PubMed Health⌕ Search

Biomedical subjects

P Simmonds

Publications and source records attributed to P Simmonds.

At least 199 records · Page 11Linked to original sources
PubMed1993

Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV): use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3.

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenicity of the NS-4 protein was investigated by epitope mapping and by enzyme-linked immunosorbent assay with branched oligopeptides. Epitope mapping of the region between amino acid residues 1679 and 1768 in the HCV polyprotein revealed two major antigenic regions (1961 to 1708 and 1710 to 1728) that were recognized by antibody elicited upon natural infection of HCV. The antigenic regions were highly variable between variants of HCV, with only 50 to 60% amino acid sequence similarity between types 1, 2, and 3. Although limited serological cross-reactivity between HCV types was detected between peptides, particularly in the first antigenic region of NS-4, type-specific reactivity formed the principal component of the natural humoral immune response to NS-4. Type-specific antibody to particular HCV types was detected in 89% of the samples from anti-HCV-positive blood donors and correlated almost exactly with genotypic analysis of HCV sequences amplified from the samples by polymerase chain reaction. Whereas almost all blood donors appeared to be infected with a single virus type (97%), a higher proportion of samples (40%) from hemophiliacs infected from transfusion of non-heat-inactivated clotting factor contained antibody to two or even all three HCV types, providing evidence that long-term exposure may lead to multiple infection with different variants of HCV.

Amino Acid Sequence↗
PubMed1993

Selection for specific sequences in the external envelope protein of human immunodeficiency virus type 1 upon primary infection.

Viral RNA was extracted from plasma samples collected from five individuals during the period of viremia before seroconversion in primary infection with human immunodeficiency virus type 1 (HIV-1) and amplified by polymerase chain reaction. Nucleotide sequence analysis of amplified DNA from the V3 and V4 hypervariable regions indicated that the initial virus population of each acutely infected individual was completely homogeneous in sequence. No intrasample variability was found among the 44,090 nucleotides sequenced in this region of env, contrasting with the high degree of variability normally found in seropositive individuals. Paradoxically, substantial sequence variability was found in the normally high conserved gag gene (encoding p17) in most of the preseroconversion samples. The diversity of p17 sequences in samples that were homogeneous in V3 and V4 can most readily be explained by the existence of strong selection for specific env sequences either upon transmission or in the interval between exposure and seroconversion in the exposed individual. Evidence that localizes the selected region upon transmission to V3 is provided by the similarity or identity of V3 loop sequences in five individuals with epidemiologically unrelated HIV-1 infections, while regions flanking the V3 loop and the V4 hypervariable region were highly divergent. The actual V3 sequences were similar to those associated with macrophage tropism in primary isolates of HIV, irrespective of whether infection was acquired by sexual contact or parenterally through transfusion of contaminated factor VIII. Proviral DNA sequences in peripheral blood mononuclear cells remained homogeneous in the V3 and V4 regions (and variable in p17gag) for several months after seroconversion. The persistence of HIV sequences in peripheral blood mononuclear cells identical to those found at primary infection in the absence of continued virus expression provides an explanation for the previously observed differences in the composition of circulating DNA and RNA populations in sequential samples from seropositive individuals.

Amino Acid Sequence↗
PubMed1992

Convergent and divergent sequence evolution in the surface envelope glycoprotein of human immunodeficiency virus type 1 within a single infected patient.

In an investigation of the evolution of the third hypervariable loop of gp120 (V3), the principal neutralization determinant of human immunodeficiency virus type 1, we have analyzed 89 V3 sequences of plasma viral RNA purified from peripheral blood samples donated over 7 years by an infected hemophiliac. Considerable sequence diversity in the V3 region was found at all time points after seroconversion. Phylogenetic analysis revealed that an important diversification had occurred by 3 years postinfection and that, subsequently, most sequences could be allocated to either one of two major lineages that persisted throughout the remainder of the infection. Rapid changes in frequency of the most common sequences and the observation that the same hexapeptide motif (GPGSAV) at the crown of the V3 loop has evolved convergently provide strong evidence that selective processes determine the evolutionary fate of sequence variants in this region.

Amino Acid Sequence↗
PubMed1992

Comparison of hepatitis B virus subtyping of d/y determinants by radioimmunoprecipitation assay and the polymerase chain reaction.

Using a double polymerase chain reaction a method was devised for detecting and subtyping hepatitis B virus DNA in serum samples. Primers from the S-gene were selected from the sequence analyses of five HBV HBsAg subtypes, to amplify HBV DNA and subtype for y specific DNA. Thirty-eight samples were subtyped for d and y determinants by radioimmunoprecipitation assay (RIPA) and the polymerase chain reaction (PCR). Subtyping by PCR and RIPA was in agreement in 100% of subtype y samples and 83.3% of subtype d, giving an overall correlation of 92.1%. As a third comparison, 12 amplified samples were digested by the restriction enzyme Sau 3A, which differentiates between subtypes y and d. The digest results agreed with PCR in 83.3% of the samples. In addition, we compared our standard phenol/chloroform extraction against a rapid one step method. The phenol/chloroform stage was found to be essential for the removal of nucleases and polymerase inhibitors present in sera.

Base Sequence↗
PubMed1992

Maternal antibodies to gp120 V3 sequence do not correlate with protection against vertical transmission of human immunodeficiency virus.

A retrospective study of sera from mothers infected with human immunodeficiency virus (HIV-1) was undertaken to investigate whether the titers or affinities of antibodies against the third hypervariable region (V3 loop) of gp120 correlated with transmission of the virus from mother to child. The cohort comprised 7 mothers who transmitted HIV-1 to their children and 20 who did not. Sera were screened for reactivity against two synthetic peptides, one encompassing the entire V3 loop of gp120 (amino acids 297-330) and the other containing an immunodominant epitope from gp41 (amino acids 596-614). Doubling dilutions of sera were tested to obtain antibody titers against both peptides: Anti-gp41 titers were used to normalize the anti-V3 titers. Maternal sera were also screened for the presence of high-affinity antibodies against the V3 peptide. No differences were observed in either titers or affinities of maternal antibodies to the V3 sequence from transmitters and nontransmitters.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Analysis of a new hepatitis C virus type and its phylogenetic relationship to existing variants.

Sequences obtained in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) were obtained from Scottish blood donors and compared with previously published HCV sequences. Phylogenetic analysis revealed the existence of three distinct groups of sequences; two of these corresponded to the recently described HCV types 1 and 2 variants, while viral sequences detected in around a third of the blood donors formed a separate phylogenetic group that probably represents infection with a novel virus species. Nucleotide sequences of this latter group differed from all previously published 5'NCR sequence variants by at least 9%. This new virus type also differed considerably from previously published variants in other regions of the viral genome (core, NS-3 and NS-5), with corrected nucleotide distances of 15, 43 and 49% respectively from the prototype HCV-1 sequence. Formal phylogenetic analysis of each of the coding regions confirmed that HCV type 1 variants could be clearly differentiated into regional variants (Far East and U.S.A./European), in contrast to the clearly overlapping geographical distributions of the main HCV types in U.K. blood donors. We discuss the evidence for and against the hypothesis that the three main phylogenetic groups identified in this study represent separate species of HCV.

Amino Acid Sequence↗
PubMed1992

Use of several second generation serological assays to determine the true prevalence of hepatitis C virus infection in haemophiliacs treated with non-virus inactivated factor VIII and IX concentrates.

To investigate the prevalence of hepatitis C virus infection in two risk groups, stored serum samples from treated haemophiliacs and intravenous drug users were tested for anti-HCV by both anti-C-100 based and second generation ELISAs (Abbott and Ortho) followed by testing in two confirmatory immunoblot assays that incorporate core as well as other non-structural antigens (Innogenetics LIA and Chiron RIBA-HCV test). Clear evidence of HCV infection was found in all but one of 78 haemophiliacs treated with non-virus inactivated clotting factor concentrates, but in none exposed only to super dry heat-treated concentrates. Only four samples gave rise to conflicting serological results between the four tests, two of these occurred in patients with advanced HIV related disease and almost certainly reflected loss of humoral immunity associated with disease progression, and the others occurred in the only two patients tested who were chronic carriers of hepatitis B infection and may reflect an interaction between the two viruses. Comparison of anti-C-100 versus second generation tests in immunocompetent drug users revealed a false negative rate of 20% using C-100 alone, indicating the advantage of using second generation assays for detection of past or current HCV infection. Of all of the antigens used in the confirmatory assay, positive sera showed strongest and most frequent reactivity with the C22 and C33c proteins (Ortho RIBA).

Drug Contamination↗
PubMed1992

Hepatitis C viraemia in United Kingdom blood donors. A multicentre study.

Of 10,633 blood donations tested in three regional blood transfusion centres with two commercial first generation screening assays for antibodies to the hepatitis C virus (HCV), 65 (0.61%) were found to be repeatedly reactive in one or both assays. Five of the 65 were confirmed positive by recombinant immunoblot assay (Ortho RIBA-2) and a further 4 were judged indeterminate. All 5 RIBA-2 positive donations and 1 of the 4 RIBA-2 indeterminates were shown to be viraemic by HCV-RNA polymerase chain reaction (PCR) assays performed at three independent reference laboratories. The remaining 56 screen test reactive donations proved negative by RIBA-2 and, with 1 exception, negative by PCR. We conclude that while first generation anti-HCV screening assays generate a high proportion of false reactions when screening low prevalence populations, results of the RIBA-2 confirmatory test correlate well with PCR findings and thus indirectly with both hepatitis C viraemia and infectivity.

Base Sequence↗
PubMed1991

Determinants of HIV disease progression: six-year longitudinal study in the Edinburgh haemophilia/HIV cohort.

Markers of immune function present before infection may determine the subsequent course of disease in HIV-infected individuals. In 1983, we measured immune function in a group of haemophiliacs in Edinburgh. In 1984, 18 of these patients became infected with HIV-1 from contaminated factor VIII. We have followed-up these patients since their seroconversion. The rate of disease progression, as assessed by the appearance or not of AIDS symptoms or signs within five years of seroconversion, was related both to the concentration of total plasma IgM before exposure to infection and to the pattern of specific IgM and IgA anti-HIV response around the time of IgG seroconversion. Disease progression also correlated with concentrations of plasma interleukin-2 receptor (a marker of lymphocyte activation) and with the number and percentage of circulating DR + ve (activated) T cells. Our findings show that the extent of host immune reactivity, which may be genetically determined, is a powerful factor in the pathogenesis of HIV-associated disease.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Detection, quantification and sequencing of HIV-1 from the plasma of seropositive individuals and from factor VIII concentrates.

A highly sensitive and reliable RNA polymerase chain reaction method has been developed which has been used to detect, quantify and sequence cell-free HIV RNA directly from the plasma of seropositive individuals. Plasma from 10 out of 12 haemophiliacs tested was found to contain detectable levels of HIV-1 RNA [log mean value: 1.2 x 10(3) copies for Centers for Disease Control (CDC) group II patients, 5.5 x 10(3) copies for CDC group IV patients]. The presence of cell-free circulating virus in both symptomatic and asymptomatic individuals suggests that viral replication continues throughout the course of infection. The same procedure has been used to detect and sequence HIV-1 RNA in two batches of unheated commercial factor VIII concentrate distributed in 1981 and 1983. The sequences obtained revealed a closer relationship to North American than to African strains of HIV-1.

Amino Acid Sequence↗
PubMed1991

Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis.

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Hepatitis C quantification and sequencing in blood products, haemophiliacs, and drug users.

The polymerase chain reaction (PCR) detected specific hepatitis C viral (HCV) RNA sequences in plasma from 15 of 21 haemophiliacs (12 HCV-antibody positive) and 7 of 27 intravenous drug users (13 HCV-antibody positive). Quantification of RNA-positive samples showed high levels of HCV (10(5) to 10(6) copies of RNA/ml) in infected patients. HCV was more frequently found in haemophiliacs infected with human immunodeficiency virus (11/11 HIV-positive and 4/10 HIV-negative patients). HCV-RNA was detected in all batches of commercially available factor VIII tested and in low concentrations in some pools of plasma donations from volunteers. Factor VIII, manufactured from volunteer donations, was uniformly negative by PCR. Phylogenetic analysis of viral sequences showed two distinct groups: one was associated with intravenous drug users and the other with haemophiliacs infected with Scottish factor VIII preparations. Both were distinct from sequences found in commercially available factor VIII.

Acquired Immunodeficiency Syndrome↗
PubMed1990

First coitus before menarche and risk of sexually transmitted disease.

The prevalence of sexually transmitted disease (STD), pelvic inflammatory disease (PID), and cervical cancer, and the relation between these conditions were studied in 2111 Ethiopian women. Early sexual activity was associated with an increase in prevalence rates of STD and PID; possible aetiological factors include physical and immunological immaturity of the female genital tract and the number of sexual partners.

Adolescent↗
PubMed1990

The polymerase chain reaction in the diagnosis of vertically transmitted HIV infection.

The presence of HIV-1 DNA sequences in DNA from peripheral blood mononuclear cells (PBMCs) was investigated in a two-stage polymerase chain reaction ('double' PCR) using four sets of nested primers. The PBMCs tested were obtained from 46 children born to HIV-seropositive mothers, seven 'control' children born to HIV-seronegative mothers and seropositive fathers, and 45 healthy adult blood donors who were HIV seronegative. Nine of the children had symptomatic HIV infection and other laboratory features characteristic of HIV infection: all nine were PCR-positive with each set of primers in each of their 22 blood samples tested. The remaining 44 children had no clinical or laboratory evidence of HIV infection, and each of their 50 samples was PCR-negative with each set of primers, as were all blood donor samples. PCR-positive samples were tested in more detail using two of the sets of primers, which spanned hypervariable regions in the env gene. Polyacrylamide gel electrophoresis of DNA amplified from these regions yielded patterns of amplified DNA length variation which were characteristic for each child, and which changed little with time (in serial samples obtained over periods of 3-7 months). This excluded contamination as a cause of PCR positivity. This is the first report of the use of a double PCR for the diagnosis of HIV infection. The results demonstrate the specificity of this PCR method in diagnosis, with failure to reveal in this cohort any cases of vertically transmitted HIV-1 infection in addition to those already confirmed by conventional laboratory techniques.

Child, Preschool↗