Microbial platform for vaccine production for low and medium income countries (LMICs): 2 case studies

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PENGARUH pH ELUEN TERHADAP HASIL DESALTING CRUDE PERTUSSIS TOXIN (PT) DAN FILAMENTOUS HEMAGGLUTININ (FHA) DARI Bordetella pertussis

Optimizing desalting crude process of PT and FHA using sephadex G-25 as intermediate purification phase for PT and FHA as acellular pertussis vaccine components has been developed. The factor influencing optimization including the impact of chemical factor such as eluation buffer pH towards protein’s concentrate were studied using 3 different pH (6, 7, and 8). The result were : Different Buffer pH has influence towards protein recoveries whereas phosphate buffer 50 mM pH 7.0 as eluation buffer (with 74,23 + 3,07 % recoveries percentage) best for desalting crude PT and FHA

Technical transfer of a rapid microbial platform for vaccine production

The limited availability and affordability of vaccines to low- and middle-income countries (LMIC) has created a need for solutions that will ensure effective, affordable vaccine production technology. To establish a rapid and economical platform for the expression of viral proteins in high yield and purity by Pichia pastoris (X33), the receptor-binding domain (RBD) protein of the SARS-CoV2 was selected in this study. After fermentation at the 5 L scale, the protein was purified by a simplified chromatography, with minimal sample treatment. The purified protein was characterized biochemically, and after its formulation, the immunogenicity was evaluated in mice. Collectively, the data suggested that the vaccine candidate is a suitable COVID-19 vaccine candidate antigen for technology transfer. Furthermore, this study creates a robust foundation for industrial production at scale

Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction

Microbial platform for vaccine production for Low-and Medium-Income Countries (LMICs): 2 case studies

Vaccination is critical for the prevention and control of infectious-disease outbreaks, being of paramount importance to global health, they are a key component of primary health care and an indisputable human right. Yet far too many people around the world have insufficient access to vaccines. The limited availability and affordability of vaccines to resource low-income countries has created a need for solutions that will ensure effective, affordable vaccine production technology. With potential for more pandemics, the urgency to expand vaccine range has become even more evident. We will present a collaborative project between UCL and PT Bio Farma in which we have developed platforms to manufacture two novel immunization candidates, a recombinant virus-like-particles vaccine against Dengue disease (case study 1) and a recombinant protein vaccine against COVID-19 disease (case study 2). Both vaccines will have a social and economic impact by reducing the number of cases, and the overall mortality and morbidity. Dengue is an emerging mosquito-borne viral infection with increasing reports of outbreaks and can be in tropical and sub-tropical areas, with Southeast Asia and the Western Pacific being the most seriously affected zones. A total of 3.8 billion people is potentially at risk, and in a scenario of global warming, this can increase to 6 billion people by 2080. To date, there is one commercialized vaccine (Sanofi-Pasteur) but is restricted to individuals aging from 9-45 years, who have been previously infected with the virus. The COVID-19 pandemic has sum approx. 400 million cases and 6 million deaths worldwide since it was first reported in 2019. Even though the production and delivery of a vaccine has significantly increased over time, the affordability, accessibility, and acceptability at individual and country levels is still a major limitation. Please click Download on the upper right corner to see the full abstract

Reverse-Transcriptase Characteristics of Hepatitis B Virus Polymerase Gene in Treatment-Naïve Asymptomatic Chronic Hepatitis B Individuals

Nucleos(t)ide analogues (NUCs) remain the main treatment for chronic hepatitis B (CHB). Long-term use of NUCs significantly reduces disease progression; however, it might lead to resistance-associated mutations. We studied characteristics of polymerase gene related to NUCs resistance in naïve hepatitis B surface antigen (HBsAg)-positive individuals. The research was done at Laboratory of Hepatitis, Eijkman Institute, Jakarta Thirty eight samples were obtained and submitted for HBV DNA detection. Identification of mutations was performed by PCR-sequencing, and analyzed to obtain NUCs resistance motifs. Genotype and subtype were determined based on HBsAg sequence. Mutation of rtQ238H/N was found in 37 (97.4%) samples. Of those, 23 (62.2%) showed rtQ238H mutation, 10 (27.0%) had rtQ238N mutation, and four (10.8%) with double mutations of rtA194T and rtQ238H. Genotype B was found in 26 (68.4%), C in 11 (28.9%), and D in one (2.6%) samples. Statistically, the mutation variant of rtQ238H was associated with genotype B (p<0.001), while rtQ238N with C (p<0.001). The ayw subtype was found in 25 (65.8%), adr in 11 (28.9%), and adw in two (5.3%) samples. No mutation associated with NUCs resistance was found in most samples. This emphasizes that NUCs are still a prospective treatment in naïve CHB patients.  Mutation of rtQ238H was a variant found to be significantly associated with HBV genotype B and rtQ238N with genotype C

Pengembangan Vaksin Hepatitis B Generasi Ke Tiga dan Vaksin Terapi Berbasis Protein Rekombinan Subunit Indonesia

Abstract In strengthening National Innovation System (Sistem Inovasi Nasional, SINas) through im- provement of synergy, productivity and utilization of national resources as well as partner- ships with industry/private sector in research and development particularly in health sector, a hepatitis B consortium has been established. This consortium is a collaboration between several institutions including PT. Bio Farma (Persero), Lembaga Eijkman, Pusat Teknologi Farmasi dan Medika BPPT, and ITB. Development and production of recombinant protein- based hepatitis B vaccines in this consortium is a multi-year program where in 2012-2014 periods it has generated the second generation of hepatitis B vaccines. As a continuation of the program, construction of clones for expression of M HBsAg and L HBsAg in Pichia pas- toris for the third generation of hepatitis B vaccines, and development of a therapeutic hep- atitis B vaccine comprising of HBsAg and HBcAg were done in 2015. Expression of M and L HBsAg in Pichia pastoris and HBcAg in E. coli and characterization of these proteins were then completed in 2016. In 2017, the purification of the proteins, formulation develop- ment involving adjuvants, in vitro and in vivo evaluations of the formulation in animal model were performed. The expected outcome of the program is to generate the second-generation vaccine and therapeutic vaccine candidates against hepatitis B, which could be transferred to industrial applications. The vaccines that are being developed are essential in enhancing Indonesian self-reliance in combating hepatitis B disease. Besides, production of these vac- cines offers enormous economical benefits since the products can be marketed to fulfill both national and global demands. Keywords: Hepatitis B vaccine, Recombinant Protein, sHBsAg, HBcAg, M HBsAg, L HBsAg, Pichia pastoris, E.coli

Reverse-Transcriptase Characteristics of Hepatitis B Virus Polymerase Gene in Treatment-Naïve Asymptomatic Chronic Hepatitis B Individuals

Nucleos(t)ide analogues (NUCs) remain the main treatment for chronic hepatitis B (CHB). Long-term use of NUCs significantly reduces disease progression; however, it might lead to resistance-associated mutations. We studied characteristics of polymerase gene related to NUCs resistance in naïve hepatitis B surface antigen (HBsAg)-positive individuals. The research was done at Laboratory of Hepatitis, Eijkman Institute, Jakarta Thirty eight samples were obtained and submitted for HBV DNA detection. Identification of mutations was performed by PCR-sequencing, and analyzed to obtain NUCs resistance motifs. Genotype and subtype were determined based on HBsAg sequence. Mutation of rtQ238H/N was found in 37 (97.4%) samples. Of those, 23 (62.2%) showed rtQ238H mutation, 10 (27.0%) had rtQ238N mutation, and four (10.8%) with double mutations of rtA194T and rtQ238H. Genotype B was found in 26 (68.4%), C in 11 (28.9%), and D in one (2.6%) samples. Statistically, the mutation variant of rtQ238H was associated with genotype B (p<0.001), while rtQ238N with C (p<0.001). The ayw subtype was found in 25 (65.8%), adr in 11 (28.9%), and adw in two (5.3%) samples. No mutation associated with NUCs resistance was found in most samples. This emphasizes that NUCs are still a prospective treatment in naïve CHB patients.  Mutation of rtQ238H was a variant found to be significantly associated with HBV genotype B and rtQ238N with genotype C