Standardization of in-house anti-IgG and IgA ELISAs for the detection of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection.

Influenza-Like Symptom Incidence, Illness-Associated Expenses, and Economic Impact Among Healthcare Personnel in Thailand: A Prospective Observational Cohort Study (2020-2021).

OBJECTIVES: We estimated influenza-like symptom (ILS) incidence among healthcare personnel (HCP) in four hospitals and the economic impact due to ILS in the Thai HCP population during July 2020-June 2021 (Thailand's expected 2020 influenza season), which also coincided with the novel coronavirus disease 2019 pandemic. METHODS: We followed HCP, in a prospective observational cohort, weekly for ≥1 of: muscle pain, cough, runny nose/nasal congestion, sore throat, or difficulty breathing. We fitted population-averaged Poisson regression models to identify factors associated with acquiring ILS and to calculate ILS incidence. We applied epidemiologic parameters to Thailand's HCP population (227 349 persons) to estimate economic impact. RESULTS: Of 2184 participants, adjusted all-cause ILS incidence was 6.1 episodes per 100 person-years (95% confidence interval 3.4-10.9). Among Thailand's HCP population, 13 909 ILS episodes were estimated to occur annually and would result in US$235 135 economic loss. Controlling for study site and calendar month, factors associated with acquiring ≥1 ILS versus no ILS included being female, having asthma, and using personal protective equipment 'frequently, but not always'. CONCLUSIONS: All-cause ILS resulted in considerable economic loss among Thai HCP workforce. These findings underscore the importance of public health interventions to reduce the risk of acquiring ILS.

Antibody Responses to the SARS-CoV-2 Ancestral Strain and Omicron Variants in Moderna mRNA-1273 Vaccinated Active-Duty US Navy Sailors and Marines.

Omicron and its subvariants have steadily gained greater capability of immune escape compared to other variants of concern, resulting in an increased incidence of reinfections even among vaccinated individuals. We evaluated the antibody response to Omicron BA.1, BA.2, and BA.4/5 in US military members vaccinated with the primary 2-dose series of Moderna mRNA-1273 in a cross-sectional study. While nearly all vaccinated participants had sustained spike (S) IgG and neutralizing antibodies (ND50) to the ancestral strain, only 7.7% participants had detectable ND50 to Omicron BA.1 at 8 months postvaccination. The neutralizing antibody response to BA.2 and BA.5 was similarly reduced. The reduced antibody neutralization of Omicron correlated with the decreased antibody binding to the receptor-binding domain. The participants' seropositivity to the nuclear protein positively correlated with ND50. Our data emphasizes the need for continuous vigilance in monitoring for emerging variants and the need to identify potential alternative targets for vaccine design.

Simultaneous detection of omicron and other SARS-CoV-2 variants by multiplex PCR MassARRAY technology.

The rapid emergence of SARS-CoV-2 variants with high severity and transmutability adds further urgency for rapid and multiplex molecular testing to identify the variants. A nucleotide matrix-assisted laser-desorption-ionization time-of-flight mass spectrophotometry (MALDI-TOF MS)-based assay was developed (called point mutation array, PMA) to identify four major SARS-CoV-2 variants of concern (VOCs) including Alpha, Beta, Delta, and Omicron (namely PMA-ABDO) and differentiate Omicron subvariant (namely PMA-Omicron). PMA-ABDO and PMA-Omicron consist of 24 and 28 mutation sites of the spike gene. Both PMA panels specifically identified VOCs with as low as 10 viral copies/µl. The panel has shown a 100% concordant with the Next Generation Sequencing (NGS) results testing on 256 clinical specimens with real-time PCR cycle threshold (Ct) values less than 26. It showed a higher sensitivity over NGS; 25/28 samples were positive by PMA but not NGS in the clinical samples with PCR Ct higher than 26. Due to the mass of nucleotide used to differentiate between wild-type and mutation strains, the co-infection or recombination of multiple variants can be determined by the PMA method. This method is flexible in adding a new primer set to identify a new emerging mutation site among the current circulating VOCs and the turnaround time is less than 8 h. However, the spike gene sequencing or NGS retains the advantage of detecting newly emerged variants.

Anti-Arbovirus Antibodies Cross-React With Severe Acute Respiratory Syndrome Coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is found in regions where dengue (DENV) and chikungunya (CHIKV) viruses are endemic. Any serological cross-reactivity between DENV, CHIKV, and SARS-CoV-2 is significant as it could lead to misdiagnosis, increased severity, or cross-protection. This study examined the potential cross-reactivity of anti-DENV and CHIKV antibodies with SARS-CoV-2 using acute and convalescent-phase samples collected before the SARS-CoV-2 pandemic. These included healthy, normal human (NHS, n = 6), CHIKV-positive (n = 14 pairs acute and convalescent), primary DENV-positive (n = 20 pairs), secondary DENV-positive (n = 20 pairs), and other febrile illnesses sera (n = 23 pairs). Samples were tested using an in-house SARS-CoV-2 and a EUROIMMUN IgA and IgG ELISAs. All NHS samples were negative, whereas 3.6% CHIKV, 21.7% primary DENV, 15.7% secondary DENV, and 10.8% febrile diseases sera resulted as anti-SARS-CoV-2 antibody positive. The EUROIMMUN ELISA using spike 1 as the antigen detected more positives among the primary DENV infections than the in-house ELISA using spike 1-receptor binding domain (RBD) protein. Among ELISA-positive samples, four had detectable neutralizing antibodies against SARS-CoV-2 reporter virus particles yet none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated the SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. IMPORTANCE SARS-CoV-2 continues to cause significant morbidity globally, including in areas where DENV and CHIKV are endemic. Reports using rapid diagnostic and ELISAs have demonstrated that serological cross-reactivity between DENV and SARS-CoV-2 can occur. Furthermore, it has been observed that convalescent DENV patients are at a lower risk of developing COVID-19. This phenomenon can interfere with the accuracy of serological testing and clinical management of both DENV and COVID-19 patients. In this study, the cross-reactivity of primary/secondary anti-DENV, CHIKV, and other febrile illness antibodies with SARS-CoV-2 using two ELISAs has been shown. Among ELISA-positive samples, four had detectable levels of neutralizing antibodies against SARS-CoV-2 reporter virus particles. However, none had detectable neutralizing antibodies against the live Wuhan strain of SARS-CoV-2. These data demonstrated SARS-CoV-2 diagnostic cross-reactivity, but not neutralizing antibody cross-reactivity, among dengue seropositive cases. The data discussed here provide information regarding diagnosis and may help guide appropriate public health interventions.

Homologous or Heterologous COVID-19 Booster Regimens Significantly Impact Sero-Neutralization of SARS-CoV-2 Virus and Its Variants.

We determined the levels of neutralizing antibodies against the SARS-CoV-2 ancestral strain, Delta and Omicron variants of concern (VOCs), in 125 healthcare workers who received CoronaVac as their primary vaccination and later received either a single ChAdOx1 or a combi-nation of two consecutive boosters using either two ChAdOx1 doses or a ChAdOx1 or BNT162b2 as the primary and second boosters, respectively, or two doses of BNT162b2. The titers 12 weeks after primary vaccination were inadequate to neutralize all strains. After a single ChAdOx1 booster, the levels of neutralization at Day 30 varied significantly, with only a small proportion of participants developing neutralizing titers against Omicron at Day 7 and 30. The two doses of ChAdOx1 as the booster induced the lowest activity. A combination ChAdOx1 and BNT162b2 induced greater neutralization than by two doses of ChAdOx1. Two doses of BNT162b2 as the booster had the maximal activity against Omicron VOC.

Chimeric Virus-like Particle-Based COVID-19 Vaccine Confers Strong Protection against SARS-CoV-2 Viremia in K18-hACE2 Mice.

Virus-like particles (VLPs) are highly immunogenic and versatile subunit vaccines composed of multimeric viral proteins that mimic the whole virus but lack genetic material. Due to the lack of infectivity, VLPs are being developed as safe and effective vaccines against various infectious diseases. In this study, we generated a chimeric VLP-based COVID-19 vaccine stably produced by HEK293T cells. The chimeric VLPs contain the influenza virus A matrix (M1) proteins and the SARS-CoV-2 Wuhan strain spike (S) proteins with a deletion of the polybasic furin cleavage motif and a replacement of the transmembrane and cytoplasmic tail with that of the influenza virus hemagglutinin (HA). These resulting chimeric S-M1 VLPs, displaying S and M1, were observed to be enveloped particles that are heterogeneous in shape and size. The intramuscular vaccination of BALB/c mice in a prime-boost regimen elicited high titers of S-specific IgG and neutralizing antibodies. After immunization and a challenge with SARS-CoV-2 in K18-hACE2 mice, the S-M1 VLP vaccination resulted in a drastic reduction in viremia, as well as a decreased viral load in the lungs and improved survival rates compared to the control mice. Balanced Th1 and Th2 responses of activated S-specific T-cells were observed. Moderate degrees of inflammation and viral RNA in the lungs and brains were observed in the vaccinated group; however, brain lesion scores were less than in the PBS control. Overall, we demonstrate the immunogenicity of a chimeric VLP-based COVID-19 vaccine which confers strong protection against SARS-CoV-2 viremia in mice.

Anti-SARS-CoV-2 IgG antibody levels among Thai healthcare providers receiving homologous and heterologous COVID-19 vaccination regimens.

BACKGROUND: We examined SARS-CoV-2 anti-spike 1 IgG antibody levels following COVID-19 vaccination (AstraZeneca [AZ], Sinovac [SV], Pfizer-BioNTech [PZ]) among Thai healthcare providers. METHODS: Blood specimens were tested using enzyme-linked immunosorbent assay. We analyzed seven vaccination regimens: (1) one dose of AZ or SV, (2) two doses of homologous (2AZ, 2SV) or heterologous (1AZ + 1PZ) vaccines, and (3) three doses of heterologous vaccines (2SV + 1AZ, 2SV + 1PZ). Differences in antibody levels were assessed using Kruskal-Wallis statistic, Mann-Whitney test, or Wilcoxon matched-pairs signed-rank test. Antibody kinetics were predicted using fractional polynomial regression. RESULTS: The 563 participants had median age of 39 years; 92% were female; 74% reported no underlying medical condition. Antibody levels peaked at 22-23 days in both 1AZ and 2SV vaccinees and dropped below assay's cutoff for positive (35.2 binding antibody units/ml [BAU/ml]) in 55 days among 1AZ vaccinees compared with 117 days among 2SV vaccinees. 1AZ + 1PZ vaccination regimen was highly immunogenic (median 2279 BAU/ml) 1-4 weeks post vaccination. 2SV + 1PZ vaccinees had significantly higher antibody levels than 2SV + 1AZ vaccinees 4 weeks post vaccination (3423 vs. 2105 BAU/ml; p-value < 0.01), and during weeks 5-8 (3656 vs. 1072 BAU/ml; p-value < 0.01). Antibodies peaked at 12-15 days in both 2SV + 1PZ and 2SV + 1AZ vaccinees, but those of 2SV + 1AZ declined more rapidly and dropped below assay's cutoff in 228 days while those of 2SV + 1PZ remained detectable. CONCLUSIONS: 1AZ + 1PZ, 2SV + 1AZ, and 2SV + 1PZ vaccinees had substantial IgG levels, suggesting that these individuals likely mounted sufficient anti-S1 IgG antibodies for possible protection against SARS-CoV-2 infection.

Coding-Complete Genome Sequences of Alpha and Delta SARS-CoV-2 Variants from Kamphaeng Phet Province, Thailand, from May to July 2021.

We report coding-complete genome sequences of 44 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains of the alpha and delta variants identified from patients in Kamphaeng Phet, Thailand. Two nonsense mutations in open reading frame 3a (ORF3a) (G254*) and ORF8 (K68*) were found in the alpha variant sequences. Two lineages of the delta variant, B.1.617.2 and AY.30, were found.

Limited and Short-Lasting Virus Neutralizing Titers Induced by Inactivated SARS-CoV-2 Vaccine.

In vitro determination of severe acute respiratory syndrome coronavirus 2 neutralizing antibodies induced in serum samples from recipients of the CoronaVac vaccine showed a short protection period against the original virus strain and limited protection against variants of concern. These data provide support for vaccine boosters, especially variants of concern circulate.

Genomic surveillance of SARS-CoV-2 in Thailand reveals mixed imported populations, a local lineage expansion and a virus with truncated ORF7a.

Coronavirus Disease 2019 (COVID-19) is a global public health threat. Genomic surveillance of SARS-CoV-2 was implemented in March of 2020 at a major diagnostic hub in Bangkok, Thailand. Several virus lineages supposedly originated in many countries were found, and a Thai-specific lineage, designated A/Thai-1, has expanded to be predominant in Thailand. A virus sample in the SARS-CoV-2 A/Thai-1 lineage contains a frame-shift deletion at ORF7a, encoding a putative host antagonizing factor of the virus.