Inger Lise Haugen

Outer membrane vesicles from group B meningococci are strongly immunogenic when given intranasally to mice

Outer membrane vesicles (OMVs) from group B meningococci induced both serum and mucosal antibodies when given as a nasal and rectal vaccine to mice. Cholera toxin (CT) enhanced the antibody responses in serum both after nasal and rectal immunizations, and the mucosal responses after rectal immunizations only. Nasal immunizations, however, were most effective, with mucosal responses which were not dependent on the use of CT. The serum bactericidal activity was similarly not enhanced by CT, indicating that the positive effect of CT on the serum IgG level was not including bactericidal activity. A small nasal booster dose induced antibody responses in serum as far as eight months after intranasal and subcutaneous immunizations, and in saliva after intranasal immunizations. Nasal vaccines may thus be favorably combined with parenteral vaccines.

Comparison of functional immune responses in humans after intranasal and intramuscular immunisations with outer membrane vesicle vaccines against group B meningococcal disease

A serogroup B meningococcal outer membrane vesicle (OMV) vaccine was delivered either intranasally or intramuscularly to 12 and 10 volunteers, respectively. The mucosal vaccine was given as four weekly doses followed by a fifth dose after 5 months; each dose consisted of OMVs equivalent to 250 microg of protein. The intramuscular (i.m.) vaccine, consisting of the same OMVs but adsorbed to Al(OH)(3), was administered as three doses each of 25 microg of protein, with 6 weeks interval between first and second doses and the third dose after 10 months. Both groups of vaccinees demonstrated significant immune responses when measured as specific IgG antibodies against live meningococci, as serum bactericidal activity (SBA) and as opsonophagocytic activity. Two weeks after the last dose, the anti-meningococcal IgG concentrations were significantly higher in the i.m. group (median IgG concentration: 43.1 microg/ml) than in the intranasal group (10.6 microg/ml) (P=0.001). The corresponding opsonophagocytic activity was 7.0 and 3.0 (median log(2) titre) (P=0.001), and the SBA was 5.0 and 2.0 (median log(2) titre) (P=0.005), for the i.m. and intranasal groups, respectively. The last immunisation induced an enhanced immune response in the i.m. group, whereas the intranasal group showed no significant booster response. Accordingly, affinity maturation of anti-OMV-specific IgG antibodies was seen only after i.m. vaccination. The IgG1 subclass dominated the responses in both groups, whereas the significant IgG3 responses observed in the i.m. group were absent in the intranasal group. Although the intranasal OMV vaccination schedule used here induced functional immune responses relevant to protection, an improved vaccine formulation and/or a modified mucosal immunisation regimen may be needed to achieve a systemic effect comparable to that seen after three doses of intramuscular vaccination.

A nasal whole-cell pertussis vaccine can induce strong systemic and mucosal antibody responses which are not enhanced by cholera toxin

The immunogenicity of formaldehyde-inactivated Bordetella pertussis (Bp) delivered by the intranasal or colonic-rectal routes in BALB/c mice was studied by immunization four times at weekly intervals with Bp alone, or with Bp mixed with cholera toxin (CT) as a mucosal adjuvant. Mice given Bp subcutaneously, and untreated mice served as controls. Antibody responses in serum, saliva, bronchoalveolar lavage (BAL) fluids and extracts of faeces were measured by enzyme-linked immunosorbent assay. Nasal immunizations with Bp alone induced high levels of IgG antibodies to Bp in serum and BAL fluids, as well as IgA antibodies in serum, saliva, BAL fluids and extracts of faeces. The IgA responses were significantly reduced, and the IgG responses were not increased, when CT was given intranasally together with Bp. However, CT increased the IgA responses to Bp in faeces when both antigens were given rectally, while rectal administration of Bp alone did not induce significant serum or secretory antibody responses. However, when mixed with Bp, the CT itself induced antibodies to CT in serum and samples representing secretions after both nasal and rectal administrations. Thus, Bp is strongly immunogenic when applied intranasally, but not when presented into the intestinal lumen via the rectal route. It appears that CT, which is known to be a mucosal adjuvant and which in itself is a strong mucosal immunogen, will inhibit the immune responses of other strong immunogens when applied on the nasal mucosa.

Induction of antigen-specific T cell responses in human volunteers after intranasal immunization with a whole-cell pertussis vaccine.

We have studied the ability of an intranasally administered whole-cell pertussis vaccine (WCP) without adjuvant to induce antigen-specific T cell responses in humans. Six adult volunteers were given a vaccine dose (corresponding to 250 microg protein) by nasal spray four times at weekly intervals, and peripheral blood mononuclear cells were assayed for antigen-specific proliferative T cell responses. All six vaccinees had a WCP-specific response, which in four of them remained elevated throughout the 2 month study. All participants also responded to the filamentous haemagglutinin (FHA) antigen, and four of them responded to inactivated pertussis toxin (PTd). A significant correlation between T cell proliferation against WCP and WCP-specific IgA antibody levels in nasal secretions was observed. This demonstrates that intranasal administration of a non-proliferating bacterial vaccine without any additional mucosal adjuvant can induce vaccine-specific T cell responses related to mucosal IgA secretion.

Oral spray immunization may be an alternative to intranasal vaccine delivery to induce systemic antibodies but not nasal mucosal or cellular immunity.

Sixty‐five healthy adult volunteers were immunized four times at 1‐week intervals with an inactivated whole‐virus influenza vaccine based on the strain A/New Caledonia/20/99 (H1N1) without adjuvant. The vaccine was administered as nasal spray with a newly developed device to secure intranasal delivery (OptiMist™, OptiNose AS, Oslo, Norway), as regular nasal spray, nasal drops or as an oral spray. Significant IgA‐antibody responses in nasal secretions were induced in volunteers immunized intranasally but not after oral spray immunization. In saliva, IgA antibodies were only marginally amplified even after oral spray immunizations. At least 73% of the volunteers belonging to any group of vaccine delivery reached serum haemagglutination inhibition titres of 40 or higher, considered protective against influenza, after only two vaccine doses. Those who had the vaccine delivered intranasally also showed evidence from in vitro secretion of granzyme B that cytotoxic T cells had been stimulated. Although immunization with the breath‐actuated OptiMist™ device and nasal drops were superior with respect to both mucosal and systemic immune responses, oral spray immunization might still be considered for studies of mucosal adjuvants that are not yet acceptable for intranasal use.

Meningococcal outer membrane vesicle vaccine given intranasally can induce immunological memory and booster responses without evidence of tolerance.

ABSTRACT We have studied the ability of outer membrane vesicle (OMV) vaccines from Neisseria meningitidis serogroup B to induce vaccine-specific antibody and spleen cell proliferative responses in mice after being administered intranasally (i.n.) and/or subcutaneously (s.c.). A series of four weekly i.n. doses (25 μg) without adjuvant or a single s.c. dose (2.5 μg) with aluminum hydroxide was followed 2 months later by secondary i.n. or s.c. immunizations. After i.n. priming, both immunoglobulin G (IgG) antibody responses in serum, measured by enzyme-linked immunosorbent assay, and IgA antibodies in saliva and extracts of feces were significantly boosted by later i.n. immunizations. The IgG antibody responses in serum were also significantly augmented by secondary s.c. immunization after i.n. as well as s.c. priming. Sera from mice immunized i.n. reached the same level of bactericidal activity as after s.c. immunizations. The s.c. immunizations alone, however, had no effect on mucosal IgA antibody responses, but could prime for booster antibody responses in secretions to later i.n. immunizations. The i.n. immunizations also led to marked OMV-specific spleen cell proliferation in vitro. Both serum antibody responses and spleen cell proliferation were higher after i.n. priming and later s.c. immunizations than after s.c. immunizations alone. There was thus no evidence that i.n. priming had induced immunological tolerance within the B- or T-cell system. Our results indicate that a nonproliferating meningococcal OMV vaccine given i.n. can induce immunological memory and that it may be favorably combined with similar vaccines for injections.

Mucosal antibodies can be measured in air-dried samples of saliva and feces

IgA antibodies reflecting airways or intestinal mucosal immune responses can be found in saliva and feces, respectively, and IgG antibodies reflecting serum antibodies can be found in saliva. In this study, antibodies were detected in samples of saliva and feces which had been air-dried at room temperature (+20 degrees C) or +37 degrees C, and stored at these temperatures for up to 6 months. In saliva the antibody levels increased, while the antibodies in feces decreased upon storage. The individual IgA antibody concentrations which were adjusted by using the ratios of specific IgA/total IgA were relatively stable in both saliva and feces, and correlated with corresponding antibody levels in samples which had been stored at -20 degrees C. The results indicate that air-dried saliva and feces can be used for semiquantitative measurements of mucosal antibodies, even after prolonged storage at high temperatures and lack of refrigeration.

Influence of Intravenous Anesthesia on Mucosal and Systemic Antibody Responses to Nasal Vaccines

ABSTRACT Inhalation of antigens may stimulate the immune system by way of the upper as well as the lower airways. We have shown that at least 1,000 times more live pneumococci were recovered from pulmonary tissue after being presented as drops of a liquid suspension onto the nares of anesthetized mice compared to the number of bacteria recovered from animals that were not anesthetized in the course of the challenge. Mice that were similarly immunized intranasally by inhalation of three different nonreplicating particulate vaccine formulations, i.e., a meningococcal outer membrane vesicle (OMV) vaccine, a formalin-inactivated whole-virus influenza (INV) vaccine, and the INV vaccine with OMVs as a mucosal adjuvant, during general intravenous anesthesia developed concentrations of vaccine-specific serum immunoglobulin G (IgG) antibodies that were four to nine times higher than in mice that were fully awake during immunizations. The concentrations of IgA antibodies in serum were also higher in anesthetized than in nonanesthetized mice and correlated positively with the corresponding levels of serum IgG antibodies in the anesthetized but not in the nonanesthetized mice. In saliva and feces, however, the concentrations of IgA antibodies were equally high whether or not the animals were dormant during immunizations. The results indicate that intrapulmonary antigen presentation, as a part of an intranasal immunization strategy, is of importance for systemic but not for mucosal antibody responses. A major portion of IgA antibodies in serum may thus be derived from nonmucosal sites.

Systemic and mucosal antibody responses to group B streptococci following immunization of the colonic-rectal mucosa

The cervico-vaginal mucosa is poorly designed for inducing a mucosal immune response, but it can effect such a response evoked at other mucosal sites. This study was undertaken to determine whether colonic-rectal immunization with group B streptococci (GBS) might induce a local cervico-vaginal immune response. Mice were immunized with either fragmented GBS rectally, whole GBS rectally, or whole GBS subcutaneously. Cholera toxin (CT) was used as an adjuvant for the rectal immunizations. Following colonic-rectal immunization with whole GBS, the mean anti-GBS IgA antibody level in vaginal secretions was 735 kU/ml, with individual values reaching 3480 kU/ml. Corresponding levels of IgA antibodies never exceeded 10 kU/ml in serum and intestinal secretions, or 90 kU/g in feces. In vaginal secretions IgA antibodies to GBS also constituted a much larger fraction of total IgA than in serum, intestinal secretions and feces. Immunizations with fragmented GBS produced much lower IgA responses. Anti-GBS IgA response at the inductive site in the colon-rectum was not significant, as opposed to a strong anti-CT IgA response. Except in serum, the anti-GBS IgG responses to colonic-rectal immunizations were generally low, or absent. The results may provide a basis for the development of mucosal vaccines against GBS-infection.

Immunoglobulin‐A antibodies in Upper Airway Secretions may Inhibit Intranasal Influenza Virus Replication in Mice but not Protect Against Clinical Illness

Mice immunized intranasally with a formalin‐inactivated A/PR/8/34 (H1N1) influenza whole virus vaccine adjuvanted with cholera toxin, outer membrane vesicles from group B meningococci or formalin‐inactivated whole cell Bordetella pertussis were protected against replication of the homologous virus in the nasal cavity. Only some mice were protected against clinical illness measured as weight loss and lowered body temperature. All mice immunized subcutaneously with one‐tenth the intranasal vaccine dose without adjuvant were protected against clinical illness but not against local mucosal viral replication. Replicating virus was primarily found in animals with low concentrations of immunoglobulin (Ig)‐A antibodies in saliva regardless of concentrations of IgG antibodies in serum. Clinical illness was seen only in those with low serum antibodies regardless of antibody levels in saliva. Nonreplicating nasal vaccines may not be sufficiently protective unless they also have a substantial influence on systemic immunity.