Finding a way to neutralize or block infection by HIV has long been pursued by viral researchers. Various treatments have been developed, driven by the need to find effective drugs to manage HIV in infected individuals. The ultimate goal is to develop a vaccine to prevent HIV from even taking hold in the body. With all of our knowledge about the cellular receptors HIV needs to enter the cell, there has to be a method to prevent a viral particle from binding and being internalized. Many researchers are pursuing neutralizing antibodies to the virus as one method. What about antibodies that target the cellular receptor recognized by the virus? In a recently published article in Proceedings of the National Academy of Sciences, antibodies to cellular receptors for rhinovirus and HIV were tethered to the plasma membrane and tested for the ability to prevent infection.
Finding a Receptor-Binding Antibody
Nonenveloped rhinovirus was used as the model system because of its simplicity and ability to be investigated in a standard research laboratory. Intercellular Adhesion Molecule (ICAM) protein is the receptor for rhinovirus A and B and became a target for this proof-of-concept experiment. Antibodies that bind ICAM-1 were selected from a human antibody library in phage. After two rounds of testing, the genes were transferred to lentivirus vectors, pooled together and used to infect HeLa cells. These infected cells expressed antibodies that bound to ICAM-1 in the plasma membrane. After two days, the cells were challenged with rhinovirus. While most of the cells died, there were colonies that grew even in the presence of virus.
After extracting genomic DNA from these resistant colonies, the antibody coding sequences were PCR amplified and cloned a second time into the lentivirus vector. This output pool of antibodies were shown to better protect cells from infection compared to the input pool that was selected based on binding to ICAM. As a control, lentivirus expressing fluorescent protein tomato also infected HeLa cells, but provided no protection from rhinovirus infection.
Identifying Individual Protective Antibodies
Individual antibodies were identified by sequencing 100 bacterial clones, packaging the clone into lentivirus, infecting HeLa cells and testing for protection against rhinovirus. Five antibodies were identified as persistently protective two and three days after rhinovirus challenge. Switching antibody expression from membrane tethered to soluble also showed inhibition of rhinovirus infection with each of the five different antibodies having different inhibition levels. However, the strongest effect of each antibody was shown when tethered to the membrane. This suggested the antibody molarity when membrane associated is greater than even the highest concentration of the same antibody in soluble form. Cells expressing membrane-tethered antibodies (MTAs) also increased in number after viral challenge and multiplied to a cell number nearly equal to uninfected cells.
Preventing Infection, Preserving Cell Function
There is more to preventing infection than having a MTA that binds the viral receptor. The antibody needs to block virus recognition, but still preserve the normal cellular functions of the receptor. Because ICAM mediates T-cell binding to endothelial cells, a T-cell attachment assay was used to assess how the most potent MTA, Ab100, affected the ICAM interaction with T cells. Not surprisingly, the strong viral protection of Ab100 also blocked T-cell binding.
Xie et al. decided to adjust the ICAM binding site on Ab100 to restore ICAM cellular function. Using known hotspot residues in complementarity-determining region 3 (CDR3), the region most responsible for antigen binding, Ab100 mutants were generated, creating a diverse library of the yeast surface expression vectors. After five rounds of screening for high-affinity antibodies, three clones of the output pool were selected for further study. These mutated MTAs had ICAM KDs 30–60% lower than the original Ab100 and similar reductions in IC50 for blocking infection. When tested in the T-cell attachment assay, two of the selected MTAs blocked attachment by 30% but the third almost completely prevented binding. Therefore, MTAs can be selected for binding cellular receptors to prevent viral infection while preserving receptor function.
Recapitulating for HIV-1
With evidence that MTA to ICAM can block rhinovirus infection, researchers wanted to test a similar system for HIV, an enveloped virus. Antibodies to CD4, the known HIV receptor, were expressed on TZM-bl cells, a cell line widely used in standardized infectivity assays to evaluate antibody neutralization. The anti-CD4 antibody leu3a, converted to an MTA format, was able to completely inhibit infection for four of six cross-clade viruses and offer protection for the remaining two viruses. Because the number of expected MTA molecules is about the same as the number of HIV receptors on the T cells, the density of available receptors is likely reduced to a point where HIV is unable to find any receptors to infect the cell.
The authors used membrane-tethered antibodies to block rhinovirus and HIV-1 from entering cells by binding to the viral receptor. This strategy not only offers a new way to block infection from HIV, but also has the potential to reverse the course of infection in an individual. MTAs work by reducing receptor density on the cell, and the results presented suggest that if a cell can block HIV from infecting it, the resistant cell could multiply and survive. This selection gives rise to a population of cells that could replace the permissive infected cells present in an individual to the point an individual may be considered cured. While this research article shows proof of concept, more research on HIV and other viruses may discover more optimal antibodies that could work tethered to the plasma membrane to block infection and preserve receptor function, all driven by cell engineering and selection.
Xie, J., Sok, D., Wu, N.C., Zheng, T., Zhang, W., Burton, D.R. and Lerner, R.A. (2017) Immunochemical engineering of cell surfaces to generate virus resistance. Proc. Natl. Acad. Sci. USA. [Epub ahead of print] doi: 10.1073/pnas.1702764114.