Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins.     

Human single-chain antibodies inhibit replication of human immunodeficiency virus type 1 (HIV-1)

The F240 human monoclonal antibody specifically recognizes the disulfide loop-bonded immunodominant epitope of gp41 spanning residues 592-606 and expressed broadly on HIV-1 primary isolates. Despite broad reactivity with native virions and HIV-infected cells, the antibody fails to neutralize infection. However, cytoplasmic expression of single-chain antibody (scFv) directed against gp41 of HIV-1 provides a rationale means to inhibit the maturation of envelope protein. The variable regions of the heavy chain and light chain of human monoclonal antibody were amplified by PCR and linked by a 15 amino acid (GGSGS)3 linker in an orientation of VL-linker-VH and retroviral expression vectors were constructed to simultaneously express F240 scFv and eGFP to facilitate selection of scFv-producing cells. Incorporation of a human immunoglobulin signal sequence directed secretion of the F240 scFv (s-scFv) while an otherwise identical vector lacked this sequence (scFv) resulting in intracellular expression of scFv. Transduced human CD4+ H9 T cells were challenged with HIV. While both secreted and nonsecreted F240 scFv inhibited viral production, secretory F240 scFv was more potent. Thus, this novel approach to direct expression of a nonneutralizing scFv using the Ig signal sequence suggests that targeted therapy using antibodies to conserved, highly expressed epitopes may result in a decrease in viral production due to a reduction of viral assembly and/or transport and expression.     

Genetic subtypes of human immunodeficiency virus type 1 (HIV-1) in Istanbul, Turkey.

BACKGROUND: Epidemiological surveillance of HIV-1 subtypes is an important and ongoing element of preparation for global antiviral interventions. OBJECTIVE: To assess the molecular epidemiology of HIV-1 in Istanbul, Turkey. STUDY DESIGN: 27 HIV/AIDS patients were investigated. Data on age, sex, country of birth, and HIV acquisition route were collected. Following amplification with PCR the sequences of the gp41 region of the env gene were determined using a 310 DNA sequencer (ABI prism, Foster City, USA) and phylogenetically analyzed. RESULTS: Among the 27 patients (26 adults and 1 infant), 22 were male, born in Turkey, and 20 infected through heterosexual contact. Two patients acquired the virus through blood and/or blood transfusion and one infant by vertical transmission. The distribution of the subtypes was as follows: four were subtype A, 19 subtype B, one subtype C, one subtype D, and two subtype F1. According to our results, although the B subtype is still predominant, non-B subtypes are also present, even though the number of registered HIV/AIDS patients is low. CONCLUSION: These are the first subtyped HIV-1 strains in Turkey where a low level of HIV prevalence has been observed since the first reported case in 1985. These findings and Turkey's specific geographic localization indicate the need for a nationwide surveillance to detect all subtypes including the new recombinant ones.     

Human immunodeficiency virus type 1 (HIV-1) gp120-specific antibodies in neonates receiving an HIV-1 recombinant gp120 vaccine.

Infants born to human immunodeficiency virus type 1 (HIV-1)-infected mothers were immunized at birth and at ages 4, 12, and 20 weeks with low-, medium-, or high-dose recombinant gp120 vaccine with MF59 adjuvant (HIV-1(SF-2); n=52) or with MF59 alone as a placebo (n=9). An accelerated schedule (birth and ages 2, 8, and 20 weeks) was used for an additional 10 infants receiving the defined optimal dose and for 3 infants receiving placebo. At 24 weeks, anti-gp120 ELISA titers were greater for vaccine-immunized than for placebo-immunized infants on both schedules, and 87% of vaccinees had a vaccine-induced antibody response. At 12 weeks, antibody titers of infants on the accelerated vaccine schedule exceeded those of infants receiving placebo (4949 vs. 551; P=.01), and 63% of the vaccinees met the response criteria. Thus, an accelerated schedule of gp120 vaccinations generated an antibody response to HIV-1 envelope distinct from transplacental maternal antibody by age 12 weeks. These results provide support for further studies of vaccine strategies to prevent mother-to-infant HIV-1 transmission.     

Identification of a high-affinity RNA-binding site for the human immunodeficiency virus type 1 Rev protein.

Expression of the structural proteins of human immunodeficiency virus type 1 requires the direct interaction of multiple copies of the viral Rev protein with its highly structured RNA target sequence, the Rev response element (RRE). Nucleotides critical for Rev monomer binding have been mapped by chemical interference to a single site flanking the base of an RNA helix (stem IIB) located within the 234-nucleotide RRE. Binding of additional Rev molecules to an RRE probe did not require any RNA primary sequence information detectable by modification interference beyond that required for binding of a single Rev protein molecule. A synthetic 29-nucleotide RNA molecule designed to incorporate nucleotides identified as critical for Rev binding retained the ability to bind Rev specifically and, therefore, represents a minimal Rev-binding site. We propose that Rev binding to the RRE initiates with the direct interaction of a Rev monomer with a high-affinity binding site located at the base of the IIB stem of the RRE. The subsequent formation of Rev multimers on the RRE appears, in contrast, primarily driven by specific protein-protein interactions.     

Preliminary evaluation of human immunodeficiency virus type 1 (HIV-1) immunogen in children with HIV-1 infection.

The safety and preliminary activity of human immunodeficiency virus type 1 (HIV-1) immunogen were evaluated in 10 HIV-1-infected children with disease stage N1,2 or A1,2. Multiple inoculations of 2. 5 or 10 units (U) of HIV-1 immunogen were safe and well tolerated without an acceleration of disease progression. When antiretroviral agents were coadministered, the 10 U dose appeared to be associated with more sustained reduction in plasma HIV-1 RNA than the 2.5 U dose (median log10 HIV-1 RNA at month 18, 3.07 vs. 4.01 copies/mL in 10 U [n=4] vs. 2.5 U [n=3], respectively; P=.034). Levels of regulated-on-activation, normal T cell-expressed and -secreted chemokine produced from HIV-1 immunogen-stimulated lymphocytes in vitro were increased in the children who had HIV-1 immunogen-specific antibody responses (P<.02) and appeared to be inversely correlated with levels of plasma HIV-1 RNA (P<.01). These preliminary data warrant larger studies to determine the effectiveness of adjunctive therapy with HIV-1 immunogen in children with HIV-1 infection.     

Trends in human immunodeficiency virus type 1 (HIV-1) load among HIV-1-infected children with hemophilia.

In human immunodeficiency virus type 1 (HIV-1)-infected persons, virus load (serum/plasma level of HIV) predicts outcome. Virus load trends have been characterized in adults and infants but not in children. Virus load trends in 22 male children with hemophilia who acquired HIV-1 postnatally (age 0.7-5.2 years at seroconversion) were studied. The mean HIV-1 load 2 years after seroconversion was 4.40 log10 copies/mL, and the mean change over time (slope) was 0.03 log10 copies/(mL x year). Significant among-children variation was apparent: a random effects model predicted that 95% of children had early virus loads 3.75-5.04 log10 copies/mL and slopes -0.07 to 0.12 log10 copies/(mL x year). Higher early virus loads and higher slopes were each associated with increased mortality (P=.006 and P=.03, respectively). In conclusion, those subjects had virus load trends similar to those in adults. Early virus loads were lower than those in vertically infected infants, which suggests that factors changing soon after birth affect viral replication.     

Hepatic encephalopathy in primary human immunodeficiency virus type 1 (HIV-1) infection

Primary infection of human immunodeficiency virus type 1 (HIV-1) is occasionally associated with common cold-like symptoms, and rarely with a self-limited illness resembling infectious mononucleosis. We report a 32-year-old man who presented with infectious mononucleosis-like blood picture on admission. Five days after admission he developed hepatic encephalopathy, which was ameliorated by administration of bolus corticosteroid. Based on the results of serologic studies, we diagnosed that he had primary HIV-1 infection. To our knowledge, this is the first published report of hepatic encephalopathy as a clinical manifestation of primary HIV-1 infection.     

Human T-cell lymphotropic virus type I (HTLV-I)-associated adult T-cell leukemia-lymphoma in a patient infected with human immunodeficiency virus type 1 (HIV-1)

A patient had adult T-cell leukemia-lymphoma in the unusual setting of coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell lymphotropic virus type I (HTLV-I). The leukemic cells were CD4 positive and showed clonal genetic rearrangement of the T-cell receptor complex. Cytogenetic analysis showed three clonal karyotypic abnormalities: trisomy 3 and two translocations [t(1;15), (X;1)]. The patient was seropositive for HIV and HTLV-I; HTLV-I and HIV-1 DNA sequences were detected in peripheral blood leukocytes by the polymerase chain reaction. The HTLV-I sequences were detected in a relatively high proportion of mononuclear cells (at least 1 in 30 cells), whereas HIV-1 sequences were detected in a smaller proportion of cells (at least 1 in 3000 cells). Clinical remission was achieved after chemotherapy. There was a decrease in the proportion of HTLV-I positive mononuclear cells (at least 1 in 1000 cells), whereas the proportion of HIV-1 positive cells was relatively unchanged (at least 1 in 1000 cells). Adult T-cell leukemia-lymphoma in the setting of HIV coinfection may become increasingly common because asymptomatic retroviral coinfections are frequent.     

Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals.     

Kinetics of human immunodeficiency virus type 1 (HIV-1) DNA and RNA synthesis during primary HIV-1 infection.

HIV-1 replication and viral burden in peripheral blood mononuclear cells (PBMC) have been reported to be high in primary infection but generally very low during the prolonged period of clinical latency. It is uncertain precisely when this transition occurs during the HIV-1 infection and what the relationship is between the changes in HIV-1 replication versus the clearance of infected cells in the overall control of viral replication. In the present study, the kinetics of viral burden (i.e., frequency of HIV-1-infected cells) and replication during primary and early-chronic infection were analyzed in PBMC of four acutely infected individuals. High frequencies of HIV-1-infected cells and high levels of virus replication were observed in PBMC after primary HIV-1 infection. Down-regulation of virus replication in PBMC was observed in all four patients coincident with the emergence of HIV-1-specific immune responses. Other parameters of virus replication, such as circulating plasma p24 antigen and plasma viremia showed similar kinetics. In contrast, a significant decline in viral burden in PBMC was observed in only one of four patients. These results indicate that the down-regulation in the levels of virus replication associated with the clinical transition from acute to chronic infection does not necessarily reflect a reduction in viral burden, thus suggesting the involvement of additional factors. Identification of these factors will be important in elucidating the host mechanisms involved in the early control of HIV-1 infection and disease.     

Human immunodeficiency virus type 1 (HIV-1) inhibitory interactions between protease inhibitor Ro 31-8959 and zidovudine, 2',3'-dideoxycytidine, or recombinant interferon-alpha A against zidovudine-sensitive or -resistant HIV-1 in vitro.

Protease inhibitor Ro 31-8959, a compound that interrupts human immunodeficiency virus (HIV)-specific formation of infectious virions, was evaluated in two-drug combined regimens with zidovudine, 2',3'-dideoxycytidine (ddC), or recombinant interferon-alpha A (rIFN-alpha A) against HIV-1 replication in vitro. By using peripheral blood mononuclear cells infected with HIV-1, drug interactions were evaluated by the median-effect principle and the isobologram technique. A zidovudine-sensitive and -resistant HIV-1 isolate pair was studied. Additive to synergistic anti-HIV-1 interactions were seen with 7.5-30 nM Ro 31-8959 and 0.005-0.02 microM zidovudine (for the zidovudine-sensitive HIV-1 isolate), 0.25-1.0 microM zidovudine (for the zidovudine-resistant HIV-1 isolate), 0.025-0.1 microM ddC, and 8-32 units/mL rIFN-alpha A, without additive toxicity. Phase I/II clinical trials of Ro 31-8959 for therapy of HIV-1 infection are in progress. If results are favorable, combined regimens including Ro 31-8959 deserve consideration for future clinical trials.     

Human immunodeficiency virus type 1 (HIV-1) and Mycobacterium leprae co-infection: HIV-1 subtypes and clinical, immunologic, and histopathologic profiles in a Brazilian cohort

Co-infections with human immunodeficiency virus (HIV) and Mycobacterium leprae represent unique opportunities to investigate the interaction of both pathogens. We determined the immunologic, virologic, and histopathologic characteristics of 22 co-infected Brazilian patients (median age = 38 years, 81.8% males, 72.2% with paucibacillary leprosy, and 95.4% with acquired immunodeficiency syndrome). The HIV-1 subtypes B and BF predominated in envelope and gag heteroduplex mobility analysis. Borderline tuberculoid (BT), tuberculoid, lepromatous, and indeterminate morphology with CD3+, CD8+, and CD68+ cell distributions compatible with leprosy patients not infected with HIV were observed. Histologic evidence of nerve damage was observed in BT lesions. IgM antibody to M. leprae-specific phenolic glycolipid I was not detected. Two of six co-infected patients monitored during highly active antiretroviral therapy (HAART) developed a leprosy type 1 reaction after an increase in CD4+ cells, suggesting an immune restoration phenomenon. Clinical, immunologic, histopathologic, and virologic features among these HIV-leprosy co-infected patients indicate that each disease progressed as in single infection. However, HAART immune reconstitution may trigger potential adverse effects, such as leprosy acute inflammatory episodes.     

Factors contributing to the lack of human immunodeficiency virus type 1 (HIV-1) transmission in HIV-1-discordant partners

Correlates of resistance to infection by human immunodeficiency virus type 1 (HIV-1) are important for defining potential therapeutic interventions and for prophylactic vaccination. In this study, 11 couples discordant in their HIV-1 infection status were prospectively evaluated for the presence of protective factors. Behavioral characteristics of all subjects entailed a high risk of transmission. Cytotoxic T lymphocyte (CTL) responses against viruses isolated from the infected partner, and against laboratory virus isolates, were detected in 5 (45%) of 11 HIV-negative partners, including a CCR5Delta32-homozygous and a heterozygous subject. No CTL responses were observed in 6 control unexposed subjects. Marked variation in lymphocyte susceptibility to viral infection was noted. Resistance attributable to major histocompatibility complex discordance or anti-major histocompatibility complex antibodies was not identified. These results suggest that a combination of factors, including cellular immunity, viral characteristics, and coreceptor integrity, may be involved in the persistent nontransmission of HIV.     

Thrombotic thrombocytopenic purpura associated with human immunodeficiency virus type 1 (HIV-1) infection

The cases of 14 patients with thrombotic thrombocytopenic purpura admitted to one institution after 1980 were reviewed. Three of the fourteen cases occurred in patients with the acquired immunodeficiency syndrome (AIDS)-related complex and one occurred in a patient with probable human immunodeficiency virus (HIV) infection. The diagnosis in all four cases had been made after 1985. The association of thrombotic thrombocytopenic purpura with HIV infection was judged to be statistically significant on the basis of the proportion of patients with AIDS among the general population of patients admitted to the same institution during the same period. The fact that this association is only now being recognized suggests that there may be a long incubation period for thrombotic thrombocytopenic purpura or that the association is a rare one recognized now only because of the increased number of persons with AIDS.