In Vivo Antiviral Efficacy of LCTG-002, a Pooled, Purified Human Milk Secretory IgA product, Against SARS-CoV-2 in a Murine Model of COVID-19 (preprint)

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosal compartments including the respiratory tract, with the secretory form of IgA (sIgA) being dominant and uniquely stable in these environments. sIgA is naturally found in human milk, which could be considered a global resource for this biologic, justifying the development of human milk sIgA as a dedicated airway therapeutic for respiratory infections such as SARS-CoV-2. In the present study, methods were therefore developed to efficiently extract human milk sIgA from donors who were either immunologically naive to SARS-CoV-2 (pooled as a control IgA) or had recovered from a PCR-confirmed SARS-CoV-2 infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA Abs in their milk (pooled together to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1.0% or greater were all associated with sIgA. None of the proteins exhibited statistically significant differences between batches. Western blot demonstrated all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher binding to Spike, and was also capable of blocking the Spike - ACE2 interaction in vitro with 6.3x greater potency compared to control IgA (58% inhibition at ~240ug/mL). LCTG-002 was then tested in vivo for its capacity to reduce viral burden in the lungs of K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 was demonstrated to significantly reduce SARS-CoV-2 titers in the lungs compared to control IgA when administered at either 250ug/day or 1 mg/day, as measured by TCID50, plaque forming units (PFU), and qRT-PCR, with a maximum reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure, efficacious, and well tolerated in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics against SARS-CoV-2 and other mucosal infections.

Molecular and Clinical Epidemiology of SARS-CoV-2 Infection Among Vaccinated and Unvaccinated Individuals in a Large Healthcare Organization from New Jersey (preprint)

New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOC) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5,007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOC, including Alpha (n =714), Delta (n =1,877), and Omicron (n =1,802). Omicron isolates were further sub-typed as BA.1 (n =899), BA.2 (n =853), and BA.4/BA.5 (n =50); the remaining 614 isolates were classified as “Other”. Approximately 31.5% (1,577/5,007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p <0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p <0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p <0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.

Assessing Vulnerability to COVID-19 in High-Risk Populations: The Role of SARS-CoV-2 Spike-Targeted Serology (preprint)

Importance: Individuals at increased risk for severe outcomes from COVID-19, due to compromised immunity or other risk factors, would benefit from objective measures of vulnerability to infection based on prior infection and/or vaccination. We reviewed published data to identify a specific role and interpretation of SARS-CoV-2 spike-targeted serology testing for such individuals. We also provide real-world evidence of spike-targeted antibody test results, identifying the seronegativity rate across the United States from March 2021 through June 2022. Analysis of antibody test results were compared between post-transplant (ie, immunocompromised) and all other patients tested in the first half of 2022. Finally, specific recommendations are provided for an evidence-based and clinically useful interpretation of spike-targeted serology to identify vulnerability to infection and potential subsequent adverse outcomes. Observations: Decreased vaccine effectiveness among immunocompromised individuals is linked to correspondingly high rates of breakthrough infections. Evidence indicates that negative results on SARS-CoV-2 antibody tests are associated with increased risk for subsequent infection. Results from widely available, laboratory-based tests do not provide a direct measure of protection but appear to correlate well with the presence of surrogate pseudovirus-neutralizing antibodies. The results of SARS-CoV-2 semiquantitative tests have also been associated with vaccine effectiveness and the likelihood of breakthrough infection. The data suggest that "low-positive" results on semiquantitative SARS-CoV-2 spike-targeted antibody tests may help identify persons at increased relative risk for breakthrough infection leading to adverse outcomes. In an analysis of data from large national laboratories during the COVID-19 Omicron-related surge in 2022, results from SARS-CoV-2 spike-targeted antibody tests were negative in 16.6% (742/4459) of solid organ transplant recipients tested compared to only 11.0% (47,552/432,481) of the remaining tested population. Conclusions and Relevance: Standardized semiquantitative and quantitative SARS-CoV-2 spike-targeted antibody tests may provide objective information on risk of SARS-CoV-2 infection and associated adverse outcomes. This holds especially for high-risk populations, including transplant recipients, who demonstrate a relatively higher rate of seronegativity. The widespread availability of such tests presents an opportunity to refine risk assessment for individuals with suboptimal SARS-CoV-2 antibody levels and to promote effective interventions. Interim federal guidance would support physicians and patients while additional investigations are pursued.

Sex differences in cardio-pulmonary pathology of SARS-CoV2 infected and Trypanosoma cruzi co-infected mice (preprint)

BackgroundCoronavirus disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV2) is a deadly contagious infectious disease. For those who survived COVID-19, post-COVID cardiac damage poses a major threat for the progression of cardiomyopathy and heart failure. Currently, the number of COVID-related cases and deaths are increasing in Latin America, where a major COVID comorbidity is Chagas heart disease (caused by the parasite Trypanosoma cruzi). Here, we investigated the effect of T. cruzi infection on the pathogenesis and severity of CoV2 infection and, conversely, the effect of CoV2 infection on heart pathology during coinfection. Methodology/findingsWe used transgenic human angiotensin-converting enzyme 2 (huACE2) mice infected with CoV2, T. cruzi, or coinfected with both in this study. Our study shows for the first time that white adipose tissue (WAT) serves as a reservoir for CoV2 and the persistence of CoV2 in WAT alters adipose tissue morphology and adipocyte physiology. Our data demonstrate a correlation between the loss of fat cells and the pulmonary adipogenic signaling (via adiponectin isomers) and pathology in CoV2 infection. The viral load in the lungs is inversely proportional to the viral load in WAT, which differs between male and female mice. Our findings also suggest that adiponectin-PPAR signaling may differently regulate Chagas cardiomyopathy in coinfected males and females. ConclusionWe conclude that adipogenic signaling may play important roles in cardio-pulmonary pathogenesis during CoV2 infection and T. cruzi coinfection. The levels of adiponectin isomers differ between male and female mice during CoV2 infection and coinfection with T. cruzi, which may differently regulate inflammation, viral load, and pathology in the lungs of both the sexes. Our findings are in line with other clinical observations that reported that males are more susceptible to COVID-19 than females and suffer greater pulmonary damage.

Emergence of multiple SARS-CoV-2 antibody escape variants in an immunocompromised host undergoing convalescent plasma treatment (preprint)

SARS-CoV-2 Variants of Concerns (VOC), e.g., B.1.351 (20H/501Y.V2) and P1 (20J/501Y.V3), harboring N-terminal domain (NTD) or the receptor-binding domain (RBD) (e.g., E484K) mutations, exhibit reduced in vitro susceptibility to convalescent serum, commercial antibody cocktails, and vaccine neutralization, and have been associated with reinfection. The accumulation of these mutations could be the consequence of intra-host viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on tacrolimus, steroids and convalescent plasma therapy, and identify the emergence of multiple NTD and RBD mutations associated with reduced antibody neutralization as early as three weeks after infection. SARS-CoV-2 genomes from the first swab (Day 0) and three tracheal aspirates (Day 7, 21 and 27) were compared at the sequence level. We identified five different S protein mutations at the NTD or RBD regions from the second tracheal aspirate sample (21 Day). The S:Q493R substitution and S:243-244LA deletion had ~70% frequency, while ORF1a:A138T, S:141-144LGVY deletion, S:E484K and S:Q493K substitutions demonstrated ~30%, ~30%, ~20% and ~10% mutation frequency, respectively. However, the third tracheal aspirate sample collected one week later (Day 27) was predominated by the haplotype of ORF1a:A138T, S:141-144LGVY deletion and S:E484K (> 95% mutation frequency). Notably, S protein deletions (141-144LGVY and 243-244LA deletions in NTD region) and substitutions (Q493K/R and E484K in the RBD region) previously showed reduced susceptibly to monoclonal antibody or convalescent plasma. The observation supports the hypothesis that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune-escape mutants.

CERC-002, a human anti-LIGHT mAb reduces respiratory failure and death in hospitalized COVID-19 ARDS patients (preprint)

Background Severe COVID-19 infection is associated with dysregulated immune response which, in a substantial minority of patients, results in cytokine release syndrome (CRS) and acute respiratory distress syndrome (ARDS). Inhibition of cytokines or cytokine-associated signal transduction is a promising strategy to ameliorate ARDS associated with CRS. We and others have previously shown that serum free LIGHT (TNFSF14) levels are markedly elevated in patients with COVID-19 pneumonia/ARDS10,11, suggesting that LIGHT neutralization may offer therapeutic benefit to COVID-19 ARDS patients. Methods We conducted a randomized, double-blind, placebo-controlled, multi-center, proof-of-concept clinical trial of CERC-002 in adults with mild to moderate ARDS associated with COVID-19 (n=83). Enrolled patients received a single dose of CERC-002 or placebo, in addition to standard of care that included high dose corticosteroids. The primary efficacy endpoint was alive and free of respiratory failure status through Day 28. Secondary outcomes included alive status at Day 28, free of invasive ventilation through Day 28, and serum free LIGHT levels. Results In patients hospitalized with COVID-19 associated pneumonia and mild to moderate (ARDS), CERC-002 increased the rate of alive and free of respiratory failure status through Day 28 as compared to placebo (83.9% vs 64.5%; p=0.044). Efficacy was highest in the prespecified subgroup of patients 60 years old and older (76.5% vs 47.1%; p=0.042), the population most vulnerable to severe complications and death with COVID-19 infection. Through both the initial 28-day and 60-day follow-up periods, reductions of approximately 50% in mortality were observed for CERC-002 compared to placebo (7.7% vs 14.3% at Day 28 and 10.8% vs 22.5% at Day 60). Importantly, this improvement was incremental to standard of care including high dose steroids and remdesivir 88.0% and 57.8%, respectively). In addition, serum LIGHT levels but not IL-6 levels were markedly reduced in patients treated with CERC-002. Conclusions The data presented herein demonstrate that CERC-002 markedly reduces the risk of respiratory failure and death incremental to standard of care including high dose corticosteroids and reduces LIGHT levels in patients with COVID-19 ARDS. (ClinicalTrials.gov number NCT04412057).

A Novel Diagnostic Test to Screen SARS-CoV-2 Variants Containing E484K and N501Y Mutations (preprint)

Spike protein mutations E484K and N501Y carried by SARS-CoV-2 variants have been associated with concerning changes of the virus, including resistance to neutralizing antibodies and increased transmissibility. While the concerning variants are fast spreading in various geographical areas, identification and monitoring of these variants is lagging far behind, due in large part to the slow speed and insufficient capacity of viral sequencing. In response to the unmet need for a fast and efficient screening tool, we developed a single-tube duplex molecular assay for rapid and simultaneous identification of E484K and N501Y mutations from nasopharyngeal swab (NS) samples within 2.5 h from sample preparation to report. Using this tool, we screened a total of 435 clinical NS samples collected from COVID patients at 8 hospitals within the Hackensack Meridian Health network in New Jersey. While B.1.351 and P.1 variants were absent from the current study, our data revealed a dramatic increase in the frequency of E484K over time, underscoring the need for continuous epidemiological monitoring.

An Observational Study of COVID-19 from A Large Healthcare System in Northern New Jersey: Diagnosis, Clinical Characteristics, and Outcomes (preprint)

BackgroundNew Jersey was an early epicenter for the COVID-19 pandemic in the United States, yet information on hospitalized COVID-19 patients from this area is scarce. This study aimed to provide data on demographics and clinical features of a hospitalized patient population who were confirmed with infection by our in-house (CDI) real-time reverse-transcription polymerase chain reaction (RT-PCR) test. MethodsWe included consecutive patients who were admitted to Hackensack Meridian Health system hospitals with laboratory-confirmed diagnoses of COVID-19 at Hackensack University Medical Center by the CDI virus test between March 12, 2020, and April 8, 2020. Clinical data and viral testing results were collected and analyzed for characteristics associated with outcomes, as well as the correlation with viral load. ResultsA total of 722 patients were included in the study, with a median age of 63 (interquartile range (IQR), 51-75) and 272 (37.7%) females. Mortality of this case series was 25.8%, with a statistically significant linear increase observed from age 40 to [≥]80 by 10-year intervals. Viral load, as indicated by the cycle of threshold (Ct) values from the RT-PCR test, was significantly higher in the oldest patient group ([≥]80), and inversely correlated with survival. ConclusionsThis is the first report to describe the clinical characteristics and outcomes in a large hospitalized COVID-19 patient series from New Jersey. Findings from this study are valuable to the ongoing response of both nationwide healthcare networks and the medical research community. SummaryWe describe the diagnosis, clinical characteristics, and outcomes of a large hospitalized patient population in northern New Jersey during the early stages of the COVID-19 pandemic.

Levels of the TNF related cytokine, LIGHT increase in hospitalized COVID-19 patients with Cytokine Release Syndrome and ARDS (preprint)

Many COVID-19 patients demonstrate lethal respiratory complications caused by cytokine release syndrome (CRS). Multiple cytokines have been implicated in CRS, but TNFSF14 (LIGHT) has not been previously measured in this setting. In this study, we observed significantly elevated serum LIGHT levels in hospitalized COVID-19 patients as compared to healthy age and gender matched control patients. The assay detected bioavailable LIGHT unbound to the inhibitor Decoy receptor-3 (DcR3). Bioavailable LIGHT levels were elevated in patients both on and off ventilatory support, with a trend toward higher levels in patients requiring mechanical ventilation. In hospitalized patients over the age of 60, who exhibited a mortality rate of 82%, LIGHT levels were significantly higher (p=0.0209) in those who died compared to survivors. As previously reported, interleukin 6 (IL-6) levels were also elevated in these patients with significantly (p=0.0076) higher levels observed in patients who died vs. survivors, paralleling the LIGHT levels. Although attempts to block IL-6 binding to its receptor have shown limited effect in COVID-19 CRS, neutralization of LIGHT may prove to be more effective owing to its more central role in regulating antiviral immune responses.