HHS Public Access Author manuscript Author Manuscript
Antiviral Res. Author manuscript; available in PMC 2017 September 01. Published in final edited form as: Antiviral Res. 2016 September ; 133: 218–222. doi:10.1016/j.antiviral.2016.08.012.
Treatment of Lassa virus infection in outbred guinea pigs with first-in-class human monoclonal antibodies Robert W Cross#1,2, Chad E Mire#1,2, Luis M Branco#3, Joan B Geisbert1,2, Megan M. Rowland3, Megan L. Heinrich3, Augustine Goba4,5, Mambu Momoh4,5,6, Donald S Grant4,5, Mohamed Fullah4,5, Sheik Humarr Khan4,5, James E Robinson7, Thomas W Geisbert1,2, and Robert F Garry3,8 for the Viral Hemorrhagic Fever Consortium
Author Manuscript
1Department
of Microbiology and Immunology, University of Texas Medical Branch at Galveston,
Texas, USA 2Galveston
National Laboratory, University of Texas Medical Branch at Galveston, Galveston, Texas, USA
3Zalgen 4Viral
Labs, LLC, Germantown, MD
Hemorrhagic Fever Program, Kenema Government Hospital, Kenema, Sierra Leone
5Ministry
of Health and Sanitation, Freetown, Sierra Leone
6Polytechnic
College, Kenema Sierra Leone
7Sections
Author Manuscript
of Infectious Disease, Departments of Pediatrics and Internal Medicine, School of Medicine, Tulane University, New Orleans, LA
8Department
#
of Microbiology and Immunology, Tulane University, New Orleans, Louisiana, USA
These authors contributed equally to this work.
Abstract Lassa fever is a significant health threat to West African human populations with hundreds of thousands of annual cases. There are no approved medical countermeasures currently available. Compassionate use of the antiviral drug ribavirin or transfusion of convalescent serum has resulted in mixed success depending on when administered or the donor source, respectively. We previously identified several recombinant human monoclonal antibodies targeting the glycoprotein of Lassa virus with strong neutralization profiles in vitro. Here, we demonstrate remarkable
Author Manuscript
*
Corresponding author: Dr. Thomas W. Geisbert, University of Texas Medical Branch, Galveston National Laboratory, 301 University Blvd, Galveston, TX 77550-0610, Phone: 409-266-6906 Fax: 409-266-6810, [email protected]. Conflict of interest statement: RWC, CEM, JBG, MMR, MLH, AG, MM, DSG, JER, and TWG declare that they have no conflicts of interest. LMB and RFG are co-founders of Zalgen Labs, a for profit biotechnology company. Presentation History: This work was presented in part at the 11th ASM Biodefense and Emerging Diseases Research Meeting,
Washington DC, February 25 – 27, 2013 (Poster 13-BIO-A-337-ASM “Efficacy of Human Monoclonal Antibodies as an Immunotherapeutic in a Guinea Pig Model of Lassa fever”) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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therapeutic efficacy using first-in-class human antibodies in a guinea pig model of Lassa infection thereby presenting a promising treatment alternative.
Author Manuscript
Lassa Fever (LF) is a major public health problem in Western Africa where hundreds of thousands of infections are estimated to occur annually. For those who present to a hospital for treatment and are referred to a ward specific for the treatment of viral hemorrhagic fevers, the mortality rate approaches 70% (Shaffer et al., 2014). Elevated mortality, environmental stability that favors artificial aerosol infection, and a dearth of medical countermeasures for LF also make Lassa virus a clear biodefense concern (Stephenson et al., 1984). The nucleoside analog ribavirin, convalescent sera, and supportive therapy are the only treatments options currently in use for LF. Safety profile concerns, lack of controlled and randomized clinical studies, and short window of efficacy for therapeutic ribavirin or convalescent sera use in LF patients support the need for development of alternative and robust therapeutic options (Bausch et al., 2010). Single monoclonal antibody therapies against viral agents have recently proven highly effective; however, only one has been approved for use in humans (Mejias and Ramilo, 2008). The 2013 – 2016 Ebola hemorrhagic fever epidemic in West Africa has, however, potentiated the accelerated development and clinical evaluation of ZMapp, a cocktail of three chimeric monoclonal antibodies against distinct epitopes on the Ebola virus glycoprotein (McCarthy, 2014).
Author Manuscript
Since 2008, the Viral Hemorrhagic Fever Consortium (http://www.VHFC.org) has concentrated efforts at characterizing protective or pathogenic roles of B cells in LF directly from human patients (Robinson et al., 2016). This work has resulted in the derivation of the largest known collection of fully human monoclonal antibodies (huMAbs) from convalescent West African LF patients. Here, we describe the protective efficacy of select LASV glycoprotein specific huMAbs in vivo where a panel of 11 huMAbs targeting the glycoprotein of Lassa virus (LASV) was evaluated in an outbred guinea pig (GP) model of LF based on their in vitro neutralization profiles. Optimized bicistronic IgG1 backbone vectors for high level, transient expression in mammalian cells were used to generate huMAbs for therapeutic evaluation in vitro and in a GP model of LF. Scaled production of antibodies for in vivo studies is detailed in the supplementary materials. Antibodies for therapeutic studies were formulated in a buffer for injection at > 20 mg/mL, and assayed for purity, concentration, identity, and endotoxin levels prior to lot release.
Author Manuscript
A panel of 125 independently derived LASV glycoprotein-specific huMAbs were extensively characterized for binding, epitope grouping, and neutralization potential in vitro (Robinson et al., 2016). Eleven huMAbs consistently registered greater than 50% neutralization in a lentiviral entry assay system pseudotyped with the LASV (Josiah strain, Clade IV) GPC, a lymphocytic choriomeningitis virus (LCMV) backbone pseudotyped with LASV GPC, or in a live LASV plaque reduction neutralization test (PRNT) (Robinson et al., 2016). Antibodies segregated into two distinct performance clusters of IC50 and IC80 values (Supplemental Table 1, Figure 1A, 1B). Neutralizing antibodies (NAbs) 37.2D, 8.9F, 12.1F,
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37.7H, 25.6A, 36.1F, and 25.10C clustered in the lowest combined Log10 IC50 (Figure 1A.) and Log10 IC80 (Figure 1B.) values range. A second cluster of NAbs comprised of 19.7E, 10.4B, 37.2G, and 2.9D recorded significantly combined higher Log10 IC50 and Log10 IC80 values range (Figure 1A, 1B). Ten of the 11 huMAbs bound mammalian cell-generated LASV glycoprotein immobilized on ELISA plates. The single exception, huMAb 8.9F was identified via a pseudovirus neutralization assay performed as previously reported (Robinson et al., 2016), and did not bind purified glycoprotein in ELISA.
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Binding efficacy to different clades of LASV was assessed using flow cytometry to detect the glycoprotein complex (GPC) genes of LASV Josiah (Clade IV), and Nigerian strains NigA19-08 (Clade III) and NigA18-237 (Clade II) which were transiently expressed on the surface of transfected HEK-293T/17 cells for 24 hours prior to flow cytometric analysis (FACS). Histograms were generated that display the log fluorescence intensity for each antibody bound to GPC on the surface of live transfected cells. All 11 huMAbs bound to HEK-293T/17 cell surface-expressed LASV GPC by flow cytometry (Figure 1C). Three LASV huMAbs (8.9F, 12.1F, and 37.2D) also bound cell surface expressed GPCs from Clade II (strain Nig A18-237) and III (strain Nig A19-08) strains (Figure 1C and 1D). These binding profiles suggest that these 3 LASV huMAbs may have therapeutic utility across LASV Clades. These data guided the identification and prioritization of leading candidate therapeutic huMAbs for in vivo studies.
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Animal studies were completed under biosafety level (BSL)-4 biocontainment at the Galveston National Laboratory and were approved by the University of Texas Medical Branch Institutional Laboratory Animal Care and Use Committee. Female outbred Hartley strain GP (~ 350-400 g, ~ 5-6 weeks old, Harlan Sprague Dawley, Houston, TX) were acclimatized for ~ 1 week prior to challenge intraperitoneally (IP) with 1000 plaque forming units of guinea pig adapted (GPA) LASV-Josiah (N=5/ treatment group). The GPA LASV infection model of outbred Hartley GP has been described recently for testing therapeutics against LASV (Safronetz et al., 2015). The advantage of using outbred animals to model human infection is inferred from the higher variability of immune responsiveness inherent in outbred populations allowing for increased countermeasure testing stringency versus those inbred for genetically tractable immune deficiencies which may, in turn, result in reduced predictive efficacy of the treatment in question by only testing a single immune phenotype rather than multiple at one time, as would be present in an outbred test population.
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Animals were administered huMAbs (30 mg/kg of body weight) IP immediately after challenge (day 0) and on days 3 and 6 post infection (PI). Control animals were not treated. Guinea pigs were monitored for clinical observations, appearance, behavior, activity, and signs of paresis. Clinical scores were determined as Bright, Alert, Responsive = 0, Rough = 4, Sick = 8, Moribund = 12, or Expired = 16 over the course of 28 days after which survivors were euthanized at the study endpoint per IACUC protocol. Animals which were scored rough had roughed fur, delayed responsiveness, and some evidence of weight loss. Sick animals had marked lack of grooming, unstable gait, and clear evidence of weight loss. Blood was collected into EDTA containing tubes on days 7 and 14 and viremia was assessed from separated plasma using classical titration methods as outlined in supplemental materials. Kaplan-Meier survival curves were generated in Prism 5 and survival curves were
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compared using Log-rank (Mantel-Cox) test (GraphPad Software, La Jolla, CA) for treated groups versus untreated controls for each study (Figure 2A). Eleven LASV huMAbs tested in a Hartley GP model of LF segregated the antibodies into three distinct protection groups. One group of huMAbs, 25.6A, 2.9D, 8.9F, 12.1F, and 37.7H conferred 100% protection and no change in clinical score in GPs. In a second group, 37.2D, 19.7E, and 37.2G protected 80 – 90% of animals. A third group comprised of 10.4B, 25.10C, and 36.1F, conferred 40, 30, and 20% protection, respectively. An irrelevant recombinant human isotype control (IgG1) antibody did not confer protection (0% survival). From the tested antibodies, no significant differences were observed between protection level and epitope groups; however, more antibodies from each respective treatment group will need to be tested in order to verify the potential importance of any one particular epitope group in regard to protection (Supplemental Table 1).
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The combined in vitro IC50 and IC80 data generally correlated well with protection in vivo for the top tier antibodies (100% protection). All but 2.9D neutralized pseudovirus in the nanomolar range, and registered a PRNT IC50 mean of 13.6nM. Despite conferring 100% protection in GP huMAb 2.9D registered high IC50 values in vitro (Supplemental Table 1). 37.2D and 19.7E generated distinct IC50 values in pseudovirus assays, with the former neutralizing in the low nanomolar range, and the latter at > 20 nanomolar. Both antibodies neutralized LASV in PRNT at IC50 ~ 18 nM, and conferred 90% overall protection in GPs. It should be noted that 19.7E and 37.2D antibodies conferred 100% protection yet showed some clinical signs in an initial study (5/5) so a repeat study was performed where 80% (4/5) survived and similar clinical values were observed.
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37.2G protected 80% of animals tested, despite high IC50 values in pseudovirus and PRNT assays (16.0 and >100 nM, respectively). 10.4B neutralized poorly in all assays and conferred only 40% protection in GPs. Surprisingly, the most potent antibody identified in vitro, 25.10C, with subnanomolar activity in all pseudovirus platforms, and 6.24 nM in PRNT, conferred an average 30% (3/10) protection in GPs. In our initial assessment, 25.10C protected 40% (2/5) of GPs, yet only 20% (1/5) in a repeat study. The difference in results could be attributed to inherent immune variability of outbred animals where differences in individual’s susceptibility to the virus contributes to disease course. Importantly, these data indicate that in vitro neutralization assays have value in selecting antibodies with protective efficacy in vivo, but are not necessarily predictive of levels of protection.
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Viremia was compared by Kruskal-Wallis test supported by Dunn’s Multiple comparison posttest (Prism 5, GraphPad Software, La Jolla, CA) to detect differences from the control group for time points relevant to onset (day 7) or peak viremia (day 14) as determined from historical data (Geisbert, T.W. unpublished data). Untreated control animals averaged 3.5 and 4.5 Log PFU/mL on days 7 and 14, respectively. Despite 100% protection at the study endpoint, some animals from treatment group 8.9F or 37.7H, 2.9D, and 25.6A had low level viremia on day 7 or 14, respectively. Treatment groups where 90% protection was afforded (37.2D and 19.7E) had reduced mean viremia titers and minimal clinical score values. Treatment groups with 80% or less survival had comparable mean viremia titers to control animals on day 7, but by day 14 mean viremia was markedly lower than control animals.
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Groups with 80-90% survival exhibited relatively low mean clinical scores and all remaining treatment groups exhibited concomitant increases in mean clinical scores with decreases in survival per group (Figure 2B, 2C, and Supplemental Figure 1). Endpoint viremia was not determined for these studies as survival was the primary metric of interest, though all surviving animals demonstrated no clinical signs to indicate disease was present at study endpoint 28 days PI. (Figure 2B and Supplemental Figure 1).
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Lassa fever is a major public health threat in West Africa. Recent importations of cases of LF to non-endemic regions of the world illustrate the global public health threat (Amorosa et al., 2010; Haas et al., 2003; Hirabayashi et al., 1989). Currently, no approved LF vaccines or therapeutics exist, yet there is a clear public health need in endemic regions. Further, LASV has been categorized as one of the World Health Organization’s top 10 emerging pathogens and has thus created increased demand for medical countermeasures(WHO, 2016). Off label use of Ribavirin can be partially effective in the treatment of acute LF; however, it has a narrow window of efficacy, generally within 6 days following the onset of fever and is often associated with marked side effects due to dosing regimen(Bausch et al., 2010). Unfortunately, the drug has not demonstrated great efficacy in treating advanced LF(Bausch et al., 2010).
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The current studies present a novel alternative for treating LF infections that relies on the use of fully human antibodies derived from convalescent donors. Early studies employing convalescent plasma to treat active LF infections met with mixed success; this was likely due to unreliable titers of protective antibodies in donors’ blood or antibody specificities that were not well matched against circulating LASV strains (Cummins et al., 1991; Jahrling et al., 1985; Leifer et al., 1970). Here we show that the majority of antibodies administered either individually or in combination prevented development of active disease in GP when administered soon after infection. These studies demonstrate the in vivo potential of these antibodies and merit further preclinical development of huMAbs as mono-therapies or cocktails for pre-exposure prophylaxis or post-exposure treatment in the gold standard animal model for LF, non-human primates. This technology may be a particularly relevant treatment for high risk scenarios such as caregivers working directly with LF patients or those who have otherwise become exposed to the virus in the community. In the absence of approved vaccines, safe and effective immunotherapeutics would provide a superior prophylactic and post-exposure treatment alternative, with well tolerated profiles, and likely few metabolic side effects.
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The ongoing outbreak of LF in Nigeria and neighboring Benin, endemicity of the disease in Sierra Leone, Guinea, and Liberia, potential for geographic expansion (Fichet-Calvet and Rogers, 2009) and threat to national security if developed as a bioweapon should prioritize development of a new and safe class of Pan-Lassa virus therapeutics (Ibekwe, 2012).
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments The authors would like to extend gratitude to Daniel J. Deer, Jessica Graber, and the University of Texas Medical Branch Animal Resource Centers for assistance with animal experiments. The authors also thank Krystle N. Agans and Kimberly Schuenke for administrative support. Finally, we wish to thank all the members of the Viral Hemorrhagic Fever Consortium for their continued support and contributions to the VHF field (www.vhfc.org). In memoriam: Tragically, two co-authors, who contributed greatly to public health and VHF research efforts in Sierra Leone, contracted EHF and lost their battle with the disease before this manuscript could be published: Mohamed Fullah and Sheik Humarr Khan. We wish to honor their memory. Funding statement This work was supported by Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119, Health and Human Services Contract HHSN272200900049C, and National Institutes of health/ national Institute of Allergy and Infectious Diseases Grant Number R01AI104621, UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory, and RC-0013-07 from the Louisiana Board of Regents.
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protects guinea pigs from lethal Lassa virus infection post-disease onset. Sci. Rep. 2015; 5:14775. [PubMed: 26456301] Shaffer JG, Grant DS, Schieffelin JS, Boisen ML, Goba A, Hartnett JN, Levy DC, Yenni RE, Moses LM, Fullah M, Momoh M, Fonnie M, Fonnie R, Kanneh L, Koroma VJ, Kargbo K, Ottomassathien D, Muncy IJ, Jones AB, Illick MM, Kulakosky PC, Haislip AM, Bishop CM, Elliot DH, Brown BL, Zhu H, Hastie KM, Andersen KG, Gire SK, Tabrizi S, Tariyal R, Stremlau M, Matschiner A, Sampey DB, Spence JS, Cross RW, Geisbert JB, Folarin OA, Happi CT, Pitts KR, Geske FJ, Geisbert TW, Saphire EO, Robinson JE, Wilson RB, Sabeti PC, Henderson LA, Khan SH, Bausch DG, Branco LM, Garry RF, Viral Hemorrhagic Fever, C. Lassa fever in postconflict sierra leone. PLoS Negl. Trop. Dis. 2014; 8:e2748. [PubMed: 24651047] Stephenson EH, Larson EW, Dominik JW. Effect of environmental factors on aerosol-induced Lassa virus infection. J. Med. Virol. 1984; 14:295–303. [PubMed: 6512508] WHO. WHO publishes list of top emerging diseases likely to cause major epidemics. World Health Organization; 2016. URL: http://www.who.int/medicines/ebola-treatment/WHO-list-of-topemerging-diseases/en/ [Accessed 31May16]
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Highlights •
125 Lassa glycoprotein-specific huMAbs were assessed for in vitro activity.
•
Eleven huMAbs were assessed in an outbred guinea pig model of Lassa fever.
•
Five huMAbs completely protected animals when given shortly after Lassa exposure.
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Figure 1. Correlation of IC50 and IC80 values among leading huMAb candidates in LASV neutralization assay platforms
Averaged, log10 transformed IC50 (A.) and IC80 (B.) values for therapeutic candidate huMAbs derived in LASV/HIV-1pp, LASV/LCMVpp, and PRNT assays. Antibodies segregated into two distinct clusters of IC50 and IC80 values. (C.) FACS binding profiles of NAbs (Red) to 293T cell surface-expressed LASV GPC (Josiah) versus irrelevant human IgG antibody isotype control (Black). LASV NAbs did not bind significantly to mock transfected 293T cells (data not shown). A potential therapeutic cocktail comprised of NAbs
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8.9F, 12.1F, and 37.2D bound to 293T cells expressing GPC from clade III (D.) and clade II (E.) strains of LASV, highlighting their relevance as potential Pan Lassa therapeutics.
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Figure 2. Protective efficacy profile of LASV GPC huMAbs in a Hartley GP model of LF
(A.) Pooled survival plots for treated and control GPs challenged with LASV from multiple independent experiments, as noted. Control GPs succumbed to LF between 14 and 22 PI (median = 15 days). In these studies a single GP survived infection through the 28-day study endpoint (1/18). Hartley GPs challenged with GPA LASV strain Josiah on day 0 ( ) were treated with LASV huMAbs, at 30 mg/kg of body weight on the same day (↓). Additional therapeutic antibody was administered at the same dose on days 3 and 6 PI (↓). Animals were monitored for clinical signs of LF throughout the 28-day study timeline. (B.) Viremia
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data from treated and control GP plasma on days 7 and 14 PI. Viremia levels for day 7 treatment groups 37.7H, 12.1F, and 25.6A as well as day 14 12.1F, 37.2D, 19.7E, and 10.4B were below the limit of detection (LOD). Error bars represent standard deviation from mean values. * denotes P≤0.05. ** denotes P≤0.001. *** denotes P≤0.0001.
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Antiviral Res. Author manuscript; available in PMC 2017 September 01. Published in final edited form as: Antiviral Res. 2016 September ; 133: 218–222. doi:10.1016/j.antiviral.2016.08.012.
Treatment of Lassa virus infection in outbred guinea pigs with first-in-class human monoclonal antibodies Robert W Cross#1,2, Chad E Mire#1,2, Luis M Branco#3, Joan B Geisbert1,2, Megan M. Rowland3, Megan L. Heinrich3, Augustine Goba4,5, Mambu Momoh4,5,6, Donald S Grant4,5, Mohamed Fullah4,5, Sheik Humarr Khan4,5, James E Robinson7, Thomas W Geisbert1,2, and Robert F Garry3,8 for the Viral Hemorrhagic Fever Consortium
Author Manuscript
1Department
of Microbiology and Immunology, University of Texas Medical Branch at Galveston,
Texas, USA 2Galveston
National Laboratory, University of Texas Medical Branch at Galveston, Galveston, Texas, USA
3Zalgen 4Viral
Labs, LLC, Germantown, MD
Hemorrhagic Fever Program, Kenema Government Hospital, Kenema, Sierra Leone
5Ministry
of Health and Sanitation, Freetown, Sierra Leone
6Polytechnic
College, Kenema Sierra Leone
7Sections
Author Manuscript
of Infectious Disease, Departments of Pediatrics and Internal Medicine, School of Medicine, Tulane University, New Orleans, LA
8Department
#
of Microbiology and Immunology, Tulane University, New Orleans, Louisiana, USA
These authors contributed equally to this work.
Abstract Lassa fever is a significant health threat to West African human populations with hundreds of thousands of annual cases. There are no approved medical countermeasures currently available. Compassionate use of the antiviral drug ribavirin or transfusion of convalescent serum has resulted in mixed success depending on when administered or the donor source, respectively. We previously identified several recombinant human monoclonal antibodies targeting the glycoprotein of Lassa virus with strong neutralization profiles in vitro. Here, we demonstrate remarkable
Author Manuscript
*
Corresponding author: Dr. Thomas W. Geisbert, University of Texas Medical Branch, Galveston National Laboratory, 301 University Blvd, Galveston, TX 77550-0610, Phone: 409-266-6906 Fax: 409-266-6810, [email protected]. Conflict of interest statement: RWC, CEM, JBG, MMR, MLH, AG, MM, DSG, JER, and TWG declare that they have no conflicts of interest. LMB and RFG are co-founders of Zalgen Labs, a for profit biotechnology company. Presentation History: This work was presented in part at the 11th ASM Biodefense and Emerging Diseases Research Meeting,
Washington DC, February 25 – 27, 2013 (Poster 13-BIO-A-337-ASM “Efficacy of Human Monoclonal Antibodies as an Immunotherapeutic in a Guinea Pig Model of Lassa fever”) Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cross et al.
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therapeutic efficacy using first-in-class human antibodies in a guinea pig model of Lassa infection thereby presenting a promising treatment alternative.
Author Manuscript
Lassa Fever (LF) is a major public health problem in Western Africa where hundreds of thousands of infections are estimated to occur annually. For those who present to a hospital for treatment and are referred to a ward specific for the treatment of viral hemorrhagic fevers, the mortality rate approaches 70% (Shaffer et al., 2014). Elevated mortality, environmental stability that favors artificial aerosol infection, and a dearth of medical countermeasures for LF also make Lassa virus a clear biodefense concern (Stephenson et al., 1984). The nucleoside analog ribavirin, convalescent sera, and supportive therapy are the only treatments options currently in use for LF. Safety profile concerns, lack of controlled and randomized clinical studies, and short window of efficacy for therapeutic ribavirin or convalescent sera use in LF patients support the need for development of alternative and robust therapeutic options (Bausch et al., 2010). Single monoclonal antibody therapies against viral agents have recently proven highly effective; however, only one has been approved for use in humans (Mejias and Ramilo, 2008). The 2013 – 2016 Ebola hemorrhagic fever epidemic in West Africa has, however, potentiated the accelerated development and clinical evaluation of ZMapp, a cocktail of three chimeric monoclonal antibodies against distinct epitopes on the Ebola virus glycoprotein (McCarthy, 2014).
Author Manuscript
Since 2008, the Viral Hemorrhagic Fever Consortium (http://www.VHFC.org) has concentrated efforts at characterizing protective or pathogenic roles of B cells in LF directly from human patients (Robinson et al., 2016). This work has resulted in the derivation of the largest known collection of fully human monoclonal antibodies (huMAbs) from convalescent West African LF patients. Here, we describe the protective efficacy of select LASV glycoprotein specific huMAbs in vivo where a panel of 11 huMAbs targeting the glycoprotein of Lassa virus (LASV) was evaluated in an outbred guinea pig (GP) model of LF based on their in vitro neutralization profiles. Optimized bicistronic IgG1 backbone vectors for high level, transient expression in mammalian cells were used to generate huMAbs for therapeutic evaluation in vitro and in a GP model of LF. Scaled production of antibodies for in vivo studies is detailed in the supplementary materials. Antibodies for therapeutic studies were formulated in a buffer for injection at > 20 mg/mL, and assayed for purity, concentration, identity, and endotoxin levels prior to lot release.
Author Manuscript
A panel of 125 independently derived LASV glycoprotein-specific huMAbs were extensively characterized for binding, epitope grouping, and neutralization potential in vitro (Robinson et al., 2016). Eleven huMAbs consistently registered greater than 50% neutralization in a lentiviral entry assay system pseudotyped with the LASV (Josiah strain, Clade IV) GPC, a lymphocytic choriomeningitis virus (LCMV) backbone pseudotyped with LASV GPC, or in a live LASV plaque reduction neutralization test (PRNT) (Robinson et al., 2016). Antibodies segregated into two distinct performance clusters of IC50 and IC80 values (Supplemental Table 1, Figure 1A, 1B). Neutralizing antibodies (NAbs) 37.2D, 8.9F, 12.1F,
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37.7H, 25.6A, 36.1F, and 25.10C clustered in the lowest combined Log10 IC50 (Figure 1A.) and Log10 IC80 (Figure 1B.) values range. A second cluster of NAbs comprised of 19.7E, 10.4B, 37.2G, and 2.9D recorded significantly combined higher Log10 IC50 and Log10 IC80 values range (Figure 1A, 1B). Ten of the 11 huMAbs bound mammalian cell-generated LASV glycoprotein immobilized on ELISA plates. The single exception, huMAb 8.9F was identified via a pseudovirus neutralization assay performed as previously reported (Robinson et al., 2016), and did not bind purified glycoprotein in ELISA.
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Binding efficacy to different clades of LASV was assessed using flow cytometry to detect the glycoprotein complex (GPC) genes of LASV Josiah (Clade IV), and Nigerian strains NigA19-08 (Clade III) and NigA18-237 (Clade II) which were transiently expressed on the surface of transfected HEK-293T/17 cells for 24 hours prior to flow cytometric analysis (FACS). Histograms were generated that display the log fluorescence intensity for each antibody bound to GPC on the surface of live transfected cells. All 11 huMAbs bound to HEK-293T/17 cell surface-expressed LASV GPC by flow cytometry (Figure 1C). Three LASV huMAbs (8.9F, 12.1F, and 37.2D) also bound cell surface expressed GPCs from Clade II (strain Nig A18-237) and III (strain Nig A19-08) strains (Figure 1C and 1D). These binding profiles suggest that these 3 LASV huMAbs may have therapeutic utility across LASV Clades. These data guided the identification and prioritization of leading candidate therapeutic huMAbs for in vivo studies.
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Animal studies were completed under biosafety level (BSL)-4 biocontainment at the Galveston National Laboratory and were approved by the University of Texas Medical Branch Institutional Laboratory Animal Care and Use Committee. Female outbred Hartley strain GP (~ 350-400 g, ~ 5-6 weeks old, Harlan Sprague Dawley, Houston, TX) were acclimatized for ~ 1 week prior to challenge intraperitoneally (IP) with 1000 plaque forming units of guinea pig adapted (GPA) LASV-Josiah (N=5/ treatment group). The GPA LASV infection model of outbred Hartley GP has been described recently for testing therapeutics against LASV (Safronetz et al., 2015). The advantage of using outbred animals to model human infection is inferred from the higher variability of immune responsiveness inherent in outbred populations allowing for increased countermeasure testing stringency versus those inbred for genetically tractable immune deficiencies which may, in turn, result in reduced predictive efficacy of the treatment in question by only testing a single immune phenotype rather than multiple at one time, as would be present in an outbred test population.
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Animals were administered huMAbs (30 mg/kg of body weight) IP immediately after challenge (day 0) and on days 3 and 6 post infection (PI). Control animals were not treated. Guinea pigs were monitored for clinical observations, appearance, behavior, activity, and signs of paresis. Clinical scores were determined as Bright, Alert, Responsive = 0, Rough = 4, Sick = 8, Moribund = 12, or Expired = 16 over the course of 28 days after which survivors were euthanized at the study endpoint per IACUC protocol. Animals which were scored rough had roughed fur, delayed responsiveness, and some evidence of weight loss. Sick animals had marked lack of grooming, unstable gait, and clear evidence of weight loss. Blood was collected into EDTA containing tubes on days 7 and 14 and viremia was assessed from separated plasma using classical titration methods as outlined in supplemental materials. Kaplan-Meier survival curves were generated in Prism 5 and survival curves were
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compared using Log-rank (Mantel-Cox) test (GraphPad Software, La Jolla, CA) for treated groups versus untreated controls for each study (Figure 2A). Eleven LASV huMAbs tested in a Hartley GP model of LF segregated the antibodies into three distinct protection groups. One group of huMAbs, 25.6A, 2.9D, 8.9F, 12.1F, and 37.7H conferred 100% protection and no change in clinical score in GPs. In a second group, 37.2D, 19.7E, and 37.2G protected 80 – 90% of animals. A third group comprised of 10.4B, 25.10C, and 36.1F, conferred 40, 30, and 20% protection, respectively. An irrelevant recombinant human isotype control (IgG1) antibody did not confer protection (0% survival). From the tested antibodies, no significant differences were observed between protection level and epitope groups; however, more antibodies from each respective treatment group will need to be tested in order to verify the potential importance of any one particular epitope group in regard to protection (Supplemental Table 1).
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The combined in vitro IC50 and IC80 data generally correlated well with protection in vivo for the top tier antibodies (100% protection). All but 2.9D neutralized pseudovirus in the nanomolar range, and registered a PRNT IC50 mean of 13.6nM. Despite conferring 100% protection in GP huMAb 2.9D registered high IC50 values in vitro (Supplemental Table 1). 37.2D and 19.7E generated distinct IC50 values in pseudovirus assays, with the former neutralizing in the low nanomolar range, and the latter at > 20 nanomolar. Both antibodies neutralized LASV in PRNT at IC50 ~ 18 nM, and conferred 90% overall protection in GPs. It should be noted that 19.7E and 37.2D antibodies conferred 100% protection yet showed some clinical signs in an initial study (5/5) so a repeat study was performed where 80% (4/5) survived and similar clinical values were observed.
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37.2G protected 80% of animals tested, despite high IC50 values in pseudovirus and PRNT assays (16.0 and >100 nM, respectively). 10.4B neutralized poorly in all assays and conferred only 40% protection in GPs. Surprisingly, the most potent antibody identified in vitro, 25.10C, with subnanomolar activity in all pseudovirus platforms, and 6.24 nM in PRNT, conferred an average 30% (3/10) protection in GPs. In our initial assessment, 25.10C protected 40% (2/5) of GPs, yet only 20% (1/5) in a repeat study. The difference in results could be attributed to inherent immune variability of outbred animals where differences in individual’s susceptibility to the virus contributes to disease course. Importantly, these data indicate that in vitro neutralization assays have value in selecting antibodies with protective efficacy in vivo, but are not necessarily predictive of levels of protection.
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Viremia was compared by Kruskal-Wallis test supported by Dunn’s Multiple comparison posttest (Prism 5, GraphPad Software, La Jolla, CA) to detect differences from the control group for time points relevant to onset (day 7) or peak viremia (day 14) as determined from historical data (Geisbert, T.W. unpublished data). Untreated control animals averaged 3.5 and 4.5 Log PFU/mL on days 7 and 14, respectively. Despite 100% protection at the study endpoint, some animals from treatment group 8.9F or 37.7H, 2.9D, and 25.6A had low level viremia on day 7 or 14, respectively. Treatment groups where 90% protection was afforded (37.2D and 19.7E) had reduced mean viremia titers and minimal clinical score values. Treatment groups with 80% or less survival had comparable mean viremia titers to control animals on day 7, but by day 14 mean viremia was markedly lower than control animals.
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Groups with 80-90% survival exhibited relatively low mean clinical scores and all remaining treatment groups exhibited concomitant increases in mean clinical scores with decreases in survival per group (Figure 2B, 2C, and Supplemental Figure 1). Endpoint viremia was not determined for these studies as survival was the primary metric of interest, though all surviving animals demonstrated no clinical signs to indicate disease was present at study endpoint 28 days PI. (Figure 2B and Supplemental Figure 1).
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Lassa fever is a major public health threat in West Africa. Recent importations of cases of LF to non-endemic regions of the world illustrate the global public health threat (Amorosa et al., 2010; Haas et al., 2003; Hirabayashi et al., 1989). Currently, no approved LF vaccines or therapeutics exist, yet there is a clear public health need in endemic regions. Further, LASV has been categorized as one of the World Health Organization’s top 10 emerging pathogens and has thus created increased demand for medical countermeasures(WHO, 2016). Off label use of Ribavirin can be partially effective in the treatment of acute LF; however, it has a narrow window of efficacy, generally within 6 days following the onset of fever and is often associated with marked side effects due to dosing regimen(Bausch et al., 2010). Unfortunately, the drug has not demonstrated great efficacy in treating advanced LF(Bausch et al., 2010).
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The current studies present a novel alternative for treating LF infections that relies on the use of fully human antibodies derived from convalescent donors. Early studies employing convalescent plasma to treat active LF infections met with mixed success; this was likely due to unreliable titers of protective antibodies in donors’ blood or antibody specificities that were not well matched against circulating LASV strains (Cummins et al., 1991; Jahrling et al., 1985; Leifer et al., 1970). Here we show that the majority of antibodies administered either individually or in combination prevented development of active disease in GP when administered soon after infection. These studies demonstrate the in vivo potential of these antibodies and merit further preclinical development of huMAbs as mono-therapies or cocktails for pre-exposure prophylaxis or post-exposure treatment in the gold standard animal model for LF, non-human primates. This technology may be a particularly relevant treatment for high risk scenarios such as caregivers working directly with LF patients or those who have otherwise become exposed to the virus in the community. In the absence of approved vaccines, safe and effective immunotherapeutics would provide a superior prophylactic and post-exposure treatment alternative, with well tolerated profiles, and likely few metabolic side effects.
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The ongoing outbreak of LF in Nigeria and neighboring Benin, endemicity of the disease in Sierra Leone, Guinea, and Liberia, potential for geographic expansion (Fichet-Calvet and Rogers, 2009) and threat to national security if developed as a bioweapon should prioritize development of a new and safe class of Pan-Lassa virus therapeutics (Ibekwe, 2012).
Supplementary Material Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments The authors would like to extend gratitude to Daniel J. Deer, Jessica Graber, and the University of Texas Medical Branch Animal Resource Centers for assistance with animal experiments. The authors also thank Krystle N. Agans and Kimberly Schuenke for administrative support. Finally, we wish to thank all the members of the Viral Hemorrhagic Fever Consortium for their continued support and contributions to the VHF field (www.vhfc.org). In memoriam: Tragically, two co-authors, who contributed greatly to public health and VHF research efforts in Sierra Leone, contracted EHF and lost their battle with the disease before this manuscript could be published: Mohamed Fullah and Sheik Humarr Khan. We wish to honor their memory. Funding statement This work was supported by Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119, Health and Human Services Contract HHSN272200900049C, and National Institutes of health/ national Institute of Allergy and Infectious Diseases Grant Number R01AI104621, UC7AI094660 for BSL-4 operations support of the Galveston National Laboratory, and RC-0013-07 from the Louisiana Board of Regents.
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Highlights •
125 Lassa glycoprotein-specific huMAbs were assessed for in vitro activity.
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Eleven huMAbs were assessed in an outbred guinea pig model of Lassa fever.
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Five huMAbs completely protected animals when given shortly after Lassa exposure.
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Figure 1. Correlation of IC50 and IC80 values among leading huMAb candidates in LASV neutralization assay platforms
Averaged, log10 transformed IC50 (A.) and IC80 (B.) values for therapeutic candidate huMAbs derived in LASV/HIV-1pp, LASV/LCMVpp, and PRNT assays. Antibodies segregated into two distinct clusters of IC50 and IC80 values. (C.) FACS binding profiles of NAbs (Red) to 293T cell surface-expressed LASV GPC (Josiah) versus irrelevant human IgG antibody isotype control (Black). LASV NAbs did not bind significantly to mock transfected 293T cells (data not shown). A potential therapeutic cocktail comprised of NAbs
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8.9F, 12.1F, and 37.2D bound to 293T cells expressing GPC from clade III (D.) and clade II (E.) strains of LASV, highlighting their relevance as potential Pan Lassa therapeutics.
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Figure 2. Protective efficacy profile of LASV GPC huMAbs in a Hartley GP model of LF
(A.) Pooled survival plots for treated and control GPs challenged with LASV from multiple independent experiments, as noted. Control GPs succumbed to LF between 14 and 22 PI (median = 15 days). In these studies a single GP survived infection through the 28-day study endpoint (1/18). Hartley GPs challenged with GPA LASV strain Josiah on day 0 ( ) were treated with LASV huMAbs, at 30 mg/kg of body weight on the same day (↓). Additional therapeutic antibody was administered at the same dose on days 3 and 6 PI (↓). Animals were monitored for clinical signs of LF throughout the 28-day study timeline. (B.) Viremia
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data from treated and control GP plasma on days 7 and 14 PI. Viremia levels for day 7 treatment groups 37.7H, 12.1F, and 25.6A as well as day 14 12.1F, 37.2D, 19.7E, and 10.4B were below the limit of detection (LOD). Error bars represent standard deviation from mean values. * denotes P≤0.05. ** denotes P≤0.001. *** denotes P≤0.0001.
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