Immune complex formation causes underdetection of p24 antigen in human immunodeficiency virus (HIV) infection. Brieflyboiling diluted plasma releases all complexed antigen, which can then be measured by some commercial assays. In a retrospective pediatric cohort study, the specificity of this procedure in 390 uninfected samples was 96.9% after initial testing and 100% after neutralization. Sensitivity among 125 postnatal infected samples was, at a detection of 2 pg/ml., 96.0% (97% neutralizable) compared with 47.7% for regular antigen (76% neutralizable), 96% for polymerase chain reaction, and 77% for viral culture. The high sensitivity and specificity of heat-denatured antigen was confirmed by prospectively testing 113 additional samples. Quantitative analysis of samples from infected infants showed low levels of p24 antigen in 29% of cord blood sera, a postnatal increase to levels that were during the first 6 months of life inversely associated with survival, and persistence of antigenemia thereafter independent of clinical status. Prevalence and antigen levels were significantly lower in mothers. The persistent antigenemia in children indicates that their immune systems cannot restrict HIV expression as efficiently as those of adults.
A previously described self-complementary oligodeoxynucleotide termed triplex-forming oligodeoxynucleotide (TFO A), 54 bases in length, designed against the polypurine tract of HIV-1 RNA, inhibited viral replication at a 1 to 3 microM concentration in acutely infected cells, whereas antisense and scrambled sequence oligodeoxynucleotides were ineffective. Three HIV-1 viral isolates from patients of clinical categories A1, B, and C3 were transmitted to peripheral blood mononuclear cells and tested for production of p24 antigen and syncytium formation in the absence and in the presence of either TFO A or a control oligodeoxynucleotide of randomized sequence. No p24 antigen or syncytia were detected for up to 30 days when TFO A was added to the cells. Viability of the cells was found not to be affected by the drugs compared to controls within 2 weeks. Analysis of viral DNA synthesis by PCR for the LTR and gag gene indicated no DNA signal, suggesting that TFO A affects viral replication before formation of a DNA provirus. Measurements of the stability of TFO A indicate a half-life of about 2 hr. A two-dimensional computer fold analysis of TFO A suggested a self-complementary hairpin-loop configuration with GC-rich stems and single-stranded 5' and 3' ends. Since intracellular triplex formation may not be an efficient process, the observed inhibitory effect may be due to a direct inhibition of the RT and RNase H enzyme activities by the oligodeoxynucleotide. However, a triple-helix effect on the incoming RNA may play a role as well.
A 54-base-long oligodeoxynucleotide (ODN) termed triple helix-forming oligonucleotide A (TFO A), designed against the 3'-polypurine tract (PPT) of the human immunodeficiency virus type 1 (HIV-1), exhibits long-term efficacy in antiretroviral treatment. Viral replication of strains propagated in this laboratory as well as primary patient isolates are inhibited by TFO A, whereas ODNs with a randomized sequence but identical base composition show no effect. TFO A inhibits proviral DNA synthesis. To learn more about the molecular mechanism of function of TFO A, three HIV-1 isolates whose reverse transcriptase (RT) exhibits resistance against RT inhibitors were analyzed. They exhibit resistance against azidothymidine, dideoxyinosine, deoxythiacytidine, and the nonnucleoside inhibitor nevirapine. HIV-1 replication in TFO A-treated T cell cultures was assessed by monitoring p24 viral core antigen production and syncytium formation. No p24 antigen or syncytia were detected for up to 30 days when cells that had been infected with wild-type virus received TFO A. Similarly, replication of all three mutant HIV-1 strains was completely inhibited by TFO A treatment during the whole duration of the culturing period. No viral breakthrough was detectable. These results indicate that TFO A interferes with functions of the replicative cycle distinct from polymerization by the RT.
Reverse transcription of retroviral RNA into double-stranded DNA is catalyzed by reverse transcriptase (RT). A highly conserved polypurine tract (PPT) on the viral RNA serves as primer for plus-strand DNA synthesis and is a possible target for triple-helix formation. Triple-helix formation during reverse transcription involves either single-stranded RNA or an RNA.DNA hybrid. The effect of triple-helix formation on reverse transcription has been analyzed here in vitro using a three-strand-system consisting of an RNA.DNA hybrid and triplex-forming oligonucleotides (TFOs) consisting either of DNA or RNA. Three strand triple-helices inhibit RNase H cleavage of the PPT-RNA.DNA hybrid and initiation of plus-strand DNA synthesis in vitro. Triple-helix formation on a single-stranded RNA target has also been tested in a two-strand-system with TFOs comprising Watson-Crick and Hoogsteen base-pairing sequences, both targeted to the PPT-RNA, on a single strand connected by a linker (T)4. TFOs prevent RNase H cleavage of the PPT-RNA and initiation of plus-strand DNA synthesis in vitro. In cell culture experiments one TFO is an efficient inhibitor of retrovirus replication, leading to a block of p24 synthesis and inhibition of syncytia formation in newly infected cells.
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