Select Agents and Toxins Exclusions:
Excluded Attenuated Strains of HHS Select Agents, Section 73.3 (e)

Clostridium botulinum Beluga Ei strain (effective 12-12-2019)
The C. botulinum Beluga Ei strain was constructed using the CRISPR-Cas9 tool. Mutations were introduced into the toxin sequence to alter the amino acid residues (E213A/R348A/Y351F) responsible for zinc-binding and hydrolytic domains localized within the light chain, resulting in a loss of toxin biological activity. Mutation stability tests confirmed that the introduced mutations were stable and unlikely to revert to wild-type genotype. BoNT/Ei produced by the Clostridium botulinum Beluga Ei strain showed no biological activity by the mouse bioassay when tested at the equivalent of 22,113 mLD₅₀ of active trypsinized BoNT/E. No signs of paralysis were observed when 25 nM of BoNT/Ei was tested by the mouse phrenic nerve hemidiaphragm assay.

Reference(s):

1. Binz T, Bade S, Rummel A, Kollewe A, Alves J. Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization. Biochemistry. 2002 Feb 12;41(6):1717-23.
2. Matsui T et al. Structural basis of the pH-dependent assembly of a botulinum neurotoxin complex. J Mol Biol. 2014 Nov 11;426(22):3773-3782.
3. Ingle et al. Generation of a fully erythromycin sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system. Sci Rep. 2019 May 31;9(1):8123.
4. Simon S et al. Recommended immunological strategies to screen for botulinum neurotoxin containing samples. Toxins (Basel). 2015 Nov 26;7(12):5011-34.
5. Duff JT, Wright GG, Yarinsky A. Activation of Clostridium botulinum type E toxin by trypsin. J Bacteriol. 1956 Oct;72(4):455-60.
6. Centers for Disease Control and Prevention: Botulism in the United States, 1899–1996. Handbook for epidemiologists, clinicians, and laboratory workers, Atlanta: Centers for Disease Control and Prevention, 1998. https://www.cdc.gov/botulism/pdf/bot-manual.pdf
7. Bigalke H and Rummel A. Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN). Toxins (Basel). 2015 Nov 25;7(12):4895-905.

Coxiella burnetii Phase II, Nine Mile Strain, plaque purified clone 4 (effective 10-15-2003)
Lipopolysaccharide (LPS) phase variation is the only confirmed virulence factor of C. burnetii. Organisms isolated from natural infections or in the laboratory are in phase I and have a smooth-type LPS. Repeated passage of phase I organisms through embryonated eggs or cultured cells resulted in the conversion to phase II and a change in the LPS to a rough-type. Based upon consultations with subject matter experts and a review of relevant published studies, HHS has determined that Coxiella burnetii, Phase II, Nine Mile Strain, plaque purified clone 4, does not pose a significant threat to human or animal health.

*Note: Coxiella burnetii Phase II, Nine Mile Strain, plaque purified clone 4 with reversion to wildtype cbu0533 is a select agent and subject to select agent regulations [5].  This revertant strain displayed an intermediate length LPS phenotype under certain growth conditions and was capable of causing infection in guinea pigs and can be isolated from their spleens.

Reference(s):

1. O’Rourke AT, Peacock M, Samuel JE, Frazier ME, Natvig DO, Mallavia LP, Baca O. Genomic analysis of phase I and II Coxiella burnetii with restriction endonucleasesexternal icon. J. Gen. Microbiol. 1985 June; 131(6):1543-46.
2. Vodkin MH, Williams JC, Stephenson EH. Genetic heterogeneity among isolates of Coxiella burnetii. J. Gen. Microbiolexternal icon. 1986 Feb; 132(2):455-63.
3. Moos A, Hackstadt T. Comparative virulence of intra- and interstrain lipopolysaccharide variants of Coxiella burnetii in the guinea pig modelexternal icon. Infect. Immun. 1987 May; 55(5):1144-50.
4. Beare PA, Jeffrey BM, Long CM, Martens CM, Heinzen RA. Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation. PLoS Pathog. 2018;14(3):e1006922. Published 2018 Feb 26. doi:10.1371/journal.ppat.1006922
5. Federal Register Notice: Select Agent Determination Concerning Coxiella burnetii Phase II, Nine Mile Strain, Plaque Purified Clone 4 With Reversion to Wildtype cbu0533. August 10, 2023: https://www.federalregister.gov/d/2023-16929

South American genotypes of Eastern Equine Encephalitis virus (effective 12-4-2012)
The final rule (77 FR 194, published on October 5, 2012) excluded the South American genotypes of Eastern Equine Encephalitis virus (SA EEEV) effective December 4, 2012. South American genotypes are typically avirulent for humans and are not clearly linked to human disease. However, North American EEE virus (NA-EEE) genotype strains, which are the strains responsible for human and equine disease, are currently regulated as select agents and can be easily distinguished from SA EEEV genotype strains by sequencing.

Reference(s):

1. Arrigo NC, Adams AP, Weaver SC. Evolutionary patterns of eastern equine encephalitis virus in North versus South America suggest ecological differences and taxonomic revision. J Virol. 2010 Jan; 84(2):1014-25.

Ebola ΔVP30 replication incompetent virus (effective 01-02-2013)
This virus lacks the gene encoding for the VP30 protein; therefore, biologically contained Ebola virus (Ebola ΔVP30 viruses) are replication incompetent and do not form infectious progeny in wild-type cells due to the lack of the VP30 gene. The genome of Ebola ΔVP30 virus is stable, infection of Vero cells and various animals with Ebola ΔVP30 virus particles did not indicate a single event of virus replication, infection of animals with Ebola ΔVP30 virus particles did not cause disease in infected animals.

Reference(s):

1. Halfmann P, Kim JH , Ebihara H , Noda T, Neumann G , Feldmann H , Kawaoka Y. Generation of biologically contained Ebola viruses. Proc Natl Acad Sci U S A. 2008 Jan 29;105(4):1129-33.
2. Halfmann P, Ebihara H, Marzi A, Hatta Y, Watanabe S, Suresh M, Neumann G, Feldmann H, Kawaoka Y. Replication-deficient ebolavirus as a vaccine candidate. J Virol. 2009 Apr;83(8):3810-5.

Francisella tularensis subspecies tularensis, SCHU S4∆clpB strain (effective November 10, 2014)
Francisella tularensis subspecies tularensis, SCHU S4ΔclpB strain contains a deletion of the clpB chaperone, an important contributor to F. tularensis intracellular growth and virulence. This strain is more attenuated than LVS in mice against respiratory challenge when compared to the wild-type SCHU S4 and fails to revert after in vivo passage or after co-culturing with a different mutant strain that possesses the clpB gene.

Reference(s):

1. Conlan JW, Shen H, Golovliov I, Zingmark C, Oyston PC, Chen W, House RV, Sjöstedt A. Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization. Vaccine. 2010 Feb 17;28(7):1824-31. doi: 10.1016/j.vaccine.2009.12.001. Epub 2009 Dec 16.
2. Golovliov I, Twine SM, Shen H, Sjostedt A, Conlan W.A ΔclpB mutant of Francisella tularensis subspecies holarctica strain, FSC200, is a more effective live vaccine than F. tularensis LVS in a mouse respiratory challenge model of tularemia. PLoS One. 2013 Nov 13;8(11):e78671. doi: 10.1371/journal.pone.0078671.
3. Meibom KL, Dubail I, Dupuis M, Barel M, Lenco J, Stulik J, Golovliov I, Sjöstedt A, Charbit A. The heat-shock protein ClpB of Francisella tularensis is involved in stress tolerance and is required for multiplication in target organs of infected mice. Mol Microbiol. 2008 Mar;67(6):1384-401. doi: 10.1111/j.1365-2958.2008.06139.x. Epub 2008 Feb 15.
4. Ryden P, Twine S, Shen H, Harris G, Chen W, Sjostedt A, Conlan W.Correlates of protection following vaccination of mice with gene deletion mutants of Francisella tularensis subspecies tularensis strain, SCHU S4 that elicit varying degrees of immunity to systemic and respiratory challenge with wild-type bacteria. Mol Immunol. 2013 May;54(1):58-67. doi: 10.1016/j.molimm.2012.10.043. Epub 2012 Nov 28.
5.  Shen H, Harris G, Chen W, Sjostedt A, Ryden P, Conlan W. Molecular immune responses to aerosol challenge with Francisella tularensis in mice inoculated with live vaccine candidates of varying efficacy. PLoS One. 2010 Oct 12;5(10):e13349. doi: 10.1371/journal.pone.0013349.
6. Su J, Yang J, Zhao D, Kawula TH, Banas JA, Zhang JR. Genome-wide identification of Francisella tularensis virulence determinants. Infect Immun. 2007 Jun;75(6):3089-101. Epub 2007 Apr 9.
7. Twine S, Shen H, Harris G, Chen W, Sjostedt A, Ryden P, Conlan W. BALB/c mice, but not C57BL/6 mice immunized with a ΔclpB mutant of Francisella tularensis subspecies tularensis are protected against respiratory challenge with wild-type bacteria: association of protection with post-vaccination and post-challenge immune responses.Vaccine. 2012 May 21;30(24):3634-45. doi: 10.1016/j.vaccine.2012.03.036. Epub 2012 Apr 3.

All Francisella tularensis subspecies novicida (also referred to as Francisella novicida) strains and F. novicida-like strains (effective November 10, 2014)
Francisella novicida species are phylogenticially distinct from Francisella tularensis species; therefore, F. novicida and F. novicida-like strains are not subject to the requirements of the select agent regulations. F. tularensis evolved independently of F. novicida, with a completely distinct ecological niche and mechanism of transmission. In addition, symptoms associated with tularemia have not been observed for F. novicida infections in healthy individuals.Based on laboratory animal model studies, significant virulence differences between F. tularensis and F. novicida were evident upon pulmonary infection of Fischer 344 rats via intratracheal instillation, where the LD50 of F. tularensis subsp tularensis is ~5 x 102 CFU while F. novicida is 5 x106 CFU.In addition, F. novicida very rarely causes human illness and infections that do occur are associated primarily with patients who are immune compromised or have other underlying health problems.

Reference(s):

1. Barns SM, Grow CC, Okinaka RT, Keim P, Kuske CR. Detection of diverse new Francisella-like bacteria in environmental samples. Appl Environ Microbiol. 2005 Sep;71(9):5494-500
2. Kingry, L.C and Petersen J.M. (2014). Comparative review of Francisella tularensis and Francisella novicida. Frontier in Cell and Infect Microbiol. 4:35
3. Larsson P., Elfsmark D., Svensson K., Wikström P., Forsman M., Brettin T., et al. (2009). Molecular evolutionary consequences of niche restriction in Francisella tularensis, a facultative intracellular pathogen. PLoS Pathog. 5:e1000472 10.1371/journal.ppat.1000472
4. Ray H. J., Chu P., Wu T. H., Lyons C. R., Murthy A. K., Guentzel M. N., et al. (2010). The Fischer 344 rat reflects human susceptibility to Francisella pulmonary challenge and provides a new platform for virulence and protection studies. PLoS ONE 5:e9952 10.1371/journal.pone.0009952

Francisella tularensis subspecies novicida (also referred to as Francisella novicida) strain, Utah 112 (ATCC 15482) (effective 2-27-2003)
The exclusion is only for the type strain, Utah 112. This strain was originally isolated from a water sample taken from Ogden Bay, Utah in 1951. It is experimentally pathogenic for mice, guinea pigs and hamsters, producing lesions similar to those of tularemia; rabbits, white rats and pigeons are resistant. The Utah 112 strain is not known to infect man.

Reference(s):

1. Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin. Microbiol.Rev. 2002 Oct; 15(4): 631-46.

Francisella tularensis subspecies holarctica LVS (live vaccine strain; includes NDBR 101 lots, TSI-GSD lots, and ATCC 29684) (effective 2-27-2003)
This strain is used for studies on the genetics, biology, and pathogenesis of F. tularensis , studies on the host response to infection, development and use in diagnostic assays, and development of vaccines and therapeutics.

Reference(s):

1. Burke DS. Immunization against tularemia : analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J Infect Dis. 1977 Jan;135(1):55-60.
2. Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin. Microbiol.Rev. 2002 Oct; 15(4): 631-46.
3. Waag DM, Galloway A, Sandstrom G, Bolt CR, England MJ, Nelson GO, Williams JC. Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis. J Clin Microbiol. 1992 Sep;30(9):2256-64.
4. Waag DM, McKee KT Jr, Sandstrom G, Pratt LL, Bolt CR, England MJ, Nelson GO, Williams JC. Cell-mediated and humoral immune responses after vaccination of human volunteers with the live vaccine strain of Francisella tularensis. Clin Diagn Lab Immunol. 1995 Mar;2(2):143-8.
5. Waag DM, Sandström G, England MJ, Williams JC. Immunogenicity of a new lot of Francisella tularensis live vaccine strain in human volunteers. FEMS Immunol Med Microbiol. 1996 Mar;13(3):205-9.

Francisella tularensis biovar tularensis strain B-38 (ATCC 6223) (effective 2-27-2003)
This strain has fastidious growth requirements and grows poorly in the laboratory. Mice are used as a model to study the pathogenesis of tularemia. The LD 50 of virulent strains of F. tularensis biovar tularensis for mice infected via the subcutaneous route is <10 CFU. However, mice infected intraperitoneally with 10⁵ CFU or intradermally with 10⁷ CFU of strain ATCC 6223 were not killed.

Reference(s):

1. Ellis J, Oyston PCF, Green M, Titball RW. Tularemia. Clin. Microbiol. Rev. 2002 Oct; 15(4):631-46.

Junín virus vaccine strain Candid No. 1 (effective 02-07-2003)
The protective efficacy of Candid No. 1, a live-attenuated vaccine against Argentine hemorrhagic fever (AHF), was evaluated in non-human primates. Twenty rhesus macaques immunized 3 months previously with graded doses of Candid No. 1 (16-127,000 PFU), as well as 4 placebo-inoculated controls, were challenged with 4.41 log 10 PFU of virulent P3790 strain Junín virus. All controls developed severe clinical disease; 3 of 4 died. In contrast, all vaccinated animals were fully protected; none developed any signs of AHF during a 105 day follow-up period. Candid No. 1 was highly immunogenic and fully protective against lethal virus challenge in rhesus macaques, even at extremely low (16 PFU) vaccine doses.

Reference(s):

1. McKee KT Jr, Oro JG, Kuehne AI, Spisso JA, Mahlandt BG. Candid No. 1 Argentine hemorrhagic fever vaccine protects against lethal Junín virus challenge in rhesus macaques. Intervirology. 1992: 34(3):154-63.

Mopeia/Lassa (MOP/LAS) arenavirus construct ML-29 (effective 03-02-2005)
The construct is not capable of encoding infectious and/or replication competent forms of any select agent viruses. Clone ML29, selected from a library of MOPV/LASV (MOP/LAS) reassortants, encodes for the major antigens (nucleocapsid and glycoprotein) of LASV and the RNA polymerase and zinc-binding protein of MOPV. Replication of clone ML29 was attenuated in guinea pigs and nonhuman primates. In murine adoptive-transfer experiments, as little as 150 PFU of clone ML29 induced protective cell-mediated immunity. All strain 13 guinea pigs vaccinated with clone ML29 survived at least 70 days after LASV challenge without either disease signs or histological lesions. Rhesus macaques inoculated with clone ML29 developed primary virus-specific T cells capable of secreting gamma interferon in response to homologous MOP/LAS and heterologous MOPV and lymphocytic choriomeningitis virus. Detailed examination of two rhesus macaques infected with this MOPV/LAS reassortant revealed no histological lesions or disease signs.

Reference(s):

1. Kiley MP, Lange JV, Johnson KM. Protection of rhesus monkeys from Lassa virus by immunization with closely related Arenavirus. Lancet. 1979 Oct 6; 314(8145):738.
2. Lange JV, Mitchell SW, McCormick JB, Walker DH, Evatt BL, Ramsey RR. Kinetic study of platelets and fibrinogen in Lassa virus -infected monkeys and early pathologic events in Mopeia virus -infected monkeys. Am J Trop Med Hyg. 1985 Sep; 34(5):999-1007.
3. Lukashevich IS, Patterson J, Carrion R, Moshkoff D, Ticer A, Zapata J, Brasky K, Geiger R, Hubbard GB, Bryant J, Salvato MS. A live attenuated vaccine for Lassa fever made by reassortment of Lassa and Mopeia viruses. J Virol. 2005 Nov; 79(22):13934-42.
4. Peters CJ , Jahrling PB , Liu CT, Kenyon RH, McKee KT Jr, Barrera Oro JG. Experimental studies of arenaviral hemorrhagic fevers. Curr Top Microbiol Immunol. 1987; 134:5-68.

West African clade of monkeypox virus (effective 12-4-2012)
Clinical and laboratory studies indicate that the Congo Basin clade is significantly more pathogenic to humans and animals than the West African clade. The 37 confirmed cases of human Monkeypox associated with the 2003 importation of a West African strain from Ghana into the United States were associated with no case-fatalities and no observed chain of human-to-human transmission. Clinically severe human disease associated with West African strains is rare and this virus clade has not been associated with human mortality.

Reference(s):

1. Chen N., et al. 2005. Virulence differences between monkeypox virus isolates from West Africa and the Congo basin. Virology 340:46–63.
2. Hutson C. L., et al. 2009. A prairie dog animal model of systemic orthopoxvirus disease using West African and Congo Basin strains of monkeypox virus. J. Gen. Virol. 90:323–333.
3. Saijo M., et al. 2009. Virulence and pathophysiology of the Congo Basin and West African strains of monkeypox virus in non-human primates. J. Gen. Virol. 90:2266–2271.
4. Sbrana E., Xiao S. Y., Newman P. C., Tesh R. B. 2007. Comparative pathology of North American and central African strains of monkeypox virus in a ground squirrel model of the disease. Am. J. Trop. Med. Hyg. 76:155–164.
5. Likos AM, Sammons SA, Olson VA, Frace AM, Li Y, Olsen-Rasmussen M, Davidson W, Galloway R, Khristova ML, Reynolds MG, Zhao H, Carroll DS, Curns A, Formenty P, Esposito JJ, Regnery RL, Damon IK. A tale of two clades: monkeypox viruses. J Gen Virol. 2005 Oct; 86(Pt 10):2661-72.

NATtrol^TM treated SARS-CoV molecular controls (effective February 8, 2013)
NATtrol^TM Coronavirus-SARS Stock is designed to evaluate the performance of nucleic acid tests for determination of the presence of Coronvavirus-SARS RNA. SARS-CoV treated with NATtrolTM, a paraformaldehyde treatment (PFA), inactivates SARS-CoV. Inactivation was verified by the absence of viral growth in validated culture based infectivity assays (data not published). This exclusion also pertains to the RNA genome from NATrolTM treated SARS-CoV.

Yersinia pestis CO92 strain ΔlppΔmsbBΔpla (effective 05-01-2019)
This mutant strain is completely deleted (in-frame) for three genes, namely lpp, msbB, and pla which code for Braun Lipoprotein (Lpp), an acetyltransferase (MsbB), and the plasminogen activator (Pla) protease. The pneumonic plague mouse model data indicated that the triple mutant is highly attenuated (100% animal survival) at high infectious doses (2.5 x 10⁶ CFU and 5.0 x 10⁶ CFU doses of the triple mutant CO92 strain, representing 5,000 and 10,000 LD₅₀ of the wild-type bacterium). This triple mutant strain remained highly attenuated even when administered by other routes (e.g., subcutaneous). Mice remained completely free of the disease.

Reference(s):

1. Agar SL, Sha J, Baze WB, Erova TE, Foltz SM, Suarez G, Wang S and Chopra AK. 2009. Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague. Microbiology. 155:3247-59. PMID:19589835
2. van Lier CJ, Sha J, Kirtley ML, Cao A, Tiner BL, Erova TE, Cong Y, Kozlova EV, Popov VL, Baze WB, Chopra AK. 2014. Deletion of Braun lipoprotein and plasminogen activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague. Infect. Immun. 82: 2485-2503; PMID:24686064
3. van Lier CJ, Tiner BL, Chauhan S, Motin VL, Fitts EC, Huante MB, Endsley JJ, Ponnusamy D, Sha J,Chopra AK. 2015 Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages. Microb Pathog.80:27-38. PMID: 25697665.
4. Tiner BL, Sha J, Cong YZ, Kirtley ML, Andersson JA, Chopra AK. 2016. Immunization of two rodent species with new live-attenuated mutants of Yersinia pestis CO92 induces protective long-term humoral and cell-mediated immunity against pneumonic plague. npj Vaccines 1: 16020 doi:10.1038/npjvaccines.2016.20

Yersinia pestis CO92 triple mutant ΔlppΔmsbBΔail (effective May 19, 2016)
Studies demonstrated that the triple mutant is significantly attenuated (100% animal survival) at high infectious doses (2.0-3.4 x10⁶ CFU which is equivalent to 4,000-6,800 LD50 of wild-type Y. pestis CO92) in a pneumonic plague mouse model.

Reference(s):

1. Tiner BL et al. Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague. Clin Vaccine Immunol. 2015 Dec;22(12):1255-68. doi: 10.1128/CVI.00499-15. Epub 2015 Oct 7
2. Tiner BL et al. Combinational deletion of three membrane protein-encoding genes highly attenuated Yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague. Infect Immun. 2015 Apr;83(4):1318-38. doi: 10.1128/IAI.02778-14. Epub 2015 Jan 20

Yersinia pestis strains which are Pgm^– due to a deletion of a 102-kb region of the chromosome termed the pgm locus (i.e., Δpgm). Examples are Y. pestis strain EV or various substrains such as EV 76 (effective 3-14-2003)
Pgm^– mutants of Yersinia pestis occur at a high frequency (ca 10^-5) and result in avirulence and Pgm^– strains such as the EV 76 strain. These strains have been used for years as live human vaccines with no significant plague-associated problems. The mutation in question is due to the excision of about 102-kb of chromosomal DNA via reciprocal recombination between adjacent IS 100 elements. The lost DNA sequence encodes the ability to synthesize and utilize the siderophore yersiniabactin, which is necessary for growth in mammalian peripheral tissue, as well as the Hms⁺ locus, which is necessary for biofilm production in the flea vector. However, PCR and/or Southern blot analysis will be required to ensure that “Pgm^– ” derivatives have undergone this deletion rather than a mutation in the hemin storage genes (hms), which also causes loss of Congo red (CR) binding, which is the most common characteristic used to evaluate the pigmentation phenotype.

Reference(s):

1. Brubaker RR. Mutation rate to nonpigmentation in Pasteurella pestis. J. Bacteriol. 1969 June; 98(3):1404-06.
2. Fetherston JD, Scheutze P, Perry RD. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol. Microbiol. 1992 Sept; 6(18):2693-04.
3. Bearden SW, Perry RD. The Yfe system of Yersinia pestis transports iron and manganese and is required for full virulence of plague. Mol. Microbiol. 1999 Apr; 32(2):403-14.
4. Une, T, Brubaker BB. In vivo comparison of avirulent Vwa – and Pgm – or Pstr phenotypes of Yersiniae. Infect. Immun.1984 Mar; 43(3):895-00.

Yersinia pestis strains (e.g., Tjiwidej S and CDC A1122) devoid of the 75 kb low-calcium response (Lcr) virulence plasmid (effective 2-27-2003)
Strains of Yersinia pestis that lack the 75 kb low-calcium response (Lcr) virulence plasmid are excluded. Strains lacking the Lcr plasmid (Lcr^–) are irreversibly attenuated due to the loss of a virulence plasmid. An Lcr^– strain of Yersinia pestis (Tjiwidej S) has been extensively used as a live vaccine in humans in Java.

Reference(s):

1. Meyer KF, Cavanaugh DC, Bartelloni PJ, Marshal JD Jr. Plague immunization. I. Past and present trends. J. Infect. Dis. 1974 May; 129 (suppl1): S13-S18.