Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes.

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

Prevalence and Significance of Substitutions in the Fusion Protein of Respiratory Syncytial Virus Resulting in Neutralization Escape From Antibody MEDI8897.

Background: Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection among infants and young children. To date, no vaccine is approved for the broad population of healthy infants. MEDI8897, a potent anti-RSV fusion antibody with extended serum half-life, is currently under clinical investigation as a potential passive RSV vaccine for all infants. As a ribonucleic acid virus, RSV is prone to mutation, and the possibility of viral escape from MEDI8897 neutralization is a potential concern. Methods: We generated RSV monoclonal antibody (mAb)-resistant mutants (MARMs) in vitro and studied the effect of the amino acid substitutions identified on binding and viral neutralization susceptibility to MEDI8897. The impact of resistance-associated mutations on in vitro growth kinetics and the prevalence of these mutations in currently circulating strains of RSV in the United States was assessed. Results: Critical residues identified in MARMs for MEDI8897 neutralization were located in the MEDI8897 binding site defined by crystallographic analysis. Substitutions in these residues affected the binding of mAb to virus, without significant impact on viral replication in vitro. The frequency of natural resistance-associated polymorphisms was low. Conclusions: Results from this study provide insights into the mechanism of MEDI8897 escape and the complexity of monitoring for emergence of resistance.

A broadly neutralizing human monoclonal antibody directed against a novel conserved epitope on the influenza virus H3 hemagglutinin globular head.

UNLABELLED: Most neutralizing antibodies elicited during influenza virus infection or vaccination target immunodominant, variable epitopes on the globular head region of hemagglutinin (HA), which leads to narrow strain protection. In this report, we describe the properties of a unique anti-HA monoclonal antibody (MAb), D1-8, that was derived from human B cells and exhibits potent, broad neutralizing activity across antigenically diverse influenza H3 subtype viruses. Based on selection of escape variants, we show that D1-8 targets a novel epitope on the globular head region of the influenza virus HA protein. The HA residues implicated in D1-8 binding are highly conserved among H3N2 viruses and are located proximal to antigenic site D. We demonstrate that the potent in vitro antiviral activity of D1-8 translates into protective activity in mouse models of influenza virus infection. Furthermore, D1-8 exhibits superior therapeutic survival benefit in influenza virus-infected mice compared to the neuraminidase inhibitor oseltamivir when treatment is started late in infection. The present study suggests the potential application of this monoclonal antibody for the therapeutic treatment of H3N2 influenza virus infection. IMPORTANCE: Recently, a few globular head-targeting MAbs have been discovered that exhibit activity against different subtypes of influenza subtypes, such as H1; however, none of the previously described MAbs showed broadly neutralizing activity against diverse H3 viruses. In this report, we describe a human MAb, D1-8, that exhibits potent, broadly neutralizing activity against antigenically diverse H3 subtype viruses. The genotypic analysis of escape mutants revealed a unique putative epitope region in the globular head of H3 HA that is comprised of highly conserved residues and is distinct from the receptor binding site. Furthermore, we demonstrate that D1-8 exhibits superior therapeutic efficacy in influenza virus-infected mice compared to the neuraminidase inhibitor oseltamivir when treatment is started late in infection. In addition to describing a novel anti-globular head of H3 HA MAb with potent broadly neutralizing activity, our report suggests the potential of D1-8 for therapeutic treatment of seasonal influenza virus H3 infection.

Natural polymorphisms and resistance-associated mutations in the fusion protein of respiratory syncytial virus (RSV): effects on RSV susceptibility to palivizumab.

Specific mutations in respiratory syncytial virus (RSV) fusion protein can cause palivizumab resistance. We assessed the incidence of sequence polymorphisms and palivizumab resistance in clinical RSV isolates collected from immunoprophylaxis-naive subjects. Polymorphisms were identified at low frequency, and only polymorphic mutations in antigenic site A (<1% of all polymorphisms) conferred palivizumab resistance.

Analysis of respiratory syncytial virus preclinical and clinical variants resistant to neutralization by monoclonal antibodies palivizumab and/or motavizumab.

BACKGROUND: Palivizumab is a US Food and Drug Administration-approved monoclonal antibody for the prevention of respiratory syncytial virus (RSV) lower respiratory disease in high-risk infants. Motavizumab, derived from palivizumab with enhanced antiviral activity, has recently been tested in humans. Although palivizumab escape mutants have been generated in the laboratory, the development of resistant RSV in patients receiving palivizumab has not been reported previously. METHODS: We generated palivizumab and motavizumab escape mutants in vitro and examined the development of resistant mutants in RSV-breakthrough patients receiving immunoprophylaxis. The effect of these mutations on neutralization by palivizumab and motavizumab and in vitro fitness was studied. RESULTS: Antibody-resistant RSV variants selected in vitro had mutations at position 272 of the fusion protein, from lysine to asparagine, methionine, threonine, glutamine, or glutamate. Variants containing mutations at positions 272 and 275 were detected in breakthrough patients. All these variants were resistant to palivizumab, but only the glutamate variant at position 272 demonstrated resistance to motavizumab. Mixtures of wild-type and variant RSV soon lost the resistant phenotype in the absence of selection. CONCLUSIONS: Resistant RSV variants were detected in a small subset (∼ 5%) of RSV breakthrough cases. The fitness of these variants was impaired, compared to wild-type RSV.

Attenuate Newcastle disease virus by codon modification of the glycoproteins and phosphoprotein genes.

A codon modification strategy was used to attenuate the avian pathogenicity of an oncolytic mesogenic Newcastle disease virus (NDV) by targeting the three major virulence factors: the fusion (F) protein, hemagglutinin neuraminidase (HN) and phosphoprotein (P). Recoding the F and HN genes with rare codons greatly reduced expression of both F and HN proteins and resulted in their low incorporation into virions. The F and HN recoded virus was partially attenuated in chickens even when the F protein cleavage site was modified. Full attenuation was achieved when the 5' portion of the P gene was recoded. The recoded P, F and HN triple gene mutant exhibited delayed cell death in human cancer cells with prolonged expression of a GFP transgene. While this engineered attenuated NDV strain has lower oncolytic potency, its capacity for prolonged transgene expression may allow its use as a vaccine or gene delivery vector.

HBeAg seroconversion is associated with a more effective PD-L1 blockade during chronic hepatitis B infection.

BACKGROUND & AIMS: Current therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade. METHODS: Expression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790). RESULTS: Chronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients' clinical or treatment status. CONCLUSION: Patients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy. LAY SUMMARY: Hepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection.

A highly potent extended half-life antibody as a potential RSV vaccine surrogate for all infants.

Prevention of respiratory syncytial virus (RSV) illness in all infants is a major public health priority. However, no vaccine is currently available to protect this vulnerable population. Palivizumab, the only approved agent for RSV prophylaxis, is limited to high-risk infants, and the cost associated with the requirement for dosing throughout the RSV season makes its use impractical for all infants. We describe the development of a monoclonal antibody as potential RSV prophylaxis for all infants with a single intramuscular dose. MEDI8897*, a highly potent human antibody, was optimized from antibody D25, which targets the prefusion conformation of the RSV fusion (F) protein. Crystallographic analysis of Fab in complex with RSV F from subtypes A and B reveals that MEDI8897* binds a highly conserved epitope. MEDI8897* neutralizes a diverse panel of RSV A and B strains with >50-fold higher activity than palivizumab. At similar serum concentrations, prophylactic administration of MEDI8897* was ninefold more potent than palivizumab at reducing pulmonary viral loads by >3 logs in cotton rats infected with either RSV A or B subtypes. MEDI8897 was generated by the introduction of triple amino acid substitutions (YTE) into the Fc domain of MEDI8897*, which led to more than threefold increased half-life in cynomolgus monkeys compared to non-YTE antibody. Considering the pharmacokinetics of palivizumab in infants, which necessitates five monthly doses for protection during an RSV season, the high potency and extended half-life of MEDI8897 support its development as a cost-effective option to protect all infants from RSV disease with once-per-RSV-season dosing in the clinic.

Analysis of human and primate CD2 molecules by protein sequence and epitope mapping with anti-human CD2 antibodies.

A panel of anti-human CD2 monoclonal antibodies (mAb) and soluble human CD58 (LFA-3) were tested for binding to human peripheral blood mononuclear cells (PBMCs), recombinant human CD2 and mononuclear cells from Cynomolgus, Rhesus and African green monkey, Stump-tail, Pig-tail and Assamese macaque, Chimpanzee and Baboon. This analysis revealed that whilst some antibodies recognized all species, there were differential binding profiles with others. Three antibodies, MEDI-507, 6F10.3 and 4B2, recognized CD2 from human and Chimpanzee but not that from the other primates. We have cloned eight of the previously unknown primate CD2 molecules and report here their sequences for the first time. This analysis revealed that 12 amino acids formed a common set of residues in the extra cellular domain of human and Chimpanzee CD2. Using a "knock-in" mutagenesis approach starting with Baboon CD2, which does not bind MEDI-507, 6F10.3 and 4B2, we have identified three residues in the adhesion domain of human CD2 which are critical for its binding to these mAbs. These residues, N18, K55 and T59 define a region located outside of the previously described binding regions on CD2. Affinity measurements of the mutants revealed a variety of degrees of binding restoration for MEDI-507, 6F10.3 and 4B2, indicating that there are fine differences within a given epitope. Furthermore, the analysis of the competition of several of the anti-human CD2 antibodies with each other and CD58 demonstrated the existence of a continuum of overlapping epitopes on human CD2, which is in contrast to the commonly held belief that epitopes on human CD2 are clearly segregated.

A multifunctional bispecific antibody protects against Pseudomonas aeruginosa.

Widespread drug resistance due to empiric use of broad-spectrum antibiotics has stimulated development of bacteria-specific strategies for prophylaxis and therapy based on modern monoclonal antibody (mAb) technologies. However, single-mechanism mAb approaches have not provided adequate protective activity in the clinic. We constructed multifunctional bispecific antibodies, each conferring three mechanisms of action against the bacterial pathogen Pseudomonas aeruginosa by targeting the serotype-independent type III secretion system (injectisome) virulence factor PcrV and persistence factor Psl exopolysaccharide. A new bispecific antibody platform, BiS4, exhibited superior synergistic protection against P. aeruginosa-induced murine pneumonia compared to parent mAb combinations or other available bispecific antibody structures. BiS4αPa was protective in several mouse infection models against disparate P. aeruginosa strains and unexpectedly further synergized with multiple antibiotic classes even against drug-resistant clinical isolates. In addition to resulting in a multimechanistic clinical candidate (MEDI3902) for the prevention or treatment of P. aeruginosa infections, these antibody studies suggest that multifunctional antibody approaches may be a promising platform for targeting other antibiotic-resistant bacterial pathogens.

Potential for palivizumab interference with commercially available antibody-antigen based respiratory syncytial virus diagnostic assays.

Palivizumab is a monoclonal antibody indicated for the prevention of serious lower respiratory tract disease caused by respiratory syncytial virus infection in infants. The potential for palivizumab to interfere with commercially available respiratory syncytial virus diagnostic tests was demonstrated. Negative test results in palivizumab-treated subjects should be interpreted with caution and confirmed by a nucleic acid amplification-based assay.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening.

Pseudomonas aeruginosa is a leading cause of hospital-associated infections in the seriously ill, and the primary agent of chronic lung infections in cystic fibrosis patients. A major obstacle to effective control of P. aeruginosa infections is its intrinsic resistance to most antibiotic classes, which results from chromosomally encoded drug-efflux systems and multiple acquired resistance mechanisms selected by years of aggressive antibiotic therapy. These factors demand new strategies and drugs to prevent and treat P. aeruginosa infections. Herein, we describe a monoclonal antibody (mAb) selection strategy on whole P. aeruginosa cells using single-chain variable fragment phage libraries derived from healthy individuals and patients convalescing from P. aeruginosa infections. This approach enabled identification of mAbs that bind three distinct epitopes on the product of the Psl. This exopolysaccharide is important for P. aeruginosa attachment to mammalian cells, and for the formation and maintenance of biofilms produced by nonmucoid and mucoid P. aeruginosa isolates. Functional screens revealed that mAbs to one epitope exhibit superior activity in opsonophagocytic killing and cell attachment assays, and confer significant protection in multiple animal models. Our results indicate that Psl is an accessible serotype-independent surface feature and promising novel protective antigen for preventing P. aeruginosa infections. Furthermore, our mAb discovery strategy holds promise for application to other bacterial pathogens.

Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo.

Human metapneumovirus (hMPV) is a recently described member of the Paramyxoviridae family/Pneumovirinae subfamily and shares many common features with respiratory syncytial virus (RSV), another member of the same subfamily. hMPV causes respiratory tract illnesses that, similar to human RSV, occur predominantly during the winter months and have symptoms that range from mild to severe cough, bronchiolitis, and pneumonia. Like RSV, the hMPV virus can be subdivided into two genetic subgroups, A and B. With RSV, a single monoclonal antibody directed at the fusion (F) protein can prevent severe lower respiratory tract RSV infection. Because of the high level of sequence conservation of the F protein across all the hMPV subgroups, this protein is likely to be the preferred antigenic target for the generation of cross-subgroup neutralizing antibodies. Here we describe the generation of a panel of neutralizing monoclonal antibodies that bind to the hMPV F protein. A subset of these antibodies has the ability to neutralize prototypic strains of both the A and B hMPV subgroups in vitro. Two of these antibodies exhibited high-affinity binding to the F protein and were shown to protect hamsters against infection with hMPV. The data suggest that a monoclonal antibody could be used prophylactically to prevent lower respiratory tract disease caused by hMPV.

Surveillance of clinical isolates of respiratory syncytial virus for palivizumab (Synagis)-resistant mutants.

Premature infants and those with chronic lung disease or congenital heart disease are at high risk of severe respiratory syncytial virus (RSV) disease. Palivizumab (Synagis), a humanized anti-RSV monoclonal antibody, has been used extensively since 1998 to prevent severe RSV disease in high-risk infants. To monitor for possible palivizumab-resistant mutants, an immunofluorescence binding assay that predicts palivizumab neutralization of RSV was developed. RSV isolates were collected at 8 US sites from 458 infants hospitalized for RSV disease (1998-2002). Palivizumab bound to all 371 RSV isolates able to be evaluated, including 25 from active-palivizumab recipients. The palivizumab epitope appears to be highly conserved, even in infants receiving prophylaxis with palivizumab.