Initial HIV-1 antigen-specific CD8+ T cells in acute HIV-1 infection inhibit transmitted/founder virus replication

Abstract

CD8-mediated virus inhibition can be detected in HIV-1-positive subjects who naturally control virus replication. Characterizing the inhibitory function of CD8(+) T cells during acute HIV-1 infection (AHI) can elucidate the nature of the CD8(+) responses that can be rapidly elicited and that contribute to virus control. We examined the timing and HIV-1 antigen specificity of antiviral CD8(+) T cells during AHI. Autologous and heterologous CD8(+) T cell antiviral functions were assessed longitudinally during AHI in five donors from the CHAVI 001 cohort using a CD8(+) T cell-mediated virus inhibition assay (CD8 VIA) and transmitted/founder (T/F) viruses. Potent CD8(+) antiviral responses against heterologous T/F viruses appeared during AHI at the first time point sampled in each of the 5 donors (Fiebig stages 1/2 to 5). Inhibition of an autologous T/F virus was durable to 48 weeks; however, inhibition of heterologous responses declined concurrent with the resolution of viremia. HIV-1 viruses from 6 months postinfection were more resistant to CD8(+)-mediated virus inhibition than cognate T/F viruses, demonstrating that the virus escapes early from CD8(+) T cell-mediated inhibition of virus replication. CD8(+) T cell antigen-specific subsets mediated inhibition of T/F virus replication via soluble components, and these soluble responses were stimulated by peptide pools that include epitopes that were shown to drive HIV-1 escape during AHI. These data provide insights into the mechanisms of CD8-mediated virus inhibition and suggest that functional analyses will be important for determining whether similar antigen-specific virus inhibition can be induced by T cell-directed vaccine strategies.

Fig 1

CD8⁺ T cells from HIV-1 donors before or during the time of seroconversion suppress T/F viruses at levels similar to suppression in elite controllers. CD8⁺ T cells isolated from 3 seronegative donors, 5 HIV-1-positive donors (Fiebig score of ≤5), and 5 HIV-1-positive elite controllers were tested for antiviral activity against a panel of 7 T/F IMC in the CD8 VIA. Dots represent the log reduction in virus replication (RLU) for each CD8 donor against a single virus at an E/T ratio of 2:1 compared to the results from a CD8-depleted infection control. Statistical significance was determined by using the mean virus inhibition across T/F viruses for each donor (Student's t test). The dotted line indicates a log inhibition cutoff for positivity of 0.96 (3 standard deviations above the mean seronegative donor T/F virus inhibition).

Fig 2

CD8⁺ T cell-mediated virus inhibition of heterologous T/F viruses is dynamic during the acute phase of infection. Antiviral activity was measured from CD8⁺ T cells isolated longitudinally from 4 HIV-1-positive patients (3 ART-naïve and CH106 who began ART at study week 1) during acute infection (shown as log reduction in virus titer [TCID₅₀] from the results for a CD8-depleted control). Mean inhibition across T/F viruses is shown for 4 patients: (A) 700010199; (B) 702010157; (C) 702010736; (D) 700010106.

Fig 3

CD8⁺ T cell-mediated inhibition of autologous virus is maintained even in the absence of high viral loads. Antiviral activities exerted by CD8⁺ T cells that were isolated longitudinally from a clade B-infected HIV-1-positive patient during acute infection are shown as log reductions in titer (TCID₅₀) compared to the results for the CD8 T cell-depleted control sample. CD8⁺ T cell-mediated inhibition of 3 heterologous T/F viruses (CH040.c, CH058.c, and CH077.t), one autologous T/F virus (CH106.c), and NL4-3 is shown over 48 weeks for patient 7000010106.

Fig 4

Virus rapidly escapes from CD8-driven pressure exerted early after infection. (A) CD8⁺ T cells isolated from 3 early time points (enrollment to week 3) were tested in the CD8 VIA for inhibitory capacity against autologous (CH077.t) and heterologous (CH058.c) T/F and 6-month viruses. Inhibition of virus replication (TCID₅₀) relative to the results for a CD8-depleted control is shown. (B) Inhibition of T/F and paired 6-month virus by CD8⁺ T cells from heterologous acute patients is shown as log reduction in virus titer (TCID₅₀) relative to the results for a CD8-depleted control. The range of inhibition for heterologous donors is shown as log reduction in virus titer at 2:1 E/T.

Fig 5

Soluble inhibition of HIV-1 correlates with cytokine expression in CD8⁺ T cells from a virus controller. Transwell CD8 VIA was used to assess the soluble-factor inhibitory capacity of PTE peptide-stimulated CD8⁺ T cells from a virus controller. Suppression of virus replication is shown as log reduction in RLU from the results for an infected TZM-bl control. Background is determined using peptide pool-stimulated CD8⁺ T cells from a seronegative donor and has been subtracted. (A) Suppression of CH040.c T/F and IIIB viruses by peptide pool-stimulated VC9 CD8⁺ T cells is shown. (B) Each panel shows the percentage of total CD8⁺ T cells expressing the indicated cytokine of CD107a as determined by flow cytometric analysis. (C) Each panel shows supernatant cytokine levels as measured by Luminex following peptide stimulation of CD8⁺ T cells and coculture with infected CD4⁺ T cell targets. Lines indicate best fit, by least squares method. Pearson's correlations and P values are shown (Prism software).

Fig 6

CD8⁺ T cells inhibit replication of T/F viruses through antigen-specific release of soluble inhibitors. Transwell CD8 VIA assay was used for longitudinal assessment of the soluble-factor inhibitory capacity of HIV-1 peptide-stimulated CD8⁺ T cells from 4 acute donors. Suppression of virus replication is shown as log reduction in RLU from the results for an infected TZM-bl control. Results for the JR-HVS CD8⁺ T cell line positive control are shown in red. Background is determined using peptide pool-stimulated CD8⁺ T cells from a seronegative donor and has been subtracted. (A) Soluble-factor suppression of CH040.c virus by 700010106 CD8⁺ T cells. (B) Soluble-factor suppression of CH040.c virus by 701010199 CD8⁺ T cells. (C) Soluble-factor suppression of CH042.c virus by 702010157 CD8⁺ T cells. (D) Soluble-factor suppression of CH042.c virus by 702010736 CD8⁺ T cells. (E) A summary of positive responses seen at enrollment or week 1 (cutoff of 2.5 standard deviations above the seronegative mean after background subtraction).

Fig 7

Soluble-factor inhibition of autologous T/F virus corresponds with rapid virus escape and correlates with cytokine release. Transwell CD8 VIA was used for longitudinal assessment of the soluble-factor inhibitory capacity of PTE peptide-stimulated CD8⁺ T cells from an acute patient. Suppression of virus replication is shown as log reduction in RLU from the results for an infected TZM-bl control. Results for the JR-HVS CD8⁺ T cell line control are shown in red. Background is determined using peptide pool-stimulated CD8⁺ T cells from a seronegative donor and was subtracted. (A) Suppression of CH077.t T/F virus by peptide pool-stimulated 700010077 acute CD8⁺ T cells is shown. (B) Each panel shows supernatant cytokine levels as measured by Luminex following peptide stimulation of CD8⁺ T cells and coculture with infected CD4⁺ T cell targets. Pearson's correlations and P values are shown (Prism software).

Fig 8

Schematic of methods for assessing CD8⁺ T cell antiviral function in acute HIV-1 infection. Four assays were utilized in concert to assess CD8⁺ T cell function. Two assays measure the ability of primary CD8⁺ T lymphocytes to inhibit HIV-1 virus replication (using full-length HIV-1 transmitted/founder infectious molecular clones) via cococulture and through soluble mechanisms: a CD8⁺ virus inhibition assay (CD8 VIA) (1) and an HIV-1 peptide stimulation and soluble-factor inhibition assay (HIV-1-specific CD8 transwell VIA) (2). The other two assays are for cell surface marker phenotype and intracellular cytokines from HIV-1 peptide-stimulated primary CD8⁺ T lymphocytes using multiparameter intracellular cytokine staining assays (3) and antigen-specific cytokine secretion in cell culture supernatants using Luminex multiplex cytokine measurements (4). The combination of the four assays using the same PBMC specimen provides a comprehensive measurement of CD8⁺ T cell antiviral function against both autologous and circulating HIV-1 isolates.