Lymphocyte-targeted Immunoantigens improve immune response to HIV Env trimers

Introduction: : A successful HIV vaccine will require the generation of broadly-neutralizing antibodies (bnAbs) targeting conserved regions of the highly mutable HIV Env antigen. Extensive somatic hypermutation (SHM) is characteristic of the few bnAbs that have been isolated from HIV+ patients, indicating a need for HIV vaccines to initiate and sustain germinal centers (GCs), where SHM takes place. One strategy to kickstart long-lasting GCs is to efficiently target antigens to follicles within draining lymph nodes (dLNs) and to keep them there using targeting moieties like nanobodies. Here, we hypothesized that appending nanobodies targeting various immune cell surface markers to HIV Env trimers would improve the adaptive immune response to these poor antigens, potentially due to more efficient trafficking to dLN, longer half-lives within GCs, and subsequent GC persistence.

Materials and

Methods: : BG18-class HIV Env trimers were engineered to express C-terminal fusions of targeting or control VHH nanobodies (hereafter, immunoantigens or Env-VHHs) and expressed in HEK cells. Pan-lymphocyte marker CD45, antigen presentation marker MHC-II, and GFP (as control) were selected for directing Env-VHHs toward immune compartments within secondary lymphoid organs. Immunoantigens were characterized for purity and formation of tertiary and quaternary structure using fast protein and size exclusion chromatography and TEM, for antigenicity using immunoassays including ELISA, and for targeting fidelity using ELISA and flow cytometry. Biodistribution and immunological profiling was conducted in C57BL/6 mice, where cellular uptake, injection site half-life, serum antibody titers, and GC responses were assessed by IVIS, ELISA, and flow cytometry, respectively.

Results, Conclusions, and Discussions:: Env-VHH fusions were successfully cloned and expressed in mammalian protein systems and found to be stable and to form Env trimers capable of engaging epitope-specific antibodies. Specificity of each immunoantigen was validated against its target by ELISA and flow cytometry. IR800-labeled anti-CD45 immunoantigens were retained 10 hours longer at the injection site compared to the aGFP control or to soluble Env trimers with no VHH domain; and cellular uptake of fluorescent immunoantigens 24 hours post injection revealed substantial improvements in antigen association with B cells, T cells, NK cells, Neutrophils, and dendritic cells compared to controls. In vivo, anti-CD45 Env-VHHs initiated earlier antigen-specific seroconversion, larger germinal centers, and more Env-specific GC B cells 21 days post-prime compared to controls. These data suggest that cell surface receptor targeting may prove a fruitful strategy to improve adaptive immune responses to protein antigens.

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