Production of rAAV by plasmid transfection induces antiviral and inflammatory responses in suspension HEK293 cells.

Recombinant adeno-associated virus (rAAV) is a clinically proven viral vector for delivery of therapeutic genes to treat rare diseases. Improving rAAV manufacturing productivity and vector quality is necessary to meet clinical and commercial demand. These goals will require an improved understanding of the cellular response to rAAV production, which is poorly defined. We interrogated the kinetic transcriptional response of HEK293 cells to rAAV production following transient plasmid transfection, under manufacturing-relevant conditions, using RNA-seq. Time-series analyses identified a robust cellular response to transfection and rAAV production, with 1,850 transcripts differentially expressed. Gene Ontology analysis determined upregulated pathways, including inflammatory and antiviral responses, with several interferon-stimulated cytokines and chemokines being upregulated at the protein level. Literature-based pathway prediction implicated multiple pathogen pattern sensors and signal transducers in up-regulation of inflammatory and antiviral responses in response to transfection and rAAV replication. Systematic analysis of the cellular transcriptional response to rAAV production indicates that host cells actively sense vector manufacture as an infectious insult. This dataset may therefore illuminate genes and pathways that influence rAAV production, thereby enabling the rational design of next-generation manufacturing platforms to support safe, effective, and affordable AAV-based gene therapies.

Vertical T cell immunodominance and epitope entropy determine HIV-1 escape.

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell-mediated in vivo control of HIV-1. Primary HIV-1-specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or "vertical" immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.

Impact of immune escape mutations on HIV-1 fitness in the context of the cognate transmitted/founder genome.

BACKGROUND: A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method. RESULTS: The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region. CONCLUSIONS: These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.

Early low-titer neutralizing antibodies impede HIV-1 replication and select for virus escape.

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC(50)) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1-V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one.

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism.

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

Quantitative analyses of RAG-RSS interactions and conformations revealed by atomic force microscopy.

During V(D)J recombination, site specific DNA excision is dictated by the binding of RAG1/2 proteins to the conserved recombination signal sequence (RSS) within the genome. The interaction between RAG1/2 and RSS is thought to involve a large DNA distortion that is permissive for DNA cleavage. In this study, using atomic force microscopy imaging (AFM), we analyzed individual RAG-RSS complexes, in which the bending angle of RAG-associated RSS substrates could be visualized and quantified. We provided the quantitative measurement on the conformations of specific RAG-12RSS complexes. Previous data indicating the necessity of RAG2 for recombination implies a structural role in the RAG-RSS complex. Surprisingly, however, no significant difference was observed in conformational bending with AFM between RAG1-12RSS and RAG1/2-12RSS. RAG1 was found sufficient to induce DNA bending, and the addition of RAG2 did not change the bending profile. In addition, a prenicked 12RSS bound by RAG1/2 proteins displayed a conformation similar to the one observed with the intact 12RSS, implying that no greater DNA bending occurs after the nicking step in the signal complex. Taken together, the quantitative AFM results on the components of the recombinase emphasize a tightly held complex with a bend angle value near 60 degrees , which may be a prerequisite step for the site-specific nicking by the V(D)J recombinase.

Supercoiling-induced DNA bending.

Local DNA bending is a critical factor for numerous DNA functions including recognition of DNA by sequence-specific regulatory binding proteins. Negative DNA supercoiling increases both local and global DNA dynamics, and this dynamic flexibility can facilitate the formation of DNA-protein complexes. We have recently shown that apexes of supercoiled DNA molecules are sites that can promote the formation of an alternative DNA structure, a cruciform, suggesting that these positions in supercoiled DNA are under additional stress and perhaps have a distorted DNA geometry. To test this hypothesis, we used atomic force microscopy to directly measure the curvature of apical positions in supercoiled DNA. The measurements were performed for an inherently curved sequence formed by phased A tracts and a region of mixed sequence DNA. For this, we used plasmids in which an inverted repeat and A tract were placed at precise locations relative to each other. Under specific conditions, the inverted repeat formed a cruciform that was used as a marker for the unambiguous identification of the A tract location. When the A tract and cruciform were placed diametrically opposite, this yielded predominantly nonbranched plectonemic molecules with an extruded cruciform and A tract localized in the terminal loops. For both the curved A tract and mixed sequence nonbent DNA, their localization to an apex increased the angle of bending compared to that expected for DNA unconstrained in solution. This is consistent with increased helical distortion at an apical bend.