Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires

Sequential Immunization Strategies to Elicit HIV-1 bNAbs in Animal Models With a Polyclonal B Cell Repertoire

Background: Immunization regimens that can elicit broadly neutralizing antibodies (bNAbs) in humans would be an effective vaccine against HIV-1. Our previous work showed that an immunization strategy involving a sequence of Env-based germline targeting immunogens that were gradually engineered to resemble the native Env protein, successfully elicited bNAb-like antibodies in a knock-in mouse carrying the inferred germline PGT121/10-1074 antibody. Despite this achievement, immunization protocols that elicit bNAbs in systems with a polyclonal B cell repertoire have not been reported to date. The low frequencies of germline bNAb precursors in polyclonal systems hinder their activation by immunization which therefore requires high affinity immunogens. In addition, competition between different epitope-specific B cells in polyclonal germinal centers may frustrate bNAb development. Methods: Based on our previous results in knock-in mice, we have aimed to optimize sequential immunization strategies to elicit bNAbs in animal models with polyclonal B cell repertoires. Results: The results of immunization experiments in several animal models will be presented

Sequential Immunization Strategies to Elicit HIV-1 bNAbs in Animal Models With a Polyclonal B Cell Repertoire

Background: Immunization regimens that can elicit broadly neutralizing antibodies (bNAbs) in humans would be an effective vaccine against HIV-1. Our previous work showed that an immunization strategy involving a sequence of Env-based germline targeting immunogens that were gradually engineered to resemble the native Env protein, successfully elicited bNAb-like antibodies in a knock-in mouse carrying the inferred germline PGT121/10-1074 antibody. Despite this achievement, immunization protocols that elicit bNAbs in systems with a polyclonal B cell repertoire have not been reported to date. The low frequencies of germline bNAb precursors in polyclonal systems hinder their activation by immunization which therefore requires high affinity immunogens. In addition, competition between different epitope-specific B cells in polyclonal germinal centers may frustrate bNAb development. Methods: Based on our previous results in knock-in mice, we have aimed to optimize sequential immunization strategies to elicit bNAbs in animal models with polyclonal B cell repertoires. Results: The results of immunization experiments in several animal models will be presented

Continually recruited naïve T cells contribute to the follicular helper and regulatory T cell pools in germinal centers

Abstract Follicular helper T cells (TFH) mediate B cell selection and clonal expansion in germinal centers (GCs), and follicular regulatory T cells (TFR) prevent the emergence of self-reactive B cells and help to extinguish the reaction. Here we show that GC reactions continually recruit T cells from both the naïve conventional and naive thymic regulatory T cell (Treg) repertoires. In the early GC, newly recruited T cells develop into TFH, whereas cells entering during the contraction phase develop into TFR cells that contribute to GC dissolution. The TFR fate decision is associated with decreased antigen availability and is modulated by slow antigen delivery or mRNA vaccination. Thus, invasion of ongoing GCs by newly developing TFH and TFR helps remodel the GC based on antigen availability

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

Background: Patients with heterozygous germline mutations in PTEN (phosphatase and tensin homologue deleted on chromosome 10) develop autoimmunity and lymphoid hyperplasia. Objectives: Since regulation of the phosphoinositol-3-kinase (PI3K) pathway is critical for maintaining regulatory T cell (Treg) functions we investigate Tregs in patients with heterozygous germline PTEN mutations (PTEN harmatoma tumour syndrome, PHTS). Methods: PHTS patients were assessed for immunological conditions, lymphocyte subsets, FOXP3+ Treg levels and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway we assessed Treg induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunological synapse. Results: Autoimmunity and peripheral lymphoid hyperplasia was found in 43% of 79 PHTS patients. Immune dysregulation in PHTS patients included lymphopenia, CD4+ T cell reduction and changes in T and B cell subsets. Although total CD4+FOXP3+ Treg numbers are reduced, frequencies are maintained in blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PHLPP downstream of PTEN is highly expressed in normal human Tregs and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Tregs in vitro and Treg mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunological synapse. Conclusion: Heterozygous loss of function of PTEN in humans has an significant impact on T and B cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T cell receptor activation, which is important for limiting PI3K hyperactivation in Tregs despite PTEN haploinsufficiency. </p

Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.

Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires

Fluorescence Development of Latent Fingerprint with Conjugated Polymer Nanoparticles in Aqueous Colloidal Solution

Poly­(<i>p</i>-phenylenevinylene) (PPV) nanoparticles in aqueous colloidal solution have been prepared via a modified Wessling method, with the addition of surfactant. The fluorescent colloidal solution was used as the developing solution to develop the fingerprints on different substrates. The developing process was accomplished simply by immersing the substrates into developing solution and then taking out, followed by rinsing with deionized water. The initial study about the fingerprints on the adhesive tapes showed that the developing solution is very effective in fluorescence development on both fresh and aged visible fingerprints; and such an effect was negligibly affected by treating the fingerprints with water or other organic solvents, whether before developing or after. Further study on latent fingerprints (LFPs) demonstrated that PPV nanoparticles in colloidal solution have high sensitivity in developing fingerprints to give very clearly fluorescent patterns. At least 6 months of storage of the colloidal solution did not reduce the developing effect; and each developing solution (3.6 mg/mL, 5.0 mL) can be used to develop at least 30 fingerprints without sacrificing the legibility of the pattern. The preliminary mechanism investigation suggested that selectivity achieved toward the ridge of the fingerprint is very likely due to the affinity between PPV molecules and oily secretions of the fingerprints. Digital magnification of the developed fingerprints provided more details about the fingerprint