Collective influencers in protein interaction networks.

Recent research increasingly shows the relevance of network based approaches for our understanding of biological systems. Analyzing human protein interaction networks, we determined collective influencers (CI), defined as network nodes that damage the integrity of the underlying networks to the utmost degree. We found that CI proteins were enriched with essential, regulatory, signaling and disease genes as well as drug targets, indicating their biological significance. Also by focusing on different organisms, we found that CI proteins had a penchant to be evolutionarily conserved as CI proteins, indicating the fundamental role that collective influencers in protein interaction networks plays for our understanding of regulation, diseases and evolution.

Induction of cytolytic T lymphocytes directed towards the V3 loop of the human immunodeficiency virus type 1 external glycoprotein gp120 by p55gag/V3 chimeric vaccinia viruses.

T cell-mediated cytotoxicity may play an important role in controlling infection by human immunodeficiency virus (HIV). In order to study the ability of rationally designed antigens to induce cytolytic T lymphocytes (CTLs) we replaced stretches of 30 to 50 amino acids at the p17-MA/p24-CA cleavage site, within the p24-CA moiety and within the p6-LI portion of the HIV type 1 p55gag precursor by the third variable domain (V3) of the external glycoprotein gp120. This site is known to be a target for CTL attack in mice and humans. The chimeric antigens were recombined into highly attenuated vaccinia viruses in order to investigate class I major histocompatibility complex (MHC)-restricted presentation of antigenic V3 peptides. Immunoprecipitation and Western blot analysis of the group-specific antigen (p55gag)/V3 chimeric proteins demonstrated significant differences in the accessibility of the V3 domain for a monoclonal antibody or polyclonal V3-specific antisera, depending on the position of the V3 loop within the p55gag carrier protein. Immunization of BALB/c mice with three variants of p55gag/V3 recombinant vaccinia virus, however, resulted in a comparable priming of CD4-CD8+ CTLs in vivo irrelevant of the position of the V3 loop within p55gag. Local conformational changes, including the V3 domain within the p55gag/V3 chimeras, did not demonstrate a significant effect on V3-specific lysis of the target cells when compared to the authentic gp120 envelope protein. Class I MHC-restricted CTLs induced by a V3 consensus sequence cross-reacted perfectly with the LAI strain-derived V3 loop sequence. These data indicate that the combination of selected epitopes (V3) with immunologically relevant complex carrier proteins (p55gag) can be accomplished without the loss of biological activity.

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.

Histopathological studies on the local reactions induced by complete Freund's adjuvant (CFA), bacterial lipopolysaccharide (LPS), and synthetic lipopeptide (P3C) conjugates.

The inflammatory reactions following subcutaneous application of adjuvants revealed characteristic pathological patterns. The injection of complete Freund's adjuvant (CFA) resulted in the formation of large lipid deposits encircled by an inflammatory reaction and concentrically arranged collagen bundles. Bacterial lipopolysaccharide (LPS) caused granulomatous aggregations of mononuclear cells with thrombotic vessel occlusions. Inoculation of the lipopeptide adjuvants induced accumulation of mononuclear cells with only minimal fibrotic changes which were resolved after day 28. Lipopeptide conjugates based on the head group tripalmitoyl-S-glyceryl-cysteinyl-serin (P3CS) can thus be used as effective immunogens and adjuvants without long-term tissue damage.

The antibody response in BALB/c mice to the Plasmodium falciparum circumsporozoite repetitive epitope covalently coupled to synthetic lipopeptide adjuvant.

The repetitive epitope of Plasmodium falciparum circumsporozoite protein (Asn-Ala-Asn-Pro)3 [(NANP)3] was coupled to tripalmitoyl-S-glyceryl-cysteine (P3C) and tripalmitoyl-S-glyceryl-cysteinyl-serine (P3CS). The lipopeptide P3CS is a potent B-cell and macrophage activator. The resulting immunogenic lipopeptides were used for immunization of the low responder mouse strain BALB/c. These low molecular weight conjugates induced specific anti-(NANP)3 IgG and IgM levels without any carrier proteins or admixed adjuvants after a single administration.

Distinction between HIV-1 and HIV-2 infection using novel synthetic lipopeptide conjugates as antigens in enzyme immunoassays.

A novel immunoassay technique using synthetic lipopeptide (Pam3Cys-Ser) linked to immunodominant peptide domains of HIV-1 and HIV-2 envelope proteins as an antigen adsorbent has been developed. Attachment of peptides to microtiter plates can be considerably improved with this method by employing the hydrophobic properties of lipopeptide. From the sera of 121 HIV-1 infected patients 117 reacted with Pam3Cys-Ser-[HIV-1(598-609)cyclic disulfide]. Five of 5 HIV-2 positive sera were positive with Pam3Cys-Ser-[HIV-2(593-603)cyclic disulfide]. Control sera failed to react with these conjugates.