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Vaccination is an effective way to control many infectious diseases in fish. Israeli fish farming has successfully used two vaccines over the last 30 years and has no problem with the side effects of vaccination. However, after introducing new species, a new problem emerged: these fish, after vaccination, demonstrated peritoneal lesions such as granulomas. At the same time, the fish did not show retarded growth or suffering during the fattening period. This study was conducted to establish the connection between vaccination and the appearance of granulomas. Evidence drawn from this research work and comparing vaccinated and non-vaccinated fish confirms that intraperitoneal granulomas do not impact the growth, performance, or fish fillet quality at harvest.

I vaccini hanno rappresentato una svolta nella medicina. Dalla loro introduzione da parte di jenner nel 1798, i vaccini hanno abbassato drammaticamente il tasso di mortalità, esteso l’aspettativa di vita media e ridotto l’incidenza di malattie come tetano, polio, morbillo e altre, ed hanno portato all’eradicazione del vaiolo. Dopo secoli di miglioramenti, i vaccini si sono evoluti: dai vaccini attenuati, molto efficaci ma non troppo sicuri in quanto usavano patogeni vivi, fino ai moderni vaccini a sottounità od ad acidi nucleici. Questi ultimi sono molto più sicuri dei vaccini attenuati, poiché la possibilità di effetti collaterali è estremamente più bassa, ma senza una formulazione adeguata sono anche meno efficienti nel portare ad una buona memoria immunologica, poiché mancano delle proprietà immunostimolatorie di un patogeno intero. In effetti, i vaccini moderni contengono molti meno antigeni che in passato, pertanto, per arrivare ad un buon tasso di efficaca, è necessaria la presenza di adiuvanti nella loro formulazione. Gli adiuvanti vaccinali sono composti chimici o biologici che inducano una risposta immunitaria tale da migliorare la capacità di un antigene di generare una buona memoria immunologica, così da accrescere l’efficacia di un vaccino mantenendo un basso rischio. Per decenni, l’unico adiuvante vaccinale approvato per l’uso umano è stato l’Alum, una miscela di alluminio idrossido ed alluminio fosfato. Negli ultimi anni, tuttavia, c’è stato un maggiore interesse in questo ambito, che ha portato all’approvazione di un certo numero di adiuvanti, fra cui l’MPLA, un agonista del Toll-Like Receptor 4 (TLR4). Il TLR4 è una proteina fondamentale nel processo dell’immunità innata: è il sensore umano della presenza batterica, grazie alla sua abilità di legare il lipopolisaccaride (LPS), un componente fondamentale della membrana batterica esterna. Dopo il legame, il TLR4 dimerizza, provocando una cascata di chinasi intracellulare che a sua vol, Vaccines have been a breakthrough in medicine. Since their introduction in 1798 by Jenner, vaccines dramatically lowered mortality rate, extended average life expectancy and reduced incidence of severe diseases such as tetanus, polio, measles and others, while totally eradicating smallpox. After centuries of improvements, vaccines evolved: from attenuated live vaccines, having high efficacy rate but low safety profile as they used alive pathogens, to modern subunit or nucleic acid vaccines. The latter are much safer than attenuated vaccines, as the possibility of adverse events is extremely lower, but without the proper formulation they’re also less efficient in eliciting a good immune memory, as they lack the immunostimulatory properties of a whole pathogen cell. Indeed, modern vaccines contains far less antigens than in the past, therefore, to reach high efficacy rates, the presence of adjuvants is required in vaccine formulation. Vaccine adjuvants are chemical or biological compounds able to induce a proper immune response which enhances antigens ability to generate a good immune memory, so they can rise the overall efficiency of a vaccine, while maintaining a low risk profile. For decades, the only vaccine adjuvant approved for human use has been Alum, a mixed salt formed by aluminium hydroxide and aluminium phosphate In recent decades, however, increased interest on this topic brought to approval of a handful of adjuvants such MPLA, a Toll-Like Receptor 4 (TLR4) agonist. TLR4 is a pivotal protein in innate immunity: it is the human sensor for bacterial presence, due to its ability to bind lipopolysaccharide (LPS), a fundamental component of bacterial outer membrane. Upon binding, TLR4 dimerizes, triggering an intracellular kinase cascade which kickstarts the inflammatory process through the release of cytokines. These cytokines also help shaping the adaptive immune response, directing it toward a Th1 or Th2 response and priming memory B cells. For its centralit

We previously demonstrated that the dendritic cell (DC)-targeting nasal double DNA adjuvant system, which consists of a DNA plasmid expressing Flt3 ligand (pFL) and CpG oligodeoxynucleotide 1826 (CpG ODN), elicits specific immune responses to various antigens in the mucosal and systemic compartments. Here, we investigated, using phosphorylcholine (PC)-conjugated keyhole limpet hemocyanin (PC-KLH) as an antigen, whether the nasal double DNA adjuvant system induces protective immunity to atherosclerosis in apolipoprotein E-deficient (ApoE KO) mice. Further, we assessed the molecular and cellular mechanisms in the induction of anti-PC-specific immune responses. Nasal immunization with PC-KLH plus pFL and CpG ODN enhanced induction of PC-specific IgM in plasma, peritoneal fluids, and nasal washes when compared with mice administered PC-KLH alone. Of importance, these antibodies exhibited highly specific binding to the PC molecule, and dose-dependent binding to anti-T15 idiotype (AB1-2). Twelve weeks after the last immunization, the nasal double DNA adjuvant system with PC-KLH resulted in a reduction of atherogenesis in the aortic arch of ApoE KO mice. Therefore, we next assessed immunocytological mechanism to induce these antibodies. The nasal double DNA adjuvant system with PC-KLH resulted not only in significantly increased frequencies of CD11c+ DCs in the spleen, peritoneal cavity (PEC), and nasopharyngeal-associated lymphoid tissues (NALT), but also significantly increased expression of a proliferation-inducing ligand and B-cell-activating factor by CD11c+ DCs. In addition, the double DNA adjuvant system induced significantly increased numbers of B-1 B cells in the spleen, PEC, and NALT, and increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor on CD5+ B220+ (B-1a) B cells. These findings demonstrated that the nasal double DNA adjuvant system with PC-KLH resulted in the induction of T15-like antibodies in the mucosal

Recent advances in the development of protein-based vaccines have expanded the opportunities for preventing and treating both infectious diseases as well as cancer. However, the development of readily and efficient antigen delivery systems capable of stimulating strong cytotoxic T-lymphocyte (CTL) responses remains a challenge. With the attempt to closely mimic the properties of viruses in terms of their size and molecular organization, we constructed RNA (which is a ligand for Toll-like receptor 7 (TLR7) and TLR8) and antigen-loaded nanoparticles resembling the structural organization of viruses. Cationic polymers containing either azide or bicyclo[6.1.0]nonyne (BCN) groups were synthesized as electrostatic glue that binds negatively charged single stranded RNA (PolyU) to form a self-crosslinked polyplex core. An azide-modified model antigen (ovalbumin, OVA) and a BCN-modified mannosylated or galactosylated polymer were sequentially conjugated to the RNA core via disulfide bonds using copper free click chemistry to form the shell of the polyplexes. The generated reducible virus mimicking particles (VMPs) with a diameter of 200 nm and negatively surface charge (−14 mV) were colloidally stable in physiological conditions. The immunogenicity of these VMP vaccines was evaluated both in vitro and in vivo. The surface mannosylated VMPs (VMP-Man) showed 5 times higher cellular uptake by bone marrow derived DCs (BMDCs) compared to galactosylated VMP (VMP-Gal) counterpart. Moreover, VMP-Man efficiently activated DCs and greatly facilitated MHC I Ag presentation in vitro. Vaccination of mice with VMP-Man elicited strong OVA-specific CTL responses as well as humoral immune responses. These results demonstrate that the modular core-shell polymeric nanoparticles described in this paper are superior in inducing strong and durable immune responses compared to adjuvanted protein subunit vaccines and offer therefore a flexible platform for personalized vaccines.

Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1β release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases., SCOPUS: ar.j, info:eu-repo/semantics/published

There are a limited number of adjuvants that elicit effective cell-based immunity required for protection against intracellular bacterial pathogens. Here, we report that STING-activating cyclic dinucleotides (CDNs) formulated in a protein subunit vaccine elicit long-lasting protective immunity to Mycobacterium tuberculosis in the mouse model. Subcutaneous administration of this vaccine provides equivalent protection to that of the live attenuated vaccine strain Bacille Calmette-Guérin (BCG). Protection is STING dependent but type I IFN independent and correlates with an increased frequency of a recently described subset of CXCR3-expressing T cells that localize to the lung parenchyma. Intranasal delivery results in superior protection compared with BCG, significantly boosts BCG-based immunity, and elicits both Th1 and Th17 immune responses, the latter of which correlates with enhanced protection. Thus, a CDN-adjuvanted protein subunit vaccine has the capability of eliciting a multi-faceted immune response that results in protection from infection by an intracellular pathogen.

Vaccines often contain adjuvants to strengthen the response to the vaccine antigen. However, their modes of action at the site of injection (SOI) are poorly understood. Therefore, we assessed the local effects of adjuvant on the innate immune system in mice. We investigated the safe, widely used adjuvants MF59 and aluminum hydroxide (alum), as well as trehalose-6,6???-dibehenate (TDB), Complete Freund's Adjuvant (CFA) and the Toll-Like-Receptor-ligands lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam3CSK4). We assessed muscle immune cell infiltration after adjuvant injection and observed 16??h post immunization (hpi) an increased influx with CFA, MF59 and TDB, but not with alum, LPS or Pam3CSK4. An elevated influx with the latter three became visible only 72??hpi. Contribution of granulocytes, macrophages and dendritic cells to the influx differed per adjuvant and in time. Adjuvants generally induced a local pro-inflammatory micro-milieu that was transient except for CFA and TDB. The gene expression of CXCL-1, CCL-2 and CCL-5, involved in recruitment of immune cells, varied per adjuvant and corresponded grossly with the observed influx of granulocytes and monocytes/macrophages. Muscles injected with CFA or MF59 (when co-injected with peptide) resulted in APC ex vivo capable to induce proliferation of peptide-specific T-cells. By adding in vitro an excess of peptide to the APC/T cell co-cultures, we observed an adjuvant-enhanced co-stimulation or antigen presentation by APC after CFA- but not MF59-injection. After TDB-injection this effect was observed only at 72??hpi, but not 24??hpi. Thus the cellular influx profile and the local cytokine and chemokine micro-milieu in the muscle were strongly influenced by the type of adjuvant. Additionally, the capacity of muscle APC to load and present antigen was affected by the adjuvant. These findings may assist the development of novel adjuvanted vaccines in a more rational manner.

BACKGROUND: A better understanding of mechanisms underlying dose-effects of probiotics in their applications as treatments of intestinal infectious or inflammatory diseases and as vaccine adjuvant is needed. In this study, we evaluated the modulatory effects of Lactobacillus rhamnosus GG (LGG) on transplanted human gut microbiota (HGM) and on small intestinal immune cell signaling pathways in gnotobiotic pigs vaccinated with an oral attenuated human rotavirus (AttHRV) vaccine. RESULTS: Neonatal HGM transplanted pigs were given two doses of AttHRV on 5 and 15 days of age and were divided into three groups: none-LGG (AttHRV), 9-doses LGG (AttHRV + LGG9X), and 14-doses LGG (AttHRV + LGG14X) (n = 3-4). At post-AttHRV-inoculation day 28, all pigs were euthanized and intestinal contents and ileal tissue and mononuclear cells (MNC) were collected. AttHRV + LGG14X pigs had significantly increased LGG titers in the large intestinal contents and shifted structure of the microbiota as indicated by the formation of a cluster that is separated from the cluster formed by the AttHRV and AttHRV + LGG9X pigs. The increase in LGG titers concurred with significantly increased ileal HRV-specific IFN-γ producing T cell responses to the AttHRV vaccine reported in our previous publication, suggesting pro-Th1 adjuvant effects of the LGG. Both 9- and 14-doses LGG fed pig groups had significantly higher IkBα level and p-p38/p38 ratio, while significantly lower p-ERK/ERK ratio than the AttHRV pigs, suggesting activation of regulatory signals during immune activation. However, 9-doses, but not 14-doses LGG fed pigs had enhanced IL-6, IL-10, TNF-α, TLR9 mRNA levels, and p38 MAPK and ERK expressions in ileal MNC. Increased TLR9 mRNA was in parallel with higher mRNA levels of cytokines, p-NF-kB and higher p-p38/p38 ratio in MNC of the AttHRV + LGG9X pigs. CONCLUSIONS: The relationship between modulation of gut microbiota and regulation of host immunity by different doses of probiotics is complex.

The T-cell adjuvanticity of mycobacterial cord factor trehalose 6,6'-dimycolate (TDM) is well established. The identification of the C-type lectin Mincle on innate immune cells as the receptor for TDM and its synthetic analogue trehalose 6,6'-dibehenate (TDB) has raised interest in development of synthetic Mincle ligands as novel adjuvants. Trehalose mono- (TMXs) and diesters (TDXs) with symmetrically shortened acyl chains [denoted by X: arachidate (A), stearate (S), palmitate (P), and myristate (M)] were tested. Upon stimulation of murine macrophages, G-CSF secretion and NO production were strongly augmented by all TDXs tested, in a wide concentration range. In contrast, the TMXs triggered macrophage activation only at high concentrations. Macrophage activation by all TDXs required Mincle, but was independent of MyD88. The superior capacity of TDXs for activating macrophages was paralleled by direct binding of TDXs, but not of TMXs, to a Mincle-Fc fusion protein. Insertion of a short polyethylene glycol between the sugar and acyl chain in TDS reduced Mincle-binding and macrophage activation. Immunization of mice with cationic liposomes containing the analogues demonstrated the superior adjuvant activity of trehalose diesters. Overall, immune activation in vitro and in vivo by trehalose esters of simple fatty acids requires two acyl chains of length and involves Mincle.

We have recently described the antiviral effect in mice of in vitro-transcribed RNAs mimicking structural domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome RNA. These small, synthetic and non-infectious RNA molecules (ncRNAs) are potent type-I interferon (IFN) inducers in vivo. In this work, the immunomodulatory effect of the ncRNA corresponding to the internal ribosome entry site (IRES) on immunization with two different FMD vaccine formulations, both based on inactivated virus, including or not a commercial adjuvant, was analyzed in the mice model. The effect of the time interval between RNA inoculation and immunization was also studied. RNA delivery consistently increased the titers of specific anti-FMDV antibodies, including neutralizing antibodies, elicited after vaccination. Moreover, at day 2 after immunization, significant differences in mean antibody titers could be detected between the groups of mice receiving either vaccine co-administered with the RNA and the control group, unlike those immunized with the vaccine alone. When vaccinated mice were challenged with FMDV, the mean values of viral load were lower in the groups receiving the RNA together with the vaccine. Our results show the enhancing effect of the IRES RNA on the immune response elicited after vaccination and suggest the potential of this molecule as an adjuvant for new FMD vaccine design. © 2013 Elsevier Ltd.

We have recently described the antiviral effect in mice of in vitro-transcribed RNAs mimicking structural domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome RNA. These small, synthetic and non-infectious RNA molecules (ncRNAs) are potent type-I interferon (IFN) inducers in vivo. In this work, the immunomodulatory effect of the ncRNA corresponding to the internal ribosome entry site (IRES) on immunization with two different FMD vaccine formulations, both based on inactivated virus, including or not a commercial adjuvant, was analyzed in the mice model. The effect of the time interval between RNA inoculation and immunization was also studied. RNA delivery consistently increased the titers of specific anti-FMDV antibodies, including neutralizing antibodies, elicited after vaccination. Moreover, at day 2 after immunization, significant differences in mean antibody titers could be detected between the groups of mice receiving either vaccine co-administered with the RNA and the control group, unlike those immunized with the vaccine alone. When vaccinated mice were challenged with FMDV, the mean values of viral load were lower in the groups receiving the RNA together with the vaccine. Our results show the enhancing effect of the IRES RNA on the immune response elicited after vaccination and suggest the potential of this molecule as an adjuvant for new FMD vaccine design. © 2013 Elsevier Ltd.

Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1), demonstrates the need for a much improved, more sophisticated influenza vaccine. Our group has developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC). Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Here we report methodologies for the construction of membrane-bound cytokine fusion constructs in which our cytokine of interest (mouse GM-CSF, mouse IL-2, mouse IL-4) was fused to the membrane anchoring regions of viral Hemagglutinin (HA). Progeny virions, produced from influenza infected MDCK cells expressing membrane-bound cytokines, readily incorporated membrane-bound cytokines during budding and these cytokines on the virus particles retained bioactivity following viral inactivation. In vivo vaccination studies in mice showed enhanced antibody titers and improved protection following lethal challenge in those mice vaccinated with IL-2 and IL-4-bearing CYT-IVAC's compared to the conventional wild-type vaccine without membrane-bound cytokines. In addition, the immune response induced by IL-2 and IL-4-bearing CYT-IVACs was skewed toward Th1 (cellular) mediated immunity compared to the Th2 (humoral) dominated response induced with wild-type vaccination. Cellular mediated immunity afforded by IL-2 and IL-4 CYT-IVACs was manifested as enhanced influenza specific T cell proliferation and activation. In conclusion, we have developed a novel methodology to introduce bioactive membrane-bound cytokines directly into virus particles in order to augment the immunogenicity of inactivated, whole virus influenza vaccines.

Starch microparticles have been shown to be effective as a particulate adjuvant carrier in oral vaccination. In mice, formulations with bacterial antigens have elicited both systemic and mucosal immune responses providing protection upon challenge with live bacteria. A vaccine formulation with formaldehyde-treated diphtheria toxin cross-reacting material, CRM197, optimised in mice, was tested in healthy volunteers in a booster design. Specific antibodies as well as toxin-neutralising antibodies in a Vero cell analysis indicated that the vaccine was not effective in man. It is obvious that the longer transit time in the human GI tract and possible unfavourable distribution of Peyer's patches and M-cells necessary for the uptake of the starch particles require a more stable formulation. It is proposed that enteric coating of the particles or particles in a gastro-resistant capsule could be a more efficacious vaccine formulation.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.