Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A.

Botulinum neurotoxin A is a category A bioterrorism agent. Current antitoxin therapies are scarce and produce adverse reactions. XOMA 3AB consists of 3 IgG1 monoclonal antibodies (MAbs), each with a distinct human or humanized variable region, which bind to distinct epitopes on botulinum neurotoxin serotype A. This first-in-human study evaluated the safety and pharmacokinetics (PK) of escalating doses of XOMA 3AB administered intravenously (i.v.) to healthy adults. In this double-blind placebo-controlled dose escalation study, 3 cohorts of 8 healthy subjects received a single intravenous dose of XOMA 3AB or placebo at a 3:1 ratio. Follow-up examinations included physical examinations, hematology and chemistry blood tests, electrocardiograms, and pharmacokinetics. Pharmacokinetic parameters were estimated using noncompartmental methods. There were no infusion discontinuations or hypersensitivity reactions. Two or more subjects experienced headache, hyperglycemia, or anemia; none was dose related. All adverse events (AEs) were mild to moderate except for an episode of exercise-induced elevation of a subject's creatine phosphokinase (CPK) level, unrelated to XOMA 3AB. Concentration-time plots demonstrated a peak in MAb concentrations 1 to 2 h after completion of the infusion, after which the levels declined in a biexponential decay pattern for all analytes. For each MAb, the maximum concentration of drug in serum (Cmax) and the area under the concentration-time curve from 0 to infinity (AUCinf) increased as the dose increased. Clearance of the humanized mouse MAb was more rapid than that of the two fully human MAbs, particularly at the lowest dose. None of the MAbs was immunogenic. At the doses administered, XOMA 3AB was well tolerated. These safety findings support further investigation of XOMA 3AB as a potential agent for botulism treatment and postexposure prophylaxis. (This study has been registered at ClinicalTrials.gov under registration no. NCT01357213.).

Stability of colistimethate sodium in aqueous solution.

Colistimethate sodium, increasingly used to treat multidrug-resistant Gram-negative infections, spontaneously hydrolyzes to form colistin A (polymyxin E1) and B (polymyxin E2/B) when mixed with water. High levels of these active breakdown products at the time of administration have been associated with nephrotoxicity and even death. In this study, reconstituted colistimethate sodium was shown to be stable (<1.0% colistin A/B formation) for up to 24 h when stored at 21, 0, -20, and -70°C.

Lipooligosaccharides (LOS) of Neisseria gonorrhoeae and Neisseria meningitidis have components that are immunochemically similar to precursors of human blood group antigens. Carbohydrate sequence specificity of the mouse monoclonal antibodies that recognize crossreacting antigens on LOS and human erythrocytes.

We have used mouse mAbs, 3F11 and 06B4, that are specific for highly conserved epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) to identify immunochemically similar structures on human erythrocytes. mAb 3F11 agglutinated erythrocytes from all randomly selected adult humans, while mAb 06B4 agglutinated only 80% of the same specimens. The antibodies had an activity with erythrocytes similar to human cold agglutinins in that agglutination occurred at 4 degrees C and decreased with increasing incubation temperature. Human infant erythrocytes were agglutinated less well, but enzymatic treatment of either infant or adult cells resulted in an increase in expression of the 3F11- and 06B4-defined epitopes. Both antibodies bound to a series of neutral glycosphingolipids from human erythrocytes and neutrophils that have a type 2 (Gal beta 1----4GlcNAc) or N-acetyllactosamine structure. Neither antibody bound to glycosphingolipids from human meconium, which have a type 1 (Gal beta 1----3GlcNAc) structure. The antibodies were unable to bind to N-acetyl-lactosamine glycosphingolipids with a nonreducing terminal sialic acid or a Gala1----3Gal disaccharide. Antibody binding also was blocked by the presence of fucose linked to the penultimate glucosamine residue of N-acetyllactosamine glycosphingolipids. Although both antibodies bound to linear and branched-chain N-acetyllactosamine glycosphingolipids, 3F11 had a higher affinity for branched structures than did 06B4. The activity of 3F11 with human adult and infant treated and untreated erythrocytes with N-acetyllactosamine glycosphingolipids, and with LOS was very similar, if not identical, in specificity to 1B2, an mAb prepared from mice inoculated with a linear N-acetyllactosamine glycosphingolipid.

IgA-dependent, monocyte-mediated, antibacterial activity.

IgA purified from the sera of patients convalescing from disseminated group C meningococcal disease induced human monocyte-mediated anti-meningococcal activity in vitro in the absence of complement. Both IgA- and IgG-dependent activity were directed against the group C meningococcal polysaccharide (Csss) capsule. The amount of IgA that was effective bound less than 1 ng of Csss. Antibacterial activity was dependent upon the length and the temperature of the test incubation and on the concentration of monocytes. The implications of this mechanism for local cell-mediated antibacterial immunity are discussed.

Expression of paragloboside-like lipooligosaccharides may be a necessary component of gonococcal pathogenesis in men.

To learn how lipooligosaccharide (LOS) phase variations affect pathogenesis, we studied two male volunteers who were challenged intraurethrally with Neisseria gonorrhoeae that make a single LOS of 3,600 daltons and sequentially followed LOS expression by gonococci as urethritis developed. LOS variation occurred in vivo. Signs and symptoms of gonorrhea began with the appearance of variants making 4,700-dalton LOS that are immunochemically similar to glycosphingolipids of human hematopoietic cells (Mandrell, R.E., J.M. Griffiss, and B.A. Macher. 1989. J. Exp. Med. 168:107) and that have acceptors for sialic acid. A variant that appeared at the onset of leukorrhoea was shed by 34/36 men with naturally acquired gonorrhea at the time they sought medical attention; the other two shed the variant associated with dysuria. None shed the challenge variant. These data show that in vivo phase shifts to higher molecular mass LOS that mimic human cell membrane glycolipids are associated with the development of gonococcal leukorrhea.

In vitro and in vivo modification of Neisseria gonorrhoeae lipooligosaccharide epitope structure by sialylation.

After growth of gonococci in the presence of cytidine monophospho-N-acetyl-neuraminic acid (CMP-NANA), their 4.5-kD lipooligosaccharide (LOS) component was increased by approximately 400 daltons, whereas the LOS of strains lacking the 4.5-kD component were unaffected. Expression of mAb-defined epitopes on the 4.5-kD component was decreased on LOS of strains grown in CMP-NANA, and treatment of the LOS with neuraminidase reversed this affect. Gonococci incubated with human PMNs also had decreased expression of the 4.5-kD+ epitopes. A detergent extract of gonococci incorporated radiolabeled NANA in the LOS, suggesting the presence of a sialyltransferase in gonococci. Exogenous sialyltransferases also could use LOS as an acceptor.

Complex of meningococcal group B polysaccharide and type 2 outer membrane protein immunogenic in man.

A noncovalent complex of meningococcal group B polysaccharide and type 2 outer membrane protein has been characterized and its potential as a vaccine against group B meningococcal disease investigated. The polysaccharide component was found to have a partition coefficient, K(d), of 0.34 on Sepharose CL-4B in the presence of sodium deoxycholate. The protein consisted of four to five major proteins including the principal outer membrane protein. Hydrophobic binding between the protein and polysaccharide was demonstrated by gel filtration and isopycnic CsCl density gradient centrifugation and found to involve all of the proteins. After demonstrating safety and immunogenicity in animals, two lots of vaccine were tested in a total of eight volunteers. Two 120-mug doses were given subcutaneously at 0 and 5 wk. Mild local reactions occurred in all eight volunteers, but no systemic reactions were observed. 2 wk after the first dose, six of the volunteers had increased levels of bactericidal antibodies against both the group B polysaccharide and the outer membrane proteins. Antibody rises to the group B polysaccharide (mean 6-fold) were confirmed by passive hemagglutination assays and rises to the proteins (mean 10-fold) by a solid phase radioimmunoassay. The second dose resulted in little or no increase in antibody titers. Antibody titers declined over a period of 14 wk but mostly remained above preimmunization levels. Bactericidal antibodies with specificity for the group B polysaccharide were mostly of the immunoglobulin (Ig)M class, and were directed against a determinant associated only with high molecular weight polysaccharides. We conclude that both the group B polysaccharide and the outer membrane protein are immunogenic in man when presented as a complex and that the complex warrants further testing and development as a vaccine against group B meningococcal disease.

Antibody-dependent mononuclear cell-mediated antimeningococcal activity. Comparison of the effects of convalescent and postimmunization immunoglobulins G, M, and A.

We have compared the abilities of immunoglobulin (Ig)G, IgM, and IgA to induce either mononuclear cell-mediated (complement-independent) or complement-mediated (cell-free) antibacterial activity against group C meningococci. In each of these assays, immunoglobulins purified from the sera of individuals immunized with meningococcal group C polysaccharide were compared with those purified from sera of patients convalescing from disseminated meningococcal disease. Our data support three conclusions. First, although nonbactericidal in cooperation with complement, IgA can induce cell-mediated antibacterial activity as well as IgG. Second, the amount of IgG required to induce cell-mediated antibacterial activity is similar to the amount required for complement-mediated killing. Third, although the amount of either postimmunization or convalescent IgM required to induce complement-mediated killing is 16- to 20-fold less than the amount of respective IgG required, IgM is inferior to IgG in its ability to induce cell-mediated antibacterial activity because in the cell-mediated system (a) postimmunization IgM is ineffective; (b) the amount of convalescent IgM required for minimal activity is eightfold more than the amount of convalescent IgG required; and (c) the maximal antibacterial index induced by convalescent IgM is 50% less than that which can be induced by IgG. These data suggest that IgG and IgA may play a greater role than IgM in mononuclear cell-mediated antibacterial host immune defense.

Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces.

One percent of circulating IgG in humans recognizes galactose alpha 1,3 galactose residues (anti-Gal) and is synthesized in response to stimulation by enteric bacteria. In this study, we found that the prevalence of binding of anti-Gal to blood isolates is significantly higher than its binding to normal stool isolates. When anti-Gal bound onto the lipopolysaccharide of a representative blood isolate, Serratia marcescens #21, it blocked its alternative complement pathway (ACP) lysis and made the organism serum resistant. In contrast, when anti-Gal bound to the capsular polysaccharide of a serum sensitive Serratia, #7, it increased ACP killing of this strain. The mechanism of blockade of ACP lysis by anti-Gal did not involve a decrease in the number of C3 molecules deposited onto Serratia #21 or an inhibition of the binding of C3b to its LPS, nor did it change the iC3b and C3d degradation products of bound C3b or prevent membrane attack complex formation on this organism. Our findings suggest that the effect of anti-Gal on immune lysis is dependent on the bacterial outer membrane structure to which it binds. We postulate that anti-Gal may play a role in the survival of selected Enterobacteriacae in Gram-negative sepsis by blocking ACP-mediated lysis of such bacteria by the nonimmune host, and that this effect depends on where anti-Gal finds its epitope on the bacterial outer membrane.

Epitope expression of gonococcal lipooligosaccharide (LOS). Importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS.

Antigenic expression of lipooligosaccharide (LOS) of strain F62 of Neisseria gonorrhoeae, was investigated with mouse monoclonal IgM antibody 3F11. F62 LOS was modified in various ways in order to understand structural requirements for expression of the 3F11-defined epitope. When the LOS was partially deacylated by treating it with 50 mM NaOH at 80 degrees C for 20 min or with anhydrous hydrazine at 80 degrees C for 20 min, the binding of 3F11 to those deacylated LOS samples decreased significantly. Removal of phosphate groups by treatment of the LOS with HF (4 days at 4 degrees C) did not affect the antigenicity at all. Neither did reduction of carboxyl groups in the LOS molecule (by activation of carboxyl groups with a carbodiimide followed by treatment with NaBH4) alter epitope expression. On oxidation with NaIO4, the LOS lost its antigenicity completely. The presence of Mg2+ did not change the circular dichroism (CD) behavior of F62 LOS. However, the partially deacylated LOS samples showed significantly different CD patterns in the 190-200 nm region compared with F62 LOS, which suggests conformational changes of F62 LOS due to the loss of fatty acids in the lipoidal moiety. Oligosaccharide (OS) and lipoidal components obtained after hydrolysis of F62 LOS with 1% acetic acid, were not recognized by the antibody. The antigenicity of OS was not retained by non-stereospecific acylation of OS with decanoyl chloride. We conclude the following: (1) 3F11-defined epitope exists in the OS moiety of F62 LOS; however, for it to be expressed, the carbohydrate moiety must be in a certain conformation that is defined by an overall structure of the LOS molecule. This structure is significantly influenced by some of the fatty acids in the lipoidal moiety of the LOS molecule; (2) the presence of phosphate or 3-deoxy-manno-2-octulosonic acid (dOclA) is not essential for expression of the 3F11-defined epitope; (3) the presence of divalent cations does not affect epitope expression.

A bactericidal microassay for testing serum sensitivity of Neisseria gonorrhoeae.

A reproducible gonococcal serum bactericidal microassay is described which has easily determined endpoints and lends itself to large scale tests. The microassay may be used to assess sensitivity to normal human serum among strains of gonococci, and to define the antigens conferring this sensitivity. It may be readily adapted to test for bactericidal activity in hyperimmune antisera and to evaluate antigens which inhibit serum bactericidal activity.

Disease due to serogroup W135 Neisseria meningitidis.

Five cases of disseminated meningococcal disease due to serogroup W135 Neisseria meningitidis are presented. The cases ranged in age from 16 months to 23 years, and spanned a clinical spectrum from mild meningitis without rash or evidence of meningococcal septicemia to severe meningoencephalitis with fulminant meningococcemia, disseminated intravascular coagulation, and death. These cases demonstrate that serogroup W135 N meningitidis is fully pathogenic for man and capable of producing the full spectrum of disseminated meningococcal disease associated with other serogroups. Since this serogroup has recently emerged as a significant cause of disease in Europe, attention should be focused on the correct serogroup designation of strains of N meningitidis isolated from clinical material and reported as "nongroupable" by clinical laboratories, so that additional clinical and epidemiologic information may be obtained.

Influence of age on serogroup distribution of endemic meningococcal disease.

The age distribution of 126 infants and children with disseminated meningococcal disease hospitalized consecutively in Houston between January 1977 and June 1979, and between January 1981 and June 1981 was analyzed and compared with that in the United States as a whole and to that during outbreaks of group B disease in North America and epidemics of group C disease in South America. Eighty-one (64.3%) isolates from Houston cases were serogroup B and 37 (29.4%) were serogroup C Neisseria meningitidis. Children with serogroup C disease were significantly older than those with group B disease (P = .017). Of the children with serogroup B infections, 33% were less than 12 months of age and 8.6% were less than 3 months of age. Of those with serogroup C disease, only 2.7% were less than 3 months of age and the majority (73%) were more than 2 years of age. These age distributions are similar to those reported for the entire United States during endemic periods. In contrast, focal outbreaks of group B meningococcal infection occurred in populations that were significantly older (0.02 greater than P less than .05). Similarly, epidemic disease in South America due to serogroup C strains also occurred in older children when compared with the occurrence of endemic group C disease in the United States (P = .02).

Correlation of Pneumocystis carinii cyst density with mortality in patients with acquired immunodeficiency syndrome and pneumocystis pneumonia.

Fifteen percent to 20% of patients with the acquired immunodeficiency syndrome and pneumocystis pneumonia do poorly despite early intervention. It is not known what distinguishes those who die, despite early intervention and aggressive therapy, from those who readily respond to therapy. We used image analysis to determine the relative abundance of cysts within aggregates of Pneumocystis carinii found in induced sputa (21 patients) and bronchoalveolar lavage fluid (14 patients) from 35 patients with pneumocystis pneumonia. We calculated a cyst density (number of cysts per area of aggregate) for each aggregate and a mean cyst density for all of the aggregates on the smear. Six patients died within 2 weeks of diagnosis; four of these six patients who had autopsies all had residual P carinii. The mean cyst density for those who died was 9.7 +/- 3.9 (range, 5 to 15 x 10(-3)). The 29 patients who survived beyond 2 weeks had a mean cyst density of 18.4 +/- 8.7 (range, 5 to 35 x 10(-3); P = .01). Mean cyst density was not influenced by the number of aggregates present in the smear, the variation in cyst density among aggregates in a smear, or the episode of pneumocystis pneumonia. Cyst density determinations alone should not be used to predict outcome for individuals with P carinii pneumonia until further study is completed. Nevertheless, the current study suggests that a low cyst density specimen, which may indirectly indicate a greater proportion of trophozoites compared with a high cyst density specimen, may be associated with an unfavorable outcome in acquired immunodeficiency syndrome-associated pneumocystis pneumonia.

Epidemiological value of lipopolysaccharide and heat-modifiable outer-membrane protein serotyping of group-A strains of Neisseria meningitidis.

The lipopolysaccharide (LPS) and heat-modifiable outer-membrane protein (P') serotypes of 39 coded strains of group-A Neisseria meningitidis isolated from patients during seven geographically and temporally separate outbreaks of infection were determined blindly. LPS serotype discriminated between strains from different outbreaks and between strains of differing sulphadiazine sensitivity within a single outbreak. Thirty-seven strains were of three separate serotypes and no strain was of multiple serotypes. In contrast, P' serotypes did not discriminate between strains. Multiple serotypes for single strains and among strains from a single outbreak were the rule. LPS serotyping appears to be a useful epidemiological tool for distinguishing group-A strains of N. meningitidis.

Meningococcal molecular mimicry and the search for an ideal vaccine.

The carbohydrates expressed on the surface of meningococcal strains of groups B and C mimic those commonly found on human cells and thus are not functionally antigenic in infancy. In order to develop an effective vaccine, it will be necessary to find ways of circumventing this molecular mimicry. Three possible ways of achieving this are discussed. (i) The surface polysaccharides can theoretically present conformationally different epitopes, some of which might be recognized as antigenic by the host. Experimental evidence is presented that such differences do indeed exist; what is needed is to determine which of these conformations are unique to the organism and hence potentially antigenic. (ii) Precursors of the surface lipooligosaccharides may be unable to mimic human antigens, and so may be potential candidates for vaccine development. (iii) Natural immunity to some strains of meningococci develops in young children who are colonized with strains of Neisseria lactamica, and it is possible that its development could be enhanced by widespread intentional colonization by N. lactamica strains that are particularly efficient inducers of broad immunity.

Pigment gallstone pathogenesis: slime production by biliary bacteria is more important than beta-glucuronidase production.

Pigment stones are thought to form as a result of deconjugation of bilirubin by bacterial beta-glucuronidase, which results in precipitation of calcium bilirubinate. Calcium bilirubinate is then aggregated into stones by an anionic glycoprotein. Slime (glycocalyx), an anionic glycoprotein produced by bacteria causing foreign body infections, has been implicated in the formation of the precipitate that blocks biliary stents. We previously showed that bacteria are present within the pigment portions of gallstones and postulated a bacterial role in pigment stone formation through beta-glucuronidase or slime production. Ninety-one biliary bacterial isolates from 61 patients and 12 control stool organisms were tested for their production of beta-glucuronidase and slime. The average slime production was 42 for biliary bacteria and 2.5 for stool bacteria (P <0.001). Overall, 73% of biliary bacteria and 8% of stool bacteria produced slime (optical density >3). In contrast, only 38% of biliary bacteria produced beta-glucuronidase. Eighty-two percent of all patients, 90% of patients with common bile duct (CBD) stones, 100% of patients with primary CBD stones, and 93% of patients with biliary tubes had one or more bacterial species in their stones that produced slime. By comparison, only 47% of all patients, 60% of patients with CBD stones, 62% of patients with primary CBD stones, and 50% of patients with biliary tubes had one or more bacteria that produced beta-glucuronidase. Most biliary bacteria produced slime, and slime production correlated better than beta-glucuronidase production did with stone formation and the presence of biliary tubes or stents. Patients with primary CBD stones and biliary tubes had the highest incidence of slime production. These findings suggest that bacterial slime is important in gallstone formation and the blockage of biliary tubes.

Pneumocystis carinii in sputum. Comparable efficacy of screening stains and determination of cyst density.

To determine whether accurate quantification of Pneumocystis carinii cysts in sputum is possible using currently available means and to determine the optimal stain for screening sputum for P carinii, we undertook a comparative evaluation of readily available stains: (1) Diff-Quik, a stain that provides tinctorial detail comparable with Giemsa, (2) toluidine blue, and (3) silver methenamine on simultaneously thawed sputum samples containing P carinii. We also studied the Diff-Quik stain in combination with (4) toluidine blue and (5) silver methenamine. The three stains were comparable for screening purposes. Each disclosed similar numbers of P carinii aggregates on similarly prepared slides. Cysts were difficult to recognize on Diff-Quik due to the negatively staining character of the cyst wall with this stain. The cyst-wall stains, toluidine blue and silver methenamine, clearly stained the cyst wall. The combination of a cyst-wall stain with Diff-Quik allowed visualization of cysts, highlighted by the cyst-wall stain, and aggregate area, delineated by the Diff-Quik, allowing expression of number of cysts per aggregate area, a measure termed cyst density.