Class Of Adjuvants Research Articles

Abstract P6-02-06: Safety and feasibility of neoadjuvant combined chemotherapy of breast cancer with paclitaxel carried in a lipid nanoemulsion (LDE) associated with adriamycin and cyclophosphamide

Abstract Background The introduction of neoadjuvant chemotherapy considerably changed the natural history of breast cancer by permitting a big tumors to be operable, permitting less aggressive surgical procedures and when achieving complete pathological response prolonging the disease-free period. However, the chemotherapy schemes bear high toxicity rates and are even life-threatening. Neoadjuvant chemotherapy aims at reducing mortality and improving surgical options and offers an in vivo chemosensitivity testing at the same time. It is the ideal setting for clinical and translational research. Compared to the classical adjuvant treatment, it offers several advantages. Methods We performed a pilot study to evaluate the safety and feasibility of neoadjuvant combined chemotherapy of breast cancer with paclitaxel carried in a lipid nanoemulsion (LDE) associated with adriamycin and cyclophosphamide in comparison to the classical neoadjuvant chemotherapy paclitaxel, adriamicyn and cyclofosfamide (TAC). All the patients were women with recently diagnosed breast cancer who needed primary chemotherapy as the first treatment selected from Perola Byington Hospital in São Paulo - Brazil. Results From April 2006 to June 2008, 39 patients from a center for breast disorders (Pérola Byington Hospital) were included in the study. These patients were randomized to the standard chemotherapy scheme (control arm – n=16) or LDE-paclitaxel (LDE arm – N=17). Six patients were excluded from the study because they did not complete at least 5 cycles of chemotherapy. We also excluded three patients because of lack of follow up. The majority of patients had stage III (37% IIIA and 27% IIIB). There was no significant difference among the groups, considering the clinical variables, so the sample was considered homogeneous. The patients were grouped in four different groups regarding the expression of ER, PR and CerbB2: Luminal subtype, Triple Negative, Her2-positive and Hybrid Luminal. We observed 37,5% of complete response in the control arm and 23,5% in the LDE arm. The partial response were 43,7% in the control group and 58,8% in the LDE group. The toxicity particularly in grade 3 and 4 events were substantially lower in LDE group: Nausea 11% vs 2,2%; Vomiting 7,3% vs 0.7% and neutropenia 8,8% vs 2,2%. Conclusion LDE scheme was effective and had lower grad 3 and 4 events than the TAC regiment. More studies are necessary to evaluate this approach. Citation Format: Andre Mattar, Luciana B Ferreira, Luiz H Gebrim, Roberto Hegg, Raul C Maranhão. Safety and feasibility of neoadjuvant combined chemotherapy of breast cancer with paclitaxel carried in a lipid nanoemulsion (LDE) associated with adriamycin and cyclophosphamide [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-02-06.

Peptide amphiphile micelles self-adjuvant group A streptococcal vaccination.

Delivery system design and adjuvant development are crucially important areas of research for improving vaccines. Peptide amphiphile micelles are a class of biomaterials that have the unique potential to function as both vaccine delivery vehicles and self-adjuvants. In this study, peptide amphiphiles comprised of a group A streptococcus B cell antigen (J8) and a dialkyl hydrophobic moiety (diC16) were synthesized and organized into self-assembled micelles, driven by hydrophobic interactions among the alkyl tails. J8-diC16 formed cylindrical micelles with highly α-helical peptide presented on their surfaces. Both the micelle length and secondary structure were shown to be enhanced by annealing. When injected into mice, J8-diC16 micelles induced a strong IgG1 antibody response that was comparable to soluble J8 peptide supplemented with two classical adjuvants. It was discovered that micelle adjuvanticity requires the antigen be a part of the micelle since separation of J8 and the micelle was insufficient to induce an immune response. Additionally, the diC16 tail appears to be non-immunogenic since it does not stimulate a pathogen recognition receptor whose agonist (Pam3Cys) possesses a very similar chemical structure. The research presented in this paper demonstrates the promise peptide amphiphile micelles have in improving the field of vaccine engineering.

Triple negative breast cancer - BCL2 in prognosis and prediction. Review.

Breast cancer (BC), the most frequent malignancy in women worldwide, is currently diagnosed in about 1.4 million female patients annually. Approximately 10-20% of BC is represented by triple negative breast cancer (TNBC) which is aggressive, the prognosis is poor and patients cannot benefit from targeted treatment based on hormonal or HER2 receptors. For this reason, search for markers that can predict the efficacy of chemotherapy in TNBC is a priority. This review focuses on BCL2 protein as a prognostic marker in TNBC and its potential as a predictor of sensitivity to chemotherapy. BCL2 protein expression is a positive prognostic factor in BC. Better survival of patients with BCL2 positivity (BCL2+) has been explained by the correlation with estrogen receptor positive (ER+) status. BCL2+ is however not simply a surrogate marker for ER+. Moreover, BCL2 protein expression is also a positive prognostic marker in the TNBC subgroup. We and others show, that low BCL2 expression was associated with good outcome of TNBC patients treated with both adjuvant and neoadjuvant anthracycline-based chemotherapy. On the other hand, recent studies have shown that a subset of TNBC patients may benefit from the classical adjuvant CMF (cyclophosphamide, methotrexate, 5-fluorouracil) regimen. Given the heterogeneity of TNBC there is an urgent need to find and validate the sensitivity predictors to these regimens making them usable in clinical practice. BCL2 enrichment has been described in the mesenchymal stem-like (MSL) TNBC subgroup.

Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice

Cholera toxin B subunit (CTB) was investigated as a classical mucosal adjuvant that can increase vaccine immunogenicity. In this study, we found out the in vitro efficacy of cholera toxin B subunit (CTB) in activating mice bone marrow-derived dendritic cells (BMDCs) through Toll-like receptor signaling pathways. In vitro RNA and transcriptional level profiling arrays revealed that CTB guides high levels of Th1 and Th2 type cytokines, inflammatory cytokines, and chemokines. Based on the robustness of these profiling results, we examined the induction of HIV Env-specific immunity by CTB co-inoculated with HIV Env DNA vaccine intramuscularly in vivo. CTB enhanced HIV-Env specific cellular immune responses in Env-specific IFN-γ ELISPOT, compared with DNA vaccine alone. Moreover, CTB induced high levels of Env specific humoral response and promoted antibody maturation after the third round of vaccination. This combination immunization strategy induced a Th2-type bias response which is indicative of a high ratio of IgG1/IgG2a. This study reports that CTB as a classical mucosal adjuvant could enhance HIV-1 DNA-based vaccine immunogenicity intramuscularly; therefore, these findings suggest that CTB could serve as an effective candidate adjuvant for DNA vaccination.

Immunomodulation of ALT-2 and TLR may collude in antigen specific T cell hyporesponsiveness: Proposed mechanism for elevated IL-10 levels in Balb/C mice

ALT-2, a novel antigen belonging to the chromadorea ALT-2 family of the filarial nematode is proved to clear filarial parasites in Jirds. In order to increase the protection efficacy by stimulating the cell mediated immunity, MPLA a detoxified derivative of LPS known to induce the cellular response, was used in this study as an adjuvant on mice models. ALT-2+MPLA formulation elicited a high titer of total IgG antibody, with profoundly increased levels of IgG2b. Reduced splenocyte proliferation was observed in immunized group when compared to control groups which could be attributed to many in vivo factors. The levels of IFN-γ were high in unstimulated MPLA group compared to ALT-2 stimulated MPLA group, suggesting that the ALT-2 antigen suppressed the IFN-γ levels. A high level of IL-10 was induced by the ALT-2+MPLA formulation, which inhibited the production of Th2 cytokines (IL-4, IL-5) and also reduced the Th1 cytokine (IFN-γ, IL-2) levels which are not in vogue with the classical MPLA adjuvant formulation. We propose a mechanism for this immunomodulation which involves a diminished expression of TLR-4, by which the filarial parasites have evolved to evade host immune mechanism.

Immunization of Mice by BCG Formulated HCV Core Protein Elicited Higher Th1-Oriented Responses Compared to Pluronic-F127 Copolymer

BackgroundA supreme vaccine for Hepatitis C virus (HCV) infection should elicit strong Th1-oriented cellular responses. In the absence of a Th1-specific adjuvant, immunizations by protein antigens generally induce Th2-type and weak cellular responses.ObjectivesTo evaluate the adjuvant effect of BCG in comparison with nonionic copolymer-Pluronic F127 (F127) as a classic adjuvant in the formulation of HCV core protein (HCVcp) as a candidate vaccine for induction of Th1 immune responses.Materials and MethodsExpression of N-terminally His-Tagged HCVcp (1-122) by pIVEX2.4a-core vector harboring the corresponding gene under the control of arabinose-inducible (araBAD) promoter was achieved in BL21-AI strain of E.coli and purified through application of nitrilotriacetic acid (Ni-NTA) chromatography. Mice were immunized subcutaneously (s.c.) in base of the tail with 100 μl of immunogen (F127+HCVcp or BCG+HCVcp; 5 μgHCVcp/mouse/dose) or control formulations (PBS, BCG, F127) at weeks 0, 3, 6. Total and subtypes of IgG, as well as cellular immune responses (Proliferation, In vivo CTL and IFN-γ/IL-4 ELISpot assays against a strong and dominant H2-d restricted, CD8+-epitopic peptide, core 39-48; RRGPRLGVRA of HCVcp) were compared in each group of immunized animals.ResultsExpression and purification of core protein around the expected size (21 kDa) was confirmed by Western blotting. The HCVcp + BCG vaccinated mice showed significantly higher lymphocyte proliferation and IFN-γ production but lower levels of cell lysis (45% versus 62% in CTL assay) than the HCVcp+F127 immunized animals. “Besides, total anti-core IgG and IgG1 levels were significantly higher in HCVcp + F127 immunized mice as compared to HCVcp + BCG vaccinated animals, indicating relatively higher efficacy of F127 for the stimulation of humoral and Th2-oriented immune responses”.ConclusionsResults showed that HCVcp + BCG induced a moderate CTL and mixed Th1/Th2 immune responses with higher levels of cell proliferation and IFN-γ secretion, indicating that BCG may have a better outcome when formulated in HCVcp-based subunit vaccines.

Matrix-M™ adjuvant: enhancing immune responses by ‘setting the stage’ for the antigen

Adjuvants belong to a diverse collection of substances or formulations that, in one way or another, increase or improve immune responses to antigen(s). Classical adjuvants target the physical prese...

Conjugation, characterization and toxicity of lipophosphoglycan-polyacrylic acid conjugate for vaccination against leishmaniasis

Research on the conjugates of synthetic polyelectrolytes with antigenic molecules, such as proteins, peptides, or carbohydrates, is an attractive area due to their highly immunogenic character in comparison to classical adjuvants. For example, polyacrylic acid (PAA) is a weak polyelectrolyte and has been used in several biomedical applications such as immunological studies, drug delivery, and enzyme immobilization. However, to our knowledge, there are no studies that document immune-stimulant properties of PAA in Leishmania infection. Therefore, we aimed to develop a potential vaccine candidate against leishmaniasis by covalently conjugating PAA with an immunologically vital molecule of lipophosphoglycan (LPG) found in Leishmania parasites. In the study, LPG and PAA were conjugated by a multi-step procedure, and final products were analyzed with GPC and MALDI-TOF MS techniques. In cytotoxicity experiments, LPG-PAA conjugates did not indicate toxic effects on L929 and J774 murine macrophage cells. We assume that LPG-PAA conjugate can be a potential vaccine candidate, and will be immunologically characterized in further studies to prove its potential.

A protocol with adjuvant chemotherapy (CT) and radiotherapy (RT) for endometrial cancer: Results.

e16549 Background: The classic adjuvant treatment for high-risk endometrial cancer is RT. Several phase III trials explored the role of CT, as single treatment or added to RT sequentially (ST) or concurrently (CR), but design heterogeneity hampers conclusions. We report the results of a single-institution protocol with adjuvant CT + RT for advanced stages or type II histologies. Key points: (1) ST and CR permitted. (2) CT used pre-, post- and/or during RT: carboplatin AUC 5 + paclitaxel 175 mg/m2 (150 during RT) Q3W, 4-6 cycles (“carbotaxol”). (3) since 2010, cisplatin 50 mg/m2 was used for CR (on day 1 and 28 of RT). CR was preceded/followed by carbotaxol. Methods: All patients (PT) included in the protocol between 1/2005 and 9/2011 were retrospectively revised. Endpoints were survival and toxicity grade 3-4. Kaplan Meier and Fisher test were used. Conclusions: CR-carbotaxol showed a longer progression-free and overall survival than ST without reaching statistical significance (log rank, p= 0.14 and p=0.33). Median survivals with CR-cisplatin were not reached because of shorter follow-up. Statistical differences in toxicities were not found. In the CR-carbotaxol arm, 5 PT had grade 3-4 acute diarrhoea vs none in the others. [Table: see text]

Collagen Type II and a Thermo-Responsive Polymer of N-Isopropylacrylamide Induce Arthritis Independent of Toll-Like Receptors : A Strong Influence by Major Histocompatibility Complex Class II and Ncf1 Genes

We established and characterized an arthritis mouse model using collagen type II (CII) and a thermo-responsive polymer, poly(N-isopropylacrylamide) (PNiPAAm). The new PNiPAAm adjuvant is TLR-independent, as all immunized TLR including MyD88-deficient mice developed an anti-CII response. Unlike other adjuvants, PNiPPAm did not skew the cytokine response (IL-1β, IFN-γ, IL-4, and IL-17), as there was no immune deviation towards any one type of immune spectrum after immunization with CII/PNiPPAm. Hence, using PNiPAAm, we studied the actual immune response to the self-protein, CII. We observed arthritis and autoimmunity development in several murine strains having different major histocompatibility complex (MHC) haplotypes after CII/PNiPAAm immunization but with a clear MHC association pattern. Interestingly, C57Bl/6 mice did not develop CII-induced arthritis, with PNiPAAm demonstrating absolute requirement for a classical adjuvant. Presence of a gene (Ncf1) mutation in the NADPH oxidation complex has a profound influence in arthritis and using PNiPAAm we could show that the high CIA severity in Ncf1 mutated mice is independent of any classical adjuvant. Macrophages, neutrophils, eosinophils, and osteoclasts but not mast cells dominated the inflamed joints. Furthermore, arthritis induction in the adjuvant-free, eosinophil-dependent Vβ12 DBA/1 mice could be shown to develop arthritis independent of eosinophils using CII/PNiPAAm. Thus, biocompatible and biodegradable PNiPAAm offers unique opportunities to study actual autoimmunity independent of TLR and a particular cytokine phenotype profile.

The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus.

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.

Cutting Edge: Intrathymic Differentiation of Adaptive Foxp3+ Regulatory T Cells upon Peripheral Proinflammatory Immunization

Thymocytes differentiate into CD4(+) Foxp3(+) regulatory T cells (T(R)) upon interaction between their TCR and peptide-MHC II complexes locally expressed in the thymus. Conversion of naive CD4(+) T cells into T(R) can additionally take place in the periphery under noninflammatory conditions of Ag encounter. In this study, making use of TCR transgenic models naturally devoid of Foxp3(+) cells, we report de novo generation of T(R) upon a single footpad injection of Ag mixed with a classic proinflammatory adjuvant. Abrupt T(R) differentiation upon immunization occurred intrathymically and was essential for robust tolerance induction in a mouse model of spontaneous encephalomyelitis. This phenomenon could be attributed to a specific feature of thymocytes, which, in contrast to mature peripheral CD4(+) T cells, were insensitive to the inhibitory effects of IL-6 on the induction of Foxp3 expression. Our findings uncover a pathway for T(R) generation with major implications for immunity and tolerance induction.

Encephalitogenicity of Complete Freund’s Adjuvant Relative to CpG Is Linked to Induction of Th17 Cells

For decades, CFA has been the classic adjuvant for the induction of experimental autoimmune encephalomyelitis (EAE). Its encephalitogenic activity has been originally linked to the induction of Th1 responses. CpG, which is also a potent Th1 inducer, has been suggested by some studies to be comparably encephalitogenic. In this study, using the SJL proteolipid protein (PLP) 139-151 peptide EAE model, we show that active immunizations using CFA but not CpG 1826/IFA as an adjuvant induced disease. Passive induction of EAE resulted in severe disease when cells were transferred from PLP in CFA-primed mice but resulted in only a mild, transient disease when cells originated from PLP in CpG 1826/IFA-primed mice. In accordance with these findings, immunizations using CFA but not CpG 1826/IFA as an adjuvant elicited a delayed-type hypersensitivity response. ELISPOT analysis revealed that CFA promoted the differentiation of much higher levels of PLP-specific, IL-17-secreting cells compared with CpG 1826/IFA. Both adjuvants induced comparable frequencies of PLP-specific, IFN-gamma-secreting cells and also induced Ag-specific proliferation to the same extent. The severity of EAE in PLP in CFA-immunized mice was reduced when IL-17 was neutralized in vivo, demonstrating the crucial role of this cytokine in disease induction. The data show that immunizations using the autoantigen in CpG 1826/IFA result in very low frequencies of Ag-specific IL-17 cells, suggesting a lower risk of Th17-mediated pathology when using this adjuvant.

Models of Antigen Receptor Activation in the Design of Vaccines

Vaccination techniques have developed rapidly over the last several decades from the immunization with live attenuated pathogens to the use of peptide and DNA subunit vaccines, from the use of classical adjuvants to cell-directed delivery. Vaccination techiques are also under investigation for the treatment of tumors and autoimmune diseases. However, profound knowledge of activation mechanisms of the immune cells on a molecular level is prerequisite for a better understanding of the immune response, and for the development of effective immunomodulatory tools. In this review we discuss the models of BCR and TCR activation, and using the example of some vacciantion technologies, we show, how the understanding of these models could help in the design of a new generation of vaccines.

Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy

Innate immune system activation is a critical step in the initiation of an effective adaptive immune response; therefore, activation of a class of innate pathogen receptors called pattern recognition receptors (PRR) is a central feature of many adjuvant systems. It has recently been shown that one member of an intracellular PRR, the NLRP3 inflammasome, is activated by a number of classical adjuvants including aluminum hydroxide and saponins [Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA. Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 2008;453(June (7198)):1122–6; Li H, Willingham SB, Ting JP, Re F. Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3. J Immunol 2008;181(July (1)):17–21]. Inflammasome activation in vitro requires signaling of both the Toll-like receptor (TLR) and NLRP3 in antigen-presenting cells. Here we present a class of nanomaterials endowed with these two signals for rapid optimization of vaccine design. We constructed this system using a simple approach that incorporates lipopolysaccharides (LPS) onto the surface of nanoparticles constructed from a biocompatible polyester, poly(lactic-co-glycolic acid) (PLGA), loaded with antigen. We demonstrate that LPS-modified particles are preferentially internalized by dendritic cells compared to uncoated nanoparticles and the system, when administered to mice, elicits potent humoral and cellular immunity against a model antigen, ovalbumin. Wild-type macrophages pulsed with LPS-modified nanoparticles resulted in production of the proinflammatory cytokine IL-1β consistent with inflammasome activation. In comparison, NLRP3-deficient and caspase-1-deficient macrophages showed negligible production of IL-1β. Furthermore, when endocytosis and lysosomal destabilization were inhibited, inflammasome activity was diminished, supporting the notion that nanoparticles rupture lysosomal compartments and behave as ‘danger signals’ [Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 2008;9(August (8)):847–56]. The generality of this vaccination approach is tested by encapsulation of a recombinant West Nile envelope protein and demonstrated by protection against a murine model of West Nile encephalitis. The design of such an antigen delivery mechanism with the ability to stimulate two potent innate immune pathways represents a potent new approach to simultaneous antigen and adjuvant delivery.

Immunostimulatory activity of haptenated proteins

Antigen recognition alone is insufficient for the activation of adaptive immune responses mediated by conventional lymphocytes. Additional signals that indicate the origin of the antigen are also required. These signals are generally provided by the innate immune system upon recognition of conserved microbial structures by a variety of pattern recognition receptors (PRRs). The Toll-like receptors (TLRs) are the best-characterized family of PRRs and control the activation of adaptive immune responses to a variety of immunizations and infections. However, recent studies have questioned the role of TLRs in the induction of antibody responses and, thus, this issue has become controversial. In contrast to earlier studies supporting a role for TLRs in antibody responses, these studies used haptenated antigens rather than native antigens for immunization, but did not consider the potential effect of antigen haptenation on immunogenicity. Here, we show that commonly used haptenated proteins, unlike native proteins, are inherently immunogenic. This immunogenicity is TLR-independent, but the T and B cell responses induced are primarily hapten-specific, rather than protein-specific. Thus, although haptens have immunostimulatory activity, it is distinct from classical adjuvants, which induce immune responses directed at the admixed antigens. Our results thus highlight an unappreciated and unique immunogenicity of haptenated proteins, and provide an experimental explanation for a seeming discrepancy between published results.

NKT-cell help to B lymphocytes can occur independently of cognate interaction

AbstractCD4+ T (Th)–cell help to B lymphocytes requires cognate interaction and CD40 engagement. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that recognize αgalactosylceramide (αGalCer) presented by CD1d, and can help B-cell responses. We asked whether αGalCer-activated iNKT cells help B lymphocytes through cognate interaction, or indirectly, via enhancement of Th-B–cell interaction. After immunization with protein Ags and αGalCer, antibody titers were assessed in wild-type or splenectomized mice, and in bone marrow radiation chimeras lacking CD1d or CD40 expression on B lymphocytes, or expressing CD1d or MHC II disjointly on antigen-presenting cells (APCs). We find that αGalCer-dependent enhancement of B-cell response (1) can occur when B cells do not express CD1d but express CD40; (2) requires that iNKT and Th cells interact with the same APCs that coexpress both CD1d and MHC-II; and (3) takes place without spleen. These findings demonstrate αGalCer-induced help for antibody responses can occur without cognate iNKT/B-cell interaction, and suggest this help entails activation of APCs by iNKT cells, which in turn activate Th cells and their helper functions for B cells. Thus, the αGalCer-induced help recapitulates the function of classical adjuvants that stimulate the innate immune system to support adaptive immune responses.

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol

A single-domain variable heavy chain (V HH) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama ( Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax™ Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V HH library was constructed from amplified cDNA by nested PCR. V HH fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V HH monomer (mNAT-267) at a final concentration of 1.3 mg mL −1. Isolated NAT-267 V HH DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V HH pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL −1. Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant ( K D) of 5 μM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC 50 values of 1.24 and 0.50 μM, respectively, for 15-AcDON hapten. These values were similar to the IC 50 value of 1.42 μM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V HH fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

GlaxoSmithKline Adjuvant Systems in vaccines: concepts, achievements and perspectives

The need for potentiating immune responses to recombinant or subunit antigens has prompted GlaxoSmithKline (GSK) Biologicals to develop various Adjuvant Systems for the design of prophylactic and therapeutic vaccines. Adjuvant Systems are formulations of classical adjuvants mixed with immunomodulators, specifically adapted to the antigen and the target population. They can activate the appropriate innate immune system and subsequently impact on adaptive immune responses. AS04 is an Adjuvant System that has demonstrated significant achievements in several vaccines against viral diseases. AS02, another Adjuvant System, is being evaluated in various contexts, where a strong T-cell response is needed to afford protection. Likewise, AS01 has been developed for vaccines where the induction of a yet stronger T-cell-mediated immune response is required. Altogether, the promising clinical results strongly support the concept of Adjuvant Systems and allow for further development of new vaccines, best adapted to the target population and the immune mechanisms of protection.

Expression of hemagglutinin-neuraminidase protein of Newcastle disease virus in transgenic tobacco

Newcastle disease is one of the most important pathogens of domestic poultry including chickens. The hemagglutinin-neuraminidase (HN) of Newcastle disease virus is the principal target of neutralizing and protective antibodies against Newcastle disease. In this paper, we transformed tobacco plants with Agrobacterium tumefaciens EHA105 to generate the plants expressing HN of Newcastle disease virus (NDV). The insertion and copy numbers of HN gene in the genomic DNA of phosphinothricin-resistant plants were confirmed by PCR and Southern blot, respectively. The presence of the HN-specific transcript in the total RNAs of the leaves of transgenic plants was verified by Northern analysis. The recombinant HN proteins were detected by Western blot analysis using a polyclonal antibody against GST–HN fusion proteins. The highest level of expression of HN in leaves of transgenic plants was approximately 0.069% of the total soluble protein. ELISA assay showed that the recombinant protein extracted from transformants has normal immunoactivity. Transgenic tobacco expressing HN of NDV with sterilized PBS was fed to 6-week-old chickens. Immunized chickens developed slightly high titers of anti-HN serum IgG compared with those of the wild type plant. These results suggest that oral immunization with HN-transgenic tobacco provides a potential means of protecting chickens from NDV. Further modified animal experiments would be needed to increase the immunity of HN by co-administration of classical adjuvants or other trials.