A bacteriophage lambda DNA vaccine expressing the small surface antigen (HBsAg) of hepatitis B was compared with Engerix B, a commercially available vaccine based on the homologous recombinant protein (r-HBsAg). Rabbits (five per group) were vaccinated intramuscularly at weeks 0, 5 and 10. Antibody responses against r-HBsAg were measured by indirect enzyme-linked immunosorbent assay, by limiting dilutions and by subtyping. Specific lymphocyte proliferation in vitro was also measured. After one vaccination, three of the five phage-vaccinated rabbits showed a strong antibody response, whereas no r-HBsAg-vaccinated animals responded. Following two vaccinations, all phage-vaccinated animals responded and antibody levels remained high throughout the experiment (220 days total). By 2 weeks after the second vaccination, antibody responses were significantly higher (P<0.05) in the phage-vaccinated group in all tests. After three vaccinations, one out of five r-HBsAg-vaccinated rabbit still failed to respond. The recognized correlate of protection against hepatitis B infection is an antibody response against the HBsAg antigen. When combined with the fact that phage vaccines are potentially cheap to produce and stable at a range of temperatures, the results presented here suggest that further studies into the use of phage vaccination against hepatitis B are warranted.
Hepatitis B virus is a major global health problem. There are thought to be 350 million chronic carriers of the virus worldwide (World Health Organisation, 2000). These chronically infected persons are at a high risk of developing cirrhosis of the liver and liver cancer, with 500 000–1.2 million dying of the virus every year (Mahoney, 1999). The disease is especially prevalent in many developing countries, including all of Africa, parts of South America and South East Asia. As a result of this significant health burden, in 1992, the World Health Organisation set a goal for all countries to incorporate childhood hepatitis B vaccination into their immunization programmes. This programme has been supported by both the Global Alliance for Vaccines and Immunization and the Vaccine Fund and has been largely successful. By 2008, 177 WHO member states (84%) included infant hepatitis B in their immunization schedules compared with 31 in 1992 (British Medical Association Web Site, accessed October 2010). However, although the recombinant hepatitis B vaccine is provided at a reduced cost in developing countries, it still costs $4.50 for a three dose schedule. This makes it more expensive than all of the other childhood vaccines recommended by the WHO Expanded Programme on Immunization combined (BCG, measles, three doses of diphtheria/tetanus/pertussis and four doses of oral polio vaccine). (World Health Organisation web site, accessed October 2010). In some countries, cost is a contributing factor that has prevented the inclusion of hepatitis B in infant immunization schedules (Mahoney, 1999; Lavanchy, 2004). Even in countries that already routinely vaccinate, reducing the significant burden of hepatitis B immunization would free up resources for other health care needs. The two most common vaccines used in the Western world are rH-B-Vax (Merck & Co.) and Engerix B (GlaxoSmithKline Biologicals, Belgium). Both of these vaccines are produced in yeast and only contain the recombinant, nonglycosylated small (or S) antigen of the virus.
In addition to the cost of the vaccine, a complete three-dose schedule is only 95% protective in healthy adults (Jilg et al., 1988), with rates of protection declining as low as 50% in older patients (World Health Organisation web site, accessed June 2010). Nonresponsiveness can be due to genetic predisposition (i.e. major histocompatibility complex haplotype), some chronic illnesses, immunosuppression brought on by concomitant infection or due to life-style (Sjogren, 2005). The degree of responsiveness is also dependent on age, gender, number of doses and dose levels (Jilg et al., 1988, 1989). There is evidence to suggest that DNA vaccination may be able to raise protective antibody responses in some cases where protein vaccination is not effective (Schirmbeck et al., 1995). However, it is recognized that standard plasmid-based DNA vaccination can give rise to relatively low antibody levels, especially in animals larger than mice (Liu & Ulmer, 2005), and there are no DNA vaccines currently available for any disease in humans. As of June 2010, http://www.clinicaltrials.gov lists three trials for hepatitis B DNA vaccines, although all are for the treatment of the chronic disease, where cellular responses are more important than in prophylactic vaccination.
Several methods have been tested for improving responses against DNA vaccines (Lemieux, 2002; Abdulhaqq & Weiner, 2008). We have shown previously that bacteriophages (or phages – viruses of bacteria) can be used to deliver DNA vaccines (Clark & March, 2004a). In this technique, a DNA vaccine expression cassette, consisting of a eukaryotic promoter, vaccine gene and polyadenylation site, can be cloned into phage λ and purified whole phage particles used to immunize the host. Using this method, we have demonstrated antibody levels significantly higher than with standard plasmid-based DNA vaccination in mice and rabbits with HBsAg and other antigens (Clark & March, 2004b; March et al., 2004, 2006). Lambda phage particles expressing heterologous genes from eukaryotic expression cassettes have also been used for tumour therapy in a mouse model (Ghaemi et al., 2010), while filamentous phages have been used as DNA vaccine delivery vehicles against human syncytial virus (Hashemi et al., 2010). To achieve a more meaningful comparison of immune responses against HBsAg, we have compared immunization with a phage vaccine (λHBs) expressing the hepatitis B surface antigen to immunization with a protein vaccine (Engerix B, GlaxoSmithKline Biologicals) containing recombinant HBsAg in rabbits. The Engerix B vaccine was used according to the manufacturer's instructions, following the accelerated vaccination schedule and compared with vaccination with λHBs following an identical timetable.
Materials and methods
Construction of vectors
The phage λHBs was based on the plasmid pRcCMV-HBs(S), obtained from Aldevron. The expression cassette contained in this plasmid expresses the small HBsAg antigen. The entire plasmid was digested with MfeI (a single cut in a noncoding region that yields EcoRI compatible ends) and cloned into the EcoRI site of purified λgt11 (Young & Davies, 1983) genomic DNA. Phage DNA was then packaged in vitro (Packagene® Lambda DNA packaging system, Promega) before standard amplification and purification.
Preparation of phage vaccines
λHBs was amplified on Escherichia coli strain LE392 (Murray et al., 1977), and then purified and concentrated, using standard microbiological techniques, as described previously (Clark & March, 2004b). Briefly, an overnight infected culture was treated with DNase and RNase, before NaCl was added, and debris were removed by centrifugation. Phages were then precipitated by polyethylene glycol (PEG), pelleted by centrifugation and resuspended. Chloroform extraction was used to remove PEG and cells debris before the aqueous phase was unltracentrifuged to pellet pure phage particles. Phage were resuspended in SM buffer (50 mM Tris-HCl, pH 7.5, 100 mM sodium chloride, 8 mM magnesium sulphate, 0.01% gelatine), the standard buffer for phage manipulations unless otherwise stated.
Immunization of rabbits
Rabbits (New Zealand White strain; n=5) treated with bacteriophage vaccines were given 200 μL λHBs intramuscularly in SM buffer at a concentration of 2 × 1011 phage mL−1 (4 × 1010 phage per rabbit). Control rabbits (n=2) were given the phage vector (lacking the vaccine insert) at the same dose. Rabbits (n=5) treated with the commercial protein vaccine (Engerix B, GlaxoSmithKline Biologicals) were given 200 μL of the vaccine per dose. A 1 mL vaccine dose is recommended for a fully grown adult. Vaccinations occurred at weeks 0, 5 (day 33) and 10 (day 68). This is in accordance with the rapid immunization schedule given in the pack insert provided with the Engerix B vaccine. Bleeds were collected on days 0, 12, 33, 47, 68, 82, 103, 124, 180, 194, 209 and 220. Throughout the course of the experiment, animals were monitored for signs of inflammation at the site of injection, fever and other signs of distress.
Measurement of antibody responses
Antibody responses against recombinant HBsAg (Aldevron) or bacteriophage λ coat proteins were measured by indirect enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated overnight in 0.05 M sodium carbonate buffer at pH 9.6 with either 100 ng of purified HBsAg or 109 bacteriophage in 100 μL volume per well. Coating buffer was then removed and 200 μL per well blocking buffer [5% Marvel dry skimmed milk in phosphate-buffered saline (PBS)–Tween (140 mM NaCl, 3 mM KCl, 0.05% Tween 20, 10 mM phosphate buffer, pH 7.4)] was added for 30 min at 37 °C. Blocking buffer was then removed and primary antibody (i.e. rabbit serum) was added at a dilution of 1 : 50 to triplicate wells in blocking buffer at 100 μL per well and plates were incubated overnight at 4 °C. Plates were then washed five times in PBS–Tween and anti-rabbit horseradish peroxidase-labelled secondary antibody (Dako) diluted in blocking buffer was added for 1 h at 37 °C at the manufacturer's recommended dilution (usually 1 : 2000). In experiments to measure antibody subtypes, the secondary antibody was immunoglobulin G (IgG), IgM or IgA specific. Plates were then washed five times in PBS–Tween and 200 μL per well substrate (Sigma Fast-OPD tablets) was added and the plates were developed for 15 min in the dark. The reaction was stopped by the addition of 50 μL per well 3 M H2SO4 and the OD was read at 492 nm.
Limiting dilutions of rabbit antisera
To quantify comparative antibody levels, serial dilutions of primary antisera from groups 1 and 2 (protein and phage vaccines) were performed. ELISAs were carried out as described in the previous section, but for the primary antibody, twofold dilutions of serum were performed in triplicate across 10 wells of an ELISA plate. For weeks −2 to 5, an initial dilution of 1 : 25 was used, yielding dilutions of 1 : 25, 50, 100, 200, 400, 800, 1600, 3200, 6400 and 12 800. For weeks 7–18, an initial dilution of 1 :100 was used, yielding dilutions of 1 : 100, 200, 400, 800, 1600, 3200, 6400, 12 800, 25 600 and 51 200. Serum from a previous rabbit experiment that was known to have high anti-HBsAg titres was used as a positive control at a 1 : 100 dilution. Serum from a prebleed, also at a dilution of 1 : 100, was used as a negative control. Controls were included on each plate and limiting dilution endpoint values were taken as two times the value of the negative control well.
Preparation of peripheral blood mononuclear cells (PBMCs)
Blood (5–10 mL) was extracted from each rabbit (two rabbits per group) into a vacutainer containing sodium heparin (10 U mL−1). This was centrifuged at 900 g for 15 min at room temperature and the white buffy coat (found at the interface) was recovered and resuspended in 5 mL complete RPMI (Sigma-Aldrich, UK) (supplemented with final concentrations of 10% foetal bovine serum, 1.5 g L−1 sodium bicarbonate, 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, 0.4 mg mL−1 G418, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin, 2.5 μg mL−1 amphotericin B and 100 μg mL−1 gentamycin). For RPMI+H heparin was added to RPMI at 10 U mL−1. Ficoll (8 mL) was then added before centrifugation at 600 g for 30 min at room temperature. The band (containing lymphocytes) was recovered and resuspended in 5 mL RPMI+H, centrifuged at 400 g for 10 min and resuspended in 10 mL RPMI+H wash medium. The wash was repeated before the final resuspension in 1 mL complete RPMI. Cells were then counted in a haemocytometer with nigrosin viability stain and diluted to 2 × 106 viable cells mL−1 in complete RPMI.
Lymphocyte stimulation assay (LSA)
Sterile antigens HBsAg (125 ng–1 μg per well) and whole phage particles (1.25 × 109–1010 per well), diluted in RPMI, were added in 100 μL volumes to 96-well tissue culture plates. PBMCs (2 × 105 cells in 100 μL volume) were then seeded onto the antigen-containing wells. PBMCs were prepared from two randomly selected rabbits in each group. To yield sufficient cells to perform the assays, PBMCs from both animals in each group were pooled. All assays were performed in triplicate, with average values and SDs calculated. As a positive control, cells were cultured with concanavalin A (Sigma), which was present at a final concentration of 2.5 μg mL−1. Medium alone was used as a negative control. Plates were incubated in a humid 5% CO2 environment at 37 °C for 96 h, and then pulsed for 18 h with 18.25 KBq (1 μCi) [3H] thymidine (Amersham Biosciences, UK) per well. The cells were harvested using a Packard Filtermate Harvester onto glass fibre filters (Packard, the Netherlands), and activity was counted in a MicroBeta TriLUX direct beta counter (Perkin Elmer). Results were expressed as average counts per minute (±SD).
All data were expressed as means±SEM SEs. Statistical significance (P-values) was determined using Student's t-test. Results were considered significant with P<0.05 and highly significant with P<0.01.
Comparison of vaccines by ELISA
Figure 1 shows a comparison of HBsAg-specific antibody responses between the groups of rabbits vaccinated with the phage vaccine, λHBs and the commercial protein vaccine, Engerix B. Rabbits in the phage vaccine group showed significantly higher (P<0.01) responses at days 33, 47, 68 and 82, when compared with the protein-vaccinated group. Additionally, three of the phage-vaccinated rabbits showed a response with an OD >1 after one vaccination and all did after two vaccinations (Fig. 1c). Only one rabbit in the protein-vaccinated group showed a response >1 OD unit after two vaccinations and it took three vaccinations until four of the five rabbits showed this level of response (Fig. 1b).
LSAs on peripheral blood
LSAs were performed on two randomly selected animals from each of the bacteriophage and commercial vaccine groups. PBMCs were extracted as described, pooled for the two animals from each group and stimulated with either recombinant HBsAg or whole phage particles. The results of these LSAs are shown in Fig. 2. The results are plotted as raw counts. The stimulation indices (SIs) were calculated by taking the raw count at a specific time and dividing it by the value from the control (i.e. no antigen) value. Lymphocytes from rabbits vaccinated with either the phage or the commercial vaccines that were then stimulated with recombinant HBsAg antigen both showed an increase in counts, with maximal SIs of 3.45 (phage vaccinated) and 3.20 (protein vaccinated) (Fig. 1b). SIs in cells to which phage were added as a stimulating antigen (Fig. 2b) were significantly higher, reaching 34 in the phage-vaccinated group and 25 in the HBsAg recombinant protein-vaccinated group. The relatively high stimulation index observed in the cells extracted from animals vaccinated with recombinant protein, when stimulated with phage antigen, is due to the nonspecific immunostimulatory properties of the phage preparation. It is important to note that due to the fact that bleeds from two animals were pooled to generate enough material to perform this experiment, statistical analysis was not possible.
Limiting dilutions of phage antisera
In order to quantify antibody responses in vaccinated animals, limiting dilutions were performed on all rabbits. A value of twice that of a standard negative control serum (serum from a naïve rabbit) was used as the cut-off value. The results are shown in Fig. 3. Limiting dilutions confirmed the results from the standard ELISA, with responses from the phage-vaccinated group being significantly higher than the recombinant protein-vaccinated group (P<0.05) on days 47 and 68.
Subtyping of antibody responses
Specific secondary antibodies were used to subtype the antibody responses against the hepatitis B small surface antigen. Because of the limited availability of reagents for rabbits, only IgG, IgM and IgA levels were determined. For all groups, no significant IgA responses were observed and these results are not shown. IgG and IgM responses are shown in Fig. 4a and b. On day 47, 2 weeks after the second vaccination, both IgG and IgM responses were significantly higher (P<0.05) in the phage vaccine group, when compared with the Engerix B-vaccinated group.
The Engerix B hepatitis B vaccine is based on a recombinant HBsAg antigen produced in yeast. However, it is recognized that this recombinant protein is relatively poorly immunogenic and even four vaccinations do not protect 100% of patients (World Health Organisation, 2000). Immune responses to the vaccine vary considerably from person to person. For example, El-Sayed et al. (2009) found a 500-fold variation in antibody levels in a study involving 200 children. These antibody responses are similar to those seen in rabbits in this study when using the recombinant protein, with limiting dilution titres measured 2 weeks after the third vaccination ranging from 81 to 8000 in the Engerix B-vaccinated group (Fig. 3b). Responses in the phage-vaccinated group ranged from 3200 to 10 400 at the same time point (Fig. 3c).
DNA vaccination with a construct expressing HBsAg has been proposed as an alternative to vaccination with a recombinant protein (Davis et al., 1993). However, despite initially promising results in mice (e.g. Davis et al., 1993, 1995), as is the case with most other DNA vaccines, relatively poor immune responses in larger animal models have meant that at the time of writing, there are still currently no hepatitis B DNA vaccines that have been approved for use in humans (http://www.hepb.org/professionals/hbf_vaccine_watch.htm). Previously, we have shown that vaccination with whole lambda phage particles containing an expression cassette for the protective HBsAg antigen yields antibody levels that are significantly higher than those produced by vaccination with a naked DNA vaccine (Clark & March, 2004b; March et al., 2004). Here, we show that vaccination with a phage construct expressing the HBsAg antigen gives rise to antibody levels that are significantly higher than those produced after vaccination with Engerix B, a commercially available recombinant HBsAg protein vaccine. In all ELISAs performed in this study, whole Ig, IgG and IgM antibody responses are significantly higher in the phage-vaccinated group than the Engerix B group 2 weeks after the second vaccination (P<0.05 –Figs 1, 3 and 4).
It is possible that the differences in immune responses observed are in part due to differences in post-translational processing of the protein. In human cells, the S-protein is naturally monoglycosylated, but Engerix B is produced in yeast cells and this glycosylation does not occur (Block et al., 2007). Additionally, when HBsAg is synthesized in mammalian cells, it naturally forms virus-like particles, which are exported from the cell by extruding through the membrane and that incorporate lipid from the host cell. In yeast cells, these HBsAg particles are also released from the cells after synthesis of the antigen, but the lipid component will be derived from the yeast cell wall and may not resemble that found in a natural infection (Sonveaux et al., 1995). However, as the recombinant HBsAg protein used as an antigen in ELISAs and LSAs was produced in yeast, it is more likely to resemble the protein present in the Engerix B vaccine (which is also produced in yeast) than that produced after vaccination with the HBsAg bacteriophage vaccine; hence, it is likely that other factors are contributing to the differences in responses.
One other potential reason for the increased antibody responses measured after vaccination with λHBs when compared with the recombinant protein vaccine could be the adjuvant effect of the bacteriophage particles themselves. Several papers have been published that report on the immunostimulatory effects of unmodified bacteriophage particles (e.g. see Miedzybrodzki et al., 2005; Gorski et al., 2003 and references therein), due to the presence of CpG motifs on the foreign phage DNA or due to the virus-like, repeating peptide structure of the phage coat. Kleinschmidt et al. (1970), also observed the stimulation of interferon production after exposure of the innate immune system to phage particles. This nonspecific stimulation is apparent in LSAs (Fig. 2b), where naïve spleen cells stimulated with phage particles show the occurrence of nonspecific stimulation. It is possible that CpG motifs on the phage DNA are responsible for the improved antibody responses seen after phage vaccination in this trial. CpG motifs have been shown to stimulate a Th1 immune response in mice when delivered in conjunction with recombinant HBsAg (Malanchèrè-Brès et al., 2001), but more generally, they have also been shown to stimulate B-cell responses (Liang et al., 1996) resulting in increased antibody responses.
One other factor to consider when interpreting the results from this study is the level of purity of the phage preparations, particularly the level of lipopolysaccharide contamination present in the phage used. Lipopolysaccharide is recognized as a strong inflammatory modulator and a strong B-cell stimulator (e.g. Andersson et al., 1972) and will probably influence the immune responses observed in this study to some extent. However, there are several reports of lipopolysaccharide-free phage also causing immune stimulation due to the virus-like structure of the phage coat (Gorski et al., 2003; Miedzybrodzki et al., 2005) and CpG motifs in the phage DNA (Klinman, 2003) and it is possible that all three factors (lipopolysaccharide, CpG motifs and the repeating peptide motif of the phage coat) will contribute to the immune responses observed. Typically, using our current purification procedures, the dose given to rabbits in this trial would contain 500–2500 EU per dose – higher than currently allowed for human vaccines. However, none of the rabbits used in this study showed any signs of inflammation at the site of injection, or fever or other distress throughout the course of the experiment. This agrees with earlier research, where phages have been given to animals by a variety of routes, with no reported adverse reactions caused (e.g. see Clark & March, 2004a). This lack of inflammatory response/fever suggests that the role of lipopolysaccharide in generating the responses observed in this trial may be relatively minor.
The results presented here are preliminary, with further work needed to quantify and qualify immune responses in more detail. It should be noted, however, that the only correlate of protection measured to test whether immunity against hepatitis B has been achieved is a serum antibody responses against the small surface antigen (Yu et al., 2004; Plotkin, 2010); hence, the highly significantly increased immune responses presented here do suggest that further trials with the phage vaccine are merited.
Phage vaccination against hepatitis B potentially has several advantages over the standard recombinant-protein-based vaccination. Because of their relatively straightforward production on a prokaryotic host, they should be relatively cheap to manufacture. Following administration with a phage vaccine, the intracellular synthesis of vaccine protein should ensure that post-translational modifications occur correctly and that the viral envelope most closely resembles that found in a natural infection. The phage particles themselves are relatively stable at a variety of temperatures and can be freeze-dried for storage and transport (Jepson & March, 2004). To expand on the results presented here, animal experiments are currently being planned to examine the effect of dose (phages given per dose and number of doses), as well as the route of administration.
Here, we have shown that bacteriophage-mediated DNA vaccination gives rise to antibody levels in rabbits that are higher than those produced after vaccination with a commercially available recombinant protein vaccine, using one of the recommended delivery schedules. It is proposed that the increased antibody responses observed in phage-mediated vaccination are due to the inherent advantages of the phage delivery system, although further studies are required to show protection against challenge and the possible immunostimulatory effects of lipopolysaccharide contamination must also be ruled out.