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Viral pathogens causing global disease burdens are often characterized by high rates of evolutionary changes. The extensive viral diversity at baseline can shorten the time to escape from therapeutic or immune selective pressure and alter mutational pathways. The impact of genotypic background on the barrier to resistance can be difficult to capture, particularly for agents in experimental stages or that are recently approved or expanded into new patient populations. We developed an evolutionary model-based counting method to quickly quantify the population genetic potential to resistance and assess population differences. We demonstrate its applicability to HIV-1 integrase inhibitors, as their increasing use globally contrasts with limited availability of non-B subtype resistant sequence data and corresponding knowledge gap. A large sequence data set encompassing most prevailing HIV-1 subtypes and resistance-associated mutations of currently approved integrase inhibitors was investigated. A complex interplay between codon predominance, polymorphisms, and associated evolutionary costs resulted in a subtype-dependent varied genetic potential for 15 resistance mutations against integrase inhibitors. While we confirm the lower genetic barrier of subtype B for G140S, we convincingly discard a similar effect previously suggested for G140C. A supplementary analysis for HIV-1 reverse transcriptase inhibitors identified a lower genetic barrier for K65R in subtype C through differential codon usage not reported before. To aid evolutionary interpretations of genomic differences for antiviral strategies, we advanced existing counting methods with increased sensitivity to identify subtype dependencies of resistance emergence. Future applications include novel HIV-1 drug classes or vaccines, as well as other viral pathogens.

The lectin actinohivin (AH) is a monomeric carbohydrate-binding agent (CBA) with three carbohydrate-binding sites. AH strongly interacts with gp120 derived from different X4 and R5 human immunodeficiency virus (HIV) strains, simian immunodeficiency virus (SIV) gp130, and HIV type 1 (HIV-1) gp41 with affinity constants ( K D ) in the lower nM range. The gp120 and gp41 binding of AH is selectively reversed by (α1,2-mannose) 3 oligosaccharide but not by α1,3/α1,6-mannose- or GlcNAc-based oligosaccharides. AH binding to gp120 prevents binding of α1,2-mannose-specific monoclonal antibody 2G12, and AH covers a broader epitope on gp120 than 2G12. Prolonged exposure of HIV-1-infected CEM T-cell cultures with escalating AH concentrations selects for mutant virus strains containing N-glycosylation site deletions (predominantly affecting high-mannose-type glycans) in gp120. In contrast to 2G12, AH has a high genetic barrier, since several concomitant N-glycosylation site deletions in gp120 are required to afford significant phenotypic drug resistance. AH is endowed with broadly neutralizing activity against laboratory-adapted HIV strains and a variety of X4 and/or R5 HIV-1 clinical clade isolates and blocks viral entry within a narrow concentration window of variation (∼5-fold). In contrast, the neutralizing activity of 2G12 varied up to 1,000-fold, depending on the virus strain. Since AH efficiently prevents syncytium formation in cocultures of persistently HIV-1-infected HuT-78 cells and uninfected CD4 + T lymphocytes, inhibits dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated capture of HIV-1 and subsequent virus transmission to CD4 + T lymphocytes, does not upregulate cellular activation markers, lacks mitogenic activity, and does not induce cytokines/chemokines in peripheral blood mononuclear cell cultures, it should be considered a potential candidate drug for microbicidal use.

Pradimicin S (PRM-S) is a highly water-soluble, negatively charged derivative of the antibiotic pradimicin A (PRM-A) in which the terminal xylose moiety has been replaced by 3-sulfated glucose. PRM-S does not prevent human immunodeficiency virus (HIV) adsorption on CD4 + T cells, but it blocks virus entry into its target cells. It inhibits a wide variety of HIV-1 laboratory strains and clinical isolates, HIV-2, and simian immunodeficiency virus (SIV) in various cell culture systems (50% and 90% effective concentrations [EC 50 s and EC 90 s] invariably in the lower micromolar range). PRM-S inhibits syncytium formation between persistently HIV-1- and SIV-infected cells and uninfected CD4 + T lymphocytes, and prevents dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-mediated HIV-1 and SIV capture and subsequent virus transmission to CD4 + T cells. Surface plasmon resonance (SPR) studies revealed that PRM-S strongly binds to gp120 in a Ca 2+ -dependent manner at an affinity constant ( K D ) in the higher nanomolar range. Its anti-HIV activity and HIV-1 gp120-binding properties can be dose-dependently reversed in the presence of an (α-1,2)mannose trimer. Dose-escalating exposure of HIV-1-infected cells to PRM-S eventually led to the isolation of mutant virus strains that had various deleted N-glycosylation sites in the envelope gp120 with a strong preference for the deletion of the high-mannose-type glycans. Genotypic resistance development occurred slowly, and significant phenotypic resistance occurred only after the sequential appearance of up to six mutations in gp120, pointing to a high genetic barrier of PRM-S. The antibiotic is nontoxic against a variety of cell lines, is not mitogenic, and does not induce cytokines and chemokines in peripheral blood mononuclear cells as determined by the Bio-Plex human cytokine 27-plex assay. It proved stable at high temperature and low pH. Therefore, PRM-S may qualify as a potential anti-HIV drug candidate for further (pre)clinical studies, including its microbicidal use.

Combination therapy can successfully suppress human immunodeficiency virus (HIV) replication in patients but selects for drug resistance, requiring subsequent resistance-guided therapeutic changes. This report describes the development and validation of a novel assay that offers a uniform method to measure susceptibility to all clinically approved HIV type 1 (HIV-1) drugs targeting reverse transcriptase (RT), protease (PR), integrase (IN), and viral entry. It is an assay in which the antiviral effect on infection within a single replication cycle is measured in triply transfected U87.CD4.CXCR4.CCR5 cells, based on homologous recombination between patient-derived amplicons and molecular proviral clones tagged with the enhanced green fluorescent protein (EGFP) reporter gene and from which certain viral genomic regions are removed. The deletions stretch from p17 codon 7 to PR codon 98 in pNL4.3-ΔgagPR-EGFP, from PR codons 1 to 99 in pNL4.3-ΔPR-EGFP, from RT codons 1 to 560 in pNL4.3-ΔRT-EGFP, from IN codons 1 to 288 in pNL4.3-ΔIN-EGFP, and from gp120 codon 34 to gp41 codon 237 in pNL4.3-Δenv-EGFP. The optimized experimental conditions enable the investigation of patient samples regardless of viral subtype or coreceptor use. The extraction and amplification success rate for a set of clinical samples belonging to a broad range of HIV-1 group M genetic forms (A-J, CRF01-03, CRF05, and CRF12-13) and displaying a viral load range of 200 to >500,000 RNA copies/ml was 97%. The drug susceptibility measurements, based on discrimination between infected and noninfected cells on a single-cell level by flow cytometry, were reproducible, with coefficients of variation for resistance ranging from 7% to 31%, and were consistent with scientific literature in terms of magnitude and specificity.

Pradimicin A (PRM-A), an antifungal nonpeptidic benzonaphtacenequinone antibiotic, is a low-molecular-weight (molecular weight, 838) carbohydrate binding agent (CBA) endowed with a selective inhibitory activity against human immunodeficiency virus (HIV). It invariably inhibits representative virus strains of a variety of HIV-1 clades with X4 and R5 tropisms at nontoxic concentrations. Time-of-addition studies revealed that PRM-A acts as a true virus entry inhibitor. PRM-A specifically interacts with HIV-1 gp120 and efficiently prevents virus transmission in cocultures of HUT-78/HIV-1 and Sup T1 cells. Upon prolonged exposure of HIV-1-infected CEM cell cultures, PRM-A drug pressure selects for mutant HIV-1 strains containing N-glycosylation site deletions in gp120 but not gp41. A relatively long exposure time to PRM-A is required before drug-resistant virus strains emerge. PRM-A has a high genetic barrier, since more than five N-glycosylation site deletions in gp120 are required to afford moderate drug resistance. Such mutated virus strains keep full sensitivity to the other known clinically used anti-HIV drugs. PRM-A represents the first prototype compound of a nonpeptidic CBA lead and, together with peptide-based lectins, belongs to a conceptually novel type of potential therapeutics for which drug pressure results in the selection of glycan deletions in the HIV gp120 envelope.

The major limitation of drug resistance genotyping for human immunodeficiency virus remains the interpretation of the results. We evaluated the concordance in predicting therapy response between four different interpretation algorithms (Rega 6.3, HIVDB-08/04, ANRS [07/04], and VGI 8.0). Sequences were gathered through a worldwide effort to establish a database of non-B subtype sequences, and demographic and clinical information about the patients was gathered. The most concordant results were found for nonnucleoside reverse transcriptase (RT) inhibitors (93%), followed by protease inhibitors (84%) and nucleoside RT inhibitor (NRTIs) (76%). For therapy-naive patients, for nelfinavir, especially for subtypes C and G, the discordances were driven mainly by the protease (PRO) mutational pattern 82I/V + 63P + 36I/V for subtype C and 82I + 63P + 36I + 20I for subtype G. Subtype F displayed more discordances for ritonavir in untreated patients due to the combined presence of PRO 20R and 10I/V. In therapy-experienced patients, subtype G displayed a lot of discordances for saquinavir and indinavir due to mutational patterns involving PRO 90 M and 82I. Subtype F had more discordance for nelfinavir attributable to the presence of PRO 88S and 82A + 54V. For the NRTIs lamivudine and emtricitabine, CRF01_AE had more discordances than subtype B due to the presence of RT mutational patterns 65R + 115 M and 118I + 215Y, respectively. Overall, the different algorithms agreed well on the level of resistance scored, but some of the discordances could be attributed to specific (subtype-dependent) combinations of mutations. It is not yet known whether therapy response is subtype dependent, but the advice given to clinicians based on a genotypic interpretation algorithm differs according to the subtype.

Limited data are available on the genotypic and phenotypic resistance profile of the α-(1-2)mannose oligomer-specific prokaryotic lectin cyanovirin (CV-N). Therefore, a more systematic investigation was carried out to obtain a better view of the interaction between CV-N and human immunodeficiency virus type 1 (HIV-1) gp120. When HIV-1-infected CEM cell cultures were exposed to CV-N in a dose-escalating manner, a total of eight different amino acid mutations exclusively located at N-glycosylation sites in the envelope surface gp120 were observed. Six of the eight mutations resulted in the deletion of high-mannose type N-glycans (i.e., at amino acid positions 230, 332, 339, 386, 392, and 448). Two mutations (i.e., at position 136 and 160) deleted a complex type N-glycan in the variable V1/V2 domain of gp120. The level of phenotypic resistance of the mutated virus strains against CV-N generally correlated with the number of glycan deletions in gp120, although deletion of the glycans at N-230, N-392, and N-448 generally afforded a more pronounced CV-N resistance than other N-glycan deletions. However, the extent of the decrease of antiviral activity of CV-N against the mutated virus strains was markedly less pronounced than observed for α(1-3)- and α(1-6)-mannose-specific plant lectins Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which points to the existence of a higher genetic barrier for CV-N. This is in agreement with a more consistent suppression of a wider variety of HIV-1 clades by CV-N than by HHA and GNA. Whereas the antiviral and in vitro antiproliferative activity of CV-N can be efficiently reversed by mannan, the pronounced mitogenic activity of CV-N on peripheral blood mononuclear cells was unaffected by mannan, indicating that some of the observed side effects of CV-N are unrelated to its carbohydrate specificity/activity.

The mannose-specific plant lectins from the Amaryllidaceae family (e.g., Hippeastrum sp. hybrid and Galanthus nivalis ) inhibit human immunodeficiency virus (HIV) infection of human lymphocytic cells in the higher nanogram per milliliter range and suppress syncytium formation between persistently HIV type 1 (HIV-1)-infected cells and uninfected CD4 + T cells. These lectins inhibit virus entry. When exposed to escalating concentrations of G. nivalis and Hippeastrum sp. hybrid agglutinin, a variety of HIV-1(III B ) strains were isolated after 20 to 40 subcultivations which showed a decreased sensitivity to the plant lectins. Several amino acid changes in the envelope glycoprotein gp120, but not in gp41, of the mutant virus isolates were observed. The vast majority of the amino acid changes occurred at the N glycosylation sites and at the S or T residues that are part of the N glycosylation motif. The degree of resistance to the plant lectins was invariably correlated with an increasing number of mutated glycosylation sites in gp120. The nature of these mutations was entirely different from that of mutations that are known to appear in HIV-1 gp120 under the pressure of other viral entry inhibitors such as dextran sulfate, bicyclams (i.e., AMD3100), and chicoric acid, which also explains the lack of cross-resistance of plant lectin-resistant viruses to any other HIV inhibitor including T-20 and the blue-green algae (cyanobacteria)-derived mannose-specific cyanovirin. The plant lectins represent a well-defined class of anti-HIV (microbicidal) drugs with a novel HIV drug resistance profile different from those of other existing anti-HIV drugs.

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120 , gp41 , and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41 . Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.