Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine

Westfall, Jennifer; Yates, Jennifer L.; Slyke, Greta Van; Ehrbar, Dylan; Measey, Thomas; Straube, Richard; Donini, Oreola; Mantis, Nicholas J.

doi:10.1016/j.vaccine.2018.08.059

Cited by 19 publications

(30 citation statements)

References 42 publications

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“…The SyH7 and IB2 capture ELISA with biotin-labeled RT. 36 Immulon 4HBX 96-well microtiter plates (ThermoFisher Scientific) were coated with SyH7 or IB2 MAbs (1 µg/ml in PBS) overnight at 4 o C. The plates were blocked with goat serum (2%), washed, and then overlaid with a mixture of biotinlabeled RT and serial dilutions of Rhesus macaque competitor serum. The amount of biotin-RT used in the competition ELISA was equivalent to the EC 90 for each capture MAb (100-150 ng/ml).…”

Section: Serum Antibody Analysismentioning

confidence: 99%

Passive immunization with an extended half-life monoclonal antibody protects Rhesus macaques against aerosolized ricin toxin

et al. 2020

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Inhalation of ricin toxin (RT), a Category B biothreat agent, provokes an acute respiratory distress syndrome marked by proinflammatory cytokine and chemokine production, neutrophilic exudate, and pulmonary edema. The severity of RT exposure is attributed to the tropism of the toxin's B subunit (RTB) for alveolar macrophages and airway epithelial cells, coupled with the extraordinarily potent ribosome-inactivating properties of the toxin's enzymatic subunit (RTA). While there are currently no vaccines or treatments approved to prevent RT intoxication, we recently described a humanized anti-RTA IgG 1 MAb, huPB10, that was able to rescue non-human primates (NHPs) from lethal dose RT aerosol challenge if administered by intravenous (IV) infusion within hours of toxin exposure. We have now engineered an extended serum half-life variant of that MAb, huPB10-LS, and evaluated it as a pre-exposure prophylactic. Five Rhesus macaques that received a single intravenous infusion (25 mg/kg) of huPB10-LS survived a lethal dose aerosol RT challenge 28 days later, whereas three control animals succumbed to RT intoxication within 48 h. The huPB10-LS treated animals remained clinically normal in the hours and days following toxin insult, suggesting that pre-existing antibody levels were sufficient to neutralize RT locally. Moreover, pro-inflammatory markers in sera and BAL fluids collected 24 h following RT challenge were significantly dampened in huPB10-LS treated animals, as compared to controls. Finally, we found that all five surviving animals, within days after RT exposure, had anti-RT serum IgG titers against epitopes other than huPB10-LS, indicative of active immunization by residual RT and/or RT-immune complexes.

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Section: Serum Antibody Analysismentioning

confidence: 99%

Passive immunization with an extended half-life monoclonal antibody protects Rhesus macaques against aerosolized ricin toxin

et al. 2020

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“…In one instance the mAbs were used as tools in competition ELISAs to demonstrate epitope use within humans and non-human primates vaccinated with RiVax [14]. More recently, the mAbs were used to evaluate the integrity of key neutralizing epitopes on RiVax during long-term storage [25]. The 21 V H Hs described here focused around RTA’s active site now add to that growing list of critical reagents.…”

Section: Discussionmentioning

confidence: 99%

“…Our long-term goal is to generate a comprehensive molecular B-cell epitope map of each of these clusters and define the specific antibody-contact points on RTA that render the toxin inactive. Such information will be invaluable in efforts to deconvolute the complex human antibody response profile to ricin toxin and RiVax [25].…”

Section: Introductionmentioning

confidence: 99%

A Collection of Single-Domain Antibodies that Crowd Ricin Toxin’s Active Site

et al. 2018

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In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot (“cluster 3”) located in close proximity to RTA’s active site. HX-MS analysis revealed that the 21 VHHs recognized four distinct epitope subclusters (3.1−3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing a-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1−3.3 physically occlude RTA’s active site cleft, while the single antibody in subcluster 3.4 associates on the active site’s upper rim.

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“…Mouse experiments were conducted in strict accordance with protocol #18-384 approved and RT challenges were carried out as reported previously [22]. Mice were immunized with RiVax ® (0.3, 1 or 3 µg) administered SC on days 0 (prime) and 21 (boost) [22]. Sera were harvested by submandibular bleeding on day 30.…”

Section: Mouse Vaccination and Rt Challenge Studiesmentioning

confidence: 99%

Endpoint and Epitope-specific Antibody Responses as Correlates of Vaccine-mediated Protection of Mice against Ricin Toxin

Slyke

Ehrbar

Doering

et al. 2020

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25The successful licensure of vaccines for biodefense is contingent upon the availability of 26 well-established correlates of protection (CoP) in at least two animal species that can then be 27 applied to humans, without the need to assess efficacy in the clinic. In this report we describe a 28 multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values 29 derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in 30 mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a 31 modified competition ELISA in which serum samples from vaccinated mice were assessed for 32 their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized 33 monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions 34 on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n=40) vaccinated with 35 suboptimal doses of the RTA subunit vaccine, RiVax ® , we identified two mAbs, PB10 and 36 SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with 37 survival following a challenge with 10 x LD50 of RT administered by intraperitoneal (IP) 38 injection. Analysis of a larger cohort of mice (n=645) revealed that a multivariate model 39 combining endpoint titers and an epitope-profiling immune-competition capture (EPICC) assay 40 values for PB10 and SyH7 as predictive variables had significantly higher statistical power than 41 any one of the independent variables alone. Establishing the correlates of vaccine-mediated 42 protection in mice represents an important steppingstone in the development of RiVax ® as a 43 medical countermeasure under the United States Food and Drug Administration's "Two Animal 44 Rule." 45 46 47 48 Abbreviations: Enzyme-linked immunosorbent assay (ELISA), epitope profiling immune-49 competition capture (EPICC); Monoclonal antibody (mAb); biotinylated (b), ricin toxin (RT), 50 RT A chain (RTA) 51 3 52 1. Introduction 53 The development of vaccines to counteract biothreats, including toxins, remains a high 54 priority in many countries, including the United States [1, 2]. There are, however, formidable 55 challenges to development and licensure [3]. Foremost is the need to assess vaccine efficacy 56 (VE) in humans in the absence of clinical outcomes, because conventional efficacy studies are 57 not ethical and field trials are not feasible for most if not all the Category A and B Biothreats. 58 Under the Two Animal Rule, however, the United States Food and Drug Administration (FDA) 59 will evaluate VE based on "adequate and well-controlled studies in animal models of the human 60 disease or condition of interest" [4]. Of utmost importance are well-established and robust 61 correlates of protection (CoP) that apply across species and and can be applied to humans. The 62 undertaking of cross-species bridging studies has been successfully applied to anthrax vaccine 63 adsorbed (AVA) to estimate survival probabili...

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Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine

Cited by 19 publications

References 42 publications

Passive immunization with an extended half-life monoclonal antibody protects Rhesus macaques against aerosolized ricin toxin

Passive immunization with an extended half-life monoclonal antibody protects Rhesus macaques against aerosolized ricin toxin

A Collection of Single-Domain Antibodies that Crowd Ricin Toxin’s Active Site

Endpoint and Epitope-specific Antibody Responses as Correlates of Vaccine-mediated Protection of Mice against Ricin Toxin

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