Current laboratory methods for biological threat agent identification.

The authors present an integrated approach for the identification of biological threat agents. The methods used have been used extensively in field exercises and during response to incidents of biological terrorism. A diagnostic system, which integrates the clinical diagnosis or medical intelligence with immunodiagnostic tests, rapid gene amplification assays, and standard culture, provides results of the highest quality and confidence. In the future, selected reagents and technologies will be distributed through a network of civilian and military laboratories.

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.

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.

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.