From small molecules to large proteins: Delivery through B family vitamin uptake pathways

Delivery of therapeutic molecules using the B family vitamin uptake pathways is presented. First a study examining targeting the folate receptor to deliver AZT was successfully conducted in vitro. Conjugation of AZT to folic acid resulted in a drug that showed an increase in cytotoxicity of ∼20-fold compared to free AZT, when tested in the A2780/AD drug resistant ovarian cell line. This compound was taken to pilot in vivo studies. Conjugates of the folic acid analogue methotrexate with nucleoside analogues AZT, IUdR and ddC were subsequently studied. The strategy was to conjugate two drugs that would work synergistically together. The individual conjugates did not show any synergism, but a cocktail of the AZT and IUdR conjugates showed a synergistic relationship with a lower IC 50 over 24 hours compared to cocktails of the free drugs. In an attempt to create an oral vaccine, conjugates of tetanus toxoid and B12 were explored. It was hypothesized that the B 12 uptake pathway could protect the toxoid in the digestive tract and facilitate crossing of the enterocyte. The pilot B 12 -tetanus toxoid conjugates were synthesized and characterized. Different ratios of B 12 /tetanus toxoid were synthesized. The conjugates were fluorescently tagged and intestinal uptake was modeled using BeWo placental carcinoma cells, followed by confocal microscopy

Assembly, Biochemical Characterization, Immunogenicity, Adjuvanticity, and Efficacy of Shigella Artificial Invaplex

ABSTRACT The native Invaplex (InvaplexNAT) vaccine and adjuvant is an ion exchange-purified product derived from the water extract of virulent Shigella species. The key component of InvaplexNAT is a high-molecular-mass complex (HMMC) consisting of the Shigella lipopolysaccharide (LPS) and the invasin proteins IpaB and IpaC. To improve product purity and immunogenicity, artificial Invaplex (InvaplexAR) was developed using recombinant IpaB and IpaC proteins and purified Shigella LPS to assemble an HMMC consisting of all three components. Characterization of InvaplexAR by various methods demonstrated similar characteristics as the previously reported HMMC in InvaplexNAT. The well-defined InvaplexAR vaccine consistently contained greater quantities of IpaB, IpaC, and LPS than InvaplexNAT. InvaplexAR and InvaplexNAT immunogenicities were compared in mouse and guinea pig dose escalation studies. In both models, immunization induced antibody responses specific for InvaplexNAT and LPS while InvaplexAR induced markedly higher anti-IpaB and -IpaC serum IgG and IgA endpoint titers. In the murine model, homologous protection was achieved with 10-fold less InvaplexAR than InvaplexNAT and mice receiving InvaplexAR lost significantly less weight than mice receiving the same amount of InvaplexNAT. Moreover, mice immunized with InvaplexAR were protected from challenge with both homologous and heterologous Shigella serotypes. Guinea pigs receiving approximately 5-fold less InvaplexAR compared to cohorts immunized with InvaplexNAT were protected from ocular challenge. Furthermore, adjuvanticity previously attributed to InvaplexNAT was retained with InvaplexAR. The second-generation Shigella Invaplex vaccine, InvaplexAR, offers significant advantages over InvaplexNAT in reproducibility, flexible yet defined composition, immunogenicity, and protective efficacy. IMPORTANCE Shigella species are bacteria that cause severe diarrheal disease worldwide, primarily in young children. Treatment of shigellosis includes oral fluids and antibiotics, but the high burden of disease, increasing prevalence of antibiotic resistance, and long-term health consequences clearly warrant the development of an effective vaccine. One Shigella vaccine under development is termed the invasin complex or Invaplex and is designed to drive an immune response to specific antigens of the bacteria in an effort to protect an individual from infection. The work presented here describes the production and evaluation of a new generation of Invaplex. The improved vaccine stimulates the production of antibodies in immunized mice and guinea pigs and protects these animals from Shigella infection. The next step in the product’s development will be to test the safety and immune response induced in humans immunized with Invaplex