Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Performance evaluation of a novel platelet count parameter, hybrid platelet count, on the BC-780 automated hematology analyzer.

OBJECTIVES: Automated hematology analysis is expected to improve the performance of platelet counting. We evaluated the performance of a new platelet counting, hybrid (PLT-H) and also impedance (PLT-I) and optical (PLT-O) on the BC-780 automated hematology analyzer compared to the international reference method (IRM) in blood samples with thrombocytopenic and platelet interference. METHODS: The basic platelet count performance of the BC-780 automated hematology analyzer was evaluated according to the requirements of the Clinical Laboratory and Standards Institute (CLSI) Document H26-A2. Additionally, the thrombocytopenic (low PLT count) blood samples and the platelet interference blood samples including fragmented red blood cells (RBCs), microcytes or small RBCs, and giant platelets were determined with the BC-780 hematology analyzer compared to the IRM. RESULTS: Blank counting and the carry-over contamination rate of platelet count using the BC-780 both met the manufacturers' claim. For both 123 thrombocytopenic and 232 platelet interference blood samples (72 fragmented RBCs, 91 microcytes and 51 giant platelets), all three platelet counting methods exhibited high comparability with the IRM (the lowest correlation (r)=0.916). Interestingly, the comparability of PLT-H (r=0.928-0.986) with the IRM was better than that of PLT-I (r=0.916-0.979). CONCLUSIONS: The performance of PLT-H in the BC-780 met the manufacturer's specifications. PLT-H exhibits better reproducibility than did PLT-I, correlates well with the PLT-O for thrombocytopenic samples and demonstrates good anti-interference ability. PLT-H counting is therefore recommended as a zero-cost alternative platelet counting method for platelet interference samples in clinical settings.

Performance evaluation of alternate ESR measurement method using BC-780 automated hematology analyzer: a comparison study with the Westergren reference method.

OBJECTIVES: Implementation of alternate erythrocyte sedimentation rate (ESR) measurement method is increasing worldwide due to its various advantages. In this study, we aim to evaluate the analytical performance of the BC-780 automated hematology analyzer in measurement of ESR value. METHODS: Analyzer performance including precision study, carryover, sample stability and potential interferences are examined. Samples with ESR values spanning the whole analytical ESR range are included for method comparison study. Samples with different hematocrit (Hct) and mean corpuscular volume (MCV) values are also analyzed and compared with the results obtained from the Westergren reference method. RESULTS: Precisions and carryover results are consistent with the manufacturers' claim. ESR values do not change significantly in the samples stored at 2-8 °C for 24 h (h) or at room temperature (RT) for 8 h, but significantly decreased (p<0.001) when stored at RT for 24 h. Significant increase in ESR value is documented in samples that are hemolyzed (hemoglobin concentration ranged from 1.28-6.01 g/L) (p=0.010) or lipemic (triglyceride above 4.75 mmol/L) (p=0.001). Method comparison study yields a proportional difference with a regression equation=3.08+ 0.98x. Bland-Altman analysis shows a mean absolute bias of 3.12 mm. The obtained absolute mean biases are below 5 mm in all analytical categories except for the group where MCV>100 fL. CONCLUSIONS: Most tested parameters met the manufacturer's specifications and were comparable to the reference method. Despite the presence of positive bias, it falls within acceptable criteria. Extensive validation against potential interferences such as hemolysis/lipemia is still necessary in future.

Untapped Pharmaceutical Potential of 4,5,4'-Trihydroxy-3,3'-dimethoxybibenzyl for Regulating Obesity: A Cell-Based Study with a Focus on Terminal Differentiation in Adipogenesis.

Obesity and its global prevalence has become a threat to human health, while its pharmacotherapy via the application of natural products is still underdeveloped. Here, we probed how 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (TDB) derived from an orchid (Dendrobium ellipsophyllum) could exert its roles on the differentiation and function of murine (3T3-L1) and human (PCS-210-010) pre-adipocytes and offer some implications to modulate obesity. Cytotoxic effects of TDB on adipocytes were 2-fold lower than those detected with pre-adipocytes, and no significant difference was detected in cytotoxic profiles between both cell lineages. TDB in a dose-dependent manner decreased cellular lipid accumulation and enhanced lipolysis of both cell lines assessed at early differentiation and during maturation. Underlining molecular mechanisms proved that TBD paused the cell cycle progression by regulating inducers and inhibitors in mitotic clonal expansion, leading to growth arrest of pre-adipocytes at the G0/G1 phase. The compound also governed adipocyte differentiation by repressing expressions of crucial adipogenic regulators and effectors through deactivating the AKT/GSK-3ß signaling pathway and activating the AMPK-ACC pathway. To this end, TDB has shown its pharmaceutical potential for modulating adipocyte development and function, and it would be a promising candidate for further assessments as a therapeutic agent to defeat obesity.

Cisplatin-induced hydroxyl radicals mediate pro-survival autophagy in human lung cancer H460 cells.

BACKGROUND: Accumulated evidence demonstrates cisplatin, a recommended chemotherapy, modulating pro-survival autophagic response that contributes to treatment failure in lung cancer patients. However, distinct mechanisms involved in cisplatin-induced autophagy in human lung cancer cells are still unclear. RESULTS: Herein, role of autophagy in cisplatin resistance was indicated by a decreased cell viability and increased apoptosis in lung cancer H460 cells pre-incubated with wortmannin, an autophagy inhibitor, prior to treatment with 50 µM cisplatin for 24 h. The elevated level of hydroxyl radicals detected via flow-cytometry corresponded to autophagic response, as evidenced by the formation of autophagosomes and autolysosomes in cisplatin-treated cells. Interestingly, apoptosis resistance, autophagosome formation, and the alteration of the autophagic markers, LC3-II/LC3-I and p62, as well as autophagy-regulating proteins Atg7 and Atg3, induced by cisplatin was abrogated by pretreatment of H460 cells with deferoxamine, a specific hydroxyl radical scavenger. The modulations in autophagic response were also indicated in the cells treated with hydroxyl radicals generated via Fenton reaction, and likewise inhibited by pretreatment with deferoxamine. CONCLUSIONS: In summary, the possible role of hydroxyl radicals as a key mediator in the autophagic response to cisplatin treatment, which was firstly revealed in this study would benefit for the further development of novel therapies for lung cancer.

Jorunnamycin A Suppresses Stem-Like Phenotypes and Sensitizes Cisplatin-Induced Apoptosis in Cancer Stem-Like Cell-Enriched Spheroids of Human Lung Cancer Cells.

It has been recognized that cancer stem-like cells (CSCs) in tumor tissue crucially contribute to therapeutic failure, resulting in a high mortality rate in lung cancer patients. Due to their stem-like features of self-renewal and tumor formation, CSCs can lead to drug resistance and tumor recurrence. Herein, the suppressive effect of jorunnamycin A, a bistetrahydroisoquinolinequinone isolated from Thai blue sponge Xestospongia sp., on cancer spheroid initiation and self-renewal in the CSCs of human lung cancer cells is revealed. The depletion of stemness transcription factors, including Nanog, Oct-4, and Sox2 in the lung CSC-enriched population treated with jorunnamycin A (0.5 µM), resulted from the activation of GSK-3ß and the consequent downregulation of ß-catenin. Interestingly, pretreatment with jorunnamycin A at 0.5 µM for 24 h considerably sensitized lung CSCs to cisplatin-induced apoptosis, as evidenced by upregulated p53 and decreased Bcl-2 in jorunnamycin A-pretreated CSC-enriched spheroids. Moreover, the combination treatment of jorunnamycin A (0.5 µM) and cisplatin (25 µM) also diminished CD133-overexpresssing cells presented in CSC-enriched spheroids. Thus, evidence on the regulatory functions of jorunnamycin A may facilitate the development of this marine-derived compound as a novel chemotherapy agent that targets CSCs in lung cancer treatment.

Colicin N Mediates Apoptosis and Suppresses Integrin-Modulated Survival in Human Lung Cancer Cells.

The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5-15 µM selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10-15 µM) for 12 h. Moreover, 5-15 µM of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin ß1 and αV in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.

Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic.

As the world is witnessing the epidemic of COVID-19, a disease caused by a novel coronavirus, SARS-CoV-2, emerging genetics and clinical evidences suggest a similar path to those of SARS and MERS. The rapid genomic sequencing and open access data, together with advanced vaccine technology, are expected to give us more knowledge on the pathogen itself, including the host immune response as well as the plan for therapeutic vaccines in the near future. This review aims to provide a comparative view among SARS-CoV, MERS-CoV and the newly epidemic SARS-CoV-2, in the hope to gain a better understanding of the host-pathogen interaction, host immune responses, and the pathogen immune evasion strategies. This predictive view may help in designing an immune intervention or preventive vaccine for COVID-19 in the near future.

Dengue vaccine: Global development update.

The first licensed dengue vaccine, CYD-TDV (Dengvaxia®), has received regulatory approval in a number of countries. However, this vaccine has some limitations. Its efficacy against DENV2 was consistently lower than other serotypes. Protective efficacy also depended on prior dengue sero-status of the vaccinees. Lower efficacy was observed in children with < 9 years old and dengue-na?ve individuals. More importantly, risk of hospitalization and severe dengue was increased in the youngest vaccine recipients (2-5 years) compared to controls. Thus, the quest of a better vaccine candidate continues. There are two live-attenuated vaccine candidates currently testing in phase III trial including DENVax, developed by US CDC and Inviragen (now licensed to Takeda) and TV003/TV005, constructed by US NIAID. In addition, there are several phase I-II as well as preclinical phase studies evaluating vaccines for safety and immunogenicity, this include other live-attenuated platform/strategy, purified-inactivated viruses formulated with adjuvants, DNA vaccine, subunit vaccine, viral vector and also heterologous prime/boost strategies. The major difficulties of dengue vaccine development are included the lack of the best animal model, various immune status of individual especially in endemic areas and clear cut off of protective immunity. Several research and development efforts are ongoing to find a better effective and accessible dengue vaccine for people needed.

Strategies to improve the immunogenicity of prM+E dengue virus type-2 DNA vaccine.

BACKGROUND: An important goal for dengue vaccines is to induce a high and durable level of neutralizing antibody. OBJECTIVE: Three strategies were investigated for improving the immunogenicity of a prM+E dengue serotype 2 (DENV-2) DNA vaccine: 1) expression in two different plasmids; 2) adjustment of dose; and, 3) introduction of the E sequence of Japanese encephalitis virus (JEV) at the carboxy-terminal portion of DENV-2 E. METHOD: Expression cassettes were designed to encode a full-length prM+E sequence of DENV-2 virus employing human-preferred codons (D2prMEopt), or a chimeric prM+E sequence in which the 100-residue carboxy-terminal region of E was derived from JEV (D2prMEJE20opt). pHIS and pCMVkan in the presence and absence of CpG motif, respectively, were used for cassette expression. The immunogenicity was compared in mice. RESULTS: Three injections of full-length-D2prMEopt in pHIS and pCMVkan induced a comparable neutralizing antibody titer at post-week-2-injection and post-week-4-injection. The 100-µg DNA dose induced a numerically but not statistically higher neutralizing antibody titer than the 10-µg dose. The chimeric-D2prMEJE20opt produced higher extracellular prM and E protein levels in transfected Vero cells, but had a tendency to induce a lower neutralizing antibody titer in mice when compared with the full-length-D2prMEopt. To optimize the immunogenicity of D2prMEopt-DNA candidate, both expression plasmids can be used to generate reproducible high neutralizing titer. A higher dose of DNA immunogen may induce a higher neutralizing antibody response. CONCLUSION: The strategy of the C-terminal region chimeric counterpart with JE20 did not improve but may have reduced the induction of neutralizing antibodies.

The immunogenicity of tetravalent dengue DNA vaccine in mice pre-exposed to Japanese encephalitis or Dengue virus antigens.

BACKGROUND: Asian countries are an endemic area for both dengue (DENV) and Japanese encephalitis viruses (JEV). While JEV vaccines have been used extensively in this region, DENV vaccines remains under development. Whether preexisting naturally acquired or vaccination-induced immunity against JEV may affect the immune response to dengue vaccine candidate is unclear. In this study we used mice previously immunized with JEV vaccines to evaluate the impact on dengue-specific neutralizing antibody responses to a tetravalent dengue DNA vaccine candidate (TDNA). METHODS: A tetravalent cocktail of plasmids encoding pre-membrane and envelope proteins from each dengue serotype was administered into mice which had been previously primed with inactivated or live-attenuated JEV vaccines, or dengue serotype2 virus (DENV-2). Neutralizing antibody response was measured employing a plaque reduction neutralization test at two weeks after the priming and at four weeks after the second dose of the dengue tetravalent plasmids. RESULTS: Inactivated or live-attenuated JEV vaccines, or DENV-2 induced low levels of neutralizing antibodies against the homologous viruses (JE and dengue virus, respectively). DENV-2 injection induced also low levels of cross-reactive antibodies against DENV-1, -3 and -4. JEV vaccines have no effect on the dengue-specific neutralizing antibody responses to the subsequent TDNA immunization. Pre-exposure to DENV-2 infection increased DENV-2 specific response neutralizing antibody to two doses of TDNA plasmids by six folds, but did not affect antibody response to other serotypes. CONCLUSIONS: Priming with JEV vaccines did not impact on dengue virus-specific neutralizing antibody response to a dengue TDNA vaccine candidate in mice.

Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice.

Respiratory syncytial virus (RSV) is a highly infectious respiratory virus that causes serious illness, particularly in young children, elderly people, and those with immunocompromised individuals. RSV infection is the leading cause of infant hospitalization and can lead to serious complications such as pneumonia and bronchiolitis. Currently, there is an RSV vaccine approved exclusively for the elderly population, but no approved vaccine specifically designed for infants or any other age groups. Therefore, it is crucial to continue the development of an RSV vaccine specifically tailored for these populations. In this study, the immunogenicity of the two plant-produced RSV-F Fc fusion proteins (Native construct and structural stabilized construct) were examined to assess them as potential vaccine candidates for RSV. The RSV-F Fc fusion proteins were transiently expressed in Nicotiana benthamiana and purified using protein A affinity column chromatography. The recombinant RSV-F Fc fusion protein was recognized by the monoclonal antibody Motavizumab specific against RSV-F protein. Moreover, the immunogenicity of the two purified RSV-F Fc proteins were evaluated in mice by formulating with different adjuvants. According to our results, the plant-produced RSV-F Fc fusion protein is immunogenic in mice. These preliminary findings, demonstrate the immunogenicity of plant-based RSV-F Fc fusion protein, however, further preclinical studies such as antigen dose and adjuvant optimization, safety, toxicity, and challenge studies in animal models are necessary in order to prove the vaccine efficacy.

Alpha and gamma mangostins inhibit wild-type B SARS-CoV-2 more effectively than the SARS-CoV-2 variants and the major target is unlikely the 3C-like protease.

Background: Anti-SARS-CoV-2 and immunomodulatory drugs are important for treating clinically severe patients with respiratory distress symptoms. Alpha- and gamma-mangostins (AM and GM) were previously reported as potential 3C-like protease (3CLpro) and Angiotensin-converting enzyme receptor 2 (ACE2)-binding inhibitors in silico. Objective: We aimed to evaluate two active compounds, AM and GM, from Garcinia mangostana for their antivirals against SARS-CoV-2 in live virus culture systems and their cytotoxicities using standard methods. Also, we aimed to prove whether 3CLpro and ACE2 neutralization were major targets and explored whether any additional targets existed. Methods: We tested the translation and replication efficiencies of SARS-CoV-2 in the presence of AM and GM. Initial and subgenomic translations were evaluated by immunofluorescence of SARS-CoV-2 3CLpro and N expressions at 16 h after infection. The viral genome was quantified and compared with the untreated group. We also evaluated the efficacies and cytotoxicities of AM and GM against four strains of SARS-CoV-2 (wild-type B, B.1.167.2, B.1.36.16, and B.1.1.529) in Vero E6 cells. The potential targets were evaluated using cell-based anti-attachment, time-of-drug addition, in vitro 3CLpro activities, and ACE2-binding using a surrogated viral neutralization test (sVNT). Moreover, additional targets were explored using combinatorial network-based interactions and Chemical Similarity Ensemble Approach (SEA). Results: AM and GM reduced SARS-CoV-2 3CLpro and N expressions, suggesting that initial and subgenomic translations were globally inhibited. AM and GM inhibited all strains of SARS-CoV-2 at EC50 of 0.70-3.05 µM, in which wild-type B was the most susceptible strain (EC50 0.70-0.79 µM). AM was slightly more efficient in the variants (EC50 0.88-2.41 µM), resulting in higher selectivity indices (SI 3.65-10.05), compared to the GM (EC50 0.94-3.05 µM, SI 1.66-5.40). GM appeared to be more toxic than AM in both Vero E6 and Calu-3 cells. Cell-based anti-attachment and time-of-addition suggested that the potential molecular target could be at the post-infection. 3CLpro activity and ACE2 binding were interfered with in a dose-dependent manner but were insufficient to be a major target. Combinatorial network-based interaction and chemical similarity ensemble approach (SEA) suggested that fatty acid synthase (FASN), which was critical for SARS-CoV-2 replication, could be a target of AM and GM. Conclusion: AM and GM inhibited SARS-CoV-2 with the highest potency at the wild-type B and the lowest at the B.1.1.529. Multiple targets were expected to integratively inhibit viral replication in cell-based system.

Phase II prefusion non-stabilised Covid-19 mRNA vaccine randomised study.

ChulaCov19 mRNA vaccine demonstrated promising phase 1 results. Healthy adults aged 18-59 years were double-blind randomised 4:1 to receive two intramuscular doses of ChulaCov19 50 µg or placebo. Primary endpoints were safety and microneutralization antibody against-wild-type (Micro-VNT50) at day 50. One hundred fifty adults with median (IQR) age 37 (30-46) years were randomised. ChulaCov19 was well tolerated, and most adverse events were mild to moderate and temporary. Geometric mean titres (GMT) of neutralizing titre against wild-type for ChulaCov19 on day 50 were 1367 IU/mL. T-cell IFN-γ-ELISpot showed the highest responses at one week (Day29) after dose 2 then gradually declined. ChulaCov19 50 µg is well tolerated and elicited high neutralizing antibodies and strong T-cell responses in healthy adults.Trial registration number: ClinicalTrials.gov Identifier NCT04566276, 28/09/2020.

Inhibitory Effect of Isopanduratin A on Adipogenesis: A Study of Possible Mechanisms.

The root of Boesenbergia rotunda, a culinary plant commonly known as fingerroot, has previously been reported to possess anti-obesity activity, with four flavonoids identified as active principles, including pinostrobin, panduratin A, cardamonin, and isopanduratin A. However, the molecular mechanisms underlying the antiadipogenic potential of isopanduratin A remain unknown. In this study, isopanduratin A at non-cytotoxic concentrations (1-10 µM) significantly suppressed lipid accumulation in murine (3T3-L1) and human (PCS-210-010) adipocytes in a dose-dependent manner. Downregulation of adipogenic effectors (FAS, PLIN1, LPL, and adiponectin) and adipogenic transcription factors (SREBP-1c, PPARγ, and C/EBPα) occurred in differentiated 3T3-L1 cells treated with varying concentrations of isopanduratin A. The compound deactivated the upstream regulatory signals of AKT/GSK3ß and MAPKs (ERK, JNK, and p38) but stimulated the AMPK-ACC pathway. The inhibitory trend of isopanduratin A was also observed with the proliferation of 3T3-L1 cells. The compound also paused the passage of 3T3-L1 cells by inducing cell cycle arrest at the G0/G1 phase, supported by altered levels of cyclins D1 and D3 and CDK2. Impaired p-ERK/ERK signaling might be responsible for the delay in mitotic clonal expansion. These findings revealed that isopanduratin A is a strong adipogenic suppressor with multi-target mechanisms and contributes significantly to anti-obesogenic activity. These results suggest the potential of fingerroot as a functional food for weight control and obesity prevention.

CHO-produced RBD-Fc subunit vaccines with alternative adjuvants generate immune responses against SARS-CoV-2.

Subunit vaccines feature critical advantages over other vaccine platforms such as stability, price, and minimal adverse effects. To maximize immunological protection of subunit vaccines, adjuvants are considered as main components that are formulated within the subunit vaccine. They can modulate adverse effects and enhance immune outcomes. However, the most suitable formulation providing the best immunological outcomes and safety are still under investigation. In this report, we combined recombinant RBD with human IgG1 Fc to create an RBD dimer. This fusion protein was expressed in CHO and formulated with alternative adjuvants with different immune activation including Montanide ISA51, Poly (I:C), and MPLA/Quil-A® as potential vaccine candidate formulations. Using the murine model, a potent induction of anti-RBD IgG antibodies in immunized mice sera were observed. IgG subclass analyses (IgG1/IgG2a) illustrated that all adjuvanted formulations could stimulate both Th1 and Th2-type immune responses in particular Poly (I:C) and MPLA/Quil-A®, eliciting greater balance. In addition, Montanide ISA51-formulated RBD-Fc vaccination provided a promising level of neutralizing antibodies against live wild-type SARS-CoV-2 in vitro followed by Poly (I:C) and MPLA/Quil-A®, respectively. Also, mice sera from adjuvanted formulations could strongly inhibit RBD:ACE2 interaction. This study offers immunogenicity profiles, forecasted safety based on Vaccine-associated enhanced disease (VAED) caused by Th1-skewed immunity, and neutralizing antibody analysis of candidates of RBD-Fc-based subunit vaccine formulations to obtain an alternative subunit vaccine formulation against SARS-CoV-2.

Blockade-of-Binding Activities toward Envelope-Associated, Type-Specific Epitopes as a Correlative Marker for Dengue Virus-Neutralizing Antibody.

Humans infected with dengue virus (DENV) acquire long-term protection against the infecting serotype, whereas cross-protection against other serotypes is short-lived. Long-term protection induced by low levels of type-specific neutralizing antibodies can be assessed using the virus-neutralizing antibody test. However, this test is laborious and time-consuming. In this study, a blockade-of-binding enzyme-linked immunoassay was developed to assess antibody activity by using a set of neutralizing anti-E monoclonal antibodies and blood samples from dengue virus-infected or -immunized macaques. Diluted blood samples were incubated with plate-bound dengue virus particles before the addition of an enzyme-conjugated antibody specific to the epitope of interest. Based on blocking reference curves constructed using autologous purified antibodies, sample blocking activity was determined as the relative concentration of unconjugated antibody that resulted in the same percent signal reduction. In separate DENV-1-, -2-, -3-, and -4-related sets of samples, moderate to strong correlations of the blocking activity with neutralizing antibody titers were found with the four type-specific antibodies 1F4, 3H5, 8A1, and 5H2, respectively. Significant correlations were observed for single samples taken 1 month after infection as well as samples drawn before and at various time points after infection/immunization. Similar testing using a cross-reactive EDE-1 antibody revealed a moderate correlation between the blocking activity and the neutralizing antibody titer only for the DENV-2-related set. The potential usefulness of the blockade-of-binding activity as a correlative marker of neutralizing antibodies against dengue viruses needs to be validated in humans. IMPORTANCE This study describes a blockade-of-binding assay for the determination of antibodies that recognize a selected set of serotype-specific or group-reactive epitopes in the envelope of dengue virus. By employing blood samples collected from dengue virus-infected or -immunized macaques, moderate to strong correlations of the epitope-blocking activities with the virus-neutralizing antibody titers were observed with serotype-specific blocking activities for each of the four dengue serotypes. This simple, rapid, and less laborious method should be useful for the evaluation of antibody responses to dengue virus infection and may serve as, or be a component of, an in vitro correlate of protection against dengue in the future.

Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 µg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

Erythrocyte sedimentation rate measurements using MIX-RATE® X20 and VISION A automated analyzers: Method validation and comparison study.

BACKGROUND: The Westergren method for erythrocyte sedimentation rate (ESR) measurement has a few drawbacks such as being a time-consuming process, poses a risk of biohazard exposure and requires high sample volume. Recent alternative methods and analyzers were developed to overcome those limitations. In this study, we validated two automated ESR analyzers, MIX-RATE® X20 and VISION A, and assessed their analytical performance against the Westergren method. METHODS: The analyzers were validated for inter-run and intra-run precision. Hemolysis interference and sensitivity to fibrinogen were also analysed. Analytical performance was performed using 177 patient samples spanning low (<40 mm/h), medium (40-80 mm/h), and high (>80 mm/h) ESR ranges. Method agreement and bias against the Westergren method were calculated. RESULTS: The highest intra-run imprecision was seen in the low ESR range for both analyzers. They showed very high agreement with the Westergren method assessed by Spearmen rank correlation coefficient analysis, r = 1.000, p < .0001 for both analyzers. Bland-Altman analysis yielded overall insignificant mean biases for all comparisons. However, systematic positive and negative bias were observed at medium and high ESR levels analysed by MIX-RATE® X20 while negative bias was evidenced in the high ESR level measured by VISION A. CONCLUSIONS: Overall, results from both automated ESR analyzers showed comparable analytical performance with the Westergren method especially for low ESR levels. However, both positive and negative systematic bias were documented in the high levels. Thus, for clinical use, it must be assessed whether these biases could affect the cut-off for significant clinical values.