Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia

J Conlan,H Gelhaus,Karen Elkins,Ronald Cobb,Kevan Mcrae,Roberto De Pascalis,Perry Fleming,Anders Sjöstedt

doi:10.3390/pathogens10070795

Abstract

Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005–2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 ΔclpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 ΔclpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 ΔclpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 ΔclpB to a degree sufficient for removal from the U.S. Select Agent list and for human use; optimizing SCHU S4 ΔclpB vaccine production, scale up, and long-term storage; and developing appropriate quality control testing approaches.

Highlights

Tularemia is the generic term for a spectrum of infectious diseases caused by the facultative intracellular bacterium, Francisella tularensis subspecies tularensis (Ftt), or F. tularensis subspecies holarctica (Fth) [1,2]
The results showed that fewer mice infected IN with ∆clpB either passaged in Chamberlain’s defined broth (CDB) or grown at scale in Chamberlains defined broth (CBD) survived infection with 104 colony forming units (CFU) as compared with the starting stocks grown at lab scale (Table 3, bottom half)
In the early half of the 20th century, Fth was responsible for large epidemics involving millions of cases in the former USSR [10]

Summary

Introduction

Tularemia is the generic term for a spectrum of infectious diseases caused by the facultative intracellular bacterium, Francisella tularensis subspecies tularensis (Ftt), or F. tularensis subspecies holarctica (Fth) [1,2]. 100 4g ND ND severe NA a Two distinct virulent strains of Ftt; b (MTD) = median time to death; c ~1 × 103 CFU of LVS IN invariably killed all BALB/c mice over a 20 year span at NRC-C; d inter- experimental range; e normal ID vaccination dose; f not always head-to-head comparisons; g score range 1–6 We evaluated the potential for ∆clpB to revert to a virulent phenotype For these experiments, death of BALB/c mice when given an IN dose of 104 CFU (the lowest LD50 reported for ∆clpB, Table 2) was considered to be to reflect gain of virulence.

Clearance of ΔclpB from Fischer Rat Tissues

Discussion

Findings

Materials and Methods