CROI 2025 Abstract eBook

Abstract eBook

Oral Abstracts

cases. Twenty of the 24 positives (83%) were detected at UVRI and near full length Mpox genomes were generated for 8 samples with a genome coverage of >99%. Phylogenetic analysis showed that Ugandan sequences were closely genetically related to clade Ib sequences from the DRC suggesting a close link to the Sudan-Kivu Mpox outbreaks (Figure 1). Differential testing using unbiased deep sequencing of suspected Mpox cases that tested PCR-negative (n=73) detected human herpesvirus 3 as the top viral hit present in the samples (genome coverage of 90-100%) (Figure 1). Conclusions: The Mpox outbreak in Uganda suggests an epidemiologic link to clade Ib MPXV associated with recent South Kivu Mpox outbreaks in the Democratic Republic of Congo (DRC). An increase in Mpox positivity is likely due to increasing community transmissions and findings from differential testing of Mpox-negative samples suggests co-circulation of Mpox and Chickenpox viruses, highlighting the need for routine surveillance of other emerging and re-emerging infections. The figure, table, or graphic for this abstract has been removed. Characterization of Mpox Cases in Uganda: Diversity of the Confirmed Cases Ritah Namusoosa, Susan N. Nabadda, Susan Nambozo Nambozo, Aidarayaan Namakula, Judith A. Kyokushaba, Viola Kasone, Godfrey Pimundu, Alisen Background: Mpox, a zoonotic disease caused by the Monkeypox virus (MPXV) within the Orthopoxvirus family, includes two major clades: Clade I, associated with severe disease and higher mortality, and Clade II. On August 14, 2024, the World Health Organization (WHO) declared Mpox a public health emergency of international concern following multi-country outbreaks since May 2022. Uganda declared a Mpox outbreak on August 2, 2024. With increasing case counts, sexually mediated clusters, and sustained human-to-human transmission, this study aimed to characterize the epidemiology, clinical presentations, and genetic diversity of Mpox cases confirmed in Uganda from July to October 2024. Methods: This cross-sectional study analyzed data from the national Results Dispatch System (RDS) for Mpox cases reported between July 22 and October 31, 2024. Extracted data included patient demographics, symptom onset dates, symptoms, sample collection dates, sample types, test results, epidemiological exposure links, and whole-genome sequencing results. Results: A total of 323 Mpox cases were confirmed from 1,991 samples tested (positivity rate: 16%) across 35 districts (24% of Uganda’s 146 districts), with Kampala reporting the highest case burden (41%). The median age was 24 years (IQR: 17), and 28% of cases involved children (0–17 years). Males accounted for 55% of cases. Symptom onset began on July 12, 2024, with a median delay of 4 days (IQR: 6) to sample collection. Common symptoms included rash (92%), fever (85%), and lymphadenopathy (78%). Atypical genital lesions were observed in 25% of cases within sexually mediated clusters. Close contact with infected individuals was reported in 60% of cases. Genomic analysis classified all positive samples as Clade 1b, with five distinct subclades, and no APOBEC3-like mutations were identified. No associations were found between clustering and variables such as district, gender, and collection batch. Conclusions: The Mpox outbreak in Uganda was primarily driven by behavioral and environmental factors, emphasizing the importance of targeted interventions to address sexual transmission dynamics and protect vulnerable populations. While viral evolution resulted in five subclades, transmission was not linked to specific demographic or geographic factors, indicating widespread dissemination. Replication-Competent HIV-Infected Cells Have Deoxyuracil-Containing Proviruses Rodrigo Matus Nicodemos 1 , David R. Ambrozak 1 , Samuel Darko 2 , Amy Henry 1 , Farida Laboune 1 , Jianfei Hu 1 , Joseph P. Casazza 1 , Fernanda Torres Ruiz 3 , Yara Andrea Luna Villalobos 3 , Mauricio González Navarro 3 , Perla Mariana Del Río Estrada 3 , Santiago Avila Rios 3 , Michael Betts 4 , Daniel C. Douek 1 , Richard A. Koup 1 1 Vaccine Research Center, Bethesda, MD, USA, 2 National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA, 3 Instituto Nacional de Enfermedades Infecciosas, Mexico City, Mexico, 4 University of Pennsylvania, Philadelphia, PA, USA Background: Persistence of the HIV reservoir during antiretroviral therapy is a major obstacle to curing the infection. This reservoir can be established in the hyperacute stage of the infection, and lymphoid tissue analysis of this stage revealed HIV infection of resting T cells. Given that the HIV reservoir resides in Ayitewala, Linda Nabitaaka Kisaakye Ministry of Health Uganda, Kampala, Uganda

resting CD4 T cells, we investigated whether direct infection of resting CD4 T cells could be involved in its establishment. Methods: We infected primary resting CD4 T cells with a CCR5-tropic replication competent GFP reporter virus. We co-measured GFP expression with activation and proliferation markers by flow cytometry over five days. We performed bulk and single-cell RNAseq of sorted GFP+ cells to measure host and virus mRNAs. We bioinformatically analyzed cellular dNTP pathways for HIV reverse transcription and read-through HIV transcripts for HIV integration. Lastly, we cross-validated our findings with peripheral and lymphoid tissue samples from people living with HIV (PLWH). Results: We detected resting GFP+ cells 3 to 5 days after infection. These GFP+ cells lacked CD69, CD25, HLA-DR, and CD154 expression. They also did not express the proliferation markers Ki-67 and phospho-H3.3. Transcriptome analysis of resting GFP+ cells revealed that infected cells expressed a cell-cycle independent pathway for dNTP synthesis. This pathway lacked expression of the genes required to convert deoxyuracil (dU) to thymidine (dT), which resulted in incorporation of dU into the provirus. We observed that dU-containing (dU+) proviruses were integrated into the host genome. We designed and ran a qPCR assay that differentiated between proviruses with dU or dT on samples from PLWH. dU+ proviruses and dT+ proviruses were detected in different CD4 T cell populations in all donors tested. Finally, after performing viral outgrowth assays, we found that cell subsets with dU+ proviruses had replication-competent virus. Conclusions: We conclude that HIV can directly infect primary resting CD4 T cells and establish the reservoir in these cells. A key feature of HIV infection of resting cells is dU incorporation during reverse transcription due to the absence of enzymes required for dT synthesis. Therefore, the HIV reservoir comprises integrated, replication-competent, deoxyuracil-containing proviruses. Gag-Mediated Control Over CARD8 Activation During HIV-1 Assembly Ivy K. Hughes, Andres Quinones-Molina, Hisashi Akiyama, Suryaram Gummuluru Boston University, Boston, MA, USA Background: The CARD8 inflammasome is a key driver of innate immune responses to HIV-1. CARD8, containing HIV-1 protease (PR) cleavage sites, responds to PR upon enforced NNRTI-induced dimerization of the viral Gag-Pol polyprotein and premature PR activation. In the absence of antiretrovirals, the apparent lack of CARD8 activation during HIV-1 assembly along with the observation that PR activation is precisely timed during virus release strongly suggests the existence of HIV-1 encoded mechanisms that prevent premature PR activation and CARD8-mediated cell death. In this study, we determined how structural domains of Gag coordinate the timing of PR activity to evade cleavage of CARD8 and facilitate innate immune escape. Methods: We established an overexpression system in HEK293T cells using transient transfection to assay CARD8 cleavage by wildtype HIV-1 (WT) or mutants with selective inactivation of MA (Δ6-125), CA oligomerization (VK181-2/P224A), NC (zinc-finger deletion, Δ15-49) or p6 (ΔPTAP) domains of Gag. We infected PMA-differentiated THP-1 cells (PMA/THP1) and primary monocyte-derived macrophages (MDM) with WT or Gag-mutant viruses and determined CARD8 inflammasome activation by measuring IL-1β secretion and LDH release. We also infected primary CD4+ T cells (CD4T) with WT or Gag mutant viruses in the presence or absence of lopinavir (PR inhibitor) to assess efficiency of PR-mediated cell killing. Results: We found that alteration of HIV-1 assembly site (MA/Δ6-125) or arrest of virus particle release (p6/ΔPTAP) but not RNA binding (NC/Δ15-49) dramatically induced CARD8 inflammasome by de novo assembling Gag in HEK293T, PMA/THP1, and MDM. In the context of MA-mutant infection, CARD8 activation was dependent on CA-CA interactions and MA myristoylation, consistent with a model where CARD8 is cleaved late in the viral life cycle if the assembly process is altered. In CD4T, NC deletion did cause enhanced PR-mediated cell death, but the effect remained less significant than infection with the MA or p6 mutants. Interestingly, replacement of the NC domain with a nonspecific RNA-binding leucine zipper domain (GagZip) was sufficient to abolish PR-mediated activation of CARD8 in CD4T. Conclusions: This work reveals important structural regulators of PR activation during the assembly of HIV-1 particles, and identifies the MA and p6 domains of Gag as potential targets to pharmaceutically enact killing of myeloid and lymphoid cells productively infected with HIV-1.

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CROI 2025