CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

chimeric plasma-PBMC Envs, early anAb targets were mapped to specific variable regions in gp120. Conclusions: At early ARTi, viruses replicating in the plasma are anAb-escape variants while the proviral reservoir sampled 24 weeks later reveals envs archived from acute HIV-1 through ARTi including the targets of initial anAb responses. Together, phylogenetic and phenotypic testing of paired ARTi plasma and archived reservoir enables tracking of virus-host evolution that can be exploited in curative immunotherapy and vaccine strategies. Structural Insight into the High- and Low-Affinity Alleles of Human and Macaque FcγRIIA (CD32A) William D. Tolbert 1 , Marek K. Korzeniowski 1 , Paula Nhan 1 , Madeleine Lee 1 , Haleigh Conley 2 , Julianna Veilleux 1 , Suneetha Gottumukkala 1 , Margaret E. Ackerman 3 , Justin Pollara 2 , Marzena Pazgier 1 1 Uniformed Services University of the Health Sciences, Bethesda, MD, USA, 2 Duke University School of Medicine, Durham, NC, USA, 3 Dartmouth College, Hanover, NH, USA Background: Rhesus macaques (RM), Macaca mulatta , are one of the main nonhuman primate models for the testing of vaccines and antibody-based therapeutics. Macaques, like humans, have high and a low affinity alleles of FcγRIIA (CD32A) which is expressed on macrophages, dendritic cells and other antigen presenting cells. The polymorphism in FcγRIIA potentially influences the ‘vaccinal effect’ via FcγRIIA-Ab mediated phagocytosis (ADCP) and the functionality of the IgG2 subclass in humans. The high affinity allele has H131 in both species, while the low affinity allele has R131 in humans and P131 in macaques. We determined the structures of the receptor variants in complex with the IgG1 Fc for both species, as well as the complex of the RM high-affinity allele with RM IgG2 Fc. These studies have enabled us to elucidate the molecular mechanisms underlying the affinity differences and specificity of the FcγRIIA receptor for the IgG subclasses. Methods: Human receptors were expressed in HEK 293 cells and purified on IgG sepharose and macaque receptors expressed in CHO cells and purified using nickel magnetic beads. IgG1 Fcs were generated by papain digest of IgG1 (human) or as Fc constructs (both IgG1 and IgG2, macaque) expressed in HEK 293 cells. IgG subclass binding to FcγRIIA was evaluated by surface plasmon resonance. Complexes were made with an excess of Fc, which was removed by purification by gel filtration, and were then used to grow protein crystals for x-ray structure determination. Results: We determined the structures of the human hFcγRIIA-H131 and hFcγRIIA-R131 in complex with hIgG1Fc to 2.4 Å and 2.8 Å, the structures of the macaque mFcγRIIA-H131 and mFcγRIIA-P131 with mIgG1Fc to 3.5 Å and 2.7 Å, and the structure of the mFcγRIIA-H131 with mIgG2Fc to 3.2 Å. In both species H131 forms an H-bond to Fc monomer A and packs against G237 stabilizing the N-terminus similar to the analogous His in FcγRIIIA. P131 and R131 instead pack against G236; R131 also forms an H-bond to the carbonyl of G236. These differences can explain the low affinity allele’s reduced affinity for IgG2 in both species. Conclusions: We show how a single amino acid polymorphism changes the FcγRIIA-Fc complex by modulating how the N-terminus of monomer A interacts with FcγR to not only affect affinity but also specificity. Our findings suggest that these structural changes can be leveraged to engineer novel variants of human and RM antibodies to modulate ADCP and thereby influence therapeutic strategies and vaccine efficacy. Antigen Affinity Ceiling for Selection of Functional Mutations and HIV-1 Neutralization Ankita Singh, Kara Anasti, Amanda Newman, Advaiti Pai Kane, Maggie Barr, Robert Parks, Elizabeth Van Itallie, Sravani Venkatayogi, Kevin Saunders, Derek W. Cain, Rory Henderson, Kevin Wiehe, Barton Haynes, S. Munir Alam Duke Human Vaccine Institute, Durham, NC, USA Background: The mechanism by which B cells sense antigen affinity and initiate signaling that leads to full B activation and differentiation remains unclear. Understanding the relationship between antigen affinity/kinetic rates and selection of functional mutations is critical for HIV-1 Envelope (Env) protein immunogens designed to target broadly neutralizing antibody (bnAb) precursors. Previously, we had reported that B cell signaling strength is significantly associated with antigen binding kinetic on-rate and not affinity (K D ). Here, we performed immunization studies in a CD4 binding-site specific bnAb (CH235) precursor knock-in (KI) mouse model to test the hypothesis that a

prime immunogen with the optimal K D will favor germinal center (GC) selection of functional mutations and induction of autologous virus neutralization. Methods: Affinity (K D ) and kinetic rates of HIV Env (CH505M5 and CH505TF) trimers were measured by SPR. B cell activation was measured using calcium flux and phosphokinase signaling assays. Each group (6) of CH235UCA KI mice (expresses CH235 unmutated common ancestor BCRs) were immunized (4x) with each of 5 trimers of varying affinities (10.6nM-23.5 µ M). Sera antibody titer and the GC and memory B cells frequency were analyzed using ELISA and flow cytometric phenotyping, respectively. Neutralization titers by pseudovirus TZM-bl neutralization assay and NGS was done to identify frequencies of key functional mutations. Results: Increasing sera IgG binding titers against each of the trimer immunogen was observed. The weaker/moderate affinity (K D =4.7/0.56 µ M) groups showed higher frequencies of Env-specific IgG+ GC B cells compared to the high affinity group (K D =16.7nM). Both the weaker/moderate affinity immunogens induced significantly higher ID 50 -neutralization titers when compared to either the high (K D =10.6-16.7nM) or the weakest (K D =23.5 µ M) affinity immunogens (p=0.0022). A significantly higher frequency of the key improbable mutation (K19T) was observed in the weaker affinity groups (p=0.0087; p=0.0043). Importantly, several previously defined key VH mutations (K19T+N52D+S57R) were significantly increased in the weaker affinity group (K D =4.7 µ M) (p=0.0022). Thus, selection of key functional mutations was favored by weaker over higher affinity immunogens. Conclusions: A lower and an upper affinity threshold set boundaries for selection of key functional mutations and the induction of higher titers of autologous HIV-1 neutralizing antibodies. Maturation of B-Cell Lineages During cART in Chronic SHIV-Infected Juvenile Rhesus Macaques Wilton Williams 1 , Yue Chen 2 , Ryan Tuck 2 , Derek W. Cain 2 , Robert Edwards 2 , Taylor Keyes 2 , Mieke van der Mescht 1 , Katayoun Mansouri 2 , Taylor Spence 2 , Francesco E. Marino 3 , Seth Rohr 2 , Madison Berry 2 , Kevin Wiehe 2 , Katharine Bar 3 , Guido Ferrari 1 1 Duke University School of Medicine, Durham, NC, USA, 2 Duke Human Vaccine Institute, Durham, NC, USA, 3 University of Pennsylvania, Philadelphia, PA, USA Background: HIV-1 infection alters the frequencies and functions of B cell subsets, which may be restored by combined antiretroviral therapy (cART). There is still a gap in our knowledge that limits our understanding of the maturation of autologous HIV-1 neutralizing antibodies (NAbs) during cART. Our goal was to interrogate the dynamics of B cell lineages pre- and post- cART in a chronic Simian-HIV infection model of juvenile rhesus macaques (RMs). Methods: Nine RMs were infected at birth with a pathogenic SHIV and untreated for ~3-4 years. Six RMs were treated with daily cART for 13 months, while 3 RMs remained therapy-naïve because viremia <1000 RNA copies/ml. Purified IgG from plasma was tested for HIV-1 neutralization in TZM-bl cells, and structurally mapped on autologous HIV-1 envelope (Env) trimer via electron microscopy-based polyclonal epitope mapping (EMPEM). We quantified peripheral blood (PBMC)-derived antigen-reactive B cells via FACS using autologous Env trimer. We generated B cell receptor (BCR) and transcriptome sequences of negatively-enriched single cells from PBMCs (10X Genomics) of SHIV-infected (N=9; ~270K B cells) and SHIV-naïve (N=3; ~16K B cells) RMs. Results: Pre-cART plasma IgG in SHIV infection mapped to NAb epitopes on autologous Env, including V3-glycan and V2-apex broadly-NAbs (bNAbs), and post-cART IgG neutralized autologous SHIV with varied potencies indicative of NAb persistence during cART. In 6/9 SHIV-infected RMs, we detected HIV-1 Env-reactive memory B cells, even at 13 months post-cART in RMs on therapy. We identified computationally-inferred clonally-related BCRs in SHIV-infected RMs (+/- cART), including persisting IgG clones and strictly expanding IgG clones with increased numbers of cells post- versus pre- cART. Transcriptomics revealed 16 unique B cell clusters, including 3 clusters with memory B cell gene signatures that had predominantly cells from SHIV-infected RMs on cART and SHIV-naïve controls, compared to therapy-naïve SHIV-infected RMs, suggestive of cART-restored functional memory B cell subsets in infection. Ongoing transcriptomics will further delineate the nature and activation states of B cell clusters. Conclusions: In this study, B cell clonal expansion underlies their maturation under cART, suggestive of a similar process in PLWH that may be driven by residual viremia within germinal centers. Future studies will evaluate the

414

416

Poster Abstracts

415

98

CROI 2025