W. Graeme Laver

Epitopes on protein antigens: misconceptions and realities.

Afin de determiner les structures des epitopes a la surface de proteines, des complexes entre anticorps monoclonal Fab et antigene sont formes et analyses par cristallisation et diffraction des rayons-X. De tels complexes Fab-lysozyme et Fab neuraminidase ont ete etudies dans cet article

The structure of a complex between the NC10 antibody and influenza virus neuraminidase and comparison with the overlapping binding site of the NC41 antibody

BACKGROUND While it is well known that different antibodies can be produced against a particular antigen, and even against a particular site on an antigen, up until now there have been no structural studies of cross-reacting antibodies of this type. One antibody-antigen complex whose structure is known is that of the influenza virus antigen, neuraminidase, in complex with the NC41 antibody. Another anti-neuraminidase antibody, NC10, binds to an overlapping site on the antigen. The structure of the complex formed by this antibody with neuraminidase is described here and compared with the NC41-containing complex. RESULTS The crystal structure of the NC10 Fab-neuraminidase complex has been refined to a nominal resolution of 2.5A. Approximately 80% of the binding site of the NC10 antibody on neuraminidase overlaps with that of the NC41 antibody. The epitope residues of neuraminidase are often engaged in quite different interactions with the two antibodies. Although the NC10 and NC41 antibodies have identical amino acid sequences within the first complementarity determining region of their heavy chains, this is not the basis of the cross-reaction. CONCLUSIONS The capacity of two different proteins to bind to the same target structure on a third protein need not be based on the existence of identical or homologous amino acid sequences within those proteins. As we have demonstrated, amino acid residues on the common target structure may be in quite different chemical environments, and may also adopt different conformations within two protein-protein complexes.

Binding of antibodies to isolated haemagglutinin and neuraminidase molecules of influenza virus observed in the electron microscope.

Highly purified preparations of 7 S immunoglobulin G antibody molecules against the “common” and “specific” antigenic determinants of the haemagglutinin and neuraminidase antigens of influenza virus were prepared. The binding of these antibodies to the isolated antigens has been studied in the electron microscope. We have shown that there are two (and probably no more) of each kind of determinant on each haemagglutinin “spike”, whether these were isolated intact by treatment with detergent or in a partially degraded state following proteolytic digestion of the virus particles. Both common and specific determinants appear to be located on the side of the haemagglutinin spike just below its tip. None appeared to be located anywhere else on the molecule. In the case of the neuraminidase, which is known to consist of four identical subunits, antibodies to the common and specific determinants bound near the four corners of the box-shaped head of the enzyme. Often two antibody molecules (either common or specific) were found bound to a single neuraminidase head. Sometimes three were seen and very rarely four, suggesting that there are four common and four specific antigenic determinants on each neuraminidase molecule. No conclusion could be drawn from these observations regarding the symmetry of the head.

A new series of C3-aza carbocyclic influenza neuraminidase inhibitors: synthesis and inhibitory activity☆

The synthesis and influenza neuraminidase inhibitory activity of a new series of C3-aza carbocyclic neuraminidase inhibitors are described. Analogues 3c and 3j, bearing a 3-pentyl group, exhibit influenza A inhibitory activities comparable to that of 1.

Structure-activity relationships of carbocyclic influenza neuraminidase inhibitors

Abstract The structure-activity relationships (SAR) for a new class of potent inhibitors (1) of influenza neuraminidase are described. Systematic modifications of substituents at the C-3, C-4, and C-5 positions of the carbocyclic ring were performed to establish fundamental SAR to assist in the design of potent inhibitors with activity against both of influenza A and B viruses.

Detection and isolation of H5N1 influenza virus from large volumes of natural water

Various species of aquatic or wetlands birds can be the natural reservoir of avian influenza A viruses of all hemagglutinin (HA) subtypes. Shedding of the virus into water leads to transmission between waterfowl and is a major threat for epidemics in poultry and pandemics in humans. Concentrations of the influenza virus in natural water reservoirs are often too low to be detected by most methods. The procedure was designed to detect and isolate low concentrations of the influenza virus in large volumes of water without the need for costly installations and reagents. The virus was adsorbed onto formalin-fixed erythrocytes and subsequently isolated in chicken embryos. Sensitivity of the method was determined using a reverse-genetic H5N1 virus. A concentration as low as 0.03 of the 50% egg infection dose per milliliter (EID50/ml) of the initial volume of water was effectively detected. The probability of detection was approximately 13%, which is comparable to that of detecting the influenza virus M-gene by PCR amplification. The method can be used by field workers, ecologists, ornithologists, and researchers who need a simple method to isolate H5N1 influenza virus from natural reservoirs. The detection and isolation of virus in embryonated chicken eggs may help epidemiologic, genetic, and vaccine studies.

The molecular basis of antigenic variation in influenza virus.

Publisher Summary This chapter summarizes recent information on the structure of the hemagglutinin (HA) and neuraminidase (NA), the way in which these glycoproteins vary, and the effects of the changes on the antigenic properties of the virus. Sequences and structures of the two surface glycoprotein antigens of influenza virus provide important information and insight into the mechanism of antigenic variation of influenza virus. Many epitopes exist over most if not all of the accessible surfaces of the HA and NA. These epitopes are critically dependent on the three-dimensional structure of the protein; no effective neutralizing antibodies have been raised when fragments of the protein have been used as immunogens, and neutralizing monoclonal antibodies made against active protein fail to recognize protein that has been denatured by procedures such as drying or methanol fixing on an ELISA plate. A single amino acid change is sufficient to completely destroy binding of a monoclonal antibody to the HA or NA. In natural drift, more than one change is usually found between antigenically distinct isolates, but not as many as there are antigenic sites. It has been demonstrated that attached carbohydrate can mask antigenic determinants, but other factors, as yet undefined, also affect induction of antibodies.

Novel α- and β-Amino Acid Inhibitors of Influenza Virus Neuraminidase

ABSTRACT In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 α- and β-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited Ki values in the 50 μM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the α-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the β-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the α- or β-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.

The Origin and Control of Pandemic Influenza

In 1918 an epidemic of influenza killed 20 million people worldwide. Spanish flu, as it was called, was a horrific disease. The flu would start with headaches, muscular pain, and fever. These would be rapidly followed by vomiting, dizziness, labored breathing, and profuse sweating. Sometimes purple blisters would appear on the skin, and often blood would spurt out of the nose from hemorrhages in the lungs. Some of the victims of this dreadful, sudden, and unexpected illness went into violent fits of coughing. Death often followed, sometimes only hours after the first symptoms appeared. Influenza viruses infect a number of different animals, and some of these viruses can cause very serious disease indeed, particularly in domesticated chickens and turkeys. Avian influenza viruses sometimes rapidly kill these birds, with 100 percent mortality, and the symptoms resemble, at least to some extent, those of the Spanish flu in 1918. You can imagine, therefore, the concern felt when, in late 1997, a virulent bird flu virus, which had never before been seen in man, started infecting and killing people in Hong Kong [1]. This virus, designated H5N1, killed six of the 18 people it infected. The virus seems to have been transmitted to people from infected chickens in the live bird markets, but so far there has been no evidence that the virus had learned how to spread from person to person. But there is also no reason to suppose that this might not happen, some time in the future. Killing off all the chickens in Hong Kong seems to have stopped the epidemic, at least for the time being. What could be done to control such a virulent influenza virus, which, if it took off, would spread through

Sialic acid is cleaved from glycoconjugates at the cell surface when influenza virus neuraminidases are expressed from recombinant vaccinia viruses.

Three different influenza virus neuraminidase (NA) genes have been subcloned into the vector pSC11 and expressed from the recombinant vaccinia viruses. These genes are from influenza viruses A/Tokyo/3/67 (N2); A/tern/Australia/G70c/75 (N9); and B/Hong Kong (HG)(NA of B/Lee/40). Cells infected with recombinants containing the NA gene express enzymatically active NA on the cell surface. The expressed protein results in the infected cells beings stripped of sialic acid, the receptor for influenza virus. This is not due to cleavage by NA from detached cells since at low multiplicity of infection only cells present at plaques are devoid of sialic acid. Thus NA is able to cleave sialic acid from neighboring glycoconjugates on the same membrane.