Molecular adjuvants for mucosal immunity

Summary: Mucosal surfaces represent the entry route of a multitude of viral pathogens. For many of these viruses, such as the herpes simplex viruses and human immunodeficiency virus, no effective vaccine exists. Hence, it is important that prospective vaccines engender maximal immunity at these susceptible sites. Genetic vaccines encoding adjuvant molecules represent one approach to optimize mucosal as well as systemic immunity. Promising candidates include various inflammatory cytokines and chemokines that might be used to enhance the primary response to a level sufficient for protection. Encouraging studies involving cytokines such as granulocyte/macrophage colony‐stimulating factor, interleukin‐2 (IL‐2), IL‐12, IL‐18, and many others are examined. Notable chemokines that may offer hope in such efforts include IL‐8, RANTES, CCL19, CCL21, and a few others. Combinatorial approaches utilizing several cytokines and chemokines will most likely yield the greatest success. In addition, as more is discovered regarding the requirements for memory development of T cells, boosters involving key cytokines such as IL‐15 and IL‐23 may prove beneficial to long‐term maintenance of the memory pool. This review summarizes the progress in the use of genetic vaccines to achieve mucosal immunity and discusses the needed strategies to maximize long‐term prospective immunity at this vulnerable entry site.     

Recent advances in mucosal vaccines and adjuvants

Mucosal vaccines may be used both to prevent mucosal infections through the activation of antimicrobial immunity and to treat systemic inflammatory diseases through the induction of antigen-specific mucosal tolerance. New, efficient mucosal adjuvants for human use have been designed based on, amongst others, bacterial toxins and their derivatives, CpG-containing DNA, and different cytokines and chemokines, with the aim of improving the induction of mucosal Th1 and Th2 responses. Mucosal delivery systems, in particular virus-like particles, have been shown to enhance the binding, uptake and half-life of the antigens, as well as target the vaccine to mucosal surfaces. DNA vaccines are currently being developed for administration at mucosal surfaces. However, there have also been failures, such as the withdrawal of an oral vaccine against rotavirus diarrhea and a nasal vaccine against influenza, because of their potential side effects.     

The potential of CpG oligodeoxynucleotides as mucosal adjuvants

The development of mucosal vaccines for humans has been hindered by the lack of safe yet effective mucosal adjuvants. Bacterial toxins are commonly used as adjuvants in animal models, but they are too toxic for use in humans. A novel class of adjuvant is CpG DNA, which contains unmethylated CpG dinucleotides in particular base contexts (CpG motifs). CpG DNA is most often coadministered with antigen in the form of synthetic oligodeoxynucleotides (CpG ODN), which are made with a nuclease-resistant phosphorothioate backbone. The vast majority of studies using CpG DNA as adjuvant have been with parenteral delivery; recently, however, mucosal immunization with CpG DNA as adjuvant has also been shown to induce both systemic (humoral and cellular) and mucosal antigen-specific immune responses. This review will highlight the recent uses of CpG DNA as an adjuvant at mucosal surfaces.     

Phosphatidylinositol Mannosides are Efficient Mucosal Adjuvants

The development of defined sub-unit vaccines requires the inclusion in the vaccine of an immunological adjuvant. The most important property of adjuvants for vaccines aimed at inducing optimal protection against intracellular bacteria such as Mycobacterium tuberculosis or M. bovis is the ability to enhance cell-mediated immunity, specifically Th1 responses. In this paper, we describe a system where transgenic mice expressing a high proportion of T cells specific for an ovalbumin (OVA) peptide are used to assess the ability of a novel class of adjuvants to positively modulate cell-mediated immune responses. Defined fractions containing purified native or synthetic phosphatidylinositol mannosides (PIMs) from mycobacteria were assessed for their adjuvant activities in response to the model antigen (OVA). Purified PIM preparations given to mice with OVA by the subcutaneous route were shown to elicit an enhanced release of interferon-gamma (IFN-γ) in cellular responses to OVA peptide in vitro. Very little interleukin-4 (IL-4) was released by cells from mice immunized with PIMs and OVA, whereas cells from animals immunized with complete Freund's adjuvant (CFA) and OVA released IL-4 as well as IFN-γ. Synthetic preparations of PIM2 and PIM4 also acted as adjuvants in the mouse model studied. In addition, PIM preparations were shown to generate an efficient cell-mediated immune response to OVA, when the antigen/adjuvant preparations were administered via the oral route or intranasal route. PIM preparations elicited substantial release of interleukin-12 (IL-12) from dendritic cells (DCs). These data suggest that purified or synthetic PIMs act as adjuvants when administered at mucosal surfaces and represent a new class of adjuvants for mucosal immunization against intracellular pathogens.     

Gamma interferon and monophosphoryl lipid A-trehalose dicorynomycolate are efficient adjuvants for Mycobacterium tuberculosis multivalent acellular vaccine

In this study, we examined the immunogenicity and protective efficacy of six immunodominant Mycobacterium tuberculosis recombinant antigens (85B, 38kDa, ESAT-6, CFP21, Mtb8.4, and 16kDa) in a multivalent vaccine preparation (6Ag). Gamma interferon (IFN-gamma) and monophosphoryl lipid A-trehalose dicorynomycolate (Ribi) adjuvant systems were used separately or in combination for immunization with the recombinant antigens. Our results demonstrate that immunization of mice with Ribi emulsified antigens in the presence of IFN-gamma (Ribi+6Ag+IFN-gamma) resulted after challenge with a virulent M. tuberculosis strain in a significant reduction in the CFU counts that was comparable to that achieved with the BCG vaccine ( approximately 0.9-log protection). Antigen-specific immunoglobulin G (IgG) titers in the Ribi+6Ag+IFN-gamma-immunized mice were lower than in mice immunized with Ribi+6Ag and were oriented toward a Th1-type response, as confirmed by elevated IgG2a levels. In addition, splenocyte proliferation, IFN-gamma secretion, and NO production were significantly higher in splenocytes derived from Ribi+6Ag+IFN-gamma-immunized mice, whereas IL-10 secretion was decreased. These findings confirm the induction of a strong cellular immunity in the vaccinated mice that correlates well with their enhanced resistance to M. tuberculosis. The adjuvant effect of IFN-gamma was dose dependent. A dose of 5 mug of IFN-gamma per mouse per immunization gave optimal protection, whereas lower or higher amounts (0.5 or 50 mug/ mouse) of IFN-gamma failed to enhance protection.     

Mutants of type II heat-labile enterotoxin LT-IIa with altered ganglioside-binding activities and diminished toxicity are potent mucosal adjuvants

The structure and function LT-IIa, a type II heat-labile enterotoxin of Escherichia coli, are closely related to the structures and functions of cholera toxin and LT-I, the type I heat-labile enterotoxins of Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. While LT-IIa is a potent systemic and mucosal adjuvant, recent studies demonstrated that mutant LT-IIa(T34I), which exhibits no detectable binding activity as determined by an enzyme-linked immunosorbent assay, with gangliosides GD1b, GD1a, and GM1 is a very poor adjuvant. To evaluate whether other mutant LT-IIa enterotoxins that also exhibit diminished ganglioside-binding activities have greater adjuvant activities, BALB/c mice were immunized by the intranasal route with the surface adhesin protein AgI/II of Streptococcus mutans alone or in combination with LT-IIa, LT-IIa(T14S), LT-IIa(T14I), or LT-IIa(T14D). All three mutant enterotoxins potentiated strong mucosal immune responses that were equivalent to the response promulgated by wt LT-IIa. All three mutant enterotoxins augmented the systemic immune responses that correlated with their ganglioside-binding activities. Only LT-IIa and LT-IIa(T14S), however, enhanced expression of major histocompatibility complex class II and the costimulatory molecules CD40, CD80, and CD86 on splenic dendritic cells. LT-IIa(T14I) and LT-IIa(T14D) had extremely diminished toxicities in a mouse Y1 adrenal cell bioassay and reduced abilities to induce the accumulation of intracellular cyclic AMP in a macrophage cell line.     

汉坦病毒经不同途径黏膜免疫效果研究

目的 以大肠埃希菌不耐热肠毒素8亚单位为佐剂研究汉坦病毒经不同黏膜途径免疫的效果.方法 以乳糖为诱导剂,在大肠埃希菌表达不耐热肠毒素B亚单位(LTB),Ni2+亲和层析进行纯化.以灭活汉坦病毒84Fli株为疫苗,LTB为佐剂,分别采用滴鼻、口服、经阴道3种黏膜途径接种C57BL/6小鼠.ELISA检测血清中特异IgG和阴道冲洗液中特异IgA.结果 免疫印迹和神经节苷脂结合活性研究,证实了LTB的表达.经滴鼻、口服、阴道免疫,均可诱导分泌型IgA抗体和血清IgG抗体反应,不同途径接种组间差异无统计学意义.结论 以LTB为佐剂的灭活汉坦病毒通过3种黏膜途径均能产生抗汉坦病毒黏膜免疫和系统免疫应答.     

Anti-complementary amines are immunological adjuvants in mice

Complement (C) inactivation by ammonia, ethylenediamine and methylamine in mouse serum was studied in relation to a possible adjuvant effect of the substances in a cell-mediated immune response. The amines caused a dose-dependent depletion of both alternative pathway (AP) and overall C activity in vitro and showed also pronounced adjuvant effects in the delayed type hypersensitivity response of mice to SRBC. A significant correlation between momentary inhibition of AP activity and adjuvanticity was observed (r = 0.9995; P approximately 0.02), suggesting a causative relationship between these two phenomena. Both effects seem to be a direct function of the number of amino-groups per molecule. Since, on the other hand, lysosomotropic activity of amines is known to decrease with the number of amino-residues, our findings exclude an important role of direct phagocyte inhibition in the immuno-adjuvanticity of these compounds. A longer persistence and improved presentation of antigen as indirect result of local C-depletion could account for the immunological adjuvant effects of amines.     

New generation of mucosal adjuvants for the induction of protective immunity

Invasion of infectious agents through mucosal surfaces can be prevented by use of the common mucosal immune system (CMIS), which interconnects inductive tissues, including Peyer's patches (PPs) and nasopharyngeal-associated lymphoreticular tissue (NALT), and effector tissues of the intestinal and respiratory tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant has been shown to be essential. When vaccine antigen is administered together with mucosal adjuvant, antigen-specific T-helper (Th) 1 and Th2 cells, cytotoxic T lymphocytes (CTLs) and IgA B cell responses are effectively induced by oral or nasal routes via the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium (FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying dendritic cells (DCs). Elucidation of the molecular/cellular characteristics of M cells and mucosal DCs will greatly facilitate the design of a new generation of effective mucosal adjuvants and of a vaccine delivery vehicle that maximises the use of the CMIS. Our recent efforts at mucosal vaccine development have focused on nasal administration of vaccine antigen together with nontoxic mutant-based or cytokine-/chemokine-based adjuvant for the induction of the protective immunity. To this end, a chimeric form of a nontoxic adjuvant combining the merits of mutant cholera toxin A subunit (mCT-A) and heat labile toxin B subunit (LT-B) was created as the second generation of detoxified toxin-based mucosal adjuvant. When a vaccine antigen was coexpressed together with an immune stimulatory/delivery molecule in crop seed, this edible vaccine is not only effective but also extremely practical in that it can be produced in huge quantities and preserved and shipped over long distances at room temperature without altering the quality of the vaccine. Because such qualities would greatly facilitate global vaccination, this new generation edible vaccines with a built-in adjuvant and/or M cell-targeted edible vaccine promises to be a powerful weapon for combating infectious diseases and bioterrorism.     

Antipeptide antibody responses following intranasal immunization: effectiveness of mucosal adjuvants

Toxicity is a major factor limiting the development and use of potent adjuvants for human mucosally delivered vaccines. Novel adjuvant formulations have recently become available, and in the present study two have been used for intranasal immunization with a synthetic peptide immunogen (MAP-M2). This peptide represents a multiple antigenic peptide containing multiple copies of a mimotope M2, a peptide mimic of a conformational epitope of the fusion protein of measles virus. MAP-M2 was administered intranasally to experimental animals together with synthetic oligodeoxynucleotides containing unmethylated CpG motifs with or without a mutant of wild-type enterotoxin of Escherichia coli (LTR72). The combination of the mutant toxin LTR72 and the CpG repeats, codelivered with a peptide immunogen, induced both local and systemic peptide- and pathogen-specific humoral and cellular immune responses comparable to those obtained after intranasal immunization with the wild-type toxin LT. In addition, this combination of adjuvants induced a predominantly immunoglobulin G2a antibody response. If both the LTR72 and CpG adjuvants are shown to be safe for use in humans, this particular combination would appear to have potential as an adjuvant for mucosally delivered vaccines in humans.     

Basidiolipids from Agaricus are novel immune adjuvants.

The primary aim of the present study was to compare the immune adjuvanticity of two different groups of glycolipids, i.e., the newly discovered basidiolipids from Basidiomycete mushrooms (Bl-1, Bl-2, Bl-3, and Bl-4), and saponin fractions from Quillaja saponaria. The basidiolipids, though with differential effectiveness of the Bl-components, stimulated the expression of serum immune globulins in mice that recognized co-injected antigens, bovine serum albumin (BSA) or a keyhole-limpet hemocyanin-ganglioside Gfpt1 conjugate (KLH-Gfpt1), respectively. The immune adjuvanticity of the basidiolipids was comparable to that of acidic (QAS2, QAS5, QAS10), and novel neutral (QNS1, QNS2, QNS3) saponin compounds isolated and purified from Quillaja saponaria bark bulk material. Basidiolipids, as well as, the Q. saponin fractions were only marginally antigenic. MPL-A, by contrast, a comparable immune adjuvant, stimulated the expression of specific antibodies that recognized this glycophospholipid. Different from the Q. saponins with restricted toxicity, the basidiolipids displayed no toxic or hemolytic properties.     

M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery

In the mucosal immune system, M cells are known as specialized epithelial cells that take up luminal antigens, although the receptors on M cells and the mechanism of antigen uptake into M cells are not well-understood. Here, we report the expression of the complement C5a receptor (C5aR) on the apical surface of M cells. C5ar mRNA expression in co-cultured Caco-2 human M-like cells was six-fold higher than in mono-cultured cells. C5aR expression was detected together with glycoprotein 2, an M-cell-specific protein, on the apical surface of M-like cells and mouse Peyer's patch M cells. Interestingly, after oral administration of Yersinia enterocolitica which expresses outer membrane protein H (OmpH) that is homologous to the Skp α1 domain of Escherichia coli, a ligand of C5aR, dense clustering and phosphorylation of C5aR were detected in M cells. Finally, targeted antigen delivery to M cells using C5aR as a receptor was achieved using the OmpH α1 of Y. enterocolitica such that the induction of ligand-conjugated antigen-specific immune responses was confirmed in mice after oral immunization of the OmpH β1α1-conjugated antigen. Collectively, we identified C5aR expression on M cells and suggest that C5aR could be used as a target receptor for mucosal antigen delivery.     

The low-toxicity versions of LPS, MPL adjuvant and RC529, are efficient adjuvants for CD4+ T cells

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less-toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long-term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon-gamma following a 21-day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long-term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low-inflammation adjuvants, such as MPL and RC529, are capable of enhancing short-term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long-term retention of antigen-specific T cells.     

Neutrophils are gatekeepers of mucosal immunity

Mucosal tissues are constantly exposed to the outside environment. They receive signals from the commensal microbiome and tissue-specific triggers including alimentary and airborne elements and are tasked to maintain balance in the absence of inflammation and infection. Here, we present neutrophils as sentinel cells in mucosal immunity. We discuss the roles of neutrophils in mucosal homeostasis and overview clinical susceptibilities in patients with neutrophil defects. Finally, we present concepts related to specification of neutrophil responses within specific mucosal tissue microenvironments.     

Enterotoxin-based mucosal adjuvants alter antigen trafficking and induce inflammatory responses in the nasal tract

The safety of nasal vaccines containing enterotoxin-based mucosal adjuvants has not been studied in detail. Previous studies have indicated that native cholera toxin (nCT) can alter antigen trafficking when applied nasally. In this study, we determined the enterotoxin-based variables that alter antigen trafficking. To measure the influence of enterotoxin-based mucosal adjuvants on antigen trafficking in the nasal tract, native and mutant enterotoxins were coadministered with radiolabeled tetanus toxoid (TT). The nCT and heat-labile enterotoxin type 1 (LTh-1) redirected TT into the olfactory neuroepithelium (ON/E). Antigen redirection occurred mainly across the nasal epithelium without subsequent transport along olfactory neurons into the olfactory bulbs (OB). Thus, no significant accumulation of the vaccine antigen TT was observed in the OB when coadministered with nCT. In contrast, neither mutant CT nor mutant LTh-1, which lack ADP-ribosyltransferase activity, redirected TT antigen into the ON/E. Thus, ADP-ribosyltransferase activity was essential for antigen trafficking across the olfactory epithelium. Accumulation of TT in the ON/E was also due to B-subunit binding to GM1 gangliosides, as was demonstrated (i) by redirection of TT by LTh-1 in a dose-dependent manner, (ii) by ganglioside inhibition of the antigen redirection by LTh-1 and nCT, and (iii) by the use of LT-IIb, a toxin that binds to gangliosides other than GM1. Redirection of TT into the ON/E coincided with elevated production of interleukin 6 (IL-6) but not IL-1beta or tumor necrosis factor alpha in the nasal mucosa. Thus, redirection of TT is dependent on ADP-ribosyltransferase activity and GM1 binding and is associated with production of the inflammatory cytokine IL-6.     

Impairment by mucosal adjuvants and cross-reactivity with variant peptides of the mucosal immunity induced by injection of the fusion peptide PADRE-ELDKWA

Secretory immunity protects against mucosal transmission of viruses, as demonstrated with the oral poliovirus vaccine. In a previous study we showed that this immunity could be induced in mice by injection of a fusion peptide consisting of an unnatural peptide-like sequence (PADRE) and a viral epitope (ELDKWASLW). PADRE is a T-helper-cell epitope able to bind most major histocompatibility complex class II molecules of different haplotypes in mice and humans and to increase antibody responses. ELDKWA is a well-known consensual sequence of gp41 involved in a key structure of human immunodeficiency virus (HIV) type 1. Here, the antibody response to the native form of ELDKWA was mainly of the immunoglobulin A isotype and selectively occurred in mucosa. Adjuvants, such as cholera toxin and cytosine polyguanine, were useless and even competed with PADRE for the response. Interestingly, these antibodies were cross-reactive with the three major variants of the epitope, as shown both by direct enzyme-linked immunosorbent assay and by inhibition. This unconventional route of mucosal immunization allows control of the administered dose. The lack of adjuvant and the cross-reactivity of the antibodies increase the safety and the spectrum of the candidate vaccine, respectively. The drug-like nature of the construct suggests further improvements by synthesis of more antigenic sequences. The reasonable cost of short peptides at the industrial level and their purity make this approach of interest for future vaccines against mucosal transmission of HIV or other pathogens.     

Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles

Mucosal immune responses are the first‐line defensive mechanisms against a variety of infections. Therefore, immunizations of mucosal surfaces from which majority of infectious agents make their entry, helps to protect the body against infections. Hence, vaccinization of mucosal surfaces by using mucosal vaccines provides the basis for generating protective immunity both in the mucosal and systemic immune compartments. Mucosal vaccines offer several advantages over parenteral immunization. For example, (i) ease of administration; (ii) non‐invasiveness; (iii) high‐patient compliance; and (iv) suitability for mass vaccination. Despite these benefits, to date, only very few mucosal vaccines have been developed using whole microorganisms and approved for use in humans. This is due to various challenges associated with the development of an effective mucosal vaccine that can work against a variety of infections, and various problems concerned with the safe delivery of developed vaccine. For instance, protein antigen alone is not just sufficient enough for the optimal delivery of antigen(s) mucosally. Hence, efforts have been made to develop better prophylactic and therapeutic vaccines for improved mucosal Th1 and Th2 immune responses using an efficient and safe immunostimulatory molecule and novel delivery carriers. Therefore, in this review, we have made an attempt to cover the recent advancements in the development of adjuvants and delivery carriers for safe and effective mucosal vaccine production.     

Neutrophils in mucosal secretion are functionally active

The leucocytes in the secretion on the tonsillar surface have been regarded as inactivated and dying cells with no essential function in the defence of the underlying mucosa. In recent studies, we showed that in acute tonsillitis there are very few, if any, bacteria in the parenchyma, whereas in the secretion on the tonsillar surface there are huge numbers of bacteria and neutrophils, and furthermore, extensive phagocytosis. The present study was performed to elucidate the functional capacity of the neutrophils in the secretion on the tonsillar surface. Secretion samples were taken from the tonsillar surface of healthy volunteers with an imprint technique which allows transfer of secretion from the mucosa to a glass slide with maintained topographic position of the cells. The capacity of the neutrophils to respond to chemotactic stimuli and to phagocytize and further process labelled yeast particles was studied in this in vitro system. The results show that the neutrophils in the secretion have the same properties as in tissue, and also when having arrived in the secretion they can identify and attack new prey.     

Maternal allergen immunisation to prevent sensitisation in offspring: Th2-polarising adjuvants are more efficient than a Th1-polarising adjuvant in mice

Background

Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period.

Results

Maternal immunization with OVA showed increased levels of FcγRIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-γ-secreting T cells and IL-4 and IL-12- secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced FcγRIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers.

Conclusion

Maternal immunization upregulates the inhibitory FcγRIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.