1 Department of Respiratory Medicine, Imperial College, St Mary's Campus, Norfolk Place, Paddington, London W2 1PG, UK
2 Institute of Medical Microbiology and Immunology, University of Copenhagen, Copenhagen, Denmark
Correspondence
Peter J. M. Openshaw
p.openshaw{at}imperial.ac.uk
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ABSTRACT |
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Present address: T-cellic, Hoersholm, Denmark.
These authors contributed equally to this work.
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INTRODUCTION |
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The mouse model has been used extensively to try to understand immunological and pathogenic effects of candidate RSV vaccines. Scarification of BALB/c mice with recombinant vaccinia virus (rVV) expressing the G, F or M2 proteins of RSV induces partial protection against RSV replication, but also leads to augmented disease during subsequent intranasal (i.n.) RSV challenge. The immune response induced and the pattern of vaccine-enhanced disease differ, depending on the protein used in immunization. rVV-G induces a TH2-biased response with enhanced disease that is characterized by an extensive pulmonary eosinophilic infiltrate; by contrast, scarification with rVV-F or rVV-M2 induces a TH1/TC1-dominated response that leads to augmented disease, characterized by a pulmonary infiltrate of mononuclear cells and PMNs (Openshaw et al., 1992; Alwan & Openshaw, 1993
).
CD8+ T cells exert antiviral effects against RSV in both humans (Isaacs, 1991) and mice (Kulkarni et al., 1993
). rVV-M2-scarified BALB/c mice mount a strong CD8+ T-cell response and develop transient antiviral immunity (Openshaw et al., 1992
; Kulkarni et al., 1995
). The induced CD8+ T cells are almost all specific for a single, H-2Kd-restricted peptide that corresponds to residues 8290 of the transcription anti-terminator protein M2 (M28290) (Openshaw et al., 1990
). RSV vaccination strategies that have been designed to induce M28290-specific CD8+ T cells include i.n. administration of a chimeric M2 peptide (Hsu et al., 1998a
), mucosal delivery of M28290 synthetic peptide with enterotoxin-based adjuvant (Simmons et al., 2001
) and minigene DNA vaccination (Hsu et al., 1998b
; Iqbal et al., 2003
). In the first two cases, a strong antiviral CD8+ T-cell response is associated with enhanced disease. Thus, as with the rVV-M2 vaccine, strong RSV-specific CD8+ T-cell responses accelerate virus clearance, but can also lead to fatal pulmonary disease enhancement. However, TH2-inducing vaccines also have immunopathogenic effects. It is therefore probable that an ideal (non-pathogenic) RSV vaccine should generate neutralizing antibodies and either a balanced TH1/TH2 response or a weak TH1/TC1 response.
DNA vaccination is an efficient way of inducing CD8+ T-cell responses, although responses are generally weaker than those induced by live vectors. However, DNA vaccines offer several advantages. They are simple to store and administer and generate endogenous synthesis of antigen, allowing encoded proteins to enter the major histocompatibility complex (MHC) class I presentation pathway and securing efficient induction of CD8+ T cells (Li et al., 1998). Moreover, the weak CD8+ responses that they induce may cause reduced immunopathology on RSV infection, but still potentially be protective. Previously tested RSV DNA vaccines include intramuscular (i.m.) immunization with DNA encoding the F or G proteins of RSV (Li et al., 1998
; Tripp et al., 1999
) and intradermal (i.d.) and i.n. immunization with DNA encoding the M28290 epitope (Hsu et al., 1998b
; Iqbal et al., 2003
). In all of these studies, the immunization regimes required large quantities of DNA (60100 µg). Gene-gun DNA administration allows effective immune responses to be induced with much smaller quantities of DNA (13 µg DNA per immunization). Gene-gun immunization with DNA encoding the F or G proteins of RSV has been shown to protect against RSV infection (Bembridge et al., 2000
). However, these authors found that gene-gun immunization was associated with an unwanted, TH2-biased response to RSV infection, particularly in mice that were immunized with an empty plasmid vector.
We have recently shown that gene-gun immunization with a single epitope induces protective immunity against lymphocytic choriomeningitis virus (LCMV) (Bartholdy et al., 2003). The constructs used encoded MHC class I-restricted epitopes that were linked covalently to human
2-microglobulin (
2m) and a murine leader of
2m inserted ahead of an LCMV epitope (to ensure translation in the endoplasmic reticulum). Notably, this construct reverses the bias towards a type 2 profile that is associated with some gene-gun immunization protocols, including those mentioned above (Pertmer et al., 1996
; Feltquate et al., 1997
; Bembridge et al., 2000
).
In the present report, we have assessed the vaccine potential of a DNA construct encoding the immunodominant epitope from the RSV transcription anti-terminator protein (M28290) linked to human 2m in BALB/c mice. We found that gene-gun administration of this DNA construct induced an M28290-specific CD8+ T-cell population that was smaller than that elicited by scarification with rVV-M2, but could be expanded rapidly after RSV infection and accelerated virus clearance from the lungs. However, gene-gun DNA vaccination also led to enhanced disease during RSV challenge. This was partly mediated by RSV-specific CD8+ T cells, but was also due to non-specific effects of gene-gun-administered DNA. These findings add to the multiplicity of mechanisms by which enhanced disease can occur in this model.
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METHODS |
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DNA vaccine construction.
The M22m and NP
2m vaccines comprised the eukaryotic expression vector pcDNA3.1/zeo+ (Invitrogen) containing the murine
2m leader followed by either the M28290 RSV peptide epitope or the NP118126 LCMV peptide epitope, respectively, tethered to human
2m by a 10 aa linker [(G3S)2GG]. The constructs were generated by using a similar construct as template, murine
2m leaderGP334110 aa linkerhuman
2m inserted as an NheI/NotI fragment in pcDNA3.1/zeo+. The GP3341 peptide sequence was exchanged for either the M28290 or NP118126 sequence by using PCR. A PCR product covering the last 28 bases of the leader (containing a HindIII site, the peptide, linker and human
2m) was generated by using the forward primer 5'-GCAGCCAAGCTTGACCGGCTTGTATGCTagttatataggatcaataaacaatataGGTGGTGGTAGTGGGGGA-3' for M28290 and 5'-GCAGCCAAGCTTGACCGGCTTGTATGCtaggccccaggcttcaggggtatatatgGGTGGTGGTAGTGGGGGA-3' for NP118126. The reverse primer 5'-GCAGCCGCGGCCGCTTACATGTCTCGATCCCACTTA-3' (containing a NotI site) was used for both constructs. PCR products, containing some of the vector, the leader sequence and the peptide sequence, the linker and human
2m terminated by a stop codon, were subsequently cloned as HindIII/NotI fragments into the template vector.
The M2 construct without human 2m was amplified from M2
2m by using a forward primer that was situated 200 bp upstream of the insert (5'-CTGCTTACTGGCTTATCGA-3') and a reverse primer that comprised some of the peptide sequence, a stop codon and an XbaI restriction site (5'-cctcgtctagattcaTATATTGTTTATTGATCC-3'). PCR products containing some of the vector, the leader sequence and the peptide sequence terminated by a stop codon were subsequently cloned as a HindIII/XbaI fragment into the template vector. Cells of Escherichia coli strain XL-1 Blue (Stratagene) were transformed with the constructs by electroporation. DNA sequencing using cycle sequencing, Big Dye Terminator and ABI 310 genetic analyser (ABI Prism) identified positive clones. Primers were obtained from Hobolth DNA Syntese. Large-scale DNA preparations were produced by using Qiagen Maxi Prep.
Gene-gun immunization.
DNA was coated on to 1·6 nm gold particles at a concentration of 2 µg DNA (mg gold)1. The DNA/gold complex was coated onto plastic tubes and 0·5 mg gold was delivered to the mouse per shot (1 µg DNA per shot). These procedures were performed according to the manufacturer's instruction (Bio-Rad). Mice were immunized on the abdominal skin by using a hand-held gene-gun device employing compressed helium (400 p.s.i.) as the particle-motive force. Unless otherwise mentioned, mice were inoculated twice at an interval of 3 weeks and then allowed to rest for 3 weeks before further challenge/investigation.
Virus infection.
Mice were lightly anaesthetized and challenged i.n. with 1x106 p.f.u. human RSV A2 strain in 100 µl. For vaccinia infection, mice were scarified on the rump with 3x106 p.f.u. (10 µl) rVV expressing the RSV M2 protein (rVV-M2) or the RSV G protein (rVV-G).
Depletion of CD8+ T cells.
Mice received 50 µl clarified ascitic fluid containing mAb CD8a 53-6.7 in 0·5 ml PBS intraperitoneally on days 1, 0, 2 and 5 relative to infection. Flow cytometry consistently showed <1 % CD8+ T cells in spleen, bronchoalveolar lavage (BAL), mediastinal lymph nodes (MLNs) and lung mash in depleted mice.
Cell recovery.
Mice were terminally anaesthetized with pentobarbitone and bled via the femoral artery. BAL was collected as described previously (Hussell et al., 1997). Lungs were inflated six times with 1·5 ml 12 mM lidocaine in Earl's balanced salts solution. Peritoneal cells were obtained by lavage with 5 ml ice-cold Hanks' balanced salts solution. Lungs and/or MLNs were removed aseptically and transferred to RPMI 1640 medium supplemented with 2-mercaptoethanol, L-glutamine and penicillin/streptomycin solution. Single-cell suspensions were obtained by pressing the organs through a fine sterile steel mesh. Cells were washed once and the cell concentration was adjusted in supplemented RPMI 1640 medium that contained 10 % fetal calf serum.
Flow cytometry.
Fluorescein isothiocyanate-conjugated rat anti-mouse CD44, peridininchlorophyllprotein complex (PerCP)- or phycoerythrin (PE)-conjugated anti-CD8a or CD8b and PE-conjugated anti-gamma interferon (IFN-) were purchased from Pharmingen. For visualization of peptide-specific, cytokine-producing CD8+ T cells, 2x106 MLN cells were incubated with MHC Kd-restricted M28290 RSV peptide or Ld-restricted NP118126 LCMV peptide at a concentration of 1 µg ml1 for 5 h in the presence of interleukin 2 (50 U ml1) and monensin (3 µM). After incubation, cells were stained for surface markers with directly labelled mAbs in staining buffer (1 % BSA, 0·1 % NaN3, 3 µM monensin in PBS) for 20 min in the dark at 4 °C, washed and fixed with 2 % formaldehyde for 30 min. Subsequently, cells were permeabilized in 0·05 % saponin, stained with cytokine-specific mAbs, washed and resuspended in staining buffer. Cells were analysed by using a FACSCalibur instrument (Becton Dickinson) and at least 104 cells were gated by using a combination of low angle and side scatter to exclude dead cells and debris. Data analysis was conducted by using Cell-Quest or WinMDI software.
Clinical severity of infection.
Mice were monitored daily for 7 days after i.n. RSV challenge. Weight loss was calculated as the weight on the indicated day relative to the initial weight.
Enumeration of lymphocytes, eosinophils and PMNs.
BAL fluid (100 µl) from each mouse was cytocentrifuged onto glass slides and stained with Giemsa's reagent for cytological analysis. Lymphocytes, eosinophils and PMNs were enumerated by microscopy. At least 300 cells per sample were counted.
Virus titration.
RSV titres were assessed in lung homogenates as described by Stott et al. (1987). Briefly, lungs were removed on day 4 post-infection (p.i.) and snap-frozen in liquid nitrogen. For analysis, lungs were thawed and homogenized. After centrifugation at 7000 r.p.m. for 2 min, supernatants were titrated in doubling dilutions on 6080 % confluent HEp-2 cell monolayers in 96-well, flat-bottomed plates. After 24 h incubation at 37 °C, monolayers were washed and fixed in absolute methanol for 20 min. The monolayer was subsequently incubated for 1 h at room temperature with biotin-conjugated goat anti-RSV antibodies (Biogenesis), washed and incubated with streptavidinhorseradish peroxidase for 30 min at room temperature. Infected cells were detected by using 3-amino-9-ethylcarbazole; infectious units were enumerated by light microscopy.
Statistical analysis.
A non-parametric MannWhitney rank sum test was used to perform comparisons between groups. P values of <0·05 were considered to be statistically significant.
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RESULTS |
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DNA vaccination induced signs of enhanced disease (ruffled fur and a hunched posture), as well as weight loss (Fig. 5) following RSV infection. We were surprised to find that disease severity in M2
2m DNA-vaccinated mice was similar to that in rVV-M2-scarified mice, despite reduced CD8+ T-cell responses. Similar disease severity was seen in M2 DNA-vaccinated, RSV-infected mice (data not shown). CD8+ T-cell depletion significantly reduced the severity of illness and weight loss in M2
2m DNA-immunized mice, but these mice still suffered more severe weight loss than non-immunized, RSV-infected mice. Even more surprisingly, NP
2m control-vaccinated mice suffered weight loss that was almost as severe as that suffered by M2
2m DNA- and rVV-M2-immunized mice, although it was delayed by 2 days.
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DISCUSSION |
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In this study, we investigated the protective capacity of a DNA vaccine-induced CD8+ T-cell response specific for the immunodominant H-2Kd-restricted epitope from the second matrix protein of RSV (M28290). This immune response was elicited by gene-gun DNA immunization using a construct encoding the M28290 epitope, linked covalently to human 2m and a similar construct without human
2m for comparison. DNA vaccination with the M2
2m construct induced an antigen-specific CD8+ T-cell memory response that was detected in the peritoneum by ICCS for IFN-
. The M2 DNA vaccine only occasionally induced vaccine-specific cells to the same extent. Splenic M28290-specific CD8+ T cells were below the detection limit when analysed by ICCS for IFN-
. However, restimulation of splenocytes with the M28290 peptide did give rise to in vitro CTL responses (data not shown). The fact that higher frequencies could be detected in the peritoneum than in the spleen is in agreement with the theory that primed cells are enriched in tertiary tissues (Masopust et al., 2001
).
Infection of M22m and M2 DNA-vaccinated mice with RSV gave rise to accelerated M28290-specific CD8+ T-cell responses when compared with control-vaccinated and unvaccinated mice. Thus, vaccine-primed cells were able to expand rapidly on encountering virus. Similarly accelerated responses were observed in the majority of mice that were infected 3 months after the last immunization, demonstrating that the vaccine-induced responses were long-lived. M2
2m DNA-vaccinated mice, but not M2 DNA-vaccinated mice, had significantly lower virus titres in the lungs 4 days after RSV infection, compared with control-vaccinated mice. This indicated that inclusion of human
2m augmented priming, consistent with findings in the LCMV model (Bartholdy et al., 2003
), and also showed that DNA vaccine-primed CD8+ T cells are capable of functioning in vivo. Thus, gene-gun immunization with very low doses of DNA managed to mediate significant control of RSV infection. The DNA vaccine-elicited, M28290-specific CD8+ T-cell response was weaker than that induced by rVV-M2 scarification. This was revealed by several observations. Firstly, lower frequencies of M28290-specific CD8+ T cells were found prior to infection in the spleen and the peritoneum, compared with rVV-M2-scarified mice. Secondly, protection against RSV infection was not complete, in contrast to that in rVV-M2-immunized mice. Thirdly, RSV-G-mediated TH2 responses measured by eosinophilia during RSV infection were not altered in M2
2m DNA-vaccinated mice, in contrast to previous findings in rVV-M2-vaccinated mice (Simmons et al., 2001
).
As a weaker CD8+ T-cell response was found after DNA vaccination than after rVV-M2 scarification, we expected to find less severe disease in DNA-vaccinated mice on subsequent RSV infection. However, similar weight loss was seen in M22m and M2 DNA- and rVV-M2-immunized mice that were infected with RSV. CD8+ T cells were only partly responsible for disease in M2
2m DNA-vaccinated mice, as anti-CD8 antibody treatment did not completely abrogate weight loss and clinical symptoms. More surprisingly, mice that were vaccinated with a DNA vaccine encoding the immunodominant H-2Ld-restricted LCMV epitope NP118126 linked to human
2m had disease symptoms that were almost as severe as those suffered by M2
2m DNA- and rVV-M2-immunized mice, although clinical symptoms and weight loss were delayed by approximately 2 days.
Analysis of the cell infiltrate in BAL revealed that M22m DNA-vaccinated mice had few eosinophils after RSV infection. Instead, lymphocytes and PMNs were present, which is indicative of a TH1-biased response. However, in the absence of CD8+ T cells, an influx of eosinophils was detected in M2
2m DNA-vaccinated mice, supporting a role for CD8+ T cells in preventing eosinophilia. The increased number of eosinophils in these mice may also account for some of the weight loss.
Notably, no early influx of lymphocytes and PMNs and no eosinophils could be detected in NP2m DNA-vaccinated, RSV-infected mice. This eliminated the theoretical possibility that the vector encoded an unknown immunogen that stimulated cross-reactive, RSV-specific cells.
It has recently been reported that memory T cells to one virus can become activated during infection with an unrelated heterologous virus and may play a role in antiviral immunity and immunopathology (Chen et al., 2001; Welsh & Selin, 2002
). However, the lack of NP118126-specific CD8+ T-cell responses on days 4 and 7 post-RSV infection and the lack of accelerated virus clearance in NP
2m DNA-vaccinated mice suggested strongly that this was not the case in our studies.
Interestingly, and contrary to the above-described hypothesis, it was recently shown that in LCMV-immune BALB/c mice challenged with RSV, bystander recruitment of memory T cells actually impaired virus clearance and enhanced immunopathology (Ostler et al., 2003). The authors suggested that the presence of irrelevant, heterologous memory T cells in some way competed with RSV-specific cells for recruitment to the lungs. However, the frequency of irrelevant memory cells induced by our DNA vaccine was very low (0·050·12 % of epitope-specific CD8+ T cells) (Bartholdy et al., 2003
) and we did not see any difference in either the number of M28290-specific CD8+ T cells and the recruitment of lymphocytes to the lungs or virus clearance in NP
2m DNA-vaccinated, RSV-infected mice, compared with unvaccinated, RSV-infected mice (Figs 2 and 6
, and data not shown). Together, this indicated that unknown, CD8-independent factors account for disease in NP
2m DNA-vaccinated mice during RSV infection.
It has been observed in several studies that i.d. DNA vaccination favours the development of an immune response that is biased towards TH2 cytokine production (Pertmer et al., 1996; Feltquate et al., 1997
; Li et al., 1998
), which in the RSV model leads to severe immunopathology. Bembridge et al. (2000)
showed that gene-gun-vaccinated mice that were immunized with empty DNA vector had a TH2-biased immune response with increased numbers of eosinophils in the lungs on RSV infection, a phenomenon that was not seen in unvaccinated, RSV-infected mice.
M22m DNA vaccination, however, resulted in a TH1-biased response, despite the fact that the vaccine was given intradermally, and this was consistent with previous findings from the LCMV model (Bartholdy et al., 2003
). Thus, in contrast to the observations made by Bembridge et al. (2000)
, disease in NP
2m DNA-vaccinated, RSV-infected mice did not seem to be the result of TH2-mediated immunopathology that may be induced non-specifically during gene-gun immunization. Rather, non-specific components in the vector prime mice systemically, e.g. to produce more inflammatory cytokines on RSV infection. CpG motifs contained in the vector may be the cause of this difference, due to their non-specific immunostimulatory effects. Co-delivery of CpG motif-containing oligodeoxynucleotides has been shown to be able to shift TH2 immunity, primed by gene-gun DNA vaccination, towards TH1 immunity (Zhou et al., 2003
). We suggest that our DNA constructs encoded sufficient CpG motifs to shift the immune response towards TH1 on infection. In addition to the pcDNA 3.1 vector itself, the human
2m gene might be a good source of CpG. The priming of NP118126-specific CD8+ T cells might also contribute to biasing antiviral immunity towards a TH1 response. It should be noted that the non-specific immunopathology that we observed is not a general phenomenon, as mice that are similarly DNAvaccinated do not develop enhanced disease on systemic LCMV infection (unpublished observations).
In conclusion, several different factors seem to be able to cause pathology during RSV infection of DNA-vaccinated mice: firstly, RSV may induce some pulmonary cell damage by itself; secondly, vaccine-specific CD8+ T cells cause immunopathology by the killing of infected cells and/or production of proinflammatory cytokines; thirdly, in the absence of CD8+ T cells, a TH2 response is not suppressed and eosinophil recruitment to the lungs is enhanced; and, lastly, unknown factors associated with gene-gun DNA immunization may prime for RSV-induced disease. Thus, even though low doses of DNA are able to induce long-lived CD8+ T-cell memory responses that reasonably mediate control of the infection, improved constructs with fewer pathogenic effects are needed for this DNA vaccine approach to be beneficial for the host.
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ACKNOWLEDGEMENTS |
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Received 8 March 2004;
accepted 21 June 2004.