Immunization with meningococcal outer-membrane protein vesicles containing lipooligosaccharide protects mice against lethal experimental group B Neisseria meningitidis infection and septic shock.

Detergent-treated group B Neisseria meningitidis outer membrane vesicles (D-OMVs) from wild-type M986 and from nonencapsulated mutant M986-non-capsule variant (NCV) were compared as immunogens. Eight weeks after 3 consecutive immunizations with the immunogens, mice were challenged with a lethal dose of purified endotoxin or heat-killed or living N. meningitidis, plus d-galactosamine (400 mg/kg). D-OMVs from M986 induced bactericidal antibodies to both M986 (B : 2a : P1.5,2 : L3,7) and 6275 (B : 2a : P1.2,5 : L3) and protected the animals against both strains, whereas D-OMVs from M986-NCV did not protect the animals against infection with 6275 even when high serum bactericidal activity was induced. Tumor necrosis factor-alpha detected after bacterial infection was high in both protected and unprotected mice; interleukin (IL)-6 was high in mice that died but low in animals that survived. Exogenous administration of recombinant mouse IL-6 reversed the immunogens' protective effects. Protection against infection in mice does not necessarily correlate with the measured levels of serum bactericidal antibody alone, opsonic antibody alone, or cytokine profile alone. A comprehensive assessment of the preclinical efficacy of group B outer-membrane protein vaccines should include monitoring humoral antibodies, cytokine response, and protective effects against lethal infection.

Modulation of the biological activities of meningococcal endotoxins by association with outer membrane proteins is not inevitably linked to toxicity.

Meningococcal sepsis results partly from overproduction of host cytokines after macrophages interact with endotoxin. To obtain less toxic and highly immunomodulatory meningococcal endotoxins for prophylactic purposes, we investigated the relationship between endotoxicity and immunomodulatory activity of several endotoxin preparations from Neisseria meningitidis group B. Using the D-galactosamine-sensitized mouse model to determine endotoxin lethality, we found that the toxicity of purified lipooligosaccharide (LOS) from M986, a group B disease strain, was three to four times higher than those of purified LOSs from the noncapsulated strains M986-NCV-1 and OP-, the truncated-LOS mutant. The LOSs of outer membrane vesicles (OMVs) and detergent-treated OMVs (D-OMVs) from the three strains were 2 to 3 and over 300 times less toxic than the purified LOSs, respectively. Intraperitoneal administration of these preparations induced production of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in serum 2 h after injections. However, repeated doses of low- and high-toxicity preparations induced lower amounts of TNF-alpha and IL-6, i.e., LOS tolerance. Injection of mice with low doses of LOS was as effective as injection with high doses in inducing tolerance. Peritoneal macrophages from tolerant mice pretreated with either high- or low-toxicity LOS preparations produced only a fraction of the amounts of TNF-alpha and IL-6 produced by control groups in response to LOS ex vivo. Despite tolerance to LOS induced by pretreatment with reduced-toxicity preparations, killing of N. meningitidis M986 by macrophages from these animals was enhanced. Protection was achieved when mice treated with LOS, and especially that of D-OMVs, were challenged with live N. meningitidis. The least toxic LOS, that in D-OMVs, was most effective in inducing hyporesponsiveness to endotoxin in mice but protected them against challenge with N. meningitidis. No inevitable link between toxicity and host immune modulation and responses was shown. Our results show that LOS is responsible for both toxicity and immunomodulation. When LOS is tightly associated with outer membrane proteins in D-OMV, it reduces toxicity but enhances beneficial effects compared to results with its purified form. Thus, systematic and critical evaluation of D-OMVs as adjuvants or as portions of group B meningococcal vaccines may help improve survival and outcome in meningococcal sepsis.

Immunization with meningococcal membrane-bound lipooligosaccharide accelerates granulocyte recovery and enhances lymphocyte proliferation in myelosuppressed mice.

Protective effects of detergent-treated outer membrane vesicles (D-OMVs) prepared from the parent group B M986 strain and the nonencapsulated M986-NCV mutant in myelosuppressed mice were investigated in models of experimental septic shock. The effects of D-OMVs on expansion of the myeloid compartment, on spleen cell proliferation to mitogen stimulation, and on cytokines induced during this period were investigated. On 3 consecutive days, mice were injected with 1 microgram/kg of lipooligosaccharide (LOS) or lipopolysaccharide, or 75 micrograms/kg D-OMV followed by a single dose of cyclophosphamide (200 mg/kg) 24 h later. Eight weeks after the last injection, animals were challenged with a combination of galactosamine (400 mg/kg) and live Neisseria meningitidis. More than 90% of control mice died within 24 h when challenged with 10(5) CFU of bacteria. Mice immunized with LOS or D-OMV were rendered neutropenic but were protected against bacterial challenge of at least 10(7) CFU. At different time intervals, peripheral blood samples were obtained to characterize changes in circulating blood cells. The rise in absolute granulocyte numbers occurred 24 h earlier with peak cell counts about 3 times higher than those seen in the placebo groups. Peripheral blood cells from D-OMV-treated animals expressed about twofold more Gr-1 antigen (myeloid surface cell marker) than placebo-treated controls. The proliferative responses to both B and T cells were reduced in all treatment groups due to the effects of cyclophosphamide. D-OMV treatment afforded the greatest protection for mitogen-activated lymphocytes from the lethal effects of cyclophosphamide and also enhanced T and B cell proliferation. Low IL-1 beta levels and increases in serum IL-6 were detected in all treatment groups. In contrast, significant IFN-gamma and IL-3 levels were only detected in D-OMV-treated groups. These results indicate that D-OMVs, which have reduced toxicity, have prophylactic potential in inducing specific cytokines that accelerate granulocyte recovery following cytoreductive therapy by promoting both proliferation and maturation of myeloid precursors as well as augmenting the immune system.