Publications by authors named "Wen-Han Yu"

30 Publications

  • Page 1 of 1

HIV Antibody Fc N-Linked Glycosylation Is Associated with Viral Rebound.

Cell Rep 2020 Dec;33(11):108502

Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA. Electronic address:

Changes in antibody glycosylation are linked to inflammation across several diseases. Alterations in bulk antibody galactosylation can predict rheumatic flares, act as a sensor for immune activation, predict gastric cancer relapse, track with biological age, shift with vaccination, change with HIV reservoir size on therapy, and decrease in HIV and HCV infections. However, whether changes in antibody Fc biology also track with reservoir rebound time remains unclear. The identification of a biomarker that could forecast viral rebound time could significantly accelerate the downselection and iterative improvement of promising HIV viral eradication strategies. Using a comprehensive antibody Fc-profiling approach, the level of HIV-specific antibody Fc N-galactosylation is significantly associated with time to rebound after treatment discontinuation across three independent cohorts. Thus virus-specific antibody glycosylation may represent a promising, simply measured marker to track reservoir reactivation.
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http://dx.doi.org/10.1016/j.celrep.2020.108502DOI Listing
December 2020

Vi-specific serological correlates of protection for typhoid fever.

J Exp Med 2021 02;218(2)

Oxford Vaccine Group, Department of Pediatrics, University of Oxford, Oxford, UK.

Typhoid Vi vaccines have been shown to be efficacious in children living in endemic regions; however, a widely accepted correlate of protection remains to be established. We applied a systems serology approach to identify Vi-specific serological correlates of protection using samples obtained from participants enrolled in an experimental controlled human infection study. Participants were vaccinated with Vi-tetanus toxoid conjugate (Vi-TT) or unconjugated Vi-polysaccharide (Vi-PS) vaccines and were subsequently challenged with Salmonella Typhi bacteria. Multivariate analyses identified distinct protective signatures for Vi-TT and Vi-PS vaccines in addition to shared features that predicted protection across both groups. Vi IgA quantity and avidity correlated with protection from S. Typhi infection, whereas higher fold increases in Vi IgG responses were associated with reduced disease severity. Targeted antibody-mediated functional responses, particularly neutrophil phagocytosis, were also identified as important components of the protective signature. These humoral markers could be used to evaluate and develop efficacious Vi-conjugate vaccines and assist with accelerating vaccine availability to typhoid-endemic regions.
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http://dx.doi.org/10.1084/jem.20201116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668386PMC
February 2021

Passive Transfer of Vaccine-Elicited Antibodies Protects against SIV in Rhesus Macaques.

Cell 2020 Oct;183(1):185-196.e14

Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. Electronic address:

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.
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http://dx.doi.org/10.1016/j.cell.2020.08.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534693PMC
October 2020

Improved permeability and antifouling properties of polyvinyl chloride ultrafiltration membrane via blending sulfonated polysulfone.

J Colloid Interface Sci 2020 Nov 27;579:562-572. Epub 2020 Jun 27.

School of Chemistry and Chemical Engineering, Xinxiang University, Xinxiang 453003, Henan, China; Department of Polymer Science and Engineering, ERC of Membrane and Water Treatment (MOE), Zhejiang University, Hangzhou 310027, Zhejiang, China.

To improve the permeability and antifouling properties of polyvinyl chloride (PVC) ultrafiltration (UF) membrane, amphiphilic sulfonated polysulfone (SPSF) was introduced into PVC matrix. Three types of PVC/SPSF blend membranes containing different SPSF with the sulfonation degree (SD) of 20%, 30%, and 50% were fabricated by non-solvent induced phase separation (NIPS) process. The excellent compatibility between PVC and SPSF was confirmed by differential scanning calorimetry (DSC). Surface chemical compositions, morphology, roughness, charge, hydrophilicity, permeability and antifouling properties of the pristine PVC membrane and the PVC/SPSF blend membranes were systematically compared and characterized. Due to the improved hydrophilicity and endowed negative charge, the blend membrane showed high water permeability (i.e. 880 L mh bar), high bovine serum albumin (BSA) rejection (i.e. 95.7%), and high flux recovery ratio (i.e. 96%), which outperformed ever reported and commercialized PVC membranes. Furthermore, the permeability and rejection properties of PVC/SPSF UF membranes were maintained after soaking in acidic and alkaline solutions for 30 days, indicating their outstanding acid and alkali tolerance. Therefore, SPSF was expected as a potential versatile modifier for fabricating high performance UF membranes.
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http://dx.doi.org/10.1016/j.jcis.2020.06.097DOI Listing
November 2020

Selective induction of antibody effector functional responses using MF59-adjuvanted vaccination.

J Clin Invest 2020 02;130(2):662-672

Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA.

Seasonal and pandemic influenza infection remains a major public health concern worldwide. Driving robust humoral immunity has been a challenge given preexisting, often cross-reactive, immunity and in particular, poorly immunogenic avian antigens. To overcome immune barriers, the adjuvant MF59 has been used in seasonal influenza vaccines to increase antibody titers and improve neutralizing activity, translating to a moderate increase in protection in vulnerable populations. However, its effects on stimulating antibody effector functions, including NK cell activation, monocyte phagocytosis, and complement activity, all of which have been implicated in protection against influenza, have yet to be defined. Using systems serology, we assessed changes in antibody functional profiles in individuals who received H5N1 avian influenza vaccine administered with MF59, with alum, or delivered unadjuvanted. MF59 elicited antibody responses that stimulated robust neutrophil phagocytosis and complement activity. Conversely, vaccination with MF59 recruited NK cells poorly and drove moderate monocyte phagocytic activity, both likely compromised because of the induction of antibodies that did not bind FCGR3A. Collectively, defining the humoral antibody functions induced by distinct adjuvants may provide a path to designing next-generation vaccines that can selectively leverage the humoral immune functions, beyond binding and neutralization, resulting in better protection from infection.
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http://dx.doi.org/10.1172/JCI129520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994146PMC
February 2020

Predicting the broadly neutralizing antibody susceptibility of the HIV reservoir.

JCI Insight 2019 09 5;4(17). Epub 2019 Sep 5.

Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA.

Broadly neutralizing antibodies (bNAbs) against HIV-1 are under evaluation for both prevention and therapy. HIV-1 sequence diversity observed in most HIV-infected individuals and archived variations in critical bNAb epitopes present a major challenge for the clinical application of bNAbs, as preexistent resistant viral strains can emerge, resulting in bNAb failure to control HIV. In order to identify viral resistance in patients prior to antibody therapy and to guide the selection of effective bNAb combination regimens, we developed what we believe to be a novel Bayesian machine-learning model that uses HIV-1 envelope protein sequences and foremost approximated glycan occupancy information as variables to quantitatively predict the half-maximal inhibitory concentrations (IC50) of 126 neutralizing antibodies against a variety of cross clade viruses. We then applied this model to peripheral blood mononuclear cell-derived proviral Env sequences from 25 HIV-1-infected individuals mapping the landscape of neutralization resistance within each individual's reservoir and determined the predicted ideal bNAb combination to achieve 100% neutralization at IC50 values <1 μg/ml. Furthermore, predicted cellular viral reservoir neutralization signatures of individuals before an analytical antiretroviral treatment interruption were consistent with the measured neutralization susceptibilities of the respective plasma rebound viruses, validating our model as a potentially novel tool to facilitate the advancement of bNAbs into the clinic.
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http://dx.doi.org/10.1172/jci.insight.130153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777915PMC
September 2019

Publisher Correction: IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure.

Nat Med 2019 Jul;25(7):1175

Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.

In the version of this article originally published, there was an error in the abstract. The word disease should not have been included in the sentence "These individuals were highly exposed to Mtb but tested negative disease by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI". The sentence should have been "These individuals were highly exposed to Mtb but tested negative by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI." The error has been corrected in the HTML and PDF versions of this article.
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http://dx.doi.org/10.1038/s41591-019-0519-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609258PMC
July 2019

IFN-γ-independent immune markers of Mycobacterium tuberculosis exposure.

Nat Med 2019 06 20;25(6):977-987. Epub 2019 May 20.

Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA.

Exposure to Mycobacterium tuberculosis (Mtb) results in heterogeneous clinical outcomes including primary progressive tuberculosis and latent Mtb infection (LTBI). Mtb infection is identified using the tuberculin skin test and interferon-γ (IFN-γ) release assay IGRA, and a positive result may prompt chemoprophylaxis to prevent progression to tuberculosis. In the present study, we report on a cohort of Ugandan individuals who were household contacts of patients with TB. These individuals were highly exposed to Mtb but tested negative disease by IFN-γ release assay and tuberculin skin test, 'resisting' development of classic LTBI. We show that 'resisters' possess IgM, class-switched IgG antibody responses and non-IFN-γ T cell responses to the Mtb-specific proteins ESAT6 and CFP10, immunologic evidence of exposure to Mtb. Compared to subjects with classic LTBI, 'resisters' display enhanced antibody avidity and distinct Mtb-specific IgG Fc profiles. These data reveal a distinctive adaptive immune profile among Mtb-exposed subjects, supporting an expanded definition of the host response to Mtb exposure, with implications for public health and the design of clinical trials.
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http://dx.doi.org/10.1038/s41591-019-0441-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559862PMC
June 2019

Prediction of VRC01 neutralization sensitivity by HIV-1 gp160 sequence features.

PLoS Comput Biol 2019 04 1;15(4):e1006952. Epub 2019 Apr 1.

Vaccine and Infectious Disease Division and Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

The broadly neutralizing antibody (bnAb) VRC01 is being evaluated for its efficacy to prevent HIV-1 infection in the Antibody Mediated Prevention (AMP) trials. A secondary objective of AMP utilizes sieve analysis to investigate how VRC01 prevention efficacy (PE) varies with HIV-1 envelope (Env) amino acid (AA) sequence features. An exhaustive analysis that tests how PE depends on every AA feature with sufficient variation would have low statistical power. To design an adequately powered primary sieve analysis for AMP, we modeled VRC01 neutralization as a function of Env AA sequence features of 611 HIV-1 gp160 pseudoviruses from the CATNAP database, with objectives: (1) to develop models that best predict the neutralization readouts; and (2) to rank AA features by their predictive importance with classification and regression methods. The dataset was split in half, and machine learning algorithms were applied to each half, each analyzed separately using cross-validation and hold-out validation. We selected Super Learner, a nonparametric ensemble-based cross-validated learning method, for advancement to the primary sieve analysis. This method predicted the dichotomous resistance outcome of whether the IC50 neutralization titer of VRC01 for a given Env pseudovirus is right-censored (indicating resistance) with an average validated AUC of 0.868 across the two hold-out datasets. Quantitative log IC50 was predicted with an average validated R2 of 0.355. Features predicting neutralization sensitivity or resistance included 26 surface-accessible residues in the VRC01 and CD4 binding footprints, the length of gp120, the length of Env, the number of cysteines in gp120, the number of cysteines in Env, and 4 potential N-linked glycosylation sites; the top features will be advanced to the primary sieve analysis. This modeling framework may also inform the study of VRC01 in the treatment of HIV-infected persons.
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http://dx.doi.org/10.1371/journal.pcbi.1006952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459550PMC
April 2019

Publisher Correction: Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

Nature 2018 12;564(7734):E8

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

In Fig. 4b of this Article, the x-axis labels 'PGT121' and 'GS-9620' were inadvertently swapped in both graphs. In Fig. 5a, b, 'TLR7' should have been 'GS-9620'. These figures have been corrected online.
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http://dx.doi.org/10.1038/s41586-018-0721-yDOI Listing
December 2018

Antibody and TLR7 agonist delay viral rebound in SHIV-infected monkeys.

Nature 2018 11 3;563(7731):360-364. Epub 2018 Oct 3.

Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

The latent viral reservoir is the critical barrier for the development of a cure for HIV-1 infection. Previous studies have shown direct antiviral activity of potent HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) administered when antiretroviral therapy (ART) was discontinued, but it remains unclear whether bNAbs can target the viral reservoir during ART. Here we show that administration of the V3 glycan-dependent bNAb PGT121 together with the Toll-like receptor 7 (TLR7) agonist vesatolimod (GS-9620) during ART delayed viral rebound following discontinuation of ART in simian-human immunodeficiency virus (SHIV)-SF162P3-infected rhesus monkeys in which ART was initiated during early acute infection. Moreover, in the subset of monkeys that were treated with both PGT121 and GS-9620 and that did not show viral rebound after discontinuation of ART, adoptive transfer studies and CD8-depletion studies also did not reveal virus. These data demonstrate the potential of bNAb administration together with innate immune stimulation as a possible strategy for targeting the viral reservoir.
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http://dx.doi.org/10.1038/s41586-018-0600-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237629PMC
November 2018

Antigen-specific antibody Fc glycosylation enhances humoral immunity via the recruitment of complement.

Sci Immunol 2018 08;3(26)

Ragon Institute of MGH, MIT and Harvard, Cambridge, MA 02139, USA.

HIV-specific broadly neutralizing antibodies (bNAbs) confer protection after passive immunization, but the immunological mechanisms that drive their development are poorly understood. Structural features of bNAbs indicate that they originate from extensive germinal center (GC) selection, which relies on persistent GC activity. However, why a fraction of infected individuals are able to successfully drive more effective affinity maturation is unclear. Delivery of antigens in the form of antibody-immune complexes (ICs), which bind to complement receptors (CRs) or Fc receptors (FcRs) on follicular dendritic cells, represents an effective mechanism for antigen delivery to the GC. We sought to define whether IC-FcR or CR interactions differ among individuals who develop bNAb responses to HIV. Enhanced Fc effector functions and FcR/CR interactions, via altered Fc glycosylation profiles, were observed among individuals with neutralizing antibody responses to HIV compared with those without neutralizing antibody activity. Moreover, both polyclonal neutralizer ICs and monoclonal IC mimics of neutralizer antibodies induced higher antibody titers, higher-avidity antibodies, and expanded GC B cell reactions after immunization of mice via accelerated antigen deposition within B cell follicles in a complement-dependent manner. Thus, these data point to a direct role for altered Fc profile/complement interactions in shaping the maturation of the humoral immune response, providing insights into how GC activity may be enhanced to drive affinity maturation in next-generation vaccine approaches.
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http://dx.doi.org/10.1126/sciimmunol.aat7796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298214PMC
August 2018

A Role for Fc Function in Therapeutic Monoclonal Antibody-Mediated Protection against Ebola Virus.

Cell Host Microbe 2018 08;24(2):221-233.e5

Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA. Electronic address:

The recent Ebola virus (EBOV) epidemic highlighted the need for effective vaccines and therapeutics to limit and prevent outbreaks. Host antibodies against EBOV are critical for controlling disease, and recombinant monoclonal antibodies (mAbs) can protect from infection. However, antibodies mediate an array of antiviral functions including neutralization as well as engagement of Fc-domain receptors on immune cells, resulting in phagocytosis or NK cell-mediated killing of infected cells. Thus, to understand the antibody features mediating EBOV protection, we examined specific Fc features associated with protection using a library of EBOV-specific mAbs. Neutralization was strongly associated with therapeutic protection against EBOV. However, several neutralizing mAbs failed to protect, while several non-neutralizing or weakly neutralizing mAbs could protect. Antibody-mediated effector functions, including phagocytosis and NK cell activation, were associated with protection, particularly for antibodies with moderate neutralizing activity. This framework identifies functional correlates that can inform therapeutic and vaccine design strategies against EBOV and other pathogens.
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http://dx.doi.org/10.1016/j.chom.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298217PMC
August 2018

Exploiting glycan topography for computational design of Env glycoprotein antigenicity.

PLoS Comput Biol 2018 04 20;14(4):e1006093. Epub 2018 Apr 20.

Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, United States of America.

Mounting evidence suggests that glycans, rather than merely serving as a "shield", contribute critically to antigenicity of the HIV envelope (Env) glycoprotein, representing critical antigenic determinants for many broadly neutralizing antibodies (bNAbs). While many studies have focused on defining the role of individual glycans or groups of proximal glycans in bNAb binding, little is known about the effects of changes in the overall glycan landscape in modulating antibody access and Env antigenicity. Here we developed a systems glycobiology approach to reverse engineer the complexity of HIV glycan heterogeneity to guide antigenicity-based de novo glycoprotein design. bNAb binding was assessed against a panel of 94 recombinant gp120 monomers exhibiting defined glycan site occupancies. Using a Bayesian machine learning algorithm, bNAb-specific glycan footprints were identified and used to design antigens that selectively alter bNAb antigenicity as a proof-of concept. Our approach provides a new design strategy to predictively modulate antigenicity via the alteration of glycan topography, thereby focusing the humoral immune response on sites of viral vulnerability for HIV.
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http://dx.doi.org/10.1371/journal.pcbi.1006093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931682PMC
April 2018

ADCC-Mediated CD56 NK Cell Responses Are Associated with Early HBsAg Clearance in Acute HBV Infection.

Pathog Immun 2018 19;3(1):2-18. Epub 2018 Feb 19.

The Ragon Institute of MGH, MIT, and Harvard, Cambridge, Massachusetts.

Background: Hepatitis B virus (HBV) affects up to 400 million people worldwide and accounts for approximately one million deaths per year from liver pathologies. Current treatment regimens are effective in suppressing viremia but usually have to be taken indefinitely, warranting research into new therapeutic approaches. Acute HBV infection in adults almost universally results in resolution of viremia, with the exception of immunocompromised persons, suggesting that the immune response can functionally cure or even eradicate HBV infection.

Methods: Because immunophenotypic and functional studies have implicated a role for Natural Killer (NK) cells in HBV clearance during acute infection, we hypothesized that a distinct NK-cell profile exists in acute HBV infection that could provide information for the mechanism of HBV clearance. Using multivariate flow cytometry, we evaluated the expression of key activating and inhibitory receptors on NK cells, and their ability to respond to classic target cell lines.

Results: Multivariate analysis revealed selective perturbation of the CD56 NK-cell subset during acute infection, displaying low levels of NKp46+, NKp30+, CD160+ and CD161+ cells. Intriguingly, the CD56 NK-cell profile predicted time to HBV surface antigen (HBsAg) clearance from the blood, and distinct NK-cell profiles predicted early (NKp30, CD94, CD161) and late clearance (KIR3DL1, CD158a, perforin, NKp46). Finally, functional analysis demonstrated that early and late clearance tracked with elevated degranulation (CD107a) or IFNγ production, respectively, in response to ADCC-mediated activation.

Conclusion: The cytolytic CD56 NK-cell subset is selectively activated in acute HBV infection and displays distinct phenotypic and functional profiles associated with efficient and early control of HBV, implicating antibody-mediated cytolytic NK-cell responses in the early control and functional cure of HBV infection.
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http://dx.doi.org/10.20411/pai.v3i1.228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5847299PMC
February 2018

A Functional Role for Antibodies in Tuberculosis.

Cell 2016 Oct 22;167(2):433-443.e14. Epub 2016 Sep 22.

Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA. Electronic address:

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control.
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http://dx.doi.org/10.1016/j.cell.2016.08.072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526202PMC
October 2016

Dissecting Polyclonal Vaccine-Induced Humoral Immunity against HIV Using Systems Serology.

Cell 2015 Nov;163(4):988-98

Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA 02139, USA. Electronic address:

While antibody titers and neutralization are considered the gold standard for the selection of successful vaccines, these parameters are often inadequate predictors of protective immunity. As antibodies mediate an array of extra-neutralizing Fc functions, when neutralization fails to predict protection, investigating Fc-mediated activity may help identify immunological correlates and mechanism(s) of humoral protection. Here, we used an integrative approach termed Systems Serology to analyze relationships among humoral responses elicited in four HIV vaccine trials. Each vaccine regimen induced a unique humoral "Fc fingerprint." Moreover, analysis of case:control data from the first moderately protective HIV vaccine trial, RV144, pointed to mechanistic insights into immune complex composition that may underlie protective immunity to HIV. Thus, multi-dimensional relational comparisons of vaccine humoral fingerprints offer a unique approach for the evaluation and design of novel vaccines against pathogens for which correlates of protection remain elusive.
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http://dx.doi.org/10.1016/j.cell.2015.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490491PMC
November 2015

Novel compound heterozygous mutations identified in ADAMTSL4 gene in a Chinese family with isolated ectopia lentis.

Acta Ophthalmol 2015 Feb 7;93(1):e91-2. Epub 2014 May 7.

Ophthalmic Laboratories, Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu, China.

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http://dx.doi.org/10.1111/aos.12399DOI Listing
February 2015

Single nucleotide polymorphism of MYOC affected the severity of primary open angle glaucoma.

Int J Ophthalmol 2013 18;6(3):264-8. Epub 2013 Jun 18.

Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China.

Aim: To detect the mutations in two candidate genes, myocilin (MYOC) and cytochrome P450 1B1 (CYP1B1), in a Chinese family with primary open angle glaucoma (POAG).

Methods: The family was composed of three members, the parents and a daughter. All members of the family underwent complete ophthalmologic examinations. Exons of MYOC and CYP1B1 genes were screened for sequence alterations by polymerase chain reaction (PCR) and direct DNA sequencing.

Results: The mother was the proband, she was diagnosed as POAG in both eyes. Her daughter was diagnosed as juvenile-onset POAG. The father was asymptomatic. One MYOC heterozygous mutation c.1150 G>A (D384N) in exon 3 was identified in the mother, another MYOC heterozygous variation c.1058 C>T (T353I) in exon 3 was identified in the father, and the daughter inherited both of the variations. Meanwhile, three single nucleotide polymorphisms (SNPs) in CYP1B1 gene were found in the family.

Conclusion: The D384N mutation of MYOC has been reported as one of disease-causing mutations in POAG, whereas T353I variation of MYOC was thought as a high risk factor for POAG. The two variations of MYOC were first reported in one juvenile-onset POAG patient who presented with more severe clinical manifestations, suggesting that T353I polymorphism of MYOC may be associated with the severity of POAG.
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http://dx.doi.org/10.3980/j.issn.2222-3959.2013.03.02DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3693003PMC
July 2013

The Mycobacterium tuberculosis regulatory network and hypoxia.

Nature 2013 Jul 3;499(7457):178-83. Epub 2013 Jul 3.

Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.
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http://dx.doi.org/10.1038/nature12337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4087036PMC
July 2013

Studies of a pedigree with limbal dermoid cyst.

Int J Ophthalmol 2012 18;5(5):641-3. Epub 2012 Oct 18.

Ophthalmic Laboratories and Department of Ophthalmology, Translational neuroscience Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China.

Aim: To study clinical features and gene mutations within the paired-like homeodomain transcription factor 2 (PITX2) gene in a pedigree of bilateral limbal dermoids.

Methods: Complete eye examinations have been performed on each individual of the family. Exons of paired-like homeodomain transcription factor 2 (PITX2) were amplified by polymerase chain reaction, sequenced, and compared with a reference database.

Results: We described the phenotype, clinic findings in a family with two affected members. The masses of the proband's eyes were excised surgically demonstrating a dermoid cyst by histopathological examination. No mutation was detected in the gene PITX2 in this pedigree.

Conclusion: A family of limbal dermoid cyst was reported. In addition, no pathogenic sequence variations were found in PITX2, indicating that this phenotype in this family is a distinctive entity.
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http://dx.doi.org/10.3980/j.issn.2222-3959.2012.05.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3484697PMC
November 2012

Comprehensive transcriptome analysis of the periodontopathogenic bacterium Porphyromonas gingivalis W83.

J Bacteriol 2012 Jan 28;194(1):100-14. Epub 2011 Oct 28.

Department of Oral Biology, University of Oslo, Oslo, Norway.

High-density tiling microarray and RNA sequencing technologies were used to analyze the transcriptome of the periodontopathogenic bacterium Porphyromonas gingivalis. The compiled P. gingivalis transcriptome profiles were based on total RNA samples isolated from three different laboratory culturing conditions, and the strand-specific transcription profiles generated covered the entire genome, including both protein coding and noncoding regions. The transcription profiles revealed various operon structures, 5'- and 3'-end untranslated regions (UTRs), differential expression patterns, and many novel, not-yet-annotated transcripts within intergenic and antisense regions. Further transcriptome analysis identified the majority of the genes as being expressed within operons and most 5' and 3' ends to be protruding UTRs, of which several 3' UTRs were extended to overlap genes carried on the opposite/antisense strand. Extensive antisense RNAs were detected opposite most insertion sequence (IS) elements. Pairwise comparative analyses were also performed among transcriptome profiles of the three culture conditions, and differentially expressed genes and metabolic pathways were identified. With the growing realization that noncoding RNAs play important biological functions, the discovery of novel RNAs and the comprehensive transcriptome profiles compiled in this study may provide a foundation to further understand the gene regulation and virulence mechanisms in P. gingivalis. The transcriptome profiles can be viewed at and downloaded from the Microbial Transcriptome Database website, http://bioinformatics.forsyth.org/mtd.
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http://dx.doi.org/10.1128/JB.06385-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3256594PMC
January 2012

Strand-specific transcriptome profiling with directly labeled RNA on genomic tiling microarrays.

BMC Mol Biol 2011 Jan 14;12. Epub 2011 Jan 14.

Department of Molecular Genetics, The Forsyth Institute, Cambridge, MA, USA.

Background: With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes this method and its application to the mapping of transcriptome profiles.

Results: RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. Five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were experimental artifacts.

Conclusions: An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is potentially more sensitive in detecting small amount of RNA compared to conventional end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment.
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http://dx.doi.org/10.1186/1471-2199-12-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3031212PMC
January 2011

Bioinformatics analysis of macrophages exposed to Porphyromonas gingivalis: implications in acute vs. chronic infections.

PLoS One 2010 Dec 23;5(12):e15613. Epub 2010 Dec 23.

Bioinformatics Graduate Program, Boston University, Boston, Massachusetts, United States of America.

Background: Periodontitis is the most common human infection affecting tooth-supporting structures. It was shown to play a role in aggravating atherosclerosis. To deepen our understanding of the pathogenesis of this disease, we exposed human macrophages to an oral bacteria, Porphyromonas gingivalis (P. gingivalis), either as live bacteria or its LPS or fimbria. Microarray data from treated macrophages or control cells were analyzed to define molecular signatures. Changes in genes identified in relevant pathways were validated by RT-PCR.

Methodology/principal Findings: We focused our analysis on three important groups of genes. Group PG (genes differentially expressed by live bacteria only); Group LFG (genes differentially expressed in response to exposure to LPS and/or FimA); Group CG (core gene set jointly activated by all 3 stimulants). A total of 842 macrophage genes were differentially expressed in at least one of the three conditions compared to naïve cells. Using pathway analysis, we found that group CG activates the initial phagocytosis process and induces genes relevant to immune response, whereas group PG can de-activate the phagocytosis process associated with phagosome-lysosome fusion. LFG mostly affected RIG-I-like receptor signaling pathway.

Conclusion/significance: In light of the fact that acute infections involve live bacteria while chronic infections involve a combination of live bacteria and their byproducts, group PG could represent acute P. gingivalis infection while group LFG could represent chronic P. gingivalis infection. Group CG may be associated with core immune pathways, triggered irrespective of the specific stimulants and indispensable to mount an appropriate immune response. Implications in acute vs. chronic infection are discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015613PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009741PMC
December 2010

TGFBI gene mutation analysis in a Chinese pedigree of Avellino corneal dystrophy.

Int J Ophthalmol 2011 18;4(3):275-9. Epub 2011 Jun 18.

Ophthalmic Laboratories & Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China.

Aim: To analyze phenotype and genotype of a Chinese pedigree with Avellino corneal dystrophy (ACD).

Methods: Complete ophthalmic examinations were performed on all the family members. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database.

Results: A single heterozygous G>A (R124H) point mutation was identified in exon 4 of TGFBI in three affected members and two unaffected children who were offsprings of the affected members, but not in the other family members.

Conclusion: Mutation R124H in TGFBI was identified in this pedigree and appeared to be the disease causing mutation. Atypical phenotype and low penetrance was observed in this pedigree.
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http://dx.doi.org/10.3980/j.issn.2222-3959.2011.03.13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3340809PMC
August 2012

The human oral microbiome.

J Bacteriol 2010 Oct 23;192(19):5002-17. Epub 2010 Jul 23.

Department of Molecular Genetics, The Forsyth Institute, 245 First St., Cambridge, MA 02142, USA.

The human oral cavity contains a number of different habitats, including the teeth, gingival sulcus, tongue, cheeks, hard and soft palates, and tonsils, which are colonized by bacteria. The oral microbiome is comprised of over 600 prevalent taxa at the species level, with distinct subsets predominating at different habitats. The oral microbiome has been extensively characterized by cultivation and culture-independent molecular methods such as 16S rRNA cloning. Unfortunately, the vast majority of unnamed oral taxa are referenced by clone numbers or 16S rRNA GenBank accession numbers, often without taxonomic anchors. The first aim of this research was to collect 16S rRNA gene sequences into a curated phylogeny-based database, the Human Oral Microbiome Database (HOMD), and make it web accessible (www.homd.org). The HOMD includes 619 taxa in 13 phyla, as follows: Actinobacteria, Bacteroidetes, Chlamydiae, Chloroflexi, Euryarchaeota, Firmicutes, Fusobacteria, Proteobacteria, Spirochaetes, SR1, Synergistetes, Tenericutes, and TM7. The second aim was to analyze 36,043 16S rRNA gene clones isolated from studies of the oral microbiota to determine the relative abundance of taxa and identify novel candidate taxa. The analysis identified 1,179 taxa, of which 24% were named, 8% were cultivated but unnamed, and 68% were uncultivated phylotypes. Upon validation, 434 novel, nonsingleton taxa will be added to the HOMD. The number of taxa needed to account for 90%, 95%, or 99% of the clones examined is 259, 413, and 875, respectively. The HOMD is the first curated description of a human-associated microbiome and provides tools for use in understanding the role of the microbiome in health and disease.
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http://dx.doi.org/10.1128/JB.00542-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944498PMC
October 2010

The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information.

Database (Oxford) 2010 Jul 6;2010:baq013. Epub 2010 Jul 6.

The Forsyth Institute, Boston, MA 02115, USA.

The human oral microbiome is the most studied human microflora, but 53% of the species have not yet been validly named and 35% remain uncultivated. The uncultivated taxa are known primarily from 16S rRNA sequence information. Sequence information tied solely to obscure isolate or clone numbers, and usually lacking accurate phylogenetic placement, is a major impediment to working with human oral microbiome data. The goal of creating the Human Oral Microbiome Database (HOMD) is to provide the scientific community with a body site-specific comprehensive database for the more than 600 prokaryote species that are present in the human oral cavity based on a curated 16S rRNA gene-based provisional naming scheme. Currently, two primary types of information are provided in HOMD--taxonomic and genomic. Named oral species and taxa identified from 16S rRNA gene sequence analysis of oral isolates and cloning studies were placed into defined 16S rRNA phylotypes and each given unique Human Oral Taxon (HOT) number. The HOT interlinks phenotypic, phylogenetic, genomic, clinical and bibliographic information for each taxon. A BLAST search tool is provided to match user 16S rRNA gene sequences to a curated, full length, 16S rRNA gene reference data set. For genomic analysis, HOMD provides comprehensive set of analysis tools and maintains frequently updated annotations for all the human oral microbial genomes that have been sequenced and publicly released. Oral bacterial genome sequences, determined as part of the Human Microbiome Project, are being added to the HOMD as they become available. We provide HOMD as a conceptual model for the presentation of microbiome data for other human body sites. Database URL: http://www.homd.org.
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http://dx.doi.org/10.1093/database/baq013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911848PMC
July 2010

A hidden Markov support vector machine framework incorporating profile geometry learning for identifying microbial RNA in tiling array data.

Bioinformatics 2010 Jun 15;26(11):1423-30. Epub 2010 Apr 15.

Department of Molecular Genetics, The Forsyth Institute, Boston, MA 02115, USA.

Motivation: RNA expression signals detected by high-density genomic tiling microarrays contain comprehensive transcriptomic information of the target organism. Current methods for determining the RNA transcription units are still computation intense and lack the discriminative power. This article describes an efficient and accurate methodology to reveal complicated transcriptional architecture, including small regulatory RNAs, in microbial transcriptome profiles.

Results: Normalized microarray data were first subject to support vector regression to estimate the profile tendency by reducing noise interruption. A hybrid supervised machine learning algorithm, hidden Markov support vector machines, was then used to classify the underlying state of each probe to 'expression' or 'silence' with the assumption that the consecutive state sequence was a heterogeneous Markov chain. For model construction, we introduced a profile geometry learning method to construct the feature vectors, which considered both intensity profiles and changes of intensities over the probe spacing. Also, a robust strategy was used to dynamically evaluate and select the training set based only on prior computer gene annotation. The algorithm performed better than other methods in accuracy on simulated data, especially for small expressed regions with lower (<1) SNR (signal-to-noise ratio), hence more sensitive for detecting small RNAs.

Availability And Implementation: Detail implementation steps of the algorithm and the complete result of the transcriptome analysis for a microbial genome Porphyromonas gingivalis W83 can be viewed at http://bioinformatics.forsyth.org/mtd.
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http://dx.doi.org/10.1093/bioinformatics/btq162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913668PMC
June 2010

Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum 'Synergistetes' isolated from the human oral cavity.

Int J Syst Evol Microbiol 2009 May;59(Pt 5):972-80

King's College London Dental Institute, Infection Research Group, London SE1 9RT, UK.

Four strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group distinct from any species with validly published names. 16S rRNA and 23S rRNA gene sequence analyses and DNA-DNA reassociation data revealed that the strains constituted a novel group within the phylum 'Synergistetes' and were most closely related to Jonquetella anthropi. Two libraries of randomly cloned DNA were prepared from strain W5455(T) and were sequenced to provide a genome survey as a resource for metagenomic studies. A new genus and novel species, Pyramidobacter piscolens gen. nov., sp. nov., is proposed to accommodate these strains. The genus Pyramidobacter comprises strains that are anaerobic, non-motile, asaccharolytic bacilli that produce acetic and isovaleric acids and minor to trace amounts of propionic, isobutyric, succinic and phenylacetic acids as end products of metabolism. P. piscolens gen. nov., sp. nov. produced hydrogen sulphide but was otherwise largely biochemically unreactive. Growth was stimulated by the addition of glycine to broth media. The G+C content of the DNA of the type strain was 59 mol%. The type strain of Pyramidobacter piscolens sp. nov. is W5455(T) (=DSM 21147(T)=CCUG 55836(T)).
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http://dx.doi.org/10.1099/ijs.0.000364-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2868594PMC
May 2009

A novel retrieval system for nearly complete microbial genomic fragments from soil samples.

J Microbiol Methods 2008 Feb 8;72(2):197-205. Epub 2007 Dec 8.

Graduate Institute of Agricultural Chemistry, National Taiwan University, No. 1 Section 4 Roosevelt Road, Taipei, Taiwan.

The construction of a complex genomic library is one of the comprehensive ways to study a complex bacterial community and to access the variety of metabolic pathways present in the rich soil environment. In this report, we developed a new protocol whereby we are able to retrieve nearly complete microbe genomic fragments from soil samples, which are employed to generate a metagenomic library for visualizing the basic scaffolding of the soil microbial community. The use of direct cell lysis within soil-embedded agarose plugs, along with a double-size selection, enabled us to successfully isolate pure and high-molecular weight DNA (0.1-1 Mb) without the need for any further purification. A metagenomic library containing 1.2 Gbp of DNA in total was constructed. Furthermore, analysis of the microbial community structure using 16S rDNA partial sequences found the dominant phylotypes to consist of alpha-Proteobacteria and Actinobacteria, which are similar to those seen in forest and agricultural soils, and numerous uncultured microbes from a wide variety of bacterial taxa as well. In conclusion, this study presents a novel protocol for generating a metagenomic library that carries much larger and diverse DNA fragments from soil bacteria that will be applied for the reconstruction of soil microbial genomes and the discovery of novel habitat-specific pathways.
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http://dx.doi.org/10.1016/j.mimet.2007.11.022DOI Listing
February 2008