Infection and Immunity Group, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland1
Division of Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UK2
Author for correspondence: Kingston Mills. Fax +353 1 7083845. e-mail kingston.mills{at}may.ie
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Abstract |
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Introduction |
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Immunization with plasmid DNA encoding influenza virus haemagglutinin (HA) is capable of inducing cell-mediated and humoral immunity and in protecting against intranasal challenge with homologous influenza virus (Fynan et al., 1993 ; Ulmer et al., 1994
; Webster et al., 1994
; Bot et al., 1997
; Deck et al., 1997
; Feltquate et al., 1997
). Furthermore, induction of class I-restricted CTL and protection of mice against heterologous virus challenge has been demonstrated with plasmid DNA encoding the nucleoprotein of influenza virus (Ulmer et al., 1993
). It appears that these vaccines selectively induce type 1 T cells, whereas protein vaccines induce Th2 or mixed Th1/Th2 responses. Although antibody responses have been reported after a single immunization with a DNA vaccine (Deck et al., 1997
), most studies with HA DNA have reported antibody responses only after a booster immunization (Fynan et al., 1993
; Ulmer et al., 1994
; Bot et al., 1997
; Feltquate et al., 1997
). Furthermore, the antibody response seen after a single immunization does not reach that observed with natural infection or with conventional influenza virus vaccines which are normally administered as a single dose (Bot et al., 1997
; Feltquate et al., 1997
). Therefore, the relative ability of DNA vaccines to generate cell-mediated and humoral immunity remains controversial, and the persistence of the immunity and the optimum dose and schedule to selectively prime distinct arms of the immune response remain to be defined.
In this study, we have examined the effect of the immunizing dose and schedule on the induction of cellular and humoral immune responses and the persistence of this response using DNA encoding the HA gene from influenza virus A/Sichuan/2/87 (H3N2). We demonstrate that HA DNA, like natural infection, selectively induces influenza virus-specific type 1 T cell responses, but the persistence of these responses and the induction of antibodies, is dependent on booster immunizations. Nevertheless, despite its limited ability to induce humoral immunity, the DNA vaccine could protect against intranasal challenge with live influenza virus.
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Methods |
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Plasmid construction and purification.
A/Sichuan/2/87 (H3N2) full-length HA was amplified by PCR and cloned into plasmid pI.17. pI.17 is a pUC-based plasmid carrying a bacterial origin of replication and ampicillin-resistance gene for growth and selection in Escherichia coli K12. It also carries a truncated enhancer region, full promoter and full Intron A gene from human CMV and a CMV terminator sequence. Plasmid DNA was purified using Qiagen endofree mega kits.
Immunization and infection of mice.
Mice were infected with approximately 5x105 50% tissue culture infective dose (TCID50) per ml of A/Sichuan/2/87 influenza virus (infectious allantoic fluid) by intranasal administration under light anaesthesia. Intramuscular (i.m.) injections of HA DNA in 125 µl PBS per mouse were given at four sites (both quadriceps and biceps). -Propiolactone inactivated A/Sichuan/2/87 influenza virus, had equivalent efficacy to commercially produced homologous influenza virus vaccines in animal and laboratory assays, and was used as the whole virus vaccine (15 µg HA per dose). This vaccine was also administered by the i.m. route. At the indicated time-points, mice were sacrificed by cervical dislocation and spleens, lymph nodes and blood samples were removed.
Generation of influenza virus-specific T cell clones.
T cell clones were generated from the spleens of individual BALB/c mice as described previously (Mills et al., 1986 ). Spleens were taken 14 days after i.m. immunization with 50 µg HA DNA. T cell lines were first established by culturing spleen cells (2x106 per ml) for 45 days with 0·8 µg/ml purified A/Sichuan/2/87 influenza virus in RPMI 1640 supplemented with 2% normal mouse serum at 37 °C with 5% CO2. After this period, 5 U/ml recombinant IL-2 (rIL-2) was added to cultures and cells were cultured for a further 7 days. Surviving T cells were then washed once and recultured at 1x105 cells per ml for 7 days with syngeneic irradiated spleen cells (2x106 per ml) as feeder cells. T cell lines were established by maintaining these cultures at 1x105 cells per ml in a 4 day feed/7 day starve cycle, by alternately culturing with 0·8 µg/ml purified influenza virus and 2x106 per ml APC (irradiated syngeneic spleen cells) or with 5 U/ml rIL-2.
T cell lines were cloned by limiting dilution at 1 cell per well in 200 µl volumes in 96-well plates in the presence of APC (2x106 per ml), virus (0·8 µg/ml) and rIL-2 (5 U/ml). A further 5 U/ml rIL-2 was added to cultures 5 days later. The clones were restimulated after a further 7 days incubation in 1 or 2 ml volumes in 24-well plates, and progressively expanded to 25 ml tissue culture flasks. T cell clones were maintained by restimulation at the initial concentration of 1x105 cells per ml with virus and irradiated APC every 10 days. The specificity of the T cell clones was tested against A/Sichuan/2/87 influenza virus using proliferation and cytokine assays.
Cytokine assays.
T cell cytokine production was assessed by culturing spleen cells or lymph node cells (2x106 per ml) or T cell clones (105 per ml) and APC (2x106 per ml) with purified inactivated virus at concentrations of 0·4, 4·0 or 12 µg/ml. Negative and positive control stimuli included medium alone and anti-CD3 (2·0 µg/ml) plus phorbol myristate acetate (PMA; 25 ng/ml), respectively. Concentrations of IFN-, IL-4 and IL-5 were measured by immunoassay using pairs of commercially available monoclonal antibodies (PharMingen) as described previously (Mills et al., 1998
).
Antibody assays.
The levels of serum antibody against influenza virus were determined by ELISA. Purified influenza virus (12·5 µg/ml) was used to coat plates. Bound antibodies were detected using alkaline phosphatase-conjugated anti-mouse IgG (Sigma). Antibody levels are expressed as the mean endpoint titre (±SD).
Haemagglutination inhibition (HI) antibodies were determined by assessing the ability of sera to inhibit agglutination of turkey red blood cells. Sera were incubated overnight at 37 °C with 4 volumes of receptor-destroying enzyme, prepared from Vibrio cholerae, in order to remove non-specific inhibitors. After inactivation of the receptor-destroying enzyme at 56 °C for 30 min, twofold dilutions of sera were incubated with approximately 106 TCID50 of A/Sichuan/2/87 virus. The assay was developed by adding 0·7% (v/v) turkey red blood cells and the HI titres read as the reciprocal of the highest dilution causing complete inhibition of agglutination.
Influenza virus challenge and virus titrations.
Mice were infected with 50 MID50 (50% mouse infectious dose) live influenza virus in PBS with 2% (w/v) BSA (approximately 105 TCID50). The virus was administered to non-anaesthetized mice in 50 µl volumes bilaterally by intranasal instillation. At intervals after challenge, nasal washes were performed using 0·5 ml PBS with 2% (w/v) BSA per mouse. Serial tenfold dilutions of nasal washes, prepared in serum-free Eagles minimal essential medium with TPCK-treated trypsin, were incubated for 4 days at 35 °C on confluent monolayers of MDCK cells in 96-well tissue culture plates. The presence of virus in each well was determined by incubation of 50 µl supernatant with an equal volume of 0·7% (v/v) turkey red blood cells. The virus titre of each specimen, expressed as TCID50 per ml, was calculated using the Karber equation (Hawkes, 1979 ).
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Results |
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Discussion |
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One approach to vaccine design is to attempt to achieve a response that mimics natural infection, without the risk associated with a live virus. We therefore investigated the type and persistence of the immune response generated with the DNA vaccine and compared these with the responses induced by natural infection with influenza virus. Our results suggest that a single intranasal challenge with influenza virus generates antigen-specific IFN--secreting T cells in the spleen and this confirms earlier reports that natural infection selectively induces Th1 responses in BALB/c mice (Feltquate et al., 1997
; Graham et al., 1998
). However, a more heterogeneous cytokine profile has been reported for T cell clones generated from spleens of influenza virus-infected C57BL/6 and CBA/Ca mice suggesting that the induction of both Th1 and Th2 cells is influenced by genetic background (Graham et al., 1998
). We observed a broadening to a mixed Th1/Th2 response in the draining lymph nodes 6 months after infection of BALB/c mice. Consistent with previous reports (Feltquate et al., 1997
; Bot et al., 1997
), we also demonstrated that immunization of BALB/c mice with HA DNA selectively primed systemic Th1 cells. Our studies extended these early reports by demonstrating that IFN-
-secreting influenza virus-specific T cells were detected in the spleen with a single dose as low as 0·01 µg DNA. In addition, CD4+ T cell clones generated from the spleens of immunized mice also secreted significant amounts of IFN-
in response to influenza virus, providing direct evidence for the generation of Th1 cells following immunization with HA DNA.
The potent induction of cell-mediated immunity may reflect endogenous expression of the antigenic protein either in muscle cells or professional APC after i.m. immunization. This may explain the induction of potent class I-restricted CTL with DNA vaccines (Donnelly, 1997 ), but not the selective induction of Th1 cells. Although there is no direct evidence of distinct processing pathways for Th2 cells, we have observed that vaccine delivery systems that allow protein antigens to enter the endogenous route of antigen processing also favour the induction of CD4+ Th1 cells (Moore et al., 1995
, 1999
). It is possible that the IFN-
produced by CD8+ T cells may exert a positive influence on Th1 induction, while suppressing the development of Th2 cells. In addition, certain bacterial immunostimulatory sequences, DNA sequences containing unmethylated CpG motifs, stimulate APC to express co-stimulatory molecules and, more importantly, to produce IL-12, which promotes the development of Th1 cells (Brazolot Millan et al., 1998
; Davis, 1998
; Klinman, 1998
; Krieg et al., 1998
; Chu et al., 1997
; Klinman et al., 1997
).
Despite a strong influenza virus-specific T cell response in the spleens 2 weeks following a single dose of HA DNA, multiple immunizations were required for persistence of the response. Furthermore, local T cell responses could not be detected in the local lymph nodes of mice immunized with HA DNA, whereas potent influenza virus-specific T cell responses were detected in the cervical lymph nodes from respiratory tract-infected mice. Recent studies have demonstrated a specific response in the draining lymph nodes following i.m. immunization with DNA (Torres et al., 1997 ; Casares et al., 1997
). However, it has also been suggested that following i.m. injection, the functional DNA appears to move as free DNA through the blood to the spleen where professional APC initiate responses (Robinson & Torres, 1997
). In a separate investigation, we found little or no response in the draining lymph nodes following immunization with HA DNA by four different routes (data not shown). However, a DNA construct encoding human immunodeficiency virus gp120 did elicit T cell responses in lymph nodes that drain the site of injection (J. Daly, P. A. Johnson and K. H. G. Mills, unpublished observations). It appears that, following i.m. immunization, the HA DNA preferentially migrates from the muscle to the spleen either as naked plasmid or within transfected APC. In support of this hypothesis, we detected plasmid DNA in the circulation and transfected APC in the spleen within 1 h of i.m. immunization with HA DNA (P. A. Johnson, M. A. Conway and K. H. G. Mills, unpublished observations). This novel finding that the site of immune activation may vary with different plasmid constructs has implications for the future design of vaccines based on this technology.
In contrast to the potent systemic cellular immune responses, only low concentrations of circulating anti-influenza virus antibodies were detected following a single immunization of HA DNA over a wide dose range. However, booster immunization with high doses of DNA did enhance serum antibody titres, and this was further augmented after a third immunization, but the levels remained below that observed following infection with influenza virus. Functional antibodies detected by HI assays were at or below the threshold of detection following two immunizations with 100 µg HA DNA, but were enhanced, compared with non-immunized mice, after influenza virus respiratory challenge. Although these data appear to contradict the conclusions on antibody induction in other reports on DNA vaccines encoding influenza virus HA, this can in part be explained on the experimental approach employed in the different studies. Previous studies have reported antibody responses after at least two and, in most cases, three immunizations. Our study, as well as that of Feltquate et al. (1997) , showed that conventional influenza virus vaccines, which are administered in a single immunization and a single exposure to the live virus induced antibody levels equal to or greater than that observed with three doses of 100 µg HA DNA.
It has already been reported that immunization of mice with HA DNA can confer protective immunity (Fynan et al., 1993 ; Ulmer et al., 1994
; Bot et al., 1997
; Deck et al., 1997
) and it has been concluded that protection is related to HI antibody induction (Deck et al., 1997
). However, it has also been suggested that memory B cells may be important in protection (Bot et al., 1997
) and our investigation is consistent with this conclusion. We observed that two doses of HA DNA vaccine provided excellent protection against challenge with live virus. Although HI antibodies were undetectable prior to challenge, the titres were higher in immunized mice compared with control mice after challenge, suggesting that the DNA vaccine had primed antigen-specific B cells in vivo which produced protective antibodies after challenge. Alternatively, the protection observed with the HA DNA vaccine may be mediated solely through the induction of cell-mediated immunity. This conclusion is consistent with reports which have demonstrated that mice that lack mature B cells and do not secrete immunoglobulin can clear an influenza virus infection from the respiratory tract (Topham et al., 1996
; Graham & Braciale, 1997
).
The present investigation has demonstrated that the humoral immunity induced with a DNA vaccine based on the HA molecule of influenza virus does not approach that observed either by influenza virus respiratory infection or immunization with a whole influenza virus vaccine. Nevertheless, HA DNA was capable of conferring high levels of protective immunity against respiratory infection with A/Sichuan/2/87 influenza virus. Although the protection induced surpassed that conferred with a whole virus vaccine, booster immunization was necessary with the DNA vaccine. Our demonstration that HA DNA was capable of selectively inducing Th1 cells, which mimics that generated following virus infection, point to a role for cellular immunity in protection.
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Acknowledgments |
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References |
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Received 28 January 2000;
accepted 10 March 2000.