Damian C. Ekiert

Antibody Recognition of a Highly Conserved Influenza Virus Epitope

Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

A highly conserved neutralizing epitope on group 2 influenza A viruses.

An antibody against a conserved epitope broadly neutralizes group 2 influenza viruses. Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of VH1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the VH1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies.

Structural Basis of Preexisting Immunity to the 2009 H1N1 Pandemic Influenza Virus

Swine Flu Neutralized The 2009 H1N1 flu virus had an unusually low infection rate in elderly people. An antibody isolated from survivors of the 1918 flu pandemic was recently shown to cross-neutralize 2009 H1N1 viruses. Xu et al. (p. 357, published online 25 March) report crystal structures of the virus envelope protein, hemagglutinin (HA) from 2009 H1N1 and of 1918 H1 HA in complex with a neutralizing antibody that cross-reacts with both pandemic viruses. These studies reveal an epitope that is conserved in the pandemic viruses, but divergent in other known H1 HAs, from the 1930s to the present. This antigenic similarity explains the age-related immunity to the 2009 H1N1 influenza. An epitope conserved between the 1918 and 2009 pandemic flu viruses explains age-related immunity to the 2009 virus. The 2009 H1N1 swine flu is the first influenza pandemic in decades. The crystal structure of the hemagglutinin from the A/California/04/2009 H1N1 virus shows that its antigenic structure, particularly within the Sa antigenic site, is extremely similar to those of human H1N1 viruses circulating early in the 20th century. The cocrystal structure of the 1918 hemagglutinin with 2D1, an antibody from a survivor of the 1918 Spanish flu that neutralizes both 1918 and 2009 H1N1 viruses, reveals an epitope that is conserved in both pandemic viruses. Thus, antigenic similarity between the 2009 and 1918-like viruses provides an explanation for the age-related immunity to the current influenza pandemic.

Computational design of proteins targeting the conserved stem region of influenza hemagglutinin.

Proteins can be designed that bind to specific patches on target proteins to alter their subsequent interactions. We describe a general computational method for designing proteins that bind a surface patch of interest on a target macromolecule. Favorable interactions between disembodied amino acid residues and the target surface are identified and used to anchor de novo designed interfaces. The method was used to design proteins that bind a conserved surface patch on the stem of the influenza hemagglutinin (HA) from the 1918 H1N1 pandemic virus. After affinity maturation, two of the designed proteins, HB36 and HB80, bind H1 and H5 HAs with low nanomolar affinity. Further, HB80 inhibits the HA fusogenic conformational changes induced at low pH. The crystal structure of HB36 in complex with 1918/H1 HA revealed that the actual binding interface is nearly identical to that in the computational design model. Such designed binding proteins may be useful for both diagnostics and therapeutics.

Highly conserved protective epitopes on influenza B viruses.

Influenza Antibodies, Part B With its ability to reassort in animal hosts like pigs and birds, and to cause pandemics, influenza A viruses are often in the spotlight. However, a substantial portion of the annual flu burden is also the result of influenza B virus, which is a single influenza type that is characterized by two antigenically and genetically distinct lineages. Dreyfus et al. (p. 1343, published online 9 August) identify three monoclonal human antibodies that are able to protect against lethal infection with both lineages of influenza B virus in mice. Two antibodies, which bind to distinct regions of the viral hemagluttinin (HA) molecule, neutralize multiple strains from both lineages of influenza B virus, whereas the third antibody binds to the stem region of HA and is able to neutralize both influenza A and B strains. The structural data from these antibodies bound to HA, together with already known antibodies targeting influenza A, may provide clues for designing a universal vaccine to protect against both influenza virus types. Three broadly neutralizing human monoclonal antibodies protect mice against influenza B. Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody–based immunotherapy and “universal” vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody–based treatments and a universal flu vaccine for all influenza A and B viruses.

A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis.

An Escherichia coli dihydrofolate reductase mutant is catalytically defective, because motions in the active site are impaired. Conformational dynamics play a key role in enzyme catalysis. Although protein motions have clear implications for ligand flux, a role for dynamics in the chemical step of enzyme catalysis has not been clearly established. We generated a mutant of Escherichia coli dihydrofolate reductase that abrogates millisecond-time-scale fluctuations in the enzyme active site without perturbing its structural and electrostatic preorganization. This dynamic knockout severely impairs hydride transfer. Thus, we have found a link between conformational fluctuations on the millisecond time scale and the chemical step of an enzymatic reaction, with broad implications for our understanding of enzyme mechanisms and for design of novel protein catalysts.

Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes

Current influenza virus vaccines protect mostly against homologous virus strains; thus, regular immunization with updated vaccine formulations is necessary to guard against the virus hallmark remodeling of regions that mediate neutralization. Development of a broadly protective influenza vaccine would mark a significant advance in human infectious diseases research. Antibodies with broad neutralizing activity (nAbs) against multiple influenza virus strains or subtypes have been reported to bind the stalk of the viral hemagglutinin, suggesting that a vaccine based on this region could elicit a broadly protective immune response. Here we describe a hemagglutinin subunit 2 protein (HA2)-based synthetic peptide vaccine that provides protection in mice against influenza viruses of the structurally divergent subtypes H3N2, H1N1, and H5N1. The immunogen is based on the binding site of the recently described nAb 12D1, which neutralizes H3 subtype viruses, demonstrates protective activity in vivo, and, in contrast to a majority of described nAbs, appears to bind to residues within a single α-helical portion of the HA2 protein. Our data further demonstrate that the specific design of our immunogen is integral in the induction of broadly active anti-hemagglutinin antibodies. These results provide proof of concept for an HA2-based influenza vaccine that could diminish the threat of pandemic influenza disease and generally reduce the significance of influenza viruses as human pathogens.

Cross-neutralization of influenza A viruses mediated by a single antibody loop

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Broadly neutralizing antibodies against influenza virus and prospects for universal therapies

Vaccines are the gold standard for the control and prevention of infectious diseases, but a number of important human diseases remain challenging targets for vaccine development. An influenza vaccine that confers broad spectrum, long-term protection remains elusive. Several broadly neutralizing antibodies have been identified that protect against multiple subtypes of influenza A viruses, and crystal structures of several neutralizing antibodies in complex with the major influenza surface antigen, hemagglutinin, have revealed at least 3 highly conserved epitopes. Our understanding of the molecular details of these antibody-antigen interactions has suggested new strategies for the rational design of improved influenza vaccines, and has inspired the development of new antivirals for the treatment of influenza infections.

A common solution to group 2 influenza virus neutralization

Significance The HA surface glycoprotein on influenza A viruses mediates viral entry into host cells. HA is highly variable and classified into 18 divergent subtypes, which cluster into two major phylogenetic groups. Antibody CR8043 has heterosubtypic neutralizing activity against group 2 viruses, including H3 viruses that currently circulate in humans. X-ray and EM structures of CR8043 Fab in complex with H3 HAs reveal that the antibody targets a conserved epitope on the HA stem. Compared with CR8020, the only other structurally characterized group 2 neutralizing antibody, CR8043 binds to HA with a different approach angle using different contact residues. The epitopes of both antibodies are very similar, which suggests that this conserved stem epitope has great potential for design of therapeutics and vaccines. The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.