Engineering B cells to improve HIV treatments

To prevent viruses from infecting cells, our immune systems depend on antibodies. Antibodies bind specific antigens present on viruses to tag them for destruction. For some antibodies, known as neutralizing antibodies, this antibody-antigen interaction can block the virus from infecting a cell or target the infected cell. Many viruses, including HIV, are very diverse, and in some cases antibodies that can neutralize one strain of the virus cannot even bind to other strains. Even within an individual, HIV evolves rapidly and often changes its antigen so that existing antibodies can no longer bind or neutralize the virus. When viral evolution outpaces antibody production, antibodies can’t do much to protect us.

Luckily, researchers have discovered broadly neutralizing antibodies (bNAbs) that target multiple strains of HIV at conserved antigens that are unlikely to evolve. Though bNAbs are only expressed by a small subset of people living with HIV, finding ways to introduce them into patients who do not express them can help control viral load and improve health outcomes.

Researchers have tried injecting HIV bNAbs into people living with HIV. While they are safe and transiently reduce HIV viral load, they are quickly cleared by the body, limiting their clinical efficacy. To overcome this problem, scientists in the Kiem lab at Fred Hutch want to use gene therapy to induce bNAb expression in people living with HIV. To do this, they decided to genetically engineer B cells to overexpress HIV bNAbs.

Why B cells? While the field of B cell engineering is still emerging with a limited number of studies, the therapeutic applications would be impactful. “Because B cells naturally produce antibodies, they are already equipped for protein secretion, and the applications are broad for infectious or non-infectious diseases requiring therapeutic protein secretion,” explains Dr. Anne-Sophie Kuhlmann, lead author of the new study in Molecular Therapy Methods & Clinical Development. One of the key functions of B cells normally is to produce antibodies against specific antigens. “With the B cells, it is more physiological bNAb expression because they will react to the detection of their viral antigen with bNAb secretion but should also be able to differentiate into persisting memory B cells and mature their antibody response, all this in an antigen-specific manner,” says Kuhlmann. The team began their study by isolating and expanding B cells from primates infected with SHIV, an engineered version of HIV that infects primates. They chose to use SHIV-infected primates treated with antiretrovirals to demonstrate that their engineering approach could allow those B cells to produce HIV-targeted bNAbs. With these cells in hand, they moved forward testing their engineering approach.

Typically, it takes two steps to deliver gene therapy products to cells in a dish. First, a ribonucleoprotein complex is electroporated into the cell. This complex contains a CRISPR-Cas9 enzyme that cuts DNA and a guide RNA that takes the complex to the correct spot in the DNA. For HIV gene therapy, this would be the DNA that encodes the antibody gene. Second, researchers use viral vectors to deliver the donor template DNA into the cell. For HIV gene therapy, the template DNA is the gene encoding the HIV bNAb. Several factors can impact the efficiency of this process. For one, the guide RNA for the Cas9 enzyme may be non-specific. Using an editing approach initially developed by their collaborator, Dr. Justin Taylor, the team electroporated two different guide RNAs and their accompanying Cas9 enzymes into B cells to test how selective their guides were. Without a donor template, the Cas9 cut triggers DNA repair that introduces random DNA insertions or deletions (indels). After inserting their guide RNAs and Cas9 enzymes, the team looked for indels at the guide RNA sites. They found that both guide RNAs efficiently edited the antibody gene, indicating that they were both good candidates for gene therapy, with one of them being truly specific to the targeted site.

With an effective editing system in hand, the team next sought to find a viral vector that could efficiently insert donor template DNA into the B cells. They tested three different viruses carrying DNA that encoded for GFP, infected B cells with them, and then measured GFP fluorescence in the B cells. They found that all their viral vectors infected B cells at similar rates, so they proceeded with one of the vectors that had been successfully used for human B cell studies by other groups.

With successful tools in hand, the team then attempted to induce expression of the bNAb VRC01 in B cells. They isolated the B cells, grew them for two days, electroporated CRISPR-Cas9 and guide RNA complex into them, and, finally, introduced a viral vector carrying the VRC01 DNA. They found that the VRC01 DNA successfully integrated into the antibody gene locus in animals with and without SHIV. Next, they analyzed VRC01 mRNA and protein expression. They found that the B cells from animals without SHIV expressed VRC01 mRNA and protein. while B cells from SHIV animals expressed VRC01 mRNA, but they did not express the protein, indicating that editing is less efficient in these cells. Despite this limitation, these results suggest that B cell gene therapy is a viable way to induce bNAb expression.

Currently, there is no cure for HIV, but the virus can be controlled with antiretroviral therapy. This therapy requires daily medication that can be difficult to access in resource-limited regions with high HIV prevalence. Kuhlmann and her team envision a future where gene therapy products can help alleviate this burden. “We are trying to develop a once-and-done approach where you wouldn’t have to take medicine every day,” says Kuhlmann. Future innovations to improve editing efficiency and deliver the gene therapy in vivo will help move the field towards this more equitable and accessible vision of HIV treatment.

This work was supported by funding from the NIDDK Cooperative Center of Excellence in Hematology, the Bill and Melinda Gates Foundation, and the National Institute of Allergy and Infectious Diseases.

Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Dr. Hans-Peter Kiem contributed to this research.

Kuhlmann A, Madkhali N, Moskovitz E, Parrott JE, Raman SS, Riker AO, Martinez-Reyes J, Gupta M, Jang RA, Nelson V, Gray MD, Taylor JJ, Peterson CW, Kiem HP. 2025. Heavy-chain immunoglobulin locus editing in rhesus macaque B cells to confer antibody production. Mol Ther Methods Clin Dev. 33(4):101598. doi: 10.1016/j.omtm.2025.101598.