Abstract
With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.
Significance Statement Here we describe the development of Ae. aegypti mosquitoes synthetically engineered to impede vector competence to multiple serotypes of DENV. Given the global significance of DENV, these mosquitoes may be used to curtail Dengue transmission by this potent vector.
Introduction
Dengue fever is a devastating viral disease caused by several antigenically distinct dengue viral (DENV) serotypes that are all dangerous to humans but cannot be readily controlled using broad-spectrum techniques1, 2. Transmitted by infected mosquitoes, DENV infection typically manifests as severe fever, headaches, and myalgia3 and can advance to the life-threatening dengue hemorrhagic fever and dengue shock syndrome4. Global incidences of DENV and its associated economic burden have increased dramatically in recent years5, 6, with over 50% of the world’s population now at risk of infection7 and 390 million documented infections per year6 for an estimated $40 billion in economic losses annually8, 9. Moreover, there are currently no specific treatments nor preventive prophylactic measures10 because the single commercially available vaccine11 is only partially effective12, and due to increased risk of severe dengue illness and hospitalization among certain groups, its use is prevented in many contexts13–15. Therefore, control of the disease-spreading mosquitoes is currently the best option for preventing DENV transmission13.
Aedes aegypti16, the main vector of DENV and other epidemiologically significant viruses such as chikungunya (CHIKV), yellow fever (YFV), and Zika (ZIKV), is a domestic endophilic mosquito17 that has expanded its habitable range in recent decades18 and will likely continue to spread19. Current control measures including the removal of standing water and the use of chemical insecticides have had limited success in reducing Aedes populations 20 and, thereby, incidences of DENV21, and can instead cause insecticide resistance and behavioral changes such as a shift in biting times22, 23. Therefore, novel vector control strategies24, like the use of genetically modified mosquitoes to either suppress mosquito populations or render mosquitoes unable to transmit pathogens25, are increasingly needed. For example, the development and deployment of a genetic Sterile Insect Technique (SIT)-like system termed Release of Insect Dominant Lethal (RIDL) has had some success in reducing Aedes mosquito populations in the wild26, 27. Moreover, releases of mosquitoes artificially infected with the intracellular endosymbiont Wolbachia, which can make infected males incapable of successfully mating with uninfected females in an SIT-like manner and can inhibit mosquito infection with pathogens such as DENV and ZIKV28, 29, have also been carried out to either suppress mosquito populations or make them less likely to transmit pathogens, and may hold promise for reducing incidence of disease30, 31. However, these technologies require releases of large numbers of insects - and must be carried out on an ongoing basis for RIDL and Wolbachia-based SIT - for continued vector control, which is laborious and expensive.
Therefore, there has been increasing interest in the development of engineered gene-drive technologies, which are able to rapidly transmit themselves and any linked “cargo” genes, such as anti-pathogen effectors, through wild disease-transmitting populations25, 32, 33 such that only a few releases of modest amounts of engineered insects could drive desirable cargo genes through wild populations, making them efficient and cost effective for vector control. To achieve disease reduction, such gene-drive systems need to be linked to useful “cargo”, such as effective anti-pathogen genes, and several approaches for engineering Ae. aegypti resistance to DENV have been attempted. For example, one study used RNA interference by employing inverted RNA repeats to target DENV-2 in a conditional and tissue-specific manner34, 35, while another described miRNA cassettes targeting DENV-3 that reduced viral transmission rates36. In addition to using synthetic small RNAs, others have taken advantage of the mosquito’s innate antiviral JAK/STAT pathway to increase resistance to DENV-2 and DENV-437. However, all previous approaches have been limited by their ability to target only one or two—not all four—major DENV serotypes. Because hyperendemicity of DENV in tropical areas is frequent5 and secondary DENV infection has been linked to severe dengue disease (SDD), refractory mosquitoes should be capable of blocking all serotypes or risk being ineffective in controlling dengue epidemics. Therefore, better anti-DENV effectors are needed.
Broadly neutralizing antibodies may be especially promising as anti-DENV effector gene candidates because of their ability to neutralize antigenically diverse viruses38. However, while engineered monoclonal antibodies that confer resistance to Plasmodium, a protozoan parasite that causes malaria, have been expressed in Anophelene mosquitoes39–41, none targeting a virus have been described in any mosquito species. Previously, a DENV-targeting 1C19 monoclonal antibody (MAb) was identified from a large panel of naturally occurring MAbs from human subjects following vaccination or natural infection42. In vitro studies demonstrated that this antibody neutralized viruses from all major DENV serotypes and was capable of significantly reducing viremia in a mouse model after DENV-1 and DENV-2 infection42. Here, we engineer Ae. aegypti to express a 1C19-based, broadly neutralizing, single-chain variable fragment (scFv) that is capable of neutralizing all four DENV serotypes42. Crucially, we demonstrate that mosquitoes expressing this anti-DENV scFv cannot be infected with or transmit any of the four DENV serotypes and have few significant fitness costs conferred by the presence of the antibody. These results provide a promising route for developing effective DENV control strategies using genetically engineered mosquitoes.
Results
Generation of DENV-resistant mosquitoes
To determine whether expressing an anti-DENV antibody in mosquitoes could confer resistance to DENV, we first needed to engineer a broadly neutralizing antibody that was compatible with mosquitoes and could be expressed in vivo in its desired form. We chose 1C19 as our model due to its ability to cross-neutralize multiple DENV serotypes in humans42. As it is a human monoclonal antibody, however, it cannot be unobtrusively expressed in mosquitoes, so a new form that is both compatible with mosquitoes and maintains its neutralization capabilities had to be designed. We then choose to engineer an scFv comprising the linked variable heavy (VH) and light (VL) chains because this format removes the human-specific constant region that could impart difficulties in a mosquito and it can be expressed in one “chunk” in an organism without the need for additional in vivo processing. To do this, sequences for the 1C19 VH and VL chains were obtained from hybridoma cells expressing the human monoclonal antibody42. We then engineered a scFv comprising the VH and VL domains of 1C19 linked using a 15-amino-acid repeating glycine-serine [G(4)S]3 linker43 that was codon-optimized for Ae. aegypti. We also engineered a version of this 1C19 scFv that was fused with a 3’ 30-amino-acid human influenza hemagglutinin (HA) epitope tag, commonly used as a general expression tag, reasoning that it might be useful in downstream expression analyses. To conditionally drive expression of the 1C19 scFvs in the midgut of female mosquitoes following a blood meal, which would ensure 1C19 expression any time the mosquito was in contact with DENV, we used the Ae. aegypti carboxypeptidase (CP) promoter44 that induces expression in the midgut following blood ingestion (Figure 1A). The engineered anti-DENV transgenes (termed plasmid OA984 for the untagged version and plasmid OA984-HA for the HA-tagged version) also contained an eye-specific 3xP3 promoter45, driving expression of tdTomato as a fluorescent transgenesis marker. Following the typical transgenesis procedure in mosquitoes, consisting of embryonic microinjection and G0 outcrossing, multiple independent transgenic lines (n = 6) were readily identified in the G1 generation via the robust expression of tdTomato fluorescence; three of the lines with the strongest marker expression (termed Transgenic Anti-DENV [TADV]-A, containing OA984; and TADV-B and C, containing OA984-HA) were selected for further experiments. We carried out inverse PCR (iPCR) on genomic DNA extracted from the transgenic mosquitoes to verify the transgene insertion site and performed backcrosses to WT for multiple generations to ensure that the transgenic lines represented single chromosomal insertions, and were able to confirm that, in all three independent lines, the anti-DENV transgenes were stably integrated into single chromosomes.
Expression of 1C19 scFv antibody
Robust expression and processing of 1C19 scFv transcripts is required for proper neutralization of DENV, and it is important to know if such expression perturbs global gene-expression patterns, which might interfere with the fitness of the mosquito. To confirm this, we sequenced total RNA populations from dissected midgut tissues isolated from both blood-fed and non-blood-fed female Higgs wildtype (WT) or TADV-A, B, or C mosquitoes using an Illumina platform. We detected robust expression of the 1C19 scFv mRNA in both non-blood-fed and 24-hour post-blood-fed transgenic mosquitoes of all three lines, with clearly increased expression levels at 24-hours post-blood meal, while no expression was observed in the midguts of female WT mosquitoes, suggesting that expression of the 1C19 scFv antibody is transgene-dependent and blood-meal-inducible (∼2.35-14.45 fold) as was intended. Importantly, there appeared to be no major shifts in global gene expression patterns between WT and TADV mosquitoes, suggesting that the 1C19 scFv expression did not globally affect gene-expression patterns (Figure S1, Supplementary Table S4).
To confirm the proper expression of the 1C19 scFv, we performed western blots on dissected midgut tissue from non-blood-fed and blood-fed WT and TADV-A as well as blood-fed TADV-B and TADV-C female mosquitoes using either a custom anti-1C19 scFv antibody, or an anti-HA antibody. Blot analyses revealed that the 1C19 scFv peptide was efficiently expressed following a blood meal exclusively in transgenic mosquitoes (Figure S2). Altogether, these results suggest that the anti-DENV transgene is stably integrated into the mosquito genome and that the DENV-targeting 1C19 antibody is expressed in an appropriate context (i.e., in the midgut following a blood meal) for DENV suppression.
Engineered mosquitoes are resistant to infection with all four DENV serotypes
To determine the functional significance of anti-DENV 1C19 scFv expression on vector competence, the DENV-2 infection rates of the three transgenic TADV lines were first compared to that of WT. To do this, adult females (WT or TADV-A, B, or C) were exposed to serotype DENV-2 (isolate ET300, Genbank EF440433) via membrane blood feeding. At 4 days post infection (dpi), midguts from blood-fed mosquitoes were dissected, and DENV RNA copies were measured using RT-qPCR. All three lines showed a significant reduction in midgut infection rate (45 to 71%) and viral RNA levels (2 to 3 log lower) compared with WT control mosquitoes (infection rate 92%) (Figure S3; Table S1). Since no significant difference in DENV-2 midgut infection levels was detected between the three transgenic lines, TADV-A, which exhibited the strongest antiviral phenotype (Figure S3; Table S1), was selected for further comprehensive characterization.
For a more detailed study of the TADV-A DENV vector competence, the effect of transgene zygosity on the infection rate was determined by exposing additional adult females (WT or TADV-A) to serotype DENV-2 and analyzing the dissected midguts at 4 dpi. Results from three biological replicates revealed that none of the TADV-A mosquitoes homozygous for the transgene (n = 35) were positive for DENV-2 infection in the midguts (Figure 1B). DENV-2 infection was detected in 85.4% (35/41) of the TADV-A mosquitoes that were heterozygous for the transgene; however, these mosquitoes had significantly (p < 0.001) lower (∼3 log10) viral RNA levels (8.20 x 101 genome equivalent [GE]) than the WT (4.25 x 104 GE) (Figure 1B, Table 1).
To assay for viral dissemination to the rest of the mosquito body, total RNA was collected from whole TADV-A mosquito carcasses (without midguts) and dissected midguts from both homozygous and heterozygous mosquitoes at 14 dpi. The results from three biological replicates indicated that none of the homozygous TADV-A mosquitoes (n = 30) were positive for viral replication (dissemination) in either the midgut or the midgut-free carcass (Figure 1B, Table 1). DENV-2 prevalence was detected in 86.6% (26/30) of heterozygous TADV-A mosquitoes in both the carcass and midgut; however, they also had significantly (p < 0.001) lower levels of viral RNA (∼3 log10) compared to the WT (Figure 1B, Table 1). Finally, as transmission occurs through the saliva, viral transmission rates were determined by collecting the saliva from individual mosquitoes at 14 dpi and measuring the DENV-2 titers using an assay for the median tissue culture infective dose (TCID50). No DENV-2 was detected in the saliva of homozygous TADV-A mosquitoes (n = 30) (Figure 1B), though it was detected in 83.3% (25/30) of heterozygous TADV-A mosquitoes; however, here again the DENV-2 titers were significantly (p < 0.001) lower (3.56 x 102 TCID50/ml/mosquito) than the WT mosquitoes (2.70 x 105 TCID50/ml/mosquito) (Figure 1B, Table 1).
To determine whether the anti-DENV 1C19 scFv is broadly inhibitory for other DENV serotypes, the vector competence of TADV-A mosquitoes was assessed using DENV-1 (isolate ET243, GenBank EF440432) , DENV-3 (isolate ET209, Genbank EF440434) , and DENV-4 (isolate ET288, Genbank EF440435). Tests for infection, dissemination, and transmission were carried out as above, and the results, presented together in Figure 1B and Table 1, were comparable to those obtained with the DENV-2 serotype. In short, the TADV-A mosquitoes homozygous for the transgene proved to be refractory to infection with all three additional serotypes also showing no infection in their midguts at 4 dpi (DENV-1 n = 28; DENV-3 n = 30; DENV-4 n = 27). Even at 14 dpi, there was no sign of viral replication in the midgut or carcass for all tested specimens, and none of the saliva samples (DENV-1 n = 28; DENV-3 n = 30; DENV-4 n = 28) were positive for the virus. As with DENV-2, the mosquitoes heterozygous for the transgene still tested positive for the virus in most specimens, though the overall DENV titers were significantly lower than compared to the WT in all cases (Figure 1B; Table 1).
Engineered anti-DENV mosquitoes outperform Wolbachia
To compare the inhibitory effect of the anti-DENV 1C19 scFv to DENV inhibition through Wolbachia47–49 infection, we challenged WT mosquitoes infected with Wolbachia (wMel) with DENV-2. Vector competence results revealed that midguts from mosquitoes infected with Wolbachia had significantly (p < 0.001) reduced DENV-2 RNA levels (4.75 x 101 GE) at 4 dpi compared with the WT (4.25 x 104 GE) (Figure 2A, Table 1). Similarly, viral dissemination at 14 dpi was also reduced (p < 0.001) in wMel mosquitoes (∼3 log10), and DENV titers in mosquito saliva at 14 dpi were significantly (p < 0.01) lower (∼3 log10) in wMel mosquitoes (4.90 x 101 TCID50/ml/mosquito) than in the WT (2.70 x 105 TCID50/ml/mosquito) (Figure 1B, Table 1). Importantly, a direct comparison revealed that the TADV-A mosquitoes are significantly (p < 0.001) more effective as homozygotes, and similarly effective as heterozygotes, at blocking DENV infection as Wolbachia-infected mosquitoes.
Transgene impact on fitness
To determine whether the anti-DENV 1C19 scFv had any significant fitness effects on transgenic mosquitoes, we assessed several fitness parameters including larval to pupal development time, male and female fecundity and fertility, male mating success, and longevity (Table 2). No significant differences were observed between WT and TADV-A mosquitoes when examining male mating success and fecundity and fertility in both males and females (p > 0.05). However, we noticed a significant difference in larval to pupal development times (p < 0.0001), with WT mosquitoes developing, on average, 0.8 days faster than TADV-A mosquitoes. When assessing mosquito survivorship, there was no significant difference between WT and TADV-A males (p > 0.05; Figure S4), though, WT female mosquitoes lived, on average, 4.5 days longer than TADV-A females (p < 0.05; Figure S4). The longevity of infected mosquitoes was also assessed. Transgenic, WT, or wMel mosquitoes were infected with four DENV serotypes and their survivorship was assessed 14 dpi (Table 2). No significant (p > 0.01) differences between WT and TADV-A longevity upon infection with serotypes DENV-2, −3, and −4 were observed. However, there was a significant difference in survival upon infection with serotype DENV-1, with a higher proportion of WT mosquitoes surviving at 14 dpi (p < 0.01; Table 2, Figure S4). In addition, a significant difference in survival between wMel mosquitoes and WT and TADV-A mosquitoes infected with serotype DENV-2 was observed (p < 0.0001; Figure S4).
Discussion
Our results demonstrate that conditional expression of the anti-DENV 1C19 scFv renders mosquitoes refractory to all four major DENV serotypes and therefore appears to be a potent viral inhibition strategy. While mosquitoes homozygous for the anti-DENV 1C19 scFv showed complete refractoriness to DENV infection, heterozygous mosquitoes were still partially refractory to DENV infection, dissemination, and transmission, with significant, several orders-of-magnitude reductions in viral titers in the saliva. Based on previous findings, it is likely that this significant reduction in viral titers would be sufficient to render heterozygous mosquitoes unable to transmit DENV to a susceptible host50. Though this remains to be demonstrated, our results show that heterozygous 1C19 scFv antibody-expressing transgenic mosquitoes are just as efficient at viral suppression as—and homozygous mosquitoes are significantly more efficient than—Wolbachia-infected mosquitoes, which are currently being released for DENV control because they are known to be refractory to DENV47.
The difference in refractory levels in the homozygous versus heterozygous mosquitos also suggests that the refractory phenotype is particularly sensitive to scFv antibody expression levels, a phenomenon previously observed with anti-malarial scFv transgenes 39 and anti-ZIKV synthetic small RNA transgenes 51. If this means that complete refractoriness is susceptible to positional effects, e.g., not refractory when the scFv antibody transgene is expressed from a different, possibly more weakly expressing genomic insertion position, the identification of more robust midgut-specific promoters may help to ensure sufficiently high expression levels from a single copy of the transgene regardless of insertion site, as can the use of multiple anti-DENV scFv antibodies in a single transgene 39, 41.
The strategy we describe here provides an efficient “cargo” gene that can be coupled with a gene-drive system to reduce or eliminate the risk of DENV transmission by mosquitoes. Given the success of previous efforts to develop Cas9-mediated homing-based gene drives in Anopheles stephensi52, the malaria vector, it should be possible to develop such drives in Ae. aegypti. Additionally, since homing-based drive systems quickly convert heterozygotes to homozygotes 25, linking the anti-DENV 1C19 scFv antibody described here to such a drive system could, in theory, rapidly convert wild mosquito populations into transgenic homozygotes that would be completely resistant to DENV transmission. Of paramount importance to the viability of such an approach is the evolutionary stability of the refractory transgene, specifically in terms of the likelihood of viral-resistant evolution. Indeed, several studies have shown that, in some contexts, DENV can rapidly evolve resistance in response to neutralizing antibodies53, 54, although this can be managed through the selection of antibodies with mechanisms/epitopes that minimize the chance of evolved resistance and the use of a combination of distinct anti-DENV antibodies, many of which have been described (e.g.,42, 53–56). The deployment of such a pan-serotype-targeting strategy could serve as an effective component of a comprehensive program to reduce the incidence and impact of DENV.
Due to similarities within viral families, this research could have far-reaching consequences for rendering mosquitoes resistant to other arboviruses like ZIKV and CHIKV by using similar genetic engineering strategies to develop scFv-based transgenes. Multiple potent antibodies that effectively neutralize these various mosquito-borne viruses have also been identified in the last decade57–61. Although not all of these will confer robust viral resistance when expressed in vivo in mosquitoes, the availability of diverse, well-characterized antibodies of this sort, largely as a result of antibody therapeutic development efforts58, should allow for the identification of those that function within the desired context. Given the increasing incidence of disease caused by these viruses and the resulting global health implications, such scFv-based transgenes (coupled with gene-drive systems) can provide an effective, sustainable, and comprehensive strategy for reducing the impact of arboviral mosquito-borne diseases.
Author Contributions
A.B., P.N.P., C.H.C., and O.S.A. conceived and designed the experiments. A.B., S.G., M.L., S-H.L., and S-W.W. performed all molecular and genetic experiments. I.A. performed sequencing and bioinformatic analysis. J.E.C. provided sequence data for the 1C19 monoclonal antibody. M.J.K., J.B.D., and P.N.P. performed and analyzed all data for DENV mosquito challenge assays. All authors contributed to the writing and approved the final manuscript.
Data Availability Statement
All sequencing data associated with this study are available from the NCBI sequence read archive (SRA) accession ID: PRJNA510258. The complete annotated plasmid sequence and DNA is publicly available at Addgene (plasmid #120362 [OA984-HA] and #120363 [OA984]). Transgenic mosquitoes will be made available by the corresponding authors upon request.
Competing interests
J.E.C. has served as a consultant for Takeda Vaccines, Sanofi Pasteur, Pfizer, and Novavax, is on the Scientific Advisory Boards of CompuVax, GigaGen, Meissa Vaccines, and is the Founder of IDBiologics, Inc. All other authors declare no competing financial interests.
Materials and Methods
Anti-DENV scFv design
Sequences for the 1C19 variable heavy and light chains were obtained from hybridoma cells expressing the human monoclonal antibody 42 that had been cloned biologically by flow cytometry. RNA was extracted using the RNeasy kit (Qiagen #74104), and RT-PCR amplification of antibody gene cDNAs was performed using primer sets designed to detect all human antibody variable gene sequences 42. The sequence of the antibody cDNAs was determined by automated Sanger sequence analysis. The sequence analysis of the antibody variable gene sequences in the cDNAs was performed using the international ImMunoGeneTics information system (IMGT).
The variable regions of 1C19 were joined by a 15-amino-acid repeating glycine-serine [G(4)S]3 linker43 to encode a scFv form of the antibody62. These chain regions were codon optimized for Ae. aegypti expression and then gene synthesized into a vector (GenScript, Piscataway, NJ). For OA984-HA, a 3’ 30-amino-acid human influenza hemagglutinin (HA) epitope tag with a G(4)S linker63 was added to the carboxy terminus of the single chain antibody for protein expression verification.
Plasmid assembly
To generate vector OA984 (the anti-DENV scFv-antibody transgene), several components were cloned into the PiggyBac plasmid pBac[3xP3-DsRed] (a kind gift from R. Harrell) using Gibson assembly/enzymatic assembly (EA) cloning 64. First, a Drosophila codon-optimized tdTomato marker was amplified with primers 984.1A and 984.1B (Supplementary Table S2 for all primers) from a gene synthesized vector (GenScript, Piscataway, NJ) and cloned into a XhoI/FseI-digested pBac[3xP3-DsRed] backbone using EA cloning. The resulting plasmid was digested with AscI, and the following components were cloned in via EA cloning: the predicted Ae. aegypti carboxypeptidase promoter 44 amplified from Ae. aegypti genomic DNA using primers 984.2A and 984.2B, a GFP sequence amplified from vector pMos[3xP3-eGFP] 65 with primers 984.3A and 984.3B, and a 677-bp p10 3’ untranslated region (UTR) amplified with primers 984.4A and 984.4B from vector pJFRC81-10XUAS-IVS-Syn21-GFP-p10 (Addgene plasmid #36432). The anti-DENV scFv was then subcloned into the final vector from a gene-synthesized plasmid (GenScript, Piscataway, NJ) using PmeI and PacI sites and traditional ligation cloning. Annotated plasmid sequences and plasmid DNA are available via Addgene (plasmid #120363).
To generate vector OA984-HA (anti-DENV scFv with HA-epitope tag), the G(4)S linker and HAx3 tag were amplified with primers 984B.C1 and 984B.C2 from the ninaE[SBP-His] vector containing these components 66 and cloned into the PacI digested OA984 backbone using EA cloning. Annotated plasmid sequences and plasmid DNA are available via Addgene (plasmid #120362). All primer sequences used to generate these plasmids are listed in Table S2.
Generation of transgenic mosquitoes
Germline transformations were carried out largely as described 67. Briefly, 0–1 hr old Higgs wildtype (WT) Ae. aegypti pre-blastoderm embryos were injected with a mixture of vector OA984 or OA984-HA (200 ng/µL) and a source of PiggyBac transposase (200 ng/µL)65; the injected embryos were hatched in deoxygenated H2O. A total of 127 surviving WT adult G0 males and 115 surviving WT adult G0 females were recovered after the injection. Microinjected WT G0 adults were assigned to 48 pools and outcrossed to WT of the opposite sex in medium-sized cages. Larvae were fed ground fish food (TetraMin Tropical Flakes, Tetra Werke, Melle, Germany) and adults were fed with 0.3 M aqueous sucrose. Adult females were blood fed three to five days after eclosion using anesthetized mice. All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as recommended by the National Institutes of Health, and the methods were supervised by the local Institutional Animal Care and Use Committee (IACUC). A total of 38,177 WT G1s were screened. G1 larvae with strong positive fluorescent signals (3xp3-tdTomato) were selected under the fluorescent stereomicroscope (Leica M165FC) and were separated into six individual groups characterized by fluorescence patterning and intensity. One single transgenic male from each group was then allowed to separately mate with 10 WT females to isolate each independent line. Three independent lines, TADV-A (vector OA984), TADV-B (vector OA984-HA), and TADV-C (vector OA984-HA) with the strongest fluorescence expression patterns were selected for further characterization. To determine whether these lines represented single chromosomal insertions, we backcrossed single individuals from each of the lines for four generations to WT stock and measured the Mendelian transmission ratios in each generation; in all cases, we observed a 50% transmission ratio, indicating single-chromosome insertion. For one of the three lines (TADV-A), transgenic mosquitoes were inbred for at least 20 generations to generate a homozygous stock. Mosquito husbandry was performed under standard conditions as previously described 68.
Characterization of insertion sites
To characterize the insertion site of vector OA984 or OA984-HA in transgenic mosquitoes, we adapted a previously described inverse polymerase chain reaction (iPCR) protocol 69 as follows. First, genomic DNA (gDNA) was extracted from 10 transgenic fourth instar larvae using the DNeasy Blood & Tissue Kit (Qiagen #69504) per the manufacturer’s protocol. Two separate restriction digests were performed on the gDNA (at 100 ng/μL) to characterize the 5’ and 3’ ends of the PiggyBac insertion using Sau3AI (5’ reaction) or HinP1I (3’ reaction) restriction enzymes. A ligation step using NEB T4 DNA Ligase (NEB #M0202S) was performed on the restriction digest products to circularize digested gDNA fragments, and two subsequent rounds of PCR were carried out per ligation using the corresponding PiggyBac primers listed in Supplementary Table S3. The final PCR products were cleaned up using the MinElute PCR Purification Kit (Qiagen #28004) in accordance with the manufacturer’s protocol and were sequenced via Sanger sequencing (Source BioScience, Nottingham, UK). To confirm the transgene insertion locus and orientation via PCR, primers were designed based on iPCR-mapped genomic regions and used in tandem with PiggyBac primers based on their location as listed in Supplementary Table S3. Sequencing data then was blasted to the AaegL5.0 reference genome 70. The sequencing data was aligned with SeqManPro (DNASTAR, Madison, WI) to determine the orientation of the transgene insertion site. Analysis of the sequencing data indicated that the insertion site for TADV-A is on chromosome 2 (approximate position 310,340,476), the insertion site for TADV-B is on chromosome 2 (approximate position 301,489,980), and the insertion site for TADV-C is on chromosome 1 (approximate position 30,451,048) when aligned to the AaegL5 assembly (GenBank assembly accession: GCA_002204515.1)46.
Total RNA extraction, isolation, and sequencing
Total RNA was extracted from the midguts of non-blood-fed and 24-hours post-blood-fed TADV-A, TADV-B, TADV-C or WT adult females using the Ambion mirVana mRNA Isolation Kit (ThermoFisher Scientific #AM1560). Following extraction, the RNA was treated with Ambion Turbo DNase (ThermoFisher Scientific #AM2238). The RNA quality was assessed using an RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies #5067-1513) and a NanoDrop 1000 UV-vis spectrophotometer (NanoDrop Technologies/Thermo Scientific, Wilmington, DE). mRNA was isolated using an NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490), and libraries were constructed using an NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770). The libraries were quantified using a Qubit dsDNA HS Kit (ThermoFisher Scientific #Q32854) and a High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies #5067-4626) and sequenced on an Illumina HiSeq2500 in single-read mode with a read length of 50 nt and sequencing depth of 30 million reads per library following the manufacturer’s instructions. Reads were mapped to the Ae. aegypti genome (AaegL5.0) supplemented with the 1C19 cDNA sequence using STAR aligner 71, and the expression levels were determined with featureCounts 72 (Supplementary Table S4). Correlation coefficients of the transcripts-per-million (TPM) values between WT and transgenic animals were calculated in R[14] and plotted with ggplot2 (Figure S1). All sequencing data can be accessed at NCBI SRA (study accession ID PRJNA510258).
Western blot assays
The general western blot protocol was adapted from CSH Protocols: SDS-PAGE of Proteins 74. Briefly, 5–7 days post eclosion, midguts from 25 non-blood-fed and 16-hour post-blood-meal heterozygous TADV-A transgenic and WT mosquitoes were dissected and collected in 1x PBS. Protein samples from dissected tissues were extracted with ice-cold radioimmunoprecipitation assay buffer (RIPA buffer; 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% Na-deoxycholate, 1% NP-40, 1 mM EDTA). The protein concentration was measured using Protein Assay Dye (Bio-Rad, Cat. No#5000006) and multi-detection microplate readers (Molecular Devices, SpectraMax M2). Next, 40 µg of total protein were run on a 12% SDS-PAGE and transferred onto a 0.45 µm Immobilon®-P Transfer Membrane (Merck Millipore, Cat. NO#IPVH00010). The membrane was hybridized with a custom antibody at a 1 µg/mL dilution (GenScript, Item number: U3233DA170_2) to directly recognize the 1c19 scFv peptide (26.3KDa) as well as a monoclonal antibody specific to the HA tag for lines TADV-B and C (Cell Signaling, #3724S) at a 1:1,000 dilution; these were subsequently detected by using rabbit IgG antibody (HRP) (GeneTex, Cat. No#GTX 213110-01) at a 1:10,000 dilution. Images were generated by applying the chemiluminescent HRP substrate (Millipore, Cat. No#WBKLS0500) to the blots.
DENV infection of mosquitoes and virus determination
All experiments were performed under biosafety level 3 (BSL-3) conditions in the insectary at the Australian Animal Health Laboratory. The following DENV strains were used for all viral challenge experiments: DENV-1 (isolate ET243, GenBank EF440432) , DENV-2 (isolate ET300, GenBank EF440433) , DENV-3 (isolate ET209, GenBank EF440434) , DENV-4 (isolate ET288, GenBank EF440435). The virus was passaged in Vero cell monolayer cultures before use for mosquito infections. WT or transgenic (confirmed by red fluorescence in the eye) mosquitoes were exposed to DENV as described previously 75. Briefly, female mosquitoes were challenged with an infected blood meal (TCID50 /mL) through membrane feeding using chicken blood and skin. For infection frequency and virus titer, mosquito midguts were collected at 4 dpi. For dissemination and transmission frequency, mosquito saliva, midguts, and carcasses were collected at 14 dpi. Mosquito saliva was used to determine viral titers using a TCID50 assay on Vero cell monolayer cultures. Midguts and carcasses were used to determine the presence of viral RNA using RT-qPCR against NS5. Mosquito viral challenge, processing, saliva testing, and molecular analyses of infection and dissemination were carried out as previously described 75. DENV infection frequency was defined by the number of midguts (day 4) found positive for viral nucleic acid. Similarly, the dissemination frequency was calculated by the number of carcasses (day 14) found positive by qPCR. Transmission frequency was defined by the number of TCID50-positive saliva samples over the number tested. These different frequencies and average TCID50 values were compared by the Student’s two-tailed t-test.
Confirmation of transgene zygosity
Both homozygous and heterozygous (generated by crossing out homozygous individuals to WT) mosquitoes were used for assays. To confirm the zygosity of tested transgenic mosquitoes, mosquito heads were homogenized using a bead-beater device for DNA extraction in 30 μL of extraction buffer (1x Tris-EDTA, 0.1 M EDTA, 1 M NaCl, and 2.5 μM proteinase K) and incubated at 56°C for 5 minutes and then at 98°C for 5 minutes. The first round of PCR was performed to test for the presence of the anti-DENV transgene using primers 991.3F1 and 1018.S19 (Supplementary Table S3). Another round of PCR then was performed using primers 1018.S19 and 1018.S21 (Supplementary Table S3) to amplify the WT insertion locus (i.e., locus lacking transgene insertion) and thus determine zygosity. For TADV-B, primer set 991.3R2 and 1018.S73 was used to amplify the anti-DENV transgene and primer set 1018.S73 and 1018.S74 were used to amplify the WT insertion site. For TADV-C, primer sets 991.3F2 and 1018.S80 and set 1018.S80 and 1018.S82 were used to amplify the anti-DENV transgene and WT insertion site, respectively. WT mosquitoes served as controls to ensure that the WT locus was successfully amplified in the genetic background. A PCR kit (ThermoFisher Scientific #F553S) with a 57°C annealing temperature was used for all PCRs following standard protocols.
Generation of wMel Wolbachia Line and Infection Assay
Eggs of Ae. aegypti infected with the Wolbachia strain wMel were obtained from the World Mosquito Program (Prof. Scott O’Neill, Monash University). WT mosquitoes infected with wMel were generated by crossing wMel+ females with males from the WT line, and the resulting offspring were used for DENV infection experiments. At the end of the experiment, the Wolbachia infection status of these mosquitoes was tested using PCR with primers specific for wMel detection 76 (Supplementary Table S3). The PCRs indicated the presence of wMel in >90% of mosquitoes, and only results from these positive mosquitoes were used for further analysis.
Fitness evaluation on transgenic anti-DENV mosquitoes
To determine if the anti-DENV transgene conferred a fitness cost, several fitness parameters were evaluated in TADV-A transgenic heterozygous and sibling WT mosquitoes. The evaluations of all experimental and control replicates were performed simultaneously. Insectary conditions were maintained at 28℃ and 70 to 80% in relative humidity with a 12 hr light/dark cycle. To assess the larval to pupal development time, the eggs were vacuum hatched, and the larvae were distributed into pans (50 larvae per pan) containing 2.5 L of ddH2O and 0.6 mL of fish food slurry. To determine the larval to pupal development time of transgenic and WT control mosquitoes, the larvae were allowed to pupate, and pupae were collected and counted every day until no pupae were left. To assess female fertility and fecundity, 90 WT and transgenic females were mated to 20 WT males in a cage. After four days, the females were blood fed and individually transferred into plastic vials filled with water and lined with egg paper. After three days, egg papers were collected, and the eggs were counted and vacuum hatched in nine-ounce plastic cups. Starting on the fourth day, the larvae were counted every day until no larvae were present. Female fecundity refers to the number of eggs laid per female, and fertility reflects the number of eggs hatching to produce larvae. To measure male mating success, fecundity, and fertility, one TADV-A transgenic or WT male was mated to five WT females in a single mesh-covered cup filled with water and lined with egg paper. Three days post blood meal, the cups were checked for the presence of eggs, which were collected, counted, and hatched. Hatched larvae were then counted every day until no larvae were present. Male mating success was calculated as the percentage of single male outcrosses that produced larvae. Fecundity was measured as the number of eggs laid per cup; fertility was determined by the number of hatching larvae in each cup. Finally, to assess mosquito longevity, equal numbers of male and female TADV-A transgenic or WT mosquitoes were placed in medium-sized cages (in triplicate). Mosquitoes that died were counted and removed daily until all mosquitoes had died. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, California, USA). The means were compared using unpaired t tests with Welch’s correction, with the exception of male mating success that did not use Welch’s correction. The analyses of mosquito survivorship used the Mantel-Cox test. P values > 0.05 were considered not significant.
Acknowledgements
This work was supported in part by a Defense Advanced Research Project Agency (DARPA) Safe Genes Program Grant (HR0011-17-2-0047) awarded to O.S.A. and a NIH Exploratory/Developmental Research Grant Award (1R21AI123937) awarded to O.S.A and CSIRO internal funding to P.N.P. We thank Prof. Scott O’Neill (Institute of Vector Borne Diseases, Monash University, Australia) and the World Mosquito Program for providing Wolbachia-infected mosquito eggs.
Footnotes
↵# Institut Pasteur de la Guyane, Cayenne, French Guiana