Cynthia C. Hager

Enhanced Production of Recombinant Mycobacterium tuberculosis Antigens in Escherichia coli by Replacement of Low-Usage Codons

ABSTRACT A major obstacle to development of subunit vaccines and diagnostic reagents for tuberculosis is the inability to produce large quantities of these proteins. To test the hypothesis that poor expression of some mycobacterial genes in Escherichia coli is due, in part, to the presence of low-usage E. coli codons, we used site-directed mutagenesis to convert low-usage codons to high-usage codons for the same amino acid in the Mycobacterium tuberculosis genes for antigens 85A and 85B and superoxide dismutase. Replacement of five codons in the wild-type gene for antigen 85B increased recombinant protein production in E. coli 54-fold. The recombinant antigen elicited proliferation and gamma interferon production by lymphocytes from healthy tuberculin reactors and was recognized by monoclonal antibodies to native antigen 85, indicating that the recombinant antigen contained T-cell and B-cell epitopes. Northern blotting demonstrated only a 1.7- to 2.5-fold increase in antigen 85B mRNA, suggesting that the enhanced protein production was due primarily to enhanced efficiency of translation. Codon replacement in the genes encoding antigen 85A and superoxide dismutase yielded four- to sixfold increases in recombinant protein production, suggesting that this strategy may be generally applicable to overexpression of mycobacterial genes in E. coli.

Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG

Background In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB. During decades of in vitro cultivation BCG not only lost some genes due to deletions of regions of the chromosome but also underwent gene duplication and other mutations resulting in increased antioxidant production. Methodology/Principal Findings To determine whether microbial antioxidants influence vaccine immunogenicity, we eliminated duplicated alleles encoding the oxidative stress sigma factor SigH in BCG Tice and reduced the activity and secretion of iron co-factored superoxide dismutase. We then used assays of gene expression and flow cytometry with intracellular cytokine staining to compare BCG-specific immune responses in mice after vaccination with BCG Tice or the modified BCG vaccine. Compared to BCG, the modified vaccine induced greater IL-12p40, RANTES, and IL-21 mRNA in the spleens of mice at three days post-immunization, more cytokine-producing CD8+ lymphocytes at the peak of the primary immune response, and more IL-2-producing CD4+ lymphocytes during the memory phase. The modified vaccine also induced stronger secondary CD4+ lymphocyte responses and greater clearance of challenge bacilli. Conclusions/Significance We conclude that antioxidants produced by BCG suppress host immune responses. These findings challenge the hypothesis that the failure of extensively cultivated BCG vaccines to prevent pulmonary tuberculosis is due to over-attenuation and suggest instead a new model in which BCG evolved to produce more immunity-suppressing antioxidants. By targeting these antioxidants it may be possible to restore BCGs ability to protect against pulmonary TB.

Detection of Rochalimaea henselae DNA by polymerase chain reaction from suppurative nodes of children with cat-scratch disease

A polymerase chain reaction (PCR) assay was designed to amplify DNA from Rochalimaea henselae, Rochalimaea quintana and Afipia felis in the purulent material from lymph nodes in three patients with clinical cat-scratch disease (CSD) and two patients with lymphadenitis from other causes. All of the patients with CSD had positive immunofluorescent antibody serology for R. henselae, while none of the controls was positive. PCR amplification confirmed the presence of R. henselae DNA and the absence of R. quintana and A. felis DNA in the purulent material from CSD patients. PCR samples from control patients were negative. The PCR amplification of R. henselae DNA was performed quickly and with great sensitivity and specificity. It confirmed the presence of R. henselae in the CSD patients and eliminated the need for more extensive diagnostic and therapeutic procedures.

Booster response to acellular pertussis vaccine in children primed with acellular or whole cell vaccines.

Few data are available on the effect of a booster dose of acellular pertussis vaccine in children primed as infants with acellular vaccine. We administered acellular pertussis vaccine (ACV) at 19 months to children immunized in infancy with ACV or whole cell vaccine. Forty-one infants had been randomly assigned to receive either ACV or whole cell vaccine at 2, 4 and 6 months of age. Antibody titers to pertussis toxin and filamentous hemagglutinin were significantly higher in ACV than whole cell vaccine recipients at 7 months; at 15 months antibody to filamentous hemagglutinin (but not pertussis toxin) remained significantly higher among those receiving ACV. At 19 months all 41 children received an ACV booster. Local and systemic reactions were few and minor and were equally distributed between the two groups. All children responded to booster with significant increases in antibody; these in-creases tended to be greater for those having been primed with ACV. ACV booster immunization appears safe and immunogenic, regardless of the vaccine given for primary immunization.

Cocaine Enhances HIV-1–Induced CD4+ T-Cell Apoptosis: Implications in Disease Progression in Cocaine-Abusing HIV-1 Patients

Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.

Synthesis and in vitro and in vivo activity of a hybrid composed of ricin B chain-barley ribosome-inactivating protein

In our continued studies on hybrid proteins for use as cytotoxins and possible suicide transport agents, we have begun to investigate the use of ribosome-inactivating proteins (RIP) isolated from grain. The RIP from barley has been purified to homogeneity by modifications of the methods of Roberts and Selitrennikoff and crosslinked to the binding subunit B of the seed toxin ricin (RTB). The resulting hybrid was purified by a combination of gel filtration and affinity chromatography on acid-washed Sepharose 4B. This model suicide transport agent was assayed in vitro against K-562 cells and was found to be cytotoxic in a dose-dependent manner (ID50 = 0.15 micrograms/ml). Lactose inhibited the toxicity of the hybrid, indicating that cytotoxicity was dependent on the cell binding property of the ricin B moiety. In addition, free RIP and free ricin B, either alone or in combination, were nontoxic over this concentration range. The in vivo effects of the RTB-RIP hybrid were assessed by pressure microinjection into the vagus nerves of rats. Injection of 0.18 to 6.5 micrograms of conjugate resulted in death of vagal sensory but not motor neurons after 3-17 days. The cytotoxic changes in vagal sensory neurons were identical to those previously observed with a variety of RIP toxins such as ricin.

A Modified Bacillus Calmette-Guérin (BCG) Vaccine with Reduced Activity of Antioxidants and Glutamine Synthetase Exhibits Enhanced Protection of Mice despite Diminished in Vivo Persistence

Early attempts to improve BCG have focused on increasing the expression of prominent antigens and adding recombinant toxins or cytokines to influence antigen presentation. One such modified BCG vaccine candidate has been withdrawn from human clinical trials due to adverse effects. BCG was derived from virulent Mycobacterium bovis and retains much of its capacity for suppressing host immune responses. Accordingly, we have used a different strategy for improving BCG based on reducing its immune suppressive capacity. We made four modifications to BCG Tice to produce 4dBCG and compared it to the parent vaccine in C57Bl/6 mice. The modifications included elimination of the oxidative stress sigma factor SigH, elimination of the SecA2 secretion channel, and reductions in the activity of iron co-factored superoxide dismutase and glutamine synthetase. After IV inoculation of 4dBCG, 95% of vaccine bacilli were eradicated from the spleens of mice within 60 days whereas the titer of BCG Tice was not significantly reduced. Subcutaneous vaccination with 4dBCG produced greater protection than vaccination with BCG against dissemination of an aerosolized challenge of M. tuberculosis to the spleen at 8 weeks post-challenge. At this time, 4dBCG-vaccinated mice also exhibited altered lung histopathology compared to BCG-vaccinated mice and control mice with less well-developed lymphohistiocytic nodules in the lung parenchyma. At 26 weeks post-challenge, 4dBCG-vaccinated mice but not BCG-vaccinated mice had significantly fewer challenge bacilli in the lungs than control mice. In conclusion, despite reduced persistence in mice a modified BCG vaccine with diminished antioxidants and glutamine synthetase is superior to the parent vaccine in conferring protection against M. tuberculosis. The targeting of multiple immune suppressive factors produced by BCG is a promising strategy for simultaneously improving vaccine safety and effectiveness.