Structural relationships and sialylation among meningococcal L1, L8, and L3,7 lipooligosaccharide serotypes.

Eighteen of 34 endemic meningococcal case strains were of the L8 lipooligosaccharide (LOS) type; four of these were both L3 and L7 (L3,7), and seven were L1. L1 structures arose by alternative terminal Gal substitutions of lactosyl diheptoside L8 structures, as determined by electrospray ionization and other mass spectrometric techniques, and enzymatic and chemical degradations (Structures L1 and L1a). [see text for structure] The more abundant molecule, designated L1, had a trihexose globosyl alpha chain; the less abundant one, designated L1a, had a beta-lactosyl alpha chain and a parallel alpha-lactosaminyl gamma chain. A P(k) globoside (Galalpha1-->4Galbeta1-->4 Glc-R) monoclonal antibody bound 9/10 L1 strains, but a P(1) globoside (Galalpha1-->4Galbeta1-->4GlcNAc-R) mAb bound none of them. alpha-Galactosidase caused loss of both L1 structures and creation of L8 structures; beta-galactosidase caused loss of the L8 determinant. The L1/P(k) glycose was partially sialylated. Some LOS also had unsubstituted basal beta-GlcNAc additions. These structural relationships explain co-expression of L8, L1, and L3,7 serotypes.

Safety and immunogenicity testing of an intranasal group B meningococcal native outer membrane vesicle vaccine in healthy volunteers.

An intranasal vaccine composed of native outer membrane vesicles (NOMV) not exposed to detergent or denaturing agents was prepared from the group B meningococcal strain 9162 SynX(-)(-:15:P1.3:P5.10,11:L3,7,9) and tested in 32 healthy adult volunteers. Four groups of 8 volunteers were vaccinated intranasally with three doses of vaccine. The vaccine was very well tolerated in all dosing groups, despite the presence of lipo-oligosaccharide in the vaccine at a level of 25% relative to protein. The antibody response as measured by ELISA in serum, saliva and nasal wash fluids was relatively low in all 4 groups, but the induced serum antibodies had strong bactericidal activity. Persistent bactericidal antibodies (> or =4-fold increase) were produced in 75% of the recipients. Some of the bactericidal antibodies were cross reactive against divergent group B strains. Most of the bactericidal antibodies appeared to be specific for PorA and L3,7,9 LOS. The vaccine also produced a local antibody response which was detected in the nasal wash fluids of volunteers. These data suggest that nasal immunization with NOMV is a safe and effective approach to induce systemic and local immunity against the group B meningococcus and deserves further study.

Redesignation of a purported P1.15 subtype-specific meningococcal monoclonal antibody as a P1.19-specific reagent.

Two reference monoclonal antibodies against the meningococcal P1.15 subtype PorA, MN3C5C and 2-1-P1.15, showed only partial concordant recognition of meningococcal isolates. Cyanogen bromide cleavage of P1.19,15 PorA, peptide mapping, and sequencing of porA regions demonstrated that 2-1-P1.15 was specific for subtype P1.19, and henceforth it is to be redesignated as 2-1-P1.19.

Immunogenicity of intranasally administered meningococcal native outer membrane vesicles in mice.

Colonization of the human nasopharyngeal region by Neisseria meningitidis is believed to lead to natural immunity. Although the presence of bactericidal antibody in serum has been correlated with immunity to meningococcal disease, mucosal immunity at the portal of entry may also play an important role. This study was undertaken to examine in mice the possibility of safely using native outer membrane vesicles (NOMV) not exposed to detergent as an intranasal (i.n.) vaccine. The mucosal and systemic responses of mice to intranasal and intraperitoneal (i.p.) vaccination with NOMV were compared over a range of doses from 0.1 to 20 microgram. Intranasal vaccination of mice with NOMV induced a strong systemic bactericidal antibody response, as well as a strong local immunoglobulin A immune response in the lung as determined by assay of lung lavage fluid by enzyme-linked immunosorbent assay and lung antibody secreting cells by enzyme-linked immunospot assay. However, 8- to 10-fold-higher doses of NOMV were required i.n. compared to i.p. to elicit an equivalent bactericidal antibody response in serum. Some NOMV vaccine was aspirated into the lungs of mice during i.n. immunization and resulted in an acute inflammatory response that peaked at 1 to 2 days postimmunization and was cleared by day 7. These results indicate that i.n. delivery of meningococcal NOMV in mice is highly effective in eliciting the production of both a mucosal immune response and a systemic bactericidal antibody response.

Immunological and molecular characterization of three variant subtype P1.14 strains of Neisseria meningitidis.

Epidemic outbreaks of group B meningococcal disease exhibit a clonal nature consisting of a common serotype-subtype. Subtype-specific monoclonal antibodies (MAbs) directed toward two variable regions (VR1 and VR2) of the class 1 protein of Neisseria meningitidis are used in this classification scheme. A new MAb was developed to classify a nonsubtypeable (NST) strain of N. meningitidis, 7967. This MAb bound to both the NST strain and the prototype subtype P1. 14 strain, S3446, by dot blot analysis. However, a MAb produced to the prototype P1.14 strain did not bind to strain 7967. Sixteen additional strains were further identified as P1.14 with the prototype MAb; of these, 15 strains bound both MAbs. Differences in the characteristics of binding of both antibodies to the three apparently diverse P1.14 strains were studied further by using outer membrane complex proteins, immobilized peptides, and soluble peptides. Deduced amino acid analysis suggested that both MAbs bind to VR2 and that single amino acid changes within VR2 (KM, NM, or KK) might explain the differences in binding characteristics. These results demonstrated that minor variations which exist within subtype variable regions may be clearly identified only by a combination of molecular and immunologic testing. The impact of subtype variation will become more evident as subtype-specific vaccines are developed and tested for efficacy.

Confirmation of suspicious cases of meningococcal meningitis by PCR and enzyme-linked immunosorbent assay.

A significant problem in efficacy trials of meningococcal vaccines has been accurate identification of all cases of meningococcal disease that occur in study populations. The accuracy of case determination would be improved by utilizing methods which confirm or disprove suspicious cases of meningococcal disease that are culture negative. A collection of serum and cerebrospinal fluid (CSF) samples from a meningococcal vaccine field trial performed in Iquique, Chile, were utilized to assess the status of patients for whom cultures, Gram stains, and clinical evaluations for meningococcal disease were available. Nested PCRs (nPCRs) for amplification of Neisseria meningitidis DNA in CSF samples and enzyme-linked immunosorbent assays (ELISAs) for quantification of serum immunoglobulin G antibodies specific for N. meningitidis were used in combination to confirm or eliminate cases classified by physicians as suspicious for meningococcal disease. Samples from 12 of 79 patients suspected of having meningococcal meningitis tested positive by both methods; specimens from 61 of the 79 were negative by both methods; and samples from 6 patients yielded ambiguous results, and these cases remained unconfirmed. Direct sequence analysis of amplified DNA from patients suspected of having meningococcal disease confirmed that 2 of the 12 newly confirmed cases were not attributable to the typical epidemic strain (B:15:P1.[7],3) while the others were due to the epidemic strain. A combination of nPCR and ELISA reduced the number of suspicious cases in this study from 79 to 6, thereby improving the potential for assessment of vaccine efficacy. Molecular identification by nPCR in conjunction with immunological assessment of patient response could be considered diagnostic of disease in future testing of meningococcal vaccines to improve efficacy analyses.

A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis.

The predicted amino acid sequence was determined for the class-1 outer membrane protein, PorA, from a B:15:P1.7,3 strain of Neisseria meningitidis that is currently causing an epidemic of meningitis in Northern Chile. The P1.7,3 PorA showed a unique sequence in the exposed loop 4 of the putative porin structure that is different from all the reported PorA sequences. Based on the nucleotide (nt) sequence of the P1.7,3 porA, we designed two sets of PCR (polymerase chain reaction) primers that specifically amplified porA from any N. meningitidis strain, and a third set of primers that amplified porA only from the P1.7,3 strain. Using these primers, we developed a sensitive double hot-start nested PCR (HNPCR) strategy that could amplify porA and generate nt sequence from as low as a single colony-forming unit. This strategy consisted of three phases of PCR. The first two phases were designed to generate amplified target DNA that could be directly visualized by ethidium bromide staining starting from one to two molecules of Neisseria genome. The third phase was designed to generate a sequence of several hundred nt directly from the amplified DNA. A number of culture-negative cerebrospinal fluid samples from individuals suspected of meningitis during a vaccine trial were analyzed by this strategy to obtain more accurate information on the actual number of cases that occurred in the study and the non-study populations. The basic HNPCR strategy described here could be applied to amplify and sequence target DNAs from any low-copy-number biological sample.

Pasteurella multocida pneumonia in a man with AIDS and nontraumatic feline exposure.

A case of acute pneumonia due to Pasteurella multocida ssp multocida occurred in a young man with AIDS and chronic sinusitis. The pneumonia was diagnosed by bronchoscopy and responded to treatment with aztreonam. Epidemiologic investigation revealed the case was temporally related to nontraumatic exposure to cat secretions that the patient presumably had acquired via an aerosol. The cat's oral cavity was cultured and an isolate of P multocida ssp multocida with identical biochemical reactions, DNA restriction patterns, and nearly identical fatty acid profile to that of the patient's isolate was obtained suggesting they were identical strains and therefore epidemiologically linked. A control strain with identical biochemical reactions and antibiotic sensitivities exhibited different patterns. To our knowledge, this is the first such reported infection in a patient infected with human immunodeficiency virus.

A rapid polymerase-chain-reaction-directed sequencing strategy using a thermostable DNA polymerase from Thermus flavus.

We have developed a polymerase chain reaction (PCR)-directed sequencing strategy for rapid sequencing of DNA from crude viral or cell preparations. The basic strategy consists of two phases. In the first phase, the target DNA is amplified by symmetric PCR with low concentrations of deoxyribonucleotide triphosphate (dNTP) and oligodeoxyribonucleotide primers. This results in exponential amplification of DNA in the initial cycles, reaching a plateau by 25 cycles due to limiting concentrations of dNTP and primers. In the second phase, a small aliquot of the PCR mixture is amplified without any purification, by asymmetric PCR in the presence of a 5'-labeled primer and one of the four dideoxyribonucleotide triphosphates. This results in the accumulation of single-stranded DNA products that are terminated at specific points by incorporation of the appropriate dideoxyribonucleotide monophosphate. The products are then analyzed by electrophoresis on a sequencing gel followed by autoradiography. The PCR conditions are optimized to generate sequence ladders of several hundred nucleotides starting from as low as 100 copies of bacteriophage or bacterial genome in one to two days.

Immunoglobulin composition of three commercially available intravenous immunoglobulin preparations.

To evaluate whether significant differences in immunoglobulin composition exist among the three commercially available intravenous immunoglobulin (IVIG) preparations, we compared three of these products in terms of their quantitative immunoglobulin concentration, IgG subclass concentration, and the presence of IgG aggregates. Three different lots were tested for each of the IVIG formulations, and the IgG subclass assays were performed by three different laboratories. Differences were found among the three formulations in subclass concentration and aggregate content. Sandoglobulin (Sandoz Pharmaceuticals, East Hanover, N.J.) contained a significantly higher amount of IgG2 (p less than 0.002) compared to the other formulations. All the formulations tested were found to be deficient in IgG4 relative to the World Health Organization standards, with Gammagard (Hyland Therapeutics, Glendale, Calif.) demonstrating only negligible amounts. Immunoglobulin aggregate content was different among manufacturers with Gamimmune N (Cutter Biological, Berkeley, Calif.) containing the highest amount of monomer IgG (99.8%). Significant differences were found in the subclass results obtained by the ICN ImmunoBiologicals assay (Lisle, Ill.), compared to the two reference laboratories. This difference among the subclass assays raises the question as to the use of these assays in the evaluation of patients with suspected subclass deficiencies. The differences in subclass concentration and aggregate content in IVIG preparations were great, and future clinical trials with these formulations would be indicated to determine the clinical significance of these findings.

Development of a human monoclonal antibody from a Graves' disease patient that identifies a novel thyroid membrane antigen.

To investigate the interaction between antibodies and the thyroid gland in Graves' disease, PBL were harvested from seven Graves' disease patients and transformed into lymphoblasts by the addition of EBV in the presence of cyclosporine A. These lymphoblasts were cloned by limiting dilution and then assayed for binding activity to human thyroglobulin, thyroid-stimulating hormone, thyroid microsome, and thyroid as well as guinea pig fat cell membranes. Four patients' cells produced antibody that bound to at least one of the Ag; a single clone from one patient that bound equally well to both thyroid and guinea pig fat cell membranes (but not to other thyroid Ag) was selected for further evaluation. Fusion of these cells with SHM-D33 heteromyeloma cells yielded three cell lines that produced genetically identical mAb. Immunostaining of human thyrocytes with this mAb demonstrated an Ag present on both nuclear and cell membranes. This Ag was identified as an 18,000 m.w. protein band on Western blots of both human thyroid and guinea pig fat cell membranes. The mAb was also able to alter thyrocyte physiology as the short term incubation of this mAb with FRTL-5 cells in vitro inhibited thyroid-stimulating hormone-mediated production of cAMP. Thus, this mAb and the Ag it identifies may be relevant to Graves' disease.

Seronegative Hashimoto thyroiditis with thyroid autoantibody production localized to the thyroid.

A patient without serologic evidence of an autoimmune disorder had a thyroidectomy for neck compression caused by her goiter. Histologic examination of the gland revealed pathologic changes compatible with Hashimoto disease, and lymphocytes isolated from her thyroid gland were transformed into lymphoblastoid cells in vitro by Epstein-Barr virus in the presence of cyclosporine. These cells produced antibodies against thyroglobulin, thyroid microsome, thyrocyte membrane, and thyrotropin, whereas lymphoblastoid cells produced from the patient's peripheral blood lymphocytes showed no antithyroid activity. Several human monoclonal antibodies were produced from the intrathyroidal lymphocytes by fusion with the SHM-D33 heteromyeloma, and the resulting cell lines produced antibodies with high-affinity binding specific for thyroid antigens. These studies show that lymphocytes capable of producing antithyroid antibodies can be present in the thyroid glands of patients with Hashimoto disease without evidence of a peripheral immune response, thus indicating that Hashimoto disease can exist as an organ-restricted autoimmune disorder.