Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component

Immunological reactivity of a human immunodeficiency virus type I derived peptide representing a consensus sequence of the GP120 major neutralizing region V3

To reduce the opportunities for human immunodeficiency virus type 1 (HIV-1) to evade vaccine induced immunity, the development of subunit vaccines must focus on the characterization of immunogenic epitopes, which are major targets for the immune system. The most dominant site for elicitation of neutralising immune response is located on the external envelope glycoprotein gp120 within the third variable domain (V3). To overcome virus type specificity of antibodies directed to the V3-domain we designed a 36 amino acids long gp120/V3-consensus peptide (V3-C36) based on published biological data and sequence comparisons of various HIV-1 virus isolates. This peptide contains a conserved core sequence which is suggested to form a surface-exposed beta-turn. This peptide also includes T-cell epitopes defined in mice and humans, an ADCC-epitope and two highly conserved cysteine residues which were oxidized to form a cystine derivate, thus allowing correct peptide folding. In ELISA-tests, this peptide reacts with at least 90% of randomly selected sera of European and African patients infected with HIV-1 and is recognized by three different HIV-1/V3 "type-specific" antisera (MN, RF, IIIB-strain). Using this peptide as immunogen in rabbits, antisera could be raised with highly cross-reactive and HIV-1/IIIB strain neutralizing properties. Moreover, HTLV/HIV-1/IIIB specific cytotoxic T-lymphocytes (CTLs) of BALB/c mice infected with a gp120 recombinant vaccinia virus recognized the central 16- and 12-mer peptides of the V3-C36 consensus peptide in cytolytic assays, indicating perfect compatibility of the consensus peptide with the IIIB-primed CTLs. The DNA-sequence encoding the V3-consensus loop region might be an important component in newly designed recombinant subunit vaccines. In addition, due to its broad serological reactivity, the V3-consensus peptide might play an important role in special diagnostic purposes

Interaction of oxygen with silver at high temperature and atmospheric pressure: A spectroscopic and structural analysis of a strongly bound surface species

X-ray photoelectron spectroscopy, ultraviolet photoelectron spectroscopy (UPS), and ion scattering spectroscopy (ISS) have been used to study the Ag(111) single-crystal surface after exposure to O2 at high temperature and at atmospheric pressure. The activated formation of a strongly bound surface layer has been observed, as identified by an asymmetry of the Ag 3d5/2 core-level peak at 367.3 eV and an O 1s peak at 529.0 eV (Oγ). In addition, oxygen was found to be dissolved in the bulk (Oβ), exhibiting an O 1s binding energy between 531 and 530 eV depending on its abundance. X-ray-excited oxygen KVV Auger electron spectroscopy revealed the presence of Oγ by additional peaks at 514.8 and 494.7 eV. UPS displayed oxygen-derived bands located above the emission from the Ag 4d band at 3.2 and 2.5 eV. Oxygen-related peaks below the Ag 4d band were identified as resulting from OH groups formed by reaction of surface oxygen (Oα) with residual hydrogen. The incorporated oxygen caused a pronounced charge separation as reflected by a 1 eV increase in the work function. ISS measurements revealed that Oγ is incorporated in the topmost surface layer, shielding underlying Ag atoms from the He+ beam. All spectroscopic data point to the presence of one monolayer of silver-embedded oxygen, which is in dynamic equilibrium with surface atomic oxygen segregated from the bulk at high temperature. The oxygen embedded in the topmost silver layer is strongly bound to the metal, with its interaction being different from adsorbed atomic oxygen and bulk Ag2O. It is stable up to 900 K, in contrast to the binary silver oxides, and relevant for high-temperature oxidation reactions catalyzed by Ag. A qualitative analysis is presented of the chemical bonding of the different surface species in comparison to the situation of a complex silver oxide reference

Electronic structure of barium-doped C₆₀

We have investigated the electronic structure of Ba-doped C60 films with Ba concentrations of up to x≈12 (BaxC60) by applying valence-band photoemission and x-ray-absorption spectroscopy. A crystal orbital (CO) formalism based on a semiempirical Hamiltonian of the intermediate-neglect-of-differential-overlap type has been employed to derive solid-state results for the Ba-doped C60 fullerides. Using x-ray diffraction, we show three distinct phases for the bulk BaxC60 system with Ba concentrations of up to x=6. In all cases, the experimental observations strongly indicate that fulleride formation leads to the occupation of hybrid bands on both sides of the Fermi level. The theoretical data indicate that the alkaline-earth atoms are essentially monovalent and hybridize strongly with the π-type functions of the C60 network. The Ba atoms in the BaxC60 fullerides deviate from the limit of complete charge transfer as a consequence of the competition between covalent Ba-C60 bonding and ionic contributions. Furthermore, it is shown that the calculated density-of-state profiles reproduce the photoemission data in the extreme outer valence-band region

Electronic Structure of the C₆₀ Fragment in Alkali- and Alkaline-earth-doped Fullerides

The electronic structure of the C60 fragment in alkali- and alkaline-earth-doped fullerides is studied theoretically. With increasing metal-to-C60 charge transfer (CT) the n electronic properties of the soccerball are changed. In the undoped solid and for not too high a concentration of doping atoms the hexagon-hexagon (6-6) bonds show sizeable double bond character while the hexagon-pentagon (6-5) bonds are essentially of single bond type. In systems with a high concentration of doping atoms this relative ordering is changed. Now the 6-5 bonds have partial double bond character and the 6-6 bonds are essentially single bonds. The high ability of the C60 unit to accomodate excess electrons prevents any sizeable weakening of the overall n bonding in systems with up to 12 excess electrons on the soccerball. A crystal orbital (CO) formalism on the basis of an INDO (intermediate neglect of differential overlap) Hamiltonian has been employed to derive solid state results for potassium- and barium-doped C60 fullerides. For both types of doping atoms an incomplete metal-to-C60 CT is predicted. In the potassium-doped fullerides the magnitude of the CT depends on the interstitial site of the dopant. The solid state data have been supplemented by INDO and ab initio calculations on molecular C60, C6-60 and C12-60. The calculated bondlength alternation in the neutral molecule is changed in C12-60 where the length of the 6-6 bonds exceeds the length of the 6-5 bonds. The geometries of the three molecular species have been optimized with a 3-21 G* basis. The theoretically derived modification of the C60 (π) electronic structure as a function of the electron count is explained microscopically in the framework of two quantum statistics accessible for π electronic ensembles. In the π ensemble of the C60 fragment so-called hard core bosonic properties are maximized where the Pauli antisymmetry principle has the character of a hidden variable only. Here the electronic degrees of freedom are attenuated only by the Pauli exclusion principle. This behaviour leads to the changes in the π electronic structure mentioned above

Second international diagnostic accuracy study for the serological detection of west nile virus infection.

Background: In recent decades, sporadic cases and outbreaks in humans of West Nile virus (WNV) infection have increased. Serological diagnosis of WNV infection can be performed by enzyme-linked immunosorbent assay (ELISA), immunoflu- orescence assay (IFA) neutralization test (NT) and by hemagglutination-inhibition assay. The aim of this study is to collect updated information regarding the performance accuracy of WNV serological diagnostics. Methodology/Principal findings: In 2011, the European Network for the Diagnostics of Imported Viral Diseases- Collaborative Laboratory Response Network (ENIVD-CLRN) organized the second external quality assurance (EQA) study for the serological diagnosis of WNV infection. A serum panel of 13 samples (included sera reactive against WNV, plus specificity and negative controls) was sent to 48 laboratories involved in WNV diagnostics. Forty-seven of 48 laboratories from 30 countries participated in the study. Eight laboratories achieved 100% of concurrent and correct results. The main obstacle in other laboratories to achieving similar performances was the cross-reactivity of antibodies amongst heterologous flaviviruses. No differences were observed in performances of in-house and commercial test used by the laboratories. IFA was significantly more specific compared to ELISA in detecting IgG antibodies. The overall analytical sensitivity and specificity of diagnostic tests for IgM detection were 50% and 95%, respectively. In comparison, the overall sensitivity and specificity of diagnostic tests for IgG detection were 86% and 69%, respectively. Conclusions/Significance: This EQA study demonstrates that there is still need to improve serological tests for WNV diagnosis. The low sensitivity of IgM detection suggests that there is a risk of overlooking WNV acute infections, whereas the low specificity for IgG detection demonstrates a high level of cross-reactivity with heterologous flaviviruse

Photophysics of polymeric carbon nitride: An optical quasimonomer

A comprehensive investigation of the luminescent properties of carbon nitride polymers, based on tri-s-triazine units, has been conducted. Steady-state temperature- and excitation-power-dependent as well as time-resolved measurements with near-UV excitation (λ=325 nm and 405 nm) yield strong photoluminescence, covering the visible spectrum. The spectral, thermal, and temporal features of the photoluminescence can be satisfactorily described by the excitation and radiative recombination of molecular excitons, localized at single tri-s-triazine units. The discussed model is in accordance with the recently reported absorption features of carbon nitride polymers. Thus, from the point of view of optical spectroscopy, the material effectively behaves as a monomer

An optical quasimonomer

A comprehensive investigation of the luminescent properties of carbon nitride polymers, based on tri-s-triazine units, has been conducted. Steady-state temperature- and excitation-power-dependent as well as time-resolved measurements with near-UV excitation (λ=325 nm and 405 nm) yield strong photoluminescence, covering the visible spectrum. The spectral, thermal, and temporal features of the photoluminescence can be satisfactorily described by the excitation and radiative recombination of molecular excitons, localized at single tri-s-triazine units. The discussed model is in accordance with the recently reported absorption features of carbon nitride polymers. Thus, from the point of view of optical spectroscopy, the material effectively behaves as a monomer