W. Edward Robinson

ANTIBODY-DEPENDENT ENHANCEMENT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION

Two components of human serum enhance human immunodeficiency virus type 1 (HIV-1) infection and mask HIV-1 neutralising antibody activity. The first is heat-stable, unique to HIV-1 seropositive sera, and is removed by protein-A chromatography. The second is heat-labile and ubiquitous; it is found in normal serum and is removed by heating at 60 degrees C for 1 h or by treatment with cobra venom anticomplementary protein. Additionally, complement component C3 deficient serum lacks the labile activity although Clq deficient serum contains the labile factor. The data suggest that the two components are antibody and the alternative pathway of complement fixation. The mechanism of action does not involve an increase in either complement-mediated cytolysis or syncytium formation. The activity has been identified in 11 of 16 patients tested to date.

New and facile method of preparation of the anti-HIV-1 agent,1,3-dicaffeoylquinic acid

A facile and inexpensive preparation of 1,3-dicaffeoylquinic acid (cynarin) from the leaves Cynara cardunculus L. (Asteraceae) without the use any chromatographic steps is described. The procedure was based on separation of the fraction rich in 1,5-dicaffeoylquinic acid, isomerisation of 1,5-dicaffeoylquinic acid to cynarin and, owing to its higher polarity, the simple isolation of cynarin from the reaction mixture. Cynarin inhibited HIV-1 replication in MT-2 cell culture at nontoxic concentrations similar to other previously tested dicaffeoylquinic acids, which have been recently established as a potent and highly selective class of HIV-1 integrase inhibitors.

l-Chicoric acid, an inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase, improves on the in vitro anti-HIV-1 effect of Zidovudine plus a protease inhibitor (AG1350)

Combinations of anti-human immunodeficiency virus (HIV) drugs, including reverse transcriptase inhibitors and protease inhibitors, have proven immensely potent in the therapy of acquired immune deficiency syndrome (AIDS). To determine whether HIV integrase is a suitable target for combination therapy, the ability of an HIV integrase inhibitor, L-chicoric acid, to work in combination with a protease inhibitor and Zidovudine was tested in vitro. The addition of L-chicoric acid to either Zidovudine or protease inhibitor improved upon the observed anti-HIV activity of either compound alone. When all three drugs were combined, the anti-HIV activity was substantially better than either of the three compounds alone or any combination of two inhibitors. Doses of both Zidovudine and protease inhibitor could be reduced by more than 33% for an equivalent anti-HIV effect if L-chicoric acid was added. The improved anti-HIV activity was observed with a tissue culture adapted strain of HIV (HIV(LAI)) and with limited passage clinical isolates of HIV (HIV(R19) and HIV(R45)). These data demonstrate that a first generation HIV integrase inhibitor, L-chicoric acid, is at least additive in combination with existing multi-drug regimens and suggest that HIV integrase will be an excellent target for combination therapy of HIV infection.

Differential Effects on Human Immunodeficiency Virus Type 1 Replication by α-Defensins with Comparable Bactericidal Activities

ABSTRACT In addition to their antibacterial activities, certain antimicrobial peptides inactivate enveloped viruses, including the human immunodeficiency virus (HIV). To determine whether peptide bactericidal activities are predictive of antiviral activity, the anti-HIV properties of recombinant human α-defensin 5, mouse α-defensins, cryptdins (Crp) 3 and 4, and rhesus macaque myeloid α-defensins (RMADs) 3 and 4 were determined in vitro. The peptides, purified to homogeneity, had equivalent bactericidal activities that were similar to those of the native molecules. Nuclear magnetic resonance spectroscopy showed RMAD-4 and Crp3 had characteristic α-defensin tridisulfide arrays. Of the peptides analyzed, only RMAD-4 inhibited HIV infectivity at 150 μg/ml, and Crp3 unexpectedly increased HIV replication. Quantitative real-time PCRs for minus-strand strong stop DNA and complete viral cDNA synthesis were used to distinguish between preentry and postentry anti-HIV effects by RMAD-4. Viral exposure to RMAD-4 for 1 h prior to infection reduced HIV minus-strand strong stop DNA and HIV cDNA by 4- to 20-fold during the first round of replication, showing that RMAD-4-exposed virions were not entering cells during the first 24 h. On the other hand, when RMAD-4 was added coincident with HIV inoculation, no anti-HIV activity was detected. Viral exposure to Crp3 resulted in a threefold increase in both HIV minus-strand strong stop DNA and HIV cDNA over the first round of replication. Therefore, two α-defensins, RMAD-4 and Crp3, inhibit or augment HIV replication, respectively, by mechanisms that precede reverse transcription.

Human immunodeficiency virus type-1 integrase containing a glycine to serine mutation at position 140 is attenuated for catalysis and resistant to integrase inhibitors.

L-chicoric acid (L-CA) is a potent inhibitor of HIV integrase (IN) in vitro. In this report, the effects of a glycine to serine mutation at position 140 (G140S) on HIV IN and its effects on IN inhibitor resistance are described. HIV containing the G140S mutation showed a delay in replication. Using real-time polymerase chain reaction, the delay was secondary to a failure in integration. The mutant protein (IN(G140S)) was attenuated approximately four-fold for catalysis under equilibrium conditions compared to wild-type IN (IN(WT)) and attenuated five-fold in steady-state kinetic analysis of disintegration. Fifty percent inhibitory concentration assays were performed with IN inhibitors against both IN proteins in disintegration and strand transfer reactions. IN(G140S) was resistant to both L-CA and L-731,988, a diketoacid. HIV containing the mutation was resistant to both inhibitors as well. The G140S mutation attenuates IN activity and confers resistance to IN inhibitors, suggesting that diketoacids and L-CA interact with a similar binding site on HIV IN.

A Human Immunodeficiency Virus type 1 (HIV-1) infection-enhancing factor in seropositive sera

While testing sera for Human Immunodeficiency Virus neutralizing antibody titers, three sera were identified which had the ability to enhance infectivity of the virus. The sera were from three different individuals residing in Nashville, TN. The enhancing factor was not removed by either filtration through 0.05 micron filters or by incubation for one hour with a stoichiometric amount of protein A sepharose. Two of the sera were able to enhance infection by two divergent isolates (HTLV-IIIB and HTLV-IIIRF) while one was only capable of enhancing infection of target cells by HTLV-IIIB. None of the sera induced syncytium formation in chronic HIV-infected cells. The findings suggest that the substance is neither a virus nor an IgG class 1 or 2.

A docking study of L-chicoric acid with HIV-1 integrase.

Human immunodeficiency virus 1 integrase (HIV-1 IN) is the enzyme responsible for integrating the viral DNA into the host genome, and is essential to the replication of the virus. L-Chicoric acid (L-CA) is a bidentate catechol that has been identified as a potent inhibitor of HIV-1 IN. Using the new Autodock 4.0 free-energy function we have obtained a L-CA binding mode that explains its observed potency and is consistent with available experimental data. Because of the alpha,beta-unsaturated ester functionality of the side arms of L-CA we first performed an extensive conformational analysis of L-CA using semiempirical and ab initio calculations. As a result we have identified two distinct L-CA binding modes, one for the s-cis/s-cis and another for the s-cis/s-trans isomers. The most stable conformer was found to be the structure with the alpha,beta-unsaturated ester in the s-cis conformation for both arms of L-CA. This conformer also gave the top-ranked docking solution. Analysis of the interactions with key IN residues, combined with results using a L-CA tetraacetylated derivative and a Q148A IN mutant, correlate well with the experimental data.

Natural killer cell infection and inactivation in vitro by the human immunodeficiency virus

Cytolytic activity of human mononuclear peripheral blood leukocytes from healthy donors, cultured in interleukin-2 conditioned medium, was abrogated by in vitro infection with the lymphadenopathy associated virus (LAV) isolate of the human immunodeficiency virus (HIV). Although viral antigens are not expressed in cultured cells until 14 days postinfection, cytolytic activity was lost as early as 3 days after infection. Loss of cytolytic function was not a result of the release of suppressive factors from either infected cells or uninfected CEM cells since supernatants from neither infected cultures nor CEM cell cultures had any inhibitory effects on the function of uninfected cells. Cultured lymphocytes expressing Leu 11b were also shown to express HIV antigens via immunofluorescence after 14 days in culture. These results suggest that natural killer (NK) cells, as defined by expression of Leu 11b, were infected by HIV in vitro and the loss of lytic function was likely a direct consequence of that infection.

Mismatched double-stranded RNA (polyI-polyC12U) is synergistic with multiple anti-HIV drugs and is active against drug-sensitive and drug-resistant HIV-1 in vitro

Although highly active anti-retroviral therapy (HAART) is successful in the treatment of HIV infection, problems with toxicity, drug-resistant variants, and therapeutic failures have compromised the long-term utility of existing combination regimens. Mismatched double-stranded RNA (polyI-polyC(12)U) is an immune modulator with inherent anti-HIV activity. Cell toxicities and anti-HIV activities of fourteen anti-HIV agents were determined alone and in combination with polyI-polyC(12)U. Combination analyses for anti-HIV activity were performed at three drug ratios. Using Mixed Dose Effect analyses and the CalcuSyn for Windows software package, combination indeces were determined for all drug combinations. In general, polyI-polyC(12)U was synergistic in combination with abacavir, zidovudine, zalcitabine, didanosine, stavudine, efavirenz, indinavir, ritonavir, nelfinavir, and amprenavir. It was synergistic to antagonistic with lamivudine, delavirdine, nevirapine, and saquinavir. Thus, polyI-polyC(12)U is synergistic with most anti-HIV agents at most drug ratios and across most effective concentrations in vitro, although, certain members of each class were exceptions. PolyI-polyC(12)U alone was equally active against wild-type HIV and HIV resistant to nevirapine, protease inhibitors, or nucleoside analogue reverse transcriptase inhibitors. These results suggest that polyI-polyC(12)U should be re-evaluated as a potential adjunct therapy in patients who have failed current anti-retroviral therapeutic regimens.

Preliminary Mapping of a Putative Inhibitor-Binding Pocket for Human Immunodeficiency Virus Type 1 Integrase Inhibitors

ABSTRACT Molecular modeling studies have identified a putative human immunodeficiency virus (HIV) integrase (IN) inhibitor-binding pocket for l-chicoric acid (l-CA) and other inhibitors of IN (C. A. Sotriffer, H. Ni, and A. McCammon, J. Med. Chem. 43:4109-4117, 2000). By using site-directed mutagenesis of several amino acid residues identified by modeling studies, a common inhibitor-binding pocket on IN was confirmed for l-CA and the diketo acid L-731,988. Specifically, the single mutations E92K, Q148A, K156A, K156R, G140S, and G149S, as well as the double mutations C65S-K156N and H67D-G140A were evaluated for their effects on enzymatic activity and inhibitor susceptibility. Each recombinant IN was attenuated for 3′-end processing and strand transfer activities. Most proteins were also attenuated for disintegration; the IN that contained K156R and C65S-K156N, however, displayed disintegration activity similar to that of IN from HIVNL4-3. All mutant IN proteins demonstrated decreased susceptibility to l-CA, while all mutant proteins except E92K and K156R demonstrated resistance to L-731,988. These data validate the computer modeling data and demonstrate that l-CA and L-731,988 share an overlapping inhibitor-binding pocket that involves amino acids Q148, C65, and H67. The resistance studies confirm that L-731,988 fills one-half of the inhibitor-binding pocket and binds to Q148 but excludes E92, while l-CA fills the entire binding groove and thus interacts with E92. These results provide “wet laboratory” evidence that molecular models of the HIV IN inhibitor-binding pocket can be used for drug discovery.