Acquired immune responses to the seasonal trivalent influenza vaccination in COPD.

Epidemiological data suggest that influenza vaccination protects against all-cause mortality in chronic obstructive pulmonary disease (COPD) patients. However, recent work has suggested there is a defect in the ability of some COPD patients to mount an adequate humoral response to influenza vaccination. The aim of our study was to investigate humoral and cell-mediated vaccine responses to the seasonal trivalent influenza vaccination (TIV) in COPD subjects and healthy controls. Forty-seven subjects were enrolled into the study; 23 COPD patients, 13 age-matched healthy controls (HC ≥ 50) and 11 young healthy control subjects (YC ≤ 40). Serum and peripheral blood mononuclear cells (PBMC) were isolated pre-TIV vaccination and at days 7 and 28 and 6 months post-vaccine for haemagglutinin inhibition (HAI) titre, antigen-specific T cell and antibody-secreting cell analysis. The kinetics of the vaccine response were similar between YC, HC and COPD patients and there was no significant difference in antibody titres between these groups at 28 days post-vaccine. As we observed no disease-dependent differences in either humoral or cellular responses, we investigated if there was any association of these measures with age. H1N1 (r = -0·4253, P = 0·0036) and influenza B (r = -0·344, P = 0·0192) antibody titre at 28 days negatively correlated with age, as did H1N1-specific CD4+ T helper cells (r = -0·4276, P = 0·0034). These results suggest that age is the primary determinant of response to trivalent vaccine and that COPD is not a driver of deficient responses per se. These data support the continued use of the yearly trivalent vaccine as an adjunct to COPD disease management.

Comparison of a novel microcrystalline tyrosine adjuvant with aluminium hydroxide for enhancing vaccination against seasonal influenza.

BACKGROUND: Vaccination against seasonal influenza strains is recommended for "high risk" patient groups such as infants, elderly and those with respiratory or circulatory diseases. However, efficacy of the trivalent influenza vaccine (TIV) is poor in many cases and in the event of an influenza pandemic, mono-valent vaccines have been rapidly developed and deployed. One of the main issues with use of vaccine in pandemic situations is the lack of a suitable quantity of vaccine early enough during the pandemic to exert a major influence on the transmission of virus and disease outcome. One approach is to use a dose-sparing regimen which inevitably involves enhancing the efficacy using adjuvants. METHODS: In this study we compare the use of a novel microcrystalline tyrosine (MCT) adjuvant, which is currently used in a niche area of allergy immunotherapy, for its ability to enhance the efficacy of a seasonal TIV preparation. The efficacy of the MCT adjuvant formulation was compared to alum adjuvanted TIV and to TIV administered without adjuvant using a ferret challenge model to determine vaccine efficacy. RESULTS: The MCT was found to possess high protein-binding capacity. In the two groups where TIV was formulated with adjuvant, the immune response was found to be higher (as determined by HAI titre) than vaccine administered without adjuvant and especially so after challenge with a live influenza virus. Vaccinated animals exhibited lower viral loads (as determined using RT-PCR) than control animals where no vaccine was administered. CONCLUSIONS: The attributes of each adjuvant in stimulating single-dose protection against a poorly immunogenic vaccine was demonstrated. The properties of MCT that lead to the reported effectiveness warrants further exploration in this and other vaccine targets - particularly where appropriate immunogenic, biodegradable and stable alternative adjuvants are sought.

Defective interfering viruses and their potential as antiviral agents.

Defective interfering (DI) virus is simply defined as a spontaneously generated virus mutant from which a critical portion of the virus genome has been deleted. At least one essential gene of the virus is deleted, either in its entirety, or sufficiently to make it non-functional. The resulting DI genome is then defective for replication in the absence of the product(s) of the deleted gene(s), and its replication requires the presence of the complete functional virus genome to provide the missing functions. In addition to being defective DI virus suppresses production of the helper virus in co-infected cells, and this process of interference can readily be observed in cultured cells. In some cases, DI virus has been observed to attenuate disease in virus-infected animals. In this article, we review the properties of DI virus, potential mechanisms of interference and progress in using DI virus (in particular that derived from influenza A virus) as a novel type of antiviral agent.

Interfering vaccine (defective interfering influenza A virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection.

Defective interfering (DI) virus RNAs result from major deletions in full-length viral RNAs that occur spontaneously during de novo RNA synthesis. These RNAs are packaged into virions that are by definition non-infectious, and are delivered to cells normally targeted by the virion. DI RNAs can only replicate with the aid of a coinfecting infectious helper virus, but the small size of DI RNA allows more copies of it to be made than of its full-length counterpart, so the cell produces defective virions in place of infectious progeny. In line with this scenario, the expected lethal disease in an influenza A virus-mouse model is made subclinical by administration of DI virus, but animals develop solid immunity to the infecting virus. Hence DI virus has been called an 'interfering vaccine'. Because interfering vaccine acts intracellularly and at a molecular level, it should be effective against all influenza A viruses regardless of subtype. Here we have used the ferret, widely acknowledged as the best model for human influenza. We show that an interfering vaccine with defective RNAs from an H3N8 virus almost completely abolished clinical disease caused by A/Sydney/5/97 (H3N2), with abrogation of fever and significant reductions in clinical signs of illness. Animals recovered fully and were solidly immune to reinfection, in line with the view that treatment converts the otherwise virulent disease into a subclinical and immunizing infection.

Complete genome sequences of Chandipura and Isfahan vesiculoviruses.

Chandipura virus (CHPV) and Isfahan virus (ISFV) are two members of the genus Vesiculovirus from Asia. Both are arthropod-transmitted and are able to infect humans, but neither causes vesicular stomatitis in livestock. The complete genome sequence for each virus has been determined. The negative-sense RNA genome comprises 11,119 nt (CHPV) or 11,088 nt (ISFV). The most variable of the non-transcribed regions is the intergenic spacer at the G-L gene junction (4 bases in ISFV, 20 in CHPV). Phylogenetic analysis of deduced protein sequences shows that although CHPV and ISFV are distinct viruses, they are more related to each other than either is to the New World vesicular stomatitis viruses (VSV). The South American virus, Piry virus, is more closely related to the Asian viruses ISFV and CHPV, than it is to VSV.

Chimeric pneumovirus nucleocapsid (N) proteins allow identification of amino acids essential for the function of the respiratory syncytial virus N protein.

The nucleocapsid (N) protein of the pneumovirus respiratory syncytial virus (RSV) is a major structural protein which encapsidates the RNA genome and is essential for replication and transcription of the RSV genome. The N protein of the related virus pneumonia virus of mice (PVM) is functionally unable to replace the RSV N protein in a minigenome replication assay. Using chimeric proteins, in which the immediate C-terminal part of the RSV N protein was replaced with the equivalent region of the PVM N protein, it was shown that six amino acid residues near the C terminus of the N protein (between residues 352-369) are essential for its function in replication and for the ability of the N protein to bind to the viral phosphoprotein, P.

Fidelity of leader and trailer sequence usage by the respiratory syncytial virus and avian pneumovirus replication complexes.

The specificity of usage of promoters for replication and transcription by the pneumoviruses human respiratory syncytial virus (HRSV) and avian pneumovirus (APV) was studied using minigenomes containing a reporter gene. When infectious HRSV or APV was used as helper virus, replication could occur only if both the leader and trailer regions (containing the replicative and transcriptional promoters) were derived from the helper virus. In contrast, when the HRSV replication complex was supplied from cDNA plasmids, a minigenome containing either the APV leader or trailer was recognized and substantial levels of replication and transcription occurred. These data suggest that in pneumovirus-infected cells, helper virus functions can discriminate between genomes on the basis of the terminal sequences and that there is an association between the leader and trailer required for productive replication. This association is required only in virus-infected cells, not when replication and transcription are mediated by plasmid-directed expression of the component proteins required for replication and transcription. The possible implications of this are discussed.

Matrix protein of Chandipura virus inhibits transcription from an RNA polymerase II promoter.

Chandipura virus (CHPV) is a Vesiculovirus, related to, but phylogenetically distinct from, vesicular stomatitis virus (VSV). The matrix protein of VSV, as well as its role in virus assembly, inhibits the transcription from promoters for host RNA polymerases I and II. Cloning and expression of the matrix protein of CHPV in human cells showed that this protein is also functional in its inhibitory effect on transcription of a reporter gene from the cytomegalovirus immediate-early promoter, despite sharing only 28% amino acid sequence identity with the matrix protein of VSV.

A single amino acid substitution in the phosphoprotein of respiratory syncytial virus confers thermosensitivity in a reconstituted RNA polymerase system.

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.

Rescue of synthetic minireplicons establishes the absence of the NS1 and NS2 genes from avian pneumovirus.

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.

Role of grey squirrels and pheasants in the transmission of Borrelia burgdorferi sensu lato, the Lyme disease spirochaete, in the U.K.

In Britain, grey squirrels (Sciurus carolinensis Gmelin) and pheasants (Phasianus colchicus Linnaeus) are important hosts of larvae and nymphs of Ixodes ricinus L., the principal European vector of the Lyme disease spirochaete, Borrelia burgdorferi sensu lato. To test whether squirrels are competent hosts of B. burgdorferi s.l., three females were trapped in the wild and then held in captivity. Following treatment, each animal was exposed to uninfected xenodiagnostic I. ricinus ticks. Squirrel A (an adult) which was inoculated experimentally with B. burgdorferi s.l., transmitted the infection to xenodiagnostic ticks. In contrast, squirrel B (a juvenile that was not inoculated)-showed no evidence of infection. Xenodiagnostic ticks that fed on control squirrel C (an adult) became infected and subsequently transmitted the infection experimentally to an uninfected hamster. The results indicated that squirrel C had a disseminated infection acquired in the wild and which persisted for at least 11 weeks. These data clearly demonstrate that grey squirrels are amplifying and reservoir hosts of B. burgdorferi s.l. The strain associated with squirrels was related to the B. afzelii genotype. Two observations implicated pheasants in a similar role: (i) a high prevalence of infection in engorged larvae collected from trapped pheasants, and (ii) the detection of B. burgdorferi s.l. (B. garinii genotype) in the wattle of 1/10 pheasants using PCR. Xenodiagnostic experiments similar to those undertaken with the squirrels are needed to confirm the role of pheasants in the transmission cycle of Lyme disease spirochaetes.

Identification of mutations contributing to the reduced virulence of a modified strain of respiratory syncytial virus.

The nucleotide sequences of the genome of the RSS-2 wild type strain of respiratory syncytial (RS) virus, which is known to induce upper respiratory tract infection in adults, and that of the attenuated ts1C candidate vaccine derived from it by three cycles of mutagenesis and selection of temperature-sensitive (ts) mutants, have been determined. Comparison of the sequences has located the genetic changes which contribute to the reduced pathogenicity in adults of the candidate vaccine. Thirty-seven nucleotide changes distinguish the wild type and ts1C, 13 of which confer amino acid substitutions; no mutations are present in extragenic regions. Partial nucleotide sequencing of the genomes of the first stage ts mutant (ts1A) and the second stage ts mutant (ts1B), which were intermediates in the derivation of the third stage mutant ts1C, established that five mutations resulting in amino acid substitutions had been induced in the first cycle of mutagenesis, one in the second cycle, and seven in the third cycle. The unique mutation differentiating ts1B from ts1A substitutes an alanine for a threonine at residue 736 in the polymerase (L) protein. The occurrence of a mutation in ts1C inducing substitution of a phenylalanine for a serine residue at an adjacent site (731) suggests that mutations in this region of the polymerase can have significant attenuating effects. The data suggest also that a mutation in the F gene may contribute to the attenuated phenotype.

Large RNA segment of Dugbe nairovirus encodes the putative RNA polymerase.

The nucleotide sequence of the large (L) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) was determined, completing the first entire genome sequence of a nairovirus. The L segment comprised 12255 nucleotides, making a total genome size of 18855 nucleotides, and the ends showed identity with the ends of the medium (M) and small (S) genomic segments. A single open reading frame (ORF) was present in the viral complementary strand, sufficient to encode a protein of 459 kDa. The predicted protein sequence showed the core polymerase motifs characteristic of the RNA-dependent RNA polymerases of segmented negative-stranded viruses. Comparison of the conserved motifs with the corresponding region of other segmented negative-strand viruses showed a closer relationship between nairoviruses and phleboviruses than with other Bunyaviridae or with other virus families. However, the core polymerase was the only function that could be assigned to a region of the DUG L gene.

Immune protection conferred by the baculovirus-related glycoprotein of Thogoto virus (Orthomyxoviridae).

The coding region of segment 4 of Thogoto (THO) virus, a tick-borne member of the Orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. This construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (MAbs). Immunization of hamsters with Spodoptera frugiperda (Sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against virus challenge; control hamsters developed clinical disease with high-titer viremia 3 days postchallenge. In contrast to hamsters, guinea pigs are comparatively resistant to THO virus infection but support nonviremic transmission between cofeeding infected and uninfected ticks. However, when uninfected ticks fed on guinea pigs immunized with the baculovirus recombinant, only 2% became infected following virus challenge of the animals compared to 26% of ticks on control nonimmune guinea pigs. Furthermore, neutralizing MAbs specific for THO viral glycoprotein protected mice against lethal challenge with THO virus; nonneutralizing MAbs specific for the glycoprotein, which inhibit THO viral agglutinating activity, did not induce a protective response. Thus at least in the murine model, protective immunity is conferred by antibodies directed against the neutralizing epitope(s) of the baculovirus-related glycoprotein of THO virus.

Detection of human antibodies to Crimean-Congo haemorrhagic fever virus using expressed viral nucleocapsid protein.

Diagnosis of Crimean-Congo haemorrhagic fever (CCHF) virus infections is hampered by the problems of handling this human pathogen, which requires the highest levels of biological containment. Recombinant antigens were examined for their potential as non-hazardous diagnostic reagents. The nucleocapsid (N) gene of the Greek AP92 isolate of CCHF virus was sequenced from cloned PCR products and the open reading frame was identified by homology to the N protein of a Chinese isolate of CCHF virus. The N protein was expressed to high levels in a baculovirus expression system. Three N protein-derived peptides were expressed in Escherichia coli as fusions with glutathione S-transferase and the antigenicities of these proteins and the baculovirus-expressed protein were tested by ELISA. When tested with laboratory animal sera representing all seven serogroups of nairoviruses, the only reactive sera were those raised to CCHF virus (Greek, Nigerian and Chinese isolates) and, more weakly, Hazara virus. When tested with a panel of known positive and negative human sera, the baculovirus-expressed N protein, and the peptide derived from the central region of the N protein, proved to be the best for identifying CCHF virus-specific IgG.

Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy.

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.

Comparison of the S RNA segments and nucleoprotein sequences of Crimean-Congo hemorrhagic fever, Hazara, and Dugbe viruses.

The S RNA segments of the nairoviruses Crimean-Congo hemorrhagic fever (CCHF) virus (Chinese isolate) and Hazara (HAZ) virus were cloned and sequenced from PCR products. The RNAs comprise 1672 and 1677 nucleotides, respectively, and each encodes a protein in the viral complementary strand (54.0 and 54.2 kDa, respectively). The deduced protein sequences show homology to each other and to the nucleoprotein of Dugbe (DUG) nairovirus, although both the CCHF and HAZ viral proteins are larger. Alignment of the nucleoprotein sequences of CCHF, HAZ, and DUG viruses show that the CCHF and HAZ sequences are somewhat more closely related to each other (60.0% identity) than either is to the DUG sequence (55.4 and 53.0% identity, respectively); 39.5% of residues are identical across all three proteins. The carboxyl-terminus of DUG N protein shows a 40-residue deletion relative to the N proteins of the other two viruses.

Expression of the nucleocapsid protein of Dugbe virus and antigenic cross-reactions with other nairoviruses.

The small (S) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) encodes a single protein, the nucleocapsid (N) protein, of M(r) 49.4 kDa. cDNA derived from the complete coding region for the N protein was cloned into Autographa californica nuclear polyhedrosis virus (AcNPV) under control of the polyhedrin promoter and used to infect Spodoptera frugiperda insect cells. Western blotting analysis using monoclonal antibodies demonstrated the production of DUG N protein in the infected cells. Monoclonal and polyclonal antibodies to the N protein of Crimean-Congo haemorrhagic fever (CCHF) virus were found to cross-react weakly with the baculovirus expressed DUG N protein by Western blotting. When used in an enzyme linked immunoassay (ELISA), the DUG N protein reacted with polyclonal mouse immune ascitic fluids raised against either CCHF or Hazara viruses (both members of the CCHF serogroup of nairoviruses). Cross-reactions between DUG virus (Nairobi sheep disease serogroup) and members of other nairovirus serogroups were not detected.

The glycoprotein of Thogoto virus (a tick-borne orthomyxo-like virus) is related to the baculovirus glycoprotein GP64.

Thogoto (THO) virus is a tick-transmitted virus which shares morphological and biochemical characteristics with members of the Orthomyxoviridae. The genome of Thogoto virus comprises six segments of single-stranded, negative sense RNA. The complete nucleotide sequence of the fourth largest RNA segment of THO virus has been determined from cDNA analyses. This RNA segment is 1574 nt long and has a coding capacity for a glycoprotein of 512 amino acids with a predicted molecular weight of 57,550 Da. The sequence of this protein has extensive homology with the putative envelope protein of Dhori (DHO) virus, the only other recorded tick-borne orthomyxo-like virus, but not with the envelope glycoproteins of the influenza viruses. A search of the translation of the available nucleotide database has produced evidence for a relationship between the glycoproteins of THO and DHO viruses and the gp64 glycoprotein of two DNA-containing insect baculoviruses, Autographa californica nuclear polyhedrosis virus and Orgyia pseudotsugata nuclear polyhedrosis virus. The baculovirus gp64 protein is a membrane protein implicated in endocytotic fusion events during infection. A multiple alignment between these four glycoproteins gave significance scores of greater than 28 standard deviations, indicating that the homologies between them are highly significant. The distribution of cysteine residues is conserved between all four proteins which also have similar hydropathy profiles, suggestive of a type I membrane protein topology.