Pichia pastoris strain GS115 as a Cost-Effective Expression Host to

Produce Recombinant Protein Target (rPstS-1) in Tuberculosis Vaccine

Development Candidates
Tiara Rahayu1, Junindra Duha2, Nabila Swarna Puspa Hermana3,4,*
1
Undergraduate Student of Biology, Faculty of Military Mathematics and Natural Sciences, Republic of Indonesia Defense University,
Indonesia
2
Graduate Student of Defense Economics, Faculty of Defense Management, Republic of Indonesia Defense University, Indonesia
3
Faculty of Military Mathematics and Natural Sciences, Republic of Indonesia Defense University, Indonesia
4
Doctoral Program in Animal Biomedical Sciences, School of Veterinary Medicine and Biomedical Sciences, IPB University, Indonesia

*correspondence E-mail: hermananabila@gmail.com

ABSTRACT

The Indonesian government refers to the National Medium-Term Development Plan 2020-2024, which aims to achieve a Tuberculosis (TB)
free world by 2035. Indonesia's TB case ranking in 2022 is the 3rd after India and China, with 93.000 deaths per year with 824 thousand
cases or 11 per hour. There is an urgency to expand the range of vaccinations required with limited vaccine availability and affordability
s for developing countries that have created the solutions to ensure a cost-effective vaccine production system. The current study review
effort to produce an inexpensive vaccine using Pichia pastoris strain GS115 as an expression host to make recombinant protein rPstS-1 .
s systematic review of the literature was conducted in five databases, including Google Scholar, PubMed, Science Direct, Scopus, and
A
Springer Link. This literature study evaluates producing a cost-effective TB vaccine by utilizing the genetics of P. pastoris as one of the
candidates for TB vaccine solutions to be able to conduct further research and can be followed up by national research agencies. The
novelty of this study that P. pastoris as the host in the TB vaccine development with strain GS115 is cost-effective expression host on a
large industrial scale, can perform genetic and molecular manipulation and increase the secretion of proteins into the medium and is
feasible to be implemented in Indonesia's national TB vaccine program.

Keywords: Cost-Effective Expression Host, Pichia pastoris, Recombinant protein, Tuberculosis Vaccine.

Introduction

Minister of Health announces partnerships to improve vaccination rates and global demand for vaccines continues to
increase, considering the development of effective and efficient vaccination strategies. Tuberculosis is one of the deadliest
diseases in the world. The death rate is much higher than the death caused by Covid-19. In 2022 TB in Indonesia ranks
third, with five hundred thousand people who have not been treated and are at risk of becoming a transmission source. An
important reason for developing a renewable TB vaccine formula is the presence of drug resistance which causes treatment
not to be completed, and mutation occurs in bacteria, so the vaccine is no longer effective (Rodrigues LC, 2005) (Leung CC,
2001). BCG (Bacillus Calmette-Guerin) is a live strain of Mycobacterium bovis attenuated to cause sensitivity to M.
tuberculosis which was first discovered in 1882. The BCG vaccine was first used for humans in 1921 in France but did not
prevent TB from becoming endemic. Until now, TB control is still considered multidrug-resistant (MDR) TB To realize a
healthy and TB-free world, WHO launches new guideline for the control and elimination of human.

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
"Stop the TB Strategy" by 2035, the strategy aims to reduce the incidence of tuberculosis and the mortality ratio by 90% and
95% respectively. The BCG vaccine is thought to be less effective. The novelty of this paper for develops a new
tuberculosis vaccine using a recombinant protein vaccine formula strategy with P. pastoris as a safer host because it does
not directly involve pathogenic bacteria has low production costs on a large industrial scale and can be administered to
immunocompromised patients. Future TB vaccine development should not alter the target organism's immunological
characteristics, leading to drug resistance. It should control TB MDR and XDR, especially given the reported resistance to
new mycobacterial drugs bedaquiline and delamanid (Bloemberg GV, 2015).

The need to develop a TB vaccine is urgent in the aftermath of Covid-19 and the world conditions experiencing an
economic crisis and recession. Countries need to strengthen health resilience, especially developing countries such as
Indonesia, to design low-cost TB vaccines that can be produced on a large industrial scale by utilizing natural resources and
biodiversity. P. pastoris is closely related to the economic value, which is very effective in vaccine production when
assessed through cost-effectiveness analysis (CEA). The method is designed to compare costs used in health for
implemented programs or interventions with other alternative results (Vogenberg, 2001). Health output is expressed in
objective and measurable terms, such as the number of the decrease in blood pressure and cases treated, and not in monetary
terms (Vogenberg, 2001). Cost-effectiveness analysis is one way to estimate and select the best program when several
programs with similar goals are on hand. The evaluation criteria for which program to elect are based on the unit cost
discount of each alternative program with the lowest unit cost discount and selected by the analyst maker (Prijona
Tjiptoherijanto & Budhi Soesetyo, 1994). The CEA method has an output element in the form of effectiveness in solving
problems, expressed in a certain measure which for the health sector is in the form of health parameters (Rainer et al.,
2000). In economic evaluation, effectiveness differs from cost savings, which refers to competing for alternative programs
that provide lower costs, while cost-effectiveness does not merely consider softer cost aspects (Grosse et al., 2008). CEA
helps provide optimal alternatives that do not necessarily mean lower costs. CEA helps identify and promote the most
efficient treatment therapies (Grosse et al., 2008).

The method used in the TB vaccine implementation based on CEA is beneficial when comparing alternative programs or
intervention alternatives where the different aspects are not only the program or intervention but also the clinical outcome or
therapy. By calculating the efficiency measures (cost-effectiveness ratio), alternatives with additional costs, efficacy rates, and
safety rates, the comparison will be carried out in a balanced manner (Grosse et al., 2008). Cost Effectiveness Analysis is
used when benefits are challenging to transform into money. Hence, CEA is perfect for measuring efficiency in the social
sector, especially in the health sector, which is a program/intervention. An intervention can be understood as any activity
using a variety of inputs that aims to improve health. CEA is often used to measure the efficiency of various programs with
the same goal.

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
 Programs Goals

Figure 1. Different programs with the same objective

Methods

Literature research

Databases: Google scholar, Science direct, Springer link, PubMed, Scopus

Literature search results: 125

Articles are filtered on the basis of titles, abstracts and keywords (cost-effective Expression host,
Pichia pastoris, recombinant protein, TB vaccine)

The article is not thoroughly accessible The article results to be Unprocessed article results (n=37)
to the aim this paper (n=19) processed back (n= 69)

Articles filtered by looking at the whole text

The article results to be processed back The article results that are not processed
back (n=15)

Filtering the article to continue processed

Previous studies relevant to this search (n= 54)

Figure 2. Diagram of systematic review stages with PRISMA

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
Result and Discussion

In economic evaluation, effectiveness differs from cost savings, which refers to competing for alternative programs that
provide lower costs. At the same time, cost-effectiveness does not merely consider lower-cost aspects. In considering the
choice of a product or type of health service to be selected, cost-effectiveness must still be considered if CEA helps provide
an optimal alternative which does not always mean lower costs. CEA helps identify and promote the most efficient
treatment therapies. CEA is beneficial when comparing alternative programs or intervention alternatives where the different
aspects are not only the program or intervention but also the clinical outcome or therapy. Health outcomes used as a
denominator in the cost-effectiveness ratio can be expressed in units such as the number of years saved or an index of utility
or need such as QALYs. Many people use QALYs as the denominator of CUA outcomes, but now many experts have
recommended that CEA use QALYs wherever possible.

Table 1 Comparison of healthcare and societal perspectives on cost expenditure

Source: (Machlaurin et al., 2020)

Figure3Displaysthecost-effectivenessratio(ICER)ofvaccinationstrategiesversusnon-vaccinationapproaches.

Source: (Machlaurin et al., 2020)

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
The bar height represents the ICER for the basic vaccine, that has 71% cost effectiveness against TB, and the error bar
(42%), and the cost-effectiveness range upper (85%). Three types of protection used to: one for ten years (base case), one
for a 20- year linear decrease effect (scenario 1), and one for an exponential decrease effect (scenario 2).

Applying the second protection for the 20-year linear decay effect in Scenario 1 nearly triples the ICER in social and health
care ($226/QALY and $233/QALY). However, when exponential overall effects were applied in Scenario 2, the ICER was not
significantly different ($113/QALY and $121/QALY for the social and health perspectives, respectively). Combined with
ICER, the above vaccination strategies can be evaluated and tend to be cost-effective as all ICERs were well below Indonesia's
GDP per capita ($3847) in 2018, as shown in Table 1.

Univariate Sensitivity Analysis

Figure 6 illustrates the tornado diagrams for the various model input parameters that affect ICER. The four most essential
activators in the analysis were the vaccine effectiveness for the cost of vaccination, disease prevention, natural case fatality
rate, and case detection rate for untreated TB, with other components having little effect on ICER.

Figure 4. The Tornado diagram from univariate analysis. TB=Tuberculosis, DSTB = drug-susceptible TB, MDRTB = drug-
resistant TB, VE = vaccine effectiveness, and QALY = quality-adjusted years of life.
Source: (Machlaurin et al., 2020)

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
 Probabilistic Sensitivity Analysis

Figure 5. Demonstrate a cost-effective strategy to comply with different float value readiness to pay levels.

With a threshold value of one GDP per capita, the price of BCG vaccination is 100% cost-effective, with a 95% chance of
being cost-effective. The monthly GDP per capita is close to USD 175.

(a)

(b)

Figure 5 (a) Acceptability curve of cost-effectiveness and (b) Field of cost-effectiveness of health care perspective
obtained from probabilistic sensitivity analysis. QALYs = years of quality-adjusted life.

Source: (Machlaurin et al., 2020)

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
It is possible to analyze BCG vaccination in general by applying TB epidemiological data and TB incidence rates by age to
the Indonesian context, which represents the various efficacy of BCG against TB infection, disease, and progression and
applies various protections and durations of waning. According to our statistics modeling results, the BCG vaccination
strategy is far more cost-effective than not vaccinating too. The vaccine, which cost $14 per vaccination, prevented 49,713
TB cases and 7,598 TB deaths (Table 1). Moreover, the strategy requires a threshold below one GDP per capita per QALY
that remains below monthly GDP per capita from both a health payment and a community objective. In the one-way
sensitivity analysis, the method's cost-effectiveness was proved to be robust for most of the input parameters. Other BCG
vaccination health economic studies (Machlaurin et al., 2019) show that the vaccine's efficacy against TB disease is the most
influential variable in the cost-effectiveness analysis. Despite the fact that the BCG vaccination was introduced decades ago,
its efficacy is still debated. The level of difference from previous exposure to non-tuberculosis mycobacteria (NTM) could
be one reason for this inconsistency (Mangtani et al., 2014). Individuals in low-income areas, as well as older children, are
more susceptible to NTM exposure (Clemens et al., 1983). Given the objective evidence in this review article, the applied
model requires relatively conservative assumptions. The VE profile study was based on a meta-analysis that discovered
three levels of BCG efficacy: protection from TB disease and infection (latent TB) and limiting progression from condition
to disease (i.e., from latent TB to active TB) (Roy et al., 2014). Although other studies have not examined VE associated
with TB disease (Rahman et al., 2001; Trunz et al., 2006), these estimates are certain to be included because recent evidence
suggests that the BCG vaccine prevents not only TB disease but also TB infection and infection. Its emergence (Abubakar et
al., 2013; Colditz et al., 1994; Mangtani et al., 2014). The cost of vaccination is another crucial component in this analysis.
Although the cost of immunization appears only once in the first year and appears modest in correlation to other health
costs, it's the second most influential variable in the analysis. This implies that vaccination prices play a crucial role in the
overall TB, cost burden, and decision-makers will be justified in relying more on the implementation, optimization, and
sustainability of TB vaccination programs. The Markov model also confirms the finding (Trunz et al., 2006) that universal
BCG vaccination costs $2-3 and is a very cost-effective intervention to prevent TB in severely ill children in countries with
high and low incidence levels of BCG coverage. Southeast Asia, Africa, and the West Pacific region are heavily affected.
These findings, however, do not support studies conducted in Japan and Taiwan, which have low to moderate TB
incidence. A study in Japan, in particular (Rahman et al., 2001), showed a significant cost of universal BCG vaccination per
prophylactic case, US$35,950-175,862, with a VE range of 40% to 80%.

Most therapeutically relevant proteins require posttranslational modifications to function normally, such as N-glycosylation
or O-glycosylation; the most abundant glycoproteins are currently expressed in mammalian cell cultures. Based on its high
volumetric productivity, low media cost, lack of retroviral contamination, and ease of stable cell creation, the P. pastoris
protein expression system is a feasible alternative. The presence of mannose-type N- and O- glycans, on the other hand,
makes glycoproteins derived from the P. pastoris expression system less desirable for human applications. Pichia has
emerged recently as a model system for protein expression, and previous research laboratories have described the
humanization of this P. pastoris protein-glycosylation pathway.

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
In fungi, O-mannosylation proteins are initiated in the endoplasmic reticulum (E.R.) by the protein-Omannosyltransferases
(PMT) family, whereby transmission of mannose from dolichol Phosphate-activated mannose (Dol-P-Man) to serine or
threonine proteins that enter the E.R. Additional mannosyl transferase in P. pastoris extends the mannoses chain through
four residues (Nett et al., 2013).

Some aspects of producing recombinant Mycobacterium (MTB) antigens are essential, including studying antigen
interactions and their effects in vivo and in vitro to develop an efficient MTB vaccine design (Bando-Campos et al., 2019).
Rv2164c, Rv3491, Rv0175, Rv1887, Rv1096, Rv2068c, Rv2744c, Rv2799, Rv3835, and Rv1860 are some protein antigens
secreted and found in MTB cultures (Smith GT, 2014). Glycoantigen PstS-1, which has posttranslational characteristics
similar to the original MTB antigen, is one of the targets of MTB phosphate binding proteins (Bando-Campos et al.,
2019). The use of preferential codons in P. pastoris is a sequence of genes encoding M. tuberculosis PstS-1 (Rv0934)
(GenBank number: P9WGU1). The secondary structure of rPstS-1 produced in P. pastoris had a conformation-sheet
corresponding to that observed for the original protein (Bando-Campos et al., 2019) conducted a western blot experiment using
polyclonal antibodies of anti-Mycobacterium-tuberculosis-PstS-1 rabbit and a human sample serum of patients with
clinically confirmed active TB Anti-PstS-1 polyclonal antibodies reacted violently to P. pastoris rPstS-1 while compared to
His-Tag- rPstS-1 expressed E. coli. MTB-derived proteins can be found in high levels in P. pastoris GS115. It is assumed
to be capable of boosting immunoreactivity in the presence of this modification (S. Wang et al., 2018). It has been proposed
that PstS-1 has three O-glycosylation sites, most likely on threonine 20, 21, and 28 as in N-terminus, as a predictive tool for
analyzing some MTB glycoproteins. Previous studies have explained the production of recombinant O-mannosylated
glycoantigen that does not mark PstS-1 to produce PstS-1 glycoantigen with post-translation characteristics similar to
MTB's original antigen and to avoid the use of harmful MTB that requires a long period of cultivation. This article discusses
the laboratory-scale production of recombinant glycoantigen O-mannosylated PstS-1 (rPstS-1, with 98.9% sequence identity
to the original protein), its purification and characterization, some modifications to its O-mannosylation, and its
immunological reactivity with serum from TB patients. This glycoprotein's production will assist in the study of its
immunological activity. The vaccine will be useful as a diagnostic tool and as a TB vaccine. The unproduction recombinant
O-mannosylated rPstS-1 antigen, which contains all predicted B-cell epitopes, had been synthesized and produced by
researchers (Bando-Campos et al., 2019). The recombinant proteins are genetically engineered synthetic proteins encoded
by recombinant DNA and expressed in systems involved in recombinant DNA transcription and translation into potential
commercial proteins (Chumnanpuen P, 2016). Based on antigenic components in polysaccharides, nucleic acids, or proteins,
recombinant DNA is modified to control high-level target protein expression promoters (Bill, 2015).

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
They have no side effects, are stable, and are immunogenic on all immunizations (M. Wang et al., 2016). Design of
strategies for manufacturing recombinant proteins in P. pastoris can be fulfilled by optimizing the target gene sequence,
stable protein engineering (al. S. D., 2013), codon optimization to help functional expression (al, 2011), and the location and
molecular weight of virtual objects necessary for P. pastoris development. It must be considered (Madhavan A, 2021), as
well as optimization in P. pastoris culture by inducing stress (methanol), which can increase productivity (al. R. C., 2013).

P. pastoris is a methylotrophic species that utilizes methanol as a carbon source. Even though alcohol oxidase has a low
affinity for O2, P. pastoris compensates by producing a large number of enzymes. Historically, Guilliermond isolated P.
pastoris from the chestnut exudate and named the Zygosaccharomyces pastoris (Zahra, 2017). The recombinant protein
Kallikrein inhibitor (Kalbitor) was first generated by P. pastoris, which was approved by the FDA in 2009 as a potential
host for biopharmaceutical production (Kim H, 2015). Ribosome gene sequence analysis shows that P. pastoris is classified
into a new phylogenetically different genus, Komagataella (C., 2005). Currently, six genus Komagataella are described (K.
pastoris, K. phaffii, K. pseudopastoris, K. poluli, K. ulmi, and K. kurtzmanii) (Naumov GI, 2013). The protein production
platform of Pichia pastoris is based on strains of K. phaffii or K. pastoris (CP, 2009). Strains of the previous type of the
species P. pastoris CBS704 (NRRL Y-1603) remain strains of the type of the genus Komagataella and the species K.
pastoris, according to biological taxonomy rules. The public version of genome is available at http://Pichiagenome.org/. P.
pastoris produces proteins that can be folded appropriately and secreted into the medium, did perform post-translational
protein modification, spreads on simple media, and produces low-level endogenous proteins (Barrett & Beasley, 2009).

The alcohol oxidase genetic factors in P. pastoris are AOX1 and AOX2 using methanol (HPLC class), to two different
phenotypic types of recombinant strains, Mut+ and MutS. The mut+ strain's chromosomes contain the AOX1 and AOX2
genes. The MutS phenotype has been due to an incorrect aox1 locus. However, belongs to the AOX2 wild type. When
evaluating Pichia transformants for target gene integration, these phenotypes are used. The rate of growth of different
strains had been derived from their Mut form (Mohammadzadeh et al., 2021). The alcohol oxidase (AOX) promoter
expressed by methanol induction regulates this expression system. The AOX1 promoter is a strong promoter that is strictly
regulated by the carbon source in the medium and is strongly inhibited by glucose and glycerol. These characteristics
facilitated P. pastoris to become a highly preferred strain for bioreactor production. The transcription factors Mxr1 and
Prm1 are activated by methanol. AOX1 will be induced by both proteins. The automatic activation of Prm1 and the
activation of Mit1 by Prm1 both enhance AOX1 activation. AOX1 encodes the alcohol oxidase enzyme, which allows
methanol metabolism to generate energy. In peroxisomes, alcohol oxidase converts methanol to formaldehyde.
Formaldehyde inserts the mitochondria and is used to generate energy in the Krebs cycle. Based on AOX regulation,
Methylotroph P. pastoris can express two phenotypes: MUT+ and MUT. MUT indicates a strain that can use methanol at a
slower rate than MUT+ and also does so on a regular basis (Lestari & Novientri, 2021).

9
Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
Figure 6. Scheme of standard methanol metabolism in methylotroph P. pastoris. It was adapted from (Yurimoto H, 2011) (Wang
X, 2016) (S, 2010)

This system has significant advantages, such as proper protein folding, post-translational modification, recombinant protein
glycosylation, and protein stability. The benefits of utilizing the P. pastoris system for protein production include proper
folding (in the endoplasmic reticulum) and secretion (by Kex2 as signal peptidase) of recombinant proteins into the cell's
outer layer. P. pastoris Strain GS115 contains two genes that encode the alcohol oxidase enzyme (AOX1 and AOX2). In the
presence of methanol, transcription of these genes is induced, resulting in a significant amount of the enzyme AOX of been
produced. T-cell activation significantly increases the occurrence of an immune response (Todryk, 2018). P. pastoris
mannosylated antigens have been shown to increase antigen percentage and T-cell activation properties. As a side effect,
glycoproteins derived from P. pastoris may be useful as auxiliary materials. Mannosylated glycoprotein immunogenicity is
associated with specific mannose-binding receptors carried by professional antigen-presenting cells such as dendritic cells
and macrophages. (Karbalaei et al., 2020).

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
 Table 2 The advantages and disadvantages of using P pastoris as a cost-effective expression host system

Advantages Disadvantages Ref

High Expression Inefficient Secretion of more (Karbalaei, Rezaee, & Farsiani,
significant protein (>30kDa) 2020)
Cost-Effective Proteolysis of secreted protein (Velez-Suberbie et al., 2018)
Relatively rapid growth Methanol safety requirements (Fischer & Glieder, 2019)
Scalability Some glycosylation patterns (Li et al., 2019)
different from mammalian (Karbalaei, Rezaee, & Farsiani,
Efficient Secretion& simple purification 2020)
(Werten, Eggink, Cohen Stuart,
& de Wolf, 2019)
Efficient protein folding

Choice of secreted/intracellular expression (Ahmad, Hirz, Pichler, &

Schwab, 2014)
N-glycosylation close to higher eukaryotes (Li et al., 2019)

Using P. pastoris strain GS115 to produce extracellularly secreted HBHA without labels and evaluating the immunogenicity of
this HBHA protein with dimethyl dioctadecyl ammonium/trehalose 6,6'-behenate (DDA / TDB) showed favorable
immunogenicity and high levels of HBHA and IFN- specific antibodies (Teng et al., 2018). Besides that, strain GS115
These results may be effective for further developing HBHA that hasn't been labeled as a candidate for a TB vaccine. P. pastoris
as a host of expression has the advantage of allowing for high cell density, allowing for controllable processes, highly
genetically stable cell lines, and high resistance to contamination. More than 500 heterologous proteins have been expressed
successfully in these hosts, with some of them approved for human and commercial use (Ahmad, Hirz, Pichler, & Schwab,
2014) (H., S.A., & M., 2020). Purifying rPstS-1 from P. pastoris bioreactor culture supernatant was accomplished via
ultrafiltration, which is regularly used as an alternative method to affinity chromatography procedures used for protein tagged
proteins and did not require refolding steps if compared to other bioprocesses (Khurshid S, 2013). Based on the research (Jacobs
et al., 2022), there were 156 study participants, 22 confirmed TB patients, and four likely TB patients. Stuntz, Mark, others and
Bielinski, Francisco. A Retrospective on Men's Health and the Nurse Practitioner Student" were HIV-positive. When the seven
antibodies (anti-Rv3019c IgA + anti-PstS1 IgA + anti-"Kit 1" IgA + anti-"Kit 2" IgA + anti-Apa IgA + anti-NarL IgA + anti-
LAM IgM) were combined, it resulted in a positive result. antibodies against seven specific MTB antigens (Apa, NarL,
Rv3019c, PstS1, LAM, "kit1"; a mixture of MTP64 and Tpx. evaluated the serodiagnostic potential of antibodies against seven
specific MTB antigens (Apa, NarL, Rv3019c, PstS1, LAM, "kit1"; a mixture of MTP64 and Tpx, and Kit2; a mixture of
MPT64, Tpx and 19 kDa), for the immunological diagnosis of TB IgA titers against Narl Rv3019c, "Kit 1," and "Kit 2"
antibodies differed significantly between TB patients and individuals with ORD, with anti-NarL IgA being the single most
promising antibody. NarL is a putative nitrate response regulator that is found in the membrane fraction of M. TB (] de Souza
GA, 2011). The model is applied to Indonesian citizens born in 2017, including 4,900,000 babies, using the static Markov
model. Using the basic assumption of protection during the first ten years of life, we calculated that a BCG vaccination strategy
costing approximately $57 million with an 87% absorption rate would result in 488,592 QALY and save approximately $51
million and
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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
$55 million in Social and Health Care costs, respectively (Table 1).

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience
 Conclusion
The vaccine candidate of the recombinant protein subunit rPstS-1 expressed by P. pastoris strain GS115 is
immunogenic and has a good manufacturing. This strategy shows that the availability of vaccination is one of the
factors that play an important role in lowering the overall cost burden of TB, and decision-makers will pay closer
attention to the implementation, optimization, and sustainability of the TB vaccination program. The rPstS-1
recombinant protein subunit vaccine with the expression host P. pastoris strain GS115 has a significant advantage over
infectious viruses. It is expected to produce a TB vaccine that has no side effects, is stable, and is immunogenic in all
immunizations.

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Tables

Table 1. Comparison of healthcare and societal perspectives in cost expenditure.

Table 2. The advantages and disadvantages of using P patoris as a cost-effective expression host system.

Figures

Figure 1. Different programs with the same objective.

Figure 2. Diagram of systematic review stages with PRISMA.

Figure 3. The cost-effectiveness ratio (ICER) compares vaccination strategies compared to non-vaccination approaches.

Figure 4. Tornado diagram from univariate analysis. VE = vaccine effectiveness, TB = TB, DSTB = drug-susceptible TB, MDRTB = drug-
resistant TB, QALY = quality-adjusted years of life.

Figure 5. Demonstrate a cost-effective strategy according to different threshold value willingness to pay levels.

Figure 6. Scheme of standard methanol metabolism in methylotroph P. pastoris. It was adapted from (Yurimoto H, 2011) (Wang X, 2016)
(S, 2010).

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Pharmaceutical Perspective: From Upstream to Downstream for Reinforcing Health Resilience