Challenges in the development and scale-up of a purification process for an attenuated live virus vaccine candidate

Prophylactic live attenuated vaccines (LAV) have been successfully developed for multiple viral disease targets, offering an advantage over subunit vaccine approaches by simultaneously stimulating innate, humoral and cellular immune responses. However, the development of manufacturing processes for robust production of LAVs at commercially viable scales can be challenging, particularly because of the need to use novel and/or adherent cell lines, the inefficient performance of conventional chromatography for processing large viral particles, and the complexity of product characterization. Further adding to these challenges, closed-system aseptic processes are required for those viruses too large for terminal sterile filtration, thereby limiting processing options and complicating process logistics at commercial scale. Highlighting these challenges, we present here on the development of a scalable, fully sterile, purification process for a large, enveloped, live attenuated virus having multiple glycoprotein complexes. During the development of this vaccine the process was changed from a static cell culture process to one that is amenable to scaling in a stirred tank single-use bioreactor. This change presented challenges for the purification process, requiring modifications of the process separation techniques including evaluation of new unit operations of various separation modes such as membrane and monolith absorbers, resin chromatography, selective precipitation, large pore tangential flow filtration, and centrifugation. Critical to this evaluation was the understanding of the adaptability of these unit operations to closed sterile processing, with a premium placed on industry ready, single-use technologies. Through this process development effort, a scalable, sterile, purification process was defined that met targets for purity and yield

Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile

Tetravalent vaccine generates high neutralizing antibody titers in rhesus macaques.

<p>Rhesus macaques (n = 5) were given 3 immunizations with the tetravalent vaccine formulated with either ISCOMATRIX™ or ISCOMATRIX^™ plus AAHS on d0, d7, and d30. Control animals were immunized on the same days with a formalin inactivated toxoid vaccine adjuvanted with Rehydragel. Serum samples were collected on days 0, 7, 21 and 45. Individual (Day 45) or pooled (days 0, 7 and 21) serum samples were preincubated with active toxin prior to being added to Vero cells. ED50 values indicate the serum dilution at which the area of the Vero cell monolayer is reduced by 50%. Strong neutralizing antibody titers were generated against (A) TcdA, (B) TcdB, and (C) binary toxin. Day 45 titers in these graphs represent the mean value of the individual neutralizing titers. Neutralizing antibody titers to D) TcdA, E) TcdB, and F) binary toxin were measured from individual rhesus macaque serum samples on day 45. Statistical analysis was performed using One-way ANOVA and Tukey multiple comparisons test. * = p<0.05, ns = no significant difference.</p

Purified vaccine components.

<p>Three micrograms 5mTcdA (lane A, 308kD), 5mTcdB (lane B, 269kD), proCDTb (lane C, 94kD) and 4mCDTa (lane D, 48kD) were loaded on a 8–16% Tris-Glycine (Invitrogen, Carlsbad, CA). GelCode Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA) was then used to stain the gel.</p

Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.

<p>Potency of recombinant and native toxins in a cytotoxicity assay and mouse toxicity model.</p