AIDS Research and Human RetrovirusesVol. 30, No. S1 B Cell Repertoires for ProtectionRole of Intestinal Microbiota in Shaping the B Cell Repertoire in HIV Infection and Env VaccinationLarry (Huaxin) Liao, A.M. Trama, W.B. Williams, M.A. Moody, Nathan Vandergrift, G.D. Tomaras, D.J. Marshall, T. Gurley, J. Whitesides, J. Eudailey, A. Foulger, R. Parks, C. Stolarchuk, K.E. Lloyd, K. Soderberg, J.R. Mascola, R. Koup, L. Corey, G.B. Nabel, P. Gilber, C. Morgan, J. Maenza, M. Keefer, S. Hammer, G. Churchyard, D.C. Montefior, B.S Graham, L.R. Baden, T.B. Kepler, and B.F. HaynesLarry (Huaxin) LiaoDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, A.M. TramaDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, W.B. WilliamsDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, M.A. MoodyDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, Nathan VandergriftDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, G.D. TomarasDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, D.J. MarshallDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, T. GurleyDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, J. WhitesidesDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, J. EudaileyDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, A. FoulgerDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, R. ParksDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, C. StolarchukDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, K.E. LloydDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, K. SoderbergDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, J.R. MascolaNational Institute of Allergy and Infectious Diseases, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, R. KoupNational Institute of Allergy and Infectious Diseases, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, L. CoreyUniversity of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA, United StatesSearch for more papers by this author, G.B. NabelNational Institute of Allergy and Infectious Diseases, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, P. GilberUniversity of Washington, SCHARP, Fred Hutchinson Cancer Research Center, Seattle, WA, United StatesSearch for more papers by this author, C. MorganUniversity of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA, United StatesSearch for more papers by this author, J. MaenzaUniversity of Washington, Fred Hutchinson Cancer Research Center, Seattle, WA, United StatesSearch for more papers by this author, M. KeeferUniversity of Rochester Medical Center, Division of Infectious Disease, Rochester, NY, United StatesSearch for more papers by this author, S. HammerColumbia University Medical Center, New York, NY, United StatesSearch for more papers by this author, G. ChurchyardThe Aurum Institute, Johannesburg, South AfricaSearch for more papers by this author, D.C. MontefiorDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this author, B.S GrahamNational Institute of Allergy and Infectious Diseases, Vaccine Research Center, Bethesda, MD, United StatesSearch for more papers by this author, L.R. BadenBrigham and Women's Hospital, Boston, MA, United StatesSearch for more papers by this author, T.B. KeplerBoston University, Boston, MA, United StatesSearch for more papers by this author, and B.F. HaynesDuke University Medical Center, Human Vaccine Institute, Durham, NC, United StatesSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5023a.abstractAboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"Role of Intestinal Microbiota in Shaping the B Cell Repertoire in HIV Infection and Env Vaccination." AIDS Research and Human Retroviruses, 30(S1), p. A19FiguresReferencesRelatedDetails Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Larry (Huaxin) Liao, A.M. Trama, W.B. Williams, M.A. Moody, Nathan Vandergrift, G.D. Tomaras, D.J. Marshall, T. Gurley, J. Whitesides, J. Eudailey, A. Foulger, R. Parks, C. Stolarchuk, K.E. Lloyd, K. Soderberg, J.R. Mascola, R. Koup, L. Corey, G.B. Nabel, P. Gilber, C. Morgan, J. Maenza, M. Keefer, S. Hammer, G. Churchyard, D.C. Montefior, B.S Graham, L.R. Baden, T.B. Kepler, and B.F. Haynes.Role of Intestinal Microbiota in Shaping the B Cell Repertoire in HIV Infection and Env Vaccination.AIDS Research and Human Retroviruses.Oct 2014.A19-A19.http://doi.org/10.1089/aid.2014.5023a.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

A successful HIV-1 vaccine must elicit immune responses that impede mucosal virus transmission, though functional roles of protective HIV-1 Envelope (Env)-specific mucosal antibodies remain unclear. Colostrum is a rich source of readily accessible mucosal B cells that may help define the mucosal antibody response contributing to prevention of postnatal HIV-1 transmission. To examine the HIV-1 Env-specific colostrum B-cell repertoire, single B cells were isolated from 17 chronically HIV-infected, lactating women, producing 51 blood and 39 colostrum HIV-1 Env-specific B-cell antibodies. All HIV-1 Env-specific colostrum-derived antibodies were immunoglobulin (Ig)G1 isotype and had mean heavy chain complementarity-determining region 3 (CDR3) lengths and mutation frequencies similar to those isolated from blood. However, variable heavy chain (VH) gene subfamily 1∼69 usage was higher among colostrum than blood HIV-1 Env-reactive antibodies (49% vs. 20%, P=0.006, Fisher's exact test). Additionally, more HIV-1 Env-specific colostrum antibodies were gp120 specific than those isolated from blood (44% vs. 16%, P=0.005, Fisher's exact test). One cross-compartment HIV-1 Env-specific clonal B-cell lineage was identified. These unique characteristics of colostrum B-cell antibodies suggest selective homing of HIV-1-specific IgG1-secreting memory B cells to the mammary gland and have implications for targeting mucosal B-cell populations by vaccination.

doi:10.1182/blood-2011-08-371203Prepublished online December 12, 2011;2012 119: e35-e44€€€€Nicholas ChiorazziHua-Xin Liao, Rosa Catera, Charles C. Chu, Xiao-Jie Yan, Micah A. Luftig, Barton F. Haynes and Kwan-Ki Hwang, Xi Chen, Daniel M. Kozink, Marietta Gustilo, Dawn J. Marshall, John F. Whitesides,€

Background HIV-1 vaccines must induce protective antibodies at mucosal surfaces; the role of IgA in protection remains unknown. The HIV-1 Env antibody response begins ~day 17 after transmission, and derives from a polyreactive memory B cell pool of gut flora-reactive IgG1 and IgA B cells. Whereas the IgG Env antibody response persists years after acute HIV-1 infection, the initial IgA response decreases over the first month. There is also selective destruction of terminal ileum germinal centers in early HIV-1 infection (EHI). To determine HIV-1 IgA responses in gut, we isolated Env-reactive antibodies from ileum from patients in EHI and chronic HIV-1 infection (CHI).

Background Antibody Dependent Cellular Cytotoxicity (ADCC) may be a contributing factor of immune responses controlling HIV-1 replication. Understanding the epitopes recognized by ADCC-mediating antibodies is likely to be important for the development of an effective AIDS vaccine. We characterized the epitope specificity and breadth of the ADCC-mediating antibody response elicited by the RV144 vaccine regimen.

The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The poly-clonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.

Open Access Oral presentation S021-04 OA. A large-scale analysis of immunoglobulin sequences derived from plasmablasts/plasma cells in acute HIV-1 infection subjects S Munshaw*1, H Liao1, A Dixon1, X Chen1, A Nagel1, K Derosa1, R Parks1, J Amos1, JF Whitesides1, DJ Marshalls1, Y Yang1, F Gao1, GD Tomaras1, MA Moody1, GH Kelsoe1, TC Shea2, DM Margolis2, M Markowitz2, P Goepfert3, G Shaw3, BF Haynes1 and TB Kepler1

Methods Blood PCs from three AHI subjects obtained approximately 17, 20 and 30 days after HIV-1 transmission were sorted into 96-well plates for amplification of VH and VL genes by RT/PCR. The isolated VH and VL genes were expressed as recombinant IgG1 mAbs in 293T cells by transfection using linear Ig expression cassettes, The specificity of produced mAbs was determined by ELISA and luminex bead immunoassays against a panel of HIV-1 and non-HIV-1 antigens.

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5 + B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts (‘low-count’ MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V H family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Aldehyde dehydrogenases are cytosolic enzymes that convert aldehydes into carboxylic acids. Human aldehyde dehydrogenase 1 (ALDH1) is highly expressed in the liver and in hematopoietic stem cells (HSCs). Although ALDH1 is a selectable marker of HSCs, its HSC-specific function is unknown. We hypothesized that ALDH might play a critical role in HSC fate determinations since it is required for the production of retinoic acids, which are broadly implicated in tissue differentiation, tissue patterning and embryonic development in vertebrates. In this study, highly purified human CD34+CD38−lin− HSCs were cultivated with early acting cytokines, thrombopoietin, stem cell factor and Flt-3 ligand (TSF) in the presence or absence of the competitive ALDH inhibitor, diethylaminobenzaldehyde (DEAB). Treatment of human BM and CB HSCs with TSF x 7 days caused a loss of CD34+CD38− cells in culture, morphologic differentiation, amplification of committed colony forming cells (CFCs) and a loss of primitive cells capable of repopulating non-obese diabetic/severe combined immunedeficient mice (SCID-Repopulating Cells, SRCs). Conversely, 7 day culture of BM and CB HSCs with TSF plus 100 μM DEAB blocked the morphologic differentiation and lineage commitment of HSCs in culture, expanded the CD34+CD38− population, and amplified SRCs 2-fold compared to input, indicating a fundamental role for ALDH in HSC differentiation. The effects of DEAB could be reversed by the co-administration of the retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), suggesting that the ability of ALDH to produce retinoic acids is important in determining HSC fate. Via screening studies of direct ligands of RAR and RXR, we also identified a selective RXR modulator which functioned similarly to DEAB by impeding HSC differentiation and causing the 4-fold expansion of SRCs. Interestingly, treatment with either DEAB or the RXR modulator reversed the down-modulation of HOXB4 transcription that was otherwise observed in CD34+CD38−lin- cells during culture with cytokines, suggesting that inhibition of ALDH or RXR modulation may promote HSC self-renewal via discrete interactions with other regulatory pathways. Modulation of ALDH activity and retinoid signaling is a novel and effective strategy to amplify human HSCs.

Chronic lymphocytic leukemia (CLL) results in the accumulation of mature immunologically defective lymphocytes in GO phase. Lymphocytes from CLL patients were exposed to UVC radiation to determine whether these cells are capable of undergoing apoptosis, as a response to DNA damage. Lymphocytes from CLL patients were found to be readily killed by ultraviolet light-C (UVC) radiation. Cells from healthy donors were minimally affected by doses of UVC ten times higher then those which caused dramatic drops in the metabolism of CLL cells. At four hours after irradiation, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) had dropped by 50% for CLL cells exposed to a dose of 10 J/m2. In contrast, there was no significant drop for healthy cells exposed to 100 J/m2. Cell death was measured by trypan blue staining, flow cytometry of Annexin V-PI stained cells, and Wright staining. By 24 hours after irradiation, significant amounts of cell death were observed in CLL cells at doses which had no significant effects on viability of healthy lymphocytes. The extreme sensitivity of CLL lymphocytes to UVC indicates that phototherapy should be explored as a potential treatment for this neoplasm.