Determining the Time of Booster Dose Based on the Half-Life and Neutralization Titers against SARS-CoV-2 Variants of Concern in Fully Vaccinated Individuals.

Although mRNA-based COVID-19 vaccines reduce the risk of severe disease, hospitalization and death, vaccine effectiveness (VE) against infection and disease from variants of concern (VOC) wanes over time. Neutralizing antibodies (NAb) are surrogates of protection and are enhanced by a booster dose, but their kinetics and durability remain understudied. Current recommendation of a booster dose does not consider the existing NAb in each individual. Here, we investigated 50% neutralization (NT50) titers against VOC among COVID-19-naive participants receiving the Moderna (n = 26) or Pfizer (n = 25) vaccine for up to 7 months following the second dose, and determined their half-lives. We found that the time it took for NT50 titers to decline to 24, equivalent to 50% inhibitory dilution of 10 international units/mL, was longer in the Moderna (325/324/235/274 days for the D614G/alpha/beta/delta variants) group than in the Pfizer (253/252/174/226 days) group, which may account for the slower decline in VE of the Moderna vaccine observed in real-world settings and supports our hypothesis that measuring the NT50 titers against VOC, together with information on NAb half-lives, can be used to dictate the time of booster vaccination. Our study provides a framework to determine the optimal time of a booster dose against VOC at the individual level. In response to future VOC with high morbidity and mortality, a quick evaluation of NAb half-lives using longitudinal serum samples from clinical trials or research programs of different primary-series vaccinations and/or one or two boosters could provide references for determining the time of booster in different individuals. IMPORTANCE Despite improved understanding of the biology of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the evolutionary trajectory of the virus is uncertain, and the concern of future antigenically distinct variants remains. Current recommendations for a COVID-19 vaccine booster dose are primarily based on neutralization capacity, effectiveness against circulating variants of concern (VOC), and other host factors. We hypothesized that measuring neutralizing antibody titers against SARS-CoV-2 VOC together with half-life information can be used to dictate the time of booster vaccination. Through detailed analysis of neutralizing antibodies against VOC among COVID-19-naive vaccinees receiving either of two mRNA vaccines, we found that the time it took for 50% neutralization titers to decline to a reference level of protection was longer in the Moderna than in the Pfizer group, which supports our hypothesis. In response to future VOC with potentially high morbidity and mortality, our proof-of-concept study provides a framework to determine the optimal time of a booster dose at the individual level.

Elevated circulating monocytes and monocyte activation in COVID-19 convalescent individuals.

Background: Monocytes and macrophages play a pivotal role in inflammation during acute SARS-CoV-2 infection. However, their contribution to the development of post-acute sequelae of SARS-CoV-2 infection (PASC) are not fully elucidated. Methods: A cross-sectional study was conducted comparing plasma cytokine and monocyte levels among three groups: participants with pulmonary PASC (PPASC) with a reduced predicted diffusing capacity for carbon monoxide [DLCOc, <80%; (PG)]; fully recovered from SARS-CoV-2 with no residual symptoms (recovered group, RG); and negative for SARS-CoV-2 (negative group, NG). The expressions of cytokines were measured in plasma of study cohort by Luminex assay. The percentages and numbers of monocyte subsets (classical, intermediate, and non-classical monocytes) and monocyte activation (defined by CD169 expression) were analyzed using flow cytometry analysis of peripheral blood mononuclear cells. Results: Plasma IL-1Ra levels were elevated but FGF levels were reduced in PG compared to NG. Circulating monocytes and three subsets were significantly higher in PG and RG compared to NG. PG and RG exhibited higher levels of CD169+ monocyte counts and higher CD169 expression was detected in intermediate and non-classical monocytes from RG and PG than that found in NG. Further correlation analysis with CD169+ monocyte subsets revealed that CD169+ intermediate monocytes negatively correlated with DLCOc%, and CD169+ non-classical monocytes positively correlated with IL-1α, IL-1ß, MIP-1α, Eotaxin, and IFN-γ. Conclusion: This study present evidence that COVID convalescents exhibit monocyte alteration beyond the acute COVID-19 infection period even in convalescents with no residual symptoms. Further, the results suggest that monocyte alteration and increased activated monocyte subsets may impact pulmonary function in COVID-19 convalescents. This observation will aid in understanding the immunopathologic feature of pulmonary PASC development, resolution, and subsequent therapeutic interventions.

Identification of severe acute respiratory syndrome coronavirus 2 breakthrough infections by anti-nucleocapsid antibody among fully vaccinated non-healthcare workers during the transition from the delta to omicron wave.

Uncontrolled transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the emergence of several variants of concern (VOC). As vaccine-induced neutralizing antibodies against VOC waned over time, breakthrough infections (BTIs) have been reported primarily among healthcare workers or in long-term care facilities. Most BTIs were identified by reverse transcription-polymerase chain reaction (RT-PCR) or antigen test for individuals experiencing symptoms, known as symptomatic BTIs. In this study, we detected seroconversion of anti-nucleocapsid (N) antibody to identify both symptomatic and asymptomatic BTIs in a cohort of COVID-19-naive university employees and students following two or three doses of mRNA vaccines. We reported 4 BTIs among 85 (4.7%) participants caused by the Omicron and Delta VOC during the transition from the Delta to Omicron wave of the pandemic; three were symptomatic and confirmed by RT-PCR test and one asymptomatic. A symptomatic reinfection two and half months after a BTI was found in one participant. Two of three symptomatic BTIs and the reinfection were confirmed by whole genome sequencing. All were supported by a >4-fold increase in neutralizing antibodies against the Delta or Omicron variant. Moreover, we found both symptomatic and asymptomatic BTIs can boost neutralizing antibodies against VOC with variable degrees ranging from 2.5- to 77.4-fold increase in neutralizing antibody titers. As BTIs continue, our findings highlight the application of anti-N antibody test to ongoing studies of immunity induced by spike-based vaccine, and provide new insights into the establishment of herd immunity in the community during the post-vaccination era.

Dynamic SARS-CoV-2 emergence algorithm for rationally-designed logical next-generation vaccines.

SARS-CoV-2 worldwide spread and evolution has resulted in variants containing mutations resulting in immune evasive epitopes that decrease vaccine efficacy. We acquired SARS-CoV-2 positive clinical samples and compared the worldwide emerged spike mutations from Variants of Concern/Interest, and developed an algorithm for monitoring the evolution of SARS-CoV-2 in the context of vaccines and monoclonal antibodies. The algorithm partitions logarithmic-transformed prevalence data monthly and Pearson's correlation determines exponential emergence of amino acid substitutions (AAS) and lineages. The SARS-CoV-2 genome evaluation indicated 49 mutations, with 44 resulting in AAS. Nine of the ten most worldwide prevalent (>70%) spike protein changes have Pearson's coefficient r > 0.9. The tenth, D614G, has a prevalence >99% and r-value of 0.67. The resulting algorithm is based on the patterns these ten substitutions elucidated. The strong positive correlation of the emerged spike protein changes and algorithmic predictive value can be harnessed in designing vaccines with relevant immunogenic epitopes. Monitoring, next-generation vaccine design, and mAb clinical efficacy must keep up with SARS-CoV-2 evolution, as the virus is predicted to remain endemic.

Algorithm for the Quantitation of Variants of Concern for Rationally Designed Vaccines Based on the Isolation of SARS-CoV-2 Hawai'i Lineage B.1.243.

SARS-CoV-2 worldwide emergence and evolution has resulted in variants containing mutations resulting in immune evasive epitopes that decrease vaccine efficacy. We acquired clinical samples, analyzed SARS-CoV-2 genomes, used the most worldwide emerged spike mutations from Variants of Concern/Interest, and developed an algorithm for monitoring the SARS-CoV-2 vaccine platform. The algorithm partitions logarithmic-transformed prevalence data monthly and Pearson's correlation determines exponential emergence. The SARS-CoV-2 genome evaluation indicated 49 mutations. Nine of the ten most worldwide prevalent (>70%) spike protein changes have r- values >0.9. The tenth, D614G, has a prevalence >99% and r -value of 0.67. The resulting algorithm is based on the patterns these ten substitutions elucidated. The strong positive correlation of the emerged spike protein changes and algorithmic predictive value can be harnessed in designing vaccines with relevant immunogenic epitopes. SARS-CoV-2 is predicted to remain endemic and continues to evolve, so must SARS-CoV-2 monitoring and next-generation vaccine design.

A real-time and high-throughput neutralization test based on SARS-CoV-2 pseudovirus containing monomeric infrared fluorescent protein as reporter.

Neutralizing antibodies to SARS-CoV-2 have been shown to correlate with protection in animals and humans, disease severity, survival, and vaccine efficacy. With the ongoing large-scale vaccination in different countries and continuous surge of new variants of global concerns, a convenient, cost-effective and high-throughput neutralization test is urgently needed. Conventional SARS-CoV-2 neutralization test is tedious, time-consuming and requires a biosafety level 3 laboratory. Despite recent reports of neutralizations using different pseudoviruses with a luciferase or green fluorescent protein reporter, the laborious steps, inter-assay variability or high background limit their high-throughput potential. In this study we generated lentivirus-based pseudoviruses containing a monomeric infrared fluorescent protein reporter to develop neutralization assays. Similar tropism, infection kinetics and mechanism of entry through receptor-mediated endocytosis were found in the three pseudoviruses generated. Compared with pseudovirus D614, pseudovirus with D614G mutation had decreased shedding and higher density of S1 protein present on particles. The 50% neutralization titers to pseudoviruses D614 or D614G correlated with the plaque reduction neutralization titers to live SARS-CoV-2. The turn-around time of 48-72 h, minimal autofluorescence, one-step image quantification, expandable to 384-well, sequential readouts and dual quantifications by flow cytometry support its high-throughput and versatile applications at a non-reference and biosafety level 2 laboratory, in particular for assessing the neutralization sensitivity of new variants by sera from natural infection or different vaccinations during our fight against the pandemic.

Genetic Characteristics and Phylogeny of 969-bp S Gene Sequence of SARS-CoV-2 from Hawai'i Reveals the Worldwide Emerging P681H Mutation.

The COVID-19 pandemic has ravaged the world, caused over 1.8 million deaths in its first year, and severely affected the global economy. Hawai'i has not been spared from the transmission of SARS-CoV-2 in the local population, including high infection rates in racial and ethnic minorities. Early in the pandemic, we described in this journal various technologies used for the detection of SARS-CoV-2. Herein we characterize a 969-bp SARS-CoV-2 segment of the S gene downstream of the receptor-binding domain. At the John A. Burns School of Medicine Biocontainment Facility, RNA was extracted from an oropharyngeal swab and a nasal swab from 2 patients from Hawai'i who were infected with SARS-CoV-2 in August 2020. Following PCR, the 2 viral strains were sequenced using Sanger sequencing, and phylogenetic trees were generated using MEGAX. Phylogenetic tree results indicate that the virus has been introduced to Hawai'i from multiple sources. Further, we decoded 13 single nucleotide polymorphisms across 13 unique SARS-CoV-2 genomes within this region of the S gene, with 1 non-synonymous mutation (P681H) found in the 2 Hawai'i strains. The P681H mutation has unique and emerging characteristics with a significant exponential increase in worldwide frequency when compared to the plateauing of the now universal D614G mutation. The P681H mutation is also characteristic of the new SARS-CoV-2 variants from the United Kingdom and Nigeria. Additionally, several mutations resulting in cysteine residues were detected, potentially resulting in disruption of the disulfide bridges in and around the receptor-binding domain. Targeted sequence characterization is warranted to determine the origin of multiple introductions of SARS-CoV-2 circulating in Hawai'i.

Genetic Characteristics and Phylogeny of 969-bp S Gene Sequence of SARS-CoV-2 from Hawaii Reveals the Worldwide Emerging P681H Mutation.

COVID-19 pandemic has ravaged the world, caused over 1.8 million deaths in the first year, and severely affected the global economy. Hawaii is not spared from the transmission of SARS-CoV-2 in the local population, including high infection rates in racial and ethnic minorities. Early in the pandemic, we described in this journal various technologies used for the detection of SARS-CoV-2. Herein we characterize a 969-bp SARS-CoV-2 segment of the S gene downstream of the receptor-binding domain. At the John A. Burns School of Medicine Biocontainment Facility, RNA was extracted from an oropharyngeal swab and a nasal swab from two patients from Hawaii who were infected with the SARS-CoV-2 in August 2020. Following PCR, the two viral strains were sequenced using Sanger sequencing, and phylogenetic trees were generated using MEGAX. Phylogenetic tree results indicate that the virus has been introduced to Hawaii from multiple sources. Further, we decoded 13 single nucleotide polymorphisms across 13 unique SARS-CoV-2 genomes within this region of the S gene, with one non-synonymous mutation (P681H) found in the two Hawaii strains. The P681H mutation has unique and emerging characteristics with a significant exponential increase in worldwide frequency when compared to the plateauing of the now universal D614G mutation. The P681H mutation is also characteristic of the new SARS-CoV-2 variants from the United Kingdom and Nigeria. Additionally, several mutations resulting in cysteine residues were detected, potentially resulting in disruption of the disulfide bridges in and around the receptor-binding domain. Targeted sequence characterization is warranted to determine the origin of multiple introductions of SARS-CoV-2 circulating in Hawaii.

Elevated Levels of Pentraxin 3 Correlate With Neutrophilia and Coronary Artery Dilation During Acute Kawasaki Disease.

Kawasaki disease (KD) is the leading cause of acquired pediatric heart disease in the developed world as 25-30% of untreated patients and at least 5% of treated patients will develop irreversible coronary artery lesions (CAL). Pentraxin-3 (PTX-3) has been well-studied in inflammatory diseases, particularly in cardiovascular diseases associated with vascular endothelial dysfunction. We hypothesized that PTX-3 plays an important role in the development of KD-associated CAL and investigated the circulating levels of PTX-3 in the serum of KD patients. Children with acute KD were followed from diagnosis through normalization of the clinical parameters of inflammation (convalescent phase). Serum samples were obtained and echocardiograms were conducted at several phases of the illness: acute [prior to intravenous immunoglobulin (IVIG) treatment], sub-acute (5-10 days after IVIG treatment), and convalescent (1-4 months after KD diagnosis). Seventy children were included in the final cohort of the study, of whom 26 (37%) presented with CAL and 18 (26%) developed IVIG resistance. The patients included in this study came from diverse ethnic backgrounds, mostly with mixed ancestry/ ethnicity. Significantly increased PTX-3 levels were observed during the acute phase of KD compared to the sub-acute and the convalescent phases. The PTX-3 levels during acute KD were significantly higher among KD patients with CAL compared to patients with normal coronary arteries (NCA). Also, the PTX-3 levels were significantly higher in patients with IVIG resistance. Furthermore, the PTX-3 levels were significantly higher in IVIG-resistant KD patients with CAL as compared to the NCA group. Moreover, the PTX-3 levels were significantly correlated to coronary artery z-score during acute KD and to neutrophil counts throughout KD progression regardless of coronary artery z-score. Elevated PTX-3 levels correlated to elevated neutrophil counts, a known source of PTX-3 in acute inflammation and an important player in the development of KD vasculitis. We, therefore, suggest PTX-3 as a novel factor in the development of KD-associated CAL and propose neutrophil-derived PTX-3 as contributing to KD vascular dysfunction.

Study design and rationale to assess Doxycycline Efficacy in preventing coronary Artery Lesions in children with Kawasaki disease (DEAL trial) - A phase II clinical trial.

BACKGROUND: Kawasaki disease (KD) is the most common acquired heart disease in children of the developed world, and triggers progressive coronary artery lesions (CAL) in 30% of cases if left untreated. Despite standard anti-inflammatory treatment for KD, CAL (dilation or aneurysm) still occurs in 5-10% of children, increasing their risk for fatal coronary artery complications. CAL is mediated by enhanced matrix metalloproteinase activity and elastin breakdown induced by the inflammatory process in the coronary artery wall. Doxycycline is an effective inhibitor of matrix metalloproteinases, and has been shown to reduce elastin breakdown and CAL in a mouse model of KD, but has not been evaluated in patients. OBJECTIVE: We aim to evaluate the efficacy of doxycycline in the prevention of CAL in children during the acute phase of KD. DESIGN: This is a phase II prospective, randomized, double-blinded, clinical trial in two steps. In Step 1, any child older than 1month with the diagnosis of KD will be included. Children with KD will be included in Step 2 if they develop coronary artery dilation (z-score≥2.5) within 20days from the onset of fever. Study subjects in Step 2 will be randomized to receive a 3-week course of doxycycline or placebo. EVALUATION: The efficacy of a 3-week doxycycline course during the acute phase of KD will be evaluated by measuring the decline in coronary artery z-scores from baseline with doxycycline treatment compared to placebo. CLINICAL TRIAL REGISTRATION: This study was registered on clinicaltrials.gov (NCT01917721).