Antigen Tetanus Toxoid Research Articles

A minority of proliferating human CD4+ T cells in antigen-driven proliferation assays are antigen specific.

Antigen-driven T-cell proliferation is often measured using fluorescent dye dilution assays, such as the CFSE-based proliferation assay. Dye dilution assays have been powerful tools to detect human CD4+ T-cell responses, particularly against autoantigens. However, it is not known how many cells within the proliferating population are specific for the stimulating antigen. Here we determined the frequency of CD4+ T cells specific for the stimulating antigen within the antigen-responsive population of CFSE-based proliferation assays. We compared CD4+ T-cell responses to a type 1 diabetes autoantigen (proinsulin C-peptide) and to a vaccine antigen (tetanus toxoid). The TCRs expressed by antigen-responsive CD4+ T cells were sequenced, and their antigen specificity was tested functionally by expressing them in a reporter T-cell line. Responses to C-peptide were weak, but detectable, in PBMC from individuals with T1D, whereas responses to tetanus toxoid were much stronger. The frequency of antigen-specific CD4+ T cells correlated with the strength of the response to antigen in the proliferation assay. However, antigen-specific CD4+ T cells were rare among antigen-responsive CD4+ T cells. For C-peptide, an average frequency of 7.5% (1%-11%, n = 4) of antigen-responsive CD4+ T cells were confirmed to be antigen specific. In the tetanus-toxoid-stimulated cultures, on average, 45% (16%-78%, n = 5) of the antigen-responsive CD4+ T cells were tetanus toxoid specific. These data show that antigen-specific CD4+ T cells are a minority of the cells that proliferate in response to antigen and have important implications for in vitro CD4+ T-cell proliferation assays.

Detection of tetanus toxoid with iron magnetic nanobioprobe

Diagnosis of diseases with low facilities, speed, accuracy and sensitivity is an important matter in treatment. Bioprobes based on iron oxide nanoparticles are a good candidate for early detection of deadly and infectious diseases such as tetanus due to their high reactivity, biocompatibility, low production cost and sample separation under a magnetic field. In this study, silane groups were coated on surface of iron oxide nanoparticles using tetraethoxysilane (TEOS) hydrolysis. Also, NH2 groups were generated on the surface of silanized nanoparticles using 3-aminopropyl triethoxy silane (APTES). Antibody was immobilized on the surface of silanized nanoparticles using TCT trichlorothriazine as activator. Silanization and stabilized antibody were investigated by using of FT-IR, EDX, VSM, SRB technique. UV/vis spectroscopy, fluorescence, agglutination test and ELISA were used for biosensor performance and specificity. The results of FT-IR spectroscopy showed that Si–O–Si and Si-O-Fe bonds and TCT chlorine and amine groups of tetanus anti-toxoid antibodies were formed on the surface of iron oxide nanoparticles. The presence of Si, N and C elements in EDX analysis confirms the silanization of iron oxide nanoparticles. VSM results showed that the amount of magnetic nanoparticles after conjugation is sufficient for biological applications. Antibody stabilization on nanoparticles increased the adsorption intensity in the uv/vis spectrometer. The fluorescence intensity of nano bioprobe increased in the presence of 10 ng ml−1. Nanobio probes were observed as agglomerates in the presence of tetanus toxoid antigen. The presence of tetanus antigen caused the formation of antigen-nanobioprobe antigen complex. Identification of this complex by HRP-bound antibody confirmed the specificity of nanobioprobe. Tetanus magnetic nanobioprobe with a diagnostic limit of 10 ng ml−1 of tetanus antigen in a short time can be a good tool in LOC devices and microfluidic chips.

Validation of Free Formaldehyde Determination Method in DT Vaccine and Tetanus Toxoid Antigen.

Validation is a Good Manufacturing Practice principle that proves any procedure, process, method, equipment, material, activity, or system actually leads to the expected results. This study validates the method for the determination of free formaldehyde in biological products (including the diphtheria-tetanus vaccine and tetanus toxoid antigen). The operatingprocedure of this method is based on pharmacopoeial monographs. It also does not require full validation, although its suitability under the actual condition of use should be verified. Performance characterizations, such as accuracy, intra-precision (repeatability), intermediate precision (inter-precision), linearity, range, and the limit of quantitation, were investigated and calculated. Accuracy and precision were studied at different concentration levels by spiking known amounts of formaldehyde in real samples. The accuracy and precision results were expressed as the recovery and the relative standard deviation (RSD), respectively. Precision was expressed as intra-precision (repeatability) and inter-precision. Intra-precision or repeatability was performed by one operator in one day by adding three levels of concentration to the products. The inter-precision was conducted by one operator in three individual days within the same laboratory at three concentration levels. Range and linearity were assessed by investigating the correlation coefficient of the regression line between different concentrations of formaldehyde and their response. The acceptance criteria and limits were defined for these validation parameters in these biological products. The RSD for intra-day and inter-day precision studies was less than 5% in a medium concentration of linear range. At this concentration level, accuracy was 90%-110%. The method's linearity ranged between 0.0000039%-0.01% w/v of formaldehyde with a correlation coefficient of 0.9999. The results exhibited sufficient linearity, accuracy, precision, and range. Therefore, this method can be used successfully to determine free formaldehyde for biological products.

Ex Vivo Blockade of the PD-1 Pathway Improves Recall IFNγ Responses of HIV-Infected Persons on Antiretroviral Therapy.

Despite antiretroviral therapy (ART), immune exhaustion persists in HIV infection and limits T cell responses to HIV or other pathogens. Moreover, HIV infection results in the loss of pre-existing immunity. Here, we investigated the effect of blocking the PD-1 pathway on recall IFNγ responses to tetanus toxoid (TT) and measles virus (MV) antigens in HIV-infected persons on ART with prior TT and MV immunity. The ex vivo treatment of lymphocytes with anti-PD-1 and anti-PD-L1 antibodies significantly increased TT- and MV-specific IFNγ responses. The responses to TT and MV antigens alone or in combination with antibodies blocking the PD-1 pathway positively correlated with CD4 T cell levels. Furthermore, T cell PD-1 expression levels inversely correlated with recall IFNγ responses in combination with antibodies blocking the PD-1 pathway but not with IFNγ responses to antigens only. Our study suggested that targeting the PD-1 pathway may boost vaccine-induced pre-existing immunity in HIV-infected persons on ART depending on the degree of immune exhaustion.

Enhanced immune responses to vaccine antigens in the corneal stroma.

To examine the widely accepted dogma that the eye is an immune-privileged organ that can suppress antigen immunogenicity, we explored systemic immune responses to a model vaccine antigen (tetanus toxoid) delivered to six compartments of the rodent eye (ocular surface, corneal stroma, anterior chamber, subconjunctival space, suprachoroidal space, vitreous body). We discovered that antigens delivered to corneal stroma induced enhanced, rather than suppressed, antigen-specific immune responses, which were 18- to 30-fold greater than conventional intramuscular injection and comparable to intramuscular vaccination with alum adjuvant. Systemic immune responses to antigen delivered to the other ocular compartments were much weaker. The enhanced systemic immune responses after intrastromal injection were related to a sequence of events involving the formation of an antigen "depot" in the avascular stroma, infiltration of antigen-presenting cells, up-regulation of MHC class II and costimulatory molecules CD80/CD86, and induction of lymphangiogenesis in the corneal stroma facilitating sustained presentation of antigen to the lymphatic system. These enhanced immune responses in corneal stroma suggest new approaches to medical interventions for ocular immune diseases and vaccination methods.

MAKING A STAT DIAGNOSIS IN A PATIENT PRESENTING WITH VILLOUS ATROPHY

<h3>Introduction</h3> Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) gene mutations are highly penetrant, resulting in enhanced STAT1-dependent responses to interferon-alpha/beta and interferon-gamma signaling, and impaired Th17 development. This manifests as recurrent infections, cytopenias, and autoimmune disease. Herein, we present a case of a child diagnosed with a STAT1 mutation. <h3>Case Description</h3> A 3-year-old female presented for secondary evaluation of persistent gastrointestinal and respiratory symptoms, recurrent sinopulmonary and skin infections, failure to thrive, and developmental delay. An esophagogastroduodenoscopy performed elsewhere showed increased duodenal intraepithelial lymphocytes with crypt hyperplasia and villous atrophy. Testing for Celiac disease and autoimmune enteritis were negative. Chest computed tomography showed bilateral bronchiectasis with mucous plugging. Bronchoalveolar lavage culture was positive for Streptococcus pneumoniae. Laboratory workup was notable for anemia and T-cell lymphopenia. Quantitative lymphocytes showed a low CD3+ count of 446 cells/mcL, low CD4+ count of 185 cells/mcL, and low CD8+ count of 194 cells/mcL. Lymphocyte proliferation to antigens showed essentially absent total CD45+ lymphocytes and CD3+ T cell responses to Candida and Tetanus toxoid antigens. Genetic testing later revealed a heterozygous, pathogenic c.1154C>T (p.Thr385Met) variant in the STAT1 gene. Additional testing for Th17 enumeration showed a decreased IL17 and Th17 cell percentage. She was treated with supportive care, antibiotics, and referred to Medical Genetics for additional education and counseling. <h3>Discussion</h3> STAT1-GOF gene mutations are rare and associated with variable phenotypes and disease severity. Genetic testing can help determine the cause of complex, multisystem diseases. Stem cell transplant, immunoglobulin replacement therapy, and JAK inhibitors are potential treatment considerations.

Controlled release vaccine implants for delivery of booster immunisations

Most current animal vaccine regimes involve a primary vaccination followed sometime later by a booster vaccination. This presents challenges when vaccinating difficult to access animals such as livestock. Mustering livestock to deliver a vaccine boost is costly and stressful for animals. Thus, we have produced a platform system that can be administered at the same time as the priming immunisation and delivers payload after an appropriate delay time to boost the immune response, without need for further handling of animals. A 30 × 2 mm osmotically triggered polymer implant device with burst-release characteristics delivered the booster dose of a tetanus vaccine. Blood samples were collected from an experimental group that received the priming vaccine and implant on day 0 and control group that received the initial vaccine (tetanus toxoid) and then a bolus dose 28 days later via subcutaneous injection. The two groups showed identical weight gain curves. T cell proliferation following in vitro stimulation with antigen was identical between the two groups at all time points. However, serum IgG antibody responses to the tetanus toxoid antigen were significantly higher in the control group at weeks 8 and 12. The implant capsules stayed at the site of implantation and at week 12 there was evidence of tissue integration. No local reactions at the implant site were observed, other than mild thickening of the skin in half of the experimental group animals and no other adverse health events were recorded in either group.

Combined assessment of S- and N-specific IL-2 and IL-13 secretion and CD69 neo-expression for discrimination of post-infection and post-vaccination cellular SARS-CoV-2-specific immune response.

BackgroundAntibody‐based tests are available for measuring SARS‐CoV‐2‐specific immune responses but fast T‐cell assays remain scarce. Robust T cell‐based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination.MethodsOne hundred seventeen individuals (COVID‐19 convalescent patients: n = 40; SARS‐CoV‐2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS‐CoV‐2‐specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation‐induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS‐CoV‐2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin).ResultsN‐peptide mix stimulation of WB identified the combination of IL‐2 and IL‐13 secretion as superior to IFN‐γ secretion to discriminate between COVID‐19‐convalescent patients and healthy controls (p < .0001). Comparable results were obtained with M‐ or S‐peptides, the latter almost comparably recalled IL‐2, IFN‐γ, and IL‐13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID‐19, but not allergic disease status, positively correlated with IL‐13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen‐induced neo‐expression of the C‐type lectin CD69 on CD4+ (p < .0001) and CD8+ (p = .0002) T cells informed best about the SARS‐CoV‐2 exposure status with additional benefit coming from CD25 upregulation.ConclusionAlong with N‐ and S‐peptide‐induced IL‐2 and CD69 neo‐expression, we suggest to include the type 2 cytokine IL‐13 as T‐cellular recall marker for SARS‐CoV‐2 specific T‐cellular immune responses after infection and vaccination.

Young but not older adults exhibit an expansion of CD45RA+CCR7+CD95+ T follicular helper cells in response to tetanus vaccine.

A subset of CD4+ T cells, known as T follicular helper (Tfh), provides co-stimulating signals required to establish long-term humoral immunity. Recent studies have shown a reduced frequency and functionality of this population in older adults in comparison to young adults, in response to vaccination. To evaluate whether memory generation of circulating Tfh (cTfh) cells contributes to this phenomenon, the memory subpopulations of cTfh, and their activation degree, were evaluated both ex-vivo and in-vitro, in response to the model antigen tetanus toxoid (TT) after the first dose of tetanus vaccine. Here, we report a lower frequency of cTfh after vaccination in older adults compared to young adults. Moreover, whereas cTfh from older adults preferably expanded with an effector memory phenotype, young adults experienced a temporal increase of CCR7+CD45RA+ cTfh cells, which also displayed higher levels of CD95, CD40L, CXCR3, and Bcl-6 upon antigen re-encounter. This phenotype was confirmed using automatized algorithm. In conclusion, our results suggest that an age-related loss of heterogeneity and an expansion of more differentiated memory cells within the cTfh compartment could affect the responsiveness of older individuals to vaccines, making this phenotype a characteristic feature of immunosenescence.

Chemically Synthesized Alcaligenes Lipid A as an Adjuvant to Augment Immune Responses to Haemophilus Influenzae Type B Conjugate Vaccine.

We previously identified Alcaligenes spp. as a commensal bacterium that resides in lymphoid tissues, including Peyer’s patches. We found that Alcaligenes-derived lipopolysaccharide acted as a weak agonist of Toll-like receptor four due to the unique structure of lipid A, which lies in the core of lipopolysaccharide. This feature allowed the use of chemically synthesized Alcaligenes lipid A as a safe synthetic vaccine adjuvant that induces Th17 polarization to enhance systemic IgG and respiratory IgA responses to T-cell–dependent antigens (e.g., ovalbumin and pneumococcal surface protein A) without excessive inflammation. Here, we examined the adjuvant activity of Alcaligenes lipid A on a Haemophilus influenzae B conjugate vaccine that contains capsular polysaccharide polyribosyl ribitol phosphate (PRP), a T-cell–independent antigen, conjugated with the T-cell–dependent tetanus toxoid (TT) antigen (i.e., PRP-TT). When mice were subcutaneously immunized with PRP alone or mixed with TT, Alcaligenes lipid A did not affect PRP-specific IgG production. In contrast, PRP-specific serum IgG responses were enhanced when mice were immunized with PRP-TT, but these responses were impaired in similarly immunized T-cell—deficient nude mice. Furthermore, TT-specific—but not PRP-specific—T-cell activation occurred in mice immunized with PRP-TT together with Alcaligenes lipid A. In addition, coculture with Alcaligenes lipid A promoted significant proliferation of and enhanced antibody production by B cells. Together, these findings suggest that Alcaligenes lipid A exerts an adjuvant activity on thymus-independent Hib polysaccharide antigen in the presence of a T-cell–dependent conjugate carrier antigen.

Pain Management in a Model of Interstitial Cystitis/Bladder Pain Syndrome by a Vaccinal Strategy

Current analgesic treatments for Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) are limited. Here, we propose a novel antinociceptive strategy exploiting the opioid-mediated analgesic properties of T lymphocytes to relieve from bladder pain. In a chronic model of IC/BPS in rats, we show that a secondary T cell response against intravesically administered ovalbumin prevents from visceral pain in OVA-primed animals. The analgesic effect is associated with the recruitment of T lymphocytes within the inflamed mucosa and is reversed by naloxone-methiodide, a peripheral opioid receptor antagonist. Similarly, intravesical instillation of BCG or tetanus toxoid antigens in vaccinated rats protects from pain in the same model. We show opioid-dependent analgesic properties of local vaccine antigen recall in a preclinical rat model of chronic cystitis. Since BCG bladder instillation is regularly used in humans (as anticancer therapy), our results open it as a new therapeutic positioning for a pain management indication for IC/BPS patients.

The dry powder formulation of mixed cross-linked dextran microspheres and tetanus toxoid-loaded trimethyl chitosan nanospheres as a potent adjuvant for nasal delivery system.

Objective(s):The present study aimed to determine the immunoadjuvant efficacy of mixed cross-linked dextran microspheres (CDM) and tetanus toxoid (TT)-loaded trimethyl chitosan (TMC) nanospheres in dry powder form. Materials and Methods:The TMC nanoparticles (NPs) containing TT were produced using the ionic gelation method. Co-administration of TT-loaded TMC NPs and CDM as an absorption enhancer was performed to improve immunity against the antigen. Dry powder formulations were delivered via the nasal route in a rabbit model. Results:Among immunization groups, mixing of CDM with TT encapsulated in TMC NPs could elicit the highest titer of systemic IgG antibody. Furthermore, the addition of CDM to TT-loaded TMC enhanced the sIgA response relative to the TT solution. Conclusion:The TMC NPs had a considerable effect on mucosal and systemic immunity against the TT antigen. Therefore, the CDM excipient can be utilized for nasal immunization to elevate systemic and mucosal responses.

Anti-bacterial antibodies in multiple myeloma patients at disease presentation, in response to therapy and in remission: implications for patient management

Multiple myeloma (MM) is associated with increased risk of infection, but little is known regarding antibody levels against specific bacteria. We assessed levels of polyclonal immunoglobulin and antibacterial antibodies in patients recruited to the TEAMM trial, a randomised trial of antibiotic prophylaxis at the start of anti-myeloma treatment. Polyclonal IgG, IgA and IgM levels were below the reference range in 71%, 83% and 90% of 838 MM patients at diagnosis. Anti-vaccine targeted tetanus toxoid antibodies were protective in 95% of 193 healthy controls but only 41% of myeloma patients. In healthy controls, protective antibodies against 6/12 pneumococcal serotypes, haemophilus and meningococcus A were present in 67%, 41% and 56% compared to just 15%, 21% and 17% of myeloma patients. By 1 year, myeloma patients IgG levels had recovered for 57% of patients whilst the proportion with protective levels of IgG against thymus-dependent protein antigen tetanus toxoid had changed little. In contrast the proportions of patients with protective levels against thymus independent polysaccharide antigens pneumococcus, haemophilus and meningococcus had fallen from 15 to 7%, 21 to 0% and 17 to 11%. Findings highlight the need for strategies to protect patients against bacterial infections during therapy and vaccination programmes during remission.

Evaluation of a capture antigen ELISA for the characterisation of tetanus vaccines for veterinary use

We previously developed an ELISA assay for detection of tetanus toxoid antigen in tetanus vaccines for human use. Tetanus vaccines for veterinary use are qualitatively different to those used in humans, often containing a larger number and variety of non-tetanus antigens in the multi-valent products, and adjuvants that are not found in human vaccines. We assessed performance of the capture ELISA with a range of veterinary tetanus vaccines as a first step towards development of an immunoassay as a potential in vivo potency substitute. Nine tetanus vaccines were tested and all produced a good dose response in the ELISA. The shape of the dose response curve for the whole vaccine compared to a matched non-adjuvanted tetanus toxoid antigen was more comparable for vaccines containing a non-aluminium adjuvant than products containing aluminium adjuvants. Elution of the antigen from aluminium adjuvant did not improve the comparability of the dose response curve but did increase the total amount of tetanus antigen available for detection. The ELISA was highly specific for tetanus with no signal obtained for a large number of non-tetanus antigens. These results suggest that a capture ELISA assay can be applied to a control strategy for veterinary tetanus vaccines.

Dissolvable Microneedle-Mediated Transcutaneous Delivery of Tetanus Toxoid Elicits Effective Immune Response.

Transcutaneous immunization using a microneedle device presents a promising alternative to syringe-based injection of vaccines. The aim of this study was to investigate the effective immune response elicited after application of tetanus toxoid antigen-loaded dissolvable microneedles (TT-MN) in mice model. Dissolvable microneedles were prepared using 20% w/v of polyvinyl alcohol and polyvinyl pyrrolidone polymer mixture by micromolding technique. TT-MN were prepared by addition of tetanus toxoid to polymer mixture before casting microneedles. TT-MN were characterized using texture analyzer, stereomicroscope, and scanning electron microscope. Tetanus toxoid loading was found to be 77 ± 2μg per microneedle array. Confocal microscopic analysis showed that the microneedles penetrated to a depth of 130μm inside mouse skin. Complete dissolution of microneedles was achieved within 1h after insertion in skin. Immunization studies in Swiss albino mice demonstrated significantly (p < 0.001) greater IgG, IgG1, and IgG2a antibody titers for TT-MN and intramuscular injection groups compared with naïve control. Splenocyte proliferation assay confirmed effective re-stimulation on exposure to tetanus toxoid in microneedle treatment groups. Taken together, TT-MN can be developed as minimally invasive system for transcutaneous delivery of tetanus toxoid antigen.

Abstract B132: A new concept of cancer immunotherapy by targeting pancreatic ductal adenocarcinoma through a childhood recall antigen

Abstract Patients with pancreatic ductal adenocarcinoma (PDAC) have a five-year survival of only 6%. Cancer immunotherapy has shown promising results for metastatic cancer but only in 20% of the patients. Our lab has developed a new concept of immunotherapy using an attenuated non-pathogenic bacterium Listeria monocytogenes for the selective delivery of a highly immunogenic recall antigen tetanus toxoid (TT) into tumor cells of pancreatic tumors and metastases. Most individuals have been exposed to TT during childhood vaccinations and memory T-cells to TT circulate in blood for live. These memory T-cells to TT can be reactivated by Listeria-TT at any time in life and will now destroy the infected tumor cells. Low doses of gemcitabine (GEM) were added to further improve T-cell responses. Panc-02 and KPC mice were immunized with the “human TT vaccine” to generate memory T-cells before development of the pancreatic cancer. After cancer development, mice were treated with Listeria-TT and GEM, and tumors (weight) and metastases (number) were analyzed by eye in the Panc-02 mice, and by SUVmax (measurement of tumor growth) in the KPC mice. T-cell responses in the spleen were analyzed by flow cytometry and ELISPOT, and KPC tumors were analyzed by RNAseq. Listeria-TT and GEM nearly completely eliminated both early and advanced pancreatic cancer in Panc-02 and KPC mice, in correlation with abundant CD4 and CD8 T-cell responses producing perforin, granzyme B, or IFNγ, in vivo or in vitro, respectively. RNAseq analysis of “treated” KPC tumors showed a strong influx of CD4 and CD8 T-cells, upregulation of multiple granzymes, perforin, co-stimulatory molecules, and cell death pathways. These results suggest the multiple synergistic effects of Listeria-TT and GEM on pancreatic cancer. This novel treatment modality of Listeria-TT and GEM warrants further investigation in a clinical setting. Citation Format: Benson C. Selvanesan, Dinesh Chandra, Wilber Quispe, Ankur Patel, Kiran Meena, Steven K. Libutti, Ziqiang Yuan, Jennifer Chuy, Claudia Gravekamp. A new concept of cancer immunotherapy by targeting pancreatic ductal adenocarcinoma through a childhood recall antigen [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B132.

Aluminum Phosphate Vaccine Adjuvant: Analysis of Composition and Size Using Off-Line and In-Line Tools

PurposeAluminum-based adjuvants including aluminum phosphate (AlPO4) are commonly used in many human vaccines to enhance immune response. The interaction between the antigen and adjuvant, including the physical adsorption of antigen, may play a role in vaccine immunogenicity and is a useful marker of vaccine product quality and consistency. Thus, it is important to study the physicochemical properties of AlPO4, such as particle size and chemical composition. Control of the vaccine adjuvant throughout the manufacturing process, including raw materials and the intermediate and final product stages, can be effectively achieved through monitoring of such key product attributes to help ensure product quality. MethodsThis study focuses on the compositional analysis of AlPO4 adjuvant at the intermediate and final manufacturing stages using the off-line methods Fourier-Transform Infrared (FTIR) and Raman spectroscopy, X-ray Photoelectron Spectroscopy (XPS), and the in-line method Attenuated Total Reflectance (ATR). Particle size distribution of AlPO4 was measured off-line using Laser diffraction (LD) and in-line using Focused Beam Reflectance Measurement (FBRM®). ResultsThere was no observable difference in size distribution between the intermediate and final stage AlPO4 by off-line and in-line analysis, in both small- or large-scale production samples. Consistent peak shifts were observed in off-line and in-line infrared (IR) spectroscopy as well as off-line XPS for both small- and large-scale AlPO4 manufacturing runs. Additionally, IR spectroscopy and FBRM® for size distribution were used as in-line process analytical technology (PAT) to monitor reaction progress in real-time during small-scale AlPO4 manufacturing from raw materials. The small-scale adsorption process of a model protein antigen (Tetanus toxoid) to AlPO4 adjuvant was also monitored by in-line ReactIR probe. ConclusionThis study demonstrated that in-line PAT can be used to monitor particle size and chemical composition for the various stages of adjuvant manufacturing from raw materials through intermediate to final adjuvant product stage. Similar approaches can be utilized to help assess lot-to-lot consistency during adjuvant manufacturing and vaccine product development. Moreover, the use of in-line PAT is highly conductive to advanced manufacturing strategies such as real-time product release testing and automated processes of the future.

HYPER IGM SYNDROME TYPE 1 CARRIER WITH RECURRENT INFECTIONS; MORE THAN JUST A CARRIER.

Introduction CD40 ligand (CD40L) deficiency is an X-linked hyper-IgM syndrome that presents with defects in humoral and cellular immunity, resulting in severe bacterial and opportunistic infections, as well as autoimmunity and malignancy. Female carriers of CD40L deficiency should be asymptomatic and have normal immunoglobulin levels. Case Description A 38-year-old female CD40L deficiency carrier presented with CMV hepatitis, several hospitalizations for pyelonephritis and recurrent upper respiratory infections. She demonstrated elevated IgM, normal IgG with an abnormal polysaccharide response. She also had low B cells with a normal proportion of B cell subsets. Lymphocyte stimulation showed a low response to Tetanus Toxoid and Candida albicans antigen. Her mutation occurred at a CD40L splice site that results in the utilization of several alternative splice sites and the expression of a wild-type protein of unknown function. Her T-cell studies demonstrated 56% CD40L expression and 57% binding to the soluble CD40L receptor making extreme lyonization unlikely. Whole exome sequencing was performed. She was started on with immunoglobulin replacement and demonstrated clinical improvement. Discussion This is the first known case describing a symptomatic carrier of CD40L deficiency without evidence of skewed X-linked inactivation. Although CD40L deficiency carriers are typically thought to be asymptomatic our case demonstrates the need for a high index of suspicion and continued thoughtful evaluation in patients with atypical presentations.

Impact of Mothers' Schistosomiasis Status During Gestation on Children's IgG Antibody Responses to Routine Vaccines 2 Years Later and Anti-Schistosome and Anti-Malarial Responses by Neonates in Western Kenya.

The potential consequences of parasitic infections on a person’s immune responsiveness to unrelated antigens are often conjectured upon in relationship to allergic responses and autoimmune diseases. These considerations sometimes extend to whether parasitic infection of pregnant women can influence the outcomes of responses by their offspring to the immunizations administered during national Expanded Programs of Immunization. To provide additional data to these discussions, we have enrolled 99 close-to-term pregnant women in western Kenya and determined their Schistosoma mansoni and Plasmodium falciparum infection status. At 2 years of age, when the initial immunization schedule was complete, we determined their children’s IgG antibody levels to tetanus toxoid, diphtheria toxoid, and measles nucleoprotein (N-protein) antigens using a multiplex assay. We also monitored antibody responses during the children’s first 2 years of life to P. falciparum MSP119 (PfMSP119), S. mansoni Soluble Egg Antigen (SEA), Ascaris suum hemoglobin (AsHb), and Strongyloides stercoralis (SsNIE). Mothers’ infections with either P. falciparum or S. mansoni had no impact on the level of antibody responses of their offspring or the proportion of offspring that developed protective levels of antibodies to either tetanus or diphtheria antigens at 2 years of age. However, children born of S. mansoni-positive mothers and immunized for measles at 9 months of age had significantly lower levels of anti-measles N-protein antibodies when they were 2 years old (p = 0.007) and a lower proportion of these children (62.5 vs. 90.2%, OR = 0.18, 95% CI = 0.04–0.68, p = 0.011) were considered positive for measles N-protein antibodies. Decreased levels of measles antibodies may render these children more susceptible to measles infection than children whose mothers did not have schistosomiasis. None of the children demonstrated responses to AsHb or SsNIE during the study period. Anti-SEA and anti-PfMSP119 responses suggested that 6 and 70% of the children acquired schistosomes and falciparum malaria, respectively, during the first 2 years of life.

Vaccine antigens modulate the innate response of monocytes to Al(OH)3.

Aluminum-based adjuvants have widely been used in human vaccines since 1926. In the absence of antigens, aluminum-based adjuvants can initiate the inflammatory preparedness of innate cells, yet the impact of antigens on this response has not been investigated so far. In this study, we address the modulating effect of vaccine antigens on the monocyte-derived innate response by comparing processes initiated by Al(OH)3 and by Infanrix, an Al(OH)3-adjuvanted trivalent combination vaccine (DTaP), containing diphtheria toxoid (D), tetanus toxoid (T) and acellular pertussis (aP) vaccine antigens. A systems-wide analysis of stimulated monocytes was performed in which full proteome analysis was combined with targeted transcriptome analysis and cytokine analysis. This comprehensive study revealed four major differences in the monocyte response, between plain Al(OH)3 and DTaP stimulation conditions: (I) DTaP increased the anti-inflammatory cytokine IL-10, whereas Al(OH)3 did not; (II) Al(OH)3 increased the gene expression of IFNγ, IL-2 and IL-17a in contrast to the limited induction or even downregulation by DTaP; (III) increased expression of type I interferons-induced proteins was not observed upon DTaP stimulation, but was observed upon Al(OH)3 stimulation; (IV) opposing regulation of protein localization pathways was observed for Al(OH)3 and DTaP stimulation, related to the induction of exocytosis by Al(OH)3 alone. This study highlights that vaccine antigens can antagonize Al(OH)3-induced programming of the innate immune responses at the monocyte level.