Treatment of CMV retinitis.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
B Wahren
Publications and source records attributed to B Wahren.
Identification of human neutralization-inducing regions of the human immunodeficiency virus type 1 envelope glycoproteins.
Four major neutralizing regions of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein were identified and characterized with a panel of 80 HIV-1 antibody-positive human sera. Levels of neutralizing antibodies against the HIV-1 strains IIIB, SF2, and RF were compared with reactivity in ELISAs against peptides that correspond to certain regions of the HIV-1 envelope. A correlation between high neutralizing activity and strong seroreactivity against specific peptides suggested that the corresponding regions might be involved in neutralization. This was further substantiated by using peptides to inhibit neutralization by a panel of 10 HIV-1 antibody-positive sera. The positions of three neutralizing sites, defined earlier mostly by antisera from animals, were confirmed in the present study. Human sera thus recognize the strain-specific third variable region of gp120 (amino acids 304-318), the C-terminal end of gp120 (amino acids 489-508), and the conserved region in the intracellular part of gp41 (amino acids 732-746). It is likely that these different regions mediate help rather than self-sufficient neutralization. Furthermore, a human neutralizing region was detected in a conserved part of gp41 (amino acids 647-671). Accordingly, neutralizing antibodies directed to this region were found to be cross-reactive between HIV-1 strains. Peptides corresponding to these four regions were able to inhibit neutralization mediated by serum from HIV-1 antibody-positive individuals. These results indicate that this conserved B-cell epitope of the HIV-1 envelope elicits a virus-neutralizing antibody response during natural infection in humans and may therefore be considered for inclusion in a vaccine against HIV-1.
Amino Acid Sequence↗
Immunoglobulin G subclasses and lymphocyte stimulatory responses to cytomegalovirus in transplant patients with primary cytomegalovirus infections.
The early activation of T- and B-cell responses to cytomegalovirus was studied in immunosuppressed patients. Primary lymphocyte stimulation to cytomegalovirus (CMV) antigen, a measure of T-helper activity, and anti-CMV IgG subclass responses were analyzed. Ten patients suffering from primary CMV infection following renal transplantation were studied. Of the ten, nine became positive for CMV induced lymphocyte proliferation 5-40 weeks after transplantation. Nine showed an almost simultaneous appearance of anti-CMV IgG1 and three at 3-32 weeks after transplantation, while one patient synthesized only low levels of anti-CMV IgG1. The lymphocyte proliferation assays have limited diagnostic value for primary CMV infection in renal transplant patients. The humoral and cellular immune responses seemed to be independent of each other.
Adolescent↗
Immunoglobulin V regions and epitope mapping of a murine monoclonal antibody against p24 core protein of HIV-1.
The nucleotide sequence of a murine monoclonal antibody (CB-mab-p24/13-5) against p24 core protein of the human immunodeficiency virus (HIV-1) was determined for variable regions of the heavy and light chain, respectively. Genetic elements encoding the VDJH- and VJL-regions of the antibody were generated from RNA by the polymerase chain reaction, cloned into the vector pICEM 19R and sequenced. Synthetic peptides, 10 amino acids overlapping served for the localization of the epitope. The residues 152-156 within the p24 sequence contain the epitope.
Amino Acid Sequence↗
Immune response to a single peptide containing an immunodominant region of hepatitis C virus core protein: the isotypes and the recognition site.
We have used one single peptide covering the 17 N-terminal amino acids of the hepatitis C virus (HCV) core protein (c) to analyse the human immune response against B-cell epitope(s) within this region. The sequence MSTNPKPQRKTKRNTNR was obtained from two sequenced HCV genomes, and the peptide was synthesized by a newly developed method. The peptide was assayed with 144 human sera which had all been assayed for antibodies to HCV (anti-HCV) using commercial assays. Forty-nine sera were found to be positive for anti-HCV using these assays; 40 of these were found to be positive with our anti-HCV IgG peptide assay. The class (IgM, IgG) and subclass (IgA1, IgG1-4) specific reactions were determined using the polyclonal and monoclonal anti-HCV peptide enzyme immunoassays. Isotypes of mainly IgG1 and IgG3, but also IgG4, IgM and IgG2, gave specific reactions with this region. Using omission peptide analogues of the region 1-18, the sequence RKTKRNTN within residues 9-16 was common to 34 out of 37 sera of which the IgG antibody binding site could be mapped. It is unusual for a single peptide assay to have such high sensitivity since B cell epitopes within a protein are often discontinuous. It seems that at least 80% of HCV infected individuals develop antibodies of various isotypes to the antigenic site RKTKRNTN, located in the N-terminal portion of the HCV core. Thus, the immune response to this peptide should be further investigated with regard to the reactive Ig isotypes developing during HCV infection.
Amino Acid Sequence↗
Early appearance of simian immunodeficiency virus (SIV) antigen and antibodies as variables in evaluating antiviral drugs in macaques.
SIV infection in macaques has become an important animal model for HIV-1 infection in humans. An antibody assay was therefore developed and compared to a commercially available antigen assay with respect to their usefulness to monitor the course of simian immunodeficiency virus (SIV) infection in cynomolgus monkeys. A peptide, JB6T, consisting of 21 amino acids with the sequence NSWGCAFRQVCHTTVPWVNDS corresponding to a segment in the env protein of human immunodeficiency virus (HIV) type 2 was used as antigen in an enzyme-linked immunosorbent assay (ELISA). JB6T was found to detect IgG and IgM antibodies to viral antigens with high specificity. The earliest anti-SIV IgM antibodies were detected at days 13-16, with a maximum at day 20 and subsequently the levels fell. Specific IgG antibody levels increased at day 16-20 after SIV infection and reached a plateau at day 60. The commercially available HIV-1 p24/26 antigen test could, due to cross-reactivity, be employed to detect SIV antigen delay, peak and duration.
Amino Acid Sequence↗
Loss of synergistic response to combinations containing AZT in AZT-resistant HIV-1.
The use of combination chemotherapy for the treatment of HIV-1 infection offers promise for overcoming the problems of toxicity and development of resistance. Primary HIV-1 isolates from three patients before and after treatment with azidothymidine (AZT) were examined for sensitivity to AZT and synergistic response to three different combinations of drugs: AZT+fluorothymidine (FLT), AZT+dideoxyinosine (ddI), and FLT+ddI. All three patients initially harbored AZT-sensitive virus, but posttherapy isolates were resistant to AZT. The pretreatment, AZT-sensitive virus from each patient showed synergistic inhibition by the combinations of AZT+FLT, AZT+ddI, and FLT+ddI. In contrast, the posttreatment, AZT-resistant virus showed only addition or antagonism by the combinations containing AZT, whereas the synergistic response to the combination of FLT+ddI was preserved. Our study argues for early intervention with combination chemotherapy, since after development of resistance, AZT no longer showed synergy but addition or antagonism in combination with other drugs. After resistance to AZT has developed, combination chemotherapy not involving AZT may offer advantages over continued mono- or combination therapy involving AZT.
Antiviral Agents↗
Cytomegalovirus infection of the central nervous system in patients with AIDS: diagnosis by DNA amplification from cerebrospinal fluid.
A nested polymerase chain reaction (PCR) was evaluated for the detection of cytomegalovirus (CMV) DNA in cerebrospinal fluid (CSF). CSF and serum samples from 19 AIDS patients with intracerebral CMV infection diagnosed at autopsy were retrospectively examined. As controls, CSF and serum samples from 15 AIDS patients with only extracerebral CMV involvement at autopsy, from 10 AIDS patients without CMV infection at autopsy, and from 10 anti-human immunodeficiency virus-negative patients without ongoing CMV infection, were studied. CMV DNA was detected from patients with intracerebral CMV infection in 9 of 9, 5 of 6, and 1 of 4 CSF samples collected, respectively, 1-30, 30-90, and 90-300 days before death. Twelve of 13 sera from these patients were CMV PCR-positive. None of the control patients had CMV DNA in CSF. PCR was positive in 6 of 8 sera from AIDS patients with only extracerebral CMV infection and in serum from 1 AIDS patient without CMV involvement at autopsy. CMV PCR on CSF is highly sensitive and specific. It should be considered a rapid and reliable diagnostic method for CMV infection of the central nervous system.
AIDS-Related Opportunistic Infections↗
Dynamic changes in HIV-1 quasispecies from azidothymidine (AZT)-treated patients.
The emergence of azidothymidine (AZT)-resistant human immunodeficiency virus (HIV) variants in clinical samples was studied by a direct genomic sequencing method. Sequential lymphocyte samples from four patients, who had been treated with AZT for up to 27 months, were shown to gradually accumulate multiple nucleotide changes, some of which are known to be associated with AZT resistance. Several samples were shown to contain mixtures of wild-type and mutated genomes, indicating gradual rather than sudden changes in the HIV-1 quasispecies. These results demonstrate for the first time that automated solid-phase DNA sequencing is a rapid and useful tool for investigation of antiviral drug resistance and suggest that DNA sequencing may be important in routine clinical diagnostics in the future.
Acquired Immunodeficiency Syndrome↗
Patterns of immunoglobulin G subclass reactivity to HIV-1 envelope peptides in children born to HIV-1-infected mothers.
OBJECTIVE: To analyse the pattern of immunoglobulin (Ig) G subclasses specific for HIV-1 envelope peptides in sera from HIV-1-infected mothers and their newborns. We sought to determine whether there was a selective transfer of antibodies from mother to child, and to establish diagnostic or prognostic properties by analysing HIV-1 peptide-specific IgG isotypes. DESIGN AND METHODS: Parallel sera from 12 HIV-1-infected mothers and their newborn children were analysed for IgG subclass responses to six HIV-1 envelope peptides by enzyme-linked immunosorbent assay. Levels of IgG were compared with levels of IgG1 and IgG3 to an immunodominant gp41 peptide in children's sera obtained during the first months of life. In a longitudinal study of 16 children born to HIV-1-seropositive mothers, of whom 11 were infected, the IgG1 and IgG3 responses to peptides representing an immunodominant epitope of gp41 and the principal neutralizing determinant of the gp120 V3 region were analysed. RESULTS: IgG1 and IgG3 were found to constitute the predominant peptide-specific antibody responses. A parallel distribution of IgG subclass reactivity was seen in maternal and paediatric sera. There was no evidence of selective antibody transfer. Comparable levels of IgG and IgG1 to the immunodominant peptide were seen in infected and uninfected children, while the IgG3 had disappeared in the majority of uninfected children. A decrease in peptide-specific IgG1 and IgG3 levels was observed in sequential sera from uninfected children, although the kinetic profile varied. Sera from infected children showed de novo synthesis of IgG1 and/or IgG3 binding to the selected HIV-1 peptides. Most children with rapid disease progression failed to produce IgG1 and/or IgG3 to the V3 peptide. CONCLUSION: Analysis of IgG subclass kinetics to selected HIV-1 peptides might be a useful additional diagnostic and prognostic tool in evaluating HIV-1 infection in children born to seropositive mothers.
Adult↗
Inhibition of viral replication by monoclonal antibodies directed against human immunodeficiency virus gp120.
Monoclonal antibodies (MAbs) were raised against the glycoprotein gp120 of human immunodeficiency virus type 1 (strain HTLV-IIIB). The reactivity of five selected MAbs was characterized in several tests: ELISA, immunostaining of Western blots, immunofluorescence, immunoprecipitation, immunoelectron microscopy, alkaline phosphatase-anti-alkaline phosphatase assay and neutralization. The binding region was delimited by sequential overlapping Escherichia coli fusion proteins of the gp120 sequence between amino acids (aa) 49 and 280. In the ELISA, when using sequential overlapping 15 aa peptides, the binding epitopes were localized between aa 64 and 78 for three MAbs and between aa 114 and 123 for the fourth Mab. The fifth Mab showed multiple reactions with different peptides possibly indicating a reaction with a discontinuous epitope. In virus growth inhibition assays, all five MAbs inhibited the spread of HIV-1 infection in cell cultures after a single or repeated treatment at a concentration of 63 micrograms/ml of the purified MAbs. All MAbs showed low but significant neutralizing activity at concentrations of 100 micrograms/ml.
Amino Acid Sequence↗
Human MoAbs produced from normal, HIV-1-negative donors and specific for glycoprotein gp120 of the HIV-1 envelope.
Human MoAbs of IgM class were developed against three regions of the HIV-1 envelope. Uninfected donor lymphocytes were immunized in vitro with recombinant protein pB1. Four out of five antibodies were directed to different parts of the V3 region, which contains a major neutralizing site. Two out of these antibodies were directed to more than one amino acid sequence, indicating reactivity to discontinuous sites. Two of the human MoAbs inhibited viral spread between cells in tissue culture, interpreted as reactivities to conserved amino acid sequences exposed during viral maturation. No MoAb neutralized virus, which may be explained by the relatively low avidity of the antibodies. One MoAb was directed to a region containing amino acids participating in CD4 binding. This technique appears to allow formation of antibodies with fine specificities other than those obtained in infected hosts.
Amino Acid Sequence↗
Penetration of zidovudine and 3'-fluoro-3'-deoxythymidine into the brain, muscle tissue, and veins in cynomolgus monkeys: relation to antiviral action.
Cynomolgus monkeys had microdialysis probes implanted under ketamine anesthesia into peripheral veins, thigh muscles, and the brain in order to sample the extracellular fluid for the concentrations of unbound nucleoside analogs. A dose of 25 mg of zidovudine or 3'-fluoro-3'-deoxythymidine (FLT) per kg was administered subcutaneously to each of three animals. Relatively high antiviral concentrations of FLT and zidovudine were present in peripheral tissues and in the brain. It was found that the concentration of zidovudine in the brain was approximately one-third of that in muscle and veins; the same relation was observed for FLT. The in vivo unbound concentrations of both drugs in the brain, muscle, and venous blood exceeded those reported to inhibit human immunodeficiency virus replication in vitro. In addition, in a correlative study we found that the appearance of p24 antigen in sera of monkeys infected with simian immunodeficiency virus was significantly delayed by both compounds (15 mg/kg three times daily for 9 days after infection). Thus, we have shown that the extracellular concentrations of unbound FLT and zidovudine in the brain and peripheral tissues attained with in vivo antiviral doses exceed in vitro antiviral concentrations.
Animals↗
Antibody responses to synthetic peptides from cytomegalovirus phosphoprotein 150.
We have identified antigenic regions within phosphoprotein 150 of human cytomegalovirus (CMV pp150) to which seroreactivity appears in patients with active CMV infection or persists in seropositive persons. A range of 8.3 to 61.6% of healthy CMV-seropositive blood donors were immunoglobulin G positive for single peptides, while 91.6% reacted to a mixture of four peptides. All convalescent-phase serum samples from 26 patients with active CMV infection reacted with either of two peptides encompassing amino acids (aa) 594 to 623 and aa 614 to 643. Patients with a primary CMV infection had patterns of reactivity to single peptides different from those of patients with reactivated CMV infection. The immunoglobulin M antibodies reacted preferentially with the peptides encompassing aa 594 to 663 of CMV pp150.
Amino Acid Sequence↗
Variations in the cytomegalovirus major immediate-early gene found by direct genomic sequencing.
An assay to detect and sequence DNA from human cytomegalovirus (HCMV) immediate-early gene region 1 has been developed; it involves in vitro amplification by the polymerase chain reaction and direct solid-phase sequencing of the amplified material. Urine samples from 16 patients tested positive for HCMV DNA in both a colorimetric assay for the detection of immobilized amplified nucleic acids and a standard polymerase chain reaction assay with agarose gel electrophoresis. Ten urine samples from healthy people tested negative in the same assays. Analysis of 106-bp fragments from seven patients and two laboratory HCMV strains (Ad 169 and Towne) demonstrated that the viral sequences were conserved in samples collected at different times from the same patient and in tissue-cultured samples. Two of the patient strains had variations in the amplified region, with a total of seven nucleotide substitutions yielding five amino acid alterations in the coding sequence.
Amino Acid Sequence↗
Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.
Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.
Antibodies, Viral↗
Immunodominant regions within the hepatitis C virus core and putative matrix proteins.
The complete amino acid sequences of hepatitis C virus (HCV) core (residues 1 to 115) and putative matrix (residues 116 to 190) proteins were synthesized as 18-residue-long peptides with an 8-amino-acid overlap. The peptides were assayed with 50 human serum samples with antibodies to HCV (anti-HCV) and 46 serum samples without anti-HCV, as determined by several commercial assays. Immunodominant regions were defined within residues 1 to 18, 11 to 28, 21 to 38, 51 to 68, and 101 to 118. The peptides that covered these regions were recognized by 40 of 50 (80%), 42 of 50 (84%), 36 of 50 (72%), 34 of 48 (68%), and 36 of 48 (72%) of the anti-HCV positive serum samples, respectively. Two anti-HCV negative serum samples were each repeatedly reactive with one peptide, but both were found to be negative by confirmatory anti-HCV assays. Four serum samples that were confirmed to be positive for anti-HCV in commercial assays did not recognize any of the peptides that cover the HCV core-matrix regions. Ninety-two percent of anti-HCV-positive serum samples reacted with a combination of peptides covering residues 1 to 18 and 11 to 28. Testing of peptides that contain the reported genotypic variations of the HCV core within the regions at residues 1 to 18, 51 to 68, and 101 to 118 showed that a change from Thr-110 to Asn-110 decreased the reactivities of eight serum samples. In conclusion, we found that human antibodies to the HCV core-matrix protein(s) are mainly directed to linear determinants and can easily be reproduced by using short synthetic peptides. We also found that such antibodies develop in more than 90% of HCV-infected people.
Amino Acid Sequence↗
Persistence of azidothymidine-resistant human immunodeficiency virus type 1 RNA genotypes in posttreatment sera.
The rate of reversion from azidothymidine (zidovudine; AZT) resistance was studied by direct sequencing of human immunodeficiency virus type 1 (HIV-1) virion RNA in sera from four patients who discontinued long-term treatment. Before cessation of treatment, all four patients harbored HIV-1 with multiple mutations reported to confer AZT resistance. In three patients, slow reversions of these mutations starting after 9, 9, and 18 months were detected. The slow reversions indicate that AZT-resistant HIV-1 variants are likely to have an unaltered replicative capacity and pathogenic potential. Furthermore, there were discrepancies between the in vivo RNA sequences and the sequences of virus isolates, indicating that the isolation procedure may select for nonrepresentative virus variants.
Acquired Immunodeficiency Syndrome↗