Preclinical development of the quadrivalent meningococcal (ACYW) tetanus toxoid conjugate vaccine, MenQuadfi®.

Bacterial capsular polysaccharide vaccines are generally poorly immunogenic in infants and older adults. The immunogenicity of capsular polysaccharide vaccines can be improved by conjugating them to immunogenic carrier proteins. One of the most recently licensed conjugate vaccines is the quadrivalent meningococcal vaccine with serogroups A, C, Y, and W conjugated to a tetanus toxoid protein carrier (MenACYW-TT; MenQuadfi, Sanofi Pasteur, Swiftwater, PA, USA). MenACYW-TT was developed to induce optimal immune responses against each of the meningococcal serogroups A, C, W, and Y, and across all age groups, especially infants and older adults (those aged ≥ 50 years). Here, we detail the early iterative vaccine development approach taken, whereby many different 'small-scale' conjugate vaccine candidates were prepared and examined for immunogenicity in a mouse model to identify the most immunogenic vaccine. Additional insights from phase I clinical studies informed further optimization of the vaccine candidates by tailoring their conjugation parameter attributes for the optimal immune response in humans. The parameters studied included: different carrier proteins [PR]; polysaccharide [PS] sizes; conjugation chemistries [linker vs. no-linker; lattice vs. neoglycoprotein; activation/derivatization levels]; conjugate size; PS:PR loading ratio; percent free PS; percent free PR; and O-acetylation content. The lead quadrivalent conjugate vaccine (polysaccharides of > 50 kDa size conjugated to TT at a high PS:PR ratio via reductive amination for serogroups C, W and Y, and carbonyldiimidazole/adipic acid dihydrazide linker chemistry for serogroup A) empirically identified from the extensive preclinical studies, was ultimately confirmed by the robust antibody responses observed in all age groups in the various clinical studies, including in the most challenging infant and older adult age groups, and subsequently led to the licensed formulation.

The Highly Productive Thermothelomyces heterothallica C1 Expression System as a Host for Rapid Development of Influenza Vaccines.

(1) Influenza viruses constantly change and evade prior immune responses, forcing seasonal re-vaccinations with updated vaccines. Current FDA-approved vaccine manufacturing technologies are too slow and/or expensive to quickly adapt to mid-season changes in the virus or to the emergence of pandemic strains. Therefore, cost-effective vaccine technologies that can quickly adapt to newly emerged strains are desirable. (2) The filamentous fungal host Thermothelomyces heterothallica C1 (C1, formerly Myceliophthora thermophila) offers a highly efficient and cost-effective alternative to reliably produce immunogens of vaccine quality at large scale. (3) We showed the utility of the C1 system expressing hemagglutinin (HA) and a HA fusion protein from different H1N1 influenza A virus strains. Mice vaccinated with the C1-derived HA proteins elicited anti-HA immune responses similar, or stronger than mice vaccinated with HA products derived from prototypical expression systems. A challenge study demonstrated that vaccinated mice were protected against the aggressive homologous viral challenge. (4) The C1 expression system is proposed as part of a set of protein expression systems for plug-and-play vaccine manufacturing platforms. Upon the emergence of pathogens of concern these platforms could serve as a quick solution for producing enough vaccines for immunizing the world population in a much shorter time and more affordably than is possible with current platforms.

Predictive modeling for assessing the long-term thermal stability of a new fully-liquid quadrivalent meningococcal tetanus toxoid conjugated vaccine.

Establishing product stability is critical for pharmaceuticals. We used a modeling approach to predict the thermal stability of a fully-liquid quadrivalent meningococcal (serogroups A, C, W, Y) conjugate vaccine (MenACYW-TT; MenQuadfi®) at potential transportation and storage temperatures. Vaccine degradation was determined by measuring the rate of hydrolysis through an increase of free polysaccharide (de-conjugated or unconjugated polysaccharide) content during six months storage at 25 °C, 45 °C and 56 °C. A procedure combining advanced kinetics and statistics was used to screen and compare kinetic models describing observed free polysaccharide increase as a function of time and temperature for each serogroup. Statistical analyses were used to quantify prediction accuracy. A two-step kinetic model described the increase in free polysaccharide content for serogroup A; whereas, one-step kinetic models were found suitable to describe the other serogroups. The models were used to predict free polysaccharide increases for each serogroup during long-term storage under recommended conditions (2-8 °C), and during temperature excursions to 25 °C or 40 °C. In both cases, serogroup-specific simulations accurately predict the respective observed experimental data. Experimental data collected to 48 months at 5 °C were within 99% predictive bands. The models described here can be used with confidence to establish shelf-life for this fully-liquid quadrivalent meningococcal conjugate vaccine; as well as, monitor in real-time free polysaccharide increase for vaccines experiencing temperature excursions during shipment/storage.

Multivalent dendrimeric compounds containing carbohydrates expressed on immune cells inhibit infection by primary isolates of HIV-1.

Specific glycosphingolipids (GSL), found on the surface of target immune cells, are recognized as alternate cell surface receptors by the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein. In this study, the globotriose and 3'-sialyllactose carbohydrate head groups found on two GSL were covalently attached to a dendrimer core to produce two types of unique multivalent carbohydrates (MVC). These MVC inhibited HIV-1 infection of T cell lines and primary peripheral blood mononuclear cells (PBMC) by T cell line-adapted viruses or primary isolates, with IC(50)s ranging from 0.1 to 7.4 µg/ml. Inhibition of Env-mediated membrane fusion by MVC was also observed using a dye-transfer assay. These carbohydrate compounds warrant further investigation as a potential new class of HIV-1 entry inhibitors. The data presented also shed light on the role of carbohydrate moieties in HIV-1 virus-host cell interactions.

LC/MS characterization of meningococcal depolymerized polysaccharide group C reducing endgroup and internal repeating unit.

Hydrogen peroxide has been used to cleave the native Neisseria meningiditis polysaccharide (PS) from mega-Dalton molecular weight to a smaller size (approximately 20 kDa) depolymerized polysaccharide. The polysaccharide was examined after partial peroxide depolymerization to verify the presence of the carboxyl group at position 1 and the intactness of the internal sialic acid repeating units. The reducing end group of meningococcal polysaccharide type C was also examined after derivatization by L-tyrosine hydrazide. Partial peroxide depolymerization did not result in loss of the position 1 carboxyl group at the reducing end of the polysaccharide. In addition, no loss of structural integrity was noted for the internal sialic repeat units.

Novel polysulfated galactose-derivatized dendrimers as binding antagonists of human immunodeficiency virus type 1 infection.

Evidence indicates that galactosyl ceramide (GalCer) and its 3'-sulfated derivative, sulfatide (SGalCer), may act as alternate coreceptors for human immunodeficiency virus type 1 (HIV-1) in CD4(-) cells. Glycosphingolipids (GSLs) may also be necessary for fusion of HIV-1 and host cell membranes. Using an enzyme-linked immunosorbent assay to determine which GSL was the best ligand for both recombinant and virus-associated gp120, we found that SGalCer was the best ligand for each rgp120 and HIV-1 isolate tested. Therefore, novel multivalent glycodendrimers, which mimic the carbohydrate clustering reportedly found in lipid rafts, were synthesized based on the carbohydrate moiety of SGalCer. Here we describe the synthesis of a polysulfated galactose functionalized, fifth generation DAB dendrimer (PS Gal 64mer), containing on average two sulfate groups per galactose residue. Its ability to inhibit HIV-1 infection of cultured indicator cells was compared to that of dextran sulfate (DxS), a known, potent, binding inhibitor of HIV-1. The results indicate that the PS Gal 64mer inhibited infection by the HIV-1 isolates tested as well as DxS.

Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120.

Multivalent neoglycoconjugates are valuable tools for studying carbohydrate-protein interactions. To study the interaction of HIV-1 gp120 with its reported alternate glycolipid receptors, galactosyl ceramide (GalCer) and sulfatide, galactose- and sulfated galactose-derivatized dendrimers were synthesized, analyzed as ligands for rgp120 by surface plasmon resonance, and tested for their ability to inhibit HIV-1 infection of CXCR4- and CCR5-expressing indicator cells. Four different series of glycodendrimers were made by amine coupling spacer-arm derivatized galactose residues, either sulfated or nonsulfated, to poly(propylenimine) dendrimers, generations 1-5. One series of glycodendrimers was prepared from the ceramide saccharide derivative of purified natural GalCer, and another was from chemically synthesized 3-(beta-D-galactopyranosylthio)propionic acid. Synthesis of 3-sulfogalactopyranosyl-derivatized dendrimers was accomplished using the novel compound, 3-(beta-D-3-sulfogalactopyranosylthio)propionic acid. The fourth series was made by random sulfation of the 3-(beta-D-galactopyranosylthio)propionic acid functionalized dendrimers. Structures of the carbohydrate moieties were confirmed by NMR, and the average molecular weights and polydispersities of the different glycodendrimers were determined using MALDI-TOF MS. Surface plasmon resonance studies found that rgp120 IIIB bound to the derivatized dendrimers tested with nanomolar affinity, and to dextran sulfate with picomolar affinity. In vitro studies of the effectiveness of these compounds at inhibiting infection of U373-MAGI-CCR5 cells by HIV-1 Ba-L indicated that the sulfated glycodendrimers were better inhibitors than the nonsulfated glycodendrimers, but not as effective as dextran sulfate.

Botulinum neurotoxin A activity is dependent upon the presence of specific gangliosides in neuroblastoma cells expressing synaptotagmin I.

Botulinum neurotoxin A (BoNT/A) is the deadliest of all known biological substances. Although its toxicity makes BoNT/A a biological warfare threat, its biologic activity makes it an increasingly useful therapeutic agent for the treatment of muscular disorders. However, almost 200 years after its discovery, the neuronal cell components required for the activity of this deadly toxin have not been unequivocally identified. In this work, neuroblastoma cells expressing synaptotagmin I, a protein shown to be bound by BoNT/A, were used to determine whether specific gangliosides were necessary for BoNT/A activity as measured by synaptosomal-associated protein of 25 kDa (SNAP-25) cleavage. Ganglioside GT1b was found to support BoNT/A activity significantly more effectively than GD1a, which was far more effective than GM1 when added to ganglioside-deficient murine cholinergic Neuro 2a or to human adrenergic SK-N-SH neuroblastoma cells. Whereas both cell lines expressed synaptotagmin I, SNAP-25 cleavage was not observed in the absence of complex gangliosides. These results indicate that 1) gangliosides are required for BoNT/A activity, 2) synaptotagmin I in the absence of gangliosides does not support BoNT/A activity, and 3) Neuro 2a cells are an efficient model system for studying the biological activity of BoNT/A.