Monoclonal antibody to fungal glucosylceramide protects mice against lethal Cryptococcus neoformans infection

Glucosylceramides (GlcCer) are involved in the regulation of Cryptococcus neoformans virulence. In the present study, we demonstrate that passive immunization with a monoclonal antibody to GlcCer significantly reduces host inflammation and prolongs the survival of mice lethally infected with C. neoformans, revealing a potential therapeutic strategy to control cryptococcosis.     

An anti-idiotype antibody which mimics the inner-core region of lipopolysaccharide protects mice against a lethal challenge with endotoxin.

Recently, we described the generation and characterization of an Armenian hamster Ab2 beta anti-idiotype monoclonal antibody (MAb4G2) specific for the binding site of a mouse monoclonal antibody, MAbY1-4A6, directed against the conserved 2-keto-3-deoxyoctulosonate (Kdo)-containing inner-core region of lipopolysaccharide (LPS) (S. K. Field, M. Pollack, and D. C. Morrison, Microb. Pathog. 15:103-120, 1993). In that study, mice and hamster immunized with MAb4G2 generated serum immunoglobulin G and M (IgG and IgM) antibodies which cross-react with Salmonella minnesota R595-chemotype rough mutant LPS (Re-LPS). In this report, we demonstrate that in C3Heb/FeJ mice, MAb4G2 elicits an immune response which is characterized by specific binding of antibody to Re-LPS, as assessed by enzyme-linked immunosorbent assay. The practical use of MAb4G2 as a potentially effective therapeutic agent against gram-negative bacterial sepsis is suggested by the demonstration that immunization of these mice with MAb4G2 results in significant protection of D-galactosamine-sensitized animals against an otherwise lethal dose of Re-LPS. Assessment of the temporal changes in Re-LPS-specific serum antibody titers from mice immunized with MAb4G2 or Re-LPS over a 40-day period indicates that immunization with Re-LPS elicits significantly higher titers of serum IgM antibodies compared with those in animals immunized with MAb4G2. Conversely, two immunizations with MAb4G2 result in an up to 10-fold increase in anti-Re-LPS-specific IgG serum antibody titers relative to those obtained in mice immunized with Re-LPS. Nineteen days after the secondary boost with MAb4G2, anti-Re-LPS-specific IgG serum antibody titers were significantly higher (three- to fourfold) compared with those in Re-LPS-treated animals. Initial immunization with the anti-idiotype antibody primes animals for enhanced secondary responses to Re-LPS, as assessed by the titers of anti-Re-LPS-specific IgG profiles. These data suggest the potential utility of MAb4G2 as a candidate vaccine against the lethal properties of gram-negative bacterial LPS.     

Iron deficiency protects inbred mice against infection with Plasmodium chabaudi.

Plasmodium chabaudi infections of NFR/N mice made anemic by dietary iron deficiency produced mortalities of 25% (male) and 7% (female) compared with 100% in iron-sufficient controls. When iron-deficient mice convalescing from the primary infection were returned to the normal diet, 100% experienced recrudescent parasitemia. No recrudescence occurred in mice maintained on the iron-deficient diet.     

Administration of antibody to the lung protects mice against pneumonic plague

Intratracheal delivery of aerosolized monoclonal antibodies with specificity for Yersinia pestis LcrV and F1 antigens protected mice in a model of pneumonic plague. These data support the utility of inhaled antibodies as a fast-acting postexposure treatment for plague.     

Oral immunization with pBsVP6-transgenic alfalfa protects mice against rotavirus infection

A critical factor in edible plant-derived vaccine development is adequate expression of the exogenous antigens in transgenic plants. We synthesized a codon-optimized gene (sVP6) encoding the VP6 protein of human group A rotavirus and inserted it into the alfalfa genome using agrobacterium-mediated transformation. As much as 0.28% of the total soluble protein of the pBsVP6-transgenic alfalfa was sVP6. Female BALB/c mice were gavaged weekly with 10 mg of transgenic alfalfa extract containing 24 microg of sVP6 protein and 10 microg of CpG-rich oligodeoxynucleotides as mucosal adjuvant. Immunized mice developed high titers of anti-VP6 serum IgG and mucosal IgA. Offspring of immunized dams developed less severe diarrhea after challenge with simian rotavirus SA-11, indicating that antibodies generated in the dams provided passive heterotypic protection to the pups. These results suggest that oral immunization with pBsVP6-transgenic alfalfa provides a potential means of protecting children and young animals from severe acute rotavirus-induced diarrhea.     

Vaccination with recombinant vaccinia viruses protects mice against Mycobacterium tuberculosis infection.

A number of subunit-based vaccine candidates have recently begun to erode the exclusive position of Mycobacterium bovis bacillus Calmette-Guérin (BCG), which gives unpredictable and highly variable protection against tuberculosis. In this paper we investigated the protective capacity of the 19,000 MW and 38,000 MW glyco-lipoproteins of M. tuberculosis expressed by recombinant vaccinia viruses in a mouse Mycobacterium tuberculosis infection model. Both proteins were expressed at high levels by recombinant vaccinia-infected cells. In addition, two inoculations of C57B1/6 mice with either recombinant vaccinia virus significantly reduced the bacterial counts in the lungs of M. tuberculosis H37Rv-infected mice, when compared with the group infected with control virus. This is the first report of protection against tuberculous infection using recombinant vaccinia viruses with results that suggest that secreted glyco-lipoproteins in conjunction with the vaccinia vector represent suitable candidates for further vaccine-related studies.     

Failure of endotoxin to increase nonspecific resistance to infection of lipopolysaccharide low-responder mice.

In vitro and in vivo responses to lipopolysaccharide (LPS) and various other bacterial immunostimulants were compared in c3H/He low-responder mice. The principal findings were as follows. (i) Their splenic lymphocytes were stimulated by various gram-negative mitogens such as an Escherichia coli peptidoglycan, a detoxified derivative of LPS, and even endotoxins extracted by trichloroacetic acid that are known to contain protein; spleen cells of these mice were also transformed by two other B-cell mitogens extracted from acid-fast organisms. (ii) Their macrophages were refractory to LPS and weakly responsive to a mycobacterial prepartion. (iii) LPS failed to elicit nonspecific resistance in these mice against Klebsiella pneumoniae infection. (iv) Endotoxin extracted by trichloroacetic acid and a mycobacterial preparation that could increase nonspecific resistance to infection in other strains did not protect C3H/He mice against a challenge by K. pneumoniae, although both prepartions could evoke nonspecific responses of B cells in this low-responder subline.     

Gelatinase B deficiency protects against endotoxin shock

Gelatinase B or matrix metalloproteinase-9 (MMP-9) is stored in the tertiary granules of polymorphonuclear leukocytes. These cells are key effectors in acute inflammatory diseases such as sepsis. Endotoxin leads to rapid release of gelatinase B from these granules in vitro and in vivo, but the role of this enzyme in bacterial sepsis and endotoxin shock remains unclear. We studied the clinical course of endotoxinemia and its relation with the expression of gelatinase B from the pool of circulating leukocytes in adult as well as in young mice in a model of endotoxin-induced shock and compared wild-type with gelatinase B-deficient mice. The gelatinase B-deficient mice were resistant to endotoxin shock, which implies that specific MMP-9 inhibition constitutes anapproach for the treatment of septic shock syndromes.     

Commensal enteric bacteria lipopolysaccharide impairs host defense against disseminated Candida albicans fungal infection

Commensal enteric bacteria maintain systemic immune responsiveness that protects against disseminated or localized infection in extra-intestinal tissues caused by pathogenic microbes. However, as shifts in infection susceptibility after commensal bacteria eradication have primarily been probed using viruses, the broader applicability to other pathogen types remains undefined. In sharp contrast to diminished antiviral immunity, we show commensal bacteria eradication bolsters protection against disseminated Candida albicans fungal infection. Enhanced antifungal immunity reflects more robust systemic expansion of Ly6G^hiLy6C^int neutrophils, and their mobilization into infected tissues among antibiotic-treated compared with commensal bacteria-replete control mice. Reciprocally, depletion of neutrophils from expanded levels or intestinal lipopolysaccharide reconstitution overrides the antifungal protective benefits conferred by commensal bacteria eradication. This discordance in antifungal compared with antiviral immunity highlights intrinsic differences in how commensal bacteria control responsiveness for specific immune cell subsets, because pathogen-specific CD8⁺ T cells that protect against viruses were suppressed similarly after C. albicans and influenza A virus infection. Thus, positive calibration of antiviral immunity by commensal bacteria is counterbalanced by restrained activation of other immune components that confer antifungal immunity.     

Vitamin E protects mice against Diplococcus pneumoniae type I infection

Vitamin E protects nonimmunized and immunized mice against fatal Diplococcus pneumoniae type I (DpI) infection. A dietary supplementation of 180 mg of dl-alpha-tocopheryl acetate per kg of diet increased survival of nonimmunized mice from 20 to 80% when challenged with 20 organisms, and of mice immunized with 0.5 ng of DpI polysaccharide from 15 to 70% when challenged with 20,000 organisms. The phagocytic index of immunized mice was four times higher in the 180-mg vitamin E group than in the control group. Both the survival and phagocytic index revealed a biphasic dose response, indicating a cause-effect relationship between phagocytosis and survival. Vitamin E also significantly increased the rate of carbon clearance from blood, indicating a general increase in phagocytic activity. The data indicated that increased macrophage activity probably aided by increased antibody production was the principal reason for increased protection.     

Lipid X protects mice against fatal Escherichia coli infection.

Lipid X, the major monosaccharide precursor of lipid A, is nontoxic and has previously been shown to protect mice and sheep from the harmful effects of endotoxin. To test the hypothesis that lipid X could be therapeutic against infections with gram-negative organisms, neutropenic ICR mice were infected by intramuscular inoculation of Escherichia coli and subsequently treated with lipid X alone or in combination with the antibiotic ticarcillin. Lipid X slightly prolonged survival; treated mice had a significantly improved rate of survival 18 h after intramuscular inoculation as compared with controls (P less than 0.025). By 24 h, however, this difference disappeared. When lipid X was combined with ticarcillin, survival differences were both significant and prolonged. Treatment of mice with one to two doses of lipid X for a total dose of 1 mg intravenously and with 1,200 mg of ticarcillin per kg every 6 h improved survival over a 48-h treatment period from 5 to 23% (P less than 0.0025). Treatment with lipid X and ticarcillin over a broad range of antibiotic dosages in 362 mice demonstrated improved survival of two- to fourfold (P less than 0.0001 at 24 h after inoculation, P less than or equal to 0.0005 at 48 h, and P less than or equal to 0.0001 at 5 days). Lipid X enabled the dose of ticarcillin necessary to protect 50% of mice from death to be reduced by two- to fivefold. Pretreatment with lipid X was not necessary to improve survival: 16 of 17 (94%) infected and visibly ill animals that received lipid X and ticarcillin 6 h after thigh inoculation survived versus 30 of 44 (68%) control animals treated with ticarcillin alone (P less than 0.0001). Lipid X had no antimicrobial activity in vitro. Lipid X is a novel agent that enhances survival in an animal model of severe infection with gram-negative organisms.     

Recombinant interleukin-6 protects mice against experimental bacterial infection.

Because of reports of high levels of interleukin-6 (IL-6) in patients during infection, we studied the role of IL-6 in experimental infection. Mice infected with the facultative intracellular pathogen Listeria monocytogenes displayed high levels of IL-6 in their sera and tissues, particularly the spleen, 1 to 3 days after infection. At this time, the IL-6 titers correlated with bacterial numbers in individual mice and in groups of mice given graded doses of Listeria organisms. However, the presence of IL-6 in serum declined after 4 days, even when a large initial dose of bacteria meant that bacterial numbers were still increasing at this time. Recombinant mouse IL-6 injected intraperitoneally before infection protected mice in a dose-dependent manner. It was effective when given 4 h before infection but not when administration was delayed for 24 h postinfection. It is therefore believed that IL-6 plays a role in early priming of the immune response to infection. Its exact function in this model is being investigated.     

Immunization with polyamine transport protein PotD protects mice against systemic infection with Streptococcus pneumoniae

The human pathogen Streptococcus pneumoniae contains genes for a putative polyamine ABC transporter which are organized in an operon and designated potABCD. Polyamine transport protein D (PotD) is an extracellular protein which binds polyamines and possibly other structurally related molecules. PotD has been shown to contribute to virulence in both a murine sepsis model and a pneumonia model with capsular type 3 pneumococci. The protective efficacy of recombinant PotD was evaluated by active immunization and intravenous challenge with capsular type 3 pneumococci in CBA/N mice. Immunized mice had 91.7% survival following lethal pneumococcal challenge, compared with 100% mortality in the control group. Immunized animals had high-titer anti-PotD antibodies following three immunizations with alum. Protection in a sepsis model was also seen after passive administration of rabbit antiserum raised against PotD (P < 0.004). These results suggest that antibodies to PotD confer protection against invasive pneumococcal disease and that this protein should be studied further as a potential vaccine candidate for protection against invasive pneumococcal infections.     

Recombinant GroES in combination with CpG oligodeoxynucleotides protects mice against Mycobacterium avium infection

The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and the recombinant GroES protein was purified by affinity chromatography. The GroES preparation showed high purity by electrophoresis and immunoblotting. Immuno-electron microscopy showed that GroES was located both in the cytoplasm and on the surface of the mycobacterial cells and thus is readily available to interact with the host immune system. BALB/c mice were immunised intranasally with recombinant GroES, alone or in combination with a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, and tested for protection against infection with M. avium. Neither GroES nor CpG alone provided any protection against subsequent challenge with M. avium, whereas a combination of the two significantly protected the lungs and spleen against colonisation by M. avium after intranasal challenge with a low dose of the organism. This indicates that intranasal administration of GroES and CpG oligodeoxynucleotides increases the resistance of BALB/c mice to M. avium infection.     

Pure paraflagellar rod protein protects mice against Trypanosoma cruzi infection.

The paraflagellar rod proteins (PAR) purified from Trypanosoma cruzi epimastigotes were shown to protect mice against an otherwise lethal challenge inoculum of 10(3) bloodstream-form trypomastigotes. The injection route used for immunization was shown to have a marked impact on the development of protective immunity. Mice receiving subcutaneous (s.c.) injections of PAR proteins had reduced bloodstream parasitemias and showed 100% survival following challenge. In contrast, mice immunized via the intraperitoneal (i.p.) route developed parasitemia levels equivalent to those of unimmunized controls and did not survive infection. Western blotting (immunoblotting) demonstrated that sera from both i.p. and s.c. immunized mice reacted specifically with PAR proteins; however, the antibody titer of the i.p. immunized mice was approximately 64-fold greater than that of the s.c. immunized mice, suggesting that the protective response in the s.c. immunized mice is cell mediated rather than humoral.     

Immunization with recombinant schistosome adenylate kinase 1 partially protects mice against Schistosoma japonicum infection

Highly effective and safe prophylactic vaccines are urgently needed to sustainably control schistosomiasis, one of the most serious endemic zoonoses in China. In this study, we characterized adenylate kinase 1 from Schistosoma japonicum (SjAK1), a phosphotransferase that regulates cellular energy and metabolism, and evaluated its potential as a recombinant vaccine. Based on real-time quantitative PCR, western blot, and immunolocalization, SjAK1 is active throughout the life of the worm, although its expression is higher in 21-day-old schistosomula, adult worms, and eggs deposited in the host liver. Further, the enzyme accumulates in the eggshell, intestinal epithelium, integument of adult worms and in the vitellaria tissue in female worms. A 594-bp full-length complementary DNA (cDNA) encoding SjAK1 was synthesized from total RNA of 3-day-old schistosomes, and immunization with recombinant SjAK1 reduced worm burden by 50%, decreased the density of eggs deposited in the host liver by 40%, and reduced the area of granulomas in the host liver by 56%. ELISA results showed that recombinant SjAK1 also stimulated Th1 cytokines such as IL-2 and IFN-γ, but not IL-5 and IL-4. Collectively, a recombinant form of the enzyme SjAK1 elicits partial protective immunity against Schistosoma japonicum infection and the induction of Th1 cytokines. Thus, the enzyme has potential as a component of a multivalent vaccine against schistosomiasis.

     

Prior genital tract infection with a murine or human biovar of Chlamydia trachomatis protects mice against heterotypic challenge infection

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar of Chlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars of C. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murine C. trachomatis induces immunity against challenge with either of two human biovars.