Profiling Humoral Immunity After Mixing and Matching COVID-19 Vaccines Using SARS-CoV-2 Variant Protein Microarrays.

In November 2022, 68% of the population received at least one dose of COVID-19 vaccines. Owing to the ongoing mutations, especially for the variants of concern (VOCs), it is important to monitor the humoral immune responses after different vaccination strategies. In this study, we developed a SARS-CoV-2 variant protein microarray that contained the spike proteins from the VOCs, e.g., alpha, beta, gamma, delta, and omicron, to quantify the binding antibody and surrogate neutralizing antibody. Plasmas were collected after two doses of matching AZD1222 (AZx2), two doses of matching mRNA-1273 (Mx2), or mixing AZD1222 and mRNA-1273 (AZ+M). The results showed a significant decrease of surrogate neutralizing antibodies against the receptor-binding domain in all VOCs in AZx2 and Mx2 but not AZ+M. A similar but minor reduction pattern of surrogate neutralizing antibodies against the extracellular domain was observed. While Mx2 exhibited a higher surrogate neutralizing level against all VOCs compared with AZx2, AZ+M showed an even higher surrogate neutralizing level in gamma and omicron compared with Mx2. It is worth noting that the binding antibody displayed a low correlation to the surrogate neutralizing antibody (R-square 0.130-0.382). This study delivers insights into humoral immunities, SARS-CoV-2 mutations, and mixing and matching vaccine strategies, which may provide a more effective vaccine strategy especially in preventing omicron.

Reverse-Phase Protein Microarrays for Overexpressed Escherichia coli Lysates Reveal a Novel Tyrosine Kinase.

Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.

Antibody Profiling of Dengue Severities Using Flavivirus Protein Microarrays.

Dengue is a viral disease transmitted by Aedes aegypti mosquitoes. According to the World Health Organization, about half of the world's population is at risk of dengue. There are four serotypes of the dengue virus. After infection with one serotype, it will be immune to such a serotype. However, subsequent infection with other serotypes will increase the risk of severe outcomes, e.g., dengue hemorrhagic fever, dengue shock syndrome, and even death. Since severe dengue is challenging to predict and lacks molecular markers, we aim to build a multiplexed Flavivirus protein microarray (Flaviarray) that includes all of the common Flaviviruses to profile the humoral immunity and cross-reactivity in the dengue patients with different outcomes. The Flaviarrays we fabricated contained 17 Flavivirus antigens with high reproducibility (R-square = 0.96) and low detection limits (172-214 pg). We collected serums from healthy subjects (n = 36) and dengue patients within 7 days after symptom onset (mild dengue (n = 21), hospitalized nonsevere dengue (n = 29), and severe dengue (n = 36)). After profiling the serum antibodies using Flaviarrays, we found that patients with severe dengue showed higher IgG levels against multiple Flavivirus antigens. With logistic regression, we found groups of markers with high performance in distinguishing dengue patients from healthy controls as well as hospitalized from mild cases (AUC > 0.9). We further reported some single markers that were suitable to separate dengue patients from healthy controls (AUC > 0.9) and hospitalized from mild outcomes (AUC > 0.8). Together, Flaviarray is a valuable tool to profile antibody specificities, uncover novel markers for decision-making, and shed some light on early preventions and treatments.

Profiling humoral responses to COVID-19 immunization in Kawasaki disease using SARS-CoV-2 variant protein microarrays.

Kawasaki disease (KD) is a form of acute systemic vasculitis syndrome that predominantly occurs in children under the age of 5 years. Its etiology has been postulated due to not only genetic factors but also the presence of foreign antigens or infectious agents. To evaluate possible associations between Kawasaki disease (KD) and COVID-19, we investigated humoral responses of KD patients against S-protein variants with SARS-CoV-2 variant protein microarrays. In this study, plasma from a cohort of KD (N = 90) and non-KD control (non-KD) (N = 69) subjects in categories of unvaccinated-uninfected (pre-pandemic), SARS-CoV-2 infected (10-100 days after infection), and 1-dose, 2-dose, and 3-dose BNT162b2 vaccinated (10-100 days after vaccination) was collected. The principal outcomes were non-KD-KD differences for each category in terms of anti-human/anti-His for binding antibodies and neutralizing percentage for surrogate neutralizing antibodies. Binding antibodies against spikes were lower in the KD subjects with 1-dose of BNT162b2, and mean differences were significant for the P.1 S-protein (non-KD-KD, 3401; 95% CI, 289.0 to 6512; P = 0.0252), B.1.617.2 S-protein (non-KD-KD, 4652; 95% CI, 215.8 to 9087; P = 0.0351) and B.1.617.3 S-protein (non-KD-KD, 4874; 95% CI, 31.41 to 9716; P = 0.0477). Neutralizing antibodies against spikes were higher in the KD subjects with 1-dose of BNT162b2, and mean percentage differences were significant for the 1-dose BNT162b2 B.1.617.3 S-protein (non-KD-KD, -22.89%; 95% CI, -45.08 to -0.6965; P = 0.0399), B.1.1.529 S-protein (non-KD-KD, -25.96%; 95% CI, -50.53 to -1.376; P = 0.0333), BA.2.12.1 S-protein (non-KD-KD, -27.83%; 95% CI, -52.55 to -3.115; P = 0.0195), BA.4 S-protein (non-KD-KD, -28.47%; 95% CI, -53.59 to -3.342; P = 0.0184), and BA.5 S-protein (non-KD-KD, -30.42%; 95% CI, -54.98 to -5.869; P = 0.0077). In conclusion, we have found that KD patients have a comparable immunization response to healthy individuals to SARS-CoV-2 infection and COVID-19 immunization.

Antibody Profiling in COVID-19 Patients with Different Severities by Using Spike Variant Protein Microarrays.

The disease progression of COVID-19 varies from mild to severe, even death. However, the link between COVID-19 severities and humoral immune specificities is not clear. Here, we developed a multiplexed spike variant protein microarray (SVPM) and utilized it for quantifying neutralizing activity, drug screening, and profiling humoral immunity. First, we demonstrated the competition between antispike antibody and ACE2 on SVPM for measuring the neutralizing activity against multiple spike variants. Next, we collected the serums from healthy subjects and COVID-19 patients with different severities and profile the neutralizing activity as well as antibody isotypes. We identified the inhibition of ACE2 binding was stronger against multiple variants in severe compared to mild/moderate or critical patients. Moreover, the serum IgG against nonstructural protein 3 was elevated in severe but not in mild/moderate and critical cases. Finally, we evaluated two ACE2 inhibitors, Ramipril and Perindopril, and found the dose-dependent inhibition of ACE2 binding to all the spike variants except for B.1.617.3. Together, the SVPM and the assay procedures provide a tool for profiling neutralizing antibodies, antibody isotypes, and reagent specificities.

Development and Application of Human Coronavirus Protein Microarray for Specificity Analysis.

Coronavirus is an enveloped RNA virus that causes mild to severe respiratory diseases in humans, including HKU1-CoV, 229E-CoV, NL63-CoV, OC43-CoV, SARS-CoV, MERS-CoV, and SARS-CoV-2. Due to the outbreak of SARS-CoV-2, it is important to identify the patients and investigate their immune responses. Protein microarray is one of the best platforms to profile the antibodies in the blood because of its fast, multiplexed, and sensitive nature. To fully understand the immune responses and biological specificities, this study developed a human coronavirus (HCoV) protein microarray and included all seven human coronaviruses and three influenza viruses. Each protein was printed in triplicate and formed 14 identical blocks per array. The HCoV protein microarray showed high reproducibility and sensitivity to the monoclonal antibodies against spike and nucleocapsid protein with detection limits of 10-200 pg. The HCoV proteins that were immobilized on the array were properly folded and functional by showing interactions with a known human receptor, e.g., ACE2. By profiling the serum IgG and IgA from 32 COVID-19 patients and 36 healthy patients, the HCoV protein microarray demonstrated 97% sensitivity and 97% specificity with two biomarkers. The results also showed the cross-reactivity of IgG and IgA in COVID-19 patients to spike proteins from various coronaviruses, including that from SARS-CoV, HKU1-CoV, and OC43-CoV. Finally, an innate immune protein named surfactant protein D showed broad affinities to spike proteins in all human coronaviruses. Overall, the HCoV protein microarray is multiplexed, sensitive, and specific, which is useful in diagnosis, immune assessment, biological development, and drug screening.

Rituximab, but not other biologics, impairs humoral immunity in patients with rheumatoid arthritis-a study using CoVariant protein arrays.

Objectives: RA is an autoimmune disease characterized by chronic inflammation and joint destruction. Biologics are crucial to achieving treat-to-target goals in patients with RA. The global spread and continuous variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitate the monitoring of variant-specific humoral responses post-vaccination. The aim of this study was to investigate how different biologic treatments for vaccinated RA patients might affect their neutralizing antibodies against multiple SARS-CoV-2 variants. Methods: We recruited RA patients who had received three doses of conventional SARS-CoV-2 vaccines and were treated with various biologics, e.g. TNF inhibitor (etanercept), IL-6 inhibitor (tocilizumab), CTLA4-Ig (abatacept) or anti-CD20 (rituximab). Serum samples were used to profile the binding and neutralizing antibodies using our own SARS-CoV-2 variant (CoVariant) protein array, developed previously. Results: Compared with healthy controls, only RA therapy with rituximab showed a reduction in neutralizing antibodies capable of targeting spike proteins in SARS-CoV-2 wild-type and most variants. This reduction was not observed in binding antibodies against SARS-CoV-2 wild-type or its variants. Conclusion: After receiving three doses of SARS-CoV-2 vaccination, RA patients who underwent rituximab treatment generated sufficient antibodies but exhibited lower neutralizing activities against wild-type and multiple variants, including current Omicron. Other biological DMARDs, e.g. TNF inhibitor, IL-6 inhibitor and CTLA4-Ig, did not show obvious inhibition.

Developing magnetic barcode bead fluorescence assay for high throughput analyzing humoral responses against multiple SARS-CoV-2 variants.

The continuous mutation of SARS-CoV-2 highlights the need for rapid, cost-effective, and high-throughput detection methods. To better analyze the antibody levels against SARS-CoV-2 and its variants in vaccinated or infected subjects, we developed a multiplex detection named Barcode Bead Fluorescence (BBF) assay. These barcode beads were magnetic, characterized by 2-dimensional edges, highly multiplexed, and could be decrypted with visible light. We conjugated 12 magnetic barcode beads with corresponding nine spike proteins (wild-type, alpha, beta, gamma, delta, and current omicrons), two nucleocapsid proteins (wild-type and omicron), and one negative control. First, the conjugated beads underwent serial quality controls via fluorescence labeling, e.g., reproducibility (R square = 0.99) and detection limits (119 pg via anti-spike antibody). Next, we investigated serums from vaccinated subjects and COVID-19 patients for clinical applications. A significant reduction of antibody levels against all variant beads was observed in both vaccinated and COVID-19 studies. Subjects with two doses of mRNA-1273 exhibited the highest level of antibodies against all spike variants compared to two doses of AZD1222 and unvaccinated. We also found that COVID-19 patients showed higher antibody levels against spike beads from wild-type, alpha, beta, and delta. Finally, the nucleocapsid beads served as markers to distinguish infections from vaccinated subjects. Overall, this study developed the BBF assay for analyzing humoral immune responses, which has the advantages of robustness, automation, scalability, and cost-effectiveness.

Development of SARS-CoV-2 variant protein microarray for profiling humoral immunity in vaccinated subjects.

SARS-CoV-2 is quickly evolving from wild-type to many variants and spreading around the globe. Since many people have been vaccinated with various types of vaccines, it is crucial to develop a high throughput platform for measuring the antibody responses and surrogate neutralizing activities against multiple SARS-CoV-2 variants. To meet this need, the present study developed a SARS-CoV-2 variant (CoVariant) array which consists of the extracellular domain of spike variants, e.g., wild-type, D614G, B.1.1.7, B.1.351, P.1, B.1.617, B.1.617.1, B.1.617.2, and B.1.617.3. A surrogate virus neutralization on the CoVariant array was established to quantify the bindings of antibody and host receptor ACE2 simultaneously to spike variants. By using a chimeric anti-spike antibody, we demonstrated a broad binding spectrum of antibodies while inhibiting the bindings of ACE2 to spike variants. To monitor the humoral immunities after vaccination, we collected serums from unvaccinated, partial, or fully vaccinated individuals with either mRNA-1273 or AZD1222 (ChAdOx1). The results showed partial vaccination increased the surrogate neutralization against all the mutants while full vaccination boosted the most. Although IgG, IgA, and IgM isotypes correlated with surrogate neutralizing activities, they behave differently throughout the vaccination processes. Overall, this study developed CoVariant arrays and assays for profiling the humoral responses which are useful for immune assessment, vaccine research, and drug development.

A Critical Review of Genome Editing and Synthetic Biology Applications in Metabolic Engineering of Microalgae and Cyanobacteria.

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi-omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value-added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.