Development of a Kinetic ELISA and Reactive B Cell Frequency Assay to Detect Respiratory Syncytial Virus Pre-Fusion F Protein-Specific Immune Responses in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants, making vaccination an attractive preventive strategy. Due to earlier reports of vaccine-enhanced disease in RSV-naive children, assessing prior RSV infection is critical for determining eligibility for future infant vaccine trials. However, this is complicated by the presence of maternally transferred maternal antibodies. We sought to develop assays that measure immune responses to RSV pre-fusion (F) protein that discriminates between maternal and infant responses. METHODS: We measured RSV-specific responses in two groups of children <3 years of age; those with laboratory-confirmed RSV (RSV-infected) and those enrolled prior to their first RSV season (RSV-uninfected). Serial blood samples were obtained and recent infections with RSV and other respiratory viruses were assessed during follow-up. An RSV pre-F-specific kinetic enzyme-linked immunosorbent assay (kELISA) and an F-specific reactive B cell frequency (RBF) assay were developed. RESULTS: One hundred two young children were enrolled between July 2015 and April 2017; 74 were in the RSV-uninfected group and 28 were in the RSV-infected group. Participants were asked to provide sequential blood samples over time, but only 53 participants in the RSV-uninfected group and 22 participants in the RSV-infected groups provided multiple samples. In the RSV-infected group, most had positive kELISA and RBF during the study. In the RSV-uninfected group, two patterns emerged: declining kELISA values without reactive B cells, due to maternal transplacental antibody transfer, and persistently positive kELISA with reactive B cells, due to asymptomatic undiagnosed RSV infection. CONCLUSIONS: A kELISA targeting RSV pre-F epitopes and an RBF assay targeting RSV F-specific B cells generally allow discrimination between maternally and infant-derived antibodies.

Neutralizing COVID-19 Convalescent Plasma in Adults Hospitalized With COVID-19: A Blinded, Randomized, Placebo-Controlled Trial.

BACKGROUND: Convalescent plasma has been one of the most common treatments for COVID-19, but most clinical trial data to date have not supported its efficacy. RESEARCH QUESTION: Is rigorously selected COVID-19 convalescent plasma with neutralizing anti-SARS-CoV-2 antibodies an efficacious treatment for adults hospitalized with COVID-19? STUDY DESIGN AND METHODS: This was a multicenter, blinded, placebo-controlled randomized clinical trial among adults hospitalized with SARS-CoV-2 infection and acute respiratory symptoms for < 14 days. Enrolled patients were randomly assigned to receive one unit of COVID-19 convalescent plasma (n = 487) or placebo (n = 473). The primary outcome was clinical status (disease severity) 14 days following study infusion measured with a seven-category ordinal scale ranging from discharged from the hospital with resumption of normal activities (lowest score) to death (highest score). The primary outcome was analyzed with a multivariable ordinal regression model, with an adjusted odds ratio (aOR) < 1.0 indicating more favorable outcomes with convalescent plasma than with placebo. In secondary analyses, trial participants were stratified according to the presence of endogenous anti-SARS-CoV-2 antibodies ("serostatus") at randomization. The trial included 13 secondary efficacy outcomes, including 28-day mortality. RESULTS: Among 974 randomized patients, 960 were included in the primary analysis. Clinical status on the ordinal outcome scale at 14 days did not differ between the convalescent plasma and placebo groups in the overall population (aOR, 1.04; one-seventh support interval [1/7 SI], 0.82-1.33), in patients without endogenous antibodies (aOR, 1.15; 1/7 SI, 0.74-1.80), or in patients with endogenous antibodies (aOR, 0.96; 1/7 SI, 0.72-1.30). None of the 13 secondary efficacy outcomes were different between groups. At 28 days, 89 of 482 (18.5%) patients in the convalescent plasma group and 80 of 465 (17.2%) patients in the placebo group had died (aOR, 1.04; 1/7 SI, 0.69-1.58). INTERPRETATION: Among adults hospitalized with COVID-19, including those seronegative for anti-SARS-CoV-2 antibodies, treatment with convalescent plasma did not improve clinical outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov; No.: NCT04362176; URL: www. CLINICALTRIALS: gov.

Greater activation of peripheral T follicular helper cells following high dose influenza vaccine in older adults forecasts seroconversion.

BACKGROUND: Influenza related morbidity and mortality disproportionately impacts older adults. The serologic response to vaccine is diminished in older adults; however, high dose inactivated influenza vaccine (HD IIV) has shown improved rates of seroconversion compared to standard dose (SD IIV). We hypothesize this may be due to the superior ability of high dose vaccine to activate T follicular helper (Tfh) cells and provide B cell dependent T cell help. METHODS: We measured peripheral Tfh (pTfh) activation in 50 community dwelling adults 65years or older who were randomly assigned to receive either the HD IIV or SD IIV. RESULTS: The HD vaccination elicited significantly higher levels of ICOS expression on pTfh cells, at day 7 compared to SD vaccination (p=0.02). The magnitude of the increase in ICOS+ pTfh cells from baseline to day 7 was predictive of seroconversion for both influenza A and B vaccination. CONCLUSION: Strong Tfh activation in response to influenza vaccination forecasts successful seroconversion in older adults, and HD IIV elicits greater Tfh activation than SD IIV. Future vaccine studies should focus on ways to further optimize the Tfh response.

H7N9 influenza virus neutralizing antibodies that possess few somatic mutations.

Avian H7N9 influenza viruses are group 2 influenza A viruses that have been identified as the etiologic agent for a current major outbreak that began in China in 2013 and may pose a pandemic threat. Here, we examined the human H7-reactive antibody response in 75 recipients of a monovalent inactivated A/Shanghai/02/2013 H7N9 vaccine. After 2 doses of vaccine, the majority of donors had memory B cells that secreted IgGs specific for H7 HA, with dominant responses against single HA subtypes, although frequencies of H7-reactive B cells ranged widely between donors. We isolated 12 naturally occurring mAbs with low half-maximal effective concentrations for binding, 5 of which possessed neutralizing and HA-inhibiting activities. The 5 neutralizing mAbs exhibited narrow breadth of reactivity with influenza H7 strains. Epitope-mapping studies using neutralization escape mutant analysis, deuterium exchange mass spectrometry, and x-ray crystallography revealed that these neutralizing mAbs bind near the receptor-binding pocket on HA. All 5 neutralizing mAbs possessed low numbers of somatic mutations, suggesting the clones arose from naive B cells. The most potent mAb, H7.167, was tested as a prophylactic treatment in a mouse intranasal virus challenge study, and systemic administration of the mAb markedly reduced viral lung titers.

Tissue-specific regulatory T cells: biomarker for acute graft-vs-host disease and survival.

Regulatory T cells (Tregs) are a subset of CD4(+) T cells that are characterized by the expression of CD25 and Foxp3 and are capable of suppressing alloimmune responses. We assessed whether high frequencies of circulating skin or gut tissue-specific Tregs at engraftment could predict acute graft-vs-host disease (aGVHD) incidence and survival in a cohort of hematopoietic cell transplant (HCT) recipients. Tregs were analyzed at engraftment in 74 patients receiving HCT. Treg skin-homing (CLA(+)) or gut-homing (α(4)ß(7)(+)) subsets were identified by flow cytometry, and patients were divided into high CLA(+) Tregs or high α(4)ß(7)(+) Tregs groups, using the 75(th) percentile of tissue-specific Treg percentages as a threshold. At day +100 post-HCT, the cumulative incidence of any stage skin or gut aGVHD was significantly lower in those patients with high CLA(+) Tregs or high α(4)ß(7)(+) Tregs at engraftment, respectively (high CLA(+) Tregs, 24.0% vs low CLA(+) Tregs, 55.1%; p = 0.011 for skin aGVHD or high α(4)ß(7)(+) Tregs, 47.3% vs low α(4)ß(7)(+) Tregs, 74.5%; p = 0.029 for gut aGVHD). The 2-year probabilities of overall survival and nonrelapse mortality were 73.4% and 7.5% among patients with high frequencies of tissue-specific Tregs vs 49.4% and 36.1% for those with both low CLA(+) Tregs and low α(4)ß(7)(+) Tregs (p = 0.039, p = 0.010). These results suggest that a threshold value for CLA(+) or α(4)ß(7)(+) Tregs could be used to predict important HCT outcomes, and to direct the rationale use of tissue-specific pre-emptive therapies to decrease clinical aGVHD and improve HCT survival.

Predicting posttransplantation diabetes mellitus by regulatory T-cell phenotype: implications for metabolic intervention to modulate alloreactivity.

Chronic inflammation and decreased frequency of regulatory T cells (Tregs) in visceral adipose tissue contribute to the propagation of insulin resistance to diabetes mellitus. We tested the hypothesis that new-onset posttransplantation diabetes mellitus (PTDM) is associated with measurable changes in Treg subsets after allogeneic hematopoietic stem cell transplantation (HSCT). PTDM before day 100 and Treg phenotype at engraftment were determined in 36 HSCT recipients without preceding history of diabetes mellitus. Among patients with new-onset PTDM (N = 24), the frequency of circulating CLA(+) (skin-homing) Tregs was decreased (1.53% vs 3.99%; P = .002) and the percentage of α(4)ß(7)(+) (gut-homing) Tregs was increased (17.9% vs 10.7%; P = .048). In multivariate analysis, patients with PTDM continued to demonstrate elevated ratios of α(4)ß(7)(+) Tregs to CLA(+) Tregs (odds ratio, 18.1; P = .020). PTDM is associated with altered immune regulation after HSCT and could represent a target to modulate alloreactivity.

Identification of potential human respiratory syncytial virus and metapneumovirus T cell epitopes using computational prediction and MHC binding assays.

Human respiratory syncytial virus (RSV) and human metapneumovirus (MPV) are two of the most common causes of serious viral lower respiratory tract illness in humans. CD8+ T cells have been shown to be important in animal models and human clinical studies for the clearance of viral infection, and they may contribute in part to protection against severe disease during reinfections. Precise enumeration and accurate phenotyping of RSV- or MPV-specific CD8+ T cells in humans is currently limited by the relatively small number of T cell epitopes that have been mapped with accompanying identification of MHC restriction patterns. We sought to expand the number of potential RSV and MPV epitopes for use in clinical and translational studies by identifying an expanded set of MHC-binding peptides based on RSV and MPV wild-type virus strain protein sequences. We interrogated the full protein sequences of all 9 or 11 proteins of MPV or RSV respectively using four established epitope prediction algorithms for human HLA A*0101, A*0201, or B*0702 binding and attempted to synthesize the top-scoring 150-152 peptides for each of the two viruses. Synthesis resulted in 442 synthesized and soluble peptides of the 452 predicted epitopes for MPV or RSV. We then determined the binding of the synthetic peptides to recombinant human HLA A*0101, A*0201 or B*0702 molecules with the predicted restriction using a commercially available plate-based assay, iTopia. A total of 230 of the 442 peptides tested exhibited binding to the appropriate MHC molecule. The binding results suggested that existing algorithms for prediction of MHC A*0201 binding are particularly robust. The binding results also provided a large benchmarking data collection for comparison of new prediction algorithms.

Antibodies recognizing protective pertussis toxin epitopes are preferentially elicited by natural infection versus acellular immunization.

Despite more than 50 years of vaccination, disease caused by the bacterium Bordetella pertussis persists, with rates increasing in industrialized countries over the past decade. This rise may be attributed to several factors, including increased surveillance, emergence of vaccine escape variants, waning immunity in adults, and the introduction of acellular subunit vaccines, which include chemically detoxified pertussis toxin (PTd). Two potently protective epitopes on pertussis toxin (PTx) are recognized by the monoclonal antibodies 1B7 and 11E6, which inhibit catalytic and cell-binding activities, respectively. In order to determine whether the PTx exposure route affects antibody responses to these epitopes, we analyzed sera from 30 adults with confirmed pertussis exposure and from 30 recently vaccinated adults for specific anti-PTx antibody responses and in vitro CHO cell neutralization titers. While overall titers against PTx and the genetically detoxified variant, PTg, containing the R9K and E129G substitutions, were similar in the two groups, titers against specific epitopes depended on the exposure route. Natural infection resulted in significantly higher titers of anti-PTx-subunit 1, 1B7-like, and 11E6-like antibodies, while acellular vaccination resulted in significantly higher titers of antibodies recognizing PTd. We also observed a correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Notably, chemical detoxification, as opposed to genetic inactivation, alters the PTx tertiary and quaternary structure, thereby affecting conformational epitopes and recognition of PTx by 1B7 and 11E6. The lower levels of serum antibodies recognizing clinically relevant epitopes after vaccination with PTd support inclusion of PTg in future vaccines.

Cellular immune responses to diluted and undiluted aventis pasteur smallpox vaccine.

BACKGROUND: Recent primary vaccine trials of diluted Aventis Pasteur smallpox vaccine (APSV) demonstrated that immunization "take" rates, defined by the presence of a vesicle or pustule ("take") at the inoculation site 6-11 days after immunization, did not differ between the dilution groups. To our knowledge, there have been no studies that examine the cellular immune response or that distinguish CD4(+) T cell responses from CD8(+) T cell responses after primary immunization with varying dilutions of APSV. METHODS: In the present study, we examined the cellular immune response in vaccinia-naive healthy adults (n=91) receiving inoculations with an undiluted or diluted (1:5 and 1:10) suspension of the APSV, using an intracellular cytokine staining assay. RESULTS: The diluted vaccine induced vaccinia virus (VV)-specific CD4(+) and CD8(+) T cell responses 1 month after primary immunization that were comparable to those induced by undiluted vaccine. The cellular immune responses were correlated with the reactogenicity profile of subjects and did not differ between dilution groups. Furthermore, expression of the interleukin-7 receptor alpha chain, which has been proposed to distinguish antigen-specific T cells that differentiate into long-lived memory T cells, did not differ among groups, suggesting that dilution of the vaccine does not affect the quantity of VV-specific memory T cells. CONCLUSIONS: APSV is an effective smallpox vaccine inducing strong humoral and cellular immune responses after a primary immunization even at diluted doses.

Differential regulation of granzyme and perforin in effector and memory T cells following smallpox immunization.

Primary immunization of healthy adults with vaccinia virus induces a local vesicle or "take" in the majority of vaccinees that previously has been shown to correlate with protection against smallpox. However, the immunologic mechanisms underlying this protective response in humans are not well characterized. We have studied human CD8+ T cells for the expression patterns of phenotypic markers and cytolytic effector molecules before and after primary smallpox immunization using nine-color polychromatic flow cytometry. One month after immunization, vaccinees developed vaccinia virus-specific CD8+ T cells with an effector cell phenotype containing both granzyme A and granzyme B. One year after immunization, we found a significant decrease in granzyme B containing cells and an increased memory cell phenotype in virus-specific CD8+ T cells. Perforin was rarely expressed directly ex vivo, but was highly expressed after Ag-specific activation in vitro. Together, these data suggest an important role for effector CD8+ T cells in controlling poxvirus infection, and have implications for our understanding of human CD8+ T cell differentiation.

Vaccination success rate and reaction profile with diluted and undiluted smallpox vaccine: a randomized controlled trial.

CONTEXT: Additional smallpox vaccine doses are needed to augment current US national stockpile. Aventis Pasteur smallpox vaccine (APSV), initially manufactured in the 1950s from the New York Board of Health vaccinia strain in a frozen preparation, appears as effective as lyophilized vaccine but the effectiveness of diluted doses of APSV is unclear. OBJECTIVE: To compare the vaccination success rate and the reaction profile of various APSV dilutions. DESIGN, SETTING, AND PARTICIPANTS: A double-blind, randomized controlled trial of 340 healthy vaccinia-naive adults aged 18 to 32 years from 3 academic medical centers who were vaccinated with 1 of 3 strengths of APSV dilutions (undiluted, 1:5, and 1:10) between October 9, 2002, and February 24, 2003. Volunteers were followed up every 3 to 5 days until the vaccination site healed for bandage changes, vaccine response assessment, and adverse event evaluation, followed by 1- and 2-month clinic evaluations and 6-month telephone interview. MAIN OUTCOME MEASURES: Successful vaccination, defined by presence of a vesicle or pustule at the inoculation site 6 to 11 days postvaccination, and local and systemic reactions to vaccination. RESULTS: A total of 340 volunteers were vaccinated (vaccine dose: undiluted, n = 113; 1:5 dilution, n = 114; and 1:10 dilution, n = 113). Following vaccination, 99.4% (95% confidence interval [CI], 97.9%-99.9%) of all volunteers had successful vaccinations. Success rates did not differ between the dilution groups (undiluted, 100.0%; 95% CI, 96.8%-100.0%; 1:5 dilution, 98.2%; 95% CI, 93.8%-99.8%; 1:10 dilution, 100.0% 95% CI, 96.8%-100.0%; P =.33). Overall, 99.7% of volunteers reported at least 1 local symptom at the vaccination site, and 61.8% had axillary lymphadenopathy, 15.0% developed satellite lesions, and 7.6% developed a rash away from the vaccination site. Fever developed in 21.5%. No differences were noted in local or systemic reactions between the 3 dilution groups (P>.05 for each comparison). A total of 25% of volunteers missed scheduled duties due to vaccine-related symptoms. CONCLUSIONS: Even at diluted doses, APSV is an effective smallpox vaccine, allowing for expansion of the current stockpile. However, reactogenicity was not reduced with dilution of the vaccine and, as with other smallpox vaccines, may impair daily activities.

Adverse events after smallpox immunizations are associated with alterations in systemic cytokine levels.

The immunization of healthy adults with vaccinia virus (VV) induces a protective response against smallpox in most individuals but is also reactogenic in a significant number of vaccinees. The immunological mechanisms underlying the protective response or adverse events in humans are not well defined. Although cytokines contribute to antiviral immunity and, in some cases, cause systemic adverse effects, their role in the human response to VV is unknown. We investigated the effect of smallpox immunization on systemic cytokine concentrations in a cohort of VV-naive individuals. We found that smallpox immunization induces an interferon (IFN)- gamma -dominant response in the systemic compartment 1 week after immunization, with concentrations returning to baseline during convalescence. The level of IFN- gamma induced was not affected by the dilution of vaccine used. We also found that particular adverse events correlated with systemic cytokine patterns, which suggests a role for these molecules in the pathogenesis of adverse events.

Role of complement in neutralization of respiratory syncytial virus.

Serum neutralizing antibody titers to respiratory syncytial virus (RSV) are higher when assayed with guinea pig complement. A number of different mechanisms have been suggested for enhancement of neutralization by complement. The most straightforward is that complement-antibody complexes present a greater steric hindrance to viral entry than with antibody alone. To define the implications of measuring serum neutralizing antibody with and without complement, sera from adults, young children, infants, and cord bloods were run in plaque neutralization assays with representative viruses of the RSV A and B subgroups. Although titers of neutralizing antibody were higher in the presence of complement, the addition of complement did not increase the ability to detect antibody rises after natural infection. Some of the complement effect may be attributable to an inhibition of RSV replication by complement alone. While these observations do not address the role for complement in the pathogenesis of RSV infection, they suggest that neutralization assays performed without complement may be most reflective of physiologic conditions in the respiratory tract.