Aberrant glucose metabolism underlies impaired macrophage differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that is responsible for transporting G6P into the endoplasmic reticulum. GSD-Ib is characterized by disturbances in glucose homeostasis, neutropenia, and neutrophil dysfunction. Although some studies have explored neutrophils abnormalities in GSD-Ib, investigations regarding monocytes/macrophages remain limited so far. In this study, we examined the impact of G6PT deficiency on monocyte-to-macrophage differentiation using bone marrow-derived monocytes from G6pt-/- mice as well as G6PT-deficient human THP-1 monocytes. Our findings revealed that G6PT-deficient monocytes exhibited immature differentiation into macrophages. Notably, the impaired differentiation observed in G6PT-deficient monocytes seemed to be associated with abnormal glucose metabolism, characterized by enhanced glucose consumption through glycolysis, even under quiescent conditions with oxidative phosphorylation. Furthermore, we observed a reduced secretion of inflammatory cytokines in G6PT-deficient THP-1 monocytes during the inflammatory response, despite their elevated glucose consumption. In conclusion, this study sheds light on the significance of G6PT in monocyte-to-macrophage differentiation and underscores its importance in maintaining glucose homeostasis and supporting immune response in GSD-Ib. These findings may contribute to a better understanding of the pathogenesis of GSD-Ib and potentially pave the way for the development of targeted therapeutic interventions.

Proof-of-Concept: Smartphone- and Cloud-Based Artificial Intelligence Quantitative Analysis System (SCAISY) for SARS-CoV-2-Specific IgG Antibody Lateral Flow Assays.

Smartphone-based point-of-care testing (POCT) is rapidly emerging as an alternative to traditional screening and laboratory testing, particularly in resource-limited settings. In this proof-of-concept study, we present a smartphone- and cloud-based artificial intelligence quantitative analysis system (SCAISY) for relative quantification of SARS-CoV-2-specific IgG antibody lateral flow assays that enables rapid evaluation (<60 s) of test strips. By capturing an image with a smartphone camera, SCAISY quantitatively analyzes antibody levels and provides results to the user. We analyzed changes in antibody levels over time in more than 248 individuals, including vaccine type, number of doses, and infection status, with a standard deviation of less than 10%. We also tracked antibody levels in six participants before and after SARS-CoV-2 infection. Finally, we examined the effects of lighting conditions, camera angle, and smartphone type to ensure consistency and reproducibility. We found that images acquired between 45° and 90° provided accurate results with a small standard deviation and that all illumination conditions provided essentially identical results within the standard deviation. A statistically significant correlation was observed (Spearman correlation coefficient: 0.59, p = 0.008; Pearson correlation coefficient: 0.56, p = 0.012) between the OD450 values of the enzyme-linked immunosorbent assay and the antibody levels obtained by SCAISY. This study suggests that SCAISY is a simple and powerful tool for real-time public health surveillance, enabling the acceleration of quantifying SARS-CoV-2-specific antibodies generated by either vaccination or infection and tracking of personal immunity levels.

Baculoviral COVID-19 Delta DNA vaccine cross-protects against SARS-CoV2 variants in K18-ACE2 transgenic mice.

After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronavirus's intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50 % survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). The AcHERV-COVID19D induced higher neutralizing antibodies against the Delta variant than the prototype or Omicron variant. On the other hand, cellular immunity was similarly high for all three SARS-CoV2 viruses. Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100 % survival upon challenge with Delta and Omicron variants and 71.4 % survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants.

Mosaic RBD nanoparticles induce intergenus cross-reactive antibodies and protect against SARS-CoV-2 challenge.

Recurrent spillovers of α- and ß-coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome-CoV, SARS-CoV-2, and possibly human CoV have caused serious morbidity and mortality worldwide. In this study, six receptor-binding domains (RBDs) derived from α- and ß-CoV that are considered to have originated from animals and cross-infected humans were linked to a heterotrimeric scaffold, proliferating cell nuclear antigen (PCNA) subunits, PCNA1, PCNA2, and PCNA3. They assemble to create a stable mosaic multivalent nanoparticle, 6RBD-np, displaying a ring-shaped disk with six protruding antigens, like jewels in a crown. Prime-boost immunizations with 6RBD-np in mice induced significantly high Ab titers against RBD antigens derived from α- and ß-CoV and increased interferon (IFN-γ) production, with full protection against the SARS-CoV-2 wild type and Delta challenges. The mosaic 6RBD-np has the potential to induce intergenus cross-reactivity and to be developed as a pan-CoV vaccine against future CoV spillovers.

Molecular mechanisms of aberrant neutrophil differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in glucose-6-phosphate transporter (G6PT). Neutropenia in GSD-Ib has been known to result from enhanced apoptosis of neutrophils. However, it has also been raised that neutrophil maturation arrest in the bone marrow would contribute to neutropenia. We now show that G6pt-/- mice exhibit severe neutropenia and impaired neutrophil differentiation in the bone marrow. To investigate the role of G6PT in myeloid progenitor cells, the G6PT gene was mutated using CRISPR/Cas9 system, and single cell-derived G6PT-/- human promyelocyte HL-60 cell lines were established. The G6PT-/- HL-60s exhibited impaired neutrophil differentiation, which is associated with two mechanisms: (i) abnormal lipid metabolism causing a delayed metabolic reprogramming and (ii) reduced nuclear transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ) in G6PT-/- HL-60s. In this study, we demonstrated that G6PT is essential for neutrophil differentiation of myeloid progenitor cells and regulates PPARγ activity.

Human endogenous retrovirus-enveloped baculoviral DNA vaccines against MERS-CoV and SARS-CoV2.

Here we report a recombinant baculoviral vector-based DNA vaccine system against Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus-2 (SARS-CoV2). A non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) was constructed as a DNA vaccine vector for gene delivery into human cells. For MERS-CoV vaccine construction, DNA encoding MERS-CoV S-full, S1 subunit, or receptor-binding domain (RBD) was inserted into the genome of AcHERV. For COVID19 vaccine construction, DNA encoding SARS-CoV2 S-full or S1 or a MERS-CoV NTD domain-fused SARS-CoV2 RBD was inserted into the genome of AcHERV. AcHERV-DNA vaccines induce high humoral and cell-mediated immunity in animal models. In challenge tests, twice immunized AcHERV-MERS-S1 and AcHERV-COVID19-S showed complete protection against MERS-CoV and SARS-CoV2, respectively. Unlike AcHERV-MERS vaccines, AcHERV-COVID19-S provided the greatest protection against SARS-CoV2 challenge. These results support the feasibility of AcHERV-MERS or AcHERV-COVID19 vaccines in preventing pandemic spreads of viral infections.

Identification and characterization of a Toll-like receptor gene from Macrobrachium nipponense.

Outbreaks of infectious disease in shrimp pose a serious threat to shrimp agriculture worldwide. Shrimp lack adaptive immunity and depend only on innate immunity as a defense system against infectious disease. Toll-like receptors (TLR) are reported to play a critical role in the innate immune system. In this study, we identified a Toll-like receptor gene of a species of freshwater shrimp, Macrobrachium nipponense, designated MnToll, for the first time. The sequence of MnToll encoded 935 residues arranged as 10 leucine-rich repeat (LRR) domains, a leucine-rich repeat C-terminal (LRR CT) domain and a Toll/interleukin-1 receptor (TIR) domain and displayed 90% amino acid similarity to previously identified TLRs (Toll 1 and 2) of Macrobrachium rosenbergii. We additionally evaluated mRNA expression of MnToll in various tissues, including heart, gills, stomach, digestive gland, ventral nerve cord, antennal gland and muscle. Following infection with a viral pathogen, white spot syndrome virus (WSSV), MnToll expression was significantly upregulated between 12 and 72 h. Our data collectively suggest that the newly identified MnToll gene belongs to the TLR family in shrimp and is potentially involved in innate host defense, especially against WSSV.

Retention of neutralizing antibodies to Japanese encephalitis vaccine in age groups above fifteen years in Korea.

BACKGROUND: The incidence of Japanese encephalitis (JE) has markedly decreased after the national immunization program in Korea, but still reported intermittently in adults. Prospective studies are required to determine whether JE virus (JEV)-neutralizing antibody (NAb) levels decline with age after the final JE vaccination. In this study, we evaluated the titers of NAbs against JEV in Korean adolescents and adults. METHODS: Specimens were collected from normal, healthy individuals aged 1 to >70 years (a total of 1,605 cases) from five regional hospitals in Korea. Neutralizing antibody (NAb) titers were determined using a pseudotyped JEV NAb assay. RESULTS: The JEV NAb-positive population was >95% in the 15-29 year age group, 89.42% in the 30-44 year age group, 75.24% in the 55-59 years age group, and 59.77% in the ≥70 year age group. NAb titers from the 20 to 70 year age groups showed a progressive decrease in proportion to age (P < 0.0001). CONCLUSIONS: The retention of NAbs after the final JE vaccination in childhood is a key indicator of the vaccination program and will have a significant impact on future JE prevention strategies.

Glucose-6-phosphate transporter mediates macrophage proliferation and functions by regulating glycolysis and mitochondrial respiration.

Glycogen storage disease type Ib (GSD-Ib), caused by a deficiency in glucose-6-phosphate transporter (G6PT), is characterized by disrupted glucose homeostasis, inflammatory bowel disease, neutropenia, and neutrophil dysfunction. The purpose of this study was to investigate the role of G6PT on macrophage functions and metabolism. Peritoneal macrophages of G6pt-/- mice were lower in number and their effector functions including migration, superoxide production, and phagocytosis were impaired. To investigate the underlying mechanisms of macrophage dysfunction, the G6PT gene was mutated in porcine alveolar macrophage 3D4/31 cells using the CRISPR/Cas9 technology. The G6PT-deficient macrophages exhibited significant decline in cell growth, bactericidal activity, and antiviral response. These phenotypes are associated with the impaired glycolysis and mitochondrial oxidative phosphorylation. We therefore propose that the G6PT-mediated metabolism is essential for effector functions of macrophage, the immune deficiencies observed in GSD-Ib extend beyond neutropenia and neutrophil dysfunction, and future therapeutic targets aimed both the neutrophils and macrophages may be necessary.

Porcine endogenous retrovirus envelope coated baculoviral DNA vaccine against porcine reproductive and respiratory syndrome virus.

PERV is a major virus concerning xenotransplantation study. However, the interesting part is that PERV is present in all kinds of pigs without pathogenicity and immune response. Furthermore, since pig cells have receptors for PERV, the gene delivery system using PERV envelope is highly likely to develop into an excellent viral vector in pigs. We developed a recombinant baculovirus with a modified surface for expressing the porcine endogenous retrovirus (PERV) envelope. Porcine reproductive and respiratory syndrome virus (PRRSV) infection is a severe concern in the porcine industry due to reproduction failure and respiratory symptoms. GP5 and M proteins are major immunogenic proteins of PRRSV. Using PERV-modified baculovirus (Ac mPERV) as a delivery vector, we constructed a dual antigen (GP5 and M)-encoding DNA vaccine system, Ac mPERV-C5/C6. Intramuscular immunization in mice and pigs, Ac mPERV-C5/C6 induced comparative high humoral and cellular immune responses. Our results support further development of Ac mPERV-C5/C6 as a potential PRRSV vaccine in the porcine industry. In addition, the Ac mPERV system may be applied to the generation of other effective DNA vaccines against porcine viral diseases.

Fusion of flagellin 2 with bivalent white spot syndrome virus vaccine increases survival in freshwater shrimp.

Despite large economic losses attributable to white spot syndrome virus (WSSV), an infectious pathogen of penaeid shrimp and other crustaceans worldwide, no efficient vaccines or antiviral agents to control the virus are available at present. Here, we designed and constructed baculovirus-based vaccines delivering genes encoding the WSSV envelope proteins, VP28 and VP19. To enhance the immunogenicity of the baculovirus-based vaccine, we fused a Salmonella typhimurium flagellin 2 (FL2) gene with VP28 or VP19 gene. Both vaccine constructs elicited similar high titlers of anti-WSSV IgG after oral immunization in mice. The protective effect of oral vaccines upon WSSV challenge was observed in Macrobrachium nipponense. Bivalent vaccine displaying WSSV envelope proteins, VP19 and VP28, led to enhanced more than 10% survival protection against WSSV infection, compared to monovalent vaccine containing WSSV envelope protein, VP19 or VP28. Furthermore, a baculovirus-based WSSV vaccine fused with FL2 gene, Ac-VP28-ie1VP19FL2, efficiently protected mice against WSSV challenge (89.5% survival rate). In support of the efficacy of FL2 in our vaccine, we verified FL2 enhanced survival rate and induced the NF-κB gene in Palaemon paucidens. The collective results strongly suggest that our recombinant baculoviral system displaying WSSV envelope protein and delivering FL2-fused WSSV envelope gene effectively induced protective responses, supporting the utility of a potential new oral DNA vaccine against WSSV.

Effect of AcHERV-GmCSF as an Influenza Virus Vaccine Adjuvant.

INTRODUCTION: The first identification of swine-originated influenza A/CA/04/2009 (pH1N1) as the cause of an outbreak of human influenza accelerated efforts to develop vaccines to prevent and control influenza viruses. The current norm in many countries is to prepare influenza vaccines using cell-based or egg-based killed vaccines, but it is difficult to elicit a sufficient immune response using this approach. To improve immune responses, researchers have examined the use of cytokines as vaccine adjuvants, and extensively investigated their functions as chemoattractants of immune cells and boosters of vaccine-mediated protection. Here, we evaluated the effect of Granulocyte-macrophage Colony-Stimulating Factor (GmCSF) as an influenza vaccine adjuvant in BALB/c mice. METHOD AND RESULTS: Female BALB/c mice were immunized with killed vaccine together with a murine GmCSF gene delivered by human endogenous retrovirus (HERV) envelope coated baculovirus (1 × 10(7) FFU AcHERV-GmCSF, i.m.) and were compared with mice immunized with the killed vaccine alone. On day 14, immunized mice were challenged with 10 median lethal dose of mouse adapted pH1N1 virus. The vaccination together with GmCSF treatment exerted a strong adjuvant effect on humoral and cellular immune responses. In addition, the vaccinated mice together with GmCSF were fully protected against infection by the lethal influenza pH1N1 virus. CONCLUSION: Thus, these results indicate that AcHERV-GmCSF is an effective molecular adjuvant that augments immune responses against influenza virus.

Enhanced Anti-inflammatory Effects of γ-irradiated Pig Placenta Extracts.

Porcine placenta extract (PPE) is known to possess anti-inflammatory properties owing to its high concentration of bioactive substances. However, the need to eliminate blood-borne infectious agents while maintaining biological efficacy raises concerns about the optimal method for sterilizing PPE. Therefore, the objective of this study was to compare the effects of the standard pressurized heat (autoclaving) method of sterilization with γ-irradiation on the anti-inflammatory effects of PPE. The anti-inflammatory actions of these two preparations of PPE were evaluated by measuring their inhibitory effects on the production of NO, the expression of iNOS protein, and the expression of iNOS, COX2, TNF-α, IL-1ß, and IL-6 mRNA in lipopolysaccharide-stimulated RAW 264.7 cells. Compared with autoclaved PPE, γ-irradiated PPE showed significantly greater inhibition of NO production and iNOS protein expression, and produced a greater reduction in the expression of iNOS, COX2, TNF-α, IL-1ß, and IL-6 mRNA. These results provide evidence that the sterilization process is crucial in determining the biological activity of PPE, especially its anti-inflammatory activity. Collectively, our data suggest that γ-irradiated PPE acts at the transcriptional level to effectively and potently suppresses the production of NO and the expression of pro-inflammatory cytokines.