A platform approach for the production of Hand, Foot, Mouth Disease vaccines

Hand, Foot and Mouth Disease (HFMD) is an endemic childhood disease in Southeast Asia, with substantial disease burden affecting millions of children each year. Occasionally the central nervous system is involved causing serious and sometimes fatal neurological complications. HFMD outbreaks are also observed outside the Asia-Pacific countries. HFMD can be caused by multiple enteroviruses of which the best known virus is EVA71. However, also other enteroviruses such as CVA6, CVA10 and CVA16 can cause the disease. Inactivated EVA71 vaccines are registered in China, but in order to prevent all HFMD cases, multivalent vaccines are warranted. Intravacc is developing an HFMD combination vaccine. Here we used our rescue platform to generate the starting materials required for vaccine production. Infectious clones from EVA71_B4, EVA71_C4, CVA6, CVA10 and CVA16 were constructed and the corresponding enteroviruses were rescued. Virus seeds were produced on Vero cells in animal component free medium. Rescued enteroviruses could efficiently replicate, resulting in seed lots with high viral titers. This rescue platform has the major advantage that clinical isolates are not required to obtain the starting material to produce a vaccine, thus mitigating the risk that other, unwanted, viruses are also present. Next to that, the virus source is pre-designed, controlled and well documented. Please click Download on the upper right corner to see the full abstract

Third generation vaccine for world eradication of poliomyelitis

Great efforts have been undertaken by the World Health Organization to achieve eradication of poliomyelitis, a paralytic disease. At present, two different vaccines are available: inactivated polio vaccine (IPV) developed by Salk based on chemical inactivation of the virus and oral polio vaccine (OPV) developed by Sabin based on live attenuated virus strains. The risks associated with IPV concern the safety of the production process as it is based on highly virulent wild type strains, and in contrast, the OPV risks are associated with the reversibility of the attenuated viruses to a transmissible paralytic form. There is therefore a need for a new generation polio vaccines capable to overcome outbreaks and manufacturing risks. With the evolution of molecular virology of Sabin vaccine strains, it is now possible to design extremely genetically stable and hyperattenuated strains without the associated reversion risks. Sabin poliovirus strains were therefore genetically modified giving rise to the third generation of polio vaccine strains [1, 2]. In the present work we have explored the possibility of using the already well-established IPV production process, developed at our site [3] and integrated worldwide [4] for the production and manufacturing of third generation of IPV strains. Specifically, we have produced third generation vaccines in animal component free medium and at 50-L pilot scale. The product obtained did show acceptable yields and was immunogenic in rats. Together, our results indicate that the third generation vaccine strains produced under the flexible platform process are potential candidates which provide increased biosafety during manufacturing which is necessary after polio eradication. In addition, the flexibility and scalability of the process constitute a platform for the production of a large range of vaccines worldwide. 1. Knowlson, S., et al., New Strains Intended for the Production of Inactivated Polio Vaccine at Low-Containment After Eradication. PLoS Pathog, 2015. 11(12): p. e1005316. 2. Macadam, A.J., et al., Rational design of genetically stable, live-attenuated poliovirus vaccines of all three serotypes: relevance to poliomyelitis eradication. J Virol, 2006. 80(17): p. 8653-63. 3. Thomassen, Y.E., et al., Scale-down of the inactivated polio vaccine production process. Biotechnol Bioeng, 2013. 110(5): p. 1354-65. 4. Wezel, v., Monolayer growth systems: Homogeneous unit processes. Spier, R. E. and Griffiths, J. B., eds., 1985: p. 266-281

DEVELOPMENT OF INACTIVATED POLIO VACCINE FROM ATTENUATED SABIN STRAINS FOR CLINICAL STUDIES AND TECHNOLOGY-TRANSFER PURPOSES

Recently, responding to WHO’s call for new polio vaccines, the development of Sabin-IPV (injectable, formalin-Inactivated Polio Vaccine, based on attenuated ‘Sabin’ polio virus strains) was initated at NVI. This activity plays an important role in the WHO polio eradication strategy. The use of Sabin instead of wild-type Salk polio strains will provide additional safety during vaccine production. Initially, the Sabin-IPV production process will be based on the scale-down model of the current, and well-established, Salk-IPV process. In parallel, process development, optimization and formulation research is being carried out to further modernize the process and reduce cost per dose. The lab-scale accelerated process development, product characterization, clinical lot production, and preparations for technology transfer will be discussed. Multivariate data analysis (MVDA) was applied on data from current IPV production (more than 60 Vero cell culture based runs) to extract relevant information, like operating ranges. Subsequently, based on the MVDA analysis, a 3-L scale-down model of the current twin 750-L bioreactors has been setup. Currently, in this lab-scale process, cell and virus culture approximate the large-scale and process improvement studies are in progress. This includes the application of increased cell densities, animal component free media, and DOE optimization in multiple parallel bioreactors. Also, results will be shown from large-scale (to prepare for future technology transfer) generation and testing of Master- and Working virus seedlots, and clinical lot (for phase I studies) production under cGMP conditions. The obtained product was used for immunogenicity studies in rats. It was shown that Sabin-IPV induces a good immune response, and a comparison will be made to regular Salk-IPV. Finally, technology transfer to vaccine manufacturers in low and middle–income countries will take place. For that, an international Sabin-IPV manufacturing course, including practical training at pilot-scale, is being setup

Rat immunogenicity (VNT against wild-type viruses).

<p>Panel A, B and C: VNT (log₂ titer) to immunization with plain sIPV (blue) and adjuvanted sIPV (red) for PV type 1, 2 and 3 respectively; Panel D, E and F: VNT of plain sIPV 20/32/64 (light blue), 10/16/32 (red), 5/8/16 (green) and plain IPV 40/8/32 (dark blue) for PV type 1, 2 and 3 respectively. Error bars in panel A-F depict standard deviation of the median (n=10 rats).</p

Stability of sIPV.

<p>Panel A: PV type 1 VNT of plain sIPV 20/32/64 (blue), 10/16/32 (red), 5/8/16 (green) in time, error bars depict standard deviation of the median (n=10 rats); Panel B: slopes of linear regression lines determined for stability based on rat immunogenicity as illustrated in panel A, error bars depict 95% confidence interval; Panel C: Stability of PV type 1 D-antigen of sIPV 20/32/64; Panel D: slopes of linear regression lines determined for stability based on D-antigen as illustrated in panel C, error bars depict 95% confidence interval.</p

Process overview for preparation of trivalent IPV.

<p>Monovalent bulks are prepared for each PV (type 1, 2 and 3) separately. During monovalent bulk preparation Vero cells are expanded using two pre-culture steps and a cell culture followed by virus culture. Virus is purified using normal flow filtration for clarification, tangential flow filtration for concentration and two chromatography units, size exclusion and ion exchange chromatography. Purified virus is subsequently inactivated using formaldehyde. Subsequently these are mixed to obtain trivalent bulk prior to formulation and filling.</p

Purification of Sabin PV.

<p>Panel A depicts a SEC chromatogram of Sabin PV type 1. The 1^st peak contained mostly large cell components; the 2^nd peak contained the majority of PV, following peaks consist of smaller components. Panel B shows a SDS-PAGE (4-20% gel); lanes represent (from left to right) the marker, the concentrated product, followed by the 1^st and 2^nd fraction of SEC and finally the IEX purified PV. Panel C shows chromatograms of Sabin PV type 1 (left) and Sabin PV type 2 (right) IEX purification. Panel D shows host cell protein (open) and DNA (solid) impurities. Panel E depicts the inactivation of PV, the gray area indicates the lower detection limit. In chromatograms A and C, the dotted and solid lines represent absorbance at respectively 254nm and 280nm. Gray dotted lines indicate peak fractioning.</p

Cell and virus culture.

<p>Panel A shows the average Vero cell growth curve (n=12; error bars represent SD) in 350-L bioreactors. Photographs are light microscopy images (size bar 200 µm). Panel B shows the average (of the three subtypes) Vero cell death during virus culture determined microscopically (n=12; error bars represent SD). Photographs show corresponding images. Panel C shows average virus titers for Sabin PV type 1, 2 and 3 (n=4; error bars represent SD). Panel D shows average D-antigen concentrations after virus culture for Sabin PV type 1, 2 and 3 (n=4; error bars represent SD).</p