Expression and scale-up production of recombinant human papillomavirus type 52 L1 protein in methylotrophic yeast Hansenula polymorpha.

BACKGROUND: Human papillomavirus (HPV) vaccination is one of the crucial national vaccination programs aimed at reducing the prevalence of the diseases associated with HPV infections, which continue to pose a global health concern. However, a significant disparity exists in the distribution of HPV vaccine, particularly in low-middle income countries where the cost of HPV vaccine becomes a major obstacle. Thus, it is essential to ensure the availability of an economically feasible HPV vaccine, necessitating immediate efforts to enhance the cost-effectiveness of vaccine production. This study aimed to develop an efficient production system for the recombinant HPV type 52 L1 protein as HPV vaccine material using methylotrophic yeast Hansenula polymorpha expression system. RESULTS: This study presents an in-depth examination of the expression and scale-up production of HPV type 52 L1 protein using DASGIP® parallel bioreactor system. The pHIPX4 plasmid, which is regulated by the MOX promoter, generates stable clones that express the target protein. Cultivation employing the synthetic medium SYN6(10) with controlled parameters (e.g. temperature, pH, feeding strategy, and aeration) produces 0.15 µg/mL of HPV type 52 L1 protein, suggesting a possibility for scaling up to a higher production level. CONCLUSION: The scale-up production of HPV type 52 L1 protein using Hansenula polymorpha expression system described in this study provides an opportunity for an economical manufacturing platform for the development of the HPV vaccine.

Molecular characterization of enterovirus-A71 in children with acute flaccid paralysis in the Philippines.

BACKGROUND: Several inactivated enterovirus-A71 (EV-A71) vaccines are currently licensed in China; however, the development of additional EV-A71 vaccines is ongoing, necessitating extensive analysis of the molecular epidemiology of the virus worldwide. Until 2012, laboratory confirmation of EV-A71 for hand, foot, and mouth disease (HFMD) and other associated diseases had not occurred in the Philippines. Because EV-A71 has been linked with cases of acute flaccid paralysis (AFP), AFP surveillance is one strategy for documenting its possible circulation in the country. To expand current knowledge on EV-A71, molecular epidemiologic analysis and genetic characterization of EV-A71 isolates were performed in this study. METHODS: A retrospective study was performed to identify and characterize nonpolio enteroviruses (NPEVs) associated with AFP in the Philippines, and nine samples were found to be EV-A71-positive. Following characterization of these EV-A71 isolates, the complete viral protein 1 (VP1) gene was targeted for phylogenetic analysis. RESULTS: Nine EV-A71 isolates detected in 2000 (n = 2), 2002 (n = 4), 2005 (n = 2), and 2010 (n = 1) were characterized using molecular methods. Genomic regions spanning the complete VP1 region were amplified and sequenced using specific primers. Phylogenetic analysis of the full-length VP1 region identified all nine EV-A71 Philippine isolates as belonging to the genogroup C lineage, specifically the C2 cluster. The result indicated a genetic linkage with several strains isolated in Japan and Taiwan, suggesting that strains in the C2 cluster identified in the Asia-Pacific region were circulating in the Philippines. CONCLUSION: The study presents the genetic analysis of EV-A71 in the Philippines. Despite some limitations, the study provides additional genetic data on the circulating EV-A71 strains in the Asia-Pacific region, in which information on EV-A71 molecular epidemiology is incomplete. Considering that EV-A71 has a significant public health impact in the region, knowledge of its circulation in each country is important, especially for formulating vaccines covering a wide variety of strains.

Multiple independent emergences of type 2 vaccine-derived polioviruses during a large outbreak in northern Nigeria.

Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5'-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that ∼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.