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Gene drives are engineered selfish genetic elements with the potential to spread throughout entire insect populations for sustainable vector control. Recently, a gene drive was shown to eliminate caged populations of the malaria mosquito by targeting the highly conserved female-specific exon of the doublesex gene. This caused females, homozygous for the gene drive, to develop as sterile intersex individuals, leading to the observed population crash. However, target site resistant alleles that block gene drive activity, whilst encoding a functional copy of the target gene, may halt gene drive spread in the wild. These may be naturally occurring or generated by the gene drive itself. This thesis presents a pipeline for the discovery, genetic engineering, and testing of putative drive-resistant variants. First, to investigate the potential for natural resistance, existing population genomics data were interrogated for the presence of natural single nucleotide polymorphisms (SNPs) at the highly conserved gene drive target region. To investigate the potential for drive-induced resistance, a high-throughput assay was designed to generate a high volume of mutations at the gene drive target site and screen them for their ability to restore dsx function. These methods yielded three putatively resistant SNPs: one natural polymorphism and two rare Cas9-induced mutations. These were engineered in the mosquito genome for testing, using a novel method termed CRISPR-mediated cassette exchange (CriMCE). It was confirmed that all three polymorphisms are functional and offer full, partial or no resistance to gene drive. Importantly, partial resistance to gene drive is being demonstrated for the first time. To mitigate observed resistance, gene drive systems targeting multiple sites simultaneously were developed. These showed improved drive dynamics and caused rapid elimination of caged mosquito populations within 7-8 generations. The experimental pipeline described here can be applied to pre-empt and mitigate resistance against any gene drive strategy, prior to field testing.

Malaria is an infectious disease caused by parasites of the genus Plasmodium which is responsible for approximately 400,000 deaths annually, primarily in sub-Saharan Africa. Malaria is transmitted by mosquitoes belonging to the Anopheles gambiae species complex. While progress has been made to reduce the incidence of malaria, the emergence of insecticide resistance necessitates the development of novel vector control strategies. Gene drive technologies have seen significant advances in recent years, providing hope for their implementation in the near future. While gene flow has been identified between sibling species of the An. gambiae species complex, they are reproductively isolated by both pre- and post-zygotic isolation mechanisms. Interspecific crosses between most member species produce sterile hybrid males, in accordance with Haldane's rule of speciation. The aim of this project was to support the development of gene drive technologies by investigating hybrid incompatibilities between two of the most significant vector species, Anopheles gambiae and Anopheles arabiensis. The potential for the introgression of genomic regions from one species into the genetic background of the other was investigated to help inform models regarding the spread of gene drives between sibling species. In addition, the identification of genetic elements involved in hybrid male sterility could provide potential targets for vector control strategies. Large autosomal regions were found to introgress and persist in interspecific genomes without a detectable fertility cost. In addition, the introduction of distinct autosomal regions of conspecific DNA into otherwise heterospecific genomes of hybrid males was found to overcome hybrid incompatibilities and partially restore fertility. While no specific genetic factors involved in hybrid incompatibilities could be identified, the results indicate that such factors are present at least on the X chromosome. Furthermore, the evidence suggests that asynapsis between interspecific homologous autosomes during gametogenesis plays a role in the manifestation of hybrid male sterility.

Discovery and adaptation of CRISPR-Cas systems for genome editing have allowed us to gain an efficient and yet simple tool for genetic manipulation in various fields of molecular biology and biotechnology. One of the most promising applications is the use of CRISPR-Cas9 endonuclease for gene drive systems as a population control strategy for various insect pests of medical and agricultural importance. Use of CRISPR-Cas9 endonuclease in gene drive applications has shown great promise in the laboratory, particularly for the control of Anopheles gambiae, the major vector of malaria. However, the performance of such gene drives can be limited by the range of available target sequences and by a propensity of existing endonuclease formulations to generate resistant mutations that hinder the gene drive's efficiency. To expand the flexibility of gene drive systems, computational analysis was performed to identify additional Cas9 orthologs and their specificities that could usefully augment the targeting range of endonuclease-based gene drives. Two alternative variants of CRISPR-Cas endonucleases found in the bacterial species Lactobacillus rhamnosus and Bacteroides fragilis were assessed for their potential to expand the targeting space in the genome Anopheles gambiae. In addition, a computational tool was developed that evaluates neighbouring sequences to the target site to measure both its likely functional constraint and its likely propensity for DNA repair that could generate in-frame alleles. Using this approach we were able to generate a prioritized list of Anopheles gambiae target sites for gene drive applications that are less likely to be compromised by resistant alleles.

Malaria is a devastating disease that causes more than 400,000 deaths each year, primarily in underprivileged regions of sub-Saharan Africa. During the last two decades, mortality caused by the disease has been reduced by half, largely driven by coordinated vector control based on the use of insecticides and bed nets. Nevertheless, the declining trend in malaria cases appears to have stalled recently and there is a growing concern that new interventions will be needed to reach widespread malaria elimination. Gene drive systems are potentially transformative in this endeavour because they allow rapid, self-sustaining and species-specific control of the mosquito vector through the limited release of genetically modified mosquitoes. While proof-of-principle studies have demonstrated the feasibility of the approach, none of them have managed to fulfil the requirements needed to progress in field or semi-field testing, largely due to strong fitness costs and genetic resistance to gene drive. This thesis describes the first gene drive system demonstrated to spread in caged populations of Anopheles gambiae mosquitoes, unimpeded by resistance or fitness constraints. By targeting an ultra-conserved locus in the gene doublesex, this strategy is able to thwart target site resistance in caged experiments whilst driving complete population suppression through the conversion of genetic females to sterile intersex. In this thesis, I demonstrate complete population elimination of caged populations from single releases of gene drive mosquitoes using a 12.5% initial allele release frequency, and crucially, I demonstrate its effectiveness in large cage semi-field conditions designed to reveal complex behaviours otherwise absent in small scale testing. The complete suppression of vector populations using a gene system is a landmark achievement and brings the gene drive technology closer to be implemented in the wild to complement current interventions against malaria.

Mating causes dramatic changes of female physiology, behaviour, and immunity in many insects, often inducing oogenesis, oviposition, and refractoriness to further mating. Females from the Anopheles gambiae species complex typically mate only once in their lifetime during which they receive sperm and seminal fluid proteins as well as a mating plug that contains the steroid hormone 20E. This hormone, also induced by bloodfeeding, plays a major role in activating vitellogenesis for egg production. In this thesis, I present data showing that the mating status of an Anopheles coluzzii female influences her midgut bacterial load and bacterial composition. Furthermore, I show that her susceptibility to Plasmodium falciparum infection is also enhanced upon mating especially when infection intensity is high. I find that mating status has a major impact on the midgut transcriptome, but only under sugar-fed conditions; once females have bloodfed, the transcriptional changes that are still observable as induced by mating are masked. To determine whether increased susceptibility to parasites could be driven by the additional 20E that mated females receive from males, I mimicked mating by injecting 20E into virgin females, finding that these females have significantly increased infection intensity compared to controls. I carried out further RNAseq to examine whether the genes that change upon 20E injection in the midgut are similar to those that change upon mating. I find that 11% of the genes upregulated by 20E are in common with genes upregulated by mating. Together, these findings suggest that male Anopheles mosquitoes might contribute to malaria transmission by influencing female midgut bacterial loads and by potentially increasing P. falciparum susceptibility in females.

Background: Malaria, a preventable and curable disease, is still one the primary causes of death worldwide. Around 90% of deaths occur in sub-Saharan Africa. Increased funding and new programmes by international agencies and national governments have led to significant progress in malaria control, but the improvement curve is levelling off. This is due to the limitations of current technologies and difficulties in deploying the interventions on the ground. There are calls for technological innovations which are able to provide interventions that are cost-effective, sustainable, area-wide and equitable. The development of selfsustaining genetically modified (GM) Anopheles mosquitoes is deemed to have these potential attributes, but this technology is not only innovative, but also highly complex and controversial. Using the introduction of GM mosquitoes for malaria eradication in Burkina Faso as an example this study helps to better understand how complex health technology innovations are adopted in low-income countries. Methods: The research uses a mixed methods approach and combines an in-depth analysis of the Burkinabé (Burkina Faso) health system and its wider context, qualitative system dynamics modelling and an econometric analysis in order to investigate the key barriers and enablers that influence the adoption of GM mosquito technology. Results: The research confirms the importance of community trust and acceptance, and health system readiness, in influencing the adoption process. It also emphasises the need for a perspective on the adoption of highly complex innovations which takes more account of the iterative nature of their development, the interrelationship between development and adoption, and the cognitive barriers in stakeholders' understanding of the technology. Conclusions: The intrinsic complexity of the technology development and local adoption processes, and the fact that the nature of the technology itself means that adoption decisions are societal rather than individual, together have important implications for policy and practice. These need to be taken into consideration both by local policy makers and technology researchers.

Genetic drive systems have the potential to modify entire insect populations in a few years through the super-Mendelian inheritance of a genetic trait. We describe a novel gene drive system called CRISPRh, which acts as a selfish genetic element in the malaria mosquito Anopheles gambiae. CRISPRh is a synthetic allele containing CRISPR/Cas9 endonuclease designed to specifically target and insert a copy of itself into its homologous chromosome in the germline of heterozygous individuals. Using a gene targeting approach, we identified three genes that confer recessive female sterility upon disruption. By inserting CRISPRh at each of these genes, we achieved super-Mendelian inheritance of CRISPRh alleles, ranging from 91 to 99.6% of the progeny of heterozygotes. Population modelling predicts that these high rates of transmission and a recessive knockout phenotype would enable CRISPRh alleles to spread rapidly through a wild mosquito population, causing population suppression through the generation of sterile females. We tested the potential of one CRISPRh allele to invade a caged population of naïve mosquitoes and observed an average increase from 50-76.4% over six generations, demonstrating the first gene drive designed to spread through a natural vector population. The CRISPRh constructs described here generate strong unintended fertility effects and are highly susceptible to target site resistance, making them unsuitable for malaria control. We expect that simple modifications can solve these issues and allow the development of gene drives for the suppression of mosquito populations to levels that do not support malaria transmission.

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. The work described in this thesis led to the characterization of an Anopheles gambiae transgenic strain obtained by transposase-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair a site-specific recombination signal was introduced onto the Y chromosome and the resulting docking line, named YAttP, was proven to allow secondary integration. To demonstrate its utility, the activity of a germline-specific promoter when located on the Y chromosome was studied. Anopheles arabiensis is another important vector of human malaria. Since active insertions onto the Y chromosome are extremely rare, a scheme based on crossing and selection was used to overcome F1 hybrid male sterility and introgress the modified Anopheles gambiae Y chromosome in the Anopheles arabiensis genetic background. Fertility of Y-introgressed males, tested after up to 10 backcross generations, was comparable to fertility of wild-type Anopheles arabiensis males. The molecular manipulation of the Y chromosome in Anopheles gambiae, opens up a number of ways to explore one of the most fascinating of evolution's upshots and its introgression in the Anopheles arabiensis genetic background may answer important questions on the similarity and differences in Y chromosome biology of closely related species. The Y-linked fluorescent transgenes allow automated sex separation of these important vector species, providing the means to generate large single-sex populations. Furthermore, the possibility of introducing genes of interest specifically onto the Y chromosome makes these strains a valuable tool for vector control strategies.

Over the last two decades concern about influenza has increased worldwide due to the rising number of human infections caused by an avian virus, H5N1, and the 2009-pandemic of swine-origin H1N1 virus. Counter-measures, for example the selective administration of antivirals to infected individuals, their hospitalization, and the culling of large numbers of infected animals require a prompt and reliable diagnosis, that ideally identifies the type and subtype of the virus and can be carried out at the Point-of-Care (POC). Nowadays instead diagnosis is still largely laboratory-bound, as the available POC-suitable tests are hampered by poor and very variable clinical sensitivity. This PhD was developed in the frame of a European joint effort (Fluarray) that aimed at constructing an automated diagnostic system for the rapid influenza diagnosis. Key technology of the system is an antibody microarray technology that permits the simultaneous analysis of hundreds of antigen-antibody interactions. This research investigated ways to detect the influenza virus using a panel of monoclonal antibodies that were purified, characterized and integrated into the microarray platform. The optimised immunoassay detects and differentiates the influenza nucleoproteins of type A and B at concentrations in the order of few nanograms/ml. A panel of influenza viruses were detected and their type identified within 1 hour hands-on time. The developed microarray platform will soon be integrated into an Automated Diagnostic Analyser for Microarrays (ADAM) developed by the collaborating partner Microtest Matrices. It is envisaged that the system will be a consistent aid in the influenza diagnosis and sensibly facilitate and speed up diagnostic procedures.

Studying gene function in the human malaria vector Anopheles gambiae is the key to understanding its biology and vector-parasite interactions. Existing tools to study gene function do not include gene targeting, which would allow insights into gene function by permitting a range of specific modifications to any gene of choice. Based on developments in Drosophila (RONG and GOLIC 2000) this PhD project proposes to establish a method for gene targeting in Anopheles by homologous recombination, using a linear targeting construct generated in vivo. To this end, a transgenic strain was generated that expresses FLP recombinase and I-SceI endonuclease under the control of the germline-specific vasa promoter. Together they were to excise and linearise a targeting molecule from a transgenic “donor” locus. Homology in the targeting construct would enable integration, via recombination in the germline, at a gene of interest, thereby permitting its targeted modification. Expression of vasa-driven I-SceI resulted in high cleavage activity in the germline. A systematic analysis using a variety of transgenic target loci revealed that homologous repair, rather than non-homologous end joining, was the predominant mechanism employed to repair the double stranded breaks generated by I-SceI. These findings offer encouraging prospects for population genetic engineering using homing endonuclease genes (BURT 2003). FLP activity was shown in Anopheles cell culture, yet in vivo excision via FLP at two independent loci in the germline was not observed, precluding the obtainment of a knock-out. After eliminating many possible sources of error, likely causes include antagonistic interference between I-SceI and FLP or unfavourable reaction kinetics of FLP. Examination of the ortholog of Drosophila yellow-g1 as a target gene suggests it is required somatically for female fertility, making it a good candidate for vector population control with a genetic drive system based on homing endonuclease genes. Furthermore, the above finding that recombination is promoted by endonuclease activity driven by the vasa promoter augurs well for its use in mediating efficient drive in such a system.

A key factor that limits the transmission of malaria is the age of its vector, Anopheles gambiae. This is due to the long extrinsic incubation period (EIP) of the parasite which can last a high proportion of the adult mosquito's life. Therefore, only a small percentage of a mosquito population is able to transmit malaria. This bottleneck could be of use in developing novel vector control strategies which are based on artificially shortening the adult life span of mosquitoes through the introduction of suitably tailored transgenic constructs. One strategy was to induce a toxic late-acting amino acid metabolism disease in mosquitoes that kills females after their amino acid rich blood meal. Phenylpyruvate, the toxic accumulation product in human Phenylketonuria disease, was found to cause increased death when fed to mosquitoes. GC-MS analysis showed that similar to PKU patients, phenylpyruvate had been converted to another toxic metabolite phenyllactate. Using RNAi, two enzymes PAH and PPO9 of the phenylalanine pathway were knocked down and their effect on survival, behaviour and melanisation immune response was investigated. While knockdown of one enzyme caused a reduced melanisation in response to parasite infection, reduced activity of the other led to a shortened adult life span but no change in melanisation. This led to the conclusion that both enzymes are regulating different processes and that melanisation is not necessary for mosquito survival and possibly mosquito immunity. Another strategy was to express long stretches of polyglutamines that are responsible for the toxicity of several neurodegenerative diseases, including Huntington's disease. Because uniform CAG repeats containing huntingtin exon 1 were highly instable in vitro and in vivo, different N-terminal huntingtin fragments with alternating CAG/CAA repeats fused to EGFP were inserted by attP/attB integration system in the model system Drosophila melanogaster and in Anopheles gambiae to characterise polyglutamine length dependent aggregation formation in the optic lobe and its effect on survival and behaviour. In both species, the polyglutamine fragments of putative toxic length tended to form aggregates in the photoreceptor cells. This led to a significant reduced adult life span in flies but not in mosquitoes. The latter showed a change in their chromatic vision ability. Alternative promoters were characterised for future HD mosquito models and could help to understand the observed difference between flies and mosquitoes. Both strategies have a potential as promising late-life acting vector control tools that merit further exploration.

Malaria is a tropical parasitic disease spread worldwide by protozoan parasites of the genus Plasmodium. Attempts to eradicate or control the disease have largely failed and the weapon-of-choice in combating malaria, a vaccine, still eludes us. Now that the Plasmodium falciparum (Pf ) genome has been revealed, it is essential to search for new potential antigens. On that basis, my project aims to select and produce putative vaccine candidates that together with protein microarray techniques will unravel possible correlates of protection against the human malaria parasite Pf, responsible for the majority (90%) of deaths from malaria. My PhD project forms part of FightMal a consortium-based study that first use bioinformatic analysis to identify those proteins within the Pf proteome that are known or anticipated to be either anchored on the parasite/infected host cell surface or secreted. Such in silico analysis produced 3 ‘candidate lists’ of interest, one for each of the 3 types of protein chips planned for production: i) current vaccine candidates (n.25), ii) putative immune targets (n.260), iii) variant surface antigens, VSA (n.74). Candidates belonging to CHIP 2 were cloned and expressed (in vivo) using customized high-throughput platforms. To date, 394 (88%) Chip 2 DNA targets have been successfully amplified by PCR, 325 (82%) of these have been inserted into the expression plasmid and sequenced. A total of 269 constructs resulted suitable for heterologous expression. Due to the complexity of soluble expression of plasmodial proteins a number of different expression conditions were tested. Both the strain of the E. coli host and the composition of the expression medium resulted crucial variables for satisfactory results. Via a multi-step optimisation phase we increased the initial number of soluble candidates from 18% to 44%, which eventually led to a 6-fold increase in the number of targets available for purification. During an early evaluation study, 77% (10/13) of the recombinant proteins produced in large-scale were purified using embedded affinity tags yielding a ≥90% purity level. Preliminary results, obtained spotting 40 in vitro expressed Pf candidates, successfully demonstrated the potentials of the protein microarray technology as an efficient serum-based multiplex assay capable of identifying new antigens. Using an optimised protocol, over 7000 antigen-antibody interactions were evaluated. 92% of the Pf protein panel was recognised by both total and cytophilic IgGs, with 6 novel candidates resulting as antigenic as some of the antigens evaluated in vaccine clinical trials for malaria. It is envisaged that my PhD will deliver multi-level technical know-how, which could improve the production and evaluation of novel plasmodial vaccine candidates. The final Pf microarray will be employed to screen clinical samples collected from protected and non-protected children enrolled in a longitudinal, case-control study in an area of Uganda where malaria is endemic. It is expected that the FightMal project overall will eventually generate novel immunological data and information pertaining to both the complexity of the plasmodial proteome and variability of the naturally-induced immune response against the malaria parasite.

Based on the published protein-protein interaction maps of five organisms and other public databases for domain-domain and protein-protein interactions, two new approaches are proposed to infer the protein-protein interaction network of the Anopheles gambiae (A. gambiae) mosquitoes. Our main contributions are: i) Adopted an orthologous protein pair transfer method that has so far not been seen in literature; ii) Proposed a new hybrid Bayes method; iii) Used voting machines at two levels of the combined classifier/predictor; iv) Used heterogeneous datasets as the training data; v) And finally, used the trained classifier to predict the protein interactions maps for A. gambiae, arguably one of a few least known organisms in terms of protein interaction mechanism. With the first method, the orthologous and in-paralogous protein clusters are extracted for both species. The relations between two peer-to-peer proteins in the two species are identified so that the interactions in the D. melanogaster protein interaction maps are transferred to pairs of interacting proteins in A. gambiae. The second strategy, namely the hybrid Bayes, is based on the domain composition of proteins, with which we utilize a probability model to build virtual domain-domain maps by integrating large-scale protein interaction data from five organisms, namely Saccharomyces cerevisiae, Caenorhabditis elegans, Escherichia coli, Mus musculus and Drosophila melanogaster. For the hybrid Bayes method, once the virtual domain-domain interaction maps are constructed, we propose two ways to predict the protein-protein interaction maps. These two methods are compared and then combined to form a voting machine to collectively decide a protein-pair's candidacy. The users could adjust the weights for different methods to flexibly control the output. Parameters are chosen through running different experiments on the training data set. While both the orthologous cluster and hybrid Bayes methods produce encouraging results the second one predicts more protein-protein interaction than the first. Yet these two data sets share a very small fraction of common interactions. We adopt a second voting machine and calibrate the parameters with the putative protein interaction data. Those parameters for the voting machine are used to predict the protein-protein interaction maps of the A. gambiae and produces reasonably good results.

Allergy, or type 1 hypersensitivity, is defined as a malfunction of immune system where a person's body is hypersensitized to react immunologically to non-immunogenic substances. These substances are usually innocuous environmental antigens such as pollen, house dust mites, animal epithelia, moulds, etc and collectively are known as allergens. Type I hypersensitivity is characterised by excessive activation of mast cells by IgE resulting in a systemic inflammatory response. Therefore, elevated levels of allergen specific IgEs in the serum is the key in the pathogenesis of allergic diseases. The worldwide prevalence of allergy is dramatically increasing and it is of particular concern in industrialized countries. Fifty million Americans suffer from allergic diseases including asthma, rhinitis, sinusitis, dermatitis and food allergy and in the UK it affects one in four people at some point in their lives. Therefore, there is an increasing need for a sensitive in vitro test capable of rapidly and accurately quantify allergen-specific IgE from patient sera. The aim of this project is to assess the potential of high throughput technology for simultaneous in vitro measurement of allergen-specific IgEs to a variety of well-known environmental and food allergens and the development of a novel diagnostic biochip for clinical use. For this purpose, a protein microarray platform based on non purified allergens has been developed, evaluated, and validated to generate proof-of- principle and to demonstrate the specificity and sensitivity of the array design. The protein microarray method developed for detection of specific IgE antibodies in human sera consist of four defined steps: 1. Printing: microspots containing total allergen extract and, antibodies are covalently immobilized onto activated glass microscope slides by means of high speed robotics. Minute volumes reagents are arrayed in 7 X 7 matrices, each spot diameter being 400 pm. 2. Processing: slides are initially developed with small volumes (100 pl_) of serum samples. A seguential series of reagents are applied to the slide including detecting antibody and amplification reagents conjugated to a fluourophore. The total assay time required is less than 3 hours. 3. Scanning slides are processed by means of a high sensitivity fluorescence-detecting scanner that is able to gather the fluorescence signal emitted by each array component. 4. Quantification and analysis: computational analysis of the image data. The studies carried out to validate the assay have shown that by using the tyramide amplification system it is possible to achieve an excellent degree of analytical sensitivity and analytical specificity. In addition, no cross-reactivity to other immunoglobulins at normal serum levels was detected. Reproducibility and repeatability are also within the standard requirements for this kind of immunoassay. Short-term stability studies have been performed to assess the stability of the assay over-time and the results are satisfactory over 90 days. Finally, the clinical validation of the microarray assay showed that the data generated with this system is substantially better than the reference method in terms of sensitivity, correlation between class score and fluorescent signal. The microarray technology has several advantages over existing diagnostic methods such as ELISA. This system offers high throughput and true parallelism; it is highly economical in the use of specimens and reagents and also provides a rapid and accurate detection method for the quantification of specific IgEs in human serum. The knowledge and experience gained in this project will be used in the evaluation of a pre-market microarray- based in vitro diagnostic device.