Endoplasmic reticulum aminopeptidases in the pathogenesis of ankylosing spondylitis

There has been significant progress in our understanding of the pathogenesis of AS. The advent of genome-wide association studies has increased the known loci associated with AS to more than 40. The endoplasmic reticulum resident aminopeptidases (ERAP) 1 and 2 were identified in this manner and are of particular interest. There appears to be a genetic as well as a functional interaction of ERAP1 and 2 with HLA-B27 based on the known functions of these molecules. Recent studies on the structure, immunological effects and the peptide-trimming properties of ERAP 1 and 2 have helped to provide insight into their pathogenic potential in AS. In this review, we explore the role of ERAP 1 and 2 in the pathogenesis of AS. © The Author 2015.

The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8(+) T Cells

We previously reported that Rv1860 protein from Mycobacterium tuberculosis stimulated CD4(+) and CD8(+) T cells secreting gamma interferon (IFN-gamma) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulent M. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latently M. tuberculosis-infected individuals dominated by CD8(+) T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8(+) PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studied M. tuberculosis antigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4(+) T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8(+) T-cell-stimulating antigens has the potential to prevent progression of latent M. tuberculosis infection to TB disease.

DNA Damage and Transcriptional Changes in the Gills of Mytilus galloprovincialis Exposed to Nanomolar Doses of Combined Metal Salts (Cd, Cu, Hg)

[ENG]Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs

Octopamine neurons mediate flight-induced modulation of visual processing in Drosophila melanogaster

Activity-dependent modulation of sensory systems has been documented in many organisms, and is likely to be essential for appropriate processing of information during different behavioral states. However, the mechanisms underlying these phenomena, and often their functional consequences, remain poorly characterized. I investigated the role of octopamine neurons in the flight-dependent modulation observed in visual interneurons in the fruit fly Drosophila melanogaster. The vertical system (VS) cells exhibit a boost in their response to visual motion during flight compared to quiescence. Pharmacological application of octopamine evokes responses in quiescent flies that mimic those observed during flight, and octopamine neurons that project to the optic lobes increase in activity during flight. Using genetic tools to manipulate the activity of octopamine neurons, I find that they are both necessary and sufficient for the flight-induced visual boost. This work provides the first evidence that endogenous release of octopamine is involved in state-dependent modulation of visual interneurons in flies. Further, I investigated the role of a single pair of octopamine neurons that project to the optic lobes, and found no evidence that chemical synaptic transmission via these neurons is necessary for the flight boost. However, I found some evidence that activation of these neurons may contribute to the flight boost. Wind stimuli alone are sufficient to generate transient increases in the VS cell response to motion vision, but result in no increase in baseline membrane potential. These results suggest that the flight boost originates not from a central command signal during flight, but from mechanosensory stimuli relayed via the octopamine system. Lastly, in an attempt to understand the functional consequences of the flight boost observed in visual interneurons, we measured the effect of inactivating octopamine neurons in freely flying flies. We found that flies whose octopamine neurons we silenced accelerate less than wild-type flies, consistent with the hypothesis that the flight boost we observe in VS cells is indicative of a gain control mechanism mediated by octopamine neurons. Together, this work serves as the basis for a mechanistic and functional understanding of octopaminergic modulation of vision in flying flies.

Estudo da resposta celular e apresentação antigênica dos fibrócitos na infecção por Leishmania (Leishmania) amazonensis

Os fibrócitos foram inicialmente identificados pelo seu recrutamento rápido para os tecidos lesionados e por atuar diretamente na resposta imune através do reconhecimento, apresentação antigênica e produção de citocinas e quimiocinas. Segundo Grab (2004) fibrócitos podem participar da resposta imune na leishmaniaose e por isso no presente estudo propomos analisar a resposta celular e apresentação antigênica dos fibrócitos na infecção por L. (L.) amazonensis. Para os experimentos in vivo camundongos C57BL/6 e knockout para o receptor TLR-2 foram inoculados na derme auricular com 105 formas promastigotas de L. (L.) amazonensis e 1, 7, 15 dias após a infecção as regiões de inóculo e os linfonodos foram retirados e processados para citometria de fluxo. Para os experimentos in vitro fibrócitos foram obtidos de mononucleares do sangue periférico de camundongos C57BL/6. Os fibrócitos foram avaliados quanto às suas características morfológicas e fenotípicas, infecção, expressão de MHC-II/B7-1/B7-2 e ativação de linfócitos T CD4+. As análises na região de inóculo mostraram o aumento no percentual de fibrócito na derme de camundongos após 15 dias de infecção tanto em animais C57BL/6 quanto em animais KO TLR-2. Neste sítio, os fibrócitos produziram citocinas e expressaram MHC-II, B7-1 e B7-2 podendo participar da resposta imune. As análises no linfonodo mostraram a existência de um alto percentual de fibrócitos nos animais controle, contudo, após infecção este percentual foi reduzido. Após infecção verificamos que os fibrócitos de animais WT C57BL/6 foram capazes de produzir as citocinas IL-4, IL-10 e IFN- durante o primeiro dia. Entretanto, na ausência de TLR-2 os fibrócitos presentes no linfonodo produziram TNF-α e IFN- que podem estar relacionadas com a ativação celular e aumento da capacidade de apresentação antigênica destas células durante a infecção. No linfonodo verificamos que os fibrócitos podem participar da apresentação antigênica após a infecção por L. (L.) amazonensis. Contudo, nos linfonodos de animais WT C57BL/6 observamos a redução significativa no percentual destas células expressando MHC-II e B7-1, o que pode estar relacionada a presença do TLR-2. Nos ensaios in vitro fibrócitos de camundongos C57BL/6 apresentaram alta capacidade endocítica, eliminaram os parasitas nas primeiras 24 horas de infecção, expressaram MHC-II/B7-1/B7-2 e foram capazes de ativar linfócitos T CD4+. Com isso, nossos resultados sugerem que os fibrócitos podem atuar na resposta celular e na apresentação antigênica durante a infecção por L. (L.) amazonensis, contudo estas funções podem ser moduladas pela participação do TLR-2 e pelo microambiente onde estes se encontram.

Influência da superexpressão da claudina-3 na radiorresposta de células de câncer de cólon

O câncer colorretal (CCR) é o terceiro tipo de câncer mais incidente no mundo para o sexo masculino, o segundo para o sexo feminino e a radioterapia é um dos tratamentos de primeira linha no combate a este tipo de câncer. Durante a progressão do CCR as células sofrem alterações morfogenéticas, sendo a desorganização do complexo juncional apical (CJA) um dos eventos iniciais desse processo. As junções oclusivas (JTs) são um dos principais componentes da CJA e desempenham papel importante no controle do fluxo paracelular, na determinação da polaridade celular e na transdução de sinais relacionados com a progressão tumoral. As claudinas são proteínas transmembrana, constituintes das JTs e cumprem um importante papel no controle desses eventos. Alterações na expressão das claudinas são observadas em tumores de diferentes órgãos e têm sido relacionadas com a progressão tumoral. No entanto os mecanismos que regulam essas alterações e sua consequência na progressão do CCR são poucos conhecidos. Desta forma, o presente estudo teve como objetivo avaliar a influência da superexpressão da claudina-3 na radiorresposta de células CCR. Nossos resultados mostraram que a superexpressão de claudina-3 minimiza alterações morfológicas causadas pela radiação, causa diminuição da resistência elétrica transepitelial e não tem efeito na permeabilidade a macromoléculas após a irradiação. Além disso, observamos que a superexpressão de claudina-3 aumenta o potencial proliferativo das células e que esta característica torna as células mais sensíveis a radiação. Porém quando avaliamos eventos celulares relacionados a progressão tumoral observamos que apesar da radiação diminuir a capacidade migratória das progênies, as células que superexpressam claudina-3 apresentam migração mais elevada. Além disso, verificamos que a superexpressão de claudina-3 diminui a invasão e a capacidade de formação de colônias frente ao tratamento com a radiação. Em seguida fomos avaliar o efeito da inibição das vias de proliferação (MEK/ERK) e sobrevivência (PI3K-Akt) na resposta das células que superexpressam claudina-3 frente a radiação. Observamos que a inibição de MEK é capaz de sensibilizar as células que superexpressam claudina-3 à radiação no ensaio de proliferação celular, no entanto a inibição de MEK e PI3K antes da exposição à radiação é capaz aumentar a migração e a capacidade de formação de colônias de células que superexpressam claudina-3 contribuindo para o aumento do potencial maligno. Em conjunto nossos resultados mostram que a superexpressão de claudina-3 contribui para um fenótipo mais maligno, no entanto frente ao tratamento com a radiação é capaz de sensibilizar as células.

Subpopulações e ativação de linfócitos T CD4+ e T CD8+ em crianças e adolescentes HIV positivos vacinados contra Neisseria meningitidis C

Neisseria meningitidis sorogrupo C (MenC) tem sido causador de surtos no Brasil, desde 2005. Vacinas conjugadas contra MenC estão disponíveis desde 1999 nos países desenvolvidos e mais recentemente no Brasil. São vacinas eficazes em pacientes imunologicamente normais, mas pouco se conhece sobre o impacto em pacientes HIV+. O objetivo principal deste estudo foi investigar se há alguma correlação entre a resposta de LT CD4+ de memória e a resposta de anticorpos específicos para o MenC, assim como conhecer as populações de memória dos LT CD8+, em crianças e adolescentes infectados pelo HIV respondedores ou não à vacina MenC conjugada. Amostras de sangue de 36 pacientes HIV+ foram coletadas antes e após imunização, para análises laboratoriais, soros e células coletadas foram congelados e enviados ao nosso laboratório para a análise da resposta imune humoral e celular. Utilizamos o ensaio bactericida para avaliar a resposta humoral e dividir a população de estudo em soroconversor positivo e soroconversor negativo. A citometria de fluxo foi aplicada para identificação das seis subpopulações de LT CD4+ e T CD8+ e avaliação do perfil de ativação. Não encontramos mudanças no perfil de distribuição das subpopulações antes e após a vacinação. A subpopulação LT CD4+ Int correlacionou positivamente com os títulos de anticorpos e a ativação, de um modo geral, estava elevada nos respondedores, conferindo certa importância para essa célula. Semelhanças foram observadas entre as subpopulações LT CD4+ e T CD8+. Em suma, este estudo revelou importantes associações entre a resposta de anticorpos bactericidas após a vacinação, o perfil de distribuição das subpopulações de LT CD4+ LT CD8+ e seu status de ativação.

Studies on advantages of heavy ions in radiotherapy compared with gamma-rays

Hepatoma and melanoma cells were exposed to C-12(6+) beams generated by HIRFL facility and gamma-rays and the cell response was studied by colony assays as well as the analysis of RBE of carbon ions was evolved. The survival curves of cells irradiated by heavy ions were different from those of cells irradiated by gamma-rays. And two kinds of cell showed the obvious discrepancy in response to the photon and ion irradiation. The results showed that heavy ions have special physical properties and mighty potency to kill cell in both single and fractional irradiation meanwhile it can kill tumor cells with high radioresistance more efficiently. When involved in clinical therapy, heavy ions will enhance the therapy efficiency and decrease the suffering of patients because it can impair the repair for sublethal damage of cells which can lead to fewer irradiation fractions.

Cloning of hypoxia-inducible factor 1 alpha cDNA from a high hypoxia tolerant mammal - plateau pika (Ochotona curzoniae)

Hypoxia-inducible factor I is a transcription factor composed of HIF-1alpha and HIF-1beta. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species livin only at 3000-5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1alpha cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1alpha cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1alpha and pSHIF-1alpha. The HIF-1alpha mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1alpha mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1alpha may play an important role in the pika's adaptation to hypoxia, especially in brain and kidney, and pika HIF-1alpha function pattern may be different from that of mouse HIF-1alpha. Further-more, for the high ratio of HIF-1alpha homology among the animals, the HIF-1alpha gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. (C) 2004 Elsevier Inc. All rights reserved.

EXPERIMENTAL INVESTIGATION OF LINEAR BUBBLE DYNAMICS IN A VISCOELASTIC XANTHAN GEL

Oceanic bubble plumes caused by ship wakes or breaking waves disrupt sonar communi- cation because of the dramatic change in sound speed and attenuation in the bubbly fluid. Experiments in bubbly fluids have suffered from the inability to quantitatively characterize the fluid because of continuous air bubble motion. Conversely, single bubble experiments, where the bubble is trapped by a pressure field or stabilizing object, are limited in usable frequency range, apparatus complexity, or the invasive nature of the stabilizing object (wire, plate, etc.). Suspension of a bubble in a viscoelastic Xanthan gel allows acoustically forced oscilla- tions with negligible translation over a broad frequency band. Assuming only linear, radial motion, laser scattering from a bubble oscillating below, through, and above its resonance is measured. As the bubble dissolves in the gel, different bubble sizes are measured in the range 240 – 470 μm radius, corresponding to the frequency range 6 – 14 kHz. Equalization of the cell response in the raw data isolates the frequency response of the bubble. Compari- son to theory for a bubble in water shows good agreement between the predicted resonance frequency and damping, such that the bubble behaves as if it were oscillating in water.

Assessment of the immunomodulatory effects of raw/farm milk and probiotic bacteria

The overall aims of this study were to investigate the differences between raw/farm milk and pasteurised milk with respect to potential immune modifying effects following consumption and investigate the bacterial composition of raw milk compared to pasteurised milk. Furthermore, in this thesis, panels of potential probiotic bacteria from the Bifidobacterium and Lactobacillus genera were investigated. The overall bacterial composition of raw milk was compared with pasteurised milk using samples obtained from commercial milk producers around Ireland using next generation sequencing technology (454 pyrosequencing). Here the presence of previously unrecognised and diverse bacterial populations in unpasteurised cow’s milk was identified. Futhermore the bacterial content of pasteurised milk was found to be more diverse than previously thought. The global response of the adenocarcinoma cell line HT-29 to raw milk and pasteurised milk exposures were also characterised using whole genome microarray technology. Over one thousand differentially expressed genes were identified which were found to be involved in a plethora of cellular functions. Interestingly a reduction in immune related activity (e.g. Major histocompatability complex class II signalling and T and B cell proliferation) was identified in cells exposed to pasteurised milk compared with raw milk exposures. Further studies comparing human cell response to raw versus pasteurised milk was performed using peripheral blood mononuclear cells (PBMC) from healthy donors. A reduction in CD14 was identified following raw milk exposures compared with pasteurised milk and the pattern of cytokine production may indicate that gram positive bacteria in the raw milk were contributing to the differences in the cellular response to raw versus pasteurised milk. Panels of potentially probiotic bacteria (comprising of lactobacilli and bifidobacteria) were further assessed for immunomodulatory capabilities using cell culture based models. Gene expression and cytokine production were used to evaluate stimulated and unstimulated (LPS) cellular responses as well as interaction mechanisms

HIV-1 envelope gp41 antibodies can originate from terminal ileum B cells that share cross-reactivity with commensal bacteria.

Monoclonal antibodies derived from blood plasma cells of acute HIV-1-infected individuals are predominantly targeted to the HIV Env gp41 and cross-reactive with commensal bacteria. To understand this phenomenon, we examined anti-HIV responses in ileum B cells using recombinant antibody technology and probed their relationship to commensal bacteria. The dominant ileum B cell response was to Env gp41. Remarkably, a majority (82%) of the ileum anti-gp41 antibodies cross-reacted with commensal bacteria, and of those, 43% showed non-HIV-1 antigen polyreactivity. Pyrosequencing revealed shared HIV-1 antibody clonal lineages between ileum and blood. Mutated immunoglobulin G antibodies cross-reactive with both Env gp41 and microbiota could also be isolated from the ileum of HIV-1 uninfected individuals. Thus, the gp41 commensal bacterial antigen cross-reactive antibodies originate in the intestine, and the gp41 Env response in HIV-1 infection can be derived from a preinfection memory B cell pool triggered by commensal bacteria that cross-react with Env.

The Ontogeny of Mucosal and Systemic Antibody Responses to HIV-1 Infection

The humoral immune system plays a critical role in the clearance of numerous pathogens. In the setting of HIV-1 infection, the virus infects, integrates its genome into the host's cells, replicates, and establishes a reservoir of virus-infected cells. The initial antibody response to HIV-1 infection is targeted to non-neutralizing epitopes on HIV-1 Env gp41, and when a neutralizing response does develop months after transmission, it is specific for the autologous founder virus and the virus escapes rapidly. After continuous waves of antibody mediated neutralization and viral escape, a small subset of infected individuals eventually develop broad and potent heterologous neutralizing antibodies years after infection. In this dissertation, I have studied the ontogeny of mucosal and systemic antibody responses to HIV-1 infection by means of three distinct aims: 1. Determine the origin of the initial antibody response to HIV-1 infection. 2. Characterize the role of restricted VH and VL gene segment usage in shaping the antibody response to HIV-1 infection. 3. Determine the role of persistence of B cell clonal lineages in shaping the mutation frequencies of HIV-1 reactive antibodies.

After the introduction (Chapter 1) and methods (Chapter 2), Chapter 3 of this dissertation describes a study of the antibody response of terminal ileum B cells to HIV-1 envelope (Env) in early and chronic HIV-1 infection and provides evidence for the role of environmental antigens in shaping the repertoire of B cells that respond to HIV-1 infection. Previous work by Liao et al. demonstrated that the initial plasma cell response in the blood to acute HIV-1 infection is to gp41 and is derived from a polyreactive memory B cell pool. Many of these antibodies cross-reacted with commensal bacteria, Therefore, in Chapter 3, the relationship of intestinal B cell reactivity with commensal bacteria to HIV-1 infection-induced antibody response was probed using single B cell sorting, reverse transcription and nested polymerase chain reaction (RT- PCR) methods, and recombinant antibody technology. The dominant B cell response in the terminal ileum was to HIV-1 envelope (Env) gp41, and 82% of gp41- reactive antibodies cross-reacted with commensal bacteria whole cell lysates. Pyrosequencing of blood B cells revealed HIV-1 antibody clonal lineages shared between ileum and blood. Mutated IgG antibodies cross-reactive with both Env gp41 and commensal bacteria could also be isolated from the terminal ileum of HIV-1 uninfected individuals. Thus, the antibody response to HIV-1 can be shaped by intestinal B cells stimulated by commensal bacteria prior to HIV-1 infection to develop a pre-infection pool of memory B cells cross-reactive with HIV-1 gp41.

Chapter 4 details the study of restricted VH and VL gene segment usage for gp41 and gp120 antibody induction following acute HIV-1 infection; mutations in gp41 lead to virus enhanced neutralization sensitivity. The B cell repertoire of antibodies induced in a HIV-1 infected African individual, CAP206, who developed broadly neutralizing antibodies (bnAbs) directed to the HIV-1 envelope gp41 membrane proximal external region (MPER), is characterized. Understanding the selection of virus mutants by neutralizing antibodies is critical to understanding the role of antibodies in control of HIV-1 replication and prevention from HIV-1 infection. Previously, an MPER neutralizing antibody, CAP206-CH12, with the binding footprint identical to that of MPER broadly neutralizing antibody 4E10, that like 4E10 utilized the VH1-69 and VK3-20 variable gene segments was isolated from this individual (Morris et al., 2011). Using single B cell sorting, RT- PCR methods, and recombinant antibody technology, Chapter 4 describes the isolation of a VH1-69, Vk3-20 glycan-dependent clonal lineage from CAP206, targeted to gp120, that has the property of neutralizing a neutralization sensitive CAP206 transmitted/founder (T/F) and heterologous viruses with mutations at amino acids 680 or 681 in the MPER 4E10/CH12 binding site. These data demonstrate sites within the MPER bnAb epitope (aa 680-681) in which mutations can be selected that lead to viruses with enhanced sensitivity to autologous and heterologous neutralizing antibodies.

In Chapter 5, I have completed a comparison of evolution of B cell clonal lineages in two HIV-1 infected individuals who have a predominant VH1-69 response to HIV-1 infection--one who produces broadly neutralizing MPER-reactive mAbs and one who does not. Autologous neutralization in the plasma takes ~12 weeks to develop (Gray et al., 2007; Tomaras et al., 2008b). Only a small subset of HIV-1 infected individuals develops high plasma levels of broad and potent heterologous neutralization, and when it does occur, it typically takes 3-4 years to develop (Euler et al., 2010; Gray et al., 2007; 2011; Tomaras et al., 2011). The HIV-1 bnAbs that have been isolated to date have a number of unusual characteristics including, autoreactivity and high levels of somatic hypermutations, which are typically tightly regulated by immune control mechanisms (Haynes et al., 2005; 2012b; Kwong and Mascola, 2012; Scheid et al., 2009a). The VH mutation frequencies of bnAbs average ~15% but have been shown to be as high as 32% (reviewed in Mascola and Haynes, 2013; Kwong and Mascola, 2012). The high frequency of somatic hypermutations suggests that the B cell clonal lineages that eventually produce bnAbs undergo high-levels of affinity maturation, implying prolonged germinal center (GC) reactions and high levels of T cell help. To study the duration of HIV-1- reactive B cell clonal persistence, HIV-1 reactive and non HIV-1- reactive B cell clonal lineages were isolated from an HIV-1 infected individual that produces bnAbs, CAP206, and an HIV-1 infected individual who does not produce bnAbs, 004-0. Single B cell sorting, RT-PCR and recombinant antibody technology was used to isolate and produce monoclonal antibodies from multiple time points from each individual. B cell sequences clonally related to mAbs isolated by single cell PCR were identified within pyrosequences of longitudinal samples of these two individuals. Both individuals produced long-lived B cell clones that persisted from 0-232 weeks in CAP206, and 0-238 weeks in 004-0. The average length of persistence of clones containing members isolated from two separate time points was 91.5 weeks both individuals. Examples of the continued evolution of clonal lineages were observed in both the bnAb and non-bnAb individual. These data indicated that the ability to generate persistent and evolving B cell clonal lineages occurs in both bnAb and non-bnAb individuals, suggesting that some alternative host or viral factor is critical for the generation of highly mutated broadly neutralizing antibodies.

Together the studies described in Chapter 3-5 show that multiple factors influence the antibody response to HIV-1 infection. The initial antibody response to HIV-1 Env gp41 can be shaped by a B cell response to intestinal commensal bacteria prior to HIV-1 infection. VH and VL gene segment restriction can impact the B cell response to multiple HIV-1 antigens, and virus escape mutations in the MPER can confer enhanced neutralization sensitivity to autologous and heterologous antibodies. Finally, the ability to generate long-lived HIV-1 clonal lineages in and of itself does not confer on the host the ability to produce bnAbs.

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

Toward understanding the essence of post-translational modifications for the Mycobacterium tuberculosis immunoproteome

CD4⁺ T cells are prominent effector cells in controlling Mycobacterium tuberculosis (Mtb) infection but may also contribute to immunopathology. Studies probing the CD4⁺ T cell response from individuals latently infected with Mtb or patients with active tuberculosis using either small or proteome-wide antigen screens so far revealed a multi-antigenic, yet mostly invariable repertoire of immunogenic Mtb proteins. Recent developments in mass spectrometry-based proteomics have highlighted the occurrence of numerous types of post-translational modifications (PTMs) in proteomes of prokaryotes, including Mtb. The well-known PTMs in Mtb are glycosylation, lipidation, or phosphorylation, known regulators of protein function or compartmentalization. Other PTMs include methylation, acetylation, and pupylation, involved in protein stability. While all PTMs add variability to the Mtb proteome, relatively little is understood about their role in the anti-Mtb immune responses. Here,we reviewMtb protein PTMs and methods to assess their role in protective immunity against Mtb. © 2014 van Els, Corbière, Smits, vanGaans-van den Brink, Poelen, Mascart, Meiring and Locht.