Decrease and dysfunction of dendritic cells correlate with impaired hepatitis C virus-specific CD4+ T-cell proliferation in patients with hepatitis C virus infection

Through the production of stimulatory and suppressive cytokines, dendritic cells (DCs) regulate virus-specific immune responses that are crucial to virus eradication. To explore a possible role of DCs in the persistence of hepatitis C virus (HCV) infection, in this study we analysed peripheral blood DCs (PBDCs) in patients with chronic hepatitis C (CHC) compared with those in both healthy seronegative (HSN) controls and a group of subjects who had spontaneously resolved infection, defined as healthy HCV-seropositive (HSP), and we evaluated the relationships between PBDCs and HCV-specific CD4(+) T-cell reactivity. The number of PBDCs, their immunophenotype and expression of regulatory cytokines were evaluated by flow cytometry on whole-blood samples. HCV-specific CD4(+) T-cell activation, proliferation and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV peptides. We found that PBDCs from CHC subjects were numerically reduced and showed lower interleukin-12 (IL-12) and higher IL-10 expression than those from HSN controls. PBDCs from HSP subjects were similar to those from HSN controls. HCV-specific CD4(+) T-cell proliferation was less frequent and vigorous in CHC than in HSP patients and was directly related to the number of PBDCs and their IL-12 production but inversely related to their IL-10 production. Taken together, these results seem to suggest that cytokines of DC origin contribute to the regulation of HCV-specific immunity in CHC patients and indicate that PBDCs may represent a novel non-invasive tool for immune monitoring of these patients.     

Poly(gamma-glutamic acid) nanoparticles as an efficient antigen delivery and adjuvant system: potential for an AIDS vaccine

Antigen delivery systems using polymeric nanoparticles are of special interest as stable protein-based antigen carriers. In the present study, novel biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles were examined for their antigen delivery and immunostimulatory activities in vitro and in vivo. The uptake of ovalbumin by dendritic cells was markedly enhanced by gamma-PGA nanoparticles, and the ovalbumin was gradually released from gamma-PGA nanoparticles into the cells. In addition, gamma-PGA nanoparticles appeared to have great potential as an adjuvant, because they could induce the maturation of dendritic cells. Although not only ovalbumin-encapsulating nanoparticles (OVA-NPs) but also a simple mixture of ovalbumin and nanoparticles induced dendritic cell maturation, the only dendritic cells exposed to OVA-NPs could strongly activate antigen-specific interferon (IFN)-gamma-producing T cells. Subcutaneous immunization of mice with human immunodeficiency virus type 1 (HIV-1) p24-encapsulating nanoparticles activated antigen-specific IFN-gamma-producing T cells in spleen cells and induced p24-specific serum antibodies, as compared to immunization with p24 alone. Like ovalbumin, a mixture of p24 and nanoparticles also induced antigen-specific serum antibodies but did not activate IFN-gamma-producing T cells in spleen cells, suggesting that nanoparticles play a critical role in inducing cellular immune responses. Furthermore, gamma-PGA nanoparticles had a capacity comparable to that of the complete Freund's adjuvant (CFA) in inducing p24-specific serum antibody. However, unlike CFA, they predominantly activated p24-specific IFN-gamma-producing T cells. Thus, gamma-PGA nanoparticles encapsulating various antigens may have great potential as novel and efficient protein-based vaccines against infectious diseases, including HIV-1 infection.     

Efficacy of interferon-based antiviral therapy in patients with chronic hepatitis C infected with genotype 5: a meta-analysis of two large prospective clinical trials

The characteristics and response rate to pegylated interferon and ribavirin (PEG-INF + RBV) of patients with chronic hepatitis C infected with genotype 5 are poorly documented. A meta-analysis of two large phase III/IV prospective randomized clinical trials conducted in Belgium in patients with chronic hepatitis C (n = 1,073 patients) was performed in order to compare the response to antiviral therapy of hepatitis C virus (HCV) genotype 5 with that of other HCV genotypes. A subset of HCV-1 infected patients selected from within the study database were selected to match the HCV-5 sample for known prognostic factors. In Belgium HCV-5 is responsible for a significant minority of cases of chronic hepatitis C CHC (4.5%) and is characterized by a more advanced age (58.4 years), a high frequency of cirrhosis (27.7%), a specific mode of HCV acquisition, and a particular geographic origin (66.7% of patients from West Flanders). The primary comparative analysis showed that response to treatment with PEG-INF + RBV of HCV-5 is similar to HCV-1 and lower compared to HCV-2/3. The analysis of the matched patient subgroup demonstrates that the HCV-5 "intrinsic sensitivity" to PEG-IFN + RBV therapy is identical to HCV-1, with a sustained virological response of 55% in both groups. In contrast to previous publications, this meta-analysis suggests that HCV-5 response to treatment is closer to HCV-1 than to HCV-2/3 and suggests that in Belgium HCV-5 infection should be treated with the same antiviral regimen as HCV-1.     

Association of phosphatidylinositol 3 kinase to protein kinase C ζ during interleukin-2 stimulation

Interleukin-2 induces a serine-phosphorylated phosphatidylinositol 3 kinase activity in the mouse T cell line TS1αβ. Moreover, protein kinase C (PKC) ζ directly or indirectly associates with the phosphatidylinositol 3 kinase and the association appears to be necessary for the serine-phosphorylated phosphatidylinositol 3 kinase activity, since release of ζPKC by competition of binding with peptides spanning the p110 sequence from amino acids 907 to 925 abolishes the serine-phosphorylated phosphatidylinositol 3 kinase activity. This kinase activity is also blocked when ζPKC expression is inhibited by antisense oligonucleotide. Inhibition of phosphatidylinositol 3 kinase activity by wortmannin does not abolish ζPKC association.     

Histological difference between complete responders and nonresponders to interferon therapy of the livers of patients with chronic hepatitis C

Liver histology was compared in patients with chronic hepatitis C to note the differences between responders and nonresponders to interferon treatment. Fifty-eight patients were administered interferon in varying doses and over various periods, and were then followed up for 1 year. According to the improvement status of serum alanine aminotransferase (ALT) levels during this period, the patients were classified into complete responders who showed complete normalization of ALT; partial responders who exhibited a significant decrease, but not complete normalization of ALT. Before application of the interferon treatment, liver biopsies were analyzed in four parameters and given scores from 0 to 5 for three groups in cord with no prior knowledge of the efficacy. The parameters included necroinflammation, fibrosis/lobular distortion, portal lymphocytic reaction and portal (or fibrous septal) outline destruction. Results indicated that there were no significant differences in the score of necroinflammation and portal lymphocytic reaction between the complete responder group and the nonresponder group. In contrast, the complete responder group exhibited weaker fibrosis/lobular distortion and less portal outline destruction than the non-responder group. The partial responder group was more akin to the former group in these parameters. Thus, it is safe to conclude that liver histology may predict the efficacy of interferon treatment.     

Effect of interferon on GB virus C and hepatitis C virus in hepatitis patients with the co-infection

Of 74 patients who were infected with hepatitis C virus (HCV) and received interferon, 12 (16%) were positive for RNA of GB virus C (GBV-C). RNA of GBV-C was determined in sera from the co-infected patients retrospectively, and the effect of interferon on GBV-C was compared with that on HCV in them. Titers of both GBV-C and HCV RNAs decreased during interferon in all of them. Two patients lost both GBV-C and HCV RNAs and remained clear until 6 months after treatment with interferon, while 2 lost RNA for GBV-C only and 2 for HCV RNA alone. Low pre-treatment RNA titers of GBV-C and HCV correlated with the efficacy of interferon in clearing. Alanine aminotransferase returned to normal only in the patients who lost HCV RNA, regardless of the persistence or loss GBV-C RNA. These results indicate that the response to interferon of GBV-C is comparable to but independent of that of HCV and that the persistence of GBV-C would not prevent the normalization of aminotransferases in response to interferon in patients with chronic hepatitis C. J. Med. Virol. 52:156–160, 1997. © 1997 Wiley-Liss, Inc.     

Interferon therapy in chronic hepatitis C virus: Evidence of different outcome with respect to different viral strains

The aim of the study was to assess the role of different viral strains of hepatitis C virus (HCV) in determining the outcome of the alpha-interferon (IFN) therapy. Fifty-seven patients (34 from Italy and 23 from Japan) with HCV-positive liver disease were enrolled in the study. The NS4 region of HCV was amplified in sera by “nested” polymerase chain reaction (PCR) using a primer pair synthesized according to the sequence of JK-1. The NS4 region was positive in 14 (41%) Italian and in 13 (56%) Japanese patients. In positive patients the sequence of the NS4 region was also obtained. Subsequently, HCV genotype was determined in all patients by PCR amplification of the core region. All patients received recombinant alpha2a-interferon (IFN), 6 million units 3 times a week for 1 month followed by 3 million units 3 times a week for 5 months. The patients were followed for 1 year after the end of treatment. At the end of the follow-up, 17 (30%) had sustained normal levels of serum alanine aminotransferase (ALT). The outcome of treatment was not correlated with race, age, sex, histology, and pretreatment ALT level, but was significantly (P < 0.00001) associated with the presence of both the NS4-JK-1 region and HCV type II. Among the 27 NS4-positive patients, only 1 patient (3.7%) achieved a complete response, whereas the remaining 26 patients (96.3%) either were non-responders or relapsed after IFN was discontinued. In contrast, among the 30 NS4-JK-1-negative patients, 15 (53%) had a sustained remission. HCV genotyping showed type I in 3 (6%), type II in 40 (74%), type III in 4 (7%), and type IV in 3 (6%) cases. Coinfection was present in 4 (7%), while in 3 cases amplification was not obtained. Patients with type II were all non-responders or relapsers, while a response to the treatment was oberved in 17 of 17 (100%) of the remaining patients. These data indicate that the presence of JK-1 variant of HCV or HCV type II is almost always predictive of a poor response rate Of IFN therapy. © 1995 Wiley-Liss, Inc.     

Efficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.     

B-cell epitopes in hypervariable region 1 of hepatitis C virus obtained from patients with chronic persistent hepatitis

The hypervariable domain (HVR1) within the N-terminus of the E2 protein of hepatitis C virus (HCV) is known to be variable antigenically during the course of persistent infection. The aim of the study was to detect B-cell epitopes in HVR1 responsible for neutralizing HCV. The B-cell epitopes were analyzed using two series of synthetic peptides: 25 heptapeptides from the most common amino acids within 73 HVR1 sequences, and 216 heptapeptides, the sequences of which cover more than 65% of the 73 HVR1 sequences. Sera from three patients with chronic hepatitis C were tested for reactivity to the synthetic peptide sequences by enzyme-linked immunosorbent assay (ELISA). The post-interferon (IFN) serum of one patient who had a long-term response to treatment reacted specifically with 13 heptapeptides of 216 variable sequences of HVR1. Some of the amino acid sequences (amino acids 398, 399, 400, 404) of the heptapeptides were also found in those deduced from the nucleotide sequences of HCV genomes in the pre-IFN serum. The sera of the other two patients who did not respond to treatment did not react with the 13 heptapeptides. It is concluded that the B-cell epitopes in HVR1 may be relevant for eliminating viremia in the case of the patient who had a good response to treatment. These results suggest that the analysis of the B-cell epitopes recognized in HVR1 may be important in understanding the mechanism of persistent infection and progression of hepatitis. © 1996 Wiley-Liss, Inc.     

Identification of a new autoantibody in patients with chronic hepatitis

To comprehensively study autoantibodies in patients with chronic hepatitis (CH), especially those with hepatitis C virus (HCV) infection, proteins extracted from HepG2 cells were separated by two-dimensional electrophoresis. Spots reacting with sera from 15 patients with CH-C were detected by Western blotting. Proteins extracted from the spots were subjected to mass spectroscopy for identification by mass fingerprinting. Antigenicity of the proteins identified was confirmed by Western blotting and enzyme-linked immunoabsorbent assay. The localization of the autoantigens so detected was investigated by immunohistochemistry. Among 20 protein spots detected, four were identified as actin, heat shock protein (HSP) 70, HSP60, and a novel protein (hepalaminin). Hepalaminin consists of two domains of laminin β-2 and a specific domain. Autoantibodies against the specific domain were detected in 60.8% of patients with CH-C, 37.7% of those with CH-B, 42.3% of those with autoimmune CH, 28.6% of nonalcoholic steatohepatitis, 10.0% of asymptomatic HCV carriers, but in no healthy volunteers. Antihepalaminin positivity in CH-C and CH-B was related to histologic grading. Immunohistochemical staining demonstrated that hepalaminin is present in the cytoplasm of hepatocytes and cholangiocytes but not of fibroblasts or the vascular epithelium. Hepalaminin is a novel protein expressed in hepatocytes and cholangiocytes. Autoimmunity to this protein may exacerbate inflammation in chronic viral hepatitis.     

暴露N端对趋化因子LTN黏膜佐剂作用的影响

我们前期构建了编码CVB3结构蛋白VP1的真核表达质粒(pVP1),通过滴鼻免疫诱导了一定水平的细胞和体液免疫应答。为了进一步增强其免疫效果,我们在该黏膜疫苗中引入新型黏膜佐剂LTN,发现当两种质粒共免疫时可显著增强pVP1诱导的CVB3特异性血清IgG和黏膜IgA水平,促进脾脏及黏膜局部IFN-γ+T细胞产生,显著减轻CVB3感染小鼠心肌病理损伤。随后我们对该疫苗体系进行了优化,使用双顺反子形式将编码VP1和LTN的基因构建在同一质粒上,通过滴鼻免疫小鼠后同样获得了免疫增强作用;当尝试将LTN N端融合至VP1蛋白,以该融合质粒pVP1-LTN滴鼻免疫小鼠后发现其增强免疫应答的能力显著下降;为排除融合蛋白空间构象改变及空间位阻对LTN佐剂功能的影响,我们在蛋白连接处引入柔性接头肽段(G4S)3,构建了融合质粒pVP1-IRES-LTN,发现该疫苗增强全身及黏膜局部特异性抗体细胞免疫应答的能力也非常有限,与pVP1-LTN相比并无明显差异,因此不能有效清除心肌病毒和减轻心肌损伤,提示融合蛋白的空间位阻或构像改变并非是影响LTN佐剂效应的关键因素,而暴露的LTN N端对其增强全身及黏膜局部免疫应答强度、提高心肌炎的免疫保护作用十分关键。     

Differential flotation centrifugation study of hepatitis C virus and response to interferon therapy

Hepatitis C virus (HCV) appears to circulate in various forms such as native virion, immune complexes, and nucleocapsids during chronic infections. To determine the association of the physicochemical properties of HCV and its response to interferon therapy in patients with chronic hepatitis C, we examined pretreatment serum samples from 43 patients with HCV RNA who had received interferon therapy, using differential flotation centrifugation in a NaCl solution with a density of 1.063 g/ml. After centrifugation, the ratio of HCV RNA in the top and bottom fractions was determined by the polymerase chain reaction and expressed as T/B. Patients with a sustained response to IFN therapy were found to have higher T/B ratios than transient responders who relapsed after treatment (P < 0.01) and nonresponders (P < 0.01). With regards to HCV genotypes, patients with genotype 1b had higher T/B ratios in the sustained response group than in the nonsustained response group (P = 0.001), but patients with genotype 2 had a similar distribution of T/B among the 3 response groups (not significant). These findings indicate that the physicochemical properties of HCV affect the effectiveness of interferon therapy, particularly in patients with genotype 1b. J. Med. Virol. 52:190–194, 1997. © 1997 Wiley-Liss, Inc.     

Evaluation of properties of chitosan as an adjuvant for inactivated influenza vaccines administered parenterally

Studies on mice showed that chitosan as an adjuvant for H5 inactivated influenza vaccines administered intramuscularly enhances significantly antibody titers and protective efficiency not only against homologous influenza viruses, but also against drift variants. Chitosan adjuvanted vaccines induced high antibody titers after a single immunization and with a low dose of antigen. High antibody titers remained for at least 6 months. Chitosan adjuvanted vaccine stored at 4°C preserves its adjuvant properties for at least 8 months. Chitosan stimulates proliferative and cytotoxic activity of splenic mononuclear leukocytes in mice and promotes an increase in the numbers of CD3, CD3/NK, I‐AK (MHC II), and H‐2Db (MHC I) cells. After intramuscular immunization, chitosan did not induce IgE antibodies and antibodies against chitosan itself. Chitosan is a promising adjuvant candidate for inactivated influenza vaccines administered parenterally. J. Med. Virol. 81:494–506, 2009. © 2009 Wiley‐Liss, Inc.     

Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels

The canonical transient receptor potential (TRPC) proteins have been recognized as key players in calcium entry pathways activated through phospholipase-C-coupled receptors. While it is clearly demonstrated that members of the TRPC3/6/7 subfamily are activated by diacylglycerol, the mechanism by which phospholipase C activates members of the TRPC1/4/5 subfamily remains a mystery. In this paper, we provide evidence for both negative and positive modulatory roles for membrane polyphosphoinositides in the regulation of TRPC5 channels. Depletion of polyphosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP₂) through inhibition of phosphatidylinositol 4-kinase activates calcium entry and membrane currents in TRPC5-expressing but not in TRPC3- or TRPC7-expressing cells. Inclusion of polyphosphatidylinositol 4-phosphate or PIP₂, but not phosphatidylinositol 3,4,5-trisphosphate, in the patch pipette inhibited TRPC5 currents. Paradoxically, depletion of PIP₂ with a directed 5-phosphatase strategy inhibited TRPC5. Furthermore, when the activity of single TRPC5 channels was examined in excised patches, the channels were robustly activated by PIP₂. These findings indicate complex functions for regulation of TRPC5 by PIP₂, and we propose that membrane polyphosphoinositides may have at least two distinct functions in regulating TRPC5 channel activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mohamed Trebak and Loic Lemonnier contributed equally to this work.     

Rapid and simple detection of IFN-neutralizing antibodies in chronic hepatitis C non-responsive to IFN-alpha

Different mechanisms have been proposed for the failure of interferon (IFN) therapy in patients with chronic hepatitis C and multiple sclerosis, for example, the presence of IFN-neutralizing antibodies. In this study, a novel assay system based on the IFN-inducible Mx-promoter was used to detect IFN-neutralizing antibodies in sera of patients with chronic hepatitis C. To monitor IFN bioactivity in IFN-treated patients, a real-time RT-PCR for MxA gene expression in PBMCs was established. Using these two methods, patients with chronic hepatitis C virus (HCV) infection receiving IFN therapy and patients with treatment induced HCV clearance were monitored. Importantly, neutralizing anti-IFN antibodies were detected in the sera of 3 of 38 chronically HCV-infected patients who failed to respond to therapy but none in sera of patients who cleared HCV after IFN therapy. Interestingly, the presence of these antibodies correlated with the lack of MxA induction in PBMCs after initiation of IFN-alpha therapy. Retrospective analysis of one patient's sera revealed that the anti-IFN-alpha antibodies had already developed after the first of four unsuccessful IFN therapies, suggesting that neutralizing antibodies may have contributed to the failure of previous IFN treatments. In summary, a novel screening assay was established that may be helpful for testing IFN non-responders for the presence of clinically relevant anti-IFN-alpha antibodies and for selecting alternative IFN preparations not neutralized by these antibodies.     

Hepatitis C virus and the controversial role of the interferon sensitivity determining region in the response to interferon treatment

The degree of variability of the interferon sensitivity determining region (ISDR) in the hepatitis C virus (HCV) genome has been postulated to predict the response to interferon therapy, mainly in patients infected with subtype 1b, although this prediction has been the subject of a long controversy. This prediction has been tested by analyzing a cohort of 67 Spanish patients infected with HCV genotype 1, 23 of which were infected with subtype 1a and 44 with subtype 1b. A sample previous to therapy with alpha-interferon plus ribavirin was obtained and several clones (between 25 and 96) including the ISDR were sequenced from each patient. A significant correlation between mutations at the ISDR and response to treatment for subtype 1b patients, but not for those infected with subtype 1a, has been detected. Although the results suggest that the same relationship holds true for subtype 1a, lack of statistical power because of the small sample size of this subtype prevented firmer conclusions. However, identical ISDR sequences were found in responder and non-responder patients, suggesting that the stability of the ISDR sequence can occasionally help HCV to evade interferon therapy, although this is not a sufficient condition. More complex interactions, including the ISDR or not, are likely to exist and govern the HCV response to interferon treatment.     

Ifi202, an IFN-inducible candidate gene for lupus susceptibility in NZB/W F1 mice, is a positive regulator for NF-kappaB activation in dendritic cells

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies and lupus nephritis. The [New Zealand black (NZB) x New Zealand white (NZW)]F1 (BWF1) mouse has been recognized as an important animal model of human SLE. The T(h)1-prone phenotype of BWF1 mice has been shown to contribute to the development of the lupus. However, the molecular basis for T(h)1 skewing in BWF1 mice has not been clarified. We noticed that IL-6, IL-12 and other proinflammatory cytokines as well as IkappaB-zeta induction were higher in mature bone marrow-derived dendritic cells (BMDCs) from NZB and BWF1 mice than those from NZW mice. The expression of an IFN-inducible gene Ifi202, a candidate gene for lupus, was almost undetectable in NZW BMDCs. Thus, we hypothesized that Ifi202 is involved in elevated IL-12 production from BWF1 BMDCs. Overexpression of Ifi202 enhanced the LPS-induced IkappaB-zeta, IL-12p40 and NF-kappaB promoter activities, while anti-sense (AS) RNA against Ifi202 strongly suppressed them in a monocytic cell line, RAW 264.7. Furthermore, overexpression of Ifi202 enhanced LPS-induced IL-12p40 and IkappaB-zeta mRNA induction while Ifi202 AS RNA suppressed these in RAW 264.7 cells. In addition, forced expression of Ifi202 enhanced IL-12p40 mRNA induction in NZW BMDCs. Thus, Ifi202 is an important NF-kappaB activator in DCs and involved in IL-12 production, which may account for a T(h)1-prone phenotype of BWF1 mice.     

A complex of lactoferrin with monophosphoryl lipid A is an efficient adjuvant of the humoral and cellular immune response in mice

Our recent investigations demonstrated adjuvant properties of lactoferrin (LF). Other studies proved efficacy and safety of monophosphoryl lipid A (MPL) as an adjuvant in humans. In an attempt to construct more efficient and safer adjuvants, we evaluated the activity of LF-MPL complex, formed by incubation of LF and MPL from Hafnia alvei at 20:1 w/w ratio, and verified its characteristics by SDS-PAGE analysis. Binding kinetics was determined by surface plasmon resonance analysis using a BIAcore^TM 1000 biosensor system. The efficiency of the complex in enhancing the humoral and cellular immune responses was analyzed in BALB/c mice. The complex stimulated the humoral immune response to ovalbumin (OVA) and sheep red blood cells significantly stronger than both components separately, used at respective doses. In addition, the complex increased the serum levels of IgG, IgG2a and IgG1 OVA-specific antibodies as compared to the actions of LF or MPL alone. In the model of delayed type hypersensitivity (DTH) the strongest immune response was demonstrated with OVA administered subcutaneously, admixed with the complex. Administration of the complex in incomplete Freund’s adjuvant, together with a sensitizing dose of antigen, was similarly effective as immunization with complete Freund’s adjuvant. The complex also significantly enhanced the DTH response to orally administered Calmette-Guérin bacilli. In summary, the new type of adjuvant, the LF-MPL complex, was described. Its activity surpassed the adjuvant action of both constituents tested separately in the humoral and cellular immune responses in mice. The plausible mode of action of the new adjuvant is discussed.