CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

307 IMPACT OF ATI ON B-CELL REGULATION BY IGG3 IN CHRONICALLY HIV- INFECTED INDIVIDUALS Valerie Melson 1 , Lela Kardava 1 , Christine Youn 1 , Jesse Justement 1 , Victoria Shi 1 , Catherine Seamon 2 , Michael Sneller 1 , Tae-Wook Chun 1 , Susan Moir 1 1 NIAID, Bethesda, MD, USA, 2 NIH, Bethesda, MD, USA Background: Numerous immunologic abnormalities have been described in HIV infection, especially in the absence of antiretroviral therapy (ART). B-cell abnormalities include loss of classical memory B cells and gain of activated, exhausted and differentiated B cells. We recently reported a role for IgG3 in regulating B-cell activation in certain individuals with chronic HIV viremia. Here, we investigated the dynamics of IgG3 regulation in a longitudinal cohort of HIV- infected individuals undergoing analytical treatment interruption (ATI). Methods: Longitudinal immunologic and virologic analyses were conducted on specimens obtained from seven individuals prior to receiving ART, after several years of ART, during ATI, and following re-initiation of ART. The dynamics of HIV plasma viremia, changes in B-cell populations and IgG3 binding in the peripheral blood were evaluated longitudinally. The immunologic assays were performed by multiparameter flow cytometry. Results: In the absence of ART (pre-ART), four of seven individuals had IgG3 bound to their IgM+ B cells. After a median of ten years on ART (pre-ATI), none of the individuals had IgG3+IgM+ B cells. Following ATI, varying degrees of IgG3 binding to B cells was observed at peak viremia in the same four individuals who had the profile pre-ART. The IgG3+IgM+ B-cell profile was again extinguished following re-initiation of ART. During ATI, for all seven HIV- infected individuals studied, total B-cell percentages decreased significantly and contained a higher percentage of plasmablasts compared to ART-naïve, pre- and post-ATI time-points. During ATI, the percentage of activated memory B cells also increased significantly compared to the pre-ATI period, while the percentage of tissue-like memory B cells did not change, remaining significantly lower compared to the pre-ART period. The percentage of classical memory B cells decreased significantly during ATI compared to pre- and post-ATI periods, but remained higher compared to the pre-ART period. Conclusion: We provide evidence that ATI elicits significant changes in B cells of HIV-infected individuals as a result of rebounding plasma viremia. The presence of circulating IgG3+IgM+ B cells is a property consistently observed in certain HIV-infected individuals during chronic plasma viremia and closely associated with active viral replication. The profile is present pre-ART, extinguishing itself during effective ART and gradually returns when individuals undergo ATI and plasma viremia rebounds. 308 PTEN OVEREXPRESSION IN ANTIGEN-SPECIFIC B CELLS FROM HIV- INFECTED INDIVIDUALS ON ART Lesley R. de Armas 1 , Suresh Pallikkuth 1 , Li Pan 1 , Stefano Rinaldi 1 , Nicola Cotugno 2 , Sarah F. Andrews 3 , Rajendra Pahwa 1 , Adrian B. McDermott 3 , Paolo Palma 2 , Savita Pahwa 1 1 University of Miami, Miami, FL, USA, 2 Bambino Gesu Children’s Hospital, Rome, Italy, 3 Vaccine Research Center, NIAID, Bethesda, MD, USA Background: Memory B cells (MBC) respond to secondary antigen challenge to protect against infection and to boost immunity following vaccinations. Despite effective treatment, chronic HIV infection disturbs MBCs by reducing numbers and altering functionality due to hyper-activation and increased apoptosis leading to suboptimal antibody responses against common infectious agents such as Influenza. We and others have shown that influenza-specific responses in B cells are impaired in HIV-infected individuals in both young and old (>60 years) individuals. However, these studies have largely been performed using bulk cell analysis from in vitro antigen-stimulated culture experiments and technological advances in single cell analysis now allow for deeper interrogation of cellular states in cell populations with diverse functions, such as MBC. Methods: We used single cell gene expression analysis to evaluate post- vaccination antigen-specific memory B cells isolated from peripheral blood of virally-suppressed HIV-infected individuals and healthy controls stratified by serum H1N1 antibody response 3 weeks post-administration of the seasonal trivalent inactivated influenza vaccine. We used a fluorescent probe to isolate influenza H1N1-specific B cells and a multiplexed and targeted RT-PCR approach (Fluidigm BioMark) to measure expression levels of 96 genes involved in B cell activation and function. H1N1-specific B cells were also analyzed for memory phenotype and Ig isotype by flow cytometry to integrate with gene profiles. Results: Single cell gene profiling revealed a 4-gene predictive signature containing IL10RA, APOBEC3G, TLR7 and the phosphoinositide-3 kinase

available for 5 participants (Spearman r=0.9, P=0.04). In 9 participants with entry PBMCs, VRC01 IC50s did not significantly correlate with time to rebound (Spearman r=-0.35, P=0.37), but IC50<0.5 µg/mL was associated with delayed time to rebound (>8 weeks) (P=0.0278). Conclusion: We found a wide range in baseline neutralization susceptibilities to clinically relevant bnAbs with highly correlated values across plasma and PBMC-derived samples over 3 years of ART. In A5340, PhenoSense nAb susceptibilities on entry PBMCs were similar to published pre-ART values and IC50<0.5 µg/mL was associated with delayed rebound after ATI. Results support the utility of screening for neutralization susceptibility prior to therapeutic bnAb use and suggest PhenoSense nAb PBMC testing may be a valid approach in suppressed individuals.

306 HIGH-THROUGHPUT ASSAY TO ASSESS ADCC ACTIVITY AGAINST CLINICAL ISOLATES OF HIV-1 Allison S. Thomas 1 , Melissa Ghulam-Smith 1 , Manish Sagar 2 1 Boston University, Boston, MA, USA, 2 Boston Medical Center, Boston, MA, USA Background: High throughput infection based antibody dependent cellular cytotoxicity (ADCC) assays are needed to gain insights into the role of ADCC in preventing transmission. Current infection based assays often use a natural killer (NK) cell resistant CD4 T cell that expresses the CCR5 receptor (CEM- NKr-CCR5) as target cells, but transmitted viruses, such as those circulating in infected individuals, often cannot replicate in these cells. Furthermore, primary cells demonstrate highly variable susceptibility to primary HIV-1 strains. Methods: Two different CD4 T cell lines, PM1 and MT4, were transduced with a CCR5 and a tat-inducible luciferase expression plasmid. Target cells were exposed to primary and lab-adapted HIV-1 strains and cultured with the NK cell line, KHYG-1, in the presence and absence of antibody. Percent ADCC was calculated as the loss of luciferase expression in the presence as compared to the absence of antibodies. Results: Incubation with NK cells, without HIV-1 antibodies, decreased luciferase in infected PM1-CCR5-Luc (43.87%, range=31.5-56.7) and MT4-CCR5- Luc (1.09%, range=-1.9-4.1), suggesting PM1 but not MT4 cells were highly susceptible to background NK cell killing. Thus, PM1-CCR5-Luc cells were not examined further and MT4-CCR5-Luc cells were deemed NK cell resistant. NL4-3 infected CEM-NKr-CCR5-Luc (68.2%, range=55.4-83.8) and MT4-CCR5-Luc (70.6%, range=60.8-78.0) yielded similar ADCC estimates in the presence of 500ug/ml HIV-1 IgG (p=0.79). While NL4-3 replicated in both CEM-NKr-CCR5- Luc and MT4-CCR5-Luc cells, fold luciferase expression over background was only elevated in the MT4-CCR5-Luc cells after infection with primary CCR5-using variants, such as CH058 (4.54 fold), CH077 (2 fold), ZM247Fv2 (29.53 fold), and BJOX2000 (42.53 fold). In MT4-CCR5-Luc cells, similar ADCC estimates were obtained in the presence of heat inactivated plasma compared to isolated IgG (p=0.31). Breast milk isolated IgG, heat inactivated infant and maternal plasmas, but not breast milk supernatant, demonstrated ADCC activity against maternal strains and reference envelope panel viruses. Conclusion: Our MT4-CCR5-Luc cell line can be used to estimate ADCC activity present in plasma, breast milk and among immunoglobulins against both primary and T/F strains. The high throughput reliable assay will be used to compare mother-infant pairs where transmission did and did not occur in order to determine if ADCC is a correlate of protection in MTCT.

Poster Abstracts

CROI 2019 112