Self-enhancement lateral flow immunoassay for COVID-19 diagnosis.

Equipment-free colorimetric-based lateral flow immunoassay (LFIA) is the most convenient and popular tool for various applications, including diagnostic tools requiring high sensitivity for the detection of pathogens. Thus, improvements and developments of LFIA are constantly being reported. Herein, we enriched the sensitivity of LFIA using the gold enhancement principle, emphasizing needlessly complicated apparatus, only one step for the strip test operation, and typical time incubation (15 min) process. Self-enhanced LFIA was then executed for subsequent flows by overlapping the additionally enhanced pad composed of gold ions and reducing agent on the conjugate pad and the sample pad. Self-enhanced LFIA was performed to detect SARS-CoV-2 antigens in saliva. The obtained result depicted that the achieved sensitivity was up to tenfold compared with that of conventional LFIA by visual measurements. The detection limits of self-enhanced LFIA detecting nucleocapsid protein antigens in the saliva sample was 0.50 and 0.10 ng/mL employed by naked eye detection and calibration curve-based calculation, respectively. When the proposed device was applied to 207 human saliva samples, the diagnostic performance presented a 96.10 % sensitivity and 99.23 % specificity. This self-enhanced LFIA could be implemented in large-scale production and demonstrates higher sensitivity with effortless use, which meets the requirements for point-of-care testing and on-field mass screening.

Comparison of the immunogenicity of ChAdOx1 nCoV-19 vaccine against the wild-type and delta variants in kidney transplant recipients and healthy volunteers.

Little is known about immunogenicity after ChAdOx1 nCov-19 vaccination after transplantation. We assessed the vaccine response by antibody testing, surrogate neutralization test (sVNT) against wild-type (WT) and delta variant (DT), and T cell assay in 83 kidney transplant recipients (KTRs) and 52 healthy volunteers (HVs). For KTRs, a positive anti-RBD antibody was seen in 2.8% after one dose and 15.7% after two doses of the vaccine. After two doses, the positivity rate by sVNT was equal (4.9% each, for WT and DT) and was 13.4% by T cell response. Post two doses, KTRs had significantly lower geometric mean titer than HVs (1.93 [95% CI: 1.39-2.69] vs. 248.3 [95% CI: 203.7-302.6] BAU/ml, respectively, p < .001). Daily mycophenolate dose of ≥1000 mg significantly associated with negative seroconversion [risk ratio (RR) of 0.33, 95% CI: 0.15-0.72, p = .005]. Compared with cyclosporine, daily tacrolimus dose of ≤3 mg and >3 mg of tacrolimus significantly associated with negative seroconversion [RR = 0.38 (95% CI, 0.17-0.85, p = .018) and RR = 0.16 (95% CI, 0.37-0.73, p = .018)], respectively. The vaccine was safe and well-tolerated but the immune response after the two doses of ChAdOx1 nCov-19 vaccine in KTRs was very low.

Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDVAVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. IMPORTANCE: Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity.

Mechanism of an ATP-independent protein disaggregase: II. distinct molecular interactions drive multiple steps during aggregate disassembly.

The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of "ATPases associated with various cellular activities" (AAA(+)) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP.

Mechanism of an ATP-independent protein disaggregase: I. structure of a membrane protein aggregate reveals a mechanism of recognition by its chaperone.

BACKGROUND: A novel chaperone, cpSRP43, recognizes and disassembles the aggregates formed by its client proteins. RESULTS: The client proteins of cpSRP43 form stable disc-shaped aggregates with the chaperone recognition motif displayed onthe surface. CONCLUSION: The surface-exposed motif on the aggregate allows it to be recognized by its chaperone. SIGNIFICANCE: Understanding the structure and energetics of protein aggregates provides insights into the mechanism of theirDISASSEMBLY.Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system in which the 38-kDa subunit of chloroplast signal recognition particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the nonamyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone.

Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation.

Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNP hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.

Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I.

BACKGROUND: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. METHODS: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. RESULTS: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. CONCLUSIONS: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor.

Crystal structure of the lambda repressor and a model for pairwise cooperative operator binding.

Bacteriophage lambda has for many years been a model system for understanding mechanisms of gene regulation. A 'genetic switch' enables the phage to transition from lysogenic growth to lytic development when triggered by specific environmental conditions. The key component of the switch is the cI repressor, which binds to two sets of three operator sites on the lambda chromosome that are separated by about 2,400 base pairs (bp). A hallmark of the lambda system is the pairwise cooperativity of repressor binding. In the absence of detailed structural information, it has been difficult to understand fully how repressor molecules establish the cooperativity complex. Here we present the X-ray crystal structure of the intact lambda cI repressor dimer bound to a DNA operator site. The structure of the repressor, determined by multiple isomorphous replacement methods, reveals an unusual overall architecture that allows it to adopt a conformation that appears to facilitate pairwise cooperative binding to adjacent operator sites.

PEDV nucleocapsid antagonizes zinc-finger antiviral protein by disrupting the interaction with its obligate co-factor, TRIM25.

The genomes of many pathogens contain high-CpG content, which is less common in most vertebrate host genomes. Such a distinct di-nucleotide composition in a non-self invader constitutes a special feature recognized by its host's immune system. The zinc-finger antiviral protein (ZAP) is part of the pattern recognition receptors (PRRs) that recognize CpG-rich viral RNA and subsequently initiate RNA degradation as an antiviral defense measure. To counteract such ZAP-mediated restriction, some viruses evolve to either suppress the CpG content in their genome or produce an antagonistic factor to evade ZAP sensing. We have previously shown that a coronavirus, Porcine epidermic diarrhea virus (PEDV), employs its nucleocapsid protein (PEDV-N) to suppress the ZAP-dependent antiviral activity. Here, we propose a mechanism by which PEDV-N suppresses ZAP function by interfering with the interaction between ZAP and its essential cofactor, Tripartite motif-containing protein 25 (TRIM25). PEDV-N was found to interact with ZAP through its N-terminal domain and with TRIM25 through its C-terminal domain. We showed that PEDV-N and ZAP compete for binding to the SPla and the RYanodine Receptor (SPRY) domain of TRIM25, resulting in PEDV-N preventing TRIM25 from interacting with and promoting ZAP. Our result also showed that the presence of PEDV-N in the complex reduces the E3 ligase activity of TRIM25 on ZAP, which is required for the antiviral activity of ZAP. The host-pathogen interaction mechanism presented herein provides an insight into the new function of this abundant and versatile viral protein from a coronavirus which could be a key target for development of antiviral interventions.

Immunogenicity of BNT162b2 in children 6 months to under 5 years of age with previous SARS-CoV-2 infection, in the era of Omicron predominance.

Background: Children 6 months to < 5 years old are recommended to receive 3-dose regimen of BNT162b2. Children previously infected with Omicron variant of SARS-CoV-2 develop immunity from natural infection, therefore may require fewer doses of vaccine. Objective: To compare immunogenicity of 1- or 2-dose BNT162b2 in healthy children post COVID-19 with 3-dose BNT162b2 in COVID-naïve children. Methods: Children aged 6 months to < 5 years who developed COVID-19 during the Omicron-predominant period were enrolled; Group A 3-6 months(N = 40) and Group B > 6 months(N = 40) prior to vaccination. Participants in Group A and B received 2-dose BNT162b2 intramuscularly 1 month apart. COVID-naïve children were enrolled as a control group (N = 40) and received 3-dose BNT162b2 at month 0,1,3. Neutralizing antibody against Omicron variant(BA.2.75 and BA.4/5) was determined by pseudovirus assays(pVNT) as reported by neutralization dilution for 50%inhibition (ID50) at 28 days after the 1st and 2nd dose. Results: From October-November 2022, 120 children with a median age of 2.8 years (IQR 1.6-4.0) were enrolled. The median duration since COVID-19 to vaccination was 4.4 months(IQR 3.8-5.4) in Group A and 7.9 months(7.0-8.5) in Group B. In Group A, the geometric means(GMs) of pVNT-BA.2.75 ID50 were 553 (95%CI 338-906) and 753(516-1098) after 1 and 2 doses, respectively, and the GMs of pVNT-BA.4/5 ID50 were 1936(1402-2673) and 1885(1414-2512), respectively. In Group B, the GMs of pVNT-BA.2.75 ID50 were 1383(1100-1742) and 1419 (1104-1823), and the GMs of pVNT-BA.4/5 ID50 were 2627(2048-3367) and 2056(1546-2735), respectively. Meanwhile in COVID-naïve group, the GMs of pVNT-BA.2.75 and pVNT-BA.4/5 ID50 were 158(98-255) and 59(31-114) after the 3rd dose, respectively. The geometric mean ratio(GMR) of pVNT-BA.2.75 ID50 after 1 dose in Group A and B compared with after 3 doses in COVID-naïve group were 3.50 (1.93-6.34) and 8.74 (4.79-15.95), respectively. The GMR of pVNT-BA.2.75 ID50 after 1 dose in Group B compared with Group A was 2.50 (1.45-4.31). Conclusions: Children previously infected with SARS-CoV-2 Omicron variant, developed robust neutralizing antibody response against Omicron variant after single-dose BNT162b2. Children with an interval of > 6 months since COVID-19 infection developed higher neutralizing antibody response compared to those with a 3-to-6-month interval.

Immunogenicity and Safety of the Third Booster Dose with mRNA-1273 COVID-19 Vaccine after Receiving Two Doses of Inactivated or Viral Vector COVID-19 Vaccine.

The changes in the severe acute respiratory syndrome coronavirus 2 and the tapering of immunity after vaccination have propelled the need for a booster dose vaccine. We aim to evaluate B and T cell immunogenicity and reactogenicity of mRNA-1273 COVID-19 vaccine (100 µg) as a third booster dose after receiving either two doses of inactivated COVID-19 vaccine (CoronaVac) or two doses of viral vector vaccine (AZD1222) in adults not previously infected with COVID-19. The anti-receptor-binding-domain IgG (anti-RBD IgG), surrogate virus neutralization test (sVNT) against the Delta variant, and Interferon-Gamma (IFN-γ) level were measured at baseline, day (D)14 and D90 after vaccination. In D14 and D90, the geometric means of sVNT were significantly increased to 99.4% and 94.5% inhibition in CoronaVac, respectively, whereas AZD1222 showed inhibition of 99.1% and 93%, respectively. Anti-RBD IgG levels were 61,249 to 9235 AU/mL in CoronaVac and 38,777 to 5877 AU/mL in AZD1222 after D14 and D90 vaccination. Increasing median frequencies of S1-specific T cell response by IFN-γ concentration were also elevated in D14 and were not significantly different between CoronaVac (107.8-2035.4 mIU/mL) and AZD1222 (282.5-2001.2 mIU/mL). This study provides evidence for the high immunogenicity of the mRNA-1273 booster after two doses of CoronaVac or AZD1222 in the Thai population.

The immunogenicity of an extended dosing interval of BNT162b2 against SARS-CoV-2 Omicron variant among healthy school-aged children, a randomized controlled trial.

OBJECTIVES: To evaluate the immunogenicity of an extended interval regimen of BNT162b2 among healthy school-age children. METHODS: A randomized-control trial conducted among healthy Thai children aged 5-11 years. Participants received two doses of BNT162b2 with an 8-week (extended dosing) vs 3-week interval. Immunogenicity was determined by neutralization test (NT) against the Omicron variant, surrogate virus NT (sVNT; BA.1, % inhibition), and pseudovirus NT (BA.2, the half-maximal inhibition dilution or ID50). The third dose was offered to participants who had sVNT <68% inhibition. The immunogenicity outcome was evaluated at 14 days after the second and third doses. RESULTS: During February to April 2022, 382 children with a median age (interquartile range) of 8.4 years (6.6-10.0) were enrolled. At 14 days, after two doses of BNT162b2, the geometric means of sVNT in 8-week vs 3-week interval groups were 49.6 (95% confidence interval [CI] 44.8-54.9) vs 16.5 (95% CI 13.0-20.9), with a geometric means ratio of 3.0 (95% CI 2.4-3.8). Among 102 participants who received the third dose at a median of 15 weeks from the second dose, the geometric means of sVNT increased to 73.3 (95% CI 69.0-77.8) and pseudovirus NT increased to 326 (95% CI 256-415). CONCLUSION: The extended 8-week interval regimen of BNT162b2 induced higher neutralizing antibodies than a standard 3-week interval regimen. The third dose induced high neutralizing antibodies against the Omicron variant.

Autoimmune Hepatitis Triggered by COVID-19 Vaccine: The First Case From Inactivated Vaccine.

We report a case of a 52-year-old woman without previous underlying liver disease, presenting with progressive jaundice and diagnosed with autoimmune hepatitis after 2 doses of an inactivated coronavirus disease 2019 (CoronaVac) vaccine. All serology and histology were compatible with autoimmune hepatitis. Symptoms were improved and liver function tests were normalized after treatment with steroids and azathioprine.

Zinc-finger antiviral protein-mediated inhibition of porcine epidemic diarrhea virus growth is antagonized by the coronaviral nucleocapsid protein.

Coronaviruses have long posed a major threat not only to human health but also to agriculture. Outbreaks of an animal coronavirus such as porcine epidemic diarrhea virus (PEDV) can cause up-to-100% mortality in suckling piglets, resulting in devastating effects on the livestock industry. Understanding how the virus evades its host's defense can help us better manage the infection. Zinc-finger antiviral protein (ZAP) is an important class of host antiviral factors against a variety of viruses, including the human coronavirus. In this study, we have shown that a representative porcine coronavirus, PEDV, can be suppressed by endogenous or porcine-cell-derived ZAP in VeroE6 cells. An uneven distribution pattern of CpG dinucleotides in the viral genome is one of the factors contributing to suppression, as an increase in CpG content in the nucleocapsid (N) gene renders the virus more susceptible to ZAP. Our study revealed that the virus uses its own nucleocapsid protein (pCoV-N) to interact with ZAP and counteract the activity of ZAP. The insights into coronavirus-host interactions shown in this work could be used in the design and development of modern vaccines and antiviral agents for the next pandemic.

Immunogenicity of an mRNA-Based COVID-19 Vaccine among Adolescents with Obesity or Liver Transplants.

There are limited data regarding the immunogenicity of mRNA-based SARS-CoV-2 vaccine BNT162b2 among immunosuppressed or obese adolescents. We evaluated the humoral immune response in adolescents with obesity and adolescent liver transplant recipients (LTRs) after receiving two BNT162b2 doses. Sixty-eight participants (44 males; mean age 14.9 ± 1.7 years), comprising 12 LTRs, 24 obese, and 32 healthy adolescents, were enrolled. Immunogenicity was evaluated by anti-SARS-CoV-2 spike protein immunoassay and surrogate viral neutralization tests (sVNT) against the Delta and Omicron (BA.1) variants. At 27.1 ± 3.2 days after the second dose, the antibody levels were 1476.6 ± 1185.4, 2999.4 ± 1725.9, and 4960.5 ± 2644.1 IU/mL in the LTRs, obese adolescents, and controls, respectively (p < 0.001). Among obese individuals, liver stiffness <5.5 kPa was associated with higher antibody levels. The %inhibition of sVNT was significantly lower for the Omicron than that for the Delta variant. Injection site pain was the most common local adverse event. Nine participants (three obese and six controls) developed COVID-19 at 49 ± 11 days after the second vaccination; four were treated with favipiravir. All infections were mild, and the patients recovered without any consequences. Our study supports the need for the booster regimen in groups with an inferior immunogenic response, including LTRs and obese individuals.

A Randomized Clinical Trial of a Fractional Low Dose of BNT162b2 Booster in Adults Following AZD1222.

In the era of globally predominant omicron strains, a COVID-19 booster vaccine is needed. Our study aimed to evaluate the immunogenicity of a half-dose BNT162b2 booster after AZD1222 in healthy adults. A randomized trial of volunteers aged 18-69 years who received two-dose AZD1222 was conducted. The participants were randomized to receive the BNT162b2 vaccine intramuscularly-half (15 µg) vs. standard dose (30 µg). The immunogenicity was evaluated by a surrogate virus neutralization test (sVNT) against omicron variants and anti-spike-receptor-binding-domain IgG (anti-S-RBD IgG). From November-December 2021, 100 adults with a median age of 59.3 years (IQR 33.4-65.5) were enrolled. A booster dose was given at median of 98 days (IQR 92-128) after AZD1222. At day 14, the geometric means (GMs) of anti-S-RBD IgG in half- vs. standard-dose group were 2329.8 vs. 2574.7 BAU/mL, with a geometric mean ratio (GMR) of 0.90 (0.77-1.06). The GMs of sVNT against the omicron variant in the half- and standard-dose groups were 74.4% inhibition (95% CI 68.8-80.5) and 67.3% inhibition (57.9-78.1), respectively, with GMR of 0.95 (0.69-1.30). At day 90, the sVNT indicated 22.3% inhibition (95% CI 14.9-33.4) and 20.4% inhibition (13.1-32.0), respectively, with GMR of 1.09 (0.60-1.98). The fractional low-dose BNT162b2 mRNA booster vaccine provided non-inferior immunogenicity responses. During a shortage of vaccine supply, a fractional low dose should be considered for a booster vaccination program.

Immunogenicity to SARS-CoV-2 Omicron variant among school-aged children with 2-dose of inactivated SARS-CoV-2 vaccines followed by BNT162b2 booster.

Background: A primary series of 2-dose SARS-CoV-2 vaccines based on an ancestral strain generate inadequate neutralizing antibodies against the SARS-CoV-2 Omicron variant. This study aimed to describe the immune response from giving healthy school-aged children who previously received 2 inactivated vaccines an mRNA BNT162b2 booster. Methods: Healthy children aged 5-11 years who received 2 doses of CoronaVac or Covilo were enrolled and received 10 µg BNT162b2 intramuscularly. Neutralizing antibody against Omicron variant was measured at pre-booster and 14-21 days post-booster by surrogate virus neutralization test (sVNT, %inhibition) and pseudovirus neutralization test (pVNT, ID50). Antibody responses were compared with a parallel cohort of children who received 2 doses of BNT162b2 3 weeks apart. Results: From April to May 2022, 59 children with a mean age (SD) of 8.5 years (1.7) were enrolled: 20 CoronaVac and 39 Covilo recipients. The median interval from the primary series was 49 days (IQR 33-51). After booster, the geometric means (GMs) of sVNT and pVNT were 72.2 %inhibition (95 %CI 67.2-77.6) and 499 (95 %CI 399-624), respectively. The proportion of children with sVNT against Omicron strain ≥68 %inhibition increased from none to 70.2 %. The geometric mean ratio (GMR) of sVNT and pVNT compared with a parallel cohort were 4.3 and 12.2, respectively. The GMR of sVNT and pVNT between children who received booster dose at >6-week interval were 1.2 (95 %CI 1.1-1.3). and 1.8 (95 %CI 1.2-2.7) compared with 4-6 weeks interval. Conclusion: A regimen of 2-dose of inactivated vaccine followed by BNT162b2 booster dose elicited high neutralizing antibody against the Omicron variants in healthy school-aged children.

Heterologous Prime-boost of SARS-CoV-2 inactivated vaccine and mRNA BNT162b2 among Healthy Thai Adolescents.

Background: Heterologous prime-boost SARS-CoV-2 vaccination is a widely accepted strategy during the COVID-19 pandemic, which generated a superior immune response than homologous vaccination strategy. Objective: To describe immunogenicity of heterologous prime-boost vaccination with inactivated vaccine, CoronaVac, followed by BNT162b2 and 5-month booster dose with BNT162b2 in healthy Thai adolescents. Methods: Adolescents aged 12-18 years were randomized 1:1:1:1 to receive CoronaVac (SV) followed by BNT162b2 (PZ) 30 or 20 µg at either 3- or 6-week interval (SV3w/PZ30µg, SV3w/PZ20µg, SV6w/PZ30µg or SV6w/PZ20µg). During the Omicron-predominant period, participants were offered a BNT162b2 booster dose 30, 15, or 10 µg. Immunogenicity was determined using IgG antibody against spike-receptor-binding domain of wild type(anti-S-RBD IgG) and surrogate virus neutralization test(sVNT) against Delta variant at 14 days and 5 months after the 2nd dose. Neutralization tests(sVNT and pseudovirus neutralization test; pVNT) against Omicron strain were tested pre- and 14 days post-booster dose. Results: In October 2021, 76 adolescents with a median age of 14.3 years (IQR 12.7-16.0) were enrolled: 20 in SV3w/PZ30µg; 17 in SV3w/PZ20µg; 20 in SV6w/PZ30µg; 19 in SV6w/PZ20µg. At day 14, the geometric mean(GM) of anti-S-RBD IgG in SV3w/PZ30µg was 4713 (95 %CI 4127-5382) binding-antibody unit (BAU)/ml, while geometric mean ratio(GMR) was 1.28 (1.09-1.51) in SV6w/PZ30µg. The GMs of sVNT against Delta variants at day 14 among participants in SV3w/PZ30µg and SV6wk/PZ30µg arm were 95.3 % and 99.7 %inhibition, respectively. At 5 months, GMs of sVNT against Delta variants in SV3w/PZ30µg were significantly declined to 47.8 % but remained at 89.0 % inhibition among SV6w/PZ30µg arm. In April 2022, 52 adolescents received a BNT162b2 booster dose. Proportion of participants with sVNT against Omicron strain > 80 %inhibition was significantly increased from 3.8 % pre-booster to 67 % post-booster. Proportion of participants with pVNT ID50 > 185 was 42 % at 14 days post 2nd dose and 88 % post booster, respectively. Conclusions: Heterologous prime-boost vaccination with CoronaVac followed by BNT162b2 induced high neutralizing titer against SARS-CoV-2 Delta strain. After 5-month interval, booster with BNT162b2 induced high neutralizing titer against Omicron strain.Thai Clinical Trials Registry (thaiclinicaltrials.org): TCTR20210923012.

Evaluation of Methylotrophic Yeast Ogataea thermomethanolica TBRC 656 as a Heterologous Host for Production of an Animal Vaccine Candidate.

Multiple yeast strains have been developed into versatile heterologous protein expression platforms. Earlier works showed that Ogataea thermomethanolica TBRC 656 (OT), a thermotolerant methylotrophic yeast, can efficiently produce several industrial enzymes. In this work, we demonstrated the potential of this platform for biopharmaceutical manufacturing. Using a swine vaccine candidate as a model, we showed that OT can be optimized to express and secrete the antigen based on porcine circovirus type 2d capsid protein at a respectable yield. Crucial steps for yield improvement include codon optimization and reduction of OT protease activities. The antigen produced in this system could be purified efficiently and induce robust antibody response in test animals. Improvements in this platform, especially more efficient secretion and reduced extracellular proteases, would extend its potential as a competitive platform for biopharmaceutical industries.

Immunogenicity and reactogenicity after booster dose with AZD1222 via intradermal route among adult who had received CoronaVac.

BACKGROUND: Currently, booster dose is needed after 2 doses of non-live COVID-19 vaccine. With limited resources and shortage of COVID-19 vaccines, intradermal(ID) administration might be a potential dose-sparing strategy. OBJECTIVE: To determine immunologic response and reactogenicity of ID ChAdOx1 nCoV-19 vaccine (AZD1222,Oxford/AstraZeneca) as a booster dose after completion of 2-dose CoronaVac(SV) in healthy adult. METHODS: This is a prospective cohort study of adult aged 18-59 years who received 2-dose SV at 14-35 days apart for more than 2 months. Participants received ID AZD1222 at fractional low dose(1×1010 viral particles,0.1 ml). Antibody responses were evaluated by surrogate virus neutralization test(sVNT) against delta variant and wild type, and anti-spike-receptor-binding-domain immunoglobulin G(anti-S-RBD IgG) at prior, day14, 28, 90, and 180 post booster. Solicited reactogenicity was collected for 7 days post-booster. Primary endpoint was the differences of sVNT against delta strain ≥ 80% inhibition at day14 and 90 compared with the parallel cohort study of 0.5-ml intramuscular(IM) route. RESULTS: From August2021, 100 adults with median age of 46 years(IQR 41-52) participated. Prior to booster, geometric mean(GM) of sVNT against delta strain was 22.4% inhibition(95 %CI 18.7-26.9) and of anti-S-RBD IgG was 109.3 BAU/ml(95.4-125.1). Post ID booster, GMs of sVNT against delta strain were 95.5% inhibition (95%CI 94.2-96.8) at day14, 73.1% inhibition (66.7-80.2) at day90, and 22.7% inhibition (14.9-34.6) at day180. The differences of proportion of participants achieving sVNT against delta strain ≥ 80% inhibition in ID recipients versus IM were + 4.2% (95 %CI -2.0to10.5) at day14, and -37.3%(-54.2to-20.3) at day90. Anti-S-RBD IgG GMs were 2037.1 BAU/ml (95%CI 1770.9-2343.2) at day14 and 744.6 BAU/ml(650.1-852.9) at day90, respectively. Geometric mean ratios(GMRs) of anti-S-RBD IgG were 0.99(0.83-1.20) at day14, and 0.82(0.66-1.02) at day90. Only 18% reported feverish, compared with 37% of IM (p = 0.003). Common reactogenicity was erythema at injection site(53%) while 7% reported blister. CONCLUSION: Low-dose ID AZD1222 booster enhanced lower neutralizing antibodies at 3 months compared with IM route. Less systemic reactogenicity occurred, but higher local reactogenicity.