Broadly neutralizing HIV antibodies (bnAbs) are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel ...phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM) and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Challenge studies following passive immunization with neutralizing antibodies suggest that an HIV vaccine could be efficacious were it able to elicit broadly neutralizing antibodies (bNAbs
4
). To ...better understand the requirements for activation of B cells producing bNAbs, we generated cell lines expressing bNAbs or their germline-reverted versions (gl-bNAbs) as BCRs. We then tested the abilities of the bNAb-expressing cells to recognize HIV pseudovirions and vaccine candidate proteins by binding and activation assays. The results suggest that HIV Env antigen-expressing, infection-competent virions are poorly recognized by high affinity bNAb-expressing cells, as measured by the inability of antigens to induce rapid increases in intracellular calcium levels. Other antigen forms appear to be highly stimulatory: in particular, soluble gp140 trimers and a multimerized, scaffolded epitope protein. Virions failed to efficiently activate bNAb-expressing B cells owing to delayed or inefficient BCR recognition, most likely caused by the low density of Env spikes. Importantly, B cells carrying gl-bNAb BCRs were not stimulated by any of the tested vaccine candidates. These data provide insight into why many HIV immunogens, and natural HIV infections, fail to rapidly stimulate bNAb responses and suggest that bNAb-expressing cell lines might be useful tools in evaluation of vaccine antigens for infectious diseases. As soluble Env trimers or multimerized scaffolded epitopes are best at activating B cell expressing bNAbs, these antigenic forms should be considered as preferred vaccine components, though they should be modified to better target naïve gl-bNAb B cells.
Abstract
Circulating tumor cells (CTCs) have been implicated as a potential population of cells that may seed metastasis in cancer patients. In order to characterize and understand this population of ...cells, an enrichment-free immunofluorescence detection method was developed by our lab. This method take whole patient blood and classifies cells based on cytokeratin (CTCs) and CD45 (WBCs) antibody staining. Using this CTC detection method, a population of cells residing in tumor cell aggregates was observed in blood draws from Stage IV breast cancer patients. It was previously observed in ex vivo mouse experiments that homotypic aggregate formation of tumor cells may increase metastatic efficiency (Glinsky, et al., Cancer Research, 2003). In this work, we seek to determine if a significant proportion of CTCs reside in homotypic aggregates in patient blood by enumerating CTC aggregates from the blood draws of 23 Stage IV breast cancer patients and 16 healthy donor patients. Preliminary analysis measuring associations of CTCs with other nucleated cells found in patient blood draws revealed that ∼43% of cytokeratin-positive (CK+) cells found in breast cancer patient samples localize with either other CK+ or white blood cells (WBCs) versus 14% in healthy donor patient samples. Furthermore, ∼19% of WBCs were found to be associated with either CK+ or other WBCs in breast cancer patient samples versus ∼7% in healthy donor patient samples. Pathological review confirming CK+ cells as CTCs revealed that of the CTCs detected in patient blood after pathological review, ∼2% of CTCs were found as homotypic aggregates versus ∼0.07% in healthy donor samples (p=0.0005). Four breast cancer patients were found to have no CTC aggregates. Two breast cancer patients had significantly higher than average CTC aggregate percentages (∼6% and ∼7%). Patients will continue to be monitored to measure any differences in disease outcome of patients with high incidence of CTC aggregates versus patients with low incidence of CTC aggregates and healthy donors.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5225. doi:10.1158/1538-7445.AM2011-5225
Antibody Engineering and Therapeutics Almagro, Juan Carlos; Gilliland, Gary L; Breden, Felix ...
mAbs,
05/2014, Letnik:
6, Številka:
3
Journal Article
Recenzirano
Odprti dostop
The 24
th
Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC ...Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.
A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env ...to unmutated precursors of bnAbs, including those of the VRC01 class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short CDRL3 (light-chain complementarity-determining region 3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets.
A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a ...soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.
The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life ...Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.
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