RNase-sensitive and RNase-insensitive protective components isolated fromListeria monocytogenes

Crude ribosomes were isolated fromListeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice againstListeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment; but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction 11, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part nonspecific, because both preparations also protected mice againstL. monocytogenes serotype 3,Streptococcus pneumoniae andPseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations fromP. aeruginosa.     

Enhancement of delayed-type hypersensitivity and induction of interferon by the lipophilic agents DDA and CP-20,961

The lipophilic amines dimethyl dioctadecyl ammonium bromide (DDA) and N,N-dioctadecyl-N′, N′-bis(2-hydroxyethyl)propanediamine (CP-20,961) are compared on their capacities to induce interferon, nonspecific protection to viral infection, and enhancement of delayed-type hypersensitivity (DH). DDA, a well-known adjuvant for the induction of DH is a moderate interferon inducer like CP-20,961. On the other hand, CP-20,961, a known interferon inducer and resembling in structure DDA, is shown to enhance DH to inactivated Semliki Forest virus (SFV). Nonspecific protection to challenge with a lethal dose of either SFV or encephalomyocarditis (EMC) virus was induced on injection of both compounds.     

Protective activities of ribosomal ribonucleic acid and lipopolysaccharide of Pseudomonas aeruginosa: a comparative study

Ribosomal ribonucleic acid (RNA) and lipopolysaccharide (LPS) from P. aeruginosa were compared with respect to their protective activities in mice against an infection with P. aeruginosa. This study is concentrated on the protective activity of RNA. RNA isolated from purified ribosomes did not contain LPS as determined with the Limulus test. Injection of RNA with the adjuvant dimethyldioctadecylammonium bromide (DDA) protected mice against P. aeruginosa without inducing LPS-specific antibodies. C₃H/HeJ mice which are relatively insensitive to the protective activity of LPS could be protected with RNA. The protective activities of RNA and LPS from a mutant strain of P. aeruginosa, PAC 605, containing defective lipopolysaccharide, were compared with the protective activities of RNA and LPS from the parent strain, PAC IR. The protective activity of LPS from PAC 605 was 1000 fold lower than the protective activity of LPS from PAC IR. RNA preparations of both strains induced similar percentages of survival. The protective activity of ribosomal RNA from P. aeruginosa was nonspecific since mice were also protected against a heterologous serotype of P. aeruginosa and against Escherichia coli. RNA from ribosomes of P. aeruginosa, E. coli and the non-lipopolysaccharide containing Saccharomyces cerevisiae had similar protective activities. No protection was obtained with the ribonucleic acid from the E. coli phage MS 2. It is concluded that ribosomal RNA has protective activities distinct from those of LPS.     

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.     

Ribonuclease-sensitive ribosomal vaccines

This paper presents an analysis of the protective properties of the components in ribonuclease (RNase)-sensitive ribosomal vaccines, in particular the ribonucleic acid (RNA). The protective activities in mice of purified ribosomes derived fromPseudomonas aeruginosa and fromListeria monocytogenes were compared. Both ribosomal vaccines had to be combined with the adjuvant dimethyldioctadecylammonium bromide (DDA) in order to be protective, and both lost their activity after RNase treatment. The ribosomal vaccines as well as RNA purified from the ribosomes induced non-specific protection. Intraperitoneal injection of RNA with DDA induced an influx of peritoneal cells. Furthermore, RNA with DDA activated macrophages as shown by, a.o., enhanced phagocytic activity and killing capacity forL. monocytogenes. The results suggest that the observed macrophage activation is probably T-cell-independent. With regard to the ribosomal vaccine ofP. aeruginosa it is concluded that RNA also contributed to the protective activity by increasing the humoral response against suboptimal concentrations of contaminating cell surface antigens. In conclusion, it is proposed that ribosomal vaccines may be considered as a combination of a non-specific immunomodulator (RNA) with pathogen-specific cell surface antigens. This concept of ribosomal vaccines is discussed in relation to the literature concerning RNase-sensitive ribosomal vaccines.     

RIG‐I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill‐defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG‐I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG‐I‐dependent IL‐1β‐production and inflammasome activation. The signalling molecule CARD9 contributed to IL‐1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG‐I provides a mechanistic explanation for efficient induction of immunity by live bacteria.     

Immunogenicity and Protective Efficacy of a Vero Cell Culture-Derived Whole-Virus H7N9 Vaccine in Mice and Guinea Pigs

BackgroundA novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models.MethodsAntibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine.ResultsThe whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic.ConclusionsThe induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.     

Stimulation of protective and cross-protective immunity against influenza B virus after adjuvant mucosal immunization of mice

Intratracheal immunization of mice with inactivated influenza B virus and delipidated Bacillus firmus as adjuvant increases protection of mice against infection with the homologous virus strain and induces cross-protection: mice immunized by influenza virus B/Yamanashi 166/98 were protected even against phylogenetically distant virus drift variant B/Lee/40 lethal for mice.     

Differential effects of concanavalin A and phytohemagglutinin on murine immunity. Suppression and enhancement of cell-mediated immunity.

To assess the effects of the selective T-cell mitogens concanavalin A (Con A) and phytohemagglutinin P (PHA) on cell-mediated immunity (CMI), the mitogens were injected before, with, or after intravenous (iv) challenge of mice with Listeria monocytogenes. Mitogenic treatment differentially influenced the CMI response to Listeria. Con A enhanced listericidal activity when given before or with Listeria challenge, but Con A suppressed the CMI response when given after infection with Listeria. In contrast, PHA enhanced listericidal activity at all intervals. Since Con A, but not PHA, affected the growth of Listeria in the spleens of mice 24 and 48 hr after infection, Con A was shown to have an immediate effect on the development of listericidal activity and PHA was shown to have a delayed effect. In addition, Con A induction of immediate nonspecific listericidal activity was short-lived, while PHA induced a longer-lasting effect on resistance to Listeria. The mitogen-induced effects in the CMI response to Listeria were shown to be dependent upon the activities of activated T cells. The enhancement and suppression of listericidal activity appears to result from the activation of different T-cell subpopulations, known to be stimulated preferentially by Con A or PHA.     

Studies of delayed hypersensitivity to L. Monocytogenes in mice: nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH.Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.     

Susceptibility of C5-deficient mice to listeriosis: modulation by Concanavalin A.

The susceptibility of normal (C5n) and C5 deficient (C5d) mice to Listeria monocytogenes infection and the effects of concanavalin A (Con A) on host resistance to Listeria were investigated. C5d mice were shown to be more susceptible to Listeria, but their morbidity was enhanced only with certain doses, 2 to 6 × 10⁴ viable Listeria. Listeria infection produced immune cells in both C5n and C5d mice as assessed in a passive transfer study, but the C5d immune cells significantly suppressed Listeria growth only when transferred into C5n mice. Con A altered the response to Listeria in both C5n and C5d mice. The growth of Listeria in the spleens of C5n and C5d mice, which were treated with Con A 1 day before infection, was clearly suppressed, and their mortality was reduced. Con A given 1 day after infection (Day +1) consistently enhanced the early (Days 1 to 3 post-infection) accumulation of Listeria in the spleens, and A/J (C5d) and CBA/J (C5n) mortality was increased but B10.D2 (C5d and C5n) mortality was reduced. Con A given 7 days before Listeria infection produced no differential effects on the early growth of Listeria in the spleens of C5n and C5d mice, but most C5n mice survived a lethal dose of Listeria and the C5d mice died. Since C5n and C5d mice differ in their resistance to Listeria, Con A (Day ?7), a selective T-cell mitogen, produced differential effects in C5n and C5d mice and Listeria is believed to induce predominantly a cell-mediated imune response, T cells and complement components independently or cooperatively appear to play a role in immunity to Listeria.     

An Inactivated Cell Culture Japanese Encephalitis Vaccine (JE-ADVAX) Formulated with Delta Inulin Adjuvant Provides Robust Heterologous Protection against West Nile Encephalitis via Cross-Protective Memory B Cells and Neutralizing Antibody

West Nile virus (WNV), currently the cause of a serious U.S. epidemic, is a mosquito-borne flavivirus and member of the Japanese encephalitis (JE) serocomplex. There is currently no approved human WNV vaccine, and treatment options remain limited, resulting in significant mortality and morbidity from human infection. Given the availability of approved human JE vaccines, this study asked whether the JE-ADVAX vaccine, which contains an inactivated cell culture JE virus antigen formulated with Advax delta inulin adjuvant, could provide heterologous protection against WNV infection in wild-type and β2-microglobulin-deficient (β2m^−/−) murine models. Mice immunized twice or even once with JE-ADVAX were protected against lethal WNV challenge even when mice had low or absent serum cross-neutralizing WNV titers prior to challenge. Similarly, β2m^−/− mice immunized with JE-ADVAX were protected against lethal WNV challenge in the absence of CD8⁺ T cells and prechallenge WNV antibody titers. Protection against WNV could be adoptively transferred to naive mice by memory B cells from JE-ADVAX-immunized animals. Hence, in addition to increasing serum cross-neutralizing antibody titers, JE-ADVAX induced a memory B-cell population able to provide heterologous protection against WNV challenge. Heterologous protection was reduced when JE vaccine antigen was administered alone without Advax, confirming the importance of the adjuvant to induction of cross-protective immunity. In the absence of an approved human WNV vaccine, JE-ADVAX could provide an alternative approach for control of a major human WNV epidemic.     

Intranasal Immunization of Mice Against Naegleria fowleri1

ABSTRACT. The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 × 10³ amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10³ amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.     

Cadmium-Induced Adaptive Resistance and Cross-Resistance to Zinc in Xanthomonas campestris

Cadmium (Cd) and zinc (Zn) are environmental pollutants affecting both soil and water. The toxicity resulting from the exposure of Xanthomonas campestris, a soil bacterium and plant pathogen, to these metals was investigated. Pretreatment of X. campestris with sub-lethal concentrations of Cd induced adaptive protection against subsequent exposure to lethal doses of Cd. Moreover, Cd-induced cells also showed cross-resistance to lethal concentrations of Zn. These induced protections required newly synthesized proteins. Unexpectedly, Zn-induced cells did not exhibit adaptive protection against lethal concentrations of Zn or Cd. These data suggested that the increased resistance to Cd and Zn killing probably involved other protective mechanisms in addition to ion efflux.Received: 4 December 2002 / Accepted: 24 January 2003     

Antigens of Subcellular Fractions of Trypanosoma cruzi. III. Humoral immune response and histopathology of immunized mice*

SYNOPSIS. The relation of humoral antibody response to protection was evaluated in mice immunized with whole homogenates of Trypanosoma cruzi or with its flagellar fraction by direct agglutination and indirect fluorescent antibody test as well as by lytic and neutralizing activity against blood trypomastigotes. The results indicated that lytic antibodies were not implicated directly in protection against these trypanosomes. It was evident from histopathologic examination that the higher the degree of protection achieved, the lower the tissue damage observed in the challenged mice. Serum-neutralizing activity was highest in the groups protected most effectively.     

Subpatent infection with nucleoside transporter 1‐deficient Plasmodium blood stage parasites confers sterile protection against lethal malaria in mice

Repeated immunizations with whole Plasmodium blood stage parasites and concomitant drug cure of infection confer protective immunity against parasite challenge in mice, monkeys and humans. Moreover, it was recently shown that infections with genetically modified rodent malaria blood stage parasites conferred sterile protection against lethal blood stage challenge. However, in these models vaccination resulted in high parasitemias and, in consequence, carries risk of vaccine‐induced pathology and death. Herein, we generated a novel, completely blood stage‐attenuated P. yoelii rodent malaria strain by targeted deletion of parasite nucleoside transporter 1 (NT1). Immunization of inbred and outbred mouse strains with a single low dose of Pynt1^‐ blood stages did not induce any patent infections and conferred complete sterile protection against lethal heterologous blood stage and sporozoite challenges. Partial protection was observed against lethal challenges with another parasite species, P. berghei. Importantly, subcutaneous immunization with Pynt1^‐ conferred sterile protection against lethal blood stage challenges. We show that cellular and humoral immune responses are both essential for sterile protection. The study demonstrates that genetic manipulation provides a platform for the designed, complete attenuation of malaria parasite blood stages and suggests testing the safety and efficacy of P. falciparum NT1 knockout strains in humans.     

Dendritic cells from mice neonatally vaccinated with modified vaccinia virus Ankara transfer resistance against herpes simplex virus type I to naive one-week-old mice

Modified vaccinia Ankara (MVA) is an attenuated virus. MVA induces the production of IFN and Flt3-L (FL), which results in the expansion of dendritic cells (DC) and enhanced resistance against viral infections. We report on the interplay among IFN, FL, and DC in the resistance against heterologous virus after injection of neonatal mice with MVA. The induction of serum FL was tested on day 2, and the expansion of DC was tested 1 wk after treatment with MVA. At this time point the resistance against infection with heterologous virus was also determined. After MVA treatment, serum FL was enhanced, and DC, including plasmacytoid cells in spleen, were increased in number. Mice that lacked functional IFN type I and II systems failed to increase both the concentration of FL and the number of DC. Treatment with MVA enhanced resistance against HSV-1 in wild-type animals 100-fold, but animals without a functional IFN system were not protected. Transfer of CD11c(+) cells from MVA-treated mice into naive animals protected against lethal infection with HSV-1. Thus, although the increased resistance could be largely attributed to the increase in activation of IFN-producing plasmacytoid cells, this, in turn, depends on a complex interplay between the DC and T cell systems involving both FL and IFNs.     

Effect of polysaccharides from Ganoderma applanatum on immune responses. I. Enhancing effect on the induction of delayed hypersensitivity in mice.

Prior intraperitoneal (i.p.) or oral administration of the polysaccharide preparation from a kind of mushroom, Ganoderma applanatum (Pers.) Pat. of Basidiomycetes, exerted an enhancing effect on the induction of delayed hypersensitivity (DH) to protein antigen as measured by the footpad reaction (FPR), and expanded the size of T cell memory for the IgG antibody response. One of the active principles was partially purified and found to be associated with a polysaccharide-rich fraction. The induction of DH was enhanced by treatment with an appropriate dose of the mushroom extract, whereas increasing the dose resulted in almost complete loss of the enhancing activity. The mechanism for the enhancing effect of the mushroom extract on the induction of DH was explored by the adoptive cell transfer technique. Although an i.p. injection of methylated bacterial α-amylase (M-BαA) in incomplete Freund's adjuvant (IFA) has been found to generate in the spleen the antigen-specific suppressor T cells capable of inhibiting the induction of DH 5 days after immunization, the same treatment of mice given prior injections of the mushroom extract did not raise the suppressor cell activity, but transfer of these spleen cells (6 × 10⁷) into syngeneic recipient mice which had been primed with a subcutaneous (s.c.) injection of M-BαA in complete Freund's adjuvant (CFA) resulted in substantial amplification of the expression of DH. The absence of effector T cells for DH in the transferred spleen cells was confirmed by the failure to transfer DH into cyclophosphamide (CY)-treated mice with the amplifying cells. The amplifying activity was antigen-nonspecific and mediated by cells sensitive to treatment with anti-θ antiserum plus complement. Therefore, the nonspecific enhancing effect of the mushroom extract could not be explained by the possibility that pretreatment with the extract eliminated the antigen-specific suppressor T cells. Other adoptive cell transfer experiments revealed that nylon wool-passed cells from mice unprimed but treated with the mushroom extract were able to exert an enhancing activity on the expression of effector T cells in DH. The results indicate that the treatment with an appropriate dose of the extract enhances the induction of DH by activation of the nonspecific amplifier T cells.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.