Good Manufacturing Practices production and analysis of a DNA vaccine against dental caries

Abstract

Aim: To prepare a clinical-grade anti-caries DNA vaccine pGJA-P/VAX and explore its immune effect and protective efficacy against a cariogenic bacterial challenge. Methods: A large-scale industrial production process was developed under Good Manufacturing Practices (GMP) by combining and optimizing common unit operations such as alkaline lysis, precipitation, endotoxin removal and column chromatography. Quality controls of the purified bulk and final lyophilized vaccine were conducted according to authoritative guidelines. Mice and gnotobiotic rats were intranasally immunized with clinical-grade pGJA-P/VAX with chitosan. Antibody levels of serum IgG and salivary SIgA were assessed by an enzyme-linked immunosorbent assay (ELISA), and caries activity was evaluated by the Keyes method. pGJA-P/VAX and pVAX1 prepared by a laboratory-scale commercial kit were used as controls. Results: The production process proved to be scalable and reproducible. Impurities including host protein, residual RNA, genomic DNA and endotoxin in the purified plasmid were all under the limits of set specifications. Intranasal vaccination with clinical-grade pGJA-P/ VAX induced higher serum IgG and salivary SIgA in both mice and gnotobiotic rats. While in the experimental caries model, the enamel (E), dentinal slight (Ds), and dentinal moderate (Dm) caries lesions were reduced by 21.1%, 33.0%, and 40.9%, respectively. Conclusion: The production process under GMP was efficient in preparing clinical-grade pGJA-P/VAX with high purity and intended effectiveness, thus facilitating future clinical trials for the anti-caries DNA vaccine.

Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors

“…Based on a clinically approved plasmid vector pVax1 (Thermo Fisher Scientific) [ 62 ], multicistronic constructions encoding various gene combinations (pVax1-VEGF-FGF2-DsRed, pVax1-VEGF-DsRed, pVax1-FGF2-DsRed, pVax1-DsRed) were designed and developed ( Figure 1 ). Nucleotide sequences of the VEGF (GeneBank AF486837.1) and FGF2 (GeneBank #DD406196.1) genes were obtained from the NCBI database and cloned under a single CMV promoter.…”

Vaccine against Dental Caries: An Update

“…However, the still high prevalence of such disease in the world population [13], justifies the search for a new preventive action, such as the development of a caries vaccine. The main target is the mechanism of adherence of S. mutans, which can be affected by active or passive immunization [14] [15], or by DNA vaccines [9]- [11].…”

“…Passive immunization consists of the topical application of antibodies (Abs) performed antigen-specific on the surface of teeth against the virulence factors of S. mutans. As for the active immunization, it involves the application of microbial antigens (Ags), inducing the mucosal immune system, by stimulating the production of specific SIgA; besides induction of the systemic immune system, stimulating the production of serum Abs [7], which may be used in DNA vaccines [9]- [11]. The main immunological studies for developing a caries vaccine are based on active immunization [7].…”

“…It also presents great potential for further modification and improvement [41]. However, the need for large amounts availability (milligrams or grams) of this vaccine to make it effective prevents it from being conventionally produced in laboratory, requiring large-scale industrial production [11].…”

“…In search of new preventive measures against caries, in addition to the consolidated ones, as the disorganization of the biofilm by brushing and using toothpaste with or without fluoride [5] [6], some researches have been conducted with the aim of developing a caries vaccine [7]- [11].…”