Red blood cells are anucleate cells, which are the most abundant type of cell in the body, being present within all tissues, with a lifespan of 120 days. They also do not have class I major histocompatibility complex molecules, and thus, type O negative RBCs can be used by all patients. This automatically makes them ideal as delivery vehicles for therapeutic molecules, for a range of conditions, including the ongoing coronavirus disease 2019 (COVID-19) pandemic.
In a new study, released on the bioRxiv* preprint server, a team of researchers explores the antiviral potential of red blood cells against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of COVID-19.
Achieving high levels of HIV-1 receptors
Engineered RBCs can be used to design viral traps, which attract viruses to bind to and infect them. This is achieved by enabling them to present viral receptors on their surface. The viruses that infect the RBCs cannot replicate because of the lack of nucleic acid, which protects the actual host target cells from infection.
In order to express a protein, the cell must have translation machinery, which is absent within mature RBCs. In order to do this, erythroid progenitor cells have to be engineered before they differentiate. During the process of maturation, transgene expression is typically prevented by silencing transcription, mechanisms that control protein synthesis from transcribed genes, and the breakdown of proteins that are not normally found in RBCs.
To achieve this, the researchers combined a transgenic expression system along with transgene codon optimization, to allow the RBCs to express HIV-1 receptors CD4 and CCR5 at high levels. This transformed the enucleated RBCs into viral traps that are powerful inhibitors of HIV-1 infection.
The researchers first applied an in vitro protocol to differentiate human CD34+ hemopoietic stem cells (HSCs) into reticulocytes (immature RBCs that still contain ribosomal RNA, and can therefore still carry out translation of proteins, but lack nuclei). The HSC proliferation was followed by the insertion of the ‘foreign’ CD4 or CCR5 genes into the erythroid progenitor cells by lentiviral vectors.
In addition, the researchers inserted a gene to express a CD4-glycophorin A (CD4-GpA) fusion protein. This protein is composed of CD4 D1D2 extracellular domains fused to the N-terminal end of the common RBC protein, GpA. This was to allow single-domain antibodies to be expressed in RBCs. CD4 is a single-pass, and CCR5 a multi-pass transmembrane protein. This protocol is suitable only to CD4, and not to CCR5
They found that the use of the CMV promoter or ubiquitous promoters led to low transgene expression. They therefore switched to using an erythroid-specific promoter using the CCL-βAS3-FB lentivirus. This vector contains elements that upregulate beta-globin expression during the development of RBCs. These vector elements include β-CD4, β-CD4-GpA, and β-CCR5.
The result was a big increase in CD4 expression, a smaller enhancement of CCR5, and no change in CD4-GpA. The reason attributed to this was the small number of ribosomal and transfer RNAs within the RNAs, which limited the expression of transgenes in the differentiating RBCs.
To address this, they optimized the transgene codons, ensuring they generated cDNA sequences that enhanced the expression of all the transgenes.
These engineered cells underwent efficient differentiation into enucleated RBCs, almost all expressing GpA, while one in three expressed high levels of CD4 and CCR5 similar to that of CD4+ T cells. About 6% of the CD4-CCR5-RBCs were infected with the test HIV-1 virus, compared to 0.3% or less of control RBCs or CD4+ RBCs. Overall, therefore, this amounts to about a fifth of all CD4-CCR5-RBCs.
With CD4-CXCR4-RBCs, higher infection rates were seen. Infection rates were low with CD4-GpA-CCR5, or CD4-GpA-CXCR4 cells. This persisted even with the addition of the CD4 D3D4 domains. The reason for such low infection frequencies could be the inability of CD4-GpA to co-localize with CCR5 and CXCR4 co-receptors, since GpA cannot localize to lipid subdomains, unlike CD4.
Engineered RBCs potently neutralized HIV-1
The potential for viral trapping was evaluated by an HIV-1 neutralization assay. This showed that it is possible to achieve therapeutic concentrations in vivo, because the half-maximal inhibitory concentration (IC50) for HIV-1 pseudoviruses was 1.7x106 RBCs/mL, which is about 0.03% of the RBC concentration in human blood. The higher level of expression of CD4-GpA enhanced neutralization activity by fourfold.
Lower neutralization activity occurred when CCR5 was co-expressed with CD4 or CD4-GpA RBCs, by 2-3 times, perhaps because CCR5 causes a small drop in the level of expression of CD4-GpA and CD4. However, in vivo studies will be required to demonstrate the potential for benefit with the co-expression of CCR5 on RBC viral traps.
Their work on virus-like nanoparticles presenting clusters of CD4 (CD4-VLPs) showed that the ability of these particles to interact with HIV-1 envelope protein trimers allowed them to neutralize a variety of strains of HIV-1, while also blocking viral escape in vivo. Interestingly, such interactions increase the potency of the CD4-VLPs by over 10,000 times compared to soluble CD4 and CD4-Ig inhibitors. The current study shows that the same level of efficacy can be expected with the use of RBC viral traps and CD4-VLPs via high-avidity interactions with HIV-1 Env antigens.
Constant generation of RBC viral traps
This strategy was then used to produce erythroid progenitor lines that continue to generate potent RBC viral traps against HIV-1 and SARS-CoV-2. To make sure that these viral traps would be produced continuously, the researchers modified an immortalized erythroblast cell line (BEL-A) to express CD4-GpA at high stable levels. They found that they underwent efficient differentiation into enucleated RBCs in over half the cells, while continuing to express the engineered antigen. These cells powerfully neutralized HIV-1 infection with an IC50 of 2.1x107 RBCs/mL.
The SARS-CoV-2 utilizes the host cell angiotensin-converting enzyme 2 (ACE2) as its entry receptor. The extracellular domain of ACE2 was fused to GpA to create a chimeric protein, and the BEL-165 A cell line was engineered to express this protein. When this was exposed to the lentivirus-based SARS-CoV-2 pseudovirus, the researchers found that the virus was highly neutralized, with an IC50 of 7x105 RBCs/mL.
These results suggest that the cell lines that are used to produce such viral traps from RBCs, expressing a defined host receptor, can be quickly generated. “RBC viral traps have the potential to become powerful antiviral agents against a range of viruses.” They can persist in the body for 120 days, thus ensuring continuous control of the HIV-1 infection.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.