Prions and protein-folding diseases.

Prions represent a group of proteins with a unique capacity to fold into different conformations. One isoform is rich in beta-pleated sheets and can aggregate into amyloid that may be pathogenic. This abnormal form propagates itself by imposing its confirmation on the homologous normal host cell protein. Pathogenic prions have been shown to cause lethal neurodegenerative diseases in humans and animals. These diseases are sometimes infectious and hence referred to as transmissible spongiform encephalopathies. In the present review, the remarkable evolution of the heterodox prion concept is summarized. The origin of this phenomenon is based on information transfer between homologous proteins, without the involvement of nucleic acid-encoded mechanisms. Historically, kuru and Creutzfeldt-Jakob disease (CJD) were the first infectious prion diseases to be identified in man. It was their relationship to scrapie in sheep and experimental rodents that allowed an unravelling of the particular molecular mechanism that underlie the disease process. Transmission between humans has been documented to have occurred in particular contexts, including ritual cannibalism, iatrogenic transmission because of pituitary gland-derived growth hormone or the use in neurosurgical procedures of dura mater from cadavers, and the temporary use of a prion-contaminated protein-rich feed for cows. The latter caused a major outbreak of bovine spongiform encephalopathy, which spread to man by human consumption of contaminated meat, causing approximately 200 cases of variant CJD. All these epidemics now appear to be over because of measures taken to curtail further spread of prions. Recent studies have shown that the mechanism of protein aggregation may apply to a wider range of diseases in and possibly also outside the brain, some of which are relatively common such as Alzheimer's and Parkinson's diseases. Furthermore, it has become apparent that the phenomenon of prion aggregation may have a wider physiological importance, but a full understanding of this remains to be defined. It may involve maintaining neuronal functions and possibly contributing to the establishment of long-term memory.

Characterization of T cell epitopes in measles virus nucleoprotein.

T cell epitopes of the measles virus (MV) nucleoprotein were studied by synthesizing overlapping 20 aa peptides over the known sequence of the protein and analysing the proliferation responses of a panel of MV-specific T cell lines and clones against these peptides. T cell lines were established from eleven healthy controls and seven multiple sclerosis patients, all with a history of past MV infection. The epitopes recognized by these lines were concentrated in a few regions of the polypeptide chain. Overlapping peptides containing aa 321-340 and 331-350 were most often recognized. Other epitopes were detected close to the amino-terminal end of the polypeptide chain as each of the peptides 1-20, 21-40, 31-50 and 51-70 contained stimulating moieties. Some responses were also detected towards peptides 151-200 and 221-250, but the carboxy-terminal end of the polypeptide was not recognized by any of the tested T cell lines. The amino acid sequences of the peptides that stimulated the T cell clones and lines, as a rule, contained binding motifs described for HLA-DR alleles found in T cell donors. The regions of protein sequence which did not reveal any T cell epitopes were, instead, relatively free of binding motifs. The results suggest that only a few epitopes of the MV nucleoprotein are important in establishing T cell immunity.

Development of human antibodies against linear antigenic and immunogenic regions of respiratory syncytial virus (RSV) nucleocapsid and phospho-proteins shows the site-directed characteristics.

BACKGROUND: The successful development of an RSV vaccine requires a better understanding of the pathogenesis of primary infection, susceptibility to reinfection, and the immunopathology of enhanced illness in children immunized with a non-replicating RSV candidate vaccine. The exact role of different immune parameters in RSV pathogenesis remains controversial. OBJECTIVES: To study the contribution of antibodies directed to the linear antigenic and immunogenic regions of the N and P proteins in the titer rise and avidity maturation of total anti-RSV antibodies. STUDY DESIGN: The occurrence of antibodies directed against three linear antigenic and immunogenic regions in each of the nucleocapsid (N): N3 (Thr11 to Gly30), N25 (Ser231 to Ala250) and N39 (Thr371 to Leu391), and the phospho-(P) proteins of respiratory syncytial virus (RSV), subgroup A: P49 (Pro91 to Asp110), P56 (Ser161 to Lys180) and P62 (Glu221 to Phe241), were analyzed in ELISA with (a) 32 paired sera from humans with recent or previous RSV subgroup A and/or B infection diagnosed by conventional ELISA, detection of antigen in nasopharyngeal aspirates and measurement of antibody avidity change; and (b) 40 RSV antibody-positive sera (HCS) obtained from patients during their convalescence from RSV infection and possessing clearly demonstrable titers of RSV IgG in conventional enzyme immunoassays (EIA) based on whole RSV antigen. RESULTS: The titer rise of antibodies directed to the combined three peptides representing the RSV N protein was well correlated with the rise in anti-RSV antibodies measured in whole antigen ELISA. Surprisingly, the rise in antibodies against a truncated main C-terminal epitope (Gln381 to Leu391) of the N protein (represented by subgroup A specific sequence of the N39/1 peptide) was inversely correlated with the titer rise of total anti-RSV antibodies. The titer rise of antibodies to the C-terminal linear site of the RSV N (N39/1) protein was subgroup-specific during the course of primary RSV infection. A titer rise in antibodies to the C-terminal linear sites of RSV N (i.e. N39/1) and P (P62) proteins had a dominating appearance in sera from newborn infants (6-7 months) and from patients with RSV reinfections. Anti-RSV antibody titers of late convalescent sera correlated with the titers of antibodies directed to the C-terminal linear site of RSV P (P62) protein. The avidity maturation of the anti-RSV immune response followed the titer rise of anti-P62 antibodies during the course of primary or secondary RSV infection.

The cellular stress response increases measles virus-induced cytopathic effect.

Plaque area is a measure of the degree of cytopathic effect and a predictor of neurovirulence for tissue culture adapted morbilliviruses. In the present work, the cellular stress response was shown to be a determinant of the expression of distinct measles virus large plaque phenotypes in Vero cells. The emergence of these large plaque phenotypes was associated with increased mean viral transcriptional activity and expression of the viral fusion glycoprotein, but not upregulation of the virus receptor CD46.

Spread of measles virus through axonal pathways into limbic structures in the brain of TAP1 -/- mice.

The spread of measles virus into the brain was studied exploiting the olfactory pathway, which represents an important route of neuroinvasion by viruses. The virus was injected into the main olfactory bulb of wild-type mice and mice with disrupted TAP1 gene (TAP refers to the Transporter associated with Antigen Presentation), which codes for products essential for the cell-mediated immune response. Virus invasion was monitored for 4 weeks by immunohistochemistry. The distribution of measles virus was found to be restricted to brain areas connected with the olfactory bulbs. However, in the wild-type mice there was a marked infiltration of lymphocytes in the infected brain structures, and the virus did not pass beyond the piriform cortex. In the TAP1 -/- mice the virus spread more extensively along olfactory projections into the limbic system and monoaminergic brainstem neurons. Infected mice of both types developed seizures, which may have been focally evoked from the piriform cortex. This study provides evidence that measles virus can spread through axonal pathways in the brain. The findings obtained in the gene-manipulated mice point out that a compromised immune state of the host may potentiate targeting of virus to the limbic system through olfactory projections.

Measles virus in the brain.

Measles virus can give three different forms of infections in the central nervous system. These are acute postinfectious encephalitis, acute progressive infectious encephalitis, and subacute sclerosing panencephalitis (SSPE). The postinfectious acute disease is interpreted to reflect an autoimmune reaction. The acute progressive form of brain disease, also referred to as inclusion body encephalitis, reflects a direct attack by the virus under conditions of yielding cellmediated immunity. The late progressive form of encephalitis (SSPE) has been extensively analyzed. Recent molecular genetic studies have unravelled a range of mechanisms by which a defective expression of either the matrix, the fusion, or the hemagglutinin proteins may lead to viral persistence in brain cells under conditions not allowing identification by immune surveillance mechanisms. Many aspects of virus-cell interactions have been examined by use of explant cultures of neuronal cells of human and animal origin. Some of the findings are reviewed. Experimental animals, in particular rodents, have been used to establish systems in which phenomena, pivotal to the evolution of acute as well as persistent measles virus infections in the brain, can be studied. A wide range of potentially important mechanisms has been highlighted and is discussed. More recently, mice with genetic defects in immune functions were used to evaluate consequences as to initiation and dissemination of virus infection in the brain.

Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells.

Human antibodies against HIV-1 have been sought to study neutralization events on the molecular level, and for possible use in passive immune intervention. The development of phage display techniques has opened the possibility of rapidly generating human monoclonal antibodies with desired specificities. We and others have isolated human HIV-1 neutralizing antibody fragments using this technique. Bacterial expression of isolated clones does, however, differ broadly both in expression levels and functional activity. In addition, intact IgG cannot be expressed in bacteria. By transferring the genes of isolated Fab clones to a mammalian expression system we could perform a comparison of functional activity between Fab expressed in bacterial and mammalian cells, as well as Fab and whole IgG. Fab fragments expressed in mammalian cells showed increased virus neutralizing activity compared to the same Fab clones expressed in Escherichia coli, underlining the inefficiency of procaryotic expression. No difference in HIV-1 neutralizing capacity was detected between monovalent (Fab) and divalent (whole antibody) reagents expressed in CHO cells. Thus, bivalency does not always confer improved neutralization efficacy.

Biological phenotype of HIV type 2 isolates correlates with V3 genotype.

The biological phenotype of HIV-2 isolates can be divided into two groups, rapid/high and slow/low, based on the ability to infect CD4+ tumor cell lines. Similar differences in the biological phenotype of HIV-1 isolates are largely determined by the charge of two specific amino acids in the V3 loop of the envelope protein gp120. In this study we have sequenced the V3 loop and flanking regions of 14 HIV-2 isolates from Guinea-Bissau and the Ivory Coast and correlated the results to the biological phenotype of the isolates. The sequences were obtained by PCR amplification of DNA from peripheral blood mononuclear cells infected with the different isolates, followed by direct sequencing of the amplified products. Eleven other HIV-2 isolates with known V3 sequence and biological phenotype were also included. Thirteen of the 14 new isolates were classified as subtype A of HIV-2 and one as subtype B. The V3 loop of rapid/high HIV-2 isolates differed significantly from slow/low isolates in that it was more heterogeneous in sequence and had higher net charge. Mutations at two specific amino acid positions (313 and 314), often to positively charged amino acids, were also significantly associated with the rapid/high phenotype. There were no sequence differences between rapid/high and slow/low isolates in the regions that flank the V3 loop. Our findings indicate that there may be a high degree of similarity in the molecular features that underlie the biological phenotypes of HIV-1 and HIV-2 isolates.

Solution conformation of an immunogenic peptide derived from the principal neutralizing determinant of the HIV-2 envelope glycoprotein gp125.

BACKGROUND: The conformational preferences of a number of peptides with sequences related to the envelope glycoproteins of HIV-1 have been investigated in the past few years. Similar studies have not been made for HIV-2, which is a distinct virus with similar physiological effects to those of HIV-1. The discovery of common structural features would be a promising route to the design of immunogens for generally effective HIV vaccines. We present the results of an NMR conformational study of a sequence deriving from the V3 loop of HIV-2. RESULTS: Three synthetic immunogenic peptides were studied, of 12, 22 and 39 amino acids in length, all containing a central Met-Ser-Gly-Arg sequence conserved among a number of HIV-2 isolates. In addition, the 39-mer contained a disulfide bond between cysteine residues close to the ends of the molecule, forming a loop that is thought to comprise an important structural and immunological component of the intact glycoprotein. All three peptides display well defined beta-turns in the Met-Ser-Gly-Arg sequence, independent of the integrity of the disulfide bond. No other conformational preferences for folded conformations were found for the peptides. CONCLUSIONS: The presence of a beta-turn in the Met-Ser-Gly-Arg sequence is strikingly similar to the behavior seen for the corresponding principal neutralizing determinant sequence from gp120 of HIV-1 and argues, in the absence of information of the three-dimensional structure of the intact proteins, for a similarity in the structure of this region that could be exploited in the design of synthetic peptide vaccines generally effective against HIV infections.

Evaluation of subgroup-specific peptides of the G protein of respiratory syncytial virus for characterization of the immune response.

Two synthetic peptides, designated peptides 12G(A) and 12G(B), representing amino acids 174-188 of the G glycoprotein of respiratory syncytial virus (RSV) subgroup A (strain A2) and subgroup B (strain CH18537) were evaluated for their properties as subgroup-specific antigens for enzyme immunoassay (ELISA). These peptides were used to characterize the immune response of children with naturally occurring RSV infection during six annual epidemics in the Huntington area, West Virginia, USA; viz. 1978-1979, 1979-1980, 1980-1981, 1983-1984, 1989-1990, and 1990-1991. The study group comprised 43 paired sera from 42 infants and children, who ranged in age between 1 month and 5.5 years of age (median age 16 months). The inclusion criteria were subgroup identification of RSV, respiratory tract illness requiring admission to hospital, and the availability of paired sera. Five of 30 children with subgroup A and 3 of 13 children with subgroup B infections developed homologous or dual fourfold or greater antibody responses to peptides 12G(A) and 12G(B) during convalescence; six of these eight children also developed antibody rises to whole virus antigens. Twenty children (14 subgroup A and 6 subgroup B) developed such responses in antibody only to whole virus (not to the peptides), and 15 children (11 subgroup A and 4 subgroup B) failed to develop a rise in antibody. Children who developed rises in antibody to the peptides were usually less than 9 months of age, suggesting that a response to peptides was more likely to occur during primary infection. Peptides 12G(A) and 12G(B) of RSV G protein lacked sufficient sensitivity and specificity to serve as antigens for ELISA for characterizing the subgroup-specific immune responses to RSV infection in infants and children.

Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein.

The fusion (F) glycoprotein of measles virus, a structural component of the virion envelope, contains four potential sites for attachment of N-linked oligosaccharides. Three are located in the F2 subunit of the protein and one in the signal peptide. Four mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Ser/Thr to Ser-X-Ser/Thr. The wild-type and altered forms of the F protein were expressed in BHK-21 and HeLa T4 cells by use of the recombinant vaccinia virus-encoding T7 polymerase system. Analysis of these proteins revealed that three (residues 29, 61 and 67) potential sites for addition of N-linked glycans in the F2 subunit are actually utilized. The functional glycosylation sites were systematically removed in all possible combinations from the F protein to form a panel of mutants from which the role of carbohydrates, singly or in various combinations, could be evaluated. One single-site mutant protein lacking the glycosylation site of Asn-67 was processed, transported to the cell surface and could induce cell fusion. However, the other two single-site mutant proteins with deletions of glycosylation sites Asn-29 or Asn-61 exhibited a defect in processing, were not transported to cell surface and thus induced no cell fusion. The absence of any two of the three or of all three glycosylation sites resulted in protein retention in the endoplasmic reticulum. Therefore, it appears that glycosylation of sites Asn-29 and Asn-61 has important roles in maintaining the native structure of the F protein.

Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus.

A collection of simian immunodeficiency virus (SIV) neutralizing recombinant Fab fragments was generated using the combinatorial antibody library approach. Functional antibody fragments efficiently expressed in Escherichia coli were identified only in the form of chimeric macaque heavy chain gamma 1 and human light chain kappa. The gamma 1 and kappa chains were derived from a clinically healthy long-term surviving SIVsm-infected cynomolgus macaque and from an asymptomatic HIV-2 seropositive individual, respectively. The combinatorial library was constructed on the surface of filamentous phage using the pComb3 phagemid vector and screened against purified SIVsm surface glycoprotein (gp148). Twelve chimeric clones reacting with the antigen were isolated. Six of these clones showed a pronounced neutralizing activity against SIVsm with effects at concentrations of 0.01-0.1 micrograms/ml. All neutralizing Fab fragments were clonally unrelated as demonstrated by nucleic acid sequencing. These potent neutralizing reagents will be used for prophylactic and therapeutic immune intervention of lentivirus infection in macaques and to map neutralizing determinants of SIV.

Linear antigenic and immunogenic regions of human respiratory syncytial virus N protein.

Three linear antigenic regions on the N protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were identified by using peptides which reacted in ELISA with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the protein: Thr11 to Gly30 (N3 peptide), Ser231 to Ala250 (N25 peptide) and Thr371 to Leu391 (N39 peptide). The site represented by the N39 peptide reacted with four subgroup A-specific MAbs. There were minor variations in the amino acid epitope dependencies of each of these MAbs. Two additional antigenic regions Ser131 to Arg150 and Ala181 to Leu200, were represented by peptides that reacted with human convalescent sera, but these peptides did not differentiate between acute and convalescent sera from RSV-infected humans. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the N protein from a nucleocapsid-containing homogenate derived from extracts of RSV-(subgroup A and/or B)-infected 35S-labelled cells in a radioimmuneprecipitation assay (RIPA); the sera were also used to demonstrate RSV infection in cells by immunofluorescent assay (IFA). Anti-N3 peptide sera precipitated N41, the full-length (M(r) 41,000) form of N protein, in a RIPA done on a soluble protein pool. Anti-N39 (C-terminal region) peptide sera precipitated both forms, suggesting that N38 (M(r) 38,000) is an N-terminally truncated (probably at position Tyr23 located inside the N3 peptide linear antigenic region) form of N41 protein.

Protection of hamsters against experimental mumps virus (MuV) infection by antibodies raised against the MuV surface glycoproteins expressed from recombinant vaccinia virus vectors.

The fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of mumps virus (MuV) have been produced in CV1 cells via vaccinia virus recombinants. Recombinant proteins accumulated in infected cells and were glycosylated. Upon reduction, the F protein product was completely converted into its subunits. Hamsters infected with vaccinia recombinants expressing either the F or HN proteins produced antibodies recognizing MuV antigens and neutralizing MuV infectivity in vitro. These antibodies provided protection against MuV-induced encephalitis in newborn hamsters.

The helping encounter--occupational therapists' perception of therapeutic relationships.

The present paper deals with the art of occupational therapy in terms of therapeutic, helping relationships. The aim was to get a picture of how occupational therapists perceive the therapeutic encounter with their patients. Sixteen qualified occupational therapists, from various occupational areas, were interviewed and a qualitative approach was employed to analyze their description of therapeutic encounters in clinical settings. The qualitative analyses were performed with sensitization as the principle objective and a tentative theoretical model was formed, developed by three content categories; 1) Basic Professional-Oriented Helping; 2) Understanding-Oriented Helping; and 3) Action-Oriented Helping. The relationships between these categories constitute a dynamic complex pattern. One conclusion drawn from this model was that the therapists might have problems finding a balance between their aspiration for becoming professionalized and their belief in egalitarian patient-therapist relationships in the rehabilitation treatment sessions.