Smaller amounts of antiretroviral drugs are needed when combined with an active ribozyme against HIV-1.

We have tested for combined anti-HIV-1 effects of a hammerhead ribozyme and antiretroviral drugs and the possibility of reducing the drug burden of patients on highly active antiretroviral therapy (HAART). The antiretroviral compounds used represent the three groups in HAART: nucleoside analogue reverse-transcriptase inhibitors, nonnucleoside reverse-transcriptase inhibitors, and protease inhibitors. A human T cell line (HUT78), stably expressing a hammerhead ribozyme targeted to nef (hhRz.nef(9016-9029)), was infected with HIV-1(SF2) in the presence of a single drug. The combined effects on HIV-1 replication were measured by p24 antigen determinations over a 2-week period. In the presence of the ribozyme, smaller amounts of antiretroviral drugs were required to reduce the HIV-1 p24 levels equally as much as when only drugs were present. The results support a strategy of combining ribozyme gene therapy with HAART to improve the long-term outcome of anti-HIV-1 therapy.

The nontoxic tripeptide glycyl-prolyl-glycine amide inhibits the replication of human immunodeficiency virus type 1.

OBJECTIVE: To determine whether short peptides corresponding to the RGPGR motif of the V3 loop of gp 120 have anti-human immunodeficiency virus type 1 (anti-HIV-1) activity. DESIGN/METHODS: Short peptides were tested against the HIV-1 laboratory strains and clinical isolates. RESULTS: The tripeptide glycyl-prolyl-glycine amide (GPG-NH2) inhibited the replication of both laboratory strains and 47 clinical isolates, including 19 strains that were resistant to other drugs or that were from patients with failing therapy. The 50% inhibitory concentrations values were 2.7 to 37 microM. Phenotypic change of two isolates from nonsyncytia-inducing to syncytia-inducing did not change their sensitivity to GPG-NH2. The tripeptide added to the antiviral effect of both zidovudine and ritonavir. CONCLUSIONS: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action. Glycyl-prolyl-glycine-NH2 might prove useful by itself or as a lead compound for the treatment of drug-resistant HIV-1. Glycyl-prolyl-glycine-NH2 is currently undergoing phase I/II human clinical trials in Sweden.

The tripeptide glycyl-prolyl-glycine amide does not affect the early steps of the human immunodeficiency virus type 1 replication.

OBJECTIVE: To determine whether the peptide glycyl-prolyl-glycine amide (GPG-NH2) corresponding to a conserved motif in the tip of the third hypervariable region of gp120 affected the early events in the human immunodeficiency virus type 1 (HIV-1) replication. DESIGN/METHODS: Glycyl-prolyl-glycine amide was tested for its effect on HIV-1 adsorption, co-receptor usage, proviral DNA synthesis, messenger RNA (mRNA) synthesis and splicing, translation, tat/TAR transactivation, and virus protease activity. RESULTS: Glycyl-prolyl-glycine amide did not appear to affect the early events of the virus replication. HIV-1 having glycine-leucine-glycine instead of GPG in the V3 loop and the mutants deleted of the GPG motif were still inhibited by the peptide. Glycyl-prolyl-glycine-NH2 had no discernible effect on any of the other steps in the virus replication cycle tested. The only effect observed was an increased sodium dodecyl sulfate polyacrylamide amide gel electrophoresis mobility of gp160/120 at high concentrations of GPG-NH2. CONCLUSIONS: The tripeptide GPG-NH2 is a nontoxic compound that inhibits the replication of HIV-1 by an apparently new mode of action.

Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays.

BACKGROUND: Real-time monitoring of cytomegalovirus (CMV) infections in transplant patients demands a rapid and high-throughput CMV DNA quantification method. METHODS: TaqMan polymerase chain reaction assays based on CMV immediate early protein exon 4 and glycoprotein B were developed and were compared with a COBAS AMPLICOR CMV MONITOR (CMM) test for quantifying CMV DNA in peripheral blood leukocytes from seven stem cell transplant patients having received antiviral treatment. RESULTS: There was a good correlation between the TaqMan assays and the CMM test for CMV DNA quantification. The throughput of the TaqMan assays was, however, about 3 times higher than that of the CMM test. The CMV DNA dynamics patterns determined by the TaqMan polymerase chain reaction were well in line with the outcome of the antiviral therapy. CONCLUSIONS: The TaqMan assays may potentially serve as a useful tool for rapid quantification of CMV infections in stem cell transplant patients.

Deletion of the GPG motif in the HIV type 1 V3 loop does not abrogate infection in all cells.

The three amino acids glycine, proline, and glycine (GPG) constitute a conserved motif at the center of the V3 loop of HIV-1 surface glycoprotein 120. It has been indicated that deletion of this GPG motif is lethal for viral infectivity and abrogates the ability of the virus to form syncytia. In the present work, we studied the effects of GPG deletion on viral infectivity, cell tropism, syncytium formation, and initiation of apoptosis by constructing a mutant provirus based on the infectious clone pBRu-2. Successful infection and replication of GPG-deleted virus were detected in MT-2 cells, although the mutant virus showed lower infectivity. Infection could also be observed in the C8166, C91-PL, Molt-3, and THP-1 cell lines, and in PBMC-derived dendritic cells (DCs), but not in CEM-SS, HUT78, H9, Jurkat, and U937 cell lines or in PBMCs. Mutant virus also induced syncytia and apoptosis in the MT-2 cells. An intact GPG motif is probably necessary for unimpaired induction of fusion in some HIV-1-permissive cells. However, once the virus enters the cells, the GPG sequence does not seem to be indispensable for syncytium formation or apoptosis induction in MT-2 cells. Our data also imply that cell surface molecules other than CD4 and CXCR4 may be involved in entry of the GPG-deleted virus.

Long terminal repeat promoter/enhancer activity of different subtypes of HIV type 1.

Transcription of the HIV-1 provirus genome is regulated by a complex interplay between viral regulatory proteins and cellular transcription factors that interact with the viral long terminal repeat (LTR) region of HIV-1. However, several cellular transcription factors have been identified that can interact with the HIV-1 LTR; the significance of all of these factors is not clearly understood. In this study we have characterized the LTR region of different subtypes of HIV-1 with regard to nucleotide sequence and promoter activity. The LTR regions of HIV-1 from peripheral blood mononuclear cells of 29 infected individuals originating from 10 different geographical regions were sequenced and further analyzed for promoter/enhancer activity in transient transfection of HeLa cells, in the context of a reporter gene and in the context of the complete virus genome. We found several subtype-specific LTR sequences of the various HIV-1 strains, such as an insertion that created a potential third NF-kappaB site in the LTR of the subtype C strains. The USF-binding site in the NRE also contained subtype-specific sequences. Interestingly, the promoter/enhancer activities of the subtype C LTRs were higher than the activities of the other subtypes analyzed here (subtypes A, B, D, E, and G), suggesting that the potential third NF-kappaB site may confer higher LTR activity or that the subtype C NRE may be less potent. Thus, our data suggest that genetic diversity of the LTR may result in HIV-1 subtypes with different replicative properties.

No association of HIV type 1 long terminal repeat sequence pattern with long-term nonprogression and in vivo viral replication levels in European subjects.

The HIV-1 long terminal repeat (LTR) promotes and modulates proviral transcription in the infected cell. It has been suggested that truncations and even point mutations in functional sites of the LTR are associated with low viral replication and attenuated pathogenesis in HIV-1-infected long-term nonprogressors (LTNPs). We performed a detailed analysis of LTR sequences from proviral DNA of 21 Italian and Swedish, well-characterized LTNPs and of 15 progressor patients. No truncation was found and no correlation was identified between specific LTR mutations and disease progression. We also failed to find a significant correlation between phylogenetic distance and clinical status. Although HIV-1 LTR interpatient heterogeneity among LTNPs and subjects with HIV-1 RNA levels <500 copies/ml tended to be lower, no sequence mutation was correlated with in vivo viral loads. Our results suggest that HIV-1 LTR defects are rare among Italian and Swedish LTNPs.

Increased serum concentrations of interleukin-2 receptor in the first trimester in women who later developed severe preeclampsia.

BACKGROUND: To determine the concentration of cytokines having an immunomodulating effect in the first trimester in women who subsequently developed preeclampsia. METHODS: The serum concentrations of IL-10, TNFalpha, IL-6 and IL-2R were determined in ten women who later developed severe preeclampsia and in ten healthy controls. The groups were compared with the Mann-Whitney U test. RESULTS: The IL-2R concentration was significantly higher in the women who later developed preeclampsia than in normal patients (p = 0.028). No significant differences were detected between the groups with respect to the other evaluated cytokines. CONCLUSION: Elevated IL-2R concentrations in maternal serum as early as in the first trimester may be a sign of immunological maladaptation and might be associated with a disturbance of trophoblastic invasion.

Nonsynonymous mutations within the human immunodeficiency virus type 1 p17 gene are clustered to sequences binding to the host human leukocyte antigen class I molecules.

We have analyzed the relation between intrapatient variabilities of the p17 gene and the location of known host p17 cytotoxic T lymphocyte (CTL) epitopes in five patients infected with human immunodeficiency virus type 1 (HIV-1). All patients were typed with respect to the human leukocyte antigen (HLA) class I type. One to seven previously fine-mapped p17 CTL epitopes corresponded to the HLA class I restriction elements of each patient. An average of 28+/-16% of the p17 gene of each patient encoded CTL epitopes corresponding to the HLA restriction elements of the host. Twenty full-length p17 gene clones were sequenced from each patient. The intrapatient homology between the p17 sequences ranged from 96.4 to 98.9%. The interpatient homology between the consensus sequences of each patient ranged from 83.1 to 91.6%. A total of 246 nucleotide differences within the 100 p17 clones was noted. Fifteen (16%) of 96 synonymous substitutions were found within host CTL epitopes, whereas 72 (48%) of 150 nonsynonymous nucleotide changes were found within CTL epitopes corresponding to the HLA restriction elements of the host (p < 0.0001; Fisher's exact test). Subsequently, variable residues indicating the evolution of at least two major p17 species (i.e., >20% of the clones) were determined to be more common at positions contained within these CTL epitopes (p < 0.01). The present data suggest that the evolution of the p17 gene is influenced by contact areas with the host HLA class I molecules.

HIV-1 nef mutations and clinical long-term nonprogression. A molecular epidemiology study.

OBJECTIVES: To analyze HIV-1 nef gene mutations in a cohort of Italian and Swedish long-term nonprogressors (LTNP) and to investigate whether particular amino acid substitutions are associated with LTNP. STUDY DESIGN/METHODS: nef alleles from 21 LTNP and 8 progressor controls were amplified by polymerase chain reaction (PCR) and sequenced. The amino acid sequences were compared with the previously reported sequences of 16 North American LTNP and of 28 patients with progressive infection. RESULTS: An untruncated intact open reading frame was observed as major sequence in all LTNP and controls. None of the amino acid substitutions in known biologically functional sites was linked to LTNP. A valine/isoleucine at the variable position 11 was associated with both European (P = .0001) and American (P = .001) LTNP. The interpatient nef variation was lower among European LTNP (P = .002) than in European progressor controls. CONCLUSIONS: Nef amino acid heterogeneity is lower among LTNP, probably reflecting the lower HIV-1 replication rate. Nef gene defects appear uncommon in both Swedish and Italian LTNP, although the presence of a valine/isoleucine at position 11 is statistically associated with a lower probability to progress to disease.

Differences in humoral responses to the p24 antigen between Ethiopian and Swedish human immunodeficiency virus type 1-infected patients may suggest influences from a T-helper 2-like phenotype.

The levels of human immunodeficiency virus type 1 p24 antibodies and p24 antigen among 256 Ethiopians and Africans and 86 Swedes were compared. The elevated levels of total anti-p24 immunoglobulin G (IgG) and anti-p24 IgG4 among the Ethiopian and African patients suggest influences from T-helper-cell type 2-like responses.

Quantification of HIV-1 proviral DNA from peripheral blood mononuclear cells using a high throughput four-competitor competitive PCR.

A multiple competitor PCR (mcPCR) was developed to quantify HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMC). DNA extracted from a mixture of HIV infected PBMC and four size-mutated DNA competitors were co-amplified. The Cy5-fluorescence labelled PCR products were denatured by heating, separated using an automated DNA sequencer and quantified by a fragment analysis computer software. An internal standard was generated by plotting the peak areas of the four competitors against their inputs. Based on the internal standard, HIV sample DNA was quantified by extrapolating the corresponding signal. The linear range of the mcPCR was three log wide and the quantitation limit was about 20 copies of HIV DNA/10(6) PBMC. Using the mcPCR, HIV DNA was quantified from 14 long-term non progressors (LTNP) and 14 patients with advanced disease. A significantly lower copy number of HIV DNA was obtained in the LTNP (p = 0.018). These data suggest that the mcPCR is sensitive, reliable and especially useful for HIV DNA quantification of a large number of clinical samples.

Identification of cross-reactive antigenic target regions for HIV type 1-specific antibody-dependent cellular cytotoxicity.

Monkey-derived hyperimmune antisera against 40 peptides, together representing the entire envelope gp120 of HIV-1LAI, were used to map antibody-dependent cellular cytotoxicity (ADCC) target regions. Four regions corresponding to amino acids 65-126, 152-230, 248-330, and 445-466 were found to contain epitopes inducing ADCC activity not only against HIV-1LAI-infected cells but also against cells infected with HIV-1SF2 and clinical isolates of HIV-1. When comparing seroreactivity to the individual peptides with ADCC titers none of the regions seemed to be dominant. These results thus describe cross-reactive regions involved in the functional immunity against HIV-1 gp120.

mtDNA variation indicates Mongolia may have been the source for the founding population for the New World.

mtDNA RFLP variation was analyzed in 42 Mongolians from Ulan Bator. All four founding lineage types (A [4.76%], B [2.38%], C [11.9%], and D [19.04%]) identified by Torroni and colleagues were detected. Seven of the nine founding lineage types proposed by Bailliet and colleagues and Merriwether and Ferrell were detected (A2 [4.76%], B [2.38%], C1 [11.9%], D1 [7.14%], D2 [11.9%], X6 [16.7%], and X7 [9.5%]). Sixty-four percent of these 42 individuals had "Amerindian founding lineage" haplotypes. A survey of 24 restriction sites yielded 16 polymorphic sites and 21 different haplotypes. The presence of all four of the founding lineages identified by the Torroni group (and seven of Merriwether and Ferrell's nine founding lineages), combined with Mongolia's location with respect to the Bering Strait, indicates that Mongolia is a potential location for the origin of the founders of the New World. Since lineage B, which is widely distributed in the New World, is absent in Siberia, we conclude that Mongolia or a geographic location common to both contemporary Mongolians and American aboriginals is the more likely origin of the founders of the New World.

A novel ribozyme target site located in the HIV-1 nef open reading frame.

We have tested the sequence UUC CAG UCA GAC CU, at position 9016--9029 within the HIV-1(SF2) nef open reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate-modified 14-nt DNA oligo (complementary to the nef9016--9029 site). A dose-dependent repression of HIV-1(SF2) growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for the nef9016--9029 site (hhRz.nef9016--9029) was constructed and transfected into the human T-cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016--9029 were infected with HIV-1(SF2). A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (10(3)-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviral pol and gag sequences in hhRz.nef9016--9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since no pol and gag PCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support the nef9016--9029 site as a strong new candidate for ribozymal gene therapy against HIV-1 infection.

Acyclovir treatment of relapsing-remitting multiple sclerosis. A randomized, placebo-controlled, double-blind study.

Acyclovir treatment was used in a randomized, double-blind, placebo-controlled clinical trial with parallel groups to test the hypothesis that herpes virus infections are involved in the pathogenesis of multiple sclerosis (MS). Sixty patients with the relapsing-remitting form of MS were randomized to either oral treatment with 800 mg acyclovir or placebo tablets three times daily for 2 years. The clinical effect was investigated by an extensive test battery consisting of neurological examinations, neuro-ophthalmological and neuropsychological tests, and evoked potentials. Results were based on "intent-to-treat" data and the primary outcome measure was the exacerbation rate. In the acyclovir group (n = 30), 62 exacerbations were recorded during the treatment period, yielding an annual exacerbation rate of 1.03. The placebo group (n = 30) had 94 exacerbations and an annual exacerbation rate of 1.57. Thus, 34% fewer exacerbations were encountered during acyclovir treatment. This difference in exacerbation rate between the treatment groups was not significant (P = 0.083). However, this trend to a lower disease activity in acyclovir-treated patients was supported in subsequent data analysis. If the patients were grouped according to exacerbation frequencies, i.e. into low (0-2), medium (3-5) and high (6-8) rate groups, the difference between acyclovir and placebo treatment was significant (P = 0.017). Moreover, in a subgroup of the population with a duration of the disease of at least 2 years providing an exacerbation rate base-line before entry, individual differences in exacerbation rates were compared between the 2-year pre-study period and the study period in acyclovir-treated (n = 19) and placebo (n = 20) patients and acyclovir-treated patients showed a significant reduction of exacerbations (P = 0.024). Otherwise, neurological parameters were essentially unaffected by acyclovir treatment and there were no convincing signs of reduced neurological deterioration in the acyclovir group. This study indicates that acyclovir treatment might inhibit the triggering of MS exacerbations and thus suggests that acyclovir-susceptible viruses might be involved in the pathogenesis of MS. This possibility warrants further investigation.

Characterization of an antigen shared by human thymic epithelium and human T cell leukemia virus p19 Gag protein.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

Emergence of precore TAG mutation during hepatitis B e seroconversion and its dependence on pregenomic base pairing between nucleotides 1858 of 1896.

The G-->A mutation of nucleotide (nt) 1896 in the precore region of hepatitis B virus (HBV) prevents production of hepatitis B e antigen (HBeAg) by creating a TAG stop codon. This mutation is often found in anti-HBe-positive patients with productive HBV infection and has been associated with severe chronic and fulminant hepatitis in some geographic areas. Emergence of the TAG mutation during HBe seroconversion was studied as was its relationship with nt 1858, which forms a base pair with nt 1896 in the pregenomic RNA loop. A TAG mutant evolved in 18 (72%) of 25 patients with a T-1858 strain but in only 1 (8%) of 13 with a C-1858 strain. Viremia with C-1858 strains was, despite their limited ability to develop the TAG mutation, as persistent as with T-1858 strains. Generally, T-1858-infected patients in whom a TAG mutant did not emerge were HBV DNA-negative by polymerase chain reaction on follow-up, whereas patients who developed the TAG mutation had prolonged viremia.