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DNA Vaccines Expressing the Duck Hepatitis B Virus Surface Proteins

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DNA Vaccines Expressing the Duck Hepatitis B Virus Surface Proteins CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Virology 351 (2006) 159–169 www.elsevier.com/locate/yviro DNA vaccines expressing the duck hepatitis B virus surface proteins lead to reduced numbers of infected hepatocytes and protect ducks against the development of chronic infection in a virus dose-dependent manner ⁎ ⁎ Darren S. Miller a, , Ieva Kotlarski b, Allison R. Jilbert a,c, a Hepatitis Virus Research Laboratory, School of Molecular and Biomedical Science, University of Adelaide, North Terrace, Adelaide, SA 5005, Australia b Faculty of Health Sciences, The University of Adelaide, Adelaide, SA 5005, Australia c Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Adelaide, SA 5005, Australia Received 29 January 2006; returned to author for revision 21 February 2006; accepted 27 February 2006 Available online 19 April 2006 Abstract We tested the efficacy of DNAvaccines expressing the duck hepatitis B virus (DHBV) pre-surface (pre-S/S) and surface (S) proteins in modifying the outcome of infection in 14-day-old ducks. In two experiments, Pekin Aylesbury ducks were vaccinated on days 4 and 14 of age with plasmid DNA vaccines expressing either the DHBV pre-S/S or S proteins, or the control plasmid vector, pcDNA1.1Amp. All ducks were then challenged intravenously on day 14 of age with 5 × 107 or 5 × 108 DHBV genomes. Levels of initial DHBV infection were assessed using liver biopsy tissue collected at day 4 post-challenge (p.c.) followed and immunostained for DHBV surface antigen to determine the percentage of infected hepatocytes. All vector vaccinated ducks challenged with 5 × 107 and 5 × 108 DHBV genomes had an average of 3.21% and 20.1% of DHBV-positive hepatocytes respectively at day 4 p.c. and 16 out of 16 ducks developed chronic DHBV infection. In contrast, pre-S/S and S vaccinated ducks challenged with 5×107 DHBV genomes had reduced levels of initial infection with an average of 1.38% and 1.93% of DHBV-positive hepatocytes at day 4 p.c. respectively and 10 of 18 ducks were protected against chronic infection. The pre-S/S and the S DNAvaccinated ducks challenged with 5 × 108 DHBV genomes had an average of 31.5% and 9.2% of DHBV-positive hepatocytes on day 4 p.c. respectively and only 4 of the 18 vaccinated ducks were protected against chronic infection. There was no statistically significant difference in the efficacy of the DHBV pre-S/S or S DNA vaccines. In conclusion, vaccination of young ducks with DNA vaccines expressing the DHBV pre-S/S and S proteins induced rapid immune responses that reduced the extent of initial DHBV infection in the liver and prevented the development of chronic infection in a virus dose-dependent manner. © 2006 Elsevier Inc. All rights reserved. Keywords: Human hepatitis B virus; Duck hepatitis B virus; DNA vaccine; DHBV surface antigen; Humoral immunity; Cell-mediated immunity Introduction Conversely, those who resolve their HBV infection mount strong, multi-HBV epitope-specific humoral and cell-mediated The human hepatitis B virus (HBV) is the prototype member immunity (CMI) (Bertoletti and Naoumov, 2003). of the family Hepadnaviridae. More than 2000 million Vaccination of adults and children using the current individuals alive today have been infected with HBV. Of recombinant HBV surface antigen (HBsAg) vaccine produced these, ∼350 million people remain chronically infected, in yeast has been implemented in many countries (Maugh, resulting in significant rates of chronic liver disease, cirrhosis 1981). Vaccination with recombinant HBsAg elicits strong and in some cases, progression to primary liver cancer or humoral immune responses but only weak CMI. Approxi- hepatocellular carcinoma (Lavanchy, 2004). Individuals chron- mately 95% of vaccinated adults sero-convert achieving anti- ically infected with HBV generally have low to undetectable HBs antibody titers of >10 mIU per ml after two booster cytotoxic T lymphocyte (CTL) responses to HBV antigens. immunizations (Maugh, 1981). These antibodies provide protection against HBV challenge in a majority of recipients ⁎ Corresponding authors. Fax: +61 8 8303 7532. but provide no therapeutic effect in chronically infected E-mail addresses: [email protected] (D.S. Miller), patients (Michel et al., 2001). Vaccination with the recombi- [email protected] (A.R. Jilbert). nant HBsAg vaccine is not likely to be effective in all areas 0042-6822/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2006.02.037 160 D.S. Miller et al. / Virology 351 (2006) 159–169 of the world, as this vaccine is expensive and labile, requiring The effect of virus dose on infection outcome was also controlled temperatures for both storage and distribution. If recently demonstrated through use of the nucleoside analogue eradication of HBV is to be successful, future vaccine entecavir (ETV; now also known as baraclude); treatment of 14- strategies need to include countries with high endemicity of day-old ducks with ETV for 14 or 49 days did not prevent initial HBV infection and to concentrate primarily on infants DHBV infection of the liver but restricted the spread of virus because they commonly develop chronic HBV infection infection and prevented the development of chronicity (Foster et (Bertoletti and Naoumov, 2003). It is certain that a simpler, al., 2005). cheaper and more effective HBV vaccine needs to be In previous DNA vaccine studies, adult ducks were developed for infection to be controlled and/or eradicated in vaccinated with DNA vaccines expressing either the large a reasonable timeframe. envelope protein, pre-S/S (Rollier et al., 1999; Triyatni et al., In theory, vaccines based on DNA technology could provide 1998)orS(Triyatni et al., 1998). However, neither study the basis for the development of effective therapeutic anti-HBV determined if pre-S/S or S DNA vaccines provided more vaccine(s). DNA vaccines are relatively cheap and simple to effective protection against the development of chronic make because they require little downstream processing, they are infection since adult ducks readily clear DHBV infection stable at ambient temperatures and provide long-lasting resulting in low rates of chronic DHBV infection, even when protective immunity to HBV, woodchuck hepatitis virus inoculated with 2 × 1011 DHBV genomes (Jilbert et al., 1998). (WHV) and DHBV (Lu et al., 1999; Prince et al., 1997; Rollier In the Triyatni study, adult ducks vaccinated with a DHBV S et al., 1999; Triyatni et al., 1998). Unlike conventional sub-unit DNA vaccine had faster rates of removal of inoculated virus vaccines that typically generate strong humoral immunity but from the bloodstream following virus challenge and reduced weak CMI, DNA vaccines elicit both humoral and CMI, numbers of DHBV-infected cells compared to ducks vaccinated including the induction of antigen-specific CTL (Davis et al., with a DHBV pre-S/S vaccine (Triyatni et al., 1998). Although 1997) stimulated by the presentation of peptide fragments, the smaller S vaccine was not tested in the Rollier study, derived from endogenously synthesized and degraded antigen, in vaccination with a pre-S/S construct provided significant context with Major Histocompatibility Complex Class I (MHC I) protection (Rollier et al., 1999). Since this time there has been products (Davis et al., 1998). Further mechanisms underlying the considerable controversy as to which of the constructs provides response to DNA vaccines are the innate immune response to the greatest protective efficacy against DHBV challenge. immunostimulatory non-methylated CpG motifs unique to The only previous study that investigated the ability of DNA bacterial and viral DNA. The response to non-methylated CpG vaccines expressing DHBV surface proteins to induce immune DNA is mediated by Toll Like Receptor-9 (TLR-9), a member of responses in young ducks demonstrated that ducks injected a family of pattern recognition receptors involved in innate intramuscularly with a single 100 μg dose of DHBV pre-S/S immunity (Medzhitov et al., 1997). Upon recognition by TLR-9, DNA vaccine at 3 days of age developed detectable anti- an intracellular signalling cascade is activated resulting in the DHBsAg (anti-DHBs) antibodies approximately 4 weeks later. stimulation of B-cells, macrophages and dendritic cells, and However, these animals were not challenged with DHBV to test production of cytokines (Akira et al., 2001). the protective efficacy of the pre-S/S vaccine (Rollier et al., DNA vaccines designed to combat HBV infection are likely 2000a). In other studies, maternally transferred antibodies from to have the greatest impact if they induce protective immune DHBV pre-S/S DNA-immunized ducks protected offspring responses in young or newborn individuals. To date little work against DHBV infection (Rollier et al., 2000b) suggesting a key has been done to investigate if DNA vaccines elicit protective role for anti-DHBs antibodies in virus neutralization and immune responses in young animals. protection. DHBV infection of Pekin Aylesbury ducks provides a good More recently, we assessed the protective efficacy of whole model for studying vaccines and developing protocols aimed at cell vaccines expressing DHBV core antigen (DHBcAg) (Miller controlling HBVinfection in humans. In our previous studies, we et al., in press). In these studies, virus challenge led to DHBV defined the age- and dose-related outcomes of DHBV infection infection of similar numbers of infected hepatocytes in both the and showed that, like humans infected with HBV, young ducks vaccinated and non-vaccinated groups, confirming that anti- are highly susceptible to the development of chronic DHBV DHBc antibodies induced by the vaccine did not restrict DHBV infection (Foster et al., 2005; Jilbert et al., 1998, 1996; Meier et infection and that DHBcAg-specific CMI was responsible for al., 2003; Miller et al., in press). However, the susceptibility of targeting of infected cells and preventing the development of newly hatched ducks to the development of chronic DHBV chronic DHBV infection.
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