Prevention of invasive bacterial diseases by immunization with polysaccharide-protein conjugates.

Covalent binding of CPS to T cell-dependent carrier proteins to form conjugates can be done by clinically acceptable methods. As a component of a conjugate, two immunologic properties of CPS are changed: 1) their immunogenicity is increased and; 2) reinjection induces a booster response in the young (T cell-dependence). Serum antibodies induced by the CPS alone, or as a component of a conjugate, are qualitatively similar: the difference between antibodies elicited by the CPS or the conjugate is quantitative. A clinical trial with a Hib-DT conjugate showed that conjugates could confer immunity in an age group not protected by the CPS alone. (table; see text) Induction of serum CPS antibodies confers protection against capsulated bacteria in the bloodstream: their role in the interaction of these pathogens on the mucous membranes has not been characterized. Preliminary in vitro experiments suggest that secretory antibodies to non-capsular structures may also exert protective immunity.

Epidemiology of capsular and surface polysaccharide in Staphylococcus aureus infections complicated by bacteraemia.

Staphylococcus aureus is a leading cause of serious hospital- and community-acquired infections. The discovery of serologically distinct capsular polysaccharides on the surface of clinical isolates has allowed the development of vaccines and passive protective immunity. We have studied patient characteristics, infection characteristics and the surface and capsular polysaccharide serotype distribution in patients with S. aureus infections complicated by bacteraemia admitted to VA hospitals in Maryland between 1995 and 2000. Nine hundred and ninety-three blood cultures from 331 patients were positive for S. aureus. Thirty-eight percent of patients had diabetes, 11% had end-stage renal failure, and 23% were injection drug users. Forty-two percent of infections were caused by methicillin-resistant strains (MRSA), and 60% were acquired during hospitalization. Serotyping of the first available isolate per patient (N=234 isolates) using polyclonal antibodies showed three major phenotypes--42%, type 8 (T8) capsule; 50%, type 5 (T5) capsule; and 8%, 336 polysaccharide. MRSA isolates were significantly more likely to be T5 than methicillin-susceptible isolates (66% vs. 39%, P<0.001). The proportion of T5 MRSA increased significantly (years 1-2: 41%; years 3-4: 65%; years 5-6: 90%, P<0.001). This large sample of patients with serious S. aureus infection confirms that capsular polysaccharides T5 and T8 cause most human infections, and together with serotype 336, account for nearly all those with bacteraemia.

Prevalence of capsular serotypes among Staphylococcus aureus isolates from cows with mastitis in the United States.

Development of an appropriate Staphylococcus aureus vaccine for bovine mastitis has eluded researchers for decades. The ability of S. aureus to form a protective exopolysaccharide capsule has posed a major obstacle because of the multiple serotypes and the poor immune response elicited by exopolysaccharides. This study characterized S. aureus serotypes isolated from cases of bovine mastitis obtained from veterinary diagnostic laboratories that service 44% of the dairy cattle in the United States. Major milk producing areas of the northeast, north central, Pacific coast and southwest were proportionately represented. Sub-samples of mastitic milk that contained S. aureus were frozen and sent to our laboratory for strain serotyping. The only other regional serotyping of S. aureus from bovine mastitis to date was done in France. The primary serotypes found were types 5 (51%) and 8 (18%) and 31% were non-typeable. In the current study, serotype 5 accounted for 18% of the isolates and serotype 8 for 23%. More importantly 59% of the isolates were not typeable with either type 5 or 8 antisera. These data indicate that S. aureus vaccines employing serotypes 5 and 8 would only be marginally effective in the United States. These data also suggest that development of a S. aureus vaccine for bovine mastitis should take into account regional variation in S. aureus serotypes.

A nicotine conjugate vaccine reduces nicotine distribution to brain and attenuates its behavioral and cardiovascular effects in rats.

Vaccination of animals to elicit drug-specific antibodies, or the passive transfer of such antibodies from other animals, can reduce the behavioral effects of drugs such as cocaine and heroin. To study the potential application of this approach to treating nicotine dependence, IgG was isolated from rabbits immunized with a nicotine-protein conjugate vaccine. Anesthetized rats received immune IgG containing nicotine-specific antibodies (Nic-IgG) or control-IgG i.v.. Thirty minutes later, rats received nicotine at 0.03 mg/kg i.v., equivalent on an mg/kg basis to the nicotine intake from two cigarettes by a smoker. Compared to control-IgG, Nic-IgG reduced the brain nicotine concentration in a dose-related manner (65% reduction at the highest IgG dose). Pretreatment with Nic-IgG also reduced the distribution to brain of five repeated doses of nicotine (equivalent to the nicotine intake from 10 cigarettes) administered over 80 min. To study blood pressure effects, rats received control-IgG or Nic-IgG 1 day prior to administering nicotine. Nicotine-induced systolic blood pressure increases were attenuated by Nic-IgG in a dose-related manner, and were almost completely blocked by the highest Nic-IgG dose. Pretreatment with Nic-IgG also completely prevented the nicotine-induced stimulation of locomotor activity observed in rats receiving control-IgG. Nic-IgG did not prevent locomotor activation from cocaine, demonstrating its specificity for nicotine. These data demonstrate that the administration of nicotine-specific antibodies can reduce or prevent some of the pharmacokinetic, cardiovascular, and behavioral consequences of nicotine in rats. Effects were observed at nicotine doses and nicotine serum concentrations equal to or exceeding those typically associated with nicotine exposure in cigarette smokers. A potential role for immunization in the treatment of nicotine dependence is suggested.

Passive immunization against nicotine prevents nicotine alleviation of nicotine abstinence syndrome.

Passive immunization against nicotine interferes with its locomotor and pressor effects. The current study determined whether immunization could prevent another nicotine action: the reversal of nicotine abstinence syndrome. IgG containing 4.4-5.6% nicotine-specific antibody was isolated from rabbits immunized with 3'-amino-methyl-nicotine conjugated to a carrier protein. Twenty rats were rendered dependent by 7 days of subcutaneous infusion of 3.15 mg/kg/day nicotine (expressed as the base). Upon termination of nicotine infusion, each rat was injected intraperitoneally with 150 mg of IgG from normal serum (n=13) or from nicotine antiserum (n=7). Twenty-two and one-half hours later, all rats were observed over 15 min for baseline nicotine abstinence signs. Two and one-half hours after baseline observations, seven of the 13 rats pretreated with control IgG and all seven rats pretreated with nicotine-specific IgG were then challenged by 0.12 mg/kg (sc) nicotine. The remaining six rats pretreated with control IgG were challenged with saline alone. All rats were then observed again for abstinence signs. Nicotine injection caused significantly less reduction of abstinence signs in the immunized rats. The nicotine effect in immunized rats was comparable to the saline effect in nonimmunized rats. Immunization also significantly reduced free serum nicotine concentration and nicotine distribution to the brain. These results raise the possibility that immunization might prevent nicotine consumption from relieving the discomforts of smoking cessation.

Inhibition of nicotine-induced seizures in rats by combining vaccination against nicotine with chronic nicotine infusion.

The ability of a nicotine vaccine to protect against nicotine-induced seizures was studied in rats. Groups of 10 rats were vaccinated with 3 doses of either a nicotine conjugate vaccine over 6 weeks to elicit high titers of nicotine-specific antibodies or with a control vaccine. Rats were then pretreated with a 1-week subcutaneous infusion of either nicotine 1 mg/kg/day or saline and then received a single 2 mg/kg ip dose of nicotine to provoke seizures. Vaccination reduced the incidence of seizures. The combination of vaccination and pretreatment with nicotine infusion was more effective than either treatment alone. These data suggest that vaccination is protective against this toxic effect of nicotine and that combining vaccination and chronic nicotine administration may provide a novel strategy for blocking some effects of nicotine.

Capsular polysaccharide serotypes of coagulase-positive staphylococci associated with tenosynovitis, osteomyelitis, and other invasive infections in chickens and turkeys: evidence for new capsular types.

To study the role of capsular polysaccharides in staphylococcal infections in chickens and turkeys, we surveyed 101 coagulase-positive isolates obtained from investigators in geographically diverse locales in the United States and Canada. Fifty-six isolates were obtained from chickens; 51 (91%) were type 5, and 5 (9%) were type 8. Forty isolates were obtained from turkeys; 13 (33%) were type 5, 15 (38%) were type 8, and 12 (29%) were untypable. Five type 5 isolates were obtained from poultry of uncertain species. Two untypable turkey isolates were studied further. Rabbit serum obtained after immunization with formalin-killed staphylococci was type-specific as assessed by a tube agglutination test and promoted opsonophagocytosis of the isolate that had been used for immunization. Chromatography of carboxy-reduced capsular polysaccharide from one of the isolates suggested that mannoseaminouronic acid was present in the unreduced polysaccharide. The spectrum obtained by 13C-nuclear magnetic resonance suggested the presence of N-acetyl sugars such as mannoseaminouronic acid and 6-deoxy sugars such as N-acetyl fucosamine. The structure of the polysaccharide differed from that of the type 5 and type 8 capsular polysaccharides. Thus, poultry isolates can be classified into at least four serotypes on the basis of their capsular polysaccharides. This observation provides a new focus for investigation into the epidemiology and virulence determinants of this important pathogen in chickens and turkeys.

Serotyping scheme for Staphylococcus aureus isolated from cows with mastitis.

OBJECTIVE: To identify the Staphylococcus aureus capsular serotypes that are not typable, using capsular serotypes 5 and 8, which are currently used to type S aureus isolated from cows with mastitis. SAMPLE POPULATION: Milk samples (n = 273) from cows with mastitis in 178 dairy herds in California, Wisconsin, Michigan, Texas, and New York that were collected by state diagnostic laboratories and S aureus-positive milk samples collected by Veterinary Health Services in the United Kingdom (15), France (22), The Netherlands (36), and Germany (21). PROCEDURE: Capsular serotyping of coded isolates was performed by use of direct cell agglutination and immunoprecipitation of cell extracts with antisera specific for capsular types 5 and 8 and a newly developed S aureus serotyping antiserum 336. RESULTS: In the United States, S aureus capsular types 5 and 8 accounted for 18 and 23% of the isolates, respectively, and type 336 accounted for 59%. Percentage of capsular serotypes in European samples were as follows: type 5 = 34%, type 8 = 34%, type 336 = 30%, and nontypable = 2%. CONCLUSIONS: Serotypes 5 and 8 accounted for only 41% of S aureus isolates from US milk samples, but accounted for 70% of isolates from European milk samples. Addition of the newly developed serotyping antiserum 336 to the typing scheme accounted for 100% of US samples and 98% of European samples and will enable development of a more comprehensive S aureus vaccine.

Hydrophobicity as an adhesion mechanism of benthic cyanobacteria.

The capacity of benthic cyanobacteria to adhere to solid substrates was examined in terms of their cell surface properties. By using a biphasic water-hydrocarbon test system, it was demonstrated that benthic cyanobacteria from divergent habitats were all hydrophobic, whereas all the planktonic cyanobacteria tested were hydrophilic. Divalent cations were found more efficient than monovalent cations in effecting the expression of hydrophobicity. Mechanical shearing of the cell surface, as well as chemical removal of the cell wall, demonstrated that the hydrophobicity was confined to the outer surface layers. The hydrophobic sites were distributed along the whole length of the cyanobacterial filament. Hydrophilic hormogonia of benthic cyanobacteria became hydrophobic within 48 h when grown in the light; chloramphenicol, 3(3,4-dichlorophenyl)1,1 dimethylurea, or incubation in the dark prevented this transition. Hydrophobicity of Phormidium filaments was masked in late stationary phase; this effect was removed by gentle washing.

Capsular polysaccharide serotyping scheme for Staphylococcus epidermidis.

A scheme for the capsular typing of Staphylococcus epidermidis that is based on direct slide agglutination between proteinase-treated bacterial cells and specific antisera is described. Antisera were prepared from serum from rabbits immunized with two selected strains of encapsulated S. epidermidis isolated from bacteremic patients. Antisera were shown to be type specific and designated type 1 and type 2. Blood isolates of S. epidermidis from hospitals in different locations within the United States and Europe were serotyped, and it was found that over 90% of all strains were of type 1 or type 2. Type-specific antibodies mediated type-specific opsonophagocytosis and killing of S. epidermidis. The specificity was shown to be due to two distinct capsular polysaccharides. The data presented in this report may open a new window on the pathogenesis of S. epidermidis which could lead to the development of new vaccines and therapies.

Staphylococcus aureus vaccination for dialysis patients--an update.

Staphylococcus aureus infections are a major cause in both hemodialysis and peritoneal dialysis patients. The availability of a safe and effective protective vaccine would be of great benefit to these patients, but attempts at using vaccines consisting of inactivated whole cells have been unsuccessful. This article discusses an alternate approach to S. aureus vaccine design using a capsular polysaccharide conjugate and preliminary results in hemodialysis and peritoneal patients.

Staphylococcal vaccines: a realistic dream.

Staphylococcus aureus, especially multidrug resistant strains, continues to be a leading cause of serious nosocomial infections. In spite of the debate among investigators in the field, the discovery of serologically distinct capsular polysaccharides on the surface of clinical isolates has renewed the prospects for development of vaccines and passive protective immunity against S. aureus infections. Capsular polysaccharide conjugate vaccines have now been produced and proven to be safe and immunogenic in both healthy and in a significant percentage of immunocompromised patients. Antibodies generated in humans against these vaccines have been shown to mediate type-specific opsonophagocytosis, and to protect animals against lethal challenge with the appropriate S. aureus isolate.

Capsular polysaccharide-protein conjugate vaccines and intravenous immunoglobulins.

Capsular polysaccharides (CPs), present on the surface of most pathogenic bacteria, have been recognised as virulence factors. Antibodies specific to these polysaccharides can mediate the killing of these bacteria by phagocytes in the presence of complement. The conjugation of polysaccharides to carrier proteins enhances their immunogenicity and renders the immune response T-cell dependent. The currently licensed capsular polysaccharide vaccines and polysaccharide-protein conjugate vaccines under development for the prevention of bacterial infections will be discussed in this review. Use of these vaccines for active vaccination and for the vaccination of healthy plasma donors to produce hyperimmune iv. immunoglobulins for the passive immunisation of appropriate patient populations is also discussed.

A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge.

The efficacy of capsular polysaccharide (CP)-specific antibodies elicited by active immunization with vaccines composed of Staphylococcus aureus types 5 and 8 CP linked to Pseudomonas aeruginosa exoprotein A or with immune immunoglobulin G (I-IgG) obtained from vaccinated plasma donors was tested in lethal and sublethal bacterial mouse challenge models. A dose of 2 x 10(5) CFU of S. aureus type 5 CP per mouse administered intraperitoneally (i.p.) with 5% hog mucin was found to cause 80 to 100% mortality in BALB/c mice within 2 to 5 days. Mice passively immunized i.p. 24 h earlier or subcutaneously 48 h earlier with 0.5 ml of I-IgG showed significantly higher average survival rates than animals receiving standard IgG or saline (P < 0.01) following the bacterial challenge. Animals actively immunized with the monovalent type 5 CP-P. aeruginosa exoprotein A conjugate showed a survival rate of 73% compared with 13% in phosphate-buffered saline-immunized animals. The prechallenge geometric mean titer of type 5 CP antibodies in animals that died was significantly (P < 0.05) lower than that of animals which survived the challenge (95.7 versus 223.6 micrograms/ml, respectively). The IgG was further evaluated in mice challenged i.p. with a sublethal dose of 5 x 10(4) CFU per mouse. Serial blood counts were performed on surviving animals at 6, 12, 24, and 48 h. Surviving animals were sacrificed at 72 h, and bacterial counts were performed on their kidneys, livers, and peritoneal lavage fluids. Animals receiving I-IgG had lower bacterial counts in blood samples and lower bacterial densities in kidneys, livers, and peritoneal lavage samples than mice immunized with standard IgG (P < 0.05). These data suggest that S. aureus type 5 CP antibodies induced by active immunization or administered by passive immunization confer protection against S. aureus infections.

Antibody-secreting peripheral blood lymphocytes induced by immunization with a conjugate consisting of Streptococcus pneumoniae type 12F polysaccharide and diphtheria toxoid.

Healthy adult volunteers were injected either with one of two conjugates composed of Streptococcus pneumoniae type 12F polysaccharide (Pn12F) covalently coupled to diphtheria toxoid or with Pn12F alone (as a component of Pnu-Imune, a 23-valent pneumococcus vaccine). The conjugates induced Pn12F-specific antibody-secreting cells in peripheral blood with numbers and isotype distribution similar to those induced by Pnu-Imune, with immunoglobulin A (IgA) as the predominant isotype. The conjugates also elicited high numbers of diphtheria toxoid-specific antibody-secreting cells of the IgG class. There was no distinct booster effect, since a second dose of the conjugates induced antibody-secreting cells at significantly lower numbers than after the first dose. In contrast to the cell numbers, the conjugate vaccines induced higher increases of IgA1 Pn12F antibodies in serum than did Pnu-Imune. However, neither the conjugates nor Pnu-Imune induced a secretory antibody response. Antibody levels in serum and saliva correlated poorly with the frequency of antigen-specific antibody-secreting cells. Circulating antibody-secreting cells present 7 days postimmunization were probably not responsible for the high increase of antibodies in serum but rather represented a population of in vivo-activated B cells with the ability to disseminate the humoral response from the antigen recognition site to distant locations of antibody production.

Protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide in a modified model of endocarditis in rats.

The protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide (CP5) was examined in a modified model of catheter-induced endocarditis. Rats were catheterized by surgically passing a polyethylene catheter through the right carotid artery and aortic valve into the left ventricle. The following day, the rats were injected by the intraperitoneal (i.p.) route with immunoglobulin G (IgG) purified from nonimmunized rabbits or from rabbits immunized with a conjugate vaccine composed of CP5 and CP8 linked covalently to recombinant Pseudomonas aeruginosa exotoxoid A. One day after passive immunization, the animals were challenged i.p. with one of three serotype 5 S. aureus isolates (strain Reynolds, Lowenstein, or VP) or nontypeable strain 521. Protection was evaluated by comparing quantitative cultures of blood, endocardial vegetations, and kidneys from control and immune animals. For experiments performed with S. aureus Reynolds and Lowenstein, rats given capsular antibodies (645 microg of CP5-specific IgG) showed a significantly (P < 0.05) lower prevalence of endocarditis than rats injected with nonimmune IgG. Similarly, quantitative cultures of the blood, kidneys, and aortic valve vegetations revealed that fewer S. aureus cells were recovered from rats given capsule-specific IgG than from rats administered nonimmune IgG. Rats challenged with strain VP were protected with 1.145 mg of CP5-specific IgG. Capsular antibodies did not protect against infection elicited by a nontypeable strain. These results demonstrate that capsular antibodies elicited by immunization with a polysaccharide-protein conjugate vaccine protect experimental animals against serotype 5 S. aureus infection in a modified model of endocarditis.

Vaccination against nicotine during continued nicotine administration in rats: immunogenicity of the vaccine and effects on nicotine distribution to brain.

Vaccination against nicotine has been proposed as a potential treatment for nicotine dependence. Because vaccination may take months to elicit satisfactory antibody levels, the clinical usefulness of this approach will be enhanced if vaccination can be accomplished during continued nicotine intake (e.g., before a smoker quits). The current study examined the immunogenicity of a nicotine conjugate vaccine during continued nicotine dosing in rats, and its effects on nicotine distribution to brain. In the first experiment, nicotine was administered over 11 weeks as 20 intra venous (i.v.) bolus injections per day during the rat's active cycle to simulate the usual pattern of nicotine intake from cigarette smoking. In the second experiment, rats received a continuous s.c. infusion of nicotine by osmotic pump for 11 weeks to provide serum nicotine concentrations equivalent to those of a heavy smoker and 24 h/day nicotine exposure. Nicotine-specific antibody titers after the third booster dose were not compromised by either regimen of concurrent nicotine administration compared to those of rats receiving saline. A single additional i.v. nicotine dose was administered at the end of each experiment. The distribution of this single nicotine dose to brain was reduced by 40-60% in vaccinated rats compared to controls. Vaccine efficacy in reducing nicotine distribution to brain was not compromised by concurrent nicotine administration. These data suggest that vaccination during concurrent nicotine administration is feasible, and that the ability of vaccination to reduce nicotine distribution to brain is preserved even after months of nicotine dosing at rates approximating cigarette smoking.