Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia
Abstract
:1. Introduction
2. Results
2.1. Studies Supporting Attenuation and Removal of SCHU S4 ΔclpB from the U.S. Select Agent List
2.2. Clearance of ΔclpB from Fischer Rat Tissues
2.3. ΔclpB Vaccine Production Optimization, Scale-Up, and Quality Control Testing
2.4. Long-Term Storage of ΔclpB
2.5. Quality Control and Lot Release Assays for Clinical Lots of ΔclpB
3. Discussion
4. Materials and Methods
4.1. Generation of ΔclpB and Evaluation of Its Potential for Reversion to Wild Type
4.2. Fermenter Growth of ΔclpB at NRC-C
4.3. Manufacturing of ΔclpB at the 25 L Scale at Ology Bioservices
4.4. Lyophilization of ΔclpB Drug Product
4.5. Analytical Assays
4.6. Biological Assays
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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ID Virulence for BALB/c Mice of 60 Distinct Single Gene Deletion Mutants of SCHU S4 | ||
---|---|---|
ID LD50 <30 CFUa ΔFtt | ID LD50 >102 but <106 CFU ΔFtt: | ID LD50 ≥ 107 CFU ΔFtt |
ahp b, capB, chiX, fadAD2, fadD2, feoB, gpx, hfq, katG, mdaB, oppD, oxyR c, pckA-L d, pdpC c,e, pdpD, pepO, PI1 c,g, PI2 b,g, PilAEV f, pilB, pilC, pilQ, PilT, pmrA c, pyrB, qseC b, RD5, RD8, recA, relA, rimK, sodC, tet, usp, 0023, 0024, 0029, 0069, 0086, fupB, 1023 c, 1149 b, 1564 b | pckA-S d, fupA c, ggt f | iglB, iglC, iglD, gplX, wbtC, wbtI, clpB f kdtA, lpcC, purF, sspA, mglA, FLT0439 g,h FTT0085, |
Criterion | LVS | ΔclpB | FSC033 a, SCHU S4 a (MTD) b |
---|---|---|---|
ID LD50 | >107 CFU | >107 CFU | <10 CFU (7) |
IN LD50 | ~103 CFU c | 104–106 CFU d | <10 CFU (6) |
% deaths following ~105 CFU ID e | 2.1 (n = 185) f | 1.4 (n = 560) f | 100 |
Necrosis score at site of injection | 2–3 g | 0–1 g | 4 g |
SCID mice MTD ID 103 CFU ID | 15 | 17 h | ND |
SCID mice MTD IN 102 CFU IN | 14 | 19 | ND |
Clinical signs given at 105 ID | Mild | Mild | severe |
Survival SCHU S4 20–100 CFU IN i | ≤20% | 60–100% i | NA |
Source of ΔclpB | % Survival after IN Inoculation with ~104 CFU | % Survival after IN Challenge with ~100 CFU SCHU S4 42 Days Later |
---|---|---|
Original stock | 100% | 100% |
10× in vitro 1 | 100% | 100% |
5× in vivo 2 | 100% | 100% |
Original stock | 100% | 100% |
5× in CDM 3,4 | 60% | 100% |
Fermenter CDM 5 | 40% | 100% |
Characteristic | |
---|---|
Time of harvest from flask | 20.75 h |
CFU/ml at harvest a | 3.9 × 109 |
Cryopreservative | Sucrose 10% w/v |
Colony morphology b | Typical of Ftt and Fth |
Gram stain c | Typical of Ftt and Fth |
Lytic phage | Negative |
Lysogenic phage | Negative |
Contaminant bacteria d | Negative |
Genomic sequence e | Identical to original mutant |
42-day survival of mice after 104 CFU IN administration (original stock/new stock) f | 100%/60% at day 42; NS h |
42-day survival of mice following 105 CFU ID administration (original stock/new stock) | 100%/100% |
Clinical signs g (original vs. new stock) | Significantly greater vs. old stock on days 3, 4, and 5 |
Skin reactogenicity g (original vs new stock) | NS h |
ΔclpB organ load (skin, spleen, liver, lung) 4 days after ID vaccination with 105 CFU of original vs. new stock. | ΔclpB lung burden was significantly higher (p = 0.016) for new vs. original stock |
Day 4 serum cytokine/chemokine levels (21-plex assay) (original vs. new stock) | NS h |
28-day survival against IN challenge with ~100 CFU SCHU S4 after IN vaccination (original/new stock) | 100%/100% |
Protection against IN challenge with ~100 CFU SCHU S4 after ID vaccination original/new stock (MTD in days) | 60% (28)/0% (16). NS h,i |
Protection against ID challenge with 105 CFU SCHU S4 or FSC033 after ID vaccination | 100%/100% |
Date of Run | Flask Starting Inoculum CFU/mL | Flask Harvest Concentration CFU/mL (Culture Time, h) | #Doublings (Doubling Time, h) | Fermenter Inoculum CFU/mL | Fermenter Harvest Time, h (CFU/mL) | #Doublings (Doubling Time, h) | Total CFU × 1014 (# Doses × 106) 1 | Biomass g |
---|---|---|---|---|---|---|---|---|
December 2014 | 4.0 × 107 | 5.2 × 109 (18.5) | 7 (2.6) | 3.90 × 106 | 22 (1.1 × 1010) | 12 (1.85) | 242 (24) | 177.3 |
September 2015 | 2.6 × 107 | 9.0 × 109 (18.25) | 8.4 (2.1) | 1.40 × 107 | 20.5 (1.1 × 1010) | 10 (2.3) | 242 (24) | 204.1 |
June 2017 | 7.8 × 106 | 9.0 × 109 (18.5) | 10.2 (1.8) | 1.43 × 107 | 26 (1.1 × 1010) | 10 (2.9) | 242 (24) | 74.9 2 |
Parameter | Test Method | Specification |
---|---|---|
clpB specific PCR | Identity confirmed a | |
Purity | Absence of contaminating organisms b | |
CFU/mL | Report result | |
Gram Stain | Gram negative coccobacillus | |
TBD c | Round, smooth and slightly mucoid single colonies | |
CFU/Ml d | Report result | |
LAL e | <5 EU/kg/dose | |
Susceptible to tetracycline, levofloxacin, gentamycin, chloramphenicol, ciprofloxacin, streptomycin, rifampin f | Susceptible | |
Karl Fisher g | Report Result | |
Appearance of cake h | Report result |
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Conlan, J.W.; Sjöstedt, A.; Gelhaus, H.C.; Fleming, P.; McRae, K.; Cobb, R.R.; De Pascalis, R.; Elkins, K.L. Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia. Pathogens 2021, 10, 795. https://doi.org/10.3390/pathogens10070795
Conlan JW, Sjöstedt A, Gelhaus HC, Fleming P, McRae K, Cobb RR, De Pascalis R, Elkins KL. Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia. Pathogens. 2021; 10(7):795. https://doi.org/10.3390/pathogens10070795
Chicago/Turabian StyleConlan, J. Wayne, Anders Sjöstedt, H. Carl Gelhaus, Perry Fleming, Kevan McRae, Ronald R. Cobb, Roberto De Pascalis, and Karen L. Elkins. 2021. "Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia" Pathogens 10, no. 7: 795. https://doi.org/10.3390/pathogens10070795
APA StyleConlan, J. W., Sjöstedt, A., Gelhaus, H. C., Fleming, P., McRae, K., Cobb, R. R., De Pascalis, R., & Elkins, K. L. (2021). Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia. Pathogens, 10(7), 795. https://doi.org/10.3390/pathogens10070795