s2fieldsofstudy: Biology Chemistry

text:
Redirecting RiPP Biosynthetic Enzymes to Proteins and Backbone-Modified Substrates

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products with potent antibiotic, antiviral, and anticancer properties. RiPP enzymes known as cyclodehydratases and dehydrogenases work together to catalyze intramolecular, inter-residue condensation and dehydrogenation reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here, we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers—including aminobenzoic acid derivatives and beta-amino acids—within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse non-α-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond—both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

■ INTRODUCTION
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products that include the FDA-approved analgesic ziconotide 1,2 as well as compounds with potent antibiotic, antiviral, and anticancer properties. 3 RIPP biosynthesis begins with a ribosomally synthesized polypeptide whose N-terminal leader sequence (∼20−110 aa) recruits one or more endogenous enzymes capable of diverse post-translational modification (PTM) of an adjacent C-terminal substrate sequence. 3,4 Researchers have leveraged this leader-dependent mechanism to direct RiPP PTM enzymes to C-terminal substrate sequences containing diverse noncanonical α-amino acids (nc-α-AAs). 5,6 Cyclodehydratases and dehydrogenases represent an exceptionally well-studied class of RiPP enzymes. 3 These enzymes work together to catalyze intramolecular cyclization 3,7 and subsequent aromatization reactions that install oxazoline/ oxazole and thiazoline/thiazole heterocycles within polypeptide chains ( Figure 1A,B). Previous work has shown that the cyclodehydratases PatD 8 and TruD 9 support leader sequencedependent oxazoline/thiazoline formation within substrates containing nc-α-AAs adjacent to 10 or at the cyclization site itself. 11 −13 In related work, it was shown that a chimeric leader peptide could direct the cyclodehydratase LynD 9 and the dehydrogenase TbtE 14 to install thiazol(in)es within substrates containing nc-α-AAs adjacent to the cyclization site. 15 Finally, reconstituted lactazole biosynthesis, 16 including the cyclodehydratase-dehydrogenase pair LazDE/LazF, was found to install oxazoles and thiazoles within polypeptide substrates containing α-hydroxy, N-methyl, cyclic α-, and β 3 -amino acids 17 at sites distal from the site of heterocyclization (>4 residues away).
Previous work has also shown that certain cyclodehydratase enzymes can process leader sequence-free substrates when the leader peptide is provided in trans ( Figure 1C). 18,19 Building on this observation and earlier work on lantibiotic synthetases by van der Donk, 20 Naismith and co-workers engineered a family of cyclodehydratases in which the leader peptide is fused to the N-terminus of the cyclodehydratase catalyst as opposed to the N-terminus of the substrate polypeptide. These constitutively activated enzymes, notably, LynD Fusion (LynD-F) 19 and MicD Fusion (MicD-F), 21 act in a leader peptide-independent manner to promote the cyclodehydration of polypeptides containing a C-terminal Ala-Tyr-Asp (AYD) recognition sequence. 7,19,21−23 In complementary work, Schmidt and co-workers demonstrated that two dehydrogenases, ArtGox and ThcOx, also accept leaderless peptide substrates. 24 Taken together, these enzymes represent a fully leader-free route toward polypeptides (and proteins, vide infra) containing mRNA-programmed thiazole and oxazole link-agesdistinct from approaches based on chimeric leader peptides 25 or leader peptide exchange. 26 Indeed, some tolerance for noncanonical α-amino acid residues has been reported: LynD-F was shown to install a thiazoline in a peptide substrate containing 3-azido-L-alanine positioned four residues away from the site of cyclization, 23 and the combination of LynD-F and ArtGox installed a thiazole in an AYD-containing peptide with a polyethylene glycol spacer two residues from the site of cyclization. 22 Here, we report that MicD-F 21,24 and ArtGox 21,24 act together to process polypeptide substrates containing diverse translation-compatible 27−32 aminobenzoic acid derivatives and β-amino acids, even at sites directly flanking the reaction site ( Figure 1D). We show further that MicD-F 21,24 and ArtGox 21,24 process substrates even when the CAYD sequence is positioned at the C-terminus of mCherry, a large β-barrel protein, or embedded within the loop of the dimeric α-helical bundle protein Rop. The products are folded, globular proteins containing a conformationally restricted, fully unnatural, heterocyclic backbone. To the best of our knowledge, these studies represent the first example of leader-free azol(in)e biosynthesis within polypeptides containing diverse non-αamino acid monomers flanking the site of cyclization and the first report of a cooperatively folded protein containing a posttranslationally installed heterocyclic ring. 33 The effects of the embedded heterocycle on local conformational flexibility are examined computationally, providing important insight into the backbone restrictions that could be leveraged to improve the physiochemical properties of therapeutic proteins. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

■ RESULTS
MicD-F and ArtGox Accept Substrates with Diverse Structures at the +1 Site. We began by exploring the tolerance of MicD-F for sequences containing non-α-amino acid monomers at the +1 site ( Figure 1A and Figure 2A,B). A series of nine potential substrates were prepared in which a non-α-amino acid preceded the reaction site (substrates 1(ai)). Monomers evaluated included arenes, aminobenzoic acid derivatives, fluorophores, and linear and cyclic β-amino acids. All substrates contained a C-terminal AYD recognition sequence 36 and were incubated with MicD-F (Supplementary Figure 1A,B) (5 mol %) under mild conditions (pH 8.0, 37°C, 4 h) that resulted in complete conversion of ICAYDG, a substrate with the natural α-amino acid Ile at the +1 site (Supplementary Figure 2). Cyclization was analyzed initially via liquid chromatography−mass spectrometry (LC-MS), and the extent of product formation was estimated by integrating the extracted ion chromatogram ( Figure 2B,C). Virtually every substrate examined underwent MicD-F-catalyzed cyclization to the corresponding thiazoline under these conditions. Substrates containing electron-withdrawing or donating aromatic rings, bulky multiring systems, and linear and cyclic β-amino acids were all cyclized efficiently by MicD-F, with yields between 91 and 99% (products 2(a-i)). UHPLC analysis of each reaction mixture confirmed that thiazolines 2a-i were the sole reaction products under these conditions (Supplementary Figure 3). It is notable that monomers with highly divergent structures are accepted almost equally by MicD-F, suggesting that the +1 residue interacts minimally if at all with the enzyme active site.
Next, we explored whether MicD-F and ArtGox could act in synergy to convert peptides containing non-α-amino acids at the +1 site directly into the corresponding thiazoles 3(a-i) ( Figure 2A). Substrates 1(a-i) were incubated with MicD-F (5 mol %) and ArtGox (Supplementary Figure 1A,C) (40 mol %) under conditions (pH 8.0, 37°C, 16 h) that resulted in complete two-step conversion of ICAYDG into the corresponding thiazole product (Supplementary Figure 4). ArtGox efficiently oxidized each thiazoline to the corresponding thiazole in yields that exceeded 97% over the two steps for every example (products 3(a-i)) ( Figure 2B,D). UHPLC analysis of each reaction mixture confirmed that thiazoles 3a-e were the sole reaction product. Coelution with excess flavin mononucleotide precluded UHPLC analysis of thiazoles 3f-i (Supplementary Figure 5). These results indicate that MicD-F and ArtGox tolerate diverse nonproteinogenic, non-α-amino acid monomers at the +1 site. Many of these non-α-amino acid monomers have been installed at the N-termini of ribosomally translated peptides in vitro, 30,31,37,38 suggesting a path toward proteins and polypeptides with highly unique N-terminal appendages.
MicD-F and ArtGox Accept Substrates with Diverse Structures at the −1 Site. Next, we explored whether MicD-F and ArtGox would accept leader-free polypeptide substrates containing non-α-amino acid monomers at the −1 site ( Figure  3). We explored a diverse array of monomers − β 3 -amino acids, β 2 -amino acids, cyclic β 2 , β 3 -amino acids, as well as substituted and unsubstituted aminobenzoic acid derivatives. Notably, inserting a single α-amino acid (Ile) residue between the site of cyclization and the C-terminal AYD motif required higher concentrations (50 mol %) of MicD-F and up to 24 h reaction time to complete the cyclodehydration reaction (Supplementary Figure 6).
All −1 site substrates (substrates 4(a-g), Figure 3A,B) contained a C-terminal AYD recognition sequence 36 and were incubated with MicD-F (50 mol %) and ArtGox (8 mol %) under conditions (pH 8.0, 37°C, 24 h) that resulted in complete conversion of a substrate with a natural α-amino acid at the −1 site to the corresponding thiazole (Supplementary Figure 7). Reactions were analyzed as described above. Under these conditions, every peptide evaluated was a substrate for MicD-F, and a few were substrates for both MicD-F and ArtGox ( Figure 3B−C). Substrates containing β 3 -amino acid-, β 2 -amino acid-, or cyclic β 2 ,β 3 -amino acids at the −1 site (substrates 4(a-d)) were fully consumed under these conditions (<1% unmodified peptide). Those with β 3 -alkyl substituents (4a, c, and d) were converted cleanly into the corresponding thiazolines 5a, c, and d, with little (4a) or no (4c,d) thiazole formation. In contrast, substrate 4b, with geminal β 2 -methyl substituents, was converted into a 30/70 mixture of thiazoline 5b and thiazole 6b. Substrates 4e-g containing aminobenzoic acid derivatives at the −1 position reacted more slowly under these conditions, producing the analogous thiazoline products in 65−85% yield after 24 h reaction at pH 8 ( Figure 3B,C). Surprisingly, while all substrates containing +1 site modifications were efficiently oxidized to the corresponding thiazole ( Figure 2B,D), only the substrate containing a geminal β2-dimethyl substituent at the −1 site was efficiently oxidized by ArtGox (70%) ( Figure  3B,C). With the exception of substrate 4c, increasing the pH to 9.0 promoted formation of the desired thiazole product (Supplementary Figure 8B,C). However, even under these conditions only substrate 4b (88%) yielded a greater than 41% thiazole product (Supplementary Figure 8B,C). These data indicate that MicD-F and ArtGox are both less tolerant of nonα-amino acid monomers at the −1 site than at the +1 site. ArtGox appears especially intolerant of substitution or sp 2 hybridization at the β 3 -position of substrates at the −1 site.
MicD-F Is Sensitive to Amino Acid Identity at the Cyclization Site. To complete the exploration of the substrate tolerance of MicD-F and ArtGox, we synthesized a set of potential substrates containing a non-α-amino acid directly at the cyclization site. Each contained a C-terminal AYD sequence preceded by either L-β 3 -or D-β 3 -threonine (Supplementary Figure 9A). Incubation of these substrates with MicD-F (50 mol %) under conditions (pH 8.0, 37°C, 24 h) that resulted in substantial cyclization of a substrate containing L-αthreonine at the cyclization site led to no detectable cyclization (<1%) (Supplementary Figure 9B). Even at pH 9.0, no cyclization occurred (Supplementary Figure 9C), indicating that MicD-F is highly sensitive to amino acid identity at the site of cyclization. This result is in line with previous work that demonstrated the cyclodehydratase PatD failed to react with substrates containing D-α-threonine at the cyclization site. 12 Redirecting RiPP Biosynthetic Enzymes to Intact Folded Proteins. Thiazolines and thiazole are replete in natural products 39−41 and synthetic drug-like small molecules, 42,43 and calculations confirm the expected decrease in conformational freedom that derives from aromatic and/or sp 2 character within the peptide backbone. 44 This finding and the leader-independent nature of MicD-F and ArtGox-mediated thiazol(in)e biosynthesis inspired us to explore substrates in which the site of cyclodehydration/dehydrogenation is embedded within a stable protein fold (Figure 4). We first asked whether MicD-F and ArtGox could install thiazol(in)e linkages within loops and/or at the termini of mCherry. mCherry is a prototypic fluorescent beta-barrel protein derived from DsRed, isolated originally from Discosoma sea anemones. 45 We cloned, expressed, and purified a set of mCherry variants in which the core sequence MCAYDG was appended to the mCherry C-terminus (mCherryC+) or inserted into a loop immediately downstream of residues D137 (mCherry137+), D174 (mCherry174+), V192 (mCherry192+), or E211 (mCherry211+) ( Figure 4A, Supplementary Table 2, Supplementary Figure 10). Although mCherry137+ and mCherry211+ were partially/completely nonfluorescent or could not be purified, mCherryC+, mCherry174+ and mCherry192+ were soluble and fluorescent. In all three of these cases, mass spectrometry of the purified proteins showed the characteristic loss of 22 Da, indicating chromophore maturation (Supplementary Figure 11).
Treatment of mCherryC+ with 50 mol % MicD-F (pH 9.0, 24 h, 37°C) led to virtually complete conversion to the thiazoline product as indicated by a loss of water in the deconvoluted mass spectrum ( Figure 4B,C). No such mass change was observed in an analogous reaction containing mCherryC-, which carries the sequence MAAYDG in place of MCAYDG at the C-terminus, providing evidence that the observed cyclodehydration demanded a Cys residue immediately upstream of the AYD recognition sequence (Supplementary Figure 12B,C). Neither mCherry174+ nor mCherry192+ displayed the loss of water characteristic of successful cyclodehydration even after 24 h at 37°C  Figure 12D,E). Nevertheless, we explored the potential for MicD-F and ArtGox to act in tandem to install an aromatic thiazole backbone in mCherryC+. Simultaneous treatment of mCherryC+ for 24 h (pH 9.0, 37°C) with MicD-F (50 mol %) and ArtGox (80 mol %) resulted in the expected −2 Da shift in the deconvoluted mass spectrum ( Figure 4D) relative to that of mCherryC+ treated with only MicD-F ( Figure 4C). This result indicates that the MicD-F/ ArGox enzyme pair can post-translationally install an aromatic thiazole backbone within a structurally unconstrained region of a well-folded beta-barrel protein.
We hypothesized that the absence of cyclodehydration reactivity for mCherry174+ and mCherry192+ at 37°C was due to neighboring structural elements that disfavor productive interaction with MicD-F and/or enzyme-promoted thiazoline formation. Therefore, we carried out a second set of cyclodehydration reactions at 42°C, the highest temperature at which MicD-F remained stable in our hands. At this elevated temperature, mCherryC+ again displayed cysteine-specific loss of water characteristic of successful cyclodehydration (Supplementary Figure 13B,C). However, again neither mCherry174+ or mCherry192+ displayed the loss of water characteristic of successful cyclodehydration after 24 h at 42°C (Supplementary Figure 13D,E). It has been reported 46 that the apparent melting temperature of mCherry is upward of 90°C. Taken together with our results, this finding suggests that there is an inherent mismatch between the temperature stability of MicD-F and the thermodynamic stabilities of the mCherry loop insertions evaluated here.
To test this hypothesis, we sought a folded, globular protein with a lower melting temperature than mCherry with the expectation that it would be more amenable to insertion of an internal thiazol(in)e linkage. Rop is a homodimeric four-helix bundle protein formed by the antiparallel association of two helix-turn-helix monomers. 47 Regan and co-workers reported many years ago that the native two-residue turn in Rop could be replaced by up to 10 glycine residues without loss of the native dimer structure. The Rop variant with the longest insertionGly 10 melted cooperatively at 50°C, 48 suggesting that it might tolerate an embedded thiazole or thiazoline heterocycle ( Figure 5A). To test this hypothesis, we expressed and purified three Rop variants containing a single CAYD sequence embedded near the N-terminus (RopN), the Cterminus (RopC), or centrally (RopM) within a 10-residue glycine-rich loop ( Figure 5A, Supplementary Table 3, Supplementary Figure 14, and Supplementary Figure 15). All three Rop variants exhibited high α-helical content at 20 μM as judged by wavelength-dependent CD measurements ( Figure  5B). RopC and RopM migrated as discrete dimers at 50 μM as judged by size-exclusion chromatography (SEC) and melted cooperatively and reversibly with T M values of 28 and 32°C (Supplementary Figure 16). RopN, by contrast, migrated as a heterogeneous mixture upon SEC and melted noncooperatively, albeit at a slightly higher apparent T M (43°C) perhaps because of disulfide formation (Supplementary Figure 16). 49 Although RopC, RopN, and RopM all contained the same CAYD recognition sequence, only oneRopCunderwent clean conversion into the corresponding thiazoline upon treatment with 50 mol % MicD-F (pH 9.0, 37°C, 16 h). RopM reacted partially under these conditions, and RopN was unreactive ( Figure 5C). Reaction of RopC to generate thiazoline RopC-U proceeded more slowly at 25°C (Supplementary Figure 17). RopC could be converted directly into the thiazole RopC-Z upon treatment with 50 mol % MicD-F and 80 mol % ArtGox ( Figure 5D, Supplementary  Figure 18). No reaction was observed when the Cys residue within the RopC reaction site was replaced with Ala or when the C-terminal AYD sequence was replaced by GGG (Supplementary Figure 19).
The products of the reaction of RopC with MicD-F (RopC-U) and with MicD-F and ArtGox (RopC-Z) were purified, confirmed via protease digest (Supplementary Figure 20), and analyzed by size-exclusion chromatography and wavelengthand temperature-dependent CD. Thiazoline-containing RopC-U was a homogeneous dimer as judged by SEC (Supplementary Figure 16) and retained a significant level of α-helical structure ( Figure 5E). It also melted cooperatively and reversibly with a T M value of 27°C, a value almost identical to that of RopC itself (28°C) ( Figure 5F). Thiazolecontaining RopC-Z displayed more complex behavior; it was less homogeneous as judged by SEC and melted cooperatively (T M = 24°C) but only after a refolding step (Supplementary Figure 21). These results indicate that the MicD-F can posttranslationally install a thiazoline within a backbone of a helical bundle protein, and that ArtGox can oxidize this substrate to install a fully aromatic thiazole unit.
Computational Analysis of the Effects of Thiazoline/ Thiazole Formation on Local Backbone Flexibility. To explore the effects of thiazoline/thiazole formation on local backbone flexibility, we examined the conformational space of the tetrapeptide Ac-AACA-NH 2 . The use of this simplified substrate allowed the inherent peptide backbone energetics to be evaluated without the complications of side chain fluctuations. Molecular mechanics methods (Macromodel, OPLS4 force field, implemented in Schrodinger Maestro software) were first used to generate and minimize large populations of conformers for cysteine-, thiazoline-, and thiazole-containing analogues ( Figure 6A). For each species, 10 000 starting structures were sampled using the mixed torsional/low-mode method. All conformers within 4 kcal/mol of each global minimum were then subjected to geometry optimization using DFT (Jaguar: B3LYP-D3/6-31G**). An SM8 method was used to determine the relative energies in aqueous media. 50 All nonredundant conformers were then ranked based on these energies and compared. The results of the conformational analysis appear in Figure  6B−E, sorted by progressive energy cutoffs relative to each global minimum. The noncyclized, cysteine-containing peptide exhibits the greatest flexibility, with 5 conformers being identified within 1.36 kcal/mol of the global minimum (91% of the population) and 16 within 2.72 kcal/mol (99%). Moreover, the identified conformers are largely non-superimposable, indicating that a high degree of conformational space is accessible within these energy ranges. In contrast, the thiazoline exhibits the most significant reduction in flexibility, with only three conformers identified at the 1.36 kcal/mol cutoff level and only seven identified at a cutoff of 2.72 kcal/ mol. Superposition of the thiazoline rings of these conformers reveals a rigid six-bond motif that is preserved in all cases ( Figure 6F). The thiazole analogue exhibits similarly reduced flexibility, with 11 conformers being identified within 2.72 kcal/mol of the global minimum. In this case, a rigid sevenbond motif can be identified ( Figure 6G). These evaluations provide the basis of models that could be used to predict the conformational effects of backbone cyclization on larger sequences and could be used to predict sequence locations in which cyclizations are more likely to be successful. In current experiments, we are combining molecular dynamics studies with experimental data to examine the longer-range effects that result from introducing thiazoline and thiazole groups in longer peptides and intact proteins. Such information could be used to apply this chemistry more generally to improve the physiochemical properties of therapeutic proteins.

■ CONCLUSIONS
One can imagine two mutually synergistic strategies to introduce non-natural monomers into polypeptide and protein oligomers. 51 One "bottom-up" approach relies on extant or engineered ribosomes to accept and process tRNAs carrying diverse noncanonical α-amino or non-α-amino acids. 52 Hundreds of noncanonical α-amino acids (as well as αhydroxy acids 53−56 ) have been introduced into proteins in cells and animals using genetic code expansion, 57,58 which usually relies on novel orthogonal aminoacyl tRNA synthetases to generate the requisite acylated tRNAs. Select noncanonical αamino acids 59 and one β-amino acid 27 have also been incorporated into proteins in vivo using endogenous αaminoacyl tRNA synthetases. Alternatively, many noncanonical α-amino acids, as well as certain non-α-amino acids, including β-amino acids 29,60 and certain polyketide precursors, 30 can be introduced into short peptides in vitro and on a small scale using genetic code reprogramming, in which a stoichiometric RNA coreagent (Flexizyme 61 ) generates the requisite acylated tRNA.
The second "top-down" approach is reminiscent of late-stage functionalization reactions used to manipulate complex small molecule natural products 62,63 and the natural biosynthetic strategy used to assemble ribosomally synthesized and posttranslationally modified peptides (RiPPs). 6 In this approach, enzymes, chemical reagents, or chemical catalysts are employed to post-translationally modify a peptide 5 or protein 51 to install a new or modified monomer. Examples of this approach include reactions of natural or noncanonical protein side chains or modification of the N-or C-terminus. 64−67 The only backbone-focused nonenzymatic reaction of which we are aware is the O-mesitylenesulfonylhydroxylamine-promoted oxidative elimination of Cys residues to generate a dehydroalanine backbone 68 that is subsequently modified. We note that the top-down and bottom-up strategies are complementary, and both have the potential to operate in vivo where very high protein titers are possible. 69 Here, we show that a constitutively active form of MicD and ArtGox, two enzymes used in the biosynthesis of cyanobactin natural products, 70 are sufficiently promiscuous to process substrates containing diverse backbone-modified monomers within substrate polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The backbone-modified monomers compatible with MicD-F and ArtGox include many accepted by extant ribosomes in small-scale in vitro reactions, including aminobenzoic acid derivatives and β 2 -and β 3 -amino acids. The products of these reactions are sequence-defined chemical polymers with multiple, diverse, non-α-amino acid monomers. We show further that cyclodehydration and dehydrogenation can install thiazoline or thiazole backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reported here reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bondboth features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. Moreover, as thiazoles and thiazoline heterocycles are replete in natural products, 71−73 small molecule drugs, 42,43 and peptide-mimetic therapeutics, 74 their installation in protein-based biotherapeutics could improve or augment performance, activity, stability, and/or selectivity. This work represents a general strategy to expand the chemical diversity of the proteome without need for genetic manipulations.


s2fieldsofstudy: Biology Medicine

text:
Nucleocapsid Antigenemia Is a Marker of Acute SARS-CoV-2 Infection

Abstract Detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is essential for diagnosis, treatment, and infection control. Polymerase chain reaction (PCR) fails to distinguish acute from resolved infections, as RNA is frequently detected after infectiousness. We hypothesized that nucleocapsid in blood marks acute infection with the potential to enhance isolation and treatment strategies. In a retrospective serosurvey of inpatient and outpatient encounters, we categorized samples along an infection timeline using timing of SARS-CoV-2 testing and symptomatology. Among 1860 specimens from 1607 patients, the highest levels and frequency of antigenemia were observed in samples from acute SARS-CoV-2 infection. Antigenemia was higher in seronegative individuals and in those with severe disease. In our analysis, antigenemia exhibited 85.8% sensitivity and 98.6% specificity as a biomarker for acute coronavirus disease 2019 (COVID-19). Thus, antigenemia sensitively and specifically marks acute SARS-CoV-2 infection. Further study is warranted to determine whether antigenemia may aid individualized assessment of active COVID-19.

Although it is the standard of care for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reverse transcription polymerase chain reaction (RT-PCR) remains an imperfect diagnostic marker for coronavirus disease 2019 (COVID-19) because SARS-CoV-2 RNA commonly persists beyond the period of acute infection [1][2][3]. Accordingly, Centers for Disease Control and Prevention (CDC) guidelines do not recommend retesting most individuals by RT-PCR within 90 days following diagnosis. Instead, isolation guidelines are based on time from symptom onset [4,5]. This creates a dilemma when screening tests detect SARS-CoV-2 RNA in a patient without welldefined onset or resolution of COVID-19-like illness. Alternative molecular markers for acute infection are not widely available [6] and low-sensitivity respiratory antigen testing may be effectively applied at a population level [7,8], but there remains a need for more sensitive and specific diagnostics to provide individualized guidance.
The presence of viral nucleocapsid protein in peripheral blood (antigenemia) has been demonstrated in SARS-CoV-1 and SARS-CoV-2 infection [9][10][11][12][13][14][15][16][17][18][19][20][21]. A blood-based antigen biomarker may have inherent advantages over upper respiratory tract antigen testing, or biomarkers such as RT-PCR cycle threshold (Ct) value and subgenomic RNA (sgRNA), because specimen quality and quantity can be standardized. Reports of antigenemia test performance as a diagnostic biomarker are inconsistent, likely due to varying assay composition and inconsistent reference standards as many studies compare against respiratory RT-PCR as a gold standard and fail to account for the persistence of RNA beyond acute infection.
In this study, we present evidence from a large serosurvey of adults in inpatient and outpatient settings to explore the hypothesis that nucleocapsid antigenemia is a sensitive and specific marker of acute infection as defined by a clinical timeline. Specifically, each blood sample was categorized through rigorous review of clinical history and respiratory SARS-CoV-2 testing in a schema that assumes a typical course of COVID-19 for all subjects. Our study is novel among others evaluating nucleocapsid antigenemia in that we were able to capture blood samples from each stage of infection to evaluate the performance of antigenemia testing for staging acuity, which we define based on onset of symptoms and timing of respiratory SARS-CoV-2 positivity. Furthermore, our evaluation of outliers in each staging category uncovered evidence of reinfection as well as persistence of antigenemia in immunocompromised individuals. Overall, we find a strong association between acute infection and nucleocapsid antigenemia, which also correlates with serostatus and disease severity. Together our findings suggest antigenemia may clarify disease timing and provide needed insight in many clinical settings.

Clinical Specimens
We collected a convenience sample of residual plasma, serum, and whole blood specimens from the clinical chemistry laboratory of Emory Medical Laboratories 1 day per week between 11 January 2021 and 12 March 2021. These specimens were originally collected for routine clinical testing from inpatient (medical/ surgical wards, intensive care, obstetrics) and outpatient settings (clinics, emergency department, infusion centers, ambulatory surgery). Samples were transferred to a −80°C repository after clinical testing was completed, but prior to being discarded. More than 1 blood sample from the same patient was permitted with a minimum time of 5 days between samples. This study was approved and granted complete Health Insurance Portability and Accountability Act (HIPAA) and consent waiver by the Emory University Institutional Review Board (STUDY00000510). This study was approved by the Institutional Review Board of Emory University for the use of residual clinical specimens. Written informed consent by the patients was not required.

Nucleocapsid Assay
Nucleocapsid antigenemia was quantified on the Quanterix HD-X platform. Residual serum and plasma samples were thawed once after storage at −80°C and diluted 1 to 3 in assay sample diluent. Diluted samples were then run using the ultrasensitive SIMOA SARS-CoV-2 N Protein Antigen assay (Quanterix) on the automated Quanterix HD-X platform (Quanterix), which has a validated limit of detection of 0.099 pg/mL in respiratory and saliva samples. Analytical validation in serum and plasma samples is reported by Quanterix for research use outside of their Emergency Use Authorization. Samples with antigen levels too high for the linear range of the assay were further diluted 1 to 20 and retested. Final antigen concentrations were determined by interpolation after sigmoidal fitting of duplicate calibration curves run on each test plate.

Serological Testing
In-house developed single-dilution serological screening assays for SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid antibodies were used to establish serological status at the time of antigenemia testing. Antibody class-specific RBD serologies were performed as previously described [22][23][24]. Nucleocapsid antibody testing was performed using an in-house developed enzyme-linked immunosorbent assay (ELISA; Supplementary Material).

Medical Record Review
Patient medical record number was recorded at the time of specimen collection. The Emory Healthcare Clinical Data Warehouse (CDW) was queried for SARS-CoV-2 nucleic acid amplification tests (NAAT) and antigen tests, clinical notes, International Classification of Diseases-Tenth Revision (ICD-10) codes, laboratory values, and, if applicable, requirement for mechanical ventilation and date of death. All Ct values were obtained directly from reports produced by the manufacturer's software (Supplementary Material).
Chart review began with automated review of SARS-CoV-2 respiratory results available in the medical record. Blood samples from a patient with a positive test more than 14 days prior to sample collection were labeled convalescent and no further review of the medical record for categorization purposes was performed. History and physical clinical notes dated within 14 days before or after the date of the blood sample were then reviewed, if available, for all patients not labeled convalescent. Date of COVID-19-like symptom onset (including fever, fatigue, malaise, myalgia, headache, dyspnea, cough, wheezing, anosmia, ageusia, congestion, rhinorrhea, or diarrhea) and earliest positive SARS-CoV-2 testing was recorded if these had been described in the history narrative or clinician's assessment and plan.
The original medical records were then reviewed for all patients (other than those labeled convalescent) with a positive SARS-CoV-2 test who did not yet have date of symptom onset recorded in our data set. The entire medical record was available during this stage, but the reviewer was blinded to antigenemia status, which was not considered in labeling of COVID-19 status or category assignment.
Given that reinfection with SARS-CoV-2 was rare at the time of this study [25], our approach assumed that no reinfection events were captured in our sample set, which spanned 3 months. Patients without any record of SARS-CoV-2 testing were excluded from analysis. Further detail is provided in Supplementary Material.

Data Analysis
Data obtained during specimen collection were stored in Microsoft Excel. CDW reports were provided in commaseparated values (csv) format. All data were then imported into MATLAB (The MathWorks, Inc) for analysis. Wilcoxon rank-sum test was used for comparisons.

Specimens and COVID-19 Status Assignments
In total, 2498 serum and plasma samples were targeted for evaluation during the study period ( Figure 1B). Eleven samples were not evaluated for antigenemia due to preanalytical factors such as insufficient sample volume. Thus, 2487 samples were available for quantification of antigenemia, of which 255 (10.2%) exhibited detectable nucleocapsid; 115 of 2487 were excluded due to lack of patient identifiers and 5 additional samples were excluded as they had been collected on the same Figure 1. A, Schematic of the process for COVID-19 status assignment. Samples from patients with no record of positive SARS-CoV-2 respiratory testing were only considered negative if corresponding negative respiratory testing occurred on the same day. Due to the lack of a gold standard for active SARS-CoV-2 infection, samples from individuals with history of positive SARS-CoV-2 testing were labeled based on earliest known positive SARS-CoV-2 respiratory test and time since symptom onset. *Samples with postwindow-positive SARS-CoV-2 testing were labeled negative if a negative SARA-CoV-2 test was available following the sample but before the positive test. Otherwise the sample was labeled unknown. B, Flow chart of categorization and labeling process indicating number of samples assigned to each group. Abbreviations: COVID-19, coronavirus disease 2019; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.
day as another blood sample from a single patient. Clinical data were examined for the remaining 2367 samples from 2101 unique patients (Table 1). Of the 2367 samples, 507 were excluded because of no record of SARS-CoV-2 testing, and 11 of these 507 (2.1%) had detectable antigenemia. The remaining 1860 samples from 1607 patients had SARS-CoV-2 testing records to guide categorization and were classified as described in Figure 1B and Table 2.

Diagnostic Performance of Antigenemia for Acute COVID-19
Nucleocapsid antigenemia was present at higher frequency and with a higher median concentration in acute COVID-19 samples compared to samples categorized as late presenting, convalescent, pre-COVID-19, or same-day negative (P , .001 for all comparisons; Figure 2A). Receiver operating characteristic (ROC) analysis demonstrated area under the curve (AUC) of 0.902 in distinguishing samples from patients experiencing acute infection from all nonacute categories, and sensitivity and specificity were 85.2% and 89.9%, respectively ( Figure 2B). Test characteristics with censoring of the potentially ambiguous late-presenting group showed AUC 0.914, sensitivity 85.8%, and specificity 93.7%, while the most stringent comparison (censoring of the convalescent and latepresenting groups) demonstrated AUC 0.972, sensitivity 85.8%, and specificity 98.6%. Sensitivity improved to 93.9% when the comparison was only made among seronegative individuals (Supplementary Figure 1).
Test characteristics were also examined when adjusting the reference standard by varying parameters of the acuity window. Sensitivity decreased as the window start period increased beyond −11 days ( Figure 2C). Meanwhile, specificity consistently increased as the period of the acuity window was lengthened. Maximum AUC was observed with a window period opening at −12 days (AUC = 0.912 with window close  at +3 days) with minimal effect of varying the postsampling period from 0 to +3 days ( Figure 2D). Ct values from positive nasopharyngeal RT-PCR were available from the same day as a blood sample for 49 specimens. Only 6 of 17 samples with corresponding to Ct values greater than 33 had antigenemia and 4 of 6 of these were from the GeneXpert assay ( Figure 2E). All except for 2 samples with corresponding Ct values less than 30 exhibited antigenemia.

Temporal Trends in Antigenemia
We analyzed the dynamics of antigen level over time in samples from the acute, late-presenting, and convalescent groups. The frequency of detectable nucleocapsid and antigen concentration decreased over time following diagnosis and reported symptom onset ( Figure 3A and 3B). Eighteen samples were identified from patients who were asymptomatic at the time of COVID-19 diagnosis, 5 (27.7%) of which had detectable nucleocapsid antigenemia. Nucleocapsid antigen was detected more frequently (50.0%) in the subset of samples available from asymptomatic patients within 3 days of their diagnosis ( Figure 3C). Among 55 samples from individuals with positive respiratory RT-PCR testing on the same day, 7 convalescent samples did not exhibit antigenemia ( Figure 3D and Supplementary Table 1) and acute infections primarily exhibited high antigenemia. Among this subset of patients, no antigenemia was observed more than 14 days after the earliest known positive test ( Figure 3D).

Examination of Outliers
We reviewed medical records for individuals with unexpected presence or absence of antigenemia based on staging category. Twenty-one of 182 (11.5%) in the convalescent group had antigenemia (Supplementary Table 2   clinical evidence that might explain persistent antigen positivity. Among these, 2 individuals had clinical history consistent with reinfection by SARS-CoV-2, 2 were highly immunocompromised, and 11 samples (median time from diagnosis 20 days; interquartile range, 16.5-28.5 days) had severe COVID-19 marked by need for high-flow oxygen, intubation, or death. End-stage renal disease or dialysis was more common among samples in the convalescent group with antigenemia compared to those without antigenemia (fraction = 0.41; 95% confidence interval [CI], .20-.61] vs 0.13; 95% CI, .07-.18) whereas other comorbidities were not significantly different (Supplementary Figure 3). Three individuals had negative respiratory SARS-CoV-2 testing and antigenemia on the same day, none of which had evidence of COVID-19-related symptoms (Supplementary Table 3). Eighteen samples had antigenemia after more than 14 days of symptoms, of which 14 were seropositive for both N IgG and RBG IgG, 2 seropositive for RBD IgG only, and 2 were seronegative for both. Thirteen had nucleocapsid level less than 46 pg/mL while the other 5 exceeded 700 pg/mL, including both N-and RBD-seronegative patients and N negative/RBD positive sample (Supplementary Table 4, Supplementary Figure 4 and Supplementary Figure 5). Twenty individuals with samples categorized in the acute COVID-19 group did not have antigenemia; 10 of these were collected 10 or more days after symptom onset (Supplementary Table 5 and Supplementary Figure 6).

Antigenemia Trends by Antibody Serostatus
Distribution of nucleocapsid levels in the acute COVID-19 group were significantly different with higher median values in seronegative samples compared to seropositive samples for nucleocapsid IgG, RBD IgG, RBD IgA, and RBD IgM (P , .001 for each comparison; Figure 4A). Seropositive samples were also more likely to have undetectable antigenemia. Similar trends were seen in the late-presenting group except for the comparison based on IgM, which was not significant (Supplementary Figure 7). In addition, we compared antigenemia levels in groups defined by patterns of serostatus consistent with vaccination (RBD positive/N negative) as well as natural infection (RBD positive/N positive). Antigenemia levels were significantly lower in acutely ill patients with serological evidence of natural infection or vaccination compared to seronegative individuals ( Figure 4B).

Association of Antigenemia With COVID-19 Severity
In the acute COVID-19 group, distribution of nucleocapsid antigen was significantly different and median value was higher in samples from patients who died or required intubation within 30 days of sampling compared to those who survived or did not require intubation ( Figure 5A-C). This observation held true for comparison based on the composite of intubation or mortality. Levels of nucleocapsid antigenemia were not significantly associated with elevated D-dimer (cutoff 500 ng/mL) but were associated with elevated C-reactive protein (P = .002 in comparison based on 40 mg/L cutoff; Figure 5D and 5E).

DISCUSSION
This analysis of blood samples from routine clinical specimens collected during the ongoing COVID-19 pandemic demonstrates the following. First, antigenemia is a sensitive and specific marker for acute SARS-CoV-2 infection, as defined by timing after clinical diagnosis and symptom onset. Second, nucleocapsid is elevated in samples without evidence of antinucleocapsid (IgG) and antispike (IgG, IgM, and IgA) seroconversion. Third, antigenemia is associated with disease severity. Evolving CDC isolation guidance during the COVID-19 pandemic reflects the difficulty of objectively defining resolution of SARS-CoV-2 infection. Underlying this is the persistence of RNA targets beyond the period of acute infection in immunocompetent individuals [1][2][3]. Meanwhile, persistence of replication-competent virus for months has been demonstrated by viral culture in immunocompromised hosts [25][26][27][28][29]. This creates a diagnostic dilemma when RT-PCR is persistently positive for weeks after diagnosis, when reinfection with SARS-CoV-2 is a consideration, or when encountering positive SARS-CoV-2 RT-PCR test results in an asymptomatic individual without history of prior objective diagnosis or prior COVID-19-like illness. Our data suggest that nucleocapsid antigenemia occurs most frequently and at higher levels in the acute stages of SARS-CoV-2 infection as defined by the timing of molecular diagnosis and symptomatology in a large serosurvey representing diverse patient encounters. While prospective studies with comparison presence of viable virus are needed to more rigorously determine the clinical utility of antigenemia testing on an individualized level, our findings are consistent with a model in which antigenemia marks acute infection.
Further, our data compels interest in whether antigenemia may provide direct evidence of active viral replication, with potential to aid in evaluation of infectiousness or guide therapeutics at an individualized level. For example, antiviral agents are not likely to benefit a patient without active SARS-CoV-2 replication. Clinical trial data therefore may be confounded by failure to stratify patients according to such a marker, as late presenters after cessation of viral replication would likely fail to show benefit or may even suffer harm from investigatory antiviral agents. In fact, recent evidence emphasizes the greatest benefit of antivirals early in infection [30]. In showing its association with acute SARS-CoV-2 infection and characterizing outliers (eg, convalescent patients with persistent antigenemia), our data suggest that nucleocapsid should be further investigated as a marker of viral activity, infectiousness, and a predictor of therapeutic response. While other studies have reported similar findings, strengths and novelty of our study include a diverse cohort that is among the largest in which nucleocapsid antigenemia has been quantified to date and rigorous assignment of COVID-19 status through medical record review. Prior studies restricting the definition of a positive case to no more than 2 weeks after symptom onset report sensitivities between 90.9% and 97.5% and specificities between 94.2% and 100% [15][16][17][18][19][20] (Supplementary Table 6), and our data are consistent with these findings. Of further interest, our data revealed detectable antigen in 11 (2.1%) of the blood samples obtained in the primary serosurvey even though these patients were never screened with nasopharyngeal RT-PCR testing in our health care system. These represent likely infectious patients who may have had a missed SARS-CoV-2 diagnosis and suggest a potential role for antigenemia screening in a population for whom blood is already being sampled to complement existing infection control measures.
We also detected antigenemia in a small number of patients with subclinical SARS-CoV-2 infection. Individuals who test positive for SARS-CoV-2 without antecedent or subsequent COVID-19-like symptoms either represent shedding of replication-competent virus during subclinical disease or persistent RNA shedding following subclinical disease. While we corroborate previous findings that levels of antigenemia are associated with disease severity [19,20], the presence of antigenemia in 5 asymptomatic individuals with SARS-CoV-2 demonstrates that antigenemia can also be present in subclinical infection. Despite the difficulty associated with identifying these cases, further investigation of the prevalence of antigenemia in acute asymptomatic infection is needed to clarify its role in screening broad populations. This study is limited by use of a convenience sampling approach and retrospective data collection. Symptom onset as recorded in the medical record can be subjective and influenced by recall bias. Because of the ubiquity of community-based testing, SARS-CoV-2 diagnosis was documented prior to evaluation in our health care system for a subset of these patients and was only known to us when documented in the clinical narrative in addition to being subject to biases and imprecision. In addition, nucleocapsid-specific immunoglobulin may interfere with quantitation of antigenemia in individuals who have seroconverted although it is currently unknown whether total (Ig-bound and unbound) antigen or free (unbound only) antigen is a more meaningful clinical indicator. The primary analysis relies on the assumption that each subject is immunocompetent, that immunocompetent hosts have similar duration of acute COVID-19, and that there are no other confounding factors which may result in prolonged antigenemia. Recognizing these limitations, we performed a post hoc investigation of outlier cases, which facilitated hypothesis generation regarding reasons for prolonged antigenemia such as reduced renal function, prolonged critical illness, and immune compromise (Supplementary Tables 1-5 and 7). Several studies have demonstrated high specificity of antigenemia by evaluation of prepandemic samples [15,17,20], suggesting many false positives in our study are likely to have active infection beyond the parameters for acute infection defined in our reference standard schema. This will be further clarified as more robust comparisons to viral culture, sgRNA, RT-PCR Ct value, and respiratory antigen testing can be achieved.
Together our data demonstrate that nucleocapsid antigenemia is a sensitive and specific biomarker of acute COVID-19, wherein COVID-19 status is defined by time since earliest positive testing and symptom onset. We propose that nucleocapsid antigenemia is a candidate biomarker for active viral replication-recognizing that the available evidence points to this being an individualized process that cannot be broadly defined based on a generic timeline. Further prospective studies with rigorous documentation of clinical course and correlation with viral culture and other potential biomarkers of viral replication are needed.


A Novel C-Type Lectin Receptor-Targeted α-Synuclein-Based Parkinson Vaccine Induces Potent Immune Responses and Therapeutic Efficacy in Mice

The progressive accumulation of misfolded α-synuclein (α-syn) in the brain is widely considered to be causal for the debilitating clinical manifestations of synucleinopathies including, most notably, Parkinson’s disease (PD). Immunotherapies, both active and passive, against α-syn have been developed and are promising novel treatment strategies for such disorders. To increase the potency and specificity of PD vaccination, we created the ‘Win the Skin Immune System Trick’ (WISIT) vaccine platform designed to target skin-resident dendritic cells, inducing superior B and T cell responses. Of the six tested WISIT candidates, all elicited higher immune responses compared to conventional, aluminum adjuvanted peptide-carrier conjugate PD vaccines, in BALB/c mice. WISIT-induced antibodies displayed higher selectivity for α-syn aggregates than those induced by conventional vaccines. Additionally, antibodies induced by two selected candidates were shown to inhibit α-syn aggregation in a dose-dependent manner in vitro. To determine if α-syn fibril formation could also be inhibited in vivo, WISIT candidate type 1 (CW-type 1) was tested in an established synucleinopathy seeding model and demonstrated reduced propagation of synucleinopathy in vivo. Our studies provide proof-of-concept for the efficacy of the WISIT vaccine technology platform and support further preclinical and clinical development of this vaccine candidate.

Introduction
Synucleinopathies are neurodegenerative disorders characterized by a progressive accumulation of α-synuclein (α-syn) in the brain, including Parkinson's disease (PD), Lewy body dementia (LBD), and multiple-system atrophy (MSA). The accumulation of α-syn is widely considered to be causal to the clinical manifestations of synucleinopathies by interfering with synaptic, mitochondrial, and lysosomal function in cortical and sub-cortical brain regions [1][2][3][4]. PD is the most common synucleinopathy, affecting an estimated 1 in 500 individuals [5]. Most people develop early symptoms of the disease in their fifties. Symptom severity generally increases with age. There are currently no disease-modifying therapies available, but both passive and active immunotherapeutic approaches have shown promise for potential disease-modifying treatment of synucleinopathies [6][7][8][9].
Passive immunization with monoclonal antibodies (Abs) has previously been shown to reduce neurodegeneration and α-syn accumulation [10][11][12], decrease cell-to-cell transmis-One of the most prominent classes of polysaccharides used as alternative adjuvants is the class of C-type lectins (CLECs). Various CLECs including mannan [33] and Nacetylglucosamines [34,35], as well as the β-glucan family, have been proposed as alternative vaccine adjuvants [36][37][38]. Members of the β-glucan family such as β-(1,3)-D-glucans, Laminarin, and Curdlan, among others, constitute a class of carbohydrate pathogenassociated molecular patterns (PAMPs), with a strong adjuvant-like function. These PAMPs play a critical role in the initiation of innate and adaptive immune responses [39] and are recognized by the innate immune system, primarily by their cognate receptor dectin-1. The binding of β-glucans to dectin-1 activates phagocytosis, respiratory burst, and the secretion of cytokines, leading to the activation of antigen-presenting cells (APCs) and the migration of activated APCs to draining lymph nodes where a sustained immune response is initiated [36,37]. Glucan particles (GPs), highly purified 2-4 µm hollow porous cell wall microspheres composed of primarily β-(1,3)-D-glucans, have already been used as experimental encapsulation agents for different antigens and drugs. Recently, Rockenstein et al. showed that GPs loaded with recombinant human α-syn and rapamycin induced a robust neuroprotective humoral immune response and regulatory iTreg (CD25 and FOXP3+) cells leading to an alleviation of α-syn triggered pathologic alteration in a murine synucleinopathy model [40].
In contrast to large micro-particular encapsulation agents such as GPs, different β-glucans have also been used to generate nanoparticles consisting of protein-glucan neoglycoconjugates for dendritic cell (DC) targeting. Several studies have demonstrated that β-glucans can be used as potential adjuvants for vaccination using model antigens such as OVA [36,37], or fusion proteins based on MUC1 [41]. The immune response cascade that is triggered in response to the recognition of β-glucans, and their strong immunomodulatory characteristics, supports the potential use of β-glucans as DC-specific vaccine delivery agents and the generation of β-glucan-based neoglycoconjugate vaccines in other diseases, including PD.
Traditional vaccination approaches target antigen into the muscle or the subcutis, which are sparsely populated by immunocompetent cell types. Thus, such vaccines, including current PD vaccines, require the use of potent adjuvants. In contrast, novel approaches deliver vaccines directly into the skin, an organ rich in DC and other immune cell types. Intradermal (i.d.) vaccination has been previously shown to generate more efficient specific B-and T cell responses compared to conventional routes of application [42,43].
Alternative vaccines and/or adjuvants specifically designed for i.d. application therefore have the potential to generate a more effective immune response compared to existing vaccines. Here, we present the novel peptide-based vaccine platform WISIT (Win the Skin Immune System Trick) using different interchangeable linear α-syn peptides with a CLEC/β-glucan vaccine backbone for the development of an α-syn specific PD vaccine. WISIT constructs are unique as the backbone itself is the DC targeting/stimulating adjuvant (β-glucan), fused to variable T helper and B cell peptide epitopes. We provide proof-of-concept for the WISIT vaccine technology platform and demonstrate that candidates targeting human α-syn are superior to conventional-type conjugate vaccines in eliciting functional, protective immune responses in vitro and in vivo.

Generation of Conjugates
All peptides were synthesized by FMOC solid-phase peptide synthesis (EMC microcollections GmbH; Tübingen, Germany). Peptides used for the individual candidate vaccines are listed in Table 1. For WISIT vaccines, an activated CLEC solution was stirred with dissolved hydrazide-modified peptides in a coupling buffer (sodium acetate buffer at pH 5.4), and the carbohydrate concentration in conjugates was estimated using the anthrone method. Peptide concentration in WISIT vaccines was determined by amino acid analysis. All WISIT vaccines were applied to animals i.d., unless stated otherwise. For conventional vaccines, peptides were coupled to the carrier CRM-197 (EcoCRM™-Maleimide, Fina Biosolutions LCC, Rockville, MD, USA). Briefly, the excess peptide was added to EcoCRM™-Maleimide for coupling in a 200 mM Na-phosphate buffer (pH 6.8) and subsequently dialyzed 3× with PBS. Ellmann reagent 5,5 -dithio-bis-(2-nitrobenzoic acid; Thermo Fisher Scientific, Waltham, MA, USA) was used for quantifying free sulfhydryl groups in solution. The conventional CRM-197 conjugates were further formulated with aluminum (aluminum oxyhydroxide adjuvant 2%; InvivoGen, San Diego, CA, USA) and applied to animals s.c. Identical amounts of conjugated peptides were injected per mouse for any comparison of the CRM-197 vaccine to WISIT vaccine candidates.

Fc-Dectin-1 Binding
Fc-dectin-1 binding was analyzed by enzyme-linked immunosorbent assay (ELISA) using a soluble murine Fc-dectin-1a receptor (InvivoGen, San Diego, CA, USA) as described in Korotchenko et al. [37]. Briefly, ELISA plates were coated with a reference CLEC and conjugates were tested in a competitive ELISA to demonstrate dectin binding. Binding efficacy was calculated as IC 50 (Half maximal inhibitory concentration) values with Prism ® 9.3 (GraphPad Inc, San Diego, CA, USA) by non-linear regression analysis (four-parameter logistic fit function).

Activation Analysis Using Bone Marrow-Derived Dendritic Cells
Immature mouse DC generated in vitro with GM-CSF were stimulated with either lipopolysaccharide (LPS) using the equivalent dose contained in WISIT-conjugate preparations, WISIT-conjugates, conventional conjugates (CRM197), or oxidized CLEC-only, for 24 h. WISIT and conventional conjugates and CLEC-only preparations were used in increasing doses starting from 62.5 µg/mL to 500 µg/mL of the respective sugar. For comparison, LPS has been used as a control starting at a concentration of 2 ng/mL. DCs were identified based on CD11b/CD11c expression, and the surface expression of CD80 and major histocompatibility complex (MHC) class II were analyzed. Preparations were measured by flow cytometry. The expression of activation markers was analyzed using FCS Express (version 7.14, De Novo Software), where the expression of WISIT-and conventional conjugate preparation-treated DCs was compared to the expression of LPS-treated DC.

Animal Experiments
Immunological assessment: Female BALB/c mice (n = 5-6 mice per group; age 8-10 weeks) were immunized three times once every two weeks and blood samples were taken one day before each vaccination and two weeks after the last application unless otherwise indicated. All animal procedures were conducted at Kineto Lab Ltd. (Budapest, Hungary) in compliance with national Hungarian legislation. Experiments were performed under approval number PE/EA/448-7/2021 and were performed according to HLASA recommendations for animal use.

Induction of Synucleinopathy in Mice
All experimental procedures were conducted at Motac neuroscience, Bordeaux, in accordance with the European Communities Council Directive (2010/63/EU) for the care of laboratory animals in an AAALAC-accredited facility. For the induction of synucleinopathy, nine-week-old male C57BL/6 mice were stereotactically injected at the level of the right substantia nigra, with preformed polymorph fibrils (PFFs; i.e., preformed sonicated τpolymorph α-syn fibrils 1B). PFFs were prepared and validated as described previously [44]. Briefly, each animal received a unilateral injection of 2 µL PFFs 1B solution (concentration: 2.5 mg/mL) into the region immediately above the right substantia nigra (coordinates from bregma: −2.9 AP, ±1.3 L and −4.5 DV) at a flow rate of 0.4 µL/min [44], and the needle was left in place for 5 min before being slowly withdrawn from the brain.
Starting on the same day as the inoculation, animals received three i.d. immunizations, once every two weeks (i.e., weeks 0, 2, 4), with either CW-type 1 (n = 5) or non-conjugated CLEC (n = 10) as the control, followed by a boost immunization in week 10. Upon termination of the study (day 126), cerebrospinal fluid (CSF) was collected by cisterna magna puncture, and brains were carefully removed and fixed in paraformaldehyde (PFA; 4%). Coronal serial sections of the entire brain (from rostral cerebral cortex anterior to striatum to the medulla-i.e., bregma −6.72 mm) using a cryostat at 50 µm intervals were collected and processed for immunohistochemistry.

Vaccine Antibody Titer Determination
Standard ELISA technology was used to measure levels of vaccine-induced antibodies in plasma. Briefly, ELISA plates (Nunc Maxisorb; Thermo Fisher Scientific, Waltham, MA, USA) were coated with recombinant human α-syn protein (1 µg/mL; Anaspec; Fremont, CA, USA) using 50 mM sodium carbonate buffer overnight at 4 • C. Plates were blocked with 1% bovine serum albumin, and plasma samples were serially diluted in the plates. The detection of target-specific antibodies was performed with biotinylated anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) and a subsequent color reaction using Streptavidin-POD (Roche, Basel, Switzerland) and TMB. Titers were calculated as EC 50 values using Prism ® 9.3 following non-linear regression analysis (four-parameter logistic fit function).

Inhibition and Avidity ELISA
Inhibition ELISA was performed as previously described [45]. Briefly, titrated amounts of α-syn monomer and α-syn fibrils (both from Abcam, Cambridge, UK), pre-incubated with vaccine-candidates-induced Abs, were added to ELISA microtiter plates densely coated with α-syn monomers (Abcam, Cambridge, UK). The detection of Abs bound to Vaccines 2022, 10, 1432 6 of 20 the immobilized antigen was analyzed and IC 50 values were calculated by Prism ® 9.3 following non-linear regression analysis (four-parameter logistic fit function).
Antibody avidity to α-syn filaments was determined in the presence of the chaotropic agent sodiumthiocyanate (NaSCN) by an adapted ELISA. Briefly, ELISA plates (Nunc Maxisorb; Thermo Fisher Scientific, Waltham, MA, USA) were coated with α-syn filaments (Abcam, Cambridge, UK) using 50 mM sodium carbonate buffer, overnight at room temperature (RT). Plates were blocked with 1% bovine serum albumin, and constant amounts of plasma samples were added and incubated for 1 h. Following plate washing, 100 µL of a chaotropic agent was added at increasing concentrations (from 0 to 3 M) for 20 min at RT. Plates were washed again, and residual antibody binding was determined. Absorbance values in the absence of NaSCN were taken as the total effective binding of the specific antibody (100% binding), and subsequent absorbance values in the presence of increasing concentrations of NaSCN were converted to the corresponding percentage of the total bound antibody. The data were fitted to a plot of (% binding) vs. (log) concentration of NaSCN, and linear regression analysis was used to estimate the avidity index. The avidity index was defined as the concentration necessary to decrease the initial absorbance by 50%.

Thioflavin T Assay for Aggregation
Monomeric α-syn samples (recombinant human α-syn protein monomer (active); Abcam, Cambridge, UK) were incubated with shaking at 1000 rpm at 37 • C in nonbinding polystyrene 96-well plates (Greiner Bio ONE, Frickenhausen, Germany). Next, sonicated preformed α-syn fibrils (sPFF; recombinant human α-syn protein aggregate (active); Abcam, Cambridge, UK) alone or together with vaccine-candidate-induced Abs, the control mouse IgG1 antibody, or untreated murine plasma were added. Titrating amounts ranging from 0 to 5 µg/mL of vaccine-candidate-induced Abs, the control mouse IgG1 antibody (Southern Biotech, Birmingham, AL, USA), or untreated murine plasma (in equivalent amount) were used. All results are mean values of at least triplicate samples.

Statistical Analysis
All statistical analyses were conducted using Prism ® 9.3. Data are presented as mean ± standard error of the mean (SEM) and were analyzed with one-way analysis of variance (ANOVA) with post-hoc Tukey's multiple comparison test unless indicated otherwise. Linear regression analysis was applied to calculate different parameters. Pearson correlation analysis was used to assess the strength and direction of the linear relationships between pairs of variables. A value of p < 0.05 was considered statistically significant.

WISIT Vaccine Design
The WISIT platform technology is specifically designed to target skin-derived DCs. The skin plays a central role in triggering immune responses and defense against infections. The major advantage of targeting skin is that its reactory nature means that no additional adjuvants are required for vaccine application. Classical conjugate vaccines normally require a target-specific B cell epitope and a carrier protein providing foreign T cell epitopes in order to induce an efficient immune response. To ensure sufficient immunogenicity, adjuvants are added to classical conjugate vaccines, which are then administered i.m. or s.c. Although these tissues can mediate immune responses, they are not specialized in doing so. WISIT vaccines include a sugar residue (CLEC/β-glucan), which allows for the direct targeting of DC without the need for a standard adjuvant ( Figure 1). ditional adjuvants are required for vaccine application. Classical conjugate vaccines normally require a target-specific B cell epitope and a carrier protein providing foreign T cell epitopes in order to induce an efficient immune response. To ensure sufficient immunogenicity, adjuvants are added to classical conjugate vaccines, which are then administered i.m. or s.c. Although these tissues can mediate immune responses, they are not specialized in doing so. WISIT vaccines include a sugar residue (CLEC/β-glucan), which allows for the direct targeting of DC without the need for a standard adjuvant ( Figure 1).

Figure 1.
Functional building blocks of the α-syn WISIT vaccine. α-syn WISIT vaccines are neoglycoconjugates. The conjugate consists of three functional elements. Linear, pan-HLA T-cell epitopes (polypeptides), which have already been previously successfully used in humans (e.g., PADRE) provide T cell help. α-syn specific B-cell epitopes define the specificity of the antibody response induced. B-cell epitopes are 7-12-mer peptides (see Table 1). Both polypeptides are covalently coupled to the CLEC. Functionally, the unit of the CLEC and the T cell epitope is the vaccine's backbone.

Production of WISIT Vaccine Candidates for α-Syn
To generate the reactive sugar backbone of the WISIT vaccines ( Figure 1) for α-syn, CLECs/β-glucans were first activated by mild periodate oxidation. WISIT PD vaccine candidates CW-type 1-6 were produced by hydrazide-based coupling of six different B-cell epitope peptides (7 to 12-mers) derived from N-and C-terminal α-syn domains and the linear non-natural pan DR epitope (PADRE) as a T-helper epitope peptide, including a Cterminal hydrazide linker for coupling. The same B cell epitope peptides were coupled to CRM197 by maleimide-thiol coupling to provide conventional peptide-carrier conjugates used as benchmark controls (CC-type 1-6), respectively (Table 1).

WISIT Vaccine Candidates Targeting α-Syn Are Biologically Active in DCs
To determine the biological activity of vaccine candidates, two assays were used. First, we analyzed binding to the cognate β-glucan pattern recognition receptor (PRR) dectin-1. Non-peptide coupled, activated CLECs as well as CW-type 1 and conventional CC-type 1 vaccines were assessed for binding to dectin-1, using a competitive ELISA. The comparison of CW-type 1 and conventional CC-type 1 vaccines showed that conventional peptide-protein conjugate vaccines do not interact with the β-glucan receptor ( Figure 2A). Activated CLEC/β-glucan displayed high dectin-1 binding capacity that was slightly reduced after peptide coupling (CW-type 1). Similarly, four additional CW-type candidate vaccines were assessed. All additional CW-type candidates retained high and comparable dectin-1 binding, albeit slightly reduced compared to non-coupled β-glucan moieties (Figure 2B).
To demonstrate that WISIT vaccines targeting α-syn are not only binding to PRRs, but also exert biological function in their target cells, in vitro DC activation was measured. Figure 1. Functional building blocks of the α-syn WISIT vaccine. α-syn WISIT vaccines are neoglycoconjugates. The conjugate consists of three functional elements. Linear, pan-HLA T-cell epitopes (polypeptides), which have already been previously successfully used in humans (e.g., PADRE) provide T cell help. α-syn specific B-cell epitopes define the specificity of the antibody response induced. B-cell epitopes are 7-12-mer peptides (see Table 1). Both polypeptides are covalently coupled to the CLEC. Functionally, the unit of the CLEC and the T cell epitope is the vaccine's backbone.

Production of WISIT Vaccine Candidates for α-Syn
To generate the reactive sugar backbone of the WISIT vaccines ( Figure 1) for α-syn, CLECs/β-glucans were first activated by mild periodate oxidation. WISIT PD vaccine candidates CW-type 1-6 were produced by hydrazide-based coupling of six different B-cell epitope peptides (7 to 12-mers) derived from Nand C-terminal α-syn domains and the linear non-natural pan DR epitope (PADRE) as a T-helper epitope peptide, including a C-terminal hydrazide linker for coupling. The same B cell epitope peptides were coupled to CRM197 by maleimide-thiol coupling to provide conventional peptide-carrier conjugates used as benchmark controls (CC-type 1-6), respectively (Table 1).

WISIT Vaccine Candidates Targeting α-Syn Are Biologically Active in DCs
To determine the biological activity of vaccine candidates, two assays were used. First, we analyzed binding to the cognate β-glucan pattern recognition receptor (PRR) dectin-1. Non-peptide coupled, activated CLECs as well as CW-type 1 and conventional CC-type 1 vaccines were assessed for binding to dectin-1, using a competitive ELISA. The comparison of CW-type 1 and conventional CC-type 1 vaccines showed that conventional peptideprotein conjugate vaccines do not interact with the β-glucan receptor (Figure 2A). Activated CLEC/β-glucan displayed high dectin-1 binding capacity that was slightly reduced after peptide coupling (CW-type 1). Similarly, four additional CW-type candidate vaccines were assessed. All additional CW-type candidates retained high and comparable dectin-1 binding, albeit slightly reduced compared to non-coupled β-glucan moieties ( Figure 2B).
To demonstrate that WISIT vaccines targeting α-syn are not only binding to PRRs, but also exert biological function in their target cells, in vitro DC activation was measured. Murine GM-CSF generated DCs were stimulated in vitro with CW-type 1 as well as with a conventional CC-type 1 protein conjugate vaccine as a benchmark. Flow cytometry-based analysis of DC activation markers revealed significantly increased expression levels of CD80 and MHCII, indicative of DC maturation and activation ( Figure 2C,D). The expression levels achieved were significantly higher than the effects observed by equivalent doses of LPS, contained within the conjugate preparations ( Figure 2C,D; p < 0.001 and p < 0.01). The comparison to benchmark vaccines also revealed a significant increase in expression for both markers following CW-type 1 exposure ( Figure 2C,D, p < 0.001 and p < 0.01). No significant increase in expression for MHCII or CD80 was observed for DCs stimulated with the CC-type 1 benchmark-type vaccine ( Figure 2C,D). pression levels achieved were significantly higher than the effects observed by equivalent doses of LPS, contained within the conjugate preparations ( Figure 2C,D; p < 0.001 and p < 0.01). The comparison to benchmark vaccines also revealed a significant increase in expression for both markers following CW-type 1 exposure ( Figure 2C,D, p < 0.001 and p < 0.01). No significant increase in expression for MHCII or CD80 was observed for DCs stimulated with the CC-type 1 benchmark-type vaccine ( Figure 2C,D). to that detected in WISIT conjugate preparations), the WISIT-conjugate vaccine CW-type 1, or the respective conventional peptide-protein conjugate CCtype 1 by flow cytometry (identification of DCs was performed based on CD11c expression) ** p < 0.01; *** p < 0.001 (exposure with CW-type 1 vs. LPS). Error bars indicate the mean ± SEM of one representative experiment performed in triplicates. Statistical differences were evaluated by oneway ANOVA and Tukey's multiple comparison test. ** p < 0.01; *** p < 0.001 (exposure with CWtype 1 vs. LPS and CW-type 1 vs. CC-type 1). ns: No significant difference after exposure to conventional vaccine CC-type 1 vs. LPS. (E) Representative histogram overlays of DC CD80 (top) and MHC II (bottom) reactivity in response to vaccines as indicated in the legend insert.

WISIT PD Vaccines Elicit Higher Immune Responses Compared to Conventional PD Vaccines
Next, we analyzed the induction of anti-α-syn protein-specific antibodies following three biweekly immunizations of BALB/c mice with WISIT and conventional PD-vaccines by plasma ELISA. WISIT vaccines were specifically designed to target DC in the skin. Consequently, a comparative analysis of the antibody response elicited by immunization

WISIT PD Vaccines Elicit Higher Immune Responses Compared to Conventional PD Vaccines
Next, we analyzed the induction of anti-α-syn protein-specific antibodies following three biweekly immunizations of BALB/c mice with WISIT and conventional PD-vaccines by plasma ELISA. WISIT vaccines were specifically designed to target DC in the skin. Consequently, a comparative analysis of the antibody response elicited by immunization using different immunization routes showed i.d. application to be the most effective route at generating a substantial antibody response for WISIT vaccine candidates (Appendix A Figure A1B). Next, i.d. immunization of WISIT vaccine candidates (CW-type 1-6) was compared to immune responses induced by the respective conventional benchmark vaccines (CC-type 1-6; applied s.c., aluminum adjuvanted). Plasma ELISA analysis showed that vaccination with CW-type vaccines resulted in the induction of significantly higher titers towards the α-syn protein compared to conventional CC-type vaccines (Figure 3 and Appendix A Figure A1A). cines (CC-type 1-6; applied s.c., aluminum adjuvanted). Plasma ELI that vaccination with CW-type vaccines resulted in the induction of titers towards the α-syn protein compared to conventional CC-type v Appendix A Figure A1A). The data show that the C-terminal α-syn region aa 110-130 wa genic region tested in the study. Other domains were slightly less imm they still induced an immune response with similar characteristics A1A). Hence, we focused on CW-type candidates 1-5 for further stud

Antibodies Elicited by WISIT PD Vaccines Show Greater Binding and Aggregates Than Conventional PD Vaccines
Besides the total amount of antibodies elicited by a vaccine, the s antigen complexes or resilience against disruptive factors, as well as t erated antibodies for aggregated α-syn, are important parameters fo tential effect of a PD vaccine.
To estimate the stability of the antigen-antibody complexes, w sistance against the chaotropic agent NaSCN [46,47]. This method is sorbent assays where the preformed antibody-antigen complex is tr high molar concentrations of a chaotropic agent. The resistance to thio used as a measure of avidity, and an index (i.e., the avidity index), effective antibody binding, was used to compare different sera. The re ysis for CW1 relative to CC1-induced antibodies showed approximat avidity for α-syn filaments ( Figure 4A). We found that WISIT PD vac type 1-5 induced antibody-α-syn complexes were significantly mo Titer (x10 3 ) Figure 3. Target-specific immune response induced by WISIT vaccines. Titers of antibodies specific to recombinant full-length α-syn were detected in plasma collected after the third immunization with indicated immunogens by ELISA. WISIT-vaccines (CW type 1-5) induced significantly higher responses than conventional vaccines (CC type 1-5) adjuvanted with alum. Error bars indicate the mean values of antibody titers ± SEM of n = 10 animals per group. Statistical differences were evaluated by one-way ANOVA and Tukey's multiple comparison test (* p < 0.05; ** p < 0.01).
The data show that the C-terminal α-syn region aa 110-130 was the most immunogenic region tested in the study. Other domains were slightly less immunogenic; however, they still induced an immune response with similar characteristics (Appendix A Figure A1A). Hence, we focused on CW-type candidates 1-5 for further studies.

Antibodies Elicited by WISIT PD Vaccines Show Greater Binding and Selectivity to α-Syn Aggregates Than Conventional PD Vaccines
Besides the total amount of antibodies elicited by a vaccine, the stability of antibodyantigen complexes or resilience against disruptive factors, as well as the selectivity of generated antibodies for aggregated α-syn, are important parameters for evaluating the potential effect of a PD vaccine.
To estimate the stability of the antigen-antibody complexes, we measured their resistance against the chaotropic agent NaSCN [46,47]. This method is based on immunosorbent assays where the preformed antibody-antigen complex is transiently exposed to high molar concentrations of a chaotropic agent. The resistance to thiocyanate elution was used as a measure of avidity, and an index (i.e., the avidity index), representing 50% of effective antibody binding, was used to compare different sera. The residual binding analysis for CW1 relative to CC1-induced antibodies showed approximately four times higher avidity for α-syn filaments ( Figure 4A). We found that WISIT PD vaccine candidates CW-type 1-5 induced antibody-α-syn complexes were significantly more stable than those formed by CC-type 1-5-induced antibodies ( Figure 4B and Table 2).  We then checked if the increased avidity associated with WISIT PD vaccine candidates is also accompanied with higher selectivity towards aggregated α-syn ( Figure  4C,D). Indeed, a higher selectivity to α-syn filaments for CW-type 1-5 vaccine-induced antibodies was confirmed by inhibition ELISA, with average IC50 values towards the αsyn filament of 8 nM ± 1.5 for CW-type vaccine candidate-induced antibodies and 198 nM ± 6.5 for CC-type vaccine-induced antibodies (see Appendix A Figure A2). Interestingly, all CW-type vaccine candidates tested induced antibodies with highly increased selectivity towards α-syn filaments compared to α-syn monomers (between 8-18-fold higher selectivity towards α-syn filaments). In contrast, none of the conventional benchmark vaccines tested induced antibodies with more than 2-fold higher selectivity towards α-syn filaments. Thus, the relative improvement in selectivity towards α-syn filaments by antibodies induced by CW-type vaccines as compared to benchmark vaccines was between 5and 12-fold. Given these differences in antibody quality, we started to characterize the immune responses of the two vaccine types with respect to the affinity maturation of the antibody response. After the first two vaccinations, the immune response was still comparable in terms of affinity. However, additional immunization revealed that the antibody response of the classical CC-type conjugate vaccines did not show a further increase in affinity,  We then checked if the increased avidity associated with WISIT PD vaccine candidates is also accompanied with higher selectivity towards aggregated α-syn ( Figure 4C,D). Indeed, a higher selectivity to α-syn filaments for CW-type 1-5 vaccine-induced antibodies was confirmed by inhibition ELISA, with average IC 50 values towards the α-syn filament of 8 nM ± 1.5 for CW-type vaccine candidate-induced antibodies and 198 nM ± 6.5 for CCtype vaccine-induced antibodies (see Appendix A Figure A2). Interestingly, all CW-type vaccine candidates tested induced antibodies with highly increased selectivity towards αsyn filaments compared to α-syn monomers (between 8-18-fold higher selectivity towards α-syn filaments). In contrast, none of the conventional benchmark vaccines tested induced antibodies with more than 2-fold higher selectivity towards α-syn filaments. Thus, the relative improvement in selectivity towards α-syn filaments by antibodies induced by CW-type vaccines as compared to benchmark vaccines was between 5-and 12-fold.
Given these differences in antibody quality, we started to characterize the immune responses of the two vaccine types with respect to the affinity maturation of the antibody response. After the first two vaccinations, the immune response was still comparable in terms of affinity. However, additional immunization revealed that the antibody response of the classical CC-type conjugate vaccines did not show a further increase in affinity, whereas the affinity of the CW-type-induced antibodies doubled with the third vaccination (Appendix A Figure A3).

WISIT Vaccine Candidates Inhibit α-Syn Aggregation In Vitro
For functional characterization of the immune response induced by α-syn WISIT vaccine candidates, we assessed the capacity of WISIT-induced antibodies to inhibit α-syn aggregation using an in vitro α-syn aggregation and propagation assay [48][49][50]. Pre-formed α-syn fibrils (sPFF, Abcam) were mixed with α-syn monomers in the presence of titrated amounts of vaccine-induced or control antibodies ( Figure 5). No effect on α-syn aggregation was observed using control IgG or mouse plasma only. In contrast, normalized amounts of CW-type vaccine-and conventional CC-type vaccine-induced antibodies could significantly inhibit or slow down aggregation ( Figure 5A).
Vaccines 2022, 10, x FOR PEER REVIEW 11 of 20 whereas the affinity of the CW-type-induced antibodies doubled with the third vaccination (Appendix A Figure A3).

WISIT Vaccine Candidates Inhibit α-Syn Aggregation In Vitro
For functional characterization of the immune response induced by α-syn WISIT vaccine candidates, we assessed the capacity of WISIT-induced antibodies to inhibit α-syn aggregation using an in vitro α-syn aggregation and propagation assay [48][49][50]. Preformed α-syn fibrils (sPFF, Abcam) were mixed with α-syn monomers in the presence of titrated amounts of vaccine-induced or control antibodies ( Figure 5). No effect on α-syn aggregation was observed using control IgG or mouse plasma only. In contrast, normalized amounts of CW-type vaccine-and conventional CC-type vaccine-induced antibodies could significantly inhibit or slow down aggregation ( Figure 5A).  CW-type 1 and CW-type 3-induced antibodies were found to inhibit further aggregation in a dose-dependent way during the 72 h testing period. In contrast, antibodies induced by conventional CC-type vaccines showed significantly weaker effects on aggregation inhibition. CW-type 1 induced highly aggregate selective antibodies, which in vitro induced a 94% reduction in aggregate formation compared to 58% induced by antibodies generated by the benchmark CC-type 1 vaccine ( Figure 5, p < 0.001; kinetic of Thioflavin T (ThT) aggregation is shown in Appendix A Figure A4). Similarly, the CW-type 3 vaccine induced fewer aggregate selective antibodies, which showed a less pronounced in vitro inhibition effect as compared to CW-type 1.
As shown in Figure 5D, the selectivity of the vaccine-induced antibodies towards aggregated α-syn strongly correlated with their capacity to inhibit α-syn aggregation in vitro (Pearson r = 0.9838; CI (95%) 0.8547 to 0.9983, p = 0.0004, and R 2 = 0.9679). In contrast, CC-type vaccine-induced antibodies showed only a weak positive correlation ( Figure 5E, Pearson r = 0.6266, CI (95%) −0.3764 to 0.9534, p = 0.1831 and R 2 = 0.3926). This analysis further supports that CC-type and WISIT CW-type vaccines have different immunological properties and induce antibody types with distinct binding characteristics towards α-syn aggregates.

WISIT Vaccine CW-Type 1 Inhibits Propagation of Synucleinopathy In Vivo
To determine if WISIT vaccine candidates were able to inhibit α-syn fibril formation in vivo, a proof-of-concept experiment for WISIT vaccine CW-type 1 was initiated using an established seeding model for synucleinopathies [44]. In this model, C57BL/6 mice are stereotactically injected with α-syn pre-formed fibrils (PFFs) at the level of the right substantia nigra, subsequently causing widespread synucleinopathy, characterized specifically by phosphosynuclein immunopositive Lewy-like neurites and intracytoplasmic aggregates along anatomical connections. Animals were immunized four times at weeks 0, 2, 4, and 10 with CW-type 1, or non-conjugated CLEC as the control, starting the first immunization on the day of PFF inoculation. Then, 126 days post-PFF-injection, animals were sacrificed, and brains were analyzed for the presence of phosphor-S129 α-syn-positive aggregates in selected brain areas including the cerebral cortex, striatum, thalamus, substantia nigra, and brainstem. Analysis of the ensuing immune response was performed using plasma and CSF obtained at the time of sacrifice. High-antibody titers against the injected peptide were detected in the plasma of CW-type 1-treated animals. In contrast, no signal above the background could be detected in the CLEC-only-treated group ( Figure 6A). Analysis of the anti-peptide titer in CSF also showed a high level of CW-type 1-induced antibodies, whereas no signal above background was detectable for the vehicle-treated animals ( Figure 6B). Immunohistochemistry of brain sections showed high numbers of phosphor-S129 α-syn-positive aggregates throughout all analyzed areas in the vehicle-treated group indicating a strong propagation of α-syn pathology. In contrast, synucleinopathy was significantly reduced in CW-type 1-vaccinated mice ( Figure 6C,E,F). Of note, there was a strong and significant reciprocal correlation between the strength of the antibody response and the level of synucleinopathy in CW-type 1 recipients ( Figure 6D). Figure 6. CW-type-induced antibodies inhibit α-syn aggregation in a PFF model in vivo. C57BL/6 mice stereotactically injected in the right substantia nigra with recombinant α-syn PFFs were immunized four times using CW-type 1 vaccine or unconjugated CLEC as control starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brains, plasma, and CSF were harvested 126 days post-PFF inoculation. Plasma titer of antibodies specific to the peptide used for immunization (A) collected two weeks after the third immunization on day 126. (B) Comparison of CSF and plasma titers of antibodies specific for the B cell peptide of the vaccine at day 126. (C) Analysis of phosphor-S129 α-syn-positive aggregates over all brain areas in CW-type 1 vaccinated and CLEC-treated mice. (D) Correlation between antibody response and the level of synucleinopathy in CW-type 1 recipients (r = −0.9391; CI (95%) −0.9961 to −0.3318, p = 0.0179, and R 2 = 0.882). (E-H) Representative pSer129 α-syn staining in the injected brain hemisphere at the level of (E,F) the substantia nigra and (G,H) the striatum. (E,G) Vehicle-treated mice and (F,H) CW-type 1 treated Figure 6. CW-type-induced antibodies inhibit α-syn aggregation in a PFF model in vivo. C57BL/6 mice stereotactically injected in the right substantia nigra with recombinant α-syn PFFs were immunized four times using CW-type 1 vaccine or unconjugated CLEC as control starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brains, plasma, and CSF were harvested 126 days post-PFF inoculation. Plasma titer of antibodies specific to the peptide used for immunization (A) collected two weeks after the third immunization on day 126. (B) Comparison of CSF and plasma titers of antibodies specific for the B cell peptide of the vaccine at day 126. (C) Analysis of phosphor-S129 α-syn-positive aggregates over all brain areas in CW-type 1 vaccinated and CLEC-treated mice. (D) Correlation between antibody response and the level of synucleinopathy in CW-type 1 recipients (r = −0.9391; CI (95%) −0.9961 to −0.3318, p = 0.0179, and R 2 = 0.882). (E-H) Representative pSer129 α-syn staining in the injected brain hemisphere at the level of (E,F) the substantia nigra and (G,H) the striatum. (E,G) Vehicle-treated mice and (F,H) CW-type 1 treated mice following PFF injection. Error bars indicate the mean ± SEM of n = 5-9 animals per group. Statistical differences were evaluated by an unpaired t-test; ** p < 0.01; * p < 0.05.

Discussion
Immunotherapy targeting α-syn is the most promising approach to slow PD disease progression to date. Specific antibodies eliminating α-syn aggregates can be either passively introduced or induced by active vaccination. Several first-generation α-synimmunotherapeutics are in early clinical development. However, substantial improvements in the core technology are needed to achieve lower toxicity, higher efficacy, and more economical production to enable scalability.
The α-syn WISIT vaccine platform is designed to effectively leverage skin DCs by i.d. vaccination to generate substantially stronger and more specific immune responses compared to conventional α-syn conjugate vaccines. The skin represents a rich source of potent DCs capable of mounting strong immune responses, which are not utilized by available PD vaccines. WISIT constructs are unique as the vaccine backbone itself comprises a DC-targeting/stimulating agent (β-glucan), fused to variable T-helper and α-syn B-cell peptide epitopes, making WISIT vaccines cheaper to produce and more flexible than existing vaccines by design. The WISIT constructs used in this study consist of a β-glucan conjugated to a promiscuous Th cell epitope, the pan DR epitope PADRE. They differ at the level of the α-syn-specific variable B cell epitopes, which define the specificity of the ensuing antibody response. Specifically, β-1,3-glucans interact with dectin-1 receptors on DC to stimulate DC migration and maturation [51]. By using β-glucan as the constant backbone whilst flexibly modifying the Th-or α-syn B cell epitopes, WISIT is constructed as a "Lego"-style vaccine platform with interchangeable units that are capable of leveraging skin immunity to improve the vaccine's immunogenicity.
Here, we show that PD WISIT vaccine candidates are superior to conventional conjugate vaccines in inducing antibodies that interfere with α-syn aggregation and propagation in vitro. Preliminary in vivo studies demonstrate the WISIT vaccine CW-type 1 to prevent the propagation of α-syn pathology in an in vivo seeding model for synucleinopathies. The efficacy of WISIT vaccines was found to rely on two main immunological features. First, they are more immunogenic than conventional conjugate vaccines and induce higher antibody titers specific to the target α-syn. Second, antibodies elicited by WISIT vaccines are characterized by a dramatically improved selectivity/avidity for α-syn aggregates, the pathological α-syn moiety in PD. The functional relevance of the high antibody selectivity is supported by our finding of a strong and direct correlation between the antibody selectivity for α-syn filaments and their ability to inhibit α-syn aggregation in vitro. These findings are further in line with the results of a recent study by Höllerhage et al. demonstrating that the protective efficacy of anti-α-syn antibodies is a function of the stability of the antibody/αsyn complexes formed [52]. Using a neuronal co-culture model, in which α-syn species are released from α-syn-overexpressing cells and induce toxicity in a priori healthy cells, they found other characteristics of the antibodies, such as their α-syn epitope-binding domain and the degree of specificity of the antibodies, to be irrelevant for the therapeutic efficacy of a given α-syn antibody.
The demonstration that WISIT vaccines bind to dectin-1 and can activate DCs supports the hypothesis that they act by targeting skin-resident DCs. This is further corroborated by our finding that their administration to the dermal compartment is more efficacious than application to subcutaneous or muscle tissue in inducing target-specific antibody responses. The view that WISIT constructs act via DCs resident to the dermal compartment is in line with previous reports providing direct evidence for DC targeting via fluorescence labeling of constructs [37,53].
DC targeting is a well-known strategy for increasing the magnitude and quality of the antibody response [54]. Indeed, the fundamental difference between available peptide conjugate vaccines and skin-DC-targeted WISIT-type vaccines with respect to their antibody response, is the higher affinity for α-syn and higher selectivity for aggregated forms of α-syn induced by the WISIT vaccines. While affinity maturation upon administration of conventional vaccines is blocked after the second vaccination, affinity doubled upon the third WISIT application. Initial dose titration studies additionally document the difference between the vaccine types as there is no plateau with WISIT, and more extensive studies with a wider dose range are currently ongoing.
Six different peptides (7-12 aa) covering different α-syn domains (N-and C-terminal) were tested in the current study using the WISIT vaccine platform. Superiority over stateof-the-art conjugate vaccines was proven to be independent of the epitope-length used for immunization. Thus, peptides that are too short to contain potential MHC-I or MHC-II epitopes can be effectively used, excluding the danger of inducing α-syn specific T-cells in vivo.

Conclusions
The data presented in this study demonstrate the α-syn WISIT vaccine technology to be superior to conventional conjugate vaccines regarding both the titer of antibodies induced to an α-syn B cell epitope and also the avidity/selectivity of the antibodies elicited. In the case of our α-syn targeting vaccines, this translates into a functional benefit since induced antibodies inhibit α-syn aggregation and propagation of pathology more efficiently than antibodies triggered by their conventional counterparts. It will be interesting to explore whether the improved immunological and functional characteristics of WISIT vaccines will translate to humans. Given the fact that both backbone elements, PADRE and β-glucans, demonstrated activity in humans [55][56][57][58], WISIT technology may deliver novel α-syn-based vaccine candidates capable of modifying the course of the disease in PD patients.