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Biomedical subjects

E A Emini

Publications and source records attributed to E A Emini.

At least 91 records · Page 5Linked to original sources
PubMed1986

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

Animals↗
PubMed1986

Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C.

Sequence analysis of the genomic RNA of interstrain guanidine-resistant and antibody-resistant variant recombinants of poliovirus type 1 mapped the resistance of mutants capable of growth in 2.0 mM guanidine hydrochloride to a region located 3' of nucleotide 4444. This region of the viral genome specifies the nonstructural protein 2C. The sequence of genomic RNA encoding 2C from six independently isolated mutants resistant to 2.0 mM guanidine was determined. All six isolates contained a mutation in 2C at the same position in all cases, resulting in two types of amino acid changes. Dependent mutants were examined and found to contain two amino acid changes each within 2C. Mutants resistant to 0.53 mM guanidine were isolated and found to lack the mutations seen in variants resistant to 2.0 mM guanidine. A comparison of the amino acid sequences of the 2C proteins of poliovirus, foot-and-mouth disease virus, rhinovirus types 2 and 14, and encephalomyocarditis virus revealed a strong homology over regions totaling 115 residues. All of the mutations observed in guanidine-selected mutants were contained within this region. The amino acid region containing the mutations observed in poliovirus mutants resistant to 2.0 mM guanidine was compared with the homologous region in the other picornaviruses; a strong correlation was found between the amino acid present at this position and the sensitivity of the virus to 2.0 mM guanidine.

Amino Acid Sequence↗
PubMed1985

Antigenic conservation and divergence between the viral-specific proteins of poliovirus type 1 and various picornaviruses.

Immuneprecipitation analyses of various picornavirus-infected cell lysates were performed using antisera to poliovirus type 1-specific structural and nonstructural proteins. The results showed differing patterns of antigenic conservation and divergence. However, the VP3 and 2C polypeptides were strongly antigenically conserved among the large majority of these viruses. This conservation was especially notable given the degree of divergence exhibited by the other viral proteins and may be due to environmental pressure exerted by interaction with the host cell. The results, furthermore, allowed for an analysis of the evolutionary relationship of the tested viruses. This analysis showed a particularly strong antigenic relationship between the proteins of the poliovirus group and coxsackievirus A21 as well as a weaker, but significant, relationship with coxsackieviruses B1 and B3.

Antigens, Viral↗
PubMed1985

The immune response to poliovirus-specific synthetic peptides: effects of adjuvants and test animal species.

Carrier protein conjugates of five synthetic peptides containing amino acid sequences specific to capsid proteins VP1 and VP2 of poliovirus type 1 were tested for their abilities to elicit an immune response in the presence of either of two adjuvants and in several animal species. Freund's adjuvant induced significantly higher level anti-peptide antibody titers than A1(OH)3. However, no difference was noted between the two adjuvants in their abilities to aid in the induction of cross-reactive virus neutralizing antibody. The latter antibody was more readily produced by rabbits than by guinea pigs in spite of equivalent anti-peptide titers. Rats failed to produce neutralizing antibodies and their anti-peptide antibody levels were generally lower. The significance of these results for studies involving the development of synthetic peptide immunogens is discussed.

Adjuvants, Immunologic↗
PubMed1985

Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide.

Comparative surface feature analyses of the VP1 sequences of hepatitis A virus (HAV) and poliovirus type 1 allowed an alignment of the two sequences and an identification of probable HAV neutralization antigenic sites. A synthetic peptide containing the HAV-specific amino acid sequence of one of these sites induced anti-HAV-neutralizing antibodies. It is concluded that a structural homology exists between the two viruses, despite minimal primary sequence conservation.

Amino Acid Sequence↗
PubMed1984

Recombinants of Mahoney and Sabin strain poliovirus type 1: analysis of in vitro phenotypic markers and evidence that resistance to guanidine maps in the nonstructural proteins.

A neutralizing monoclonal antibody-resistant variant of the Sabin vaccine strain of poliovirus type 1 and a guanidine-resistant variant of the virulent parent Mahoney strain were derived. The two variants were used, in a coinfection, to generate recombinant virus containing both resistance markers. Recombinants appeared on the order of 1.0 PFU for every 10(4) total PFU. Two independently derived recombinant viruses were isolated. Each isolate contained the P1 (structural protein) gene region of the Sabin strain virus and the P3 (nonstructural replicase protein) gene region of the Mahoney strain virus. The recombinant virus phenotypes were compared with certain characteristic in vitro phenotypes of both parents. It was found that the slow growth in cell culture, temperature sensitivity, and virion surface charge characteristics of the Sabin virus mapped entirely to the structural protein gene region whereas the phenotype of the actinomycin D sensitivity of the Sabin virus mapped to the gene region specifying the nonstructural replication proteins. Sequence analysis of the recombinant RNA revealed that the crossover occurred 3' of nucleotide 3919. This result showed that the resistance of poliovirus mutants to growth in 2 mM guanidine hydrochloride maps in the 3'-terminal region of the viral genome specifying the nonstructural proteins.

Antigens, Viral↗
PubMed1984

Identification of a new neutralization antigenic site on poliovirus coat protein VP2.

Major neutralization antigenic sites have been previously mapped by us on VP1, the largest capsid protein of poliovirus type 1. Here we report the first identification of the primary sequence of a neutralization antigenic site on capsid protein VP2. Inspection of the amino acid sequence of VP2 led to the selection and synthesis of a peptide (n = 12) that, after linking to a carrier protein, induced an antiviral neutralizing antibody response in rabbits. The response was augmented by a single subsequent inoculation of intact virus; thus, the peptide was also capable of priming the production of neutralizing antibodies. These antibodies were directed only against the site specified by the synthetic peptide. Although the VP2-specific neutralization antigenic site appears not to be strongly immunogenic in the intact virion, it can nevertheless contribute to neutralization of poliovirus. This observation may be important for the development of peptide vaccines.

Amino Acid Sequence↗
PubMed1983

Identification of a poliovirus neutralization epitope through use of neutralizing antiserum raised against a purified viral structural protein.

VP4, one of the poliovirus structural proteins, was purified and used to prepare rabbit anti-VP4 serum. In addition to the anti-VP4 activity, this serum was also found to contain significant anti-VP3 and antivirion activities. The serum also effectively neutralized viral infectivity. The ease with which nonneutralizable variants were obtained indicated that neutralization was due to an antibody population which bound to a single virion epitope. Antigen saturation and immunoprecipitation experiments demonstrated that antibody to this epitope was also responsible for the serum's antivirion and anti-VP3 activities, as well as for a part of the anti-VP4 activity. The identification of a neutralization epitope most probably present on VP3, which cross-reacts with a site on denatured VP4, is the first report of such an epitope on a poliovirus structural protein other than VP1.

Antibodies, Viral↗
PubMed1983

Functional basis of poliovirus neutralization determined with monospecific neutralizing antibodies.

Antibody-mediated poliovirus neutralization was studied by using a series of 13 monospecific neutralizing antibodies. These antibodies were found to recognize seven individual viral epitopes, several of which functionally overlap one another. Each epitope was capable of undergoing variation so that the variant virus was no longer capable of being neutralized by antibody directed against that epitope. The measured degree of variation for each site varied from -3.1 to -4.2 log10 variant PFU per wild-type PFU. Under nonsaturating but neutralizing conditions, the antibodies, with the exception of those directed to one specific epitope, failed to completely inhibit the virion's binding to the cell. Similarly, none of the neutralizing antibodies completely inhibited viral penetration, but all prevented virus-specific transcription. A strong correlation was established between the binding of each of the neutralizing antibodies, with one exception, to the virion and a significant shift in the virion's pI from 7.0 to ca. 4.0.

Antibodies, Monoclonal↗
PubMed1983

Bivalent attachment of antibody onto poliovirus leads to conformational alteration and neutralization.

The treatment of nonsaturating, neutralizing antibody-poliovirus complexes with papain generally led to the loss of viral neutralization and to the loss of the neutralization-associated change in the isoelectric point (pI) of the virion. Subsequent treatment with anti-immunoglobulin G antibodies restored the neutralization of the virus and the alteration of the viral pI. It appears that, under nonsaturating conditions, poliovirus neutralization by an antibody is dependent upon the ability of the antibody to bivalently attach to the virion. Exceptions are monospecific neutralizing antibodies with an affinity for capsid protein VP3.

Antibodies, Monoclonal↗
PubMed1982

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.

Amino Acid Sequence↗
PubMed1982

Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites.

Animals↗
PubMed1981

Primary structure, gene organization and polypeptide expression of poliovirus RNA.

The primary structure of the poliovirus genome has been determined. The RNA molecule is 7,433 nucleotides long, polyadenylated at the 3' terminus, and covalently linked to a small protein (VPg) at the 5' terminus. An open reading frame of 2,207 consecutive triplets spans over 89% of the nucleotide sequence and codes for the viral polyprotein NCVPOO. Twelve viral polypeptides have been mapped by amino acid sequence analysis and were found to be proteolytic cleavage products of the polyprotein, cleavages occurring predominantly at Gln-Gly pairs.

Amino Acid Sequence↗
PubMedDate not supplied

A candidate vaccine for hepatitis B containing the complete viral surface protein.

The entire surface protein of hepatitis B virus serotype ayw containing the preS (preS1+preS2) and S domains has been expressed in the yeast Saccharomyces cerevisiae. Yeast containing a recombinant plasmid utilizing a constitutive promoter did not express this gene successfully due to the toxicity of the protein. A plasmid using a regulatable promoter directed expression which initiated late in the exponential phase of growth and resulted in the accumulation of high intracellular levels of the complete surface protein. The purified polypeptide aggregates into a form which, although not comprised of typical 20 nm particles, displays antigenic determinants encoded by the preS1, preS2 and S domains. Immunization of rabbits elicited the formation of antibodies directed against all three domains. This candidate vaccine will be useful for studying the contributions to viral immunity of the host response to the preS1 and preS2 domains.

Animals↗
PubMedDate not supplied

Priming for and induction of anti-poliovirus neutralizing antibodies by synthetic peptides.

Five peptides containing amino acid sequences from the type 1 poliovirus structural protein VP1 have been synthesized. Each of the peptides was found capable of priming the immune system of rabbits for a long-lasting, virus-neutralizing IgG antibody response following a single inoculation of intact virus. One peptide directly induced the production of neutralizing antibody.

Amino Acid Sequence↗