Red blood cells are nuclei with a nucleus, which are the most widespread type of cells in the body, present in all tissues, with a lifespan of 120 days. They also lack molecules of a large class I histocompatibility complex, and therefore, type O negative erythrocytes can be used by all patients. This automatically makes them ideal as carriers of therapeutic molecules for a number of conditions, including the ongoing coronavirus disease pandemic 2019 (COVID-19).
In a new study published on bioRxiv* overprint server, a team of researchers is investigating the antiviral potential of red blood cells against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19.
Achieving high levels of HIV-1 receptors
Constructed erythrocytes can be used to design viral traps, which attract viruses to bind and infect. This is achieved by enabling them to present viral receptors on their surface. Viruses that infect erythrocytes cannot replicate due to a lack of nucleic acid, which protects the actual target cells of the host from infection.
In order to express a protein, a cell must have a translational machinery that is absent in mature erythrocytes. To achieve this, erythroid progenitor cells must be constructed before they can differentiate. During the maturation process, transgene expression is usually prevented by silencing transcription, mechanisms that control protein synthesis from transcribed genes, and degradation of proteins not normally found in erythrocytes.
The term transgene
To achieve this, the researchers combined the transgenic expression system together with the optimization of transgenic codons, to allow erythrocytes to express HIV-1 receptors CD4 and CCR5 at a high level. This transformed enucleated erythrocytes into viral traps that are potent inhibitors of HIV-1 infection.
The researchers first applied an in vitro protocol to differentiate human CD34 + hematopoietic stem cells (HSCs) into reticulocytes (immature erythrocytes that still contain ribosomal RNA and therefore can still translate proteins but lack nuclei). HSC proliferation was monitored by inserting ‘foreign’ CD4 or CCR5 genes into erythroid progenitor cells with lentiviral vectors.
In addition, the researchers inserted a gene for the expression of the fusion protein CD4-glycophorin A (CD4-GpA). This protein consists of CD4 D1D2 extracellular domains fused to the N-terminal end of a common RBC protein, GpA. This was to allow single-domain antibodies to be expressed in erythrocytes. CD4 is a single-pass and CCR5 is a multi-pass transmembrane protein. This protocol is only suitable for CD4 and not for CCR5
They found that the use of CMV promoters or ubiquitous promoters led to low transgene expression. Therefore, they switched to the use of an erythroid-specific promoter, using CCL-βAS3-FB lentivirus. This vector contains elements that increase beta-globin expression during erythrocyte development. These vector elements include β-CD4, β-CD4-GpA and β-CCR5.
The result was a large increase in CD4 expression, less improvement in CCR5, and no change in CD4-GpA. The reason for this is attributed to the small number of ribosomal and transfer RNAs within the RNA, which limited the expression of transgenes in differentiating erythrocytes.
To address this problem, they optimized transgene codons, ensuring that they generate cDNA sequences that enhance the expression of all transgenes.
These projected cells underwent efficient differentiation into enucleated erythrocytes, almost all expressing GpA, while one in three expressed high levels of CD4 and CCR5 similar to those of CD4 + T cells. About 6% of CD4-CCR5-RBCs were infected with the HIV-1 test virus, compared with 0.3% or less of control RBCs or CD4 + RBCs. Overall, therefore, this accounts for about one-fifth of all CD4-CCR5-RBCs.
CD4-CXCR4-RBC showed a higher infection rate. Infection rates were low in CD4-GpA-CCR5 or CD4-GpA-CXCR4 cells. This was maintained even with the addition of the CD4 D3D4 domain. The reason for such low incidence of infection could be the inability of CD4-GpA to localize with CCR5 and CXCR4 co-receptors, because GpA cannot be localized to lipid subdomains, unlike CD4.
The constructed erythrocytes strongly neutralized HIV-1
The potential for viral capture was assessed by an HIV-1 neutralization test. This has shown that it is possible to achieve therapeutic concentrations in vivo, because half the maximum inhibitory concentration (IC50) for HIV-1 pseudoviruses was 1.7×106 RBC / ml, which is about 0.03% of the RBC concentration in human blood. Higher levels of CD4-GpA expression quadrupled the neutralization activity.
Lower neutralization activity occurred when CCR5 was coexpressed with CD4 or CD4-GpA RBC, 2-3 times, perhaps because CCR5 causes a small decrease in the expression level of CD4-GpA and CD4. However, in vivo studies will be needed to demonstrate the potential benefit of CCR5 coexpression on RBC virus traps.
Their work on virus-like nanoparticles that represent CD4 aggregates (CD4-VLPs) has shown that the ability of these particles to interact with HIV-1 envelope protein trimmers allows them to neutralize various strains of HIV-1, while blocking viral escape in vivo. Interestingly, such interactions increase the potency of CD4-VLPs by over 10,000-fold compared with soluble CD4 and CD4-Ig inhibitors. The current study shows that the same level of efficacy can be expected with the use of RBC and CD4-VLP viral traps through high avidity interactions with HIV-1 Env antigens.
Continuous generation of erythrocyte viral traps
This strategy was then used to produce lines of erythroid progenitors that continue to create potent RBC viral traps against HIV-1 and SARS-CoV-2. To ensure that these viral traps will be continuously produced, the researchers modified the immortalized erythroblast cell line (BEL-A) to express CD4-GpA at high stable levels. They found that they underwent efficient differentiation into enucleated erythrocytes in more than half of the cells, continuing to express the constructed antigen. These cells strongly neutralized HIV-1 infection with a 2.1×10 IC507 RBC / ml.
SARS-CoV-2 uses the host cell conversion enzyme angiotensin 2 (ACE2) as its input receptor. The extracellular domain of ACE2 was fused with GpA to form a chimeric protein, and the BEL-165 A cell line was designed to express this protein. When this was exposed to lentivirus-based pseudovirus SARS-CoV-2, the researchers found that the virus was highly neutralized, with an IC50 of 7×105 RBC / ml.
These results suggest that cell lines used to produce such viral traps from erythrocytes, which express a defined host receptor, can be rapidly generated. “RBC virus traps can become powerful antiviral agents against a range of viruses.” They can exist in the body for 120 days, thus ensuring continuous control of HIV-1 infection.
* Important notice
bioRxiv publishes preliminary scientific reports that have not been reviewed and should therefore not be considered final, guide clinical practice / health-related behavior, or treat it as established information.