Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy

Abstract

Background: In this study we successfully created a new approach to ART in SIVmac251 infected nonhuman primates. This drug regimen is entirely based on drugs affecting the pre-integration stages of replication and consists of only two nucleotidic/nucleosidic reverse transcriptase inhibitors (Nt/NRTIs) and raltegravir, a promising new drug belonging to the integrase strand transfer inhibitor (INSTI) class.

Results: In acutely infected human lymphoid CD4+ T-cell lines MT-4 and CEMx174, SIVmac251 replication was efficiently inhibited by raltegravir, which showed an EC90 in the low nanomolar range. This result was confirmed in primary macaque PBMCs and enriched CD4+ T cell fractions. In vivo monotherapy with raltegravir for only ten days resulted in reproducible decreases in viral load in two different groups of animals. When emtricitabine (FTC) and tenofovir (PMPA) were added to treatment, undetectable viral load was reached in two weeks, and a parallel increase in CD4 counts was observed. In contrast, the levels of proviral DNA did not change significantly during the treatment period, thus showing persistence of this lentiviral reservoir during therapy.

Conclusions: In line with the high conservation of the three main amino acids Y143, Q148 and N155 (responsible for raltegravir binding) and molecular docking simulations showing similar binding modes of raltegravir at the SIVmac251 and HIV-1 IN active sites, raltegravir is capable of inhibiting SIVmac251 replication both in tissue culture and in vivo. This finding may help to develop effective ART regimens for the simian AIDS model entirely based on drugs adopted for treatment in humans. This ART-treated AIDS nonhuman primate model could be employed to find possible strategies for virus eradication from the body.

Figure 1

SIVmac251 susceptibility to raltegravir in tissue culture. The effective concentrations at 50%, 90% and 95% (respectively, EC₅₀, EC₉₀, and EC₉₅) are presented (means ± SEM from at least two independent experiments) for inhibition of: lentiviral cythopathogenicity in MT-4 cells (Panel A), viral core antigen release in supernatants of acutely infected MT-4 cells (Panel B), syncytium formation in acutely infected CEMx174 cells (Panel C), viral core antigen release in supernatants of acutely SIVmac251-infected rhesus peripheral blood mononuclear cells (PBMCs) and enriched CD4⁺ T-cell fractions (Panel D). In panel A, the inhibitory concentrations were determined by the methyl tetrazolium (MTT) method when the majority of control infected cells (in the absence of drug treatment) were dead at light microscopy examination. In panel B, values were derived by quantifying, using antigen-capture ELISA assays, SIVmac251 p27 and HIV-1 p24 in supernatants from five-day old cultures. In panel C, values were calculated on the basis of the numbers of syncytia per well at five days post-infection, Syncytia were counted in triplicate on three different occasions by light microscopy. In panel D, values are representative for supernatants of primary cells from three different donors at Day 5 post-infection.

Figure 2

Effect of raltegravir (RAL), alone and in combination with PMPA and FTC, on viral load (panel A) and CD4 counts (panel B) in SIVmac251-infected macaques (Group 1). SIVmac251-infected rhesus macaques (Macaca mulatta) were randomized to receive 50 (marked by the blue symbols) or 100 (red symbols) mg of raltegravir twice daily with food (bid). Monotherapy was continued for ten days. At day 11, nonhuman primates treated with 50 mg of raltegravir bid were switched to the 100 mg regimen, and two RT inhibitors, i.e. the NtRTI, tenofovir (PMPA) and the NRTI emtricitabine (FTC), were added to treatment (henceforth referred to as ART) in all animals. Viral load values positioning on the dotted line parallel to the x axis should read as undetectable.

Figure 3

Association of viral load decrease with raltegravir treatment of SIVmac251-infected animals (Group 2). SIVmac251-infected rhesus macaques (Macaca mulatta) received 100 mg of raltegravir twice daily with food (bid). Monotherapy was continued for ten days. Comparison between pre- and post-raltegravir viral load measurements was done. Viral load values at Day 0, Day 7 and Day 10 were compared with viral loads at 27 and 166 days prior to treatment start. Significant differences (P < 0.05; Bonferroni's test following repeated-measures ANOVA; shown in the graph by the red asterisks) were found between both the values at 166 and 27 days prior to treatment start and the values at Day 7 and Day 10 of treatment. No significant differences, instead, were found between the values at 166 days, or 27 days, prior to treatment, and the values at Day 0. The dashed line parallel to the x axis marks the detection threshold of the technique adopted.

Figure 4

Persistence of proviral DNA during therapy (Group 1). Proviral DNA was measured by a quantitative PCR technique at start of treatment with antiretroviral drugs, and at 52 days of therapy.

Figure 5

Sequence alignment of the integrase catalytic core domains of HIV-1 subtype B (PDB: 1BL3_C), HIV-2 (PDB: 3F9K_A), SIVmac251 (PDB: 1C6V_A), prototype foamy virus/PFV (PDB: 3DLR_A), and Rous Sarcoma virus/RSV (PDB: 1ASU_A). The sequence alignment is based on a structural alignment performed using the VAST algorithm. Regions showing significant structural alignment are presented in blue, with the highly conserved residues shown in red. Above the alignments are shown the mutations found in HIV-1 infected individuals failing raltegravir-based drug regimens (the green arrows indicate the primary resistance mutations Y143H, Q148H/K/R, and N155H; black arrows indicate secondary resistance mutations). Other drug resistance mutations induced by other integrase strand transfer inhibitors are shown below the alignments. The mutations shown by site-directed mutagenesis to confer resistance to raltegravir are underlined. Note that the structure for HIV-1 subtype B integrase catalytic core domain (PDB: 1BL3_C) presents the secondary drug resistance mutation V151I.

Figure 6

Phylogenetic tree of lentiviral integrase core domains. Sequences adopted: human immunodeficiency virus type-1 (HIV-1) [PDB: 1BL3C]; human immunodeficiency virus type-2 (HIV-2) [PDB: 3F9K]; simian immunodeficiency virus, host: macaque (SIVmac251) [PDB: 1C6VC]; simian immunodeficiency virus, host: chimpanzee (Pan troglodytes) (SIVcpz) [accession: AAF18575]; simian immunodeficiency virus, host: gorilla (Gorilla gorilla) (SIVgor) [accession: ACM63211]; simian immunodeficiency virus, host: African green nonhuman primate (Chlorocebus sp.) (SIVagm) [accession: CAA30658]; simian immunodeficiency virus, host: mandrill (Mandrillus sphinx) (SIVmnd) [accession: AAB49569]; simian immunodeficiency virus, host: Cercopithecus lhoesti (SIVlhoest) [accession: AAF07333]; simian immunodeficiency virus, host: Skyes' nonhuman primate (Cercopithecus albogularis) (SIVsyk) [accession: AAS97874]; simian immunodeficiency virus, host: Colobus nonhuman primate (Colobus guereza) (SIVcol) [accession: AAK01033]; prosimian immunodeficiency virus, host: Microcebus murinus (pSIV) [see: additional material in Ref. [46]]; feline immunodeficiency virus, host: domestic cat (Felis sylvestris) (FIV-Pet) [accession: AAB59937]; lion lentivirus, host: lion (Panthera leo) [accession: ABX25835]; puma lentivirus, host: mountain lion (Puma concolor) [accession: AAA67168]; caprine arthritis-encephalitis virus (CAEV), host: Capra hircus [accession: NP_040939]; visna lentivirus, host: sheep (Ovis aries) [PDB: 3HPG_A]; equine infectious anemia virus (EIAV) host: horse (Equus caballus) [accession: NP_056902]; bovine immunodeficiency virus (BIV) host: wild banteng (Bos javanicus) [accession: Q82851]. Relationships between proteins were reconstructed using Phylogeny.fr. Approximate likelihood ratios > 70% are shown. This tree is not intended to reconstruct the phylogeny of primate lentiviruses, but rather to highlight the degree of similarity of the IN CCDs derived from different viruses. The similarities shown are in line with previous phylogenetic analyses based on DNA sequences corresponding to other portions of the lentiviral genome [74].

Figure 7

A three-dimensional model of SIVmac251 IN catalytic core domain colored by amino acid similarity with wild-type HIV-1 IN. The enzyme is coloured by sequence similarity with its HIV-1 orthologue [PDB:1BL3]. The level of similarity was calculated by the Swiss PDB Viewer (SPDBV) software. The colour scale is that adopted by SPDBV. Similarity is maximal at the level of the INSTI/cellular DNA binding site (indicated by a semi-transparent grey circle), as calculated by some of us in previous works [35]. Image obtained using PyMOL [73].

Figure 8

In silico docking of raltegravir at the SIVmac251 integrase (IN) active site. Panel A: An overview of the interaction between SIVmac251 integrase (in grey), 3' processed proviral DNA (green and blue cartoons) and raltegravir (in orange). The three terminal nucleotides of the 5' DNA strand (in blue) have been removed for better clarity. Metal (Mg²⁺) ions are shown in magenta. Panel B: Interaction of raltegravir (shown in CPK) and the integrase amino acids susceptible to primary drug resistance mutations (cyan sticks). The protein backbone is shown by cartoons. Metal ions are presented in magenta. The catalytic triad (D64, D116 and E152) is shown in yellow. Ligand-interacting nucleotides, dC25 and dA20, are shown as thin lines. A full three-dimensional view of the complex can be obtained using the 3D coordinates provided as additional material [see Additional file 4]. Image obtained using PyMOL [73].