Haruyo Mori

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.

Nucleotide and Amino Acid Polymorphisms at Drug Resistance Sites in Non-B-Subtype Variants of Human Immunodeficiency Virus Type 1

ABSTRACT We have compared nucleotide substitutions and polymorphisms at codons known to confer drug resistance in subtype B strains of human immunodeficiency virus type 1 (HIV-1) with similar substitutions in viruses of other subtypes. Genotypic analysis was performed on viruses from untreated individuals. Nucleotide and amino acid diversity at resistance sites was compared with a consensus subtype B reference virus. Among patients with non-subtype B infections, polymorphisms relative to subtype B were observed at codon 10 in protease (PR). These included silent substitutions (CTC→CTT, CTA, TTA) and an amino acid mutation, L10I. Subtype A viruses possessed a V179I substitution in reverse transcriptase (RT). Subtype G viruses were identified by silent substitutions at codon 181 in RT (TAT→TAC). Similarly, subtype A/G viruses were identified by a substitution at position 67 in RT (GAC→GAT). Subtype C was distinguished by silent substitutions at codons 106 (GTA→GTG) and 219 (AAA→AAG) in RT and codon 48 (GGG→GGA) in PR. Variations relative to subtype B were seen at RT position 215 (ACC→ACT) for subtypes A and A/E. These substitutions and polymorphisms reflect different patterns of codon usage among viruses of different subtypes. However, the existence of different subtypes may only rarely affect patterns of drug resistance-associated mutations.

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.

Recent diversity of human immunodeficiency virus type 1 in individuals who visited sexually transmitted infection-related clinics in Osaka, Japan

By human immunodeficiency virus type 1 (HIV-1) antibody screening of people who visited sexually transmitted infection (STI)-related clinics (venereology, urology, and gynecology) and were considered to conduct high-risk sexual activities for HIV-1 infection in Osaka, Japan, during 1992 to 2004, a total of 54 HIV-1 infected individuals (51 Japanese males and 3 non-Japanese females) were identified. Based on the sequencing at env-C2V3 and pol regions, Japanese males were mostly of subtype B (50/51 cases), with the one remaining case being a recombinant circulating form, CRF01_AE, while 3/3 viruses in non-Japanese females were of CRF01_AE. Analysis of subtype B cases since 2001 showed that these viruses became wider in their genetic variation, including amino acid insertions and also deletions, than that of the cases before 2000. Thus, it was suggested that HIV-1 spreading in Osaka has been increasing in genetic variability. Although all these infected individuals were first recognized to be infected with HIV-1 by our screening, some of them were carriers of HIV-1 with drug-resistant pol sequences, indicating that they could be infected with drug-resistant HIV-1 mutants.

Characteristics of Transmitted Drug-Resistant HIV-1 in Recently Infected Treatment-Naive Patients in Japan.

Objectives:Progress in antiretroviral treatment has led to fewer virological failure cases, but 10%–20% of treatment-naive HIV/AIDS cases are reported to harbor drug-resistant strains, suggesting transmission of drug-resistant HIV. We aimed to determine the trend in prevalence of transmitted drug-resistant (TDR) HIV in Japan, particularly in recently infected patients. Methods:Drug-resistance test was performed on 3904 HIV-1–infected cases newly diagnosed between 2007 and 2012. The number of cases infected within 6 months [recent seroconverters (RS)] was estimated by BED assay of 2700 plasma samples. Characteristics of RS cases were further analyzed. Results:The overall prevalence of TDR was 9.1%, ranging from 7.3% in 2008% to 12.5% in 2010. Among 1403 subtype B/E/D cases with >50 CD4+ T cell counts and >1000 HIV copies per milliliter, 468 (33.4%) were estimated to be RS. The prevalence of RS was significantly higher among cases who were male, Japanese, and men who have sex with men. The prevalence of TDR did not differ significantly between recent and long-term seroconverters (8.5% vs. 9.2%, respectively, P = 0.68). Common mutations in both groups were M46I/L and T215 revertants. Furthermore, sequences with these mutations, K103N and D30N/N88D formed clusters on phylogenetic trees. Conclusion:Our study clarified an increase in prevalence of TDR in Japan from 2007 to 2012. The phylogenetic clustering of cases with M46I/L or T215 revertants suggests that HIV with these mutations have become circulating strains. Furthermore, detailed analyses showed that Japanese men who have sex with men are more aware of their risk of HIV infection.

Low or no antibody responses to human immunodeficiency virus type 1 Nef in infected carriers with subtype E, in contrast to subtype B that showed antibodies preferentially recognizing subtype-specific Nef epitopes.

The viral accessory gene product Nef has been shown to play an important role in human immunodeficiency virus type 1 (HIV-1)-induced pathogenesis. Only little information is available regarding the differences in the host immune responses against Nef protein and its function in vivo among different subtypes of HIV-1. In the present study, we showed marked differences in the immune responses to Nef protein between subtypes B and E. The amino acid sequence in subtype E Nef showed 72% homology with that in subtype B. Most murine monoclonal antibodies obtained by immunization with subtype B or E Nef protein showed cross-reactivity with both Nef proteins (80 and 67%, respectively). Next, we focused on the immune responses among infected Japanese and Thai individuals. Subtyping of the individuals into B and E was carried out by enzyme-linked immunosorbent assay (ELISA) using synthetic peptides corresponding to the V3 loop representing the principal neutralizing domain. Most of the sera from these individuals reacted strongly with Gag p24 proteins derived from subtypes B and E at similar levels. However, the immune responses among these individuals to Nef protein were markedly different. Some subtype B-infected Japanese and Thai individuals (40 and 35%, respectively) showed higher levels of anti-Nef antibodies, although these antibodies preferentially recognized epitopes specific to subtype B. On the other hand, most of the subtype E-infected Japanese and Thai individuals showed low or no antibody responses to Nef proteins. Thus, immune responses to Nef were markedly different between subtypes B- and E-infected carriers, suggesting different function(s) for Nef in AIDS pathogenesis. Further, vaccine design must take into account the different subtypes of HIV-1.

A cluster of rapid disease progressors upon primary HIV-1 infection shared a novel variant with mutations in the p6gag/pol and pol/vif genes.

Few studies have described the etiologic factors associated with rapid AIDS onset during primary HIV-1 infection. Our molecular epidemiological study identified a cluster of individuals infected with HIV-1 variants characterized by novel mutations in the p6gag/pol and pol/vif genes during 2011 and 2013 in Osaka, Japan. Individuals positive for the novel HIV-1 variant showed rapid disease progression, suggesting a role of viral mutations in the fostering of the clinical course of HIV-1 infection.

Cases of HIV type 1 acute infection at STI-related clinics in Osaka.

Since 1992 we have investigated HIV antibodies in persons with a venereal disease who worked for a sex business or engaged in sexual behavior with a high risk of HIV infection at five clinics of venereology, urology, and gynecology in Osaka Prefecture. Starting at the end of 2000, we performed a nucleic acid-amplification test (NAT) on antibody-negative samples with the aim of detecting cases in the window period covering the early phase of infection. Three cases that were thought to be in this phase were found in 2006. All were confirmed to be positive in an NAT, although they were negative in initial screening for HIV performed using the IC method. Gene analysis of the env-C2V3 region of HIV-1 showed that the three samples had different origins.

ビオチン化dUTPとプライマー固相化マイクロプレートを使用した非放射性逆転写酵素活性測定法: HIV-1分離同定への応用

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.

Comparative evaluation of the GeeniusTM HIV 1/2 Confirmatory Assay and the HIV-1 and HIV-2 Western blots in the Japanese population

Accurate diagnosis of earlier HIV infection is essential for treatment and prevention. Currently, confirmation tests of HIV infection in Japan are performed using Western blot (WB), but WB has several limitations including low sensitivity and cross-reactivity between HIV-1 and HIV-2 antibodies. To address these problems, a new HIV testing algorithm and a more reliable confirmation and HIV-1/2 differentiation assay are required. The Bio-Rad Geenius™ HIV-1/2 Confirmatory Assay (Geenius) has recently been approved and recommended for use in the revised guidelines for diagnosis of HIV infection by the Center for Disease Control and Prevention (USA). We made comprehensive comparison of the performance of Geenius and the Bio-Rad NEW LAV BLOT 1 and 2 (NLB 1 and 2) which are WB kits for HIV-1 and HIV-2, respectively, to examine if Geenius is a suitable alternative to these WB assays which are now being used in HIV testing in Japan. A total of 166 HIV-1 positive samples (146 from patients with established HIV-1 infection and 20 from patients with acute infection), five HIV-1 seroconversion panels containing 21 samples and 30 HIV-2 positive samples were used. In addition, a total of 140 HIV negative samples containing 10 false-positives on screening tests were examined. The sensitivity of Geenius and NLB 1 for HIV-1 positive samples was 99.3% and 98.6%, respectively. Geenius provided more positive results in the samples from acute infections and detected positivity 0 to 32 days earlier in seroconversion panels than NLB 1. NLB 2 gave positive results in 12.3% of HIV-1 positive samples. The sensitivity of both Geenius and NLB 2 for HIV-2 positive samples was 100%. The specificity of Geenius, NLB 1 and NLB 2 was 98.5%, 81.5% and 90.0%, respectively. Geenius is an attractive alternative to WB for confirmation and differentiation of HIV-1 and HIV-2 infections. The adaptation of Geenius to the HIV testing algorithm may be advantageous for rapid diagnosis and the reduction of testing costs.