Despite the enormous reduction in morbidity and mortality that protective vaccines have afforded, there are many infections for which vaccines have yet to successfully stimulate protective antibody responses. One such pathogen is respiratory syncytial virus (RSV), a respiratory infection that is common but extremely dangerous for infants, the elderly, and susceptible populations such as those undergoing stem cell transplant. Passive administration of antibodies against RSV has afforded protection in high-risk patients, but this treatment requires monthly infusions and is an unfeasible preventative option for the general population. Because of this, research has focused on bypassing the need for recurring reinfusion, primarily by transducing a subset of cells to secrete antibodies against RSV. However, the transduction targets—usually muscle cells—can produce only a fixed amount of antibody that does not expand upon infection stimulus. In contrast, true B cells which differentiate into long-lived memory cells and antibody-secreting plasma cells respond to infection by proliferating and producing more antibody. In order to merge the existing gene editing techniques with the inherent capabilities of B cells, Dr. Howell Moffett, along with Carson Harms and colleagues from the Taylor lab (Vaccine and Infectious Disease Division), developed a gene editing technique that creates B cells that can express protective antibodies against RSV and other viruses in mouse and human cells. They recently published this work in Science Immunology.
The authors began by designing a strategy for B cell engineering. Focusing on a 2,600-nucleotide region within the heavy chain B cell receptor—the portion that recognizes pathogens and causes the secretion of antibodies with the same specificity—they inserted a transgene under the control of the upstream endogenous heavy chain promoter, allowing for physiological expression of their engineered monoclonal antibody (emAb) in response to pathogen. The inserted transgene consists of an emAb cassette containing the heavy chain promoter followed by the complete light chain gene linked to the recombined heavy chain VDJ, which could be expressed in the form of the membrane-bound B cell receptor or a secreted antibody. Harms said that this approach is “exciting because by allowing the cell to retain full control over the expression of our engineered antibody, the cell retains all the tools it needs to function properly as a part of the immune system.” They then tested the expression of this cassette in a B cell line called RAMOS where they designed guide RNAs to target and cut the appropriate genomic region with CRISPR-Cas9 gene editing, and then transduced RAMOS cells with adeno-associated virus encoding an engineered RSV-emAb cassette. Using an RSV antigen and flow cytometry, the authors confirmed that the emAb was expressed on the surface of the B cell, bound antigen, and silenced the endogenous B cell receptor, demonstrating that emAb engineering could reprogram B cells to express an antibody against RSV.
After creating a functional emAb in a cell line, the authors began to apply this engineering strategy to primary cells. They isolated B cells from human blood and used CRISPR/Cas9 guide to create emAbs for RSV as well as HIV, influenza, and EBV, all of which successfully bound their cognate antigens. Harms explained that this result demonstrated the “modularity of our approach to hypothetically allow us to target any virus,” suggesting that this engineering strategy could be applied to infections other than RSV. Unlike in the cell line, they found that if the cassette inserted into the unproductive heavy chain locus—which is normally silenced in allelic exclusion during B cell development— human primary B cells could express the emAb on one heavy chain while expressing the endogenous sequence on the other, which creates the potential for autoimmunity. However, they were able to exploit this feature by producing dual-emAb by inserting different cassettes into each locus, and successfully engineered B cells that expressed both RSV- and influenza-emAb casssettes. These experiments showed that primary B cells could be engineered to effectively bind RSV and other viruses and that a potentially deleterious flaw in the system could be harnessed to simultaneously express two different emAbs, increasing the potential for this technique.
Finally, the authors sought to test the protective capabilities of emAbs against RSV in vivo.They created mouse emAb B cells, which could bind RSV antigen and secrete engineered antibodies in culture. They transferred these B cells into wild type mice and found that RSV-specific antibodies remained elevated in the blood for 25 days before returning to control frequencies. To test the protection afforded by the elevated levels RSV-emAbs, the authors intranasally challenged mice with RSV at day seven after B cells transfer, and found that mice that received RSV-emAbs had nearly undetectable viral loads while those that had received control B cells had substantial virus in their lungs, demonstrating the protective capacity of the engineered antibodies. However, stem cell transplant patients are highly susceptible to RSV due to their immunocompromised state, so the authors tested whether the RSV-emAbs could provide long-term protection in a mouse model lacking endogenous T and B cells. They found that the transferred emAbs persisted at high titers through day 40, where they declined to baseline titers by day 72. When they challenged mice with RSV after antibody decline, the mice given RSV-emAb were nearly fully protected, suggesting that low titers of long-lived engineered B cells in immunocompromised mice are sufficient to provide protection from RSV infection.
Proving highly successful thus far, Moffett said that their work “emphasizes the importance of genome editing” and that he “hope[s] that it will help both to provide a new avenue for cellular therapy, as well as a research tool to investigate B cell biology and antibody responses from defined starting points.” Looking forward, Harms explained that the largest uncertainty is “how scalable this process can be. Initially, cost could be prohibitive and would necessitate targeting the most vulnerable populations first. Our approach would benefit greatly from other genetic engineering technologies that allow for creation of ‘universal cells’ that can be taken from one individual but given to anyone.” Moffett also pointed out that their work complements core Fred Hutch clinical research, as “emAb B cells link back to a founding technology of the FHCRC, bone marrow transplantation, which remains a crucial tool for cancer therapy. Viral infection early after transplant is devastating, with few clinical tools available to counter it. If we can safely engineer donor's B cells to provide broad, lasting antiviral protection during this vulnerable period would be a great advance for transplant.”
This work was supported by the National Institutes of Health, a Sponsored Research Agreement from Vir Biotechnology, the Hartwell Foundation, and a donation from E. Averett.
UW/Fred Hutch Cancer Consortium member Justin Taylor contributed to this work.
Moffett H, Harms CK, Fitzpatrick KS, Tooley MR, Boonyaratanakornkit J, Taylor JJ. 2019. B cells engineered to express pathogen-specific antibodies protects against infection. Science Immunology. Doi: 10.1126/sciimmunol.aax0644.