Anti-HIV-1 activity of myo-inositol hexaphosphoric acid (IP6) and myo-inositol hexasulfate(IS6).

It is known that polysulfates have some anti-HIV-1 activity. We investigated the anti-HIV-1 activity of myo-inositol hexaphosphoric acid (IP6) and myo-inositol hexasulfate(IS6), low molecular weight carbohydrates. IP6 and IS6 inhibited the replication of HIV-1 in a T cell line as well as that of a freshly isolated strain in peripheral blood mononuclear cells. Neither substance inhibited HIV-1-induced giant cell formation, but addition of IS6 when infecting cells with HIV-1 inhibited the replication of HIV-1. Neither substance inhibited HIV-1 reverse transcriptase activity in vitro and no influence on late stage replication was noted. Although the mechanisms of IP6 and IS6 action remain unclear, it can be speculated that they act on HIV-1 early replicative stage. Although it is not possible to develop IP6 and IS6 themselves as anti-AIDS drugs, studies of these anti-HIV agents might be expected to provide seed for eventual production of superior drugs for AIDS treatment.

Anti-HIV-1 activity of herbs in Labiatae.

The anti-HIV-1 activity of aromatic herbs in Labiatae was evaluated in vitro. Forty five extract from among 51 samples obtained from 46 herb species showed significant inhibitory effects against HIV-1 induced cytopathogenicity in MT-4 cells. In particular, the aqueous extracts of Melissa officinalis, a family of Mentha x piperita "grapefruit mint," Mentha x piperita var. crispa, Ocimum basilicum cv "cinnamon," Perilla frutescens var. crispa f. viridis, Prunella vulgaris subsp. asiatica and Satureja montana showed potent anti-HIV-1 activity (with an ED of 16 microg/ml). The active components in the extract samples were found to be water-soluble polar substances, not nonpolar compounds such as essential oils. In addition, these aqueous extracts inhibited giant cell formation in co-culture of Molt-4 cells with and without HIV-1 infection and showed inhibitory activity against HIV-1 reverse transcriptase.

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.

Protective effect of astragali radix by oral administration against Japanese encephalitis virus infection in mice.

We investigated the protective effect of Astragali Radix (AR) by oral administration against Japanese encephalitis virus (JEV) infection in mice, the pharmacological effects of AR extracts (AE) in different origin, and the chemical composition of the AEs. A protective effect was demonstrated in all four AEs used, however, the effective grade for each one was different. In the control group, an increase of hemagglutination inhibition (HI) antibody titer was observed in all mice surviving 25 d after JEV inoculation. However, the increase of HI antibody titer was not observed in some animals administered an AE. In the control group, the rate of HI antibody positive mice was 90% 3 d after JEV inoculation, while the four groups which received the AE had a 30-60% positive rate. In mice which received the AE, the peritoneal exudate cell (PEC) numbers increased significantly compared to the control group. The predominant cell population of PECs in mice receiving the AE was macrophages, and in the PEC, the active oxygen (AO) production was high. From these results, we propose that the protective effect of AE by oral administration is based on a non-specific mechanism during the early stage of infection, before shifting to antibody production, and that macrophages play an important role in this resistance to JEV infection, e.g., by inducing the production of AO. In the chemical composition of each AE, carbohydrate was the major component.

Protective effect of Astragali Radix by intraperitoneal injection against Japanese encephalitis virus infection in mice.

We examined the protective effect of Astragali Radix extracts (AE) by intraperitoneal injection against Japanese encephalitis virus (JEV) infection in mice. A protective effect was observed by all four samples of AE used. However, the degree of effectiveness for each AE was different. The observed survival rates of the groups injected with sample A (from Shanhsi, Japanese name Sansei-syo) and sample D (from Hokkaido) extracts were higher than 80% at 21 d after JEV inoculation. The groups injected with sample B (from Hopei, Japanese name Kahoku-syo) and sample C (from Hsiahsi, Japanese name Sensei-syo) extracts had a 60% survival rate. The increase in hemagglutination inhibition antibody titer was negligible in mice that survived 21 d after JEV inoculation. The antiviral effect of AE was examined by plaque assay in vitro, but no antiviral effect was shown. In mice injected with AE, the peritoneal exudate cell (PEC) numbers increased significantly, compared to the control. In these PEC, active oxygen production was also high. Also the group as a whole displayed a high survival rate against JEV infection, these were so strong. From these results, we propose that the protective effect of AE is dependent on a non-specific mechanism during the early stage of infection, before it shifts to antibody production, and that PEC plays an important role.

Boromycin, an anti-HIV antibiotic.

The polyether-macrolide antibiotic, boromycin, was isolated as a potent anti-human immunodeficiency virus (HIV) antibiotic from a fermentation broth of Streptomyces sp. A-3376. Boromycin was found to strongly inhibit the replication of the clinically isolated HIV-1 strain as well as the cultured strain in in vitro laboratory experiments. The mechanism for the anti-HIV activity of boromycin is suggested to involve blocking the later stage of HIV infection, and probably the maturity step for replication of the HIV molecule.

[Follow-up of HIV carriers by a laboratory method. I: Virus isolation is useful for prognostication of the onset of symptoms].

We followed 54 HIV-1 carriers (44 asymptomatic carriers and 10 AIDS patients) by virus isolation and immunological examination and evaluated their usefulness for prognostication of the onset of symptoms. From 37 carriers (27 asymptomatic carriers and 10 symptomatic), 132 HIV-1 strains were isolated; the virus isolation rate was 60% in the asymptomatic carriers (AC) but 100% in the symptomatic. In the AC, the isolation rate was 54.5% in the group showing stable in the CD4+ level but 95.5% in the group showing a decrease in the CD4+ level. With progression of the disease, the culture time required for virus isolation was shortened, and the percentage of isolates showing infectivity to the T-cell line (MT-4 cells) increased. These findings suggest that the virus in the body is changed with progression of the disease to that showing rapid replication, T-cell tropic, and high pathogenicity. Indeed, progression of the disease was observed in all carriers in whom a highly pathogenic virus was detected; some developed the disease within 1 year, some showed temporary recovery in the CD4+ level after AZT administration followed by progression to ARC, and others showed a rapid decrease in the CD4+ level. In contrast, in carriers with only slightly pathogenic virus, the CD4+ level was maintained for a long period. These results suggest that the detection of a highly pathogenic virus is one of the most reliable marker for the prognostication of the onset of the disease. The detection of HIV-1 antigen in the plasma and a decrease in the gag antibody were also associated with the progression of the disease. However, the reliability of these markers seems to be lower than that of virus isolation.

[Prognosis and evaluation of drug therapy by V3 and RT gene analysis of HIV-1].

We have reported on the investigation of 54 HIV-1 patients concerning the isolation and clinical marker in the preceding paper. We have attempted the analysis of the V3 and RT genes. HIV-1 from a patient who had rapidly taken a turn for the worse had basic amino acid at position 11 (Arg) and lost an acidic amino acid at position 25 (Gln) of V3. This sequence pattern was a distinguished feature of a virus with a rapid-high, syncytium inducing (SI) and T-cell-line tropic phenotype. In contrast, patients with no or mild clinical symptoms had sequences characterized as slow-low, non-synsytium inducing (NSI) and macrophage tropic. We then investigated the appearance of resistant to AZT and ddI. It was shown that the virulent virus obtained drug resistant variants faster than the wild type by analysis of the RT gene. We consider that these data concerning virus isolation and gene analysis are useful for prognosis and strategy for clinical therapy.

Mutations in rpoB gene of rifampicin resistant clinical isolates of Mycobacterium tuberculosis in Japan.

In Mycobacterium tuberculosis, involvement of alterations of the RNA polymerase beta subunit in resistance to rifampicin has been described by Telenti et al. To determine if the same correlation could be observed between the mutation of the rpoB gene and clinically isolated M. tuberculosis of the rifampicin-resistant phenotype in Japan, 47 strains of M. tuberculosis of the rifampicin-resistant phenotype, 17 of the rifampicin-susceptible phenotype, and 4 type strains were examined. A 411-base pair (bp) rpoB fragment was amplified by the polymerase chain reaction and subjected to solid phase direct sequencing. By comparing the nucleotides, mutation involving 8 conserved amino acids were identified in 44 of the 47 (93.6%) rifampicin-resistant isolates, but in none of the 17 sensitive isolates and 4 type strains. All mutations found were clustered within a region of 23 amino acids. Thus, similar to the results reported by Telenti et al., substitution of a limited number of highly conserved amino acids encoded by the rpoB gene appears to be the molecular mechanism responsible for resistance to rifampicin in Japanese clinical isolates of M. tuberculosis. Our results suggest that direct DNA sequencing of the rpoB gene may be a reliable method for identifying rifampicin-resistant M. tuberculosis strains among Japanese clinical isolates.

[Screening test of crude drug extract on anti-HIV activity].

The anti-HIV-1 effects of 204 crude drugs of common use in Japan were evaluated in vitro. As a result, 45 samples inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In particular, the hot water extracts of Lithospermum erythrorhizon (root) and Prunella vulgaris (spike) showed the strongest anti-HIV-1 activities. Their IC100 values were both 16 micrograms/ml. In general, the hot water extracts of the crude drug suppressed the replication of HIV-1 growth more strongly than the cold water extracts.

Use of the recombinant chimera proteins, LacZ-Env and Gag-Env, for immunological studies on HIV-1 infection.

To use Env proteins as antigens for detection of the human immunodeficiency virus type-1 (HIV-1) antibodies, we attempted to overexpress the Env proteins in Escherichia coli. To study the epitopes in the Env proteins recognized by the sera of HIV carriers, various regions of the proviral DNA encoding the Env region were fused to the 3' end of the lacZ gene. The immunoblotting analysis of the LacZ-Env(512-611) and LacZ-Env(721-826) proteins with the 41 positive sera revealed that the former and the latter immunologically reacted with 100 and 78% of the sera, respectively. To avoid rare false-positive reactions due to the LacZ moiety of the fusion protein, we attempted to express the Env(512-611) alone or Gag-Env(512-611) under the control of bacteriophage T7 promoter. Although we could express only a low level of the Env(512-611) peptide in E. coli, we succeeded in producing large amounts of the Gag(121-406)-Env(512-611) and Gag(308-406)-Env(512-611) proteins as chimeric proteins. Both of these chimera proteins strongly reacted with the 41 positive sera. We purified these proteins and analyzed the immunological reactivity by dot blot with the 60 positive sera and the 84 normal sera. As little as 20 ng of the dotted proteins was enough for the reaction with the positive sera, whereas as much as 320 ng of them did not show false-positive reactions with the normal sera. We obtained highly purified Gag-Env proteins with highly specific seroreactivity, which should be useful for diagnosis and prognosis.

[Preliminary screening for antiviral AIDS drugs. III. Report on fiscal year 1990].

Preliminary screening for antiviral AIDS drugs has been carried out using three different in vitro assay systems. Among 106 samples tested, five were found to inhibit the growth of HIV in vitro. Two of five have a hopeful sign, as the range of effective doses of the samples is wide.

[Detection of human immunodeficiency virus type-1 (HIV-1) DNA by polymerase chain reaction using nonradioactive probe and virus isolation].

We compared the results obtained with the polymerase chain reaction (PCR) and virus isolation from peripheral blood mononuclear cells (PBMC) in HIV seropositive and seronegative persons. Three primer pairs of SK38/39 (gag). SK29/30 (LTR) and SK68/69 (env) were used in the amplification of the HIV DNA sequences, and KM29/38 (beta-globin) was used as the inner control. The PCR-positive rate among the virus-isolation-positive persons was SK38/39:100% (22/22), SK29/30:95.5% (21/22) and SK68/69:90.0% (20/22). The PCR-positive rate among the virus-isolation-negative persons was SK38/39:60% (6/10), SK29/30:60% (6/10) and SK68/69:80% (8/10), and two subjects were PCR-negative with all primer pairs. We could not detect HIV DNA from seronegative samples, and all subjects were positive with the inner control. Each primer pair expressed a different PCR-positive rate. There are possible explanations for the low PCR-negative rate on virus-isolation negative-subjects that the number of infected cell was rare or infected HIV contained genetic variations or deletions. We considered that the results of PCR correlated with the character of HIV as infectivity.

Anti-HIV-1 activity of sulfated amphotericin B in vitro.

To reduce the toxicity of amphotericin B methyl ester (AME), which shows some anti-HIV-1 activity, sulfated amphotericin B (SAB) was prepared from amphotericin B (AB), and its anti-HIV-1 activity was examined in vitro. SAB at concentration of 7.8 micrograms/ml completely suppressed the HIV-1-induced cytopathic effect in MT-4 cells, at 3.9 micrograms/ml inhibited the expression of HIV-1 antigen in peripheral blood mononuclear cells infected with freshly isolated HIV-1 and at 22 micrograms/ml completely suppressed formation of giant cells in cocultures of MOLT-4 with MOLT-4/HIV-1 cells. Reverse transcriptase activity was inhibited by SAB, but only at higher concentrations (0.2-1 mg/ml). Furthermore, the toxicity of SAB was lower than that of AME or AB, and SAB did not affect the proliferation of MT-4 cells at concentrations up to 0.5 mg/ml. The anti-coagulant effect of SAB was 10-fold less than that of dextran sulfate (MW = 8000). The anti-HIV-1 effect of SAB is attributed to inhibition of binding of virions to target cells.

In vitro anti-human immunodeficiency virus type 1 activity of biliverdin, a bile pigment.

Biliverdin (BV) is a bile pigment having anti-allergic properties. We examined the effect of BV on human immunodeficiency virus type 1 (HIV-1) in vitro. BV completely inhibited the cytopathic effect of HIV-1 in MT-4 cells at concentrations of 22.2 micrograms/ml or more. This inhibitory effect was also observed when BV was present during the adsorption period of HIV-1. However, BV was cytotoxic to MT-4 cells at concentrations above 800 micrograms/ml. At a concentration of 66.7 micrograms/ml, BV completely inhibited syncytia formation by HIV-infected and uninfected MOLT-4 cells. Moreover, after exposure of HIV-1 particles to BV for 2 h, the infectivity of the virus was reduced in a dose-dependent manner. It is speculated that the anti-HIV activity of BV is due to direct inactivation of virions and inhibition of virus binding to target cells.

Inhibitory effect of tachyplesin I on the proliferation of human immunodeficiency virus in vitro.

An antimicrobial peptide, tachyplesin I, isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus) was examined for its inhibitory effects on human immunodeficiency virus (HIV) infection in vitro. At a concentration of 7.5 micrograms/ml, tachyplesin I suppressed the development of cytopathic effects (CPE) by more than 70% in MT-4 cells infected with HIV (lymphadenopathy-associated virus). This inhibitory effect was observed only when the drug was added during the adsorption period of the virus to the cells. In cocultures of MOLT-4 and persistently HIV-infected cells (MOLT-4/HIV), tachyplesin I at the same concentration completely inhibited multinucleated giant cell formation. Infectivity of HIV was reduced by 10(-2.5) in medium free from fetal calf serum containing tachyplesin I at a concentration of 200 micrograms/ml. Tachyplesin I did not show any inhibitory effect on reverse transcriptase activity of HIV at concentrations of 9-80 micrograms/ml at which tachyplesin I inhibited HIV infection. These results suggest that the anti-HIV action of tachyplesin I was due to the inhibition of virus adsorption.