Distinct disease features in chimpanzees infected with a precore HBV mutant associated with acute liver failure in humans.

Transmission to chimpanzees of a precore hepatitis B virus (HBV) mutant implicated in acute liver failure (ALF) in humans did not cause ALF nor the classic form of acute hepatitis B (AHB) seen upon infection with the wild-type HBV strain, but rather a severe AHB with distinct disease features. Here, we investigated the viral and host immunity factors responsible for the unusual severity of AHB associated with the precore HBV mutant in chimpanzees. Archived serial serum and liver specimens from two chimpanzees inoculated with a precore HBV mutant implicated in ALF and two chimpanzees inoculated with wild-type HBV were studied. We used phage-display library and next-generation sequencing (NGS) technologies to characterize the liver antibody response. The results obtained in severe AHB were compared with those in classic AHB and HBV-associated ALF in humans. Severe AHB was characterized by: (i) the highest alanine aminotransferase (ALT) peaks ever seen in HBV transmission studies with a significantly shorter incubation period, compared to classic AHB; (ii) earlier HBsAg clearance and anti-HBs seroconversion with transient or undetectable hepatitis B e antigen (HBeAg); (iii) limited inflammatory reaction relative to hepatocellular damage at the ALT peak with B-cell infiltration, albeit less extensive than in ALF; (iv) detection of intrahepatic germline antibodies against hepatitis B core antigen (HBcAg) by phage-display libraries in the earliest disease phase, as seen in ALF; (v) lack of intrahepatic IgM anti-HBcAg Fab, as seen in classic AHB, but at variance with ALF; and (vi) higher proportion of antibodies in germline configuration detected by NGS in the intrahepatic antibody repertoire compared to classic AHB, but lower than in ALF. This study identifies distinct outcome-specific features associated with severe AHB caused by a precore HBV mutant in chimpanzees, which bear closer resemblance to HBV ALF than to classic AHB. Our data suggest that precore HBV mutants carry an inherently higher pathogenicity that, in addition to specific host factors, may play a critical role in determining the severity of acute HBV disease.

Role of humoral immunity against hepatitis B virus core antigen in the pathogenesis of acute liver failure.

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen.

Next-generation sequencing of the intrahepatic antibody repertoire delineates a unique B-cell response in HBV-associated acute liver failure.

Hepatitis B virus (HBV) is a major cause of acute liver failure (ALF) worldwide. While liver damage in classic acute hepatitis B is believed to be T-cell mediated, the pathogenesis of HBV-associated ALF remains largely unknown. Access to liver specimens from well-characterized patients with HBV-associated ALF provided us with the opportunity to perform next-generation sequencing (NGS) of the entire VH repertoires of IgM and IgG from the livers of four ALF patients, a control liver donor and a patient with chronic HBV infection. We found that ALF is not associated with expansion of specific B-cell lineages. However, NGS showed that the intrahepatic VH repertoires from ALF patients were characterized by the abundant presence of antibodies in germline configuration in contrast to their marginal prevalence in controls. Moreover, NGS identified a large number of VH genes in germline configuration with identical VDJ sequences in the IgM and IgG repertoires in all four ALF patients, indicating that isotype switch from IgM to IgG had occurred without somatic hypermutation. The results of this study indicate that the presence of intrahepatic antibodies in unmutated germline configuration is a broad phenomenon in the global antibody repertoire generated from total RNA derived from whole-liver tissue that is strongly associated with ALF, suggesting a major role of T cell-independent humoral immunity in the pathogenesis of ALF.

Rapid Discovery and Characterization of Synthetic Neutralizing Antibodies against Anthrax Edema Toxin.

Anthrax, a lethal, weaponizable disease caused by Bacillus anthracis, acts through exotoxins that are primary mediators of systemic toxicity and also targets for neutralization by passive immunotherapy. The ease of engineering B. anthracis strains resistant to established therapy and the historic use of the microbe in bioterrorism present a compelling test case for platforms that permit the rapid and modular development of neutralizing agents. In vitro antigen-binding fragment (Fab) selection offers the advantages of speed, sequence level molecular control, and engineering flexibility compared to traditional monoclonal antibody pipelines. By screening an unbiased, chemically synthetic phage Fab library and characterizing hits in cell-based assays, we identified two high-affinity neutralizing Fabs, A4 and B7, against anthrax edema factor (EF), a key mediator of anthrax pathogenesis. Engineered homodimers of these Fabs exhibited potency comparable to that of the best reported neutralizing monoclonal antibody against EF at preventing EF-induced cyclic AMP production. Using internalization assays in COS cells, B7 was found to block steps prior to EF internalization. This work demonstrates the efficacy of synthetic alternatives to traditional antibody therapeutics against anthrax while also demonstrating a broadly generalizable, rapid, and modular screening pipeline for neutralizing antibody generation.

Zeptomole per milliliter detection and quantification of edema factor in plasma by LC-MS/MS yields insights into toxemia and the progression of inhalation anthrax.

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.

Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor.

Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus.

Development of Norwalk virus-specific monoclonal antibodies with therapeutic potential for the treatment of Norwalk virus gastroenteritis.

Passive immunoprophylaxis or immunotherapy with norovirus-neutralizing monoclonal antibodies (MAbs) could be a useful treatment for high-risk populations, including infants and young children, the elderly, and certain patients who are debilitated or immunocompromised. In order to obtain antinorovirus MAbs with therapeutic potential, we stimulated a strong adaptive immune response in chimpanzees to the prototype norovirus strain Norwalk virus (NV) (genogroup I.1). A combinatorial phage Fab display library derived from mRNA of the chimpanzees' bone marrow was prepared, and four distinct Fabs reactive with Norwalk recombinant virus-like particles (rVLPs) were recovered, with estimated binding affinities in the subnanomolar range. Mapping studies showed that the four Fabs recognized three different conformational epitopes in the protruding (P) domain of NV VP1, the major capsid protein. The epitope of one of the Fabs, G4, was further mapped to a specific site involving a key amino acid residue, Gly365. One additional specific Fab (F11) was recovered months later from immortalized memory B cells and partially characterized. The anti-NV Fabs were converted into full-length IgG (MAbs) with human γ1 heavy chain constant regions. The anti-NV MAbs were tested in the two available surrogate assays for Norwalk virus neutralization, which showed that the MAbs could block carbohydrate binding and inhibit hemagglutination by NV rVLP. By mixing a single MAb with live Norwalk virus prior to challenge, MAbs D8 and B7 neutralized the virus and prevented infection in a chimpanzee. Because chimpanzee immunoglobulins are virtually identical to human immunoglobulins, these chimpanzee anticapsid MAbs may have a clinical application.

Pre- and postexposure protection against virulent anthrax infection in mice by humanized monoclonal antibodies to Bacillus anthracis capsule.

One of the two essential virulence factors of Bacillus anthracis is the poly-γ-D-glutamic acid (γDPGA) capsule. Five γDPGA-specific antibody antigen-binding fragments (Fabs) were generated from immunized chimpanzees. The two selected for further study, Fabs 11D and 4C, were both converted into full-length IgG1 and IgG3 mAbs having human IgG1 or IgG3 constant regions. These two mAbs had similar binding affinities, in vitro opsonophagocytic activities, and in vivo efficacies, with the IgG1 and IgG3 subclasses reacting similarly. The mAbs bound to γDPGA specifically with estimated binding affinities (K(d)) of 35-70 nM and effective affinities (effective K(d)) of 0.1-0.3 nM. The LD(50) in an opsonophagocytic bactericidal assay was ≈10 ng/mL of 11D or 4C. A single 30-µg dose of either mAb given to BALB/c mice 18 h before challenge conferred about 50% protection against a lethal intratracheal spore challenge by the virulent B. anthracis Ames strain. More importantly, either mAb given 8 h or 20 h after challenge provided significant protection against lethal infection. Thus, these anti-γDPGA mAbs should be useful, alone or in combination with antitoxin mAbs, for achieving a safe and efficacious postexposure therapy for anthrax.

Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure.

Previously, the livers of patients suffering from acute liver failure (ALF), a potentially fatal syndrome arising from infection by Hepatitis B Virus (HBV), were found to contain massive amounts of an antibody specific for the core antigen (HBcAg) capsid. We have used cryo-electron microscopy and molecular modeling to define its epitope. HBV capsids are icosahedral shells with 25Å-long dimeric spikes, each a 4-helix bundle, protruding from the contiguous "floor". Of the anti-HBcAg antibodies previously characterized, most bind around the spike tip while one binds to the floor. The ALF-associated antibody binds tangentially to a novel site on the side of the spike. This epitope is conformational. The Fab binds with high affinity to its principal determinants but has lower affinities for quasi-equivalent variants. The highest occupancy site is on one side of a spike, with no detectable binding to the corresponding site on the other side. Binding of one Fab per dimer was also observed by analytical ultracentrifugation. The Fab did not bind to the e-antigen dimer, a non-assembling variant of capsid protein. These findings support the propositions that antibodies with particular specificities may correlate with different clinical expressions of HBV infection and that antibodies directed to particular HBcAg epitopes may be involved in ALF pathogenesis.

B cell gene signature with massive intrahepatic production of antibodies to hepatitis B core antigen in hepatitis B virus-associated acute liver failure.

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. The mechanisms whereby HBV induces ALF are unknown. Here, we show that liver tissue collected at the time of liver transplantation in two patients with HBV-associated ALF is characterized by an overwhelming B cell response apparently centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. We demonstrate that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg); that these anti-bodies display a restricted variable heavy chain (V(H)) repertoire and lack somatic mutations; and that these two unrelated individuals with ALF use an identical predominant V(H) gene with unmutated variable domain (IGHV1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human V(H) gene. These data suggest that humoral immunity may exert a primary role in the pathogenesis of HBV-associated ALF.

Structural basis of anthrax edema factor neutralization by a neutralizing antibody.

Fine epitope mapping of EF13D, a highly potent neutralizing monoclonal antibody specific for the anthrax edema factor (EF), was accomplished through random mutagenesis and yeast surface display. A yeast-displayed library of single point mutants of an EF domain III (DIII), comprising amino acids 624-800, was constructed by random mutagenesis and screened for reduced binding to EF13D. With this method, residues Leu 667, Ser 668, Arg 671, and Arg 672 were identified as key residues important for EF13D binding. They form a contiguous patch on a solvent-exposed surface at one end of the four-helix bundle of DIII. Computational protein-protein docking experiments between anEF13D model and a crystal structure of EF indicate that the EF13D heavy chain complementarity-determining region 3 (HCDR3) is deeply buried within a hydrophobic cleft between two helices of DIII and interacts directly with residues Leu 667, Ser 668, Arg 671 and Arg 672, providing an explanation for the high binding affinity. In addition, they show that the HCDR3 binding site overlaps with the binding site of the N-terminal lobe of calmodulin (CaM), an EF enzymatic activator, consistent with a previous finding showing direct competition with CaM that results in neutralization of EF. Identifying the neutralization epitope of EF13D on EF improves our understanding of the neutralization mechanism and has implications for vaccine development.

Chimpanzee-human monoclonal antibodies for treatment of chronic poliovirus excretors and emergency postexposure prophylaxis.

Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 µg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case.

Potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding.

This study describes the isolation and characterization of a neutralizing monoclonal antibody (mAb) against anthrax edema factor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic AMP (cAMP) responses in cells and protected mice from both ET-induced footpad edema and systemic ET-mediated lethality. The antibody epitope was mapped to domain IV of EF. The mAb was able to compete with calmodulin (CaM) for EF binding and displaced CaM from EF-CaM complexes. EF-mAb binding affinity (0.05-0.12 nM) was 50- to 130-fold higher than that reported for EF-CaM. This anti-EF neutralizing mAb could potentially be used alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection.

Extremely potent monoclonal antibodies neutralize Omicron and other SARS-CoV-2 variants.

The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity 1 . This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies 1, 2 . Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe 3 . Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants.

The emergence and global spread of the SARS-CoV-2 Omicron variants, which carry an unprecedented number of mutations, raise serious concerns due to the reduced efficacy of current vaccines and resistance to therapeutic antibodies. Here, we report the generation and characterization of two potent human monoclonal antibodies, NA8 and NE12, against the receptor-binding domain of the SARS-CoV-2 spike protein. NA8 interacts with a highly conserved region and has a breadth of neutralization with picomolar potency against the Beta variant and the Omicron BA.1 and BA.2 sublineages and nanomolar potency against BA.2.12.1 and BA.4. Combination of NA8 and NE12 retains potent neutralizing activity against the major SARS-CoV-2 variants of concern. Cryo-EM analysis provides the structural basis for the broad and complementary neutralizing activity of these two antibodies. We confirm the in vivo protective and therapeutic efficacies of NA8 and NE12 in the hamster model. These results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants.

Mouse monoclonal antibodies to anthrax edema factor protect against infection.

Bacillus anthracis is the causative agent of anthrax, and the tripartite anthrax toxin is an essential element of its pathogenesis. Edema factor (EF), a potent adenylyl cyclase, is one of the toxin components. In this work, anti-EF monoclonal antibodies (MAb) were produced following immunization of mice, and four of the antibodies were fully characterized. MAb 3F2 has an affinity of 388 pM, was most effective for EF detection, and appears to be the first antibody reported to neutralize EF by binding to the catalytic C(B) domain. MAb 7F10 shows potent neutralization of edema toxin activity in vitro and in vivo; it targets the N-terminal protective antigen binding domain. The four MAb react with three different domains of edema factor, and all were able to detect purified edema factor in Western blot analysis. None of the four MAb cross-reacted with the lethal factor toxin component. Three of the four MAb protected mice in both a systemic edema toxin challenge model and a subcutaneous spore-induced foreleg edema model. A combination of three of the MAb also significantly delayed the time to death in a third subcutaneous spore challenge model. This appears to be the first direct evidence that monoclonal antibody-mediated neutralization of EF alone is sufficient to delay anthrax disease progression.

Delayed treatment with W1-mAb, a chimpanzee-derived monoclonal antibody against protective antigen, reduces mortality from challenges with anthrax edema or lethal toxin in rats and with anthrax spores in mice.

OBJECTIVE: W1-mAb is a chimpanzee-derived monoclonal antibody to protective antigen that improved survival when administered before anthrax lethal toxin challenge in rats. To better define W1-mAb's efficacy for anthrax, we administered it after initiation of 24-hr infusions of edema toxin and lethal toxin either alone or together in rats or following anthrax spore challenge in mice. INTERVENTIONS: W1-mAb or placebo treatment. METHODS AND MAIN RESULTS: In toxin-challenged rats treated with placebo, survival rates were lower with edema toxin (500 µg/kg) compared to lethal toxin either alone (175 µg/kg) or with edema toxin (175 µg/kg each) (8%, 33%, and 32%, respectively), but the median time to death was longer (36, 11, and 9 hrs, respectively) (p ≤ .01 for all comparisons). W1-mAb administered up to 12 hrs after edema toxin and 6 hrs after lethal toxin increased survival and reduced hypotension (p ≤ .01). However, only administration of W1-mAb at 0 hrs improved these variables with lethal toxin and edema toxin together (p ≤ .0002). In C57BL/6J mice challenged with anthrax spores subcutaneously, compared to placebo treatment (0 of 15 animals survived), W1-mAb administered beginning 24 hrs after challenge increased survival (13 of 15 survived) (p ≤ .0001). CONCLUSION: While rapidity of lethality may influence the effectiveness of delayed W1-mAb treatment, these rat and mouse studies provide a basis for further exploring this agent's usefulness for anthrax.

Distinct Cytokine Profiles Correlate with Disease Severity and Outcome in Longitudinal Studies of Acute Hepatitis B Virus and Hepatitis D Virus Infection in Chimpanzees.

Historical studies conducted in chimpanzees gave us the opportunity to investigate the basis for the different severities of liver damage and disease outcome associated with infection with wild-type hepatitis B virus (HBV) versus a precore HBV mutant, HBV/hepatitis D virus (HDV) coinfection, and HDV superinfection. Weekly samples from 9 chimpanzees were studied for immune responses by measuring plasma levels of 29 cytokines in parallel with alanine aminotransferase (ALT) levels and viral kinetics. Comparison of classic acute hepatitis B (AHB) with severe or progressive AHB and HBV/HDV coinfection or superinfection identified distinct cytokine profiles. Classic AHB (mean ALT peak, 362 IU/liter) correlated with an early and significant induction of interferon alpha-2 (IFN-α2), IFN-γ, interleukin-12 p70 (IL-12 p70), and IL-17A. In contrast, these cytokines were virtually undetectable in severe AHB (mean ALT peak, 1,335 IU/liter), characterized by significant elevations of IL-10, tumor necrosis factor alpha (TNF-α), and MIP-1ß. In progressive AHB (mean ALT peak, 166 IU/liter), there was a delayed and lower-magnitude induction of cytokines. The ALT peak was also delayed (mean, 23.5 weeks) compared to those of classic (13.5 weeks) and severe AHB (7.5 weeks). HBV/HDV coinfection correlated with significantly lower levels of IFN-α2, IFN-γ, and IL-17A, associated with the presence of multiple proinflammatory cytokines, including IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-15. Conversely, HDV superinfection induced the highest ALT peak (1,910 IU/liter) and was associated with a general suppression of cytokines. Our data demonstrate that the most severe liver damage, caused by an HBV precore mutant and HDV, correlated with restricted cytokine expression and lack of Th1 response, raising the question of whether these viruses are directly cytopathic.IMPORTANCE Studies performed in chimpanzees at the National Institutes of Health (NIH) demonstrated a significant difference in ALT levels during acute hepatitis of different viral etiologies, with a hierarchy in the extent of liver damage according to the infecting virus: the highest level was in HDV superinfection, followed by infection with a precore HBV mutant, HBV/HDV coinfection, and, lastly, wild-type HBV infection. Our study demonstrates that both the virus and host are important in disease pathogenesis and offers new insights into their roles. We found that distinct cytokine profiles were associated with disease severity and clinical outcome. In particular, resolution of classic acute hepatitis B (AHB) correlated with a predominant Th1 response, whereas HBV/HDV coinfection showed a predominant proinflammatory response. Severe AHB and HDV superinfection showed a restricted cytokine profile and no evidence of Th1 response. The lack of cytokines associated with adaptive T-cell responses toward the precore HBV mutant and HDV superinfection argues in favor of a direct cytopathic effect of these viruses.

Novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody.

Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC(50)) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC(50) of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.

A single chimpanzee-human neutralizing monoclonal antibody provides post-exposure protection against type 1 and type 2 polioviruses.

BACKGROUND: Development of anti-poliovirus therapies to complement vaccination is an urgent priority. A number of antiviral drugs are in development. Recently we have developed human monoclonal antibodies that could be used for treatment of chronically infected individuals and emergency response to potential reappearance of polioviruses after eradication. OBJECTIVE: The aim of this study was to characterize neutralizing activity of anti-poliovirus monoclonal antibody A12 against wild type, vaccine-derived, and drug-resistant poliovirus strains, evaluate in vivo pre- and post-exposure protective properties of the antibody against polioviruses of serotypes 1 and 2, and to determine whether it interferes with response to immunization with poliovirus vaccine. STUDY DESIGN: Immunogenicity studies were performed in CD1 mice. Poliovirus neutralizing titers were determined in poliovirus microneutralization assay. Poliovirus immunization-challenge experiments were performed in poliovirus-susceptible TgPVR21 mice. RESULTS: We show that monoclonal antibody A12 effectively neutralizes in vitro a broad range of type 1 and type 2 wild and vaccine-derived polioviruses, provides effective pre- and post-exposure protection of TgPVR21 mice from challenge with a lethal dose of poliovirus. Treatment of animals with the antibody concurrent with IPV immunization does not prevent immune response to the vaccine. CONCLUSIONS: Anti-poliovirus antibody A12 effectively neutralizes a range of wild and VDPV strains and protectstransgenic mice susceptible to poliovirus against lethal challenge upon pre- and post-exposure administration. This suggests that the antibodies could be used in combination with drugs and/or vaccine to improve their efficacy and prevent emergence of resistant variants, and provides a justification for initiating their clinical evaluation.