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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

Influenza DNA vaccines have been widely studied in experimental animal models and protection documented after lethal viral challenge. In this study, we have investigated the humoral response after a non-lethal viral challenge of mice vaccinated with plasmids encoding the influenza haemagglutinin (HA) or nucleoprotein (NP) genes. BALB/c mice were immunized intramuscularly with three doses (100 µg) of HA, NP or backbone plasmid at 3-week intervals, or alternatively infected intranasally, before being challenged with homologous virus 13 weeks later. Mice were then sacrificed at weekly intervals and the antibody-secreting cell response was examined systemically (spleen and bone marrow) and in the respiratory tract (nasal associated lymphoid tissue (NALT) and lungs). Sera were collected after each dose of vaccine and at sacrifice and analyzed by ELISA, haemagglutination inhibition and virus neutralization assays. We found that previous viral infection apparently elicits sterilizing immunity. Vaccination with HA or NP DNA significantly reduced viral replication in the nasal cavity after viral challenge, however, increases in serum antibody titres were observed after challenge. Prior to challenge, specific antibody-secreting cells were observed in the systemic compartment after HA or NP DNA vaccination but were also found in the NALT after viral challenge. In conclusion, intramuscular DNA vaccination resulted in immunological memory in the systemic compartment, which was rapidly reactivated upon viral challenge.


Introduction

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

Influenza virus undergoes frequent antigenic changes in its surface glycoproteins, which results in frequent influenza outbreaks. Vaccination is the cornerstone of influenza prophylaxis and the efficacy of current influenza vaccines depends upon the antigenic match between the vaccine strains and those circulating in the community. Annual vaccination is required to combat antigenic drift and influenza vaccines are generally under-utilized in the high-risk groups. Thus, there is a continuing need for improved influenza vaccines to provide a broader and longer lasting immunity.

DNA vaccines are a promising new approach to vaccination, eliciting a full spectrum of immune responses including antibody, cytotoxic T cell (CTL) and helper T (Th)-cell responses [reviewed in 1]. DNA vaccination involves the introduction of a nonreplicating plasmid encoding an antigen into the host, which results in endogenous antigen synthesis producing an antigen that is appropriately processed and presented to the immune system. During the last decade, extensive development has occurred in the field of DNA vaccines with immune responses and/or protection demonstrated to many different pathogens in both small and large animal models [reviewed in 2] and a number of DNA vaccines have recently entered human clinical trials. Influenza DNA vaccines have been extensively studied and constructs encoding the surface glycoproteins, the HA and the neuraminidase (NA), the NP, the matrix proteins (M1 and M2) and the non-structural protein (NS1) have all been tested for their ability to induce immune responses [3–7].

Immunization with HA-encoding plasmids is thought to provide protection by humoral immunity involving neutralizing antibodies [7], although a recent report found that Th1 cells appear to mediate protective immunity in the absence of detectable antibody [9]. Both cytotoxic CD8+ and cytokine-producing CD4+ T cells provide protective immunity after intramuscular NP DNA vaccination [10,11]. Many studies of influenza DNA vaccines in mice have used a lethal pulmonary infection and reported protection from death as an end-point [3–8]. In this study, we have examined the immune response in the systemic and local compartments after viral challenge with a non-lethal influenza strain in mice vaccinated intramuscularly (i.m.) with HA or NP DNA vaccines.

Materials and methods

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

Plasmids Three plasmids were used in this study; pI.17 HA, pI.18 NP and pI.18 control plasmid. The pI.17 HA contains the A/Sichuan/2/87 (H3N2) full-length HA and the pI.18 NP contains the full length A/Sichuan/2/87 (H3N2) NP gene. The relevant gene was amplified by polymerase chain reaction (PCR) and cloned into the appropriate plasmid backbone (either pI.17 and pI.18). The backbone plasmids are pUC-based plasmids encoding a bacterial origin of replication and ampicillin-resistance gene for growth and selection in Escherichia coli K12. They also carry a truncated enhancer region, full promoter and full Intron A gene from human cytomegalovirus (CMV) and a CMV terminator sequence. All plasmids were purified using Qiagen QIAfilter mega plasmid kits (Qiagen, Hilden, Germany).

Viruses and antigens A/Sichuan/2/87 (H3N2) virus was propagated in the allantoic cavity of 10-day-old-embyronated hens' eggs. A/Sichuan/2/87 (H3N2) virus was adapted to mice by three skim passages and the virus in the final lung wash was propagated in embryonated hens' eggs. Allantoic fluid was aliquoted and stored at −70 °C until required. Two antigens were used in the immunological assays; concentrated influenza A/Sichuan/2/87 (H3N2) virus (10 mg/ml) (kindly supplied by Diane Major, NIBSC, South Mimms, UK) and a NP preparation (kindly provided by Medeva, Liverpool, UK). Both antigens were titrated by ELISA to find the optimum coating concentration.

Mice Eighty BALB/c mice (6 weeks old) were purchased from Bomholt Gaard (Copenhagen, Denmark) and housed according to the Norwegian law on the use of experimental animals. The experimental design is presented in Fig. 1. Mice were divided into four groups of 20 mice. Three groups of 20 animals per group were immunized three times i.m. into the quadricep muscles with 100 µg of HA, NP or control plasmid (50 µg per leg) diluted in sterile phosphate-buffered saline (PBS). Serum samples were collected pre- and 20–21 days after each dose of vaccine from the sapenous vein in the lateral side of the hind limb. Thirteen weeks after the third dose of vaccine, the mice were challenged by intranasal instillation of 25 µl mouse adapted A/Sichuan/2/87 (H3N2) virus (105.1 50% tissue culture infectious doses (TCID50)). An additional group of 20 mice was infected with 25 µl mouse adapted A/Sichuan/2/87 (H3N2) virus (106.1 TCID50) and was challenged 13 weeks after the initial infection with 25 µl mouse adapted A/Sichuan/2/87 (H3N2) virus (105.1 TCID50). Nasal wash samples were collected from each group of 16 mice at 2, 4 and 6 days post infection. Awake mice (nonanaesthetized) were restrained by holding the skin of the neck and the nasal cavity washed by administering 400 µl PBS containing 0.05% (w/v) bovine serum albumin (BSA) in a steady stream. The exhaled PBS–BSA was collected and stored at −70 °C until assayed for infectious virus in Madin Darby Canine Kidney (MDCK) cells. Mice were sacrificed (four individuals per group) on days 0, 7, 14, 21 and 28 days post viral challenge. Mice were anaesthetized using vival/hypnorm, exsanguinated by cardiac puncture and the blood was collected. The spleen, lung, femur and tibia bones of the hind limbs and palate were collected and lymphocytes were harvested from each tissue for use in the ELISPOT assay. Lymphocytes were isolated from the spleen on Lymphoprep (Nycomed Pharma, Oslo, Norway) by density gradient centrifugation at 800 × g. Lymphocytes were also isolated from the bone marrow [12], lungs [13, 14] and the nasal associated lymphoid tissue (NALT) in the palate [15].

image

Figure 1.  The experimental design. Twenty mice per group were immunized with 100 µg of haemagglutinin (HA), nucleoprotein (NP) or control DNA at 3-week intervals (black arrows) or alternatively infected (dashed arrow) at the same time as the third dose of vaccine. Serum samples were collected prior to vaccination and at 20–21 days after each dose of vaccine (lines). Mice were challenged intranasally with 25 µl mouse adapted A/Sichuan/2/87 (H3N2) 13 weeks after the third dose of DNA or infection. Four mice per group were euthanized at days 0, 7, 14, 21 and 28 after the viral challenge. Serum, spleen, bone marrow, lungs and nasal associated lymphoid tissue (NALT) were collected after euthanization. In addition, nasal wash samples were collected from 16 mice per group on days 2, 4 and 6 post challenge (not shown).

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Haemagglutination inhibition test (HI) Non-specific inhibitors were removed by treating the serum overnight at 37 °C with receptor destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) and then inactivated at 56 °C for 1 h. HI assays were carried out using eight haemagglutinating units of virus and 0.7% turkey red blood cells as previously described [16].

Enzyme linked immunospot assay (ELISPOT) The ELISPOT assay was used to investigate the influenza and NP-specific antibody secreting cell (ASC) response in the spleen, bone marrow, lungs and NALT as previously described [16], with the following exceptions. Plates were coated with 100 µl/well 10 µg/ml concentrated influenza virus or 1/1600 NP diluted in PBS overnight at 4 °C. The detector antibodies were biotinylated goat antimouse class (immunoglobulin (Ig)G; 1030–08, IgA; 1040–08, IgM; 1020–08, Southern Biotechnologies, Birmingham, AL, USA) and IgG subclass (IgG1; 1070–08, IgG2a; 1080–08, IgG2b; 1090–08, IgG3; 1100–08, Southern Biotechnologies) antibodies (2 µg/ml). The mean number of specific ASC per 500 000 lymphocytes was calculated for each individual mouse and the mean data of each group used to compile Table 1.

Table 1.   The systemic and local influenza and NP specific ASC after viral challenge
CompartmentTissueVaccineAntigenDays post challenge
07142128
  1.  Mice were immunized three times with 100 µg of HA, NP or control DNA at 3-week intervals or alternatively infected intranasally (infected) 13 weeks prior to viral challenge. The table refers to the mean number of total (IgG, IgA and IgM) influenza-specific ASC per 500 000 lymphocytes for each group where; (−) no detected ASC, (*) 1–5 influenza-specific ASC, (+) 5–20 influenza-specific ASC, (+ +) 20–50 influenza-specific ASC and (+ + +) > 50 influenza-specific ASC. Only influenza-specific IgA ASC were measured in the NALT. There were 3–4 mice in each group at each time point.

SystemicSpleenHAInfluenza++ ++ + +++ +
NP ++ ++ ++ ++ +
Control ++++
Infected +++++
HANP*+*+
NP ++ ++ ++ ++ +
Control ++**
Infected +++*+
Bone marrowHAInfluenza+++++
NP ++++ +
Control **
Infected + +++ ++ ++
HANP**+*+
NP +++ + +++
Control ****
Infected + ++ ++*+
LocalLungHAInfluenza*++**
NP *++ ++ ++
Control ++**
Infected *****
HANP++*
NP +*++
Control +*
Infected *
NALTHAInfluenza+ + ++ ++ + +
NP + + ++ ++
Control + + ++ ++
Infected ++

Virus neutralization assay Two-fold dilutions of serum were incubated with 100 TCID50 influenza virus for 1 h at 20 °C and residual virus infectivity was tested on MDCK cell monolayers prepared in 96-well tissue culture plates. After 3 days incubation at 35 °C in 5% CO2, the presence of virus in the supernatant was tested in a HA assay using 0.7% turkey red blood cells. The virus neutralization titres are expressed as the reciprocal of the titre required to neutralize 50% of infectious virus, calculated by the method of Reed and Muench [17].

Titration of nasal wash samples Nasal wash samples were collected 2, 4 and 6 days after viral challenge. Serial 10-fold dilutions of nasal washes were incubated for 72 h at 35 °C on confluent MDCK cell monolayers. The presence of replicative virus in the supernatants was determined by the HA assay and the virus titre of each nasal wash samples, expressed as TCID50 per ml, was calculated using the Karber equation [18].

Detection of influenza and NP-specific serum antibody titres by ELISA Ninety-six well ELISA plates were coated with 5 µg/ml concentrated A/Sichuan/2/87 (H3N2) or 1/1600 NP extract overnight at 4 °C. The ELISA was carried out as previously described by Cox et al. [16], with the exception of the use of 0.5 µg/ml goat antimouse class and IgG subclass biotinylated antibodies (as detailed under ELISPOT assay) for 1 h 45 min at room temperature and subsequent detection with 1/1000 dilution of Extravidin peroxidase antibody for 45 min. The antibody titre was calculated by subtracting the background absorbance and using a linear regression of the log10 serum dilution against log10 absorbance. An absorbance reading of 0.200 was used as the cut-off point.

Statistics The students t-test was performed using SPSS version 10.0 for Apple Macintosh computer.

Results

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

The mice were divided into four groups each containing 20 mice per group. Animals were immunized three times at 3-week intervals with 100 µg HA, NP or control DNA, or alternatively infected with mouse adapted A/Sichuan/2/87 (H3N2) virus (10 times challenge dose of virus) (Fig. 1). These mice were subsequently challenged intranasally 13 weeks later with a low volume (25 µl) of mouse adapted A/Sichuan/2/87 (H3N2) (105.1 TCID50) whilst awake, which aimed to produce a limited upper respiratory tract infection.

Nasal wash virus

Nasal wash samples were collected from 16 mice per group at 2, 4 and 6 days post challenge and assayed for virus infectivity in MDCK cells. Virus was detected in all of the control mice at all time points, except no detectable virus was found in five mice (day 2) and one mouse (day 6) (Fig. 2). Significantly higher levels of virus were detected in the control group than in the other groups at 4 days post challenge (HA, P= 0.0023 and NP, P= 0.0028). No virus was detected in the nasal washes of any of the previously infected mice. Very low levels of nasal wash virus were detected in the NP- and HA-immunized groups, with one mouse and three mice, respectively, completely protected from infection.

image

Figure 2.  The nasal wash viral titres detected in DNA immunized or infected mice after viral challenge. Twenty mice per group were immunized three times with 100 µg of HA (▪), NP (▴) or control (•) DNA at 3-week intervals or alternatively intranasally infected (I, ×) 13 weeks prior to viral challenge. Nasal wash samples were collected from 16 mice per group at 2, 4 and 6 days post challenge and the presence of replicative virus tested in MDCK cells. The scatter plot shows the nasal wash viral titres of individual mice and the horizontal bar represents the mean. No nasal wash virus was detected in the previously infected group at any time point.

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Serum HI and virus-neutralizing (VN) antibody titres after viral challenge

Prior to viral challenge, serum HI and VN antibodies were only detected in the previously infected group and no increase in antibody titres was observed after viral challenge in this group (Fig. 3). In the HA-immunized group, no HI or VN antibodies were detected pre challenge (except HI antibodies in one mouse) but were found in two of the four mice at 7 days post challenge. The HI and VN antibody titres continued to increase up to 28 days post challenge in the HA-immunized group, and remained higher than in the NP vaccinated and control groups throughout the study period. The HI antibody was detected at 14 days post challenge in the NP and control mice; however, only one mouse in the NP group had a low VN antibody titre at this time point. The HI and VN antibody titres continued to increase up to 28 days post challenge in the control group. In contrast in the NP immunized mice, the highest HI titres were detected 21 days post challenge and only very low VN antibody titres were found throughout the study.

imageimage

Figure 3.  The HI titre (A) and VN (B) antibody titres detected after intranasal viral challenge.Twenty mice per group were immunized three times with 100 µg of HA □), NP ▪) or control bsl00036) DNA at 3 week intervals or alternatively infected intranasally bsl00077) 13 weeks prior to viral challenge. The HI titre is presented as the geometric mean titre (GMT) ± standard error of the mean (SEM). The mean VN titre ± SEM is shown. Four mice per group were euthanized at days 0, 7, 14, 21 and 28 after viral challenge. nd: not detected.

The class and IgG subclass of serum antibody induced after viral challenge

The class and IgG subclass of serum antibody titres were examined by ELISA using two different antigenic formulations, whole influenza virus (Figs 4A and 5A) and purified NP (Figs 4B and 5B).

imageimage

Figure 4.  The influenza (A) and NP (B) specific serum IgG antibody response after vaccination and viral challenge. Mice were immunized three times with 100 µg of HA (□), NP (▪) or control (bsl00036 DNA at 3-week intervals or alternatively infected intranasally (bsl00077 13 weeks prior to viral challenge. Serum was collected from 20 mice per group pre, post 1st, 2nd and 3rd doses of DNA vaccine and then from 4four mice per group at days 0, 7, 14, 21 and 28 after viral challenge. The bars are the mean titre ± SEM. nt: not tested.

image

Figure 5.  The serum IgG1 and IgG2a antibody response to influenza virus (A) and NP (B) after viral challenge. Mice were immunized three times with 100 µg of HA, NP or control DNA at 3-week intervals or alternatively infected intranasally 13 weeks prior to viral challenge. Serum was collected from four mice per group at days 0 (□), 7 bsl00170), 14 bsl00077), 21 bsl00036) and 28 bsl00001) after viral challenge. The bars are the mean titre ± SEM. nt: not tested.

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No specific antibody was detected prior to vaccination in any of the groups (Fig. 4). ELISA antibody titres increased after each dose of vaccine in the HA- and NP-immunized groups to influenza and both influenza and NP, respectively, but no antibody was detected in the control group until after challenge. Higher specific IgG titres than IgM were detected after vaccination in the HA (influenza specific) and NP (influenza and NP specific) DNA-vaccinated groups (results not shown). The previously infected mice had significantly higher IgG antibody titres at days 0 and 7 than the HA (P = 0.05 and P= 0.001, respectively) and NP DNA immunized mice (P = 0.001 and 0.022, respectively). After viral challenge, no increase in antibody titres was found in the previously infected mice, whereas the antibody titres continued to increase in the DNA-vaccinated mice.

The specific IgG response consisted predominantly of IgG2a in the HA and NP DNA immunized mice after vaccination (Fig. 5A,B), but in the previously infected mice similar IgG1 and IgG2a antibody titres were detected. After viral challenge both subclasses increased in the HA and NP DNA immunized mice, however, the antibody response continued to be skewed to IgG2a to both the influenza and NP antigens. In the control mice, similar titres of IgG1 and IgG2a were observed to both antigens from 14 days post viral challenge.

Antibody-secreting cell (ASC) response after viral challenge

The ELISPOT assay was used to investigate the ASC response after viral challenge to whole influenza virus and purified NP (Table 1). The ASC response was investigated in the systemic compartment (spleen and bone marrow) and the local compartment (NALT and lungs, representing the upper respiratory tract [19] and lower respiratory tract, respectively). The total (IgG, IgA and IgM) response was examined in the spleen, bone marrow, and lungs whereas only the IgA response was investigated in the NALT owing to the low numbers of cells harvested from the NALT.

Systemic response

In the previously infected group higher numbers of bone marrow and splenic ASC were found prior to challenge, but no increase in numbers of systemic ASC was observed after viral challenge (Table 1). Low numbers of systemic influenza and NP-specific ASC were detected prior to challenge in HA- and NP-vaccinated groups and an increase in the number of splenic ASC was observed after viral challenge in both groups. The only increase in bone marrow specific ASC was observed on day 14 in the NP-immunized group. No specific ASC were detected prior to challenge in the control group and only low numbers were found after viral challenge. The number of influenza-specific ASC was highest in the HA-immunized group and higher numbers of NP-specific ASC were detected in the NP-immunized group, whereas similar numbers of influenza and NP-specific ACS were found in the previously infected and control groups.

Local response

Low numbers of influenza-specific IgA ASC were only found prior and at 7 days post viral challenge in the NALT of previously infected mice (Table 1). In all other groups, the number of ASC peaked at day 7 in the NALT, with one mouse in the HA-immunized group having a very high number of ASC (575 specific ASC per 500 000 lymphocytes). The DNA immunized and control groups followed a similar time course of appearance of specific ASC in the NALT, and generally the numbers of influenza-specific ASC were similar in the NP-immunized and control groups. No, or very low, numbers of specific ASC were detected in the lungs pre- and post-viral challenge, except influenza-specific ASC on days 14 and 21 after challenge in the NP-immunized group (Table 1). The low numbers of lung ASC observed after viral challenge confirms that the challenge dose of virus resulted in an upper respiratory tract infection.

Class and IgG subclass distribution of ASC

Prior to viral challenge, the ASC were mainly specific IgM in the HA and NP DNA-immunized groups whereas similar numbers of IgG, IgA and IgM ASC were found in the infected group (results not shown). After viral challenge, influenza-specific IgG and IgA predominated in the HA and NP DNA-vaccinated mice, in contrast control mice had mainly IgM. In the NP immunized and the previously infected groups, higher numbers of NP-specific IgG and IgA ASC were found. In contrast, a mainly IgM response was detected to NP in the control and HA-immunized groups. In the previously infected and control groups the numbers of IgG1 and IgG2a were similar, whereas in the HA and NP DNA-vaccinated mice IgG2a was the main IgG subclass.

Discussion

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

Influenza DNA vaccines encoding a number of antigens (including HA and NP) have been shown to protect against lethal viral challenge [3–8]. In our study we have examined the humoral response after viral challenge using a nonlethal dose of virus that produced a self-limiting upper respiratory tract infection. We aimed to produce an upper respiratory tract infection by using a low challenge volume of a virus (25 µl) using a nonlethal mouse adapted viral strain.

We immunized mice three times at 3-week intervals in DNA encoding the HA or NP genes. We originally conducted dose-response studies (results not shown) and found that 100 µg HA or NP DNA induced the highest antibody titres, in agreement with others [5, 9]. The antibody response increased after each dose of HA or NP vaccine and consisted predominantly of IgG characterized by an IgG2a subclass response. Both the route of DNA immunization and the nature of the antigen have been shown to influence the Th1/Th2 response and consequently the IgG subclass distribution [20]. Intramuscular vaccination generates a predominant Th1 response with production of Th1 cytokines (IL-2 and interferon (IFN)-γ) and IgG2a [9, 10, 20, 21], whereas use of the gene gun results in production of IgG1 and a more mixed Th1/Th2 cytokine profile [20, 21]. The presence of unmethylated CpG motifs in the plasmid backbone also promotes a Th1 response [22–24]. In contrast, conventional protein vaccination generally results in a Th2 response [9] and natural viral infection activates the Th1 arm [9, 25]. The inclusion of relevant cytokine genes in combination with the plasmids expressing antigen has been used to modify the Th response [26–29]. In our study, the IgG subclass was further driven towards IgG2a in the HA- and NP-vaccinated groups after viral challenge, suggesting an increased polarization towards the Th1 arm after challenge. In contrast, the IgG2a to IgG1 ratio remained constant after viral challenge in the control and previously infected groups.

We found that previous infection with homologous virus produced sterilizing immunity, manifested by complete protection from viral challenge (no nasal wash virus, no increase in numbers of ASC and no boost in serum antibody titres after viral challenge). The generation of sterilizing immunity in these mice is probably due to the short time period between infection and challenge. Priming with HA or NP DNA resulted in virtually no viral replication in the upper respiratory tract. The previously infected mice were the only group who had HI and VN antibody titres prior to challenge and no increase in these titres was observed after the challenge. Despite the fact that no HI antibody titres (except for one mouse) were found before the viral challenge in the HA-immunized group, much higher HI and VN titres were observed in this group than in the NP or control groups indicating an activation of memory B cells generated by HA DNA vaccination. Similarly, vaccination of mice [9] or pigs [30] with HA DNA primed for higher HI antibody responses after viral challenge despite no or very low HI titres prior to challenge. Repeated immunization with HA DNA has been found necessary in order to generate a persistent Th1 response [9]. In agreement with our results, no or only very low HI titres were detected after two doses of 100 µg DNA vaccine, whereas significantly higher HI antibody titres were observed after infection or a single dose of inactivated protein vaccine [9]. In contrast, Deck et al. [5] have found high HI titres induced after immunization with HA DNA constructs probably as a result of differences in the constructs and perhaps more effective immunization protocols.

Specific ASC were maintained systemically in the bone marrow and spleen after intranasal infection or intramuscular immunization with HA or NP DNA and were reactivated, particularly in the spleen, upon viral challenge of HA or NP DNA-immunized mice. Gene gun immunization with HA DNA has previously been shown to result in both initial and long-term maintenance of influenza-specific ASC in these tissues [31, 32]. In contrast, we found no reactivation of ASC in the systemic compartment after viral challenge of previously infected mice.

The nasal mucosa is the initial site of contact of airborne pathogens with the immune system of the host. Secretory IgA (SIgA) plays a pivotal role in protecting the respiratory tract from influenza infection [33, 34]. In rodents the only well organized mucosal associated lymphoid tissue in the respiratory tract is the paired lymphoid aggregates in the epithelium of the nasal tract, namely the NALT [15, 19, reviewed in 35]. The appearance of IgA ASC in the NALT is associated with the production of SIgA and viral clearance at the portal of entry of influenza infection [19]. We found low numbers of ASC in the NALT of the previously infected mice before challenge. No increase in ASC was found after viral challenge in these completely protected mice. In contrast, upon viral challenge of HA, NP or control DNA-immunized mice, the highest numbers of ASC were found at the site of the viral challenge (in the NALT) and the appearance of ASC followed a similar time course in all three groups. Generally, only very low numbers of ASC were found in the lungs after viral challenge in all groups, which is probably due to the viral infection being limited to the upper respiratory tract.

The viral challenge resulted in the control mice having a primary immune response producing mainly IgM ASC, whereas IgG and IgA were commonly found in the HA- and NP-vaccinated mice and the previously infected mice. The class switch from IgM to IgG has been previously been found to require CD4+ cells [32]. Both CD4+ and CD8+ T cells have a role to play in protection from lethal challenge after vaccination with NP constructs [10, 11]. Protection is mediated by humoral immunity after immunization with HA constructs [7] although recently a role for Th1 cells has been described in the absence of HI serum antibody [9].

An interesting recent development to enhance the immune response to DNA vaccines is the prime-boost strategy, which uses DNA as the priming vehicle and an attenuated virus vector as the boost. This strategy has been very effective when attenuated viruses are used as the boost, but has not been so effective when boosting with protein vaccines owing to the induction of much stronger humoral responses. However, in pigs priming with HA DNA vaccine followed by boosting with inactivated vaccine results in significantly enhanced protection from influenza challenge [30]. This potentially represents an interesting approach for the development of future influenza vaccines.

In conclusion, immunization with HA or NP DNA vaccines resulted in priming which upon viral challenge further resulted in a significant reduction in the viral replication. Our results, and those of others, show the possible role that DNA vaccines can have as a priming vehicle in the control of influenza virus, particularly NP DNA vaccines in a pandemic scenario.

Acknowledgments

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

We wish to thank Carolyn Nicolson and Diane Major at the National Institute of Biological Standards and Control (UK), Medeva (UK), Karl Brokstad at the University of Bergen, as well as the staff at the animal house (University of Bergen, Norway) for care of the mice. This work was supported by the EU Biotechnology programme (EU Bio4-CT96-0637).

This work was presented in part at the conference ‘Options for the Control of Influenza IV’, 23–28 September 2000, Hersonissos, Crete, Greece, as abstract number W62-5.

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