Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19.

It has been revealed that SARS-CoV-2 can be efficiently isolated from clinical specimens such as nasal/nasopharyngeal swabs or saliva in cultured cells. In this study, we examined the efficiency of viral isolation including SARS-CoV-2 mutant strains between nasal/nasopharyngeal swab or saliva specimens. Furthermore, we also examined the comparison of viral isolation rates by sample species using simulated specimens for COVID-19. As a result, it was found that the isolation efficiency of SARS-CoV-2 in the saliva specimens was significantly lower than that in the nasal/nasopharyngeal swab specimens. In order to determine which component of saliva is responsible for the lower isolation rate of saliva specimens, we tested the abilities of lactoferrin, amylase, cathelicidin, and mucin, which are considered to be abundant in saliva, to inhibit the infection of SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Lactoferrin and amylase were found to inhibit SARS-CoV-2pv infection. In conclusion, even if the same number of viral genome copies was detected by the real-time RT-PCR test, infection of SARS-CoV-2 present in saliva is thought to be inhibited by inhibitory factors such as lactoferrin and amylase, compared to nasal/nasopharyngeal swab specimens.

Neutralizing antibody levels and epidemiological information of patients with breakthrough COVID-19 infection in Toyama, Japan.

Breakthrough infection (BI) after coronavirus disease 2019 (COVID-19) vaccination has exploded owing to the emergence of various SARS-CoV-2 variants and has become a major problem at present. In this study, we analyzed the epidemiological information and possession status of neutralizing antibodies in patients with BI using SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Analysis of 44 specimens diagnosed with COVID-19 after two or more vaccinations showed high inhibition of infection by 90% or more against the Wuhan strain and the Alpha and Delta variants of pseudotyped viruses in 40 specimens. In contrast, almost no neutralizing activity was observed against the Omicron BA.1 variant. Many cases without neutralizing activity or BI were immunosuppressed individuals. The results of this study show that contact with an infected person can result in BI even when there are sufficient neutralizing antibodies in the blood. Thus, even after vaccination, sufficient precautions must be taken to prevent infection.

Neutralization of Omicron subvariants BA.1 and BA.5 by a booster dose of COVID-19 mRNA vaccine in a Japanese nursing home cohort

The sustained epidemic of Omicron subvariants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide concern, and older adults are at high risk. We conducted a prospective cohort study to assess the immunogenicity of COVID-19 mRNA vaccines (BNT162b2 or mRNA-1273) in nursing home residents and staff between May 2021 and December 2022. A total of 335 SARS-CoV-2 naïve individuals, including 141 residents (median age: 88 years) and 194 staff (median age: 44 years) participated. Receptor-binding domain (RBD) and nucleocapsid (N) protein IgG and neutralizing titer (NT) against the Wuhan strain, Alpha and Delta variants, and Omicron BA.1 and BA.5 subvariants were measured in serum samples drawn from participants after the second and third doses of mRNA vaccine using SARS-CoV-2 pseudotyped virus. Breakthrough infection (BTI) was confirmed by a notification of COVID-19 or a positive anti-N IgG result in serum after mRNA vaccination. Fifty-one participants experienced SARS-CoV-2 BTI during the study period. The RBD IgG and NTs against Omicron BA.1 and BA.5 were markedly increased in SARS CoV-2 naïve participants 2 months after the third dose of mRNA vaccine, compared to those 5 months after the second dose, and declined 5 months after the third dose. The decline in RBD IgG and NT against Omicron BA.1 and BA.5 in SARS-CoV-2 naïve participants after the second and the third dose was particularly marked in those aged ≥ 80 years. BTIs during the BA.5 epidemic period, which occurred between 2 and 5 months after the third dose, induced a robust NT against BA.5 even five months after the booster dose vaccination. Further studies are required to assess the sustainability of NTs elicited by Omicron-containing bivalent mRNA booster vaccine in older adults.

Neutralizing Antibody Response of the Wild-Type/Omicron BA.1 Bivalent Vaccine as the Second Booster Dose against Omicron BA.2 and BA.5.

In addition to the original monovalent vaccines available for SARS-CoV-2, bivalent vaccines covering wild-type (WT) and Omicron BA.1 are also available. However, there is a lack of real-world data on the immunogenicity of bivalent vaccines as second boosters against the dominant Omicron sublineages, including BA.2 and BA.5. Healthcare workers (n = 565) who received the first booster vaccination were followed for 2 weeks after the second booster dose of the monovalent mRNA-1273 (WT group, n = 168) and bivalent BNT162b2 (WT+BA.1 group, n = 23) vaccines. Participants with previous SARS-CoV-2 infections were excluded from the study. The anti-receptor binding domain (RBD) antibody levels after the second booster dose in the WT and WT+BA.1 group were similar (median [interquartile range], 26,262.0 [16,951.0 to 38,137.0] U/mL versus 24,840.0 [14,828.0 to 41,460.0] U/mL, respectively). Although the neutralization activities of the pooled sera were lower against BA.5 than against other variants in both groups, the activities against BA.2 and BA.5 in the WT+BA.1 group were higher than those of the WT group in both pseudotyped and live virus assays. Vaccine-related symptoms, including systemic and local symptoms, were strongly correlated with anti-RBD antibody levels and neutralizing titers. In conclusion, the second booster dose of the bivalent (WT/Omicron BA.1) vaccine induced higher neutralizing activity against BA.2 and BA.5 than that of the original monovalent vaccine. IMPORTANCE Although Omicron BA.1-containing bivalent vaccines have been authorized, real-world data validating their safety and antibody responses remain scarce. We conducted a prospective longitudinal study to assess the safety, immunogenicity, and reactogenicity of the second booster dose with the Omicron BA.1 bivalent vaccine in health care workers. Compared with the original monovalent vaccine, the bivalent (WT+BA.1) vaccine elicited higher levels of neutralizing antibodies against the Omicron BA.2 and BA.5 subvariants. The frequency of adverse events after the second booster dose was similar to that of the monovalent vaccine. BA.5-neutralizing antibodies induced by the bivalent Omicron BA.1-containing vaccine were expected to decline. A prospective longitudinal study should be performed to determine the persistence of the humoral immunity.

Neutralization of Omicron subvariants BA.1 and BA.5 by a booster dose of COVID-19 mRNA vaccine in a Japanese nursing home cohort.

The sustained epidemic of Omicron subvariants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide concern, and older adults are at high risk. We conducted a prospective cohort study to assess the immunogenicity of COVID-19 mRNA vaccines (BNT162b2 or mRNA-1273) in nursing home residents and staff between May 2021 and December 2022. A total of 335 SARS-CoV-2 naïve individuals, including 141 residents (median age: 88 years) and 194 staff (median age: 44 years) participated. Receptor-binding domain (RBD) and nucleocapsid (N) protein IgG and neutralizing titer (NT) against the Wuhan strain, Alpha and Delta variants, and Omicron BA.1 and BA.5 subvariants were measured in serum samples drawn from participants after the second and third doses of mRNA vaccine using SARS-CoV-2 pseudotyped virus. Breakthrough infection (BTI) was confirmed by a notification of COVID-19 or a positive anti-N IgG result in serum after mRNA vaccination. Fifty-one participants experienced SARS-CoV-2 BTI during the study period. The RBD IgG and NTs against Omicron BA.1 and BA.5 were markedly increased in SARS CoV-2 naïve participants 2 months after the third dose of mRNA vaccine, compared to those 5 months after the second dose, and declined 5 months after the third dose. The decline in RBD IgG and NT against Omicron BA.1 and BA.5 in SARS-CoV-2 naïve participants after the second and the third dose was particularly marked in those aged ≥ 80 years. BTIs during the BA.5 epidemic period, which occurred between 2 and 5 months after the third dose, induced a robust NT against BA.5 even five months after the booster dose vaccination. Further studies are required to assess the sustainability of NTs elicited by Omicron-containing bivalent mRNA booster vaccine in older adults.

A novel hamster model of SARS-CoV-2 respiratory infection using a pseudotyped virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a biosafety level (BSL)-3 pathogen; therefore, its research environment is limited. Pseudotyped viruses that mimic the infection of SARS-CoV-2 have been widely used for in vitro evaluation because they are available in BSL-2 containment laboratories. However, in vivo application is inadequate. Therefore, animal models instigated with animal BSL-2 will provide opportunities for in vivo evaluation. Hamsters (6-10-week-old males) were intratracheally inoculated with luciferase-expressing vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus. The lungs were harvested 24-72 h after inoculation and luminescence was measured using an in vivo imaging system. Lung luminescence after inoculation with the SARS-CoV-2 pseudotyped virus increased in a dose-dependent manner and peaked at 48 h. The VSV-G (envelope G) pseudotyped virus also induced luminescence; however, a 100-fold concentration was required to reach a level similar to that of the SARS-CoV-2 pseudotyped virus. The SARS-CoV-2 pseudotyped virus is applicable to SARS-CoV-2 respiratory infections in a hamster model. Because of the single-round infectious virus, the model can be used to study the steps from viral binding to entry, which will be useful for future research on SARS-CoV-2 entry without using live SARS-CoV-2 or transgenic animals.

Effectiveness of the third dose of BNT162b2 vaccine on neutralizing Omicron variant in the Japanese population.

INTRODUCTION: The vaccine against SARS-CoV-2 provides humoral immunity to fight COVID-19; however, the acquired immunity gradually declines. Booster vaccination restores reduced humoral immunity; however, its effect on newly emerging variants, such as the Omicron variant, is a concern. As the waves of COVID-19 cases and vaccine programs differ between countries, it is necessary to know the domestic effect of the booster. METHODS: Serum samples were obtained from healthcare workers (20-69 years old) in the Pfizer BNT162b2 vaccine program at the Toyama University Hospital 6 months after the second dose (6mA2D, n = 648) and 2 weeks after the third dose (2wA3D, n = 565). The anti-SARS-CoV-2 antibody level was measured, and neutralization against the wild-type and variants (Delta and Omicron) was evaluated using pseudotyped viruses. Data on booster-related events were collected using questionnaires. RESULTS: The median anti-SARS-CoV-2 antibody was >30.9-fold elevated after the booster (6mA2D, 710.0 U/mL [interquartile range (IQR): 443.0-1068.0 U/mL]; 2wA3D, 21927 U/mL [IQR: 15321.0->25000.0 U/mL]). Median neutralizing activity using 100-fold sera against wild-type-, Delta-, and Omicron-derived variants was elevated from 84.6%, 36.2%, and 31.2% at 6mA2D to >99.9%, 99.1%, and 94.6% at 2wA3D, respectively. The anti-SARS-CoV-2 antibody levels were significantly elevated in individuals with fever ≥37.5 °C, general fatigue, and myalgia, local swelling, and local hardness. CONCLUSION: The booster effect, especially against the Omicron variant, was observed in the Japanese population. These findings contribute to the precise understanding of the efficacy and side effects of the booster and the promotion of vaccine campaigns.

Bovine lactoferrin increases the poly(I:C)-induced antiviral response in vitro.

Bovine lactoferrin (bLF) is a naturally occurring glycoprotein with antibacterial and antiviral activities. We evaluated whether bLF can prevent viral infections in the human intestinal epithelial cell line Caco-2. To assess antiviral responses, we measured the levels of interferon (IFN) expression, IFN-stimulated gene expression, and infection with a pseudotyped virus bearing either severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein or vesicular stomatitis virus (VSV)-G protein after treatment of cells with both bLF and polyinosinic-polycytidylic acid, an analog of double-stranded RNA that mimics viral infection. Combination treatment of cells with both bLF and polyinosinic-polycytidylic acid increased mRNA and protein expression of several IFN genes (IFNB, IFNL1, and IFNL2) and IFN-stimulated genes (ISG15, MX1, IFITM1, and IFITM3) in Caco-2 cells. However, treatment with bLF alone did not induce an antiviral response. Furthermore, combination treatment suppressed infection of the SARS-CoV-2 pseudotyped virus more efficiently than did bLF treatment alone, even though combination treatment increased the expression of mRNA encoding ACE2. These results indicate that bLF increases the antiviral response associated with the double-stranded RNA-stimulated signaling pathway. Our results also suggest that bLF and double-stranded RNA analogs can be used to treat viral infections, including those caused by SARS-CoV-2.

Effectiveness of the third dose of BNT162b2 vaccine on neutralizing Omicron variant in the Japanese population☆

Introduction The vaccine against SARS-CoV-2 provides humoral immunity to fight COVID-19;however, the acquired immunity gradually declines. Booster vaccination restores reduced humoral immunity;however, its effect on newly emerging variants, such as the Omicron variant, is a concern. As the waves of COVID-19 cases and vaccine programs differ between countries, it is necessary to know the domestic effect of the booster. Methods Serum samples were obtained from healthcare workers (20–69 years old) in the Pfizer BNT162b2 vaccine program at the Toyama University Hospital 6 months after the second dose (6mA2D, n = 648) and 2 weeks after the third dose (2wA3D, n = 565). The anti-SARS-CoV-2 antibody level was measured, and neutralization against the wild-type and variants (Delta and Omicron) was evaluated using pseudotyped viruses. Data on booster-related events were collected using questionnaires. Results The median anti-SARS-CoV-2 antibody was >30.9-fold elevated after the booster (6mA2D, 710.0 U/mL [interquartile range (IQR): 443.0–1068.0 U/mL];2wA3D, 21927 U/mL [IQR: 15321.0–>25000.0 U/mL]). Median neutralizing activity using 100-fold sera against wild-type-, Delta-, and Omicron-derived variants was elevated from 84.6%, 36.2%, and 31.2% at 6mA2D to >99.9%, 99.1%, and 94.6% at 2wA3D, respectively. The anti-SARS-CoV-2 antibody levels were significantly elevated in individuals with fever ≥37.5 °C, general fatigue, and myalgia, local swelling, and local hardness. Conclusion The booster effect, especially against the Omicron variant, was observed in the Japanese population. These findings contribute to the precise understanding of the efficacy and side effects of the booster and the promotion of vaccine campaigns.

Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants.

Many potent neutralizing SARS-CoV-2 antibodies have been developed and used for therapies. However, the effectiveness of many antibodies has been reduced against recently emerging SARS-CoV-2 variants, especially the Omicron variant. We identified a highly potent SARS-CoV-2 neutralizing antibody, UT28K, in COVID-19 convalescent individuals who recovered from a severe condition. UT28K showed efficacy in neutralizing SARS-CoV-2 in an in vitro assay and in vivo prophylactic treatment, and the reactivity to the Omicron strain was reduced. The structural analyses revealed that antibody UT28K Fab and SARS-CoV-2 RBD protein interactions were mainly chain-dominated antigen-antibody interactions. In addition, a mutation analysis suggested that the emergence of a UT28K neutralization-resistant SARS-CoV-2 variant was unlikely, as this variant would likely lose its competitive advantage over circulating SARS-CoV-2. Our data suggest that UT28K offers potent protection against SARS-CoV-2, including newly emerging variants.

Age-Dependent Reduction in Neutralization against Alpha and Beta Variants of BNT162b2 SARS-CoV-2 Vaccine-Induced Immunity.

Vaccines against severe acute respiratory syndrome coronavirus-2 have been introduced. To investigate the relationship between vaccine-induced humoral immunity and patient age, we measured antibody levels and neutralization in vaccinated sera. Sera from 13 to 17 days after the second dose of the BNT162b2 vaccine were collected from health care workers at the University of Toyama (n = 740). Antibody levels were measured by the anti-receptor binding domain antibody test (anti-RBD test), and neutralization against wild-type (WT), α- and ß-variant pseudotyped viruses were assayed using a high-throughput chemiluminescent reduction neutralizing test (htCRNT; positivity cutoff, 50% neutralization at serum dilution 1:100). Basic clinical characteristics were obtained from questionnaires. Antibodies were confirmed in all participants in both the anti-RBD test (median, 2,112 U/ml; interquartile range [IQR], 1,275 to 3,390 U/ml) and the htCRNT against WT (median % inhibition, >99.9; IQR, >99.9 to >99.9). For randomly selected sera (n = 61), 100.0% had positive htCRNT values against the α- and ß-derived variants. Among those who answered the questionnaire (n = 237), the values of the anti-RBD test were negatively correlated with age in females (P < 0.01). An age-dependent decline in neutralization was observed against the variants but not against the wild-type virus (wild type, P = 0.09; α, P < 0.01; ß, P < 0.01). The neutralizing activity induced by BNT162b2 was obtained not only against the wild-type virus, but also against the variants; however, there was an age-dependent decrease in the latter. Age-related heterogeneity of vaccine-acquired immunity is a concern in preventive strategies in the era dominated by variants. IMPORTANCE Since mRNA vaccines utilize wild-type SARS-CoV-2 spike protein as an antigen, there are potential concerns about acquiring immunity to variants of this virus. The neutralizing activity in BNT162b2-vaccinated individuals was higher against the wild-type virus than against its variants; this effect was more apparent in older age groups. This finding suggests that one of the weaknesses of the mRNA vaccine is the high risk of variant infection in the elderly population. Because the elderly are at a higher risk of SARS-CoV-2 infection, the age-dependent decline of neutralization against viral variants should be considered while planning vaccination programs that include boosters.

Correlation of the Commercial Anti-SARS-CoV-2 Receptor Binding Domain Antibody Test with the Chemiluminescent Reduction Neutralizing Test and Possible Detection of Antibodies to Emerging Variants.

Serological tests are beneficial for recognizing the immune response against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT) is critical. Whether commercial antibody tests have comparable accuracy is unknown. Serum samples were obtained from COVID-19 patients (n = 74), SARS-CoV-2 PCR-negative (n = 179), and suspected healthy individuals (n = 229) before SARS-CoV-2 variants had been detected locally. The convalescent phase was defined as the period after day 10 from disease onset or the episode of close contact. The CRNT using pseudotyped viruses displaying the wild-type (WT) spike protein and a commercial anti-receptor-binding domain (RBD) antibody test were assayed. Serology for the B.1.1.7 and B.1.351 variants was also assayed. Both tests concurred for symptomatic COVID-19 patients in the convalescent phase. They clearly differentiated between patients and suspected healthy individuals (sensitivity: 95.8% and 100%, respectively; specificity: 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titers (r = 0.31, 95% confidence interval [CI] 0.22-0.38). Compared with the WT, lower CRNT values were observed for the variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the B.1.1.7 and the B.1.351 variants, respectively. Exceeding 100 U/mL in the anti-RBD antibody test was associated with neutralization of variants (P < 0.01). The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. Strong positive results with the anti-RBD antibody test can reflect neutralizing activity against emerging variants. IMPORTANCE This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues. The correlation of the quantitative anti-receptor-binding domain antibody test, which is widely available, with neutralizing test indicates that we can know indirectly the state of acquisition of functional immunity against wild and variant-type viruses in the clinical laboratory.

Viral isolation analysis of SARS-CoV-2 from clinical specimens of COVID-19 patients.

Genetic testing using reverse transcriptase real-time polymerase chain reaction (rRT-PCR) is the mainstay of diagnosis of COVID-19. However, it has not been fully investigated whether infectious viruses are contained in SARS-CoV-2 genome-positive specimens examined using the rRT-PCR test. In this study, we examined the correlation between the threshold Cycle (Ct) value obtained from the rRT-PCR test and virus isolation in cultured cells, using 533 consecutive clinical specimens of COVID-19 patients. The virus was isolated from specimens with a Ct value of less than 30 cycles, and the lower the Ct value, the more efficient the isolation rate. A cytopathic effect due to herpes simplex virus type 1 contamination was observed in one sample with a Ct value of 35 cycles. In a comparison of VeroE6/TMPRSS2 cells and VeroE6 cells used for virus isolation, VeroE6/TMPRSS2 cells isolated the virus 1.7 times more efficiently than VeroE6 cells. There was no significant difference between the two cells in the mean Ct value of the detectable sample. In conclusion, Lower Ct values in the PCR test were associated with higher virus isolation rates, and VeroE6/TMPRSS2 cells were able to isolate viruses more efficiently than VeroE6 cells.

SARS-CoV-2 RNAemia with a higher nasopharyngeal viral load is strongly associated with disease severity and mortality in patients with COVID-19.

This study aimed to determine the frequency of SARS-CoV-2 RNA in serum and its association with the clinical severity of COVID-19. This retrospective cohort study performed at Toyama University Hospital included consecutive patients with confirmed COVID-19. The prevalence of SARS-CoV-2 RNAemia and the strength of its association with clinical severity variables were examined. Fifty-six patients were included in this study. RNAemia was detected in 19.6% (11/56) patients on admission, and subsequently in 1.0% (1/25), 50.0% (6/12), and 100.0% (4/4) moderate, severe, and critically ill patients, respectively. Patients with RNAemia required more frequent oxygen supplementation (90.0% vs. 13.3%), ICU admission (81.8% vs. 6.7%), and invasive mechanical ventilation (27.3% vs. 0.0%). Among patients with RNAemia, the median viral loads of nasopharyngeal (NP) swabs that were collected around the same time as the serum sample were significantly higher in critically ill (5.4 log10 copies/µl; interquartile range [IQR]: 4.2-6.3) than in moderate-severe cases (2.6 log10 copies/µl; [IQR: 1.1-4.5]; p = 0.030) and were significantly higher in nonsurvivors (6.2 log10 copies/µl [IQR: 6.0-6.5]) than in survivors (3.9 log10 copies/µl [IQR: 1.6-4.6]; p = 0.045). This study demonstrated a relatively high proportion of SARS-CoV-2 RNAemia and an association between RNAemia and clinical severity. Moreover, among the patients with RNAemia, the viral loads of NP swabs were correlated with disease severity and mortality, suggesting the potential utility of combining serum testing with NP tests as a prognostic indicator for COVID-19, with higher quality than each separate test.

Delayed neutralizing antibody response in the acute phase correlates with severe progression of COVID-19.

Adaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dynamics remain largely unknown. The neutralizing antibody (NAb) levels in patients with coronavirus disease 2019 (COVID-19) are helpful for understanding the pathology. Using SARS-CoV-2 pseudotyped virus, serum sample neutralization values in symptomatic COVID-19 patients were measured using the chemiluminescence reduction neutralization test (CRNT). At least two sequential serum samples collected during hospitalization were analyzed to assess NAbs neutralizing activity dynamics at different time points. Of the 11 patients, four (36.4%), six (54.5%), and one (9.1%) had moderate, severe, and critical disease, respectively. Fifty percent neutralization (N50%-CRNT) was observed upon admission in 90.9% (10/11); all patients acquired neutralizing activity 2-12 days after onset. In patients with moderate disease, neutralization was observed at earliest within two days after symptom onset. In patients with severe-to-critical disease, neutralization activity increased, plateauing 9-16 days after onset. Neutralization activity on admission was significantly higher in patients with moderate disease than in patients with severe-to-critical disease (relative % of infectivity, 6.4% vs. 41.1%; P = .011). Neutralization activity on admission inversely correlated with disease severity. The rapid NAb response may play a crucial role in preventing the progression of COVID-19.

Gold nanoparticle-adjuvanted S protein induces a strong antigen-specific IgG response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs.

The spike (S) protein of coronavirus, which binds to cellular receptors and mediates membrane fusion for cell entry, is a candidate vaccine target for blocking coronavirus infection. However, some animal studies have suggested that inadequate immunization against severe acute respiratory syndrome coronavirus (SARS-CoV) induces a lung eosinophilic immunopathology upon infection. The present study evaluated two kinds of vaccine adjuvants for use with recombinant S protein: gold nanoparticles (AuNPs), which are expected to function as both an antigen carrier and an adjuvant in immunization; and Toll-like receptor (TLR) agonists, which have previously been shown to be an effective adjuvant in an ultraviolet-inactivated SARS-CoV vaccine. All the mice immunized with more than 0.5 µg S protein without adjuvant escaped from SARS after infection with mouse-adapted SARS-CoV; however, eosinophilic infiltrations were observed in the lungs of almost all the immunized mice. The AuNP-adjuvanted protein induced a strong IgG response but failed to improve vaccine efficacy or to reduce eosinophilic infiltration because of highly allergic inflammatory responses. Whereas similar virus titers were observed in the control animals and the animals immunized with S protein with or without AuNPs, Type 1 interferon and pro-inflammatory responses were moderate in the mice treated with S protein with and without AuNPs. On the other hand, the TLR agonist-adjuvanted vaccine induced highly protective antibodies without eosinophilic infiltrations, as well as Th1/17 cytokine responses. The findings of this study will support the development of vaccines against severe pneumonia-associated coronaviruses.

Evaluation of SARS-CoV-2 neutralizing antibodies using a vesicular stomatitis virus possessing SARS-CoV-2 spike protein.

BACKGROUND: SARS-CoV-2 is a novel coronavirus that emerged in 2019 and is now classified in the genus Coronavirus with closely related SARS-CoV. SARS-CoV-2 is highly pathogenic in humans and is classified as a biosafety level (BSL)-3 pathogen, which makes manipulating it relatively difficult due to its infectious nature. METHODS: To circumvent the need for BSL-3 laboratories, an alternative assay was developed that avoids live virus and instead uses a recombinant VSV expressing luciferase and possesses the full length or truncated spike proteins of SARS-CoV-2. Furthermore, to measure SARS-CoV-2 neutralizing antibodies under BSL2 conditions, a chemiluminescence reduction neutralization test (CRNT) for SARS-CoV-2 was developed. The neutralization values of the serum samples collected from hospitalized patients with COVID-19 or SARS-CoV-2 PCR-negative donors against the pseudotyped virus infection evaluated by the CRNT were compared with antibody titers determined from an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescence assay (IFA). RESULTS: The CRNT, which used whole blood collected from hospitalized patients with COVID-19, was also examined. As a result, the inhibition of pseudotyped virus infection was specifically observed in both serum and whole blood and was also correlated with the results of the IFA. CONCLUSIONS: In conclusion, the CRNT for COVID-19 is a convenient assay system that can be performed in a BSL-2 laboratory with high specificity and sensitivity for evaluating the occurrence of neutralizing antibodies against SARS-CoV-2.