Human antibodies responsible for binding inhibition and polymerization inhibition of human immunodeficiency virus type 1 reverse transcriptase.

Using a solid-phase non-radioisotopic (non-RI) reverse transcriptase (RT) assay, antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) RT activity (RTI antibody) were investigated for their ability to inhibit binding of RT to a template-primer and DNA polymerization. The RTI antibody inhibited the binding of RT to the template-primer (BI antibody), and directly reacted with the RT-template-primer complex and inhibited enzymatic activity (PI antibody). The RTI antibody interfered with formation of the RT-template-primer complex suggesting that it recognized the antigenic site involved in template-primer binding of RT molecules. Since deoxynucleotide triphosphates (dNTPs) blocked inhibition of the RT activity by the PI antibody, the antigenic site recognized by the PI antibody may be closely related to the dNTP binding site. The seropositivities of the BI and PI antibodies were 84.6% and 91.2%, respectively, in HIV-1-infected individuals; healthy individuals, HTLV-I-positive individuals, autoimmune disease patients and leukemia patients were all seronegative. No significant correlation of residual RT activities was observed when BI and PI antibodies were compared (r = 0.688). It is possible that the epitopes recognized by the BI antibody differs from those recognized by the PI antibody. The assays described are able to detect BI and PI antibodies in the sera of HIV-1-infected individuals.

Expression of human metallothionein-2 in Escherichia coli: cadmium tolerance of transformed cells.

A genetic approach was undertaken to investigate the physiological roles of human metallothionein-2. A constructed expression plasmid, pEXPMTII, in which human metallothionein-IIA cDNA was inserted downstream of a tryptophan-lactose promoter, was used to transform Escherichia coli JM105 strain. Cadmium-binding metallothionein was successfully expressed in E. coli in the medium containing cadmium, while copper and zinc-metallothioneins were scarcely observed in copper- or zinc-containing medium. The amino acid composition and sequence of the biosynthesized cadmium-metallothionein were analyzed. The selectivity of metals bound to metallothionein and the stability of metal-binding forms of metallothionein in E. coli were discussed. In addition, cadmium, zinc, or copper resistance of the cells expressing metallothionein was examined. Cells transformed with the plasmid pEXPMTII and cultured in a medium containing cadmium exhibited tolerance only to cadmium. It was demonstrated that human metallothionein-2 functioned for cadmium detoxification in E. coli.

Comparison of the sensitivities of two non-isotopic reverse transcriptase (RT) assays for human immunodeficiency virus type 1 RT.

The sensitivities of two reverse transcriptase (RT) assays, an enzyme-linked oligonucleotide sorbent assay (ELOSA)-RT assay and a non-radioisotopic (non-RI) RT assay were compared. For measuring recombinant HIV-1 RT, the ELOSA-RT assay was 8 times less sensitive in dilution endpoint and 16 times less sensitive in measurement of RT from pelleted HIV-1 than the non-RI RT assay. Higher level of interference by an RNA-DNA hybrid observed in the former assay may indicate that the reduction in sensitivity was due to the presence of viral RNA in the sample of pelleted virus. The ELOSA-RT assay was interfered with to a great extent than the non-RI RT assay by fetal bovine serum and thus may be unsuitable for measuring RT from HIV-1 in a culture supernatant.

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

Synthesis and assignment of novel [125I]-labeled 1 alpha,25-dihydroxyvitamin D3 derivatives.

This paper describes the synthesis and structural assignment of noval 125I-1 alpha,25-dihydroxyvitamin D3 derivatives (1a) and (1b) labeled with 125I-Bolton-Hunter reagent [N-succinimidyl 3-(4-hydroxy-3-iodo[125I]phenyl)propionate] (1), which is known as a protein-labeling reagent, as tracers for radioimmunoassay (RIA). The radiospecific activities of these tracers (1a) and (1b) were calculated as 2,200 Ci/mmol (81.4 TBq/mmol).

[Studies for dynamics of reverse transcriptase inhibiting antibody in sera from HIV-1 infected individuals].

Antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) reverse transcriptase activity (RTI-antibody), Binding inhibition antibody (BI-antibody) and polymerization inhibition antibody (PI-antibody) were investigated for their ability to inhibiting RT activity in 248 HIV-1 infected individuals and 99 healthy individuals. In BI-antibody, high titer samples were determined more in than in RTI- and PI-antibodies. No significance was indicated between AC, ARC and AIDS is any antibody, however, progression from AC to AIDS was poled to high titer and low titer in RTI- and BI-antibodies. Moreover, time course of each antibody levels in the same infected individuals were resulted in no change, going up or down through all the experimental term, though all samples were collected in AC. These results were suggested that the determination factor of each stage in HIV progression would be multiple, and that the various dynamics of RTI-, BI- and PI-antibodies in the same infected individuals might be caused in the term from HIV infection to AIDS progression, prognosis or appearing of the drug resistant strain but stages of the disease.

[Clinical isolates of group B streptococci from elderly patients].

The importance of Group B streptococcal infection in elderly patients has not been clearly defined. We studied the annual incidence of Group B streptococci (GBS) isolated from sputum and urine of elderly patients who were admitted to Nagoyashi Koseiin Geriatric Hospital. The percentage of GBS isolated has increased since 1988. The type distribution of GBS isolates shows predominantly types Ia, Ib and JM9. The major clinical factors in patients from whom GBS was isolated from sputum were bedridden status, feeding tubes and tracheostomy. We also investigated the antibody to GBS in these elderly patients. The titers of GBS antibody were significantly higher in the group with GBS isolates than in the group without these pathogens. These results suggest that GBS is one of the causative organisms of nosocomial infections in elderly patients.

[Study of bacterial flora in the oral cavity and stomach of elderly patients receiving nasogastric tube feeding].

To investigate the significance of oropharyngeal flora and gastric flora in elderly patients receiving nasogastric tube feeding, throat secretions and gastric aspirates were cultured and the pH of the latter was measured. Of 116 bacterial isolates from throat secretions of 27 elderly patients, 30 were beta-streptococci and 28 were Pseudomonas aeruginosa. Bacteria isolated from gastric aspirates numbered 86 and 24 (27.9%) of them were the same species as those found in the throat secretions. Patients with gastric pH were below 3.5 had significantly lower concentrations of gram-negative bacili in gastric aspirates. We also studied oropharyngeal flora in 33 elderly patients who were admitted to Nagoyashi Koseiin Geriatric Hospital. The major bacterial isolates from throat swabs of bedridden patients were gram-negative bacilli and beta-streptococci, especially group B streptococci (GBS). We measured the level of antibody to GBS in these patients. Those from whom GBS were isolated had high titers. These results suggest that in elderly patients receiving enteral nasogastric) tube feeding, large numbers of bacteria colonize the oral cavity and stomach. The measurement of type-specific antibody to GBS may be useful in managing such patients.

An improved non-radioisotopic reverse transcriptase assay and its evaluation.

We developed an improved, highly sensitive non-radioisotopic (non-RI) reverse transcriptase (RT) assay (RTA). While the original non-RI method previously reported made use of primer immobilization, our improved method was based on a primer-template immobilization procedure. We tested the template specificity, reproducibility and linearity of the new method in assays of human immunodeficiency virus type-1 (HIV-1) RT. The sensitivities of the method previously reported, the improved method and the sensitive radioisotopic (RI-) RTA were compared in assays of recombinant HIV-1 RT, partially purified HIV-1 particles, and the culture supernatant derived from HIV-1-infected cells. For each of these samples except the culture supernatant the improved method was the most sensitive. It appeared that the fetal bovine serum presented in the culture medium interfered with the assay reaction. The curve describing inhibition of the assay reaction by fetal bovine serum showed that the highest degree of sensitivity in the assay was obtained when the culture supernatant sample was diluted four times. With this degree of dilution, the sensitivity of the new method for assay of culture supernatant sample was still half that of the sensitive RI-RTA. Culture supernatants of five peripheral blood mononuclear cell samples obtained from HIV-1-seropositive carriers were assayed by both the improved method and the sensitive RI-RTA; and with each of the methods, however all virus-positive cultures could be detected. The improved non-RI RTA was considered especially useful for assay of culture supernatants for purposes of virus isolation because of its advantages of excellent sensitivity and lack of requirement for radioisotopes.

[Non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridine-triphosphate and a primer-immobilized microtiter plate: application for detection and identification of isolated retroviruses from HIV-1-seropositive hemophiliac patients].

Non-radioisotopic reverse transcriptase assay (Non-RTA) was successfully applied for detection and identification of the retroviruses isolated from peripheral mononuclear cells from eight HIV-1-seropositive hemophiliac patients. Of 40 samples, 36 (90%) were consistent in detection between Non-RI and RI RTA. Four samples which showed RT activities slightly above the cutoff level of RI RTA were not detected by Non-RI RTA. Non-specific RT-inhibitors in the culture supernatant decreased the sensitivity of Non-RI RTA more significantly than that of RI RTA. It was demonstrated that higher concentrations (> 20%) of fetal calf serum in RPMI-1640 culture medium inhibited the hybridization of poly rA template with immobilized primer, resulting in reducing the sensitivity of Non-RI RTA. Then we identified the isolated retroviruses using specific RT-inhibiting (RTI) antibodies against HIV-1 and HIV-2. HIV-1 RTI antibody specifically inhibited RTs of isolated retroviruses and HIV-1 strain, LAV-1, but not HIV-2 and Rous-associated virus 2 (RVA-2) RT. Conversely, HIV-2 RTI antibody specifically inhibited HIV-2 RT, but not HIV-1 and RAV-2 RT. These findings agreed with previously reported results showing the type-specificity of HIV-1 and -2 RTI antibodies, and suggest the possibility that isolated retroviruses could be identified by these antibodies.

Differentiation between human immunodeficiency virus type 1 (HIV-1) and HIV-2 isolates by nonradioisotopic reverse transcriptase-typing assay.

We tested whether human immunodeficiency virus type 1 (HIV-1) could be differentiated from HIV-2 by a reverse transcriptase (RT)-typing assay that measured the reduction of enzyme activity owing to specific antibody. RT-inhibiting antibody was examined for HIV type specificity by a new nonradioisotopic RT assay. Antibodies from four rabbits immunized with recombinant HIV-1 RT and from 23 HIV-1-seropositive individuals all specifically inhibited the enzyme activities of two HIV-1 strains (LAV-1 and GH-3), three zidovudine-resistant HIV-1 mutants, and a recombinant HIV-1 RT. However, none of these antisera affected the activities of six HIV-2 strains (GH-1, GH-2, GH-4, GH-5, GH-6, LAV-2ROD), Rous-associated virus type 2, and DNA polymerase I from Escherichia coli. In contrast, HIV-2 antibody from a rabbit immunized with disrupted GH-1 virions blocked the enzyme activities of the six HIV-2 strains but not those of the three HIV-1 strains, Rous-associated virus type 2, or DNA polymerase I. These results indicate that the antigenic domains of HIV-1 and HIV-2 RTs recognized by their inhibiting antibodies are distinct from each other and are highly conserved. Clinical HIV isolates from 18 HIV-1-seropositive individuals and 3 HIV-2-seropositive Ghanaian individuals were identified as HIV-1 and HIV-2, respectively, by the nonradioisotopic RT-typing assay.