Journal of Microbiology

Research article

Epitope-guided optimization improves the protective efficacy of NA-based mRNA vaccines against seasonal influenza A(H3N2) virus: Hyunbeen Kim, Sangyi Lee, Seunghye Cho, Woojin Shin, Soyoung Lee, Atanas V. Demirev, Taeyoung Lee, You-Jin Kim, Dokeun Kim, Sae Am Song, Kyung Ran Jun, Jin Il Kim; J. Microbiol. 2026;64(10):e2609004. Published online October 31, 2026; DOI: https://doi.org/10.71150/jm.2609004

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Abstract PDF Supplementary Material: Neuraminidase (NA) is increasingly recognized as a promising target antigen for improving the breadth and durability of influenza vaccines, yet NA-based mRNA vaccines remain comparatively unexplored relative to hemagglutinin-based approaches. Here, we applied a computationally guided NA antigen-design strategy to seasonal H3N2 influenza viruses and investigated whether epitope-guided optimization could improve the immunogenicity and protective efficacy of NA-based mRNA vaccines. NA-D3, an NA antigen designed based on the evolutionary characteristics of circulating H3N2 viruses, was first evaluated in an mRNA vaccine. This vaccine provided partial protection against H3N2 challenge and reduced viral replication in the lungs, although detectable neuraminidase inhibition (NAI) activity was not observed. To further improve NA antigen immunogenicity, predicted B- and T-cell epitopes were incorporated into the antigen design process, resulting in three sequentially optimized NA candidates, F0, F1, and F2. In a subsequent challenge experiment, F1 and F2 conferred complete protection, achieving 100% survival and significantly lower viral RNA levels and infectious viral titers in the lungs compared to controls. The optimized NA mRNA vaccines also induced detectable NAI activity against heterologous H3N2 viruses, albeit at relatively modest levels. Collectively, these findings demonstrate the protective potential of NA-based mRNA vaccination against H3N2 influenza viruses and establish iterative, epitope-guided strategy in NA antigen optimization – integrating computational prediction with experimental validation – as an effective approach for developing broadly protective NA-based mRNA vaccines.

Research Support, Non-U.S. Gov't

Biochemical Characteristization of Propionyl-Coenzyme A Carboxylase Complex of Streptomyces toxytricini: Atanas V. Demirev , Anamika Khanal , Nguyen Phan Kieu Hanh , Kyung Tae Nam , Doo Hyun Nam; J. Microbiol. 2011;49(3):407-412. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1122-1

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Acyl-CoA carboxylases (ACC) are involved in important primary or secondary metabolic pathways such as fatty acid and/or polyketides synthesis. In the 6.2 kb fragment of pccB gene locus of Streptomyces toxytricini producing a pancreatic inhibitor lipstatin, 3 distinct subunit genes of presumable propionyl-CoA carboxylase (PCCase) complex, assumed to be one of ACC responsible for the secondary metabolism, were identified along with gene for a biotin protein ligase (Bpl). The subunits of PCCase complex were α subunit (AccA3), β subunit (PccB), and auxiliary ε subunit (PccE). In order to disclose the involvement of the PCCase complex in secondary metabolism, some biochemical characteristics of each subunit as well as their complex were examined. In the test of substrate specificity of the PCCase complex, it was confirmed that this complex showed much higher conversion of propionyl-CoA rather than acetyl-CoA. It implies the enzyme complex could play a main role in the production of methylmalonyl-CoA from propionyl-CoA, which is a precursor of secondary polyketide biosynthesis.

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Current trends and future prospects of lipstatin: a lipase inhibitor and pro-drug for obesity
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Research Support, Non-U.S. Gov't

Identification and Characterization of Acetyl-CoA Carboxylase Gene Cluster in Streptomyces toxytricini: Atanas V. Demirev , Ji Seon Lee , Bhishma R. Sedai , Ivan G. Ivanov , Doo Hyun Nam; J. Microbiol. 2009;47(4):473-478. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0135-5

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Abstract PDF

The gene locus for acetyl-CoA carboxylase (ACC) involved in the primary metabolism was identified from the genomic library of Streptomyces toxytricini which produces a lipase inhibitor lipstatin. The 7.4 kb cloned gene was comprised of 5 ORFs including accD1, accA1, hmgL, fadST1, and stsF. In order to confirm the biochemical characteristics of AccA1, the gene was overexpressed in Escherichia coli cells, and the recombinant protein was purified through Ni2+ affinity chromatography. Because most of the expressed AccA1 was biotinylated by host E. coli BirA in the presence of D-biotin, the non-biotinylated apo-AccA1 was purified after gene induction without D-biotin, followed by exclusion of holo-AccA1 using streptavidin beads. The separated apo-AccA1 was post-translationally biotinylated by S. toxytricini biotin apo-protein ligase (BPL) in a time- and enzyme-dependent manner. This result supports that this gene cluster of S. toxytricini encodes the functional ACC enzyme subunits to be biotinylated.

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Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains
Michal Híreš, Nora Rapavá, Martin Šimkovič, Ľudovít Varečka, Dušan Berkeš, Svetlana Kryštofová
Current Microbiology.2018; 75(5): 580. CrossRef
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Current trends and future prospects of lipstatin: a lipase inhibitor and pro-drug for obesity
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Tingheng Zhu, Lingfei Wang, Weixia Wang, Zhongce Hu, Meilan Yu, Kun Wang, Zhifeng Cui
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Biochemical characteristization of propionyl-coenzyme a carboxylase complex of Streptomyces toxytricini
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The role of acyl-coenzyme A carboxylase complex in lipstatin biosynthesis of Streptomyces toxytricini
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Research Support, Non-U.S. Gov't

Molecular Cloning of the Phospholipase D Gene from Streptomyces sp. YU100 and Its Expression in Escherichia coli: Ji-Seon Lee , Munkhtsetseg Bat-Ochir , Atanas V. Demirev , Doo Hyun Nam; J. Microbiol. 2009;47(1):116-122. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0161-8

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Abstract PDF

The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and heterologously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.

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European Journal of Lipid Science and Technology.2016; 118(5): 803. CrossRef
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Research Support, Non-U.S. Gov't

Cloning and Analysis of a Type II Polyketide Synthase Gene Cluster from Streptomyces toxytricini NRRL 15,443: Anna Yoo , Atanas V. Demirev , Ji Seon Lee , Sang Dal Kim , Doo Hyun Nam; J. Microbiol. 2006;44(6):649-654.; DOI: https://doi.org/2462 [pii]

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Abstract PDF: A standard type II polyketide synthase (PKS) gene cluster was isolated while attempting to clone the biosynthetic gene for lipstatin from Streptomyces toxytricini NRRL 15,443. This result was observed using a Southern blot of a PstI-digested S. toxytricini chromosomal DNA library with a 444 bp amplified probe of a ketosynthase (KS) gene fragment. Four open reading frames [thioesterase (TE), β-ketoacyl systhase (KAS), chain length factor (CLF), and acyl carrier protein (ACP)], were identified through the nucleotide sequence determination and analysis of a 4.5 kb cloned DNA fragment. In order to confirm the involvement of a cloned gene in lipstatin biosynthesis, a gene disruption experiment for the KS gene was performed. However, the resulting gene disruptant did not show any significant difference in lipstatin production when compared to wild-type S. toxytricini. This result suggests that lipstatin may not be synthesized by a type II PKS.

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