Administration in vivo of recombinant interleukin 2 protects mice against septic death.

Administration in vivo of recombinant interleukin 2 (rIL-2) to mice induces a polyclonal IgM response. When co-administered with a specific antigen, rIL-2 can enhance concentrations of murine IgM antibodies specific for the antigen by fivefold within 7 d of initial treatment. IgM antibodies that are induced after injection of rIL-2 include antibodies specific for J5, a cell wall core lipopolysaccharide (LPS) antigen that is shared by the different members of the Enterobactericeae family. We report here that mice pretreated with rIL-2 or immunized with J5 antigen 7 d before bacterial challenge were protected from septic death that is caused by intraperitoneal challenges with Escherichia coli. Optimal protection was provided by a combined J5 antigen and rIL-2 treatment. Acquisition of the rIL-2 and J5 antigen-induced protection against lethal bacterial infection coincided temporally with maximal serum IgM titers that also contained IgM antibodies specific for the J5 antigen. In passive immunization experiments, the affinity-purified IgM fraction in sera of rIL-2-treated animals was identified as necessary and sufficient for protection. The IgM-depleted serum had no protective effect. The nonspecific augmentation of host-defense mechanisms without the induction of endotoxin manifestations makes rIL-2 a potential candidate to any alternative LPS-containing vaccines for the prevention of bacterial infections by gram-negative organisms since the core LPS antigen is shared among gram-negative bacteria.     

Neutralization of meningococcal endotoxin by antibody to core glycolipid

Antibodies to Escherichia coli J5, a uridine 5'-diphosphate-galactose epimerase-less mutant of E. coli 0111, neutralized meningococcal endotoxemia from all three major capsular serogroups. We chose the dermal necrosis of the local Shwartzman phenomenon and the renal cortical necrosis of the general Shwartzman phenomenon as assays because these are the hallmarks of meningococcemia, and because meningococcal lipopolysaccharide (LPS) is a uniquely potent cause of dermal purpura and necrosis. Meningococcal antisera raised against LPS from MGC A, B, and C also provided good protection against endotoxemia from the homologous capsular groups, but it was inconsistent against the heterologous serogroups. The superiority of J5 antibodies (purified IgG as well as antiserum) is probably due to the fact that J5 LPS contains only the endotoxin core. Consequently, immunization with this mutant stimulates production of antibodies to core LPS without interference by the "0" antigenic determinants of the side chains. These observations indicate that the endotoxin core is the toxic moiety of meningococcal LPS, that the core LPS of meningococcus (MGC) is immunologically similar to enteric LPS, and that the antigenically variable "0" side chains of MGC LPS interfere with antibody production against the common core. They also suggest that antibodies prepared against this E. coli mutant could interrupt the devastating course of meningococcal endotoxemia in man, regardless of the capsular serogroup of the infecting strain.     

Association between protective efficacy of anti-lipopolysaccharide (LPS) antibodies and suppression of LPS-induced tumor necrosis factor alpha and interleukin 6. Comparison of O side chain-specific antibodies with core LPS antibodies

Two-core LPS antibodies, the rabbit J5 polyclonal antiserum and the human anti-lipid A IgM mAb HA-1A, did not improve the survival of mice challenged with E. coli O111 or P. aeruginosa 3, or with the LPS extracted from them, and did not decrease the incidence of Shwartzman reactions in rabbits challenged with O111 LPS. In contrast, O side chain-specific rabbit antisera were protective in these models. The protection afforded by O side chain-specific antisera against endotoxin lethality was associated with decreased LPS-induced serum TNF and IL-6 levels, whereas core LPS antibodies had no effect on TNF or IL-6 levels. The absence of reduction of LPS-induced cytokines levels by core LPS antibodies suggests that these antibodies are not able to prevent the interactions between LPS and target cells.     

Human Antiserum for Prevention of the Local Shwartzman Reaction and Death from Bacterial Lipopolysaccharides

Bacterial lipopolysaccharides from dead bacteria have been blamed for the continuing high mortality from gram-negative infections despite antibiotic treatment. Because animal antiserum against these lipopolysaccharides has been shown to protect against several of the effects of endotoxin, we undertook the development of antiserum in human subjects. 21 men were immunized with a single injection of Salmonclla typhimurium or Escherichia coli 0:111 heat-killed cells and immune serum was collected at 2 wk. Preimmune serum was obtained as a control in all animal experiments. 1 ml antiserum given intravenously protected mice against a lethal intravenous dose of homologous endotoxin (P < 0.005 for both antisera). E. coli antiserum reduced the incidence of positive local Shwartzman reactions with E. coli endotoxin from 100 to 38%; S. typhimurium antiserum reduced the incidence from 92 to 35%. (P < 0.0005 for both antisera). There was no protection against heterologous endotoxin in either animal model. These experiments demonstrate for the first time that human antiserum confers exceedingly potent passive immunity to the effects of endotoxin.     

Differentiation factor/leukemia inhibitory factor protection against lethal endotoxemia in mice: synergistic effect with interleukin 1 and tumor necrosis factor

Differentiation factor (D factor), also called leukemia inhibitory factor (LIF), is a glycoprotein that has been increasingly recognized to possess a wide range of physiological activities. We examined the possibility that the administration of D factor may confer beneficial effects and enhance host resistance against lethal endotoxemia. A single intravenous dose of recombinant human D factor completely protected C57/Bl6 mice from the lethal effect of Escherichia coli endotoxin (lipopolysaccharide [LPS]). The protective effects were dose dependent and observed when administered 2-24 h before LPS. Previous work has shown that interleukin 1 (IL-1) and tumor necrosis factor (TNF) also protect against a subsequent LPS challenge in a dose-dependent manner. When human D factor was combined with sub-protective doses of IL-1 beta or TNF-alpha, there was dramatic synergistic protection against a subsequent lethal LPS challenge.     

Active Immunization with Lipopolysaccharide Pseudomonas Antigen for Chronic Pseudomonas Bronchopneumonia in Guinea Pigs

Chronic respiratory infection with Pseudomonas aeruginosa is a leading clinical problem among patients with cystic fibrosis. Because antimicrobial agents are usually ineffective in eradicating these infections, additional therapeutic or prophylactic measures should be considered. In this study, an experimental guinea pig model of chronic Pseudomonas aeruginosa bronchopneumonia was utilized to determine whether active immunization with lipopolysaccharide (LPS) P. aeruginosa antigen may favorably influence the course of this infection. Experimental pneumonia was established by tracheobronchial instillation of suspensions of microscopic agar beads, which were impregnated with viable P. aeruginosa. After 4 wk of infection, the geometric mean (reciprocal) passive hemagglutinating Pseudomonas antibody titer was 185±1.3, and lungs contained 16.8±4 × 10³ colony-forming units Pseudomonas/ml of lung homogenate. Pseudomonas immunization, given prior to a 4-wk infection, resulted in significantly higher passive hemagglutinating titers (474±1.4; P < 0.05), lower numbers of viable Pseudomonas in lung tissues (2.4±0.6 × 10³; P < 0.01), and reduced histopathology in lungs. In contrast, providing Pseudomonas immunization to animals 2 wk after pulmonary infection was established, offered no apparent benefit. Likewise, no protection was afforded by prophylactic immunization with a non-Pseudomonas LPS antigen (Escherichia coli J5 vaccine). Using a Raji cell assay, modified to detect circulating immune complexes in vaccinated and infected guinea pig sera, there was no evidence that active immunization increased the frequency of circulating immune complexes in infected guinea pigs.It is concluded that prophylactic immunization with Pseudomonas LPS antigen may confer protection from subsequent Pseudomonas bronchopneumonia, but that immunization during established infection is not beneficial.     

Protection against group B meningococcal disease. III. Immunogenicity of serotype 2 vaccines and specificity of protection in a guinea pig model

Protein vaccines were prepared from the serotype antigen of group B Neisseria meningitidis strain M986. The detergents Triton X-100, Emulphogene BC-720, and deoxycholate were used to removed the toxic lipopolysaccharide (LPS) portion of the serotype antigen. The LPS was most preferentially solubilized by Emulphogene. Guinea pigs were immunized with one or two doses of vaccine given intramuscularly without adjuvants and the antibody response quantitated by an enzyme-linked immunosorbant assay. Immunization with graded doses of vaccine between 25 to 200 microgram protein indicated a wide range of effective dosage and that a two-dose immunization schedule was superior to a single immunization. The vaccines elicited peak mean serum antibody levels of approximately 30 microgram/ml with bactericidal titers of 1:1,600-1:6,400. The peak antibody levels occurred 5-6 wk after immunization and persisted above preimmune levels for several months. To evaluate the protective effects of immunization, stainless steel springs were implanted subcutaneously into the guinea pigs. The resulting chambers, in unimmunized animals, could be infected with less than 100 type 2 organisms. A single 25-50 microgram dose of vaccine protected 50% of animals from challenge by 5 X 10(5) type 2 meningococci, and as little as 1 microgram vaccine significantly reduced the severity of infection. A two-dose immunization schedule was best and provided nearly complete protection for at least 4 mo against type 2 strains of meningococcal groups B, C, and Y.     

Vaccination against hepatitis delta virus infection: studies in the woodchuck (Marmota monax) model

Hepatitis delta virus (HDV) superinfection of hepatitis B virus carriers causes severe liver disease and results in a high rate of chronicity. So far, neither sufficient therapy nor vaccines to prevent HBV carriers from superinfection are available. A good model to test vaccine candidates is the woodchuck chronically infected with the woodchuck hepatitis virus (WHV); the woodchuck can be superinfected with HDV and shows a course of infection similar to that of patients. Different strategies have been investigated to establish a protective vaccine against HDV superinfection. Both proteins of HDV (HDAg p24 and p27), which differ only in the C-terminal amino acid sequence, have been used as vaccine candidates. Synthetic peptides derived from B cell epitopes of HDAg and HDAg p24 expressed in Escherichia coli, yeast, or baculovirus have been used to immunize woodchucks. The protein immunization induced a specific antibody response, however, no protection from HDV superinfection was achieved. Vaccinations with vaccinia virus expressing HDAg p24 or p27 and DNA immunization with vectors expressing p24 were also not able to induce a protective immune response, but seemed to modulate the course of HDV superinfection. Thus, new strategies to develop a vaccine to prevent HDV superinfection are needed.     

Effects of vaccination in urinary infections caused by Escherichia coli in rats

The preventive effects of the immunomodulator P40 on the experimental E. coli infection of the lower urinary tract of the rat have been reported previously. In the present paper are presented the results obtained with the aid of vaccination using the same experimental model of infection in the rat. Active specific immunization of the animals with a vaccine consisting of the same strain as that used for infecting the rats afforded significant protection toward the infection. It is concluded that such vaccines could also be used in humans, either alone or in combination with other therapies with the aim at preventing recurrencies or relapses of infections of the lower urinary tract which were resistant to antibiotherapy.     

Efficacy of oral immunization with Pseudomonas aeruginosa lipopolysaccharide

Oral administration of extracted lipopolysaccharide (LPS) from Pseudomonas aeruginosa was used to achieve serotype specific protection against P. aeruginosa in an ironloaded mouse model and a burned mouse model of infection. A significant delay to death could also be achieved using a neutropenic mouse model. In the iron-loaded mouse infection model protection could be achieved against three serotypes but not a fourth (serotype G). Protection against two serotypes could be achieved by simultaneous but not sequential feeding with both serotypes. Several oral immunization protocols were examined in the iron-loaded mouse model for efficacy and for toxicity. In protection studies, concentrations of LPS in the drinking water ranging from 2 to 200 μg/ml were used for periods from 1 to 40 days resulting in total intakes of 145 μg to 5·8 mg LPS. All protocols were successful in achieving protection provided that challenge occurred later than 2 days after the initial exposure to LPS. Toxicity studies were performed with similar amounts of LPS including an additional high dose (2 mg/ml LPS for 1 day, 14 mg total intake). No evidence of toxicity was observed for any dosage using a variety of criteria including pathological examination of tissue except for small temperature elevations in all but the lowest dose. Aerosol administration of LPS was also shown to provide protection against infection with the homologous strain. Passive transfer experiments demonstrated that protection was associated with specific antibody and not by cell mediated immunity.     

Prevention of indigenous infection of mice with Escherichia coli by nonspecific immunostimulation.

We have previously reported that the lethal toxicity of 5-fluorouracil (5-FU) in specific-pathogen-free mice is due to an intestinal infection with indigenous Escherichia coli induced by the drug (K. Nomoto, T. Yokokura, Y. Yoshikai, M. Mitsuyama, and K. Nomoto, Can J. Microbiol. 37:244-247, 1991). In the present study we demonstrate that nonspecific immunostimulation is effective in the protection of mice from the lethal indigenous infection induced by 5-FU. Intravenous or subcutaneous injection of a preparation of heat-killed Lactobacillus casei YIT 9018, a potent nonspecific immunostimulant, into BALB/c mice reduced the lethal toxicity of 5-FU at doses ranging from 338 to 800 mg/kg of body weight if YIT 9018 was injected 7 to 40 days before administration of 5-FU. Systemic infection with E. coli developed in all of the 5-FU-treated control mice 7 days or more after administration of 5-FU in large doses and was accompanied by overgrowth of the bacteria in the intestinal tract. Pretreatment of mice with YIT 9018 resulted in a decreased occurrence of systemic infection with E. coli to levels of 0 to 20% and no significant changes in the population levels of E. coli in the intestinal tract during the 14 days after administration of 5-FU. The levels of leukopenia in the spleen and peripheral blood were lower, and recovery of granulocyte-macrophage precursor cells in the spleen and femur began earlier in the treated animals than in the 5-FU-treated controls. Intravenous transfusion of syngeneic normal bone marrow cells or spleen cells into the mice at an early period after administration of 5-FU diminished markedly the occurrence of the lethal indigenous infection, suggestion that an earlier recovery from chemotherapy-induced myelosuppression is important in the mechanisms of protection of the host from the infection.     

Mechanism of Protection Induced by Group A Streptococcus Vaccine Candidate J8-DT: Contribution of B and T-Cells Towards Protection

Vaccination with J8-DT, a leading GAS vaccine candidate, results in protective immunity in mice. Analysis of immunologic correlates of protection indicated a role of J8-specific antibodies that were induced post-immunization. In the present study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved in J8-DT-mediated immunity. These approaches included the passive transfer of mouse or rabbit immune serum/antibodies in addition to selective depletion of T-cell subsets prior to bacterial challenge. Passive transfer of J8-DT antiserum/antibodies from mice and rabbits conferred significant resistance against challenge to mice. To exclude the possibility of involvement of other host immune factors, the studies were repeated in SCID mice, which highlighted the need for an ongoing immune response for long-lived protection. Depletion of CD4⁺ and CD8⁺ T-cell subsets confirmed that an active de novo immune response, involving CD4⁺ T-helper cells, is required for continued synthesis of antibodies resulting in protection against GAS infection. Taken together these results indicate an involvement of CD4⁺ T-cells in J8-DT-mediated protection possibly via an ability to maintain antibody levels. These results have considerable relevance to the development of a broad spectrum passive immunotherapy for GAS disease.     

Low-density lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections.

Lipoproteins can bind lipopolysaccharide (LPS) and decrease the LPS-stimulated production of pro-inflammatory cytokines. We investigated the effect of increased plasma concentrations of low-density-lipoproteins (LDL) on survival and cytokine production after a lethal challenge with either LPS or live Gram-negative bacteria in LDL receptor deficient mice (LDLR-/-). The LDLR-/- mice challenged with LPS had an eightfold increased LD50 when compared with the wild type controls (C57Bl/6J), while tumor necrosis factor alpha (TNFalpha) and interleukin-1 alpha (IL-1 alpha) plasma concentrations were decreased twofold. LDLR-/- mice had significantly lower and delayed mortality than control mice after infection with Klebsiella pneumoniae. No differences in the outgrowth of bacteria in the organs were present between the two groups, while circulating cytokine concentrations were decreased twofold in LDLR-/- mice. In contrast, the LPS-stimulated in vitro production of cytokines by peritoneal macrophages of LDLR-/- mice was significantly increased compared with controls. This increase was associated with enhanced specific binding of LPS to the macrophages of LDLR-/- mice. In conclusion, endogenous LDL can protect against the lethal effects of endotoxin and Gram-negative infection. At least part of this protection is achieved through decreased in vivo production of pro-inflammatory cytokines, in spite of increased cytokine production capacity.     

Prepandemic H5N1 influenza vaccine adjuvanted with AS03: a review of the pre-clinical and clinical data

Background: Universal and timely administration of a prepandemic vaccine is considered to be one of the most effective measures to reduce the incidence of pandemic influenza infection and consequently its morbidity and mortality. Objectives: To provide the reader with basic insights into influenza virus infections, the threat of a pandemic and the challenges it poses for vaccine development. Methods: This review summarizes the reported preclinical and clinical data obtained with the prepandemic H5N1 vaccine adjuvanted with AS03. Results: The AS03-adjuvanted prepandemic H5N1 influenza vaccine allows for antigen sparing, has a good safety and acceptable reactogenicity profile, induces an immune response that not only meets all European Committee for Medicinal Products (CHMP) and FDA requirements for the vaccine strain but also generates neutralizing antibodies that broadly cross-react against H5N1 drift strains, and finally conveys protection in a ferret model against lethal challenges with homologous and heterologous H5N1 viruses.     

Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8+ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8+ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans.     

An antidote for Staphylococcus aureus pneumonia?

Methicillin-resistant Staphylococcus aureus (MRSA) is the leading cause of bacterial infections in the United States. Severe invasive MRSA infections, which include pneumonia, are difficult to treat because the bacteria are resistant to antibiotics. A new report now shows that immunization against alpha-hemolysin (Hla), a cytolytic toxin secreted by most S. aureus strains, protects mice against lethal pneumonia. This finding represents the first successful vaccine strategy for the treatment of staphylococcal pneumonia.     

Supplementation of Influenza Split Vaccines with Conserved M2 Ectodomains Overcomes Strain Specificity and Provides Long-term Cross Protection

Current influenza vaccines do not provide good protection against antigenically different influenza A viruses. As an approach to overcome strain specificity of protection, this study demonstrates significantly improved long-term cross protection by supplementing split vaccines with a conserved molecular target, a repeat of the influenza M2 ectodomain (M2e) expressed on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Intramuscular immunization with H1N1 split vaccine (A/California/07/2009) supplemented with M2e5x VLPs induced M2e-specific humoral and cellular immune responses, and shaped the host responses to the vaccine in the direction of T-helper type 1 responses inducing dominant IgG2a isotype antibodies as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge, M2e5x VLP-supplemented vaccination lowered lung viral loads and induced long-term cross protection against H3N2 or H5N1 subtype influenza viruses over 12 months. M2e antibodies, CD4 T cells, and CD8 T cells were found to contribute to improving heterosubtypic cross protection. In addition, improved cross protection by supplemented vaccination with M2e5x VLPs was mediated via Fc receptors. The results support evidence that supplementation with M2e5x VLPs is a promising approach for overcoming the limitation of strain-specific protection by current influenza vaccination.     

Enzyme immunoassay for the quantitation of immunoglobulin M class antibodies to Salmonella minnesota R595 and Escherichia coli J5 lipopolysaccharides

The level of human immunoglobulin (IgM) in plasma specific for the lipopolysaccharide of Salmonella minnesota R595 (R595 LPS) and Escherichia coli J5 (J5 LPS) was quantitated by an enzyme immunoassay (EIA) in which purified antigen is adsorbed directly onto polystyrene-acrylic copolymer cuvettes. Highly purified anti-J5 and R595 LPS specific IgM prepared by ion-exchange resin, gel filtration, and affinity resin chromatography were used as standards. The levels of specific IgM were determined in 200 plasma samples obtained from normal donors. Anti-R595 IgM levels varied from less than 30 micrograms/ml (91%), from 30 to 100 micrograms/ml (8.5%), and greater than 100 micrograms/ml (0.5%). Anti-J5 IgM levels in 68% of the donor plasmas were less than or equal to 5 micrograms/ml. The levels in 30.5% of the donor plasmas ranged from 6 to 100 micrograms/ml; the remaining 1.5% had greater than 100 micrograms/ml anti-J5 IgM. Specific IgM levels in four lots of normal pooled plasma each consisting of about 10,000 L averaged 12.7 micrograms/ml and 13.3 micrograms/ml for R595 and J5, respectively. The assay was modified to quantitate rabbit plasma as well. For this purpose, the EIA has been performed on microtiter plates, and the core LPS was fixed onto the wells by chemical treatment with glutaraldehyde which results in higher stability and retention of the antigen in the wells. Specificity of the EIA was demonstrated by the absence of significant cross reactivity between R595 IgM and J5 LPS and between J5 IgM and R595 LPS, furthermore, we only observed partial adsorption (approximately 25%-33%) of the R595 and J5 IgM by Pseudomonas aeruginosa LPS, a wild type endotoxin. The described quantitative assay is useful for both scientific studies and clinical investigations.     

Peroral immunization with Helicobacter pylori adhesin protein genetically linked to cholera toxin A2B subunits

Helicobacter pylori is a major cause of gastric-associated diseases. To evaluate the efficacy of a possible vaccine antigen against H. pylori infection, the chimaeric construct adhesin--CTXA2B, derived from H. pylori adhesin genetically coupled to cholera toxin (CTX) subunits A2 and B (CTXA2B), was expressed in Escherichia coli as an insoluble recombinant chimaeric protein. The protein was then purified by denaturation, renaturation and size-exclusion chromatography. The composition of purified adhesin--CTXA2B was verified by SDS/PAGE and Western blotting with antibodies to antigenic components of adhesin and CTXB, and confirmed as a chimaeric protein with G(M1)-ganglioside binding activity and adhesin epitopes by a G(M1)-ELISA developed using antibodies to adhesin. Oral immunization of mice with adhesin--CTXA2B induced higher levels of mucosal IgA and serum IgG antibodies to H. pylori adhesin and to CTXB than in mice immunized with adhesin or CTXA2B alone. Adhesin--CTXA2B was also demonstrated to be a potential protective antigen in a mouse model of H. pylori infection. The immunization of mice with adhesin--CTXA2B protected 62.5% of mice infected with H. pylori SS1 strain, whereas adhesin immunization was not able to confer protection to mice. This protection may be correlated with high levels of mucosal IgA and serum IgG antibodies against H. pylori adhesin. Taken together, the results indicate that the genetically linked CTXA2B acts as a useful mucosal adjuvant, and that the adhesin-CTXA2B chimaeric protein could be a potential component in future H. pylori vaccine development.     

Nonspecific resistance to bacterial infections. Enhancement by ubiquinone-8

A lipid fraction from Escherichia coli was extracted with apolar solvents and was found to protect mice from a number of experimental bacterial infections. The benzoquinone, ubiquinone-8, was isolated from this extract by high pressure liquid chromatography and identified as such by nuclear magnetic resonance and mass spectrometry. At a dose of 25 mg/kg this substance was found to provide complete protection against otherwise lethal infections with gram-negative and gram-positive bacteria in mice. Treatment was most effective when given intravenously 24 h before infection. In comparative studies, ubiquinone-8 had a clearly higher activity than ubiquinones-4, Q6, and Q10. A highly significant increase in the clearance rate of bacteria from the blood by the spleen and the liver of treated animals, correlated well with the protective effect of ubiquinone-8. The compound stimulated the ability of mouse macrophages to incorporate sheep erythrocytes and significantly increased the number of antibody-producing cells in spleens of mice.