Cross-reacting Material 197 Research Articles

Adjuvanted Vaccine Induces Functional Antibodies against Pseudomonas aeruginosa Filamentous Bacteriophages.

Pseudomonas aeruginosa (Pa), a WHO priority 1 pathogen, resulted in approximately 559,000 deaths globally in 2019. Pa has a multitude of host-immune evasion strategies that enhance Pa virulence. Most clinical isolates of Pa are infected by a phage called Pf that has the ability to misdirect the host-immune response and provide structural integrity to biofilms. Previous studies demonstrate that vaccination against the coat protein (CoaB) of Pf4 virions can assist in the clearance of Pa from the dorsal wound model in mice. Here, a consensus peptide was derived from CoaB and conjugated to cross-reacting material 197 (CRM197). This conjugate was adjuvanted with a novel synthetic Toll-like receptor agonist (TLR) 4 agonist, INI-2002, and used to vaccinate mice. Mice vaccinated with CoaB-CRM conjugate and INI-2002 developed high anti-CoaB peptide-specific IgG antibody titers. Direct binding of the peptide-specific antibodies to whole-phage virus particles was demonstrated by ELISA. Furthermore, a functional assay demonstrated that antibodies generated from vaccinated mice disrupted the replicative cycle of Pf phages. The use of an adjuvanted phage vaccine targeting Pa is an innovative vaccine strategy with the potential to become a new tool targeting multi-drug-resistant Pa infections in high-risk populations.

CRM197-conjugated peptides vaccine of HCMV pp65 and gH induce maturation of DC and effective viral-specific T cell responses

ABSTRACT Human cytomegalovirus (HCMV) infection is prevalent worldwide, and there is currently no licenced HCMV vaccine to control it. Therefore, developing an effective HCMV vaccine is a significant priority. Because of their excellent immunogenicity, the crucial components of HCMV, phosphoprotein 65 (pp65) and glycoproteins H (gH) are potential target proteins for HCMV vaccine design. In this study, we predicted and screened the dominant antigenic epitopes of B and T cells from pp65 and gH conjugated with the carrier protein cross-reacting material 197 (CRM197) to form three peptide-CRM197 vaccines (pp65-CRM197, gH-CRM197, and pp65-CRM197+gH-CRM197). Furthermore, the immunogenicity of the peptide-CRM197 vaccines and their effects on dendritic cells (DCs) were explored. The results showed that three peptide-CRM197 vaccines could induce maturation of DCs through the p38 MAPK signalling pathway and promote the release of proinflammatory factors, such as TNF-α and interleukin (IL) −6. Meanwhile, the peptide-CRM197 vaccines could effectively activate T cell and humoral immunity, which were far better than the inactivated HCMV vaccine. In conclusion, we constructed three peptide-CRM197 vaccines, which could induce multiple immune effects, providing a novel approach for HCMV vaccine design.

High-Level Production of Soluble Cross-Reacting Material 197 in Escherichia coli Cytoplasm Due to Fine Tuning of the Target Gene’s mRNA Structure

Cross-reacting material 197 (CRM197) is a non-toxic mutant of the diphtheria toxin and is widely used as a carrier protein in conjugate vaccines. This protein was first obtained from the supernatant of the mutant Corynebacterium diphtheriae strain. This pathogenic bacteria strain is characterized by a slow growth rate and a relatively low target protein yield, resulting in high production costs for CRM197. Many attempts have been made to establish high-yield protocols for the heterologous expression of recombinant CRM197 in different host organisms. In the present work, a novel CRM197-producing Escherichia coli strain was constructed. The target protein was expressed in the cytoplasm of SHuffle T7 E. coli cells without any additional tags and with a single potential mutation-an additional Met [-1]. The fine tuning of the mRNA structure (the disruption of the single hairpin in the start codon area) was sufficient to increase the CRM197 expression level several times, resulting in 150-270 mg/L (1.1-2.0 mg/g wet biomass) yields of pure CRM197 protein. Besides the high yield, the advantages of the obtained expression system include the absence of the necessity of CRM197 refolding or tag removal. Thus, an extensive analysis of the mRNA structure and the removal of the unwanted hairpins in the 5' area may significantly improve the target protein expression rate.

Assembly of Immunogenic Protein Particles toward Advanced Synthetic Vaccines.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

A Bacterially Expressed SARS-CoV-2 Receptor Binding Domain Fused With Cross-Reacting Material 197 A-Domain Elicits High Level of Neutralizing Antibodies in Mice.

The Coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented public health crisis worldwide. Although several vaccines are available, the global supply of vaccines, particularly within developing countries, is inadequate, and this necessitates a need for the development of less expensive, accessible vaccine options. To this end, here, we used the Escherichia coli expression system to produce a recombinant fusion protein comprising the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; residues 319–541) and the fragment A domain of Cross-Reacting Material 197 (CRM197); hereafter, CRMA-RBD. We show that this CRMA-RBD fusion protein has excellent physicochemical properties and strong reactivity with COVID-19 convalescent sera and representative neutralizing antibodies (nAbs). Furthermore, compared with the use of a traditional aluminum adjuvant, we find that combining the CRMA-RBD protein with a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH-002C-Ac) leads to stronger humoral immune responses in mice, with 4-log neutralizing antibody titers. Overall, our study highlights the value of this E. coli-expressed fusion protein as an alternative vaccine candidate strategy against COVID-19.

The recombinant protein combined vaccine based on the fragment C of tetanus toxin and the cross-reacting material 197.

Diphtheria and tetanus toxoids and acellular pertussis (DTaP) vaccines were widely used since 1940s. The exceptional success of childhood vaccination is undisputed. However, the anti-diphtheria and tetanus antibody will decrease with the increase of age in human body. A boosting vaccine for tetanus and diphtheria in adult is recommended by WHO. Recombinant protein vaccine has the advantages of single component and high safety, which is one of the directions to develop boosting vaccines. Therefore, in this study, we evaluated a recombinant TTc and CRM197 combination vaccine (RTCV) that uses the fragment C (TTc) of tetanus toxin and the cross-reacting material 197 (CRM197) of the diphtheria toxin mutant. Our results displayed that RTCV (composed of 10 μg/mL TTc, 20 μg/mL CRM197 antigens, and 500 μg/mL aluminum adjuvants) could induce high levels of IgG and IgG1 antibody in mice, which were similar as those induced by DTaP. These results will provide technical support for a novel boosting vaccine against diphtheria and tetanus. KEY POINTS: • We successfully expressed CRM197 protein in E. coli BL21 (DE3) using pET26b (+) vector. • The anti-TTc and anti-CRM197 antibody titer (IgG) of RTCV was similar with DTaP.

Super-resolution microscopy unveils transmembrane\xa0domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197’s transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197.

CRM197-Coupled Der p 2 Peptides Suppress Allergic Airway Inflammation in a Der p 2-Induced Asthma Mouse Model

Allergic diseases affect more than 25% of the global population. Der p 2 is the major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus. Allergen-specific immunotherapy is the only treatment to change the course of allergic diseases. In this study, two synthesized Der p 2 peptides coupled to cross-reacting material 197 (CRM197) showed reduced IgE reactivity and allergenic activity. CRM197-coupled Der p 2 peptides induced rDer p 2-specific IgG1 antibodies in mice, which could inhibit HDM-allergic patients’ IgE binding to rDer p 2. The immunity effects of CRM197-coupled Der p 2 peptides were studied in an rDer p 2-induced asthma mouse model. CRM197-coupled Der p 2 peptides can suppress asthmatic airway inflammation in this model. Analysis of IL-4, IL-5, and IFN-γ levels in bronchoalveolar lavage fluid revealed that the suppression was associated with a shift from a Th2 to a Th1 response. Thus, CRM197-bound Der p 2 peptides exhibited less allergenic activity than the rDer p 2 allergen, which preserved immunogenicity and may be candidates for mite allergy vaccines.

CRM197 reverses paclitaxel resistance by inhibiting the NAC-1/Gadd45 pathway in paclitaxel-resistant ovarian cancer cells.

Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is a new promising target for the treatment of ovarian cancer. Our previous study showed that cross‐reacting material 197 (CRM197), a specific HB‐EGF inhibitor, significantly reverses resistance against paclitaxel in paclitaxel‐resistant ovarian cancer cells. However, the mechanism of the effect of CRM197 on the reversion of paclitaxel resistance was unclear. In this study, in vitro and in vivo data suggested that CRM197 treatment sensitized paclitaxel‐resistant ovarian cancer cells to paclitaxel, at least in part, via nucleus accumbens‐1 (NAC‐1) and its downstream pathway, DNA damage‐inducible 45‐γ interacting protein (Gadd45gip1)/growth arrest and DNA damage‐inducible 45 (Gadd45), in A2780/Taxol and SKOV3/Taxol cells. The results also showed that CRM197 activated the proapoptotic JNK/p38MAPK pathway to enhance caspase‐3 activity and apoptosis by downregulation of the NAC‐1/Gadd45gip1/Gadd45 pathway, leading to reversion of paclitaxel resistance in A2780/Taxol and SKOV3/Taxol cells. This study provides the first mechanism through which CRM197 significantly reverses resistance against paclitaxel by modulating the NAC‐1/Gadd45gip1/Gadd45 pathway in paclitaxel‐resistant ovarian cancer cells, and the mechanism of HB‐EGF inhibition as a novel therapeutic strategy for patients with paclitaxel‐resistant ovarian cancer.

Enhanced protective efficacy of Borrelia burgdorferi BB0172 derived-peptide based vaccine to control Lyme disease

Lyme disease (LD) accounts for over 70% of tick-borne disease reported in the United States. The disease in humans is characterized by skin rash, arthritis, cardiac and neurological signs. Vaccination is the most efficient preventive measure that could be taken to reduce the incidence of the LD worldwide; however, at present no vaccine is available. In this study, evaluation of the Borrelia burgdorferi BB0172-derived peptide (PepB) in conjugated formulations was investigated as a vaccine candidate in murine model of LD. In brief, PepB was conjugated to the Cross-Reacting Material 197 (CRM197) and to Tetanus Toxoid heavy chain (TTHc) molecules, and subsequently used to immunize C3H/HeN mice. Following the challenge with 105 spirochetes/mouse via subcutaneous inoculation, TTHc:PepB construct showed protection in 66% of the immunized animals. Hence, to further evaluate the efficacy of TTHc:PepB, immunized mice were challenged with B. burgdorferi using the tick model of infection. The outcome of this experiment revealed that serum from TTHc:PepB immunized mice was borrelicidal. After tick infection, bacterial burden was significantly reduced (over 70%) in vaccinated animals when compared with the control groups regardless of whether the mice were infested 8 or 12-weeks post-priming. Therefore, we conclude that PepB conjugated antigens can serve as an alternative to prevent LD; nevertheless, further studies will be needed to dissect the mechanisms by which anti-PepB IgG antibodies are able to kill B. burgdorferi in vitro and in vivo to further advance in the development of formulations and delivery alternative to generate a safe anti-LD vaccine.

Hepatitis E vaccine candidate harboring a non-particulate immunogen of E2 fused with CRM197 fragment A

The Hepatitis E vaccine (Hecolin, licensed in China) harbors a potent particulate immunogen, p239, designed from a 26-aa N-terminal extension of its poorly immunogenic parental protein, E2. Although an effective vaccine, we sought to design a fusion protein in a non-particulate form that could improve the delivery and immunogenicity of E2 epitopes. The non-toxic mutant of diphtheria toxin, CRM197 (Cross-Reacting Material 197) has been successfully used as a carrier protein for conjugated vaccines to enhance the immunogenicity of polysaccharides. Here, we designed a fusion non-particulate protein of E2 and the catalytic domain (fragment A) of CRM197 and evaluated its antigenicity, immunogenicity and disease prevention efficacy in primates. This fusion protein, named CRM197(A)-E2, was bacterially expressed and purified by chromatography. CRM197(A)-E2 presented as a homodimer in solution, similar to its parental E2 protein, and exhibited excellent antigenicity against representative neutralizing monoclonal antibodies, like E2 and p239. However, CRM197(A)-E2 manifested higher immunogenicity in mice compared with that achieved by the particulate p239, as indicated by the 10-times lower ED50 value and 2-log higher HEV-specific antibody level that could persist for at least 28 weeks. In addition, both the 1 μg and 10 μg doses of CRM197(A)-E2 adjuvanted with aluminum could protect vaccinated monkeys against HEV challenge, matching that achieved with only the higher (10 μg) dose of the p239 vaccine. These results suggest that the CRM197 fragment A alone serves as an intra-molecular adjuvant to remarkably enhance the immunogenicity of the target of interest in a non-particulate form. These findings may pave the way for rational vaccine design, especially in cases where particulates are not accessible.

Adenovirus-Mediated CRM197 Sensitizes Human Glioma Cells to Gemcitabine by the Mitochondrial Pathway.

The cross-reacting material 197 (CRM197) is a mutation of the diphtheria toxin. The protein of CRM197 was used successfully for the therapy of various tumors in the recent studies. In this study, the recombinant adenoviruses containing the CRM197gene(AdCRM197) were used to enhance the cellar toxicity of gemcitabine in human glioma U87, U251, and H4 cells. MTT assay and flow cytometric analysis were performed to test the apoptosis of the U87, U251 and H4 cells with the combined treatment of AdCRM197 plus gemcitabine. Western blotting analyses were carried out to detect the cell apoptosis of the mitochondrial pathway. And the xenograft nude mice were used to observe the enhanced antitumor effect of AdCRM197 in vivo. AdCRM197 sensitizes human glioma cells to gemcitabine in vitro by the mitochondrial pathway. Tumor volume was inhibited and survival time was prolonged in the U251 or U87 xenografted nude mice with gemcitabine plus AdCRM197. The enhanced antitumor effect of AdCRM197 was also detected by the immunohistochemical analyses and TUNEL staining. The authors found that AdCRM197 sensitized the human glioma to gemcitabine not only in vitro but also in vivo. They provide the first evidence that adenovirus-mediated CRM197 may be a potential chemosensitizing agent for the treatment of cancer. The diphtheria toxin is of great toxicity that even one molecule of diphtheria toxin is enough to kill one cell. However, because of the high toxicity, the diphtheria toxin would kill the packing cells when it is being packaged into the recombinant viruses. Therefore, the diphtheria toxin is hard to be used in the gene therapy for virus vectors. The cross-reacting material 197 (CRM197) is a mutation of the diphtheria toxin. Unlike DTA, CRM197 exhibit a weak toxicity. The week toxicity of CRM197 is a good feature for the virus packaging. In the present study, we used a recombinant adenovirus which carried a CRM197 gene (AdCRM197) to enhance the cellar toxicity of gemcitabine in human glioma cells.

Expression, purification and characterization of diphtheria toxin mutant CRM197 in Eschrichia coli

CRM197 (cross-reacting material 197), a non-toxic mutant of diphtheria toxin, has wide application potential in biopharmaceuticals. However, it is difficult to express CRM197 in bacteria other than Corynebacterium diphtheriae. Here we proposed a new alternative method to produce soluble CRM197 without label in Escherichia coli. In particular, a synthetic gene coding for CRM197, optimized for E. coli codon usage, was cloned in the pET32a (+) vector. Accordingly, the over-expression of the protein was simply induced with IPTG in E. coli BL21 (DE3). The target protein was soluble and accounted for about 40% of the total protein in the supernatant. Following an ultrasonic cytolysis step, the recombinant protein was purified by anion exchange, affinity and desalting chromatography and the purity of the final preparation reached 95%. Cytotoxicity tests showed that the IC₅₀ value of CRM197 was 2.1×10⁷ times the IC₅₀ value of diphtheria toxin, and 9.6 times the IC50 value of diphtheria toxoid, telling that the target protein is safe and non-toxic. Subsequently, we found that both the high dose (20 μg) and the low dose (2 μg) of CRM197 were equally efficient in inducing an immune response against diphtheria toxiod in mice, and the antibodies titer of mice after three immunizations with low dose could reach 1:409 600. In conclusion, our findings provide a highly efficient strategy for the rapid production and purification of unlabeled and soluble recombinant CRM197 in E. coli, with good immunogenicity and safety.

P53 Plays a Key Role in the Apoptosis of Human Ovarian Cancer Cells Induced by Adenovirus-Mediated CRM197.

Cross-reacting material 197 (CRM197) is a mutant form of the diphtheria toxin. Recent studies have found that CRM197 exerts an experimental antitumor effect on several types of tumors. This study applied a novel treatment of adenovirus-mediated CRM197 (AdCRM197) to human ovarian cancer cells. Interestingly, it was found that A2780 cells were sensitive to AdCRM197, but SKOV3 cells were resistant to it. Since SKOV3 cells are p53 deletion cells, while A2780 cells are p53 wild-type cells, it was postulated that p53 might play a key role in AdCRM197-induced apoptosis. This presumption was demonstrated by means of knockdown of p53 of the A2780 cells through lentivirus-mediated RNA interference. This knockdown resulted in the A2780 cells becoming resistant to AdCRM197. To verify this presumption further, the wild-type p53 gene in the SKOV3 cells was replaced with adenovirus-mediated p53 (Adp53). As expected, AdCRM197 plus Adp53 resulted in apoptosis of the SKOV3 cells. The combined treatment of AdCRM197 plus Adp53 also showed a good antitumor effect in the in vivo experiment on nude mice with xenograft tumors. Taking these results together, it is concluded that AdCRM197 induces apoptosis of human ovarian cancer cells via the p53 pathway. Moreover, it was found that Adp53 can reverse the resistance of p53-deletion human ovarian cancer cells to AdCRM197. The combination of AdCRM197 and Adp53 may be a potentially effective method for overcoming the resistance of p53-deficient human ovarian cancer to AdCRM197.

Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems

Cross-reacting material 197 (CRM197), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM197 molecules demonstrate that recombinant CRM197's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM197 expressed in alternative sources have the potential to replace CRM197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM197 carrier proteins for use in various conjugate vaccines.

NMR study of mutations of glycine-52 of the catalytic domain of diphtheria toxin

Cross-reacting-material 197 (CRM197) is a naturally occurring non-toxic mutant of diphtheria toxin (DT) that is one of the few carrier protein used in the manufacture of polysaccharide vaccines targeting bacterial pathogens such as Neisseria meningitidis, Streptococcus pneumaniae and Haemophilus influenzae. A detailed explanation in structural terms for the lack of toxicity has started to emerge with the report of the X-ray structure of CRM197. Here, we present an NMR spectroscopy study of the wild-type catalytic domain of diphtheria toxin and the effects of mutations at residue 52 on its conformation. We utilized a strategy that consisted of gradually inducing steric perturbations by increasing the side chain size of the residue. Results show that the catalytic domain does not tolerate even the smallest perturbation, such as a glycine to alanine substitution, resulting in the destabilization of domain fold leading to protein aggregation. The observed behavior is further exacerbated with the substitution of amino acids with larger side chains. These findings support the concept that the lack of toxicity observed for CRM197 is the result of a highly unstable conformation of its catalytic domain that, upon insertion into the cell, cannot properly refold and perform its catalytic activity responsible for the arrest of all cellular protein synthesis.

Conjugation of the CRM197-inulin conjugate significantly increases the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein

Mycobacterium tuberculosis (Mtb) is a serious fatal pathogen that causes tuberculosis (TB). Effective vaccination is urgently needed to deal with the serious threat from TB. Mtb-secreted protein antigens are important virulence determinants of Mtb with poor immunogenicity. Adjuvants and antigen delivery systems are thus highly desired to improve the immunogenicity of protein antigens. Inulin is a biocompatible polysaccharide (PS) adjuvant that can stimulate a strong cellular and humoral immunity. Bacterial capsular PS and haptens have been conjugated with cross-reacting material 197 (CRM197) to improve their immunogenicity. CFP10 and TB10.4 were two Mtb-secreted immunodominant protein antigens. A CFP10-TB10.4 fusion protein (CT) was used as the antigen for covalent conjugation with the CRM197-inulin conjugate (CRM-inu). The resultant conjugate (CT-CRM-inu) elicited high CT-specific IgG titers, stimulated splenocyte proliferation and provoked the secretion of Th1-type and Th2-type cytokines. Conjugation with CRM-inu significantly prolonged the systemic circulation of CT and exposure to the immune system. Moreover, CT-CRM-inu showed no apparent toxicity to cardiac, hepatic and renal functions. Thus, conjugation of CT with CRM-inu provided an effective strategy for development of protein-based vaccines against Mtb infection.

Targeted delivery of rosmarinic acid across the blood–brain barrier for neuronal rescue using polyacrylamide-chitosan-poly(lactide-co-glycolide) nanoparticles with surface cross-reacting material 197 and apolipoprotein E

Rosmarinic acid-loaded polyacrylamide-chitosan-poly(lactide-co-glycolide) nanoparticles (RA-PAAM-CH-PLGA NPs) were grafted with cross-reacting material 197 (CRM197) and apolipoprotein E (ApoE) for targeting of the blood–brain barrier (BBB) and rescuing degenerated neurons. The polymeric nanocarriers were prepared by microemulsion, solvent diffusion, grafting, and surface modification, and CRM197-ApoE-RA-PAAM-CH-PLGA NPs were used to treat human brain-microvascular endothelial cells, RWA264.7 cells, and Aβ-insulted SK-N-MC cells. Experimental results revealed that an increase in the weight percentage of PAAM decreased the particle size, zeta potential, and grafting efficiency of CRM197 and ApoE. In addition, surface DSPE-PEG(2000) could protect CRM197-ApoE-RA-PAAM-CH-PLGA NPs against uptake by RWA264.7 cells. An increase in the concentration of CRM197 and ApoE decreased the transendothelial electrical resistance and increased the ability of propidium iodide and RA to cross the BBB. The order in the viability of apoptotic SK-N-MC cells was CRM197-ApoE-RA-PAAM-CH-PLGA NPs > CRM197-RA-PAAM-CH-PLGA NPs > RA. Thus, CRM197-ApoE-RA-PAAM-CH-PLGA NPs can be a promising formulation to deliver RA to Aβ-insulted neurons in the pharmacotherapy of Alzheimer’s disease.

Co-expression of sulphydryl oxidase and protein disulphide isomerase in Escherichia coli allows for production of soluble CRM197.

To investigate the production of soluble cross-reacting material 197 (CRM197 ) in Escherichia coli, a safe and effective T-cell-dependent protein carrier for polysaccharides used in the manufacture and application of multivalent conjugate vaccines. The use of co-expression of a sulphydryl oxidase (SOX) and protein disulphide isomerase for the production of soluble CRM197 in E.coli is described. CRM197 contains two disulphide bonds, which are normally unable to form in the reducing environment of the E.coli cytoplasm. It was found that co-expression yielded soluble CRM197 , at a production rate ~10% of the production of insoluble CRM197 , in equivalent small-scale cultures. Structural analysis of the purified CRM197 compared to CRM197 commercially produced in cultures of recombinant Pseudomonas fluorescens indicated that the E.coli soluble protein compares favourably on all structural levels. SOX and protein disulphide isomerase are enzymes involved in the formation of intra-protein disulphide bonds, and can influence the tertiary structure of the protein being produced, resulting in increased solubility due to the correct folding of the protein. Their use enabled the production of soluble untagged CRM197 in E.coli, which was previously unachievable. Previous literature reports have shown that CRM197 can be expressed in E.coli, though only in an insoluble form, or in soluble form as a fusion protein. It is currently commercially produced in cultures of recombinant P. fluorescens. The use of a widely used, well-characterized expression host such as E.coli, rather than P. fluorescens broadens the applicability of the production technology, and the production system described here is worthy of further investigation for scaled up manufacture of CRM197 .

The impact of administration of conjugate vaccines containing cross reacting material on Haemophilus influenzae type b antibody responses in infants: A systematic review and meta-analysis of randomised controlled trials

BackgroundProtein-polysaccharide conjugate vaccines such as Haemophilus influenzae type b (Hib), meningococcal, and pneumococcal vaccine, induce immunological memory and longer lasting protection than plain polysaccharide vaccines. The most common proteins used as carriers are tetanus toxoid (TT) and cross reacting material-197 (CRM), a mutant form of diphtheria toxoid. CRM conjugate vaccines have been reported to suppress antibody responses to co-administered Hib-TT vaccine. MethodsWe conducted a systematic review and meta-analysis of randomised controlled trials in which infants were randomised to receive meningococcal or pneumococcal conjugate vaccines along with Hib-TT. Trials of licensed vaccines with different carrier proteins were included for group C meningococcal (MenC), quadrivalent ACWY meningococcal (MenACWY), and pneumococcal vaccines. ResultsTwenty-three trials were included in the meta-analyses. Overall, administration of MenC-CRM in a 2 or 3 dose schedule resulted in a 45% reduction in Hib antibody concentrations (GMR 0.55, 95% CI 0.49–0.62). MenACWY-CRM boosted Hib antibody responses by 22% (GMR 1.22, 95% CI 1.06–1.41) whilst pneumococcal CRM conjugate vaccines had no impact on Hib antibody responses (GMR 0.91, 95% CI 0.68–1.22). ConclusionsThe effect of CRM protein-polysaccharide conjugate vaccines on Hib antibody responses varies greatly between vaccines. Co-administration of a CRM conjugate vaccine can produce either positive or negative effects on Hib antibody responses. These inconsistencies suggest that CRM itself may not be the main driver of variability in Hib responses, and challenge current perspectives on this issue.