We have characterized a glycosylated, 31 amino‐acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus ...glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino‐acid precursor containing an N‐terminal signal peptide and Arg‐Lys at the C‐terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C‐terminal parts of mature DUP99B and D. melanogaster sex‐peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex‐peptide (increased oviposition, reduced receptivity). These effects are also induced by de‐glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex‐peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex‐peptide gene family.
Non-human primates, such as the rhesus macaques, are the preferred model for down-selecting human malaria vaccine formulations, but the rhesus model is expensive and does not allow for direct ...efficacy testing of human malaria vaccines. Transgenic rodent parasites expressing genes of human Plasmodium are now routinely used for efficacy studies of human malaria vaccines. Mice have however rarely predicted success in human malaria trials and there is scepticism whether mouse studies alone are sufficient to move a vaccine candidate into the clinic.
A comparison of immunogenicity, fine-specificity and functional activity of two Alum-adjuvanted Plasmodium falciparum circumsporozoite protein (CSP)-based vaccines was conducted in mouse and rhesus models. One vaccine was a soluble recombinant protein (CSP) and the other was the same CSP covalently conjugated to the Qβ phage particle (Qβ-CSP).
Mice showed different kinetics of antibody responses and different sensitivity to the NANP-repeat and N-terminal epitopes as compared to rhesus. While mice failed to discern differences between the protective efficacy of CSP versus Qβ-CSP vaccine following direct challenge with transgenic Plasmodium berghei parasites, rhesus serum from the Qβ-CSP-vaccinated animals induced higher in vivo sporozoite neutralization activity.
Despite some immunologic parallels between models, these data demonstrate that differences between the immune responses induced in the two models risk conflicting decisions regarding potential vaccine utility in humans. In combination with historical observations, the data presented here suggest that although murine models may be useful for some purposes, non-human primate models may be more likely to predict the human response to investigational vaccines.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Interleukin-1β (IL-1β) is a key cytokine involved in inflammatory illnesses including rare hereditary diseases and common chronic inflammatory conditions as gout, rheumatoid arthritis, and type 2 ...diabetes mellitus, suggesting reduction of IL-1β activity as new treatment strategy. The objective of our study was to assess safety, antibody response, and preliminary efficacy of a novel vaccine against IL-1β. The vaccine hIL1bQb consisting of full-length, recombinant IL-1β coupled to virus-like particles was tested in a preclinical and clinical, randomized, placebo-controlled, double-blind study in patients with type 2 diabetes. The preclinical simian study showed prompt induction of IL-1β-specific antibodies upon vaccination, while neutralizing antibodies appeared with delay. In the clinical study with 48 type 2 diabetic patients, neutralizing IL-1β-specific antibody responses were detectable after six injections with doses of 900 µg. The development of neutralizing antibodies was associated with higher number of study drug injections, lower baseline body mass index, improvement of glycemia, and C-reactive protein (CRP). The vaccine hIL1bQb was safe and well-tolerated with no differences regarding adverse events between patients receiving hIL1bQb compared to placebo. This is the first description of a vaccine against IL-1β and represents a new treatment option for IL-1β-dependent diseases such as type 2 diabetes mellitus (ClinicalTrials.gov NCT00924105).
Adeno‐associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and ...Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G1 and G2, with elevated CDK inhibitor p21 and reduced cyclin E‐, A‐ and B1‐associated kinase activity. Rep78‐expressing cells were also impaired in S‐phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C‐terminal zinc finger domain of Rep78. Rep78‐induced S‐phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb−/− primary mouse embryonic fibroblasts displayed a strongly reduced S‐phase arrest when challenged with Rep78, compared with matched Rb+/+ controls. These results suggest that physiological levels of active pRb can interfere with S‐phase progression. We propose that the AAV Rep78 protein arrests cells within S‐phase by a novel mechanism involving the ectopic accumulation of active pRb.
Protective Ab levels can be maintained for years upon infection or vaccination. In this study, we studied the duration of Ab responses as a function of the life span of plasma cells and tested the ...role of persisting Ag in maintaining B cell memory. Our analysis of B cell responses induced in mice immunized with virus-like particles demonstrates the following: 1) Ab titers are long-lived, but decline continuously with a t(1/2) of approximately 80 days, which corresponds to the life span of plasma cells; 2) the germinal center (GC) reaction, which lasts for up to 100 days, is dependent on Ag associated with follicular dendritic cells; and 3) early GCs produce massive numbers of plasma and memory B cell precursors, whereas the late Ag-dependent GCs are dispensable for the maintenance of Ab levels and B cell memory.
Innate stimuli, such as TLR ligands, are known to greatly facilitate cross-priming. Currently it is unclear whether innate stimuli enhance cross-priming at the level of cross-presentation or at the ...level of T-cell priming. In this study, we addressed this question by measuring cross-presentation as well as cross-priming by virus-like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus-like particles or using mice deficient in two key molecules of TLR-signaling, namely the adaptor molecule MyD88 as well as IFN-α/β receptor. While efficient cross-presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand-mediated signals, T-cell priming was abolished. Thus, innate stimuli regulate cross-priming at the level of DC licensing for T-cell activation and not antigen presentation.
Circumsporozoite protein (CSP) of Plasmodium falciparum is a promising malaria vaccine target. RTS,S, the most advanced malaria vaccine candidate consists of the central NANP repeat and ...carboxy-terminal region of CSP displayed on a hepatitis B virus-like particle (VLP). To build upon the success of RTS,S, we produced a near full-length Plasmodium falciparum CSP that also includes the conserved amino-terminal region of CSP. We recently showed that this soluble CSP, combined with a synthetic Toll-like-receptor-4 (TLR4) agonist in stable oil-in-water emulsion (GLA/SE), induces a potent and protective immune response in mice against transgenic parasite challenge. Here we have investigated whether the immunogenicity of soluble CSP could be further augmented by presentation on a VLP. Bacteriophage QBeta VLPs can be readily produced in E.coli, they have a diameter of 25 nm and contain packaged E. coli RNA which serves as a built in adjuvant through the activation of TLR7/8. CSP was chemically conjugated to QBeta and the CSP-QBeta vaccine immunogenicity and efficacy were compared to adjuvanted soluble CSP in the C57Bl/6 mouse model. When formulated with adjuvants lacking a TLR4 agonist (Alum, SE and Montanide) the QBeta-CSP induced higher anti-NANP repeat titers, higher levels of cytophilic IgG2b/c antibodies and a trend towards higher protection against transgenic parasite challenge as compared to soluble CSP formulated in the same adjuvant. The VLP and soluble CSP immunogenicity difference was most pronounced at low antigen dose, and within the CSP molecule, the titers against the NANP repeats were preferentially enhanced by QBeta presentation. While a TLR4 agonist enhanced the immunogenicity of soluble CSP to levels comparable to the VLP vaccine, the TLR4 agonist did not further improve the immunogenicity of the QBeta-CSP vaccine. The data presented here pave the way for further improvement in the QBeta conjugation chemistry and evaluation of both the QBeta-CSP and soluble CSP vaccines in the non-human primate model.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The contribution of Toll-like receptor (TLR) signaling to T cell-dependent (TD) antibody responses was assessed by using mice lacking the TLR signaling adaptor MyD88 in individual cell types. When a ...soluble TLR9 ligand was used as adjuvant for a protein antigen, MyD88 was required in dendritic cells but not in B cells to enhance the TD antibody response, regardless of the inherent immunogenicity of the antigen. In contrast, a TLR9 ligand contained within a virus-like particle substantially augmented the TD germinal center IgG antibody response, and this augmentation required B cell MyD88. The ability of B cells to discriminate between antigens based the physical form of a TLR ligand likely reflects an adaptation to facilitate strong anti-viral antibody responses.
The cover shows a colour series of mouse footpads injected with small fluorescent nanoparticles. The image depicts the afferent lymphatic vessels connecting the injection site with the draining ...popliteal lymph node and the more distal sciatic lymph node. Lymphatic vessels (in green in the left row) run together with the veins (darker curves) and carry free draining antigen to the lymph nodes. This image was provided by the authors of Manolova (pp.
1404–1413)
, in which it is demonstrated that particle size critically influences the mode of antigen transport to the draining lymph nodes; DC are strictly required for transport of large (500–2000 nm) particles, whereas smaller particles (20–200 nm), such as virus‐like particles (30 nm), drain freely.
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