Development and preclinical evaluation of a trivalent, formalin-inactivated Shigella whole-cell vaccine.

Studies were undertaken to manufacture a multivalent Shigella inactivated whole-cell vaccine that is safe, effective, and inexpensive. By using several formalin concentrations, temperatures, and incubation periods, an optimized set of inactivation conditions was established for Shigella flexneri 2a, S. sonnei, and S. flexneri 3a to produce inactivated whole cells expressing a full repertoire of Ipa proteins and lipopolysaccharide (LPS). The inactivation conditions selected were treatment with 0.2% formalin (S. flexneri 2a and 3a) or 0.6% formalin (S. sonnei) for 48 h at 25°C. Vaccine formulations prepared under different inactivation conditions, in different doses (10E5, 10E7, and 10E9 cells), and with or without the inclusion of double-mutant heat-labile toxin (dmLT) were evaluated in mice. Two intranasal immunizations with ≥10E7 inactivated whole cells resulted in high levels of anti-Invaplex and moderate levels of LPS-specific IgG and IgA in serum and in lung and intestinal wash samples. Addition of dmLT to the vaccine formulations did not significantly enhance humoral immunogenicity. Minimal humoral responses for IpaB, IpaC, or IpaD were detected after immunization with inactivated whole Shigella cells regardless of the vaccine inactivation conditions. In guinea pigs, monovalent formulations of S. flexneri 2a of 3a or S. sonnei consisting of 10E8, 10E9, or 10E10 cells were protective in a keratoconjunctivitis assay. A trivalent formulation provided protection against all three serotypes (S. flexneri 2a, P = 0.018; S. flexneri 3a, P = 0.04; S. sonnei, P < 0.0001). The inactivated Shigella whole-cell vaccine approach incorporates an uncomplicated manufacturing process that is compatible with multivalency and the future development of a broadly protective Shigella vaccine.

Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial.

Orally delivered, inactivated whole-cell vaccines are safe methods of inducing local and systemic immunity. To increase surface proteins associated with adherence and invasion, Shigella sonnei were grown in BHI broth containing deoxycholate. A whole-cell vaccine (SsWC) was then produced by formalin inactivation. In pre-clinical studies, the SsWC vaccine was immunogenic and protected against S. sonnei-induced keratoconjunctivitis in the guinea pig model. In a randomized, double-blind, placebo-controlled, Phase I study, 10 evaluable subjects received either three doses of SsWC on Days 0, 14, and 28 (N = 3); five doses of SsWC on Days 0, 2, 4, 6, and 28 (N = 4); or placebo (N = 3). Each dose contained 2.0 x 10(10) inactivated cells. Serum and fecal antibodies against SsWC, LPS, and IpaC were measured by ELISA. A > or = 4-fold increase in titer was considered significant. Both SsWC dosing regimens were well tolerated. No fever or severe gastrointestinal symptoms were noted by any of the vaccinated subjects. Antibody responses were similar in the two dosing groups. Serum IgG or IgA responses to SsWC were seen in six of seven vaccinees (86%), to LPS in four of seven (57%), and to IpaC in five of seven (61%). Fecal IgA responses to these three antigens developed in five of five, three of five, and three of five subjects, respectively. Among the seven vaccinees, geometric mean rises in serum IgA levels to all three immunogens were significant; IgG increases trended toward significance (paired one-tailed t-test). We conclude that SsWC was immunogenic and protective in animal studies and well tolerated and immunogenic in a Phase I trial.

Case report: pulmonary blastoma: presentation of two atypical cases and review of the literature.

Pulmonary blastoma is a relatively rare aggressive adult malignancy. The clinical and radiographic features of two cases are described and the literature reviewed. Pulmonary blastoma usually presents radiologically as a well-defined mass lesion on chest radiography, which may be large enough to completely opacify the hemithorax and cause mediastinal shift. On CT, pulmonary blastoma is seen as a mixed solid and cystic lesion with variable contrast enhancement and a necrotic centre. Pleural effusion may be present but is not the predominant abnormality. Our cases are unusual as recurrent pleural effusions in the absence of a significant lung mass were the presenting findings.

Safety and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine with adjuvant among volunteers with or without subclinical infection.

Helicobacter pylori infection of the gastric mucosa can be found in approximately 50% of the world's population and is associated with a range of pathology, including peptic ulcer, atrophic gastritis, and gastric cancer. To explore immunization as a strategy for preventing and treating H. pylori-associated disease, we assessed the safety and immunogenicity in healthy adults of a formalin-inactivated, oral H. pylori whole-cell (HWC) vaccine, administered with or without mutant Escherichia coli heat-labile toxin (LT(R192G)) as a mucosal adjuvant. In a dose-response study, 23 subjects with or without H. pylori infection were vaccinated with either 2.5 x 10(6) HWC, 2.5 x 10(8) HWC, or 2.5 x 10(10) HWC, plus 25 microg of LT(R192G). Thereafter, a randomized study was conducted in which 18 H. pylori-infected subjects were assigned, in a double-blind fashion, to receive either 2.5 x 10(10) HWC plus placebo-adjuvant, placebo-vaccine plus 25 microg of LT(R192G), placebo-vaccine plus placebo-adjuvant, or 2.5 x 10(10) HWC plus 25 microg of LT(R192G). Diarrhea (six subjects), low-grade fever (five subjects), and vomiting (two subjects) were observed, usually after the first dose. Significant rises in geometric mean mucosal (fecal and salivary) anti-HWC immunoglobulin A antibodies occurred among H. pylori-infected and uninfected subjects following inoculation with 2.5 x 10(10) HWC plus 25 microg of LT(R192G). Moreover, among H. pylori-negative volunteers, this regimen induced significant lymphoproliferative responses in 5 of 10 subjects and gamma interferon production responses to H. pylori sonicate in 7 of 10 subjects. There was no evidence that vaccination eradicated H. pylori in infected volunteers. These results suggest that it is possible to stimulate mucosal and systemic immune responses in humans to H. pylori antigens by using an HWC vaccine.

Campylobacter vaccine development:a key to controlling enteric diseases.

Worldwide, Campylobacter jejuni is a major cause of diarrhoea and dysentery, with approximately 400 million cases occurring annually. Control of the disease through public health and antibiotic measures is insufficient, and vaccination offers the most promising solution. Infection produces immunity from disease, suggesting that vaccination may produce similar protection. Epidemiological data suggest that there are conserved antigens among serotypes; immunity against which protects against disease. Therefore a monovalent serotype vaccine seems practical. Several antigens on Campylobacter have been found to be immunogenic and, in some cases, associated with virulence. However, none of these proteins have been produced recombinantly in the proper conformation, nor have they been protective in preclinical models. For this reason, live attenuated or inactivated Campylobacter whole cell (CWC) vaccines may be the best approach. Development of an attenuated strain of Campylobacter has been complicated by the fact that the organism is highly transformable. Rec A mutants of Campylobacter have recently been constructed in an attempt to avoid this trait. Such mutants have been made defective in various virulence properties and are being evaluated for safety in preclinical models. Antex Biologics has made an inactivated CWC vaccine using the Company's patented NST (nutriment signal transduction) technology, whereby the cells are grown using physiologically logical conditions to maximise the expression of antigens associated with in vivo virulence. This vaccine has been shown to be safe, immunogenic, and protective in preclinical models of infection. Inclusion of a mucosal adjuvant in the vaccine increases the immunological response to the antigen. Recent Phase I and Phase II clinical trials, using the CWC vaccine formulated with an adjuvant and orally administered, show that the vaccine is safe and immunogenic in human volunteers. The approach used to develop this inactivated whole cell Campylobacter vaccine is also applicable for the rapid development of new vaccines against a variety of mucosal pathogens.

Inactivated whole-cell bacterial vaccines: current status and novel strategies.

Inactivated bacterial whole-cell vaccines have been the most widely studied prophylactic treatment for infectious diseases. They offer an economical, and potentially safe, effective means of preventing disease. The disadvantages of these vaccines have been that parenteral administration, while effective in some instances, may have caused adverse reactions in vaccinees, while oral administration often required high doses and resulted in short-term immunity. More recent studies describing new approaches for improving antigenicity of inactivated whole-cell vaccines and the enhancement of immune responses to oral immunization offer great hope for improving the efficacy of these agents. Promising whole cell vaccines include those against Vibrio cholerae, enterotoxigenic Escherichia coli, and more recently Campylobacter jejuni.

Automated fluorescent analysis procedure for enzymatic mutation detection.

The Enzymatic Mutation Detection (EMD) assay detects mutations or polymorphisms in DNA. The assay procedure takes <1 h and is followed by electrophoretic detection. We report an automated procedure, using fluorescently labeled probe and quantitative analysis on the ABI Prism 377 DNA Sequencer, that improves on earlier methods (1, 2) by eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. The EMD assay uses the bacteriophage resolvase T4 endonuclease VII, which cleaves the heteroduplex molecules at the mismatch site, forming two shorter fragments that are resolved by gel electrophoresis. Unlike existing mutation techniques, the EMD method uses a single protocol to identify point mutations, deletions, and insertions for all DNA fragments. Test DNA samples are assayed directly from PCR reactions, and fragments up to 4 kb in size have been assayed successfully. A independent analysis on the p53 tumor suppressor gene from clinical samples has shown 100% sensitivity and 94% specificity. Because the fluorescent EMD assay has been optimized for high signal-to-noise ratios, mutations can be identified in mixed samples containing up to a 20-fold excess of normal DNA.

Problems and priorities for controlling opportunistic pathogens with new antimicrobial strategies; an overview of current literature.

An International Study Group on New Antimicrobial Strategies (ISGNAS) has been formed in response to the recognition that development of microbial resistance to antibiotics is becoming a serious, world-wide problem. The group met in 1993 for the first time to discuss the feasibility of developing rational alternatives to the use of antibiotics and prepared, as a result, a comprehensive overview of normal (physiological) mechanisms involved in the control of potentially pathogenic (oppotunistic) microorganisms. One objective of ISGNAS is to understand the conditions which allow opportunistic microbes present among the symbionts to cause an infection. There is a need for more coherent information concerning the habitat, growth requirements and host and pathogen properties which allow opportunistic pathogens to cause life-threatening infections. In particular, information is urgently being sought to understand the complexity of the interactions between the vast number of microbial species, and the interactions between the microbes and their host. Another goal is to inspire and enable basic and clinical research that will lead to the development of new therapies for regulating colonization, translocation and infection by opportunistic micro-organisms in patients during periods of decreased resistance. With a sufficient amount of knowledge of how healthy individuals keep opportunistic micro-organisms under control, it may become feasible for physicians to maintain host resistance and inter-microbial factors involved in the containment of opportunistic microbes. Therapies aimed at boostering natural resistance mechanisms will be of critical importance to individuals whose resistance has been compromised as a result of another clinical condition.

Safety and immunogenicity of a prototype oral whole-cell killed Campylobacter vaccine administered with a mucosal adjuvant in non-human primates.

The safety and immunogenicity of two prototype oral Campylobacter killed whole-cell (CWC) vaccines were tested in rhesus monkeys. Animals were immunized with a primary two-dose series (days 0 and 14) of vaccine consisting of CWC (10(10) particles/dose) given alone or in combination with 0.5-1000 micrograms of the heat-labile enterotoxin of Escherichia coli as an oral adjuvant (OA). A booster vaccination, 4 weeks after primary immunization, was given to animals receiving CWC alone or supplemented with 0.5, 5 or 50 micrograms of OA. Both CWC and CWC-OA were well tolerated, with no adverse side-effects noted. Campylobacter-specific as well as adjuvant-specific antibody-secreting cells (ASCs) were determined in peripheral blood collected 7 days after each vaccine dose. Campylobacter-specific IgA ASC responses were enhanced by OA in a dose-dependent manner (p = 0.025), while IgG ASC responses were not. Seroconversions (both IgA and IgG) to Campylobacter antigens were also enhanced in monkeys receiving adjuvanted vaccine. No significant booster vaccination effect was observed in circulating ASCs in any of the immunization groups. In vitro T-cell proliferative responses to Campylobacter jejuni antigens were somewhat enhanced in both the CWC and CWC-OA immunization groups. These results demonstrate that CWC-OA is safe and superior to CWC alone in its ability to stimulate both local and systemic Campylobacter-specific IgA and IgG responses in primates and they support its further evaluation in human clinical studies.

New strategies for using mucosal vaccination to achieve more effective immunization.

Future progress in vaccination will be significantly advanced by application of emerging technologies for immunization of mucosal surfaces. It should now be possible to maximize the antigenicity of many vaccines and facilitate their interaction with appropriate lymphoid tissues to induce protective cellular and humoral responses. Mucosal vaccines requiring no more than two doses are achievable with current technologies. Living vaccines have been among the most promising candidates for mucosal vaccination, but with few exceptions their promise is still to be realized. Development of new microencapsulated delivery systems and adjuvants has made non-living vaccines reasonable options for mucosal immunization. To be practical, such vaccines should be developed as combined agent vaccines, possibly deliverable by multiple mucosal routes. Although strategies to be used for specific mucosal vaccines will depend upon a number of factors pertinent to the disease agent, in concept an adjuvant administered with inactivated but maximally antigenic pathogens or their recombinant adhesive subcomponents could prove to be among the more practical mucosal vaccine options for use globally.

Killed Campylobacter elicits immune response and protection when administered with an oral adjuvant.

The heat-labile toxin (HLT) of enterotoxigenic Escherichia coli (ETEC) is a potent oral adjuvant. We determined whether the ETEC HLT could be mixed with killed campylobacter to induce an immune response protective upon subsequent challenge with live pathogens. Mice were immunized orally three times with 10(9) sonicated campylobacter with or without 25 micrograms of ETEC HLT, and humoral immune responses in intestinal lavage fluids measured by ELISA. Whereas 10(9) live bacteria induced strong intestinal IgA responses, killed bacteria did not unless ETEC HLT was also added. The magnitude of the antibody response was dependent on the amount of antigen given. The ETEC HLT given with bacteria also induced a potent cross-reaction with cholera toxin. The latter had an adjuvant effect in mice similar to that of ETEC HLT. Protection against colonization was studied in mice and rabbits. In contrast to non-immune animals, those given live organisms or sonicated cells mixed with ETEC HLT quickly cleared homologous, but not heterologous, Lior serotypes of Campylobacter upon challenge. These data show for the first time that ETEC HLT can potentiate an immune response to killed campylobacter that promotes a rapid clearance of live pathogens from the intestine.

Therapies for radiation injuries: research perspectives.

Exposure to radiation damages the immune, hematopoietic, and gastrointestinal components of the host defense system. This may lead to serious endogenous or exogenous infections. When radiation injury is combined with other physical trauma, e.g., burn or wound, the resulting damage to these systems is synergistic, and treatment for infection requires multiple approaches. This paper reviews successful single and combined therapeutic modalities for infections in irradiated mice and irradiated mice inflicted with trauma that are currently conducted at the Armed Forces Radiobiology Research Institute. The models of endogenous and exogenous infection and combined injury are described. The management of wounds infected with bacteria, exogenous systemic infection due to gram-negative enteric bacteria, and the chemoprophylaxis of enteric-derived systemic infection with quinolones is described. Infections can be treated successfully with proper antimicrobial therapy. In gamma- and neutron-irradiated mice, the immunomodulator trehalose dimycolate (TDM) was effective in treating endogenous infection. TDM with the antimicrobial ceftriaxone was effective in treating exogenous infection due to Klebsiella pneumoniae. Improvement in managing infection in irradiated and injured hosts will require further research using these diagnostic and therapeutic modalities. Accurate biological dosimetry is critical in determining if victims are at risk of developing infection. We found that radiation induced changes in plasma diamine oxidase activity; monitoring these changes was a useful indicator of the severity of radiation injury.

Significance of flagella in colonization resistance of rabbits immunized with Campylobacter spp.

Cross-protection among different Lior and Penner serogroups of Campylobacter spp. was studied. Rabbits were orally immunized by gastric feeding with Campylobacter spp., and 27 to 30 days later, they were challenged with matched or unmatched serogroups by the removable intestinal tie adult rabbit diarrhea (RITARD) procedure. When immunized animals were challenged with different Lior serotypes, no protection against colonization was seen; however, when challenged with homologous Lior serogroups, protection was demonstrated. Immune animals were colonized for an average of 1 day or less versus at least 6 days for nonimmune animals. Rabbits challenged with matched Penner-unmatched Lior strains showed only marginal protection. Our study also demonstrated that flagella are important in initiating colonization and eliciting protective immunity. Campylobacter coli VC167B3, an isogenic, nonflagellated mutant, did not colonize rabbits regardless of the route of administration. Single feeding of the mutant strain did not protect the host, whereas three feedings, 48 h apart, resulted in complete protection against the flagellated parent strain. When mutant strain immunized rabbits were challenged with other strains of the same Lior serotype, marginal protection was obtained. Immunogold labeling indicated that there is one or more antigens on the cell surface of the nonflagellated mutant which reacts with a polyclonal antiserum from organisms of the same Lior serogroup. These data implicated the flagellum as the cross-strain protective component of the Lior antigen complex.

The effect of footrot on body weight and wool growth of sheep.

Body weight and traits associated with production of wool were measured over a 2-year period between 1985 and 1987 in south-western New South Wales in a flock of Merino wethers experimentally infected with footrot. The disease was allowed to spread freely amongst 150 of the flock but kept at very low prevalence in the remaining 50 by preventive footbathing during transmission periods. Severe, underrunning footrot had a significant adverse effect on body weight, for each year of the trial. Body weight was most severely reduced at times of the year when footrot was spreading among animals and lesions were severe. The mean body weight of the infected group at the end of the 2 years of observation was 7.3 kg (11.6%) below that of the control group. Footrot also depressed wool growth, with the mean clean fleece weight of the infected group being 0.4 kg (8%) lighter than that of the controls at each of the 2 annual shearings. There were no consistent differences between the groups for the other wool characteristics measured.

Serum antibody responses in sheep after natural infection with Bacteroides nodosus.

Soluble outer membrane protein of Bacteroides nodosus extracted with potassium thiocyanate (KSCN) was employed as antigen in an enzyme linked immunosorbent assay (ELISA) to detect serum antibody in sheep naturally infected with a heterologous serogroup. Serum antibody responses in 55 sheep were monitored for 2 years and maximum levels were directly related to the severity of clinical foot lesions. Serum antibody levels rose 2 weeks after foot lesions developed and declined within several months of resolution of lesions. After the first footrot transmission period, antibody levels persisted significantly (P less than 0.001) longer in sheep that did not become affected in the next transmission period compared with sheep in which footrot recurred. Antibody response did not appear to result in resolution of foot lesions. ELISA using KSCN antigen gave similar results to whole cell ELISA where cells prepared from an homologous serogroup were used as antigen. Both these assays were more sensitive than ELISA in which heterologous whole cell antigen was used. Proteins extracted from the outer membrane of B. nodosus, which are known to be immunogenic in natural infection and common to different serogroups of B. nodosus, appear to be useful antigens for serological investigations of ovine footrot.

Intestinal barriers to bacteria and their toxins.

Immunologic and nonimmunologic processes work together to protect the host from the multitude of microorganisms residing within the intestinal lumen. Mechanical integrity of the intestinal epithelium, mucus in combination with secretory antibody, antimicrobial metabolites of indigenous microorganisms, and peristalsis each limit proliferation and systemic dissemination of enteric pathogens. Uptake of microorganisms by Peyer's patches and other intestinal lymphoid structures and translocation circumvent the mucosal barrier, especially in immunosuppressed individuals. Improved understanding of the composition and limitation of the intestinal barrier, coupled with advances in genetic engineering of immunogenic bacteria, development of oral delivery systems, and immunomodulators, now make enhancement of mucosal barriers feasible.

Trehalose dimycolate enhances resistance to infection in neutropenic animals.

Bacterial infections are lethal complications of neutropenia, and antibiotics alone are inadequate therapy for these infections. Irradiated mice become severely neutropenic and remain susceptible to infection for 2 to 3 weeks, depending on the dose and quality of radiation. Some bacterial cell wall derivatives stimulate nonspecific host defense mechanisms against a variety of microbes which might cause postirradiation infection. In this study we determined if the cell wall glycolipid trehalose dimycolate (TDM), derived from Mycobacterium phlei, or a synthetic preparation of TDM was able to (i) enhance survival in mice when given before or after lethal doses of 60Co radiation and (ii) increase nonspecific resistance to postirradiation infection. Treatment with TDM oil-in-water emulsions and with synthetic TDM significantly enhanced survival before and after lethal doses of 60Co irradiation. This result correlated with the ability of TDM to reduce the translocation of intestinal bacteria and to stimulate hematopoiesis. With respect to nonspecific resistance to infection, TDM injected 1 h after sublethal irradiation increased resistance to a lethal Klebsiella pneumoniae challenge (10 50% lethal doses of K. pneumoniae in 30 days [LD50/30]) 4 or 14 days later. Increasing the dose of K. pneumoniae to 5,000 LD50/30 on day 4 overwhelmed the ability of TDM-treated mice to overcome infection. However, TDM treatment 1 h postirradiation combined with ceftriaxone antibiotic therapy (days 5 through 14) enhanced survival, even when the higher dose of bacteria (5,000 LD50/30) was used. These results indicate that in irradiated mice, TDM can be used to enhance survival and, as a potent stimulant of nonspecific resistance to infection in neutropenic mice, can act synergistically with antibiotic therapy to reduce sepsis and mortality.

Role of endotoxemia in cardiovascular dysfunction and mortality. Escherichia coli and Staphylococcus aureus challenges in a canine model of human septic shock.

Using different types of bacteria and a canine model simulating human septic shock, we investigated the role of endotoxin in cardiovascular dysfunction and mortality. Either Escherichia coli (a microorganism with endotoxin) or Staphylococcus aureus (a microorganism without endotoxin) were placed in an intraperitoneal clot in doses of viable or formalin-killed bacteria. Cardiovascular function of conscious animals was studied using simultaneous radionuclide heart scans and thermodilution cardiac outputs. Serial plasma endotoxin levels were measured. S. aureus produced a pattern of reversible cardiovascular dysfunction over 7-10 d that was concordant (P less than 0.01) with that of E. coli. Although this cardiovascular pattern was not altered by formalin killing (S. aureus and E. coli), formalin-killed organisms produced a lower mortality and less myocardial depression (P less than 0.01). S. aureus, compared to E. coli, produced higher postmortem concentrations of microorganisms and higher mortality (P less than 0.025). E. coli produced significant endotoxemia (P less than 0.01), though viable organisms (versus nonviable) resulted in higher endotoxin blood concentrations (P less than 0.05). Significant endotoxemia did not occur with S. aureus. Thus, in the absence of endotoxemia, S. aureus induced the same cardiovascular abnormalities of septic shock as E. coli. These findings indicate that structurally and functionally distinct microorganisms, with or without endotoxin, can activate a common pathway resulting in similar cardiovascular injury and mortality.