Viral DNA Research Articles

Role of the membrane-associated accessory protein (MAAP) in adeno-associated virus (AAV) infection.

Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.

Essential oil mediated synthesis and application of highly stable copper nanoparticles as coatings on textiles and surfaces for rapid and sustained disinfection of microorganisms

Rampant pathogenesis induced by communicable microbes has necessitated development of technologies for rapid and sustained disinfection of surfaces. Copper nanoparticles (CuNPs) have been widely reported for their antimicrobial properties. However, nanostructured copper is prone to oxidative dissolution in the oil phase limiting its sustained use on surfaces and coatings. The current study reports a systematic investigation of a simple synthesis protocol using fatty acid stabilizers (particularly essential oils) for synthesis of copper nanoparticles in the oil phase. Of the various formulations synthesized, rosemary oil stabilized copper nanoparticles (RMO CuNPs) were noted to have the best inactivation kinetics and were also most stable. Upon morphological characterization by TEM and EELS, these were found to be monodispersed (φ 5–8 nm) with copper coexisting in all three oxidation states on the surface of the nanoparticles. The nanoparticles were drop cast on woven fabric of around 500 threads per inch and exposed to gram positive bacteria (Staphylococcus aureus), gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa), enveloped RNA virus (phi6), non-enveloped RNA virus (MS2) and non-enveloped DNA virus (T4) to encompass the commonly encountered groups of pathogens. It was possible to completely disinfect 107 copies of all microorganisms within 40 min of exposure. Further, this formulation was incorporated with polyurethane as thinners and used to coat non-woven fabrics. These also exhibited antimicrobial properties. Sustained disinfection with less than 9% cumulative copper loss for upto 14 washes with soap water was observed while the antioxidant activity was also preserved. Based on the studies conducted, RMO CuNP in oil phase was found to have excellent potential of integration on surface coatings, paints and polymers for rapid and sustained disinfection of microbes on surfaces.

Diverse amdoparvoviruses infection of farmed Asian badgers (Meles meles).

Amdoparvoviruses infect various carnivores, including mustelids, canids, skunks, and felids. Aleutian mink disease virus (AMDV) belongs to the prototypical species Amdoparvovirus carnivoran1. Here, we identified a novel amdoparvovirus in farmed Asian badgers (Meles meles), and we named this virus "Meles meles amdoparvovirus" (MMADV). A total of 146 clinical samples were collected from 134 individual badgers, and 30.6% (41/134) of the sampled badgers tested positive for amdoparvovirus by PCR. Viral DNA was detected in feces, blood, spleen, liver, lung, and adipose tissue from these animals. Viral sequences from eight samples were determined, five of which represented nearly full-length genome sequences (4,237-4,265 nt). Six serum samples tested positive by PCR, CIEP, and IAT, four of which had high antibody titers (> 512) against AMDV-G. Twenty-six of the 41 amdoparvovirus-positive badgers showed signs of illness, and necropsy revealed lesions in their organs. Sequence comparisons and phylogenetic analysis of the viral NS1 and VP2 genes of these badger amdoparvoviruses showed that their NS1 proteins shared 62.6%-88.8% sequence identity with known amdoparvoviruses, and they clustered phylogenetically into two related clades. The VP2 proteins shared 76.6%-97.2% identity and clustered into two clades, one of which included raccoon dog and arctic fox amdoparvovirus (RFAV), and the other of which did not include other known amdoparvoviruses. According to the NS1-protein-based criterion for parvovirus species demarcation, the MMADV isolate from farm YS should be classified as a member of a new species of the genus Amdoparvovirus. In summary, we have discovered a novel MMADV and other badger amdoparvoviruses that naturally infect Asian badgers and are possibly pathogenic in badgers.

Singapore grouper iridovirus VP146 modulates the cGAS-STING signaling pathway to escape the interferon immune response

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.

Formulation of next-generation polyvalent vaccine candidates against three important poxviruses by targeting DNA-dependent RNA polymerase using an integrated immunoinformatics and molecular modeling approach

BackgroundPoxviruses comprise a group of large double-stranded DNA viruses and are known to cause diseases in humans, livestock animals, and other animal species. The Mpox virus (MPXV; formerly Monkeypox), variola virus (VARV), and volepox virus (VPXV) are among the prevalent poxviruses of the Orthopoxviridae genera. The ongoing Mpox infectious disease pandemic caused by the Mpox virus has had a major impact on public health across the globe. To date, only limited repurposed antivirals and vaccines are available for the effective treatment of Mpox and other poxviruses that cause contagious diseases. MethodsThe present study was conducted with the primary goal of formulating multi-epitope vaccines against three evolutionary closed poxviruses i.e., MPXV, VARV, and VPXV using an integrated immunoinformatics and molecular modeling approach. DNA-dependent RNA polymerase (DdRp), a potential vaccine target of poxviruses, has been used to determine immunodominant B and T-cell epitopes followed by interactions analysis with Toll-like receptor 2 at the atomic level. ResultsThree multi-epitope vaccine constructs, namely DdRp_MPXV (V1), DdRp_VARV (V2), and DdRp_VPXV (V3) were designed. These vaccine constructs were found to be antigenic, non-allergenic, non-toxic, and soluble with desired physicochemical properties. Protein-protein docking and interaction profiling analysis depicts a strong binding pattern between the targeted immune receptor TLR2 and the structural models of the designed vaccine constructs, and manifested a number of biochemical bonds (hydrogen bonds, salt bridges, and non-bonded contacts). State-of-the-art all-atoms molecular dynamics simulations revealed highly stable interactions of vaccine constructs with TLR2 at the atomic level throughout the simulations on 300 nanoseconds. Additionally, the outcome of the immune simulation analysis suggested that designed vaccines have the potential to induce protective immunity against targeted poxviruses. ConclusionsTaken together, formulated next-generation polyvalent vaccines were found to have good efficacy against closely related poxviruses (MPXV, VARV, and VPXV) as demonstrated by our extensive immunoinformatics and molecular modeling evaluations; however, further experimental investigations are still needed.

Embracing Complexity: What Novel Sequencing Methods Are Teaching Us About Herpesvirus Genomic Diversity

The arrival of novel sequencing technologies throughout the past two decades has led to a paradigm shift in our understanding of herpesvirus genomic diversity. Previously, herpesviruses were thought to exist as a family of DNA viruses with mostly identical genomes that evolved at much slower rates than RNA viruses. However, a growing body of evidence now suggests that herpesviruses exist as dynamic populations that possess standing variation and evolve at much faster rates than previously assumed. In this review, we explore how strategies such as deep sequencing, long-read sequencing, and haplotype reconstruction are allowing scientists to dissect the genomic composition of herpesvirus populations. We also discuss the challenges that need to be addressed before a detailed picture of herpesvirus diversity can emerge.

Lower HBV DNA level is associated with more severe liver fibrosis in HBeAg-positive chronic hepatitis B with normal alanine transaminase

BackgroundThe association of hepatitis B virus (HBV) DNA levels and liver fibrosis in chronic hepatitis B (CHB) patients with immune-tolerant phase remains unclear. We explored the association between liver fibrosis and HBV DNA levels in HBeAg-positive CHB patients with normal alanine transaminase (ALT) with relatively high HBV DNA.MethodsSix hundred and twenty-two HBeAg-positive CHB patients with normal ALT were included. Patients were divided into three categories: low (6 log10 IU/mL ≤ HBV DNA < 7 log10 IU/mL), moderate (7 log10 IU/mL ≤ HBV DNA < 8 log10 IU/mL), and high (HBV DNA ≥ 8 log10 IU/mL). APRI, FIB-4, transient elastography, or liver biopsy were used to assess liver fibrosis.ResultsThe median age of patients was 33.0 years and 57.9% patients were male. 18.8%, 52.1%, and 29.1% of patients had low, moderate, and high HBV DNA levels, respectively. The APRI (0.33 vs. 0.26 vs. 0.26, P < 0.001), FIB-4 (1.03 vs. 0.71 vs. 0.68, P < 0.001), and LSM values (7.6 kPa vs. 5.6 kPa vs. 5.5 kPa, P = 0.086) were higher in low HBV DNA group than other two groups. Low HBV DNA group had higher proportions of significant fibrosis (24.8% vs. 9.9% vs. 3.3%, P < 0.001) and cirrhosis (7.7% vs. 2.5% vs. 1.1%, P = 0.004) than moderate and high HBV DNA groups. Moderate (OR 3.095, P = 0.023) and low (OR 4.968, P = 0.003) HBV DNA were independent risk factors of significant fibrosis.ConclusionLower HBV DNA level was associated with more severe liver fibrosis in HBeAg-positive CHB patients with ALT.

The roles of nuclear orphan receptor NR2F6 in anti-viral innate immunity.

Proper transcription regulation by key transcription factors, such as IRF3, is critical for anti-viral defense. Dynamics of enhancer activity play important roles in many biological processes, and epigenomic analysis is used to determine the involved enhancers and transcription factors. To determine new transcription factors in anti-DNA-virus response, we have performed H3K27ac ChIP-Seq and identified three transcription factors, NR2F6, MEF2D and MAFF, in promoting HSV-1 replication. NR2F6 promotes HSV-1 replication and gene expression in vitro and in vivo, but not dependent on cGAS/STING pathway. NR2F6 binds to the promoter of MAP3K5 and activates AP-1/c-Jun pathway, which is critical for DNA virus replication. On the other hand, NR2F6 is transcriptionally repressed by c-Jun and forms a negative feedback loop. Meanwhile, cGAS/STING innate immunity signaling represses NR2F6 through STAT3. Taken together, we have identified new transcription factors and revealed the underlying mechanisms involved in the network between DNA viruses and host cells.

Epitranscriptomic cytidine methylation of the hepatitis B viral RNA is essential for viral reverse transcription and particle production

Epitranscriptomic RNA modifications have emerged as important regulators of the fate and function of viral RNAs. One prominent modification, the cytidine methylation 5-methylcytidine (m5C), is found on the RNA of HIV-1, where m5C enhances the translation of HIV-1 RNA. However, whether m5C functionally enhances the RNA of other pathogenic viruses remains elusive. Here, we surveyed a panel of commonly found RNA modifications on the RNA of hepatitis B virus (HBV) and found that HBV RNA is enriched with m5C as well as ten other modifications, at stoichiometries much higher than host messenger RNA (mRNA). Intriguingly, m5C is mostly found on the epsilon hairpin, an RNA element required for viral RNA encapsidation and reverse transcription, with these m5C mainly deposited by the cellular methyltransferase NSUN2. Loss of m5C from HBV RNA due to NSUN2 depletion resulted in a partial decrease in viral core protein (HBc) production, accompanied by a near-complete loss of the reverse transcribed viral DNA. Similarly, mutations introduced to remove the methylated cytidines resulted in a loss of HBc production and reverse transcription. Furthermore, pharmacological disruption of m5C deposition led to a significant decrease in HBV replication. Thus, our data indicate m5C methylations as a critical mediator of the epsilon elements' function in HBV virion production and reverse transcription, suggesting the therapeutic potential of targeting the m5C methyltransfer process on HBV epsilon as an antiviral strategy.

HHV6-Associated Hydrocephalus in a Pediatric Hematopoietic Stem Cell Transplant Recipient: An Unusual Presentation.

Human herpesvirus 6 (HHV-6) is a widely spread DNA virus that is ubiquitous and persistent with primary infection occurring in early childhood, with reactivation of the infection a common phenomenon in severely immunocompromised hosts, including hematopoietic stem cell transplant (HSCT) patients, influencing morbidity and mortality. A wide spectrum of clinical presentations is reported in the literature with HHV-6 reactivation including post-transplant limbic encephalitis (PALE). We report the unusual case of a 6-year-old female 107 days postallogenic HSCT due to transfusion dependent beta thalassemia major who developed acute cerebellitis with secondary supratentorial hydrocephalus that required invasive surgical intervention. In addition to accompanying imaging findings, the patient tested positive for HHV-6 by PCR from both serum and CSF samples and demonstrated dramatic improvement with the institution of steroid therapy in addition to ganciclovir treatment. The availability of rapid diagnostic measures in addition to a multidisciplinary approach is crucial to manage HHV-6 encephalitis and associated complications in HSCT patients.

Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.

Multi-cancer early detection (MCED) in circulating tumor DNA (ctDNA) using an ultrasensitive mutation-based amplicon next-generation sequencing (NGS) assay.

e15042 Background: Cancer remains a leading cause of mortality globally. For most cancers, routine screening options are still unavailable. Blood-based MCED has emerged as a promising strategy to bridge this gap. Mutation-based NGS in ctDNA detects highly specific cancer signals, an attribute critical for MCED assays. We developed an ultrasensitive mutation-based NGS assay optimized to detect cancer signals in 50 cancers and predict tissue-of-origin (TOO) for up to 21 cancer classes. Methods: An amplicon-based NGS assay incorporating mirror barcodes for error correction was designed to detect cancer-associated alterations (CAAs), including single nucleotide variants (SNVs), insertion-deletion mutations (indels), and viral DNA (EBV and 20 genotypes of HPV), in 50 cancers. Analytical validation was performed using reference standards and clinical validation was performed in a retrospective case-control cohort of 601 subjects comprising cell-free DNA (cfDNA) from 313 self-declared healthy donors and 288 cancer patients (30.9% early-stage, 50.3% late-stage and 9.4% hematolymphoid cancers). Tissue-of-origin (TOO) was predicted for 21 cancer classes using a weighted sum model. Where relevant, matched buffy coat genomic DNA (gDNA) was analyzed to rule out clonal hematopoiesis. Results: Analytical validation demonstrated a limit of detection (LoD) of 0.05% variant allele frequency (VAF) for indels and 0.07% VAF for SNVs in cfDNA. LoD was matched in gDNA (0.04% and 0.05% VAF for indels and SNVs respectively). In clinical validation, the assay demonstrated a specificity of 99.0% (310/313) and an overall sensitivity of 80.9% (233/288), with sensitivities of 64.0%, 91.7%, and 85.2% in early-stage, late-stage, and hematolymphoid cancers respectively. The mean number of CAAs detected in early-stage, late-stage, and hematolymphoid CAA-positive cancers was 2.1, 3.1, and 4.6. Inclusion of viral targets aided the detection of nasopharyngeal (NPC), cervical, and p16+ oropharyngeal (oPSCC) cancers. EBV DNA was detected in 91.7% of NPC cases. In cervical cancer and p16+ oPSCC, HPV DNA (genotypes 16, 18, 33 and 39) was detected in 71.4% and 90.9% of cases respectively. Conversely, none of the p16- oPSCC cancers had detectable HPV DNA. Across the 21 TOO classes, a high confidence prediction could be made in 62.4% (mean 73.6%, range 26.7%-100%) of cases with an overall accuracy of 87.7% (mean 84.9%, range 33.3%-100%). Overall TOO prediction performance was modeled in a simulated cohort based on cancer incidence in Singapore and Hong Kong, giving an accuracy of 87.3% and 88.7% respectively. Conclusions: The mutation-based NGS assay sensitively detects cancer signals and accurately predicts the tumor origin for early-stage, late-stage, and hematolymphoid cancers. Inclusion of viral targets additionally enables early detection of virus-associated cancers.

Longitudinal cell-free DNA characterization by low-coverage whole-genome sequencing in patients undergoing high-dose radiotherapy

Background and PurposeCurrent radiotherapy guidelines rely heavily on imaging-based monitoring. Liquid biopsy monitoring promises to complement imaging by providing frequent systemic information about the tumor. In particular, cell-free DNA (cfDNA) sequencing offers a tumor-agnostic approach, which lends itself to monitoring heterogeneous cohorts of cancer patients. MethodsWe collected plasma cfDNA from oligometastatic patients (OMD) and head-and-neck cancer patients (SCCHN) at six time points before, during, and after radiotherapy, and compared them to the plasma samples of healthy and polymetastatic volunteers. We performed low-pass (on average 7x) whole-genome sequencing on 93 plasma cfDNA samples and correlated copy number alterations and fragment length distributions to clinical and imaging findings. ResultsWe observed copy number alterations in 4/7 polymetastatic cancer patients, 1/7 OMD and 1/7 SCCHN patients, these patients’ imaging showed progression following radiotherapy. Using unsupervised learning, we identified cancer-specific fragment length features that showed a strong correlation with copy number-based tumor fraction estimates. In 4/4 HPV-positive SCCHN patient samples, we detected viral DNA that enabled the monitoring of very low tumor fraction samples. ConclusionsOur results indicate that an elevated tumor fraction is associated with tumor aggressiveness and systemic tumor spread. This information may be used to adapt treatment strategies. Further, we show that by detecting specific sequences such as viral DNA, the sensitivity of detecting cancer from cell-free DNA sequencing data can be greatly increased.

Metaproteomic analysis from cervical biopsies and cytologies identifies proteinaceous biomarkers representing both human and microbial species

The detection of HPV infection and microbial colonization in cervical lesions is currently done through PCR-based viral or bacterial DNA amplification. Our objective was to develop a methodology to expand the metaproteomic landscape of cervical disease and determine if protein biomarkers from both human and microbes could be detected in distinct cervical samples. This would lead to the development of multi-species proteomics, which includes protein-based lateral flow diagnostics that can define patterns of microbes and/or human proteins relevant to disease status. In this study, we collected both non-frozen tissue biopsy and exfoliative non-fixed cytology samples to assess the consistency of detecting human proteomic signatures between the cytology and biopsy samples. Our results show that proteomics using biopsies or cytologies can detect both human and microbial organisms. Across patients, Lumican and Galectin-1 were most highly expressed human proteins in the tissue biopsy, whilst IL-36 and IL-1RA were most highly expressed human proteins in the cytology. We also used mass spectrometry to assess microbial proteomes known to reside based on prior 16S rRNA gene signatures. Lactobacillus spp. was the most highly expressed proteome in patient samples and specific abundant Lactobacillus proteins were identified. These methodological approaches can be used in future metaproteomic clinical studies to interrogate the vaginal human and microbiome structure and metabolic diversity in cytologies or biopsies from the same patients who have pre-invasive cervical intraepithelial neoplasia, invasive cervical cancer, as well as in healthy controls to assess how human and pathogenic proteins may correlate with disease presence and severity.

Detection of viral RNA and DNA and immune response following administration of live attenuated measles and varicella vaccines in children with chronic liver disease

Infections preventable by live virus vaccines are surging in the setting of decreased herd immunity. Many children with chronic liver diseases (CLDs) are unimmunized and at increased risk for infection due to guidelines recommending against live vaccines within 4 weeks pretransplant. This prospective study of 21 children with CLD and 13 healthy controls defined the timing of measles virus and varicella-zoster virus (VZV) RNA- and DNA-emia following vaccination and compared immune responses to measles and varicella vaccines in both groups. Measles virus RNA and VZV DNA real-time PCR were measured weekly following vaccination; measles virus RNA was undetectable in all by 14 days postvaccination, but VZV DNA, which can be managed with antivirals, was detected in 1 child in the CLD group at 21 days and 1 control at 28 days postvaccination. Humoral or cell-mediated vaccine response was 100% to measles virus and 94% to VZV in the CLD group postvaccination, whereas it was 100% to both vaccines in controls. Our pilot study suggests that both live vaccines can be safely and effectively administered up to 14 days prior to transplantation in children with CLD. We anticipate this will improve vaccination rates and thus decrease rates of vaccine-preventable infections in vulnerable children with CLD.

Can TTMV clearance predict recurrence in HPV HNSCC?

6066 Background: Patients with locally advanced (LA) HPV HNSCC are treated with various types of induction chemotherapy (IC) followed by standard (sd) or reduced (rd) chemoradiation (CRT). Tumor tissue-modified viral HPV DNA (TTMV) pairs with PET-CT in predicting an early detection of treatment failure in HPV-related HNSCC. Mid-treatment TTMV assessment in HPV-related HNSCC can help identify patients who are eligible to receive a lower dose of IC and/or CRT or those who may require close follow-up. Methods: Patients with LA HPV+ HNSCC with high-risk features (radiographic ECE, T4 primary, &gt;N2c) and &lt;20 pack-year (py) smoking history were treated with IC followed by rdCRT (56 Gy); sdCRT (70 Gy) was used in non-responders. 16/23 received IC with TPF, 1/23 TP, and 6/23 TP-PD1, and all patients received carboplatin weekly with their CRT. Regeneron trial patients who received TP-Cemiplimab (PD1) IC followed by sdCRT are also continuing adjuvant Cemiplimab alone for 6 months (NCT05376553). PET-CT-confirmed residual, recurrent, or metastatic diseases were defined as treatment failures; TTMV + patients only if PET-CT confirmed. Results: 23 subjects treated with IC followed by CRT between 2/4/21-1/26/24, who had pre- and post-treatment TTMV were included in our analysis. The median pre-IC TTMV was 8082 (27 – 90770). The median follow-up time from treatment start was 21 months. 9/23 patients had full clearance TTMV after 1 cycle of IC defined as rapid responders (RR). 11/23 patients still had positive TTMV after 1st cycle of IC were defined as slow responders (SR). 1 patient had no TTMV done after 1 cycle of IC but was elevated after 2nd IC; he was defined as SR. 2 patients had a positive pre-IC TTMV, but no TTMV was drawn during IC to see if RR. 1 of 2 patients was TTMV negative prior to CRT, and 2/2 had a 1st post IC negative TTMV done 1 week after CRT started. They were both included in the analysis but defined as unknown response status. TTMV testing was done for all 23 subjects after CRT; all were negative. All subjects have TTMV testing done during their surveillance visits. Among 9/23 RR, none developed a failure. Among 12/23 SR, 3 patients developed a failure: 1/3 was TTMV+ but PET-, being followed with TTMV and PET-CT; and 2/3 were confirmed failures, TTMV+ and PET+; 1 locoregional, 1 metastatic. 2 patients were not known to be a SR or RR and had no recurrence. Among 23 patients, NPV of TTMV to detect failure was 100%, but PPV was 96% (1/23). Conclusions: IC followed in rapid clearance of TTMV in 39% of patients, and all remained disease-free. 3/11 (27%) with persistently elevated TTMV after cycle 1 relapsed as of this time point. This small study was not large enough to be practice-changing. However, this is significantly hypothesis-generating and should be tested in a larger trial to help identify patients for de-escalation (RR) or closer surveillance (SR).

Association of Baseline Hepatitis B Virus DNA and On-Treatment Risk of Cirrhosis and Hepatocellular Carcinoma.

Recent studies suggest an inverse relationship between baseline levels of hepatitis B virus (HBV) DNA and on-treatment risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). However, data are limited to Asian cohorts, and it is unclear if similar associations hold true for non-Asians with CHB. We aimed to evaluate association of baseline HBV DNA with long-term risks of cirrhosis and HCC among a predominantly non-Asian cohort of CHB patients in the USA. Using longitudinal data from the national Veterans Affairs database, we evaluated the risk of cirrhosis or HCC among adults with non-cirrhotic CHB who are on continuous antiviral therapy, stratified by moderate levels of baseline HBV DNA (4.00 - 6.99 log10 IU/mL) vs. high levels of baseline HBV DNA (7.00 log10 IU/mL or higher). Propensity score weighting was applied, and competing risks cumulative incidence functions and Cox proportional hazards models were utilized. Among 1,129 non-cirrhotic CHB patients (41% non-Hispanic White, 36% African American, mean age 57.0 years, 62.2% hepatitis B e antigen (HBeAg) positive), 585 had moderate levels of baseline HBV DNA and 544 had high HBV DNA. After propensity score weighting, no significant difference in risk of cirrhosis was observed between moderate vs. high baseline HBV DNA (4.55 vs. 5.22 per 100 person-years, hazard ratio (HR): 0.87, 95% confidence interval (CI): 0.69 - 1.09, P = 0.22), but risk of HCC was significantly higher in patients with moderate vs. high baseline HBV DNA (0.84 vs. 0.69 per 100 person-years, HR: 1.33, 95% CI: 1.09 - 1.62, P < 0.01). Among a national cohort of predominantly non-Asian US veterans with non-cirrhotic CHB on antiviral therapy, moderate levels of baseline HBV DNA was associated with higher risk of HCC than high HBV DNA.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties

HIV-1 integration into the human genome is dependent on 3’-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3’-processed DNA remained resistant. Here, we describe the mechanism by which the 3’-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3’-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3’-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼ 2-fold) for the 3’-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3’-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3’-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3’-processed HIV-1 DNA.