A Longitudinal Analysis of Memory Immune Responses in Convalescent Crimean-Congo Hemorrhagic Fever Survivors in Uganda.

Evaluating the adaptive immune responses to natural infection with Crimean-Congo hemorrhagic fever (CCHF) virus (CCHFV) in human survivors is critical to the development of medical countermeasures. However, the correlates of protection are unknown. As the most prevalent tick-borne human hemorrhagic fever virus with case fatality rates of 5%-30% and worldwide distribution, there is an urgent need to fill these knowledge gaps. Here, we describe adaptive immune responses in a cohort of Ugandan CCHF survivors via serial sampling over 6 years. We demonstrate persistent antibodies after infection and cross-neutralization against various clades of authentic CCHFV, as well as potent effector function. Moreover, we show for the first time persistent, polyfunctional antigen-specific memory T-cell responses to multiple CCHFV proteins up to 9 years after infection. Together, this data provides immunological benchmarks for evaluating CCHFV medical countermeasures and information that can be leveraged toward vaccine immunogen design and viral target identification for monoclonal antibody therapies.

Crimean-Congo hemorrhagic fever survivors elicit protective non-neutralizing antibodies that target 11 overlapping regions on glycoprotein GP38.

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, exclusive to Nairoviridae, is a target of protective antibodies and is a key antigen in preclinical vaccine candidates. Here, we isolate 188 GP38-specific antibodies from human survivors of infection. Competition experiments show that these antibodies bind across 5 distinct antigenic sites, encompassing 11 overlapping regions. Additionally, we show structures of GP38 bound with 9 of these antibodies targeting different antigenic sites. Although these GP38-specific antibodies are non-neutralizing, several display protective efficacy equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and may inform the development of broadly effective CCHFV antibody therapeutics.

ZMapp reduces diffusion of Ebola viral particles in fresh human cervicovaginal mucus.

Vaginal transmission from semen of male Ebola virus (EBOV) survivors has been implicated as a potential origin of Ebola virus disease (EVD) outbreaks. While EBOV in semen must traverse cervicovaginal mucus (CVM) to reach target cells, the behaviour of EBOV in CVM is poorly understood. CVM contains substantial quantities of IgG, and arrays of IgG bound to a virion can develop multiple Fc-mucin bonds, immobilizing the IgG/virion complex in mucus. Here, we measured the real-time mobility of fluorescent Ebola virus-like-particles (VLP) in 50 CVM specimens from 17 women, with and without ZMapp, a cocktail of 3 monoclonal IgGs against EBOV. ZMapp-mediated effective trapping of Ebola VLPs in CVM from a subset of women across the menstrual cycle, primarily those with Lactobacillus crispatus dominant microbiota. Our work underscores the influence of the vaginal microbiome on IgG-mucin crosslinking against EBOV and identifies bottlenecks in the sexual transmission of EBOV.

Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38.

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, unique to Nairoviridae, is a target of protective antibodies, but extensive mapping of the human antibody response to GP38 has not been previously performed. Here, we isolated 188 GP38-specific antibodies from human survivors of infection. Competition experiments showed that these antibodies bind across five distinct antigenic sites, encompassing eleven overlapping regions. Additionally, we reveal structures of GP38 bound with nine of these antibodies targeting different antigenic sites. Although GP38-specific antibodies were non-neutralizing, several antibodies were found to have protection equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and inform the development of broadly effective CCHFV antibody therapeutics.

Long-term Prophylaxis Against Aerosolized Marburg Virus in Nonhuman Primates With an Afucosylated Monoclonal Antibody.

Marburg virus (MARV) causes a hemorrhagic fever disease in human and nonhuman primates with high levels of morbidity and mortality. Concerns about weaponization of aerosolized MARV have spurred the development of nonhuman primate (NHP) models of aerosol exposure. To address the potential threat of aerosol exposure, a monoclonal antibody that binds MARV glycoprotein was tested, MR186YTE, for its efficacy as a prophylactic. MR186YTE was administered intramuscularly to NHPs at 15 or 5 mg/kg 1 month prior to MARV aerosol challenge. Seventy-five percent (3/4) of the 15 mg/kg dose group and 50% (2/4) of the 5 mg/kg dose group survived. Serum analyses showed that the NHP dosed with 15 mg/kg that succumbed to infection developed an antidrug antibody response and therefore had no detectable MR186YTE at the time of challenge. These results suggest that intramuscular dosing of mAbs may be a clinically useful prophylaxis for MARV aerosol exposure.

Structural and mechanistic basis of neutralization by a pan-hantavirus protective antibody.

Emerging rodent-borne hantaviruses cause severe diseases in humans with no approved vaccines or therapeutics. We recently isolated a monoclonal broadly neutralizing antibody (nAb) from a Puumala virus-experienced human donor. Here, we report its structure bound to its target, the Gn/Gc glycoprotein heterodimer comprising the viral fusion complex. The structure explains the broad activity of the nAb: It recognizes conserved Gc fusion loop sequences and the main chain of variable Gn sequences, thereby straddling the Gn/Gc heterodimer and locking it in its prefusion conformation. We show that the nAb's accelerated dissociation from the divergent Andes virus Gn/Gc at endosomal acidic pH limits its potency against this highly lethal virus and correct this liability by engineering an optimized variant that sets a benchmark as a candidate pan-hantavirus therapeutic.

LALAPG variant of the Human Contraception Antibody (HCA) reduces Fc-mediated effector functions while maintaining sperm agglutination activity.

High rates of unintended pregnancies worldwide indicate a need for more accessible and acceptable methods of contraception. We have developed a monoclonal antibody, the Human Contraception Antibody (HCA), for use by women in vaginal films and rings for contraception. The divalent F(ab')2 region of HCA binds to an abundant male reproductive tract-specific antigen, CD52g, and potently agglutinates sperm. Certain other antibody activities mediated by the Fc region such as mucus trapping, complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) could have beneficial or negative effects. The purpose of this study was to document HCA Fc effector functions and determine whether an engineered variant of HCA with a modified Fc region, HCA-LALAPG, retains desirable contraceptive activity while minimizing Fc-mediated effects. Fab and Fc functions were compared between HCA and HCA-LALAPG. Fab activity was assessed using sperm agglutination and modified swim-up ("sperm escape") assays. Fc functions were assessed by CDC (sperm immobilization), ADCP, and cervical mucus penetration assays. HCA and HCA-LALAPG showed equivalent activity in assays of Fab function. In the assays of Fc function, HCA supported strong CDC, ADCP, and sperm trapping in cervical mucus whereas HCA-LALAPG demonstrated little to no activity. HCA and the HCA-LALAPG variant were both highly effective in the sperm agglutination assays but differed in Fc mediated functions. Use of the HCA-LALAPG variant for contraception in women could reduce antibody-mediated inflammation and antigen presentation but may have reduced contraceptive efficacy due to much weaker sperm trapping in mucus and complement-dependent sperm immobilization activity.

Detection of biomarkers for filoviral infection with a silicon photonic resonator platform.

This protocol describes the use of silicon photonic microring resonator sensors for detection of Ebola virus (EBOV) and Sudan virus (SUDV) soluble glycoprotein (sGP). This protocol encompasses biosensor functionalization of silicon microring resonator chips, detection of protein biomarkers in sera, preparing calibration standards for analytical validation, and quantification of the results from these experiments. This protocol is readily adaptable toward other analytes, including cytokines, chemokines, nucleic acids, and viruses. For complete details on the use and execution of this protocol, please refer to Qavi et al. (2022).

Rapid detection of an Ebola biomarker with optical microring resonators.

Ebola virus (EBOV) is a highly infectious pathogen, with a case mortality rate as high as 89%. Rapid therapeutic treatments and supportive measures can drastically improve patient outcome; however, the symptoms of EBOV disease (EVD) lack specificity from other endemic diseases. Given the high mortality and significant symptom overlap, there is a critical need for sensitive, rapid diagnostics for EVD. Facile diagnosis of EVD remains a challenge. Here, we describe a rapid and sensitive diagnostic for EVD through microring resonator sensors in conjunction with a unique biomarker of EBOV infection, soluble glycoprotein (sGP). Microring resonator sensors detected sGP in under 40 min with a limit of detection (LOD) as low as 1.00 ng/mL in serum. Furthermore, we validated our assay with the detection of sGP in serum from EBOV-infected non-human primates. Our results demonstrate the utility of a high-sensitivity diagnostic platform for detection of sGP for diagnosis of EVD.

Reversion of Ebolavirus Disease from a Single Intramuscular Injection of a Pan-Ebolavirus Immunotherapeutic.

Intravenous (IV) administration of antiviral monoclonal antibodies (mAbs) can be challenging, particularly during an ongoing epidemic, due to the considerable resources required for performing infusions. An ebolavirus therapeutic administered via intramuscular (IM) injection would reduce the burdens associated with IV infusion and allow rapid treatment of exposed individuals during an outbreak. Here, we demonstrate how MBP134, a cocktail of two pan-ebolavirus mAbs, reverses the course of Sudan ebolavirus disease (Gulu variant) with a single IV or IM dose in non-human primates (NHPs) as late as five days post-exposure. We also investigate the utility of adding half-life extension mutations to the MBP134 mAbs, ultimately creating a half-life extended cocktail designated MBP431. When delivered as a post-exposure prophylactic or therapeutic, a single IM dose of MBP431 offered complete or significant protection in NHPs challenged with Zaire ebolavirus. In conjunction with previous studies, these results support the use of MBP431 as a rapidly deployable IM medical countermeasure against every known species of ebolavirus.

Combination therapy with remdesivir and monoclonal antibodies protects nonhuman primates against advanced Sudan virus disease.

A major challenge in managing acute viral infections is ameliorating disease when treatment is delayed. Previously, we reported the success of a 2-pronged mAb and antiviral remdesivir therapeutic approach to treat advanced illness in rhesus monkeys infected with Marburg virus (MARV). Here, we explored the benefit of a similar combination therapy for Sudan ebolavirus (Sudan virus; SUDV) infection. Importantly, no licensed anti-SUDV therapeutics currently exist, and infection of rhesus macaques with SUDV results in a rapid disease course similar to MARV with a mean time to death of 8.3 days. When initiation of therapy with either remdesivir or a pan-ebolavirus mAb cocktail (MBP431) was delayed until 6 days after inoculation, only 20% of macaques survived. In contrast, when remdesivir and MBP431 treatment were combined beginning 6 days after inoculation, significant protection (80%) was achieved. Our results suggest that combination therapy may be a viable treatment for patients with advanced filovirus disease that warrants further clinical testing in future outbreaks.

Human antibody recognizing a quaternary epitope in the Puumala virus glycoprotein provides broad protection against orthohantaviruses.

The rodent-borne hantavirus Puumala virus (PUUV) and related agents cause hemorrhagic fever with renal syndrome (HFRS) in humans. Other hantaviruses, including Andes virus (ANDV) and Sin Nombre virus, cause a distinct zoonotic disease, hantavirus cardiopulmonary syndrome (HCPS). Although these infections are severe and have substantial case fatality rates, no FDA-approved hantavirus countermeasures are available. Recent work suggests that monoclonal antibodies may have therapeutic utility. We describe here the isolation of human neutralizing antibodies (nAbs) against tetrameric Gn/Gc glycoprotein spikes from PUUV-experienced donors. We define a dominant class of nAbs recognizing the "capping loop" of Gn that masks the hydrophobic fusion loops in Gc. A subset of nAbs in this class, including ADI-42898, bound Gn/Gc complexes but not Gn alone, strongly suggesting that they recognize a quaternary epitope encompassing both Gn and Gc. ADI-42898 blocked the cell entry of seven HCPS- and HFRS-associated hantaviruses, and single doses of this nAb could protect Syrian hamsters and bank voles challenged with the highly virulent HCPS-causing ANDV and HFRS-causing PUUV, respectively. ADI-42898 is a promising candidate for clinical development as a countermeasure for both HCPS and HFRS, and its mode of Gn/Gc recognition informs the development of broadly protective hantavirus vaccines.

Reproducibility and flexibility of monoclonal antibody production with Nicotiana benthamiana.

The ongoing SARS-CoV-2 coronavirus pandemic of 2020-2021 underscores the need for manufacturing platforms that can rapidly produce monoclonal antibody (mAb) therapies. As reported here, a platform based on Nicotiana benthamiana produced mAb therapeutics with high batch-to-batch reproducibility and flexibility, enabling production of 19 different mAbs of sufficient purity and safety for clinical application(s). With a single manufacturing run, impurities were effectively removed for a representative mAb product (the ZMapp component c4G7). Our results show for the first time the reproducibility of the platform for production of multiple batches of clinical-grade mAb, manufactured under current Good Manufacturing Practices, from Nicotiana benthamiana. The flexibility of the system was confirmed by the results of release testing of 19 different mAbs generated with the platform. The process from plant infection to product can be completed within 10 days. Therefore, with a constant supply of plants, response to the outbreak of an infectious disease could be initiated within a matter of weeks. Thus, these data demonstrated that this platform represents a reproducible, flexible system for rapid production of mAb therapeutics to support clinical development.

Emerging antibody-based products for infectious diseases: Planning for metric ton manufacturing.

This review focuses on the emerging monoclonal antibody market for infectious diseases and the metric ton scale manufacturing requirements to meet global demand. Increasing access to existing antibody-based products coupled with the unmet need in infectious disease will likely exceed the current existing global manufacturing capacity. Further, the large numbers of individuals infected during epidemics such as the ongoing COVID-19 pandemic emphasizes the need to plan for metric ton manufacturing of monoclonal antibodies by expanding infrastructure and exploring alternative production systems.

Manufacturing plant-made monoclonal antibodies for research or therapeutic applications.

Monoclonal antibodies (mAbs) hold great promise for treating diseases ranging from cancer to infectious disease. Manufacture of mAbs is challenging, expensive, and time-consuming using mammalian systems. We describe detailed methods used by Kentucky BioProcessing (KBP), a subsidiary of British American Tobacco, for producing high quality mAbs in a Nicotiana benthamiana host. Using this process, mAbs that meet GMP standards can be produced in as little as 10 days. Guidance for using individual plants, as well as detailed methods for large-scale production, are described. These procedures enable flexible, robust, and consistent production of research and therapeutic mAbs.

Cross-Strain Neutralizing and Protective Monoclonal Antibodies against EEEV or WEEV.

The three encephalitic alphaviruses, namely, the Venezuelan, eastern, and western equine encephalitis viruses (VEEV, EEEV, and WEEV), are classified by the Centers for Disease Control and Prevention (CDC) as biothreat agents. Currently, no licensed medical countermeasures (MCMs) against these viruses are available for humans. Neutralizing antibodies (NAbs) are fast-acting and highly effective MCMs for use in both pre- and post-exposure settings against biothreat agents. While significant work has been done to identify anti-VEEV NAbs, less has been done to identify NAbs against EEEV and WEEV. In order to develop anti-EEEV or -WEEV NAbs, mice were immunized using complementary strategies with a variety of different EEEV or WEEV immunogens to maximize the generation of NAbs to each of these viruses. Of the hybridomas generated, three anti-EEEV and seven anti-WEEV monoclonal antibodies were identified with in vitro neutralization activity. The most potent neutralizers (two anti-EEEV NAbs and three anti-WEEV NAbs) were further evaluated for neutralization activity against additional strains of EEEV, a single strain of Madariaga virus (formerly South American EEEV), or WEEV. Of these, G1-2-H4 and G1-4-C3 neutralized all three EEEV strains and the Madariaga virus strain, whereas G8-2-H9 and 12 WA neutralized six out of eight WEEV strains. To determine the protective efficacy of these NAbs, the five most potent neutralizers were evaluated in respective mouse aerosol challenge models. All five NAbs demonstrated various levels of protection when administered at doses of 2.5 mg/kg or 10 mg/kg 24 h before the respective virus exposure via the aerosol route. Of these, anti-EEEV NAb G1-4-C3 and anti-WEEV NAb 8C2 provided 100% protection at both doses and all surviving mice were free of clinical signs throughout the study. Additionally, no virus was detected in the brain 14 days post virus exposure. Taken together, efficacious NAbs were developed that demonstrate the potential for the development of cross-strain antibody-based MCMs against EEEV and WEEV infections.

Hexavalent sperm-binding IgG antibody released from vaginal film for development of potent on-demand nonhormonal female contraception.

Nonhormonal products for on-demand contraception are a global health technology gap; this unmet need motivated us to pursue the use of sperm-binding monoclonal antibodies to enable effective on-demand contraception. Here, using the cGMP-compliant Nicotiana-expression system, we produced an ultrapotent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF). The Nicotiana-produced FIF had at least 10-fold greater sperm-agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulated the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film and evaluated its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm were recovered from the vaginas of sheep receiving FIF Film. Our work supports the potential of multivalent contraceptive antibodies to provide safe, effective, on-demand nonhormonal contraception.

Cooperativity mediated by rationally selected combinations of human monoclonal antibodies targeting the henipavirus receptor binding protein.

Hendra virus and Nipah virus (NiV), members of the Henipavirus (HNV) genus, are zoonotic paramyxoviruses known to cause severe disease across six mammalian orders, including humans. We isolated a panel of human monoclonal antibodies (mAbs) from the B cells of an individual with prior exposure to equine Hendra virus (HeV) vaccine, targeting distinct antigenic sites. The most potent class of cross-reactive antibodies achieves neutralization by blocking viral attachment to the host cell receptors ephrin-B2 and ephrin-B3, with a second class being enhanced by receptor binding. mAbs from both classes display synergistic activity in vitro. In a stringent hamster model of NiV Bangladesh (NiVB) infection, antibodies from both classes reduce morbidity and mortality and achieve synergistic protection in combination. These candidate mAbs might be suitable for use in a cocktail therapeutic approach to achieve synergistic potency and reduce the risk of virus escape.