Measurement Of Serum Antibodies Research Articles

Direct Inoculation and Human Transferrin Supplementation Increases the Survivability of Neisseria gonorrhoeae in the Upper Reproductive Tract of Estrogen‐Treated Female BALB/c Mice

ObjectiveThis study intended to investigate methods of increasing Neisseria gonorrhoeae (Gonococcus; Gc) survivability in the upper reproductive tract (URT) of female mice with the aim of establishing a previously undescribed murine model of Gc URT infections ‐ also known as pelvic inflammatory disease (PID) ‐ in women. Such a model would allow for further investigations into the natural history and pathogenesis of ascending Gc infections.MethodsTwo main experimental arms were used in this study: 1. Gc‐infected mice treated with estrogen alone vs estrogen and human transferrin (hTF) and 2. Gc‐infected mice treated with long‐acting progesterone (Medroxyprogesterone) alone vs progesterone and hTF. Appropriate uninfected, negative controls were used in all experiments. All mice were pre‐treated with antibiotics to suppress commensal flora and then given either estrogen or progesterone prior to inoculation, according to their study groups. Diestrous or anestrous stage BALB/c mice were then inoculated with Neisseria gonorrhoeae (strain FA1090) using a novel, trans‐cervical catheterization technique and the appropriate groups were treated with hTF up to 5 days post‐inoculation. Daily vaginal swabs were taken for polymorphonuclear white blood cell (PMN WBC) enumeration and quantitative Gc cultures up to day 5. In addition, part of each study group was sacrificed at days 3 and 5 post‐inoculation for Gc quantitative cultures of endometrial scrapings. Lastly, whole URTs from a portion of each group were fixed and stained for WBCs and Gc on days 3 and 5 post‐inoculation.ResultsDirect inoculation via trans‐cervical catheter proved successful in introducing and establishing Gc infection in the URT of all groups of estrogen‐treated mice for at least five days as determined by culture of endometrial scrapings and Gc‐specific IHC staining at day 5 post‐inoculation. Mice treated with progesterone showed no infection at any time point by either culture of daily vaginal swabs or of endometrial scrapings. Higher numbers of Gc were recovered from vaginal swabs (p<.05) and endometrial tissue on day 3 and 5 post‐inoculation from mice treated with estrogen and hTF compared to those treated with estrogen alone (comparisons made by geometric mean of Gc colonies recovered on each day).Conclusions and Future DirectionsThis project established a novel method of direct, trans‐cervical inoculation of Gc into the female mouse URT thereby creating a usable model of Gc URT infection (PID) in women for further study. Future projects will utilize this model to better understand the natural history of Gc infections and to investigate potential methods for treating these infections. Currently, this laboratory is investigating host immune responses by measuring serum antibodies and vaginal chemokines/cytokines using this model. In addition, GFP‐labeled FA1090 strain Gc have been created and will be used for better visualization of Gc in histologic samples, which in turn could be used to track Gc movement through the mouse URT, as well as cellular responses to infection using sequentially fixed tissue samples.Support or Funding InformationThe Uniformed Services University of the Health Sciences and NIH/NIAID grant # R01 AI42053.

Egg-specific IgE and basophil activation but not egg-specific T-cell counts correlate with phenotypes of clinical egg allergy

Egg allergy is phenotypically heterogeneous. Asubset of patients with egg allergy can tolerate egg in an extensively heated form. Inclusion of baked egg (BE) into the diet accelerates resolution of egg allergy. Conversely, BE reactivity is associated with persistent disease. The immune basis of this clinical heterogeneity is unknown. We sought to study egg-specific antibody, basophil, and T-cell responses in children with reactivity or tolerance to BE. All participants underwent double-blind, placebo-controlled challenges to BE, and those who tolerated BE were challenged with unheated egg white protein to confirm clinical egg reactivity. Laboratory studies included serum antibody measurements, basophil activation tests, and CD154-based detection of egg-responsive T cells by using flow cytometry. Of the 129 children studied, BE-reactive participants had significantly greater levels of egg-, ovalbumin-, and ovomucoid-specific IgE; lower ratios of egg-specific IgG4/IgE; and increased basophil activation in response to egg. Among all participants, CD154-based profiling revealed egg-responsive T cells producing IL-4 and IL-13 but little IL-10 or IFN-γ, as well as the presence of egg-responsive Foxp3+CD25+CD127low regulatory T cells. Egg-responsive T cells expressed CCR4, CCR6, and CXCR5, indicating capacity for homing to the skin, mucosa, and B-cell follicles. However, neither the frequency nor phenotype of egg-responsive T cells was different in those with tolerance or reactivity to BE. Egg-specific antibody and basophil responses, but not T-cell responses, are greater in those with reactivity versus tolerance to BE. Egg-specific antibody and T-cell responses were highly heterogeneous in this cohort. The clinical implications of this immune heterogeneity will need to be studied longitudinally.

Bivalent oral cholera vaccine in participants aged 1 year and older in the Dominican Republic: A phase III, single-arm, safety and immunogenicity trial

ABSTRACTThe Dominican Republic, historically non-endemic for cholera, is experiencing an ongoing cholera epidemic. We assessed the safety and immunogenicity of two doses of the killed bivalent (O1 and O139) whole-cell oral cholera vaccine (OCV) on day (D)0 and D14 in healthy participants aged ≥1 year. Immediate unsolicited systemic adverse events (AEs) were monitored up to 30 minutes and solicited systemic reactions, up to 7 days after each vaccination. Unsolicited AEs were recorded up to D14 (post-dose 1) and 30 days post-dose 2. A vibriocidal antibody assay with microtiter technique was used to measure serum antibodies to V. cholerae strains (O1 El Tor Inaba, O1 El Tor Ogawa, O139) on D0, D14 and D28. Geometric mean titers (GMTs) and seroconversion (≥4-fold increase from D0) rates were calculated. We recruited 336 participants; 112 in three age groups (1–4, 5–14 and ≥15 years). No safety concerns were observed. GMTs increased from baseline for all serotypes, with marked increases for O1 Inaba and Ogawa post-dose 1. Post-dose 2 GMTs tended to be equal or slightly lower, with ranges: O1 Inaba, 283 (95% confidence interval 191–419) to 612 (426–880); O1 Ogawa, 346 (223–536) to 754 (553–1028); and O139, 20.3 (13.5–30.6) to 43.8 (30.1–63.7). Seroconversion rates post-dose 2 for O1 Inaba and Ogawa were high (≥87%) for all age groups. OCV demonstrated an acceptable safety profile and robust immunogenicity in these participants, in-line with previous observations in epidemic and endemic settings.This study is registered on www.clinicaltrials.gov (NCT02434822).

Elevation of Autoantibody in Patients with Ischemic Stroke.

Recent clinical research has revealed a significant correlation between atherosclerosis, one of the primary etiologies of ischemic stroke, and the immune system. Assuming that “disease-specific autoantibodies are produced in the sera of patients with ischemic stroke,” we investigated multiple arteriosclerosis-related antibodies using the serological identification of antigens by recombinant cDNA expression cloning (SEREX), an established method for identifying antigenic proteins. We either screened a human aortic endothelial cell cDNA library or conducted protein array screening using the sera from patients with ischemic stroke, such as carotid artery stenosis or transient ischemic attack (TIA). Next, we measured serum antibody levels using amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in patient/healthy donor (HD) cohorts and identified several antigens, the antibody levels of which were significantly higher in patients with ischemic stroke than in HDs. This review introduced the method of identifying antigens by the SEREX and protein microarray and summarized antigenic proteins. In particular, it focused on anti-replication protein A2 antibody and anti-programmed cell death 11 antibody, which are significantly related to atherosclerotic plaque and ischemic brain tissue, respectively, and proposed the mechanism of elevated autoantibody levels against them. Furthermore, this review suggests a possibility of clinical application as an atherosclerotic disease diagnostic marker for TIA or cerebral infarction.

Serological survey of Australian native reptiles for exposure to ranavirus.

Ranaviruses have been isolated from many ectothermic vertebrates, and serological surveys of both amphibians and reptiles have shown the presence of ranaviral antibodies in a proportion of these populations. An enzyme-linked immunosorbent assay (ELISA) was developed to measure serum antibodies against ranavirus in Australian reptiles. The ELISA was validated with serum from challenge trials with Bohle iridovirus (BIV) in 6 reptilian species. A preliminary sero-survey of northern Queensland riparian reptile fauna (saw-shelled turtles Myuchelys latisternum, Krefft's river turtles Emydura macquarii krefftii, freshwater crocodiles Crocodylus johnstoni, as well as the snakes Boiga irregularis, Dendrelaphis punctulatus, Tropidonophis mairii, Morelia spilota, Liasis childreni and L. fuscus) revealed evidence of past exposure to Bohle iridoviral antigens in part of the population at several locations sampled. Furthermore, in Krefft's river turtles and freshwater crocodiles, a statistically significant trend was apparent for larger reptiles to be more likely to have BIV-reactive sera than smaller individuals. The use of adult tortoise populations as sentinels can assist in monitoring the presence of BIV in northern Australian freshwater streams, and thereby the potential dangers to native fauna from this agent.

Modeling specific antibody responses to natural immunization to predict a correlate of protection against infection before commencing a clinical vaccine trial

ABSTRACT Background Clinical trials of vaccines for children to prevent acute otitis media (AOM) infections caused by the bacteria Streptococcus pneumonia (Spn) are in Phase I. The objective of this study was to use serum antibody measurements to pneumococcal purified protein candidate antigens that occurred after natural “immunization” to predict a correlate of protection response needed following an injectable vaccine against AOM in children. Methods 590 nasal and serum samples were collected from 129 healthy children at 6, 9, 12, 15, 18, 24 and 30–36 months of age and when the child developed AOM. Middle ear fluid to detect Spn was collected at every episode of AOM. Quantitative ELISA was used to determine serum IgG against 7 Spn vaccine antigens: PspA clade 3, PspA clade 5, PhtD, PhtE, LytB, PcpA and Ply. A correlate of protection (COP) was estimated by regressing AOM events against age adjusted antibody levels induced by nasopharyngeal colonization and AOM infections, using logistic regression and generalized estimating equation methods. Results A significant COP was found for Spn PhtD (p = 0.0015), PhtE (p = 0.00034), LytB (p = 0.004), PcpA (p = 0.002), and Ply (p = 0.007) between higher antibody levels and reduced frequency of AOM. We estimated that a 2-fold higher antibody level in a child than the mean antibody level induced by NP colonization (after adjusting for subject age) to PhtD, LytB, PcpA, PhtE or Ply reduced the risk of AOM by 14–21%, a 4-fold higher level reduced it by 25–38% and a 10-fold higher level reduced it by 39–54%. Conclusion We developed a model to predict the necessary level of serum antibody and fold higher above a threshold to PhtD, PhtE, LytB, PcpA and Ply that would correlate with a reduced likelihood of AOM in children age 6–24 months old if enrolled in a Phase III clinical efficacy trial.

Polyvalent immunoglobulin binding is an obstacle to accurate measurement of specific antibodies with ELISA despite inclusion of blocking agents

Specific antibody concentrations are frequently measured in serum (and plasma and intravenous immunoglobulin) samples by enzyme-linked immunosorbent assay (ELISA). The standard negative control involves incubation of buffer alone on antigen-coated wells. The immunoreactivity that develops in antigen-coated wells in which diluted serum has been incubated is assumed to represent specific antibody binding. This approach can result in marked overestimation of specific antibody levels, because serum contains specific polyvalent antibodies which bind, primarily with low affinity, to multiple antigens (including those on ELISA plates) despite the use of blocking agents. Non-denaturing purification of serum IgG, followed by assessment of the antigen binding or antigen-binding affinity of this purified IgG, can reduce but not eliminate the problem of polyvalent antibody binding in indirect ELISAs. Alternatively, polyvalent antibody binding can be estimated by incubating a diluted serum sample on wells coated with an irrelevant protein (such as bovine serum albumin or a scrambled peptide sequence) or buffer alone, then subtracting this reactivity from the sample's binding to wells coated with the antigen of interest. Polyvalent binding of immunoglobulins must be accounted for in order to obtain accurate ELISA measurements of serum, plasma, or intravenous immunoglobulin antibodies.

Annals for Educators - 15 August 2017

Annals for Educators - 15 August 2017

Antigenic and immunogenic evaluation of Helicobacter pylori FlaA epitopes.

Objective(s):Helicobacter pylori are among most common human pathogens affecting at least half of the world’s population. Mobility is one of the important primary factors in bacterial colonization and invasion. The purpose of this research is cloning, expression, and purification of FlaA protein specific epitopes in order to evaluate their antigenicity and immunogenicity.Materials and Methods:The antigenic region of the flaA gene was bioinformatically predicted using Epitope mapping software’s and the predicted epitopes were expressed in a prokaryotic expression vector. The antigen was injected into the animal model (mice BALB/c) and some indicators including IgG1, IgG2a, IgA, IFN-γ, and IL 5 were measured.Results:The immunogenicity studies in animal models by measuring serum antibodies (IgG1, IgG2a, and IgA) and cytokines (IFN-γ and IL5) revealed that the rFlaA induces a proper immune response in animal models.Conclusion:The recombinant FlaA protein is antigenic and immunogenic. Therefore, it might be used in order to design of specific diagnostic kits and recombinant vaccines against H. pylori.

Optimizing Direct Membrane and Direct Skin Feeding Assays for Plasmodium falciparum Transmission-Blocking Vaccine Trials in Bancoumana, Mali.

Malaria transmission-blocking vaccines (TBV) have been evaluated in field trials in Mali since 2013. However, the assays currently used to measure serum antibody TB activity (TBA) after vaccination are highly variable, in part due to the lack of optimization and standardization for field assays in which mosquitoes feed on gametocytemic blood. Herein, we report a study conducted in Bancoumana village, Mali, where we identify and optimize the parameters that contribute to successful mosquito feeding outcomes in both direct skin feeds (DSFs) and direct membrane feeding assays (DMFA). These parameters include: 1) mosquito age, 2) duration of mosquito starvation prior to feeding, 3) membrane selection for DMFA, 4) anatomical location of DSF feeding (arm, calf, and ankle), and 5) time of day for DSF (dawn or dusk). We found that younger mosquitoes were significantly associated with higher feeding, survival, and infection rates. Longer starvation times were positively, but not significantly, associated with higher infection rates, but were negatively associated with feeding and survival. Membrane type and body location did not affect infection outcome significantly. Although dusk was found to be associated with higher infection rates, this may be confounded by the time from positive blood smear. Based on these findings, we make specific recommendations for optimal feeding parameters in the different assay types to maximize the chance of detecting parasite transmission in a standardized manner.

Evaluation of protective immunity induced by DNA vaccination with genes encoding Toxoplasma gondii GRA17 and GRA23 against acute toxoplasmosis in mice

Toxoplasma gondii, an obligatory intracellular protozoan, can cause serious public health problems and economic losses worldwide. Two novel dense granule proteins (GRA17 and GRA23) were recently identified as T. gondii-secreted proteins which are localized to the parasitophorous vacuole membrane (PVM) and can mediate the movement of small molecules between the host cell and parasitophorous vacuole (PV). In the present study, we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice. Eukaryotic expressing plasmids pVAX-TgGRA17 and pVAX-TgGRA23 were constructed. Then, BALB/c mice were intramuscularly immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 followed by challenge infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were examined by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival time after challenge. Our results showed that mice immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 induced specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-γ and IL-12 (p70), and CD3+CD4+CD8− and CD3+CD8+CD4− T cells, as well as prolonged survival time (9.1 ± 0.32 days for pVAX-TgGRA17, 10.8 ± 0.79 days for pVAX-TgGRA23, and 12.6 ± 2.55 days for pVAX-TgGRA17 + pVAX-TgGRA23) compared to the blank control (7.11 ± 0.33 days), PBS control (7.22 ± 0.44 days), and pVAX I control (7.11 ± 0.33 days). These results demonstrated that both TgGRA17 and TgGRA23 are potential vaccine candidates, TgGRA23 has a better immunogenicity, and co-immunization of pVAX-TgGRA17 and pVAX-TgGRA23 induces better protective efficacy.

Tests for Serum Transglutaminase and Endomysial Antibodies Do Not Detect Most Patients With Celiac Disease and Persistent Villous Atrophy on Gluten-free Diets: a Meta-analysis

Tests to measure serum endomysial antibodies (EMA) and antibodies to tissue transglutaminase (tTG) were developed to screen for celiac disease in patients consuming gluten. However, they are commonly used to monitor patients on a gluten-free diet (GFD). We conducted a meta-analysis to assess the sensitivity and specificity of tTG IgA and EMA IgA assays in identifying patients with celiac disease who have persistent villous atrophy despite a GFD. We searched PUBMED, EMBASE, BIOSIS, SCOPUS, clinicaltrials.gov, Science Citation Index, and Cochrane Library databases through November 2016. Inclusion criteria were studies of subjects with biopsy-confirmed celiac disease, follow-up biopsies, and measurement of serum antibodies on a GFD, biopsy performed on subjects regardless of symptoms, or antibody test results. Our analysis excluded subjects with refractory celiac disease, undergoing gluten challenge, or consuming a prescribed oats-containing GFD. Tests were considered to have positive or negative findings based on manufacturer cut-off values. Villous atrophy was defined as a Marsh 3 lesion or villous height:crypt depth ratio below 3.0. We constructed forest plots to determine the sensitivity and specificity of detection for individual studies. For the meta-analysis, a bivariate random effects model was used to jointly model sensitivity and specificity. Our search identified 5408 unique citations. Following review of abstracts, 442 articles were reviewed in detail. Only 26 studies (6 of tTG assays, 15 of EMA assays, and 5 of tTG and EMA assays) met our inclusion criteria. The most common reason studies were excluded from our analysis was inability to cross-tabulate histologic and serologic findings. The serum assays identified patients with persistent villous atrophy with high levels of specificity: 0.83 for the tTG IgA assay (95% CI, 0.79-0.87) and 0.91 for the EMA IgA assay (95% CI, 0.87-0.94). However, they detected villous atrophy with low levels of sensitivity: 0.50 for the tTG IgA assay (95% CI, 0.41-0.60) and 0.45 for the EMA IgA assay (95% CI, 0.34-0.57). The tests had similar levels of performance inpediatric and adult patients. In a meta-analysis of patients with biopsy-confirmed celiac disease undergoing follow-up biopsy on a GFD, we found that tests for serum tTG IgA and EMA IgA levels had low sensitivity (below 50%) in detection of persistent villous atrophy. We need more-accurate non-invasive markers of mucosal damage in children and adults with celiac disease who are following aGFD.

What is the Clinical Role of Oral Liquid Preparations in the Treatment of Patients With Hypothyroidism?

Fallahi et al ( 1. Fallahi P. Ferrari S.M. Antonelli A. In patients with subclinical hypothyroidism while in therapy with tablet LT4, the liquid LT4 formulation is more effective in restoring euthyroidism. Endocr Pract. 2017; 23: 170-174 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar ) have performed an interesting observational, prospective study assessing the ability of comparable levothyroxine doses of tablet and liquid preparations in lowering serum thyroid-stimulating hormone (TSH) concentration in patients with subclinical hypothyroidism who are not ingesting potentially interfering medications and do not have gastrointestinal disorders that might potentially interfere with levothyroxine absorption. The authors ( 1. Fallahi P. Ferrari S.M. Antonelli A. In patients with subclinical hypothyroidism while in therapy with tablet LT4, the liquid LT4 formulation is more effective in restoring euthyroidism. Endocr Pract. 2017; 23: 170-174 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar ) studied hypothyroid individuals who were taking stable levothyroxine doses and had serum TSH concentrations ≤3 μU/mL on at least two separate determinations within 2 years prior to the study or serum TSH level at least 4 μU/mL or higher (assessed within 1 month prior to the study) in conjunction with normal serum free thyroxine (FT4) and free triiodothyronine (FT3) on two different occasions. They ( 1. Fallahi P. Ferrari S.M. Antonelli A. In patients with subclinical hypothyroidism while in therapy with tablet LT4, the liquid LT4 formulation is more effective in restoring euthyroidism. Endocr Pract. 2017; 23: 170-174 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar ) appropriately excluded patients who had Helicobacter pylori or atrophic gastritis, previous gastric or intestinal surgery, or bariatric surgery. Fallahi et al ( 1. Fallahi P. Ferrari S.M. Antonelli A. In patients with subclinical hypothyroidism while in therapy with tablet LT4, the liquid LT4 formulation is more effective in restoring euthyroidism. Endocr Pract. 2017; 23: 170-174 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar ) did an excellent job of examining and detailing the inclusion and exclusion criteria, even investigating unexplained anemia or dyspepsia and also performed serum antibody measurements to help exclude undiagnosed celiac disease and measured H. pylori antigen in feces. Patients taking medications that are known to interfere with levothyroxine absorption were also excluded.

Immune responses and protection after DNA vaccination against Toxoplasma gondii calcium-dependent protein kinase 2 (TgCDPK2).

Toxoplasma gondii, an intracellular zoonotic protozoan parasite, is possibly the most widespread parasite of warm-blooded animals and can cause serious public health problems and economic losses worldwide. TgCDPK2, a member of the T. gondii calcium-dependent protein kinase family, was recently identified as an essential regulator for viable cyst development in T. gondii. In the present study, we evaluated the protective immunity induced by DNA vaccination based on a recombinant eukaryotic plasmid, pVAX-TgCDPK2, against acute toxoplasmosis in mice. BALB/c mice were intramuscularly immunized with pVAX-TgCDPK2 plasmid and then challenged by infection with the highly virulent RH strain of T. gondii. The specific immune responses and protective efficacy against T. gondii were analyzed by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometric on lymphocytes and the survival time of mice after challenge. Our results showed that mice immunized with pVAX-TgCDPK2 could elicit special humoral and cellular responses, with higher levels of IgG antibody, and increased levels of Th1-type cytokines IFN-γ, IL-12(p70), and CD3 + CD4 + CD8 − and CD3 + CD8 + CD4 − T cells, and had a prolonged survival time (14.0 ± 2.32 days) compared to control mice. These results demonstrate that pVAX-TgCDPK2 is a potential vaccine candidate against acute toxoplasmosis.

Modeling Specific Antibody Responses to Natural Immunization to Predict a Correlate of Protection Against Infection Before Commencing a Clinical Vaccine Trial

ABSTRACTBackground: Clinical trials of vaccines for children to prevent acute otitis media (AOM) infections caused by the bacteria Streptococcus pneumonia (Spn) are in Phase I. The objective of this study was to use serum antibody measurements to pneumococcal purified protein candidate antigens that occurred after natural “immunization” to predict a correlate of protection response needed following an injectable vaccine against AOM in children.Methods: 590 nasal and serum samples were collected from 129 healthy children at 6, 9, 12, 15, 18, 24 and 30–36 months of age and when the child developed AOM. Middle ear fluid to detect Spn was collected at every episode of AOM. Quantitative ELISA was used to determine serum IgG against 7 Spn vaccine antigens: PspA clade 3, PspA clade 5, PhtD, PhtE, LytB, PcpA and Ply. A correlate of protection (COP) was estimated by regressing AOM events against age adjusted antibody levels induced by nasopharyngeal colonization and AOM infections, using logistic regression and ge...

Immune-modulating properties of horse milk administered to mice sensitized to cow milk.

The aim of this study was to examine immune adaptive changes, the expression of innate biomarkers and variations in intestinal microbiota composition after horse-milk administration in BALB/c mice, which were sensitized intraperitoneally using cow β-lactoglobulin and α-casein with aluminum adjuvant. We measured serum antibody IgE levels and the expression of MCP-1, IL-4, and TNF-α in duodenal samples. Changes in immune cell populations in peripheral blood were quantified using flow cytometry, and intestinal microbiota composition was assessed using real-time PCR. We found that horse-milk administration decreased serum IgE levels in sensitized mice. The groups that received horse milk showed an increased population of regulatory T cells (CD4+Foxp3+). Horse-milk administration decreased the mRNA levels of IL-4 and resulted in higher transcripts of TLR-4 in all treatment groups; however, the levels of MCP-1, TNF-α, and TLR-2 were unaltered. After horse-milk treatment, we observed a positive effect, with increased numbers of intestinal Bifidobacterium spp. We observed immune-modulating properties of horse milk, but future studies should focus on testing horse-milk processing, such as fermentation and destroying most allergenic epitopes to continue research under clinical conditions.

Abstract CT096: Clinical activity, immune and viral correlates of PD-1 blockade with pembrolizumab as first systemic therapy in patients with advanced Merkel cell carcinoma

Abstract Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer linked to UV exposure and the Merkel cell polyomavirus (MCPyV). Cytotoxic chemotherapy is associated with high response rates in patients (pts) with metastatic MCC, although responses are seldom durable and median progression-free survival is only 13 weeks. MCC may be responsive to PD-1 pathway blockade as tumors often express PD-L1 and MCPyV-specific T cells express the inhibitory co-receptor PD-1. Methods: In this open-label, phase II trial by the Cancer Immunotherapy Trials Network (NCT02267603), adults with advanced/metastatic MCC and no prior systemic therapy were enrolled. Pembrolizumab, a humanized IgG4 anti-PD-1 monoclonal antibody, was given IV every 3 weeks (2 mg/kg). Response was assessed by RECIST 1.1 every 9-12 weeks. Presence of MCPyV was assessed by measuring serum antibody titers to the MCPyV oncoprotein and/or quantifying tumor oncoprotein expression by immunohistochemistry (IHC). Results: Twenty-six pts were treated (median duration 22 weeks, range 4 to 49 weeks as of 1/8/16 dataset). Twenty-three pts had at least one documented radiologic or clinical assessment of response. Fifteen of 23 evaluable pts (65%) responded (4 complete responses and 11 partial responses); 2 additional pts had stable disease and 6 had progressive disease as best response. Fourteen of 15 responses (93%) were ongoing at last follow-up. Ten responders had at least one follow-up evaluation with response duration ranging from 8+ to 27+ weeks beyond initial documented response. Treatment was generally well tolerated with mostly grade 1 or 2 adverse events. Two pts developed severe drug-related toxicities including grade 4 myocarditis (n = 1) and grade 4 transaminase elevation (n = 1), both of which improved with corticosteroid treatment. Both pts have ongoing tumor responses despite discontinuation of pembrolizumab. Sixteen of 26 pts (62%) were classified as having virus-positive MCC. Among the 23 evaluable pts, 9 of 14 (64%) of those with virus positive tumors responded, while 6 of 9 (67%) of pts with virus negative tumors responded. Analyses of T cells specific to MCPyV and titers of oncoprotein antibodies during therapy, as well as IHC characterization of tumor biopsies including PD-L1 expression are ongoing. Conclusions: First-line therapy with pembrolizumab in pts with advanced MCC is associated with frequent (65%) and durable responses. Notably, the response rate was similar among pts with virus-positive and virus-negative tumors. While virus-positive tumors are known to express highly antigenic MCPyV oncoproteins, virus-negative tumors are typically UV-induced and display strikingly elevated mutational burdens generating putative neo-antigens. Thus, potentially through distinct mechanisms, both virus-positive and virus-negative MCC tumors appear to be immunogenic and frequently responsive to anti-PD-1 therapy. Citation Format: Paul Nghiem, Shailender Bhatia, Evan Lipson, Ragini Kudchadkar, Natalie Miller, Adil Daud, Steven Fling, Philip Friedlander, Harriet Kluger, Holbrook Kohrt, Lisa Lundgren, Kim Margolin, Thomas Olencki, Sunil Reddy, Erica Shantha, William Sharfman, Elad Sharon, Michi Shinohara, John Thompson, Jennifer Yearley, Suzanne Topalian, Martin Cheever. Clinical activity, immune and viral correlates of PD-1 blockade with pembrolizumab as first systemic therapy in patients with advanced Merkel cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT096.

Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus.

High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

Development of three-valent vaccine against streptococcal infections in olive flounder, Paralichthys olivaceus

Streptococcus iniae and S. parauberis types I and II are major bacterial pathogens affecting the olive flounder, Paralichthys olivaceus. With the introduction of formalin-killed vaccine for S. iniae, recent epidemiological studies have indicated that S. parauberis infections are becoming increasingly severe and frequent in the olive flounder farming industry. Here, the formalin-killed bacterins were used to develop a three-valent vaccine against three streptococcal infections, and its efficacy was assessed in laboratory- and field-challenge trials. In the laboratory-challenge test, it obtained 75%, 75% and 90% relative percent survival (RPS) values in vaccinated olive flounders challenged with S. iniae, S. parauberis type I, and S. parauberis type II, respectively. For the field-challenge trial, the three-valent vaccine was administered to over 100,000 olive flounders, which were compared to non-injected control fish. An RPS of 74% was observed when S. parauberis infection naturally occurred at 11 fish farms. Serum antibody measurements using olive flounder immunoglobulin-specific antibodies indicated that immunized fish had significantly higher serum antibody levels than control fish up to 6months post-vaccination. These results demonstrate that the three-valent vaccine using formalin-killed bacterins could effectively protect olive flounders against three major streptococcal infections. Statement of relevanceThe olive flounder, P. olivaceus, is an economically important fish species in Northeast Asia and the most valuable fish species in South Korea, corresponding to 56.5% of the total mariculture production in 2010 (Baeck et al., 2006). However, streptococcal infections often break out among farmed olive flounders subjected to certain conditions (e.g., high stocking density, poor water quality and high water temperature), leading to mass mortality and severe economic losses (Baeck et al., 2006; Cho et al., 2008) Antibiotic treatments are usually recommended to address such outbreaks, but the prevalence of multi-drug-resistant bacteria means that antibiotic compounds are not always able to eradicate the infections (Creeper and Buller, 2006).The Gram-positive bacteria, Streptococcus iniae and S. parauberis, cause streptococcosis and have severe effects on aquaculture of the olive flounder, barramundi, channel catfish, European sea bass, rainbow trout, striped bass, tilapia, turbot and yellow tail (Gudmundsdóttir and Björnsdóttir, 2007; Han et al., 2011). In the olive flounder, fish infected with S. iniae usually show severe pathological changes, such as darkening, exophthalmia, rectal hernia, abdominal distension, ascites and congestion of intestinal organs, whereas fish infected with S. parauberis rarely exhibited any remarkable pathological finding except for darkening of the skin (Kim et al., 2006).Some vaccines have been reported to prevent S. iniae infection, showing high relative percent survival (RPS) among olive flounders in laboratory trials; such vaccines are now commercially available (Cheng et al., 2010a). However, recent epidemiological studies have suggested that S. parauberis infection has increased and become more severe and frequent as much as infections of S. iniae, which was previously considered the major bacterial pathogen among olive flounder (Park et al., 2012; Park, 2009; Perera et al., 1998). S. parauberis can be divided into two serotypes (types I and II) based on serological testing of a distinctive capsular polysaccharide layer (Bromage and Owens, 2002; Plant and Lapatra, 2011). Since the two serotypes possess specific antigenic characteristics, a vaccine should be able to address both types in order to effectively prevent S. parauberis infection.The present study was undertaken to develop a three-valent vaccine against both serotypes of S. parauberis plus S. iniae using formalin-killed vaccine preparation method. The efficacy of this vaccine against the major streptococcal infections of olive flounder was calculated as RPS and conducted in both laboratory trials and large-scale field trial. This vaccine will be highly useful and more advantageous since the use of this does not require several antibiotics which means safer food production and more sustainable environment.

Correlates of Immunity to Influenza as Determined by Challenge of Children with Live, Attenuated Influenza Vaccine.

Background. The efficacy of live, attenuated live attenuated influenza vaccine(LAIV) and inactivated influenza vaccine(IIV) is poorly explained by either single or composite immune responses to vaccination. Protective biomarkers were therefore studied in response to LAIV or IIV followed by LAIV challenge in children.Methods. Serum and mucosal responses to LAIV or IIV were analyzed using immunologic assays to assess both quantitative and functional responses. Cytokines and chemokines were measured in nasal washes collected before vaccination, on days 2, 4, and 7 after initial LAIV, and again after LAIV challenge using a 63-multiplex Luminex panel.Results. Patterns of immunity induced by LAIV and IIV were significantly different. Serum responses induced by IIV, including hemagglutination inhibition, did not correlate with detection or quantitation of LAIV on subsequent challenge. Modalities that induced sterilizing immunity seen after LAIV challenge could not be defined by any measurements of mucosal or serum antibodies induced by the initial LAIV immunization. No single cytokine or chemokine was predictive of protection.Conclusions. The mechanism of protective immunity observed after LAIV could not be defined, and traditional measurements of immunity to IIV did not correlate with protection against an LAIV challenge.