Iyadh Douagi

Soluble HIV-1 Env trimers in adjuvant elicit potent and diverse functional B cell responses in primates

Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoproteins (Envs) have proven difficult to elicit by immunization. Therefore, to identify effective Env neutralization targets, efforts are underway to define the specificities of bNAbs in chronically infected individuals. For a prophylactic vaccine, it is equally important to define the immunogenic properties of the heavily glycosylated Env in healthy primates devoid of confounding HIV-induced pathogenic factors. We used rhesus macaques to investigate the magnitude and kinetics of B cell responses stimulated by Env trimers in adjuvant. Robust Env-specific memory B cell responses and high titers of circulating antibodies developed after trimer inoculation. Subsequent immunizations resulted in significant expansion of Env-specific IgG-producing plasma cell populations and circulating Abs that displayed increasing avidity and neutralization capacity. The neutralizing activity elicited with the regimen used was, in most aspects, superior to that elicited by a regimen based on monomeric Env immunization in humans. Despite the potency and breadth of the trimer-elicited response, protection against heterologous rectal simian-HIV (SHIV) challenge was modest, illustrating the challenge of eliciting sufficient titers of cross-reactive protective NAbs in mucosal sites. These data provide important information for the design and evaluation of vaccines aimed at stimulating protective HIV-1 immune responses in humans.

Human B Cell Responses to TLR Ligands Are Differentially Modulated by Myeloid and Plasmacytoid Dendritic Cells

Selected TLR ligands are under evaluation as vaccine adjuvants and are known to activate dendritic cells (DCs) and B cells to affect vaccine-induced Ab responses. However, the relative contribution of the two main human DC subsets, myeloid (MDCs) and plasmacytoid (PDCs), in supporting B cell responses to TLR ligands remains poorly understood. We found that PDCs but not MDCs markedly enhanced B cell proliferation in response to TLR7/8-L, an imidazoquinoline derivative, and to a lesser extent to TLR9 ligands (CpG ODN classes A, B, and C). PDCs strongly enhanced TLR7/8-L-induced proliferation of both memory and naive B cells but were only able to support memory cells to differentiate to CD27high plasmablasts. In response to TLR7/8 stimulation, PDCs mediated the up-regulation of transcription factors B lymphocyte-induced maturation protein 1 and X-box binding protein 1 and enhanced differentiation of B cells into IgM-, IgG-, and IgA-producing cells. Type I IFN produced to high levels by PDCs was the principal mediator of the effects on TLR7/8 stimulation. Although MDCs expressed higher levels of the known B cell growth factors IL-6, IL-10, and B cell-activating factor in response to TLR7/8 stimulation, they were unable to enhance B cell responses in this system. These data help decipher the different roles of PDCs and MDCs for modulating human B cell responses and can contribute to selection of specific TLR ligands as vaccine adjuvants.

Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein

ABSTRACT Currently there is limited information about the quality of immune responses elicited by candidate human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-based immunogens in primates. Here we describe a comprehensive analysis of neutralizing antibody and T-cell responses obtained in cynomolgus macaques by three selected immunization regimens. We used the previously described YU2-based gp140 protein trimers administered in an adjuvant, preceded by two distinct priming strategies: either alphavirus replicon particles expressing matched gp140 trimers or gp120 core proteins stabilized in the CD4-bound conformation. The rationale for priming with replicon particles was to evaluate the impact of the expression platform on trimer immunogenicity. The stable core proteins were chosen in an attempt to expand selectively lymphocytes recognizing common determinants between the core and trimers to broaden the immune response. The results presented here demonstrate that the platform by which Env trimers were delivered in the priming (either protein or replicon vector) had little impact on the overall immune response. In contrast, priming with stable core proteins followed by a trimer boost strikingly focused the T-cell response on the core sequences of HIV-1 Env. The specificity of the T-cell response was distinctly different from that of the responses obtained in animals immunized with trimers alone and was shown to be mediated by CD4+ T cells. However, this regimen showed limited or no improvement in the neutralizing antibody responses, suggesting that further immunogen design efforts are required to successfully focus the B-cell response on conserved neutralizing determinants of HIV-1 Env.

Early Alpha/Beta Interferon Production by Myeloid Dendritic Cells in Response to UV-Inactivated Virus Requires Viral Entry and Interferon Regulatory Factor 3 but Not MyD88

ABSTRACT Alpha/beta interferons (IFN-α/β) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-α/β are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-α/β production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-α/β in mDCs while fusion-defective virus did not induce IFN-α/β. The response to replication-defective virus was largely intact in MyD88−/− mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-α/β in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-α/β in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

Selective Expansion of HIV-1 Envelope Glycoprotein-Specific B Cell Subsets Recognizing Distinct Structural Elements Following Immunization

The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine. Furthermore, serological analyses do not provide information regarding how repeated Ag inoculation impacts the expansion and maintenance of Env-specific B cell subpopulations. Therefore, we developed a highly sensitive Env-specific B cell ELISPOT system, which allows the enumeration of Ab-secreting cells (ASC) from diverse anatomical compartments directed against different structural determinants of Env. In this study, we use this system to examine the evolution of B cell responses in mice immunized with engineered Env trimers in adjuvant. We demonstrate that the relative proportion of ASC specific for defined structural elements of Env is altered significantly by homologous booster immunizations. This results in the selective expansion of ASC directed against the variable regions of Env. We suggest that the B cell specificity and compartment analysis described in this study are important complements to serological mapping studies for the examination of B cell responses against subspecificities of a variety of immunogens.

Analysis of the DNA methylome and transcriptome in granulopoiesis reveals timed changes and dynamic enhancer methylation.

In development, epigenetic mechanisms such as DNA methylation have been suggested to provide a cellular memory to maintain multipotency but also stabilize cell fate decisions and direct lineage restriction. In this study, we set out to characterize changes in DNA methylation and gene expression during granulopoiesis using 4 distinct cell populations ranging from the oligopotent common myeloid progenitor stage to terminally differentiated neutrophils. We observed that differentially methylated sites (DMSs) generally show decreased methylation during granulopoiesis. Methylation appears to change at specific differentiation stages and overlap with changes in transcription and activity of key hematopoietic transcription factors. DMSs were preferentially located in areas distal to CpG islands and shores. Also, DMSs were overrepresented in enhancer elements and enriched in enhancers that become active during differentiation. Overall, this study depicts in detail the epigenetic and transcriptional changes that occur during granulopoiesis and supports the role of DNA methylation as a regulatory mechanism in blood cell differentiation.

Crimean-Congo hemorrhagic fever virus activates endothelial cells

ABSTRACT Crimean-Congo hemorrhagic fever virus (CCHFV) causes viral hemorrhagic fever with high case-fatality rates and is geographically widely distributed. Due to the requirement for a biosafety level 4 (BSL-4) laboratory and the lack of an animal model, knowledge of the viral pathogenesis is limited. Crimean-Congo hemorrhagic fever (CCHF) is characterized by hemorrhage and vascular permeability, indicating the involvement of endothelial cells (ECs). The interplay between ECs and CCHFV is therefore important for understanding the pathogenesis of CCHF. In a previous study, we found that CCHFV-infected monocyte-derived dendritic cells (moDCs) activated ECs; however, the direct effect of CCHFV on ECs was not investigated. Here, we report that ECs are activated upon infection, as demonstrated by upregulation of mRNA levels for E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Protein levels and cell surface expression of ICAM1 responded in a dose-dependent manner to increasing CCHFV titers with concomitant increase in leukocyte adhesion. Furthermore, we examined vascular endothelial (VE) cadherin in CCHFV-infected ECs by different approaches. Infected ECs released higher levels of interleukin 6 (IL-6) and IL-8; however, stimulation of resting ECs with supernatants derived from infected ECs did not result in increased ICAM1 expression. Interestingly, the moDC-mediated activation of ECs was abrogated by addition of neutralizing tumor necrosis factor alpha (TNF-α) antibody to moDC supernatants, thereby identifying this soluble mediator as the key cytokine causing EC activation. We conclude that CCHFV can exert both direct and indirect effects on ECs.

Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

ABSTRACT The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.

Increased fat cell size: a major phenotype of subcutaneous white adipose tissue in non-obese individuals with type 2 diabetes

Aims/hypothesisWe aimed to elucidate the impact of fat cell size and inflammatory status of adipose tissue on the development of type 2 diabetes in non-obese individuals.MethodsWe characterised subcutaneous abdominal adipose tissue by examining stromal cell populations by 13 colour flow cytometry, measuring expression of adipogenesis genes in the progenitor cell fraction and determining lipolysis and adipose secretion of inflammatory proteins in 14 non-obese men with type 2 diabetes and 13 healthy controls matched for age, sex, body weight and total fat mass.ResultsIndividuals with diabetes had larger fat cells than the healthy controls but stromal cell population frequencies, adipose lipolysis and secretion of inflammatory proteins did not differ between the two groups. However, in the entire cohort fat cell size correlated positively with the ratio of M1/M2 macrophages, TNF-α secretion, lipolysis and insulin resistance. Expression of genes encoding regulators of adipogenesis and adipose morphology (BMP4, CEBPα [also known as CEBPA], PPARγ [also known as PPARG] and EBF1) correlated negatively with fat cell size.Conclusions/interpretationWe show that a major phenotype of white adipose tissue in non-obese individuals with type 2 diabetes is adipocyte hypertrophy, which may be mediated by an impaired adipogenic capacity in progenitor cells. Consequently, this could have an impact on adipose tissue inflammation, release of fatty acids, ectopic fat deposition and insulin sensitivity.

Crimean Congo hemorrhagic fever virus infects human monocyte-derived dendritic cells.

For some patients infection with Crimean Congo hemorrhagic fever virus (CCHFV) causes a severe disease characterized by fever, vascular leakage and coagulopathy. Knowledge of CCHF pathogenesis is limited and today there is no information about the specific target cells of CCHFV. In this study we analyzed the permissiveness of human peripheral blood mononuclear cells (PBMCs) including monocyte-derived dendritic cells (moDCs) to CCHFV infection. Interestingly, we found that moDCs are the most permissive to CCHFV infection and this infection induced cytokine release from moDCs. Furthermore, supernatants from infected moDCs were found to activate human endothelial cells.