Low Doses Of Antigen Research Articles

Effects of FcμR expression on B cells activation

Abstract FcμR is present on the cell surface of B cells as well as other antigen presenting cells where they bind the secreted form of IgM (sIgM). Our previous studies have shown that follicular B cells internalize sIgM continuously in vivo and that lack of sIgM or the FcμR on B cells results in diminished IgG responses following influenza virus infection. We aim to determine the mechanisms underlying the need for sIgM in the development of IgG responses. For this we generated HELB-FcmR−/− and HELB-FcmR+/+ mice. In vitro stimulation of purified B cells from these mice with HEL-OVA conjugate in the presence of OVA-specific OTII CD4 T cells showed an antigen-dose-dependent stimulation of both types of B cells. However, FcmR−/− B cells proliferated less at intermediate and low doses of antigen compared to controls, while they proliferated similar as wild type B cells following high-dose antigen stimulation. Intravenous injection of CD45.1-expressing C57BL/6 mice with B cells from either FcmR-deficient or -sufficient CD45.2+ HELB mice together with sheep red blood cells conjugated to HEL showed no significant differences in HEL-specific serum IgM or IgG, or frequencies of HEL-specific B cells between mice receiving either type of B cell, as assessed by ELISA and FACS, respectively. Ongoing studies are aimed at determining whether antigen-dose affects the need for FcmR expression in vivo. For that we have created mixed bone marrow (BM) irradiation chimeras with 2% BM from CD45.2 HELB or HELB-FcmR−/− mice, and 98% CD45.1 wild type BM. To-date our studies suggest that FcmR-IgM interaction enhances B cell proliferation when antigen is limiting. Future studies are aimed at testing this conclusion and explore how sIgM-FcmR interaction enhanced B cell responses.

Design of a novel vaccine nanotechnology-based delivery system comprising CpGODN-protein conjugate anchored to liposomes

Although the well-known Toll like receptor 9 (TLR9) agonist CpGODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, its in vivo stability and potential systemic toxicity remain a concern. In an effort to circumvent these issues, different strategies have been employed to increase its stability, localise action and reduce dosage. These include conjugation of CpGODN with proteins or encapsulation/adsorption of CpGODN into/onto liposomes, and have resulted in enhanced immunopotency compared to co-administration of free CpGODN and antigen.Here, we designed a novel delivery system of CpGODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate the Group B Streptococcus (GBS) GBS67 protein antigen with the CpGODN TLR9 agonist. This treatment did not alter protein's ability to be recognised by specific antibodies or the CpGODN to function as a TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA). The novel cationic liposomes-protein conjugate complex (GBS67-CpGODN+L) shared similar vesicle characteristics (size and charge) compared to free liposomes but exhibited different structure and morphology. Following intramuscular immunisation, GBS67-CpGODN+L formed a vaccine depot at the injection site and induced a remarkable increase of functional immune responses against GBS compared to the simple co-administration of GBS67, CpGODN and liposomes. This work demonstrates that the conjugation of CpGODN to GBS67 in conjunction with adsorption on cationic liposomes, can promote co-delivery leading to the induction of a multifaceted immune response at low antigen and CpGODN doses. Our findings highlight the potential for harnessing the immunostimulatory properties of different adjuvants to develop more effective nanostructure-based vaccine platforms.

Combination therapy using human papillomavirus L1/E6/E7 genes and archaeosome: a nanovaccine confer immuneadjuvanting effects to fight cervical cancer

Cancer is a leading cause of death worldwide. Cervical cancer caused by human papillomavirus (HPV) is a major health problem in women. DNA vaccines are a perfect approach to immunization, but their potency in clinical trials has been insufficient for generating effective immunity, which may be related to the degradation of the DNA via nucleases, poor delivery to antigen-presenting cells (APCs), and insufficient uptake of DNA plasmids by cells upon injection. Archaeosome is a nano-delivery systems based on liposomes with their immunological role have been developed for gene delivery. In this study, human papillomavirus type 16 genes, containing truncated L1, E6, and E7, were simultaneously used in combination therapy with archaeosome and assessed in vivo. Findings supported that archaeosomes promotes immune responses to DNA vaccines and a long-term CTL response was generated with a low antigen dose. Combination therapy with archaeosome/L1/E6/E7 vaccines exhibited a strong cytolytic activity against tumor cells and induced prophylactic and therapeutic effect against the development of tumor in the animal model.

Bacteriophage Qβ virus-like particles displaying Chikungunya virus B-cell epitopes elicit high-titer E2 protein antibodies but fail to neutralize a Thailand strain of Chikungunya virus

Chikungunya virus (CHIKV) is a mosquito-borne virus associated with arthritis and musculoskeletal pains. More than 2.9 million people worldwide have been infected with the virus within the last 1.5 decades; currently, there are no approved vaccines to protect against CHIKV infection. To assess the potential of using CHIKV peptides as vaccine antigens, we multivalently displayed CHIKV peptides representing B-cell epitopes (amino acids 2800–2818, 3025–3058, 3073–3081, 3121–3146, and 3177–3210), from E2 glycoprotein (Singapore strain), on the surface of a highly immunogenic bacteriophage Qβ virus-like particle (VLP). We assessed the immunogenicity of CHIKV E2 amino acid 3025–3058 (including the other epitopes) displayed on Qβ VLPs in comparison to the same peptide not displayed on VLPs. Mice immunized with the E2 peptides displayed on Qβ VLPs elicited high-titer antibodies compared with the group immunized just with the peptide. However, sera from immunized mice did not neutralize CHIKV AF15561 (isolated from Thailand). The data suggest that Qβ VLPs is an excellent approach to elicit high-titer CHIKV E2-protein antibodies at a lower dose of antigen and future studies should assess whether Qβ-CHIKV E2 aa 2800–2818 VLPs and Qβ-CHIKV E2 aa 3025–3058 VLPs can neutralize a Singapore Strain of CHIKV.

The detection of canine anti-sperm antibody following parenteral immunization of bitches against homogenized whole sperm

BackgroundsThe development of a canine-specific method of immunocontraception is one of the non-invasive controlling strategies for humanely decreasing the dog population. This study was aimed to investigate the potential of whole sperm in stimulating the immune system and producing specific anti-sperm antibodies (ASAs) in female dogs. Mature, mixed-breed bitches were subcutaneously immunized with high (200 × 106 cells/mL) and low (100 × 106 cells/mL) doses of sperm vaccine, emulsified with Freund’s adjuvants. Booster immunizations were given at weeks 1, 2, 4, and 6, and serum samples were collected at days 0, 14, 28, 42, 63, and 84 prior to each immunization. Reproductive tract samples, including vaginal and uterine lavages, were also collected by flushing each section with sterile PBS at the end of the experiment. Canine anti-sperm antibody titer and specificity in sera and genital secretions were measured using an enzyme-linked immunosorbent assay technique.ResultsSpecific anti-sperm antibodies were detected in the serum of both high and low dose groups and were significantly higher than those observed in the controls. A high dose of sperm induced elevated immune responses over the low dose antigen. Immunization with a high dose of sperm increased the level of ASAs in the uterine secretions and vaginal secretions significantly. Higher ASAs were observed to have transduced to the uterine lumen compared to the vagina.ConclusionsBased on the results obtained in this study, parenteral immunization with whole sperm can induce a high level of specific antibodies in the serum and genital secretions of female dogs and the response would be dose-dependent.

Synthesis of protein conjugates adsorbed on cationic liposomes surface

The well-known Toll like receptor 9 (TLR9) agonist CpG ODN has shown promising results as vaccine adjuvant in preclinical and clinical studies, however its in vivo stability and potential systemic toxicity remain a concern. In an effort to overcome these issues, different strategies have been explored including conjugation of CpG ODN with proteins or encapsulation/adsorption of CpG ODN into/onto liposomes. Although these methods have resulted in enhanced immunopotency compared to co-administration of free CpG ODN and antigen, we believe that this effect could be further improved. Here, we designed a novel delivery system of CpG ODN based on its conjugation to serve as anchor for liposomes. Thiol-maleimide chemistry was utilised to covalently ligate model protein with the CpG ODN TLR9 agonist. Due to its negative charge, the protein conjugate readily electrostatically bound cationic liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA) in a very high degree. The novel cationic liposomes-protein conjugate complex shared similar vesicle characteristics (size and charge) compared to free liposomes. The conjugation of CpG ODN to protein in conjunction with adsorption on cationic liposomes, could promote co-delivery leading to the induction of immune response at low antigen and CpG ODN doses.•The CpG ODN Toll-like receptor (TLR) 9 agonist was conjugated to protein antigens via thiol-maleimide chemistry.•Due to their negative charge, protein conjugates readily electrostatically bound cationic liposomes composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and dimethyldioctadecylammonium bromide (DDA) resulting to the design of novel cationic liposomes-protein conjugate complexes.•The method is suited for the liposomal delivery of a variety of adjuvant-protein conjugates.

Off-target effects of an insect cell-expressed influenza HA-pseudotyped Gag-VLP preparation in limiting postinfluenza Staphylococcus aureus infections

Clinical and historical data underscore the ability of influenza viruses to ally with Staphylococcus aureus and predispose the host for secondary bacterial pneumonia, which is a leading cause of influenza-associated mortality. This is fundamental because no vaccine for S. aureus is available and the number of antibiotic-resistant strains is alarmingly rising. Hence, this leaves influenza vaccination the only strategy to prevent postinfluenza staphylococcal infections. In the present work, we assessed the off-target effects of a Tnms42 insect cell-expressed BEI-treated Gag-VLP preparation expressing the HA of A/Puerto Rico/8/1934 (H1N1) in preventing S. aureus superinfection in mice pre-infected with a homologous or heterologous H1N1 viral challenge strain. Our results demonstrate that matched anti-hemagglutinin immunity elicited by a VLP preparation may suffice to prevent morbidity and mortality caused by lethal secondary bacterial infection. This effect was observed even when employing a single low antigen dose of 50 ng HA per animal. However, induction of anti-hemagglutinin immunity alone was not helpful in inhibiting heterologous viral replication and subsequent bacterial infection. Our results indicate the potential of the VLP vaccine approach in terms of immunogenicity but suggest that anti-HA immunity should not be considered as the sole preventive method for combatting influenza and postinfluenza bacterial infections.

Review of Grippol Family Vaccine Studies and Modern Adjuvant Development

Relevance. Vaccination remains the basis for the prevention of the most severe infectious diseases, including the prevention of influenza. The large number of vaccines have been developed and applied currently. The role of adjuvant vaccines is growing. Aims: To look back to the history of adjuvant vaccines and review the literature on clinical trials of influenza vaccines Grippol family. Conclusions: This review summarized principal data on adjuvanted vaccines containing Polyoxidonium as adjuvant which has been used for the production of Grippol family vaccines for more than 22 years. Grippol family vaccines show a high safety profile and good immunogenicity for vaccineers, including those with low responsiveness. In 2008 the vaccine Grippol® plus was approved in Russia. It contains a reduced antigen doses and adjuvant Polyoxidonium, but unlike the vaccine Grippol® is free from preservative thiomersal. There are no serious post-vaccination events, unexpected reactions, post-vaccinal complications to the vaccine were reported for Grippol regardless of the examined contingent. The Grippol® plus vaccine effectively induces the immune response. Adjuvanted lowdosed vaccines Grippol® and Grippol® plus are proved to be safe and effective and to have a good prophylactic efficacy being applied for mass immune prophylaxis. In 2018 new tetravalent influenza vaccine of Grippol family – Grippol® Quadrivalent was approved in Russia. Clinical studies (Phase I-II-III) have shown that the vaccine meet the immunogenicity criteria for inactivated influenza vaccines. Phase 3 clinical trials to expand the indications are continuing. The polymer adjuvant in the vaccine is shown to provide a high immunogenicity of low doses of subunit antigens; all Grippol family vaccines have a high safety profile; the dose reduction of viral antigens reduces the risk of adverse reactions for immunized. The adjuvant in the vaccine effectively stimulates an immune response and has no negative impact on the human health. The physicochemical properties of Polyoxidonium (INN: Azoximer bromide) let to consider Polyoxidonium as an universal adjuvant for different antigens.

CD4+ T Cells Drive Lung Disease Enhancement Induced by Immunization with Suboptimal Doses of Respiratory Syncytial Virus Fusion Protein in the Mouse Model.

Respiratory syncytial virus (RSV) infection of seronegative children previously immunized with formalin-inactivated (FI) RSV has been associated with serious enhanced respiratory disease (ERD). The phenomenon was reproduced in the cotton rat and the mouse, and both preclinical models have been routinely used to evaluate the safety of new RSV vaccine candidates. More recently, we demonstrated that immunizations with suboptimal doses of the RSV fusion (F) antigen, in its post- or prefusion conformation, and in the presence of a Th1-biasing adjuvant, unexpectedly led to ERD in the cotton rat model. To assess if those observations are specific to the cotton rat and to elucidate the mechanism by which vaccination with low antigen doses can drive ERD post-RSV challenge, we evaluated RSV post-F antigen dose de-escalation in BALB/c mice in the presence of a Th1-biasing adjuvant. While decreasing antigen doses, we observed an increase in lung inflammation associated with an upregulation of proinflammatory cytokines. The amplitude of the lung histopathology was comparable to that of FI-RSV-induced ERD, confirming the observations made in the cotton rat. Importantly, depletion of CD4+ T cells prior to viral challenge completely abrogated ERD, preventing proinflammatory cytokine upregulation and the infiltration of T cells, neutrophils, eosinophils, and macrophages into the lung. Overall, low-antigen-dose-induced ERD resembles FI-RSV-induced ERD, except that the former appears in the absence of detectable levels of viral replication and in the context of a Th1-biased immune response. Taken together, our observations reinforce the recent concept that vaccines developed for RSV-naïve individuals should be systematically tested under suboptimal dosing conditions.IMPORTANCE RSV poses a significant health care burden and is the leading cause of serious lower-respiratory-tract infections in young children. A formalin-inactivated RSV vaccine developed in the 1960s not only showed a complete lack of efficacy against RSV infection but also induced severe lung disease enhancement in vaccinated children. Since then, establishing safety in preclinical models has been one of the major challenges to RSV vaccine development. We recently observed in the cotton rat model that suboptimal immunizations with RSV fusion protein could induce lung disease enhancement. In the present study, we extended suboptimal dosing evaluation to the mouse model. We confirmed the induction of lung disease enhancement by vaccinations with low antigen doses and dissected the associated immune mechanisms. Our results stress the need to evaluate suboptimal dosing for any new RSV vaccine candidate developed for seronegative infants.

Akt phosphorylation of hnRNP A1 controls CD4+ T cell fate decisions

Abstract The interaction between T-cell receptor (TCR) and a specific peptide-bound major histocompatibility complex (MHC) class II molecule on an antigen-presenting cell stimulates naive CD4+ T cells to differentiate into either effector T (Teff) or regulatory T cells (Treg). Previous studies have shown that CD4+ T cell fate decision is controlled by TCR signal strength. High dose of antigen promotes the differentiation into Teff cells, while low dose of antigen favors the Treg fate. We previously reported that the Akt/mTOR signaling pathway is involved in this process of alternate fate decision. High or low dose of antigen results in differential Akt phosphorylation and subsequent Akt substrate specificity. Upon high dose of antigen, dually phosphorylated Akt (T308 and S473) phosphorylates FoxO1, which excludes it from the nucleus; while under low dose of antigen, phosphorylated Akt (T308) activates RNA processing factors hnRNP L and hnRNP A1. Our lab has shown that both hnRNP L and A1 are necessary for the induction of Tregs in vitro. Other studies reported the identification of S199 as the phosphorylation site of hnRNP A1 by Akt. However, the relationship between Akt phosphorylation on S199 of hnRNP A1 and Treg induction is unclear. In order to investigate this, we are performing RIP-Seq on RNAs bound by hnRNP A1 under low or high stimulatory condition. We have generated a mutant mouse in which the hnRNP A1 S199 site has been mutated to Alanine. We are currently examining the impact of this mutation on the nature of RNAs bound by hnRNP A1 in T cells stimulated by low or high dose of antigen. In conclusion, our goal is to determine the mechanism by which Akt phosphorylation of hnRNP A1 is implicated in the CD4+ T cell differentiation programs.

Adjuvanted Immunotherapy Approaches for Peanut Allergy.

Food allergies are a growing public health concern with an estimated 8% of US children affected. Peanut allergies are also on the rise and often do not spontaneously resolve, leaving individuals at-risk for potentially life-threatening anaphylaxis throughout their lifetime. Currently, two forms of peanut immunotherapy, oral immunotherapy (OIT) and epicutaneous immunotherapy (EPIT), are in Phase III clinical trials and have shown promise to induce desensitization in many subjects. However, there are several limitations with OIT and EPIT, such as allergic side effects, daily dosing requirements, and the infrequent outcome of long-term tolerance. Next-generation therapies for peanut allergy should aim to overcome these limitations, which may be achievable with adjuvanted immunotherapy. An adjuvant can be defined as anything that enhances, accelerates, or modifies an immune response to a particular antigen. Adjuvants may allow for lower doses of antigen to be given leading to decreased side effects; may only need to be administered every few weeks or months rather than daily exposures; and may induce a long-lasting protective effect. In this review article, we highlight examples of adjuvants and formulations that have shown pre-clinical efficacy in treating peanut allergy.

Transcriptomics of the Vaccine Immune Response: Priming With Adjuvant Modulates Recall Innate Responses After Boosting.

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In this study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. Mice were primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. mRNA sequencing was performed on blood samples collected 1, 2, and 7 days after priming and after boosting. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared with the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared with mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen-presenting cells, and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody-secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly reprograms the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response.

High Antigen Dose Is Detrimental to Post-Exposure Vaccine Protection against Tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), causes 1.8M deaths annually. The current vaccine, BCG, has failed to eradicate TB leaving 25% of the world’s population with latent Mtb infection (LTBI), and 5–10% of these people will reactivate and develop active TB. An efficient therapeutic vaccine targeting LTBI could have an enormous impact on global TB incidence, and could be an important aid in fighting multidrug resistance, which is increasing globally. Here we show in a mouse model using the H56 (Ag85B-ESAT-6-Rv2660) TB vaccine candidate that post-exposure, but not preventive, vaccine protection requires low vaccine antigen doses for optimal protection. Loss of protection from high dose post-exposure vaccination was not associated with a loss of overall vaccine response magnitude, but rather with greater differentiation and lower functional avidity of vaccine-specific CD4 T cells. High vaccine antigen dose also led to a decreased ability of vaccine-specific CD4 T cells to home into the Mtb-infected lung parenchyma, a recently discovered important feature of T cell protection in mice. These results underscore the importance of T cell quality rather than magnitude in TB-vaccine protection, and the significant role that antigen dosing plays in vaccine-mediated protection.

Low antigen dose formulated in CAF09 adjuvant Favours a cytotoxic T-cell response following intraperitoneal immunization in Göttingen minipigs.

The relationship between the antigen dose and the quality of an immune response generated upon immunization is poorly understood. However, findings show that the immune system is indeed influenced by the antigen dose; hence underlining the importance of correctly determining which dose to use in order to generate a certain type of immune response. To investigate this area further, we used Göttingen minipigs asan animal model especially due to the similar body size and high degree of immunome similarity between humans and pigs. In this study, we show that both a humoral and a cell-mediated immune (CMI) response can be generated following intraperitoneal immunization with tetanus toxoid (TT) formulated in the CAF09 liposomal adjuvant. Importantly, a low antigen dose induced more TT-specific polyfunctional T cells, whereas antigen-specific IgG production was observed upon high-dose immunization. Independent of antigen dose, intraperitoneal administration of antigen increased the amount of TT-specific cytotoxic CD8β+ T cells within the cytokine-producing T-cell pool when compared to the non-cytokine producing T-cell compartment. Taken together, these results demonstrate that a full protein formulated in the CAF09 adjuvant and administered to pigs via the intraperitoneal route effectively generates a cytotoxic T-cell response. Moreover, we confirm the inverse relationship between the antigen dose and the induction of polyfunctional T cells in a large animal model. These finding can have implications for the design of upcoming vaccine trials aiming at establishing a cytotoxic T-cell response.

Book Review: The Foundations of Immunology and their Pertinence to Medicine

AbstractWe are in the era cursed with overspecialization. Charles Darwin's foundational Origin of Species, published in 1859, was accessible to the broadly educated individual. Starting with this statement, Peter Bretscher in his new book aimed at explaining foundational concepts of immunology to a broad public. His standpoint is that foundational ideas usually can be simply expressed. This book describes first how foundational concepts of immunology have evolved over the last two and a half centuries. The importance of self–non‐self‐discrimination by the immune system is acknowledged, and models for immunological tolerance are compared. A main focus concerns the understanding of immune class regulation. According to Bretscher's Threshold Hypothesis, a low antigen dose or low degree of foreignness results in Th1 immunity whereas a high antigen dose or high degree of foreignness induces Th2 responses. A novel vaccination strategy is proposed with an ultra‐low number of live, attenuated organisms. It is described in details how this approach may potentially be used to prevent or treat HIV infection, tuberculosis and cancer. The book is very well written in a clear, simple and stimulating style which makes it accessible not only to immunologists but also non‐specialists, teachers, medical students and clinicians. It will convince many that the immune system is not too complex to be understood. By Peter Bretscher. FriesenPress, 2016. 200 pp. ISBN: 978‐1‐4602‐9656‐1.

Novel immunotherapy and treatment modality for severe food allergies.

In recent years, many studies on oral immunotherapy (OIT) have been conducted; however, few have focused on severe food allergies. The purpose of this review was to assess the efficacy and safety of oral immunotherapies for patients with severe food allergy. We reviewed multiple immunotherapy reports published within a few years or reports focusing on severe food allergies. We also investigated recent studies on OIT and novel food allergy management. Immunotherapies targeting low-dose antigen exposure and oral food challenges using low-dose target volumes may be safer than conventional OIT. It is necessary to consider which immunotherapy regimen is appropriate based on allergy severity of the patient.

Low antigen dose in adjuvant-based vaccination selectively induces CD4 T cells with enhanced functional avidity and protective efficacy

Abstract T cells of high functional avidity sense and respond to low levels of antigen (Ag), and their superior protective efficacy was first described using CD8 T cells cultured in vitro with low levels of Ag resulting in higher avidity and anti-tumor/viral efficacy compared to low avidity T cells cultured with high Ag concentrations. Selectively inducing high avidity by low Ag concentrations has required in vitro expansion so far, as in vivo attempts with low-dose vaccination gave no response. We show for the first time that functionally high avidity CD4, but not CD8, T cells are selectively primed in vivo by low concentrations of Ag in the strong crosspriming liposomal cationic adjuvant formulations (CAF) DDA/TDB or DDA/MMG/pIC from SSI. Increased functional avidity observed after low-dose vaccinations correlated with lower surface expression of PD-1 and higher per cell IFN-gamma production compared to low avidity CD4 T cells. We further show that IL-15, known to increase functional avidity of CTLs, is essential for high avidity of vaccine specific CD4 T cells. Importantly, high avidity CD4 T cells offered superior protection in a murine model of latent infection with Mycobacterium tuberculosis compared to low avidity T cells. Furthermore, adoptive transfers of HIV-IIIB gp140-specific CD4 T cells of high functional avidity resulting from low-dose immunizations in combination with TCR transgenic HIV-IIIB gp120 specific CTLs conferred superior protection against viral (vaccinia-HIV-IIB-gp140) challenge compared to TCR-Tg CTLs alone or in combination with low avidity CD4 T cells induced by high-dose immunization. Thus, inducing high avidity CD4 T cells by low-dose immunization is important in clearing viral and bacterial infections.

Low Antigen Dose in Adjuvant-Based Vaccination Selectively Induces CD4 T Cells with Enhanced Functional Avidity and Protective Efficacy.

T cells with high functional avidity can sense and respond to low levels of cognate Ag, a characteristic that is associated with more potent responses against tumors and many infections, including HIV. Although an important determinant of T cell efficacy, it has proven difficult to selectively induce T cells of high functional avidity through vaccination. Attempts to induce high-avidity T cells by low-dose in vivo vaccination failed because this strategy simply gave no response. Instead, selective induction of high-avidity T cells has required in vitro culturing of specific T cells with low Ag concentrations. In this study, we combined low vaccine Ag doses with a novel potent cationic liposomal adjuvant, cationic adjuvant formulation 09, consisting of dimethyldioctadecylammonium liposomes incorporating two immunomodulators (monomycolyl glycerol analog and polyinosinic-polycytidylic acid) that efficiently induces CD4 Th cells, as well as cross-primes CD8 CTL responses. We show that vaccination with low Ag dose selectively primes CD4 T cells of higher functional avidity, whereas CD8 T cell functional avidity was unrelated to vaccine dose in mice. Importantly, CD4 T cells of higher functional avidity induced by low-dose vaccinations showed higher cytokine release per cell and lower inhibitory receptor expression (PD-1, CTLA-4, and the apoptosis-inducing Fas death receptor) compared with their lower-avidity CD4 counterparts. Notably, increased functional CD4 T cell avidity improved antiviral efficacy of CD8 T cells. These data suggest that potent adjuvants, such as cationic adjuvant formulation 09, render low-dose vaccination a feasible and promising approach for generating high-avidity T cells through vaccination.

Autoreactive T cells display altered CD25 expression that changes responses to low dose IL-2 and antigen delivery.

Abstract Dendritic cells (DCs) are important for T cell tolerance induction and treatment of autoimmune diseases such as type 1 diabetes. Chimeric antibodies that deliver self-antigens to DCIR2+ DCs can delay but not completely prevent diabetes development in non-obese diabetic (NOD) mice. These DCIR2 antibodies induce T cell tolerance via antigen-specific deletion and anergy, but do not increase antigen-specific Foxp3+ regulatory T cells (Tregs). Because low dose (LD) IL-2 administration can preferentially expand Tregs, we asked if delivering antigens to tolerogenic DCIR2+ DCs along with IL-2 would boost antigen-specific Tregs and block autoimmunity. But, surprisingly, adding IL-2 did not increase efficacy of DC-targeted antigens to inhibit diabetes. To date, the effect of LD-IL-2 on autoreactive Treg and effector T cells has not been fully elucidated. By using tetramer staining to identify islet-specific CD4+ T cells in NOD mice, we now show the effects of LD-IL-2, with or without specific antigen delivery to DCIR2+ DCs, on both polyclonal and autoreactive effector and regulatory T cells. As expected, LD-IL-2 increased total Tregs, but autoreactive Tregs required addition of antigen and LD-IL-2 to elicit significant expansion. We found that islet-specific Tregs had lower CD25 expression compared to polyclonal Tregs, but islet-specific Foxp3− cells had higher CD25 expression. IL-2 increased activation and expansion of Foxp3− cells, and this effect was more pronounced for autoreactive cells after treatment with IL-2 + islet antigens. Therefore, IL-2, especially when combined with self-antigens, may not be an effective treatment for chronic autoimmunity, and increased Foxp3+ cells may not be a good biomarker for treatment efficacy.

Rational design of protamine nanocapsules as antigen delivery carriers

Current challenges in global immunization indicate the demand for new delivery strategies, which could be applied to the development of new vaccines against emerging diseases, as well as to improve safety and efficacy of currently existing vaccine formulations. Here, we report a novel antigen nanocarrier consisting of an oily core and a protamine shell, further stabilized with pegylated surfactants. These nanocarriers, named protamine nanocapsules, were rationally designed to promote the intracellular delivery of antigens to immunocompetent cells and to trigger an efficient and long-lasting immune response. Protamine nanocapsules have nanometric size, positive zeta potential and high association capacity for H1N1 influenza hemagglutinin, a protein that was used here as a model antigen. The new formulation shows an attractive stability profile both, as an aqueous suspension or a freeze-dried powder formulation. In vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without eliciting significant toxicity. In vivo studies indicate that antigen-loaded nanocapsules trigger immune responses comparable to those achieved with alum, even when using significantly lower antigen doses, thus indicating their adjuvant properties. These promising in vivo data, alongside with their versatility for the loading of different antigens and oily immunomodulators and their excellent stability profile, make these nanocapsules a promising platform for the delivery of antigens. Chemical compoundsProtamine sulphate (PubChem SID: 7849283), Sodium Cholate (PubChem CID: 23668194), Miglyol (PubChem CID: 53471835), α tocopherol (PubChem CID: 14985), Tween® 20(PubChem CID: 443314), Tween® 80(PubChem CID: 5281955), TPGS (PubChem CID: 71406).