ABSTRACT
PBCC211, an aroA aroD derivative of S. typhi strain CDC10-80, was tested in phase I trials as a single dose typhoid fever vaccine. Three different vaccine preparations, reconstituted lyophilized bacteria, freshly grown bacteria or lyophilized bacteria reconstituted from sachets, were orally administered to a total of 86 adult volunteers. An aroA aroD htrA strain, PBCC222, was also tested in 38 volunteers. Formulation impacted on the determination of a safe and immunogenic dose; reconstituted lyophilized cultures required higher doses than the broth cultures to stimulate seroconversion. In general, doses which seroconverted the majority of group members produced undesirable symptoms regardless of attenuation or formulation. The inability to separate the presence of symptoms from achieving significant immunogenicity in these aroA aroD or aroA aroD htrA strains precludes their use as single dose typhoid vaccines in the formulations tested. Multiple doses of these strains at a lower, safe level may be effective as vectors for foreign antigens.
Subject(s)
Bacterial Vaccines/administration & dosage , Cell Cycle Proteins/administration & dosage , Heat-Shock Proteins , Periplasmic Proteins , Salmonella typhi/immunology , Serine Endopeptidases/genetics , Adolescent , Adult , Bacterial Vaccines/adverse effects , Bacterial Vaccines/immunology , Cell Cycle Proteins/immunology , Freeze Drying , Humans , Middle Aged , Osmolar Concentration , Salmonella typhi/growth & development , Vaccination , Vaccines, Attenuated/immunologyABSTRACT
An outer membrane PIA protein from Neisseria gonorrhoeae strain FA19 was expressed in Escherichia coli and refolded in vitro in the presence of zwitterionic detergent. Its proper folding and subunit organization was confirmed by comparison with the native counterpart. The unfolding of PIA has been investigated using fluorescence spectroscopy and analytical size-exclusion chromatography methods. Analysis of the denaturation pathway of the PIA revealed that it forms an unusually labile quaternary structure. In the presence of 1 M guanidinium chloride (GdmCl) or upon heating up to 50 degrees C, dissociation of the PIA oligomer was observed resulting in the formation of folded monomeric intermediates. Unfolding of monomers occurs at 80 degrees C or in the presence of 4.3 M GdmCl, indicating high intrinsic stability toward both GdmCl and elevated temperatures. Both oligomeric and monomeric forms of PIA exhibited affinity to the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) and bind with Kd=80 and 130 microM, respectively. Denaturation of the PIA completely abolished affinity to ANS, suggesting that hydrophobicity is a property of the folded state of the porin.
Subject(s)
Neisseria gonorrhoeae , Porins/chemistry , Chromatography, Gel , Porins/genetics , Protein Conformation , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Spectrometry, Fluorescence , TemperatureSubject(s)
Antigens/administration & dosage , Bacteria/genetics , Administration, Oral , Amino Acid Sequence , Animals , Antigens/genetics , Epitopes/genetics , Genetic Vectors , Humans , Molecular Sequence Data , Proteins/genetics , Proteins/immunology , Salmonella/genetics , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/isolation & purificationABSTRACT
Human papillomavirus type 1 (HPV-1) causes plantar warts. On the basis of previously mapped mRNAs and sequence homologies of HPV-1 to other papillomaviruses, we designed oligonucleotide primers and employed the polymerase chain reaction to recover HPV-1 cDNAs from plantar warts. Seven spliced RNA species were characterized, including three not previously detected, and the coding potentials of each were deduced. The most abundant viral mRNA encodes an E1i--E4 protein. One new species is predicted to encode the full-length E2 protein, and another can, theoretically, encode the E2-C or E1-M proteins, three products that regulate mRNA transcription and DNA replication. One RNA species originating from a novel HPV promoter in the upstream regulatory region has the potential to encode the minor capsid protein L2. A newly recognized E5a open reading frame (ORF) is contained in all mRNAs that are polyadenylated at the E-region poly(A) site and also in a putative L2 mRNA. Three distinct species, two of which are derived from the upstream regulatory region promoter, have the potential to encode the L1 protein; the third species also contains the entire coding region of the E1i--E4 protein 5' to the L1 ORF. Both the E1i--E4 mRNA and the potentially bicistronic L1 mRNA are derived from a promoter located in the E7 ORF. We uncovered no evidence of alternatively spliced mRNAs that could account for the multiple, abundant E4 proteins in plantar warts, suggesting that posttranslational modification is mainly responsible for the observed protein heterogeneity.
Subject(s)
Foot Diseases/microbiology , Genes , Papillomaviridae/genetics , RNA, Messenger/genetics , Warts/microbiology , Base Sequence , Capsid/genetics , DNA, Viral/genetics , Exons , Humans , Keratinocytes/microbiology , Male , Molecular Sequence Data , Oligonucleotide Probes , Papillomaviridae/isolation & purification , Polymerase Chain Reaction , RNA Splicing , RNA, Messenger/isolation & purificationABSTRACT
A plasmid, pTME6, containing Neisseria gonorrhoeae lipopolysaccharide biosynthesis genes was used as a probe to analyze DNA from strains of N. gonorrhoeae, N. meningitidis and various commensal Neisseria by Southern blotting. Chromosomal DNA from 26 gonococcal strains probed with 32P-labeled pTME6 produced five different hybridization patterns. No correlation between hybridization pattern and auxotype, serotype, serum sensitivity or SDS-urea-PAGE migration of LPS was observed. DNA from strains of N. meningitidis, N. lactamica and N. cinerea, but not other commensal Neisseria species, hybridized strongly to pTME6.
