Functional modulation of CD8+ T cell by approved novel immune enhancer: Nocardia rubra Cell-Wall Skeletons (Nr-CWS).

Nocardia rubra cell wall skeleton (Nr-CWS) has been reported to have innate immunostimulating and anti-tumor activities. However, the immunomodulatory effects of Nr-CWS on CD8+ T cells and their related mechanisms are still unknown. In this work, our team purified CD8+T cells from spleen cells and explored the phenotype and function of NR-CWS in vitro on CD8+T cells. We observed that Nr-CWS can significantly up-regulate the expression of CD69 and CD25 on CD8+T cells, with no significant effect on apoptosis or cell death of CD8+T cells that occurs in vitro during culture. In addition, the effect of perforin and granzyme B was increased after Nr-CWS treatment, but did not substantially alter the expression of TRAIL and FasL. A variety of cytokine analyses have shown that of the cytokines examined (IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6 and IL-10), only IFN-γ and TNF The increase in -α was more pronounced, and the effect of Nr-CWS in CD8+T cell culture medium on CD8+ T cells was independent of Th cells. Our results demonstrated that Nr-CWS could up-regulate CD69 and CD25 expression on CD8+T cells, promoting IFN-γ and TNF-α secretion, and enhancing perforin and granzyme B production. Thus Nr-CWS may have Immunoaugmenting therapeutic activity via an increase in CD8+T cells response.

Immune adjuvant therapy using Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) in advanced malignancies: A phase 1 study of safety and immunogenicity assessments.

The cell wall skeleton of Bacillus Calmette-Guérin (BCG-CWS) is a bioactive component that is a strong immune adjuvant for cancer immunotherapy. BCG-CWS activates the innate immune system through various pattern recognition receptors and is expected to elicit antigen-specific cellular immune responses when co-administered with tumor antigens. To determine the recommended dose (RD) of BCG-CWS based on its safety profile, we conducted a phase I dose-escalation study of BCG-CWS in combination with WT1 peptide for patients with advanced cancer.The primary endpoint was the proportion of treatment-related adverse events (AEs) at each BCG-CWS dose. The secondary endpoints were immune responses and clinical effects. A BCG-CWS dose of 50, 100, or 200 µg/body was administered intradermally on days 0, 7, 21, and 42, followed by 2 mg of WT1 peptide on the next day. For the escalation of a dose level, 3 + 3 design was used.Study subjects were 18 patients with advanced WT1-expressing cancers refractory to standard anti-cancer therapies (7 melanoma, 5 colorectal, 4 hepatobiliary, 1 ovarian, and 1 lung). Dose-limiting toxicity occurred in the form of local skin reactions in 2 patients at a dose of 200 µg although no serious treatment-related systemic AEs were observed. Neutrophils and monocytes transiently increased in response to BCG-CWS. Some patients demonstrated the induction of the CD4 T cell subset and its differentiation from the naïve to memory phenotype, resulting in a tumor response.The RD of BCG-CWS was determined to be 100 µg/body. This dose was well tolerated and showed promising clinical effects with the induction of an appropriate immune response.

Application of BCG-CWS as a Systemic Adjuvant by Using Nanoparticulation Technology.

The intravesical instillation of live Bacillus Calmette-Guerin (BCG) for treating bladder cancer is a powerful cancer immunotherapy. The BCG cell wall skeleton (BCG-CWS) is the main component of the adjuvant, leading to the induction of antitumor immunity. However, the use of live BCG and BCG-CWS is currently limited to local administration because of the infectiousness of live BCG and the insolubility of BCG-CWS. We previously developed a water-dispersible nanoparticle (NP) formulation of BCG-CWS (CWS-NP), which could be used to apply BCG components for use as a systemically injected adjuvant for the treatment of cancers other than bladder cancer. In the present study, we examined the possible use of CWS-NP for cancer immunotherapy, when intravenously administered. The CWS-NP was a highly uniform dispersion and showed no aggregation in serum. The intravenously injected CWS-NP accumulated in the spleen and was efficiently taken up by dendritic cells, leading to their maturation. The coadministration of CWS-NP and ovalbumin (OVA) loaded NP resulted in the generation of OVA-specific cytotoxic T cells and inhibited the growth of E.G7-OVA tumors. These results represent the first findings related to the use of systemically injected CWS-NP as an adjuvant for cancer immunotherapy.

Nanoparticulation of BCG-CWS for application to bladder cancer therapy.

The Mycobacterium bovis Bacille Calmette-Guerin cell wall skeleton (BCG-CWS) could be used to replace live BCG as a bladder cancer drug. However, because BCG-CWS is poorly soluble, has a strong-negative charge, very high molecular weight and heterogeneity in size of tens of µm, it cannot be used in such an application. We report herein on the development of a novel packaging method that permits BCG-CWS to be encapsulated into 166nm-sized lipid particles. The BCG-CWS encapsulated nano particle (CWS-NP) has a high uniformity and can be easily dispersed. Thus, it has the potential for use as a packaging method that would advance the scope of applications of BCG-CWS as a bladder cancer drug. In a functional evaluation, CWS-NP was efficiently taken up by mouse bladder tumor (MBT-2) cells in vitro and inhibited tumor growth in mice bearing MBT-2 tumors. Moreover, intravesically administered CWS-NP showed significant antitumor effects in a rat model with naturally developed bladder cancer. An enhancement in Th1 differentiation by CWS-NP was also confirmed in human T cells. In conclusion, CWS-NP represents a promising delivery system for BCG-CWS for clinical development as a potent bladder cancer drug.

Enhancement of Th1 immune responses to recombinant influenza nucleoprotein by Ribi adjuvant.

A broad coverage influenza vaccine against multiple viral strains based on the viral nucleoprotein (NP) is a goal pursued by many laboratories. If the goal is to formulate the vaccine with recombinant NP it is essential to count on adjuvants capable of inducing cellular immunity. This work have studied the effect of the monophosphoryl lipid A and trehalose dimycolate, known as the Ribi Adjuvant System (RAS), in the immune response induced in mice immunized with recombinant NP. The NP was formulated with RAS and used to immunize BALB/c mice. Immunizations with NP-RAS increased the humoral and cellular immune responses compared to unadjuvanted NP. The predominant antibody isotype was IgG2a, suggesting the development of a Th1 response. Analysis of the cytokines from mice immunized with NP-RAS showed a significant increase in the production of IFN-g and a decreased production of IL-10 and IL-4 compared to controls without RAS. These results are similar to those usually obtained using Freund's adjuvant, known to induce Th1 and CTL responses when co-administered with purified proteins, and suggest that a similar approach may be possible to enhance the performance of a T-cell vaccine containing NP.

Epitope selection to male specific antigens for sex selection in swine.

Immunological approaches to gender selection have been contemplated since the discovery of the family of male-specific H-Y antigens found only on the surface of male cells. H-Y antigens are able to elicit an immune reaction when cells or tissues from a male donor are grafted to a female recipient. We describe here the development and testing of an inexpensive approach using polyclonal antibodies against four specific H-Y outer membrane proteins male enhanced antigen 1 (MEA 1), male enhanced antigen 2 (MEA 2), sex determining region Y (SRY) and testis determining factor (TDF). Epitopes based on hydrophilic primary sequences of the proteins were synthesized, N-terminal biotin-labeled, linked to streptavidin and mixed with a Ribi adjuvant prior to immunization in rabbits. The antiserum was tested to determine affinity to swine spermatozoa using anti-motility, flow cytometry and motility and sedimentation chambers. Fluorescent microscopy and fluorescent in situ hybridization (FISH) was used to identify the percentage of motile spermatozoa that contained the Y chromosome. We found that the polyclonal antibodies had high affinity to the spermatozoa leading to a cessation of motility. Furthermore, the majority of these non-motile spermatozoa contained the Y chromosome. We conclude that the use of polyclonal antiserum against synthetic H-Y peptide antigens may be an inexpensive and simple means to inhibit the motility of swine spermatozoa bearing the Y chromosome.

Inactivation of respiratory syncytial virus by zinc finger reactive compounds.

BACKGROUND: Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins. RESULTS: Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease. CONCLUSIONS: This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.

Mice with pulmonary tuberculosis treated with Mycobacterium vaccae develop strikingly enhanced recall gamma interferon responses to M. vaccae cell wall skeleton.

Whole heat-killed Mycobacterium vaccae is used as an immunotherapeutic agent in tuberculosis (TB), but the compound(s) that triggers its immunostimulatory ability is not known. Here, we show that among different subcellular fractions, the cell wall skeleton induced a prominent expression of gamma interferon in splenocytes from both non-TB and TB M. vaccae-treated mice.

Active immunization of hamsters against Clostridium difficile infection using surface-layer protein.

Clostridium difficile is the leading cause of infectious antibiotic-associated diarrhoea, particularly among the elderly. Its surface-layer protein (SLP) was tested as a vaccine component in a series of immunization and challenge experiments with Golden Syrian hamsters, combined with different systemic and mucosal adjuvants. Some regimens were also tested in a nonchallenge BALB/c mouse model, enabling closer monitoring of the immune response. None of the regimens conferred complete protection in the hamster model, and antibody stimulation was variable within regimens, and generally modest or poor. Mice displayed stronger antibody responses to SLP compared with hamsters. Two hamsters of five given SLP with Ribi (monophosphoryl lipid A and synthetic trehalose dicorynomycolate) survived the challenge, as did two of three given SLP with Ribi and cholera toxin. This modest trend to protection is interpreted with caution, because the survivors had low anti-SLP serum antibody titres. The hamsters were an outbred line, and subject to more genetic variability than inbred animals; however, BALB/c mice also showed strongly variable antibody responses. There is a clear need for better adjuvants for single-component vaccines, particularly for mucosal delivery. The hamster challenge model may need to be modified to be useful in active immunization experiments with SLP.

Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis.

Burkholderia pseudomallei is the causative agent of melioidosis, which is a major cause of morbidity and mortality in endemic regions. Currently there is no human vaccine against melioidosis. In this study, LPS or capsular polysaccharide was used to immunize BALB/c mice. The different polysaccharide antigens induced antibody responses. Mice vaccinated with LPS developed predominantly IgM and IgG3 responses. Contrastingly, mice vaccinated with capsular polysaccharide developed a predominantly IgG2b response. After immunization, mice were challenged by the intra-peritoneal route and an increased mean time to death was observed compared with unvaccinated controls. Immunization with LPS provided an optimal protective response. Mice challenged by the aerosol route showed a small increase in the mean time to death compared with the unvaccinated controls. The passive transfer of antigen from immunized into naive mice provided protection against a subsequent challenge. This study is the first time antigens protective by active immunization have been identified and suggests that polysaccharides have potential as vaccine candidates against melioidosis.

Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions.

Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-microg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.

Enhancement of antigen-specific immunity via the TLR4 ligands MPL adjuvant and Ribi.529.

MPL (Corixa) adjuvant is a chemically modified derivative of lipopolysaccharide that displays greatly reduced toxicity while maintaining most of the immunostimulatory activity of lipopolysaccharide. MPL adjuvant has been used extensively in clinical trials as a component in prophylactic and therapeutic vaccines targeting infectious disease, cancer and allergies. With over 33,000 doses administered to date, MPL adjuvant has emerged as a safe and effective vaccine adjuvant. Recently, scientists at Corixa Corporation have developed a library of synthetic lipid A mimetics (aminoalkyl glucosaminide 4-phosphates) with demonstrated immunostimulatory properties. Similar to MPL adjuvant, these synthetic compounds signal through Toll-like receptor 4 to stimulate the innate immune system. One of these compounds, Ribi.529 (RC-529), has emerged as a leading adjuvant with a similar efficacy and safety profile to MPL adjuvant in both preclinical and clinical studies.

Reduced cerebral infection of Neospora caninum-infected mice after vaccination with recombinant microneme protein NcMIC3 and ribi adjuvant.

C57BL/6 mice were vaccinated with a bacterially expressed and purified polyhistidine-tagged full-length version of the microneme protein NcMIC3 (recNcMIC3) emulsified in Ribi Adjuvant System (RAS). Subsequently, they were challenged by intraperitoneal inoculation of 2 x 10(6) live Neospora caninum tachyzoites. As controls, groups of mice received phosphate-buffered saline (PBS)-RAS alone (adjuvant control) or were treated with PBS before infection (infection control). The protective effect of vaccination was assessed by Neospora-specific polymerase chain reaction (PCR), immunohistochemical investigation of brain tissue, and serological means (enzyme-linked immunosorbent assay). Assessment by PCR performed on DNA from different organs revealed that in all treatment groups parasite DNA could only be detected in brain tissue. According to the PCR results. the recNcMIC3 vaccine conferred protection to 75% of mice (n = 16 in 2 independent experiments), whereas application of PBS-RAS and of PBS alone resulted in protection of 12.5% and 0% of mice, respectively (n = 16 as above). Mice in the PBS-treated infection control group were affected by evident clinical signs of neosporosis starting on day 6 postinfection (p.i.). Conversely, none of the animals treated with either PBS-RAS or recNcMIC3 exhibited any symptoms until day 21 p.i. Immunohistochemical staining of paraffin-embedded brain tissue sections confirmed the protective effect of recNcMIC3 vaccination. Quantitative Neospora-specific real-time PCR revealed that infection intensities were lower in the brain tissues of recNcMIC3-vaccinated mice compared with PBS-RAS-treated adjuvant control mice. Serological analysis showed that the protective effect observed in recNcMIC3-vaccinated mice was associated with a Th2-type IgG1 antibody response directed against native NcMIC3 and a mixed IgG1-IgG2a antibody response directed against the recombinant antigen itself. Taken together, these results demonstrated that recombinant NcMIC3 vaccine confers a significant protectivity against experimentally induced cerebral neosporosis in mice.

Antigenicity of Mycobacterium paratuberculosis superoxide dismutase in mice.

Mycobacterium paratuberculosis (MPT) is the etiologic agent of paratuberculosis. The disease is prevalent in cattle worldwide, and exacts a heavy financial toll. Effective control requires the development of acellular vaccines offering a better protection than the current available vaccines without side effects and allowing the discrimination between infected and vaccinated animals. We studied the immune response of mice to the MPT superoxide dismutase (SOD) alone or adjuvanted by Ribi. We cloned, overexpressed and purified this antigen in Escherichia coli. Spleen cells from immunized mice, after exposure to recombinant MPT SOD (MPT rSOD), produced significant levels of IFNgamma, TNFalpha and IL-6. IFNgamma and TNFalpha production was increased by the addition of Ribi. In contrast, low levels of NO, IL-4 and IL-10 were secreted by spleen cells culture from immunized mice. The immunoglobulin isotype distribution analysis showed that Ribi adjuvant clearly induced a significantly higher anti-MPT rSOD antibody production of all classes tested and decreased the IgG1/IgG2a ratio thus improving the Th1 response. Delayed-type hypersensitivity responses in mice footpads were observed only in mice immunized with MPT rSOD emulsified in Ribi. Vaccination of MPT rSOD emulsified with Ribi induced both a Th2 and Th1 type of immune response with the later slightly more pronounced. The results presented here on the immunogenicity of MPT SOD suggest that this antigen should be further tested as a candidate antigen for a future acellular vaccine against paratuberculosis.

Immune responses and side effects of five different oil-based adjuvants in mice.

In this study, five different oil based adjuvants were compared to assess efficacy and side effects. Mice were injected subcutaneously (s.c.) or intraperitoneally (i.p.) with a weak immunogen (synthetic peptide) emulsified in Freund's adjuvant (FA), Specol, RIBI, TiterMax or Montanide ISA50. Efficacy of adjuvants was evaluated based on their properties to induce peptide specific IgG1, IgG2a and total IgG antibodies, native protein cross-reactive antibodies and cytokine production. Side effects were evaluated based on clinical and behavioural abnormalities, and (histo)pathological changes. Although marked differences in isotype profile and height of titre are observed among the different adjuvants used, we found that FA, Montanide ISA50 and Specol worked equally well in the s.c. and i.p. route, TiterMax functioned only when given i.p. and RIBI also did not perform up to par. The number of cytokine (interferon-gamma and interleukin-4) producing spleen cells was significantly higher after injection of RIBI compared with other adjuvants. Injection of FA or TiterMax resulted in severe pathological changes while after RIBI injection minimal changes were observed. In conclusion, high peptide specific antibody levels with limited side effects can be obtained by s.c. injection of peptide combined with Montanide ISA50 or Specol as alternatives to FA.

Former poison gas workers and cancer: incidence and inhibition of tumor formation by treatment with biological response modifier N-CWS.

Mustard gas is known to have mutagenic and carcinogenic effects on animal and human cells. In this report, 1,632 male Japanese who worked in poison gas factories at some time between the years 1927 and 1945 were studied to determine comparative risk for development of cancer, the reference population being data on Japanese males overall. The standardized mortality ratio (SMR) for lung cancer in workers directly and indirectly involved in the production of mustard gas was significantly elevated. In addition, SMR for lung cancer in worker who had worked for more than 5 years was also significantly elevated. Thus, poison gas workers who had engaged in the production of mustard gas or related work for more than 5 years are a high-risk group for lung cancer. Under the cancer preventive program, Nocardia rubra cell-wall skeleton (N-CWS) was administered to 146 former poison gas workers. During a 4.5 year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of smoking level, a significant suppression of development of cancers was noted in the N-CWS-treated workers as compared to the untreated controls. Although the molecular mechanisms of carcinogenesis in former poison gas workers remains unclear, our study proposes the possible effect of biological response modifiers in the prevention of cancer development in high-risk human subjects.

Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits.

In three experiments we evaluated several types of adjuvants as an alternative to Freund's adjuvant (FA). In the first experiment three adjuvant preparations (a water-in-oil emulsion (Specol), a combination preparation of monophosphoryl lipid A + trehalose dimycolate + cell wall skeleton and a non-ionic block polymer surfactant (TiterMax)) were evaluated. The adjuvants were combined with three different types of weak immunogenic antigens (synthetic peptide, glycolipid and particulate antigen) and administered following the intramuscular and subcutaneous route. The evaluation was based on clinical, pathological and immunological parameters. The animals did not appear to be severely or chronically impaired by the experiment. After injection of the RIBI adjuvant, side effects of the same severity as with FA were induced, while low antibody titers were produced. TiterMax caused few side effects, while antibody responses were very low. In comparing Specol and FA, Specol had far fewer adverse effects than FA. However, Specol had immunostimulating properties of the same level as FA. In the second experiment, the effect of injected volume of FA on side effects and antibody titer was studied. Immunization of rabbits with a total of 0.5 ml FA at different sites does not seem to increase the immune response when compared with the immune response seen after injection of 0.5 ml FA at one site. However side effects were seen in all the animals. In the third experiment, the side effects following intradermal (i.d.) injection of the adjuvants were studied. After i.d. injection of FA or RIBI, undesirable effects were found. No side effects occurred after i.d. injection of Specol or TiterMax. From the studies it is concluded that Specol is an alternative to FA for hyperactivation of the immune response in rabbits.

Clinical usefulness of continuous administration of Nocardia rubra cell wall skeleton (N-CWS) in diffuse panbronchiolitis (DPB).

Eight patients with diffuse panbronchiolitis (DPB) who had repeated intractable airway infections were continuously treated with Nocardia rubra cell wall skeleton (N-CWS), a biological response modifier. As a result, subjective symptoms were reduced in 6 patients. Antibiotics therapy could be discontinued completely in two patients and the dose of antibiotics could be reduced considerably in two other patients. No adverse reactions in relation to N-CWS were observed. These results suggest that N-CWS is effective in treating erythromycin-resistant DPB.