Preliminary Evaluation of QuantiFERON SARS-CoV-2 and QIAreach Anti-SARS-CoV-2 Total Test in Recently Vaccinated Individuals.

INTRODUCTION: There is an increasing body of evidence surrounding the importance of a T cell-mediated response to SARS-CoV-2 infection and after COVID-19 vaccination. In this internal feasibility study, we evaluated both the total antibody (IgA, IgM, and IgG) and T cell responses in a cohort of COVID-19 convalescents and vaccinated individuals. METHODS: Whole blood specimens were collected weekly from 12 subjects at different time points within/after the COVID-19 mRNA vaccination regimen, and from 4 PCR-confirmed convalescent donors to measure durability of humoral and cell-mediated immune response. T cell and antibody responses were evaluated via the QuantiFERON SARS-CoV-2 research use only (QFN SARS-CoV-2) assay which is an interferon gamma release assay (IGRA) and QIAreach Anti-SARS-CoV-2 total (Anti-CoV-2) test, respectively. RESULTS: In a cohort of recently vaccinated individuals, subjects demonstrated robust total antibody and CD4+/CD8+ T cell response to SARS-CoV-2 mRNA vaccines when followed for 2 months post-2nd dose. In most individuals, T cell response declined between the 1st and 2nd doses suggesting a need for a booster or the completion of the 2-dose vaccine series. In a group of convalescent donors tested with QFN SARS-CoV-2 and Anti-CoV-2 tests, all patients had an antibody and T cell response up to 1 year after natural infection. CONCLUSION: This small feasibility study demonstrates that the QFN-SARS-CoV-2 test is able to identify CD4+ and CD8+ T cell-mediated responses in SARS-CoV-2-vaccinated subjects and those recovered from COVID-19, alongside a qualitative antibody response detectable via the QIAreach Anti-CoV2 test.

Direct Diagnostic Tests for Lyme Disease.

Borrelia burgdorferi was discovered to be the cause of Lyme disease in 1983, leading to seroassays. The 1994 serodiagnostic testing guidelines predated a full understanding of key B. burgdorferi antigens and have a number of shortcomings. These serologic tests cannot distinguish active infection, past infection, or reinfection. Reliable direct-detection methods for active B. burgdorferi infection have been lacking in the past but are needed and appear achievable. New approaches have effectively been applied to other emerging infections and show promise in direct detection of B. burgdorferi infections.

Advances in Serodiagnostic Testing for Lyme Disease Are at Hand.

The cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Detection of IFN-γ Secretion by T Cells Collected Before and After Successful Treatment of Early Lyme Disease.

BACKGROUND: Current serodiagnostics for Lyme disease lack sensitivity during early disease, and cannot determine treatment response. We evaluated an assay based on QuantiFERON technology utilizing peptide antigens derived from Borrelia burgdorferi to stimulate interferon-gamma (IFN-γ) release as an alternative to serodiagnosis for the laboratory detection of Lyme disease. METHODS: Blood was obtained from patients with erythema migrans before (n = 29) and 2 months after (n = 27) antibiotic therapy. IFN-γ release was measured by enzyme-linked immunosorbent assay (ELISA) following overnight stimulation of whole blood with the peptide antigens, and compared to the results of standard serological assays (C6, ELISA, and Western blot). RESULTS: IFN-γ release was observed in pretreatment blood of 20 of 29 (69%) patients with Lyme disease. Following antibiotic treatment, IFN-γ was significantly reduced (P = .0002), and was detectable in only 4 of 20 (20%) initially positive patients. By contrast, anti-C6 antibodies were detected in pretreatment sera from 17 of 29 (59%) subjects, whereas only 5 of 29 (17%) patients had positive Western blot seroreactivity. Antibody responses persisted and expanded following treatment. CONCLUSIONS: Our findings suggest that measurement of IFN-γ after incubating blood with Borrelia antigens could be useful in the laboratory diagnosis of early Lyme disease. Also, after antibiotic treatment, this response appears to be short lived.

ISCOMATRIX vaccines mediate CD8+ T-cell cross-priming by a MyD88-dependent signaling pathway.

Generating a cytotoxic CD8(+) T-cell response that can eradicate malignant cells is the primary objective of cancer vaccine strategies. In this study we have characterized the innate and adaptive immune response to the ISCOMATRIX adjuvant, and the ability of vaccine antigens formulated with this adjuvant to promote antitumor immunity. ISCOMATRIX adjuvant led to a rapid innate immune cell response at the injection site, followed by the activation of natural killer and dendritic cells (DC) in regional draining lymph nodes. Strikingly, major histocompatibility complex (MHC) class I cross-presentation by CD8α(+) and CD8α(-) DCs was enhanced by up to 100-fold when antigen was formulated with ISCOMATRIX adjuvant. These coordinated features enabled efficient CD8(+) T-cell cross-priming, which exhibited prophylactic and therapeutic tumoricidal activity. The therapeutic efficacy of an ISCOMATRIX vaccine was further improved when co-administered with an anti-CD40 agonist antibody, suggesting that ISCOMATRIX-based vaccines may combine favorably with other immune modifiers in clinical development to treat cancer. Finally, we identified a requirement for the myeloid differentiation primary response gene 88 (MyD88) adapter protein for both innate and adaptive immune responses to ISCOMATRIX vaccines in vivo. Taken together, our findings support the utility of the ISCOMATRIX adjuvant for use in the development of novel vaccines, particularly those requiring strong CD8(+) T-cell immune responses, such as therapeutic cancer vaccines.

Development of prophylactic and therapeutic vaccines using the ISCOMATRIX adjuvant.

Adjuvants are components that when added to subunit antigen (Ag) vaccines boost their immunogenicity and thus immune efficacy. However, there are few adjuvants that are approved for clinical use resulting in a critical need for the development of safe and effective adjuvants for use in both prophylactic and therapeutic vaccines. The paucity of appropriate adjuvants is more chronic for the development of therapeutic vaccines for cancer and chronic infectious disease, which need to induce cytotoxic T-cell responses via cross-presentation of the vaccine Ag by dendritic cells. The ISCOMATRIX adjuvant represents a unique adjuvant system that facilitates Ag delivery and presentation as well as immunomodulation to provide enhanced and accelerated immune responses. The immune responses generated are of broad specificity to the vaccine Ag, and include robust antibody responses of multiple subclasses as well as both CD4(+) and CD8(+) T-cell responses. Here we discuss our understanding of the mechanisms of action by which ISCOMATRIX adjuvant may facilitate these integrated immune responses and touch on insights gained through its clinical experience.

Risk factors for medical withdrawals in United States tennis association junior national tennis tournaments: a descriptive epidemiologic study.

BACKGROUND: There have been no large-scale epidemiologic studies designed to determine the risk factors related to medical withdrawal in United States Tennis Association junior national tennis tournaments. HYPOTHESIS: Risk of medical withdrawal will increase on the basis of cumulative match volume within a tournament. METHODS: A retrospective, cross-sectional analysis was performed of data collected for every match of all 4 United States Tennis Association supernational tournaments (spring, summer, fall, winter) for the boys and girls divisions and for all age divisions (12, 14, 16, 18) during a single year (2005). Logistic regression analysis was performed to determine relative risk of all risk factors. Additional analysis was performed to determine the threshold (ie, match number) at which a player would substantially increase his or her risk of medical withdrawal. RESULTS: A total of 28336 match exposures were analyzed, with an even distribution with respect to sex and age. The total medical withdrawal rate was 15.6 per 1000 match exposures. Every factor of interest was highly significant in predicting a higher rate of medical withdrawal per 1000 match exposures. The medical withdrawal rate was significantly higher in the fifth match or greater (26.3/1000) versus the first 4 matches (12.7/1000; P < .0001), even when analyzing main draw and singles matches. CONCLUSION: In United States Tennis Association national junior tennis tournaments, there is a significant increase in risk of medical withdrawals directly related to older age divisions, boys, singles matches, and main draw matches. Specifically, there is a significant increase in medical withdrawal rate beyond the fourth match in the tournament regardless of whether it is a main draw, consolation, or singles match. CLINICAL RELEVANCE: Recommendations can be made to exercise caution in tournaments that involve boys, older age divisions, and singles. In addition, there may be sufficient evidence to suggest intervention within a tournament when players play beyond their fourth match.

Functional recovery after anterior cruciate ligament reconstruction: a longitudinal perspective.

OBJECTIVE: To evaluate functional hop performance in subjects with an anterior cruciate ligament (ACL) reconstruction with a bone-patellar tendon-bone graft during 12, 18, 26, 39, and 52 weeks. DESIGN: A longitudinal comparative study. SETTING: University research laboratory. PARTICIPANTS: Patients (N=19) were evaluated at 12, 18, 26, 39, and 52 weeks after ACL reconstruction surgery. INTERVENTION: Testing on 5 separate occasions. MAIN OUTCOME MEASURES: The Cincinnati Knee Rating System and analog scales, the 6-meter timed hop, crossover hop, stair hop, and vertical hop, and limb symmetry indices. RESULTS: The uninjured and injured legs and test order were randomized. There was a significant test occasion main effect for both the Cincinnati and analog scores (P=.001). Subjective rating scores improved over the 5 testing occasions. For all 4 hop tests, test occasion and limb main effects were significant (P=.001). Paired t test comparisons at each testing occasion indicated a significant difference between the reconstructed and uninjured limb (P<.05). Furthermore, significant test occasion main effects were noted for limb symmetry indices for the 4 hop tests (P=.001). Using a score of greater than or equal to 85% as a criterion for normative limb symmetry, normative scores were recorded in the 6-m timed hop at the week 18 test occasion, the stair hop and vertical hop at the week 26 test occasion, and the crossover hop at the week 39 test occasion. CONCLUSIONS: These hop tests showed different levels of imposed demands on the knee that could be used to assess functional recovery and readiness to resume sport.

Normal proportion and expression of maturation markers in migratory dendritic cells in the absence of germs or Toll-like receptor signaling.

Dendritic cells (DCs) play major roles in immunosurveillance. In peripheral tissues, 'immature' DCs are dedicated to capturing antigens. Detection of pathogens through Toll-like receptors (TLRs) triggers DC migration to the lymph nodes (LNs), where they acquire a 'mature' phenotype specialized at presenting antigens. However, DCs migrate from tissues and mature even in the absence of overt infections. This has been attributed to detection of commensal flora in the skin, the gut or other peripheral tissues in the steady state. To test this assumption, we have analyzed the DCs contained in the lymphoid organs of germ-free mice and of mice lacking the TLR adapter molecules, MyD88 and TRIF. We show that the proportion and expression of maturation markers in DC immigrants in the LNs of these mice are similar to those in normal mice. These results suggest that DC migration from tissues, followed by their phenotypic maturation, is regulated in the steady state by an inherent program of DC differentiation or by the release of low levels of inflammatory signals from normal tissues.

ISCOMATRIX adjuvant for prophylactic and therapeutic vaccines.

The ISCOMATRIX adjuvant has antigen-delivery and -presentation properties, as well as immunomodulatory capabilities that combine to provide enhanced and accelerated immune responses. The responses are broad, including a range of subclasses of antibodies as well as both CD4+ and CD8+ T cells. A range of ISCOMATRIX vaccines (ISCOMATRIX adjuvant combined with antigen) have been evaluated in clinical trials. The results of these completed and ongoing studies indicate that the ISCOMATRIX adjuvant is safe and generally well tolerated and increases the vaccine immune responses.

The utility of ISCOMATRIX adjuvant for dose reduction of antigen for vaccines requiring antibody responses.

The capacity of an adjuvant to reduce the amount of antigen required in vaccines would be beneficial in a variety of settings, including situations where antigen is difficult or expensive to manufacture, or in situations where demand exceeds production capacity, such as pandemic influenza. The ability to reduce antigen dose would also be a significant advantage in combination vaccines, and vaccines that by necessity must contain multiple antigens to accommodate variability between strains or genotypes. ISCOMATRIX adjuvant was compared to aluminium hydroxide adjuvant (Al(OH3)) for induction of antibody responses and dose sparing of a recombinant HIV gp120 vaccine. Neutralising antibody responses were significantly greater, at the same protein dose, when the gp120 protein was formulated with ISCOMATRIX adjuvant compared to Al(OH3). Moreover, strong responses were achieved with up to 100-fold lower doses of gp120 using ISCOMATRIX adjuvant. Therefore, ISCOMATRIX adjuvant has the potential to substantially reduce the dose of antigen required in human vaccines, without compromising the immune response.

Gene gun immunization in a preclinical model is enhanced by B7 targeting.

DNA vaccines have great potential but despite the promise shown in rodent models, responses in large animals, including humans, have been disappointing. Furthermore, gene gun delivery of DNA has been used to improve these responses. However, most cells that are transfected are not the professional antigen presenting cells (APC) which are critical for generating the primary immune response. Here, we show that in the large animal model of the pig, the combination of the use of gene gun delivery and a DNA vector that targets antigen presenting cells by expressing a CTLA4-ovalbumin (OVA) fusion antigen, leads to enhanced ovalbumin specific serum IgG, IgA, IgG1 and IgG2 immune responses.