Isolation of Anti-SARS-CoV-2 Natural Products Extracted from Mentha canadensis and the Semi-synthesis of Antiviral Derivatives.

Traditional herbal medicine offers opportunities to discover novel therapeutics against SARS-CoV-2 mutation. The dried aerial part of mint (Mentha canadensis L.) was chosen for bioactivity-guided extraction. Seven constituents were isolated and characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS). Syringic acid and methyl rosmarinate were evaluated in drug combination treatment. Ten amide derivatives of methyl rosmarinate were synthesized, and the dodecyl (13) and 3-ethylphenyl (19) derivatives demonstrated significant improvement in the anti-SARS-CoV-2 plaque reduction assay, achieving IC50 of 0.77 and 2.70 µM, respectively, against Omicron BA.1 as compared to methyl rosmarinate's IC50 of 57.0 µM. Spike protein binding and 3CLpro inhibition assays were performed to explore the viral inhibition mechanism. Molecular docking of compounds 13 and 19 to 3CLpro was performed to reveal potential interaction. In summary, natural products with anti-Omicron BA.1 activity were isolated from Mentha canadensis and derivatives of methyl rosmarinate were synthesized, showing 21- to 74-fold improvement in antiviral activity against Omicron BA.1.

The Src-ZNRF1 axis controls TLR3 trafficking and interferon responses to limit lung barrier damage.

Type I interferons are important antiviral cytokines, but prolonged interferon production is detrimental to the host. The TLR3-driven immune response is crucial for mammalian antiviral immunity, and its intracellular localization determines induction of type I interferons; however, the mechanism terminating TLR3 signaling remains obscure. Here, we show that the E3 ubiquitin ligase ZNRF1 controls TLR3 sorting into multivesicular bodies/lysosomes to terminate signaling and type I interferon production. Mechanistically, c-Src kinase activated by TLR3 engagement phosphorylates ZNRF1 at tyrosine 103, which mediates K63-linked ubiquitination of TLR3 at lysine 813 and promotes TLR3 lysosomal trafficking and degradation. ZNRF1-deficient mice and cells are resistant to infection by encephalomyocarditis virus and SARS-CoV-2 because of enhanced type I interferon production. However, Znrf1-/- mice have exacerbated lung barrier damage triggered by antiviral immunity, leading to enhanced susceptibility to respiratory bacterial superinfections. Our study highlights the c-Src-ZNRF1 axis as a negative feedback mechanism controlling TLR3 trafficking and the termination of TLR3 signaling.

SARS-CoV-2 infection among healthcare workers whom already received booster vaccination during epidemic outbreak of omicron variant in Taiwan.

BACKGROUND/PURPOSE: Healthcare workers (HCWs) are at risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection due to occupational exposure. We aim to investigate the prevalence and risk factors of SARS-CoV-2 infection among HCWs during epidemic outbreak of omicron variant in Taiwan. METHODS: Sequential reserved serum samples collected from our previous study during December 2021 and July 2022 were tested for antibodies against SARS-CoV-2 nucleocapsid protein (NP). Diagnosis of SARS-CoV-2 infection was defined as positive either of anti-SARS-CoV-2 nucleoprotein, rapid antigen test or polymerase chain reaction. Retrospective chart review and a questionnaire were used to access the symptoms and risk factors for SARS-CoV-2 infection. RESULTS: Totally 300 participants (69.3% female) with a median age of 37.9 years were enrolled. A significant increase incidence of SARS-CoV-2 infection was found before and during community outbreak (11.91 versus 230.93 per 100,000 person-days, P < 0.001), which was a trend paralleling that observed in the general population. For 61 SARS-CoV-2 infected participants, nine (14.8%) were asymptomatic. Multivariate analysis revealed recent contact with a SARS-CoV-2 infected household (odds ratio [OR], 7.01; 95% confidence interval [95% CI], 3.70-13.30; P < 0.001) and co-existed underlying autoimmune diseases (OR, 4.46; 95% CI, 1.28-15.51; P = 0.019) were significant risk factors associated with acquisition of SARS-CoV-2 infection among HCWs. CONCLUSION: Community factors, such as closely contact with SARS-CoV-2 infected individuals and underlying immune suppression status, were significant factors for acquisition of SARS-CoV-2 infection among HCWs. We suggest the application of appropriate infection control measures for HCWs should be maintained to reduce risk of SARS-CoV-2 infection.

Correlation of adverse effects and antibody responses following homologous and heterologous COVID19 prime-boost vaccinations.

BACKGROUND: Studies correlating reactogenicity and immunogenicity of COVID-19 vaccines are limited to BNT162b2, with inconsistent results. We investigated whether adverse reactions after other COVID-19 vaccines reliably predict humoral responses. METHODS: Adult volunteers were recruited for homologous or heterologous prime-boost vaccinations with adenoviral (ChAdOx1, AstraZeneca) and/or mRNA (mRNA-1273, Moderna) vaccines administered either 4 or 8 weeks apart. Adverse effects were routinely solicited and recorded by subjects in a standard diary card for up to 84 days post booster vaccination. Anti-SARS-CoV-2 IgG titers were measured pre- (visit 1), and post-booster dose at days 14 (visit 2) and 28 (visit 3). RESULTS: A total of 399 participants (75% women) with a median age of 41 (interquartile range, 33-48 IQR) years were included. Vaccine-induced antibody titers at days 14 and 28 were significantly higher among subjects who reported local erythema, swelling, pain, as well as systemic fever, chills, headache, myalgia, arthralgia, fatigue compared to those who did not experience local or systemic reactogenicity. Post-vaccination humoral responses did not correlate with the occurrence of skin rash and correlated weakly with gastrointestinal symptoms. A significant correlation between post-vaccination peak body temperature and anti-SARS-CoV-2 spike IgG at Day 14, independent of vaccine type and schedule, was found. CONCLUSION: Specific symptoms of reactogenicity such as post-vaccination injection site pain, swelling, erythema and fever, myalgia and fatigue are significantly predictive of the magnitude of the anti-SARS-CoV-2 antibody response.

Elicitation of potent neutralizing antibodies in obese mice by ISA 51-adjuvanted SARS-CoV-2 spike RBD-Fc vaccine.

Vaccination is considered to be the most effective countermeasure to prevent and combat the global health threats of COVID-19. People with obesity are at a greater risk of hospitalization, life-threatening illness, and adverse outcomes after having COVID-19. Therefore, a safe and effective COVID-19 vaccine for obese individuals is urgently needed. In the study, the vaccine composed of the ISA 51 adjuvant and the SARS-CoV-2 spike (S) receptor-binding domain (RBD) in conjugation with the human IgG1 Fc fragment (named as ISA 51-adjuvanted RBD-Fc vaccine) was developed and inoculated in the regular chow diet (RCD) lean mice and the high-fat diet (HFD)-induced obese mice. The S protein-specific IgG titers were largely induced in an increasing manner along with three doses of ISA 51-adjuvanted RBD-Fc vaccine without causing any harmful side effect. In the HFD mice, the S protein-specific IgG titers can be quickly observed 2 weeks post the first inoculation. The antisera elicited by the ISA 51-adjuvanted RBD-Fc vaccine in the RCD and HFD mice exhibited potent SARS-CoV-2 neutralizing activities in the plaque reduction neutralization test (PRNT) assays and showed similar specificity for recognizing the key residues in the RBD which were involved in interacting with angiotensin-converting enzyme 2 (ACE2) receptor. The immune efficacy of the ISA 51-adjuvanted RBD-Fc vaccine in the HFD mice can be sustainably maintained with the PRNT50 values of 1.80-1.91×10-3 for at least 8 weeks post the third inoculation. Collectively, the RBD-Fc-based immunogen and the ISA 51-adjuvanted formulation can be developed as an effective COVID-19 vaccine for obese individuals. KEY POINTS: • The ISA 51-adjuvanted RBD-Fc vaccine can induce potent SARS-CoV-2 neutralizing antibodies in the obese mouse • The antibodies elicited by the ISA 51-adjuvanted RBD-Fc vaccine can bind to the key RBD residues involved in interacting with ACE2 • The immune efficacy of the ISA 51-adjuvanted RBD-Fc vaccine can be sustainably maintained for at least 8 weeks post the third inoculation.

Factors associated with viral rebound among COVID-19 patients receiving oral antivirals.

BACKGROUND: COVID-19 rebound is usually reported among patients experiencing concurrent symptomatic and viral rebound. But longitudinal viral RT-PCR results from early stage to rebound of COVID-19 was less characterized. Further, identifying the factors associated with viral rebound after nirmatrelvir-ritonavir (NMV/r) and molnupiravir may expand understanding of COVID-19 rebound. METHODS: We retrospectively analyzed clinical data and sequential viral RT-PCR results from COVID-19 patients receiving oral antivirals between April and May, 2022. Viral rebound was defined by the degree of viral load increase (ΔCt ≥ 5 units). RESULTS: A total of 58 and 27 COVID-19 patients taking NMV/r and molnupiravir, respectively, were enrolled. Patients receiving NMV/r were younger, had fewer risk factors for disease progression and faster viral clearance rate compared to those receiving molnupiravr (All P < 0.05). The overall proportion of viral rebound (n = 11) was 12.9%, which was more common among patients receiving NMV/r (10 [17.2%] vs. 1 [3.7%], P = 0.16). Of them, 5 patients experienced symptomatic rebound, suggesting the proportion of COVID-19 rebound was 5.9%. The median interval to viral rebound was 5.0 (interquartile range, 2.0-8.0) days after completion of antivirals. Initial lymphopenia (<0.8 × 109/L) was associated with viral rebound among overall population (adjusted odds ratio [aOR], 5.34; 95% confidence interval [CI], 1.33-21.71), and remained significant (aOR, 4.50; 95% CI, 1.05-19.25) even when patients receiving NMV/r were considered. CONCLUSION: Our data suggest viral rebound after oral antivirals may be more commonly observed among lymphopenic individuals in the context of SARS-CoV-2 Omicron BA.2 variant.

Endo-Lysosomal Cation Channels and Infectious Diseases.

Among the infectious diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi, the most prevalent ones today are malaria, tuberculosis, influenza, HIV/AIDS, Ebola, dengue fever, and methicillin-resistant Staphylococcus aureus (MRSA) infection, and most recently Covid-19 (SARS-CoV2). Others with a rather devastating history and high fatality rates such as plague, cholera, or typhus seem less threatening today but have not been eradicated, and with a declining efficacy of current antibiotics they ought to be watched carefully. Another emerging issue in this context is health-care associated infection. About 100,000 hospitalized patients in the USA ( www.cdc.gov ) and 33,000 in Europe ( https://www.ecdc.europa.eu ) die each year as a direct consequence of an infection caused by bacteria resistant to antibiotics. Among viral infections, influenza is responsible for about 3-5 million cases of severe illness, and about 250,000 to 500,000 deaths annually ( www.who.int ). About 37 million people are currently living with HIV infection and about one million die from it each year. Coronaviruses such as MERS-CoV, SARS-CoV, but in particular the recent outbreak of Covid-19 (caused by SARS-CoV2) have resulted in large numbers of infections worldwide with an estimated several hundred thousand deaths (anticipated fatality rate: <5%). With a comparatively low mortality rate dengue virus causes between 50 and 100 million infections every year, leading to 50,000 deaths. In contrast, Ebola virus is the causative agent for one of the deadliest viral diseases. The Ebola outbreak in West Africa in 2014 is considered the largest outbreak in history with more than 11,000 deaths. Many of the deadliest pathogens such as Ebola virus, influenza virus, mycobacterium tuberculosis, dengue virus, and cholera exploit the endo-lysosomal trafficking system of host cells for penetration into the cytosol and replication. Defects in endo-lysosomal maturation, trafficking, fusion, or pH homeostasis can efficiently reduce the cytotoxicity caused by these pathogens. Most of these functions critically depend on endo-lysosomal membrane proteins such as transporters and ion channels. In particular, cation channels such as the mucolipins (TRPMLs) or the two-pore channels (TPCs) are involved in all of these aspects of endo-lysosomal integrity. In this review we will discuss the correlations between pathogen toxicity and endo-lysosomal cation channel function, and their potential as drug targets for infectious disease therapy.

Safety and antibody response of recipients who unexpectedly received undiluted prime dose of BNT162b2 COVID-19 vaccine in Taiwan.

We reported 25 recipients (14 females and 11 males) aged from 18 to 65 years who unexpectedly received a primary dose of undiluted BNT162b2 vaccine (180 µg). The most common adverse reactions included injection site pain (n = 22), followed by fever (9), fatigue (8), chest tightness (6), and dizziness (6). The most common laboratory abnormalities were anemia (n = 4) and elevated liver transaminase level (4), followed by abnormal leukocyte counts (3) and elevated D-dimer level (3). The adverse reactions and laboratory abnormalities of these recipients were mild and spontaneously recovered within a few weeks. Significant elevations of anti-SARS-CoV-2 spike IgG titers after a booster dose of the BNT162b2 were found. Similar to reports of BNT162b2 clinical trials, the adverse reactions and laboratory abnormalities of these recipients were mild, and they spontaneously recovered within a few weeks. These results provide clinical and immunological effects of undiluted BNT162b2 vaccine inoculation.

Immunogenicity and safety of third-dose mRNA COVID-19 vaccines in healthy adults previously vaccinated with two doses of the ChAdOx1 vaccine.

BACKGROUND/PURPOSE: The efficacy and safety of coronavirus disease 2019 (COVID-19) booster vaccines remain limited. We investigated the immunogenicity and adverse events of the third dose of mRNA vaccines in healthy adults. METHODS: Volunteers vaccinated with two doses of the adenoviral vaccine (ChAdOx1) 12 weeks before were administered with an mRNA COVID-19 vaccine. These were divided into three groups, full-dose mRNA-1273 (group 1); half-dose mRNA-1273 (group 2); and full-dose BNT-162b2 (group 3). Primary outcomes included serum anti-SARS-CoV-2 spike immunoglobulin G (IgG) titers and neutralizing antibody titers against B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron) variants. Secondary outcomes included the evaluation of humoral and cellular immunity and vaccine-associated adverse events after the boost. RESULTS: Totally 300 participants were recruited, and 298 participants were enrolled. For all three groups, an increase in anti-SARS-CoV-2 spike IgG geometric mean titers (30.12- to 71.80-fold) and neutralizing antibody titers against the alpha variant (69.80- to 173.23-folds), delta variant (132.69- to 324.63-folds), and omicron variant (135.36- to 222.37-folds) were observed on day 28. All groups showed robust T- and B-cell responses after boosting. Adverse events were overall mild and transient but with higher prevalence and severity in group 1 participants than in other groups. CONCLUSIONS: Third dose mRNA COVID-19 vaccines markedly enhanced cellular and humoral responses and were safe. Immunological responses and adverse events were higher in individuals receiving the full-dose mRNA-1273 vaccine, followed by a half-dose mRNA-1273 vaccine and BNT-162b2 vaccine.

Serological response and safety of heterologous ChAdOx1-nCoV-19/mRNA-1273 prime-boost vaccination with a twelve-week interval.

The appropriate interval between heterologous prime adenoviral vectored vaccination and boost mRNA vaccination remains unclear. We recruited 100 adult participants to receive a prime adenoviral vectored vaccine (ChAdOx1, AstraZeneca) and a boost mRNA vaccine (mRNA-1273, Moderna) 12 weeks apart and checked their serum SARS-CoV-2 anti-spike IgG titers and neutralizing antibody titers against B.1.1.7 (alpha) and B.1.617.2 (delta) variants on the 28th day after the boost dose. Results were compared with our previous study cohorts who received the same prime-boost vaccinations at 4- and 8-week intervals. Compared to other heterologous vaccination groups, the 12-week interval group had higher neutralizing antibody titers against SARS-CoV-2 variants than the 4-week interval group and was similar to the 8-week interval group at day 28. Adverse reactions after the boost dose were mild and transient. Our results support deploying viral vectored and mRNA vaccines in a flexible schedule with intervals from 8 to 12 weeks.

Serological response after COVID-19 mRNA-1273 booster dose in immunocompromised patients, Taiwan, July to August 2021.

BACKGROUND: Whether immunocompromising conditions affect the immunogenicity of COVID-19 booster vaccination remains a concern, which impedes the vaccination campaign in people most vulnerable to COVID-19-associated morbidity and mortality. We aimed to evaluate the effect of immune dysfunction on immunogenicity of homologous and heterologous prime-boost COVID-19 vaccination. METHODS: Between July and August, 2021, 399 participants were randomized to receive ChAdOx1/ChAdOx1 8 weeks apart, ChAdOx1/mRNA-1273 8 weeks apart, ChAdOx1/mRNA-1273 4 weeks apart, and mRNA-1273/mRNA-1273 4 weeks apart. The anti-SARS-CoV-2 spike IgG antibody titers on the day before booster vaccination and 4 weeks after booster vaccination were compared between participants with and without immunocompromising conditions. RESULTS: Among ChAdOx1-primed participants, a trend of lower anti-SARS-CoV-2 spike IgG titers before booster vaccination were found in participants with autoimmune diseases (geometric means, 34.76 vs. 84.25 binding antibody units [BAU]/mL, P = 0.173), compared to those without. Participants receiving immunosuppressants and/or immunomodulators had significant lower anti-SARS-CoV-2 spike IgG titers before booster vaccination than those without (geometric means, 36.39 vs. 83.84 BAU/mL; P = 0.001). Among mRNA-1273-boosted participants, anti-SARS-CoV-2 spike IgG titers 4 weeks after booster vaccination were similar across all the strata. Participants with autoimmune diseases and receiving immunosuppressants and/or immunomodulators, had numerically lower anti-SARS-CoV-2 spike IgG titers 4 weeks after booster vaccination compared to those without (geometric means, 1474.34 vs. 1923.23 and 1590.61 vs. 1918.38 BAU/mL; P > 0.05). CONCLUSION: The immunogenicity of prime vaccination with ChAdOx1 decreased by immune dysfunction, but enhanced after receiving boost vaccination with mRNA-1273. Our study results support the efficacy of mRNA-1273 booster dose among immunocompromised hosts.

Evolution of anti-SARS-CoV-2 spike protein titers after two-dose of COVID-19 vaccination among people living with HIV.

Background: A community COVID-19 outbreak caused by the B.1.1.7 SARS-CoV-2 variant occurred in Taiwan in May 2021. High-risk populations such as people living with HIV (PLWH) were recommended to receive two doses of COVID-19 vaccines. While SARS-CoV-2 vaccines have demonstrated promising results in general population, real-world information on the serological responses remains limited among PLWH. Methods: PLWH receiving the first dose of SARS-CoV-2 vaccine from 2020 to 2021 were enrolled. Determinations of anti-SARS-CoV-2 spike IgG titers were performed every one to three months, the third dose of the SARS-CoV-2 vaccine or confirmed SARS-CoV-2 infection. All serum samples were tested for anti-nucleocapsid antibody and those tested positive were excluded from analysis. Results: A total of 1189 PLWH were enrolled: 829 (69.7%) receiving two doses of the AZD1222 vaccine, 232 (19.5%) of the mRNA-1273 vaccine, and 128 (10.8%) of the BNT162b2 vaccine. At all time-points, PLWH receiving two doses of mRNA vaccines had consistently higher antibody levels than those receiving the AZD1222 vaccine (p <0.001 for all time-point comparisons). Factors associated with failure to achieve an anti-spike IgG titer >141 BAU/mL within 12 weeks, included type 2 diabetes mellitus (DM) (adjusted odds ratio [aOR], 2.24; 95% CI, 1.25-4), a CD4 T cell count <200 cells/mm3 upon receipt of the first dose of vaccination (aOR, 3.43; 95% CI, 1.31-9) and two homologous AZD1222 vaccinations (aOR, 16.85; 95%CI, 10.13-28). For those receiving two doses of mRNA vaccines, factors associated with failure to achieve an anti-spike IgG titer >899 BAU/mL within 12 weeks were a CD4 T cell count <200 cells/mm3 on first-dose vaccination (aOR, 3.95; 95% CI, 1.08-14.42) and dual BNT162b2 vaccination (aOR, 4.21; 95% CI, 2.57-6.89). Conclusions: Two doses of homologous mRNA vaccination achieved significantly higher serological responses than vaccination with AZD1222 among PLWH. Those with CD4 T cell counts <200 cells/mm3 and DM had consistently lower serological responses.

IFN-stimulated metabolite transporter ENT3 facilitates viral genome release.

An increasing amount of evidence emphasizes the role of metabolic reprogramming in immune cells to fight infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that the expression of equilibrative nucleoside transporter 3 (ENT3, encoded by solute carrier family 29 member 3, Slc29a3) is part of the innate immune response, which is rapidly upregulated upon pathogen invasion. The transcription of Slc29a3 is directly regulated by type I interferon-induced signaling, demonstrating that this metabolite transporter is an interferon-stimulated gene (ISG). Suprisingly, we unveil that several viruses, including SARS-CoV-2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of Slc29a3 expression is sufficient to significantly decrease viral replication in vitro and in vivo. Our study reveals that ENT3 is a pro-viral ISG co-opted by some viruses to gain a survival advantage.

Correction: Lee et al. The Synergistic Inhibition of Coronavirus Replication and Induced Cytokine Production by Ciclesonide and the Tylophorine-Based Compound Dbq33b. Pharmaceutics 2022, 14, 1511.

In the original publication [...].

Clinical assessment of SARS-CoV-2 infectivity by rapid antigen test compared with virus isolation.

Although real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) remains as a golden standard for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it can not be easily expanded to large-scaled screening during outbreaks, and the positive results do not necessarily correlate with infectious status of the identified subjects. In this study, the performance of Vstrip® RV2 COVID-19 Antigen Rapid Test (RAT) and its correlation with virus infectivity was examined by virus culture using 163 sequential respiratory specimens collected from 26 SARS-CoV-2 infected patients. When the presence of cytopathic effects (CPE) in cell culture was used as a reference method for virus infectivity, the sensitivity, specificity and accuracy of Vstrip® RV2 COVID-19 Antigen Rapid Test was 96.43%, 89.63%, and 90.8%, respectively. The highest Ct value was 27.7 for RdRp gene and 25.79 for E gene within CPE-positive samples, and the highest Ct value was 31.9 for RdRp gene and 29.1 for E gene within RAT positive samples. When the Ct values of specimens were below 25, the CPE and RAT results had high degree of consistency. We concluded that the RAT could be a great alternative method for determining the infectious potential of individuals with high viral load.

Simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens by a Western blot serological assay.

The nucleic acid test is still the standard assessment for the diagnosis of coronavirus disease 2019 (COVID-19), which is caused by human infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In addition to supporting the confirmation of disease cases, serological assays are used for the analysis of antibody status and epidemiological surveys. In this study, a single Western blot strip (WBS) coated with multiple Escherichia coli (E. coli)-expressed SARS-CoV-2 antigens was developed for comprehensive studies of antibody profiles in COVID-19 patient sera. The levels of specific antibodies directed to SARS-CoV-2 spike (S), S2, and nucleocapsid (N) proteins were gradually increased with the same tendency as the disease progressed after hospitalization. The signal readouts of S, S2, and N revealed by the multi-antigen-coated WBS (mWBS)-based serological assay (mWBS assay) also demonstrated a positive correlation with the SARS-CoV-2 neutralizing potency of the sera measured by the plaque reduction neutralization test (PRNT) assays. Surprisingly, the detection signals against the unstructured receptor-binding domain (RBD) purified from E. coli inclusion bodies were not observed, although the COVID-19 patient sera exhibited strong neutralizing potency in the PRNT assays, suggesting that the RBD-specific antibodies in patient sera mostly recognize the conformational epitopes. Furthermore, the mWBS assay identified a unique and major antigenic epitope at the residues 1148, 1149, 1152, 1155, and 1156 located within the 1127-1167 fragment of the S2 subunit, which was specifically recognized by the COVID-19 patient serum. The mWBS assay can be finished within 14-16 min by using the automatic platform of Western blotting by thin-film direct coating with suction (TDCS WB). Collectively, the mWBS assay can be applied for the analysis of antibody responses, prediction of the protective antibody status, and identification of the specific epitope. KEY POINTS: • A Western blot strip (WBS) coated with multiple SARS-CoV-2 antigens was developed for the serological assay. • The multi-antigen-coated WBS (mWBS) can be utilized for the simultaneous detection of antibody responses to multiple SARS-CoV-2 antigens. • The mWBS-based serological assay (mWBS assay) identified a unique epitope recognized by the COVID-19 patient serum.

Evolution of neutralizing antibodies and cross-activity against different variants of SARS-CoV-2 in patients recovering from COVID-19.

BACKGROUND: Patients recovering from COVID-19 may need vaccination against SARS-CoV-2 because acquired immunity from primary infection may wane, given the emergence of new SARS-CoV-2 variants. Understanding the trends of anti-spike IgG and neutralizing antibody titers in patients recovering from COVID-19 may inform the decision made on the appropriate interval between recovery and vaccination. METHODS: Participants aged 20 years or older and diagnosed with COVID-19 between January and December, 2020 were enrolled. Serum specimens were collected every three months from 10 days to 12 months after the onset of symptom for determinations of anti-spike IgG and neutralizing antibody titers against SARS-CoV-2 Wuhan strain with D614G mutation, alpha, gamma and delta variants. RESULTS: Of 19 participants, we found a decreasing trend of geometric mean titers of anti-spike IgG from 560.9 to 217 and 92 BAU/mL after a 4-month and a 7-month follow-up, respectively. The anti-spike IgG titers declined more quickly in the ten participants with severe or critical disease than the nine participants with only mild to moderate disease between one month and seven months after SARS-CoV-2 infection (-8.49 vs - 2.34-fold, p < 0.001). The neutralizing activity of the convalescent serum specimens collected from participants recovering from wild-type SARS-CoV-2 infection against different variants was lower, especially against the delta variants (p < 0.01 for each variant with Wuhan strain as reference). CONCLUSION: Acquired immunity from primary infection with SARS-CoV-2 waned within 4-7 months in COVID-19 patients, and neutralizing cross-activities against different SARS-CoV-2 variants were lower compared with those against wild-type strain.

Tag-Free SARS-CoV-2 Receptor Binding Domain (RBD), but Not C-Terminal Tagged SARS-CoV-2 RBD, Induces a Rapid and Potent Neutralizing Antibody Response.

Recombinant proteins are essential in the development of subunit vaccines. In the design of many recombinant proteins, polyhistidine residues are added to the N- or C-termini of target sequences to facilitate purification. However, whether the addition of tag residues influences the immunogenicity of proteins remains unknown. In this study, the tag-free SARS-CoV-2 RBD and His-tag SARS-CoV-2 RBD proteins were investigated to determine whether there were any differences in their receptor binding affinity and immunogenicity. The results showed that the tag-free RBD protein had a higher affinity for binding with hACE2 receptors than His-tag RBD proteins (EC50: 1.78 µM vs. 7.51 µM). On day 21 after primary immunization with the proteins, the serum ELISA titers of immunized mice were measured and found to be 1:1418 for those immunized with tag-free RBD and only 1:2.4 for His-tag RBD. Two weeks after the booster dose, tag-free-RBD-immunized mice demonstrated a significantly higher neutralizing titer of 1:369 compared with 1:7.9 for His-tag-RBD-immunized mice. Furthermore, neutralizing antibodies induced by tag-free RBD persisted for up to 5 months and demonstrated greater cross-neutralization of the SARS-CoV-2 Delta variant. Evidence from Western blotting showed that the serum of His-tag-RBD-immunized mice recognized irrelevant His-tag proteins. Collectively, we conclude that the addition of a polyhistidine tag on a recombinant protein, when used as a COVID-19 vaccine antigen, may significantly impair protein immunogenicity against SARS-CoV-2. Antibody responses induced were clearly more rapid and robust for the tag-free SARS-CoV-2 RBD than the His-tag SARS-CoV-2 RBD. These findings provide important information for the design of antigens used in the development of COVID-19 subunit vaccines.

Contrasting Patterns in the Early Stage of SARS-CoV-2 Evolution between Humans and Minks.

One of the unique features of SARS-CoV-2 is its apparent neutral evolution during the early pandemic (before February 2020). This contrasts with the preceding SARS-CoV epidemics, where viruses evolved adaptively. SARS-CoV-2 may exhibit a unique or adaptive feature which deviates from other coronaviruses. Alternatively, the virus may have been cryptically circulating in humans for a sufficient time to have acquired adaptive changes before the onset of the current pandemic. To test the scenarios above, we analyzed the SARS-CoV-2 sequences from minks (Neovision vision) and parental humans. In the early phase of the mink epidemic (April to May 2020), nonsynonymous to synonymous mutation ratio per site in the spike protein is 2.93, indicating a selection process favoring adaptive amino acid changes. Mutations in the spike protein were concentrated within its receptor-binding domain and receptor-binding motif. An excess of high-frequency derived variants produced by genetic hitchhiking was found during the middle (June to July 2020) and late phase I (August to September 2020) of the mink epidemic. In contrast, the site frequency spectra of early SARS-CoV-2 in humans only show an excess of low-frequency mutations, consistent with the recent outbreak of the virus. Strong positive selection in the mink SARS-CoV-2 implies that the virus may not be preadapted to a wide range of hosts and illustrates how a virus evolves to establish a continuous infection in a new host. Therefore, the lack of positive selection signal during the early pandemic in humans deserves further investigation.

Hepatitis C microelimination among people living with HIV in Taiwan.

To reach the WHO target of hepatitis C virus (HCV) elimination by 2025, Taiwan started to implement free-of-charge direct-acting antiviral (DAA) treatment programme in 2017. Evaluating the progress of HCV microelimination among people living with HIV (PLWH) is a critical step to identify the barriers to HCV elimination. PLWH seeking care at a major hospital designated for HIV care in Taiwan between January 2011 and December 2021 were retrospectively included. For PLWH with HCV-seropositive or HCV seroconversion during the study period, serial HCV RNA testing was performed using archived samples to confirm the presence of HCV viremia and estimate the prevalence and incidence of HCV viremia. Overall, 4199 PLWH contributed to a total of 27,258.75 person-years of follow-up (PYFU). With the reimbursement of DAAs and improvement of access to treatments, the prevalence of HCV viremia has declined from its peak of 6.21% (95% CI, 5.39-7.12%) in 2018 to 2.09% (95% CI, 1.60-2.77%) in 2021 (decline by 66.4% [95% CI, 55.4-74.7%]); the incidence has declined from 25.94 per 1000 PYFU (95% CI, 20.44-32.47) in 2019 to 12.15% per 1000 PYFU (95% CI, 8.14-17.44) (decline by 53.2% [95% CI, 27.3-70.6%]). However, the proportion of HCV reinfections continued to increase and accounted for 82.8% of incident HCV infections in 2021. We observed significant declines of HCV viremia among PLWH with the expansion of the DAA treatment programme in Taiwan. Further improvement of the access to DAA retreatments is warranted to achieve the goal of HCV microelimination.