Multicenter evaluation of a rapid and convenient method for determination of cytomegalovirus immunoglobulin G avidity.

An easy, rapid, and reproducible test to distinguish residual cytomegalovirus (CMV) immunoglobulin M (IgM) antibodies from antibodies produced in primary infection could be useful, especially for pregnant women. The CMV avidity of IgG antibodies with the VIDAS automated enzyme-linked fluorescent assay and 6 M urea was evaluated in a multicenter study to differentiate between primary CMV infections and past infections or reactivations. A total of 416 serum specimens were tested: 159 specimens were from follow-up of primary infections, and 257 were from past infections. All of the specimens from primary infections collected within 4 months (17 weeks) after the onset of the infection had an avidity index lower than 0.8. An avidity index higher than 0.8 excludes a recent primary infection of less than 4 months. However, an avidity index higher than 0.8 cannot confirm all past infections, since 48 specimens (18%) from past infections had an avidity index lower than 0.8 (between 0.5 and 0.8). The exclusion capacity could be improved (96.9%) by using a cutoff of 0.7, but this index would decrease the specificity of the technique, since the avidity index was found to be between 0.7 and 0.8 in two patients with recent primary infection. All specimens from primary infections obtained more than 4 months after the onset of infection had an avidity index more than 0.2. In this study, an avidity index less than 0.2 confirms the presence of a recent primary infection of less than 4 months. The VIDAS CMV IgG avidity test is a rapid, reproducible test with very good performance.

The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore.

Viral protein R (Vpr) encoded by HIV-1 is a facultative inducer of apoptosis. When added to intact cells or purified mitochondria, micromolar and submicromolar doses of synthetic Vpr cause a rapid dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), as well as the mitochondrial release of apoptogenic proteins such as cytochrome c or apoptosis inducing factor. The same structural motifs relevant for cell killing are responsible for the mitochondriotoxic effects of Vpr. Both mitochondrial and cytotoxic Vpr effects are prevented by Bcl-2, an inhibitor of the permeability transition pore complex (PTPC). Coincubation of purified organelles revealed that nuclear apoptosis is only induced by Vpr when mitochondria are present yet can be abolished by PTPC inhibitors. Vpr favors the permeabilization of artificial membranes containing the purified PTPC or defined PTPC components such as the adenine nucleotide translocator (ANT) combined with Bax. Again, this effect is prevented by addition of recombinant Bcl-2. The Vpr COOH terminus binds purified ANT, as well as a molecular complex containing ANT and the voltage-dependent anion channel (VDAC), another PTPC component. Yeast strains lacking ANT or VDAC are less susceptible to Vpr-induced killing than control cells yet recover Vpr sensitivity when retransfected with yeast ANT or human VDAC. Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC.

Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death.

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.