IL-1 is an effective adjuvant for mucosal and systemic immune responses when coadministered with protein immunogens

Mucosal immunization with soluble protein Ag alone may induce Ag-specific tolerance, whereas mucosal immunization with Ag in the presence of a mucosal adjuvant may induce Ag-specific systemic and mucosal humoral and cell-mediated immune responses. The most widely used and studied mucosal adjuvant is cholera toxin (CT). Although the mechanism of adjuvanticity of CT is not completely understood, it is known that CT induces mucosal epithelial cells to produce the proinflammatory cytokines IL-1, IL-6, and IL-8 and up-regulates macrophage production of IL-1 and the costimulatory molecule B7.2. Because IL-1 may duplicate many of the activities of CT, we evaluated IL-1alpha and IL-1beta for their ability to serve as mucosal adjuvants when intranasally administered with soluble protein Ags. IL-1alpha and IL-1beta were as effective as CT for the induction of Ag-specific serum IgG, vaginal IgG and IgA, systemic delayed-type hypersensitivity, and lymphocyte proliferative responses when intranasally administered with soluble protein Ag. Our results indicate that IL-1alpha and IL-1beta may be useful as mucosal vaccine adjuvants. Such an adjuvant may be useful, and possibly required, for vaccine-mediated protection against pathogens that infect via the mucosal surfaces of the host such as HIV.     

Effective mucosal immunity to anthrax: neutralizing antibodies and Th cell responses following nasal immunization with protective antigen

Mucosal, but not parenteral, immunization induces immune responses in both systemic and secretory immune compartments. Thus, despite the reports that Abs to the protective Ag of anthrax (PA) have both anti-toxin and anti-spore activities, a vaccine administered parenterally, such as the aluminum-adsorbed anthrax vaccine, will most likely not induce the needed mucosal immunity to efficiently protect the initial site of infection with inhaled anthrax spores. We therefore took a nasal anthrax vaccine approach to attempt to induce protective immunity both at mucosal surfaces and in the peripheral immune compartment. Mice nasally immunized with recombinant PA (rPA) and cholera toxin (CT) as mucosal adjuvant developed high plasma PA-specific IgG Ab responses. Plasma IgA Abs as well as secretory IgA anti-PA Abs in saliva, nasal washes, and fecal extracts were also induced when a higher dose of rPA was used. The anti-PA IgG subclass responses to nasal rPA plus CT consisted of IgG1 and IgG2b Abs. A more balanced profile of IgG subclasses with IgG1, IgG2a, and IgG2b Abs was seen when rPA was given with a CpG oligodeoxynucleotide as adjuvant, suggesting a role for the adjuvants in the nasal rPA-induced immunity. The PA-specific CD4(+) T cells from mice nasally immunized with rPA and CT as adjuvant secreted low levels of CD4(+) Th1-type cytokines in vitro, but exhibited elevated IL-4, IL-5, IL-6, and IL-10 responses. The functional significance of the anti-PA Ab responses was established in an in vitro macrophage toxicity assay in which both plasma and mucosal secretions neutralized the lethal effects of Bacillus anthracis toxin.     

Interleukin-1 family cytokines as mucosal vaccine adjuvants for induction of protective immunity against influenza virus

A safe and potent adjuvant is needed for development of mucosal vaccines against etiological agents, such as influenza virus, that enter the host at mucosal surfaces. Cytokines are potential adjuvants for mucosal vaccines because they can enhance primary and memory immune responses enough to protect against some infectious agents. For this study, we tested 26 interleukin (IL) cytokines as mucosal vaccine adjuvants and compared their abilities to induce antigen (Ag)-specific immune responses against influenza virus. In mice intranasally immunized with recombinant influenza virus hemagglutinin (rHA) plus one of the IL cytokines, IL-1 family cytokines (i.e., IL-1α, IL-1β, IL-18, and IL-33) were found to increase Ag-specific immunoglobulin G (IgG) in plasma and IgA in mucosal secretions compared to those after immunization with rHA alone. In addition, high levels of both Th1- and Th2-type cytokines were observed in mice immunized with rHA plus an IL-1 family cytokine. Furthermore, mice intranasally immunized with rHA plus an IL-1 family cytokine had significant protection against a lethal influenza virus infection. Interestingly, the adjuvant effects of IL-18 and IL-33 were significantly decreased in mast cell-deficient W/W(v) mice, indicating that mast cells have an important role in induction of Ag-specific mucosal immune responses induced by IL-1 family cytokines. In summary, our results demonstrate that IL-1 family cytokines are potential mucosal vaccine adjuvants and can induce Ag-specific immune responses for protection against pathogens like influenza virus.     

A single intradermal administration of soluble leishmanial antigen and plasmid expressing interleukin-12 protects BALB/c mice from Leishmania major infection.

In murine leishmaniasis, the induction of the T-helper type 1 (Th1) response contributes to infection resistance, whereas the establishment of the Th2 response makes the mice susceptible to infection. Interleukin-12 (IL-12) plays a pivotal role in the diversification of immune responses to the Th1 type. In this study, we tested whether the co-administration of IL-12 expression plasmid which compose p35 and p40 subunits and soluble leishmanial antigen (SLA) will skew the susceptible BALB/c mice to Th1 response and protect from leishmaniasis. When the mice were intradermally injected with the combination of IL-12 plasmid and SLA 7 days prior to the challenge with 1x10(6) promastigotes of Leishmania major, the local lesions completely healed and the parasite burden in the local lymph nodes significantly decreased. The cured mice attained long-term immunity, and were resistant to any subsequent rechallenge of the lethal dose of the parasite. The protective effect was associated with the development of a Th1 response, as demonstrated by the enhanced level of antigen-specific interferon-gamma (IFN-gamma) and dominant production of IgG2a in the serum. In contrast, the administration of empty plasmid plus SLA or IL-12 plasmid alone failed to protect the disease and shape the Th1 response. Furthermore, the protective efficiency induced by the vaccination was clearly prevented by the injection of either neutralizing anti-IL-12 mAb or anti-IFN-gamma mAb. The IL-12 expression plasmid is thus an effective adjuvant for the elicitation of a protective Th1 response against leishmaniasis and is therefore, considered to be appropriate for vaccinations that require the induction of Th1 type immunity.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

Alternative diphtheria, tetanus and whooping cough immunization schedule to evoke a Th2 tetanus and a Th1 pertussis immune response

In a previous study, using BALB/c mice, we found that while diphtheria (D), tetanus (T) and whooping cough (Pw, whole-cell Bordetella pertussis) immunization induces a Th1/Th2 tetanus response and memory T cells able to proliferate in response to in vitro stimulation with B. pertussis, DTPa immunization induces a Th2 tetanus immune response and no memory T cells that recognize B. pertussis as stimulus. Considering that a pro-inflammatory cytokine production is not necessary for protection against tetanus and therefore should be avoided, an alternative DTP immunization schedule with minimal Pw exposure was assessed in order to obtain a Th2 tetanus response and a Th1 pertussis response. BALB/c mice were primed with DT vaccine at day 0, with Pw vaccine at day 14 and boosted with DTPa vaccine at days 21 and 28. A control group was inoculated with saline. Antibodies against B. pertussis surface antigens, tetanus and diphtheria toxoids were produced by mice. Spleen cells stimulated in vitro with B. pertussis produced IL-6 and IFNgamma. Only IL-5 was produced by cells in response to tetanus toxoid stimulation. These results are in line with the low IgG1/IgG2a ratio for pertussis antibodies compared with those corresponding to tetanus and diphtheria. The immunization protocol presented herein succeeded in producing tetanus and pertussis immune responses of Th2 and Th1 type, respectively. In contrast to previous results obtained with DTPw immunization, no IL-12 production was observed. Our findings provide direct evidence that an immunization protocol with an interval of 14 days between DT and Pw primings, followed by DTPa boosters, can induce appropriate immune responses against DTP vaccine antigens.     

Interleukin-10 enhances immune responses to pneumococcal polysaccharides and sheep erythrocytes in young and aged mice.

Antibody responses to pneumococcal polysaccharides are decreased in aged mice. Using a system to measure murine antibody responses to the Pnu-Imune vaccine, here we demonstrate that interleukin-10 (IL-10) has an adjuvant effect in enhancing the vaccine response in the aged. IL-10 increased the vaccine responses of B cells from aged mice in vitro only if either T cells or macrophages were also present. The need for T cells or macrophages could be substituted by cytokines such as IL-1 or IL-5, which are normally made by these accessory cells. Thus, IL-10 appeared to act on B cells directly but it worked in conjunction with other cytokines to induce an antigen specific response. In vivo studies showed that IL-10 administration enhanced antibody responses not only to thymic independent antigens but also to thymic-dependent antigens such as sheep erythrocytes. These data suggest that IL-10 may be useful in enhancing vaccine-specific responses in situations in which the host is immunocompromised.     

Relative Contribution of Th1 and Th17 Cells in Adaptive Immunity to Bordetella pertussis: Towards the Rational Design of an Improved Acellular Pertussis Vaccine

Whooping cough caused by Bordetella pertussis is a re-emerging infectious disease despite the introduction of safer acellular pertussis vaccines (Pa). One explanation for this is that Pa are less protective than the more reactogenic whole cell pertussis vaccines (Pw) that they replaced. Although Pa induce potent antibody responses, and protection has been found to be associated with high concentrations of circulating IgG against vaccine antigens, it has not been firmly established that host protection induced with this vaccine is mediated solely by humoral immunity. The aim of this study was to examine the relative contribution of Th1 and Th17 cells in host immunity to infection with B. pertussis and in immunity induced by immunization with Pw and Pa and to use this information to help rationally design a more effective Pa. Our findings demonstrate that Th1 and Th17 both function in protective immunity induced by infection with B. pertussis or immunization with Pw. In contrast, a current licensed Pa, administered with alum as the adjuvant, induced Th2 and Th17 cells, but weak Th1 responses. We found that IL-1 signalling played a central role in protective immunity induced with alum-adsorbed Pa and this was associated with the induction of Th17 cells. Pa generated strong antibody and Th2 responses, but was fully protective in IL-4-defective mice, suggesting that Th2 cells were dispensable. In contrast, Pa failed to confer protective immunity in IL-17A-defective mice. Bacterial clearance mediated by Pa-induced Th17 cells was associated with cell recruitment to the lungs after challenge. Finally, protective immunity induced by an experimental Pa could be enhanced by substituting alum with a TLR agonist that induces Th1 cells. Our findings demonstrate that alum promotes protective immunity through IL-1β-induced IL-17A production, but also reveal that optimum protection against B. pertussis requires induction of Th1, but not Th2 cells.     

Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4- or IL-13-mediated signaling

Previous studies demonstrate that aluminium hydroxide adjuvant (alum) produces increased Th1 responses in IL-4-deficient mice compared with wild-type animals, although the continued production of IL-5 by spleen cells from these mice also indicates that Th2 responses are induced. In the present study, we demonstrate that alum can induce Th2-associated IL-4 and IL-5 production in the absence of IL-4 signaling in mice deficient in either IL-4Ralpha or Stat6. The Th2 responses observed could not be due to IL-13 as IL-13 responses are also impaired in IL-4Ralpha- and Stat6-deficient mice. We also detected higher levels of IL-4 in IL-4Ralpha gene-deficient, though not Stat6-deficient, mice compared with their wild-type counterparts. The increased levels of IL-4 could be explained by the IL-4R being unavailable to neutralize this cytokine in IL-4Ralpha-deficient mice. While levels of IL-5 production in IL-4Ralpha- or Stat6-deficient mice were similar to IL-4-deficient and wild-type mice, other type 2-associated responses, which are largely or wholly IL-4 dependent, such as the production of IgG1 or IgE Abs, were either reduced or absent. We conclude that alum adjuvants can induce IL-4 production and Th2 responses independently of IL-4 or IL-13, negating the requirement for an early source of IL-4 in the Th2 response induced by this adjuvant.     

TNF superfamily member, TL1A, is a potential mucosal vaccine adjuvant

The identification of cytokine adjuvants capable of inducing an efficient mucosal immune response against viral pathogens has been long anticipated. Here, we attempted to identify the potential of tumor necrosis factor superfamily (TNFS) cytokines to function as mucosal vaccine adjuvants. Sixteen different TNFS cytokines were used to screen mucosal vaccine adjuvants, after which their immune responses were compared. Among the TNFS cytokines, intranasal immunization with OVA plus APRIL, TL1A, and TNF-α exhibited stronger immune response than those immunized with OVA alone. TL1A induced the strongest immune response and augmented OVA-specific IgG and IgA responses in serum and mucosal compartments, respectively. The OVA-specific immune response of TL1A was characterized by high levels of serum IgG1 and increased production of IL-4 and IL-5 from splenocytes of immunized mice, suggesting that TL1A might induce Th2-type responses. These findings indicate that TL1A has the most potential as a mucosal adjuvant among the TNFS cytokines.     

The Adjuvant Double Mutant Escherichia coli Heat Labile Toxin Enhances IL-17A Production in Human T Cells Specific for Bacterial Vaccine Antigens

The strong adjuvant activity and low enterotoxicity of the novel mucosal adjuvant double mutant Escherichia coli heat labile toxin, LT(R192G/L211A) or dmLT, demonstrated in mice, makes this molecule a promising adjuvant candidate. However, little is known about the mechanisms responsible for the adjuvant effect of dmLT or whether dmLT also has an adjuvant function in humans.We investigated the effect of dmLT on human T cell responses to different bacterial vaccine antigens: the mycobacterial purified protein derivative (PPD) antigen, tested in individuals previously vaccinated with Bacillus Calmette-Guérin, the LT binding subunit (LTB), evaluated in subjects immunised with oral inactivated whole cell vaccines against enterotoxigenic Escherichia coli, and Streptococcus pneumoniae whole cell vaccine antigens, tested in subjects naturally exposed to pneumococci. We found that dmLT enhanced the production of IL-17A by peripheral blood mononuclear cells in response to all antigens tested. dmLT had comparable effects on IL-17A responses to PPD as the single mutant LT(R192G) adjuvant, which has demonstrated clinical adjuvant activity in humans. Neutralisation of IL-1β and IL-23, but not IL-6, suppressed the IL-17A-enhancing effect of dmLT. Furthermore, CD4+ T cells produced higher levels of IL-17A when stimulated with monocytes pulsed with PPD and dmLT compared to PPD alone, supporting an important role of antigen presenting cells in enhancing IL-17A responses. dmLT also potentiated mitogen-induced IL-17A and IL-13 production. However, dmLT had variable influences on IFN-γ responses to the different stimuli tested.Our demonstration of a potent ability of dmLT to enhance human Th17 type T cell responses to bacterial vaccine antigens encourages further evaluation of the adjuvant function of dmLT in humans.     

CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice

A murine monoclonal anti-idiotype (Id) antibody, 3H1 has been developed and characterized previously. Anti-Id 3H1 mimics a specific epitope of carcinoembryonic antigen (CEA) and can be used as a surrogate antigen for CEA. 3H1 induced anti-CEA immunity in different species of animals as well as humans and showed promise as a potential vaccine candidate in phase I/II clinical trials for colon cancer patients. One area of interest to us has been the development of new immune adjuvants that may augment the potency of 3H1 as a tumor vaccine. Oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. In this study, we have evaluated the efficacy of 3H1 as a tumor vaccine when admixed with a select CpG ODN 1826 in transgenic mice that express human CEA. The vaccine potential of 3H1 was also assessed in the presence of another widely used adjuvant, QS-21. 3H1 coupled to keyhole limpet hemocyanin (KLH) and mixed with Freund’s adjuvant (FA) was used as a gold standard in this system. 3H1 vaccination with different adjuvants induced both humoral and cellular anti-3H1, as well as anti-CEA immunity in CEA transgenic mice. The immune sera could lyse CEA-transfected murine colon carcinoma cells, C15 effectively in an antibody-dependent cellular cytotoxicity assay. The anti-CEA antibody responses were somewhat comparable in each adjuvant-treated group of mice, whereas cellular immune responses were significantly greater when CpG was used as an adjuvant. Splenocytes obtained from 3H1–CpG-immunized mice showed an increased proliferative CD4⁺ Th1-type T-cell response when stimulated in vitro with 3H1 or CEA and secreted elevated levels of Th1 cytokines (IL-2, IFN-γ). This vaccine also induced MHC class I antigen-restricted CD8⁺ T-cell responses. In a solid tumor model, C15 tumor growth was significantly inhibited by 3H1 vaccinations. In 3H1–CpG-vaccinated mice, the duration of survival was, however, longer compared to the 3H1–QS21-vaccinated mice. These findings suggest that 3H1-CpG vaccinations can break peripheral tolerance to CEA and induce protective antitumor immunity in this murine model transgenic for human CEA.     

Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells

The receptor-binding domain (RBD) of SARS coronavirus (SARS-CoV) spike (S) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. Here we used CHO-K1 cells to establish a cell line for stable expression of a 193-mer (residues 318-510) RBD (RBD193-CHO) and determined its antigenicity and immunogenicity. We found that RBD193-CHO reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in RBD, suggesting that this recombinant protein maintains intact conformation and good antigenicity. Immunization of mice with RBD193-CHO resulted in induction of high titers of RBD-specific neutralizing antibodies and potent IL-4-expressing T cell responses. RBD193-CHO induced immunity that protected a majority of the vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD produced in an established stable cell line maintains strong immunogenicity with high potential for use as an effective and economic subunit SARS vaccine.     

T cell vaccination in mice with invasive pulmonary aspergillosis

Aspergillus fumigatus, an opportunistic fungal pathogen, is responsible for multiple airway diseases of an allergic and a nonallergic nature. In a murine model of invasive pulmonary aspergillosis, resistance is associated with a decreased lung inflammatory pathology and the occurrence of an IL-12-dependent Th1-type reactivity that are both impaired by IL-4. In the present study we assess the ability of Aspergillus crude culture filtrate Ags and the recombinant allergen Asp f 2 to induce protective antifungal responses in mice with invasive pulmonary aspergillosis. Similar to what occurred upon nasal exposure to viable A. fumigatus conidia, treatment of immunocompetent mice with Aspergillus crude culture filtrate Ags resulted in the development of local and peripheral protective Th1 memory responses, mediated by Ag-specific CD4+ T cells producing IFN-gamma and IL-2 capable of conferring protection upon adoptive transfer to naive recipients. Protective Th1 responses could not be observed in mice deficient of IFN-gamma or IL-12 and did not occur in response to Asp f 2, which, on the contrary, elicited high level production of inhibitory IL-4. The results show that Ags of Aspergillus exist with the ability to induce both Th1- and Th2-type reactivity during infection, a finding that suggests a possible mechanism through which potentially protective immune responses are inhibited in mice with the infection. However, the occurrence of Th1-mediated resistance upon vaccination with Aspergillus crude culture filtrate Ags, suggests the existence of fungal Ags useful as a candidate vaccine against invasive pulmonary aspergillosis.     

Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.     

Immune responses of mice to influenza subunit vaccine in combination with CIA07 as an adjuvant

CIA07 is an immunostimulatory agent composed of E. coli DNA fragments and modified LPS lacking the lipid A moiety. In this study, we investigated whether CIA07 promotes immune responses as an adjuvant to the influenza subunit vaccine. Balb/c mice were immunized intramuscularly once or twice at a 4-week interval with the trivalent influenza subunit vaccine antigen alone or in combination with CIA07 as adjuvant. Antigen-specific serum antibody titers and hemagglutination-inhibition (HI) antibody titers were assessed. At 4 weeks after each immunization, the antigen-specific total serum IgG antibody titer in mice receiving CIA07 was 2 to 3 times higher than that in animals administered antigen alone (P<0.05). The CIA07-treated group additionally displayed higher HI antibody titers against each of the 3 vaccine strains, compared to the antigen group. Animals receiving antigen alone displayed barely detectable antigen-specific serum IgG2a antibody titers. In contrast, coadministration of CIA07 with antigen led to significantly enhanced IgG2a antibody responses, suggesting that CIA07 stimulates a Th1-type immune response. Moreover, the CIA07-treated group displayed a marked increase in the number of interferon gamma-producing CD8(+) T cells in splenocytes. These data collectively demonstrate that CIA07 has the ability to induce both Th1-type cellular and Th2-type humoral immune responses to the influenza subunit vaccine, and support its potential as an effective adjuvant to the influenza vaccine.     

Adjuvant costimulation during secondary antigen challenge directs qualitative aspects of oral tolerance induction, particularly during the neonatal period

In this report we demonstrate that although passive feeding of specific Ag to mice as neonates or adults can induce oral tolerance in both the cellular and humoral arms of the immune response, quantitative and, in particular, qualitative aspects of the tolerance process are determined by the nature of the inflammatory costimuli provided at the time of secondary Ag challenge. Moreover, this dependency upon nonspecific costimulation is more profound in Ag-fed neonates than in their adult counterparts. Thus, administration of Ag in the Th1-selective adjuvant CFA to prefed animals resulted in significant inhibition of IgG2a, IL-2, and IFN-gamma responses, whereas IL-5 responses were increased. In contrast, rechallenge with Ag in the Th2-selective adjuvant aluminum hydroxide resulted in significant inhibition of IgG1, IgE, IL-2, and IL-5 responses, whereas IFN-gamma responses were increased. Additionally, although soluble Ag challenge of prefed adults revealed marginal tolerogenic effects, the same challenge protocol in animals prefed as neonates elicited enhanced Th2-dependent IgG1 production. These results suggest that inflammatory stimulation at the time of Ag challenge is obligatory to trigger oral tolerance mechanisms, particularly in animals fed as neonates and also that the type of adjuvant used at the time of challenge selects for the type of Th cell population to be inhibited.     

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern

Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.     

IgG1 is pathogenic in Leishmania mexicana infection

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.     

Construction and immunogenicity of a new Fc-based subunit vaccine candidate against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

As an ancient disease, tuberculosis (TB) is a major global health threat. Therefore, there is an urgent need for an effective and safe anti-TB vaccine. In the current study, a delivery system of Fc domain of mouse IgG2a and early secreted antigenic target protein 6 (ESAT-6) was evaluated for the selective uptake of antigens by antigen-presenting cells (APCs). Thus, it was based on the immunogenicity of a fusion protein. The study was initiated by the transfer of recombinant expression vectors of pPICZαA-ESAT-6:Fcγ2a and pPICZαA-ESAT-6: His into Pichia pastoris (P. pastoris). Recombinant proteins were assessed for immunogenicity following the immunoblotting analysis. High levels of IFN-γ and IL-12 were produced to induce Th1-type cellular responses through vaccination with both recombinant proteins [ESAT-6:Fcγ2a (EF) and ESAT-6:His (EH)]. The Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a low increment in IL-4 compared to PBS, BCG, and EH groups. Although in all the immunized groups, the ratio of IFN-γ/IL-4 was in favor of Th1 responses, the highest Th1/Th2 balance was observed in EF immunized group. Fc fragment of mouse IgG2a may induce a selective uptake of APCs towards the cross-presentation and formation of Th1 responses in favor of an appropriate protective anti-tuberculosis reaction. Thus, further research on Fc-fusion proteins is required to develop Fc-based TB vaccines.