Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.     

Immune-stimulating complexes induce an IL-12-dependent cascade of innate immune responses

The development of subunit vaccines requires the use of adjuvants that act by stimulating components of the innate immune response. Immune-stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are potential vaccine vectors that induce a wide range of Ag-specific responses in vivo encompassing both humoral and CD4 and CD8 cell-mediated immune responses. ISCOMS are active by both parenteral and mucosal routes, but the basis for their adjuvant properties is unknown. Here we have investigated the ability of ISCOMS to recruit and activate innate immune responses as measured in peritoneal exudate cells. The i.p. injection of ISCOMS induced intense local inflammation, with early recruitment of neutrophils and mast cells followed by macrophages, dendritic cells, and lymphocytes. Many of the recruited cells had phenotypic evidence of activation and secreted a number of inflammatory mediators, including nitric oxide, reactive oxygen intermediates, IL-1, IL-6, IL-12, and IFN-gamma. Of the factors that we investigated further only IL-12 appeared to be essential for the immunogenicity of ISCOMS, as IL-6- and inducible nitric oxide synthase knockout (KO) mice developed normal immune responses to OVA in ISCOMS, whereas these responses were markedly reduced in IL-12KO mice. The recruitment of peritoneal exudate cells following an injection of ISCOMS was impaired in IL-12KO mice, indicating a role for IL-12 in establishing the proinflammatory cascade. Thus, ISCOMS prime Ag-specific immune responses at least in part by activating IL-12-dependent aspects of the innate immune system.     

In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.     

Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine.

The adjuvant activity of a single highly purified saponin from the soap bark tree Quillaja saponaria was evaluated by using it as a component in an experimental vaccine containing rHIV-1 envelope protein (HIV-1 160D) adsorbed to alum. BALB/c mice immunized with experimental vaccine formulations containing the saponin adjuvant QS-21 produced significantly higher titers of antibodies than mice vaccinated with only the alum-adsorbed HIV-1 160D. Potent amnestic antibody responses to HIV-1 viral proteins were also induced. Ag-specific proliferative responses to recombinant proteins and to three variants of HIV-1 were significantly increased using QS-21 as an adjuvant. Alum-adsorbed HIV-1 160D failed to induce measurable proliferative responses to inactivated HIV-1 viruses, but group-specific proliferative responses were raised when the QS-21 adjuvant was used in the vaccine formulation. MHC class I restricted CTL specific for the immunodominant V-3 loop were induced but only when the QS-21 adjuvant was included in the vaccine formulation. The production of serine esterase by Ag-activated splenic mononuclear cells, indicating the maturation of precursor CTL, was used as a secondary measure of CTL activity, and this response was also increased. The specificity of antibody responses was not significantly broadened using QS-21; the adjuvant increased the immune recognition of epitopes throughout the HIV-1 glycoprotein 160. However, the specificity of the proliferation and serine esterase responses was broadened, suggesting that the QS-21 augmented cell-mediated immune responses specific for epitopes outside of the V-3 loop. Additionally, the QS-21 adjuvant appeared to induce recognition of weakly immunogenic epitopes that were not recognized using only alum-adsorbed HIV-1 160D. The ability of QS-21 to augment both antibody and cell-mediated immune responses suggests that this adjuvant could be a valuable component in subunit vaccines.     

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.     

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses

Recombinant Norwalk virus-like particles (rNV VLPs) were administered to BALB/c mice by the intranasal (i.n.) route to evaluate the induction of mucosal antibody responses. The results were compared to systemic and mucosal responses observed in new and previous studies (J. M. Ball, M. E. Hardy, R. L. Atmar, M. E. Connor, and M. K. Estes, J. Virol. 72:1345-1353, 1998) after oral administration of rNV VLPs. Immunizations were given in the presence or absence of a mucosal adjuvant, mutant Escherichia coli heat-labile toxin LT(R192G). rNV-specific immunoglobulin G (IgG) and fecal IgA were evaluated by enzyme-linked immunosorbent assay. The i.n. delivery of rNV VLPs was more effective than the oral route at inducing serum IgG and fecal IgA responses to low doses of rNV particles. Vaginal responses of female mice given VLPs by the i.n. and oral routes were also examined. All mice that received two immunizations with low doses i.n. (10 or 25 microg) of rNV VLPs and the majority of mice that received two high doses orally (200 microg) in the absence of adjuvant had rNV-specific serum IgG, fecal, and vaginal responses. Additional experiments evaluated whether rNV VLPs can function as a mucosal adjuvant by evaluating the immune responses to two soluble proteins, keyhole limpet hemocyanin and chicken egg albumin. Under the conditions tested, rNV VLPs did not enhance the serum IgG or fecal IgA response to these soluble proteins when coadministered by the i.n. or oral route. Low doses of nonreplicating rNV VLPs are immunogenic when administered i.n. in the absence of adjuvant, and addition of adjuvant enhanced the magnitude and duration of these responses. Recombinant NV VLPs represent a candidate mucosal vaccine for NV infections in humans.     

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.     

Escherichia coli heat-labile enterotoxin promotes protective Th17 responses against infection by driving innate IL-1 and IL-23 production

Escherichia coli heat-labile enterotoxin (LT) is a powerful mucosal adjuvant; however, it is associated with toxic effects when delivered intranasally, and its mechanism of action is poorly understood. In this article, we demonstrate that LT acts as a highly effective adjuvant when administered parenterally, promoting Ag-specific IL-17, as well as IFN-γ, IL-4, and IL-10 production in response to coadministered Ags. We found that the adjuvant activity of LT was mediated in part by inducing dendritic cell (DC) activation; LT promoted CD80 and CD86 expression by DCs and enhanced IL-1α, IL-1β, and IL-23 production. An LT mutant, LTK63, that lacks enzyme activity was less effective than the wild-type toxin in promoting DC maturation and the development of Ag-specific Th17 cells. LT enhanced IL-23 and IL-1α production from DCs via activation of ERK MAPK and IL-1β secretion through activation of caspase-1 and the NLRP3 inflammasome. These cytokines played a major role in promoting Th17 responses by LT and LTK63. The induction of Th17 cells in vivo in response to LT and LTK63 as adjuvants was significantly reduced in IL-1RI-deficient mice. Finally, using a murine respiratory infection model, we demonstrated that LT can act as a highly effective adjuvant for a pertussis vaccine, promoting Ag-specific Th17 cells and protection against Bordetella pertussis challenge, which was significantly reduced in IL-17-defective mice. Our findings provide clear evidence that LT can promote protective immune responses in part through induction of innate IL-1 and, consequently, Th17 cells.     

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.     

No Adjuvant Effect of Bacillus thuringiensis-Maize on Allergic Responses in Mice

Genetically modified (GM) foods are evaluated carefully for their ability to induce allergic disease. However, few studies have tested the capacity of a GM food to act as an adjuvant, i.e. influencing allergic responses to other unrelated allergens at acute onset and in individuals with pre-existing allergy. We sought to evaluate the effect of short-term feeding of GM Bacillus thuringiensis (Bt)-maize (MON810) on the initiation and relapse of allergic asthma in mice. BALB/c mice were provided a diet containing 33% GM or non-GM maize for up to 34 days either before ovalbumin (OVA)-induced experimental allergic asthma or disease relapse in mice with pre-existing allergy. We observed that GM-maize feeding did not affect OVA-induced eosinophilic airway and lung inflammation, mucus hypersecretion or OVA-specific antibody production at initiation or relapse of allergic asthma. There was no adjuvant effect upon GM-maize consumption on the onset or severity of allergic responses in a mouse model of allergic asthma.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

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.     

Influence of adjuvants in inducing immune responses to different epitopes included in a multiepitope,multivalent, multistage Plasmodium falciparum candidate vaccine (FALVAC-1) in outbred mice

FALVAC-1, a vaccine against Plasmodium falciparum was developed by joining 21 epitopes from P. falciparum vaccine antigens and an universal T helper epitope from tetanus toxoid. Since adjuvants influence different aspects of immune responses, in this study we investigated the effect of four adjuvants aluminum hydroxide (alum), nonionic copolymer adjuvant P1005 (water-in-oil emulsion), CpG oligodeoxynucleotides (ODN), and QS-21 in eliciting immune responses in outbred mice. QS-21 and copolymer adjuvants were the best formulations in inducing higher and long-lasting antibody titers to the whole vaccine compared to alum and CpG. QS-21 was the only adjuvant to elicit predominantly IgG2a response and antibodies reactive with all epitopes incorporated in the vaccine construct. Vaccine elicited antibodies recognized sporozoites and asexual blood-stage parasites. FALVAC-1 immunized mice induced lymphoproliferative and IFN-gamma response to the vaccine. QS-21 and CpG adjuvants were able to elicit T proliferative responses to 20 of the 22 epitopes in the vaccine. In conclusion, this study demonstrated that with suitable adjuvant such as QS-21, it is possible to elicit immune responses to most of the epitopes included in the FALVAC-1 vaccine.     

Chemical conjugate TMV-peptide bivalent fusion vaccines improve cellular immunity and tumor protection

Chemical conjugation of CTL peptides to tobacco mosaic virus (TMV) has shown promise as a molecular adjuvant scaffold for augmentation of cellular immune responses to peptide vaccines. This study demonstrates the ease of generating complex multipeptide vaccine formulations using chemical conjugation to TMV for improved vaccine efficacy. We have tested a model foreign antigen target-the chicken ovalbumin-derived CTL peptide (Ova peptide), as well as mouse melanoma-associated CTL epitopes p15e and tyrosinase-related protein 2 (Trp2) peptides that are self-antigen targets. Ova peptide fusions to TMV, as bivalent formulations with peptides encoding additional T-help or cellular uptake via the integrin-receptor binding RGD peptide, showed improved vaccine potency evidenced by significantly enhanced numbers of antigen-reactive T cells measured by in vitro IFNgamma cellular analysis. We measured the biologically relevant outcome of vaccination in protection of mice from EG.7-Ova tumor challenge, which was achieved with only two doses of vaccine ( approximately 600 ng peptide) given without adjuvant. The p15e peptide alone or Trp2 peptide alone, or as a bivalent formulation with T-help or RGD uptake epitopes, was unable to stimulate effective tumor protection. However, a vaccine with both CTL peptides fused together onto TMV generated significantly improved survival. Interestingly, different bivalent vaccine formulations were required to improve vaccine efficacy for Ova or melanoma tumor model systems.     

TRIF is a critical negative regulator of TLR agonist mediated activation of dendritic cells in vivo

Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNγ production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy.     

Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen

The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

A novel adjuvant, a mixture of alum and the general opioid antagonist naloxone, elicits both humoral and cellular immune responses for heat-killed Salmonella typhimurium vaccine

In the current study, we tested the efficacy of the mixture of naloxone, an opioid receptor antagonist, and alum, as a new adjuvant, in the induction of humoral and cellular immunity in response to heat-killed Salmonella typhimurium (HKST) as a model vaccine. BALB/c mice were divided into five groups. Mice in the experimental groups received either the HKST vaccine alone or in combination with the adjuvant alum, naloxone or the alum-naloxone mixture. Mice in the negative control group received phosphate-buffered saline. All mice were immunized two times on days 0 and 14. Two weeks after the last immunization, immune responses to S. typhimurium were assessed. Our results indicated that the administration of the alum-naloxone mixture as an adjuvant increased the ability of the HKST vaccine to enhance lymphocyte proliferation, shifted the immune response towards a T-helper 1 (Th1) pattern and increased S. typhimurium-specific immunoglobulin G (IgG), IgG2a, IgG1 and the ratio of IgG2a to IgG1. This resulted in improved protective immunity against S. typhimurium. In conclusion, the administration of the alum-naloxone mixture as an adjuvant, in combination with the HKST vaccine, can enhance both humoral and cellular immunity and shift the immune responses to a Th1 pattern.     

Vaccination with a DNA vaccine based on human PSCA and HSP70 adjuvant enhances the antigen-specific CD8+ T-cell response and inhibits the PSCA+ tumors growth in mice

BACKGROUND: DNA vaccines have been shown to be an effective approach to induce antigen-specific cellular and humoral immunity. However, the lower immune intensity in clinical trials limits the application of DNA vaccine. Here we intend to develop a new DNA vaccine based on prostate stem-cell antigen (PSCA), which has been suggested as a potential target for prostate cancer therapy, and enhance the DNA vaccine potency with heat shock proteins (HSPs) as adjuvant. METHODS: A series of DNA plasmids encoding human PSCA, human HSP70 and their conjugates was constructed and injected into male mice intramuscularly (i.m.). To evaluate the immune responses and therapeutic efficacy of these plasmids, major histocompatibility complex (MHC)-restricted PSCA and HSP70-specific epitopes were predicted and a mouse model with a human PSCA-expressing tumor was constructed. RESULTS: The result showed that mice vaccinated with PSCA-HSP plasmids generated the strongest PSCA-specific CD8+ T-cell immune response, but the CD4+ TH1 and TH2 cell immune responses were similar with those vaccinated with other HSP-adjuvant PSCA plasmids or only PSCA DNA. The immunity of HSP70 was also observed and the mice i.m. injected with PSCA+ HSP mixed plasmids generated the lowest anti-HSP antibodies. Furthermore, these vaccinations inhibited the growth of PSCA-expressing tumors and prolonged mouse survival. CONCLUSIONS: These observations emphasize and extend the potential of the human HSP70 gene as adjuvant for DNA vaccines, and the vaccine based on PSCA and HSP70 is of potential value for treating prostate cancer.