HIV-1 tropism determination using a phenotypic Env recombinant viral assay highlights overestimation of CXCR4-usage by genotypic prediction algorithms for CRRF01_AE and CRF02_AG

Background: Human Immunodeficiency virus type-1 (HIV) entry into target cells involves binding of the viral envelope (Env) to CD4 and a coreceptor, mainly CCR5 or CXCR4. The only currently licensed HIV entry inhibitor, maraviroc, targets CCR5, and the presence of CXCX4-using strains must be excluded prior to treatment. Co-receptor usage can be assessed by phenotypic assays or through genotypic prediction. Here we compared the performance of a phenotypic Env-Recombinant Viral Assay (RVA) to the two most widely used genotypic prediction algorithms, Geno2Pheno([coreceptor]) and webPSSM. Methods: Co-receptor tropism of samples from 73 subtype B and 219 non-B infections was measured phenotypically using a luciferase-tagged, NL4-3-based, RVA targeting Env. In parallel, tropism was inferred genotypically from the corresponding V3-loop sequences using Geno2Pheno([coreceptor]) (5-20% FPR) and webPSSM-R5X4. For discordant samples, phenotypic outcome was retested using co-receptor antagonists or the validated Trofile (R) Enhanced-Sensitivity-Tropism-Assay. Results: The lower detection limit of the RVA was 2.5% and 5% for X4 and R5 minority variants respectively. A phenotype/genotype result was obtained for 210 samples. Overall, concordance of phenotypic results with Geno2Pheno([coreceptor]) was 85.2% and concordance with webPSSM was 79.5%. For subtype B, concordance with Geno2pheno([coreceptor]) was 94.4% and concordance with webPSSM was 79.6%. High concordance of genotypic tools with phenotypic outcome was seen for subtype C (90% for both tools). Main discordances involved CRF01_AE and CRF02_AG for both algorithms (CRF01_AE: 35.9% discordances with Geno2Pheno([coreceptor]) and 28.2% with webPSSM; CRF02_AG: 20.7% for both algorithms). Genotypic prediction overestimated CXCR4-usage for both CRFs. For webPSSM, 40% discordance was observed for subtype A. Conclusions: Phenotypic assays remain the most accurate for most non-B subtypes and new subtype-specific rules should be developed for non-B subtypes, as research studies more and more draw conclusions from genotypically-inferred tropism, and to avoid unnecessarily precluding patients with limited treatment options from receiving maraviroc or other entry inhibitors

Generation of isogenic control DJ-1-delP GC13 for the genetic Parkinson's disease-patient derived iPSC line DJ-1-delP (LCSBi008-A-1)

We describe the generation of an isogenic control cell line DJ-1-delP GC13 from an induced pluripotent stem cell (iPSC) line DJ-1-delP LCSBi008-A that was derived from fibroblasts obtained from a Parkinson’s disease (PD) patient. Using CRISPR/Cas9 technology, we corrected the disease causing c.471_473delGCC homozygous mutation in the PARK7 gene leading to p.158P deletion in the encoded protein DJ-1. The generated isogenic pair will be used for phenotypic analysis of PD-patient derived neurons and astrocytes

Codon-Optimization of Wild-Type Adeno-Associated Virus Capsid Sequences Enhances DNA Family Shuffling while Conserving Functionality

Adeno-associated virus (AAV) vectors have become one of the most widely used gene transfer tools in human gene therapy. Considerable effort is currently being focused on AAV capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. Selection strategies based on directed evolution rely on the generation of highly variable AAV capsid libraries using methods such as DNA-family shuffling, a technique reliant on stretches of high DNA sequence identity between input parental capsid sequences. This identity dependence for reassembly of shuffled capsids is inherently limiting and results in decreased shuffling efficiency as the phylogenetic distance between parental AAV capsids increases. To overcome this limitation, we have developed a novel codon-optimization algorithm that exploits evolutionarily defined codon usage at each amino acid residue in the parental sequences. This method increases average sequence identity between capsids, while enhancing the probability of retaining capsid functionality, and facilitates incorporation of phylogenetically distant serotypes into the DNA-shuffled libraries. This technology will help accelerate the discovery of an increasingly powerful repertoire of AAV capsid variants for cell-type and disease-specific applications. Keywords: AAV, library, directed evolution, codon optimization, DNA shuffling, capsi

Lower replication of C-Env recombinants in CD4+ T-cells is not associated with lower viral gene expression.

<p><b>A and B. Western Blot analyses of intracellular Env and Gag in CD4+ T-cells exposed to subtype B and subtype C recombinant virus pairs at day 5 post infection (A) and 40 hours post infection (B).</b> CD4+ T-cells (10⁵ cells/well) infected in triplicate wells for 5 days (A) or for 40 hours (B) were washed, pooled and lysed in 75μl of reducing Lämmli buffer. Polybrene was added at the time of infection of C-EnvEC and C-Env virions for WB detection in 40-hour lysates. Proteins were resolved by SDS-PAGE and immunoblotted with a rabbit polyclonal antibody recognizing the immature p55Gag immature precursor and the mature p17 and p24 proteins, and a goat polyclonal antibody against gp120. β-actin was immunoblotted to control for loading. The corresponding supernatant (30 μl) were immunoblotted using a mouse monoclonal antibody against p24. One representative experiment of three is shown. Samples are arranged by subtype to ease visual comparison. C and B Env samples were also run on the same gel, and similar subtype and gp41CT-related differences were observed (not shown).</p

Viruses with subtype C gp41CT have lower replication than viruses with subtype B gp41CT in CD4+ T-cells but not in MDMs.

<p><b>A and B. Replication of subtype B and subtype C recombinants in CD4+ T-cells from two different donors.</b> 10⁵ CD4+ T-cells were infected with equivalent amounts (1 ng/ml) of Env or EnvEC recombinant viruses. Infection was monitored by measuring p24 in culture supernatants at days 2 or 3, 4 or 5, 7 and 9 or 10. Examples of replication in CD4+ T cells of two different donors are shown after infection with subtype B (left panels) and subtype C (right panels) Env and EnvEC recombinant viruses. All infections were performed in triplicate wells. <b>(C and D) P24 in supernatants of 10</b>^<b>5</b> <b>CD4+ T-cells 5 days post-infection.</b> The same results at day 5 post-infection are presented and statistically significant means (paired t-test for viral pairs, unpaired t-test for inter-subtype comparisons) are indicated (<b>C</b>). In panel (<b>D</b>), the results are shown per pair. The ratio of p24 released in supernatants of cells exposed to Env recombinant viruses divided by p24 in the supernatants of cells exposed to the corresponding EnvEC recombinant virus is reported. A positive ratio indicates that the full Env recombinant replicates better than its EnvEC counterpart containing the gp41CT of NL4.3. A negative ratio implies the EnvEC recombinant virus replicates better than the Env recombinant. Because of inter-donor variability, results are presented as individual experiments performed with CD4+ T-cells from independent donors. <b>E. Replication of subtype B and subtype C recombinants in MDMs.</b> 10⁵ MDMs were infected with equivalent amounts (3 ng/ml) of subtype B (left panel) and subtype C (right panel) Env and EnvEC recombinant viruses. Infection was monitored by measuring p24 in culture supernatants at days 3, 7, 10 and 14. All infections were performed in triplicate or quadruplicate wells. <b>(F and G) p24 in supernatants of 3x10</b>^<b>5</b> <b>MDMs 7 days post-infection.</b> All infections were performed in triplicate or quadruplicate wells. Standard deviation is indicated. In panel (<b>G</b>), the same results as in panel (F) are represented for each individual strain. The ratio of p24 released in supernatants of cells exposed to Env recombinant viruses divided by p24 in the supernatants of cells exposed to the corresponding EnvEC recombinant virus is reported. Tropism is indicated as follows: R5: blue circle; X4: red circle; R5X4: purple circle.</p

Subtype C MA decreases viral production, only partially rescues viral replication in CD4+ T-cells but does not restore infectivity.

<p><b>A and B. Production of viruses containing subtype C MA.</b> The <i>matrix</i> sequences of two subtype C strains with intermediate replication capacity (MA₁₆₇₁) and with poor replication capacity (MA₀₂₆₆) were cloned into the EnvEC and Env backbones. Recombinant viruses with subtype C MAs were generated by transfecting HEK 293T cells with these backbones and the corresponding subtype C EnvEC and Env amplicons from patient samples. p24 produced was measured by ELISA 48 hours post transfection. At least three independent productions with MA_NL4.3 and two with MA₁₆₇₁ and MA₀₂₆₆ are averaged and error bars show standard deviation. Results are shown for all viruses as groups (A) and per pairs (B). <b>C and D. Replication of C-EnvEC and C-Env recombinant viruses containing subtype C MA</b>_<b>1671</b> <b>in CD4+ T-cells.</b> CD4+ T-cells were infected with C-EnvEC and C-Env recombinants containing the MA of NL4.3 or of strain 1671, and p24 was measured in the supernatants 5 days post-infection. The average and standard deviation of four independent experiments performed with CD4+ T-cells from four different donors are shown. Results are shown for all viruses as groups (C) and per pairs (D) In (D), relative infectivity with respect to the corresponding C-EnvEC value is reported. <b>E and F. Infectivity of virions produced by CD4+ T-cells in a TZM-bl single round assay.</b> TZM-bl cells were infected with supernatants from infected CD4+ T-cells collected 5 days after infection and adjusted to equivalent p24 contents (2 ng/well). Infection was monitored 48h post-infection as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161596#pone.0161596.g003" target="_blank">Fig 3</a>. The mean of two independent experiments performed with CD4+ T-cell supernatants from two independent donors are shown and error bars report standard deviation. <b>G and H. Cell-to-cell transmission of viruses with subtype C MA</b>_<b>1671.</b> CD4+ T-cells were infected with C-EnvEC, C-Env or C-EnvMA₁₆₇₁ viral particles for 40 hours, extensively washed, then co-cultured with TZM-bl cells for a further 48 hours in the presence of IDV. MVC and AMD3100 were added the next morning. The mean of two independent experiments is shown. Error bars represent standard deviation. Panel <b>G</b> shows the same data as panel <b>F</b> detailed for each individual pair.</p

Infectivity of C-Env virions, Env incorporation in virions produced by CD4+ T-cells and cell-to-cell transmission are lessened.

<p><b>(A and B) Infectivity of virions produced by HEK 293T cells (A) and CD4+ T-cells (B) in a single round TZM-bl assay.</b> TZM-bl cells (2x10⁴ cells/well) were infected with 2 ng/ml equivalent p24 of Env or EnvEC recombinant viruses produced from HEK 293T cells <b>(A)</b> or from CD4+ T-cells 5 days post infection <b>(B)</b>. Viral entry was monitored in cell lysates 48h post-infection. Infections were performed in triplicated wells. The means of three independent experiments with HEK293T cells and of 4 independent infections with CD4+ T cells from 4 different donors are shown. Error bars report standard deviation <b>C</b>. <b>Env incorporated in virions produced by CD4+ T-cells.</b> P24 and gp120 in day 5 supernatants of CD4+ T-cells were quantified by Western Blot. One representative experiment of two is shown. <b>D and E. Cell-to-cell transmission.</b> CD4+ T-cells were infected as above with 1 ng/ml equivalent p24. After 48 hours, cell were cocultured with TZM-bl cells in the presence of IDV to avoid any contribution of free virus. The next morning, MVC and AMD3100 were added to ensure single-round infection. Luciferase in TZM-bl cells was measured after 48 hours. <b>(D)</b> The mean and standard deviation of four independent experiments performed with CD4+ T-cells from four different donors are reported. <b>(E)</b> The same data is shown for each individual virus pair.</p