Dissection-independent production of a protective whole-sporozoite malaria vaccine

Complete protection against human malaria challenge has been achieved using infected mosquitoes as the delivery route for immunization with Plasmodium parasites. Strategies seeking to replicate this efficacy with either a manufactured whole-parasite or subunit vaccine, however, have shown only limited success. A major roadblock to whole parasite vaccine progress and understanding of the human infective sporozoite form in general, is reliance on manual dissection for parasite isolation from infected mosquitoes. We report here the development of a four-step process based on whole mosquito homogenization, slurry and density-gradient filtration, combined with free-flow electrophoresis that is able to rapidly produce a pure, aseptic sporozoite inoculum from hundreds of mosquitoes. Murine P. berghei or human-infective P. falciparum sporozoites produced in this way are 2-3-fold more infective with in vitro hepatocytes and can confer sterile protection when immunized intravenously with subsequent challenge using a mouse malaria model. Critically, we can also demonstrate for the first time 60-70% protection when the same parasites are administered via intramuscular (i.m.) route. In developing a process amenable to industrialisation and demonstrating efficacy by i.m. route these data represent a major advancement in capacity to produce a whole parasite malaria vaccine at scale. One-Sentence Summary A four-step process for isolating pure infective malaria parasite sporozoites at scale from homogenized whole mosquitoes, independent of manual dissection, is able to produce a whole parasite vaccine inoculum that confers sterilizing protection.

1 isolated from infected mosquitoes via manual salivary gland dissection (SGD). As well as the obvious 2 challenges this presents to vaccine development, difficulties with sporozoite isolation have held back 3 general understanding of sporozoite biology when compared to blood-stages of development [16,17]. 4 Salivary gland dissection (SGD) requires in vivo parasite development in the mosquito followed by 5 manual dissection of the salivary glands 15 to 21 days post infected-blood feed. Originally described in 6 1964 [18] with only minor variations since [16,[19][20][21][22][23][24][25], the dissection method involves mosquito 7 decapitation, gland removal and homogenisation to release sporozoites. Dissection in this way is time-8 consuming, taking a skilled technician over an hour to dissect 40-60 glands to a reasonable standard. 9 With total extraction time being a critical factor for subsequent sporozoite viability [26], there is a 10 relatively low upper limit for attaining live, infectious sporozoites. Furthermore, SGD sporozoites 11 retain a considerable amount of mosquito-originating debris [27], known to inhibit sporozoite motility 12 (critical for liver cell infection) [28] and act as immune modulators in vivo [29], potentially influencing 13 infection progression and immune responses. Likewise, the time taken, and contamination carried over, 14 places limits on the infectivity and development of Plasmodium sporozoites in vitro 15 [17,22,26,28,31,32]. Rates of cell infection with in vitro hepatocyte (primary or hepatoma) cultures 16 using SGD are typically less than 1% using the murine malaria model Plasmodium berghei 17 [19,20,24,30] and less than 2% for human P. falciparum sporozoites [16,25,27,[31][32][33]. This has been a 18 major impediment to in vitro studies and screens reliant on high numbers of infection. 19 20 Several previous attempts have sought to improve throughput and purity of whole sporozoite 21 preparation. Methods aimed at bypassing SGD have included centrifugation through glass wool [34] 22 and compression between glass plates [35]. These alternative methods, however, have consistently 23 yielded poor parasite purity, necessitating combination with density gradients [33,[36][37][38][39][40][41]. Whilst the addition of gradients increases sporozoite yield, lengthy centrifugation times were required and final 1 output was still contaminated with mosquito material [28,43]. Other methods trialled for sporozoite 2 isolation have included ion exchange chromatography [42,43], and later free-flow electrophoresis 3 (FFE) [44]. FFE is a liquid form of electrophoresis commonly used to separate organelles under native 4 conditions based on net surface charge [45]. The poor yields or complexity of both methods, however, 5 has limited interest in their scaled usage, despite recent developments in FFE technology in particular 6 [46]. To date, the only scaled means for manufacture of a clinical grade vaccine has therefore relied on 7 enlisting hundreds of skilled dissectors combined with rearing of parasites within aseptic mosquitoes. Obtaining malaria sporozoites therefore remains a major roadblock to improving understanding of 10 basic parasitic biology, and the scalable and reproducible production of whole sporozoite vaccines. As 11 a response, we have developed a process that rapidly purifies both murine P. berghei and human P. 12 falciparum sporozoites from whole mosquitoes, based on an optimized combination of 13 homogenization, size exclusion, density and charge. Our stepwise approach facilitates the processing of 14 hundreds of mosquitoes per hour and can be adapted to produce entirely aseptic, effectively 15 contaminant-free sporozoites, all by a single person. The sporozoites isolated by this process show 16 significantly improved infectivity both in vitro and in vivo. Critically, sporozoites isolated by this 17 process offer sterile protection when given as a live-attenuated vaccine in a mouse model of infection, 18 including, for the first time, via intra-muscular delivery. Being dissection-independent, this process will 19 facilitate the rapid and scalable manufacture of Plasmodium sporozoites and could form a key enabling 20 technology for delivery of a future effective malaria vaccine. The challenges of obtaining sporozoites for malaria research by salivary gland dissection (SGD) are a 1 major impediment to improving understanding of the liver stages and development of effective 2 interventions [12,24,27,30,31,[51][52][53][54]. Sporozoite isolation by SGD is a low throughput and labour-3 intensive procedure which produces sporozoites of low purity, contaminated with mosquito-associated 4 material. To address this need, we developed a dissection-independent stepwise process for the 5 purification of sporozoites from whole mosquitoes (Figure 1a).

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Our process consists of 4-steps, with capacity to process up to 1000 mosquitoes at a time by a single 8 individual. Whole mosquitoes were homogenised to release sporozoites and filtered sequentially 9 through 100µm to 10μm filters. The filtered mosquito homogenate/Mash (M) was then pre-purified by 10 density centrifugation using Accudenz (MA), as previously described [27], to remove larger mosquito-11 associated debris from sporozoites. The sporozoite layer was subsequently purified by Free-flow Sporozoites produced according to this 4-step process, demonstrated reproducible separation, 18 independent of initial sporozoite quantity. The majority of sporozoites separated into a single fraction, 19 with a characteristic tail in the distribution that lengthened as sporozoite dose load increased ( Figure   20 S1b-c). This peak fraction was used for all subsequent experiments. A single FFE run, resulted in an 21 average loss of yield of ~30% of MA input, with approximately 500-1000 mosquitoes processed by one 22 individual in 2 hours (compared to SGD ~100 mosquitoes processed by a skilled dissector in a similar 23 Page 7 of 30 timeframe). This represents a minimum 5-10-fold increase in yield that can be run continuously or in 1 parallel using multiple units.

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Given the established potential of whole sporozoites as an effective vaccine [7] we next sought to 4 establish the purity of MAF sporozoites compared to SGD sporozoites. Initial assessment of brightfield 5 images clearly showed that our approach successfully removed all visible mosquito-associated debris 6 (Figure 1d). To quantitively assess contaminants, samples were normalised by mosquito equivalents 7 (MEQ); based on the number of mosquitoes (mq) homogenised and volume (units: mq/mL) as opposed 8 to sporozoite dose, which can vary between batches. To assess the sequential reduction in protein 9 contaminants during each step of the purification, uninfected mosquitoes were purified to determine the 10 contributing mosquito protein contaminants. MAF samples were run at three different MEQs (300, 100, 11 50mq/mL) to determine an optimal purification condition. MAF purified sporozoites showed a ~100% 12 drop in detectable protein by silver stain with <100mq/mL doses by FFE in contrast to a 63.1% 13 reduction with SGD sporozoites (Figure 1e). The differences in the FFE separation profile of 14 mosquito-associated protein at the three MEQs demonstrated that at 100mq/mL or less all detectable 15 protein could be effectively removed from the peak sporozoite positive FFE fractions (Figure 1f). 16 17 Analysis of the FFE output demonstrated our ability to remove abundant mosquito proteins, such as 18 actin, as well as enriching for sporozoite proteins in the sporozoite fraction ( Figure S1d-f). In addition, 19 an identical protein purification profile was obtained when using dissected salivary glands as starting 20 homogenate rather than whole mosquitoes (referred to as Dissected-Accudenz-FFE; DAF) ( Figure   21 S1d), demonstrating the flexibility of our stepwise process for different sub-populations of sporozoite 22 within the infected mosquito.

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Given that bacterial contamination is a major problem for in vitro work, we next assessed the ability of 1 our stepwise process to separate mosquito-associated bacteria. Serial dilutions of samples normalised to 2 equal MEQ from each stage of purification were grown for 16hr at 37 o C in a non-selective tryptic soya 3 broth (TSB) medium [47] (Figure 1g). A marked reduction in bacterial growth, assessed by measuring 4 OD600, was observed with MAF purified sporozoites. This was further confirmed by measuring 5 bacteria colony forming units per mL (cfu/mL) on blood-agar plates, which showed a significant 8.1 6 log reduction in total bacterial load compared to a 5.9 log reduction by SGD (Figure 1h, S1g, S2). This 7 translates to a 173-fold reduction in the bacterial load when compared to equivalent numbers of 8 sporozoites obtained by SGD. Repeating the process with DAF produced sporozoites (dissected 9 salivary glands used as input for FFE processing), demonstrated the successful removal of all 10 detectable bacteria, effectively producing aseptic sporozoites (Figure 1i).

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As an alternative to density gradients, we also tested whether rapid gel filtration with a Sephadex-based 13 spin-column (5 minutes) could replace Accudenz, mirroring a method used with bovine sperm 14 purification [48]. In parallel, we tested whether an interval zone electrophoresis (iZE) FFE method (see 15 Methods) could add further improvements to our overall process ( Figure S3). Combining Sephadex 16 with iZE, we were able to produce completely sterile sporozoites from whole mosquitoes ( Figure S3). 17 Since purity was associated with an additional loss in yield (~80%), for the remainder of the 18 development of the process (and experiments described below), MAF purification using an FFE input 19 of 100mq/mL with the cZE method was used. 20

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Having developed a robust pipeline for high-throughput purification of sporozoites from whole 23 mosquitoes with enhanced scale and purity, we next sought to assess in vitro infectivity. Sporozoite motility is often used as a primary indicator of sporozoite viability [24,26]. Comparisons of the motility 1 patterns of SGD versus MAF sporozoites on a glass surface revealed no significant differences in the 2 2D motion patterns displayed (static, attached/waving or gliding) [49], either in terms of mean velocity 3 or overall ratios of motion pattern (Figure 2a-c).

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Extending infectivity analysis to in vitro infection of hepatoma or primary hepatocytes, RT-PCR 6 analysis of P. berghei copy number, 24 hours post infection (p.i.), showed a 1.5-and 2.1-fold increase 7 in MAF sporozoite infectivity in HepG2 and primary rat hepatocytes compared to that of SGD 8 sporozoites respectively (Figure 2d). More significantly, the proportion of P. berghei exo-erythrocytic 9 forms (EEFs) developing in primary rat hepatocytes 24 hours p.i. was 13.5-fold increased when MAF 10 sporozoites were used rather than their SGD counterparts (infection rate of 5.4% versus 0.4%) ( Figure   11 2e). At 52 hours p.i., MAF sporozoites had completed maturation into late schizonts, as indicated by 12 the presence of liver-stage merozoites (Figure 2f). These sporozoites also fully developed into late-13 stage exoerythrocytic schizonts when infected hepatocytes were extracted from rats and cultured ex 14 vivo ( Figure S4). Corroborating these results, assessment of MAF infected rat primary hepatocytes by 15 flow cytometry, showed infection rates of 10.37% (302 out of 2808 cells) ( Figure S5). Critically, 16 mirroring observations in P. berghei, P. falciparum-derived MAF sporozoites were shown to have a 3-17 fold increased rate of invasion into HC-04 cells when compared to sporozoites isolated by SGD 18 (Figure 2g-h). The absence of a robust human primary hepatocyte model for long-term in vitro 19 development precluded our ability to take these to late-stage schizogony. 20 21 Overall, these results clearly show that both P. berghei and P. falciparum sporozoites purified by our 22 method exhibit superior infectivity in vitro, making them preferable for in vitro experimentation.

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MAF sporozoites from segmented mosquitoes are more infectious than SGD in vivo 1 As MAF sporozoites showed enhanced infectivity in vitro compared to SGD counterparts, further in 2 vivo studies were performed to confirm this trend. Mice were inoculated with P. berghei sporozoites by 3 intravenous (i.v.) injection and infectivity determined by measuring the time to reach 1% blood stage 4 parasitaemia (prepatent period). Mice were inoculated i.v. with escalating doses of MAF purified P. 5 berghei sporozoites, demonstrating that, independent of the inoculum size, MAF sporozoites were able 6 to develop and establish a successful blood-stage infection (Figure 3a).
We next compared infectivity of MAF sporozoites compared to SGD sporozoites. Initially, 5000 9 sporozoites obtained from either SGD, MA or MAF were given i.v. to mice with resulting blood stage 10 parasitaemia monitored. Surprisingly, partially purified MA sporozoites and fully purified MAF 11 sporozoites showed a modest but significant delay in time to 1% parasitaemia compared to SGD (0.66 12 days longer for MA, **p=0.0049; 0.59 days longer for MAF, **p=0.0031; Mantel-Cox Test) ( Figure   13 3b). This was in contrast to in vitro infections which were significantly increased with MAF 14 sporozoites. As sporozoites purified from whole mosquitoes will necessarily include a proportion of 15 immature sporozoites that have yet to reach the salivary glands, we reasoned that this modest reduction 16 in in vivo infectivity could be due to the presence of immature sporozoites in the MA and MAF We subsequently sought to determine whether subtraction of immature sporozoites from the initial 4 inoculum could revert the delay in time to patency. Mosquito abdomens were discarded prior to the 5 initial homogenisation step in our purification platform (MAF-No Abdomen; MAF with abdomens 6 removed prior to homogenisation) and, as a measure of sporozoite in vivo infectivity, the time to 1% 7 blood parasitaemia was monitored as before. Notably, in mice infected with MAF-No Abdomen 8 sporozoites there was now a significant reduction in the time to 1% parasitaemia compared to those 9 injected with SGD sporozoites (*p = 0.0415; Mantel-Cox Test; Figure 3d). This increase in infectivity 10 was only patent when sporozoites went through all steps of the purification pipeline, as no differences 11 were observed between mice challenged with SGD and partially purified MA-No Abdomen sporozoites 12 (MA with abdomens removed prior to homogenisation) (Figure 3e). Increasing the dose of whole 13 mosquito origin MA mosquitoes (180%, 1.8-fold increase) caused a clear reduction in the pre-patent 14 period, indicating an increase in infectious dose. These findings correlate with in vitro data and 15 demonstrate that our stepwise purification process is associated with a marked increase in sporozoite 16 infectivity in vivo. 17 18 19 Having developed a process that produces sporozoites with high purity and improved infectivity over 20 SGD sporozoites, we next sought to assess the potential of MAF sporozoites as a radiation-attenuated 21 sporozoite vaccine (RASv). Prior to immunisation, the effective irradiation dose was determined to be 22 60Gy by i.v. inoculation with varying doses of gamma irradiated sporozoites (Figure 4a). Mice were 23 immunised i.v. using a three-immunisation regime of 40,000 irradiated sporozoites, two weeks apart. In parallel, cohorts of control mice were immunised with plain medium as controls. Immunisation 1 efficacy was assessed by challenging with 5 infectious mosquito bites (Figure 4b) [51]. Immunisation 2 with P. berghei (wildtype PbANKA) or P. falciparum (wildtype NF54) MAF-RASv sporozoites i.v. 3 achieved complete protection against native P. berghei or chimeric P. berghei expressing P. falciparum 4 CSP (PbANKA-PfCSP) respectively (Figure 4c-d). The level of protection (sterile protection) was 5 comparable to that offered by SGD-RASv. Equivalent total IgGs measured against whole sporozoites 6 pre-challenge were found in the serum from immunized animals irrespective of the source of P. berghei 7 sporozoites (Figure 4e).  For the first time we present an accessible, robust process for the high yield isolation of large quantities 2 of pure malaria sporozoites without requiring manual salivary gland dissection (SGD). Sporozoites 3 isolated by this dissection-independent process exhibited improved sterility and infectivity in vitro and 4 in vivo and could be shown to confer sterile protection in a mouse challenge model. With demonstrated 5 application to both murine P. berghei and human-infective P. falciparum, sporozoite production using 6 this process represents a transformative technology that will advance a multitude of applications, not 7 least advancing development of efficacious whole-parasite malaria vaccines. Given the challenges of current sporozoite purification methods we set out to develop a versatile 10 process for purification of sporozoites from whole mosquitoes; a process independent of the need for 11 dissection. By combining bulk mosquito homogenization, Sephadex filtration (or density 12 centrifugation) and free-flow electrophoresis separation (abbreviated to MAF), sporozoites purified 13 using this stepwise process could be obtained 8-10 times faster than SGD and with all detectable 14 mosquito-associated protein removed. SGD sporozoites in comparison retained ~40% of the mosquito 15 associated protein material. In addition to the improved levels of purity, MAF sporozoites also showed 16 a markedly improved infectivity in vitro for both rodent and human malaria parasites. The reduced 17 overall time required, and consistency of production are likely to be key factors in determining in vitro 18 infectivity. However, several additional factors likely account for this improvement in infectivity.

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When MAF and SGD sporozoites were added to primary human hepatocytes, SGD treatment was 20 found to be associated with abnormal human cell morphology and reduced cell numbers (Figure S6), 21 suggesting mosquito contaminants may be detrimental to host-cell growth, reducing overall viability of 22 hepatocytes. In addition, we noted that MAF sporozoites had 4.3 times more cleaved CSP on their 23 surface than SGD sporozoites ( Figure S1d) [53,54]. Previous work has explored the importance of 24 Page 14 of 30 CSP processing on P. berghei sporozoite invasion. Of note, genetically altered sporozoites expressing a 1 pre-cleaved CSP showed greater levels of in vitro infectivity [55]. Therefore, the acceleration of CSP 2 processing seen following MAF purification may be a contributing factor to improved infectivity of 3 sporozoites isolated in this way. Finally, a recent study identified the mosquito salivary protein, 4 mosGILT as negatively modulating sporozoite motility [28]. Other mosquito-associated factors that 5 reduce hepatocyte infectivity may be forthcoming and would be likely purified away from sporozoites 6 using the MAF protocol, which may further contribute to improved hepatocyte infectivity. Of key 7 interest, these factors will likely combine to help advance the relatively limited success that has been 8 seen with in vitro liver stage systems that yield low numbers of infected hepatocytes following 9 exposure to "viable" sporozoites [16,21,[56][57][58]] when compared to comparable asexual blood-stage or 10 sexual stage high throughput in vitro platforms [59][60][61][62]. Further work is clearly now warranted towards 11 this.

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Although MAF sporozoites showed the same motility patterns as SGD, this was somewhat unexpected 14 given that MAF sporozoites originate from the entire mosquito. Sporozoites originating from less 15 mature stages, such as those in oocysts or the haemolymph, are typified by reduced gliding motility 16 compared to mature salivary-gland resident sporozoites [24,49,50]. Indeed, we found that 70% of Page 15 of 30 that use of whole mosquito homogenate was indeed associated with a significant delay in blood-stage 1 parasitaemia when compared to production via our process but with prior abdomen removal (which 2 reduced this delay), increasing time to patency compared to SGD (Figure 3d). Of note, the fact that 3 removal of abdomens but without FFE (MA) does not advance infectivity, indicates that it is the FFE 4 step that is critical to improving the viability of MAF sporozoites over SGD. Thus, efforts seeking to 5 enrich specifically for highly infectious sporozoites may require prior sectioning of the mosquito to 6 remove the abdomen but will critically still rely on the FFE step for maximal infectivity. to ~5 minutes to administer in children [64]. Future work testing decreasing doses of sporozoites will be required to accurately assess comparative immunogenicity of different sporozoite sources and routes 1 of immunization.

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In conclusion, the work presented here shows the development of a complete stepwise process for 4 purification of large numbers of highly infectious sporozoites in a rapid and scalable approach that is 5 entirely compatible with basic biological, drug-screening and whole-parasite vaccine studies. Our 6 process yields sporozoites at higher purity compared to those from dissected preparations alone and is show markedly reduced levels of contaminants, can be produced aseptically and, critically, can be used 10 to demonstrate high protective efficacy following both intravenous and intramuscular immunization.

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For basic sciences, this stepwise process will be an important step towards single cell -omic studies that 12 require large amounts of highly pure sporozoites, free of the mosquito-associated contaminants that      Immunization of mice against the ANKA strain using the unaltered sporozoite as an antigen. Exp. 25 Parasitol. 1977, 42, 1-5.  Exp. Parasitol. 1980, 49, 420-429. 34 42. Mack, S. R.; Vanderberg, J. P.; Nawrot, R. Column separation of Plasmodium berghei sporozoites.  Immunisation i.v. or i.m. of Balb/c mice with irradiated P. berghei sporozoites from either manual salivary gland (SGD) dissection or 5 MAF-No Abdomen. Mice given three immunisations of 40k sporozoites, two weeks apart followed by challenge with five infectious 6 mosquito bites. Ten mice per group. Endpoint classed as 1% parasitaemia. D) Immunisation i.v. or i.m. of Balb/c mice with irradiated P. 7 falciparum sporozoites from MAF-No Abdomen. Mice given three immunisations of 40k sporozoites, two weeks apart followed by 8 challenge with five infectious mosquito bites. Six mice per group. Endpoint classed as 1% parasitaemia. E) Total titres of IgG antibodies 9 against P. berghei sporozoite lysate in mouse serum prior to challenge F) Total titres of IgG antibodies against P. falciparum sporozoite 10 lysate in mouse serum prior to challenge. Squares indicate mice not protected.