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Drug resistance of Klebsiella pneumoniae severely threatens human health. Overcoming the mechanisms of K. pneumoniae resistance to develop novel vaccines against drug-resistant K. pneumoniae is highly desired. Here, we report a technology platform that uses high pressure to drive drug-resistant K. pneumoniae to pass through a gap, inducing the formation of stable artificial bacterial biomimetic vesicles (BBVs). These BBVs had little to no bacterial intracellular protein or nucleic acid and had high yields. BBVs were efficiently taken up by dendritic cells to stimulate their maturation. BBVs as K. pneumoniae vaccines had the dual functions of inducing bacteria-specific humoral and cellular immune responses to increase animals' survival rate and reduce pulmonary inflammation and bacterial loads. We believe that BBVs are new-generation technology for bacterial vesicle preparation. Establishment of this BBV vaccine platform can maximally expand preparation technology for vaccines against drug-resistant K. pneumoniae .

This study dealt with the toxicity of inactivated bacteria intended for veterinary autogenous vaccines toward a suitable control assay. Two in vitro methods were used. The [3-(4, 5 -dimethylthiazol-2-yl) -2,5 -diphenyltetrazolium bromide] (MTT) test, based on the metabolic reaction of a tetrazolium salt in vital cells, was adopted on the basis of previous positive results. The Interleukin (IL)-1 beta release assay on monocyte-derived pig macrophages was carried out for comparative purposes, to evaluate the possible role of the inflammatory response. MTT and IL-1 beta responses showed a significant correlation (P < 0.05) at defined test dilutions of bacterial antigens, whereas no correlation was demonstrated using MTT responses normalized on bacterial cell concentration. Furthermore, the toxic effects shown in the MTT test were positively correlated to the extracellular protein content. On the whole, the above results could be a useful basis for the development of a toxicity assay on inactivated bacterial vaccines. Also, our data point at bacterial autolysis as a major component underlying toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Approximately one-third of the reportable USDA Category D and E laboratory animals in the United States are expended on the potency testing of leptospiral vaccines by the codified hamster vaccination-challenge assay. Valid tests require ≥80% of challenge controls to succumb to disease and an LD50 between 10 and 10,000. This work evaluates the risk associated with the removal of LD50 limits; thereby, eliminating back-titration hamsters from in vivo potency assays for Leptospira (L.) serogroups Canicola and Icterohaemorrhagiae. The impact was assessed through 1) retrospective analysis of industry and CVB serial release data from July 2011-April 2015 and 2) evaluation through vaccination-challenge assays. For the initial vaccination-challenge assays (n = 3/serogroup), one group received potent bacterin (PB) and six groups received subpotent bacterins (SB1-SB6). PB and SB1 were challenged with a single dilution of Leptospira between 10 and 10,000 LD50. SB2-SB6 received serial dilutions of more concentrated challenge. Based on the retrospective analysis and in vivo assays, 80% of the challenge controls succumbing to disease reasonably ensured the minimal LD50 was administered. Subpotent vaccines were not at increased risk for being deemed potent when challenged with >10,000 LD50, but potent vaccines were at risk of being deemed subpotent when challenged with >10,000 LD50., (Published by Elsevier Ltd.)

Background: Pasteurella multocida B:2 causes bovine haemorrhagic septicaemia (HS), leading to rapid fatalities in cattle and buffaloes. An attenuated derivative of P. multocida B:2 GDH7, was previously constructed through mutation of the gdhA gene and proved to be an effective live attenuated vaccine for HS. Currently, only two potential live attenuated vaccine candidates for HS are being reported; P. multocida B:2 GDH7 and P. multocida B:2 JRMT12. This study primarily aims to investigate the potential of P. multocida B:2 GDH7 strain as a delivery vehicle for DNA vaccine for future multivalent applications., Results: An investigation on the adherence, invasion and intracellular survival of bacterial strains within the bovine aortic endothelial cell line (BAEC) were carried out. The potential vaccine strain, P. multocida B:2 GDH7, was significantly better (p ≤ 0.05) at adhering to and invading BAEC compared to its parent strain and to P. multocida B:2 JRMT12 and survived intracellularly 7 h post treatment, with a steady decline over time. A dual reporter plasmid, pSRGM, which enabled tracking of bacterial movement from the extracellular environment into the intracellular compartment of the mammalian cells, was subsequently transformed into P. multocida B:2 GDH7. Intracellular trafficking of the vaccine strain, P. multocida B:2 GDH7 was subsequently visualized by tracking the reporter proteins via confocal laser scanning microscopy (CLSM)., Conclusions: The ability of P. multocida B:2 GDH7 to model bactofection represents a possibility for this vaccine strain to be used as a delivery vehicle for DNA vaccine for future multivalent protection in cattle and buffaloes.

An efficient technique for evaluation of the quality control of vaccines against clostridiosis is described in this study. This technique is capable of quantifying the toxoid of the bacterium Clostridium perfringens Type D, which is commonly found within these vaccines. The described method is performed in vivo to quantify the toxoid, replacing the current predominant approaches that use the titration of toxins before the inactivation process. This method is based on the partial neutralization of a determined dose of antitoxin by testing different doses of the toxoid. In order to guarantee its reliability, it is essential for the technique to be validated. Thus, the technique was tested using the following validation parameters: specificity and selectivity, detection limit, linear correlation, precision and robustness, in agreement with the requirements of regulatory agencies and international committees from around the world. The method was found to be specific, selective, robust, precise, and linear inside a specific concentration range. Therefore, it could be applied to the quality control of clostridiosis vaccines with satisfactory results., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis., Results: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model., Conclusion: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis.

The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bioassays play central role in evaluation of biological products and those derived from bacterial toxins often rely exclusively on in vivo models for assurance of safety and potency. This chapter reviews existing regulatory approved methods designed to provide information on potency and safety of complex biological medicines with an insight into strategies considered for alternative procedures., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this work, we produced and evaluated a vaccine based on a β toxoid of Clostridium perfringens type C produced in Escherichia coli (rBT). The non-toxic rBT was innocuous for mice and induced 14 IU mL(-1) of β antitoxin in rabbits, complying with the European Pharmacopeia and CFR9 - USDA guidelines., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A recombinant botulinum vaccine (rBV A/B) is being developed to protect adults 18-55 years of age from fatal botulism caused by inhalational intoxication with botulinum neurotoxin complex (BoNT) serotype A, subtype A1 (BoNT/A1) and BoNT serotype B, subtype B1 (BoNT/B1). Fundamental to the advanced development process is an initial demonstration of product safety in animals. A comprehensive series of studies was conducted to evaluate the general toxicity, neurobehavioral toxicity and local reactogenicity of the rBV A/B vaccine prior to first use in humans. Toxicity was evaluated in CD-1 mice vaccinated with control material and three dosages of rBV A/B with or without Alhydrogel(®) by intramuscular (IM) injection on Study Days 0, 28, 56 and 70 in a volume of 100μL. Total immunizing protein given in each dose was either 0, 2, 4 or 8 μg/animal. Local reactogenicity was evaluated in mice at the dosages given and in New Zealand white (NZW) rabbits using the same injection volume (0.5 mL) and formulations (10, 20 and 40 g/mL total antigen with 0.2% (w/v) Alhydrogel(®)) intended for human use. The rBV A/B vaccine produced no apparent systemic or neurobehavioral toxicity and only transient mild inflammation at the injection site. Together these results indicated a favorable safety profile for rBV A/B and supported its use in a Phase 1 clinical trial., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Tumor-targeting bacteria have been developed as powerful anticancer agents. Salmonella typhimurium VNP20009, a representative tumor-targeting strain, has been systemically administered as a single-agent therapy at doses of 1 x 10(6) to 3 x 10(6) colony-forming unit (cfu)/mouse, or in combination with other antitumor agents at doses of 1 x 10(4) to 2 x 10(5) cfu/mouse. Recently, we reported that oral delivery of VNP20009 at the dose of 1 x 10(9) cfu/mouse induced significant anticancer effects comparable to that induced by systemic administration of this strain at 1 x 10(4) cfu/mouse. To further address the efficacy and safety of oral administration of bacteria, here we performed a systemically comparative analysis of anticancer efficacy and toxicity of VNP20009 administered: (i) orally at a dose of 1 x 10(9) cfu/mouse (VNP9-oral); (ii) intraperitoneally at a dose of 1 x 10(4) cfu/mouse (VNP4-i.p.); or (iii) intraperitoneally at a dose of 1 x 10(6) cfu/mouse in tumor-free and tumor-bearing murine models. The results showed that VNP9-oral, similar to VNP4-i.p., induced significant tumor growth inhibition whereas VNP6-i.p. induced better anticancer effect in the B16F10 melanoma model. Among three treatments, VNP9-oral induced the mildest and reversible toxicity whereas VNP6-i.p. resulted in the most serious and irreversible toxicities when compared to other two treatments. Moreover, the combination of VNP9-oral with a low dose of chemotherapeutics produced comparable antitumor effects but displayed significantly reduced toxicity when compared to VNP6-i.p. The findings demonstrated that oral administration, as a novel avenue in the application of bacteria, is highly safe and effective. Moreover, the present preclinical study should facilitate the optimization of bacterial therapies with improved anticancer efficacy and reduced adverse effects in future clinical trials.

Botulinum neurotoxins are the most potent toxins known and causative agents of human botulism. Treatment comprises of administering purified polyclonal antitoxin or the prophylactic use of a vaccine containing formaldehyde inactivated toxoid. Whilst formaldehyde inhibits toxin activity, it induces so many structural changes in the molecule that immunisation often results in low levels of neutralising antibodies. We describe here for the first time a simple, less time consuming, novel method for producing a non-toxic toxoid that is structurally and antigenically more similar to the native toxin. Toxin is chemically inactivated by alkylation with iodoacetamide in the presence of reversibly denaturing conditions. This reduces neurotoxic activity by at least 7-orders of magnitude to undetectable levels. Following immunisation, in vivo neutralising antibody levels were 600-times higher than those produced with formaldehyde toxoid, despite generating equivalent ELISA antitoxin binding titres. These studies demonstrate that the new toxoid retains more of the native toxins structure and critical epitopes responsible for inducing life-saving neutralising antibody. Toxoid produced by the new method should substantially improve both antitoxin and vaccine production and be applicable to other toxins and immunogens.

Coxiella burnetii is a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuated C. burnetii phase II (having a truncated "O" chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-gamma(-/-)), BALB/c, Toll-like receptor 2 knockout (TLR2(-/-)), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-gamma(-/-) and TLR2(-/-) mice, which showed elevated rectal temperatures that were sustained for approximately 15 days with transient increases in splenic weights. Reinfection of IFN-gamma(-/-) and TLR2(-/-) mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2(-/-) mouse spleens was observed, greater colonization was evident in the IFN-gamma(-/-) mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-gamma(-/-) macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2(-/-) macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.

A vaccine inducing protection against most of the circulating variants of serogroup B meningococcal strains is not yet available. A number of plausible options are currently under investigation. A conjugate vaccine based on a modified capsular polysaccharide might well work, but has safety concerns from molecular mimicry between group B sialic acid and human tissue. Recently, however, the group B capsule has been shown to contain de-N-acetyl sialic acid residues that do not cross react with normal host tissues and can then be the target of bactericidal antibodies. Potentially, this polysaccharide structure could form the basis of a safe and protective group B vaccine. Outer membrane vesicles (OMVs) from Neisseria lactamica avoid the immunodominant and highly strain specific immune response against the PorA protein, and are reported to elicit cross reactive protection in mice against lethality from challenge with meningococcal group B bacteria. However, the serum antibody responses lack bactericidal activity, and the mechanisms of protection are unknown. A number of universal, cross-reactive antigens have been identified through "reverse vaccinology" and successfully tested as recombinant protein vaccines. Promising results have also been demonstrated using OMV vaccines prepared from strains engineered for upregulation of conserved, cross-reactive antigens. This approach takes advantage of experience gained with conventional wild-type OMV vaccines and the large number of new antigens identified through sequencing the genome of N. meningitidis. Initial studies show that the traditional use of detergents to decrease toxicity by extraction of lipopolysaccharides (LPS) should, if possible, be omitted in order to avoid extraction of important lipoproteins. In the absence of detergent extraction, clinical OMV formulations with acceptable toxicity may still be achieved by constructing vaccine production strains with genetically detoxified LPS. Thus, a MenB vaccine might be designed based on non-cross-reactive capsular antigens, OMV vaccines from genetically modified strains, recombinant proteins or a combination of these approaches. Given all of the recent data available and experience gained, the possibility for development of a universal vaccine for prevention of group B meningococcal disease looks promising. For evaluation of vaccine formulations relying on cross-reactive proteins, selection of strains for representation of the global epidemiological situation will be of outmost importance. Defining criteria for establishing and revising such strain collections is currently ongoing and will be a key element in developing and evaluating new protein based vaccines in the time to come.

Data on influence of Francisella tularensis C-complex preparations on formation of immunity against tularemia are presented. Study of cellular immunity characteristics as well as dynamics of antibody response was carried out on white mice and guinea pigs models. Absence of toxicity, pyrogenicity, and negative effects on immunocompetent cells in combination with protective activity points to possibility of use the C-complex as a component of a subunit vaccine.

Single-stranded RNA stimulates immune cells and induces the secretion of pro-inflammatory cytokines and type I IFN. As adjuvant RNA can induce a T(h)2 type of humoral response, however, its potency in the induction of cytotoxic T cells in vivo has not been analyzed. Here we show that immunization with the antigen ovalbumin (OVA) and the adjuvant phosphodiester RNA complexed to the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) induced a Toll-like receptor-7-dependent cytotoxic T cell and humoral response. Staining with SIINFEKL-K(b) tetramers demonstrated the induction of antigen-specific T cells that were functional in in vivo cytotoxic T cell assays against SIINFEKL-loaded target cells. In infection experiments with OVA-secreting Listeria monocytogenes, the cytotoxic T cell response strongly reduced the bacterial load in liver and spleen. The RNA-driven humoral response was characterized by OVA-specific antibodies of the IgG1 isotype whereas CpG-DNA induced antigen-specific antibodies of the IgG2a (BALB/c) or IgG2c (C57BL/6) isotype. Furthermore, stimulation with RNA did not induce splenomegaly, a common feature of CpG-DNA-driven immune activation in mice. Taken together, our data confirm that RNA can be used as a safe adjuvant and induces a strong antibody response of the IgG1 isotype. Additionally, we demonstrate that RNA induces an antigen-specific immunity characterized by a potent cytotoxic T cell response to infection.

Avian botulism is a paralytic disease caused by a toxin produced by Clostridium botulinum type C. Since type C isolates from cases of avian botulism produced a neurotoxin consisting of a mosaic form of parts of type C and D neurotoxins, we examined the antitoxin titers in the convalescent sera of botulism-affected birds which belonged to family Anatidae. ELISA using the C/D mosaic neurotoxin as an antigen revealed that the antibody was detected in the sera at 2 weeks, but not at 5 weeks after the onset, suggesting that the antibody only appeared for a short period in the convalescent phase. However, we failed to detect the antibody titers with anti-chicken IgG instead of anti-duck IgG. We therefore examine the immunological properties of IgG among different families and species. The results revealed that different species of IgG in the same family exhibited strong cross-reactivity. Ducks immunized once with the toxoid together with a commercial oil-adjuvanted vaccine were found to develop sufficient antibody to protect against a challenge with a lethal toxin dose. The ELISA titers did not correspond to the neutralization titers in the sera of immunized ducks at the early stage during immunization. These findings suggest that the neutralizing titer was more useful than the ELISA titer for evaluating the protection against the toxin, but the ELISA technique may be applicable for detecting the occurrence of botulism.

The safety and immunological activity of P. aeruginosa vaccine were experimentally evaluated. The vaccine was prepared on the basis of the antigens of P. aeruginosa extracellular slime which was accumulated in medium K-4, obtained with the use of original technology. The immunization of animals with P. aeruginosa vaccine induced the synthesis of antibodies. The introduction of the vaccine in 2 or 3 injections resulted in a high level of antibody formation, differing with the use of various strains. Hyperimmune sera, obtained by the multiple immunization of rabbits with P. aeruginosa vaccine, ensured high protection of mice from P. aeruginosa infection. The vaccine proved to be safe when evaluated in experiments of acute and chronic toxicity, made on laboratory animals.

The threat posed by botulism, classically a food- and waterborne disease with a high morbidity and mortality, has increased exponentially in an age of bioterrorism. Because botulinum neurotoxin (BoNT) could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. Although clearly the development of a safe and effective mucosal vaccine against this toxin should be a high priority, essentially no studies to date have assessed mucosal immune responses to this disease. To bridge this gap in our knowledge, we immunized mice weekly for 4 wk with nasal doses of BoNT type A toxoid and a mutant of cholera toxin termed E112K. We found elevated levels of BoNT-specific IgG Abs in plasma and of secretory IgA Abs in external secretions (nasal washes, saliva, and fecal extracts). When mice given nasal BoNT vaccine were challenged with 4 x 10(3) LD50 of BoNT type A (BoNT/A) via the i.p. route, complete protection was seen, while naive mice given the same dosage died within 2 h. To further confirm the efficacy of this nasal BoNT vaccine, an oral LD50 was determined. When mice were given an oral challenge of 5 microg (2 x oral LD50) of progenitor BoNT/A, all immunized mice survived beyond 5 days, while nonimmunized mice did not. The fecal extract samples from nasally vaccinated mice were found to contain neutralizing secretory IgA Abs. Taken together, these results show that nasal BoNT/A vaccine effectively prevents mucosal BoNT intoxication.

Objective: To construct a genetically modified nontoxigenic Pasteurella multocida toxin (PMT) and examine its immunoprotective activity against challenge exposure with wild-type PMT in pigs., Animals: 5 healthy pigs., Procedure: A nontoxigenic PMT was created by replacing the serine at position 1164 with alanine (S1164A) and the cysteine at position 1165 with serine (C1165S). Toxic activity was determined by use of the guinea pig skin test and mouse lethality test. Three pigs were vaccinated twice with the modified PMT, and the remaining 2 pigs served as nonvaccinated control animals. Vaccinated and control pigs were challenge exposed with wild-type PMT. Pigs were euthanatized and necropsied on day 14 after challenge exposure. Turbinate atrophy was examined macroscopically and assigned a score. Serum anti-PMT antibodies were determined by use of an ELISA., Results: The genetically modified PMT was characterized by a total lack of toxic activity. Pigs vaccinated with the modified PMT became seropositive; in contrast, control pigs remained seronegative. Necropsy revealed that the 2 control pigs had moderate and severe turbinate atrophy, respectively, whereas the 3 vaccinated pigs did not have any lesions in the turbinates or abnormalities in other organs., Conclusions and Clinical Relevance: Modification by use of S1164A and C1165S leads to a complete loss of toxic effects of PMT without impairment of the ability to induce protective immunity in pigs. Analysis of these results suggests that genetically modified PMT may represent a good candidate for use in developing a vaccine against progressive atrophic rhinitis in pigs.

Two different potency tests were evaluated, consisting in one-dilution used to determine 50% lethal dose (LD50) and four--dilution used to calculate 50% effective dose (ED50), respectively. Our experience has led to propose the four-dilution test for routine lot releasing.

The single-dose toxicological test for the leptospirosis vaccine vax-SPIRAL was made in rats. No clinical toxicity symptoms were found. The differences between the evaluated variables were not important from the biological point of view.

The inhalation of aerozolized botulinum toxin may represent a potential significant hazard to both military and civilian personnel. Since the lung is the primary target organ for inhaled toxin, the investigation reported herein was conducted to examine lung function in mice exposed to botulinum toxin A complex by intranasal route. Data includes lethality, symptomatology, measurement of respiratory function (minute ventilation, respiratory frequency, and tidal volume), and histopathology of the lungs. The clinical signs of intoxication are similar to those observed in foodborne botulism. Plethysmography revealed severe impairment of all respiratory parameters tested from 7 hours postexposure. Severe lung lesions, possibly secondary to the intoxication, were observed in mice who survived 14 days after the toxin challenge. These included intra-alveolar hemorrhage and interstitial edema. Mice immunized by the pentavalent (ABCDE) toxoid were protected against the neurotoxin (4 LD50) as revealed by the decrease of lethality and severity of nervous signs of intoxication, but not against histopathological changes in the lungs. These effects are nonspecific and require further experiments in order to specify the relationships between the pathology and the inflammatory process in the lung due to mediators such as cytokines,and possibly permanent physiological sequelae.

Mice immunized by the intranasal route with dendritic cells harvested from the lungs and then pulsed with Ag85 (LDC-Ag85) were able to prime naive CD4(+) T cells in vivo. As a result splenic CD4(+) T cells from these immunized mice were able to produce IFNgamma following culture with Mycobacterium tuberculosis-infected antigen presenting cells. Hematoxylin and eosin stained lung sections from LDC-Ag85 immunized mice after they had been exposed to aerosol challenge with M. tuberculosis showed a florid infiltration of macrophages and lymphocytes into granulomas and parenchymal tissues when compared to lung sections from control groups implanted with dendritic cells pulsed with ovalbumin. In addition, using immunohistochemistry, these tissues appeared to have more CD4(+) and CD8(+) cells than the control groups. This was confirmed by flow cytometric analysis which showed that lung cell digests contained increased numbers of CD4 and CD8 interferongamma secreting cells. Despite this increase however, no evidence was seen that indicated that the LDC-Ag85 immunized mice were more resistant to M. tuberculosis infection than mice immunized with LDC pulsed with an irrelevant protein. Instead, the potent inflammatory response in the LDC-Ag85 resulted in serious consolidation of the lung tissue.

Listeria monocytogenes (Lm) is an attractive vector to elicit T cell immunity because it infects antigen-presenting cells and because infection originates at the mucosa. Lm expressing HIV gag elicits sustained high levels of gag-specific CTL in mice. Since Lm causes disease in immunocompromised hosts, a highly attenuated strain of Lm that requires D-Ala for viability was produced. Attenuated bacteria expressing HIV-1 gag (Lmdd-gag) are as efficient as wild-type recombinants at stimulating gag-specific murine CTL when administered with D-Ala and at boosting human CTL in vitro. Lmdd-gag immunization protects mice from vaccinia-gag challenge and induces mucosal CTL, even after systemic immunization.

Animal experiments still play a central role in the quality control of vaccines. Generally, performance of these experiments is provided by law and laid down in the European Pharmacopoeia. Classical vaccines, the efficacy of which is calculated in International Units, require a very high number of experimental animals for quality control testing. The testing mainly consists of infection and intoxication experiments causing extreme suffering of the animals involved. This classical product group includes clostridial vaccines which are used to a great extent in veterinary medicine in particular. Within the last years, considerable efforts have been made to reduce the number of animal experiments in this field, lower the number of animals, and decrease the suffering of the animals during testing. Several research projects for the development and validation of alternative methods have been initiated. Furthermore, the 3R Concept (Replacement, Reduction and Refinement) is increasingly taken into consideration when developing or revising legal provisions. This led to various improvements regarding animal welfare in the quality control of clostridial vaccines.

Our objective is to develop a prophylactic vaccine strategy that can be evaluated for surgical and other high-risk hospitalized patients. In this paper, we describe the preparation and preclinical evaluation of a liposomal complete-core lipopolysaccharide (LPS) vaccine that is nontoxic and broadly antigenic. Complete-core (Ra-chemotype) LPSs were isolated from four gram-negative bacterial strains (Escherichia coli K-12, E. coli R1, Pseudomonas aeruginosa PAC608, and Bacteroides fragilis), mixed together to form a cocktail of complete-core LPSs, and then incorporated into multilamellar liposomes consisting of dimyristoyl phosphatidyl choline, dimyristoyl phosphatidylglycerol, and cholesterol in a 4:1:4 molar ratio. The endotoxic activities of these LPS-containing liposomes were less than 0.1% of the endotoxicities of the original free LPSs as measured by the Limulus amoebocyte lysate assay. In vivo administration of liposomal complete-core LPS mixed with Al(OH)(3) to rabbits resulted in no pyrogenicity or overt toxicity over a 7-day period. In immunoblots, sera from rabbits following active immunization elicited cross-reactive antibodies to a large panel of rough and smooth LPSs from numerous clinically relevant gram-negative bacteria, including E. coli (serotypes O1, O4, O6, O8, O12, O15, O18, O75, O86, O157, and O111), P. aeruginosa (Fisher-Devlin serotypes 1, 2, and 3, which correspond to International Antigenic Typing Scheme types 6, 11, and 2, respectively), Klebsiella pneumoniae (serotypes O1, O2ab, and O3), B. fragilis, and Bacteroides vulgatus. Active immunization of mice with liposomal complete-core LPS provided protection against a lethal challenge with E. coli O18 LPS. The vaccine tested was nontoxic, nonpyrogenic, and immunogenic against a wide variety of pathogens found in clinical settings.

The effect of immunization with Burkholderia pseudomallei, (Pur- and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2, carrying a plasmid with fragments of B. pseudomallei chromosome, was studied on four species of experimental animals, essentially differing by their sensitivity to melioidosis. B. pseudomallei mutants formed the statistically significant level of protection in subcutaneously challenged animals, moderately sensitive to melioidosis, but were not effective when tested, under the same conditions, in animals, highly sensitive to melioidosis. The effect produced by the experimental vaccines under study in animals of all species, subjected to aerogenic challenge, was leveled. The study showed good prospects for the use of tularemia vaccine with a view to create heterologous immunity to melioidosis and the possibility of its use as the basis of bivalent gene engineering vaccine.

The International Veterinary Industry Test Replacement Organisation (In-VITRO) was established in 1995 with the aim of developing, validating and harmonising in vitro alternatives to replace in vivo methods for in-process and potency testing of veterinary clostridial vaccines. The emphasis has been on the reduction of animal usage in the Clostridium chauvoei potency assay and its eventual replacement by an in vitro assay. Replacement of the toxin neutralisation assay for Cl. tetani by an internationally validated indirect ELISA has already started. A validation programme involving a collaboration organised through EDQM which could ultimately lead to the standardisation of in vitro tests for all clostridial vaccines is in progress. In addition In-VITRO is now considering the setting up of a programme for Erysipelas vaccines. The collaboration between manufacturers of veterinary vaccines in the development and validation of in vitro tests is a major step towards the reduction and replacement of animal tests.

Coxiella burnetii phase I whole cell vaccine (WCV) is associated with risk of severe local delayed-type hypersensitivity (DTH) reactions in previously immunized individuals or those sensitized by natural exposure. We compared this vaccine to another investigational vaccine derived by chloroform-methanol extraction of phase I whole cells (chloroform-methanol residue vaccine, CMRV). Hairless guinea pigs, sensitized with either WCV or CMRV, were given 60,600 and 6,000 ng of WCV or CMRV in an intradermal (i.d.) skin test. The i.d. administration of WCV consistently caused more host reactions than comparable doses of CMRV in guinea pigs sensitized with either WCW or CMRV, suggesting that CMRV may be a safer vaccine. However, the CMRV was not innocuous and caused significant indurated lesions and micro-abscesses at the 600 ng and 6,000 ng skin test sites.

A wide variety of bacterial vaccines is in various stages of preclinical and clinical development. These products range from whole killed or live attenuated bacterial organisms to purified proteins, peptides and plasmid DNA. Although preclinical strategies may be directed by a set of common guidelines focused on demonstrating safety and biological activity, the exact developmental scheme will depend on product-specific characteristics. In general, preclinical data should support the proposed clinical formulation and include detailed information on the source and quality of starting materials, manufacturing processes, characterization of bacterial seed stocks, potency, general safety, purity, and identity. Data describing product validation and testing may be appropriate depending on the type of product, e.g., genetic stability for recombinant constructs, details on inactivation or attenuation methods for organisms or toxins, demonstration of potency of combination products, and safety and toxicology studies of plasmid DNA vaccines or vaccines with novel adjuvants. The choice of dose, route, and formulation to be used clinically may be greatly affected by rigorous preclinical developmental strategies.

The comparative study of the immunogenic and protective properties of V. cholerae, grown in vivo and in vitro, and cell walls and lysates obtained from these organisms. In the mouse protection test the efficacy of preparations obtained from vibrios grown in vivo did not exceed that of the preparations obtained from V. cholerae agar cultures.

Gram-negative bacterial ghosts produced by controlled expression of the plasmid-encoded lysis gene E offers a promising approach in non-living vaccine technology. Bacterial cell wall complex and hence the antigenic determinants of the living cells are not affected by denaturation due to cell killing. However, the endotoxin content of the Gram-negative cell wall has been discussed as a potential problem for this kind of whole cell or envelope vaccines. Here we show that bacterial ghosts prepared from Escherichia coli O26:B6 and Salmonella typhimurium C5 induce dose-dependent antibody responses against bacterial cells or their corresponding lipopolysaccharides (LPS) in doses 25 ng kg-1 when administered intravenously to rabbits in a standard immunization protocol. No differences between the immune responses of the rabbits were observed when comparing equivalent doses of bacterial ghosts and antibiotic-treated whole cells. The results indicate that the bacterial ghosts exhibit all the antigenic properties of the living cells. No significant fever responses in rabbits have been recorded in doses of < 250 ng kg-1 E. coli O26:B6 ghosts and up to doses of 250 ng kg-1 S. typhimurium C5 ghosts when applying test methods recommended by the US pharmacopoeia. These findings correlate with cell culture experiments where doses 100 ng ml-1 of bacterial ghosts were needed for the release of tumour necrosis factor alpha (TNF alpha) and prostaglandin E2 (PGE2) from RAW mouse macrophage cultures. Free LPS of Salmonella abortus equi commonly used as a LPS-standard, however, stimulated TNF alpha and PGE2 synthesis of RAW cells in doses of 1 ng ml-1. The endotoxic activity of our bacterial preparations analysed by a standard limulus amoebocyte lysate and 2-keto-3-deoxyoctonate assay correlated with the capacity to stimulate the release of PGE2 and TNF alpha in RAW mouse macrophage cultures and the endotoxic responses in rabbits. It can be concluded that these in vitro systems can be used as easy predictive test systems for preparations of bacterial vaccines, particularly for bacterial ghosts.

After expression in Escherichia coli and purification by Ni++ chelate-affinity chromatography, the outer membrane protein I (OprI) of Pseudomonas aeruginosa was tested in experimental animals for its safety and pyrogenicity. Four groups of 7 adult human volunteers were then vaccinated 3 times at four-weekly intervals with either 500 micrograms, 200 micrograms, 50 micrograms or 20 micrograms of OprI adsorbed onto aluminum hydroxide. The vaccinations were well tolerated and without systemic side effects, but a significant rise of antibody titers against OprI was measured in the serum of those who had received the 500 micrograms, 200 micrograms or 50 micrograms doses. Raised antibody titers against OprI were still present 30 weeks after the final vaccination. It was possible to demonstrate binding of the complement component C1q to the elicited antibodies, and this confirms their ability to promote antibody-mediated complement-dependent opsonization.

CFC-101, a Pseudomonas vaccine, was administered to beagle dogs by intramuscular injection for 4 weeks (5 days/week) at 0.05, 0.15 and 0.45 mg/kg/d. Clinical signs considered to be related to treatment were restricted to swelling at the injection sites, being apparent 1-2 h after treatment. There was no effect on body weight, food consumption, ophthalmoscopy, electrocardiography, hematology, biochemical and urinary parameters. The histopathological examination revealed treatment-related changes at the injection sites at all dosages, particularly in the hindlimbs where both perivascular and intramuscular aggregations of inflammatory cells were seen. Thus, the only treatment-related changes seen in this study were local reactions to the test substance at the injection sites; furthermore these changes seem to represent a pharmacological response to the test material. Because no evidence of any systemic toxicity was observed at any dosage level, it is concluded that dosages of CFC-101 up to and including 0.45 mg/kg/d were well tolerated over a period of 4 weeks in the beagle dog.

A vaccine preparation consisting of tetanus toxoid (TT) encapsulated in biodegradable poly (D,L-Lactide) glycolide polymer has been developed. The toxicity of the preparation was evaluated in adult male and female Wistar rats injected IM with 20, 100 and 200 Lf tetanus toxoid, in polymer in 0.5 ml vehicle. No adverse effect of the vaccine could be seen, except for a granulomatous reaction at the site of injection. The vaccine was considered safe based on the findings in rats of both sexes.

Conditions for the submerged cultivation of a strain of V. cholerae O139, were worked out. These conditions ensured a high yield of biomass, soluble O-antigen and exoenzymes (proteinase, phospholipase A) into the culture medium, which exceeded their production by strains of serovar O1, respectively, 2, 3, 4 and 8 times. The preparation, isolated from the culture fluid and lyophilized, contained up to 50% of O-antigen and exoenzymes. In experiments on white mice the preparation exhibited low toxicity (LD50 was equal, on the average, to 1.2 mg) and immunogenicity (ED50 was equal to 3-5 micrograms) with respect to V. cholerae O139, which corresponded to the protective potency of commercial vaccine against V. cholerae O1 infection.

The safety and immunogenicity of a recombinant outer surface protein A (OspA) Lyme vaccine in patients previously diagnosed with Lyme disease was assessed in a dose-ranging, prospective study. Thirty healthy volunteers were consecutively assigned to receive three doses of 3, 10, or 30 micrograms of OspA vaccine at 0, 1, and 2 months. Subjects were seen 3 days after each vaccine dose and 1 month after completion of the three-dose schedule. Local side effects included soreness, induration, swelling, and redness. Transient systemic side effects occurred in 21 subjects, the majority of which (81%) were characterized as mild. Solicited symptoms included migratory mild arthralgias that lasted 24 h in 3 subjects. Side effects were not more evident after the second or third dose. Of the patients, 93% developed high-titer OspA antibodies. Thus, an OspA vaccine may be safe and immunogenic in patients with a history of Lyme disease.

Since October 1992, epidemics of cholera associated with Vibrio cholerae O group 139 have occurred in India, Bangladesh, and much of the rest of Asia. A volunteer model was used to determine the safety, immunogenicity, and efficacy of an attenuated delta ctxA delta zot delta ace delta cep V. cholerae O139 vaccine strain, designated CVD 112. Six volunteers received 10(6) cfu and 6 received 10(8) cfu of CVD 112. No subject who received the 10(6) dose had diarrhea or other severe symptoms after vaccination; 3 vaccinees developed mild diarrhea (mean stool volume, 648 mL) after receiving the higher dose. Five weeks after vaccination, 8 vaccinees and 15 unvaccinated control subjects underwent challenge with 10(6) cfu of wild type V. cholerae O139 AI1837. One vaccinee (13%) and 12 control subjects (80%) developed diarrhea after challenge (P = .003). The short-term protective efficacy conferred by vaccine strain CVD 112 was 84% and was remarkably similar to that conferred by primary wild type clinical infection (80%).

We are studying processes leading to protective immunity in murine Lyme borreliosis in order to derive a suitable vaccine candidate for clinical use. Our work focuses on the role of the two main outer surface lipoproteins A and B (OspA and OspB) of the causative agent of Lyme disease, the spirochete Borrelia burgdorferi, as targets for its specific elimination. We could show that native and recombinant LipOspA induce monospecific immune sera able to passively transfer protection in SCID mice against experimental and tick-borne infection and disease. Recent results for phase I and II safety and efficacy trails are promising in demonstrating that the recombinant LipOspA vaccine candidate is safe and immunogenic and elicits borreliacidal antibodies.

We tested the ability of recombinant outer membrane proteins of Pseudomonas aeruginosa to serve as a protective vaccine against this gram negative pathogen under two main pathophysiological events leading to P. aeruginosa sepsis. i) systemic infection during immunosuppression, and ii) bacterial translocation. A hybrid vaccine was cloned combining protective epitopes of outer membrane protein F (OprF) and outer membrane protein I (OprI). This vaccine proved to be highly protective against an intraperitoneal challenge with P. aeruginosa in immunosuppressed mice. Oral immunization of mice, with recombinant Salmonella dublin expressing OprI induced s-IgA antibodies in the gut mucosa against OprI and provided protection against translocation of P. aeruginosa in an immunosuppressed mouse model. To test whether OprI is safe for use in humans, recombinant OprI was purified and used for immunization of volunteers. Vaccination was well tolerated and no major side effects were observed. The induction of serum antibodies against OprI was found to be dose-dependent and was observed in total in 65% of the volunteers.

Since a limited number of O serogroups account for nearly 70% of bacteremic and meningitic Escherichia coli isolates, a polyvalent vaccine was made by conjugating a Pseudomonas aeruginosa exotoxin A carrier protein to the O polysaccharide of 12 serogroups of E. coli (O1, O2, O4, O6-O8, O12, O15, O16, O18, O25, O75). No serious reactions occurred in 88 vaccinees. Four-fold or greater increases in ELISA antibody levels over baseline were greatest (> 60% of vaccinees) for O1, O2, O6-O8 and O15; intermediate (approximately 50%) for O18 and O75, and poorest (> or = 45%) for O4, O12, O16, and O25. Responses with functionally active opsonophagocytic antibody generally paralleled ELISA antibody responses. With the availability of a safe, immunogenic E. coli vaccine, active and passive immunization strategies merit further development as adjunctive treatment for E. coli bacteremia and neonatal meningitis.

We investigated the chemical and immunologic properties of a heptavalent vaccine composed of high-molecular-weight polymers of the lipopolysaccharide (LPS) O polysaccharides representative of the most common clinical isolates of Pseudomonas aeruginosa. We also evaluated the serum antibody response to nonvaccine strains of P. aeruginosa, including strains expressing structural variants (subtype strains) of the O side chain of the vaccine strains. The polyvalent vaccine, prepared under conditions suitable for human use, contained low levels of contaminants and passed preclinical safety and toxicity tests required for human use. Chemical analyses indicated that individual polysaccharides were composed of both O-side chain and core sugars. Following immunization of C3H/HeN mice and New Zealand White rabbits, antibody titers against vaccine components increased between 32- and 200-fold. Antibodies reactive with LPS isolated from smooth and rough nonvaccine strains were also elicited. Analysis of the opsonic activity against the known LPS subtype variants of the vaccine strains revealed a variable pattern of killing, which ranged from opsonic killing of > or = 69% of bacterial cells representing all subtype variants within a serogroup to opsonization of only a minority of the subtype variant strains. Mouse and rabbit immune sera showed different patterns of opsonic activity against subtype strains, indicating that different epitopes on these antigens are immunodominant in the representatives of these two animal species tested. The polyvalent vaccine was effective at eliciting antibodies to vaccine components in mice and rabbits, but it remains to be determined if the current heptavalent formulation contains sufficient components to provoke human antibodies reactive with a majority of clinical strains of P. aeruginosa.

The toxicity, immunogenic properties and protective activity of the live culture of E. coli M17 and antigenic preparations obtained from cell suspensions of this strain have been studied under experimental conditions. As revealed in experiments on mice, E. coli M17 live culture has low virulence, moderate toxicity and provides the protection of immunized mice from challenge with homologous and highly virulent E. coli strains. E. coli M17 live culture, when introduced orally or intravenously into rabbits, ensures the synthesis of 02 and H6 antibodies. Blood sera taken from immunized rabbits yield better results than initial sera in experiments on the passive protection of mice. The results of our experiments show the expediency of the clinical trials of Colibacterin as a perspective Escherichia live oral vaccine.

A comparison of therapeutic effects of bacterial preparations (vaccinum dysenteriae, colibacterinum, bifidumbacterinum, bificolum) after gamma-irradiation was carried out. Bificolum showed the maximum and bifidumbacterinum the minimum effect, while colibacterianum had a therapeutic effect only within a narrow range of doses. Vaccinum dysenteriae was also effective.

Rats were vaccinated with saline (control) or one of the two commercially available Pasteurella haemolytica vaccines Presponse or Precon-PH. Animals were killed 3 days later and thoracic aorta removed for evaluation of the ex vivo biophysical responses to carbachol (CCh). In some experiments, vascular endothelium was mechanically removed. Vaccination of rats impairs the endothelial-dependent relaxation to CCh. In vessels with endothelium removed, the contractile response to CCh is converted into a relaxation following vaccination. Treatment of endothelial-denuded vascular rings ex vivo with methylene blue, a guanylate cyclase inhibitor, reduced the vaccination effect. Treatment of vascular rings with the superoxide dismutase inhibitor diethyldithiocarbamate, impairs the relaxant response of de-endothelialized vessels to CCh in Presponse vaccinated rats while enhancing the relaxation response of vessels from Precon-PH vaccinated rats. De-endothelialized vessels from vaccinated rats, but not control rats, relaxed in the presence of N-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthetase. Furthermore, in the presence of L-NMMA, the relaxant response to CCh is significantly enhanced by Precon-PH but not Presponse. The normal relaxant response to hydrogen peroxide is converted into a contraction following vaccination. Results suggest that exposure to commercially available P. haemolytica vaccines alters vascular smooth muscle reactivity to CCh and that several independent pathways may be altered.