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G1 - Challenges to Immunological Responses to Therapeutic Interventions (Includes host responses, therapy/prevention of infectious diseases, nucleic acid therapy, RNA-based Therapies; excludes cancer immunotherapy and cancer vaccines)

1295: B cell Production of the eCD4-Ig-Knob-in Hole-Reversed HIV-1 Immunoadhesin in Humanized NBSGW Mice

Type: Poster Session

Poster Board Number: 1295
Presentation Details
Session Title: Thursday Posters: Challenges to Immunological Responses to Therapeutic Interventions

B cells have the capacity to produce high levels of IgG proteins and may persist for years as long-lived plasma cells. Moreover, B cell-produced exogenous proteins have been reported to be immune system tolerant. These properties support the concept that B cells are an attractive cellular target for gene therapy. The HIV-1 immunoadhesin molecule, eCD4-Ig-Knob-in-Hole-Reversed (KiHR), is a CD4- and CCR5/CXCR4-mimetic antibody-like protein which we have engineered so as not to interact with B cell immunoglobulin Fc domains, and it has been shown to have nanomolar HIV-1 neutralizing potency against primary isolates. We have previously shown that the B cell-specific enhancer and promoter, EµB29, promotes eCD4-Ig-KiHR expression in B cells ex vivo (PMID-36879849). We hypothesized that the presence of the eCD4-Ig-KiHR transgene in CD34+ HSPCs could provide for long-term generation of eCD4-Ig-KiHR protein-expressing B cells. To evaluate whether eCD4-Ig-KiHR could be produced long-term from B cells in humanized NBSGW mice, we transduced adult CD34+ hematopoietic stem and progenitor cells (HSPCs) with a VSV-G-pseudotyped EμB29-GFP-P2A-eCD4-Ig-KiHR lentiviral (LV) vector. CD34+ HSPCs were transduced to achieve viral copy numbers (VCN) of 1, 3 or 8 and then HSPCs were adoptively transferred to busulfan-treated NBSGW mice. Mouse peripheral blood human CD45+/GFP+ cells and serum eCD4-Ig-KiHR levels varied depending on the transduction levels in CD34+ HPSCs. VCNs of 1, 3 and 8 resulted in 10 - 15%, 25 - 35% and 50 - 70% human CD45+/GFP+ cells in peripheral blood of humanized mice and 1 - 2 ng/mL, 3 - 4 ng/mL, and 15 - 20 ng/mL of eCD4-Ig-KiHR in sera, respectively. eCD4-IgKiHR levels were maintained until week 24 post-transfer. Since B cell maturation is restricted in humanized mice to IgM-producing B cells, we evaluated whether differentiation ex vivo of IgM+ B cells isolated from spleens of humanized mice would result in greater eCD4-Ig-KiHR protein production. Indeed, differentiation ex vivo of IgM+ B cells resulted in IgG+ class-switched CD138+ plasmablasts after 10 days. Importantly, differentiated B cells produced higher amounts of eCD4-Ig-KiHR (up to 100 ng/million cells/day). We also quantified eCD4-Ig-KiHR production at the single cell level by eCD4-Ig enzyme-linked immunosorbent spot (ELISpot), and we detected a 90-fold increase in the proportion of eCD4-Ig-KiHR-expressing B cells during culture. These data suggest that the B cell population derived from transferred eCD4Ig-KiHR-transduced CD34+ HSPCs is capable of expressing high amounts of transgene from a B cell-specific enhancer/promoter when allowed to mature. Our findings indicate that at all levels of B cell GFP-marking we observed, eCD4-Ig-KiHR could be detected in humanized mouse sera at levels sufficient for HIV neutralization (> 1 ng/mL). The findings support the promising strategy that LV-mediated delivery of the HIV-1 immunoadhesin eCD4-Ig-KiHR under the control of a B-cell specific enhancer/promoter to CD34+ HSPCs may lead to the production of high levels of eCD4-Ig-KiHR by mature B cells in vivo.

Chet Ojha1, Craig Schindewolf1, Anna Helmers1, Amalia Icreverzi1, Neele Thom1, Gregory Asher1, Meagan Neu1, Andrea Repele1, Richard James2, David J. Rawlings2, Bruce E. Torbett2

1Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA,2Department of Pediatrics, University of Washington School of Medicine, Seattle, WA"

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