Blocking Antibody Titers Research Articles

Norovirus Infection in Young Nicaraguan Children Induces Durable and Genotype-Specific Antibody Immunity.

There are significant challenges to the development of a pediatric norovirus vaccine, mainly due to the antigenic diversity among strains infecting young children. Characterizing human norovirus serotypes and understanding norovirus immunity in naïve children would provide key information for designing rational vaccine platforms. In this study, 26 Nicaraguan children experiencing their first norovirus acute gastroenteritis (AGE) episode during the first 18 months of life were investigated. We used a surrogate neutralization assay that measured antibodies blocking the binding of 13 different norovirus virus-like particles (VLPs) to histo-blood group antigens (HBGAs) in pre- and post-infection sera. To assess for asymptomatic norovirus infections, stools from asymptomatic children were collected monthly, screened for norovirus by RT-qPCR and genotyped by sequencing. Seroconversion of an HBGA-blocking antibody matched the infecting genotype in 25 (96%) of the 26 children. A subset of 13 (50%) and 4 (15%) of the 26 children experienced monotypic GII and GI seroconversion, respectively, strongly suggesting a type-specific response in naïve children, and 9 (35%) showed multitypic seroconversion. The most frequent pairing in multitypic seroconversion (8/12) were GII.4 Sydney and GII.12 noroviruses, both co-circulating at the time. Blocking antibody titers to these two genotypes did not correlate with each other, suggesting multiple exposure rather than cross-reactivity between genotypes. In addition, GII titers remained consistent for at least 19 months post-infection, demonstrating durable immunity. In conclusion, the first natural norovirus gastroenteritis episodes in these young children were dominated by a limited number of genotypes and induced responses of antibodies blocking binding of norovirus VLPs in a genotype-specific manner, suggesting that an effective pediatric norovirus vaccine likely needs to be multivalent and include globally dominant genotypes. The duration of protection from natural infections provides optimism for pediatric norovirus vaccines administered early in life.

Time Since Last Anti-CD20 Treatment Is a Major Determinant of Sars-Cov-2 Vaccine Response in a Large Cohort of Patients with B-Cell Lymphoma

Time Since Last Anti-CD20 Treatment Is a Major Determinant of Sars-Cov-2 Vaccine Response in a Large Cohort of Patients with B-Cell Lymphoma

SARS-CoV-2 elicits robust adaptive immune responses regardless of disease severity.

BackgroundThe SARS-CoV-2 pandemic currently prevails worldwide. To understand the immunological signature of SARS-CoV-2 infections and aid the search and evaluation of new treatment modalities and vaccines, comprehensive characterization of adaptive immune responses towards SARS-CoV-2 is needed.MethodsWe included 203 recovered SARS-CoV-2 infected patients in Denmark between April 3rd and July 9th 2020, at least 14 days after COVID-19 symptom recovery. The participants had experienced a range of disease severities from asymptomatic to severe. We collected plasma, serum and PBMC's for analysis of SARS-CoV-2 specific antibody response by Meso Scale analysis including other coronavirus strains, ACE2 competition, IgA ELISA, pseudovirus neutralization capacity, and dextramer flow cytometry analysis of CD8+ T cells. The immunological outcomes were compared amongst severity groups within the cohort, and 10 pre-pandemic SARS-CoV-2 negative controls.FindingsWe report broad serological profiles within the cohort, detecting antibody binding to other human coronaviruses. 202(>99%) participants had SARS-CoV-2 specific antibodies, with SARS-CoV-2 neutralization and spike-ACE2 receptor interaction blocking observed in 193(95%) individuals. A significant positive correlation (r=0.7804) between spike-ACE2 blocking antibody titers and neutralization potency was observed. Further, SARS-CoV-2 specific CD8+ T-cell responses were clear and quantifiable in 95 of 106(90%) HLA-A2+ individuals.InterpretationThe viral surface spike protein was identified as the dominant target for both neutralizing antibodies and CD8+ T-cell responses. Overall, the majority of patients had robust adaptive immune responses, regardless of their disease severity.FundingThis study was supported by the Danish Ministry for Research and Education (grant# 0238-00001B) and The Danish Innovation Fund (grant# 0208-00018B)

Oral immunization of a rAd vector expressing norovirus VP1 elicits a potent mucosal immune response without an increase in anti-vector immunity

Background: Noroviruses are the leading cause of epidemic acute gastroenteritis and foodborne diarrheal disease in humans. Potential correlates of protection identified through human challenge studies include mucosal IgA, memory B cells, and serum blocking (BT50) antibody titers. We hypothesized that fecal and memory IgA responses would be significantly elevated following oral immunization. Methods & Materials: We conducted a single-site, randomized, double-blind, placebo-controlled trial to determine the safety and immunogenicity of a norovirus vaccine consisting of a non-replicating adenovirus vector expressing VP1 delivered orally by tablet. Subjects that met inclusion/exclusion criteria were enrolled and randomized 2:1 to receive either a single dose of vaccine or placebo, respectively. Two dose levels were evaluated. The primary objective was to assess safety. The secondary objective was to determine immunogenicity, primarily assessed by measuring serum BT50. Exploratory immunological assessments included serum, fecal and saliva antibody titers measured by and the frequency of memory and antibody secreting B cells (ASCs). Anti-vector immunity was assessed by measuring Ad5 neutralizing antibody responses pre and post immunization (ClinicalTrials.gov number NCT02868073). Results: Sixty-six subjects were enrolled between July and Sept 2016. The vaccine was well-tolerated. The primary immunological endpoint (BT50 titers) was met in low and high dose groups (P = 0.0014, P < 0.0001). In the high-dose group, 18/23 (78%) subjects had a two-fold or greater increase in BT50 titer after a single vaccine dose. An average of 561 IgA ASCs x106 PBMC were induced post-immunization in the high dose group. Post immunization, a 15 fold increase in the geometric mean was observed, along with a greater than 10 fold average increase in the fecal IgA VP1 specific antibody response. Anti-Ad5 neutralizing antibody responses did not significantly increase compared to the placebo group. Conclusion: The norovirus vaccine was well-tolerated (with no dose limiting toxicities), and generated immune responses to norovirus VP1, without a corresponding increase in anti-Ad5 neutralizing antibody responses. It particular, the vaccine generated memory and local effector IgA + B cells. Because mucosal IgA is the front-line defense against enteric pathogens, vaccine-derived norovirus-specific intestinal IgA may translate to superior efficacy. This is a very promising advance in the development of an oral norovirus vaccine.

Bivalent Norovirus VLP Vaccine Candidate in Older Adults: Impact of MPL and a Second Dose in a Randomized, Controlled, Double-Blind Clinical Trial

BackgroundAcute norovirus (NoV) gastroenteritis may cause significant morbidity in healthy adults and can prove fatal in older subjects. We investigated the safety and immunogenicity in older adults of one or two doses of an intramuscular bivalent virus-like particle (VLP) vaccine candidate (genotypes GI.1 and multivalent consensus GII.4c) formulated with alum and with and without MPL (3-O-deacyl-4′-monophosphoryl lipid A) adjuvant.MethodsIn a phase II, double-blind, controlled trial, 294 healthy adults ≥ 60 years of age randomized to 4 equal groups received one or two immunizations 28 days apart. One dose groups received placebo (saline) on Day 1. Vaccine formulations contained 500μg Al(OH)3adjuvant with 15μg GI.1 and 50μg GII.4c VLP antigens, with or without 15μg MPL adjuvant. A fifth group of 26 healthy 18–49 year-olds received one dose of MPL-free vaccine. Humoral immunity was assessed as ELISA pan-Ig and histo-blood group antigen blocking (HBGA) antibody titers at Days 1, 8, 29 and 57. Cell-mediated immunity (CMI) and avidity indices (AI) were also measured. Safety was assessed as solicited local and systemic adverse events (AE) for 7 days, and unsolicited AEs until Day 28 after each vaccination.ResultsMarked increases in pan-Ig and HBGA to both genotypes occurred by Day 8 after first vaccination. Geometric mean titers were similar in magnitude in all groups and persisted at similar levels through Day 56. No increases were observed with a second vaccine dose on Day 29 or with the formulations containing MPL. Responses were similar in magnitude when assessed by age groups (60–74, 75–84 and ≥ 85 years of age) and when compared with those to a single vaccine dose in 18–49 year-olds. No clinically relevant differences in CMI responses or changes in antibody avidity were observed between formulations. Both formulations were generally well tolerated, the most frequent reaction being mild pain at the injection site. No vaccine-related SAEs were reported.ConclusionOlder adults aged over 60 years displayed immune responses to NoV VLP vaccines that were similar to those in younger adults with no apparent signs of immunosenescence. These data support the further development of the MPL-free vaccine candidate in older adults.Disclosures J. Sherwood, Takeda Pharmaceuticals International AG: Employee, Salary; J. Cramer, Takeda Pharmaceuticals International AG: Employee, Salary; N. Bouveret Le Cam, Takeda Vaccines Inc.: Employee, Salary; S. Lin, Takeda Vaccines Inc.: Employee, Salary; F. Baehner, Takeda Pharmaceuticals International AG: Employee, Salary; P. Mendelman, Takeda Vaccines Inc.: Employee, Salary; A. Borkowski, Takeda Pharmaceuticals International AG: Employee, Salary

Expression and purification of norovirus virus like particles in Escherichia coli and their immunogenicity in mice

Norovirus (NoV) virus like particles (VLPs) produced in Spodoptera frugiperda (Sf9) cells have been tested in human volunteers as vaccine candidate and were shown to be protective against NoV induced acute gastroenteritis. In this study, prevailing Sydney-2012-like NoV major capsid protein gene with or without N-terminal deletions (N26 and N38, 26 and 38 amino acids deleted from N terminus，respectively) were sub-cloned into prokaryotic expression vector, pCold III and pCold IV. Soluble and insoluble proteins were detected for both vectors after induction and higher levels of protein expression were observed for constructs pCold III-N26 and pCold III-N38. Electron microscopy observation of unpurified and purified lysates indicated in vivo assembly of VLPs with two sizes in accordance with those observed in Sf9 cells. In vitro salivary HBGA-VLP binding assay demonstrated that VLPs assembled in Escherichia coli (E. coli) exhibited the same binding pattern as that of VLPs assembled in Sf9 cells. Immunization of mice with purified VLPs derived from pCold III-N38 demonstrated higher IgG antibody titers and blocking antibody titers when compared with full-length capsid protein assembled VLPs from recombinant baculovirus expression system. In conclusion, NoV VLPs produced in E. coli using pCold expression vector might be used for the development of NoV vaccine.

Seroprevalence of antibodies against GII.4 norovirus among children in Pune, India.

This study reports the seroprevalence of antibodies against GII.4 norovirus among children (≤5 years) in Pune, India. Of 191 serum specimens, 98 (51.3%) tested positive with 61, 34 and 3 having IgG, IgG-IgA and IgG-IgA-IgM, respectively. Histoblood group antigen (HBGA)-blocking antibodies were detected in 33 of the 54 tested positive specimens. IgG and blocking antibody prevalence and titer varied with age and was lowest among children aged 6-23 months. Antibody-positive children, suggesting past norovirus exposure, showed significantly lower faecal norovirus RNA detection rate than antibody-negative children. Further investigation of the seroepidemiology of norovirus infections in India is warranted. J. Med. Virol. 88:1636-1640, 2016. © 2016 Wiley Periodicals, Inc.

Transmission blocking malaria vaccines: Assays and candidates in clinical development

Stimulated by recent advances in malaria control and increased funding, the elimination of malaria is now considered to be an attainable goal for an increasing number of malaria-endemic regions. This has boosted the interest in transmission-reducing interventions including vaccines that target sexual, sporogenic, and/or mosquito-stage antigens to interrupt malaria transmission (SSM-VIMT). SSM-VIMT aim to prevent human malaria infection in vaccinated communities by inhibiting parasite development within the mosquito after a blood meal taken from a gametocyte carrier. Only a handful of target antigens are in clinical development and progress has been slow over the years. Major stumbling blocks include (i) the expression of appropriately folded target proteins and their downstream purification, (ii) insufficient induction of sustained functional blocking antibody titers by candidate vaccines in humans, and (iii) validation of a number of (bio)-assays as correlate for blocking activity in the field. Here we discuss clinical manufacturing and testing of current SSM-VIMT candidates and the latest bio-assay development for clinical evaluation. New testing strategies are discussed that may accelerate the evaluation and application of SSM-VIMT.

High serum levels of norovirus genotype-specific blocking antibodies correlate with protection from infection in children.

Norovirus is a common cause of acute gastroenteritis in children. Serum immunoglobulin G (IgG) antibodies have been implicated in protection against norovirus-associated gastroenteritis, but the level, specificity, and functionality necessary for protection remain to be elucidated. Norovirus-specific IgG antibodies to genogroup II (GII)-4-2010 New Orleans (NO), GII-4-1999, GII-12-1998, GI-1-2001, and GI-3-2002 virus-like particles (VLPs) were determined by enzyme-linked immunosorbent assay in serum samples collected from children who presented to the hospital because of acute norovirus gastroenteritis in 2009-2011. The blocking activity of the antibodies was tested in a surrogate neutralization assay. Most norovirus infections (62.8%) in the study population were caused by a GII-4 NO variant. Children who acquired GII-4 NO infection had a low preexisting type-specific IgG level and blocking activity of the sera, in contrast to children infected with other GII genotypes. Following GII-4 NO infection, genotype-specific seroconversion and a corresponding increase in blocking antibody potential was observed. Although seroconversion to the heterologous GII-4-1999 variant was observed, there was no corresponding increase in the specific blocking antibody titer. There was no concomitant seroconversion against GI VLPs, indicating a highly genogroup-specific antibody response. High preexisting norovirus genotype-specific serum IgG titers and blocking activity in children indicate protection from norovirus infection in a strain-specific manner.

GIP005 Pharmacological study of the effect of Metronidazol

Norovirus (NoV) virus like particles (VLPs) produced in Spodoptera frugiperda (Sf9) cells have been tested in human volunteers as vaccine candidate and were shown to be protective against NoV induced acute gastroenteritis. In this study, prevailing Sydney-2012-like NoV major capsid protein gene with or without N-terminal deletions (N26 and N38, 26 and 38 amino acids deleted from N terminus，respectively) were sub-cloned into prokaryotic expression vector, pCold III and pCold IV. Soluble and insoluble proteins were detected for both vectors after induction and higher levels of protein expression were observed for constructs pCold III-N26 and pCold III-N38. Electron microscopy observation of unpurified and purified lysates indicated in vivo assembly of VLPs with two sizes in accordance with those observed in Sf9 cells. In vitro salivary HBGA-VLP binding assay demonstrated that VLPs assembled in Escherichia coli (E. coli) exhibited the same binding pattern as that of VLPs assembled in Sf9 cells. Immunization of mice with purified VLPs derived from pCold III-N38 demonstrated higher IgG antibody titers and blocking antibody titers when compared with full-length capsid protein assembled VLPs from recombinant baculovirus expression system. In conclusion, NoV VLPs produced in E. coli using pCold expression vector might be used for the development of NoV vaccine.

Adsorption column for myasthenia gravis treatment: Medisorba MG-50.

Adsorption column Medisorba MG-50 (Kuraray Medical Inc.) for the treatment of myasthenia gravis (MG) is introduced. The adsorbent in this column is composed of cellulose beads as carrier material and covalent-bound synthetic peptide as a ligand that has a specific affinity to the pathogenic anti-acetylcholine receptor antibody of MG. The amino acid sequence of the peptide is modified from the segment of alpha 183-200 of the torpedo acetylcholine receptor (AChR) protein, and the segment is the acetylcholine binding site on AChR and the target site of anti-AChR antibody. The adsorbent showed specific adsorption characteristics to the anti-ACHR antibody (blocking antibody) in vitro. Clinically, MG-50 is used in plasma-perfusion therapy, and it is recognized that MG-50 specifically reduces blocking antibody titer and improves MG symptoms. MG-50 is approved in Japan.

Competition ELISA, using monoclonal antibodies to the transmissible gastroenteritis virus (TGEV) S protein, for serologic differentiation of pigs infected with TGEV or porcine respiratory coronavirus

SUMMARY Monoclonal antibodies (MAB) to subsite A (25C9) and subsite D (44C11) of the S protein of transmissible gastroenteritis virus (tgev) were used in a blocking elisa on fixed tgev-infected swine testis cells to differentiate sera from pigs experimentally inoculated with either tgev or porcine respiratory coronavirus (prcv). Serum samples were obtained from pigs at various intervals from postinoculation day (pid) 0 through at least PID 22 to 40. Eleven-day-old pigs, seronegative for tgev-neutralizing antibodies at the time of inoculation, were inoculated orally and nasally with either the virulent Miller (M5C) strain or the attenuated Purdue (P115) strain of tgev, or with the ISU-1 strain of prcv. Gastroenteritis was observed in 100% of the M5C-tgev-inoculated pigs; but clinical signs of disease were not observed in either the PI 15-tgev- or prcv-inoculated pigs. Virus-neutralization (vn) antibody titer in sera was determined by use of a plaque-reduction assay. Blocking ELISA antibody titer for subsites A and D was determined from the serum dilution that produced 50% reduction in the absorbance values when it competed with biotinylated MAB 25C9 and 44C11, respectively. In sera from the inoculated pigs, the VN antibody titer began to increase by pid 7 and reached maximum by PID 15 to 16. For pigs inoculated with tgev M5C, subsite A and subsite D blocking antibody titers in the serum paralleled the vn antibody titer, began to increase after pid 7, and reached maximum by pid 15 to 16. The blocking antibody titer to sub-sites A and D began to increase in the PI 115- tgev-inoculated pigs after pid 15 to 16 and reached maximum by PID 22 to 26. Blocking antibody titer to subsite A in prcv-inoculated pigs behaved similarly to blocking antibody titer to subsite A in the M5C-tgev-inocuIated pigs, reaching maximum by PID 15 to 16; however, blocking antibody titer was not detected for subsite D up to PID 24 (the latest time point examined) in sera from the prcv-inoculated pigs. Serum antibody responses and clinical signs of disease were monitored in pigs initially inoculated with either M5C-tgev or -prcv and challenge-exposed with M5C-tgev on PID 24. Clinical signs of gastroenteritis were not observed in the M5C-tgev-inoculated pigs after challenge-exposure with M5C-tgev. Low increases in vn antibody titer and in sub-site A or D blocking antibody titer were detected in the M5C-tgev-inoculated and challenge-exposed pigs. Of the 12 pigs initially inoculated with prcv then challenge-exposed with M5C-tgev, 5 pigs developed diarrhea, the vn and subsite A antibody blocking titers began to increase by postchallenge-cxposure day (PCD) 2 and reached maximal titer by PCD 9, increasing approximately 100-fold above the prechallenge-exposure titer. Subsite D antibody-blocking titer began to appear after PCD 9 and, by PCD 12, had reached nearly the same level as that for the primary response to the M5C-tgev inoculation.

Myasthenia gravis: long-term correlation of binding and bungarotoxin blocking antibodies against acetylcholine receptors with changes in disease severity.

Sixty patients with myasthenia gravis were examined prospectively by measuring serial titers of antibodies against human acetylcholine receptor, and these were correlated with a quantitative clinical score. Serial titers of antibodies detected by the standard immunoprecipitation assay (binding antibodies) correlated with the clinical score in most patients. Antibodies blocking the binding of alpha-bungarotoxin to receptors (blocking antibodies) were detected in 29 patients. Serial blocking antibody titers correlated with changes in muscle weakness less often than binding antibody titers. Titers of both classes of antibodies often followed a divergent course, suggesting that the autoimmune B-cell clones that formed these classes of antibodies may have been activated asynchronously.

Efficacy and specificity of immunotherapy with laboratory animal allergen extracts

The clinical and immunologic response to immunotherapy with laboratory animal allergens was evaluated. There were 23 patients; 11 had received immunotherapy with 12 different extracts (five mouse, six rat, one rabbit), and 12 were matched untreated patients. As a group, nine of 23 had seasonal hay fever. Among the treated patients nine of 11 subjectively improved with immunotherapy. Blocking antibody titers were determined by serum inhibition of allergen-induced histamine release. Treated patients had mean blocking antibody levels of G 30 = 59.3 ± 38.7. In the untreated patients the antibody level was low (G 30 = 4.6 ± 3.6). The difference between the two groups was highly significant (t test p < 0.001). The blocking antibody level correlates with both the final weekly allergen dose and also the cumulative allergen dose received during immunotherapy. When pretreatment sera were available a temporal rise in blocking antibody was demonstrated during immunotherapy. In patients allergic to several laboratory animals and treated with one allergen, the blocking antibody response was predominantly specific to the allergen used in immunotherapy. This indicates a lack of cross-reactivity in the IgG response to the major animal allergens. When immunotherapy was discontinued in four patients there was a dramatic decrease in the blocking titer, and after 24 mo the levels were the same as those of untreated patients.

Measurement of IgG-Blocking Antibodies: Development and Application of a Radioimmunoassay

A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r8 = 0.96, p less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective.

Allergic Diseases in Upper Respiratory Tract

Three allergic disorders manifested on the nose were reported in this session. First of all, an immediate type (Type I) allergy was examplified by the so-called nasal allergy of Dermatophagoides farinae. In addition to the expression of their clinical picture, effectiveness of the hyposensitization therapy was precisely described, including changes of nasal provocation threshold, PK titer, blocking antibody titer, IgE and IgE antibody value during the period of the therapy. Secondarily, it was speculated that Arthus reaction (Type III) induced by the bacterial infection to the paranasal sinuses was one of the main factors producing chronic sinusitis. This theory was mainly proved by immunohistological examination which showed that antibodies to Staphylococcus aureus or complements were distributed in the infiltrated cells, perivascular regions, basement membrane so on. On the third, clinical pictures and differentiation of Wegener's Granulomatosis (WG) and malignant granuloma of the nose (MGN) were presented. It was concluded that the both were clearly different, since, from immunological and histological findings, WG has characteristics of autoimmune disease and MGN could be classified in malignant lymphoma.

Induction of both reaginic and blocking antibodies with a low molecular weight fraction of timothy pollen extract

In preliminary studies to evaluate the potential of the nonprecipitating antigen D for treatment of timothy-sensitive patients, a group of 8 nonallergic individuals was given a series of intracutaneous injections of antigen D at weekly intervals for 4 weeks. Each of the persons had no clinical history of allergy and gave negative skin reactions to antigen D, crude pollen extract (WST), and allergens A and B of timothy pollen. Four weeks after the last injection of antigen D, 6 of the 8 volunteers gave positive skin reactions when skin tested with various timothy allergens. Passive transfer studies with sera obtained from these volunteers not only showed significant blocking antibody titers but also varying amounts of reaginic antibody titers up to 1/25. The successful induction of homocytotropic antibodies in rhesus monkeys by repeated intracutaneous injections with antigen D was demonstrated by both direct skin reactions and histamine release from passively sensitized monkey lung fragments. The homocytotropic antibodies produced in both man and rhesus monkeys are heat labile, persist at sensitized sites for at least 48 hours, and can be passively transferred by sera.

Clinical Studies on Asthmatic Patients in Taiwan : Report2. The relationship between the efcect of hyposensitization and the threshold value of skin test and P-K reaction, as well as the blocking antibody titer.

Clinical Studies on Asthmatic Patients in Taiwan : Report2. The relationship between the efcect of hyposensitization and the threshold value of skin test and P-K reaction, as well as the blocking antibody titer.

Reagin in tears

Skin-sensitizing antibody in low titer was present in the lacrimal secretion of 2 out of 7 atopic individuals whose sera showed a high skin-sensitizing antibody titer. Blocking antibody to ragweed was not found in the lacrimal secretion of one treated patient who had an elevated serum blocking antibody titer. Serum protein components found in tears were albumin and beta-2A globulin.

The Effects of Treatment with the Emulsions of Ragweed Extract on Antibody Titers

Summary 1.The skin-sensitizing and hemagglutinating antibody titers of most ragweed hay fever patients rise after an injection of emulsified ragweed extract reaching a peak at 3 to 6 weeks and showing a gradual decline by the end of 12 weeks. 2.The least sensitive group of patients who received the highest dosage (10,000 PNU) showed no significant changes in skin-sensitizing antibody titer, but did respond with increased hemagglutinating and blocking antibody titers. 3.All groups of patients showed a rise in blocking antibody after treatment reaching a peak at approximately 6 weeks. The group that received 10,000 PNU remained essentially unchanged from the 6th to 12th weeks whereas those that received 1000, 2500, or 5000 PNU showed a decrease in titer. 4.Patients who received two injections of emulsified ragweed extract showed further increases of all antibody titers after the second injection.