Tryptophan-like side chain holding aptamers inhibit respiratory syncytial virus infection of lung epithelial cells

Abstract

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.

Figure 1

Schematics of RSV F protein selective aptamer generation and characterization. Created with BioRender.com.

Figure 2

Determining the ability of modified aptamers to distinguish between RSV’s prefusion and postfusion F protein by AlphaScreen. Biotin labelled aptamers were mixed with either the prefusion or postfusion form of F protein and Palivizumab. Relative AlphaScreen signal was calculated by forming a ratio of the sample fluorescence and the aptamer-free background fluorescence. The increased values indicate selective binding of aptamers to the different F protein forms. Error bars represent the standard deviation of three technical replicates.

Figure 3

Stability of a modified and a non-modified aptamer was demonstrated by incubating A549 cells with aptamer infused growth media. The oligonucleotide concentration was determined by real-time PCR at the indicated time points. The degradation of aptamers was remarkably different, the modified aptamer seems to possess an approx. 8 times longer half-life in comparison to its non-modified variant. Error bars represent the standard deviation of three individual experiments (N = 3).

Figure 4

Modified aptamers exhibit antiviral effect upon RSV infection. Total fluorescence (TF) was measured of the rgRSV infected A549 cell culture (MOI of 1). Palivizumab or the aptamers (A2, B1, D4, G6, H8, H9, non-modified and non-relevant aptamers) were pre-incubated with rgRSV prior to infection. (A) Lower TF is measured when rgRSV is pre-treated with modified aptamers or palivizumab in comparison to infection with mock-treated RSV. The non-modified aptamer or a non-relevant aptamer had marginal effect on virus neutralization. (B) Area under the curve (AUC) was calculated, mean and standard deviations of three replicates are shown. (C) Rt-qPCR of the viral RNA (performed 48 h post-infection) in infected A549 cells verifies the antiviral effect of modified aptamers (D, G). The most promising aptamer candidates, D4 and H9, demonstrate the highest virus neutralizing capability. (E, H) Percent inhibition (calculated from the AUC) compared to the mock-treated rgRSV control. RgRSV infection is reduced by palivizumab and D4 or H9 in a very similar, concentration dependent manner. Error bars indicate the standard deviation of three individual experiments (N = 3), the dashed lines indicate 50% inhibition. (F, I) Reduction in the amount of viral genome detected in the infected A549 cells also signifies the antiviral effect of the modified aptamers. P-values were calculated using unpaired t-test by comparing the “RSV only” group to the other groups (*P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001, ns = not significant).

Figure 5

(A) Viability of A549 cells treated with aptamers and palivizumab. Cells were incubated with 10 nM of aptamers or palivizumab for 48 h, then their viability was assessed. Neither Palivizumab, nor any of the aptamers had a significant detrimental effect on the cell viability after 48 h. (B) Dose-dependent effect of one of the most promising modified aptamer candidates and a non-relevant aptamer on the viability of A549 cells. Cells were incubated with 50, 25 and 10 nM of modified aptamer or non-relevant aptamer for 48 h, then their viability was assessed. Neither the modified aptamer, nor the non-relevant aptamer had a detrimental effect on the cell viability after 48 h. Error bars indicate the standard deviation of three individual experiments (N = 3).