Evaluation of Molecularly Imprinted Polymers as Synthetic Virus Neutralizing Antibody Mimics

Abstract

Rapid development of antibody-based therapeutics are crucial to the agenda of innovative manufacturing of macromolecular therapies to combat emergent diseases. Although highly specific, antibody therapies are costly to produce. Molecularly imprinted polymers (MIPs) constitute a rapidly-evolving class of antigen-recognition materials that act as synthetic antibodies. We report here on the virus neutralizing capacity of hydrogel-based MIPs. We produced MIPs using porcine reproductive and respiratory syndrome virus (PRRSV-1), as a model mammalian virus. Assays were performed to evaluate the specificity of virus neutralization, the effect of incubation time and MIP concentration. Polyacrylamide and N-hydroxymethylacrylamide based MIPs produced a highly significant reduction in infectious viral titer recovered after treatment, reducing it to the limit of detection of the assay. MIP specificity was tested by comparing their neutralizing effects on PRRSV-1 to the effects on the unrelated bovine viral diarrhea virus-1; no significant cross-reactivity was observed. The MIPs demonstrated effective virus neutralization in just 2.5 min and their effect was concentration dependent. These data support the further evaluation of MIPs as synthetic antibodies as a novel approach to the treatment of viral infection.

Keywords: molecularly imprinted polymer; neutralization; porcine reproductive and respiratory syndrome virus; synthetic antibody mimic; virus.

Figure 1

Assessment of PRRSV-1 neutralization by virus-imprinted hydrogel MIPs. Template virus was prepared by purification of PRRSV-1 virions by continuous density ultracentrifugation. Pools of gradient fractions (A) and individual fractions (B) containing the highest infectious titers were selected and virion purity and integrity post-dialysis and inactivation confirmed by electron microscopy (C). Three independently produced batches of MIPs were tested for their ability to neutralize PRRSV-1 infectivity in vitro (D). A suspension of PRRSV-1 was incubated for 1 h with pAA, pNHMA or pNIPAM MIPs (closed bars). Non-imprinted polymers (NIPs; open bars) and PBS (gray bars) were included as negative controls. Post-incubation, MIP/NIPs were removed by centrifugation and the infectious viral titers in supernatants determined by immunoperoxidase staining of inoculated cell monolayers. The mean infectious PRRSV titers, expressed as 50% tissue culture infectious doses (TCID₅₀), for technical triplicates of each treatment condition are presented and error bars represent the standard error of the means. The limit-of-detection of the assays are indicated by dashed horizontal lines.

Figure 2

Characterization of the virus neutralizing properties of hydrogel MIPs. The specificity of PRRSV-1 virus-imprinted MIPs was assessed by evaluating neutralization of BVDV-1 (A). A suspension of BVDV-1 was incubated for 1 h with pAA or pNHMA MIPs (closed bars). Non-imprinted polymers (NIPs; open bars) and PBS (gray bars) were included as negative controls. To assess the effect of incubation time on PRRSV-1 neutralization by MIPs, a suspension of PRRSV-1 was incubated for 60, 30, 15, 7.5, 5, 2.5, and 1.5 min with pNHMA MIPs (closed bars) (B). Sixty minute incubation with NIPs (open bar) and PBS (gray bar) were included as negative controls. To assess the effect of MIP concentration PRRSV-1 neutralization by MIPs, a suspension of PRRSV-1 was incubated for 1 h with a 5-fold serial dilution of pAA or pNHMA MIPs, a neat suspension of NIPs or PBS (C). After incubation, MIPs/NIPs were removed and the infectious viral titers in supernatants determined by immunoperoxidase staining of inoculated cell monolayers. The mean infectious titers, expressed as 50% tissue culture infectious doses (TCID₅₀), for technical triplicates of each treatment condition are presented and error bars represent the standard error of the means. The limit-of-detection of the assays are indicated by horizontal dashed lines.