Hepatitis C infection in plasmapheresis donors: an overlooked risk factor for transmission?

Hepatitis C virus infection is the leading cause of chronic liver disease worldwide and puts significant burden on health resources. Traditionally, risk factors are well recognized. Despite this, a significant percentage of people infected with hepatitis C have no identifiable mode of transmission. However, it is known that a significant number of donors at plasmapheresis have positive hepatitis C serology, thereby potentially contaminating the equipment and putting both donors and recipients at risk. Here, we describe two patients who acquired hepatitis C infection following voluntary donation at plasmapheresis abroad and suggest that this is an unusual and overlooked mode of transmission.

Omp85 proteins of Neisseria gonorrhoeae and Neisseria meningitidis are similar to Haemophilus influenzae D-15-Ag and Pasteurella multocida Oma87.

The genes encoding homologous 85 kDa outer membrane proteins of Neisseria gonorrhoeae and Neisseria meningitidis have been cloned and sequenced. The gonococcal gene, omp85, was identified by screening a genomic library with an antiserum raised against purified gonococcal outer membranes. The gene encoded a 792 amino acid protein, Omp85, having a typical signal peptide and a carboxyl-terminal phenylalanine characteristic of outer membrane proteins. The amino acid sequence was similar to that of the D15 protective surface antigen (D-15-Ag) of Haemophilus influenzae, and the Oma87 of Pasteurella multocida. Southern analysis demonstrated that omp85 was present as a single copy in N. gonorrhoeae and N. meningitidis. PCR amplification was used to obtain a clone of the N. meningitidis omp85 homologue. Sequence analysis revealed that the N. meningitidis Omp85 was 95% identical to the N. gonorrhoeae Omp85.

Generation of antiserum to specific epitopes.

The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific epitopes of an outer membrane protein of Neisseria gonorrhoeae. The MtrC AP1 sequence from the invariant MtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared to bind native MtrC protein in situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein, including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to activate complement protein can then be tested.

A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines.

Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.

The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein.

Cell-free translational analysis of the processing of infectious pancreatic necrosis virus polyprotein.

A cDNA clone of the large genomic segment of infectious pancreatic necrosis virus (IPNV) was inserted into transcription vectors and used for production of RNA transcripts of various lengths. These RNA transcripts were used to prime the synthesis of virus-specific polypeptides in a rabbit reticulocyte translation system. Full-length transcripts resulted in the synthesis of processed viral proteins pVP2, NS, and VP3. Transcripts which were deleted in the VP2 coding region did not affect the processing of NS or VP3 and similarly, deletions in VP3 did not affect the formation of NS or VP2. However, deletions which extended into the NS coding region resulted in a loss of protease activity and the production of truncated precursor polypeptides. The virus-specific proteolytic activity could not be inhibited by specific antisera and a trans activity for the viral proteases could not be demonstrated.

Expression in Escherichia coli of the large genomic segment of infectious pancreatic necrosis virus.

A complete cDNA clone of the larger A segment of the genome of infectious pancreatic necrosis virus (IPNV) was expressed in Escherichia coli in an effort to develop a vaccine for IPNV in fish. When the cDNA insert was positioned in the correct orientation to the pUC19 lacZ promoter, the viral proteins VP2, NS, and VP3 were synthesized and processed as observed in infected cells. When the insert was placed in the opposite orientation, VP3 and a 38-kDa virus-specific polypeptide were also synthesized. In addition, specific deletions made from the 3' end into the NS gene of the cloned A segment led to inactivation of the NS proteolytic activity and subsequently, the synthesis of an unprocessed VP2-NS polyprotein precursor. Antiserum to this polyprotein distinguished NS (28.5 kDa) from VP3 (31 kDa) and led to the identification of a previously undescribed 38-kDa virus-specific polypeptide in infected cells. Thus, both internal translational initiation and proteolytic cleavage could lead to the synthesis of VP2, NS, and VP3 from a single mRNA with a single open reading frame. A trpE expression vector, pATH2, was used to synthesize large quantities of the A-segment-encoded proteins in bacteria. The resulting bacterial lysate was very effective in inducing protective immunity in rainbow trout fry.

Multiple tandem promoters of the major outer membrane protein gene (omp1) of Chlamydia psittaci.

The transcription of omp1, the gene encoding the major outer membrane protein, was studied for two strains of Chlamydia psittaci, guinea pig inclusion conjunctivitis (GPIC) and mouse pneumonitis (Mn). The transcriptional initiation sites for the omp1 of each strain were mapped by S1 nuclease and primer extension analyses. Three different sizes of omp1 transcripts were observed for GPIC and four were observed for Mn. The production of these transcripts appeared to be the consequence of multiple tandem promoters. The order in which the omp1 RNA transcripts appeared during the growth cycle of the C. psittaci strains was found to differ from that of C. trachomatis.

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.