Jerry M. Keith

Optochin Resistance in Streptococcus pneumoniae: Mechanism, Significance, and Clinical Implications

Traditionally, Streptococcus pneumoniae is identified in the laboratory by demonstrating susceptibility to optochin. Between 1992 and 1998, 4 pneumococcal isolates exhibiting optochin resistance were recovered from patients at Childrens National Medical Center. Three of the 4 isolates consisted of mixed populations of optochin-resistant and -susceptible organisms. Both subpopulations had identical antibiograms, serotypes, and restriction fragment profiles. The other isolate was uniformly resistant to optochin. Resistant strains had MICs of optochin 4-30-fold higher than susceptible strains, belonged to different serotypes, and had dissimilar restriction fragment profiles, indicating clonal unrelatedness. Resistance arose from single point mutations in either the a-subunit (W206S) or the c-subunit (G20S, M23I, and A49T) of H(+)-ATPase. There is speculation of a possible association between exposure to antimalarial drugs and evolution of optochin resistance. alpha-Hemolytic streptococci resistant to optochin, particularly invasive isolates, should be tested for bile solubility or with an S. pneumoniae DNA probe before identification as viridans streptococci.

Delivery of antigens to the MHC class I pathway using bacterial toxins.

Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway. This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway. Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated. Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol. Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway. Of the toxins studied-diphtheria, pertussis, Pseudomonas, and anthrax-the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation.

Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus: Comparison with homologous genes from four strains of human and animal rotaviruses

A full-size cloned cDNA copy of the rotavirus gene encoding the structural neutralization glycoprotein (VP7) of Nebraska calf diarrhea virus (NCDV), a strain recently shown to be effective as a vaccine in children, has been sequenced. Comparison of the deduced amino acid sequence of NCDV (serotype 6) VP7 with that of four other rotavirus strains (human WA serotype 1, human HU-5 serotype 2, simian SA-11 serotype 3, and bovine UK serotype 6) indicates that the degree of amino acid homology among VP7 neutralization proteins of these serotypes ranges from 75 to 86%. Four hydrophilic regions at amino acid residues 174-183, 248-256, 287-294, and 310-317 exhibit significant homology and hence may represent common antigenic determinants, while one hydrophilic area at amino acid residues 83-102 exhibits sufficient divergence to suggest it may be involved in serotype specificity.

INTERNALIZATION OF A BACILLUS ANTHRACIS PROTECTIVE ANTIGEN-C-MYC FUSION PROTEIN MEDIATED BY CELL SURFACE ANTI-C-MYC ANTIBODIES

BackgroundAnthrax toxin, secreted by Bacillus anthracis, consists of protective antigen (PA) and either lethal factor (LF) or edema factor (EF). PA, the receptor-binding component of the toxin, translocates LF or EF into the cytosol, where the latter proteins exert their toxic effects. We hypothesized that anthrax toxin fusion proteins could be used to kill virus-infected cells and tumor cells, if PA could be redirected to unique receptors found only on these cells.Materials And MethodsTo test this hypothesis in a model system, amino acids 410–419 of the human p62c-myc epitope were fused to the C-terminus of PA to redirect PA to the c-Myc-specific hybridoma cell line 9E10.ResultsThe PA-c-Myc fusion protein killed both mouse macrophages and 9E10 hybridoma cells when administered with LF or an LF fusion protein (FP59), respectively. Similar results were obtained with PA, which suggests that PA-c-Myc used the endogenous PA receptor to enter the cells. By blocking the endogenous PA receptors on 9E10 cells with the competitive inhibitor PA SNKEΔFF, the PA-c-Myc was directed to an alternate receptor, i.e., the anti-c-Myc antibodies presented on the cell surface. The c-Myc IgG were proven to act as receptors because the addition of a synthetic peptide containing the c-Myc epitope along with PA SNKEΔFF further reduced the toxicity of PA-c-Myc + FP59.ConclusionThis study shows that PA can be redirected to alternate receptors by adding novel epitopes to the C-terminus of PA, enabling the creation of cell-directed toxins for therapeutic purposes.

A Conservative Amino Acid Mutation in the Chromosome-Encoded Dihydrofolate Reductase Confers Trimethoprim Resistance in Streptococcus pneumoniae

Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.

Preparation, characterization, and immunogenicity in mice of a recombinant influenza H5 hemagglutinin vaccine against the avian H5N1 A/Vietnam/1203/2004 influenza virus.

Production of influenza vaccines requires a minimum of 6 months after the circulating strain is isolated and the use of infectious viruses. The hemagglutinin (protective antigen) of circulating influenza viruses mutates rapidly requiring reformulation of the vaccines. Our goal is to eliminate the risk of working with infectious virus and reduce significantly the production time. A cDNA fragment encoding the influenza virus A/Vietnam/1203/2004 (H5N1) HA gene was prepared using RT-PCR with viral RNA as a template. Recombinant HA (rHA) protein was produced in Escherichia coli and purified from isolated inclusion bodies by urea solubilization and Ni(+)-ion column chromatography. Vaccine candidates were prepared by treating the rHA with formalin, adsorption onto alum or with both. Mice were injected subcutaneously with candidate vaccines two or three times 2 weeks apart. Sera were collected 1 week after the last injection and antibody measured by ELISA and hemagglutination inhibition (HI). The highest antibody response (GM 449EU) was elicited by three injections of 15microg alum-adsorbed rHA. Dosages of 5microg of rHA formulated with formalin and alum, and 5microg alum-adsorbed rHA elicited IgG anti-HA of GM 212 and 177EU, respectively. HI titers, >or=40 were obtained in >or=80% of mice with three doses of all formulations. We developed a method to produce rHA in a time-frame suitable for annual and pandemic influenza vaccination. Using this method, rHA vaccine can be produced in 3-4 weeks and when formulated with alum, induces HA antibody levels in young outbred mice consistent with the FDA guidelines for vaccines against epidemic and pandemic influenza.

The Diphtheria and Pertussis Components of Diphtheria-Tetanus Toxoids-Pertussis Vaccine Should Be Genetically Inactivated Mutant Toxins

Replacement of cellular with acellular pertussis (aP) vaccines has considerably reduced the systemic reactions observed with diphtheria-tetanus toxoids-pertussis vaccine but has not eliminated the extensive swelling (sometimes involving an entire limb) observed after the fifth injection of diphtheria-tetanus toxoids-aP (DTaP) vaccine. This local reaction, which is likely an Arthus hypersensitivity reaction caused by high levels of antibodies reacting with DTaP vaccine, could discourage its use in adults, who serve as the major reservoir of pertussis for infants. That a critical level of antibodies to pertussis toxin is both essential and sufficient to prevent infection with Bordetella pertussis is derived from data from animal and clinical studies, including data showing the similarities between the immunity induced by diphtheria and pertussis toxoids. The genetically inactivated diphtheria and pertussis mutant toxins are more immunogenic and, therefore, induce comparable levels of antitoxin at lower protein levels than do the formalin-treated native toxins. Replacement of the diphtheria and aP components with these improved antigens will reduce the amount of protein in DTaP vaccine and, most likely, the incidence and severity of local reactions in teenagers and adults.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells treated with an anthrax toxin fusion protein

As a novel and safe vaccine strategy, the anthrax toxin-mediated antigen delivery system composed of lethal factor (LF) fusion protein and protective antigen (PA) has been studied to prime hepatitis C virus (HCV) core-specific cytotoxic T lymphocytes (CTLs) in vivo. The core epitope fused to LF (LF-core) together with PA induces a negligible core-specific CTL response in mice, whereas core-specific CTL are effectively primed in mice by injecting dendritic cells (DCs) treated in vitro with LF-core and PA. These findings imply that LF fusion protein plus PA in combination with dendritic cells may be useful for a novel T cell vaccine against HCV infection.

Toward a new vaccine for pertussis

To overcome the limitations of the current pertussis vaccines, those of limited duration of action and failure to induce direct killing of Bordetella pertussis, a synthetic scheme was devised for preparing a conjugate vaccine composed of the Bordetella bronchiseptica core oligosaccharide with one terminal trisaccharide to aminooxylated BSA via their terminal ketodeoxyoctanate residues. Conjugate-induced antibodies, by a fraction of an estimated human dose injected into young outbred mice as a saline solution, were bactericidal against B. pertussis, and their titers correlated with their ELISA values. The carrier protein is planned to be genetically altered pertussis toxoid. Such conjugates are easy to prepare, stable, and should add both to the level and duration of immunity induced by current vaccine-induced pertussis antibodies and reduce the circulation of B. pertussis.

Oligosaccharide conjugates of Bordetella pertussis and bronchiseptica induce bactericidal antibodies, an addition to pertussis vaccine

Pertussis is a highly contagious respiratory disease that is especially dangerous for infants and children. Despite mass vaccination, reported pertussis cases have increased in the United States and other parts of the world, probably because of increased awareness, improved diagnostic means, and waning vaccine-induced immunity among adolescents and adults. Licensed vaccines do not kill the organism directly; the addition of a component inducing bactericidal antibodies would improve vaccine efficacy. We investigated Bordetella pertussis and Bordetella bronchiseptica LPS-derived core oligosaccharide (OS) protein conjugates for their immunogenicity in mice. B. pertussis and B. bronchiseptica core OS were bound to aminooxylated BSA via their terminal Kdo residues. The two conjugates induced similar anti-B. pertussis LPS IgG levels in mice. B. bronchiseptica was investigated because it is easier to grow than B. pertussis. Using B. bronchiseptica genetically modified strains deficient in the O-specific polysaccharide, we isolated fractions of core OS with one to five repeats of the terminal trisaccharide, having at the nonreducing end a GlcNAc or GalNAc, and bound them to BSA at different densities. The highest antibody levels in mice were elicited by conjugates containing an average of 8–17 OS chains per protein and with one repeat of the terminal trisaccharide. Conjugate-induced antisera were bactericidal against B. pertussis, and the titers correlated with ELISA-measured antibody levels (r = 0.74). Such conjugates are easy to prepare and standardize; added to a recombinant pertussis toxoid, they may induce antibacterial and antitoxin immunity.