Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

Development of a qualitative real-time RT-PCR assay for the detection of SARS-CoV-2: A guide and case study in setting up an emergency-use, laboratory-developed molecular assay

Developing and deploying new diagnostic tests is difficult, but the need to do so in response to a rapidly emerging pandemic such as COVID-19 is crucially important for an effective response. In the early stages of a pandemic, laboratories play a key role in helping health care providers and public health authorities detect active infection, a task most commonly achieved using nucleic acid-based assays. While the landscape of diagnostics is rapidly evolving, polymerase chain reaction (PCR) remains the gold-standard of nucleic acid-based diagnostic assays, in part due to its reliability, flexibility, and wide deployment. To address a critical local shortage of testing capacity persisting during the COVID-19 outbreak, our hospital set up a molecular based laboratory developed test (LDT) to accurately and safely diagnose SARS-CoV-2. We describe here the process of developing an emergency-use LDT, in the hope that our experience will be useful to other laboratories in future outbreaks and will help to lower barriers to fast and accurate diagnostic testing in crisis conditions.

Phylogenetic analysis of SARS-CoV-2 in the Boston area highlights the role of recurrent importation and superspreading events

SARS-CoV-2 has caused a severe, ongoing outbreak of COVID-19 in Massachusetts with 111,070 confirmed cases and 8,433 deaths as of August 1, 2020. To investigate the introduction, spread, and epidemiology of COVID-19 in the Boston area, we sequenced and analyzed 772 complete SARS-CoV-2 genomes from the region, including nearly all confirmed cases within the first week of the epidemic and hundreds of cases from major outbreaks at a conference, a nursing facility, and among homeless shelter guests and staff. The data reveal over 80 introductions into the Boston area, predominantly from elsewhere in the United States and Europe. We studied two superspreading events covered by the data, events that led to very different outcomes because of the timing and populations involved. One produced rapid spread in a vulnerable population but little onward transmission, while the other was a major contributor to sustained community transmission, including outbreaks in homeless populations, and was exported to several other domestic and international sites. The same two events differed significantly in the number of new mutations seen, raising the possibility that SARS-CoV-2 superspreading might encompass disparate transmission dynamics. Our results highlight the failure of measures to prevent importation into MA early in the outbreak, underscore the role of superspreading in amplifying an outbreak in a major urban area, and lay a foundation for contact tracing informed by genetic data.

Dynamics and significance of the antibody response to SARS-CoV-2 infection

BACKGROUNDCharacterizing the humoral immune response to SARS-CoV-2 and developing accurate serologic assays are needed for diagnostic purposes and estimating population-level seroprevalence. METHODSWe measured the kinetics of early antibody responses to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of 259 symptomatic North American patients infected with SARS-CoV-2 (up to 75 days after symptom onset) compared to antibody levels in 1548 individuals whose blood samples were obtained prior to the pandemic. RESULTSBetween 14-28 days from onset of symptoms, IgG, IgA, or IgM antibody responses to RBD were all accurate in identifying recently infected individuals, with 100% specificity and a sensitivity of 97%, 91%, and 81% respectively. Although the estimated median time to becoming seropositive was similar across isotypes, IgA and IgM antibodies against RBD were short-lived with most individuals estimated to become seronegative again by 51 and 47 days after symptom onset, respectively. IgG antibodies against RBD lasted longer and persisted through 75 days post-symptoms. IgG antibodies to SARS-CoV-2 RBD were highly correlated with neutralizing antibodies targeting the S protein. No cross-reactivity of the SARS-CoV-2 RBD-targeted antibodies was observed with several known circulating coronaviruses, HKU1, OC 229 E, OC43, and NL63. CONCLUSIONSAmong symptomatic SARS-CoV-2 cases, RBD-targeted antibodies can be indicative of previous and recent infection. IgG antibodies are correlated with neutralizing antibodies and are possibly a correlate of protective immunity.

SARS-CoV-2 Viral Load is Associated with Increased Disease Severity and Mortality

The relationship between SARS-CoV-2 viral load and risk of disease progression remains largely undefined in coronavirus disease 2019 (COVID-19). We quantified SARS-CoV-2 viral load from participants with a diverse range of COVID-19 severity, including those requiring hospitalization, outpatients with mild disease, and individuals with resolved infection. SARS-CoV-2 plasma RNA was detected in 27% of hospitalized participants and 13% of outpatients diagnosed with COVID-19. Amongst the participants hospitalized with COVID-19, higher prevalence of detectable SARS-CoV-2 plasma viral load was associated with worse respiratory disease severity, lower absolute lymphocyte counts, and increased markers of inflammation, including C-reactive protein and IL-6. SARS-CoV-2 viral loads, especially plasma viremia, were associated with increased risk of mortality. SARS-CoV-2 viral load may aid in the risk stratification of patients with COVID-19 and its role in disease pathogenesis should be further explored.

Clinical Sensitivity and Interpretation of PCR and Serological COVID-19 Diagnostics for Patients Presenting to the Hospital

IntroductionThe diagnosis of COVID-19 requires integration of clinical and laboratory data. SARS-CoV-2 diagnostic assays play a central role in diagnosis and have fixed technical performance metrics. Interpretation becomes challenging because the clinical sensitivity changes as the virus clears and the immune response emerges. Our goal was to examine the clinical sensitivity of two most common SARS-CoV-2 diagnostic test modalities, polymerase chain reaction (PCR) and serology, over the disease course to provide insight into their clinical interpretation in patients presenting to the hospital. MethodsA single-center, retrospective study. To derive clinical sensitivity of PCR, we identified 209 PCR-positive SARS-CoV-2 patients with multiple PCR test results (624 total PCR tests) and calculated daily sensitivity from date of symptom onset or first positive test. To calculate daily clinical sensitivity by serology, we utilized 157 PCR- positive patients with a total of 197 specimens tested by enzyme-linked immunosorbent assay for IgM, IgG, and IgA anti-SARS-CoV-2 antibodies. ResultsClinical sensitivity of PCR decreased with days post symptom onset with >90% clinical sensitivity during the first 5 days after symptom onset, 70-71% from days 9-11, and 30% at day 21. In contrast, serological sensitivity increased with days post symptom onset with >50% of patients seropositive by at least one antibody isotype after day 7, >80% after day 12, and 100% by day 21. ConclusionPCR and serology are complimentary modalities that require time- dependent interpretation. Superimposition of sensitivities over time indicate that serology can function as a reliable diagnostic aid indicating recent or prior infection.

Ultra-Sensitive High-Resolution Profiling of Anti-SARS-CoV-2 Antibodies for Detecting Early Seroconversion in COVID-19 Patients

The COVID-19 pandemic continues to infect millions of people worldwide. In order to curb its spread and reduce morbidity and mortality, it is essential to develop sensitive and quantitative methods that identify infected individuals and enable accurate population-wide screening of both past and present infection. Here we show that Single Molecule Array assays detect seroconversion in COVID-19 patients as soon as one day after symptom onset using less than a microliter of blood. This multiplexed assay format allows us to quantitate IgG, IgM and IgA immunoglobulins against four SARS-CoV-2 targets, thereby interrogating 12 antibody isotype-viral protein interactions to give a high resolution profile of the immune response. Using a cohort of samples collected prior to the outbreak as well as samples collected during the pandemic, we demonstrate a sensitivity of 86% and a specificity of 100% during the first week of infection, and 100% sensitivity and specificity thereafter. This assay should become the gold standard for COVID19 serological profiling and will be a valuable tool for answering important questions about the heterogeneity of clinical presentation seen in the ongoing pandemic.

Test performance evaluation of SARS-CoV-2 serological assays

BackgroundSerological tests are crucial tools for assessments of SARS-CoV-2 exposure, infection and potential immunity. Their appropriate use and interpretation require accurate assay performance data. MethodWe conducted an evaluation of 10 lateral flow assays (LFAs) and two ELISAs to detect anti-SARS-CoV-2 antibodies. The specimen set comprised 128 plasma or serum samples from 79 symptomatic SARS-CoV-2 RT-PCR-positive individuals; 108 pre-COVID-19 negative controls; and 52 recent samples from individuals who underwent respiratory viral testing but were not diagnosed with Coronavirus Disease 2019 (COVID-19). Samples were blinded and LFA results were interpreted by two independent readers, using a standardized intensity scoring system. ResultsAmong specimens from SARS-CoV-2 RT-PCR-positive individuals, the percent seropositive increased with time interval, peaking at 81.8-100.0% in samples taken >20 days after symptom onset. Test specificity ranged from 84.3-100.0% in pre-COVID-19 specimens. Specificity was higher when weak LFA bands were considered negative, but this decreased sensitivity. IgM detection was more variable than IgG, and detection was highest when IgM and IgG results were combined. Agreement between ELISAs and LFAs ranged from 75.7-94.8%. No consistent cross-reactivity was observed. ConclusionOur evaluation showed heterogeneous assay performance. Reader training is key to reliable LFA performance, and can be tailored for survey goals. Informed use of serology will require evaluations covering the full spectrum of SARS-CoV-2 infections, from asymptomatic and mild infection to severe disease, and later convalescence. Well-designed studies to elucidate the mechanisms and serological correlates of protective immunity will be crucial to guide rational clinical and public health policies.