The use of plasmids is critical in the gene therapy viral vector workflow: plasmids are widely used for creation of the viral vector construct, along with helper and packaging plasmids for final ...product creation. While the construct insert is universally sequenced during initial design, sequencing of the backbone of the plasmid is not frequently assessed. Sequence errors within the plasmid backbone, helper and packaging plasmids can have major impacts in downstream packaging and viral vector production. Traditional Sanger approaches can be used for fast and frequent characterization, however limitations within the technology necessitate many sequencing reactions to span the full plasmid, with a known reference sequence. Traditional Next Generation Sequencing (NGS) approaches can overcome the need for multiple reactions and a reference sequence but are historically slower and cost-prohibitive.
Here we describe a fast and cost-effective sequencing approach for sequencing circular plasmid DNA to generate a highly accurate and annotated full-length consensus sequences without the need for primer design or a known reference sequence. This long-read sequencing method is comparable in cost and time to Sanger sequencing but unlike Sanger sequencing, overcomes common issues within hard to sequence regions like long nucleotide repeats or inverted terminal repeat (ITR) domains of adeno-associated viruses (AAVs). When used effectively, it can be harnessed to characterize gene therapy viral vector plasmid and associated packaging plasmids required throughout the gene therapy development life cycle without delay, ultimately accelerating the viral vector design and development process.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background. The extent to which drug-resistant human immunodeficiency virus type 1 (HIV-1) acquired through mother-to-child transmission (MTCT) or failed chemoprophylaxis populates viral reservoirs ...and limits responses to antiretroviral treatment in infants is unknown. Methods. We evaluated the presence, type, and persistence of drug-resistant HIV-1 in pretreatment plasma and resting CD4+ T cells from US infants enrolled in a multicenter, open-label, phase 1/2 treatment trial of lopinavir/ritonavir (Pediatric AIDS Clinical Trials Group + Results. Twenty-two consecutively enrolled infants initiating highly active antiretroviral therapy at a median age of 9.7 weeks and treated for up to 96 weeks were studied. Drug-resistant HIV-1 was present in 5 (23.8%) of 21 infants analyzed; 4 (80.0%) had nonnucleoside reverse transcriptase inhibitor (NNRTI)—resistant HIV-1, only 1 of whom had a history of receiving nevirapine chemoprophylaxis. All 4 infants had NNRTI-resistant variants other than the K103N mutation. The fifth infant had the M184V mutation. Drug-resistant virus was archived in the resting CD4+ T cell latent reservoir in all 5 infants. Conclusions. The high rate, types, and early archiving of drug-resistant HIV-1 suggests that resistance testing be considered for infants, especially when an NNRTI-based regimen is planned. Furthermore, drug-resistance outcomes in infants should be an important secondary end point in MTCT trials.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Nevirapine resistance mutations arise commonly following single or extended-dose nevirapine (ED-NVP) prophylaxis to prevent mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV), ...but decay within 6-12 months of single-dose exposure. Use of ED-NVP prophylaxis in infants is expected to rise, but data on decay of nevirapine resistance mutations in infants in whom ED-NVP failed remain limited. We assessed, in Ethiopian infants participating in the Six-Week Extended Nevirapine (SWEN) Trial, the prevalence and persistence of nevirapine resistance mutations at 6 and 12 months following single-dose or up to 6 weeks of ED-NVP, and correlated their presence with the timing of infection and the type of resistance mutations. Standard population genotyping followed by high-throughput cloning were done on dried blood spot samples collected during the trial. More infants who received ED-NVP had nevirapine resistance detected by standard population genotyping (high frequencies) at age 6 months compared with those who received single-dose nevirapine (SD-NVP) (58% of 24 vs. 26% of 19, respectively; p = 0.06). Moreover, 56% of ED-NVP-exposed infants with nevirapine resistance at age 6 months still had nevirapine resistance mutations present at high frequencies at age 1 year. Infants infected before 6 weeks of age who received either SD- or ED-NVP were more likely to have Y181C or K103N; these mutations were also more likely to persist at high frequencies through 1 year of age. HIV-infected infants in whom ED-NVP prophylaxis fails are likely to experience delayed clearance of nevirapine-resistant virus in the first year of life, which in turn places them at risk for early selection of multidrug-resistant HIV after initial therapy with nonnucleoside reverse transcriptase inhibitor-based regimens.
Objectives: Identification of HIV infection in exposed infants facilitates early therapy, which may limit viral reservoirs that maintain HIV infection under HAART. Methods: The dynamics of the ...resting CD4 super(+) T-cell latent HIV reservoir was determined over the first 2 years of life in 17 HIV-infected infants initiating lopinavir/ritonavir-based HAART at a median age of 8.1 weeks and achieving adequate suppression of plasma viral load by 24 weeks. Results: The resting CD4 super(+) T-cell latent HIV reservoir was detected in 12 of 14 (86%) infants tested at 24 weeks of HAART median frequency 1.88 infectious units per million (IUPM); range <0.22 to 81.7), and remained measurable (median IUPM = 0.32; range <0.22 to 3.25) in six of 10 (60%) children retested at 96 weeks. The reservoir declined, from 24 to 96 weeks of HAART, at an estimated mean rate of 0.028 log sub(10) IUPM/month, corresponding to a half-life of 11 months (95% confidence interval 6-30 months. A strong relationship was found between the frequency of latently infected CD4 super(+) T cells at 96 weeks of HAART and time to first undetectable plasma viral load (Spearman r = 0.91, P < 0.001). Conclusion: Although the resting CD4 super(+) T-cell latent reservoir remains detectable over the first 2 years of HAART in a substantial proportion of infants, its size is associated with time to first undetectable viral load. To minimize HIV reservoirs in infants, rapid curtailment of viremia may limit HIV reservoirs and should be a therapeutic goal of early HAART in infants.
Exposure to antiretroviral drugs in resource-constrained settings is likely to result in the emergence of drug-resistant HIV-1 variants, limiting treatment options. Genotypic drug resistance testing ...assists clinical management and outcomes assessment, but a sensitive and reproducible genotypic assay feasible for resource-constrained settings is needed. A sensitive, reproducible genotyping assay to detect HIV-1 drug resistance mutations in reverse transcriptase and protease was developed and validated using blood stored as dried blood spots or as frozen RNA extracts from Zambian children infected with subtype C. HIV-1 genotypes derived from samples stored as dried blood spots were compared to those derived from paired liquid plasma samples in American young adults infected with HIV-1 subtype B. The method reproducibly amplified patient-specific sequences and detected drug resistance mutations from all of the dried blood spots or excess frozen RNA extracts with detectable viremia over a broad range of viral loads (193–3 million HIV-1 RNA copies/mL) in both HIV-1 subtypes B and C infection. This method captured the genetic variation typical of HIV-1 infection, including mutations at usual sites of drug resistance, polymorphisms, and mixtures. This sensitive and reproducible genotypic assay is feasible for detection of antiretroviral resistance in resource-constrained settings.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Objectives: Therapeutic HIV vaccinations may alter the size of the resting memory CD4 super(+) T-cell latent HIV reservoir as HIV establishes latency when memory responses are formed, including those ...toward HIV. Alternatively, latently infected CD4 super(+) T cells maybe killed, while exiting the reservoir upon activation. Methods: The effect of therapeutic immunization with modified vaccinia Ankara and Fowlpox-based HIV vaccines on the latent reservoir was examined in 19 young adults who were receiving effective antiretroviral therapy. Correlations between size of the reservoir measured in infectious units per million (IUPM) resting CD4 super(+) T cells and HIV-specific immune responses, including immune activation were examined. Decay of the reservoir was assessed using random-effects model. Results: A modest transient decrease in the size of the reservoir was observed at week 40 mean -0.31 log sub(10) IUPM (95% confidence interval: -0.60 to -0.03; P=0.03 following HIV vaccinations. The estimated half-life (T sub(1/2)) of the reservoir during the 40 weeks following vaccination was 9.8 months and statistically different from zero (P=0.02), but 35.3 months and not different from zero (P=0.21) over 72 weeks of study. Latent reservoir size at baseline was not correlated with HIV-specific CD4 super(+), CD8 super(+) responses or immune activation, but became correlated with CD4 super(+) IFN gamma (r=0.54, P=0.02) and IL-2 responses at 6 weeks after immunization (r=0.48, P=0.04). Conclusion: Therapeutic HIV vaccinations led to a transient increase in decay of latently infected CD4 super(+) T cells. Further studies of therapeutic HIV vaccines may provide important insights into facilitating decay of the latent reservoir.
The integration of three hydraulic GIS (Geographic Information System) applications is presented which represent the water infrastructures of cities and urban areas and US streams and rivers. The ...water infrastructures include drinking water distribution systems, wastewater collection systems and source water. The National Research Council states that problems dealing with the collective behavior of networks such as river systems, water distribution systems and waste water collection systems are complex because they include feedback loops, produce counter-intuitive behaviors and exhibit behaviors that cannot be predicted from the attributes of individual components. A complex system includes all of the above individual components, yet also exhibits emergent collective behavior caused by the interactions among its features. The integration of these applications have been developed for use in planning, response, training and development of monitoring strategies to address potential deliberate or accidental toxic contamination events.
The corrosion rate of electropolished 304 stainless steel surfaces (UNS S30400) is found to be lower by more than a factor of three relative to that determined previously for machined surfaces in ...mildly alkaline, hydrogenated water at 260
°C. This favorable result is attributed to significant changes in nanocrystallinity of the corrosion oxide layer caused by the removal of surface microstrain, which had been imparted during the machining process. In the absence of microstrain, a low-porosity, protective, corrosion layer forms that is composed of extremely small and uniformly-sized spinel oxide crystals. Application of scanning electron microscopy (FEG-SEM), X-ray diffraction and X-ray photoelectron spectroscopy (XPS) in conjunction with ion milling and target factor analyses, found the corrosion layer to consist of micrometer-size crystals of a ferrite-based spinel oxide (non-protective) over-laying nanometer-size crystals of a chromite-based spinel oxide (protective). Composition of both phases is unchanged from that previously observed on corroded, machined surfaces and is representative of solvus phases in the immiscible Fe(Fe
1−
n
Cr
n
)
2O
4 spinel binary. The smaller size (10 vs. 26
nm) and greater surface density (∼10,000 vs. 835
μm
−2) of the chromite-based crystals relative to those formed on machined (i.e., cold-worked) surfaces, however, is consistent with the absence of preferred high energy nucleation sites on strain-free surfaces. Therefore, electropolishing, which removes surface microstrain induced by cold-working, represents a preferred reference surface condition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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