Michelle E. Gahan

Exposure to dideoxynucleosides is reflected in lowered mitochondrial DNA in subcutaneous fat.

Objectives: Nucleoside reverse transcriptase inhibitors (NRTIs), particularly dideoxynucleoside analogs (ddNs), used in the treatment of HIV, inhibit mitochondrial DNA polymerase &ggr; in vitro. Mitochondrial DNA (mtDNA) depletion is proposed as the underlying mechanism of many of the in vivo side effects of these agents. A reliable and valid laboratory test to detect this is not yet available. The objective of this study was to correlate tissue mtDNA quantification in HIV‐infected patients with exposure to nucleoside analogs. Methods: 60 HIV‐infected adults underwent detailed clinical assessment and blood and tissue sampling. Clinical and antiretroviral treatment details were correlated with results of plasma lactate assays, and real‐time polymerase chain reaction quantification of mtDNA in peripheral blood mononuclear cells (PBMCs) and subcutaneous fat from the lower limb. Results: Forty‐nine (82%) subjects were on combination antiretroviral therapy. Of these, 33 (55%) were currently receiving one or more ddNs (stavudine, didanosine, or zalcitabine). mtDNA in subcutaneous fat was lower in subjects currently on ddNs than in those not taking ddNs (mean [log10] 2.47 vs. 2.74, p = .002). Plasma lactate was somewhat higher in subjects currently taking ddNs than those on no antiretroviral treatment (median 1.5 vs. 1.0, p = .03), but was not significantly different in either of these groups compared with subjects on other NRTIs. mtDNA in PBMCs did not vary with treatment status. Conclusions: mtDNA in subcutaneous fat was significantly reduced in patients currently taking ddNs. mtDNA in PBMCs was independent of patient exposure to NRTIs.

Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction

BACKGROUND With decreased rates of HIV mortality and disease progression attributable to treatment with nucleoside analogue reverse transcriptase inhibitors (NRTIs), attention has now become focused on the toxicities of these forms of treatment. It is believed NRTIs cause a decrease in mitochondrial DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. This hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought to manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial toxicity is needed to assess toxicity and optimise HIV treatment strategies. Peripheral blood mononuclear cells (PBMC) and subcutaneous fat could be ideal and accessible sources of mtDNA for examining toxicity. OBJECTIVES The objectives of this study were (a) to develop an assay to quantify the mtDNA copy number of PBMC and obtain reproducible results and (b) to establish the utility of subcutaneous fat as a source of mtDNA for quantification. STUDY DESIGN PBMC were isolated from blood by centrifugation over Ficoll-Paque and subcutaneous fat was obtained from two 3 mm punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantified by real-time polymerase chain reaction (PCR). RESULTS The real-time PCR assay was found to generate consistent and reproducible results with replicates of samples undertaken within the same run, and in two or more different runs, having a mean coefficient of variation of 11.3 and 17.2%, respectively. PBMC and subcutaneous fat contained 409+/-148 and 2042+/-391 copies of mtDNA per cell, respectively. CONCLUSIONS From the work carried out it can be concluded that firstly, the real-time PCR assay generates consistent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat.

Tissue-specific associations between mitochondrial DNA levels and current treatment status in HIV-infected individuals.

Background: Tissue mitochondrial DNA (mtDNA) levels have been proposed as a marker of nucleoside analouge reverse transcriptase inhibitor (NRTI) toxicity. However, clinical studies have yielded conflicting data regarding possible associations with mtDNA levels. This study examined mtDNA levels in matched samples of peripheral blood mononuclear cells (PBMCs) and subcutaneous fat from a large Australian cohort to examine treatment, clinical, and demographic associations with mtDNA depletion. Methods: mtDNA was quantified by real-time polymerase chain reaction. Results were compared across patient treatment and demographic details using linear mixed models. Results: One hundred sixty-three PBMCs and 161 fat samples were available from 61 individuals. Current NRTI exposure was the major determinant of mtDNA levels. Both ddI (didanosine) and d4T (stavudine) exposures were associated with mtDNA depletion in fat (P ≤ 0.0001 vs. those not on NRTIs). DdI exposure (P = 0.003), but not d4T exposure (P = 0.5), was associated with mtDNA depletion in PBMCs. No association between patient demographics or time on current therapy and mtDNA was observed. Conclusions: Current NRTI exposure is the major determinant of tissue mtDNA, but the precise determinants are tissue specific. Both ddI and d4T exposure are associated with fat mtDNA depletion, whereas ddI exposure was the only observed association with mtDNA depletion in PBMCs.

Assessment of precision and concordance of quantitative mitochondrial DNA assays: a collaborative international quality assurance study.

BACKGROUND A number of international research groups have developed DNA quantitation assays in order to investigate the role of mitochondrial DNA depletion in anti-retroviral therapy-induced toxicities. OBJECTIVES A collaborative study was undertaken to evaluate intra-assay precision and between laboratory concordance of measurements of mitochondrial DNA quantity, as a component of a comprehensive quality assurance project. STUDY DESIGN Four laboratories were asked to measure and report mitochondrial DNA and nuclear DNA genome copy number, as well as mitochondrial DNA copy number/cell, for 17 coded aliquots of DNA derived from serial dilutions of pooled DNA from a lymphoblastoid cell line. Samples included masked replicates and five standards. All samples had similar mitochondrial DNA/nuclear DNA ratios. Precision within laboratories was assessed by determining the coefficient of variation of replicates. Concordance between laboratories was assessed by determining the average coefficient of variation of the mean replicate values for each sample. The effect of standardising the assay for these three measurements was also assessed for laboratories A, B and C. RESULTS Measurements of mitochondrial DNA and nuclear DNA content for replicate samples varied by an average of less than 6% (based on log(10) values, 72% non-logged values), and measurements of mitochondrial DNA/cell for replicates varied by less than 12% (based on log(10) values, 32% non-logged values), with no improvement of precision after standardisation. Standardisation did significantly improve the concordance of results for measurements of mitochondrial DNA content and mitochondrial DNA/cell. Non-standardised measurements of mitochondrial DNA content for the same sample set varied by 19% between laboratories (based on log(10) values, 96% non-logged values), and after standardisation results varied by less than 3% (based on log(10) values, 54% non-logged values). There was no significant improvement for concordance of measures of nuclear DNA content after standardisation, with results varying by 4.56% between laboratories (based on log(10) values, 45% non-logged values) before standardisation, and by 2.49% (based on log(10) values, 50% non-logged values) after standardisation. Derived values of mitochondrial DNA/cell varied between laboratories by an average of 91% (non-logged, 56% log(10) values) before and by 56% (non-logged, 13% log(10) values) after standardisation. CONCLUSION All assays demonstrated good precision. The use of common standards is an important step in improving the comparability of data between laboratories.

Changes in mitochondrial DNA in peripheral blood mononuclear cells from HIV-infected patients with lipoatrophy randomized to receive abacavir

It has been suggested that lipoatrophy associated with exposure to nucleoside analogues is caused by depletion of mitochondrial DNA (mtDNA). The aim of the present study was to determine whether switching treatment from a thymidine analogue to abacavir was associated with an increase in the mtDNA copy number in peripheral blood mononuclear cells (PBMCs). Of 111 patients with lipoatrophy who were randomized to have treatment switched to abacavir or to continue treatment with thymidine analogues, 94 patients had PBMCs obtained at baseline and at weeks 4, 12, and 24, for quantification of the mtDNA copy number. During the 24-week study, there was no significant change in mtDNA copy numbers in PBMCs in either treatment group, despite improvement in peripheral lipoatrophy among patients whose treatment was switched to abacavir.

Decreased Neurotropism of nef Long Terminal Repeat ( nef /LTR)-Deleted Simian Immunodeficiency Virus

Simian immunodeficiency virus (SIV) infection of macaques results in neurological abnormalities similar to those of human immunodeficiency virus (HIV)-associated dementia in humans and is a valuable system for the identification of viral neurotropic and neurovirulence factors. The authors recently established an SIV-macaque model where macaques can be infected with wild-type or nef/LTR-deleted SIVmac239 via administration of purified proviral DNA. In this study, the ability of wild-type and nef/LTR-deleted SIV infections to enter the cerebral spinal fluid (CSF) and brain was analyzed. In situ polymerase chain reaction (PCR) readily detected SIV gag DNA-positive cells in the mid-frontal gyrus and basal ganglia of the wild-type SIV-infected macaques, but not in nef/LTR-deleted SIV-infected or SIV-uninfected macaques. PCR on extracted DNA confirmed the in situ results, with multiple brain regions of the wild-type SIV-infected macaques positive for both gag and wild-type nef, whereas in the nef/LTR-deleted SIV-infected macaques, nef/LTR and gag DNA were undetectable. Further, macaques infected with nef/LTR-deleted SIV, which later became superinfected with wild-type SIV, also remained negative for SIV DNA in the brain by both in situ and extracted DNA techniques, despite having high levels of SIV RNA both in the CSF and plasma. This study provides evidence of the inability of nef/LTR-deleted SIV to initiate central nervous system (CNS) infection and suggests that, in the brain regions examined, nef/LTR-deleted viruses have either diminished neurotropism or insufficient systemic viral replication for entry into the CNS.

Advances in oral vaccine delivery options

Vaccines have been one of the most far-reaching and important public health initiatives of the 20th century. Yet as we move into the 21st century, millions of people still die from vaccine-preventable diseases such as measles and tetanus, and complex diseases, such as malaria and HIV, for which we have no vaccines. New vaccines that can be administered orally, are stable at ambient temperature, and can be produced cheaply, have the potential to transform health policy and practice in both developed and developing countries.Although oral delivery is the preferred route of administration, it is inefficient for the delivery of ‘naked’ antigens. Delivery of a vaccine via the oral route in a sufficient dose to induce a protective immune response depends on overcoming the loss of antigen integrity that occurs during intestinal passage. Several strategies have been employed to prevent this loss of antigen(icity) and improve the delivery of vaccinogens by the oral route. These include live vectors, transgenic plants, and particulate formulations such as microparticles, liposomes, and virus-like particles. Although many of these systems have progressed to clinical trials, the most promising results are seen where strategies are combined. Prime-boost schedules and combined technologies are likely to be a critical component of future oral vaccination schedules. Further technological breakthroughs may also be required before strong protective immune responses can be consistently induced in humans. Nonetheless, the potential of future oral vaccination strategies is readily apparent.

Bacterial Antigen Expression Is an Important Component in Inducing an Immune Response to Orally Administered Salmonella-Delivered DNA Vaccines

Background The use of Salmonella to deliver heterologous antigens from DNA vaccines is a well-accepted extension of the success of oral Salmonella vaccines in animal models. Attenuated S. typhimurium and S. typhi strains are safe and efficacious, and their use to deliver DNA vaccines combines the advantages of both vaccine approaches, while complementing the limitations of each technology. An important aspect of the basic biology of the Salmonella/DNA vaccine platform is the relative contributions of prokaryotic and eukaryotic expression in production of the vaccine antigen. Gene expression in DNA vaccines is commonly under the control of the eukaryotic cytomegalovirus (CMV) promoter. The aim of this study was to identify and disable putative bacterial promoters within the CMV promoter and evaluate the immunogenicity of the resulting DNA vaccine delivered orally by S. typhimurium. Methodology/Principal Findings The results reported here clearly demonstrate the presence of bacterial promoters within the CMV promoter. These promoters have homology to the bacterial consensus sequence and functional activity. To disable prokaryotic expression from the CMV promoter a series of genetic manipulations were performed to remove the two major bacterial promoters and add a bacteria transcription terminator downstream of the CMV promoter. S. typhimurium was used to immunise BALB/c mice orally with a DNA vaccine encoding the C-fragment of tetanus toxin (TT) under control of the original or the modified CMV promoter. Although both promoters functioned equally well in eukaryotic cells, as indicated by equivalent immune responses following intramuscular delivery, only the original CMV promoter was able to induce an anti-TT specific response following oral delivery by S. typhimurium. Conclusions These findings suggest that prokaryotic expression of the antigen and co-delivery of this protein by Salmonella are at least partially responsible for the successful oral delivery of C-fragment DNA vaccines containing the CMV promoter by S. typhimurium.

Impact of prior immunological exposure on vaccine delivery by Salmonella enterica serovar Typhimurium

Recombinant Salmonella have been employed as vaccine vectors to deliver DNA and protein vaccines of viral, bacterial and parasitic origin. However, the effectiveness of Salmonella delivery may be hampered by prior immunological exposure to Salmonella. We investigated the effects of prior exposure to the Salmonella typhimurium aroAD strain BRD509 on its ability to deliver the non-toxic C fragment of tetanus toxin (TT). BALB/c mice were orally immunised with BRD509 containing the C fragment expression plasmid pTETtac4, C fragment DNA vaccine pAT153/Cfrag or BRD509 alone and, along with age matched un-vaccinated controls, were immunised again 6 months later with BRD509 (pTETtac4). Prior exposure to Salmonella was found to significantly reduce the ability of the bacteria to colonise the Peyers patches and mesenteric lymph nodes, spread systemically to the liver and spleen and significantly impair antibody responses to Salmonella LPS and TT. Results show that prior exposure to Salmonella significantly compromises its efficacy as a vaccine vector and these negative effects do not diminish with time. In addition, findings suggest Salmonella vaccine vectors cannot be employed to deliver multiple doses of a vaccine antigen.