Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID50) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 g of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease.

Relative COVID-19 viral persistence and antibody kinetics

ImportanceThe COVID-19 antibody response is a critical indicator for evaluating immunity and also serves as the knowledge base for vaccine development. The picture is still not clear because of many limitations including testing tools, time of sampling, and the unclear impact of varying clinical status. In addition to these problems, antibody levels may not be equivalent to protective capacity. ObjectiveTo define the key factor for the different patterns of COVID-19 antibody response. DesignWe elucidated the antibody response with time-series throat and serum samples for viral loads and antibody levels, then used a neutralization test to evaluate protectiveness. SettingA medical center that typically cares for patients with moderate to severe diseases. Because of the low prevalence of COVID-19 in Taiwan and local government policy, however, we also admit COVID-19 patients with mild disease or even those without symptoms for inpatient care. ParticipantsRT-PCR-confirmed COVID-19 patients. ResultsWe found that only patients with relative persistence of virus at pharynx displayed strong antibody responses that were proportional to the pharyngeal viral load. They also had proportional neutralization titers per unit of serum. Although antibody levels decreased around 2 weeks after symptom onset, the neutralization efficacy per unit antibody remained steady and even continued to increase over time. The antibody response in patients with rapid virus clearance was weak, but the neutralization efficacy per unit antibody in these patients was comparable to those with persistent presence of virus. The deceased were with higher viral load, higher level of antibody, and higher neutralization titers in the serum, but the neutralization capacity per unit antibody is relatively low. Conclusions and RelevanceStrong antibody response depends on the relative persistence of the virus, instead of the absolute virus amount. The antibody response is still weak if large amount of virus is cleared quickly. The neutralization efficacy per unit antibody is comparable between high and low antibody patterns. Strong antibody response contains more inefficient and maybe even harmful antibodies. Low antibody response is also equipped with a capable B cell pool of efficient antibodies, which may expand with next virus encounter and confer protection. Key pointsO_ST_ABSQuestionC_ST_ABSThe key factor for the different "patterns" of COVID-19 antibody response. FindingsStrong antibody response depends on the relative persistence of the virus, instead of the absolute virus amount. The antibody response is still weak if large amount of virus is cleared quickly. The neutralization efficacy per unit antibody is comparable between high and low antibody patterns. High antibody level contains more inefficient antibodies. MeaningStrong response contains inefficient and maybe harmful antibodies. Low antibody response is also equipped with a capable B cell pool of efficient antibodies, which may expand with next virus encounter and confer protection.

Sequence variation among SARS-CoV-2 isolates in Taiwan

Taiwan experienced two waves of imported cases of coronavirus disease 2019 (COVID-19), first from China in January to late February, followed by those from other countries starting in early March. Additionally, several cases could not be traced to any imported cases and were suspected as sporadic local transmission. Twelve full viral genomes were determined in this study by Illumina sequencing either from virus isolates or directly from specimens, among which 5 originated from clustered infections. Phylogenetic tree analysis revealed that these sequences were in different clades, indicating that no major strain has been circulating in Taiwan. A deletion in open reading frame 8 was found in one isolate. Only a 4-nucleotide difference was observed among the 5 genomes from clustered infections.

A Correlation between the Severity of Lung Lesions on Radiographs and Clinical Findings in Patients with Severe Acute Respiratory Syndrome

OBJECTIVE: The purpose of this study was to quantify lesions on chest radiographs in patients with severe acute respiratory syndrome (SARS) and analyze the severity of the lesions with clinical parameters. MATERIALS AND METHODS: Two experienced radiologists reviewed chest radiographs of 28 patients with SARS. Each lung was divided into upper, middle, and lower zones. A SARS-related lesion in each zone was scored using a four-point scale: zero to three. The mean and maximal radiographic scores were analyzed statistically to determine if the scorings were related to the laboratory data and clinical course. RESULTS: Forward stepwise multiple linear regression showed that the mean radiographic score correlated most significantly with the number of hospitalized days (p < 0.001). The second most significant factor was the absolute lymphocyte count (p < 0.001) and the third most significant factor was the number of days of intubation (p = 0.025). The maximal radiographic score correlated best with the percentage of lymphocytes in a leukocyte count (p < 0.001), while the second most significant factor was the number of hospitalized days (p < 0.001) and the third most significant factor was the absolute lymphocyte count (p = 0.013). The mean radiographic scores of the patients who died, with comorbidities and without a comorbidity were 11.1, 6.3 and 2.9, respectively (p = 0.032). The corresponding value for maximal radiographic scores were 17.7, 9.7 and 6.0, respectively (p = 0.033). CONCLUSION: The severity of abnormalities quantified on chest radiographs in patients with SARS correlates with the clinical parameters.