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Biomedical subjects

B Wahren

Publications and source records attributed to B Wahren.

At least 181 records · Page 10Linked to original sources
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

Induction of MHC class I antigen expression following infection of a human esthesioneuroblastoma cell line with cytomegalovirus and human immunodeficiency virus.

Productive infections with cytomegalovirus (CMV) and human immunodeficiency virus (HIV) were established in the Tp41ON cell line derived from a human esthesioneuroblastoma. HIV antigen expression was highest in cultures coinfected with CMV and HIV. Viral infection caused increased MHC class I antigen expression while class II and CD4 antigens remained undetectable using immunofluorescence methods. Uninfected cultures showed 10% and coinfected cultures 80% class I antigen positive cells. In coinfected cultures, CMV and HIV antigens were detected in 4% and 8% of the cells, respectively. The detection of CMV antigens in some multinucleated cells suggests coinfection with both viruses in these cells, as multinucleated cells were not found in cultures infected with CMV only. The study shows that a cell line showing neuronal differentiation in vitro can be infected with CMV and HIV and that this infection increases MHC class I antigen expression.

Antigens, Neoplasm↗
PubMed1992

Identification of B-cell antigenic sites on HIV-2 gp125.

Synthetic peptides were used to identify continuous antigenic sites on the external envelope glycoprotein gp125 of human immunodeficiency virus (HIV)-2. Initially, seven HIV-2-positive human serum samples were screened with 172 sequential nonapeptides containing a six-amino-acid overlap. This represents the entire gp125 molecule of HIV-2ISY. The antibody reactivity was found to be mainly restricted to 14 regions within gp125. Following these results, 33 longer peptides, 15-24 amino acids in length, were synthesized and tested against a larger number of samples. Eleven antigenic regions were thus identified. Two of these were detected within a region corresponding to the C1 region and four others within a region corresponding to the C2 region of HIV-1. The highest frequency of reactivity (90%) of 31 HIV-2 seropositive human serum samples was elicited by three peptides from a region corresponding to the V3 region of HIV-1. The C-terminal portion of this region was recognized by almost 80% of the samples. Reactive regions corresponding to the V4, V5, and N-terminal portion of V4 were also identified. A mouse monoclonal antibody reacting with gp125 was mapped to the N-terminal region of the molecule and was found to react with the sequence DVWNLFETS. The peptides were used to evaluate the antibody response of monkeys immunized with whole killed HIV-2 or simian immunodeficiency virus (SIV). The monkeys showed a pattern of reactivity similar to HIV-2-infected human serum samples. Postinfection samples from monkeys inoculated with HIV-2 or SIV reacted mainly to peptides from the V3 region. Two peptides were used to detect the seroconversion of two SIV-infected monkeys. Thus, we have demonstrated that human seroreactivity to HIV-2 gp125 occurs at a few distinct linear antigenic sites distributed at similar positions on the molecule as those in HIV-1 gp120.

Amino Acid Sequence↗
PubMed1992

Cooperativity of neutralizing antibodies directed against the V3 and CD4 binding regions of the human immunodeficiency virus gp120 envelope glycoprotein.

Human immunodeficiency virus type 1 (HIV-1) infection elicits neutralizing antibodies directed against two discrete regions of the gp120 exterior envelope glycoprotein: the third variable (V3) loop and the CD4 binding region. Monoclonal antibodies directed against these two regions demonstrated additive or, in some cases, weakly synergistic neutralization of HIV-1 infection. Cooperativity in virus neutralization was also observed for some gp120 mutants that, in the absence of anti-V3 loop antibodies, were relatively resistant to neutralization by antibodies directed against the CD4 binding region. Although the binding of some anti-V3 region monoclonal antibodies increased the recognition of the multimeric envelope glycoproteins by anti-CD4 binding antibodies, this enhanced binding was not predictive of the degree of cooperativity observed in virus neutralization. These results suggest that elicitation of both types of neutralizing antibodies should increase the efficacy of vaccine preparations.

Animals↗
PubMed1992

Lack of association between maternal antibodies to V3 loop peptides and maternal-infant HIV-1 transmission.

We investigated the association between maternal antibodies to HIV-1 peptides in pregnant women and the acquisition of HIV-1 infection by their offspring. Pregnant HIV-1-infected Haitian women were tested for the presence of antibodies against peptides of 14-17 amino acid length from the V3 loop region of strains IIIb and MN. Antibody testing was performed in two separate laboratories by enzyme-linked immunosorbent assay (ELISA). Peptides from four regions of the V3 loop were synthesized in several different laboratories and the purity confirmed by high performance liquid chromatography (HPLC). The mothers of infants who acquired HIV-1 infections did not differ significantly from the mothers of uninfected infants in the prevalence or concentration of antibodies to any of the 15 peptides evaluated. Additional studies are indicated to determine if neutralizing antibodies or other immunologic parameters are associated with maternal-infant HIV-1 transmission.

Amino Acid Sequence↗
PubMed1991

A cyclized peptide for studies of human parvovirus B19 infection.

Synthetic peptides corresponding to human parvovirus B19 sequences were evaluated for immunoreactivity with the sera of infected persons. A cyclized peptide deduced from the N terminus of viral protein VP2 and containing the amino acids 284-307 showed a high reactivity with IgM class antibodies when comparing seropositive and seronegative sera.

Antibodies, Viral↗
PubMed1991

Production of a murine monoclonal antibody with anti-HIV-1 neutralizing properties.

A mouse monoclonal hybridoma cell line producing IgG 1k to human immunodeficiency virus (HIV-1) gp120 envelope protein was cultured in several systems. A small-scale flask culture was essential for characterizing the culture variables of the hybridoma. A dialysis tubing culture appeared to be an excellent alternative to in vivo cultures of ascitic fluid, and gave high mouse monoclonal antibody (Mab) concentrations. Two continuous culture systems were both very effective in producing large amounts of Mabs. The hollow fiber system has the advantage of giving a concentrated product in the harvest. The ceramic core system, on the other hand, allows excellent monitoring of the cellular growth and production phases and gave the highest HIV antigen reactivity/micrograms of the produced IgG. Twelve grams of HIV-1 neutralizing Mabs were produced. The Mab was purified with a yield of 61%. The neutralizing capacity of the Mab was studied in vitro and shown to be excellent with 50% neutralizing titers using 5 ng Mab. The biological half-life of the Mab given intravenously to an HIV-infected individual was shown to be around 30 h.

Animals↗
PubMed1991

Characterisation of a linear binding site for a monoclonal antibody to hepatitis B core antigen.

The complete amino acid (aa) sequence of the hepatitis B virus (HBV) core protein (HBcAg), ayw subtype, was synthesized as decapeptides with five overlapping aas. The peptides were tested for reactivity with monoclonal antibodies (mAbs) to HBcAg (35/312, 37/275, and 7/275). All the mAbs specifically inhibited human anti-HBc by cross competition in assays for anti-HBc and anti-HBe. The mAb 35/312 recognised a peptide covering residues 76-85 of the HBcAg sequence. The other two mAbs did not react specifically with any linear peptide, suggesting discontinuous epitopes for these mAbs. The linear sequence EDPASR at residues 77-82 was found to constitute the epitope for mAb 35/312 when fine mapping the binding site. The most essential aas for mAb 35/312 were found to be the DP at residues 79-80, when peptides were synthesized where the aas at 77-83, were substituted by the other 19 aas. Since the mAb 35/312 inhibits the binding of human anti-HBc positive sera, which are known to recognise an SDS labile epitope, the sequence 77-82 might be a part of a larger discontinuous epitope. Alternatively the mAb 35/312 blocks the binding of human anti-HBc by steric hindrance.

Amino Acid Sequence↗
PubMed1991

Synergistic combinations and peptides in the inhibition of human immunodeficiency virus.

Various combinations of inhibitors of HIV reverse transcriptase were tested for inhibition of HIV replication in order to reveal any potential synergism or antagonism. PFA, a pyrophosphate analogue, gave synergistic inhibition of HIV replication in combination with both of the thymidine analogues AZT and FLT. The combination of PFA and AZT-TP gave only additive or weakly synergistic inhibition in a reverse transcriptase enzyme assay. The combination of AZT and FLT also gave synergistic inhibition of HIV replication, whilst the combination of AZT-TP and FLT-TP gave only additive or weakly synergistic inhibition of reverse transcriptase. Thus, the synergy does not arise from effects on reverse transcriptase alone but must be owing to other, cellular factors, such as effects on nucleoside metabolism or metabolism of the analogues. The results are consistent with the hypothesis that AZT may have an alternative mechanism of inhibition other than inhibition of reverse transcriptase. The diminished cytotoxicity observed in addition to the synergistic inhibition makes these combinations attractive from the point of view of combination chemotherapy. The inhibition of HIV replication by peptides from various parts of the V3 region of gp120 whose sequences were homologous with the tryptase inhibitor trypstatin was tested. Inhibitory activity was displayed by two peptides containing cysteine in their sequence. Antibodies to two peptides containing the two conserved cysteine residues from opposite sides of the neutralizing loop of gp120 were previously associated with protection from vertical transmission of HIV. The V3 region thus seems to be important for the function of gp120 and the transmission of HIV.

Alpha-Globulins↗
PubMed1991

Human and murine B-cells recognize the HBeAg/beta (or HBe2) epitope as a linear determinant.

The complete amino acid (aa) sequence of the hepatitis B virus (HBV) core protein (HBcAg), deduced from the genome of the HBV ayw subtype, was synthesized as decapeptides with five overlapping aas. The peptides were tested for reactivity with monoclonal antibodies (mAbs) to the beta (or HBe2) epitope of hepatitis B e antigen (HBe/b mAbs; 57/8, 78/3, 141/158 and 141/207). Cross-competition between the mAbs with a mAb to the HBe/alpha epitope (or HBe1) and an anti-HBc mAb showed that all the HBe/b mAbs specifically inhibited human anti-HBe/b binding. Screening the HBc/e peptides showed that all anti-HBe/b mAbs recognized a peptide covering the residues 126-135. Three of the mAbs, 78/3, 141/152 and 141/207, had a less restricted reactivity than the other two, suggesting the recognition of the HBe/b as a discontinuous determinant. Fine mapping of the region aa 126-135 was performed by synthesizing decapeptides with nine overlapping aas, covering residues aa 121-140. All mAbs, except 78/3, reacted with the linear sequence TPPAYR, at residues 128-133. An additional set of peptides was synthesized, where the six aas within the epitope 128-133 were substituted in turn by the other 19 possible aas. By this approach, the essential aas for mAb 57/8 were found to be the sequence of PPA at residues 129-131, and for mAb 141/158 the sequence PP-Y, at residues 129, 130 and 132, respectively. Human recognition of the linear HBe/b epitope was investigated by using a peptide covering residues 121-140 (p 33). Thirty-one sera from chronic carriers of HBsAg, of which seven were positive for HBeAg and the remaining 24 for anti-HBe, were investigated. Of the sera with HBeAg, two had low levels of anti/-HBe/b in the p 33 assay. Out of the sera with anti-HBe, eight were positive in the p 33 EIA. Thus, murine monoclonals and human sera may recognize the HBe/b epitope as a linear determinant residing around aa 130.

Amino Acid Sequence↗