Katsuhiko Kitamura

Net positive charge of HIV-1 CRF01_AE V3 sequence regulates viral sensitivity to humoral immunity.

The third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope gp120 subunit participates in determination of viral infection coreceptor tropism and host humoral immune responses. Positive charge of the V3 plays a key role in determining viral coreceptor tropism. Here, we examined by bioinformatics, experimental, and protein modelling approaches whether the net positive charge of V3 sequence regulates viral sensitivity to humoral immunity. We chose HIV-1 CRF01_AE strain as a model virus to address the question. Diversity analyses using CRF01_AE V3 sequences from 37 countries during 1984 and 2005 (n = 1361) revealed that reduction in the V3s net positive charge makes V3 less variable due to limited positive selection. Consistently, neutralization assay using CRF01_AE V3 recombinant viruses (n = 30) showed that the reduction in the V3s net positive charge rendered HIV-1 less sensitive to neutralization by the blood anti-V3 antibodies. The especially neutralization resistant V3 sequences were the particular subset of the CCR5-tropic V3 sequences with net positive charges of +2 to +4. Molecular dynamics simulation of the gp120 monomers showed that the V3s net positive charge regulates the V3 configuration. This and reported gp120 structural data predict a less-exposed V3 with a reduced net positive charge in the native gp120 trimer context. Taken together, these data suggest a key role of the V3s net positive charge in the immunological escape and coreceptor tropism evolution of HIV-1 CRF01_AE in vivo. The findings have molecular implications for the adaptive evolution and vaccine design of HIV-1.

A fluorescence sandwich ELISA for detecting soluble and cell-associated human interleukin-2

A fluorescence sandwich ELISA was developed for the quantitation of human interleukin-2 (IL-2) using a combination of two anti-human IL-2 antibodies, L61 and biotinylated anti-IL-2 rabbit IgG. The minimum level of IL-2 detectable by this ELISA was 0.3 pg/ml, indicating that this method is about 100 times more sensitive than the usual colorimetric human IL-2 ELISA. The ELISA specifically detected human IL-2 in cell culture supernatants and serum, and also in extracts from mitogen-stimulated peripheral blood mononuclear cells. All of the IL-2 species detected in the supernatants, sera and cell lysates were shown to have a molecular weight of 15,000, as estimated by size exclusion high performance liquid chromatography.

Current HIV type 1 molecular epidemiology profile and identification of unique recombinant forms in Jakarta, Indonesia.

HIV infection is a major problem in Indonesia. The number of people living with HIV has been increasing from year to year, especially among injecting drug users (IDUs). Since there were only limited data about molecular epidemiology profiles of HIV/AIDS in Indonesia, a cross-sectional study involving 208 HIV-1-seropositive individuals was conducted in 2007 in Jakarta. The majority of participants were 16-30 years of age (64.9%) and 74.5% were male. The most frequent risk factor was injecting drug use (IDU) (45.7%) followed by heterosexual transmission (34.1%). Phylogenetic analysis of gag (p17 and p6) and env C2V3 regions showed 200 (96.2%) of 208 DNA samples were CRF01_AE and only 3 (1.4%) were subtype B. Five samples (2.4%) indicated discordant subtypes between the three aforementioned regions: three of them showed unique CRF01_AE/B recombination patterns in 2.3-kbp nucleotide sequences (from p17 to part of RT), including one sample showing similarity to CRF33_01B, reported previously in Malaysia. This study shows the current predominant subtype is CRF01_AE in every risk group, with a decreasing number of pure subtype B, and the first identification of CRF01_AE/B recombinant forms among HIV-1-seropositive Indonesians.

Infection of macaques with an R5-tropic SHIV bearing a chimeric envelope carrying subtype E V3 loop among subtype B framework.

Summary. To establish simian/human immunodeficiency virus (SHIV) clones bearing a chimeric envelope carrying subtype E V3 loop among subtype B envelope, four subtype E V3 sequences were substituted into SHIVMD14, a SHIV clone bearing an envelope derived from a CXCR4 (X4)/CCR5 (R5)-dual tropic subtype B HIV-1 strain. SHIV-TH09V3, an only V3-chimera clone capable of replicating in human and macaque peripheral blood mononuclear cells (PBMCs), was propagated in pig-tailed macaque PBMCs and in cynomolgus macaque splenic mononuclear cells. The propagated virus stocks were intravenously inoculated into respective macaque species. SHIV-TH09V3 infected both macaque species as shown by plasma RNA viremia, isolated viruses from PBMCs and plasma, and antibody production against viral proteins. To assess how the substituted V3 sequence affected coreceptor usage, SHIV-TH09V3 stocks propagated in vitro and after isolation from macaques were verified for their corecepor usage by GHOST cells assay. SHIV-TH09V3 maintained R5-tropic phenotype both in vitro and after isolation from macaques, in contrast to the X4/R5-dual tropic SHIVMD14. This indicates the substituted V3 sequence among the backbone of SHIVMD14 governs coreceptor usage. Future study of infecting macaques with SHIV-TH09V3 and SHIVMD14 will focus on differences of the outcome caused by the different V3 sequences in connection with coreceptor usage.

Regulation of the susceptibility of HIV-1 to a neutralizing antibody KD-247 by nonepitope mutations distant from its epitope.

Objective:A humanized neutralizing antibody, KD-247, targets the V3 loop of HIV-1 Env. HIV-1 bearing the GPGR sequence at the V3 loop is potentially susceptible to KD-247. However, not all GPGR-positive HIV-1 isolates are neutralized by KD-247. We examined the potential mechanism by which the susceptibility of HIV-1 to KD-247-mediated neutralization is regulated. Design:We searched for nonepitope neutralization regulatory (NNR) mutations that sensitize GPGR-bearing HIV-1AD8 to KD-247 and mapped the locations of such mutations relative to the V3 loop. Methods:We generated a functional HIV-1AD8 Env library, and evaluated the viral susceptibility to KD-247 by measuring the half-inhibitory concentration (IC50) to KD-247 on TZM-bl cell assay. Results:We identified nine KD-247-sensitizing NNR mutations from 30 mutations in various regions of gp120, including the V1/V2 loop, C2, V3 loop, C4, and C5. They specifically affected KD-247-mediated neutralization, as they did not affect the b12-mediated neutralization. When combined, the KD-247-sensitizing NNR mutations additively sensitized the virus to KD-247 by up to 10 000 folds. The KD-247-sensitizing NNR mutations increased KD-247 binding to the virion. Notably, the NNR mutation in C4 coincides with the CD4-binding site of gp120. Conclusion:Given that most of the KD-247-sensitizing NNR mutations are remote from V3 loop, it is reasonable to hypothesize that the steady-state, local conformation of the V3 loop is regulated by the interdomain contact of gp120. Our mutational analysis complements crystallographic studies by helping provide a better understanding of the steady-state conformation and the functional geometry of Env.

Detection of an HTLV‐II‐Seropositive Blood Donor in Japan

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