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1.
Allergy ; 60(8): 1040-5, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15969685

ABSTRACT

BACKGROUND: Basophils of some individuals do not release histamine upon activation of their high-affinity immunoglobulin E (IgE) receptor (Fc(epsilon)RI), but do so if this receptor is circumvented for cell activation. This so-called nonresponder phenomenon is clinically relevant, because in various studies atopy was less frequent or absent in nonresponder individuals. So far, it is unknown if this phenomenon is acquired during adulthood or exists from birth on. METHODS: Histamine release was determined from isolated leucocytes stimulated with anti-IgE or calciumionophor. Also, random primed cDNA was synthesized and the open reading frame (ORF) of the Fc(epsilon)RI beta-subunit amplified and sequenced. RESULTS: In the first part of our study, we examined the role of atopic status, type of atopy, and age in a random population of 95 children of whom we found 22% to be nonresponder. None of these parameters correlated with the nonresponder status. Except for food allergy, no specific type of atopy correlated with histamine release. The mechanism underlying the nonresponder phenomenon is assumed to occur early in the signalling cascade. We hypothesized that mutations in the Fc(epsilon)RI beta-chain may be associated with the nonresponder status, and in the second part of our study sequenced the beta-subunit in 20 responders and 20 nonresponders. Two conservative and two nonconservative heterozygous one base mutations (Thr179Thr, Asp216Asp, Ile147Leu and Glu237Gly) were found in two nonresponders and one responder. Three of these mutations have not been described so far. CONCLUSION: The nonresponder phenomenon is present from birth on and genetically determined. In our population, it was not associated with age or the presence of atopy, and appeared not to be caused by mutations in the Fc(epsilon)RI beta-chain.


Subject(s)
Histamine Release , Hypersensitivity/genetics , Hypersensitivity/metabolism , Mutation , Receptors, IgE/genetics , Receptors, IgE/metabolism , Age Factors , Amino Acid Sequence , Base Sequence , Binding, Competitive , Child , Female , Food Hypersensitivity/metabolism , Heterozygote , Humans , Male , Molecular Sequence Data
2.
Biol Chem ; 381(3): 231-43, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10782994

ABSTRACT

The hepatitis B core antigen is a widely accepted carrier particle to enhance the immunogenicity of foreign epitopes. From electron cryomicroscopy, the immunodominant region between amino acid positions 79 to 81 is known to protrude from the surface of the shells. It can be replaced by heterologous sequences without interfering with the particle-forming capacity in many cases. Here we have introduced various V3 sequences of the envelope protein of different subtypes (A, B, O) of HIV-1/gp120 in order to enhance their immunogenicity and broaden the immune response against the virus. To improve purification efficiency and solubility of the E. coli-expressed hybrids, six histidine residues were fused to amino acid 156. An adjustable purification scheme was utilised including denaturation, Ni(2+)-NTA affinity chromatography and particle renaturation under high salt conditions, resulting in highly pure antigen preparations. The hybrids reacted specifically with sera of HIV-1-infected patients. They further induced an autologous, subtype-specific anti-HIV-1 antibody response superior to that of Keyhole limpet-haemocyanine-coupled peptides.


Subject(s)
HIV Envelope Protein gp120/immunology , HIV-1/classification , Hepatitis B Core Antigens/immunology , Immunodominant Epitopes/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/biosynthesis , Escherichia coli , Gene Expression , HIV Envelope Protein gp120/biosynthesis , HIV Envelope Protein gp120/genetics , HIV Infections/immunology , HIV-1/immunology , Histidine , Humans , Mice , Mice, Inbred BALB C , Microscopy, Electron , Molecular Sequence Data , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Structure-Activity Relationship
3.
J Mol Biol ; 289(5): 1163-78, 1999 Jun 25.
Article in English | MEDLINE | ID: mdl-10373359

ABSTRACT

Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPcand the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein. Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds. Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found. Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPcand of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3.


Subject(s)
Prions/genetics , Amino Acid Sequence , Animals , Base Sequence , Birds , Cats , Conserved Sequence , DNA, Complementary , Dogs , Genetic Variation , Humans , Mammals , Molecular Sequence Data , Prions/classification , Rodentia , Sequence Homology, Amino Acid
4.
J Virol Methods ; 77(2): 189-97, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10092142

ABSTRACT

Hepatitis B virus is a major cause of human liver disease. In the case of chronic infection the virus can lead to liver cancer and cirrhosis. The virion consists of an outer envelope containing lipids of the endoplasmic reticulum and virally-encoded surface proteins. This lipoprotein shell encloses the nucleocapsid or core antigen (HBcAg), which contains the viral genome. The capsid consists of dimers of a 183-residue protein, which can be divided into an assembly (residues 1-149) and a protamin-like domain (residues 150-183), responsible for polymerization into particles and RNA packaging, respectively. Upon expression of the core gene in bacteria the products are assembled into capsids resembling those of wild type particles. A purification protocol was developed for unpolymerised (dimeric) and polymerized HBcAg by fusion of six histidine residues to a C-terminal deletion mutant of the core protein allowing the isolation of the respective antigens after denaturing Ni2+-chelate affinity chromatography and renaturing dialysis. The possible incorporation of E. coli proteins during the assembly process and the inclusion of nucleic acids can be avoided. The method might be an attractive alternative to common purification protocols of hybrid virus-like particles (VLPs) for vaccine use.


Subject(s)
Chromatography, Affinity/methods , Hepatitis B Core Antigens/isolation & purification , Cloning, Molecular , Dimerization , Escherichia coli , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/ultrastructure , Histidine/analysis , Histidine/chemistry , Humans , Hydrogen-Ion Concentration , Nickel , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
5.
J Viral Hepat ; 4(5): 339-49, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9310933

ABSTRACT

A descriptive study was performed to evaluate the relative frequencies and molecular epidemiological features of viral hepatitis types A to E among the Inuit population in West Greenland. Serum samples were collected from 503 Inuits (186 males and 317 females; mean age 35 years; range 7-79 years) and were tested for markers of viral hepatitis infection. The hepatitis A prevalence averaged 54%, with a significant rise from 9% to 50% between the second and third decade of life. As for hepatitis B, 42% of the total study population showed serological evidence of current or past hepatitis B virus (HBV) infection and 7% were hepatitis B surface antigen (HBsAg) carriers. Among the carriers, 6% were also positive for hepatitis B e antigen (HBeAg), and HBV DNA could be detected in 49% of carriers by polymerase chain reaction. Typing of the HBV isolates revealed genomic group D in 83% (serotype ayw2) and group A in 17% (serotype adw 2). Less than 1% of the study population had antibodies to the hepatitis C virus. None were positive for HCV RNA. Serological evidence of hepatitis D infection was found in 7% of those with hepatitis B helper virus infection markers and in 40% of the HBsAg carriers. As for hepatitis E, 3% of the Inuits showed reactivity in an enzyme immunoassay that detected hepatitis E virus antibody. HEV RNA could not be detected.


Subject(s)
Hepacivirus/genetics , Hepatitis B virus/genetics , Hepatitis Delta Virus/genetics , Hepatitis E virus/genetics , Hepatovirus/genetics , Inuit , Molecular Epidemiology , Adolescent , Adult , Age Factors , Aged , Amino Acid Sequence , Child , DNA, Viral/blood , Female , Greenland/epidemiology , Greenland/ethnology , Hepacivirus/immunology , Hepatitis A/epidemiology , Hepatitis A/ethnology , Hepatitis A/genetics , Hepatitis Antibodies/blood , Hepatitis B/epidemiology , Hepatitis B/ethnology , Hepatitis B/genetics , Hepatitis B Surface Antigens/blood , Hepatitis B virus/classification , Hepatitis B virus/immunology , Hepatitis C/epidemiology , Hepatitis C/ethnology , Hepatitis C/genetics , Hepatitis D/epidemiology , Hepatitis D/ethnology , Hepatitis D/genetics , Hepatitis Delta Virus/immunology , Hepatitis E/epidemiology , Hepatitis E/ethnology , Hepatitis E/genetics , Hepatitis E virus/immunology , Hepatovirus/immunology , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Middle Aged , Molecular Sequence Data , Prevalence , RNA, Viral/blood , Sequence Homology, Amino Acid
7.
AIDS Res Hum Retroviruses ; 11(12): 1459-65, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8679289

ABSTRACT

Recombinant peptides from Escherichia coli encoding the principal neutralizing domain (PND) and surrounding sequences of gp120 of human immunodeficiency virus type 1 (HIV-1) with a C-terminal polyhistidine tag were expressed and purified on Ni(2+)-nitrilotriacetate agarose. High yields of more than 99% pure protein were obtained. Their serological reactivity with anti-HIV-positive and -negative human sera was compared to chemically synthesized V3 loop peptides. Overall the genetic PND peptides of the HIV-1MN isolate showed higher and broader reactivity patterns (84%) with HIV-positive sera from German patients than the chemically synthesized peptides (74%). By their higher reactivity and easy way of production and purification, recombinant peptides seem to be highly preferable for the determination of antibody titers to the PND of HIV-infected patients.


Subject(s)
Acquired Immunodeficiency Syndrome/immunology , HIV Antibodies/blood , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Peptide Fragments/immunology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Cloning, Molecular , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Rabbits , Recombinant Proteins/immunology , Viral Envelope Proteins/immunology
8.
AIDS Res Hum Retroviruses ; 11(9): 1135-7, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8554912

ABSTRACT

PIP: HIV-1 consists of eight subtypes, A through H, and group O. The HIV epidemic has only recently come to Paraguay. According to available data from the National AIDS Control Program of Paraguay, there were only 95 individuals known to have had AIDS in the country by the end of 1994. The authors report their findings from the study of HIV-1 from ten people with AIDS living in Asuncion. The subjects were male, with AIDS-related symptoms, and largely contracted HIV through homosexual contact. Some, however, contracted HIV through IV drug use or heterosexually. The nucleic acid sequences obtained from the collected viruses grown in tissue culture have been deposited in GenBank under accession numbers U28949 through U28959. All of the isolated viruses are of subtype B. Virus PY.3616, however, had a V3 loop with the rare motif APGR. The individual from whom this virus was obtained acquired his HIV infection through IV drug use, most probably in Argentina. Two viruses were obtained with V3 loop crown motifs of GPRR and GWRR (PY.12838 and PY.12839), motifs which have not been previously described, but which come close to the V3 loops of HIV-1 isolates found in Brazil with a crown motif GWGR, and also GMGR and GFGR. A motif with an arginine at another position, GRGQ, has been found in HIV-1 subtype H in Cameroon. The different motifs found in the sequences of the Paraguayan patients show greater homogeneity than those of African patients in the Central African Republic and in Paris. The observed diversity reflects the connection of Paraguayans with Brazil and other countries where HIV-1 subtype B prevails. The authors note that their findings are most likely representative of the ongoing, young HIV epidemic in Paraguay.^ieng


Subject(s)
Acquired Immunodeficiency Syndrome/virology , HIV Envelope Protein gp120/genetics , HIV-1/genetics , HIV-1/isolation & purification , Peptide Fragments/genetics , Acquired Immunodeficiency Syndrome/epidemiology , Amino Acid Sequence , Base Sequence , Cluster Analysis , DNA Primers/genetics , DNA, Viral/genetics , Humans , Male , Molecular Sequence Data , Paraguay/epidemiology , Sequence Homology, Amino Acid
9.
AIDS Res Hum Retroviruses ; 11(1): 183-4, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7734191

ABSTRACT

PIP: HIV-1 V3 loop sequences from Ugandan patients include motifs from subtypes A, B, and D. To characterize further HIV isolates, V3 loop sequences were amplified from HIV-1 isolated in 1987 from peripheral blood mononuclear cells (PBL) of three patients with full-blown AIDS from Kampala, Uganda. The PBL were separated by Ficoll Paque gradients and cocultivated with noninfected donor lymphocytes for two weeks. The HIV was then transferred to HUT-78 cells. From extracted DNA of the permanently-infected HUT-78 cells, nested polymerase chain reaction (PCR) was conducted, with V3 loop sequencing performed directly upon PCR fragments derived from two independent DNA preparations and on cloned fragments. Isolates MVP-9801, -9802, and -9803 show 35.6%, 32.4%, and 29.7% nucleotide sequence divergence from the ELI subtype D sequence; 31.5%, 25.7%, and 18.9% divergence from the Z2Z6 subtype D sequence; and 21.9%, 12.2%, and 12.2% divergence from the subtype D consensus sequence. All three deduced amino acid sequences fit into the subtype D consensus sequence rather than into other V3 loop sequences described for Ugandan subtype A isolates. MVP-9802 and MVP-9803 contain the GSGQA pentapeptide motif at the tip of the V3 loop, while MVP-9801 contains GGRA. This may be explained by a deletion of proline codon between the codons for the two glycine residues. The authors believe that this deletion has not been previously reported. They also note that the deletion does not appear to be associated with a growth difference in vitro or with a difference in pathogenicity in vivo. The immunogenic implications of this altered V3 loop crest remain unclear. The Western blot profiles for the gp160, gp120, and gp41 proteins of the three Ugandan isolates manifest normal molecular weights.^ieng


Subject(s)
Acquired Immunodeficiency Syndrome/virology , HIV-1/genetics , Acquired Immunodeficiency Syndrome/epidemiology , Acquired Immunodeficiency Syndrome/genetics , Amino Acid Sequence , Base Sequence , Humans , Molecular Sequence Data , Uganda
10.
J Virol ; 68(3): 1581-5, 1994 Mar.
Article in English | MEDLINE | ID: mdl-8107219

ABSTRACT

A new subtype (MVP-5180) of human immunodeficiency virus type 1 (HIV-1) was isolated from a Cameroonian AIDS patient. MVP-5180 was grown in several human T-cell lines and the monocytic U937 line. MVP-5180 DNA could not be amplified by nested primer PCR with conventional env primers and could be only very faintly amplified with gag and pol primers. Most German, Ivoirian, and Malawian anti-HIV-1 sera reacted faintly or moderately with Env proteins in an MVP-5180 immunoblot, whereas some Cameroonian sera reacted strongly. Of HIV-1-infected Cameroonians, 8% were identified by serological methods as infected with MVP-5180; 7% were positive when MVP-5180-specific PCR env primers were used. DNA sequence analysis of MVP-5180 showed that its genetic organization was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 had similarities to HIV-1 and HIV-2 of 53 and of 49%, respectively. V3 loop analysis identified a crown of Gly-Pro-Met-Arg by using cloned DNA and Gly-Pro-Leu-Arg by using PCR-amplified DNA, neither of which configuration has been described for other HIV strains. In an analysis of relationships, MVP-5180 occupied a position distant to all other HIV-1 strains, including the chimpanzee simian immunodeficiency virus type 1 SIVcpz and the Uganda virus U455, and closer to the HIV-1/HIV-2 divergence node. MVP-5180, together with another Cameroonian isolate, ANT-70, constitutes a group subtype O of the most divergent HIV-1 isolates yet identified. Characterization of MVP-5180 is important for understanding the natural history of the primate immunodeficiency viruses and for the development of vaccines and diagnostics.


Subject(s)
HIV-1/classification , Acquired Immunodeficiency Syndrome/epidemiology , Acquired Immunodeficiency Syndrome/microbiology , Amino Acid Sequence , Base Sequence , Cameroon/epidemiology , Cell Line , HIV Envelope Protein gp120/genetics , HIV-1/genetics , HIV-1/isolation & purification , HIV-2/genetics , Humans , Immunoblotting , Molecular Sequence Data , Polymerase Chain Reaction , Prevalence , Sequence Analysis, DNA , Sequence Homology, Amino Acid , T-Lymphocytes/cytology , T-Lymphocytes/microbiology
11.
J Immunol Methods ; 169(1): 135-6, 1994 Feb 28.
Article in English | MEDLINE | ID: mdl-8133071

ABSTRACT

Immunoprecipitation is a powerful technique for the immunochemical characterization of antigens. In combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) a number of features, e.g., presence of antigen, rate of synthesis, relative molecular weight of the polypeptide chain or post-translational modifications can be determined. Four different steps are basically involved in the immunoprecipitation procedure: (1) metabolic labelling of the antigen by incubation of viable cells with a radioactive precursor, (2) harvesting of the labelled antigen from the cells by lysis or in the case of secretory proteins from the supernatant, (3) formation and (4) purification of antibody-antigen complexes. The last step relies on secondary agents which bind to the antibody.


Subject(s)
Enzyme-Linked Immunosorbent Assay , Radioimmunoprecipitation Assay/methods , Animals , Antigen-Antibody Complex/isolation & purification , Cell Line , Electrophoresis, Polyacrylamide Gel , Fibroblasts , Rabbits , Radioimmunosorbent Test
12.
Vaccine ; 11(8): 817-24, 1993.
Article in English | MEDLINE | ID: mdl-7689283

ABSTRACT

The development of subunit vaccines against HIV requires the identification of immunologically relevant antigens and a suitable method of antigen delivery. Ideally, defined epitopes with neutralizing activity should be included in a vaccine preparation. The carrier for such peptide sequences should enhance the immunogenicity of the selected epitopes. In this study hepatitis B virus core antigen (HBcAg) was used as a carrier moiety for the principal neutralizing domain (PND, V3-loop) of HIV-1. A 25 amino acid V3-loop sequence was fused to HBcAg at various positions by genetic engineering. The resulting hybrid HBcAg/HIV polypeptides were analysed for particle formation and immunogenicity. Fusion of the PND to an internal position replacing an immunodominant antibody-binding region of HBcAg or to a C-terminally truncated HBcAg resulted in the formation of hybrid particles with biochemical and biophysical properties similar to those of wild-type HBcAg particles. Both types of hybrids are recognized by monoclonal and polyclonal antisera raised against PND peptides of various HIV-1 isolates. Hybrid particles with a C-terminal fusion but not an internal fusion are also recognized by a polyvalent anti-HBcAg serum. In both cases the V3 domain is surface accessible. Immunization of mice with hybrid particles induces an enhanced antibody response against the V3 sequence. The internal fusion is more immunogenic than the C-terminal fusion.


Subject(s)
HIV Antigens/genetics , HIV Antigens/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/immunology , AIDS Vaccines/genetics , AIDS Vaccines/immunology , Amino Acid Sequence , Animals , Base Sequence , Chimera/genetics , Chimera/immunology , Epitopes/genetics , Epitopes/immunology , Gene Expression/genetics , HIV-1/genetics , Immunization , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology
13.
Zentralbl Bakteriol ; 277(2): 219-23, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1520981

ABSTRACT

Heat inactivation of parvovirus B19 (B19) was studied in a culture of hematopoietic progenitor cells generated in vitro from peripheral human blood. After inoculating cell cultures with identical volumes of plasma (MII) containing B19 (B19-MII) heat-treated (60 degrees C) for various periods of time, a time-dependent inactivation of the input virus was determined by a decrease of viral DNA replication. No B19 DNA was detected after infection with B19-MII heat-treated for 20 min or more by Southern blot. Viral B19 protein production decreased time-dependently and was not detected after infection with samples treated for 12 min at 60 degrees C or more determined by the enzyme immunoassay. This study indicates that infectivity of B19 virus in plasma can be reduced in vitro by heat-treatment (60 degrees C). However, this does not mean that the heat treatment completely inactivated B19 virus.


Subject(s)
Hot Temperature , Parvovirus B19, Human/physiology , Cells, Cultured , DNA Replication/physiology , DNA, Viral/physiology , Humans , Kinetics , Viral Proteins/biosynthesis , Virus Replication/physiology
14.
J Virol ; 66(2): 1273-6, 1992 Feb.
Article in English | MEDLINE | ID: mdl-1731104

ABSTRACT

Erythroid progenitor cells generated in vitro from peripheral human blood in the presence of interleukin-3 and erythropoietin were infected with human parvovirus B19. B19 virus DNA replication was highest 48 to 72 h after infection, and maximum levels of B19 virus proteins were detected in culture supernatants at 72 to 96 h after infection. B19 virus propagated in vitro was infectious. This cell culture system with peripheral blood cells facilitates studies in vitro of B19 virus replication.


Subject(s)
Hematopoietic Stem Cells/physiology , Parvovirus B19, Human/physiology , Virus Replication , Blood , Blotting, Southern , Cells, Cultured , DNA, Viral/genetics , DNA, Viral/isolation & purification , Enzyme-Linked Immunosorbent Assay , Humans , Kinetics , Molecular Weight , Parvovirus B19, Human/genetics , Parvovirus B19, Human/isolation & purification , Viral Proteins/analysis
15.
Arch Virol ; 127(1-4): 117-37, 1992.
Article in English | MEDLINE | ID: mdl-1456888

ABSTRACT

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.


Subject(s)
AIDS Vaccines/immunology , Gene Products, gag/genetics , Gene Products, gag/immunology , HIV-1/genetics , Protein Precursors/genetics , Baculoviridae/genetics , Base Sequence , Capsid/genetics , Capsid/immunology , Cloning, Molecular , Escherichia coli/genetics , Gene Products, gag/metabolism , Genes, Viral , Genes, gag , Genetic Vectors , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Protein Precursors/immunology , Protein Precursors/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/immunology , Vaccines, Synthetic/immunology , Vaccinia virus/genetics , Viral Structural Proteins/genetics
16.
Arch Virol ; 127(1-4): 139-52, 1992.
Article in English | MEDLINE | ID: mdl-1456889

ABSTRACT

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.


Subject(s)
Acquired Immunodeficiency Syndrome/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , AIDS Vaccines/immunology , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Consensus Sequence/immunology , Cross Reactions , Immunity, Cellular , Molecular Sequence Data , Neutralization Tests , Peptides/immunology , Sequence Alignment
17.
Br J Haematol ; 79(1): 6-13, 1991 Sep.
Article in English | MEDLINE | ID: mdl-1654995

ABSTRACT

Parvovirus B19 exerts a highly selective cytopathic effect on erythroid progenitor cells. Studies so far on the pathogenesis of B19-infection have been performed using bone marrow samples providing large amounts of erythroid progenitor cells. Extensive study, however, has been hampered by the limited access to bone marrow samples. We have designed a liquid culture method allowing the generation of large numbers of erythroid progenitor cells, initiating cultures with CD3- and CD14-poor peripheral blood mononuclear cells. Following a 12 d preincubation in liquid cultures containing recombinant human interleukin 3 (rhIl-3) and recombinant human erythropoietin (rhEpo), cells harvested from the liquid cultures were exposed to B19-containing plasma, followed by a further cultivation in liquid culture for up to 96 h. Cells expressing the CD13 and the glycophorin A (GlyA) antigens, respectively, were monitored sequentially by flow-cytometry, demonstrating a selective inhibition of GlyA-positive cells following B19-inoculation. Typical morphological changes were observed on cytocentrifuge-spots, and typical giant-cells were identified as staining for GlyA. Productive infection by B19 was demonstrable, as B19-DNA increased by about x 100 after 72 h of culture. The liquid culture method generating erythroid target cells for effective infection by B19 virus promises to be a useful and easily accessible tool for further research on B19 infection of haemopoietic cells.


Subject(s)
Hematopoietic Stem Cells/pathology , Parvoviridae Infections/pathology , Cells, Cultured , DNA, Viral/analysis , Flow Cytometry , Humans , Immunoenzyme Techniques , Nucleic Acid Hybridization , Parvoviridae/genetics , Time Factors
18.
Biochem Biophys Res Commun ; 179(3): 1479-84, 1991 Sep 30.
Article in English | MEDLINE | ID: mdl-1656954

ABSTRACT

To examine the possible role of lymphocytes in the course of hepatitis B virus infection, we studied peripheral blood lymphocytes and Epstein-Barr virus transformed B-cells for their capability to produce hepatitis B virus gene products. Infection of these cells with a recombinant vaccinia virus containing the hepatitis B virus surface antigen resulted in the production and secretion of hepatitis B virus surface antigen shortly after infection reaching a peak after three days. When the supernatants of the cells were analyzed on density gradients, a peak of reactivity for hepatitis B virus surface antigen was reached at 1.21 g/cm3. Electron microscopy revealed the presence of spherical and filamentous HBsAg particles. These findings show that human lymphocytes are capable of producing hepatitis B virus surface antigen.


Subject(s)
B-Lymphocytes/physiology , Hepatitis B Surface Antigens/genetics , Hepatitis B virus/genetics , Herpesvirus 4, Human/genetics , Cell Transformation, Viral , Genes, Viral , Hepatitis B Surface Antigens/analysis , Humans , Kinetics , Plasmids , Restriction Mapping , Vaccinia virus/genetics
19.
Vaccine ; 9(7): 477-84, 1991 Jul.
Article in English | MEDLINE | ID: mdl-1716808

ABSTRACT

The development of recombinant subunit vaccines against pathogenic organisms requires not only the identification of epitopes eliciting a protective immune response but also suitable carriers with adjuvant function. B- and T-cell epitopes of the malaria vaccine candidate gp190 were selected on the basis of a systematic search along the gp190 molecule and by computer prediction based on the amino acid sequence. Using some of the epitopes identified, we have redesigned the surface of the hepatitis B surface antigen lipoprotein particles by replacing the major antigenic determinants with malaria-specific sequences of up to 61 amino acids in length. Upon expression via vaccinia virus the hybrid particles elicit an anti-gp190 immune response in animals. Monoclonal antibodies derived from such infections recognize the native parasite.


Subject(s)
Antibodies, Protozoan/biosynthesis , Antigens, Protozoan/immunology , Hepatitis B Surface Antigens/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Protozoan Vaccines/immunology , Vaccines, Synthetic/immunology , Amino Acid Sequence , Animals , Blotting, Western , Cell Line , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Escherichia coli/metabolism , Fluorescent Antibody Technique , Haplorhini , Hepatitis B Antibodies/biosynthesis , Humans , Molecular Sequence Data , Rabbits , Recombinant Fusion Proteins/immunology , Vaccines, Synthetic/administration & dosage , Vaccinia virus/genetics , Vaccinia virus/immunology
20.
Hepatology ; 13(1): 57-61, 1991 Jan.
Article in English | MEDLINE | ID: mdl-1846347

ABSTRACT

The relevance of the recently described membrane-bound form of the HBe protein for the antiviral immune response was examined. The data show that antibodies in anti-HBe, but not in anti-HBc-positive human sera efficiently bind to the membrane expressed HBe. No evidence was obtained that the HBc can reach the cell surface in a form that can be detected with human antibodies. The findings suggest that the decline of virus titer that is usually observed after seroconversion from HBe to anti-HBe might be the result of an antibody-mediated elimination of infected cells.


Subject(s)
Carcinoma, Hepatocellular/immunology , Hepatitis B Antibodies/metabolism , Hepatitis B e Antigens/immunology , Hepatitis B/immunology , Liver Neoplasms/immunology , Biological Transport , Hepatitis B/microbiology , Hepatitis B Antibodies/analysis , Hepatitis B Antibodies/immunology , Hepatitis B Core Antigens/immunology , Hepatitis B Core Antigens/metabolism , Hepatitis B e Antigens/metabolism , Humans , T-Lymphocytes, Cytotoxic/immunology
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