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Infection and Immunity, October 2002, p. 5479-5484, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5479-5484.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

Libuse Janakova,1 Hilde Bakke,1 Inger Lise Haugen,1 Aud K. H. Berstad,1 E. Arne Høiby,1 Ingeborg S. Aaberge,1 and Bjørn Haneberg1,2*

Division for Infectious Disease Control, Norwegian Institute of Public Health, N-0403 Oslo,1 Department of Microbiology, Institute of Pharmacy, University of Oslo, N-0316 Oslo, Norway2

Received 13 March 2002/ Returned for modification 16 May 2002/ Accepted 12 July 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Intranasal administration of vaccines can effectively induce mucosal as well as systemic antibody responses (5, 9). Studies in mice with a vaccine consisting of heat-killed Streptococcus pneumoniae have shown that the nasal route was more effective than the oral and gastric routes of presentation, even for the induction of mucosal antibodies in the intestinal tract (17). Such nasal vaccines based on simple formulations of particles derived from bacteria and viruses seem to be effective without the use of traditional mucosal adjuvants, such as cholera toxin or the heat-labile toxin from Escherichia coli (3, 5, 8, 9). It is also noteworthy that nasal vaccines consisting of outer membrane vesicles (OMVs) from Neisseria meningitidis group B seemed to induce systemic antibodies with remarkably high bactericidal activity in humans (11, 14). Nonreplicating nasal vaccines may thus be developed as an alternative to corresponding vaccines for injection.

Lymphoid tissue of potential importance for the generation of immune responses in mice is found just beneath the mucosal surfaces of both the nasal and bronchial areas (20, 28). It has also been demonstrated that M-cells, or cells similar to M-cells, are interspersed among epithelial cells overlying such mucosa-associated lymphoid tissue (18, 25). Vaccine particles intended to mimic the natural infectious particles might therefore be taken up by M-cells within these mucosal linings, in much the same way as the infectious organisms themselves (19). It is not known, however, to what degree antigens delivered into the pulmonary tissue might lead to mucosal or systemic immune responses.

In fully awake mice, when reflexes are active, fluid applied to the nares is not easily inhaled, whereas volumes of 20 to 30 µl are inhaled rapidly during general anesthesia. With the use of radiolabeled protein in solution, it has been shown that pentobarbital anesthesia leads to fluid accumulation in the lungs, whereas the radioactivity was largely confined to the nasal epithelium of nonanesthetized mice (29). It has likewise been demonstrated that intranasal delivery of an influenza subunit vaccine mixed with negatively charged liposomes during light ether or pentobarbital anesthesia increased the amount of fluid in the lungs (10). Presentation of antigens in this way to the upper as well as the lower airways has been referred to as total respiratory tract immunization (10, 13, 29). Recent studies indicate, however, that vaccines consisting of various bacterially derived components. e.g., lipopolysaccharide, native outer membrane vesicles, and tetanus toxoid combined with cholera toxin, might actually do more harm if they reach the lungs instead of being confined to the upper airways (23, 24, 26).

The present study in mice was undertaken to determine the effect of anesthesia on local mucosal and systemic antibody responses to nonreplicating vaccines administered intranasally. Three different vaccine formulations were used; one was based on outer membrane vesicles (OMVs) from group B meningococci, another consisted of formalin-inactivated group A influenza virus, and the third consisted of the same influenza virus preparation in combination with the OMVs as a mucosal adjuvant. In order to avoid a direct influence of anesthesia on pulmonary functions, we used an anesthetic with brief duration to be given intravenously. A preliminary study was first carried out with live pneumococci to confirm that this type of anesthesia would actually increase the number of bacteria reaching the lungs after being deposited in a liquid formulation onto the nasal openings.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals. Inbred female BALB/c mice, 8 to 10 weeks old, were obtained from Bomholtgård Breeding and Research Center, Ry, Denmark.

Intranasal challenge with pneumococci. A human isolate of Streptococcus pneumoniae, serotype 4, was used for intranasal challenge. The bacterial inoculum for challenge was made as previously described (1). Briefly, small aliquots of pneumococci in the mid-logarithmic growth phase were prepared by a standard method and kept frozen at -70°C, ready for challenge experiments after thawing and appropriate dilution. Six mice were exposed to live pneumococci corresponding to 2.5 x 106 CFU in volumes of 30 µl per mouse, either with anesthesia (see below) or without. Three hours after exposure, the mice were sacrificed, blood was sampled by cardiac puncture, and lungs were taken out and homogenized. Tenfold dilutions of homogenates and blood were plated on blood agar plates. Colonies of bacteria were counted after incubation at 36°C in a 5% CO2 atmosphere for 18 h.

Vaccine preparation. Three different vaccines were prepared. The first vaccine contained OMVs from the epidemic group B meningococcal strain 44/76 (15:P1.7,16). The OMVs were prepared by extraction of bacteria with 0.5% deoxycholate in 0.1 M Tris-HCl buffer (pH 8.6) containing 10 mM EDTA, followed by purification by differential centrifugation (12). The nasal vaccine was made from an original pool of OMVs, without aluminum hydroxide. The second vaccine consisted of influenza virus (A/Shanghai/11/87, H3N2) which was propagated by incubation for 2 days at 34°C in the allantoic cavity of 12-day embryonated hen's eggs. After harvesting, the virus-containing allantoic fluid was filtered through eight layers of gauze, virus was separated by continuous-flow centrifugation at 40,000 to 45,000 x g and dissolved in phosphate-buffered saline (PBS) containing 0.01% (wt/vol) merthiolate. Afterwards, the remaining debris and aggregated virus were removed by centrifugation at 1,500 x g for 45 min, virus was inactivated in 0.01% (wt/vol) formaldehyde for 1 week at 4°C, and the concentration of viral protein was adjusted to 10 mg ml-1 by the method of Lowry. The third vaccine contained the same influenza virus in combination with the OMVs as a mucosal adjuvant (8). The formalin-inactivated whole-virus influenza (INV) vaccine and bacterial OMV vaccine were mixed immediately before immunizations.

Immunizations. Groups of six to eight mice were immunized intranasally either under general anesthesia or while fully awake with the same vaccine four times at 1-week intervals. Doses of 30 µl of the vaccines consisted of 25 µg of OMVs, 125 µg of INV, and 125 µg of INV mixed with 70 µg of OMVs; all doses were measured as total protein. As an anesthetic agent, 0.02 ml of propofol (Diprivan; Seance Ltd., Macclesfield, Cheshire, United Kingdom) at a concentration of 10 mg/ml was used intravenously. Mice were placed in a special holder and given an injection of anesthetic into the tail vein. Nasal immunizations were provided while the mice were sleeping, being held in a supine position with their heads down. Antigen solution was delivered slowly with a micropipette onto the nares so that the mouse could sniff it in. The mice recovered completely 1 to 2 min after anesthesia administration. The nonanesthetized mice were vaccinated similarly, as described above. A group of six nonimmunized and nonanesthetized mice served as controls.

Collection of samples. Samples of feces, saliva, and serum were collected 1 week after the fourth intranasal dose. Saliva samples were collected with the help of absorbent wicks consisting of synthetic fibers and cellulose (Polyfiltronics Group, Inc., Rockland, Mass.) after a single intraperitoneal injection of 0.1 mg of pilocarpine-HCl (Sigma Chemical Co., St. Louis, Mo.) in 200 µl of PBS, and the net weight was recorded. Two wicks saturated with saliva obtained from each mouse were frozen at -20°C in 1.5-ml microcentrifuge tubes and subsequently extracted with 400 µl of PBS with 0.05% Tween and protease inhibitors, as described previously (15). Three to five pieces of freshly voided feces were collected into 1.5-ml microcentrifuge tubes, frozen at -20°C, and subsequently vacuum dried before their net dry weights were recorded. Extracts of feces were made by adding 20 µl of PBS with 0.05% Tween 20 and protease inhibitors per mg of dry feces, as described previously (15). The extraction buffer contained the following protease inhibitors: 0.2 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (Boehringer Mannheim GmbH, Mannheim, Germany), 1 µg of aprotinin (Sigma) per ml, 10 µM leupeptin (Sigma), and 3.25 µM bestatin (Sigma). Blood samples were taken by cardiac puncture during CO2 anesthesia. Blood was separated and stored at -20°C until it was analyzed.

Quantitation of antibody responses. OMV- and INV-specific immunoglobulin A (IgA) antibodies in saliva and extracts of feces and OMV- and INV-specific IgG and IgA antibodies in serum were analyzed by enzyme-linked immunosorbent assay (ELISA) with Nunc Immuno Plates (MaxiSorp F96; Nunc A/S, Roskilde, Denmark). ELISA plates were coated for at least 1 week at 4°C with OMVs at 4 µg/ml in 0.1 M Tris-HCl buffer (pH 8.6) or with INV at 5.4 µg/ml in 0.01 M PBS (pH 7.2). Nonspecific protein-binding sites were blocked with PBS (pH 7.2) containing 5% nonfat dry milk (Oxoid, Hampshire, United Kingdom), and after 1 h of incubation at 37°C and 30 min at room temperature, the plates were washed with PBS containing 0.05% Tween 20. Serum, extracts of saliva and feces, and standard solutions were applied to the ELISA plates (100 µl per well), serially diluted twofold in the blocking solution, and incubated at 4°C overnight. The plates were then washed with PBS containing 0.05% Tween 20 before they were incubated for 1 h at room temperature with peroxidase-conjugated goat antibodies directed against mouse IgA or IgG, both diluted 1:1,000 in blocking buffer (100 µl per well).

After washing, bound antibodies were detected with o-phenylenediamine (Sigma) in 0.05 M phosphate-citrate buffer, pH 5.0. Optical densities were read at 492 nm in a Titertek Multiscan Plus MK II (Labsystems, Helsinki, Finland). Standard curves were generated, and anti-OMV and -INV antibody concentrations (in arbitrary units) in the samples were determined based on a defined pool of serum samples or secretions. The extracted samples were corrected for the weights of the original samples and dilutions were made during extraction from wicks and preparation for ELISA.

The serum bactericidal activity assay was performed with an agar overlay method on microtiter plates as described previously (16, 21). Briefly, twofold dilutions of serum samples starting at 1:2 were tested with a meningococcal inoculum of about 80 to 100 CFU (per well) of the 44/76-SL variant of a B:15:P1.7,16 strain (22), first grown overnight on brain heart infusion agar with 1% horse serum and then grown for 4 h in a 5% CO2 atmosphere at 37°C on a new plate. Human plasma from an individual without bactericidal antibodies to the strain was used as a complement source (21). Agar was added to the plates after 30 min of incubation of the reaction mixture at 37°C. The numbers of CFU were counted after overnight incubation in 5% CO2 at 37°C. The titers are given as the highest reciprocal final dilution of serum killing more than 50% of the inoculum.

Statistical analyses. The significance of differences between groups of animals by two-tailed Mann-Whitney U test and Spearman's rank correlation was determined by the use of Prism software (GraphPad Software, San Diego, Calif.).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anesthesia led to more bacteria reaching the lungs. In mice that had been put to sleep with an intravenous anesthetic, 30-µl volumes of a suspension of pneumococci in saline were inhaled by deep breaths within 10 to 15 s after being applied as small drops onto the nares. The mice recovered completely within 3 min after having received anesthesia. In fully awake mice, equal volumes of the pneumococcal suspension were inhaled but with shallow breaths, and administration took up to 1 min to complete because the animals resisted being held in a supine position with head down to avoid having the fluid seep into the mouth. They seemed fully restored, however, as soon as they were freed.

In a study on six mice that were challenged intranasally with live pneumococci, bacteria were recovered in lung homogenates 3 h later, whether or not they had been anesthetized. After receiving 2.5 x 106 CFU by inhalation, however, two anesthetized animals had about 1,000 times higher numbers of bacteria in lung homogenates than animals that had been fully awake during inhalations, i.e., 3 x 107 and 1 x 108 CFU as opposed to 1 x 104 to 8 x 104 CFU in three nonanesthetized animals and 5 x 104 CFU in one incompletely anesthetized animal. There was no evidence that bacteria had for one reason or another gained access to the bloodstream, e.g., via the lung. A nasal vaccine containing bacteria or bacterially derived particles might thus be easily presented to the lower airways of anesthetized mice.

Anesthesia led to higher levels of antibodies in serum but not in secretions. All the immunized animals responded with marked increases in serum IgG antibodies specifically directed against the vaccine antigen in question. The responses in the anesthetized animals, however, were stronger than in those that were fully awake during immunizations, with median antibody concentrations 9, 4, and 5 times higher than in nonanesthetized mice for the OMV vaccine (P = 0.002), the INV vaccine alone (0.004), and the INV vaccine with OMV as a mucosal adjuvant (P = 0.0012), respectively (Fig. 1, 2, and 3).



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  		FIG. 1. IgG antibodies in serum and IgA antibodies in saliva and feces against meningococcal OMVs in groups of mice immunized (V+) intranasally with an OMV vaccine during general anesthesia (A+) or while fully awake (A-). Unimmunized mice (V-) served as controls. The results, measured by ELISA, are given as individual values and medians in arbitrary kilounits or units per milliliter or per gram of dry feces.

 
When testing serum samples from animals that had received the meningococcal OMV vaccine for bactericidal activity against meningococci of the same strain that had been used for making the OMVs, it appeared that the anesthetized mice had a fourfold-higher median titer than those that were awake during immunizations (P = 0.002) (results not shown). Moreover, the bactericidal activity of serum samples from all animals that received the OMV vaccine, whether or not they were anesthetized, and from nonimmunized control mice correlated positively with the corresponding IgG antibody concentrations as measured by ELISA (R = 0.88, P = 0.0001) (results not shown).

The concentrations of IgA antibodies in serum after immunizations were influenced by anesthesia largely to the same degree as were IgG antibodies, with median values 9, 3, and 4 times higher than in nonanesthetized mice with the use of the OMV vaccine, the INV vaccine alone, and the INV vaccine with OMV as an adjuvant, respectively (Fig. 4). However, only the OMV vaccine induced significantly higher serum IgA antibodies with anesthesia than without (P = 0.0002). Moreover, in the mice that had received the OMV vaccine during general anesthesia, the concentrations of IgA antibodies in serum correlated positively with the serum IgG concentrations, whereas the corresponding concentrations in nonanesthetized mice did not correlate (Fig. 5). This may have implications for understanding the origin of IgA antibodies in serum.



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  		FIG. 4. IgA antibodies in serum against meningococcal OMVs and influenza virus (INV) in groups of mice immunized (V+) intranasally with either an OMV vaccine, an INV vaccine, or an INV vaccine with OMVs as a mucosal adjuvant (INV + OMV) during general anesthesia (A+) or while fully awake (A-). Unimmunized (V-) mice served as controls. The results, measured by ELISA, are given as individual values and medians in arbitrary kilounits per milliliter.

 


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  		FIG. 5. Correlation between IgG and IgA antibodies in serum of mice after being immunized intranasally with an OMV vaccine during general anesthesia or while fully awake. The results, measured by ELISA, are given as individual values in arbitrary kilounits per milliliter.

 
In samples of saliva and feces, representing airway and intestinal secretions, respectively, we found high levels of IgA even in mice that had not been anesthetized (Fig. 1 to 3). As opposed to the findings in serum, there were no significant differences in concentrations between animals that had been and those that had not been anesthetized (P >= 0.05). Thus, there was no indication that antigen in the lungs had had any marked influence on the induction of mucosal antibodies.



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  		FIG. 3. IgG antibodies in serum and IgA antibodies in saliva and feces against influenza virus (INV) in groups of mice immunized (V+) intranasally with an INV vaccine together with OMVs as mucosal adjuvant during general anesthesia (A+) or while fully awake (A-). Unimmunized mice (V-) served as controls. The results, measured by ELISA, are given as individual values and medians in arbitrary kilounits or units per milliliter or per gram of dry feces.

 
Effect of mucosal adjuvant on serum antibodies may equate to effect of anesthesia. In animals that received the INV vaccine with OMV as a mucosal adjuvant, the concentrations of serum IgG antibodies were higher (Fig. 3, upper panel) than in those that got the INV vaccine alone (Fig. 2, upper panel), with median values 4 times higher in anesthetized (P = 0.008) and 3.5 times higher in nonanesthetized (P = 0.004) mice. This effect of OMVs when used in conjunction with the INV vaccine led to IgG concentrations in serum samples from nonanesthetized mice of the same magnitude (Fig. 3, upper panel) as from mice immunized with INV alone under general anesthesia (Fig. 2, upper panel) (P = 0.4). In order to obtain high levels of antibodies in serum, there is thus an alternative to the use of general anesthesia.



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  		FIG. 2. IgG antibodies in serum and IgA antibodies in saliva and feces against influenza virus in groups of mice immunized (V+) intranasally with an INV vaccine during general anesthesia (A+) or while fully awake (A-). Unimmunized mice (V-) served as controls. The results, measured by ELISA, are given as individual values and medians in arbitrary kilounits or units per milliliter or per gram of dry feces.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It was evident from this study that a much larger fraction of pneumococci reached the lungs of mice after being presented in saline and inhaled during general anesthesia than when presented to fully awake mice. This is in accordance with results obtained by others showing that general anesthesia, whether or not given by inhalation, would lead to more of an inhaled liquid's accumulating in the lungs of experimental mice (10, 29). In addition to making intranasal immunization easier to carry out, it was previously considered an advantage to have the vaccine also reach the lungs in order to achieve so-called total respiratory tract immunization (10, 13, 29). Moreover, it has been suggested that effective mucosal immunity in human airways will best be achieved by combined stimulation of both the upper and lower respiratory tract, requiring that mucosal vaccines for humans be presented as aerosols (27).

Our finding that animals that had been immunized during anesthesia-induced sleep developed markedly higher concentrations of vaccine-specific serum IgG antibodies than did those that were immunized while fully awake indicates that antigens in the lungs will stimulate the systemic immune system more effectively than when mainly confined to the upper airways. Possibly, this can be ascribed to vaccine components having leaked through the thin alveolar membrane or to more of the antigen having been taken up by the bronchus-associated lymphoid tissue (BALT), which is covered by epithelium also containing M-cells (2, 19). In secretions, however, the development of IgA antibodies was not influenced by anesthesia, suggesting that antigens in the lungs have little effect on production of mucosal antibodies. It is tempting to speculate, therefore, that the additional effect on serum antibody concentrations obtained with the use of general anesthesia might be ascribed to part of the vaccines having evaded the mucosal immune system.

The high concentrations of mucosal antibodies in saliva as well as in feces, with all three vaccines tested, whether or not anesthesia had been given, indicate that induction of antibody responses had taken place in the upper airway mucosa. The present finding underlines the superiority of this area for development of mucosal antibodies, as has been demonstrated before in comparison with the oral, intragastric, and rectal routes of antigen delivery, even for antibodies of the distant intestinal tract (17).

The high concentrations of IgA antibodies in the serum of mice which had been immunized during anesthesia compared to serum IgA antibodies in nonanesthetized mice suggest that the mechanism for induction of IgA in serum was the same as for IgG antibodies in serum. Furthermore, the positive correlation of IgA antibodies with IgG antibodies in serum in mice that had been immunized with a meningococcal OMV vaccine during anesthesia, whereas there was no correlation in serum values for mice that were fully awake during immunizations, may indicate that most IgA in serum is actually derived from nonmucosal sites. This has previously been claimed by others (7).

Since some vaccines based on components of bacterial origin may lead to pulmonary inflammation (23, 24, 26), nasal vaccines should be presented in a way to avoid their being deeply inhaled. In humans, this may be achieved by giving them as nasal drops (4, 14; H. H. Samdal, A. H. Berstad, F. Oftung, H. Bakke, I. L. Haugen, G. E. Korsvold, A-C. Kristoffersen, G. Krogh, K. Nord, and B. Haneberg, 2001, unpublished data) or as a spray of particles or droplets with diameters not smaller than 5 µm (2, 6). Even though the pulmonary changes induced by such nasal vaccines seemed to be transient (23), efforts should nevertheless be made to avoid toxic derivatives as part of vaccine formulations or to find alternatives to using the lower mucosal airways for induction of sizable systemic immune responses.

When meningococcal OMVs were added as a mucosal adjuvant to the INV vaccine in the present study, the serum IgG antibody concentrations increased to the same level as when anesthesia was given to mice during immunization with the influenza vaccine alone. Thus, it appeared that the high levels of serum antibodies induced by the nasal vaccines' reaching the lungs can be compensated for by the use of proper mucosal adjuvants. Future development of effective nasal vaccines may therefore depend on the identification of nontoxic mucosal adjuvants as well as proper delivery systems.

In humans, it has been demonstrated that nasal vaccines administered as nasal drops may lead to strong systemic antibody responses that are considered correlates of protection against disease (14; H. H. Samdal, A. H. Berstad, F. Oftung, H. Bakke, I. L. Haugen, G. E. Korsvold, A-C. Kristoffersen, G. Krogh, K. Nord, and B. Haneberg, 2001, unpublished data). It has also been questioned whether BALT is of less importance for creating immune response in humans than in experimental animals (20). Even though the three nasal vaccines used in the present study induced higher levels of serum antibodies with than without anesthesia and the serum bactericidal activity of the meningococcal OMV vaccine was higher with anesthesia than without, it remains to be seen whether anesthesia would make a difference in future animal studies of protection against disease. Such studies may also shed light on the relative importance of mucosal antibodies as opposed to systemic immunity.


   ACKNOWLEDGMENTS
 
We thank Bente Møgster, Mona I. Skullerud, and Kari E. Løken for excellent technical assistance.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division for Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, N-0403 Oslo, Norway. Phone: 47 22 04 23 56. Fax: 47 22 04 23 01. E-mail: bjorn.haneberg{at}fhi.no. Back

Editor: E. I. Tuomanen


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, October 2002, p. 5479-5484, Vol. 70, No. 10
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.10.5479-5484.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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