A Δ42PD1 fusion-expressing DNA vaccine elicits enhanced adaptive immune response to HIV-1: the key role of TLR4.

Since its discovery in the 1990s, the DNA vaccine has been of great interest because of its ability to elicit both humoral and cellular immune responses while showing relative advantages regarding producibility, stability and storage. However, when applied to human subjects, inadequate immunogenicity remains as the greatest challenge for the practical use of DNA vaccines. In this study, we generated a DNA vaccine Δ42PD1-P24 encoding a fusion protein comprised of the HIV-1 Gag p24 antigen and the extracellular domain of murine Δ42PD1, a novel endogenous Toll-like receptor 4 (TLR4) agonist. Using a mouse model, we found that Δ42PD1-P24 DNA vaccine elicited a higher antibody response and an increased number of IFN-γ-producing CD4 and CD8 T cells. Moreover, mice with Δ42PD1-P24 DNA vaccination were protected from a subcutaneous challenge with murine mesothelioma cells expressing the HIV-1 p24 antigen. Importantly, the Δ42PD1-mediated enhancement of immune responses was not observed in TLR4 knockout mice. Collectively, these data demonstrate that the immunogenicity and efficacy of DNA vaccines could be improved by the fusion of the extracellular domain of Δ42PD1 to target the immunogen to dendritic cells.

The Positive Rate of Nucleic Acid Testing and the Epidemiological Characteristics of COVID-19 in Chongqing.

OBJECTIVE: The purpose of this study is to analyze the positive rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid testing (NAT), cases of and deaths due to SARS-CoV-2, and the epidemiological characteristics of SARS-CoV-2 to identify high-risk populations. METHODS: A retrospective study in Jiulongpo district of Chongqing was conducted by performing continuous observations of the frequency of SARS-CoV-2 NAT, analyzing the data of close contacts of patients and asymptomatic carriers, and collecting epidemiological data. Data were collected from January 20, 2020, when the first case of SARS-CoV-2 infection was reported, to March 26, 2020. Descriptive statistical analysis and Cochrane-Mantel-Haenszel analysis were used to compare the positive detection rates and positive diagnostic rates of different exposure groups. RESULTS: A total of 7,118 people received 10,377 SARS-CoV-2 nucleic acid tests in one district, and the SARS-CoV-2 positive rates were 0.40% (18/4446) and 0.15% (4/2672) in people receiving one and ≥ two nucleic acid tests (p = 0.06), respectively. Those with suspected cases (12.35%) and close contacts (8%) had higher positive rates than people tested at fever clinics (0.39%) (p < 0.001). The median latency (range) of cases was 5 (2, 9) days, and the median time from diagnosis to recovery was 22 (14, 25) days. One recovered patient received a positive test result at 28 days after recovery when she attempted to donate blood. Six clustered cases, including one patient who died, indicated persistent human-to-human transmission. One patient who was diagnosed after death was found to have infected 13 close contacts. People working in catering and other public service departments (36.36%) and people who are unemployed and retirees (45.45%) have an increased risk of infection compared with technical staff (9.09%) and farmers (9.09%). CONCLUSION: The total positive rate was low in the tested population, and more effective detection ranges should be defined to improve precise and differentiated epidemic control strategies. Moreover, in asymptomatic carriers, SARS-CoV-2 tests were positive after recovery, and patients with suspected SARS-CoV-2 infection who die may pose serious potential transmission threats.

Dysregulated hematopoiesis in bone marrow marks severe COVID-19.

Severe coronavirus disease 2019 (COVID-19) is often indicated by lymphopenia and increased myelopoiesis; however, the underlying mechanism is still unclear, especially the alteration of hematopoiesis. It is important to explore to what extent and how hematopoietic stem cells contribute to the impairment of peripheral lymphoid and myeloid compartments in COVID-19 patients. In this study, we used single-cell RNA sequencing to assess bone marrow mononuclear cells from COVID-19 patients with peripheral blood mononuclear cells as control. The results showed that the hematopoietic stem cells in these patients were mainly in the G1 phase and prone to apoptosis, with immune activation and anti-viral responses. Importantly, a significant accumulation of immature myeloid progenitors and a dramatic reduction of lymphoid progenitors in severe cases were identified, along with the up-regulation of transcription factors (such as SPI1, LMO4, ETS2, FLI1, and GATA2) that are important for the hematopoietic stem cell or multipotent progenitor to differentiate into downstream progenitors. Our results indicate a dysregulated hematopoiesis in patients with severe COVID-19.

Suppressive Monocytes Impair MAIT Cells Response via IL-10 in Patients with Severe COVID-19.

Immune cell responses are strikingly altered in patients with severe coronavirus disease 2019 (COVID-19), but the immunoregulatory process in these individuals is not fully understood. In this study, 23 patients with mild and 22 patients with severe COVID-19 and 6 asymptomatic carriers of COVID-19 were enrolled, along with 44 healthy controls (HC). Peripheral immune cells in HC and patients with COVID-19 were comprehensively profiled using mass cytometry. We found that in patients with severe COVID-19, the number of HLA-DRlow/- monocytes was significantly increased, but that of mucosal-associated invariant T (MAIT) cells was greatly reduced. MAIT cells were highly activated but functionally impaired in response to Escherichia coli and IL-12/IL-18 stimulation in patients with severe COVID-19, especially those with microbial coinfection. Single-cell transcriptome analysis revealed that IFN-stimulated genes were significantly upregulated in peripheral MAIT cells and monocytes from patients with severe COVID-19. IFN-α pretreatment suppressed MAIT cells' response to E. coli by triggering high levels of IL-10 production by HLA-DRlow/--suppressive monocytes. Blocking IFN-α or IL-10 receptors rescued MAIT cell function in patients with severe COVID-19. Moreover, plasma from patients with severe COVID-19 inhibited HLA-DR expression by monocytes through IL-10. These data indicate a unique pattern of immune dysregulation in severe COVID-19, which is characterized by enrichment of suppressive HLA-DRlow/- monocytes associated with functional impairment of MAIT cells through the IFN/IL-10 pathway.

The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET.

The ongoing global pandemic of coronavirus disease 2019 (COVID-19) resulted from the outbreak of SARS-CoV-2 in December 2019. Currently, multiple efforts are being made to rapidly develop vaccines and treatments to fight COVID-19. Current vaccine candidates use inactivated SARS-CoV-2 viruses; therefore, it is important to understand the architecture of inactivated SARS-CoV-2. We have genetically and structurally characterized ß-propiolactone-inactivated viruses from a propagated and purified clinical strain of SARS-CoV-2. We observed that the virus particles are roughly spherical or moderately pleiomorphic. Although a small fraction of prefusion spikes are found, most spikes appear nail shaped, thus resembling a postfusion state, where the S1 protein of the spike has disassociated from S2. Cryoelectron tomography and subtomogram averaging of these spikes yielded a density map that closely matches the overall structure of the SARS-CoV postfusion spike and its corresponding glycosylation site. Our findings have major implications for SARS-CoV-2 vaccine design, especially those using inactivated viruses.

Acute Stroke Presentation, Care, and Outcomes in Community Hospitals in Northern California During the COVID-19 Pandemic.

BACKGROUND AND PURPOSE: Shelter-in-place (SIP) orders implemented to mitigate severe acute respiratory syndrome coronavirus 2 spread may inadvertently discourage patient care-seeking behavior for critical conditions like acute ischemic stroke. We aimed to compare temporal trends in volume of acute stroke alerts, patient characteristics, telestroke care, and short-term outcomes pre- and post-SIP orders. METHODS: We conducted a cohort study in 21 stroke centers of an integrated healthcare system serving 4.4+ million members across Northern California. We included adult patients who presented with suspected acute stroke and were evaluated by telestroke between January 1, 2019, and May 9, 2020. SIP orders announced the week of March 15, 2020, created pre (January 1, 2019, to March 14, 2020) and post (March 15, 2020, to May 9, 2020) cohort for comparison. Main outcomes were stroke alert volumes and inpatient mortality for stroke. RESULTS: Stroke alert weekly volume post-SIP (mean, 98 [95% CI, 92-104]) decreased significantly compared with pre-SIP (mean, 132 [95% CI, 130-136]; P<0.001). Stroke discharges also dropped, in concordance with acute stroke alerts decrease. In total, 9120 patients were included: 8337 in pre- and 783 in post-SIP cohorts. There were no differences in patient demographics. Compared with pre-SIP, post-SIP patients had higher National Institutes of Health Stroke Scale scores (P=0.003), lower comorbidity score (P<0.001), and arrived more often by ambulance (P<0.001). Post-SIP, more patients had large vessel occlusions (P=0.03), and there were fewer stroke mimics (P=0.001). Discharge outcomes were similar for post-SIP and pre-SIP cohorts. CONCLUSIONS: In this cohort study, regional stroke alert and ischemic stroke discharge volumes decreased significantly in the early COVID-19 pandemic. Compared with pre-SIP, the post-SIP population showed no significant demographic differences but had lower comorbidity scores, more severe strokes, and more large vessel occlusions. The inpatient mortality was similar in both cohorts. Further studies are needed to understand the causes and implications of care avoidance to patients and healthcare systems.

Effects of policies and containment measures on control of COVID-19 epidemic in Chongqing.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an emerging, rapidly evolving disease that spreads through the respiratory system and is highly contagious. In March 2020, the World Health Organization declared the COVID-19 outbreak a pandemic. In China, the pandemic was controlled after 2 mo through effective policies and containment measures. Describing the detailed policies and containment measures used to control the epidemic in Chongqing will provide a reference for the prevention and control of COVID-19 in other areas of the world. AIM: To explore the effects of different policies and containment measures on the control of the COVID-19 epidemic in Chongqing. METHODS: Epidemiological data on COVID-19 in Chongqing were prospectively collected from January 21 to March 15, 2020. The policies and prevention measures implemented by the government during the epidemic period were also collected. Trend analysis was performed to explore the impact of the main policy measures on the effectiveness of the control of COVID-19 in Chongqing. RESULTS: As of March 15, the cumulative incidence of COVID-19 in Chongqing was 1.84/100000 (576 cases) and the infection fatality rate was 1.04% (6/576). The spread of COVID-19 was controlled by effective policies that involved establishing a group for directing the COVID-19 epidemic control effort; strengthening guidance and supervision; ensuring the supply of daily necessities and medical supplies and equipment to residents; setting up designated hospitals; implementing legal measures; and enhancing health education. Medical techniques were implemented to improve the recovery rate and control the epidemic. Policies such as "the lockdown of Wuhan", "initiating a first-level response to major public health emergencies", and "implementing the closed management of residential communities" significantly curbed the spread of COVID-19. Optimizing the diagnosis process, shortening the diagnosis time, and constructing teams of clinical experts facilitated the provision of "one team of medical experts for each patient" treatment for severe patients, which significantly improved the recovery rate and reduced the infection fatality rate. CONCLUSION: The prevention policies and containment measures implemented by the government and medical institutions are highly effective in controlling the spread of the epidemic and increasing the recovery rate of COVID-19 patients.

Human neutralizing antibodies elicited by SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a global health emergency that is in urgent need of intervention1-3. The entry of SARS-CoV-2 into its target cells depends on binding between the receptor-binding domain (RBD) of the viral spike protein and its cellular receptor, angiotensin-converting enzyme 2 (ACE2)2,4-6. Here we report the isolation and characterization of 206 RBD-specific monoclonal antibodies derived from single B cells from 8 individuals infected with SARS-CoV-2. We identified antibodies that potently neutralize SARS-CoV-2; this activity correlates with competition with ACE2 for binding to RBD. Unexpectedly, the anti-SARS-CoV-2 antibodies and the infected plasma did not cross-react with the RBDs of SARS-CoV or Middle East respiratory syndrome-related coronavirus (MERS-CoV), although there was substantial plasma cross-reactivity to their trimeric spike proteins. Analysis of the crystal structure of RBD-bound antibody revealed that steric hindrance inhibits viral engagement with ACE2, thereby blocking viral entry. These findings suggest that anti-RBD antibodies are largely viral-species-specific inhibitors. The antibodies identified here may be candidates for development of clinical interventions against SARS-CoV-2.