Designing a Novel Multiepitope Vaccine from the Human Papilloma Virus E1 and E2 Proteins for Indonesia with Immunoinformatics and Molecular Dynamics Approaches.

One of the deadliest malignant cancer in women globally is cervical cancer. Specifically, cervical cancer is the second most common type of cancer in Indonesia. The main infectious agent of cervical cancer is the human papilloma virus (HPV). Although licensed prophylactic vaccines are available, cervical cancer cases are on the rise. Therapy using multiepitope-based vaccines is a very promising therapy for cervical cancer. This study aimed to develop a multiepitope vaccine based on the E1 and E2 proteins of HPV 16, 18, 45, and 52 using in silico. In this study, we develop a novel multiepitope vaccine candidate using an immunoinformatic approach. We predicted the epitopes of the cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) and evaluated their immunogenic properties. Population coverage analysis of qualified epitopes was conducted to determine the successful use of the vaccine worldwide. The epitopes were constructed into a multiepitope vaccine by using AAY linkers between the CTL epitopes and GPGPG linkers between the HTL epitopes. The tertiary structure of the multiepitope vaccine was modeled with AlphaFold and was evaluated by Prosa-web. The results of vaccine construction were analyzed for B-cell epitope prediction, molecular docking with Toll like receptor-4 (TLR4), and molecular dynamics simulation. The results of epitope prediction obtained 4 CTL epitopes and 7 HTL epitopes that are eligible for construction of multiepitope vaccines. Prediction of the physicochemical properties of multiepitope vaccines obtained good results for recombinant protein production. The interaction showed that the interaction of the multiepitope vaccine-TLR4 complex is stable based on the binding free energy value -106.5 kcal/mol. The results of the immune response simulation show that multiepitope vaccine candidates could activate the adaptive and humoral immune systems and generate long-term B-cell memory. According to these results, the development of a multiepitope vaccine with a reverse vaccinology approach is a breakthrough to develop potential cervical cancer therapeutic vaccines.

Immunogenicity studies of recombinant RBD SARS-CoV-2 as a COVID-19 vaccine candidate produced in Escherichia coli.

The severe acute respiratory syndrome coronavirus 2 -related global COVID-19 pandemic has been impacting millions of people since its outbreak in 2020. COVID-19 vaccination has proven highly efficient in reducing illness severity and preventing infection-related fatalities. The World Health Organization has granted emergency use approval to multiple, including protein subunit technology-based, COVID-19 vaccines. Foreseeably, additional COVID-19 subunit vaccine development would be essential to meet the accessible and growing demand for effective vaccines, especially for Low-Middle-Income Countries (LMIC). The SARS-CoV-2 spike protein receptor binding domain (RBD), as the primary target for neutralizing antibodies, holds significant potential for future COVID-19 subunit vaccine development. In this study, we developed a recombinant Escherichia coli-expressed RBD (rRBD) as a vaccine candidate and evaluated its immunogenicity and preliminary toxicity in BALB/c mice. The rRBD induced humoral immune response from day 7 post-vaccination and, following the booster doses, the IgG levels increased dramatically in mice. Interestingly, our vaccine candidate also significantly induced cellular immune response, indicated by the incrased IFN-É£-producing cell numbers. We observed no adverse effect or local reactogenicity either in control or treated mice. Taken together, our discoveries could potentially support efficient and cost-effective vaccine antigen production, from which LMICs could particularly benefit.

The SARS-CoV-2 Mpro Dimer-Based Screening System: A Synthetic Biology Tool for Identifying Compounds with Dimerization Inhibitory Potential.

The COVID-19 endemic remains a global concern. The search for effective antiviral candidates is still needed to reduce disease risk. However, the availability of high biosafety level laboratory facilities for drug screening is limited in number. To address this issue, a screening system that could be utilized at lower biosafety levels remains essential. This study aimed to develop a novel SARS-CoV-2 main protease (Mpro) dimer-based screening system (DBSS) utilizing synthetic biology in Escherichia coli BL21(DE3). We linked the SARS-CoV-2 Mpro with the DNA-binding domain of AraC regulatory protein, which regulates the reporter gene expression. Protein modeling and molecular docking showed that saquinavir could bind to AraC-Mpro both in its monomer and dimer forms. The constructed DBSS assay indicated the screening system could detect saquinavir inhibitory activity at a concentration range of 4-10 µg/mL compared to the untreated control (P ≤ 0.05). The Vero E6 cell assay validated the DBSS result that saquinavir at 4-10 µg/mL exhibited antiviral activity against SARS-CoV-2. Our DBSS could be used for preliminary screening of numerous drug candidates that possess a dimerization inhibitor activity of SARS-CoV-2 Mpro and also minimize the use of a high biosafety level laboratory.

Development of Adenovirus-Based Covid-19 Vaccine Candidate in Indonesia.

Covid-19 pandemic has struck worldwide by end of 2019 and the use of various vaccine platforms was one of the main strategies to end this. To meet the needs for vaccine technology equality among many countries, we developed adenovirus-based Covid-19 vaccine candidate in Indonesia. SARS-CoV-2 Spike gene (S) was constructed into pAdEasy vector. The recombinant serotype 5 Adenovirus (AdV_S) genome was transfected into AD293 cells to produce recombinant adenovirus. Characterization using PCR confirmed the presence of spike gene. Transgene expression analysis showed the expression of S protein in AdV_S infected AD293 and A549 cells. Optimization of viral production showed the highest titer was obtained at MOI of 0.1 and 1 at 4 days. The in vivo study was performed by injecting Balb/c mice with 3.5 × 107 ifu of purified adenovirus. The result showed that S1-specific IgG was increased up to 56 days after single-dose administration of AdV_S. Interestingly, significant increase of S1 glycoprotein-specific IFN-γ ELISpot was observed in AdV_S treated Balb/c mice. In conclusion, the AdV_S vaccine candidate was successfully produced at laboratory scale, immunogenic, and did not cause severe inflammation in Balb/c mice. This study serves as initial step towards manufacturing of adenovirus-based vaccine in Indonesia.

Curcumin-derived carbon-dots as a potential COVID-19 antiviral drug.

Even entering the third year of the COVID-19 pandemic, only a small number of COVID-19 antiviral drugs are approved. Curcumin has previously shown antiviral activity against SARS-CoV-2 nucleocapsid, but its poor bioavailability limits its clinical uses. Utilizing nanotechnology structures, curcumin-derived carbon-dots (cur-CDs) were synthesized to increase low bioavailability of curcumin. In-silico analyses were performed using molecular docking, inhibition of SARS-CoV-2 nucleocapsid C-terminal domain (N-CTD) and antiviral activity were assessed in dimer-based screening system (DBSS) and in vitro respectively. Curcumin bound with the N-CTD at ΔG = -7.6 kcal/mol, however modifications into cur-CDs significantly improved the binding affinity and %interaction. Cur-CDs also significantly increased protection against SARS-CoV-2 in both DBSS and in vitro at MOI = 0.1. This study demonstrated the effect of post-infection treatment of curcumin and novel curcumin-derived carbon-dots on SARS-CoV-2 N-CTD dimerization. Further investigation on pre-infection and in-vivo treatment of curcumin and cur-CDs are required for a comprehensive understanding on the carbon-dots enhanced antiviral activity of curcumin against SARS-CoV-2.

Porphyrin-derived carbon dots for an enhanced antiviral activity targeting the CTD of SARS-CoV-2 nucleocapsid.

BACKGROUND: Since effective antiviral drugs for COVID-19 are still limited in number, the exploration of compounds that have antiviral activity against SARS-CoV-2 is in high demand. Porphyrin is potentially developed as a COVID-19 antiviral drug. However, its low solubility in water restricts its clinical application. Reconstruction of porphyrin into carbon dots is expected to possess better solubility and bioavailability as well as lower biotoxicity. METHODS AND RESULTS: In this study, we investigated the antiviral activity of porphyrin and porphyrin-derived carbon dots against SARS-CoV-2. Through the in silico analysis and assessment using a novel drug screening platform, namely dimer-based screening system, we demonstrated the capability of the antivirus candidates in inhibiting the dimerization of the C-terminal domain of SARS-CoV-2 Nucleocapsid. It was shown that porphyrin-derived carbon dots possessed lower cytotoxicity on Vero E6 cells than porphyrin. Furthermore, we also assessed their antiviral activity on the SARS-CoV-2-infected Vero E6 cells. The transformation of porphyrin into carbon dots substantially augmented its performance in disrupting SARS-CoV-2 propagation in vitro. CONCLUSIONS: Therefore, this study comprehensively demonstrated the potential of porphyrin-derived carbon dots to be developed further as a promisingly safe and effective COVID-19 antiviral drug.

Development of Chimeric Hepatitis B (HBV) - Norovirus (NoV) P particle as candidate vaccine against Hepatitis B and norovirus infection.

Introduction: Hepatitis B remains a global problem with no effective treatment. Here, a mucosal vaccine candidate was developed with HBsAg and HBcAg, to provide both prophylactic and therapeutic protection against hepatitis B. The antigens were presented using the P particle of human norovirus (HuNov). As a result, the chimeric HBV - HuNoV P particle can act as a dual vaccine for hepatitis B and HuNoV. Methods: The vaccine candidate was expressed and purified from Escherichia coli BL21 (DE3) cells. HBV-HuNoV chimeric P particles were successfully expressed and isolated, with sizes of approximately 25.64 nm. Then, the HBV-HuNoV chimeric P particles were evaluated for safety and immunogenicity in mice and gnotobiotic (Gn) pigs. After three doses (5 µg/dose in mice and 200 µg/dose in Gn pigs) of intranasal immunization, humoral and cellular immune responses, as well as toxicity, were evaluated. Results: The vaccine candidate induced strong HBV-HuNoV specific IFN-γ producing T-cell responses in the ileum, spleen, and blood of Gn pigs. Serum IgG and IgA antibodies against HBV-HuNoV chimeric P particles also increased significantly in Gn pigs. Increased HBsAg- and HuNoV-specific serum IgG responses were observed in mice and Gn pigs, although not statistically significant. The vaccine candidate did not show any toxicity in mice. Conclusions: In summary, the chimeric HBV-HuNoV P particle vaccine given intranasally was safe and induced strong cellular and humoral immune responses in Gn pig. Modifications to the vaccine structure and dosage need to be evaluated in future studies to further enhance immunogenicity and induce more balanced humoral and cellular responses.

Infectious Disease Modeling with Socio-Viral Behavioral Aspects-Lessons Learned from the Spread of SARS-CoV-2 in a University.

When it comes to understanding the spread of COVID-19, recent studies have shown that pathogens can be transmitted in two ways: direct contact and airborne pathogens. While the former is strongly related to the distancing behavior of people in society, the latter are associated with the length of the period in which the airborne pathogens remain active. Considering those facts, we constructed a compartmental model with a time-dependent transmission rate that incorporates the two sources of infection. This paper provides an analytical and numerical study of the model that validates trivial insights related to disease spread in a responsive society. As a case study, we applied the model to the COVID-19 spread data from a university environment, namely, the Institut Teknologi Bandung, Indonesia, during its early reopening stage, with a constant number of students. The results show a significant fit between the rendered model and the recorded cases of infections. The extrapolated trajectories indicate the resurgence of cases as students' interaction distance approaches its natural level. The assessment of several strategies is undertaken in this study in order to assist with the school reopening process.

RNA sequence analysis of nasopharyngeal swabs from asymptomatic and mildly symptomatic patients with COVID-19.

OBJECTIVES: The characterization of asymptomatic and mildly symptomatic patients with COVID-19 by observing changes in gene expression profile and possible bacterial coinfection is relevant to be investigated. We aimed to identify transcriptomic and coinfection profiles in both groups of patients. METHODS: A ribonucleic acid (RNA) sequence analysis on nasopharyngeal swabs were performed using a shotgun sequencing pipeline. Differential gene analysis, viral genome assembly, and metagenomics analysis were further performed using the retrieved data. RESULTS: Both groups of patients underwent a cilia modification and mRNA splicing. Modulations in macroautophagy, epigenetics, and cell cycle processes were observed specifically in the asymptomatic group. Modulation in the RNA transport was found specifically in the mildly symptomatic group. The mildly symptomatic group showed modulation in the RNA transport and upregulation of autophagy regulator genes and genes in the complement system. No link between viral variants and disease severity was found. Microbiome analysis revealed the elevation of Streptococcus pneumoniae and Veillonella parvula proportion in symptomatic patients. CONCLUSION: A reduction in the autophagy influx and modification in the epigenetic profile might be involved in halting the disease progression. A global dysregulation of RNA processing and translation might cause more severe outcomes in symptomatic individuals. Coinfection by opportunistic microflora should be taken into account when assessing the possible outcome of SARS-CoV-2 infection.

Knowledge, Attitude, and Practice towards Hepatitis B Infection Prevention and Screening among Indonesians.

Hepatitis B has been one of the most prevalent infectious diseases in the world and specifically in Indonesia. Although the total conversion of hepatitis B virus (HBV) to chronic disease in Indonesia was reduced by 50%, the total number of hepatitis B cases increased by 2.5 times in 2021. Ineffective HBV immunization program in Indonesia prior to 1997 was addressed by the Ministry of Health through a more comprehensive HBV control, which, among others, involved Health Promotion to increase people's knowledge and awareness towards hepatitis B infection prevention. In this regard, this study aims to identify the level of knowledge, attitude, and practice/behavior of the Indonesian population towards hepatitis B infection prevention and their willingness for screening, particularly in areas with high prevalence of hepatitis B. This study used a quantitative approach in looking at correlations between this set of knowledge, attitude, and practice, mainly by using Structural Equation Model (SEM) and SmartPLS 3.3.3 toolkit in SPSS. Through an analysis of online questionnaire results from over 400 respondents in four provinces (DKI Jakarta, West Java, DI Yogyakarta, and South Sulawesi), this study shows that respondents have a very high level of knowledge about hepatitis B; high level of positive attitude; and very high level of positive practice towards hepatitis B infection prevention. However, we also noticed some dissonances between the key aspects, namely that knowledge on hepatitis B correlates negatively with the behavior of the respondents and that the behavior also correlates negatively with their willingness for screening. In conclusion, we suggest that factors such as socio-economic context and prior informed knowledge on hepatitis B be considered to build a better strategy of Health Promotion and hepatitis B diagnostic screening among the population.

Enhancement of the Immunostimulatory Effect of Phosphodiester CpG Oligodeoxynucleotides by an Antiparallel Guanine-Quadruplex Structural Scaffold.

Guanine-quadruplex-based CpG oligodeoxynucleotides (G4 CpG ODNs) have been developed as potent immunostimulatory agents with reduced sensitivity to nucleases. We designed new monomeric G4 ODNs with an antiparallel topology using antiparallel type duplex/G4 ODNs as robust scaffolds, and we characterized their topology and effects on cytokine secretion. Based on circular dichroism analysis and quantification of mRNA levels of immunostimulatory cytokines, it was found that monomeric antiparallel G4 CpG ODNs containing two CpG motifs in the first functional loop, named G2.0.0, could maintain antiparallel topology and generate a high level of immunostimulatory cytokines in RAW264 mouse macrophage-like cell lines. We also found that the flanking sequence in the CpG motif altered the immunostimulatory effects. Gc2c.0.0 and Ga2c.0.0 are monomeric antiparallel G4 CpG ODNs with one cytosine in the 3' terminal and one cytosine/adenine in the 5' terminal of CpG motifs that maintained the same resistance to degradation in serum as G2.0.0 and improved interleukin-6 production in RAW264 and bone marrow-derived macrophages. The immunostimulatory activity of antiparallel G4 CpG ODNs is superior to that of linear natural CpG ODNs. These results provide insights for the rational design of highly potent CpG ODNs using antiparallel G4 as a robust scaffold.

Complete Genome Sequence of Bordetella pertussis Pelita III, the Production Strain for an Indonesian Whole-Cell Pertussis Vaccine.

PT Bio Farma, the sole World Health Organization-approved Indonesian vaccine producer, manufactures a whole-cell whooping cough vaccine (wP) that, as part of a pentavalent diphtheria-tetanus-pertussis/hepatitis B/Haemophilus influenzae b (DTP/HB/Hib) vaccine, is used in Indonesia and many other countries. We report here the whole-genome sequence for Bordetella pertussis Pelita III, PT Bio Farma's wP production strain.