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The ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 g of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 g or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-{gamma}, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.
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BackgroundEstimated seroprevalence of Coronavirus Infectious Disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) is a critical evidence for a better evaluation of the virus spread and monitoring the progress of the COVID-19 pandemic in a population. In the Kingdom of Saudi Arabia (KSA), SARS-CoV-2 seroprevalence has been reported in specific regions, but an extensive nationwide study has not been reported. Here, we report a nationwide study to determine the prevalence of SARS-CoV-2 in the population of KSA during the pandemic, using serum samples from healthy blood donors, non-COVID patients and healthcare workers (HCWs) in six different regions of the kingdom, with addition samples from COVID-19 patients. MethodsA total of 11703 serum samples were collected from different regions of the KSA including; 5395 samples from residual healthy blood donors (D); 5877 samples from non-COVID patients collected through residual sera at clinical biochemistry labs from non-COVID patients (P); and 400 samples from consented HCWs. To determine the seroprevalence of SARS-CoV-2, all serum samples, in addition to positive control sera from RT-PCR confirmed COVID-19 patients, were subjected to in-house ELISA with a sample pooling strategy, which was further validated by testing individual samples that make up some of the pools, with a statistical estimation method to report seroprevalence estimates ResultsOverall (combining D and P groups) seroprevalence estimate was around 11% in Saudi Arabia; and was 5.1% (Riyadh), 1.5% (Jazan), 18.4% (Qassim), 20.8% (Hail), 14.7% (ER; Alahsa), and 18.8% in Makkah. Makkah samples were only D group and had a rate of 24.4% and 12.8% in the cities of Makkah and Jeddah, respectively. The seroprevalence in Saudi Arabia across the sampled areas would be 12 times the COVID-19 infection rate. Among HCWs, 7.5% (4.95-10.16 CI 95%) had reactive antibodies to SARS-CoV-2 without reporting any previously confirmed infection. This was higher in HCWs with hypertension. The study also presents the demographics and prevalence of co-morbidities in HCWs and subset of non-COVID-19 population. ConclusionOur study estimates the overall national serological prevalence of COVID-19 in Saudi Arabia to be 11%, with an apparent disparity between regions.
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The Coronavirus Disease 2019 (COVID-19), caused by the novel SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Immunological surrogate markers, in particular antigen-specific responses, are of unquestionable value for clinical management of patients with COVID-19. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized patients with RT-PCR confirmed COVID-19 infection. Our data show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Notably, anti-S and -N IgG, peaked 20-40 day after disease onset, and were still detectable for at least up to 70 days, with nAbs observed during the same time period. Moreover, nAbs titers were strongly correlated with IgG antibodies. Significantly higher levels of nAbs as well as anti-S1 and N IgG and IgM antibodies were found in patients with more severe clinical presentations, patients requiring admission to intensive care units (ICU) or those with fatal outcomes. Interestingly, lower levels of antibodies, particularly anti-N IgG and IgM in the first 15 days after symptoms onset, were found in survivors and those with mild clinical presentations. Collectively, these findings provide new insights into the characteristics and kinetics of antibody responses in COVID-19 patients with different disease severity.
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We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.Competing Interest StatementThe authors have declared no competing interest.View Full Text
