Intermediate Avidity Research Articles

Abstract 3992: Tandem CAR-T cells against mesothelin and MUC16ectoto overcome tumor-antigen heterogenicity

Abstract Introduction: Antigen heterogeneity is a challenge for CAR-T cell therapy in solid tumors due to antigen escape when targeting a single antigen. In ovarian cancer (OC), mesothelin (meso) and Mucin16 (MUC16) are two of the most overexpressed antigens. Moreover, CA125, which is part of MUC16, is lost in some OC patients, but the ectodomain remains on the cell surface. Targeting both antigens (Meso and MUC16ecto) with a tandem-CAR configuration could overcome antigen heterogeneity to improve CAR-T cell efficacy in OC. Material and Methods: We designed a library of different CARs with tandem Meso and MUC16ecto scFv combinations with varying G4S linker lengths, fused to a CD8 hinge/transmembrane domain and 41BBz intracellular domain. A series of constructs was then tested in an NFAT-GFP reporter system in Jurkat cells to determine which configuration induced maximal T cell activation and antigen-binding capacity via flow cytometry. We identified the top 2 activators and binders, produced primary human tandem CAR-T (TanCAR) cells by LV transduction, and tested their cytotoxicity in vitroagainst dual antigen-expressing or single antigen-expressing tumor cells using luciferase killing assays and Incucyte. We measured the cytokine production with a Luminex® Multiplex Assay and used a z-Movi® Cell Avidity Analyzer to test the tandem scFv avidity towards the antigens. Also, we examined TanCAR activity against a mixed tumor model in NSG mice. Their in vitro and in vivo activity were compared to CAR T cells targeting either single antigen (SS1-anti-Meso or 4H11-anti-MUC16ecto). Results: We found that TanCAR1 and TanCAR3 as the configurations with the best activation and binding capacity in response to cognate antigen. TanCAR1 and TanCAR3 had superior in vitro cytotoxic capacities compared to SS1 or 4H11 in tumor models expressing both antigens and in mixed cultures of tumor cells expressing one, both, or neither target antigen. From this mixed culture, we analyzed the remaining tumor cell populations by flow cytometry and observed an overgrowth of the population not targeted by CAR. In response to tumor cells, tandem CARs also produced INFg, IL6, IL-18, IL-13, and GM-CSF regardless of whether one or two antigens were expressed. For further experiments, we pursued TanCAR1 due to its slightly better functionality compared to TanCAR3. TanCAR1 had the same avidity for cells expressing the single antigens as SS1 or 4H11 CARs, but an intermediate avidity for the double Ag-expressing cell line compared to the single CARs. Finally, we tested TanCAR1-T cells in an in vivo mixed tumor model, showing that they better controlled and reduced tumor growth compared to either SS1 or 4H11. Conclusion: Targeting Meso and MUC16ecto through a tandem CAR design allows for a better anti-tumor response compared to CAR T cells targeting a single antigen. This approach may combat antigen heterogeneity by preventing antigen escape. Citation Format: Diego Salas-Benito, Filippo Birocchi, Alexander Armstrong, Amanda A. Bouffard, Tamina Kienka, Felix Korell, Mark B. Leick, Trisha Berger, Marcela V. Maus. Tandem CAR-T cells against mesothelin and MUC16ectoto overcome tumor-antigen heterogenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3992.

Toxoplasmosis in pregnancy: test, treatment and outcome

Objectives: The aim of this study was to share the results, follow-up, and treatment characteristics of our pregnant women who were followed-up with anti-Toxoplasma gondii Immunoglobulin (Ig) M positivity during pregnancy. Methods: Anti-T. gondii IgM- and IgG-positive pregnant women were evaluated between 2014-2018. Demographic characteristics, treatment, and information about pregnancy were obtained from the electronic database. Pregnant women were divided into three groups; primary infection, no infection, and suspected infection in pregnancy. Primary and suspected infection in pregnancy were followed up congenital toxoplasmosis risky pregnancy. Fetal ultrasonography (USG), T. gondii DNA polymerase chain reaction (PCR) result in amniotic fluid were recorded. Results: Twenty-four pregnant women with a mean age of 27.9 years were followed up. IgG avidity results were low in 37.5% (n = 9), intermediate avidity in 8.3% (n = 2), and high avidity in 54.2% (n = 13) of pregnant women. Eleven (45.9%) pregnant women had congenital toxoplasmosis risky pregnancy. Fetal USG was performed on ten pregnant women, and no signs of congenital toxoplasmosis were found. Amniocentesis was performed in 72.7% (n = 8) of the participants, and the amniotic fluid T. gondii DNA-PCR result was negative in all of them. Ten (90.9%) pregnancies resulted in mature birth and one (9.1%) resulted in miscarriage. Conclusions: Anti-T. gondii IgM positivity is an indication of acute infection. But IgM can persist for years, and be false-positive in pregnancy. Therefore, additional tests are required, and leading to emotional distress and unnecessary interventions in pregnacy women. These results can aid in developing an approach to screening and diagnosis of T. gondii infection in pregnancy.

Engineering of an Avidity-Optimized CD19-Specific Parallel Chimeric Antigen Receptor That Delivers Dual CD28 and 4-1BB Co-Stimulation.

Co-stimulation is critical to the function of chimeric antigen receptor (CAR) T-cells. Previously, we demonstrated that dual co-stimulation can be effectively harnessed by a parallel (p)CAR architecture in which a CD28-containing second generation CAR is co-expressed with a 4-1BB containing chimeric co-stimulatory receptor (CCR). When compared to linear CARs, pCAR-engineered T-cells elicit superior anti-tumor activity in a range of pre-clinical models. Since CD19 is the best validated clinical target for cellular immunotherapy, we evaluated a panel of CD19-specific CAR and pCAR T-cells in this study. First, we generated a panel of single chain antibody fragments (scFvs) by alanine scanning mutagenesis of the CD19-specific FMC63 scFv (VH domain) and these were incorporated into second generation CD28+CD3ζ CARs. The resulting panel of CAR T-cells demonstrated a broad range of CD19 binding ability and avidity for CD19-expressing tumor cells. Each scFv-modified CAR was then converted into a pCAR by co-expression of an FMC63 scFv-targeted CCR with a 4-1BB endodomain. When compared to second generation CARs that contained an unmodified or mutated FMC63 scFv, each pCAR demonstrated a significant enhancement of tumor re-stimulation potential and IL-2 release, reduced exhaustion marker expression and enhanced therapeutic efficacy in mice with established Nalm-6 leukemic xenografts. These data reinforce the evidence that the pCAR platform delivers enhanced anti-tumor activity through effective provision of dual co-stimulation. Greatest anti-tumor activity was noted for intermediate avidity CAR T-cells and derived pCARs, raising the possibility that effector to target cell avidity is an important determinant of efficacy.

Maternal Facial Nerve Palsy and a Perinatal Infection.

Maternal Facial Nerve Palsy and a Perinatal Infection.

Incomplete IgG avidity maturation after seasonal coronavirus infections.

In classical viral infections, the avidity of immunoglobulin G (IgG) is low during acute infection and high a few months later. As recently reported, SARS-CoV-2 infections are not following this scheme, but they are rather characterized by incomplete avidity maturation. This study was performed to clarify whether infection with seasonal coronaviruses also leads to incomplete avidity maturation. The avidity of IgG towardthe nucleoprotein (NP) of the seasonal coronaviruses 229E, NL63, OC43, HKU1 and of SARS-CoV-2 was determined in the sera from 88 healthy, SARS-CoV-2-negative subjects and in the sera from 70 COVID-19 outpatients, using the recomLine SARS-CoV-2 assay with recombinant antigens. In the sera from SARS-CoV-2-negative subjects, incomplete avidity maturation (persistent low and intermediate avidity indices) was the lowest for infections with the alpha-coronaviruses 229E (33.3%) and NL63 (61.3%), and the highest for the beta-coronaviruses OC43 (77.5%) and HKU1 (71.4%). In the sera from COVID-19 patients, the degree of incomplete avidity maturation of IgG towardNP of 223E, OC43, and HKU1 was not significantly different from that found in SARS-CoV-2-negative subjects, but a significant increase in avidity was observed for IgG towardNP of NL63. Though there was no cross-reaction between SARS-CoV-2 and seasonal coronaviruses, higher concentrations of IgG directed towardseasonal coronaviruses seemed to indirectly increase avidity maturation of IgG directed towardSARS-CoV-2. Our data show that incomplete IgG avidity maturation represents a characteristic consequence of coronavirus infections. This raises problems for the serological differentiation between acute and past infections and may be important for the biology of coronaviruses.

Vaccination versus infection with SARS‐CoV‐2: Establishment of a high avidity IgG response versus incomplete avidity maturation

Avidity is defined as the binding strength of immunoglobulin G (IgG) toward its target epitope. Avidity is directly related to affinity, as both processes are determined by the best fit of IgG to epitopes. We confirm and extend data on incomplete avidity maturation of IgG toward severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) nucleoprotein (NP), spike protein‐1 (S1), and its receptor‐binding domain (RBD) in coronavirus disease 2019 (COVID‐19) patients. In SARS‐CoV‐2‐infected individuals, an initial rise in avidity maturation was ending abruptly, leading to IgG of persistently low or intermediate avidity. Incomplete avidity maturation might facilitate secondary SARS‐CoV‐2 infections and thus prevent the establishment of herd immunity. Incomplete avidity maturation after infection with SARS‐CoV‐2 (with only 11.8% of cases showing finally IgG of high avidity, that is, an avidity index > 0.6) was contrasted by regular and rapid establishment of high avidity in SARS‐CoV‐2 naïve individuals after two vaccination steps with the BioNTech messenger RNA (mRNA) Vaccine (78% of cases with high avidity). One vaccination step was not sufficient for induction of complete avidity maturation in vaccinated SARS‐CoV‐2 naïve individuals, as it induced high avidity only in 2.9% of cases within 3 weeks. However, one vaccination step was sufficient to induce high avidity in individuals with previous SARS‐CoV‐2 infection.

Immunogenicity of antigens from outer membrane vesicles of Neisseria meningitidis associated with bilayer fragment of dioctadecyldimethylammonium in Swiss adult mice.

PurposeIn the present study, meningococcal serogroup B outer membrane vesicles (OMVs) were associated with bilayer fragments of a cationic lipid, dioctadecyldimethylammonium (DDA-BF), used as adjuvant, in an antigenic preparation tested in adult female outbred mice. This adjuvant was compared to the traditional adjuvant aluminum hydroxide.Materials and MethodsThe potential in generating humoral response was evaluated by enzyme-linked immunosorbent assay (ELISA). Individual serum was collected and immunoglobulin G (IgG), IgG1, IgG2a, and IgG2b were quantified. Analyses were carried out 15 and 60 days after immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and dot-ELISA were carried out to evaluate specific reaction for homologous strains and cross-reactive antigens present in other meningococcal strains isolated in 2011–2012 year, in Brazil. Delayed type hypersensitivity was used as indicative of cellular immunity and compared between two experimental groups, 24 hours after homologous strain challenge.ResultsThe OMVs of Neisseria meningitidis, and N. lactamica (related species) were characterized by electrophoretic separation of proteins in 13% polyacrylamide gel. The strains presented antigens in the range of 8 to 130 kDa, showing a heterogeneous protein migration pattern. In the group immunized with OMVs/DDA-BF, we found no significant production of total IgG 15 days after the first immunization. On the other hand, 60 days after first immunization both adjuvants act benefiting total IgG production similarly. The antibodies of the IgG isotype produced by animals immunized after one or two doses after first immunization, showed intermediate and high avidity, independent on the adjuvant used. In both experimental groups the swelling of the footpads was significantly higher than those of the controls, suggesting that only one dose was enough to stimulate the generation of cellular immunity.ConclusionThe use of this cationic adjuvant for N. meningitidis OMVs preparation revealed good potential for future new antigen preparation for N. meningitidis vaccine.

Determination the Time of Toxoplasmosis among Pregnant Women by using IgG Avidity to Various Toxoplasma Gondii Antigens

Toxoplasmosis is caused by Toxoplasma gondii, which is an intracellular protozoan parasite. Its main host is the cat. It is one of the most common human parasites. The main objective of this study is to determine the time of T. gondii infection among pregnant women by using avidity test of Toxoplasma IgG to various Toxoplasma antigens. The study period was from August 2017 to February 2018 to study the time of toxoplasmosis among 180 pregnant women and 100 non pregnant married women as control attending to hospitals, primary health care centers and some private medical laboratories. The pregnant women were examined for Toxo-IgG seroprevalence by using ELISA technique, and then examined their avidity of antibodies for specific Toxoplasma antigens by using line immune assay. The rates of Toxo-IgG seropositive were64 (35.56 %) among pregnant women. Regarding the reactivity of determined Toxo-IgG against various Toxoplasma antigens, the rates were 40(62.50%), 39 (60.93%), 63(98.43%) ,55(85.93%) ,63(98.43%), 62(96.87%) ,41(64.04%) and 42(65.62) positive for Toxoplasma ROP1C, MIC3, GRA7, GRA8, p30, MAG1,GRA1,rSAG1 antigens, respectively. Considering the avidity of Toxo-IgG for these antigens, the rates of high avidity were higher than low and intermediate avidity. So, the highest rate of high avidity was 85.0% for ROP1c antigen. It is concluded that the highest rate of predicting Toxoplasma infection among pregnant women was for a period of more than 6 months, which makes it less dangerous for maternal and fetal health..

The seroprevalence of Rubella in pregnant women in Turkey: a meta-analysis research of 90988 Rubella IgM, 84398 Rubella IgG, and 522 avidity results.

Objective:Rubella infection prevalence in pregnant women can vary from country to country, or even across regions in the same country. In this metaanalysis, the seroprevalence Rubella among pregnant women in Turkey in the last decade was evaluated.Materials and Methods:Studies conducted in Turkey between 2007 and 2017 were analyzed, and differences in seroprevalence between provinces were compared by evaluating Rubella immunoglobulin (Ig)-G, IgM, and IgG avidity results in pregnancy in this period. A data search was performed using the search terms Rubella, kızamıkçık, gebe, hamile, pregnancy, Türkiye, Turkey in Google Scholar, PubMed, Web of Science, Türk Medline, and the YÖK thesis database center.Results:A total of 26 articles associated with the seroprevalence of Rubella among pregnant women in Turkey were enrolled in the meta-analysis. As a result of an analysis of 84398 Rubella IgG, and 90988 Rubella IgM serology tests among pregnant women in 26 studies; Rubella IgG and IgM seroprevalence rates in pregnant woman in Turkey were found as 93.47% (95% CI: 91.72 to 95.03) and 0.783% (95% CI: 0.505 to 1.120), respectively. Rubella IgG low, intermediate, and high avidity rates were 4.66% (95% CI: 0.969 to 10.906), 7.51% (95% CI: 5.101 to 10.345), and 93.55% (95% CI: 82.584 to 99.311), respectively.Conclusion:The Rubella IgG seropositivity rate in Turkey among pregnant woman is high, whereas it is low for IgM. These rates may be considered as the result of successful immunization policies and practices. In a few provinces, it is necessary to revise the Rubella immunization procedures and adult vaccination strategies should be developed in order to control Rubella infections in adults, including pregnant women.

Interpretation of very low avidity indices acquired with the Liaison XL Toxo IgG avidity assay in dating toxoplasmosis infection.

Congenital toxoplasmosis is an important cause of complications in pregnancy. Toxoplasmosis is often asymptomatic and thus serological tests are usually performed to screen for it. A first serum which exhibit both IgG and IgM may be due to nascent toxoplasmosis seroconversion, non-specific IgM reaction, or residual IgM. The IgG avidity test has been proposed to identify latent infections. A high index excludes recent toxoplasmosis whereas an intermediate or low index only suggests a recent infection, the caveats being that some people with latent Toxoplasma gondii infection show IgG with low or intermediate avidity. In this study, we investigated the ability of the Liaison XL Toxo IgG avidity (DiaSorin, Saluggia, Italy) assay to confirm recent infection when IgG avidity index is very low (≤ 0.1). Four thousand two hundred ninety-seven sera exhibiting both IgG and IgM were included and avidity was performed on the Liaison device according to the manufacturer's recommendations. One hundred twenty-six sera on the 297 sera which exhibited very low IgG avidity indices (≤ 0.1) could be exploited: 97% of sera with IgG avidity indices < 0.05 actually corresponded to recent infection (less than 3months). A similar but less pronounced trend was observed for the sera exhibiting indices between 0.05 and 0.1 (69% corresponded to recent infections). The IgG avidity index data we obtained with the Liaison XL Toxo device are similar to those obtained with other devices. This body of consistent results underlines the interest of very low IgG avidity indices as a sign of probable recent toxoplasmosis.

Naturally acquired immune responses to thrombospondin-related adhesion protein (TRAP) of Plasmodium vivax in patients from areas of unstable malaria transmission

A key tool for the control, elimination, and eradication of Plasmodium vivax is the development of an effective vaccine. The thrombospondin-related adhesion protein (TRAP) is one of the major sporozoite antigens that plays an important role in the invasion of mosquito salivary glands and hepatocytes by sporozoites. The main goal of this study was to evaluate the naturally acquired antibodies to the P. vivax TRAP (PvTRAP) in patients from malaria-endemic areas of Iran (n=116), Afghanistan (n=50), and Pakistan (n=50). The PvTRAP gene was expressed in Escherichia coli Rosetta (DE3)-pET23a and used as antigen in enzyme-linked immunosorbent assay (ELISA). The profile of immunoglobulin G (IgG) isotype and the avidity of IgG, IgG1, and IgG3 to PvTRAP, as well as the association between anti-PvTRAP isotype responses and host age were evaluated. Only 42.24% of Iranian, 38% of Afghani, and 44% of Pakistani patients infected with P. vivax had positive anti-PvTRAP IgG, and the prevalence of responders in the three countries did not differ significantly (P>0.05). Moreover, the prevalence of IgG1 and IgG3 antibody responses to PvTRAP showed no significant correlation with age (P>0.05). Individuals exposed to vivax malaria in the unstable malaria transmission areas are able to produce antibodies to the TRAP antigen at all ages in response to P. vivax infections. Finally, the presence of mature IgG1 and IgG3 antibodies with high to intermediate avidity against PvTRAP antigen (>60%) provide more information to understand the interactions between the host and P. vivax parasite. In summary, the present study provides data that support the rational development of an effective pre-erythrocytic stage vaccine based on PvTRAP antigen.

Value of immunoglobulin G avidity for primary cytomegalovirus diagnosis in pregnant women compared to nested polymerase chain reaction

Primary human cytomegalovirus (HCMV) infection in pregnancy especially in the first term carries high risks to foetus. Diagnosis depends mainly on laboratory tools as clinical diagnosis is non specific. The aim of the present study was to detect recent infection with HCMV in cohort pregnant Egyptian women in the first trimester by serological detection of specific IgM, IgG and IgG avidity test compared to nested PCR method. This study was performed on consecutive pregnant women. Blood samples were obtained and sera were separated from 120 pregnant women in their first trimester of pregnancy and were screened for anti-CMV IgG, IgM and IgG avidity by ELISA and for HCMV-DNA by PCR. Positive IgG for HCMV was 62.5% and positive IgM was 15%. IgG avidity results showed that low, intermediate and high avidity represented 33.3%, 16.7% and 50%, respectively of IgM positive patients and 42.1%, 38.6% and 19.3%, respectively of IgM negative patients. Detection of HCMV DNA by PCR was positive in one of 24 IgM negative/low IgG avidity, in three of 22 IgM negative/intermediate IgG avidity and in all IgM positive/low IgG avidity patients. Confirmatory testing for primary infection with the combined use of IgG avidity with IgM antibody test in pregnant women during the first trimester is an accurate screening tool. The use of PCR as a diagnostic method can be applied to pregnant women with IgM antibodies and low IgG avidity results or in presence of isolated low or intermediate avidity IgG.

Frequency of Neospora caninum infections in beef cow–calf operations under extensive management

The aim of this study was to evaluate the frequencies of Neospora caninum horizontal and vertical transmissions in beef cow–calf operations under three different extensive management systems: group A: 0.75 head per hectare pasturing on natural grass; group B: 1.1 head per hectare on natural grass and improved cultured pastures; and group C: 2 head per hectare on natural grass, improved cultured pasture and whole corn silage. Serum samples from 72 multiparous cows assigned to each beef cow–calf operations were obtained every 3 months during 2 years. A group of 30 replacement heifers from each group were tested similarly since they were 10–21 months old. Twenty four, 20 and 34 calves from groups A, B and C respectively, were bled before colostrum intake and again 6 months later. The samples were analyzed by indirect fluorescence antibody test (IFAT) for detection of total IgG against N. caninum at a serological titre ≥200 for multiparous cows and replacement heifers, and a serological titre ≥25 for calves. Serum samples from seropositive cows were assessed by ELISA to evaluate the avidity of their specific antibodies. There were no differences in the proportion of seropositive cows from groups A, B and C at the beginning of the trial (p>0.05). Interestingly, the lowest serological titres in seropositive cows from all groups were observed during the first trimester (p<0.05). Although seropositive cows had medium to high avidity antibodies, suggesting chronic infection; seroconversion associated with low antibody avidity was found in 2, 3 and 3 seropositive cows from groups A, B and C. All replacement heifers remained seronegative. No abortions were recorded but 2, 1, and 2 calves from groups A, B and C were seropositive before colostrum intake, respectively. Seropositive calves born from cows having intermediate or high avidity remained with the same serostatus at 6 months of age. Even under varying extensive management conditions, both N. caninum horizontal and vertical transmission methods do occur in beef cow–calf operations.

Comparison of the LIAISON® CMV IgG Avidity II and the VIDAS® CMV IgG Avidity II assays for the diagnosis of primary infection in pregnant women

BackgroundThe accuracy of IgG avidity assays is crucial for the diagnosis of primary CMV infection in pregnancy. ObjectiveTo compare the performance of the second generation avidity assays LIAISON® CMV IgG Avidity II and VIDAS® CMV IgG Avidity II for the diagnosis of primary infection in pregnant women. Study designIn our laboratory, in sera of pregnant women presenting with positive CMV IgG and positive CMV IgM, the two avidity assays were run in parallel from January 2013 to April 2015. ResultsThe results of the 2 avidity assays were analyzed in 280 sera. The correlation between the second generation LIAISON® and VIDAS® avidity results was significantly higher than between the results of the first generation assays (77% versus 49%, p<0.001). Discrepant results were mainly explained by the difficulty of the VIDAS® assay to reach intermediate and high avidity status over time, suggesting the superiority of the LIAISON® assay to achieve high avidity levels. In 4 sera from 3 pregnant women (1.4%) with documented primary infection the LIAISON® avidity was falsely high. In these cases the level of CMV IgG was low (<50U/mL). ConclusionThe LIAISON® CMV IgG Avidity II assay reached more rapidly higher avidity status than the VIDAS® CMV IgG Avidity II. However, with the LIAISON® CMV IgG Avidity II, we identified rare sera with high positive avidity values in documented recent seroconversion. The advantages and drawbacks of each assay must be known for a sound interpretation of the results.

Tumor Associated Antigen Reactive Donor T Cells As Elementary Components of Streptamer Isolated Multi-Antigen Specific T Cells

▪Patients with hematological malignancies can be successfully treated with allogeneic stem cell transplantation (alloSCT). The therapeutic potential of alloSCT in the HLA-matched setting is primarily mediated by donor T cells directed against minor histocompatibility antigens (MiHA) presented in self-HLA. In addition, tumor associated antigens (TAA) represent a diversity of proteins overexpressed by various malignancies (self-peptide recognized in self-HLA) that may be recognized by donor T cells. Early application of donor T cells after alloSCT often results in graft versus host disease (GVHD) besides graft versus leukemia (GVL) responses. After T cell depleted transplantation, patients are vulnerable to opportunistic viral infections and disease relapses. Adoptive transfer of a multi-antigen specific T cell product containing T cells directed against viruses, TAA and MiHA may restore anti-viral immunity and support the GVL effect early after alloSCT. Detectable frequencies of high avidity memory virus specific T cells can be easily enriched from blood of seropositive healthy donors. However, due to the extremely low precursor frequencies of TAA and MiHA specific T cells, the isolation of these cells is more complicated. Moreover, high avidity, self-HLA restricted TAA specific T cells may have been deleted during thymic selection.In this study (T-control, EU FP7), we aim to treat patients with a prophylactic infusion of a multi-antigen specific T cell product consisting of T cells directed against MHC class I restricted peptides of CMV, EBV and adenovirus, HLA-A2 restricted peptides from the TAA WT1, RHAMM, NY-eso and the peptide PR1 derived from proteinase 3, and the MiHA HA-1h. The reversible streptamer technology allows purification of minimally manipulated T cells from donor peripheral blood mononuclear cells (PBMC) and is based on strep-tag labeled peptide/MHC complexes coupled to Strep-Tactin coated magnetic nanobeads, followed by purification using the CliniMACS under Good Manufacturing Practice (GMP) conditions.In large scale validation runs (n=3) we purified up to 12 viral, TAA and MiHA specificities out of ≥500*10^6 donor PBMC in one procedure. This resulted in an immediately clinical applicable multi-antigen specific T cell product with a purity of ≥98% within the T cell compartment. As expected the the different viral specificities comprised the main portion of the product (98%). To confirm that the remaining part of the product contains T cells directed against TAA and MiHA, we performed subsequent individual streptamer enrichments. After each enrichment the isolated cells were non-specifically expanded with allogeneic feeders, IL-2 and PHA.Starting with 100*10^6 PBMC (n=4), we could detect in every donor ≥ 3 different TAA and/or MiHA specific T cell populations after 2 or 3 enrichments. Moreover, when starting with 500*10^6 PBMC (n=3) all TAA and MiHA specific T cells could be enriched to detectable frequencies (1-78%) after 2 or 3 enrichments. This indicates that TAA and MiHA specific T cells are commonly present in healthy donors, although at very low frequencies.The TAA and MiHA specific T cell populations were clonally isolated and expanded, evaluated for CD8 and tetramer positivity and screened for antigen specific reactivity measured by cytokine release (IFN-g and GM-CSF) after 24 hours of stimulation with TAP deficient T2 cells loaded with 10^-5M of the relevant peptide.From the tetramer positive T cell clones 8/132 NY-eso clones, 8/17 RHAMM clones, 31/47 WT1 clones and 13/16 PR1 clones showed peptide recognition. Among these were TAA clones of intermediate avidity recognizing up to 10^-7M peptide, despite the anticipated self-tolerance. As expected, the HA-1h clones recognizing a foreign MiHA in self HLA displayed high functional avidity reflected by recognition of peptide-loaded T2 cells in the pM range. This illustrates that intermediate avidity TAA-specific and high avidity HA-1h specific T cell clones can be enriched from healthy donors and that these cells may have therapeutic potential.In conclusion, we are able to generate from donor PBMC a highly purified multi-antigen specific T cell product for clinical application. Besides the main component of MHC class I restricted viral specific T cells, we have demonstrated that this product also contains highly proliferative high avidity MiHA specific T cells and TAA specific T cells of intermediate avidity. DisclosuresGermeroth:Stage Cell Therapeutics: Lothar Germeroth is shareholder at Stage Cell Therapeutics which develops Cell therapeutics based on the Streptamer technology. Other.

Detailed investigation of ongoing subclinical hepatitis E virus infections; occurring in outbreak settings of North India.

Every year globally WHO reports 20 million Hepatitis E virus (HEV) infections. The disease occurs as sporadic cases or focused outbreaks and has potentials to cause massive epidemics. The reservoir of HEV during inter-epidemic period is not well characterized. The sporadic cases usually lack history of contact with clinically overt HEV patients. In the present context we evaluated the occurrence of subclinical HEV as a possible reservoir in endemic region. Blood samples were collected from 67 apparently healthy individuals and 10 acute viral hepatitis (AVH) patients during two HEV outbreaks in North India. The serum samples were tested for anti-HEV IgM, IgG, HEV-IgG avidity index, HEV viral load and conventional-PCR followed by sequencing and phylogenetic analysis. A total of 14 (20.89%) apparently healthy individuals showed the presence of anti-HEV IgM and IgG. Of 14 based on HEV-IgG avidity index, 9 (64.28%) had secondary-exposure, 4 (28.57%) had primary exposure, while one patient had intermediate avidity. Subclinical subjects with primary exposure had significantly higher anti-HEV IgM index as compared to secondary-exposure (P=0.0028). Viral load in clinically jaundiced patients was significantly higher as compared to subclinical subjects (P<0.0001). Phylogenetic analysis showed HEV sequences retrieved from subclinical individuals clustered along with AVH patients, suggesting matched source. The significantly low viral load in subclinical subjects hints towards the dose dependency for progression of clinical manifestation. We document subclinical HEV with low level viremia occurs during outbreak settings and goes un-noticed, which helps maintaining the virus in nature possibly leading to its endemicity.

The change of T cell repertoire due to central tolerance determines the antigenic requirement and strategy for inducing effective anti-tumor CD8 T cell response (TUM2P.888)

Abstract Tumor antigens are mainly low-avidity antigens to T cells and unable to elicit effective anti-tumor immunity. Little knowledge on how low-avidity antigens elicit T cell response in vivo further hinders designing strategies to improve anti-tumor immune response. Using a unique mouse tumor model, we found that neither native tumor antigen nor majority of altered peptide ligands (APLs) derived from tumor antigen but only intermediate avidity APL was able to elicit robust cross-reactive tumor-specific CD8 T cell response. Central tolerance shifted T cell repertoire to allow host T cells only respond to low avidity APLs and spare the lower cross-reactivity to self antigens (tumor antigens). In addition, the functional avidity of cross-reactive tumor-specific CD8 T cells was over 1000 times less than that of pathogen-type antigen-specific CD8 T cells. These results indicate that the extremely low functional avidity and low cross-reactivity to native tumor antigen are two key reasons why APLs are ineffective for eliciting cytotoxic activity of CD8 T cells to kill tumor cells. Our further studies revealed that two distinct mechanisms control the functionality and responsiveness of tumor-specific CD8 T cells. This study also discovered a novel immunization strategy to enhance the functionality and responsiveness of tumor-specific CD8 T cells. Altogether, our study will shed light on strategies to enhance the efficacy of both immunotherapy and vaccination against cancers.

Performance of a Cytomegalovirus IgG Enzyme Immunoassay Kit Modified To Measure Avidity

The measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r = 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity.

Primary Cytomegalovirus Infection in Pregnant Egyptian Women Confirmed by Cytomegalovirus IgG Avidity Testing

Objective: To determine the frequency of primary cytomegalovirus (CMV) infection in pregnant Egyptian women using CMV IgG avidity testing. Subjects and Methods: A cross-sectional study was conducted at Suez Canal University Hospital, Ismailia, Egypt. A total of 546 pregnant women, presenting for routine antenatal screening, were tested for CMV IgG and IgM using a commercially available enzyme-linked immunosorbent assay (ELISA). Sera from CMV IgM-positive women were tested by CMV IgG avidity assay. Results: All the 546 pregnant women were seropositive for anti-CMV IgG. Of the 546 women, 40 (7.3%) were positive or equivocal for IgM antibodies. All sera from the 40 women (IgG+/IgM+) showed a high or intermediate CMV IgG avidity index. Of the 40 women, 23 (57.5%) were in the second or third trimesters of pregnancy and had their first-trimester blood retrieved, and the tested CMV IgG avidity assay showed a high avidity index. Conclusion: Women who were IgM positive had no primary CMV infection in the index pregnancy as evidenced by the high CMV IgG avidity testing.

Prevalence and risk factors for Toxoplasma gondii infection among pregnant and postpartum women attended at public healthcare facilities in the City of Niterói, State of Rio de Janeiro, Brazil

To determine the prevalence of immunoglobulin G (IgG) and immunoglobulin M (IgM) anti-Toxoplasma gondii antibodies among pregnant and postpartum women attended within the public healthcare system in Niterói, State of Rio de Janeiro,and to detect possible exposure factors associated with T. gondii infection in this population. IgM and IgG anti- T. gondii antibodies were investigated in 276 pregnant and 124 postpartum women by using the indirect immunofluorescence (IFAT) and immunoenzymatic assay (ELISA) techniques. The participants were selected by convenience sampling. All these 400 patients filled out a free and informed consent statement, answered an epidemiological questionnaire and were informed about the disease. Among the 400 samples analyzed, 234 (58.5%) were reactive to IgG anti-T. gondii antibodies, according to the IFAT and/or ELISA assay. One pregnant woman was found to be reactive to IgM anti- T. gondii antibodies, with an intermediate IgG avidity test. Risk factor analysis showed that seropositivity was significantly associated (p<0.05) with age, contact with cats and presence of rodents at home. Through a logistic regression model, these associations were confirmed for age and contact with cats, while education at least of the high school level was found to be a protective factor. The prevalence rate of IgG anti-T. gondii antibodies in the city of Niterói was high and the risk factors for infection detected after multivariate analysis were: age over 30 years, contact with cats and education levels lower than university graduate level.