Nanoscale hydration dynamics of DNA-lipid blend dry films: DNA-size dependency.

In this study, nanoscale hydration dynamics of DNA-lipid blend dry films are investigated via small angle X-ray diffraction. Compared to the hydration of lipid films, fragmented short DNA strands and counterions in stacked lipid layers dramatically accelerate both the relaxation of the lamellar distance to a metastable interval and the subsequent peeling-off process of lipid bilayers. Moreover, genome-sized long DNA and counterions accelerate the relaxation process, but suppress the peeling-off process and simultaneously induce a damped-oscillation of the lamellar interval; this is probably due to the viscoelastic properties of the entangled long DNA dissolved in hydrated water between the stacked lipid bilayers. This study's findings can pave the way for producing cell-sized liposomes, which efficiently encapsulate any arbitrary sized DNA through natural swelling.

Polymorphism of CCR5 affecting HIV disease progression in the Japanese population.

Among several factors associated with HIV-1 disease progression, genetic polymorphism of CCR2, CCR5, and CXCR4 in HIV-1 infection has been found. Single-nucleotide polymorphisms (SNPs) in the CCR2, CCR5, and CXCR4 genes as well as a 32-base pair deletion in the open reading frame of the CCR5 gene are associated with HIV disease progression among Caucasians and African-Americans in North America and Europe. However, in populations other than Caucasians and African-Americans, SNPs have not been fully examined. In our study SNPs in CCR2 coding and CCR5 regulatory regions have been examined in 98 Japanese HIV-positive individuals. The alleles of CCR5 regulatory regions at -2135T and -2086G are associated with late onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 0.502 and 0.404, respectively). In contrast to this, the allele of CCR5 at -2086A is associated with the early onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 2.133). A haplotype including two alleles at -2135G and -2086G is associated with the late onset of AIDS (p < 0.05; odds ratio for the early onset of AIDS, 0.372). Thus we found that a CCR5 SNP and haplotype polymorphism affect HIV disease progression even in the Japanese population. This indicates that the CCR5 genetic polymorphism affecting disease progression should be studied in a wider range of population.

Seronegative HIV-2 carriers in India.

The discordant cases of seronegative, but culture and proviral HIV-2 DNA positive were found in Mumbai, India. This was corroborated by the successful isolation of HIV-2-RNA in culture medium, HIV-2 cDNA sequence determination and the detection of the antigen. The sequence of the isolated HIV-2 genomic RNA does not seem to be altered to the extent that the change will alter antibody binding. Furthermore, antibody from the same individual (even at 8 months from initial sampling) from whom HIV-2 was isolated did not react with the antigen of this strain. Those evidences imply that extremely low or non-production of the antibody may be due to suboptimal immune stimulation due to extremely slow HIV-2 replication. This low virus-load may be responsible for the negative antibody results in the HIV-2 carriers.

PIP: This paper describes the characteristics of HIV-2 seropositive and seronegative cases in Mumbai, India, and characterizes the differences between HIV-1 and HIV-2. More than 200 outpatients considered to be at high risk of HIV infection were screened for HIV-1 and HIV-2 antibody and proviral DNA. The study found 11 cases that were discordant for antibody test and HIV proviral DNA (i.e., negative for anti-HIV but positive for HIV-2 proviral DNA). The presence of this provirus was further corroborated by the detection of HIV-2 RNA in the culture medium upon HIV isolation, HIV-2 cDNA sequencing, and antigen detection. The sequence of the isolated HIV-2 genomic RNA did not seem to be altered to the extent that the change would affect antibody binding. Moreover, antibody from the same person in whom HIV-2 was detected did not react with the antigen of this strain even 8 months after the initial sampling. These findings indicate that extremely low production or non-production of the antibody may be brought about by suboptimal immune stimulation due to very low HIV-2 replication speed. This low virus load may account for the negative antibody results in the HIV-2 carriers in India.

Conservation of the central proline-rich (PxxP) motifs of human immunodeficiency virus type 1 Nef protein during the disease progression in two hemophiliac patients.

The nef gene is considered to play a crucial role in the development of acquired immunodeficiency syndrome (AIDS). In this study, we analyzed the sequence of nef quasispecies obtained from replication-competent HIV-1 isolates from two Japanese hemophiliac patients infected with HIV-1. At least 10 nef clones were isolated at each time point and a total of 75 individual nef quasispecies were sequenced. We observed a gradual increase in genetic diversity of the nef gene over time. Among the various functional regions of Nef protein, myristoylation site and the central PXXP (SH3 ligand) motifs were well conserved. The scattered regions responsible for downregulation of CD4 and class I MHC were also conserved. These data suggest that these functions of Nef may be involved throughout the disease process.

Human mannan-binding lectin inhibits the infection of influenza A virus without complement.

Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen-like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.

Characterization of human immunodeficiency virus type 1 resistant to modified cyclodextrin sulphate (mCDS71) in vitro.

Drug resistance of human immunodeficiency virus type 1 (HIV) to modified cyclodextrin sulphate (mCDS71) has been analysed with respect to both the in vitro appearance of resistance to the compound and the mechanism of the acquisition of resistance. Resistant strains could be obtained in all three strains (NL432, KK-1 and A018) tested after serial passages in MT-4 cells with a gradual increase of the concentration of mCDS71. Cross-resistance both to mCDS71 and dextran sulphate 8000 was observed. As a result of sequencing analysis of the gp120 V3-C5 region of resistant strains, the mechanism of resistance can be explained in several ways: (i) substitution of sugar chain-binding amino acids, N and S; (ii) three to five amino acid deletion in V4 loop; and (iii) several mutations in V3 and V4 regions. The real cause of the resistance may be a combination of these three mechanisms. The results suggest that the target of mCDS71 is relatively widely distributed on the viral surface glycoprotein.

Evaluation of a rapid immunochromatographic test for detection of antibodies to human immunodeficiency virus.

A new immunochromatographic rapid test, Determine HIV-1/2, for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 in human whole blood, serum, and plasma was evaluated. Determine HIV-1/2 is a sandwich immunoassay and uses a nitrocellulose strip with a capture site for the patient's results and a procedural control site to confirm the validity of the assay. The results can be read visually, and a positive result is indicated by the formation of a red line within 15 min after sample application. The test showed 100% sensitivity for HIV-1 with 102 whole-blood, 152 serum, and 144 plasma samples obtained from Ramathibodi Hospital, Bangkok, Thailand. The sensitivity of the test for HIV-2 was 100% with 100 serum or plasma samples obtained from Ivory Coast. The sensitivity of the test with 4 anti-HIV-1 seroconversion panels from Boston Biomedica Inc. was equivalent to or better than those of another agglutination assay with serum or plasma and the enzyme immunoassay licensed by the U.S. Food and Drug Administration. The specificity was 100% with 367 sets of whole-blood, serum, and plasma samples from Ramathibodi Hospital. This method had an analytical sensitivity for the detection of HIV-1 equivalent to or better than that of another agglutination assay with serum or plasma. This test had an analytical sensitivity for the detection of HIV-1 better than that of another immunochromatographic test with whole blood. This evaluation demonstrated the excellent performance of this immunochromatographic test with EDTA-anticoagulated whole-blood, serum, and plasma samples. We conclude that this test is suitable for use in emerging countries and is an excellent alternative to HIV antibody testing at remote sites, as well as in traditional laboratories.

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.

Difference in susceptibility to CC-chemokines among HIV-1 isolates.

In this study, we examined the difference in susceptibility to anti-HIV activity of the CC-chemokines (RANTES, MIP-1 alpha and MIP-1 beta) among HIV-1 isolates and analysed its relation with phenotype (syncytium inducibility) and V3 domain of gp120 of the HIV-1 isolates. Of 11 cases tested in endogenous assay, at a concentration of 200 ng/ml, RANTES, MIP-1 alpha, and MIP-1 beta showed more than 80% suppression of HIV-1 replication in 10, 8, and 7 cases, respectively. HIV-1 isolates sensitive to more than one CC-chemokine showed non-syncytium-inducing phenotype, whereas HIV-1 isolates resistant to all of the 3 CC-chemokines showed syncytium-inducing phenotype. HIV-1 isolates resistant to all of the 3 CC-chemokines contained more positively charged amino acid residues in the V3 domain of the gp120. These results indicated that utilization of the CC-chemokine receptors as co-receptors for virus entry could vary among HIV-1 isolates.

Slight difference in primary amino acid sequence of p17 matrix protein of HIV-1 exerts profound influence on its antigenicity.

HIV-1 p17 antigen has been studied for its biological significance in vitro as well as its immunological roles in vivo. By immunological approach of antibody-binding to HIV-1 p17 antigens of several subtypes in combination with computerized analysis of those tertial structures, it became evident that, irrelevant of similarity of linear amino acid sequence of different HIV-1 subtypes, a few amino acid substitutions close to or distant from specified epitope(s) affected their tertial structure resulting in change in ability of its binding to selected antibody. ELISA employing two monoclonal antibodies, A144 and C415, could detect p17 of subtypes A and B, but not of subtypes C, D, and E. Since the epitope site corresponding to A144 has been reported to be important for biological activity of p17 of HIV-1, change in tertial structure around this epitope may explain some difference in biology of HIV-1, such as infectivity of subtypes B and E.

Interleukin-9 receptor alpha chain mRNA formation in CD8+ T cells producing anti-human immunodeficiency virus type 1 substance(s).

A search for gene(s) associated with anti-human immunodeficiency virus type 1 (HIV-1) activity of CD8+ T cells was attempted using molecular cloning and the relation between the anti-HIV activity of CD8+ T cells and the interleukin-9 receptor alpha chain (IL-9R-alpha) mRNA expression from the cDNA clones obtained was examined. The anti-HIV-1 activity of CD8+ T cell culture supernatants was assessed by measuring the level of HIV-1 replication of a CD4+ T cell line transfected with an infectious HIV-1 DNA clone. IL-9R-alpha mRNA was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 5 cases showing high level of anti-HIV-1 activity (more than 80% suppression of HIV-1 replication), the mRNA was detected in 4 cases. Of 10 cases showing low level of anti-HIV-1 activity (less than 80% suppression of HIV-1 replication), the mRNA was detected in one case. Soluble recombinant human IL-9 receptor (rhIL-9sR) did not suppress HIV-1 replication at a concentration of 1 microgram/ml. These data suggest that the IL-9R-alpha mRNA formation in CD8+ T cells may correlate with and play some role in the anti-HIV-1 activity of CD8+ T cells from HIV-1-infected individuals.

Genetic subtypes of HIV-1 in the Philippines.

OBJECTIVES: To determine the genetic variability of HIV-1 amongst infected Filipinos and to analyze phylogenetic relationships, temporal introductions and transmission dynamics of identified variants. METHODS: Polymerase chain reaction amplification and direct sequencing of a 204 base-pair fragment of the env C2-V3 region from uncultured peripheral blood mononuclear cells obtained from 51 HIV-1-positive Filipinos infected from 1987 to mid-1996. Evolutionary distance and phylogenetic relationships among the DNA sequences were estimated. RESULTS: The 51 Philippine strains were classified into five env V3 subtypes, namely subtype B (n = 37), subtype E (n = 8), subtype A (n = 3), subtype C (n = 2) and subtype D (n = 1). The overall env nucleotide divergence ranged from 11.7 to 32.2%. The nucleotide variation appeared to be random and no temporal ordering was observed. The variation of the sequences at the tip of the V3 loop was very broad. Subtypes B and C isolates did not show close genetic relationship to other Asian variants. Only three of the subtype E strains had close affinity to known Asian sequences. The majority (94%) of the subjects acquired the infection by sexual transmission. About two-thirds were presumably infected outside the Philippines, whereas the remaining were infected indigenously. Information was limited to allow segregation of the identified subtypes by mode of transmission or risk groups. CONCLUSION: Our findings demonstrate the presence of multiple genetic subtypes of HIV-1 in the Philippines. The apparent geographic range of previously reported genotypes in South and South-east Asia was extended and has obvious implications for env-based antiviral interventions.

Difference of virus populations in HIV carriers in relation to AZT treatment.

To analyse the appearance of AZT-resistant HIV in HIV carriers after AZT treatment and compare the mutations responsible for resistance employing cloned HIV DNA derived from provirus and free virions in plasma, serial blood specimens were taken before and after AZT treatment. RNA in virions in plasma, proviral DNA and RNA from virus isolates by coculture of PBMCs of HIV carriers and healthy blood donors were cloned and sequenced. DNA clones were compared for their nucleotide sequences responsible for AZT resistance. AZT resistance was acquired as early as 2 months after the start of the treatment and follow-up study was performed for 16 months of the treatment. Population of DNA clones was different according to the origin of the DNA or RNA, which indicated that the provirus population in PBMC was different from that in virions in plasma. These data demonstrated the possibility of selective activation of provirus or activation of provirus in organs other than peripheral blood, although the number of cases was small.

Suppression of HIV replication in vitro by CD8+ T-cells from HIV-infected and HIV-seronegative individuals.

The inhibitory effect of CD8+ T-cells from HIV-infected or HIV-seronegative individuals on HIV replication in the naturally-infected CD4+ T-cells in vitro was examined. Not only autologous CD8+ T-cells from HIV-infected individuals but also allogeneic CD8+ T-cells from HIV-seronegative individuals prevented or delayed HIV replication, even in transwell cocultures using a semi-permeable 0.45 micron filter. The level of the inhibitory effect of allogeneic CD8+ T-cells from the HIV-seronegative individuals on the HIV replication was varied among CD4+ T-cells obtained from HIV-infected individuals used. The results suggested that CD8+ T-cells from HIV-seronegative individuals as well as HIV-infected individuals could produce some cytokine(s) which suppress HIV replication in vitro. The sensitivity to the cytokine(s) might be variable among HIV strains, depending on differences in the nucleotide sequence of different HIV-1 strains. Further studies of control of HIV replication by CD8+ anti-HIV cytokine(s) should provide new strategies for the therapy of HIV infection.

Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus.

Surfactant protein D (SP-D) is a lung-specific protein that is synthesized and secreted by lung epithelial cells and is believed to play an important role in lung host defence. This protein belongs to the C-type lectin family, which is characterized by an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain and a carbohydrate recognition domain (CRD). To elucidate the biological actions of this animal lectin against such pathogens as micro-organisms, the biological activities of a recombinant partial SP-D lacking a collagen-like domain were examined. A recombinant human SP-D, consisting of a short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain and the CRD, was expressed in Escherichia coli. The recombinant SP-D was purified on a nickel column and then on a maltose-agarose column. This protein can form a trimeric structure owing to the neck domain and exhibits sugar-binding activity and specificity similar to those of native human SP-D. The recombinant SP-D caused dose-dependent and calcium-dependent agglutination of E. coli Y1088. The agglutination titre (the concentration required to achieve a 50% decrease in light transmission by agglutination) of recombinant SP-D was approx. 6-fold that of native SP-D. As for conglutination, the recombinant trimeric conglutinin required 8-16-fold higher concentrations than the native counterpart. In haemagglutination inhibition (HI) of influenza A virus, although native and recombinant conglutinin showed similar levels of HI activity, the recombinant SP-D was unable to inhibit haemagglutination, even at a concentration approx. 120-fold that of the native SP-D. The lectin precipitation and lectin blot assays showed that the truncated SP-D could bind to influenza A virus as well as native SP-D did. These results indicate that the agglutination activity of trimeric collectins can be largely retained, and furthermore that the oligomeric structure with several hands at opposite sites can enhance agglutination activity. The difference in HI activity against influenza A virus between native and recombinant SP-D suggests that SP-D uses a different mechanism from that of conglutinin to inhibit viral haemagglutination.

Contribution to the regulation of virus replication in cells latently infected with human immunodeficiency virus 1.

Monocytes/macrophages have been known to play an important role in the initiation and propagation of human immunodeficiency virus 1 (HIV-1) infection. To analyze the function of these cells during the clinical asymptomatic period of infection, we examined the effect of murine peritoneal macrophages and human peripheral blood macrophages on two cell lines latently infected with HIV-1, a promonocytic cell line, U1, and a T-cell line, ACH-2. Monokines of the murine peritoneal macrophages induced significant viral expression in U1, but not in ACH-2 cells. Experiments employing transient transfection of U937 and CEM cells with HIV long terminal repeat (LTR)-chloramphenicol acetyl transferase (CAT) plasmids indicated that the effect of these monokines was due to specific activation of the HIV LTR. In contrast, supernatants of human macrophages induced viral expression in both ACH-2 and U1 cells. These results suggest that several monokines are active in regulating the transition from the clinical asymptomatic period of HIV infection to progression to acquired immunodeficiency syndrome (AIDS).

Genotypes of Japanese encephalitis virus isolated in three states in Malaysia.

Two hundred forty nucleotides from the pre-membrane gene region of 12 Japanese encephalitis virus (JEV) strains isolated from three different regions of Malaysia from 1993 to 1994 were sequenced and compared with each other and with the JEV strains from different geographic areas in Asia. These 12 Malaysian isolates were classified into two genotypes. The four JEV strains isolated from Sarawak in 1994 and the four JEV strains isolated from Sepang, Selangor in 1993 were classified into one genotype that included earlier isolated strains from Malaysia (JE-827 from Sarawak in 1968 and WTP/70/22 from Kuala Lumpur in 1970). The four JEV strains from Ipoh, Perak in 1994 were classified into another genotype that included JEV strains isolated from northern Thailand and Cambodia. In an earlier report, 10 JEV strains from Sabak Bernam, Selangor in 1992 were classified into the largest genotype that included strains isolated in temperate regions such as Japan, China, and Taiwan. The data indicate that at least three genotypes of JEV have been circulating in Malaysia.

Cloning and characterization of a cDNA encoding bovine mannan-binding protein.

To identify the bovine mannan-binding protein (MBP), a search for the cDNA homologue of human MBP was carried out. cDNA clones encoding bovine MBP were isolated from a bovine liver cDNA library using a cDNA fragment encoding a short collagen region, neck domain and carbohydrate recognition domain of human MBP. The cDNA carried an insert of 747 bp encoding a protein of 249 amino acid (aa) residues with a signal peptide of 19 aa. The mannan-binding protein fraction of bovine serum that eluted with 100 mM mannose from a mannan-Sepharose column was analyzed under reducing conditions by SDS-PAGE. The major band of 33 kDa obtained reacted with anti-human MBP rabbit serum. The partial aa sequence of the purified 33-kDa protein was identical to the aa sequence deduced from the obtained cDNA. Results of the passive hemolysis experiment using sheep erythrocytes coated with yeast mannan suggest that this MBP has the ability to activate complement. Northern blot analysis showed a 1.8-kb mRNA that was expressed only in the liver. Based on results of genomic analysis, this bovine MBP is likely to be a homologue of human MBP and to also have homology to rat and mouse MBP-C which are localized in liver cells rather than to rat and mouse MBP-A found in serum. Alignments of bovine collectins show that bovine MBP cannot be included among the other bovine collectins, such as bovine SP-D, conglutinin and CL-43. Finally, these genomic and biological analyses indicate that the cDNA obtained here encoded a bovine serum MBP.