Results of a Multi-Disciplinary and Multi-Institutional Pilot Creating High-Yield Physics Educational Content (Hi-Phy).

Purpose: The quality of medical physics education is heterogenous across training programs, despite its importance in radiation oncology (RO) residency training. We present the results of a pilot series of free high-yield physics educational videos covering 4 topics chosen from the American Society for Radiation Oncology core curriculum. Methods and materials: Scripting and storyboarding of videos were iterative processes performed by 2 ROs and 6 medical physicists, with animations created by a university broadcasting specialist. Current RO residents and those who had graduated after 2018 were recruited through social media and e-mail with an aim of 60 participants. Two validated surveys were adapted for use and were completed after each video as well as a final overall assessment. Content was released sequentially after completion of the survey instruments for each prior video. All videos were created and released within 1 year of project initiation with a duration of 9 to 11 minutes. Results: There were 169 enrollees for the pilot from across the world, 211% of the targeted cohort size. Of these, 154 met eligibility criteria and received the first video. One hundred eight enrollees initiated the series and 85 completed the pilot, resulting in a 78% completion rate. Participants reported improved understanding and confidence applying the knowledge learned in the videos (median score 4 out of 5). All participants reported that the use of graphic animation improved understanding across all videos. Ninety-three percent agreed with a need for additional resources geared specifically toward RO residents and 100% would recommend these videos to other residents. Use metrics revealed the average watch time was 7 minutes (range, 6:17-7:15). Conclusions: The high-yield educational physics video pilot series was successful in developing videos that were effective in teaching RO physics concepts.

Persistence of Human Immunodeficiency Virus-1 Drug Resistance Mutations in Proviral Deoxyribonucleic Acid After Virologic Failure of Efavirenz-Containing Antiretroviral Regimens.

BACKGROUND: Efavirenz (EFV)-based regimens select broad drug resistance to nonnucleoside reverse-transcriptase inhibitors (NNRTIs), limiting the effectiveness of EFV and other NNRTIs. The duration, persistence, and decay of drug resistance mutations (DRMs) in the proviral reservoir is not well defined. METHODS: Participants with virologic failure of EFV-based regimens and drug-resistant viremia with the K103N mutation in plasma ribonucleic acid (RNA) were identified from AIDS Clinical Trials Group (ACTG) studies A364 and A5095. These individuals received a second-line, boosted protease inhibitor-based regimen with suppression of viremia for up to10 years during long-term follow-up (median = 3.6 years; interquartile range, 2.1-6.9 years). Proviral deoxyribonucleic acid (DNA) from cryopreserved peripheral blood mononuclear cells was sequenced to identify the persistence of DRM. RESULTS: Twenty-eight participants from ACTG 364 and ACTG 5095 were evaluated. Sanger sequencing of proviral DNA detected K103N as well as additional reverse-transcriptase inhibitor (RTI) mutations. Ultradeep sequencing confirmed persistence of K103N in 71% of participants with minimal decay over time. In an adjusted model including years since suppression, persistent proviral K103N was 2.6 times more likely (95% confidence interval, 1.0-6.4) per log10 higher human immunodeficiency virus RNA at EFV failure. CONCLUSIONS: Persistence of RTI mutations in proviral DNA after virologic failure has implications for the effectiveness of future drug regimens and the recycling of RTI drugs.

CD8+ Cytotoxic T Lymphocyte Responses and Viral Epitope Escape in Acute HIV-1 Infection.

Epitope escape from HIV-1-targeted CD8+ cytotoxic T lymphocyte (CTL) responses occurs rapidly after acute infection and contributes to the eventual failure of effective immune control of HIV-1 infection. Because the early CTL response is key in determining HIV-1 disease outcome, studying the process of epitope escape is crucial for understanding what leads to failure of immune control in acute HIV-1 infection and will provide important implications for HIV-1 vaccine design. HIV-1-specific CD8+ T lymphocyte responses against viral epitopes were mapped in six acutely infected individuals, and the magnitudes of these responses were measured longitudinally during acute infection. The evolution of autologous circulating viral epitopes was determined in four of these subjects. In-depth testing of CD8+ T lymphocyte responses against index and all autologous-detected variant epitopes was performed in one subject. Among the four individuals examined, 10 of a total of 35 CD8+ T cell responses within Gag, Pol, and Nef showed evidence of epitope escape. CTL responses with viral epitope variant evolution were shown in one subject, and this evolution occurred with and without measurable CTL responses against epitope variants. These results demonstrate a dynamic period of viral epitope evolution in early HIV-1 infection due to CD8+ CTL response pressure.

Short communication: HIV-1 gag genetic variation in a single acutely infected participant defined by high-resolution deep sequencing.

Acute HIV-1 infection is characterized by the rapid generation of highly diverse genetic variants to adapt to the new host environment. Understanding the dynamics of viral genetic variation at this stage of infection is critical for vaccine design efforts and early drug treatment. Here, using a high-resolution deep sequencing approach targeting the HIV-1 gag region, we reveal very early immune pressure with dramatic subpopulation shifts in a single acutely infected participant providing further insight into the genetic dynamics of acute HIV-1 infection.

HIV-1 quasispecies delineation by tag linkage deep sequencing.

Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼ 2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼ 0.005% to ∼ 0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.

Ineffectual targeting of HIV-1 Nef by cytotoxic T lymphocytes in acute infection results in no functional impairment or viremia reduction.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8(+) T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection. All but one individual had a single founder strain. Nef-specific CTL responses were detected in all subjects and declined in magnitude over time. These responses were associated with mutations, but none of the mutations were detected in important functional motifs. Nef-mediated downregulation of CD4 and major histocompatibility complex (MHC) class I molecules was better preserved in acute infection than in chronic infection. Finally, Nef-specific CTL responses were not associated with a reduction in viremia from its acute-phase peak. Our results indicate that CTLs targeting Nef epitopes outside critical functional domains have little effect on the pathogenic functions of Nef, rendering these responses ineffective in acute infection. Importance: These data indicate that using the whole Nef protein as a vaccine immunogen likely allows immunodominance that leads to targeting of CTL responses that are rapidly escaped with little effect on Nef-mediated pathogenic functions. Pursuing vaccination approaches that can more precisely direct responses to vulnerable areas would maximize efficacy. Until vaccine-induced targeting can be optimized, other approaches, such as the use of Nef function inhibitors or the pursuit of immunotherapies such as T cell receptor gene therapy or adoptive transfer, may be more likely to result in successful control of viremia.

CD40 ligand (CD154) incorporated into HIV virions induces activation-induced cytidine deaminase (AID) expression in human B lymphocytes.

Most AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) arises from errors in immunoglobulin heavy-chain gene (IgH) class switch recombination (CSR) or somatic hypermutation (SHM), events that occur in germinal center (GC) B cells and require the activity of activation induced cytidine deaminase (AID). Several oncogenic viruses (EBV, HCV, HPV) can induce AID gene (AID) expression, and elevated AID expression is seen in circulating lymphocytes prior to AIDS-NHL diagnosis. Here, we report that HIV produced in peripheral blood mononuclear cells (PBMC) induced AID expression in normal human B cells. Since HIV produced in PBMC contains host cell CD40 ligand (CD40L) incorporated into the viral membrane, and CD40L is known to induce AID expression in human B cells, the role of virion-associated CD40L in HIV-induced AID expression was examined. Only viruses expressing functional CD40L were seen to induce AID expression; CD40L-negative HIV did not induce AID expression. The induction of AID expression by CD40L+ HIV was abrogated by addition of blocking anti-CD40L antibody. AID protein was detected in B cells exposed to CD40L+ HIV using intracellular multicolor flow cytometry, with most AID producing B cells expressing the CD71 activation marker on their surface. Therefore, HIV virions that express CD40L induce AID expression in B cells, and this induction appears to be due to a direct interaction between CD40L on these viruses and CD40 on B cells. These findings are consistent with a role for HIV in the direct stimulation of B cells, potentially leading to the accumulation of molecular lesions that have the potential to contribute to the development of NHL.

Mobilization-competent Lentiviral Vector-mediated Sustained Transcriptional Modulation of HIV-1 Expression.

Current anti-HIV-1 strategies reduce replication through targeting of viral proteins and RNA; meanwhile, targeting at the level of the integrated provirus has been less explored. We show here that mobilization-competent vectors containing small noncoding RNAs targeted to transcriptionally active regions of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) can take advantage of integrated virus and modulate HIV-1 replication. Transcriptional silencing of HIV-1 correlates with an increase in silent-state epigenetic marks including histone and DNA methylation, a loss of nuclear factor-kappaB (NF-kappaB) recruitment, and requires Argonaute 1 (Ago-1), histone deacetylase 1 (HDAC-1), and DNA methyltransferase 3a (DNMT3a) localization to the LTR. Long-term suppression of the virus was observed for 1 month with no evidence of viral resistance. These data show that RNA-directed transcriptional silencing of HIV-1 can be delivered by a mobilization-competent vector, suggesting that this system could be used to target long-term selective pressures on conserved promoter elements to evolve less pathogenic variants of HIV-1.