Influenza vaccination can induce new-onset anticardiolipins but not ß2-glycoprotein-I antibodies among patients with systemic lupus erythematosus.

BACKGROUND: Antiphospholipid syndrome is characterized by autoantibodies against cardiolipins (aCL), lupus anticoagulant, and independent ß2-glycoprotein (ß2GPI). Controversy exists as to whether vaccination triggers the development of antiphospholipid antibodies (aPL) in patients with systemic lupus erythematosus (SLE). METHODS: Patients with SLE (101) and matched controls (101) were enrolled from 2005-2009 and received seasonal influenza vaccinations. Sera were tested by ELISA for aCL at baseline, 2, 6, and 12 weeks after vaccination. Vaccine responses were ranked according to an overall anti-influenza antibody response index. Individuals with positive aCL were further tested for ß2GPI antibodies. RESULTS: Patients with SLE and healthy controls can develop new-onset aCL post vaccination, although at rates which do not differ between patients and controls (12/101 cases and 7/101 controls, OR 1.81, p = 0.34). New-onset moderate aCL are slightly enriched in African American SLE patients (5/36 cases; p = 0.094). The optical density measurements for aCL reactivity in patients were significantly higher than baseline at 2 weeks (p < 0.05), 6 weeks (p < 0.05), and 12 weeks (p < 0.05) post vaccination. No new ß2GPI antibodies were detected among patients with new aCL reactivity. Vaccine response was not different between patients with and without new-onset aCL reactivity (p = 0.43). CONCLUSIONS: This study shows transient increases in aCL, but not anti-ß2GPI responses, after influenza vaccination.

Apoptosis by influenza viruses correlates with efficiency of viral mRNA synthesis.

A mutant influenza virus, A/NWS-Mvi, grows well in the presence of exogenous sialidase activity sufficient to remove all cell surface sialic acids. Related wild-type viruses grow very poorly under these conditions, although mutant and wild-type viruses bind to desialylated cells with similar efficiency and show similar reduction of binding to sialidase-treated cells compared to native cells. Here we examine entry, transcription, translation, and RNA replication and find that, although the viruses appear to utilize the same entry pathway, the mutant NWS-Mvi transcribes and replicates RNA to higher levels than the wild-type strains. The kinetics of replication in multi-cycle infection show that this enhancement of RNA synthesis facilitates growth where entry is restricted. The hemagglutinin (HA) protein of NWS-Mvi lyses red blood cells 0.1 pH unit higher than wild-type viruses. This higher fusion pH may allow more efficient release of nucleocapsids from endosomes and contribute to the enhanced RNA synthesis. The efficient RNA synthesis assists virus survival at low inocula or under stringent growth conditions, such as the presence of antiviral agents. NWS-Mvi induces apoptosis in infected cells more readily than wild-type viruses, apparently as a consequence of enhanced production of viral mRNA. Since growth of NWS-Mvi is more efficient, apoptosis may play a positive role in viral replication by removing cells that have already been infected from those capable of making more virus.

Influenza virus infection of desialylated cells.

Sialic acid has long been considered to be the sole receptor for influenza virus. The viral hemagglutinin (HA) is known to bind cell surface sialic acid, and sialic acids on viral glyco-proteins are cleaved by the viral neuraminidase (NA) to promote efficient release of progeny virus particles. However, NWS-Mvi, a mutant virus completely lacking NA, grows well in MDCK cells continuously treated with exogenous neuraminidase (sialidase). Exogenous sialidase quantitatively releases all sialic acids from purified glycoproteins and glycolipids of MDCK cells and efficiently removes surface sialic acid from intact cells. Binding of NWS-Mvi and parent influenza viruses to MDCK cells is indistinguishable, and is only partially reduced by sialidase treatment of the cells. Both mutant and wild-type viruses enter enzymatically desialylated cells and initiate transcription. The ability of influenza A reassortant viruses to infect desialylated cells is shared by recent H3N2 clinical isolates, suggesting that this may be a general property of influenza A viruses. We propose that influenza virus infection can result from sialic acid-independent receptors, either directly or in a multistage process. When sialic acid is present, it may act to enhance virus binding to the cell surface to increase interaction with secondary receptors to mediate entry. Understanding virus entry will be critical to further efforts in infection control and prevention.

Influenza type B neuraminidase can replace the function of type A neuraminidase.

Influenza A and B viruses do not form reassortants with each other, presumably due to selection at either the RNA or protein level. Although differences in the promoter sequences of type A and B viruses have been studied, selection at the protein level has not been addressed. In this paper we describe experiments to determine whether differences in structure and/or function of the neuraminidase (NA) protein preclude formation of A/B NA reassortants. Influenza type A (N9) NA or B/Lee/40 NA expressed from plasmids can support multicycle growth of a NA-deficient type A virus (NWS-Mvi), indicating that their function in tissue culture is similar. To determine whether the type A or B NA supplied in trans can be incorporated into the virion of NWS-Mvi, the virus grown in NA-expressing cells was purified by sucrose gradient centrifugation. In each case there was a peak of NA activity coincident with the virus peak, indicating that some NA protein is packaged into the virion. The experiments suggest that, in spite of large sequence differences, the functions of the head, stalk, signal-anchor, and cytoplasmic domains of type A and B NAs are similar in tissue culture. Thus, lack of formation of A/B NA reassortant viruses is not due to restriction at the protein level.

Novel aromatic inhibitors of influenza virus neuraminidase make selective interactions with conserved residues and water molecules in the active site.

The active site of type A or B influenza virus neuraminidase is composed of 11 conserved residues that directly interact with the substrate, sialic acid. An aromatic benzene ring has been used to replace the pyranose of sialic acid in our design of novel neuraminidase inhibitors. A bis(hydroxymethyl)pyrrolidinone ring was constructed in place of the N-acetyl group on the sialic acid. The hydroxymethyl groups replace two active site water molecules, which resulted in the high affinity of the nanomolar inhibitors. However, these inhibitors have greater potency for type A influenza virus than for type B influenza virus. To resolve the differences, we determined the X-ray crystal structure of three benzoic acid substituted inhibitors bound to the active site of B/Lee/40 neuraminidase. The investigation of a hydrophobic aliphatic group and a hydrophilic guanidino group on the aromatic inhibitors shows changes in the interaction with the active site residue Glu275. The results provide an explanation for the difference in efficacy of these inhibitors against types A and B viruses, even though the 11 active site residues of the neuraminidase are conserved.

Design of benzoic acid inhibitors of influenza neuraminidase containing a cyclic substitution for the N-acetyl grouping.

A 2-pyrrolidinone ring containing a single hydroxymethyl side chain effectively replaces the N-acetylamino group of 4-(N-acetylamino)-3-guanidinobenzoic acid, a low micromolar inhibitor of influenza neuraminidase. This novel structural template affords new opportunities to evolve more potent benzoic acid inhibitors.

Potent inhibition of influenza sialidase by a benzoic acid containing a 2-pyrrolidinone substituent.

On the basis of the lead compound 4-(N-acetylamino)-3-guanidinobenzoic acid (BANA 113), which inhibits influenza A sialidase with a Ki of 2.5 microM, several novel aromatic inhibitors of influenza sialidases were designed. In this study the N-acetyl group of BANA 113 was replaced with a 2-pyrrolidinone ring, which was designed in part to offer opportunities for introduction of spatially directed side chains that could potentially interact with the 4-, 5-, and/or 6-subsites of sialidase. While the parent structure 1-(4-carboxy-2-guanidinophenyl)pyrrolidin-2-one (8) was only a modest inhibitor of sialidase, the introduction of a hydroxymethyl or bis(hydroxymethyl) substituent at the C5' position of the 2-pyrrolidinone ring resulted in inhibitors (9 and 12, respectively) with low micromolar activity. Crystal structures of these inhibitors in complex with sialidase demonstrated that the substituents at the 5'-position of the 2-pyrrolidinone ring interact in the 4- and/or 5-subsites of the enzyme. Replacement of the guanidine in 12 with a hydrophobic 3-pentylamino group resulted in a large enhancement in binding to produce an inhibitor (14) with an IC50 of about 50 nM against influenza A sialidase, although the inhibition of influenza B sialidase was 2000-fold less. This represents the first reported example of a simple, achiral benzoic acid with potent (low nanomolar) activity as an inhibitor of influenza sialidase.

Hydrophobic benzoic acids as inhibitors of influenza neuraminidase.

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A new benzoic acid inhibitor (11) containing a lipophilic side chain at C-3 and a guanidine at C-5 was synthesized. The X-ray structure of 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid in complex with NA revealed that the lipophilic side chain binds in a newly created hydrophobic pocket formed by the movement of Glu 278 to interact with Arg 226, whereas the guanidine of 11 interacts in a negatively charged pocket created by Asp 152, Glu 120 and Glu 229. Compound 11 was highly selective for type A (H2N2) influenza NA (IC50 1 microM) over type B (B/Lee/40) influenza NA (IC50 500 microM).

Critical interactions in binding antibody NC41 to influenza N9 neuraminidase: amino acid contacts on the antibody heavy chain.

Antibody NC41 binds to the subtype N9 neuraminidase (NA) of influenza virus A/tern/Australia/ G70c/75 and inhibits its enzyme activity. To address the molecular mechanisms by which antibodies interact with neuraminidase and the requirements for successful escape from antibody inhibition, we made amino acid substitutions in heavy chain CDRs of NC41. Antibody proteins expressed as a single-chain Fv (scFv) fused with maltose-binding protein were assayed for binding to NA by ELISA. Association constants (Ka) for wild-type and mutant scFvs are as follows: wild type, 2 x 10(7) M-1; Asn31-->Gln, 2 x 10(7) M-1; Glu96-->Asp, 1 x 10(7) M-1; Asp97-->Lys, 6 x 10(6) M-1; and Asn98-->Gln, 8 x 10(6) M-1. The Ka for intact NC41 antibody was 4 x 10(8) M-1 in the same assay, reflecting increased stability compared to that of the scFv. Mutations in the scFv antibody had less of an effect on binding than mutations in their partners on the NA, and modeling studies suggest that interactions involving the mutant antibody side chains occur, even without taking increased flexibility into account. Asp97 forms a salt link with NA critical contact Lys434; of the four mutants, D97K shows the largest reduction in binding to NA. Mutant N98Q also shows reduced binding, most likely through the loss of interaction with NA residue Thr401. Substitution N31Q had no effect on Ka. NC41 residue Glu96 interacts with NA critical contact Ser368, yet E96D showed only a 2-fold reduction in binding to NA, apparently because the H bond can still form. Asp97 and Asn98 provide the most important interactions, but some binding is maintained when they are mutated, in contrast to their partners on the NA. The results are consistent with maturation of the immune response, when the protein epitope is fixed while variation in the antibody paratope allows increasing affinity. Influenza viruses may exploit this general mechanism since single amino acid changes in the epitope allow the virus to escape from the antibody.

Generation and characterization of a mutant of influenza A virus selected with the neuraminidase inhibitor BCX-140.

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

Site-directed mutagenesis of catalytic residues of influenza virus neuraminidase as an aid to drug design.

The neuraminidase of influenza virus is a surface glycoprotein that catalyzes the hydrolysis of glycosidic linkages between terminal sialic acids and adjacent sugar moieties. Neuraminidase function is critical for the spread of virus to new cells, and if the enzyme activity is inhibited, then virus infection is abrogated. The neuraminidase active site is conserved in all influenza type-A and type-B isolates, which makes it an excellent target for drug design. To determine the potential for resistance to develop against neuraminidase inhibitors, we have constructed mutations in seven of the conserved active-site residues of a type B (B/Lee/40) neuraminidase and analyzed the effect of the altered side chains on enzyme activity. There is a reduction in k(cat) in all our mutants. A transition-state analogue inhibitor shows variation in Ki with the mutant neuraminidases, allowing interpretation of the effects of mutation in terms of transition-state binding and product release. The results show that Tyr409 is the most critical residue for enzyme activity, but that Asp149, Arg223, Glu275 and Arg374 also play important roles in enzyme catalysis. Based on the pH profile of neuraminidase activity of the D149E mutant protein, we conclude that Asp149 is not a proton donor, but is involved in stabilizing the transition state. If designed inhibitors are targeted to these residues where mutations are highly deleterious, particularly Tyr409, Glu275 and Asp149, the virus is unlikely to generate resistance to the drug.

Design of aromatic inhibitors of influenza virus neuraminidase.

Structure-based drug design, a terminology used to describe rational drug design by complementing the structure, spatially and chemically, of the target macromolecule, is rapidly developing as one of the innovative approaches to drug discovery. A growing volume of protein structure data and new techniques of protein structure determination make this all possible. The method of structure-based drug design and a specific example of the design of influenza virus neuraminidase is briefly presented. A whole new class of influenza virus neuraminidase inhibitors has been designed that can potentially be developed as antiinfluenza drugs.

Selection of influenza A and B viruses for resistance to 4-guanidino-Neu5Ac2en in cell culture.

The reassortant influenza viruses, A/NWS-G70c with N9 neuraminidase (NA) and B/HK/8/73 (HG) with B/Lee/40 NA, were selected for resistance to 4-guanidino-Neu5Ac2en (4-GuDANA) by passaging the virus in stepwise increases in the concentration of 4-GuDANA. In the NA of resistant viruses, the absolutely conserved Glu 119, which lies in a pocket beneath the active site of the enzyme and interacts with the guanidinium moiety of 4-GuDANA, was changed to Gly. The mutant NA was >200-fold more resistant to 4-GuDANA than was the wild-type enzyme. During 72 h in cell culture, resistant A and B viruses displayed much less NA activity than did wild-type viruses but did undergo multicycle replication. While emergence of resistance to 4-GuDANA has not been observed in vivo, these results demonstrate that the development of resistance is possible and can be mediated by a single amino acid change in the active site of the viral NA.

Guanidinobenzoic acid inhibitors of influenza virus neuraminidase.

The active site of influenza virus neuraminidase (NA) is formed by 11 universally conserved residues. A guanidino group incorporated into two unrelated NA inhibitors was previously reported to occupy different negatively charged sites in the NA active site, A new inhibitor containing two guanidino groups was synthesized in order to utilize both sites in an attempt to acquire a combined increase in affinity. The X-ray crystal structures of the complexes show that the expected increase in affinity could not be achieved even though the added guanidino group binds to the negatively charged site as designed. This suggests that the ligand affinity to the target protein is contributed both from ligand-protein interactions and solvation/conformation energy of the ligand.

Hemagglutinin specificity and neuraminidase coding capacity of neuraminidase-deficient influenza viruses.

Neuraminidase (NA)-deficient mutant virus stocks have been obtained by passaging A/NWS/33HA-tern/Australia/G70c/75NA (H1N9) influenza virus in medium containing neuraminidase from Micromonospora viridifaciens and antiserum against the influenza NA. Growth of the resulting mutants is dependent on addition of bacterial neuraminidase to the medium. Nucleotide sequence analysis showed large single deletions in the NA genes, with both ends of the NA gene segments conserved. These RNA fragments all have the capacity to code for a peptide that contains the N-terminal "tail" and membrane-anchoring region of the NA, but the presence of this peptide has not been demonstrated in virions or infected cells. In contrast to the ease of selection of NA-deficient mutants from the H1N9 virus, no mutants were selected from three other viruses. The HA-coding segments of parental H1N9 and mutant NWSc-Mvi predict a change of Pro to His at residue 227 (H3 numbering), close to the receptor-binding site of H3 HA, compared to the HA of an H1N2 reassortant that contains the NWS/33 HA gene. This change may contribute to an altered HA specificity that allows selection of mutants that can infect cells in the presence of high levels of NA activity. It appears that the role of NA in influenza infection is to remove sialic acid from the HA rather than to destroy receptors on cells.

Molecular basis for the resistance of influenza viruses to 4-guanidino-Neu5Ac2en.

We report the selection and characterization of influenza A/NWS-G70c and B/HK/8/73 (HG) viruses which are resistant to the potent influenza neuraminidase inhibitor, 4-guanidino-Neu5Ac2en. Viruses were selected which replicated in MDCK cells in the presence of 20 micrograms/ml inhibitor. The neuraminidase of resistant viruses was > 200-fold more resistant to 4-guanidino-Neu5Ac2en than was the neuraminidase of the parent viruses. Although amounts of neuraminidase protein were similar in resistant and parent viruses, the enzyme activity of the resistant neuraminidase heads was reduced by > 95% for the substrates used. Relative to parent viruses, the resistant viruses replicated to equal or greater titers in tissue culture and in embryonated chicken eggs. Sequence analysis revealed a single nucleotide mutation in the neuraminidase gene of each virus resulting in the change of the conserved Glu 119 (which lies in a pocket beneath the active site of the enzyme) to Gly thus eliminating an electrostatic interaction with the C-4 guanidinium moiety of the inhibitor. Mutations (Asn-->Ser) at amino acids 145 and 150 were also found in the hemagglutinin gene of the B/HK/8/73 (HG) virus resistant to 4-guanidino-Neu5Ac2en. No changes were found in the hemagglutinin gene of the resistant A/NWS-G70c virus.

A strategy for theoretical binding constant, Ki, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase.

Neuraminidase (NA) is one of the two major surface antigens of influenza virus. It plays an indispensable role in the release and spread of progeny virus particles during infection. NA inhibitors reduce virus infection in animals. To improve the clinical efficacy of NA inhibitors, we have begun the design of non-carbohydrate inhibitors based on the active site structure of NA. The approach is an iterative process of ligand modeling and electrostatic calculations followed by chemical synthesis of compounds, biological testing, and NA-inhibitor complex structure determination by X-ray crystallography. A strategy has been developed to calculate Ki for newly designed inhibitors. The calculations using the DelPhi program were performed for carbohydrate inhibitors and three preliminary benzoic acid inhibitors of neuraminidase (BANA) that have been synthesized and shown to bind to the active site of NA in the crystal structure. The calculated Kis of these inhibitors have an enlightening agreement with their in vitro biological activities. This demonstrates that the calculations produce informative results on the affinity of modeled inhibitors. GRID maps were also calculated and several pockets were identified for accepting possible new ligands. The calculated Kis for newly designed ligands suggest that these potential compounds will have high inhibitory activities.

4-(Acetylamino)-3-hydroxy-5-nitrobenzoic acid.

The 4-(acetylamino)-3-hydroxy-5-nitrobenzoic acid molecule, C9H8N2O6, a designed inhibitor for the influenza virus neuraminidase protein, crystallizes as hydrogen-bonded dimers. The dihedral angles of the substituent groups with respect to the planar phenyl moiety are 5.0 (3) degrees for the carboxyl group, 45.0 (2) degrees for the nitro group and 37.3 (1) degrees for the acetylamino substituent. The crystal structure is stabilized by intermolecular hydrogen bonding.

Red cells bound to influenza virus N9 neuraminidase are not released by the N9 neuraminidase activity.

Influenza virus neuraminidase (NA) of the N9 subtype also possesses hemagglutinin activity and the hemagglutinating, or hemabsorbing (HB), site is distinct from the catalytic site. Previous results suggested that the NA was binding to sialic acid on the red cell surface, but we now report that the HB receptor is not sensitive to N9 influenza neuraminidase activity. Cell lines that constitutively express N9 or N2 neuraminidase have been used to further investigate the specificity of red blood cell binding to the HB site. The results suggest that the ligand is N-acetylneuraminic acid in a linkage or environment that is not sensitive to influenza virus neuraminidase, but which is released by the broadly specific bacterial sialidases from Micromonospora viridifaciens or Arthrobacter ureafaciens.