A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design.

Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35 novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to understanding epitope selection from non-HIV-1 antigens.

Design and Testing of Vector-Producing HEK293T Cells Bearing a Genomic Deletion of the SV40 T Antigen Coding Region.

The use of the human embryonic kidney (HEK) 293T cell line to manufacture vectors for in vivo applications raises safety concerns due to the presence of SV40 T antigen-encoding sequences. We used CRISPR-Cas9 genome editing to remove the SV40 T antigen-encoding sequences from HEK293T cells by transfecting them with a recombinant plasmid expressing Cas9 and two distinct single guide RNAs (sgRNAs) corresponding to the beginning and end of the T antigen coding region. Cell clones lacking T antigen-encoding sequences were identified using PCR. Whole-genome (WG) and targeted locus amplification (TLA) sequencing of the parental HEK293T cell line revealed multiple SV40 T antigen-encoding sequences replacing cellular sequences on chromosome 3. The putative T antigen null clones demonstrated a loss of sequence reads mapping to T antigen-encoding sequences. Western blot analysis of cell extracts prepared from the T antigen null clones confirmed that the SV40 large and small T antigen proteins were absent. Lentiviral vectors produced using the T antigen null clones exhibited titers up to 1.5 × 107 transducing units (TU)/mL, while the titers obtained from the parent HEK293T cell line were up to 4 × 107 TU/mL. The capacity of the T antigen-negative cells to produce high titer adeno-associated virus (AAV) vectors was also evaluated. The results obtained revealed that the lack of T antigen sequences did not impact AAV vector titers.

Different human resting memory CD4+ T cell subsets show similar low inducibility of latent HIV-1 proviruses.

The latent reservoir of HIV-1 in resting CD4+ T cells is a major barrier to cure. It is unclear whether the latent reservoir resides principally in particular subsets of CD4+ T cells, a finding that would have implications for understanding its stability and developing curative therapies. Recent work has shown that proliferation of HIV-1-infected CD4+ T cells is a major factor in the generation and persistence of the latent reservoir and that latently infected T cells that have clonally expanded in vivo can proliferate in vitro without producing virions. In certain CD4+ memory T cell subsets, the provirus may be in a deeper state of latency, allowing the cell to proliferate without producing viral proteins, thus permitting escape from immune clearance. To evaluate this possibility, we used a multiple stimulation viral outgrowth assay to culture resting naïve, central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells from 10 HIV-1-infected individuals on antiretroviral therapy. On average, only 1.7% of intact proviruses across all T cell subsets were induced to transcribe viral genes and release replication-competent virus after stimulation of the cells. We found no consistent enrichment of intact or inducible proviruses in any T cell subset. Furthermore, we observed notable plasticity among the canonical memory T cell subsets after activation in vitro and saw substantial person-to-person variability in the inducibility of infectious virus release. This finding complicates the vision for a targeted approach for HIV-1 cure based on T cell memory subsets.

The Landscape of Persistent Viral Genomes in ART-Treated SIV, SHIV, and HIV-2 Infections.

Evaluation of HIV cure strategies is complicated by defective proviruses that persist in ART-treated patients but are irrelevant to cure. Non-human primates (NHP) are essential for testing cure strategies. However, the persisting proviral landscape in ART-treated NHPs is uncharacterized. Here, we describe viral genomes persisting in ART-treated, simian immunodeficiency virus (SIV)-infected NHPs, simian-human immunodeficiency virus (SHIV)-infected NHPs, and humans infected with HIV-2, an SIV-related virus. The landscapes of persisting SIV, SHIV, and HIV-2 genomes are also dominated by defective sequences. However, there was a significantly higher fraction of intact SIV proviral genomes compared to ART-treated HIV-1 or HIV-2 infected humans. Compared to humans with HIV-1, SIV-infected NHPs had more hypermutated genomes, a relative paucity of clonal SIV sequences, and a lower frequency of deleted genomes. Finally, we report an assay for measuring intact SIV genomes which may have value in cure research.