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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Secretory immunoglobulin A (SIgA) provides the first line of defence against pathogens initiating infection via the mucosal route, e.g. the influenza virus. The aim of this study was to examine the basal level of influenza-specific antibody-secreting cell (ASC) in the local mucosa of the upper respiratory tract. Nineteen patients scheduled for tonsillectomy were enrolled for the study, and they had not experienced influenza during the previous year. Tonsils, blood, oral fluid and a nasal biopsy were sampled, and the basal levels of ASC and antibodies (Abs) were determined. We found low numbers of influenza-specific ASC in the blood and tonsils, but there were about 10–100 times higher numbers of specific ASC in the nasal mucosa tissue despite no recent influenza exposure. Thus, the basal level of influenza-specific ASC in the mucosa of the respiratory tract may be important in the protection against influenza infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The majority of human pathogens initiate infection via the mucosal surfaces. Mucosal immunity [1] in the upper respiratory tract confers the first line of defence against a number of pathogens of both viral and bacterial origin. Concurrently, this local immune system is also central in the development of tolerance, allergic reactions and in some instances autoimmune diseases [1]. The quantity of foreign antigens and micro-organisms passing daily through the mouth and upper respiratory tract is vast and has to be dealt with by an effective natural and acquired immune system. The tonsils and nasal-associated lymphoid tissue (NALT) play an important role in trapping foreign antigens, neutralising them and inducing local immune reactions. After immune stimulation a high number of activated cells leave the lymph nodes and home to the mucosal surfaces in the upper respiratory tract to fight invading pathogens [1]. The mucosal surfaces contain a lymphocyte rich layer between the upper epithelial cells and the lower connective tissue called the lamina propria [1]. Altogether, the lamina propria probably contains more lymphocytes than blood, lymph and lymphoid tissue collectively. This points to the very important role of lamina propria in modulating immune responses.

The influenza virus is a major respiratory pathogen causing high morbidity in the general population and mortality in high-risk groups [2]. Parenterally administered vaccine is the most common method of influenza prophylaxis. In the absence of true efficacy markers, the serum haemagglutination inhibition titre has been correlated with protection against influenza infection [2]. Serum IgG is an indicator of previous exposure to influenza infection and/or immunization, and may be important in limiting the spread of the infection [3]. However, SIgA at mucosal surfaces confers the first line of defence against influenza [4].

We have previously used parenterally administered influenza vaccine as a model to investigate the systemic and local immune responses induced after influenza vaccination in healthy human volunteers [5–12]. Our results showed a rapid homing of influenza-specific memory B cells to the tonsils after influenza vaccination [6]. In addition, parenteral vaccination induced a significant IgA response in the peripheral blood, which correlated with the appearance of IgA-producing B cells in the tonsils and SIgA in the oral fluid of adults [5–7]. In primed subjects, IgG and IgA dominated the systemic responses, whereas IgM dominated in unprimed subjects [9,10,12]. The peak in numbers of ASC occurred at 7 days post vaccination in both the systemic and the local compartments.

The aim of this study was to examine the basal level of influenza-specific ASC in blood (circulation, periphery), tonsils (local lymphoid organ) and nasal tissue (local mucosa).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients and samples Nineteen patients (9 males and 10 females, with a mean age of 28 years) that were scheduled for tonsillectomy at the Department of Otolaryngology, Haukeland University Hospital, Bergen were enrolled in the study. All patients fulfilled the medical criteria for tonsillectomy, but were otherwise healthy with no acute bacterial infection of the tonsils, no history of allergy and no known exposure to influenza (infection or vaccination) in the previous year. Tonsils, a biopsy sample from the mid portion of the caudal medial part of the inferior turbinate, peripheral blood and oral fluid (Orasure, Epitope, Beaverton, USA) were collected. The study was approved by the Regional Ethical Committee and all volunteers signed an informed consent form.

Influenza virus antigens Purified surface glycoproteins (a generous gift from Medeva Pharma Ltd, Surrey, UK) from the current seasons (2000–2001) virus strains; A/New Caledonia/20/99 (H1N1); A/Panama/2007/99 (H3N2) and B/Yamanashi/166/98 (B) were used as antigens in the ELISA and ELISPOT assays. The previous seasons virus strains (1999–2000); A/Beijing/262/95 (H1N1); A/Sydney/5/97 (H3N2) and B/Yamanashi/166/98 were propagated in embryonated hens' eggs and used as virus antigens in the haemagglutination inhibition (HI) test.

Immunological tests Lymphocytes were isolated from the blood, tonsils and nasal tissue using Lymphoprep (Nycomed Pharma, Oslo, Norway) and were analyzed by the ELISPOT method [5]. Antibodies in the oral fluid and serum were quantified by the ELISA method [6]. All capture and detector Abs were purchased from Sigma (St. Louis, MI, USA). In addition, the level of serum antibody to the viruses was analyzed by the HI test [5].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The 19 subjects who participated in this study were chosen from patients scheduled for tonsillectomy. The subjects had not experienced tonsillitis recently (not in the 3 months prior to the operation), had no history of allergy and had not been infected or vaccinated during the previous year. Blood samples from the subjects were analyzed for Ig levels and allergy markers, confirming the healthy status of the volunteers (data not shown).

Serum antibody levels

The HI test is a conventional method for testing influenza-specific serum Abs. A serum titer equal or greater than 40 HI units has been correlated with protection against influenza infection [13]. All subjects in our study had HI titers below the level of detection (< 10) to the H1N1 and B viruses (Table 1). Nine subjects had detectable HI titers (> 10) to the H3N2 virus, but only two of these subjects had protective levels. These results indicate that the subjects had not been infected with influenza recently, which may otherwise have had an adverse effect on this study.

Table 1.   The serum antibody HI titres in healthy individuals
Subject numberH1N1H3N2B
  1. < indicates an HI titer of < 10. HI titres of ≥40 are considered protective.

1<20<
2<30<
3<20<
4<40<
5<<<
6<40<
7<<<
8<<<
9<10<
10<<<
11<<<
12<<<
13<<<
14<<<
15<10<
16<20<
17<20<
18<<<
19<<<

The concentration of the total influenza-specific Abs in the serum was quantified by the ELISA method (Fig. 1). All subjects had detectable levels of Abs towards the three vaccine strains, mean titres of 34, 40 and 32 μg/ml to H1N1, H3N2 and B, respectively. Most subjects had Ab concentrations between 1 and 10 µg/ml, but there were three subjects who had very low Ab titres (one subject to H1N1 and two volunteers to B virus). These results are comparable with the prevaccination concentrations reported in our previous work [6,9,12].

image

Figure 1.  The concentration (ng/ml) of influenza antibodies (Abs) in serum and oral fluid. The three columns to the left are the serum levels, and the other three columns are the oral fluid levels toward the three circulating influenza types (see Materials and methods for details). Each individual data point is represented by a cross (n = 19), and the vertical bar immediately to the right of these points, indicates the mean and standard deviation values.

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Oral fluid Ab levels

We found a low but detectable basal level of anti-influenza antibodies (Fig. 1), range 10 and 47 ng/ml, with one exception of 75 ng/ml, with an average of 28–30 ng/ml against the three vaccine strains. These results are also comparable with previously reported prevaccination levels [6,7]. Despite considerably lower Ab concentrations in the oral fluid than in serum, the oral fluid Abs reflects the production in a short time frame, whereas the serum levels represent an accumulated production over several days.

Influenza-specific ASC

Figure 2 shows the level of influenza-specific ASC in the blood (circulation), the tonsils (lymphoid organ) and the nasal tissue (mucosa). The level of total influenza-specific ASC in the blood and the tonsils is low and similar to our previous reports [6,9]. The number of influenza-specific ASC detected in the blood varied from 2 to 100 per million lymphocytes, with an average of 7–12. The levels of influenza-specific activated B cells are slightly higher in the tonsils, with an average of about 12–26 cells per million lymphocytes. The number of specific ASC in the nasal tissue are generally 1–2 log10 higher than in the blood or the tonsils, with an average of 327–410 cells per million lymphocytes. If the number of influenza-specific ASC in the nasal tissue is related to the large total area of mucosa surface in the upper respiratory tract, this may represent an incredible army of anti-influenza B cells.

image

Figure 2.  The basal level of influenza-specific antibody-secreting cell (ASC) in peripheral blood (left), tonsils (middle) and nasal mucosa biopsy (right). The graph shows the individual number of specific ASC per 106 lymphocytes to the current influenza viruses (n = 18). The vertical bars represent the mean and standard deviation values.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In previous studies [5–12] we examined the systemic and local immune response to influenza vaccination. We found that parenterally administered vaccination induced a rapid and strong immune response both systemically and locally in the upper respiratory tract (oral fluid and tonsils). We have thus established a link between the systemic influenza vaccination and the specific local mucosal immune response.

For many years the serum Ab levels and specificity to circulating viruses have been considered important factors in the protection against the influenza infection, despite the fact that influenza is a pathogen of the mucosa of the respiratory tract. The HI test has become a gold standard, with a serum HI titre of greater than or equal to 40 correlated with protection against influenza infection [13].

The methodological difficulties involved in assessing the immune response in the upper airway mucosa have resulted in a paucity of data on the local Ab-secreting cell response to influenza virus vaccination or infection. However, in our previous studies, we observed a low but detectable level of influenza-specific B cells and Igs prior to vaccination in the blood and the tonsils. In this study we specifically addressed the basal level of influenza-specific ASC in the local mucosa, in subjects who have not experienced influenza during the preceding year. Although the prevaccination levels of influenza-specific Abs and ASC were low we were still able to quantify them. It was also possible to detect a considerable number of activated influenza-specific ASC in the nasal mucosa, which were 10–100 times higher than in blood and tonsils, during a period of no influenza activity in the local community.

The discrete production of secreted Abs in the upper respiratory tract probably has an important role in protecting an individual from influenza infection. Low concentrations of Abs were found in the oral fluid, but the accumulated production of secreted anti influenza Abs represents an impressive protection mechanism against infection. This is in line with reports documenting the correlation between the level of influenza-specific SIgA in nasal lavage and protection [14,15].

Our findings support the hypothesis that the levels of influenza-specific ASC and SIgA in the local mucosa are important factors in the protection against influenza infection. These levels may drop gradually after years without a proper local stimulation (e.g. infection), leaving the subject more vulnerable to influenza infection. Hence, to obtain a prolonged and effective immune response, the influenza vaccine must be engineered to stimulate the local immune system resembling a natural infection.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We would like to thank Lars R. Haaheim for a valuable discussion, Turid Tynning and Hilde Garberg for their technical assistance, and the Norwegian Research Council for financial support. We would like to specially thank Medeva Pharma Ltd for providing the purified influenza virus antigens.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR. (eds) Mucosal Immunology. 2nd edn. San Diego: Academic Press, 1999.
  • 2
    Nicholson KG, Webster RG, Hay AJ. (eds). Textbook of Influenza. London: Blackwell Science Ltd., 1998.
  • 3
    Murphy BR & Clements ML. The systemic and mucosal immune response of humans to influenza A virus. Curr Top Microbiol Immunol 1989;146:10716.
  • 4
    Renegar KB & Small Pa Jr. Passive transfer of local immunity to influenza virus infection by IgA antibody. J Immunol 1991;146:19728.
  • 5
    Cox RJ, Brokstad KA, Zuckerman MA, Wood JM, Haaheim LR, Oxford JS. An early humoral immune response in peripheral blood following parenteral inactivated influenza vaccination. Vaccine 1994;12:9939.
  • 6
    Brokstad KA, Cox RJ, Olofsson J, Jonsson R, Haaheim LR. Parenteral influenza vaccination induces a rapid systemic and local immune response. J Infect Dis 1995;171:198203.
  • 7
    Brokstad KA, Cox RJ, Oxford JS, Haaheim LR. IgA, IgA subclasses and secretory component levels in oral fluid collected from subjects after parenteral influenza vaccination. J Infect Dis 1995;171:10724.
  • 8
    Brokstad KA, Cox RJ, Major D, Wood JM, Haaheim LR. Cross-reaction but no avidity change of the serum antibody response after influenza vaccination. Vaccine 1995;13:15228.DOI: 10.1016/0264-410x(95)00095-i
  • 9
    El-Madhun AS, Cox RJ, Søreide A, Olofsson J, Haaheim LR. Systemic and mucosal immune responses in young children and adults after parenteral influenza vaccination. J Infect Dis 1998;178:9338.
  • 10
    El-Madhun AS, Cox RJ, Seime A, Sovik O, Haaheim LR. Systemic and local immune responses after parenteral influenza vaccination in juvenile diabetic patients and healthy controls: results from a pilot study. Vaccine 1998;16:15660.DOI: 10.1016/s0264-410x(97)88328-4
  • 11
    Cox RJ & Brokstad KA. The post-vaccination antibody response to influenza virus proteins. APMIS 1999;107:28996.
  • 12
    El-Madhun AS, Cox RJ, Haaheim LR. Effect of age and natural priming on the IgG and IgA subclass responses after parenteral influenza vaccination. J Infect Dis 1999;180:135660.
  • 13
    Hobson D, Curry RL, Beare AS, Ward-Gardner A. The role of serum haemagglutination-inhibiting antibody in protection against challenge infection with influenza A2 and B viruses. J Hyg (Lond) 1972;70:7677.
  • 14
    Beyer WE, Van der Logt JT, Van Beek R, Masurel N. Immunoglobulin G, A and M response to influenza vaccination in different age groups: effects of priming and boosting. J Hyg (Lond) 1986;96:51322.
  • 15
    Clements ML & Murphy BR. Development and persistence of local and systemic antibody responses in adults given live attenuated or inactivated influenza A virus vaccine. J Clin Microbiol 1986;23:6672.