Search

Background Limited therapeutic options exist for coronavirus disease 2019 (COVID-19). COVID-19 convalescent plasma (CCP) is a potential therapeutic, but there is limited data for patients with moderate-to-severe disease. Research question What are outcomes associated with administration of CCP in patients with moderate-to-severe COVID-19 infection? Study design and methods We conducted a propensity score-matched analysis of patients with moderate-to-severe COVID-19. The primary endpoints were in-hospital mortality. Secondary endpoints were number of days alive and ventilator-free at 30 days; length of hospital stay; and change in WHO scores from CCP administration (or index date) to discharge. Of 151 patients who received CCP, 132 had complete follow-up data. Patients were transfused after a median of 6 hospital days; thus, we investigated the effect of convalescent plasma before and after this timepoint with 77 early (within 6 days) and 55 late (after 6 days) recipients. Among 3,217 inpatients who did not receive CCP, 2,551 were available for matching. Results Early CCP recipients, of whom 31 (40%) were on mechanical ventilation, had lower 14-day (15% vs 23%) and 30-day (38% vs 49%) mortality compared to a matched unexposed cohort, with nearly 50% lower likelihood of in-hospital mortality (HR 0.52, [95% CI 0.28–0.96]; P = 0.036). Early plasma recipients had more days alive and ventilator-free at 30 days (+3.3 days, [95% CI 0.2 to 6.3 days]; P = 0.04) and improved WHO scores at 7 days (-0.8, [95% CI: -1.2 to -0.4]; P = 0.0003) and hospital discharge (-0.9, [95% CI: -1.5 to -0.3]; P = 0.004) compared to the matched unexposed cohort. No clinical differences were observed in late plasma recipients. Interpretation Early administration of CCP improves outcomes in patients with moderate-to-severe COVID-19, while improvement was not observed with late CCP administration. The importance of timing of administration should be addressed in specifically designed trials.

Background: Coronavirus disease 2019 (COVID-19), a multi-system disorder resulting from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected 5.0 million individuals in the United States (US) resulting in more than 160,000 deaths. There is currently no proven effective therapy for SARS-CoV-2 infection beyond supportive care. Convalescent plasma (CP), collected from individuals who have recovered from SARS-CoV-2 infection, has been safely used to transfer virus-neutralizing antibodies to patients hospitalized with COVID-19. Herein we report on the clinical course and outcomes of patients who received CP for the treatment of COVID-19 across our health system. Methods: Hospitalized patients ≥ 18 years of age at 5 hospitals in the Yale-New Haven Health System with confirmed SARS-CoV-2 infection and severe or life-threatening disease (Figure 1) were enrolled in the US COVID-19 Expanded Access Program (EAP) and transfused 1 unit of CP. Patient outcomes were assessed at 7, 14, and 28 days after CP transfusion. Furthermore, we sought to determine mortality predictors in this patient cohort. Results: 105 patients enrolled in the EAP were transfused CP from 4/12/2020 to 6/14/2020. Median age was 62 years (28-88 years). 62.9% of patients were male. Individuals of a non-white race comprised 56.2% of our cohort and 33.3% identified as Hispanic or Latinx. 47.6% of patients had a body mass index greater than 30, with 13.3% classified as extremely obese. Diabetes mellitus, hypertension, and hyperlipidemia were highly prevalent in our population. Concomitant therapy for COVID-19 included hydroxychloroquine (79.0%), tocilizumab (79.0%), and remdesivir (23.8%) (Table 1). 91 patients required the intensive care unit (ICU) during their hospital course, with 87 patients in the ICU at the time of CP transfusion. 42.9% of patients had an improvement in their World Health Organization (WHO) Ordinal Scale score by day 7 post-CP transfusion (median 4 days to improvement), while 9.5% demonstrated an increase in their WHO score indicating worsening oxygenation during the 7 days following CP (Figure 2). Mortality was 10.48% at 7 days, 20.95% at 14 days, and 28.57% at 28 days post-CP transfusion (Figure 2). Of the patients in the ICU at the time of CP, 56 (64.4%) de-escalated to non-ICU care after a median of 8 days from transfusion and 55 (63.2%) were ultimately discharged from the hospital. The median time from CP transfusion to discharge in survivors was 14 days (2-103 days), while median time from CP to death in non-survivors was 10 days (1-76 days). The mean time in days both from hospital admission to CP and from positive SARS-CoV-2 polymerase chain reaction (PCR) to CP trended slightly longer in non-survivors when compared to survivors (8.6 vs 6.9 days, p=0.218 and 9.7 vs 7.3 days, p=0.85, respectively). D-dimer levels >5 milligrams per liter fibrinogen equivalent units (mg/L FEU) were associated with increased mortality at 24 (OR 2.79, 95% CI 1.18-6.72), 48 (OR 3.64, 95% CI 1.44-9.70), and 72 hours (OR 2.99, 95% CI 1.23-7.66) post-CP transfusion (Figure 3). Twelve patients (11.4%) on prophylactic or intermediate dose anticoagulation were subsequently escalated to therapeutic dosing during the 7 days after CP transfusion. Furthermore, a ferritin level of >3000 nanograms per milliliter (ng/mL) on the day of CP was associated with increased mortality (OR 5.14, 95% CI 1.34-19.68). Conclusions: CP therapy was administered through the EAP to COVID-19 patients with severe disease and with advanced age, high frequency of obesity, and extensive co-morbidity. In this COVID-19 population with high expected mortality, 42.9% of patients improved by WHO Ordinal Scale score within 7 days of CP transfusion and 52.4% were alive and discharged at 28 days. Baseline ferritin and d-dimer levels at 24, 48, and 72 hours post-CP were predictors of mortality, the latter raising the possibility that the increased thrombotic risk associated with COVID-19 may be exacerbated by administration of blood products from donors potentially in a residual procoagulant state. Further studies by our group are underway to assess hemostatic balance in donor CP, to estimate associations with outcomes by comparing patients who received CP with a matched control cohort of COVID-19 patients who did not receive CP, and to evaluate efficacy of CP in a randomized, placebo-controlled clinical trial. Disclosures McLeod: Gilead Pharmaceuticals: Other: Medical Advisor . OffLabel Disclosure: We have described in this abstract patients across our health system who received investigational convalescent plasma therapy through enrollment in the "Expanded Access to Convalescent Plasma for the Treatment of Patients with COVID-19" protocol that is funded by BARDA and run by the Mayo Clinic, where primary IRB protocol is held. Previous guidance issued by the FDA regarding investigational use of COVID-19 convalescent plasma included recommendations for the expanded access program as a method by which to facilitate access to this blood product across the nation. Safety data reported thus far from the expanded access program has demonstrated no concerning findings, with a very low rate of serious adverse events. Therefore given the overall tolerability of convalescent plasma and the lack of other proven therapies for COVID-19, we believe it is important to report these findings even in the absence of current approval for convalescent plasma transfusion in the management of COVID-19.

Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm’s pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate., Author summary Human whipworm (Trichuris trichiura) is pervasive intestinal parasite that causes malnutrition and inflammatory bowel disease (IBD), predominately among the world’s most impoverished billion people. Available treatment strategies have had little impact on the disease prevalence or morbidity. A vaccine against human whipworm would provide a low-cost and long-term solution, yet the identification of candidates is lacking. Here we characterize a stichosome-derived whey acidic protein (WAP), dominant among the worm’s excretory and secretory (ES) products, as a potential vaccine candidate. When delivered as a subcutaneous recombinant protein formulated with the clinically relevant adjuvant, Montanide ISA 720, mice were significantly protected against challenge infection. We describe the induced protection as a combined WAP-specific type 2 cellular and humoral immune response. In summary, we have identified an immunogenic whipworm protein that is effective in inducing protective efficacy in an animal model and is a promising human vaccine candidate.

Background Ascariasis remains the most common helminth infection in humans. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development targeting Ascaris, hookworm, and Trichuris infections. As16 and As14 have previously been described as two genetically related proteins from Ascaris suum that induced protective immunity in mice when formulated with cholera toxin B subunit (CTB) as an adjuvant, but the exact protective mechanism was not well understood. Methodology/Principal findings As16 and As14 were highly expressed as soluble recombinant proteins (rAs16 and rAs14) in Pichia pastoris. The yeast-expressed rAs16 was highly recognized by immune sera from mice infected with A. suum eggs and elicited 99.6% protection against A. suum re-infection. Mice immunized with rAs16 formulated with ISA720 displayed significant larva reduction (36.7%) and stunted larval development against A. suum eggs challenge. The protective immunity was associated with a predominant Th2-type response characterized by high titers of serological IgG1 (IgG1/IgG2a > 2000) and high levels of IL-4 and IL-5 produced by restimulated splenocytes. A similar level of protection was observed in mice immunized with rAs16 formulated with alum (Alhydrogel), known to induce mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both known to induce a Th1-type biased response, were not significantly protected against A. suum infection. The rAs14 protein was not recognized by A. suum infected mouse sera and mice immunized with rAs14 formulated with ISA720 did not show significant protection against challenge infection, possibly due to the protein’s inaccessibility to the host immune system or a Th1-type response was induced which would counter a protective Th2-type response. Conclusions/Significance Yeast-expressed rAs16 formulated with ISA720 or alum induced significant protection in mice against A. suum egg challenge that associates with a Th2-skewed immune response, suggesting that rAS16 could be a feasible vaccine candidate against ascariasis., Author summary Roundworms (Ascaris) infect more than 700 million people living in poverty worldwide and cause malnutrition and physical and mental developmental delays in children. As an alternative or complementary approach to global deworming, a pan-anthelminthic vaccine is under development that targets ascariasis in addition to other human intestinal nematode infections. Towards this goal, two Ascaris suum antigens, As16 and As14, were expressed in Pichia pastoris as recombinant proteins. Mice immunized with rAs16 formulated with ISA720 adjuvant produced significant larva reduction (36.7%) and stunted larval development against A. suum egg challenge. The protection was associated with predominant Th2-type responses characterized by high levels of serological IgG1 (IgG1/IgG2a > 2,000) and Th2 cytokines, IL-4 and IL-5. A similar level of protection was observed in mice immunized with rAs16 formulated with alum that induces mainly a Th2-type immune response, whereas mice immunized with rAs16 formulated with MPLA or AddaVax, both inducing major Th1-type responses, were not significantly protected against A. suum infection. High-yield expression of rAs16 in yeast will allow for large-scale manufacture, and its protective efficacy when formulated with alum suggests its suitability as a vaccine candidate.

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity.

OBJECTIVES/SPECIFIC AIMS: Trichuris trichiura, is a leading cause of chronic colitis worldwide, resulting in growth stunting, anemia, and cognitive deficits, predominately in children. Our long-term goal is to develop a vaccine against T. trichiura. Both T. trichiura and the closely related Trichuris muris release excretory/secretory (ES) macromolecules from the stichosome organ, which facilitates intracellular invasion into the cecum. We exploited the high degree of genetic sequence homology between T. trichiura and T. muris to evaluate T. muris stichosome proteins as vaccine candidates. METHODS/STUDY POPULATION: Through immunological screening of the T. muris stichosome cDNA library and T. muris ES macromolecules generated in culture, we identified, cloned, and expressed several vaccine candidates. In ranking these antigens, we selected the most promising recombinant proteins, WAP and CAP-1, and vaccinated AKR mice to evaluate the immunogenicity and efficacy of our candidate. In addition, we collected 240 serum samples in the T. trichiura endemic region of Honduras to measure the recognition of WAP and CAP-1 in naturally infected human. RESULTS/ANTICIPATED RESULTS: We measured a statistically significant reduction in larval worm burden in WAP and ES vaccinated mice, but not CAP-1, by microscopy and real-time PCR of T. muris DNA. We found a significant relationship between antigen-specific IgG1:IgG2a ratio in the mouse serum and degree of protection. Mouse splenocytes, vaccine-sensitized draining lymph nodes, and mesenteric lymph nodes were antigen-stimulated and their secreted Th1, Th2, and Th17 cytokines were measured by Luminex®. Stimulated mouse lymphoid cells were further analysed for T helper, T cytotoxic, and central/effector memory T cells by Flow Cytometry. Human IgG1, IgG2, and IgE antibody titers against WAP and CAP-1 were measured by ELISA. DISCUSSION/SIGNIFICANCE OF IMPACT: In our study, we describe the T cell dependent mechanism of humoral immunity of 2 promising ES-derived vaccines recombinant proteins, WAP and CAP-1. We evaluated the immune response, indicating a driving a Th2-induced humoral response necessary for protection. We further predict protection and allergenicity of WAP in humans using serum from a cohort in an T. trichiura endemic region.

Ascaris lumbricoides The Sabin Vaccine Institute Product Development Partnership is developing a Pan-anthelmintic vaccine that simultaneously targets the major soil-transmitted nematode infections, in other words, ascariasis, trichuriasis and hookworm infection. The approach builds off the current bivalent Human Hookworm Vaccine now in clinical development and would ultimately add both a larval Ascaris lumbricoides antigen and an adult-stage Trichuris trichiura antigen from the parasite stichosome. Each selected antigen would partially reproduce the protective immunity afforded by UV-attenuated Ascaris eggs and Trichuris stichosome extracts, respectively. Final antigen selection will apply a ranking system that includes the evaluation of expression yields and solubility, feasibility of process development and the absence of circulating antigen-specific IgE among populations living in helminth-endemic regions. Here we describe a five year roadmap for the antigen discovery, feasibility and antigen selection, which will ultimately lead to the scale-up expression, process development, manufacture, good laboratory practices toxicology and preclinical evaluation, ultimately leading to Phase 1 clinical testing.

Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.

Author Summary Current iterations of the hygiene hypothesis suggest an adaptive role for helminth parasites in shaping the proper maturation of the immune system. However, aspects of this hypothesis are based on assumptions that may not fully account for realities about human helminth infections. Such realities include evidence of causal associations between helminth infections and asthma or inflammatory bowel disease as well as the fact that helminth infections remain widespread in the United States, especially among populations at greatest risk for inflammatory and autoimmune diseases.

Soil-transmitted helminth (STH) infections, specifically Ascaris, Trichuris, and Hookworm, compose three of the most prevalent Neglected Tropical Diseases, infecting over a billion people worldwide. Recurrent childhood STH infections have been shown to cause stunted physical growth, reduced physical fitness, and decreased school performance. Ascaris and Trichuris are of notable interest, given their abrupt decline from a peak prevalence and intensity in pre-adolescence to adulthood, with no known published works evaluating the possible underlying immunological mechanism. We used modern molecular techniques to (a) determine the burden of disease and (b) evaluate the immune profile of 30 adolescents and 51 adults between the ages 13-45, in rural Honduras, endemic for Ascaris and Trichuris. Using quantitative PCR of DNA extracted from stool we quantified the burden of disease of both Ascaris and Trichuris. Positive samples were stratified into groups based on the degree of infection, and controlled for with samples positive for 6 other common gastrointestinal parasites. Each group was immunologically characterized for serum Th1, Th2, and Th17 cytokines and associated antibodies. Results from this pilot study identify the basic serum immune profile associated with protection against Ascaris and Trichuris and serve as the foundation for a second, more comprehensive study that will guide vaccine antigen selection against each parasite by mimicking endemic host protective responses.