruses , rhmbl therapy may offer a novel broad - spectrum antiviral approach . this study was supported by grant u01-ai070330 to e.v.s . from the national institutes of health ( nih ) . was additionally supported by the defense threat reduction agency medical biological defense research program , therapeutic research program 4.10007_08_rd_b . this study was supported by grant u01-ai070330 to e.v.s . from the national institutes of health ( nih ) . was additionally supported by the defense threat reduction agency medical biological defense research program , therapeutic research program 4.10007_08_rd_b .
mannose - binding lectin ( mbl ) targets diverse microorganisms for phagocytosis and complement - mediated lysis by binding specific surface glycans . 
 although recombinant human mbl ( rhmbl ) trials have focused on reconstitution therapy , safety studies have identified no barriers to its use at higher levels . 
 ebola viruses cause fatal hemorrhagic fevers for which no treatment exists and that are feared as potential biothreat agents . 
 we found that mice whose rhmbl serum concentrations were increased 7-fold above average human levels survived otherwise fatal ebola virus infections and became immune to virus rechallenge . 
 because ebola glycoproteins potentially model other glycosylated viruses , rhmbl may offer a novel broad - spectrum antiviral approach .