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A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication. Graphical Abstract

SUMMARYHIV-1 broadly neutralizing antibodies (bnAbs) require high levels of activation-induced cytidine deaminase (AID) catalyzed somatic mutations for optimal neutralization potency. Probable mutations occur at sites of frequent AID activity, while improbable mutations occur where AID activity is infrequent. One bottleneck for induction of bnAbs is the evolution of viral envelopes (Envs) that can select bnAb B cell receptors (BCR) with improbable mutations. Here we define the probability of bnAb mutations and demonstrate the functional significance of key improbable mutations in three bnAb B cell lineages. We show that bnAbs are enriched for improbable mutations, implying their elicitation will be critical for successful vaccine induction of potent bnAb B cell lineages. We outline a mutation-guided vaccine strategy for identification of Envs that can select B cells with BCRs with key improbable mutations required for bnAb development. Our analysis suggests that through generations of viral escape, Env trimers evolved to hide in low probability regions of antibody sequence space.

Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions. IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in nonhuman primates is critical for understanding how to improve upon immunogen design to inform further testing in human clinical trials. Our results demonstrate that structural modifications of Env that aim to mimic the CD4-bound conformation can result in earlier antibody elicitation, altered epitope specificity, and increased antiviral function postimmunization.

We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls ( P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia ( P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4 + effector memory, and postchallenge CD8 + transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and protein-based vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.

A patient presented with shortness of breath and pleuritic pain shortly after bilateral knee synovial injections with sodium hyaluronate (HA). He was discharged after a brief hospitalization without a diagnosis when no Doppler or radiologic evidence of deep vein thrombosis or pulmonary emboli was found. Radiologic studies found patchy ground glass opacities that were predominantly peripheral in disposition, with prominent septal lines in the lungs; a subsequent pulmonary function test showed a reduced diffusing capacity of the lung for carbon monoxide (D(LCO)). These results prompted a lung biopsy that revealed multiple emboli composed of HA and fibrin in medium size pulmonary arteries, enlarged lymphatic vessels, and a bone marrow embolus. This is the first report of HA emboli following therapeutic HA injections and demonstrates that pulmonary function tests can be used to infer the reduction in pulmonary vascular area consequent to pulmonary emboli, and so can contribute to the detection of pulmonary emboli in unusual presentations.

HIV-1-specific immunoglobulin G (IgG) subclass antibodies bind to distinct cellular Fc receptors. Antibodies of the same epitope specificity but of a different subclass therefore can have different antibody effector functions. The study of IgG subclass profiles between different vaccine regimens used in clinical trials with divergent efficacy outcomes can provide information on the quality of the vaccine-induced B cell response. We show that HIV-1-specific IgG3 distinguished two HIV-1 vaccine efficacy studies (RV144 and VAX003 clinical trials) and correlated with decreased risk of HIV-1 infection in a blinded follow-up case-control study with the RV144 vaccine. HIV-1-specific IgG3 responses were not long-lived, which was consistent with the waning efficacy of the RV144 vaccine. These data suggest that specific vaccine-induced HIV-1 IgG3 should be tested in future studies of immune correlates in HIV-1 vaccine efficacy trials.

Twenty-seven temporal bone fractures in 25 pediatric patients were evaluated over a 6-year period. The diagnosis was confirmed with otoscopy and high-resolution computed tomography scans (HRCT). Three-dimensional reconstruction of high-resolution computed tomography scans were used to aid in the diagnosis. The most common fracture was an oblique fracture which is oriented in an axial or horizontal plane with the temporal bone. Facial nerve paralysis was found in 6 of our patients, which is less than the expected incidence when compared to adults. Hearing loss was found in 24 patients, the most common of which was conductive hearing loss, which had a higher incidence than expected when compared with adults.

UnlabelledIn the RV144 HIV-1 vaccine efficacy trial, IgG antibody (Ab) binding levels to variable regions 1 and 2 (V1V2) of the HIV-1 envelope glycoprotein gp120 were an inverse correlate of risk of HIV-1 infection. To determine if V1V2-specific Abs cross-react with V1V2 from different HIV-1 subtypes, if the nature of the V1V2 antigen used to asses cross-reactivity influenced infection risk, and to identify immune assays for upcoming HIV-1 vaccine efficacy trials, new V1V2-scaffold antigens were designed and tested. Protein scaffold antigens carrying the V1V2 regions from HIV-1 subtypes A, B, C, D or CRF01_AE were assayed in pilot studies, and six were selected to assess cross-reactive Abs in the plasma from the original RV144 case-control cohort (41 infected vaccinees, 205 frequency-matched uninfected vaccinees, and 40 placebo recipients) using ELISA and a binding Ab multiplex assay. IgG levels to these antigens were assessed as correlates of risk in vaccine recipients using weighted logistic regression models. Levels of Abs reactive with subtype A, B, C and CRF01_AE V1V2-scaffold antigens were all significant inverse correlates of risk (p-values of 0.0008-0.05; estimated odds ratios of 0.53-0.68 per 1 standard deviation increase). Thus, levels of vaccine-induced IgG Abs recognizing V1V2 regions from multiple HIV-1 subtypes, and presented on different scaffolds, constitute inverse correlates of risk for HIV-1 infection in the RV144 vaccine trial. The V1V2 antigens provide a link between RV144 and upcoming HIV-1 vaccine trials, and identify reagents and methods for evaluating V1V2 Abs as possible correlates of protection against HIV-1 infection.Trial registrationClinicalTrials.gov NCT00223080.

new approach to teaching the practice of medicine. JAMA. 1992;268:24202425. 5. Parkes J, Hyde C, Deeks J, Milne R. Teaching critical appraisal skills in health care settings [database on CD-ROM]. Oxford, England: Cochrane Library, Update Software; 2002;issue 2. 6. Taylor R, Reeves B, Ewings P, et al. A systematic review of the effectiveness of critical appraisal skills training for clinicians. Med Educ. 2000;34:120-125. 7. Norman GR, Shannon SI. Effectiveness of instruction in critical appraisal (evidencebased medicine) skills: a critical appraisal. CMAJ. 1998;158:177-181. 8. Linzer M, Brown JT, Frazier LM, DeLong ER, Siegel WC. Impact of a medical journal club on house-staff reading habits, knowledge, and critical appraisal skills: a randomized controlled trial. JAMA. 1988;260:2537-2541. 9. Stern DT, Linzer M, O’Sullivan PS, Weld L. Evaluating medical residents’ literatureappraisal skills. Acad Med. 1995;70:152-154. 10. Bennett KJ, Sackett DL, Haynes RB, Neufeld VR, Tugwell P, Roberts R. A controlled trial of teaching critical appraisal of the clinical literature to medical students. JAMA. 1987;257:2451-2454. 11. Grimes DA. Introducing evidence-based medicine into a department of obstetrics and gynecology. Obstet Gynecol. 1995;86:451-457. 12. Murray E. Challenges in educational research. Med Educ. 2002;36:110-112. 13. Campbell M, Fitzpatrick R, Haines A, et al. Framework for design and evaluation of complex interventions to improve health. BMJ. 2000;321:694-696. 14. Bogdan R, Biklen SK. Qualitative Research for Education: An Introduction to Theory and Methods. Boston, Mass: Allyn & Bacon; 1982. 15. Green ML, Ellis PJ. Impact of an evidence-based medicine curriculum based on adult learning theory. J Gen Intern Med. 1997;12:742-750. 16. Cabell CH, Schardt C, Sanders L, Corey GR, Keitz SA. Resident utilization of information technology. J Gen Intern Med. 2001;16:838-844. 17. Taylor R, Reeves B, Mears R, et al. Development and validation of a questionnaire to evaluate the effectiveness of evidence-based practice teaching. Med Educ. 2001;35:544-547. 18. Flynn C, Helwig A. Evaluating an evidence-based medicine curriculum. Acad Med. 1997;72:454-455. 19. Dobbie AE, Schneider FD, Anderson AD, Littlefield J. What evidence supports teaching evidence-based medicine? Acad Med. 2000;75:1184-1185.

Electrophoretic patterns for cereal-grain prolamins were coded in numerical form to permit computer-based comparison, either for facilitating the identification of cultivars or for the study of genotypic relationships. In a group of 80 cultivars, including wheats grown in countries other than Australia, gliadin patterns reflected both pedigree and taxonomic relationships, particularly in the separate grouping of a rye, and of durum and club wheats. Certain gliadin bands were also found to be significantly associated with the quality characteristics of hardness and dough strength.

SUMMARY 1.The effect of selected drugs and various inorganic ions on ventricular fibrillation and mortality following experimental coronary artery occlusion has been investigated. 2.Control mortality of 80 per cent and early fibrillation of 30 per cent was obtained following ligation of the left anterior descending coronary artery immediately at its origin. 3.When coronary occlusion was delayed for 20 days post-pericardotomy in three animals there was no mortality and two of the three animals failed to demonstrate any change in the pre and postocclusion electrocardiogram. 4.Mortality of 80 per cent and fibrillation of 40 per cent resulted when acidosis was produced by hydrochloric acid. 5.Mortality of 80 per cent and fibrillation of 60 per cent resulted when alkalosis was produced by the infusion of sodium bicarbonate. 6.A mortality rate of 60 per cent and a fibrillation rate of 60 per cent resulted when hyperkaliemia was produced. 7.Hypercalcemia produced a mortality of 80 per cent and fibrillation of 60 per cent. 8.Following the infusion of a relatively small amount of sodium chloride an unexpected 100 per cent mortality and 80 per cent fibrillation rate resulted. 9.A mortality rate of 100 per cent and a fibrillation rate of 100 per cent followed the infusion of procaine. 10.The intravenous administration of papaverine produced mortality and fibrillation rates of 60 per cent. 11.Quinidine administered intravenously yielded a mortality rate of 60 per cent and fibrillation rate of 60 per cent. 12.The influence of infusion of certain ions and drugs upon the concentration of other ions was determined. 13.It would appear from this study that if potassium is the excitatory agent for ventricular fibrillation that myocardial potassium in ischemia is unaffected by the plasma concentration of potassium and is on a metabolic basis. 14.If potassium is the agent inciting ventricular fibrillation it would appear from this study that myocardial metabolism of potassium in myocardial ischemia is unaffected by blood pH insofar as decreasing early fibrillation is concerned. 15.None of the drugs or ions employed in this study showed any beneficial effect on reducing mortality or ventricular fibrillation following coronary artery occlusion. We gratefully acknowledge the electrocardiographic interpretation of Dr. Thomas M. Blake.

To determine the etiology of acute conjunctivitis in children seen in pediatric practice, 99 patients with conjunctivitis and 102 age-and season-matched controls were cultured for aerobic bacteria including Haemophilus influenzae, and for viruses, Chlamydia trachomatis, and mycoplasmas. Agents statistically associated with conjunctivitis included H. influenzae (42% vs 0%), Streptococcus pneumoniae (12% vs 3%), and adenoviruses (20% vs 0%). One of these three etiologic agents was isolated from 71 (72%) of the patients. Simultaneous infection with two pathogens was uncommon. Staphylococcus aureus was equally prevalent in diseased and control eyes; one strain of C. trachomatis was isolated from a control eye. Although there were variations in the clinical features of viral and bacterial conjunctivitis, differentiation in an individual patient was difficult. An adenovirus was isolated from 11 (65%) of 17 patients who had pharyngitis in addition to conjunctivitis. H. influenzae was isolated from 14 (74%) of 19 children who had both otitis and conjunctivitis. Adenovirus conjunctivitis was common in the fall and H. influenzae in winter.

1. 1. Hibernation in ground squirrels (Spermophilus mexicanus) results in: decreased RBC count, decreased WBC count, increased plasma LDH levels and increased RBC LDH levels. 2. 2. Hibernation in ground squirrels along with hyperoxia compared to hibernation alone results in: decreased hematocrit, decreased hemoglobin, decreased total RBC LDH levels, increased RBC G-3-P-DH levels and increased RBC GSH levels.

The effect of drugs and ions upon the normal heart and upon isolated myocardium has been thoroughly investigated and adequately documented. Increased interest in the application of these substances in a manner which approaches the abnormal clinical situation-that is, following coronary artery occlusion and in various arrythmias-has been demonstrated during the past few years. The stimulus for this recent investigative trend has come primarily from three sources: (1) Fibrillation following coronary artery occlusion and the efforts to devise means of permanently increasing collateral blood supply to the myocardium; (2) Cardiac arrest or ventricular fibrillation during surgery; and (3) Ventricular fibrillation during hypothermia. This report concerns experimental myocardial ischemia produced by acute coronary artery occlusion, the influence of drugs and inorganic ions on survival rates and early fibrillation and the influence on certain ions upon other ions. Materials and Methods Adult mongrel dogs were used as the experimental animals. Following anesthetization with intravenous nembutal, endotracheal intubation was accomplished and the chest opened by an incision in the fourth inter-space. The left coronary, circumflex coronary, and left anterior descending coronary arteries were carefully demonstrated. A heavy silk ligature was placed beneath the anterior descending branch immediately adjacent to its origin. The loose ligature was threaded through a small plastic tube which was anchored to the pericardium at one end and brought out through the chest wall at the other end. Following closure of the chest wall, the ligature ends and plastic tube were placed subcutaneously, the animal was given penicillin and returned to his cage.