ReviewBiochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: Use as a conjugation protein in vaccines and other potential clinical applications
Highlights
► Cross-reacting material 197 is a nontoxic mutant of diphtheria toxoid with clinical applications. ► CRM197 is a common carrier for polysaccharide vaccines against various pathogens. ► This protein also has applications in cancer and other clinical settings.
Introduction
During the last 30 years, the development and manufacture of many routine childhood and adult vaccines has included protein carriers to improve the immunogenicity of capsular polysaccharides of bacterial pathogens such as Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis serogroups A, C, W-135, and Y. The capsular polysaccharides of these organisms are either poorly immunogenic or unable to induce a response in the immature immune systems of infants and young children. With conjugate vaccines, the use of a carrier protein greatly enhances the immunogenicity of polysaccharide antigens enabling host defense against disease caused by encapsulated pathogens. As opposed to classical polysaccharide vaccines, these conjugate formulations induce T cell-dependent responses and hence have provided immunogenicity in infants as well as anamnestic responses and herd effects, or herd protection, on a population basis [1], [2], [3], [4], [5], [6].
The first carrier proteins were diphtheria and tetanus toxoids, each of which had been shown to protect individuals against disease when used as vaccine antigens. Consequently, extensive information about the immune potential and safety profile of these carriers in clinical use was well-known before the development of the first conjugate vaccines [7]. At least five carrier proteins or protein complexes are currently in use, including four purified proteins: tetanus toxoid (TT), diphtheria toxoid (DT), cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin, and protein D, which is derived from non-typeable H. influenzae—as well as the outer membrane protein complex (OMPC) derived from N. meningitidis [7] (Table 1). We describe the biochemical and biological properties of CRM197 and the implications of its use in vaccines and as a therapy in other clinical settings.
Glycoconjugate vaccines are semi-synthetic products created through multiple chemical reactions. The glycoconjugates themselves can differ based on the chemistry used in their preparation, including attachment positions. Other factors, such as saccharide chain length and the saccharide-to-protein ratio vary from product to product as do pH and the composition of storage buffer in final formulations [8]. Biochemical formulation (Fig. 1) may involve different lengths of polysaccharides or oligosaccharides, different procedures for downsizing, de-O-acetylation, direct or indirect linkage by a spacer molecule such as adipic acid, and/or the addition of adjuvants [9], [10]. The detailed physicochemical characterization of the saccharide components and carrier proteins permits consistent production of well-defined glycoconjugates [8]. Appropriate release tests have been developed by the manufacturers to guarantee reliable immunogenicity of the vaccines, and ongoing studies have attempted to evaluate the potential clinical differences between protein carriers [7], [11].
Section snippets
Basic characteristics
The diphtheria toxin gene is carried in the family of lysogenic phages known as corynebacteriophages [12]. The gene coding for diphtheria toxin, an acidic, globular, 58,350 Da protein, is contained in a 1850 base pair EcoRI-HindIII DNA fragment, and DNA sequences of tox genes from different phages show a high level of conservation of the diphtheria toxin molecule. As previously reviewed, integration of the bacteriophage genome into the otherwise non-virulent C. diphtheriae converts it to a
Genealogy of CRM197
The isolation of various non-toxic or partially toxic immunologically cross-reacting forms of diphtheria toxin (CRMs) began in 1971 before the development of recombinant DNA technology. The mutant CRM197 was isolated in 1973 [23], [24] and further optimization and conversion to a production strain were completed in the mid 1980s.
Characteristics of CRM197
The lack of toxicity of CRM197 has been evaluated using lethality tests in guinea pigs, cytotoxic activity assays on HeLa cells and Vero cells, and an ADP-ribosyltransferase enzymatic assay [18], [29]. An assay to quantify particular gene alleles in DNA mixtures was developed to estimate the relative amount of the CRM197 allele in C. diphtheriae culture samples using a quantitative restriction fragment length polymorphism (RFLP) assay in combination with polymerase chain reaction (PCR) [28].
Experience with CRM197
CRM197 has a variety of clinical applications [3], [10], [32], [34], [41], [42], [43] (Table 3).
Discussion
We described the general features of CRM197, a commonly used carrier protein in marketed conjugate vaccines. When choosing a suitable carrier protein for the development of a new conjugate vaccine, CRM197 should be considered as a useful candidate that can provide several potential benefits. Its high level of purity, homogeneous structure and availability of lysine residues, in conjunction with a specific conjugation chemistry, allow the production of well-defined and characterized
Contributors
All authors contributed to drafting the manuscript. MB substantially developed the first draft of the manuscript. Critical feedback was provided by all authors. All authors have approved the final manuscript.
Conflicts of interest
All authors are employed by Novartis Vaccines and Diagnostics.
Acknowledgments
This work was funded by Novartis Vaccines and Diagnostics. Sarah Angus of Alpharmaxim Healthcare Communications provided referencing and graphics support and drafted an early version of several sections of the manuscript. Theodore Tsai, MD provided guidance on the conceptual design of the manuscript.
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