Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.

Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.

Direct ITR-to-ITR Nanopore Sequencing of AAV Vector Genomes.

Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.

Human and Insect Cell-Produced Recombinant Adeno-Associated Viruses Show Differences in Genome Heterogeneity.

In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing "inaccurate" vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in Spodoptera frugiperda insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these "mutated" ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.

Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs for segmental deletions, or a single sgRNA with a homology-directed repair (HDR) template. We also use anti-CRISPR proteins to enable production of vectors that self-inactivate via Nme2Cas9 cleavage. We further introduce a nanopore-based sequencing platform that is designed to profile rAAV genomes and serves as a quality control measure for vector homogeneity. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice by HDR-based correction of the disease allele. These results will enable the engineering of single-vector AAVs that can achieve diverse therapeutic genome editing outcomes.

Epidemiology of dengue fever in Gabon: Results from a health facility-based fever surveillance in Lambaréné and its surroundings.

BACKGROUND: In Africa, information on dengue is limited to outbreak reports and focused on some countries with continuing transmission in West and East Africa. To estimate the proportion of dengue-positive cases among febrile patients and identify clinical indicators of dengue cases, we conducted passive facility-based fever surveillance in a catchment area population of 70,000 residents of Lambaréné and its surroundings in Gabon. METHODS: Non-malarial febrile patients with current fever or history of fever (≤7 days) between 1 and 55 years of age, were enrolled at Albert Schweitzer Hospital (ASH). Acute (visit 1, day of enrollment) and convalescent blood samples were collected between 10 and 21 days after enrollment. Acute/convalescent samples were tested with IgM/IgG ELISA, and a selected subset of acute samples with RT-PCR. RESULTS: Among 682 non-malarial febrile patients enrolled, 119 (17.4%) were identified as dengue-positive (94 dengue-confirmed and 25 dengue-probable cases). Of these dengue-positive cases, 14 were confirmed with PCR, and based on serotyping, two infections were identified to be DENV-2 and two were DENV-3. The majority of our enrolled patients were <25 years of age and close to 80% of our dengue-positive cases were <15 years of age. In adjusted analyses, retro-orbital pain and abdominal pain were 2.7 and 1.6 times more frequently found among dengue-positive cases, compared to non-dengue cases. CONCLUSION: Lambaréné is not considered dengue-endemic. However, one in six non-malarial febrile episodes was found to be dengue-positive in the study period. Dengue should be considered more frequently in clinicians' diagnosis among non-malarial febrile patients in Lambaréné. Given the lack of data on dengue in Gabon, additional prospective and longitudinal studies would help to further define the burden and patterns of dengue for improved case detection.

Estimating the Force of Infection for Dengue Virus Using Repeated Serosurveys, Ouagadougou, Burkina Faso.

Because of limited data on dengue virus in Burkina Faso, we conducted 4 consecutive age-stratified longitudinal serologic surveys, ≈6 months apart, among persons 1-55 years of age, during June 2015-March 2017, which included a 2016 outbreak. The seroconversion rate before the serosurvey enrollment was estimated by binomial regression, taking age as the duration of exposure, and assuming constant force of infection (FOI) over age and calendar time. We calculated FOI between consecutive surveys and rate ratios for potentially associated characteristics based on seroconversion using the duration of intervals. Among 2,897 persons at enrollment, 66.3% were IgG-positive, and estimated annual FOI was 5.95%. Of 1,269 enrollees participating in all 4 serosurveys, 438 were IgG-negative at enrollment. The annualized FOI ranged from 10% to 20% (during the 2016 outbreak). Overall, we observed high FOI for dengue. These results could support decision-making about control and preventive measures for dengue.

Clinical and epidemiologic characteristics associated with dengue fever in Mombasa, Kenya.

OBJECTIVES: Information on dengue in Africa is limited. To estimate the proportion of dengue-positive cases among febrile patients and describe clinical indicators of dengue, we conducted passive health facility-based fever surveillance in Mombasa, Kenya. METHODS: Non-malarial febrile patients between one and 55 years were enrolled at three health facilities between March 2016 and May 2017. Acute and convalescent blood samples were collected with an interval of 10-21 days. Acute samples were tested with dengue RDT and a selected subset with RT-PCR, and acute/convalescent samples with IgM/IgG ELISA. RESULTS: Among 482 enrollees, 295 (61.2%) were dengue-positive based on laboratory results. The surveillance covered the beginning of a dengue outbreak in April-May 2017, during which 73.9% of enrollees were dengue-positive. By contrast, during the non-outbreak period, 54.6% were dengue-positive. Dengue case status was positively associated with rash, fatigue, headache, retro-orbital pain, nausea/vomiting, nose bleeding, gum bleeding, loss of appetite, myalgia, and arthralgia. Dengue-positive cases in our study had mostly mild disease, with only two requiring observation, and no DHF. CONCLUSIONS: The clinical response was generally mild relative to what was observed in SE Asia and the Americas. Given the high level of DENV transmission in Mombasa, more data would be needed to further understand the disease burden and improve case detection for surveillance/monitoring of outbreaks.

Clinical and epidemiologic characteristics associated with dengue during and outside the 2016 outbreak identified in health facility-based surveillance in Ouagadougou, Burkina Faso.

BACKGROUND: In Africa, the magnitude of dengue virus (DENV) transmission is largely unknown. In Burkina Faso, several outbreaks have been reported and data are often based on findings from outbreak investigations. METHODS: To better understand dengue epidemiology and clinical characteristics in Burkina Faso, a fever surveillance study was conducted among patients aged 1-55 years, who presented with non-malarial febrile illness at five primary healthcare facilities in Ouagadougou, Burkina Faso from December 2014 to February 2017, encompassing a 3-month dengue outbreak in September-November 2016. Acute and convalescent blood samples were collected within an interval of 10-21 days between visits. Acute samples were tested with dengue rapid diagnostic tests (RDT) and a selected subset with RT-PCR, and all acute/convalescent samples with IgM/IgG ELISA. RESULTS: Among 2929 non-malarial febrile patients, 740 (25%) were dengue-positive based on RT-PCR and/or IgM/IgG ELISA; 428 out of 777 patients (55%) and 312 out of 2152 (14%) were dengue-positive during outbreak and non-outbreak periods, respectively. There were 11% (316/2929) and 4% (129/2929) patients showing positive for NS1 and IgM, on the RDT, respectively. DENV 2 predominated during the outbreak, whereas DENV 3 predominated before the outbreak. Only 25% of dengue-positive cases were clinically diagnosed with suspected dengue. The odds of requiring observation for ≤3 days (versus routine outpatient care) were 11 times higher among dengue-positive cases than non-dengue cases. In adjusted analyses, dengue-positivity was associated with rash and retro-orbital pain (OR = 2.6 and 7.4, respectively) during the outbreak and with rash and nausea/vomiting (OR = 1.5 and 1.4, respectively) during the non-outbreak period. CONCLUSION: Dengue virus is an important pathogen in Burkina Faso, accounting for a substantial proportion of non-malarial fevers both during and outside outbreak, but is only infrequently suspected by clinicians. Additional longitudinal data would help to further define characteristics of dengue for improved case detection and surveillance.

A multi-country study of the economic burden of dengue fever based on patient-specific field surveys in Burkina Faso, Kenya, and Cambodia.

BACKGROUND: Dengue fever is a rapidly growing public health problem in many parts of the tropics and sub-tropics in the world. While there are existing studies on the economic burden of dengue fever in some of dengue-endemic countries, cost components are often not standardized, making cross-country comparisons challenging. Furthermore, no such studies have been available in Africa. METHODS/PRINCIPAL FINDINGS: A patient-specific survey questionnaire was developed and applied in Burkina Faso, Kenya, and Cambodia in a standardized format. Multiple interviews were carried out in order to capture the entire cost incurred during the period of dengue illness. Both private (patient's out-of-pocket) and public (non-private) expenditure were accessed to understand how the economic burden of dengue is distributed between private and non-private payers. A substantial number of dengue-confirmed patients were identified in all three countries: 414 in Burkina Faso, 149 in Kenya, and 254 in Cambodia. The average cost of illness for dengue fever was $26 (95% CI $23-$29) and $134 (95% CI $119-$152) per inpatient in Burkina Faso and Cambodia, respectively. In the case of outpatients, the average economic burden per episode was $13 (95% CI $23-$29) in Burkina Faso and $23 (95% CI $19-$28) in Kenya. Compared to Cambodia, public contributions were trivial in Burkina Faso and Kenya, reflecting that a majority of medical costs had to be directly borne by patients in the two countries. CONCLUSIONS/SIGNIFICANCE: The cost of illness for dengue fever is significant in the three countries. In particular, the current study sheds light on the potential economic burden of the disease in Burkina Faso and Kenya where existing evidence is sparse in the context of dengue fever, and underscores the need to achieve Universal Health Coverage. Given the availability of the current (CYD-TDV) and second-generation dengue vaccines in the near future, our study outcomes can be used to guide decision makers in setting health policy priorities.

Evaluating dengue burden in Africa in passive fever surveillance and seroprevalence studies: protocol of field studies of the Dengue Vaccine Initiative.

INTRODUCTION: Dengue is an important and well-documented public health problem in the Asia-Pacific and Latin American regions. However, in Africa, information on disease burden is limited to case reports and reports of sporadic outbreaks, thus hindering the implementation of public health actions for disease control. To gather evidence on the undocumented burden of dengue in Africa, epidemiological studies with standardised methods were launched in three locations in Africa. METHODS AND ANALYSIS: In 2014-2017, the Dengue Vaccine Initiative initiated field studies at three sites in Ouagadougou, Burkina Faso; Lambaréné, Gabon and Mombasa, Kenya to obtain comparable incidence data on dengue and assess its burden through standardised hospital-based surveillance and community-based serological methods. Multidisciplinary measurements of the burden of dengue were obtained through field studies that included passive facility-based fever surveillance, cost-of-illness surveys, serological surveys and healthcare utilisation surveys. All three sites conducted case detection using standardised procedures with uniform laboratory assays to diagnose dengue. Healthcare utilisation surveys were conducted to adjust population denominators in incidence calculations for differing healthcare seeking patterns. The fever surveillance data will allow calculation of age-specific incidence rates and comparison of symptomatic presentation between patients with dengue and non-dengue using multivariable logistic regression. Serological surveys assessed changes in immune status of cohorts of approximately 3000 randomly selected residents at each site at 6-month intervals. The age-stratified serosurvey data will allow calculation of seroprevalence and force of infection of dengue. Cost-of-illness evaluations were conducted among patients with acute dengue by Rapid Diagnostic Test. ETHICS AND DISSEMINATION: By standardising methods to evaluate dengue burden across several sites in Africa, these studies will generate evidence for dengue burden in Africa and data will be disseminated as publication in peer-review journals in 2018.

Prospects for dengue vaccines for travelers.

Travel-acquired dengue cases have been increasing as the overall global dengue burden has expanded. In Korea, imported dengue cases have been reported since 2000 when it first became a notifiable disease. During the first four months of 2016, three times more dengue cases were reported in Korea than during the same period the previous year. A safe and efficacious vaccine for travelers would be beneficial to prevent dengue disease in individual travelers and potentially decrease the risk of virus spread to non-endemic areas. Here, we summarize the characteristics of dengue vaccines for travelers and review dengue vaccines currently licensed or in clinical development.

Molecular evidence for an old world origin of Galapagos and Caribbean band-winged grasshoppers (Acrididae: Oedipodinae: Sphingonotus).

Patterns of colonization and diversification on islands provide valuable insights into evolutionary processes. Due to their unique geographic position and well known history, the Galapagos Islands are an important model system for evolutionary studies. Here we investigate the evolutionary history of a winged grasshopper genus to infer its origin and pattern of colonization in the Galapagos archipelago. The grasshopper genus Sphingonotus has radiated extensively in the Palaearctic and many species are endemic to islands. In the New World, the genus is largely replaced by the genus Trimerotropis. Oddly, in the Caribbean and on the Galapagos archipelago, two species of Sphingonotus are found, which has led to the suggestion that these might be the result of anthropogenic translocations from Europe. Here, we test this hypothesis using mitochondrial and nuclear DNA sequences from a broad sample of Sphingonotini and Trimerotropini species from the Old World and New World. The genetic data show two distinct genetic clusters representing the New World Trimerotropini and the Old World Sphingonotini. However, the Sphingonotus species from Galapagos and the Caribbean split basally within the Old World Sphingonotini lineage. The Galapagos and Caribbean species appear to be related to Old World taxa, but are not the result of recent anthropogenic translocations as revealed by divergence time estimates. Distinct genetic lineages occur on the four investigated Galapagos Islands, with deep splits among them compared to their relatives from the Palaearctic. A scenario of a past wider distribution of Sphingonotus in the New World with subsequent extinction on the mainland and replacement by Trimerotropis might explain the disjunct distribution.