CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusions: X4 variants can emerge as minor variants early during the course of HIV-1 infection. X4 viruses co-exist with CCR5-using viruses, suggesting that the evolution of X4 variants is not driven by the lack of CCR5+ CD4+ T cells. We propose that the emergence of X4 viruses serves as a biomarker for a state of virus-specific immunodeficiency, allowing the virus to proliferate in inactivated CXCR4+ CD4+ T cells, leading to a more rapid loss of CD4. Droplet Single Genome Amplification Allows for Characterization of Autologous HIV Envelope Sequences Joseph McWhirter 1 , Thomas Murphy 1 , Xianbao He 1 , Yan Mei Liang 2 , Frida Avila 1 , Stepan Levin 2 , Manish Sagar 1 1 Boston University, Boston, MA, USA, 2 Boston Medical Center, Boston, MA, USA Background: Understanding and identifying HIV-1 neutralization escape requires isolating envelopes that have undergone antibody selection pressure. However, recombinant artifacts are generated during bulk reverse transcription (RT) and subsequent PCR, which can affect neutralization phenotype. To avoid this, amplification from a single genome is essential. We addressed this by developing a novel droplet-based one step RT-PCR method which can amplify full length envelopes with less recombination and significantly higher throughput than other single genome amplification methods. Methods: Droplet-based genome amplification (DBGA) partitions starting templates into approximately 20,000 droplets, in which around 30% or less of the droplets contain a single genome. We validated our approach by generating a sequence pool containing either 6 different full-length HIV-1 RNAs or 8 HIV-1 DNA. Envelopes in these sequence pools were amplified using one-step DBGA, bulk RT followed by DBGA (bulk-RT-DBGA), or bulk RT followed by bulk PCR. Envelope sequences were obtained using single molecule PacBio technology. Recombination was quantified using Los Alamos Recombination Identification Program (RIP) and a genetic error quantification (GEQ) method. Differences were compared using Chi-square tests. Results: Recombinant frequency was significantly lower in the RNA sequence pools amplified with our one-step DBGA (2.6% RIP) as compared to bulk RT followed by bulk PCR (25.5% RIP, p < 0.0001). Recombinant frequency was similar between one-step DBGA and bulk-RT-DBGA (2.6% and 1.5% respectively, RIP). Recombination rates in DBGA scaled with increased genomic input; as expected, because the frequency of droplets containing two or more templates increases with higher inputs amounts. Conclusions: DGBA reduces recombinant artifacts similarly to established RT-SGA protocols while being high throughput and less laborious compared to SGA. Our approach provides a platform to characterize HIV-1 envelope sequences which have undergone antibody selection pressure with significantly more depth than previous methods. High Resistance of HIV-1CH040 Envelope Glycoproteins to bNAbs Without Loss of Efficient Viral Entry Durgadevi Parthasarathy 1 , Alon Herschhorn 1 , Stephanie Pickthorn 1 , Shamim Ahmed 1 , Rajni Kant Shukla 2 , Jeffy Jeffy 1 , Amit Sharma 2 , Dmitrii Mazurov 3 1 University of Minnesota, Minneapolis, MN, USA, 2 The Ohio State University, Columbus, OH, USA, 3 Yale University, New Haven, CT, USA Background: A transmitter/founder (T/F) HIV-1 strain can establish a productive infection in healthy individuals. HIV-1 envelope glycoproteins (Envs) mediate viral entry and have to maintain a delicate balance between evasion from neutralizing antibodies and entry compatibility. Escape from broadly neutralizing antibodies (bnAbs), which target highly conserved regions in HIV Envs, pose significant challenges to HIV-1 Envs. Methods: We evaluated the impact of the T/F HIV-1 CH040 resistance to multiple bnAbs on efficient viral entry by comparing the infectivity of recombinant luciferase-expressing viruses that display CH040 Envs with the infectivity of recombinant viruses displaying Envs from different HIV-1 strains. We also introduced rationally designed engineered changes to disrupt bnAbs epitopes using Stratagene site-directed mutagenesis and tested the effects on susceptibility. We studied the efficiency of HIV-1 entry, mediated by engineered variants of CH040 Envs, by cell-cell fusion, western blotting analysis, cell-cell transmission, and cold sensitivity. We measured sensitivity to multiple bnAbs and known Env susceptibilities by neutralizing assays. In addition, viral replication kinetics and viral-cell fusion activity were tested in the context of recombinant simian-immunodeficiency viruses (SHIVs) in macaque lymphocytes.

Results: We identified high resistance of a transmitted/founding Envs CH040 to most bnAbs without a fitness cost associated with loss of efficient entry. HIV 1 CH040 , which mediates viral entry as efficiently as other primary Envs, represents a resistant viral strain of high public health concern. Unexpectedly, expression of CH040 Envs is relatively low, but HIV-1 CH040 replicates well in human T cells and HIV-1 CH040 replicates well in vitro in pig-tailed macaque lymphocytes. These results highlight the potential ability of some T/F strains to evolve high bnAb resistance and replication fitness in parallel. Conclusions: Our findings highlight several aspects of HIV strains of public health concern and provide additional considerations for the development of HIV-1 prevention and therapeutic strategies. Sex Differences in Replication Kinetics of Primary HIV-1 Isolates in Monocyte-Derived Macrophages Jake Robinson, Gregory D. Whitehill, Hannah Schrader, Ryan Krause, Frederic Bibollet-Ruche, Katharine Bar University of Pennsylvania, Philadelphia, PA, USA Background: Recent studies characterizing primary isolates of HIV-1 from acute timepoints, rebound after treatment interruption, chronic viremia, and the persistent reservoir have identified key phenotypic differences between these viruses when replicating in primary CD4 T cells. Here, we leverage these HIV-1 isolates to probe effects of virus clade, isolate origin, and cell donor sex on replication kinetics in peripheral monocyte derived macrophages (MDMs). Methods: MDMs from 4 male and 5 female healthy donors were infected at an MOI of 0.1 with primary HIV-1 isolates: Clade B/C transmitted/founder (TF) viruses (n=4), Clade B/C rebound viruses (n=4), Clade B/C chronic viruses (n=5), and Clade D TF viruses (n=4). We assessed longitudinal area under the curve of HIVp24 (total HIVp24) for the 17 primary isolates in MDMs over 20 days of infection, segregating total HIVp24 into quartiles to compare replication kinetics. Results: Across MDM from all donors, we found minimal differences in replication between Clade B/C TF viruses, Clade B/C rebound, and Clade B/C chronic viruses, except for the well-characterized brain-derived chronic virus YU2, which replicated in the highest quartile. All four Clade D TF viruses showed robust replication in MDMs, falling in the upper two quartiles. When infections in MDMs were segregated by cell donor sex, primary viruses replicated significantly better in male MDMs than female MDMs across all viruses (Two-way ANOVA, Interaction P=0.0013). In post-test comparison, the average percent difference in replication kinetics comparing male to female MDMs was 39.5% for 7 of 17 primary isolates (FDR, Q<0.05). Conclusions: In MDM, the sex of cell donor was a significant determinant of HIV-1 replication, while virus origin (TF, rebound, or chronic) was not. As previously shown, brain-derived and Clade D viruses replicated efficiently in MDMs. Studies to better understand the mechanism of biological sex as a determinate of HIV-1 replication in myeloid cells are needed. Ongoing studies will build upon these results by characterizing growth kinetics of primary HIV-1 isolates in in vitro models of resident myeloid cells, such as tissue macrophages and microglia. Our results suggest biological sex should be considered in future translational studies of HIV persistence and cure strategies. ScRNA-seq of HRSV/SARS-CoV-2 Co-Infection Reveals Unique Cellular Responses and lncRNA Signatures Claudia Vanetti 1 , Luca Piacentini 2 , Lorenzo Citterio 3 , Micaela Garziano 1 , Irma Saulle 1 , Jacopo Aiello 1 , Martina Broggiato 1 , Claudio Fenizia 1 , Diego Bottan 3 , Daria Trabattoni 1 , Mario Clerici 1 , Mara Biasin 1 1 University of Milan, Milan, Italy, 2 IRCCS Centro Cardiologico Monzino, Milano, Italy, 3 University of Pavia, Pavia, Italy Background: Respiratory co-infections caused by HRSV and SARS-CoV-2 often result in severe respiratory disease, but the underlying cellular/molecular mechanisms are poorly understood. Using an in vitro model of HRSV/SARS-CoV 2 co-infection, we investigated how individual cells respond to simultaneous or single infections, providing insights into synergistic/antagonistic viral interactions. Methods: Single-cell RNA sequencing (scRNA-seq) data of the A549-ACE2 expressing cell line, co-infected with HRSV and SARS-CoV-2 (MOI: 0.01) were analyzed 72 hours post infection. The expression of SARS-CoV-2 and HSRV specific genes was examined simultaneously with cellular genes to determine the presence or co-presence of the viruses in each profiled cell. Gene expression

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CROI 2025