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.     

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.     

Cytokines as Adjuvants for the Induction of Anti-Human Immunodeficiency Virus Peptide Immunoglobulin G (IgG) and IgA Antibodies in Serum and Mucosal Secretions after Nasal Immunization

Safe and potent new adjuvants are needed for vaccines that are administered to mucosal surfaces. This study was performed to determine if interleukin-1alpha (IL-1alpha) combined with other proinflammatory cytokines provided mucosal adjuvant activity for induction of systemic and mucosal anti-human immunodeficiency virus (HIV) peptide antibody when intranasally administered with an HIV peptide immunogen. Nasal immunization of BALB/c mice with 10 microg of an HIV env peptide immunogen with IL-1alpha, IL-12, and IL-18 on days 0, 7, 14, and 28 induced peak serum anti-HIV peptide immunoglobulin G1 (IgG1) and IgA titers of 1:131,072 and 1:7,131, respectively (P = 0.05 versus no adjuvant). The use of cholera toxin (CT) as a mucosal adjuvant induced serum IgG1 and IgA titers of 1:32,768 and 1:776, respectively. The adjuvant combination of IL-1alpha, IL-12, and IL-18 induced anti-HIV peptide IgA titers of 1:1,176, 1:7,131, and 1:4,705 in saliva, fecal extracts and vaginal lavage, respectively. Titers induced by the use of CT as an adjuvant were 1:223, 1:1,176, and 1:675, respectively. These results indicate that the proinflammatory cytokines IL-1alpha, IL-12, and IL-18 can replace CT as a mucosal adjuvant for antibody induction and are important candidates for use as mucosal adjuvants with HIV and other vaccines.     

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.     

Novel Mucosal DNA-MVA HIV Vaccination in Which DNA-IL-12 Plus Cholera Toxin B Subunit (CTB) Cooperates to Enhance Cellular Systemic and Mucosal Genital Tract Immunity

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.     

Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV-specific CTL

We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3-18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.     

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.     

MHC class I-restricted cytotoxic lymphocyte responses induced by enterotoxin-based mucosal adjuvants

The ability of enterotoxin-based mucosal adjuvants to induce CD8+ MHC class I-restricted CTL responses to a codelivered bystander Ag was examined. Escherichia coli heat-labile toxin (LT), or derivatives of LT carrying mutations in the A subunit (LTR72, LTK63), were tested in parallel with cholera toxin (CT) or a fusion protein consisting of the A1 subunit of CT fused to the Ig binding domain of Staphylococcus aureus protein A (called CTA1-DD). Intranasal (i.n.) immunization of C57BL/6 mice with CT, CTA1-DD, LT, LTR72, LTK63, but not rLT-B, elicited MHC class I-restricted CD8+ T cell responses to coadministered OVA or the OVA CTL peptide SIINFEKL (OVA257-264). CT, LT, and LTR72 also induced CTL responses to OVA after s.c. or oral coimmunization whereas LTK63 only activated responses after s.c. coimmunization. rLT-B was unable to adjuvant CTL responses to OVA or OVA257-264 administered by any route. Mice treated with an anti-CD4 mAb to deplete CD4+ T cells mounted significant OVA-specific CTL responses after i.n. coadministration of LT with OVA or OVA257-264. Both 51Cr release assays and IFN-gamma enzyme-linked immunospot assays indicated that IFN-gamma-/- and IL-12 p40-/- gene knockout mice developed CTL responses equivalent to those detected in normal C57BL/6 mice. The results highlight the versatility of toxin-based adjuvants and suggest that LT potentiates CTL responses independently of IL-12 and IFN-gamma and probably by a mechanism unrelated to cross-priming.     

Cytokine,chemokine, and costimulatory molecule modulation to enhance efficacy of HIV vaccines

Understanding key intervention points in developing immune responses may allow the rational inclusion of biological adjuvants into vaccines that could potentiate the immune response both quantitatively and qualitatively and enhance effective memory responses. Cytokine and chemokine combinations can potentially help target antigen to the appropriate antigen presenting cell and initiate maturation of these presenting cells, attract cells expressing different chemokine receptors, steer cellular immune responses toward Th1 and CD8 CTL, and enhance systemic and mucosal IgG and secretory IgA antibodies and determine their isotype balance. Animal protection studies suggest that synergistic combinations of cytokines and immunomodulating molecules may be required to protect from a viral challenge. For example, GM-CSF has been shown to be synergistic with IL-12 or CD40 ligand for induction of CTL and for antiviral protection, and the triple combination of GM-CSF, IL-12, and TNF alpha appears to induce the most effective protection in some mouse models. Chemokine-antigen fusions have also been shown to enhance immunogenicity of the antigen. Combinations of costimulatory molecules have been found to be synergistic when incorporated in a vaccine. Combined use of newer more potent vaccine constructs, containing codon optimized epitopes, relevant CpG motifs, cytokines, costimulatory molecules and chemokines, used in heterologous prime-boost strategies with viral vector vaccines or recombinant proteins, might afford the most potent vaccine approaches yet developed. In this review we will discuss the application and delivery of cytokines, costimulatory molecules, and chemokines toward improving current vaccine strategies.     

Intranasal vaccination using interleukin-12 and cholera toxin subunit B as adjuvants to enhance mucosal and systemic immunity to human immunodeficiency virus type 1 glycoproteins

We have investigated the induction of protective mucosal immunity to human immunodeficiency virus type 1 (HIV-1) isolate 89.6 by intranasal (i.n.) immunization of mice with gp120 and gp140 together with interleukin-12 (IL-12) and cholera toxin subunit B (CTB) as adjuvants. It was found that both IL-12 and CTB were required to elicit mucosal antibody responses and that i.n. immunization resulted in increased total, immunoglobulin G1 (IgG1), and IgG2a anti-HIV-1 antibody levels in serum; increased total, IgG1, IgG2a, and IgA antibody expression in bronchoalveolar lavage fluids; and increased IgA antibody levels in vaginal washes. Levels of anti-HIV-1 antibodies in both sera and secretions were higher in groups immunized with gp140 than in those immunized with gp120. However, only gp120-specific mucosal antibodies demonstrated neutralizing activity against HIV-1 89.6. Taken together, the results show that IL-12 and CTB act synergistically to enhance both systemic and local mucosal antibody responses to HIV-1 glycoproteins and that even though gp140 induces higher antibody titers than gp120, only gp120-specific mucosal antibodies interfere with virus infectivity.     

Mucosal and systemic immune responses of mice to tetanus toxoid coadministered nasally with AFCo1

Mucosal immune responses are an early and important line of defense against pathogens. The current understanding of the mucosal immune system allows us to consider the use of nasal immunization for induction of antigen-specific immune responses at the mucosal surface and the systemic compartment. Mucosal adjuvants are key for developing novel mucosal vaccines and represent 1 approach to improving mucosal and systemic immunity. However, few mucosal vaccine adjuvants are currently approved for human use. Neisseria meningitidis B proteoliposome-derived cochleate (AFCo1 - Adjuvant Finlay Cochleate 1) has been demonstrated to be a potent mucosal adjuvant. The present work demonstrates that intranasal immunization of 3 doses of tetanus toxoid (TT) coadministered with AFCo1 in mice promotes high systemic and mucosal responses. The anti-TT IgG serum titers and the mucosal anti-TT IgA in saliva and vaginal wash were significantly higher than TT alone. The analysis of antibody subclasses showed that intranasal administration of AFCo1 + TT induced not only IgG1 but also IgG2a anti-TT antibodies at levels comparable to those obtained with TT vaccine (vax-TET). These data support the fact that AFCo1 is a potent mucosal adjuvant in nasal immunization to a coadministered protein antigen.     

Nasopharyngeal-associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production

To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 micrograms) and CT (1 microgram) induced Ag-specific Th1/Th2-type response in CD4+ T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P. gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.     

Evidence for early aging in the mucosal immune system

Despite recent advances in the cellular and molecular analysis of induction and regulation of mucosal immune responses, little is yet known about differences which occur in aging. To address this important issue, we have compared the mucosal and systemic immune responses of aged (12- to 14-mo- or 2-year-old) and young adult (6- to 8-wk-old) C57BL/6 mice. Both aged and young mice were immunized weekly with three oral doses of 1 mg of OVA and 10 microg of cholera toxin (CT) as mucosal adjuvant. Both groups of mice over 1 or 2 years of age showed reduced levels of Ag-specific mucosal or systemic immune responses at day 21. An Ag-specific B cell enzyme-linked immunospot assay confirmed these results at the cellular level. When the Ag-induced cytokine responses were examined at both protein and mRNA levels, CD4(+) T cells from spleen and Peyer's patches of young adult mice revealed elevated levels of IL-4 production; however, these cytokine responses were significantly diminished in aged mice. In contrast to mucosal immunization, mice s. c. immunized with OVA plus CT resulted in impaired OVA-specific but intact CT B subunit-specific immune responses in 12- to 14-mo-old mice although the responses to both Ags were depressed in 2-year-old mice. These results provide the first evidence that the development of age-associated alterations possibly occurs earlier in the mucosal immune system than in the systemic immune compartment.     

Sendai virus fusion protein mediates simultaneous induction of MHC class I/II-dependent mucosal and systemic immune responses via the nasopharyngeal-associated lymphoreticular tissue immune system

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4(+) Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.     

Influence of DNA encoding cytokines on systemic and mucosal immunity following genetic vaccination against herpes simplex virus

The aim of our investigation was to improve the effectiveness of DNA vaccines against herpes simplex virus (HSV) infection. We chose coimmunization with DNA encoding cytokines known to emphasize components of immune defense that best correlate with immune protection. These include interferon-producing T and NK cells and the IgG2a isotype immunoglobulin. Our results show that the coadministration of plasmid DNA encoding IL-12 or IL-18 along with glycoprotein B (gB) DNA improves immune induction. Recipients of the coimmunization procedure had elevated humoral as well as IFN-gamma-producing T cell responses and showed greater resistance to vaginal challenge with a lethal dose of HSV-1. The adjuvant effects were observed when the vaccines were administered either systemically or mucosally. By most assays, the adjuvant effect of IL-18 was superior to IL-12, although gB DNA plus IL-18 failed to induce levels of immunity achieved by UV-inactivated HSV immunization. Mucosal immunization proved as an effective means of inducing systemic immunity, but was less effective than the systemic route for inducing protection from vaginal challenge. Our results also demonstrated that protection from such challenges was mainly a property of IFN-gamma. Thus, immunized IFN-gamma-/- mice remained susceptible to challenges even while generating readily measurable immune responses. The approach of using DNA vaccines combined with DNA encoding cytokines holds promise and represents a potentially useful approach for vaccines.     

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.     

The pulmonary environment promotes Th2 cell responses after nasal-pulmonary immunization with antigen alone, but Th1 responses are induced during instances of intense immune stimulation

The purpose of this study was to determine the nature of the CD4(+) Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4(+), and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-gamma, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-gamma) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4(+) Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1beta expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.     

Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice

The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-gamma) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-gamma and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-gamma and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.     

Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 agonist, promotes potent systemic and mucosal responses to intranasal immunization with HIVgp140

Successful vaccine development against HIV will likely require the induction of strong, long-lasting humoral and cellular immune responses in both the systemic and mucosal compartments. Based on the known immunological linkage between the upper-respiratory and urogenital tracts, we explored the potential of nasal adjuvants to boost immunization for the induction of vaginal and systemic immune responses to gp140. Mice were immunized intranasally with HIV gp140 together with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA) and responses were compared to R848, a TLR7/8 agonist, or chitosan, a non TLR adjuvant. GLA and chitosan but not R848 greatly enhanced serum immunoglobulin levels when compared to antigen alone. Both GLA and chitosan induced high IgG and IgA titers in nasal and vaginal lavage and feces. The high IgA and IgG titers in vaginal lavage were associated with high numbers of gp140-specific antibody secreting cells in the genital tract. Whilst both GLA and chitosan induced T cell responses to immunization, GLA induced a stronger Th17 response and chitosan induced a more Th2 skewed response. Our results show that GLA is a highly potent intranasal adjuvant greatly enhancing humoral and cellular immune responses, both systemically and mucosally.