Frederick W. Holtsberg

Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses

ABSTRACT The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site

Summary Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.

Macaque monoclonal antibodies targeting novel conserved epitopes within filovirus glycoprotein

ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.

A "Trojan horse" bispecific antibody strategy for broad protection against ebolaviruses.

Treating Ebola with a Trojan horse The recent major Ebola virus outbreak in West Africa high-lighted the need for effective therapeutics against this and other filoviruses. Neutralizing ebolaviruses with antibodies is a challenge because the viruses bind their entry receptor, NPC1, inside the cell within endosomes rather than on the cell surface. Furthermore, enzymes in endosomes cleave the Ebola virus surface glycoprotein (GP) to reveal its receptor binding site. Wec et al. now report a bispecific antibody strategy targeting all known ebolaviruses that overcomes this problem (see the Perspective by Labrijn and Parren). They coupled an antibody specific for a conserved, surface-exposed epitope of GP to antibodies that recognize either NPC1 or the NPC1 binding site on GP. Treating mice therapeutically with these antibodies allowed them to survive otherwise lethal ebolavirus infection. Science, this issue p. 350; see also p. 284 Bispecific antibodies show therapeutic efficacy against ebolaviruses in mice. There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor–binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such “Trojan horse” bispecific antibodies have potential as broad antifilovirus immunotherapeutics.

Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.

Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice

Direct‐acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance‐associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity‐matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. Conclusion: The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922‐1933).

Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel “wing” feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.

A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin

Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these agents are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge.

Antibodies to S. aureus LukS-PV Attenuated Subunit Vaccine Neutralize a Broad Spectrum of Canonical and Non-Canonical Bicomponent Leukotoxin Pairs.

S. aureus vaccine development has proven particularly difficult. The conventional approach to achieve sterile immunity through opsonophagocytic killing has been largely unsuccessful. S. aureus is highly toxigenic and a great body of evidence suggests that a successful future vaccine for this organism should target extracellular toxins which are responsible for host tissue destruction and immunosuppression. Major staphylococcal toxins are alpha toxin (a single subunit hemolysin) along with a group of bicomponent pore-forming toxins (BCPFT), namely Panton-Valentine leukocidin (PVL), gamma hemolysins (HlgCB and AB), LukAB and LukED. In our previous report, an attenuated mutant of LukS-PV (PVL- S subunit) named as “LukS-mut9” elicited high immunogenic response as well as provided a significant protection in a mouse sepsis model. Recent discovery of PVL receptors shows that mice lack receptors for this toxin, thus the reported protection of mice with the PVL vaccine may relate to cross protective responses against other homologous toxins. This manuscript addresses this issue by demonstrating that polyclonal antibody generated by LukS-mut9 can neutralize other canonical and non-canonical leukotoxin pairs. In this report, we also demonstrated that several potent toxins can be created by non-canonical pairing of subunits. Out of 5 pairs of canonical and 8 pairs of non-canonical toxins tested, anti-LukS-mut9 polyclonal antibodies neutralized all except for LukAB. We also studied the potential hemolytic activities of canonical and noncanonical pairs among biocomponent toxins and discovered that a novel non-canonical pair consisting of HlgA and LukD is a highly toxic combination. This pair can lyse RBC from different species including human blood far better than alpha hemolysin. Moreover, to follow-up our last report, we explored the correlation between the levels of pre-existing antibodies to new sets of leukotoxins subunits and clinical outcomes in adult patients with S. aureus bacteremia. We found that there is an inversed correlation between the antibody titer to sepsis for leukotoxins LukS-mut9, LukF-PV, HlgC, LukE and LukAB, suggesting the risk of sepsis was significantly lower in the patients with higher antibody titer against those toxins.

Safety and Immunogenicity of a Parenterally Administered, Structure-Based Rationally Modified Recombinant Staphylococcal Enterotoxin B Protein Vaccine, STEBVax

ABSTRACT Staphylococcus aureus produces several enterotoxins and superantigens, exposure to which can elicit profound toxic shock. A recombinant staphylococcal enterotoxin B (rSEB) containing 3 distinct mutations in the major histocompatibility complex class II binding site was combined with an alum adjuvant (Alhydrogel) and used as a potential parenteral vaccine named STEBVax. Consenting healthy adult volunteers (age range, 23 to 38 years) participated in a first-in-human open-label dose escalation study of parenteral doses of STEBVax ranging from 0.01 μg up to 20 μg. Safety was assessed by determination of the frequency of adverse events and reactogenicity. Immune responses to the vaccination were determined by measurement of anti-staphylococcal enterotoxin B (anti-SEB) IgG by enzyme-linked immunosorbent assay and a toxin neutralization assay (TNA). Twenty-eight participants were enrolled in 7 dosing cohorts. All doses were well tolerated. The participants exhibited heterogeneous baseline antibody titers. More seroconversions and a faster onset of serum anti-SEB IgG toxin-neutralizing antibodies were observed by TNA with increasing doses of STEBVax. There was a trend for a plateau in antibody responses with doses of STEBVax of between 2.5 and 20 μg. Among the participants vaccinated with 2.5 μg to 20 μg of STEBVax, ∼93% seroconverted for SEB toxin-neutralizing antibody. A strong correlation between individual SEB-specific serum IgG antibody titers and the neutralization of gamma interferon production was found in vitro. STEBvax appeared to be safe and immunogenic, inducing functional toxin-neutralizing antibodies. These data support its continued clinical development. (This study has been registered at ClinicalTrials.gov under registration no. NCT00974935.)