Biomedical subjects
R G Webster
Publications and source records attributed to R G Webster.
Potential advantages of DNA immunization for influenza epidemic and pandemic planning.
Immunization with purified DNA is a powerful technique for inducing immune responses. The concept of DNA immunization involves insertion of the gene encoding the antigen of choice into a bacterial plasmid and injection of the plasmid into the host where the antigen is expressed and where it induces humoral and cellular immunity. The most effective routes and methods for DNA immunization are bombardment with particles coated with DNA ("gene gun" technique), followed by the intramuscular and intradermal routes. DNA immunization technology has the potential to induce immunity to all antigens that can be completely encoded in DNA, which therefore include all protein, but not carbohydrate, antigens. DNA immunization results in presentation of antigens to the host's immune system in a natural form, like that achieved with live-attenuated vaccines. The DNA immunization strategy has the potential to rapidly provide a new vaccine in the face of an emerging influenza pandemic.
Influenza B virus encephalitis.
Acute encephalitis and postinfectious encephalopathy have been reported infrequently in association with influenza A and B virus infections. We report herein a case of a 6-year-old girl with acute influenza B virus encephalitis resulting in neurological sequelae. The diagnosis was made by isolation of influenza B virus from the nasopharynx, seroconversion to influenza B, and reverse transcription polymerase chain reaction (RT-PCR) identification of the virus from the patient's cerebrospinal fluid. Direct sequencing of viral RNA from the patient's nasopharynx and cerebrospinal fluid revealed identical nucleotide sequences in the HA1 region of the hemagglutinin gene. This is the first report of influenza B virus encephalitis diagnosed by use of RT-PCR and illustrates the need for increased awareness of influenza virus as a cause of acute encephalitis. PCR may be a useful tool for diagnosing future cases.
Transmission of Eurasian avian H2 influenza virus to shorebirds in North America.
Influenza A virus of the H2 subtype caused a serious pandemic in 1957 and may cause similar outbreaks in the future. To assess the evolution and the antigenic relationships of avian influenza H2 viruses, we sequenced the haemagglutinin (HA) genes of H2 isolates from shorebirds, ducks and poultry in North America and derived a phylogenetic tree to establish their interrelationships. This analysis confirmed the divergence of H2 HA into two geographical lineages, American and Eurasian. One group of viruses isolated from shorebirds in North America had HA belonging to the Eurasian lineage, indicating an interregional transmission of the H2 gene. Characterization of HA with a monoclonal antibody panel revealed that the antigenicity of the Delaware strains differed from the other avian strains analysed. The data emphasizes the importance of avian influenza surveillance.
Genetic reassortment of avian, swine, and human influenza A viruses in American pigs.
In late summer through early winter of 1998, there were several outbreaks of respiratory disease in the swine herds of North Carolina, Texas, Minnesota, and Iowa. Four viral isolates from outbreaks in different states were analyzed genetically. Genotyping and phylogenetic analyses demonstrated that the four swine viruses had emerged through two different pathways. The North Carolina isolate is the product of genetic reassortment between H3N2 human and classic swine H1N1 influenza viruses, while the others arose from reassortment of human H3N2, classic swine H1N1, and avian viral genes. The hemagglutinin genes of the four isolates were all derived from the human H3N2 virus circulating in 1995. It remains to be determined if either of these recently emerged viruses will become established in the pigs in North America and whether they will become an economic burden.
Protection against a lethal avian influenza A virus in a mammalian system.
The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig+/+) and Ig-/- mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8(+) T-cell memory in the Ig-/- mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig+/+ mice, generating secondary CD8(+) and CD4(+) T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.
Independence of evolutionary and mutational rates after transmission of avian influenza viruses to swine.
In 1979, an H1N1 avian influenza virus crossed the species barrier, establishing a new lineage in European swine. Because there is no direct or serologic evidence of previous H1N1 strains in these pigs, these isolates provide a model for studying early evolution of influenza viruses. The evolutionary rates of both the coding and noncoding changes of the H1N1 swine strains are higher than those of human and classic swine influenza A viruses. In addition, early H1N1 swine isolates show a marked plaque heterogeneity that consistently appears after a few passages. The presence of a mutator mutation was postulated (C. Scholtissek, S. Ludwig, and W. M. Fitch, Arch. Virol. 131:237-250, 1993) to account for these observations and the successful establishment of an avian H1N1 strain in swine. To address this question, we calculated the mutation rates of A/Mallard/New York/6750/78 (H2N2) and A/Swine/Germany/2/81 (H1N1) by using the frequency of amantadine-resistant mutants. To account for the inherent variability of estimated mutation rates, we used a probabilistic model for the statistical analysis. The resulting estimated mutation rates of the two strains were not significantly different. Therefore, an increased mutation rate due to the presence of a mutator mutation is unlikely to have led to the successful introduction of avian H1N1 viruses in European swine.
DNA vaccine encoding hemagglutinin provides protective immunity against H5N1 influenza virus infection in mice.
In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine's protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.
Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong.
The H5N1 avian influenza virus that killed 6 of 18 persons infected in Hong Kong in 1997 was transmitted directly from poultry to humans. Viral isolates from this outbreak may provide molecular clues to zoonotic transfer. Here we demonstrate that the H5N1 viruses circulating in poultry comprised two distinguishable phylogenetic lineages in all genes that were in very rapid evolution. When introduced into new hosts, influenza viruses usually undergo rapid alteration of their surface glycoproteins, especially in the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large proportion of amino acid changes in all gene products except in the HA. These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of "internal" virion proteins reveal amino acids previously found in human strains. These human-specific amino acids may be important factors in zoonotic transmission.
Reassortment and insertion-deletion are strategies for the evolution of influenza B viruses in nature.
The evolution of influenza B viruses is poorly understood. Reassortment of influenza B viruses in nature as a means of genetic variation has not been considered to be a major contributor to their evolution. However, the current practice of assigning evolutionary relationships by antigenic analysis of the hemagglutinin of influenza B viruses would fail to detect reassortants. In this study, influenza B viruses isolated within the past 10 years from sites in the United States and China were studied by nucleotide sequencing of the hemagglutinin and neuraminidase genes and construction of phylogenetic trees to assess evolutionary relationships. A group of viruses represented by B/Houston/1/92 possess a hemagglutinin derived from a B/Yamagata/16/88-like strain and a neuraminidase derived from a B/Victoria/2/87-like strain. A second reassortment event between the hemagglutinin of a B/Yamagata/16/88-like virus closely related to the B/Beijing/184/93 strain and the neuraminidase of a B/Victoria/2/87-like strain is represented by a single virus, B/Memphis/3/93. The neuraminidase of the reassortant viruses is most closely related to that of B/Victoria/2/87-like viruses currently circulating in Nanchang, China. A pattern of insertions and deletions in the hemagglutinin and the neuraminidase of different strains of influenza B viruses is observed. Reassortment plays a role in the evolution of influenza B viruses and may necessitate a change in the methods used to assess and identify new influenza viruses.
Growth and immunogenicity of influenza viruses cultivated in Vero or MDCK cells and in embryonated chicken eggs.
Vero cells, MDCK cells and embryonated chicken eggs (eggs) were used to evaluate influenza virus growth characteristics and immunogenicity induced by inactivated influenza B vaccines. Both cell lines produced comparable quantities of total viral and haemagglutinin (HA) proteins. Sequence analysis indicated genetic identity of the HA of Vero- and MDCK-grown virus counterparts with maintenance of antigenic characteristics of viruses derived from humans. The egg-grown influenza B/Memphis/1/93 variant differed from cell-grown counterparts at amino acid position 198 (Pro-Thr) and lost a glycosylation site. The level of neuraminidase (NA) activity was the highest in egg-grown virus, while MDCK- and Vero cell-grown viruses possessed 70% and 90% less NA activity respectively when fetuin was used as a substrate. Although each of the vaccines induced high and comparable levels of serum antibodies, mammalian cell-derived vaccines induced antibodies that were more cross reactive, and those antibodies induced by egg-derived vaccine were more specific to the homologous antigen. ELISPOT analysis indicated that the mammalian cell-grown vaccines induced high frequencies of IgG-producing cells directed against both cell- and egg-grown antigens, while egg-grown vaccine induced high frequencies of IgG and IgM-producing cells reacting with homologous antigen and low levels of IgG-producing cells reactive with cell-grown virus antigen. Taken together, our results suggest that mammalian cells are a viable option for the production of influenza virus vaccines.
Characterization of avian H5N1 influenza viruses from poultry in Hong Kong.
The transmission of avian H5N1 influenza viruses to 18 humans in Hong Kong in 1997 with six deaths established that avian influenza viruses can transmit to and cause lethal infection in humans. This report characterizes the antigenic and biological properties of the H5N1 influenza viruses isolated from chickens, ducks, and geese from farms and poultry markets in Hong Kong during 1997 and compares them with those of virus isolated from the index human case. Each of the H5N1 viruses from Hong Kong poultry markets that were tested were lethal in chickens, possessed polybasic amino acids at the carboxy-terminus of HA1, and by definition were highly pathogenic in poultry. The available nonpathogenic H5 influenza viruses and the pathogenic H5N1 virus from Hong Kong were analyzed with monoclonal antibodies prepared to A/chicken/Pennsylvania/1370/83 (H5N2). The analysis revealed limited antigenic drift in 15 years and established that monoclonal antibodies are useful reagents for identification and antigenic analysis of avian strains that may transmit to humans in the future. One of the monoclonal antibodies permitted separation of the H5N1 influenza viruses from poultry into two groups that correlated with the presence or absence of a carbohydrate at residue 158 adjacent to the receptor binding site on HA. The H5N1 viruses examined replicated in geese, pigs, rats, and mice, but to only a very limited extent in ducks. It is noteworthy that all infected geese shed virus and that the H5N1 viruses caused disease signs and death in a portion (3 of 16) of the geese, with evidence of systemic spread to the brain. The tropism for geese is unusual and may provide insight into the origin of these viruses. In mice, the H5N1 virus caused lethal pneumonia and spread systemically to the brain. Mice would thus provide an ideal model system for studying immune responses and pathogenesis. Transmission experiments in chickens revealed that the H5N1 viruses are spread by fecal-oral transmission rather than by aerosol, and that the viruses are inactivated by drying of feces at ambient temperature. However, infectivity is maintained for at least 4 days in wet feces at 25 degreesC. There were differences in the morphology of the H5N1 viruses isolated from birds and humans. The perpetuation of H5N1 influenza viruses in the poultry markets in Hong Kong and the transmission of these viruses to humans emphasize the importance of these markets in the epidemiology of influenza. The poultry markets are of critical importance in the perpetuation and transmission of influenza viruses to other avian species and to mammals, including humans.
Induction of CD8+ T cell responses to dominant and subdominant epitopes and protective immunity to Sendai virus infection by DNA vaccination.
While recent studies have demonstrated that DNA vaccination induces potent CD8+ T cell memory in vivo, it is unclear whether this memory is qualitatively and quantitatively comparable with that induced by natural viral infection. In the current studies, we have investigated the induction of CD8+ memory CTL responses to Sendai virus nucleoprotein (NP) in C57BL/6 mice following gene gun vaccination. The data demonstrate that this mode of vaccination induces potent long-lived memory CTL precursors (CTLp) specific for both the dominant (NP324-332/Kb) and the subdominant (NP324-332/Db) epitopes of NP. The frequencies of T cells specific for each of these epitopes in the spleen is about 1:2000 CD8+ T cells, similar to those induced by intranasal infection with Sendai virus. Moreover, the induction of memory CTLp by DNA vaccination is independent of MHC class II molecules or Ab, as is the case for memory CTLp induction by live Sendai virus infection. CTLp specific for both epitopes are capable of migrating to the lung following Sendai virus infection and express potent cytotoxic activity at the site of infection. Consistent with this activity, DNA vaccination with Sendai virus NP induced a substantial degree of Ab-independent protection from a challenge with a lethal dose of Sendai virus. Taken together, these data demonstrate that for the parameters tested, DNA vaccination is indistinguishable from live virus infection in terms of priming functional memory CTLp with broad specificity for both dominant and subdominant T cell epitopes.
Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus.
BACKGROUND: In May, 1997, a 3-year-old boy in Hong Kong was admitted to the hospital and subsequently died from influenza pneumonia, acute respiratory distress syndrome, Reye's syndrome, multiorgan failure, and disseminated intravascular coagulation. An influenza A H5N1 virus was isolated from a tracheal aspirate of the boy. Preceding this incident, avian influenza outbreaks of high mortality were reported from three chicken farms in Hong Kong, and the virus involved was also found to be of the H5 subtype. METHODS: We carried out an antigenic and molecular comparison of the influenza A H5N1 virus isolated from the boy with one of the viruses isolated from outbreaks of avian influenza by haemagglutination-inhibition and neuraminidase-inhibition assays and nucleotide sequence analysis. FINDINGS: Differences were observed in the antigenic reactivities of the viruses by the haemagglutination-inhibition assay. However, nucleotide sequence analysis of all gene segments revealed that the human virus A/Hong Kong/156/97 was genetically closely related to the avian A/chicken/Hong Kong/258/97. INTERPRETATION: Although direct contact between the sick child and affected chickens has not been established, our results suggest transmission of the virus from infected chickens to the child without another intermediate mammalian host acting as a "mixing vessel". This event illustrates the importance of intensive global influenza surveillance.
How to overcome resistance of influenza A viruses against adamantane derivatives.
We tested two approaches to overcoming resistance of influenza A viruses against adamantane derivatives. First, adamantane derivatives that interfere with the ion channel function of the variant M2 protein of amantadine-resistant viruses may prevent drug resistance, if they are used in mixture with amantadine. Second, amantadine acts on the M2 protein (at low concentrations) and indirectly on the hemagglutinin (at concentrations at least 100 times higher). Identifying and using a drug that reacted with both targets at the same concentration might reduce development of resistance, since, in this case, two mutations, one in each target protein would be necessary at once. Such a double mutation is assumed to be a rare event. We evaluated forty adamantane derivatives and two related compounds to determine whether they interfered with plaque formation by influenza A strains, including A/Singapore/1/57 (H2N2). Variants resistant to drugs that interfered at low concentrations (approximately 1 microg/ml; e.g. amantadine) were cross-resistant with each other, but were sensitive to those agents effective at high concentrations (8 microg/ml; e.g. memantine). The former group of compounds act on the ion channel; the corresponding escape mutants tested had amino acid replacements at positions 27, 30 or 31 of the M2 protein. Hemagglutinin was the indirect target of the latter group of compounds. Variants resistant to these agents lacked amino acid replacements within the ion channel of the M2 protein and the mutants tested had amino acid replacements in the hemagglutinin. Although we failed to identify compounds that interacted with the ion channel of amantadine-resistant variants and inhibited their replication, we were able to construct at least two compounds that interfered with both the ion channel and the hemagglutinin at about the same concentration. After passage in the presence of these compounds, we either failed to obtain any drug-resistant mutants or those obtained had amino acid replacements in the ion channel of the M2 protein and the hemagglutinin.
Long-term stability of the anti-influenza A compounds--amantadine and rimantadine.
Amantadine and rimantadine hydrochloride were tested for stability after storage at different temperatures and under different conditions for extended periods of time. Both compounds were quite stable after storage for at least 25 years at ambient temperature; they both retained full antiviral activity after long-term storage or after boiling and holding at 65-85 degrees C for several days. Thus, amantadine and rimantadine could be synthesized in large quantities and stored for at least one generation without loss of activity in preparation for the next influenza A pandemic in humans.
Influence of host species on the evolution of the nonstructural (NS) gene of influenza A viruses.
The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode two overlapping proteins. In the M gene, evolution of one protein affects that of the other. To determine whether or not this evolutionary influence operating between the two M proteins also occurs in the NS gene, we sequenced the NS genes of 36 influenza A viruses isolated from a broad spectrum of animal species (wild and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them phylogenetically, together with other previously published sequences. These analyses enabled us to conclude the following host species-related points that are not found in the other influenza A virus genes and their gene products. (1) The evolution of the two overlapping proteins encoded by the NS gene are lineage-dependent, unlike the M gene where evolutionary constraints on the Ml protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991) 5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the non-gull avian lineage contained H13 gull viruses, indicating that the gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike findings with other genes. (3) The branching topology of the recent equine lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction of the NS, M, and PB2 genes into horses from avian sources by genetic reassortment.
Profound abnormality of the B/T lymphocyte ratio during chemotherapy for pediatric acute lymphoblastic leukemia.
The fluorescence-activated cell sorter (FACS) was utilized to phenotype lymphocyte compartments in children receiving intensive chemotherapy for acute lymphoblastic leukemia (ALL). Sixteen patients (eight males and eight females) of diverse ages, risks of relapse, and within weeks 7-53 of maintenance/continuation chemotherapy treatment were arbitrarily selected for study. All 16 patients had profound B cell lymphopenia. In contrast, T cell numbers were often normal or marginally low, and accounted for up to 98% of the lymphocyte populations. No abnormality in T cell phenotypes could be demonstrated. Due to the highly skewed B/T lymphocyte ratios in these ALL patients, the absolute white blood cell counts and lymphocyte percentages were not predictive of the underlying B cell lymphopenia. Patients were also tested for serum immunoglobulin levels and most had abnormally low IgG and IgM. None of four patients immunized with the 1996-1997 influenza virus vaccine seroconverted to at least two vaccine antigens as compared to 10 of 10 healthy, age-matched controls. In total, these data highlight for the first time the profound abnormality of the B/T lymphocyte ratio in patients during treatment for ALL, and argue for consideration of B cell-targeted immunotherapy.