Fig 1.
Classical methods used to calculate viral output contain advantages and disadvantages in evaluation of infectivity.
Experiments are performed in vivo, ex vivo, and/or in vitro with HIV-1 and samples are collected on different days post infection including supernatants, protein, and RNA lysates. Indirect viral quantification methods are broken down into 3 categories: protein secretion, viral transcription, and viral maturation. Indirect viral quantification methods include ELISA-based protein secretion detection, RT activity, and qPCR measurements of viral gene expression. Direct viral quantification methods include: plaque assays and TCID50, as well as infectivity assays using permissive cell lines. Created with BioRender.com.
Table 1.
High Content Imaging Parameters: Conditions used in HCS software (Cellomics) analysis of images taken by the Cellomics CX7 platform. A Y denotes that this condition was a checkbox.
Fig 2.
GHOST cells containing a CCR5 receptor can be used to evaluate the presence of infectious virions of HIV-1 from R5-tropic strains.
Ghost (3) cells of the genotypes CD4/CCR5+/+(Hi5) and CD4/CCR5-/- (Parental) were plated in 6 well plates at 100,000 cells per well and then infected with 0 to 20 μL of HIV-1ADA and incubated for 48hrs at 37oC, as represented in (A.). Cells were washed with PBS and stained with Hoechst (1:1000) and imaged as seen in (B) at 10x magnification with 50 fields per well. Quantification in (C) shows that Hi5 cells are infectable, and increase in viral infection with increasing amounts of virus in an n = 6 independent experiments, (range 1 μL 132-718, 2.5 μL 420-951 5 μL, 517-778, 10 μL 591-951, 20 μL 618-1370, Std. Dev. 1 μL 199.2, 2.5 μL 212.2 5 μL, 100.2, 10 μL 129.5, 20 μL 282.9, one way ANOVA Treatment, *p-value = 0.0002, F = 21.48, one way ANOVA runs p = 0.3697 F = 1.131).
Fig 3.
The viral titer/ GHOST cell assay can be optimized and used in a high throughput manner in 96-well plates based on initial cell density.
(A) Hi-5 cells were plated at 500 to 11,000 cells/well and infected in triplicate with 5 μL or 10 μL of HIV-1ADA. Infection was observed at all densities, with minimal signal in vehicle controls. (B) A strong linear correlation was found between cell density and infectious units (n = 4, Pearson r = 0.9915, R² = 0.9831, ****p = 3.421 × 10 ⁻ ¹⁰). Variability was highest at the lowest (≤1,000 cells/well) and highest (≥5,000 cells/well) densities. Infectious titers did not differ significantly between 2,000 to 5,000 cells/well (One-way ANOVA, ****p < 0.0001, F(11,36) = 17.06; Tukey’s post hoc).
Fig 4.
The viral titer assay window of reliability is comparable to measurement of p24.
(A) Representative images (B) quantification of an n = 5 independent experiments of infecting Hi5 cells with 0-100ng/mL p24 concentration of HIV-1ADA showed that as p24 increases infectious unit production also significantly increases with a limit of reliability (dashed line) at 0.5ng/mL (One-way ANOVA p-value <0.0001; Tukey’s multiple comparisons p-values in S3 Table). (C) Pearson correlations of infectious units and p24 showed a strong positive linear correlation between the two variables (Pearson r = 0.9308, p-value 3.192 x 10−5, r2 = 0.8664). (D) Quantification of biological replicates from two HIV-1ADA stocks using viral titer (315.9 IU/μL, Std. Dev. = 105.1, CoV = 33.26%) and p24 AlphaLISA (2.593 × 10³ ng p24/mL Std. Dev. = 939.3, CoV = 36.23%).
Fig 5.
The viral titer assay can evaluate infectious virion production in hMDMs.
(A) Representative images of viral titer assay from cell treated with supernatants from HIV-1ADA - infected hMDM (1 ng p24/mL) at 3, 5, 7, and 9 dpi. (B) analysis of p24 secretion (C) infectious titer (D) IU:p24 ratio.
Fig 6.
Infectious unit ratio highlights differences between viral stocks of the same strain.
(A) Schematic overview of lentiviral transfection to generate to R5-tropic strains of HIV-1. (B) Representative images of viral titer assay from two stocks of HIV-1YU2 that are genetically identical but obtained from distinct sources. (C) Quantification of p24 levels (ng/mL) (D) Measurement of infectious titer (IU/μL) in YU2B compared to YU2A (E) The IU:p24 ratio was calculated to assess the relative efficiency of infectious virus production (F) hMDMs from matched donors were infected with equivalent p24 concentrations (20 ng/mL) from YU2A or YU2B stocks. Longitudinal measurement of p24 secretion.
Fig 7.
Characterizing viral output from infection of primary human monocyte derived macrophages.
(A) Representative images from viral titer assay of participant-derived viral stocks generated through lentiviral transfections (B) Quantification of p24 (ng/mL) in viral supernatants demonstrated detectable levels in all stocks (C) Infectious titer (IU/μL) varied between stocks (D) Calculation of the IU:p24 (E) hMDMs were infected with equal p24 input from each viral stock (20 ng/mL), and infection was assessed longitudinally by p24 secretion.