s2fieldsofstudy: Biology Medicine Psychology

text:
Genetic silencing of striatal CaV1.3 prevents and ameliorates levodopa dyskinesia

Abstract Background Levodopa‐induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well‐tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa‐induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa‐induced dyskinesias. Methods To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high‐potency, target‐selective, mRNA‐level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa‐induced dyskinesias in severely parkinsonian rats. Results We demonstrate that vector‐mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa‐induced dyskinesias, and this antidyskinetic benefit persists long term and with high‐dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa‐induced dyskinesias. Importantly, motoric responses to low‐dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability. Discussion The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa‐induced dyskinesia side effect. © 2019 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

approximately 95% of striatal neurons, express both CaV1.3 and CaV1.2 voltage-gated L-type calcium channels. In PD and animal models of PD, striatal DA depletion results in loss of dendritic spines on MSNs, [3][4][5][6][7][8][9][10] an aberrant feature accompanied by secondary loss of glutamate synapses from corticostriatal projections. 3,[5][6][7]10,11 Introduction of levodopa in this environment results in restoration of dendritic spines 12 and reestablishment of glutamate input, but in an aberrant pattern of apparent "miswiring." 5,8,10,13 Retraction of dendritic spines on MSNs associated with loss of striatal DA has been linked to dysregulation of intraspinous CaV1.3 channels. 3 Blockade of these channels with CaV1.2/1.3 channel antagonists (ie, nimodipine, isradipine) prevents spine retraction despite severe loss of DA in the parkinsonian striatum. 9,14 As such, it has been hypothesized that preventing initial spine loss and associated synaptopathology in PD and models of PD may prevent abnormal rewiring of glutamate inputs and diminish liability for LID despite loss of DA. Indeed, we 9 and others 14 have shown that CaV1.2/1.3 channel antagonists can prevent induction of LID produced by low-dose levodopa (6 mg/kg 14 ) and high-dose levodopa (12.5 mg/kg 9 ); however, this effect is partial and lost over time, and this paradigm is incapable of reversing established LID. 14 The transient nature of the antidyskinetic effect of currently available CaV1. 3 antagonists is speculated to be because of pharmacologic limitations of these drugs, including lack of specificity and potency for CaV1.3 channels. 15,16 To provide the first unequivocal proof-of-principle evidence, devoid of pharmacological limitations, on the ability of CaV1.3 silencing to provide meaningful and lasting functional protection against LID, we developed a recombinant adeno-associated virus (rAAV)-mediated short hairpin RNA (shRNA) to provide continuous, high-potency, and target-selective mRNA-level silencing of striatal CaV1.3 channels. We present here functional data demonstrating that delivery of rAAV-CaV1.3-shRNA to the DA-depleted striatum of unilaterally parkinsonian rats prior to the introduction of levodopa provides robust and lasting prevention of LIDs and, to a lesser extent, also can reverse LID in parkinsonian rats with established severe LID behavior.

Methods Experimental Subjects
All procedures were performed on adult male Sprague-Dawley (SD) rats (250 g; Envigo RMS Inc., Indianapolis, IN) in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines and following approval of the Michigan State University Institutional Animal Care and Use Committee.

Vector Design and Production
Portions of the CaV1.3 gene (Cacna1d) were cloned from rat cDNA and sequenced. shRNA sequences (CaV1.3 [5 0 -GA AGAGGCGCGGCCAAGAC-3 0 ] or scrambled [5 0 -CAACA AGATGAAGAGCACC-3 0 ]) were designed using standard algorithms as described previously 17 and compared against available rat genome data to ensure specificity. Particular attention was paid to ensure that the shRNA contained negligible overlap with CaV1.2. Then shRNA was cloned into a rAAV genome under control of the H1 promoter. The same genome contained green fluorescent protein (GFP) under control of the hybrid chicken b-actin/cytomegalovirus promoter as a transduction marker. 17 The rAAV was packaged as described previously 18 into AAV9 capsids using triple transfection in HEK293T cells. Viral capsids were purified using an iodixanol step gradient and concentrated using buffer exchange. Virus titers were determined using dot-blot and normalized to 1 × 10 13 vector genomes (vg)/mL. 19

Dyskinesia Rating
LIDs refer here to abnormal involuntary behaviors including dystonia, hyperkinesia, and/or stereotypies noted in the presence of levodopa in parkinsonian rats. Injections were administered to allow assessment of LID behaviors for 1 minute every 30 minutes beginning 20 minutes after injection and continued up to 170 or 200 minutes. Rats were randomized and rated by the same blinded investigator throughout the experiment. Each rat was given a total LID severity score for each point assessed based on a rating system developed with a clinical movement disorders specialist (R.K. 20 ) as previously detailed. 20,21

LID Prevention Studies
All stereotaxic surgeries (prevention and reversibility studies) were performed as previously detailed. 22 To test the hypothesis that shRNA-mediated silencing of striatal CaV1.3 channels prior to levodopa would prevent the development and provide sustained amelioration of LIDs, rats received unilateral injections of either rAAV-CaV1.3-shRNA or the control rAAV-Scrambled (Scr)-shRNA (1.0 × 10 13 vg/mL) into 2 dorsolateral sites within the left striatum (AP0.0, ML+3.0, DV-5.2; and AP+1.6, ML+2.7, DV-4.9). Vector surgery preceded by 1 week 6-hydroxydopamine (6-OHDA) neurotoxin surgery used to induce unilateral parkinsonism (Fig. 1A). Parkinsonism was induced using stereotaxic injection of 6-OHDA into substantia nigra (SN) and medial forebrain bundle per our usual protocol. 10,23 Vector delivery prior to 6-OHDA was used to minimize spine loss associated with CaV1.3 dysregulation, which occurs secondary to striatal DA depletion. However, because AAV is retrogradely transported to nigral DA neurons in which CaV1.3 silencing could interfere with 6-OHDA-induced cell death, 24 timing for these proofof-principle experiments was systematically worked out in pilot feasibility studies. Three weeks following 6-OHDA (4 weeks postvector surgery), rats began receiving daily levodopa injections given at escalating doses ranging from low (6 mg/kg) to moderate (9 mg/kg) to high (12 mg/kg) to what is referred to here as "extreme" (18 mg/kg); see Figure 1A. Each dose was given daily (Monday through Friday) for 2 weeks with a constant dose of carbidopa (12 mg/kg), a peripheral decarboxylase inhibitor. LID behaviors were rated on days 1, 6, and 10 of each dose.

LID Reversibility Studies
To test the hypothesis that in rats with established LID behaviors, ameliorating dysfunctional calcium signaling in an environment of established "miswiring" would lessen LIDs, rats were first rendered unilaterally parkinsonian per referenes 10 and 23. As depicted in Figure 2A, 3 weeks after 6-OHDA rats began receiving daily high-dose levodopa (12 mg/kg, Monday through Friday; levodopa:carbidopa 1:1). After 3 weeks of treatment, all rats exhibiting stable, high levels of LIDs were assigned to either a rAAV-CaV1.3-shRNA or control rAAV-Scr-shRNA group. Groups were assigned to ensure the average peak-dose LID severity between groups was not different. Vector surgeries were identical to those for the LID prevention study.

Cylinder Motor Test
A modified cylinder task was used to examine motor response in the absence of drug (prelevodopa [pre-LD]) and in response to low-dose levodopa (6 mg/kg; 12 mg/kg benserazide) in all rats in both prevention and reversibility studies. Rats were placed in a clear plexiglass cylinder (16 cm in diameter, 25 cm in height) and videotaped for 5 minutes prior to and again 50 mins postlevodopa injection. The number of 360 rotations and rears were quantified by a blinded investigator.
The degree of nigral DA neuron depletion in each animal was confirmed by total enumeration of TH-positive neurons. 27 Nigral TH neuron loss of ≥95% in the lesioned hemisphere compared with the unlesioned hemisphere was used as a final inclusion criterion. This magnitude of SN DA neuron depletion is required in this model to produce reliable LIDs. 9,21,23,28,29 The striatal volume of vector transduction in GFPimmunostained sections was determined with the Cavalieri estimator using StereoInvestigator software (MicroBrightField, Williston, VT). Briefly, contours were defined for both the striatum and striatal region of GFP immunoreactivity. The outlines of each structure were traced at 1× in approximately 6 coronal sections along the entire rostral-caudal extent of the striatum.
Prior to undertaking our functional studies, a small cohort of rats received either rAAV-CaV1.3-shRNA (n = 4) or rAAV-Scr-shRNA (n = 4, as described above) to ensure the absence of nonspecific shRNA or GFP-associated toxicity. 30,31 The number of NeuN+ cells was quantified in the GFP+ region of striatum employing stereological techniques according to our previously reported method. 26
For RNAscope ISH, 3 images in the GFP+ dorsolateral vector-injected or noninjected striatum were acquired on an OlympusBX51 light microscope at 20×. For dual-label IHC, 2 images in the vector-injected or noninjected striatum in the same general striatal regions as the ISH micrographs were acquired at 20× on a Nikon ElipseTi confocal using NIS Elements. The CaV immunoreactive ISH signal, which is sensitive enough to detect single transcripts, or CaV1.3 immunostaining analyses were performed using ImageJ software (NIH) using the threshold function. All microscope and camera settings were identical for all images within a given transcript or antibody. Data are represented as mean area or percent total area above the threshold. CaV1.3 transcript silencing is expressed as relative level of transcript in the rAAV-CaV-shRNA versus rAAV-Scr-shRNA striatum.

Statistical Analyses
All LID behavioral data were analyzed with nonparametric statistics (Freidman [FM] or Kruskal-Wallis [H] with Dunn's multiple-comparisons test). Percent nigral lesion comparisons was analyzed with a nonparametric Mann-Whitney test. CaV1.3 mRNA expression in the rAAV-CaV-shRNA versus rAAV-Scr-shRNA striatum was compared using an unpaired, 2-tailed t test. Statistical evaluation of CaV1.2 mRNA expression in the injected and noninjected striata of rAAV-CaV-shRNA and rAAV-Scr-shRNA was done using 1-way ANOVA with Tukey's multiple-comparisons post hoc test. Nonparametric Spearman correlation test was used for correlation of LID severity with CaV1.3 levels. We 10 and others 32 have previously reported that in the outbred SD rat strain there is a small subset (15%-20%) of subjects that despite having an equal degree of striatal DA depletion and levodopa treatment remains resistant to LID. We characterize these rats as LID negative and having a peak LID severity of ≤3.0. In the LID prevention study we identified 2 statistical outliers in the rAAV-Scr-shRNA group, verified with the ROUT method (robust regression and outlier removal test; 3-step test automated within Prism; suggested Q coefficient detects outliers with false discovery rate <1%) and having a cumulative mean (across all 4 doses of levodopa) of 0.5 AE 0.3 and 2.5 AE 0.5; these 2 rats were omitted from final LID analyses. In addition, 1 rat in the AAV-CaV1.3-shRNA group in the LID prevention study was not adequately lesioned (ie, 21.6%) and was excluded from final behavioral analyses. Final subject numbers for the LID prevention study were: rAAV-CaV1.3-shRNA, n = 10; rAAV-Scr-shRNA, n = 7; for the LID reversibility study were: rAAV-CaV1.3-shRNA, n = 11; rAAV-Scr-shRNA,

Results
Striatal rAAV-CaV1.3-shRNA Potently Prevents Induction of LID Levodopa was administered using a dose-escalation paradigm beginning 4 weeks postvector (3 weeks post-6OHDA; Fig. 1A). As demonstrated in Figure 1B, rats receiving the control rAAV-Scr-shRNA displayed a typical escalation in LID severity over time that remained stable and severe with increasing doses of levodopa (Scr [black line]: day 1, 6 mg/kg versus day 6, 10-12 and 10-18 mg/kg; FM, 39.63; P = 0.0002; Dunn's post hoc P ≤ 0.0326; Fig. 1B). In contrast, rats treated with rAAV-CaV1.3-shRNA showed significant suppression of the development of LIDs compared with rAAV-Scr-shRNA rats (red lines, CaV; Fig. 1B,C). This notable prevention of LID escalation continued to persist as a near-complete absence of dyskinetic behavior in the rAAV-CaV-shRNA rats, which showed a significant difference from control rAAV-Scr-shRNA rats beginning on day 1 of 9 mg/kg levodopa (H, 159.6; P < 0.0001; Dunn's post hoc: Scr vs CaV, 50 minutes; P = 0.0027; Scr vs CaV, 80 minutes; P = 0.0010; Scr vs CaV, 110 minutes; P = 0.0109). This potent antidyskinetic effect was noted in all rAAV-CaV-shRNA parkinsonian rats despite continuing elevation in dose of levodopa over 2 months (Fig. 1C, lower graphs illustrate uniform individual subject responses; detailed statistics provided for each day/dose in figure legends).
As depicted in Figure 2B,C, rAAV-mediated CaV1.3 silencing in parkinsonian rats with established high-level LIDs can result in progressive and significant amelioration of existing LIDs. Specifically, the ability of CaV1.3 silencing to reverse peak-dose LIDs showed a nonsignificant trend of decreasing LID severity in the rAAV-CaV1.3-shRNA group (Fig. 2B,C) compared with the rAAV-Scr-shRNA group beginning on day 20 postvector. This trend continued to be nonsignificant through day 25. As such, we examined whether a short, 5-day withdrawal of levodopa (ie, "drug holiday"), once popular for improving patient response to levodopa including potentially decreasing LIDs, 33,34 would provide an environment in which vector-mediated reduction in aberrant calcium signaling might translate into reduction of LIDs.
As presented in Figure 2B,C on day 31 postvector, the first day immediately following the 1-week levodopa-free drug holiday, LID severity in rAAV-CaV1.3-shRNA rats was significantly reduced compared with that seen in the control rAAV-Scr-shRNA group that predictably maintained a sustained high level of LID severity from the prevector through the entire postvector time frame (day 31, F = 135.7, P < 0.0001; post hoc Dunn's test P = 0.0009; Fig. 2B; F = 97.2, P < 0.0001; post hoc Dunn's test P ≤ 0.0094; Fig. 2C). The benefit from this first drug holiday for rAAV-CaV1.3 rats was sustained over the next 2 weeks (Fig. 2B,C, day 31 through day 40); however, there was no further diminution of LIDs with continued daily high-dose levodopa. Given this plateau of response together with the apparent benefit of the first drug holiday, we employed a second 1-week drug holiday to determine whether further enhancement of LID reversal was possible. However, there was no further dampening of LID severity for the rAAV-CaV1.3 vector group after a second 1-week drug holiday when levodopa was maintained at this high dose of 12 mg/kg (day 40 vs day 46, P > 0.999).
Quantification of TH-positive SN neuron loss in the lesioned hemisphere of all rats revealed nearly identical levels of unilateral lesioning between CaV and Scr groups (CaV, 99.1% AE 0.3%; Scr, 98.4% AE 0.6%; cell loss compared with the intact side, P = 0.9394 Mann-Whitney).

Striatal CaV1.3 Silencing Does Not Interfere With Levodopa Motor Response
Quantification of exploratory rearing and contralateral rotational behavior using a modified cylinder test revealed no significant difference in either behavior under baseline drug-free (pre-LD) conditions (2-way ANOVA with post hoc Sidak's multiple-comparisons test; rearing, P = 0.8503 for LID prevention, P = 0.9999 for LID reversibility; rotations, P = 0.9984 for LID prevention, P = 0.9999 for LID reversibility). However, following low-dose levodopa (6 mg/kg), rAAV-CaV1.3-shRNA-but not control rAAV-Scr-shRNA-treated rats showed a significant increase in both exploratory rearing and rotational behavior compared with baseline levels (Fig. 3 contains detailed statistics), suggesting that knockdown of CaV1.3 channels in the DA-depleted striatum may enhance motor response to levodopa. Additional evidence demonstrating that motor effects of levodopa are readily apparent and not impaired by gene-level CaV1.3 silencing can be seen in the accompanying videos (Supplemental Fig. 1).
mRNA Knockdown, Specificity of Knockdown, and Transduction Spread ISH analyses using probes generated against CaV1.3 (Cacna1d) and CaV1.2 (Cacna1c) mRNA revealed that our rAAV-CaV1.3-shRNA vector resulted in an average 84.77% reduction in CaV1.3 mRNA in the GFP-transduced region of the striatum compared with that seen with rAAV-Scr-shRNA (Fig. 4A,C). A similar degree of CaV1.3 protein knockdown was found (87.3%; Fig. 4Cii). The range of mRNA silencing was 43.6% to 99.1% (Fig. 4D). However, there was no significant correlation of degree of CaV1.3 silencing with final LID severity scores (Spearman correlation, r = 0.0886, 2-tailed P = 0.4116), suggesting that even partial silencing of overactive CaV1.3 channels in the parkinsonian striatum is capable of completely preventing LID induction. Importantly, in contrast to an 84.77% knockdown of CaV1.3 mRNA, we detected no significant change in CaV1.2 mRNA (Fig. 4Ciii), demonstrating the specificity of our genetic approach.
There was no difference in the volume of striatal transduction in rAAV-CaV1.3-shRNA rats between the LID prevention and LID reversibility studies, nor between these 2 studies in rats injected with rAAV-Scr-shRNA (Kruskal-Wallis, post hoc Dunn's multiple comparisons: P > 0.999; Fig. 4Aii). Although not of functional significance, there was less volume of the striatum transduced from the inert control rAAV-Scr-shRNA vector compared with the rAAV-CaV1.3-shRNA in rats in the LID prevention study (P = 0.0056; Fig. 4Aii). We found no evidence of toxicity related to our vectors, as demonstrated by equal numbers of NeuNpositive cells in intact versus vector-injected striatum regardless of the vector (ANOVA P = 0.182, F = 1.920; Fig. 4Aiii).
As depicted in Figure 4A, GFP expression was prominent in the target region of the striatum, but could also be seen as a result of retrograde transduction to striatal input areas including various cortical regions. In some but not all animals sparse and highly variable patterns of GFP+ fibers were also noted in the globus pallidus, medial and/or lateral geniculate nuclei, and thalamic and/or hypothalamic regions. Despite the variable presence of extrastriatal GFPimmunoreactive fibers, little GFP was observed in cell bodies (eg, Fig. 4Aii). Visual examination of CaV1.3 ISH in these extrastriatal regions confirmed that despite some transduction, there was no apparent reduction in cellular CaV1.3 mRNA levels (Fig. 4Aiii). Our findings are in agreement with the relatively low retrograde transduction efficacy seen with wild-type AAV capsids. 35,36 Although involvement of CaV1.3 silencing in extrastriatal regions cannot definitively be ruled out as a contributing factor to the LID prevention/amelioration in the current studies, the highly variable nature of its expression in such regions, in the face of uniform LID reduction suggests that extrastriatal CaV1.3 silencing is not the principal mechanism of LID suppression.

Discussion
The current genetic-based studies, developed to provide high-potency striatal CaV1.3 channel silencing, were derived from a set of exploratory preclinical studies aimed at demonstrating the novel application of common US Food and Drug Administration-approved antihypertensive dihydropyridine (DHP) drugs for a new therapeutic application in PD, specifically prevention and/or reversal of LIDs. 9,14 Indeed, although initial studies provided evidence that pharmacological antagonism of CaV1.3 channels could dampen LID in parkinsonian rats, the efficacy of these DHP drugs (ie, isradipine and nimodipine) was partial and transient.
Supported by existing data, which includes evidence that CaV1.3 channels are incompletely inhibited even by high concentrations of current DHP drugs, 15,37 we hypothesized that if other methods to block CaV1.3 channels were available, this target would provide an optimal anti-LID response. The current study has confirmed this to be true. Using the approach of continuous, high-potency, target-specific genetic silencing of striatal CaV1.3 calcium channels with rAAV-CaV1.3-shRNA, we have demonstrated that mRNA-level silencing can provide potent, uniform, and long-term (>2 months) prevention of LIDs, even with extreme doses of daily levodopa. In addition, this approach was capable of reversing preexisting severe LIDs, which was not possible with pharmacological CaV silencing. 14 Importantly, motor benefit from levodopa was maintained with the knockdown of CaV1.3. We contend that these findings provide some of the strongest preclinical data to date demonstrating the amelioration of LIDs without compromise of motor benefit.

LID Reversibility and Clinical Relevance
It is perhaps most compelling, and surprising, based on the presumed mechanism of LID prevention being the prevention of PD-associated spine changes, that the approach of CaV1.3 silencing employed in these studies could significantly reverse severe LID behavior in parkinsonian rats. Although we observed that this method was capable of significant reversal of severe LID behavior associated with high-dose levodopa, the effect was nonetheless incomplete. In addition, our experimental design in these proofof-principle studies included a short-term withdrawal of levodopa that coincided with a significant reduction of LIDs in rAAV-CaV1.3-shRNA rats when high-dose (12 mg/kg) treatment was reintroduced, which contrasted with maintenance of severe LIDs in rAAV-Scr-shRNA control rats. It is unclear whether levodopa withdrawal at this early point after vector surgery (ie, days 26-30 postvector) was necessary for this significant amelioration of LIDs. Because there was no additional benefit with a second drug withdrawal, it could be suggested that shortterm drug withdrawal in the presence of CaV1.3 silencing may not enhance or be necessary for LID amelioration. The mechanism(s) by which reduction in CaV1.3 expression can significantly dampen existing severe LIDs, whether or not a short drug withdrawal is indicated, remains to be determined.
Although beyond the scope of this report to detail such potential mechanisms, it is clear that there are adaptations in MSNs unrelated to dendritic spine density that may impact LID in the presence of CaV1.3 silencing. Specifically, these channels are linked directly to signaling cascades that not only impact, for example, long-term alterations in synaptic strength and short-term dendritic excitability, but also impact synaptic function through selective signaling to the nucleus altering transcriptional activity (eg, references 3,38-40 ). The loss of DA tone (specifically D2 receptor tone) after DA depletion disinhibits CaV1.3 channels, leading to structural (eg, spine retraction, synaptic pruning) and functional adaptations. 3 Accordingly, it is reasonable to suggest that silencing dysfunctional CaV1.3 channel activity in the parkinsonian striatum may ameliorate LIDs through functional adaptations distinct from altered dendritic spine density per se.
Despite the current studies demonstrating that the antidyskinetic potency of CaV1.3 channel silencing is most robust in a prevention paradigm, the ability to reverse LIDs holds more immediate clinical relevance. Specifically, it is unlikely that any gene therapy approach prior to induction of LIDs would be considered despite the current statistics of its occurrence in up to 90% of patients over the lifetime of the disease. 1,2 Accordingly, we posit that future studies are warranted to extend an understanding of the capacity, limitations, and mechanism(s) of striatal CaV1.3 silencing to reverse a behavioral phenomenon that is often considered steadfast. Such studies should be directed not only at understanding mechanisms of action but also the necessity of drug withdrawal and the relationship of levodopa dose and/or LID severity with the ability to maximally reverse this aberrant behavior. Indeed, it could be anticipated that if gene therapy were undertaken when LID severity and/or levodopa dose were low (eg, early in the disease), complete reversal and stable suppression of LIDs could be achieved. This idea is supported by our LID prevention studies demonstrating that once expression of the CaV1.3 channel and LIDs are maximally suppressed, the underlying mechanism(s) allowing for this near-complete amelioration is maintained despite the escalation of levodopa doses to very high levels.

Beyond LID Prevention and Future Directions
Although we interpret our motor behavior findings with due prudence, they appear to suggest that knockdown of CaV1.3 channels in the DA-depleted striatum does not impede and may benefit motor response to levodopa. Indeed, when a "subtherapeutic" dose of levodopa was administered to our severely parkinsonian rats, only those with CaV1.3 silencing showed a significant motor response. The undeniable maintenance of the motor effect of levodopa is clearly evident in the videos of the rAAV-CaV1.3-shRNAexpressing rats (Supplemental Fig. 1). Although our current findings in the rat model of PD are highly encouraging, the approach of high potency, target-selective silencing of CaV1.3 channels on LIDs and motor response to DA agonists needs to be validated in other models of PD including nonhuman primates.
A common debate about preclinical models of LID is that because rats require significant DA depletion and because in rodent and NHP models LIDs develop more rapidly than in patients, these models do not reflect PD. However, it is often underappreciated that regardless of species, nearcomplete loss of striatal DA innervation is generally required for LIDs to manifest (eg, references 10,32,[41][42][43]. Indeed, as reported by Kordower and colleagues, 44 nearcomplete loss of striatal DA innervation occurs during the first 3-5 years following a PD diagnosis, a time frame that correlates well with onset of LIDs in patients. 1,2 Future studies aimed at understanding the mechanisms of this therapy, especially in the reversal paradigm, and whether the direct versus indirect pathway of MSN silencing has similar efficacy, as well as its impact on neurophysiological and molecular indices of LIDs, are warranted to allow a more complete understanding of the safety and efficacy of this approach in PD.

Summary
Gene therapy-mediated silencing of striatal CaV1.3 expression provides some of the most profound antidyskinetic benefit reported to date. The spatial control achieved with a gene therapy approach importantly circumvents potential off-target side effects, whose relevance to DHP drugs could include cardiovascular, learning and memory, and neuroendocrine effects. [45][46][47][48] If the current findings can be translated into a clinical application with a similar magnitude, this would provide a much-needed breakthrough in the treatment of individuals with PD and has the potential to allow the most powerful antiparkinsonian therapy identified (ie, levodopa) to work unabated through the duration of the disease.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Validation of a cell-based colorimetric reporter gene assay for the evaluation of Type I Interferons

Highlights • The HEK-Blue IFN-α/β system was validated to comply international guidelines.• This system complied in accuracy, precision, specificity and, system suitability.• The validated system showed robustness to evaluate different commercial type I IFN.• This bioassay could be a complementary method to evaluate type I IFN activity.• The system met the validation criteria to be used in pharmaceutical environments.

Introduction
Type I Interferons (IFN-I) are cytokines composed mainly by IFN-α and β [1], which have important roles in innate and adaptive immune responses with effects over a broad range of mammalian cells [2,3]. The type I IFN display broad biological activity, including control of cell proliferation, induction of genes responsible for protecting cells against the effects of viral infection, regulation of the differentiation state of immune cells and modulation of their function, accordingly these cytokines have been used in the treatment of viral and proliferative disease [4][5][6][7][8][9].
Type I IFN for therapeutic use are produced by recombinant DNA technology using either eukaryotic or prokaryotic expression systems. For instance, recombinant IFN-β1a is produced in Chinese hamster ovary (CHO) cells containing a glycosylation pattern like human IFN-β, while recombinant IFN-β1b and IFN-α are produced in Escherichia coli as a non-glycosylated form with minor modifications in its original amino acid sequence [10,11]. Despite the physicochemical differences between the type I IFN, these proteins exhibit the same function as the native [12] IFN. However, it is well known that the glycosylated isoforms have a longer halflife than the non-glycosylated [10,13].
Biological characterization of recombinant type I IFN is required to confirm their identity and evaluate their strength. These attributes can be assessed by any of the following in vitro methodologies: 1) receptor binding assays, 2) antiviral activity in cell culture and/or 3) antiproliferative effects in tumor cell lines [14]. This characterization must be performed throughout different stages of their life cycle (i.e. development, stability tests, and batch release).
Several assays for the evaluation of IFN biological activity require the use of viruses to measure antiviral activity [15]. This implies the compliance of special biosafety measures to handle these pathogens. As an alternative, reporter gene assays is an indirect approach to evaluate the activation of signal transduction to measure the function of target genes using fluorescent proteins or colorimetric enzymatic reactions as detection systems [16]. Assays that employ reporter genes have demonstrated to be highly robust in terms of their specificity, sensitivity, precision, and easiness to perform [17][18][19][20][21][22] than the classical antiviral assays used to evaluate the strength of type I IFN [20,[18][19][20][21][22].
HEK-Blue IFN-α/β cells system from InvivoGen 1 is a commercial gene reporter-based bioassay kit designed to evaluate the activation of the type I IFN pathway. This system is based on stable transfection of human embryonic kidney 293 (HEK293) cells that express signal transducer and activator of transcription 2 (STAT2) and IFN regulatory factor (IRF9) human genes to enrich a fully type I IFN signaling pathway. Once IFN-α or IFN-β bind to common receptor IFNAR1/2, allow the recruitment of JAK1 (Janus kinases 1) and Tyk2 (Tyrosine kinase 2), which induce the phosphorylation and dimerization of STAT (Signal Transducer activator of transcription) 1 and STAT 2. This dimer interacts with IRF9 forming the ISGF3 (IFN stimulated gene factor 3) complex. Finally, this transcriptional complex binds to the promoter of ISG54 (IFN-stimulated gene 54) and induce the production of SEAP (secreted embryonic alkaline phosphatase) as a reporter gene [23].
The HEK-Blue IFN-α/β system has been employed for research purposes using in vitro infection models, mainly to detect or quantify the production of bioactive IFN-I during viral or bacterial infections [24][25][26][27][28][29][30]. This system has also been proposed as a diagnostic tool for the indirect identification of neutralizing autoantibodies against IFN-I in some autoimmune diseases [31].
In this work, we validated the HEK-Blue IFN-α/β gene reporter cell-based bioassay according to the international standard guidelines; International Conference for Harmonization (ICH) Q2 (R1) [32] and the <1033> United States Pharmacopeia (USP) chapter [33] that establish the types and characteristics that should be evaluated during validation execution. This validation was intended to demonstrate that the assay is appropriate to be used under a pharmaceutical quality control environment for the in vitro biological activity assessment of biopharmaceutical products containing recombinant IFN-α and IFN-β.

HEK-Blue
TM IFNα/β cells were grown in DMEM medium with 10% FBS and selective antibiotics: blasticidin, zeocin and normocin (supplemented medium) and incubated at 37 C, 5% CO 2 . The cells were harvested with trypsin when confluence reached 80%. Then cells were centrifuged, washed and resuspended in PBS. Viable cells were counted by the trypan blue exclusion method and cell concentration was adjusted to 2.8 Â 10 5 cells/mL in a supplemented medium.

Evaluation of type I IFN response by SEAP activity
HEK-Blue cells were seeded in 96 well plates at a concentration of 50,400 cells/well. Aliquots of IFN-α and IFN-β were prepared independently at different concentrations (from 5 Â 10 À2 to 5 Â 10 5 IU and 1 Â10 -1 to 1 Â10 6 IU, respectively). Twenty mL of each IFN at different concentrations were added to corresponding wells and incubated at 37 C and 5% CO 2 for 24 h. After the incubation period, 20mL of supernatant was transferred into a 96-well plate with 180 mL of QUANTI-Blue TM ready to use solution (prepared as indicated by manufacturer).

Validation
Parameters for validation were established according to the ICH guideline Q2 (R1) and the USP <1033> Biological Assay Validation Chapter [32,33] considering the parameters described below in each characteristic evaluated: dose-response curve (4 PL model fitting), specificity, precision, accuracy (dilutional linearity), and system suitability. The acceptance criteria for each evaluated parameter of validation exercise were established according to its intended use that evaluates the biological activity of IFN-α/β in vitro for quality control (QC) and biosimilarity assays using IFNα-2b and IFNβ-1a international standards. In this sense, it is expected a relative potency specification for batch release analysis establish in the biopharmaceutical industry between 80-125%. Therefore, the %CV should be established at 20%.

Dose-response curve (4 PL model fitting)
Independent triplicates were prepared at 12 dilution levels in a range from 5 Â 10 À2 to 5 Â 10 5 IU and 1 Â10 -1 to 1 Â10 6 IU for IFN-α2b and IFN-β1a respectively, in order to obtain the sigmoidal curve from the biological activity of these molecules fitted to the four parameters logistic (4 PL) model: bottom and top plateaus, the EC 50 , and the slope factor (Hill s slope). The acceptance criteria were established by the correlation coefficient (r 2 ) >0.90.

Specificity
The specific response was given by the fitting of data curve through a non-linear regression model (4 PL). As specificity negative control, we used IFN-g

Precision
Repeatability was estimated through the percentage of coefficient of variation (CV) from three independent replicates at the slope factor concentrations in the dose-response curve and intermediate precision as the %CV of three independent assays; acceptance criteria were CV 20% among replicates at all the evaluated levels.

Accuracy (dilutional linearity)
Accuracy was evaluated as dilutional linearity at all dilution levels of the dose-response curve in a concentration range from 70% to 130% for IFN-α2b and 40% to 120% for IFNβ-1b. Acceptance criteria for linearity were r 2 ! 0.90 and slope in a range from 0.80 to 1.25. The nominal value is theoretical according to the dilution of the sample at 70-130% for IFN-α2b whereas 40-120% for IFN-β1a.
While the measured potency was estimated as percentages of relative potency were obtained from EC 50 values.

System suitability
System suitability was determined considering the capacity to get a sigmoidal dose-response curve. The evaluated parameters were the %CV (from precision), r 2 of the curve within the concentration levels range, the relationship between the upper and lower asymptotes and the distribution of experimental data.

Performance of HEK-Blue system with commercial therapeutic IFN
The validated HEK-Blue system was tested with medical use type I IFN: Intron A1, Betaferon1, Avonex1, and Rebif1. The assay was performed using the established IFN-α/β concentrations and the methodology described.

Statistical analysis
Data were analyzed with Graph Pad Prism 6.0 software (La Jolla, USA) to evaluate the fit to the 4 PL model and the %CV for replicates.

Optimization of the HEK-Blue IFN-α/β cell system
To optimize the HEK-Blue IFN-α/β cell system we identified the critical steps of the assay and divided into the following three: 1) cell preparation, 2) IFN-α/β binding to its receptor and 3) detection of SEAP activity.
Under our laboratory conditions, the cell preparation considered the number of passages below 12 to guarantee the reproducibility of response, also the adjustment of cell concentration.
In the next step, the type I IFN concentration range was established: from 5 Â 10 À2 to 5 Â 10 5 IU and 1 Â10 -1 to 1 Â10 6 IU for IFN-α and IFN-β, respectively. The kit's manufacturer established a range between 20-24 hours as incubation of the reaction system; we set 24 h for our assay. To measure the SEAP activity the incubation time was set on 3 h for type I IFN, after testing 0.5, 1.0, 2.0, 3.0 h, it allowed to determine the signal in the low concentration levels and a suitable correlation coefficient with the 4 PL model.
Following these steps, we found that the assay was reproducible to evaluate the response of bioactive IFN-α/β form.   Once the assay was optimized in our laboratory, the parameters for its validation were established.

Dose-response curve fitting
As shown in Fig. 1, the biological assay presented a sigmoidal dose-response curve, which fit 4 PL showing an r 2 = 0.97 for IFN-α2b and r 2 = 0.98 for IFN-β1a. The curve showed at least two points in each asymptote and three points in the slope for both IFN-α/β that is a pharmacopeical suggestion to make a suitable fitting for 4 PL model according to obtained results of the correlation coefficient (r 2 ). We included two concentration levels higher than recommended by the manufacturer, these two added points allowed to obtain a better sigmoidal behavior in the curve.

Specificity
In order to test the specificity of the HEK-Blue cell system, we use IFN-g as a negative control as was previously reported [31]. We are shown that the responses of IFN-α2b and IFN-β1a were fitted to 4 PL, in contrast, IFN-g at the same concentration range did not fit to 4 PL (r 2 = 0.4047) demonstrating the characteristic response of these cells to type I IFN, complying with specificity parameter (Fig. 2). Despite IFN-g is a cytokine related to IFN-α and IFN-β do not share the same signaling pathway [31]. Although HEK-Blue cells are able to respond to different stimulus included type II IFN such as IFN-g, the reporter gene under control of ISG54 promoter is induced by type I IFN only, therefore it requires a different transcriptional factor complex, and this explains the high specificity of the assay.

Precision
The precision of the HEK-Blue IFN-α/β cell system showed CV lower than 20% obtained from triplicates of each level of the curve (intra-assay) and EC 50 at different days (inter-assay) ( Table 1 and 2). Both assays fulfill repeatability and reproducibility.   The bioassays have intrinsic variability however, the HEK-Blue IFN-α/β cell system showed a CV lower than 20% among replicates in a range of concentrations 5 -5 Â 10 5 and 10 -1 Â10 6 UI/mL for IFN-α2b and IFN-β1a respectively (intra-assay), meeting with established criteria validation (Table 1 and 2). As indicated in the sample preparation, we tested two lower concentrations than recommended by InvivoGen 1 , those not fulfilled with CV due to the low detection level; our results demonstrated that if these two points were or not considered, the sigmoidal curve was preserved.
The CV of EC 50 at three different days (inter-assay) was precise with a CV lower than 20%.  In summary, this assay satisfies repeatability and intermediate precision for its validation.

Accuracy
In Fig. 3 we demonstrated the accuracy of IFN-α2b in a dilution range from 70 to 130% with a linear correlation between nominal and measured potencies, r 2 = 0.99, and a slope value of 1.09. In the same way in Fig. 4, IFN-β1a demonstrated dilutional linearity (r 2 = 0.97 and slope = 1.140) in a range from 40 to 120% and was able to distinguish two more dilution levels than IFN-α. Both parameters showed to be within the established acceptance criteria.

System suitability
The system suitability was predefined according to 4 L P model fitting and precision parameters in order to verify equipment, analytical operations and samples in a routine analysis to guarantee the reliability of results as recommended the ICH Q2R1 [32]. For system suitability, we defined the same rigor in the acceptance criteria mentioned in the summary of the validation of assay (Table 3).

HEK -Blue system performance with commercial therapeutic IFN
At present, there are several commercially available IFNα/β for medical uses. Here we proved if the validated HEK-Blue IFN-α/β system is useful to determinate bioactive form of some of this commercial type I IFN through IFNAR signal transduction. Once the assay was validated with international standards, we tested this system with IFN-α2b (Intron A 1 ), IFN-β-1a (Avonex 1 and Rebif 1 ) and IFN-β-1b (Betaferon 1 ). As it was shown in Fig. 5, the HEK-Blue IFN-α/β system was suitable to detect the bioactive IFNα/β contained in these biotherapeutics, complying with the fit to 4 PL.
In summary, the HEK-Blue IFN-α/β system could be useful to evaluate the bioactive type I IFN inside biotherapeutic products despite their origin.

Discussion
In this work, we optimized and validated the commercial HEK-Blue IFN-α/β system, which complies with precision, accuracy, specificity and system suitability according to established parameters. As expected, being a biological system, it shows a classical dose-response sigmoidal curve, besides our IFN-α/β results fitted 4 PL model.
Both IFN-α/β are recognized by the same receptor IFNAR, differences in their structure might affect cell response [7]. In our results, we observed similar sigmoidal behavior by adjusting concentration range of IFN-α/β however, IFN-β shows higher potency than IFN-α as previously reported [24]. Potency differences could be explained by different molecular mechanisms as the magnitude of the signal by ligand affinity to each IFNAR subunits, response to different type I IFN concentrations and membrane receptor exchange [25][26][27][28][29].
In addition, we proved that the HEK-Blue IFN-α/β system could be used to evaluate bioactive IFNα/β of commercial biotherapeutics: IFN-α2b, IFN-β-1a, and IFN-β-1b. We found a similar response that fit to 4 PL, despite the different source of type I IFN.
Moreover, the recommended use of the HEK-Blue IFN-α/β system in the research field, our results supported that this could be extended for pharmaceutical industry use because it showed to be robust evaluating identity and potency of therapeutic type I IFN, regardless their source.

Conclusions
We validated the commercial HEK-Blue IFN-α/β system complying with precision, accuracy, specificity and system suitability according to established parameters. Our results suggest that this bioassay could be implemented as a complementary method to the classical anti-proliferative and anti-viral assays under a quality control environment because it was robust and easy to perform to evaluate type I IFN biological activity.

Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. LP is supported by INPRFM projects: NC150048 SECITI, SECITI 0048/ 2014, and NC16044.0.


extfieldsofstudy: Biology Medicine

text:
Oral immunization with a probiotic cholera vaccine induces broad protective immunity against Vibrio cholerae colonization and disease in mice

Oral cholera vaccines (OCVs) are being increasingly employed, but current killed formulations generally require multiple doses and lack efficacy in young children. We recently developed a new live-attenuated OCV candidate (HaitiV) derived from a Vibrio cholerae strain isolated during the 2010 Haiti cholera epidemic. HaitiV exhibited an unexpected probiotic-like activity in infant rabbits, preventing intestinal colonization and disease by wild-type V. cholerae before the onset of adaptive immunity. However, it remained unknown whether HaitiV would behave similarly to other OCVs to stimulate adaptive immunity against V. cholerae. Here, we orally immunized adult germ-free female mice to test HaitiV’s immunogenicity. HaitiV safely and stably colonized vaccinated mice and induced known adaptive immune correlates of cholera protection within 14 days of administration. Pups born to immunized mice were protected against lethal challenges of both homologous and heterologous V. cholerae strains. Cross-fostering experiments revealed that protection was not dependent on vaccine colonization in or transmission to the pups. These findings demonstrate the protective immunogenicity of HaitiV and support its development as a new tool for limiting cholera.

Introduction
The bacterial pathogen Vibrio cholerae causes the severe human diarrheal disease cholera, a potentially fatal illness characterized by rapid-onset of fluid loss and dehydration. Recent estimates place the global burden of cholera at~3 million cases per year, and over 1.3 billion people are at risk of this disease [1]. V. cholerae proliferates in the small intestine and produces cholera toxin (CT), which leads to water and electrolyte secretion into the intestinal lumen [2].
The O1 serogroup of V. cholerae causes virtually all epidemic cholera. This serogroup includes two serotypes, Inaba and Ogawa, whose LPS structures differ by a single methyl group on the terminal O-antigen sugar [3]. Serologic and epidemiologic studies have established the existence of extensive serotype cross-reactivity and -protectivity, although immunogenicity and protection is highest to the homologous serotype [4][5][6][7]. Toxigenic O1 strains are divided into two major biotypes, classical and El Tor, but the former has not been isolated in over a decade and is thought to be extinct [8]. Ongoing evolution of El Tor V. cholerae has given rise to variant El Tor strains, which are distinguishable from earlier strains by a variety of features, including the expression of non-canonical ctxB alleles that may impact disease severity in afflicted patients [4,9,10]. These contemporary strains, such as the ctxB7-expressing V. cholerae strain responsible for the 2010 Haitian cholera epidemic, are thought to be the globally dominant cause of cholera [10][11][12]. Currently, serogroup O139 isolates only cause sporadic disease [13]. Notably, antibodies (or immune responses) targeting the O1 O-antigen do not protect against O139 challenge and vice versa [14][15][16].
Oral cholera vaccines (OCVs) have recently become widely accepted as a tool for cholera control [17]. Vaccines are a potent method to combat cholera due to their ability to both directly and indirectly reduce disease and transmission [18]. Killed multivalent whole-cell OCVs, such as Shanchol, have shown promise both to prevent disease in endemic regions and as reactive agents to limit cholera during epidemics [19]. However, killed OCVs tend to be less effective at eliciting protective immunity in young children (<5 years old), who are most susceptible to cholera [20,21]. Additionally, these vaccines typically require two doses over the span of several weeks, although recent studies suggest that a single dose may still lead to moderate protection [20,22,23].
There is no live-attenuated OCV licensed for use in cholera-endemic regions. The only clinically available live-attenuated OCV is Vaxchora (CVD103-HgR), which is derived from a classical O1 Inaba V. cholerae strain and was approved by the US FDA in 2017 for use in travelers [24]. In contrast to killed OCVs, live vaccines, such as CVD103-HgR and the El Tor-derived vaccine Peru-15, elicit more potent immune responses in young children [25,26], potentially because they more closely mimic natural infection than killed OCVs. In particular, live vaccines can produce antigens in vivo that are not expressed in the in vitro growth conditions used to prepare killed vaccines; furthermore, the inactivation processes used to formulate killed vaccines can destroy antigenic epitopes [27].
In addition to the requirement for multiple doses of some OCVs for optimal protection, all current live and killed OCVs are thought to be accompanied by a post-vaccination lag in protection during induction of anti-V. cholerae adaptive immunity. The shortest reported time to protective efficacy is 8 days post-vaccination, a delay that could hamper reactive vaccination campaigns designed to limit the spread of cholera outbreaks [28]. We recently created HaitiV, a new live-attenuated OCV candidate derived from a variant El Tor O1 Ogawa V. cholerae clinical isolate from the 2010 Haiti cholera outbreak. HaitiV harbors many genetic alterations that render it avirulent and resistant to reversion while preserving its robust capacity for colonization of the small intestine [29]. In an infant rabbit model of cholera [30], intestinal colonization with HaitiV conferred protection against lethal wild-type (WT) V. cholerae challenge within 24 hours of vaccination, a timescale inconsistent with the development of adaptive immunity and suggestive of a "probiotic"-like mechanism of protection. Here, using a mouse model of V. cholerae intestinal colonization, we show that oral administration of HaitiV to female mice elicits serum vibriocidal antibodies and protects their pups from lethal challenge with virulent V. cholerae. Thus, HaitiV has the potential to provide rapid probiotic-like protection as well as to elicit long-lasting immune protection from cholera.

Construction of ΔctxAB Haiti V. cholerae
The CT deletion strain in the H1 V. cholerae background (HaitiWT) was generated by allelic exchange as previously described, with an additional selection step to enhance the efficiency of obtaining a stable single crossover strain [29]. Briefly, HaitiWT was conjugated with SM10λpir E. coli bearing the suicide plasmid pCVD442-ctxAB-KmR, containing sacB as well as a kanamycin resistance cassette from pKD4 sandwiched by homology arms targeting the ctxAB operon (locus tags N900_RS07040 -N900_RS07045). Single crossovers were selected on LB+Sm/Cb/Km agar plates. To select for a double crossover, verified single crossovers were grown in LB + Cb/Km for 4 hours at 37˚C and then passaged in LB+10% sucrose overnight at room temperature. Sucroseresistant (sacB-negative), Km R and Cb S colonies were then conjugated with SM10λpir E. coli bearing pCVD442-ctxAB (no Km R cassette) and clean Km S double crossovers generated via an identical protocol. The ΔctxAB deletion was verified by colony PCR with internal and flanking primers. experiment. On Day 0, 2, 4, 6, 14, 28, 42 and 56, mice were anesthetized with isoflurane and orally gavaged with 10 9 CFU of an overnight culture of either HaitiV or CVD103-HgR in 100μL 2.5% Na 2 CO 3 . Mice were weighed at every immunization and once every 4-5 days between boosts. At each weighing, fresh fecal pellets were plated on LB + Sm to enumerate shed bacteria. At Day 7, 14, 28 and 42 post first immunization, blood samples were obtained from each mouse by tail vein incision. A Day 1 blood sample was collected from the Swiss-Webster cohort and the single-dose C57BL/6 cohort. Blood was clotted at room temperature for 1 hour, centrifuged at 20000 x g for 5 minutes and the supernatant (serum) stored at -20˚C for analysis.

Quantification of vibriocidal antibody responses
Vibriocidal antibody quantification was performed by complement-mediated cell lysis using PIC018 (Inaba) or PIC158 (Ogawa) V. cholerae as the target strain as previously described [31]. Seroconversion was defined as �4x increase in titer over the baseline measurement. The characterized mouse monoclonal antibody 432A.1G8.G1.H12 targeting V. cholerae O1 OSP was used as a positive control for the vibriocidal assay. Titers are reported as the dilution of serum causing a 50% reduction in target optical density compared to no serum control wells.

Quantification of antigen-specific immune responses
Anti-cholera toxin B subunit (CtxB) and anti-O-specific polysaccharide (OSP) responses were measured by previously described isotype-specific ELISAs [32,33]. Briefly, 96-well plates (Nunc) were coated with 1 μg/mL solution of bovine GM1 monosialoganglioside (Sigma) in 50mM carbonate buffer overnight. Next, 1μg/mL CtxB in 0.1% BSA/PBS purified from the classical Inaba strain 569B (List Biological Laboratories) was layered onto the GM1-coated wells. Wells were blocked with a 1% BSA/PBS mixture after which 1:50 dilutions of the mouse serum samples were loaded into each well. Goat anti-mouse IgA, IgG or IgM secondaries conjugated to HRP (Southern Biotechnology) were then added at a concentration of 1μg/mL in 0.1% BSA/0.05% Tween/PBS and incubated for 90 minutes. Detection was performed by adding an ABTS/H 2 O 2 mixture to the wells and taking an absorbance measurement at 405nm with a Vmax microplate kinetic reader (Molecular Devices Corp., Sunnyvale, CA). Plates were read for 5 min at 30 s intervals, and the maximum slope for an optical density change of 0.2 U was reported as millioptical density units per minute (mOD/min). Results were normalized using pooled control serum from mice previously immunized against V. cholerae and reported as ELISA Units as previously described [32]. Anti-OSP responses were measured and reported similarly to anti-CtxB responses, only instead of CtxB, purified OSP:BSA from either PIC018 or PIC158 (1 μg/mL) was used to coat plates as previously described [34]. Additionally, OSP ELISAs were carried out with 1:25 dilutions of the serum samples.

Infant mouse challenge assay
The infant mouse survival challenge was adapted from previous reports to optimize the dosage for HaitiWT and to include more frequent monitoring intervals [31,32]. Pregnant dams were singly housed at E18-19 for delivery. At P3 (third day of life), pups were orally inoculated with 50μL LB containing 10 7 CFU of a directly diluted 30˚C overnight culture of V. cholerae and returned to their dam. Infected pups were monitored every 4-6 hours for onset of diarrhea and reduced body temperature. Once symptoms appeared, monitoring was increased to every 30 minutes until moribundity was reached, at which point pups were removed from the nest and euthanized by isoflurane inhalation followed by decapitation for dissection and CFU plating of the small intestine on LB + Sm/X-gal. Pups that were alive at 48 hpi were deemed protected from the challenge. Cross-fostering was performed by transferring up to half of a litter between dams on the first day of life (P1). Fostering was maintained for at least 48 hours before infection to fully replace the milk from the original dam. We excluded rejected pups from analyses due to our inability to attribute mortality to infection alone.

Statistical analysis
Statistical analyses were performed with Prism 8 (Graphpad). To analyze whether immune responses were significantly changed over time, a one-way ANOVA was performed. Due to missing values from paired measurements as a result of insufficient serum sample volumes, antibody titers were analyzed with a mixed-effects model one-way ANOVA using the earliest sample (Day 1 or Day 7) as the control and post hoc tests performed with a Dunnett's multiple comparison test. Survival curves were analyzed with the log-rank (Mantel-Cox) test and CFU burdens were compared with the Mann Whitney U test. A p-value <0.05 was considered statistically significant.

Animal use statement
This study was performed in accordance with the NIH Guide for Use and Care of Laboratory animals and was approved by the Brigham and Women's Hospital IACUC (Protocol #2016N000416). Infant (P14 or younger) mice were euthanized by isoflurane inhalation followed by decapitation. When required, adult mice were euthanized by isoflurane inhalation followed by cervical dislocation.

Experimental design and vaccine colonization
While the infant rabbit model enables investigation of the progression of a V. cholerae-induced diarrheal disease that closely mimics human cholera [30], it is not appropriate to study vaccine immunogenicity because newborn animals lack a fully developed immune system. Instead, we used adult GF mice to study HaitiV immunogenicity. In contrast to normal adult mice, which are resistant to V. cholerae intestinal colonization, oral inoculation of GF mice with V. cholerae results in stable intestinal colonization without adverse effects [35][36][37]. In the GF model, serum markers of immunity, such as vibriocidal titers, can be measured, but challenge studies are not possible due to the persistent colonization of the vaccine strain and the resistance of adult mice to diarrheal disease. Here, we further developed a variation of the GF model [38]. Besides measuring serum markers in the orally vaccinated adult mice, neonatal pups (which are sensitive to V. cholerae induced diarrheal disease) born to these mice were subjected to challenge studies to evaluate vaccine protective efficacy.
We established two cohorts of orally immunized adult female GF mice. In the first cohort, a small pilot study was set up to compare the immunogenicity of HaitiV and a streptomycinresistant derivative of CVD-103HgR. This cohort consisted of 4-week-old Swiss-Webster GF mice that were immunized with either vaccine strain (n = 3 per group). Cohort 2 consisted of a set of 4-week-old C57BL/6 mice that were all immunized with HaitiV (n = 7). We generally followed the multi-dose oral immunization scheme previously used in this model, which included eight doses of 1x10 9 CFU vaccine over eight weeks [36,37]. After this vaccination regimen, the mice in cohort 2 were mated and vaccine-induced protective immunity was assessed in the progeny (Fig 1A).
Based on fecal CFU, all animals in both cohorts were stably colonized with high levels of either vaccine strain (Fig 1B). No adverse effects of long-term colonization with HaitiV or CVD103-HgR were noted, and all mice gained weight over the course of the study (Fig 1B). Fecal shedding and presumably intestinal colonization of HaitiV in cohort 2 was eliminated after these dams were used to cross-foster pups born to specified-pathogen free (SPF) control mice (described below), suggesting that a normal microbiota can outcompete HaitiV.

HaitiV immunization elicits robust serum antibodies targeting V. cholerae
Serum samples from the immunized mice were used to quantify antibodies targeting several V. cholerae factors thought to play roles in protection from cholera. One of these metrics, the vibriocidal antibody titer, is a validated correlate of protection in vaccinated humans [39][40][41][42].
In cohort 2, HaitiV immunization of C57BL/6 mice also induced high vibriocidal titers to Ogawa and Inaba target strains (Fig 2C). Isotype-specific levels of antibodies targeting Ogawa and Inaba OSP, and CtxB were also measured since they also likely contribute to immunity to cholera [39]. Although we did not measure Day 1 titers in cohort 2, measurements from naïve GF C57BL/6 mice and baseline measurements from cohort 1, and Day 1 of HaitiV-inoculated C57BL/6 mice in a later cohort (S2 Fig) showed undetectable levels of vibriocidal antibodies (Figs 2A and S2). The cohort 2 mice developed strong anti-Ogawa and anti-Inaba OSP responses (Fig 3, S2 Table). The anti-Ogawa OSP titers were generally higher than those targeting Inaba OSP, likely reflecting the fact that HaitiV is an Ogawa strain. All mice in cohort 2 also developed high levels of anti-CtxB IgA, IgG and IgM antibodies (Fig 4, S1 Table). The 100% seroconversion rate and general increase over time of all three humoral immune responses measured (vibriocidal, anti-CtxB and anti-OSP antibodies) reveals that orally delivered HaitiV can elicit V. cholerae-specific immune responses.

Pups born to HaitiV immunized dams are protected from lethal V. cholerae challenge
To assess the protective efficacy of HaitiV in this model, we challenged the neonatal progeny of HaitiV-immunized or control dams with lethal doses of different wild type V. cholerae strains. This assay has been used to study passive immunity elicited by cholera vaccines, but has not been characterized in vaccinated GF mice [31,32,44]. Initially, we optimized this assay with litters from SPF C57BL/6 control mice. Three or four-day old pups were inoculated with 10 7 or 10 8 CFU of HaitiWT, the virulent strain from which HaitiV was derived, and returned to their dams for monitoring (Fig 5A). Infected pups from both groups rapidly developed signs of dehydrating diarrheal disease, including accumulation of nest material on their anogenital regions, lethargy, skin tenting and hypothermia. All infected pups died by 48 hours post inoculation (hpi), with a median time to moribundity of~23-26 hpi (Fig 5B). At the time of death, all pups were heavily colonized, with >10 7 CFU/small intestine (Fig 5C), and had swollen ceca, another hallmark of productive cholera infection in mammalian models [30,45]. Since there were no significant differences in survival or bacterial loads in mice challenged with either 10 7 or 10 8 CFU, the smaller dose was used in subsequent experiments (Fig 5B). Diarrhea and death in this model were entirely dependent on CT; infant mice inoculated with 10 7 CFU HaitiWT ΔctxAB or HaitiV were completely healthy at 48 hpi, despite sustained intestinal colonization (Fig 5C).
We next mated HaitiV-immunized animals from cohort 2 with age-matched GF male mice, thereby preserving their colonization with HaitiV. When challenged with HaitiWT, none of the 16 pups born to HaitiV-immunized dams developed signs of diarrhea or died by 48 hpi; in stark contrast, all pups born to non-immunized dams died within~30 hpi (Fig 6A,  left). There was a marked~5,000-fold reduction in the intestinal load of HaitiWT in pups born to immunized versus control dams (Fig 6A, right). The pups of the immunized dams remained healthy for at least 2 weeks post-challenge, even though there were still detectable but very low  Pups of HaitiV-immunized dams were similarly challenged with heterologous V. cholerae strains, to test the serotype and serogroup specificity of protection engendered by oral immunization with HaitiV. The additional challenge strains included an O1 Inaba strain (N16961) that has been used as the challenge strain in several human volunteer cholera studies [43,46] and the serogroup O139 strain MO10, which was isolated during the 1992 O139 outbreak in India. Most pups from HaitiV-immunized dams were protected from N16961 V. cholerae challenge (7/10 survival at 48 hpi, Fig 6B). Despite the clinical protection, there was a much less dramatic reduction in the intestinal burden of N16961 (~20-fold) compared to that observed with HaitiWT challenge, indicating serotype-specific responses play an important role in limiting colonization. Surprisingly, pups challenged with MO10 also exhibited some protection, but there was no concomitant reduction in the intestinal burden of this O139 strain (Fig 6C). Together, these observations demonstrate that animals can exhibit protection from death despite relatively robust colonization, suggesting that protection from disease may result from immunity targeting factors such as CtxB, in addition to those that impede colonization.

Pups fostered by HaitiV-immunized dams are protected from lethal V. cholerae challenge
Since our earlier studies indicated that HaitiV itself can mediate rapid protection against cholera independent of an adaptive immune response, it was important to investigate whether pups nursed by HaitiV immunized dams were colonized with the vaccine strain. Extensive plating of intestinal samples from the >50 pups used for survival assays (limit of detection = 50 CFU/small bowel) did not reveal any HaitiV CFU in the pups reared by HaitiV-shedding dams. Thus, vaccine strain transmission and its probiotic effects are almost certainly not the explanation for the potent protection observed in nursing pups.
Cross-fostering experiments were undertaken to investigate the likely passive nature of the protection. P1 pups born to SPF dams were transferred to and reared by HaitiV-immunized dams and then challenged 2 days later with HaitiWT ( Fig 5A, between P1-P3). All pups crossed-fostered by immunized dams were protected (100% survival at 48 hpi) and nearly all had marked reductions (~1,000 fold) in their intestinal HaitiWT burdens (Fig 7A). These observations mirror the challenge studies presented above (Fig 6A), indicating passive immunity from milk accounts for the protection that HaitiV-immunized dams bestow to their progeny. Conversely, when pups born to HaitiV-immunized dams were cross-fostered by SPF (non-vaccinated) dams, all succumbed to HaitiWT challenge, albeit with an increase in median survival time (by~6-hour) and had high HaitiWT intestinal burdens (Fig 7B). The modest extension in survival time in these mice may be due to trans-placentally derived immunity or residual milk from the HaitiV-immunized dam.

A single oral dose of HaitiV is sufficient to elicit protective immunity
Although prior work with OCVs in GF mice suggested that multiple boosts were required to maximally induce immune responses, our observation of the prolonged colonization in the lowest dilution at which vibriocidal activity was observed and the height of the bars represent geometric mean titers in each group. Ogawa-specific responses, measured using strain PIC158, are shown in white and Inaba-specific responses, measured using strain PIC018, are shown in gray. Serum samples with undetectable vibriocidal activity were assigned a titer of 1. multiply-vaccinated animals led us to test whether a single oral dose of HaitiV could also stimulate protective immune responses [37]. A singly-vaccinated group of female GF C57BL/6 mice (n = 4) was established to investigate this possibility. Like our studies with the multi-dose regimen, a single dose of HaitiV led to sustained colonization in the mice (S2 Fig). HaitiV   Fig 3. Ogawa-and Inaba-specific anti-OSP titers in C57BL/6 mice immunized with HaitiV. (A) Anti-Ogawa OSP titers with specific isotype data for IgA (top), IgG (middle) and IgM (bottom). (B) Anti-Ogawa OSP titers with specific isotype data for IgA (top), IgG (middle) and IgM (bottom). The mean ± SEM is indicated. Mixed-effect p-values were determined by one-way ANOVA analysis. Multiple comparison p-values for this data are reported in S1 Table. Although there were 7 mice per group, some samples were of insufficient volume for analysis. https://doi.org/10.1371/journal.pntd.0007417.g003 Immunogenicity of HaitiV in germfree mice induced vibriocidal antibody titers comparable in magnitude to those from serially immunized mice (Fig 2C). Litters from singly-immunized mice were also completely protected from disease resulting from HaitiWT challenge, phenocopying pups from the first cohorts (S2 Fig).

Discussion
Vaccines for cholera are being increasingly embraced as public health tools for prevention of endemic cholera and limiting the spread of cholera epidemics [17]. Killed OCVs are efficacious in endemic populations, but live OCVs promise to be more potent, particularly in young children [19]. Here, we showed that the live OCV candidate HaitiV induces vibriocidal antibodies and other immunological correlates of protection against cholera in GF mice and leads to protection against disease in their offspring. Protection in this model was dependent on passively acquired factors in the milk of immunized dams and not transmission or colonization of Hai-tiV. Although our relatively small cohort sizes precluded rigorous statistical comparisons of immune responses in the immunized mice, oral administration of even a single dose of HaitiV elicited detectable vibriocidal antibodies in all animals. These observations provide strong data establishing HaitiV's immunogenicity. Additionally, the comparable vibriocidal titers elicited by HaitiV and CVD-103HgR, a live OCV licensed by the FDA for travelers, bodes well for Hai-tiV's immunogenicity in humans. Combining the immunogenicity data presented here with our finding that HaitiV can protect from cholera prior to the induction of adaptive immunity [29], suggests that HaitiV may function as a dual-acting agent, providing both rapid-onset short-term protection from disease while eliciting stable and long-lasting immunity against cholera.
Data from the challenge experiments (Fig 6) are consistent with the prevailing notion that serogroup, and to a lesser extent serotype are major determinants of protection against V. cholerae challenge [39,47]. Although it is thought that exposure to Inaba strains is more cross-protective than exposure to Ogawa strains, the relative potency of Inaba versus Ogawa vaccines in eliciting dual protection against both O1 serotypes requires further definition, as it has been suggested that both Ogawa and Inaba vaccine strains are good candidates for development [4][5][6][7]. A mixture of Ogawa and Inaba serotypes either as distinct strains or one bivalent strain (serotype Hikojima) may be beneficial in broadening the breadth of the immune response to HaitiV [48,49].
The modest protection that HaitiV immunization provided against V. cholerae O139 was unexpected. The epidemiology of the original O139 outbreak and experimental studies in rabbits demonstrate a lack of cross-protection between the two serogroups [14,15,39]. Notably, although pups born to HaitiV-immunized dams and challenged with MO10 survived longer than pups born to non-immunized dams, there was little difference in the MO10 intestinal colonization between these groups (Fig 6C). The discrepancy between clinical protection and relatively robust colonization suggests that HaitiV stimulates immune responses to V. cholerae factors, like CT, that may contribute to disease but not directly to colonization. The capacity of live OCVs to induce immune responses to in vivo-expressed antigens, including CtxB, is a property that heightens the appeal of live vs killed OCVs [27,50].
Although GF mice enabled us to test the protective efficacy of a candidate live OCV, their absence of the microbiota and resulting improper immune development are important caveats to consider. The GF model does not recapitulate the competitive microbial environment that indicated. Mixed-effect p-values were determined by one-way ANOVA analysis. Multiple comparison p-values for this data are reported in S1 Table. Although there were 7 mice per group, some samples were of insufficient volume for analysis.
https://doi.org/10.1371/journal.pntd.0007417.g004 live OCVs will encounter in the human host. We observed similar prolonged shedding patterns for both CVD-103HgR and HaitiV in the GF mice (Fig 1), yet CVD-103HgR is known to (black) were challenged with either the O1 Ogawa strain HaitiWT (A), the O1 Inaba strain N16961 (B) or the O139 strain MO10 (C). Panels on the left show survival curves for infected pups up to 48 hours post inoculation with the indicated strains; the number of pups challenged and the median survival time in hours is shown. P-values were determined by the Mantel-Cox method. Panels on the right show the intestinal WT V. cholerae burden in pups at the time of death (open circles) or at 48 hpi (closed circles).
Burden is plotted as CFU/SI. The dotted line marks the limit of detection (50 CFU/SI). P-values were determined by the Mann-Whitney U test.
https://doi.org/10.1371/journal.pntd.0007417.g006 be shed by human volunteers at a low frequency for a short period [25,51]. Thus, our findings likely overestimate HaitiV's capacity to colonize the human intestine. The observation that exposure of HaitiV-immunized dams to SPF-derived pups during the cross-fostering experiments led to the elimination of detectable HaitiV in feces supports the prediction that this vaccine will not stably colonize humans. Since we employed a multi-dose vaccination schedule here, it remains an open question whether transient exposure of naïve mice to HaitiV will also stimulate protective immunity, as has been shown in the context of vaccination with V. cholerae outer membrane vesicles [52,53]. The streptomycin-treated mouse model of V. cholerae colonization, which allows for temporary intestinal colonization, may also be useful to investigate the duration of colonization required for immunity [54]. Ultimately, the capacity of Hai-tiV to colonize the intestine and the relationship between colonization and protective immunity will need to be defined in human volunteers.
The immunogenicity of live OCVs in mice has only been investigated in GF animals because adult mice with intact microbiota are refractory to intestinal V. cholerae replication and colonization. However, previous studies of live OCVs in GF mice only analyzed immune correlates of protection and not protection against challenge [36,37]. The combination of the neonatal survival assay with the oral GF vaccination model builds on existing knowledge of these mice to assay both the immunogenicity and protective efficacy of live OCV candidates [38]. This model may be a useful addition to existing approaches that probe the molecular bases of vaccine-mediated mucosal protection against pathogens, a topic with significant translational potential that remains poorly understood [53,55]. A recent report employing a similar maternal-infant transmission model in the context of intraperitoneally-delivered heat-killed Citrobacter rodentium highlights the versatility of assessing vaccine protective efficacy using the infant progeny of immunized animals as readouts [56]. The broad availability of genetically engineered mice and the relative ease of GF-derivation provides a powerful opportunity to leverage both host and bacterial genetics to explore how live-OCVs can be optimized to better defend against this ancient pathogen.  (DOCX) S1 References. Supplemental references for S1 Table. (DOCX)