Low Zika virus seroprevalence among pregnant women in North Central Nigeria, 2016.

BACKGROUND: Zika virus (ZIKV) has been known for decades in Africa but contemporary data is lacking at large. OBJECTIVES: To describe the seroepidemiology of ZIKV in North Central Nigeria. STUDY DESIGN: We performed a cross-sectional study at six health care facilities in North Central Nigeria from January to December 2016. Detection of ZIKV antibodies was done using an anti-ZIKV recombinant non-structural protein 1 (NS1)-based ELISA. A colorimetric assay to detect ZIKV neutralizing antibodies was used on ELISA reactive and randomly selected ELISA non-reactive samples. ZIKV real-time RT-PCR was done on a subset of samples. RESULTS: A total of 468 individual samples were included with almost 60% from pregnant women. Using NS1-based ELISA, an anti-ZIKV positive rate of 6% for IgM and 4% for IgG was found. Pregnant women showed anti-ZIKV positive rates of 4% for IgM and 3% for IgG. None of the ZIKV antibody positive samples tested ZIKV RT-PCR positive. An association with male sex was found for anti-ZIKV IgG ELISA positivity (prevalence ratio 3.49; 95% confidence interval: 1.48-8.25; p = .004). No association with pregnancy, yellow fever vaccination or malaria was found for anti-ZIKV IgM or IgG positivity. ZIKV neutralizing antibodies were detected in 17/18 (94%) anti-ZIKV NS1 positive/borderline samples and in one sample without detectable ZIKV NS1 antibodies. Partial ZIKV E gene sequence was retrieved in one sample without ZIKV antibodies, which clustered within the West African ZIKV lineage. CONCLUSIONS: Our results show a largely ZIKV immunologically naïve population and reinforce the importance of ZIKV surveillance in Africa.

Enhanced immunogenicity of pneumococcal surface adhesin A (PsaA) in mice via fusion to recombinant human B lymphocyte stimulator (BLyS).

BACKGROUND: B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of ligands that mediates its action through three known receptors. BLyS has been shown to enhance the production of antibodies against heterologous antigens when present at elevated concentrations, supporting an immunostimulatory role for BLyS in vivo. METHODS: We constructed a fusion protein consisting of human BLyS and Pneumococcal Surface Adhesin A (PsaA) and used this molecule to immunize mice. The immunostimulatory attributes mediated by BLyS in vivo were evaluated by characterizing immune responses directed against PsaA. RESULTS: The PsaA-BLyS fusion protein was able to act as a co-stimulant for murine spleen cell proliferation induced with F(ab')2 fragments of anti-IgM in vitro in a fashion similar to recombinant BLyS, and immunization of mice with the PsaA-BLyS fusion protein resulted in dramatically elevated serum antibodies specific for PsaA. Mice immunized with PsaA admixed with recombinant BLyS exhibited only modest elevations in PsaA-specific responses following two immunizations, while mice immunized twice with PsaA alone exhibited undetectable PsaA-specific serum antibody responses. Sera obtained from PsaA-BLyS immunized mice exhibited high titers of IgG1, IgG2a, IgG2b, and IgG3, but no IgA, while mice immunized with PsaA admixed with BLyS exhibited only elevated titers of IgG1 following two immunizations. Splenocytes from PsaA-BLyS immunized mice exhibited elevated levels of secretion of IL-2, IL-4 and IL-5, and a very modest but consistent elevation of IFN-γ following in vitro stimulation with PsaA. In contrast, mice immunized with either PsaA admixed with BLyS or PsaA alone exhibited modestly elevated to absent PsaA-specific recall responses for the same cytokines. Mice deficient for one of the three receptors for BLyS designated Transmembrane activator, calcium modulator, and cyclophilin ligand [CAML] interactor (TACI) exhibited attenuated PsaA-specific serum antibody responses following immunization with PsaA-BLyS relative to wild-type littermates. TACI-deficient mice also exhibited decreased responsiveness to a standard pneumococcal conjugate vaccine. CONCLUSION: This study identifies covalent attachment of BLyS as a highly effective adjuvant strategy that may yield improved vaccines. In addition, this is the first report demonstrating an unexpected role for TACI in the elicitation of antibodies by the PsaA-BLyS fusion protein. REVIEWERS: This article was reviewed by Jonathan Yewdell, Rachel Gerstein, and Michael Cancro (nominated by Andy Caton).

Cell stress proteins: novel immunotherapeutics.

Heat shock proteins (HSPs) play important roles in the immune system as carriers of tumour antigens and inflammatory agents. The HSPs are abundantly expressed stress proteins intrinsic to all cellular life, permitting proteins to carry out essential enzymic, signalling and structural functions within the tightly crowded milieu of the cell. To carry out these tasks, HSPs are equipped with a domain that binds unstructured sequences in polypeptides and a N-terminal ATPase domain that controls the opening and closing of the peptide-binding domain. HSPs can, using these domains, capture antigens processed by partial proteolysis in the cytoplasm of cancer cells. HSP/peptide complexes formed in the cytoplasm can then be secreted to take part in immune surveillance. Extracellular Hsp70 interacts with receptors on antigen presenting cells (APCs) either during episodes of cell death and lysis in vivo or during vaccination. A number of candidate receptors for Hsp70 on APCs have been proposed to take part in the antitumour immune function including the alpha2 macroglobulin receptor CD91, Toll-like receptors, the signalling receptor CD40 and a number of scavenger receptors. Finally, Hsp70 complexes are able to deliver antigens to MHC class I and II molecules on the APC cell surface and lead to the presentation of tumour antigens to T lymphocytes. HSP-antigen complexes have proven effective in the treatment of rodent tumours in preclinical studies and are now undergoing clinical trials for treatment of human cancer.

Extracellular heat shock proteins in cell signaling and immunity.

Extracellular stress proteins including heat shock proteins (Hsps) and glucose-regulated proteins (Grps) are emerging as important mediators of intercellular signaling and transport. Release of such proteins from cells is triggered by physical trauma and behavioral stress as well as exposure to immunological "danger signals." Stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis. After release into the extracellular fluid, Hsp or Grp may then bind to the surfaces of adjacent cells and initiate signal transduction cascades as well as the transport of cargo molecules, such as antigenic peptides. In addition, Hsp60 and Hsp70 are able to enter the bloodstream and may possess the ability to act at distant sites in the body. Many of the effects of extracellular stress proteins are mediated through cell-surface receptors. Such receptors include toll- like receptors (TLRs) 2 and 4, CD40, CD91, CCR5, and members of the scavenger receptor family, such as LOX-1 and SREC-1. The possession of a wide range of receptors for the Hsp and Grp family permits binding to a diverse range of cells and the performance of complex multicellular functions particularly in immune cells and neurons.

Mechanisms for Hsp70 secretion: crossing membranes without a leader.

Heat shock protein 70 (Hsp70) is released from cells of many types and plays a significant signaling role, particularly in the inflammatory and immune responses. However, Hsp70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. However, Hsp70 can be released from cells by active mechanism that are independent of de novo Hsp70 synthesis or cell death. This pathway is similar to one utilized by the leaderless protein interleukin 1beta. Hsp70 release involves transit through an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of Hsp70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of Hsp70 into this secretory compartment appears to involve the ABC-family transporter proteins. While the cell signals involved in triggering Hsp70 release through this lysosomal pathway are largely unknown, recent data suggest a regulatory role for extracellular ATP. These mechanisms are also shared by interleukin 1beta secretion. Following release it has been shown that Hsp70 binds to adjacent cells, suggesting that the secreted protein participates in paracrine or autocrine interactions with adjacent cell surfaces. Thus an outline is beginning to of the mechanisms of Hsp70 secretion. Much further study will be required to fully elucidate mechanisms involved in targeting Hsp70 towards the non-canonical secretion pathways and its regulation.

Extracellular heat shock proteins in cell signaling.

Extracellular stress proteins including heat shock proteins (Hsp) and glucose regulated proteins (Grp) are emerging as important mediators of intercellular signaling and transport. Release of such proteins from cells is triggered by physical trauma and behavioral stress as well as exposure to immunological "danger signals". Stress protein release occurs both through physiological secretion mechanisms and during cell death by necrosis. After release into the extracellular fluid, Hsp or Grp may then bind to the surfaces of adjacent cells and initiate signal transduction cascades as well as the transport of cargo molecules such as antigenic peptides. In addition Hsp60 and hsp70 are able to enter the bloodstream and may possess the ability to act at distant sites in the body. Many of the effects of extracellular stress proteins are mediated through cell surface receptors. Such receptors include Toll Like Receptors 2 and 4, CD40, CD91, CCR5 and members of the scavenger receptor family such as LOX-1 and SREC-1. The possession of a wide range of receptors for the Hsp and Grp family permits binding to a diverse range of cells and the performance of complex multicellular functions particularly in immune cells and neurones.

Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes.

Heat shock protein (HSP)70 can be released from tumor cells and stimulate a potent antitumor immune response. However, HSP70 does not contain a consensus secretory signal and thus cannot traverse the plasma membrane by conventional mechanisms. We have observed HSP70 release from intact human prostate carcinoma cell lines (PC-3 and LNCaP) by a mechanism independent of de novo HSP70 synthesis or cell death. This pathway is similar to one used by the leaderless protein IL-1beta. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1beta secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system.

Heat induced release of Hsp70 from prostate carcinoma cells involves both active secretion and passive release from necrotic cells.

PURPOSE: Heat shock protein 70 (HSP70) is released from tumour cells and stimulates a potent anti-tumour immune response. METHODS: This study examined the role of hyperthermia, including heating conditions from the fever range, the hyperthermia range and the thermal ablation range, in HSP70 release from prostate carcinoma cells. It has observed HSP70 release from human prostate carcinoma cell lines (PC-3 and LNCaP) treated with hyperthermia. RESULTS: The effects of hyperthermia were complex and appeared to involve at least two mechanisms for HSP70 release. Hyperthermia at 40 degrees C strongly stimulated HSP70 release by an active secretion pathway. However, as temperatures were increased, this rapid secretion pathway became progressively inhibited and by a temperature of 55 degrees C, active secretion was abolished. However, when cells exposed to these heating conditions were allowed to recover at 37 degrees C for 24 h after heating, HSP70 release was observed at the high ablation temperature range and this appeared to be related to a concomitant damage to the plasma membrane. CONCLUSIONS: Thus, at least two mechanisms contribute to HSP70 release during hyperthermia and the relative contribution from each pathway depends on the temperature conditions.

Evaluation of antibody responses elicited by immunization of mice with a pneumococcal antigen genetically fused to murine HSP70 and murine interleukin-4.

The heat shock (stress) protein HSP70 has been shown to be a potent stimulator of cellular immune responses. In order to determine whether HSP70 has the ability to stimulate antibody responses, we constructed and expressed fusion proteins consisting of murine HSP70 or murine interleukin (IL)-4 covalently linked to a pneumococcal cell wall-associated protein antigen designated PpmA. Immunization of mice with the PpmA-HSP70 fusion protein (PpmA-70) failed to elicit an increased PpmA-specific serum antibody response. In contrast, mice immunized with PpmA fused to IL-4 (PpmA-IL4), or PpmA fused to both IL-4 and HSP70 (PpmA-IL4-70) fusion proteins elicited high levels of PpmA-specific antibody responses. These data suggest that HSP70 has a limited capacity to stimulate immune responses to heterologous antigens in vivo.

Extracellular HSP70 binding to surface receptors present on antigen presenting cells and endothelial/epithelial cells.

Extracellular HSP70 has been found to participate in both innate and adaptive immune responses. However, little is known about the molecular mechanisms that mediate this process. Previous reports suggest that HSP70 interacts with antigen presenting cells (APC) through a plethora of surface receptors. In this study, we have examined the relative binding of potential HSP70 receptors and found high affinity binding to LOX-1 but not other structures with a role in HSP70-APC interactions such as LRP/CD91, CD40, TLR2, TLR4 or another c-type lectin family member (DC-SIGN) closely related to LOX-1. In addition to APC, HSP70 can avidly bind to non-APC cell lines, especially those from epithelial or endothelial background.

The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo.

In vitro studies have indicated the importance of Toll-like receptor (TLR) signaling in response to the fungal pathogens Candida albicans and Aspergillus fumigatus. However, the functional consequences of the complex interplay between fungal morphogenesis and TLR signaling in vivo remain largely undefined. In this study we evaluate the impact of the IL-1R/TLR/myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway on the innate and adaptive Th immunities to C. albicans and A. fumigatus in vivo. It was found that 1) the MyD88-dependent pathway is required for resistance to both fungi; 2) the involvement of the MyD88 adapter may occur through signaling by distinct members of the IL-1R/TLR superfamily, including IL-1R, TLR2, TLR4, and TLR9, with the proportional role of the individual receptors varying depending on fungal species, fungal morphotypes, and route of infection; 3) individual TLRs and IL-1R activate specialized antifungal effector functions on neutrophils, which correlates with susceptibility to infection; and 4) MyD88-dependent signaling on dendritic cells is crucial for priming antifungal Th1 responses. Thus, the finding that the innate and adaptive immunities to C. albicans and A. fumigatus require the coordinated action of distinct members of the IL-1R/TLR superfamily acting through MyD88 makes TLR manipulation amenable to the induction of host resistance to fungi.

The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism.

Amphotericin B is the most effective drug for treating many life-threatening fungal infections. Amphotericin B administration is limited by infusion-related toxicity, including fever and chills, an effect postulated to result from proinflammatory cytokine production by innate immune cells. Because amphotericin B is a microbial product, we hypothesized that it stimulates immune cells via Toll-like receptors (TLRs) and CD14. We show here that amphotericin B induces signal transduction and inflammatory cytokine release from cells expressing TLR2 and CD14. Primary murine macrophages and human cell lines expressing TLR2, CD14, and the adapter protein MyD88 responded to amphotericin B with NF-kappaB-dependent reporter activity and cytokine release, whereas cells deficient in any of these failed to respond. Cells mutated in TLR4 were less responsive to amphotericin B stimulation than cells expressing normal TLR4. These data demonstrate that TLR2 and CD14 are required for amphotericin B-dependent inflammatory stimulation of innate immune cells and that TLR4 may also provide stimulation of these cells. Our results provide a putative molecular basis for inflammatory responses elicited by amphotericin B and suggest strategies to eliminate the acute toxicity of this drug.

Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening infections in patients with qualitative and quantitative defects in phagocytic function. Here, we examined the contribution of Toll-like receptor (TLR)-2, TLR4, the adapter protein MyD88, and CD14 to signaling in response to the three forms of A. fumigatus encountered during human disease: resting conidia (RC), swollen conidia (SC), and hyphae (H). Compared with elicited peritoneal macrophages obtained from wild-type and heterozygous mice, TLR2(-/-) and MyD88(-/-) macrophages produced significantly less tumor necrosis factor-alpha (TNFalpha) following A. fumigatus stimulation. In contrast, following stimulation with RC, SC, and H, TLR4(-/-) and CD14(-/-) macrophages exhibited no defects in tumor necrosis factor-alpha release. TLR2(-/-), TLR4(-/-), MyD88(-/-), and CD14(-/-) macrophages bound similar numbers of RC and SC compared with wild-type macrophages. RC, SC, and H stimulated greater activation of a nuclear factor kappa B (NFkappaB)-dependent reporter gene and greater release of tumor necrosis factor-alpha from the human monocytic THP-1 cell line stably transfected with CD14 compared with control cells stably transfected with empty vector. A. fumigatus stimulated NFkappaB-dependent reporter gene activity in the human embryonic kidney cell line, HEK293, only if the cells were transfected with TLR2. Moreover, activity increased when TLR2 and CD14 were co-transfected. Taken together, these data suggest that optimal signaling responses to A. fumigatus require TLR2 in both mouse and human cells. In contrast, a role for CD14 was found only in the human cells. MyD88 acts as a central adapter protein mediating signaling responses following stimulation with RC, SC, and H.