Highly active antiretroviral therapies are effective against HIV-1 cell-to-cell transmission

Abstract

HIV-1 cell-to-cell transmission allows for 2-3 orders of magnitude more efficient viral spread than cell-free dissemination. The high local multiplicity of infection (MOI) observed at cell-cell contact sites may lower the efficacy of antiretroviral therapies (ART). Here we test the efficacy of commonly used antiretroviral inhibitors against cell-to-cell and cell-free HIV-1 transmission. We demonstrate that, while some nucleoside-analog reverse transcriptase inhibitors (NRTI) are less effective against HIV-1 cell-to-cell transmission, most non-nucleoside-analog reverse transcriptase inhibitors (NNRTI), entry inhibitors and protease inhibitors remain highly effective. Moreover, poor NRTIs become highly effective when applied in combinations explaining the effectiveness of ART in clinical settings. Investigating the underlying mechanism, we observe a strict correlation between the ability of individual drugs and combinations of drugs to interfere with HIV-1 cell-to-cell transmission, and their effectiveness against high viral MOIs. Our results suggest that the ability to suppress high viral MOI is a feature of effective ART regimens and this parameter should be considered when designing novel antiviral therapies.

Figure 1. Experimental design for comparing cell-free to cell-to-cell transmission of HIV-1.

(A) To measure cell-free HIV-1 infections, we generated HIV-1_GLuc by transfecting plasmids encoding for HIV-1_NL4-3 as well as the HIV-1_inGLuc reporter plasmids into HEK293 cells and infected primary CD4+ T cells by spinoculation. HIV-1 infection was determined by measuring GLuc activity 36 hr later. HIV-1 infection by cell-to-cell transmission was assessed by co-culture of HIV-1-infected Jurkat-inGLuc donor cells with target primary CD4+ T cells. Specifically, Jurkat-inGLuc cells were inoculated with HIV-1_NL4-3, washed, stimulated with 6.25 ng/mL of PMA for 2 hr at 37°C, washed again, and incubated for 18 hr at 37°C. HIV-1_GLuc-generating donor cells were then washed and co-cultured with target primary CD4+ T cells at a ratio of 1∶1. Infection was determined by measuring GLuc activity 36 hr post-infection.

Figure 2. Most NNRTIs Ent-Is and PIs potently inhibit HIV-1 cell-to-cell transmission.

(A) The efficacy of antiretroviral NRTIs, NNRTIs, Ent-Is and PIs was tested in cell-free or co-culture infection of primary CD4+ T cells. The data is displayed as a double log plot of the ratio of the relative GLuc light units measured for the inhibitor over the DMSO-only control versus increasing inhibitor concentration. Plateau signals above and below the effective drug concentrations are excluded from the figure and from analysis. The gray area represents the drug concentrations detected in the serum of treated patients (C_ave to C_Max for NRTIs and C_Min to C_Max for other drugs). Error bars represent the standard deviation from the combination of at least two individual experiments each done in triplicate. Asterisks indicate statistical significance of the difference between HIV-1 cell-to-cell transmission and cell-free infection at each drug concentration. It was calculated using the Mann-Whitney U test: * p≤0.05, ** p≤0.01, *** p≤0.001. The IC₉₀ calculated for cell-free or co-culture infection is given beneath each graph. (B) The change in IC₉₀ for co-culture over cell-free infection was plotted for each drug and grouped based on drug family. The dashed red line indicates no change. The complete drug inhibition curves for stavudine (d4T), abacavir (ABC), rilpivirine (RPV), nevirapine (NVP), etravirine (ETR), plerixafor (AMD3100), enfurvitide (T20), the indicated BMS inhibitors, indinavir (IDV), darunavir (DRV), lopinavir (LPV), and saquinavir (SQV) are shown in Supplementary Fig. S2 and S3. (C) The efficacy of a subset of antiretroviral inhibitors was tested in cell-free or co-culture infection of primary CD4+ T cells with the founder virus clone pTRJO.c . All curves are shown in Supplementary Fig. S5.

Figure 3. Most NNRTI and Ent-Is maintain their instantaneous inhibitory potential against HIV-1 cell-to-cell transmission.

(A) The average instantaneous inhibitory potential (IIP) was calculated for each drug of Fig. 2A and Supplementary Fig. S2. All curves are shown in Supplementary Fig. S6. (B) The change in average IIP at the top inhibitor dose (IC_Max) for co-culture over cell-free infection was plotted for each drug and grouped based on drug family. (C) The average IIP was calculated for the drugs tested against HIV-1_TRJO.c and the change in the average IIP at IC_Max was grouped based on drug family. All IIP curves are shown in Supplementary Fig. S7.

Figure 4. Combinations of NRTIs are highly effective against HIV-1 cell-to-cell transmission.

(A, B) An experiment as in Fig. 2 was performed for the combinations (black lines) of (A) AZT and TFV and (B) 3TC and ABC and compared to each single inhibitor treatment (red lines, left and right panel). The X-axis represents the drug concentration of the drug within the combination that is being compared to the single drug treatment (in red). See Supplementary Fig. S8A for additional inhibitor combinations. (C) The change in IC₉₀ for co-culture over cell-free infection for the single inhibitors was compared to all the inhibitor combinations tested. (D) The change in average IIP at IC_Max for co-culture over cell-free infection for the single inhibitors was compared to all the inhibitor combinations tested. See Supplementary Fig. S8B for complete set of average IIP data. (E) Cell-free and co-culture infection of primary cells with HIV-1_NL4-3 carrying the M184V mutation of reverse transcriptase (black line) compared to wild-type HIV-1_NL4-3 (red line) in the presence of increasing concentrations of AZT. Error bars represent the standard deviation from the combination of at least two individual experiments each done in triplicate.

Figure 5. Antiretroviral inhibitors and combinations that are effective against HIV-1 cell-to-cell transmission are effective against high viral MOI.

(A) An experiment as in Fig. 2 was performed for cell-free HIV-1 infection of MT-4 cells with increasing MOI in the presence of indicated single or combined antiretroviral inhibitors. Error bars represent the standard deviation of 3 measurements. (B) Changes in IC₉₀ relative to MOI = 1 were plotted for each viral dose to display the MOI-dependence of NRTIs. (C) Changes in IC₉₀ relative to MOI = 1 were plotted for increasing viral MOI to display the MOI-independence of NNRTIs and inhibitor combinations. (D) Graph displays the correlation between the effectiveness of ART inhibitors and combinations against HIV-1 cell-to-cell transmission and MOI-independence. Specifically, the comparison of the change in IC₉₀ of co-culture over cell-free infection is plotted over the change in IC₉₀ of MOI = 25 over MOI = 1.