Display of malaria transmission-blocking antigens on chimeric DHBV-derived virus-like particles produced in Hansenula polymorpha

Background Malaria caused by Plasmodium falciparum is one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates. Methods We developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens. Results In this study, a novel platform for the display of Plasmodium falciparum antigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinant Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90 % on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP. Conclusion The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.


Abstract
fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated 55 from 100 g dry cell weight with a maximum protein purity of 90 % on the protein level. 56 Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP 57 with higher purity. VLP showed reactivity with transmission-blocking antibodies and 58 supported the surface display of the malaria antigens on the native VLP. 59

Conclusion: 60
The incorporation of leading Plasmodium falciparum transmission-blocking antigens into the 61 dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled 62 particles. Competitive processes for efficient production and purification were established in 63 this study. 64 Unfortunately, subunit vaccine candidates often suffer from weak immunogenicity that has to 87 be compensated by smart formulation and/or delivery strategies [12] such as virus-like 88 particles (VLP [13,14] Recombinant H. polymorpha strains co-producing the dS and a fusion protein were 165 generated by the "staggered transformation approach" and screened as previously described 166 Darmstadt Germany), 10 g L -1 glycerol, 11 g L -1 glucose-monohydrate, and 0.1 g L -1 adenine. 189

Background
Aqueous solutions of NH 3 (12.5 % (w/w), sterile filtered) and H 3 PO 4 (28 % (w/w), Merck,190 Darmstadt, Germany) were used as corrective media to keep pH constant (set point 6.5) 191 throughout fermentation and Struktol J 673 (10 % (v/v) aqueous solution, Schill+Seilacher, 192 Hamburg, Germany) was utilized as antifoam agent. Aeration was adjusted to 1 vvm 193 (2.5 NL min -1 ) and the medium was inoculated to an optical density (OD 600 ) of 0.6 using 194 shake flask pre-cultures. For specific detection of P. falciparum antigen Pfs230, plates were coated with indicated 292 concentrations of VLP (4°C, over-night) and subsequently blocked with 1% casein in PBS 293 (Sigma-Aldrich) for 2h at 37°C before primary antibodies were added (polyclonal mouse anti-294 Pfs230 or monoclonal 1B3 antibody, 10 µg mL -1 ). Secondary HRP-conjugated antibodies 295 (polyclonal goat anti-mouse IgG at 1/1000 from Millipore) were used to detect antibody 296 binding. Color detection was developed using ABTS liquid substrate (Sigma-Aldrich), which 297 was subsequently stopped using 1 % SDS. PBS was used as a negative control and plates 298 were washed thrice using PBS with 0.05 % Tween in between antibody incubation steps. 299 The level of antibody binding was measured as absorption at 405nm (A 405nm ). 300 Analysis of VLP were performed as essentially described previously described [   Literature was screened for promising targets and the following antigens were chosen to be 321 displayed on the surface of the membranous VLP. Additional details on the fusion protein 322 construction are given in Table 2.   strains that were used for production of VLP are indicated in Table 3.   Tween 20 was applied as blocking reagent (Fig S1 in the supplementary material). Pfs25-dS/dS VLP preparation was demonstrated with two Pfs25-specific antibodies 32F81 381 [9] and 4B7 [23] having transmission-blocking activity (Fig 1 C). Just as in the anti-Pfs25 382 Western blot, cross reactivity to the dS was observed. However, the Pfs25-dS/dS VLP were 383 substantially more reactive. 384 385 Analysis by negative staining TEM and DLS (Fig 1 B and D) confirmed the formation of 386 homogeneous particles. TEM imaging indicated particles of predominantly 20-40 nm 387 according to manual evaluation. DLS showed a monomodal size distribution and a 388 monodisperse particle population characterized by a hydrodynamic diameter of 64 nm 389 (PDI 0.11). A summary of the production process and the composition of the final Pfs25-390 dS/dS VLP preparation can be found in Table 4. 391 392 N-glycosylation of the Pfs25-dS fusion protein was analyzed in crude cell lysates by 393 treatment with EndoH (Fig 2). The Pfs25-dS fusion protein construct "M" was described in 394 Table 2 and was chosen for chimeric VLP production because its recombinant expression in 395 H. polymorpha resulted in a homogeneous product (Fig 2, lanes 1 and 7). Despite its three 396 potential N-glycosylation sites within the Pfs25 aa sequence, the "M" construct was not 397 sensitive to EndoH treatment. The fusion protein was detected at ~33 kDa MW without and 398 after EndoH treatment indicating it was not N-glycosylated. However, in the initial 399 experiments two additional Pfs25-dS constructs were included ("CL" and "QQ" , Fig 2). The 400 "CL" construct analyzed in lanes 3/4 and 9/10 contained the leader sequence of the chicken 401 lysozyme at its N-terminus instead of the artificial start-methionine of the "M" construct. The 402 third construct ("QQ") analyzed in lanes 5/6 and 11/12 was like the "CL" construct but

437
From 73.4±8 g DCW of strain RK#114, 14.5±2.6 mg chimeric Pfs230c-dS/dS VLP composed 438 of wild-type dS and the fusion protein Pfs230c-dS were isolated (Y P/X = 0.2±0.06 mg g -1 ). At 439 different stages during processing of Pfs230c-dS/dS VLP substantial losses of product were 440 observed due to precipitation. Therefore, the purification protocol was adjusted compared to 441 the purification of Pfs25-dS/dS VLP: the PEG and NaCl concentrations were reduced for 442 clarification of the crude cell lysate, the Capto Core 700 matrix was used instead of the 443 Mustang Q membrane adsorber and the dialysis procedure was modified. To reach higher 444 product purity with the modified process, an additional wash step during the Aerosil batch 445 procedure and a second Capto Core 700 run were added to the purification process. 446 447 Purified Pfs230c-dS/dS VLP were analyzed in native and non-native assays (Fig 3). VLP 448 forming proteins were identified by anti-dS Western blot (Fig 3 A, lane 1) (Fig 3 A). All bands detected 452 between the fusion protein and the ds were considered as impurities. A subset of these 453 bands was reactive in an anti HCP Western blot (data not shown). The most prominent host 454 cell protein band beside the two product proteins (dS and Pfs230c-dS) was detected at 455 32 kDa apparent MW and represented 12 % of the total band volume of Coomassie stained 456 lane 3. The Pfs230c-dS specific signal appears diffuse in lanes 3 and 4 (Fig 3 A). Upon 457 treatment with EndoH, the diffuse smear disappeared and the main band became intensified 458 by factor 2.6 according to analysis by densitometry. This revealed that the six potential N- Formation of VLP was confirmed for both samples by TEM and DLS (Fig 3 B and D). DLS 468 indicated a monomodal and monodisperse (PDI 0.09) particle population characterized by 469 hydrodynamic diameter of 91 nm. However, the appearance of the Pfs230c-dS/dS VLP in 470 TEM imaging was rather heterogeneous (Fig 3 B). The dominating species of detected 471 objects were in the range of 44-60 nm diameter but also larger structures (>120 nm) were 472 observed frequently and this could be due to particle aggregation.  (Fig S 2). 480 Cross reactivity of the anti-Pfs230 polyclonal antibody with the dS VLP scaffold in form of 481 plain dS VLP [42] was not observed in this native assay (Fig 4). Pfs230D1M-dS/dS VLP (Y P/X = 0.64±0.1 mg g -1 ) that were composed of wild-type dS and the 501 fusion protein Pfs230D1M-dS. Processing of Pfs230D1M-dS/dS VLP was easier than 502 processing of Pfs230c-dS/dS VLP. No unexpected product losses during downstream 503 processing (DSP) were observed and thus a less complex DSP could be chosen. The 504 specific yield Y P/X of Pfs230D1M-dS/dS VLP was about three times as much as for the 505 Pfs230c-dS/dS VLP. 506 507 Both VLP-forming proteins were detected by anti-dS Western blot (Fig 5 A, lane 2). The 508 fusion protein Pfs230D1M-dS was specifically detected by anti- Pfs230 Western blot (lane 3). 509 Judging by their MW, the additional high MW signals in this lane likely correspond to 510 oligomeric forms (dimers, trimers, etc.) of the fusion protein. Most likely, these forms were not 511 detected by the anti-dS mAb in lane 2 because the signals were below the detection limit. 512 Analysis of a Coomassie stained PAA gel (Fig 5 A, lane 1) by densitometry indicated 72 % 513 purity on protein level and a composition of 24 % fusion protein and 76 % wild-type dS. 514 Formation of VLP was confirmed by TEM (Fig 5 B) and indicated 42 -62 nm diameter for the 515 VLP. Size distribution analyzed by DLS (Fig 5 D) indicated monomodal size distribution and 516 a monodisperse particle population characterized by a hydrodynamic diameter of 84 nm 517 (PDI 0.09). N-SIM was performed as for Pfs230c-dS/dS VLP with a similar result (Fig 5 C). 518 The Pfs230D1M-specific and the dS-specific signals co-localized in nano-scaled particles 519 (circled spots). The production processes and the compositions of the three different VLP preparations are 533 summarized in Table 4. The purification of chimeric Pfs25-dS/dS VLP from strain RK#097 534 was the most productive process and yielded 1.0±0.1 mg VLP per g DCW or 39±5 mg VLP 535 per L cell culture with 90 % purity on protein level. However, the fusion protein content in the 536 VLP was lowest (~3 %) in comparison to the other VLP preparations. 537

538
Despite the size of the Pfs230c antigen and difficulties due to product precipitation, isolation 539 of chimeric Pfs230c-dS/dS VLP was successful. However, the VLP yield was considerably 540 lower (0.2±0.06 mg g -1 or 8±2 mg L -1 ) than for the chimeric Pfs25-dS/dS VLP but the fusion 541 protein content of the VLP was approximately 10-times higher than for Pfs25. The purity of 542 Pfs230c-dS/dS VLP on the protein level was substantially lower (64 % on protein level) and 543  constructs were constructed and it was shown that depending on the design of the construct, 572 the degree of N-glycosylation varied (Fig 2). Due to product homogeneity and therewith 573 potential regulatory advantages, the non-glycosylated variant was chosen for chimeric Pfs25-574 dS/dS VLP production. The purified Pfs25 containing VLP were reactive with transmission-575 blocking monoclonal antibodies 32F81 and 4B7 in ELISA [56,57] and could be identified as 576 particulate structures in TEM imaging (Fig 1)These can be considered as a promising 577 findings for upcoming immunization studies. However, cross reactivity of the dS with the 578 antibodies 32D81 and 4B7 was observed in Western blot ( Fig 1A) and ELISA (Fig 1C) which 579 complicates interpretation of the results. In case of the antibody 4B7, the difference in 580 reactivity with the chimeric Pfs25-dS/dS VLP or with plain dS VLP not containing the Pfs25 581 antigen was only about factor 3. This difference in reactivity could be expected to be greater 582 and may be caused by misfolded Pfs25 antigen e.g. due to incomplete formation of disulfide 583 bonds or because of the low Pfs25 fusion protein content in the isolated chimeric VLP. 584 Depletion of Pfs25-dS relative to dS in the course of the DSP was not observed and thus this 585 low inclusion rate could have two possible reasons. 586 (1) The ratio of Pfs25-dS to wild-type dS produced by RK#097 was too low to 587 facilitate isolation of chimeric VLP with higher fusion protein content. 588 (2) The chosen fusion protein construct Pfs25-dS is suboptimal for high fusion protein 589 content in this kind of VLP. 590 To overcome possibility 1, recombinant H. polymorpha strains producing improved fusion 591 protein to dS ratios were generated by applying alternative strain generation approaches 592 described in [42]. Solubility of the VLP forming proteins in homogenates of these new strains 593 was however reduced compared to strain RK#097 (data not shown). As a next step, 594 solubilization of the VLP proteins would need to be optimized and purified chimeric VLP 595 should then be compared head-to-head with purified particles from strain RK#097. To 596 address possibility 2, the fusion protein construct may be modified e.g. by truncation of the 597 protein. This may also be an explanation for missing response in ELISA applying the 1B3 607 monoclonal antibody (Fig S 2). The Pfs230c-dS aa sequence contains 16 cysteine residues 608 which could be linked incorrect via disulfide bonds. In our experiments, overexpression of a 609 recombinant protein disulfide isomerase did not result in detectable reduction of product loss native ELISA (Fig 4) is a promising result. 618 619 A remaining challenge regarding the chimeric Pfs230c-dS/dS VLP purification is the relatively 620 low purity of 64 % of the final preparation. It can be speculated that a contributor to the sub-621 optimal purity is that the residual HCP impurities were tightly associated with the particles or 622 the Pfs230c antigen. Separation of product from contaminative proteins was not possible by 623 CsCl density gradient separation, ultrafiltration (MWCO 100 kDa), multimodal 624 chromatography (Capto Core 700 matrix, GE Healthcare) or HP-SEC (data not shown). A 625 revised purification protocol may need to be developed addressing reduction of the most 626 prominent, persisting protein contaminants already present in earlier purification steps. have transmission-blocking activity. Regarding VLP yield, their purity and fusion protein 681 content, the chimeric Pfs230D1M-dS/dS VLP appears to be the most promising candidate 682 that emerged from this study. The obtained product yields in combination with the versatility 683 and reliability of the described VLP production platform makes it a competitive system and 684 should be considered for future malaria vaccine development. However, the potential of the 685 three developed, chimeric VLP as effective vaccine candidates cannot be disclosed unless 686 studies to assess their immunogenicity and transmission-blocking performance are 687 completed. This represents together with improving the product purity especially for the 688