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Original Paper

Intervirology 2006;49:144–151 DOI: 10.1159/000089375

Testing of CpG-Optimized Protein and DNA Vaccines against the Hepatitis B Virus in Chimpanzees for Immunogenicity and Protection from Challenge

Paul J. Payette

a Xiaoying Ma

a Risini D. Weeratna

a Michael J. McCluskie

a Max Shapiro

b Ron E. Engle

c Heather L. Davis

a Robert H. Purcell

c

a Coley Pharmaceutical Canada, Ottawa , Canada; b

Bioqual Inc., Rockville, Md. , and c National Institute of Allergy

and Infectious Disease, Bethesda, Md. , USA

sidered protective ( 6 10 mIU/ml) and were indeed pro-tected from challenge at 8 weeks with 10 3.5 50% chimp infectious doses (CID 50 ) of intravenous HBV. The second approach was a DNA vaccine with a plasmid vector op-timized for content of immunostimulatory CpG motifs. Despite the fact that earlier studies had shown four dos-es of a similar DNA vaccine (except not optimized for CpG content) to induce strong humoral responses in 1 of 2 chimpanzees, in this study two doses of DNA vac-cine (at 0 and 4 weeks) did not generate any detectable anti-HBs in either of 2 chimpanzees, although it did pro-tect 1 that rapidly developed anti-HBs during the incuba-tion period, suggesting priming of an antibody response. The poor results may be due to an inadequate number of doses or amount of plasmid DNA in these larger ani-mals, but nevertheless point to the need to improve de-livery methods for DNA vaccines for use in larger ani-mals such as primates.

Copyright © 2006 S. Karger AG, Basel

Introduction

Infection with the hepatitis B virus (HBV) remains an important worldwide health concern despite the availabil-ity of effective prophylactic vaccines against hepatitis B

Key Words HBV vaccines � Plasmid DNA � CpG motifs � CpG ODN � Chimpanzees

Abstract Despite the existence for some time of effective prophy-lactic vaccines, hepatitis B virus (HBV) infection remains an important global concern. Improvements on existing vaccines could be benefi cial, especially in situations where it is desirable or necessary to induce protective immunity more rapidly or with fewer doses. We have compared, in chimpanzees, a current HBV vaccine that contains recombinant hepatitis B surface antigen HBsAg) adsorbed to alum, with two novel vaccine strat-egies that have proven superior to the current vaccine in mice. The fi rst approach was the use of oligodeoxynu-cleotides containing CpG motifs (CpG ODN) as an adju-vant to Engerix-B ® , a commercial HBV vaccine. The ad-dition of CpG ODN to Engerix-B greatly improved the kinetics and magnitude of the humoral response, sug-gesting that CpG ODN might allow induction of protec-tive immunity in humans more quickly and with fewer vaccine doses. All animals receiving either control or CpG-containing subunit vaccines at 0 and 4 weeks at-tained titers of HBsAg-specifi c antibody (anti-HBs) con-

Received: August 2, 2004 Accepted after revision: May 23, 2005

Dr. Heather L. DavisColey Pharmaceutical Canada340 Terry Fox Drive, Suite 200Kanata (Ottawa), Ontario K2K 3A2 (Canada)Tel. +1 613 254 5622, ext. 3120, Fax +1 613 254 5625, E-Mail [email protected]

© 2006 S. Karger AG, Basel0300–5526/06/0493–0144$23.50/0

Accessible online at:www.karger.com/int

CpG-Optimized HBV Vaccines Intervirology 2006;49:144–151 145

for over two decades [1] . Owing to the signifi cant morbid-ity associated with this disease, especially in those who become chronically infected, the World Health Assembly in 1992 endorsed the recommendations by the World Health Organization to implement universal immuniza-tion against HBV in all countries by 1997 [2] . Current prophylactic HBV vaccines are highly effective for vac-cination of healthy individuals, with a three-dose course of vaccination typically administered at 0, 1 and 6 months resulting in 90–95% of infants and young adults achieving protective immunity. However, there is still a need for improvement, particularly in cases where it is desirable to induce protective immunity more quickly or with fewer doses, as is the case for health care workers and those liv-ing in endemic areas [1, 3] . As well, the possibility to use fewer doses would make HBV vaccination more afford-able and practical to deliver in less developed areas of the world. Finally, a more immunogenic vaccine may also help reduce the incidence of hypo- or nonresponse to the current vaccines; this being 5–10% of young healthy indi-viduals and up to 50% or more of immune compromised populations such as renal dialysis patients [1, 3] . Adju-vants are frequently added to vaccines to make them more immunogenic. Current HBV vaccines contain hepatitis B surface antigen (HBsAg) adsorbed to an aluminum salt (alum) as an adjuvant. While alum has been shown to be a safe and generally very well tolerated adjuvant, it is rel-atively weak. A number of new adjuvants are being devel-oped based on mimicking pathogen-associated molecular patterns that activate the immune system through Toll-like receptors (TLR). CpG motifs are a type of pathogen-associated molecular patterns found in bacterial but not mammalian DNA that activate human B cells and plas-macytoid dendritic cells (pDC, DC2) through activation of TLR9 [4] . CpG motifs, which are most often adminis-tered within synthetic oligodeoxynucleotides (CpG ODN), also indirectly activate a number of other types of immune cells, and when combined with antigens, they strongly augment responses against a wide range of antigens [5] , including HBsAg [6–8] . The CpG effects that promote antigen-specifi c responses include the direct activation of B cells to secrete more antibodies, improved antigen pre-sentation by DC and secretion of Th1 cytokines that pro-vide CD-40 ligand-independent T help [4] .

Another novel vaccine approach that has shown prom-ise in animal models for rapid induction of strong Th1 immunity is DNA-based immunization. In this case, plasmid DNA encoding an antigenic protein is adminis-tered and an immune response occurs in response to in vivo antigen synthesis as well as to the adjuvant effects

of CpG motifs found in the plasmid backbone [9] . It is possible to increase the immunogenicity of such DNA vaccines in mice by molecularly inserting additional CpG motifs.

Herein we have evaluated two types of novel HBV vac-cines that had previously been shown to induce rapid and strong immunity in mice: (a) recombinant HBsAg/alum vaccine with CpG ODN added as a second adjuvant [8, 10] and (b) HBsAg-expressing DNA vaccines [10, 11] .

Materials and Methods

Protein Vaccine The protein-based vaccine used in this study was Engerix-B ®

(GlaxoSmithKline, Rixensart, Belgium), a commercially available HBV vaccine consisting of yeast-derived recombinant HBsAg (HBV Sad) protein adsorbed to alum, provided by aluminum hy-droxide (25 � g Al 3+ / � g antigen).

CpG ODN 2006, containing the primate-specifi c immuno-stim-ulatory sequence 5 � -TCGTCGTTTTGTCGTTTTGTCGTT-3 � synthesized with a nuclease-resistant phosphorothioate backbone to render it nuclease resistant (Coley Pharmaceutical Group Inc., Wellesley, Mass., USA), was suspended at a concentration of 10 mg/ml in sterile endotoxin-free PBS (Sigma-Aldrich, Oakville, Canada) and stored at 4 ° .

DNA Vectors The DNA vaccine used in this study, pCG-S, consisted of an

HBV S ( adw2 subtype)-expressing DNA vector based on a back-bone which had 52 CpG-neutralizing motifs removed by site-di-rected mutagenesis and 16 copies of the same 24-mer primate-spe-cifi c CpG ODN sequence as CpG 2006 (see above), for a total of 64 additional CpG motifs. This vector has previously been shown to induce strong HBsAg-specifi c antibody (anti-HBs) responses and cytotoxic T lymphocytes (CTL) in mice, and furthermore be more immunogenic than the same plasmid without extra CpG motifs added [10] . Plasmid DNA was produced by standard procedures and purifi ed on Qiagen anion-exchange chromatography columns (Qiagen Inc., Valencia, Calif., USA) with endotoxin removal step as per the manufacturer’s instructions, and redissolved in 0.15 M saline for injection. The DNA was verifi ed for purity and tested for endotoxin levels, then was stored at –20 ° .

Animals Nine juvenile chimpanzees (Pan troglodytes) derived from a

HBV-negative colony and weighing 24–33 kg were used in this study. The animals were located at Bioqual Inc. (Rockville, Md., USA), where they were housed individually in glass biocontain-ment suites and maintained according to American Association for Accreditation of Laboratory Animal Care (AAALAC) guidelines. The studies performed with these animals were approved by the animal care and use committees of Bioqual Inc. and of the Nation-al Institute of Allergy and Infectious Diseases (Bethesda, Md., USA), which operate under the guidelines of AAALAC, and of the Ottawa Hospital Research Institute, which operates under the guidelines of the Canadian Council on Animal Care.

Payette /Ma /Weeratna /McCluskie /Shapiro /Engle /Davis /Purcell

Intervirology 2006;49:144–151 146

Immunization Eight animals received i.m. injections at 0 and 4 weeks of:

(a) Engerix-B alone (10 � g); (b) Engerix-B combined with CpG ODN 2006 (1 mg), or (c) pCG-S (2 mg) at weeks 0 and 4, or(d) pCG-S (2 mg) at week 0 and Engerix-B plus CpG ODN 2006(1 mg) at week 4. Engerix-B was administered as a pediatric dose (0.5 ml containing 10 � g HBsAg, adw subtype) to better detect the adjuvant effects of CpG in these animals, which weighed less than half a typical adult human. There were two animals per group, with an additional single animal that received only saline injections at 0 and 4 weeks and served as a positive control for productive infec-tion following challenge of all of the animals at 8 weeks.

The protein-based vaccines and saline were injected into the right quadriceps muscle using a hypodermic syringe with a 23-gauge needle, and the DNA vaccines were injected through shaved skin into both quadriceps using the Biojecter needleless injection system (Bioject, Portland, Oreg., USA).

HBV Challenge At 8 weeks after the fi rst vaccine dose, each animal was chal-

lenged with 10 3.5 50% chimp infectious doses (CID 50 ) of MS-2, an HBV strain of ayw subtype [12] , administered intravenously as a single bolus. It should be noted that this is heterologous to the ad subtype used for both the subunit and DNA vaccines, thus any protection would be dependent on the dominant epitopes found in the ‘a’ region. Following challenge, the animals were evaluated for the development and clearance of a productive HBV infection.

Assay for Anti-HBs Titers Plasma levels of anti-HBs were assessed using the Monolisa 3.0

anti-HBs ELISA kit (Sanofi Diagnostics Pasteur, Montreal, Cana-da), which is based on HBsAg of the same isotype as used for both DNA and protein-based immunization, namely adw . Anti-HBs lev-els were expressed in mIU/ml by comparison with human-derived standards defi ned by the World Health Organization (Monolisa Anti-HBs Standards, Sanofi Diagnostics Pasteur). Titers consid-ered seroprotective have been defi ned as 6 10 mIU/ml.

Assay for HBsAg and HBeAg HBsAg in serum recovered from each animal was assessed using

the Auszyme Monoclonal enzyme-linked immunoassay kit (Abbott Laboratories, Abbott Park. Ill., USA). HBeAg was assessed using the Abbott HBe (rDNA) enzyme-linked immunoassay kit (Abbott Laboratories). The assays were performed as directed by the man-ufacturer.

Real-Time PCR Assay for HBV DNA Viral DNA was extracted from 200 � l of serum with a QIAamp

DNA Mini Kit (Qiagen) following the manufacturer’s recommen-dations. Nucleic acid was eluted with 200 � l of the kit AE buffer and stored at –80 ° .

The primer/probe set, located near the 5 � end of the S gene, was based on that described by Pas et al. [13] , and verifi ed with Primer Express (Applied Biosystems, Foster City, Calif., USA) software and the BioEdit suite of sequence alignment tools [14] . Each 50 � l reaction mixture contained 45 pmol of forward (5 � -GGA CCC CTG CTC GTG TTA CA-3 � , nucleotides 184–203) and reverse (5 � -GAG AGA AGT CCA CCA CGA GTC TAG A-3 � , nucleotides 273–249) primers, 12.5 pmol of probe (6FAM-TGT TGA CAA AAA TCC TCA CAA TAC CGC AGA-TAMRA, nucleotides 218–

247), 2X TaqMan Universal Master Mix (Applied Biosystems) and 10 � l of extracted sample.

PCR was performed in an ABI PRISM ® 7900HT Sequence De-tection System (Applied Biosystems). Conditions included 5-min incubation at 50 ° , 10-min incubation at 90 ° and 47 PCR cycles of 15 s at 95 ° with 1 min at 60 ° .

The Acrometrix Optiquant (Acrometrix, Benicia, Calif., USA) HBV Viral DNA panel was used to assess the performance and dynamic range of the TaqMan HBV DNA assay. In addition, the panel was used to construct a standard line from which quantities were determined. It consisted of 6 serum samples containing 2 ! 10 2 to 2 ! 10 7 IU/ml of HBV plus a negative control. The quanti-ties of HBV DNA were calibrated to the WHO international stan-dard (97/746) described by Saldanha et al. [15] .

Each test included duplicate, positive and negative and no-tem-plate controls. The threshold value was set at 0.20. The inter-assay precision (%CV) of standards ranged from 1–7% and the intra-as-say precision mean was 4%. The standard line R 2 fi t values ranged from 0.975 to 1.0 (%CV = 1). The quantifi able linear range of the assay is 10 2.3 to 10 7.3 .

Results

Anti-HBs Responses As mentioned above, an anti-HBs titer of at least

10 mIU/ml is considered necessary to confer protection against HBV infection [1] . The Engerix/CpG vaccine combination was the most immunogenic, achieving pro-tective antibody levels of 94 and 127 mIU/ml in the two animals only 2 weeks following the fi rst vaccine dose ( fi g. 1 ). In contrast, Engerix-B alone achieved antibody levels of only 11 and 6 mIU/ml at 4 weeks following the fi rst vaccine dose. All of these antibody levels were boosted by a second dose of vaccine at 4 weeks, but those with CpG remained higher (16,800 and 9,640 mIU/ml) than those vaccinated with the commercial vaccine alone (4,706 and 3,712 mIU/ml).

The animals receiving two doses of the HBsAg-express-ing DNA vaccine did not develop any detectable anti-HBs prior to challenge. The animals that received the DNA vac-cine prime followed by Engerix/CpG developed detectable anti-HBs antibodies only after the boost with the protein vaccine, to levels that were similar to those of animals re-ceiving their fi rst dose of Engerix/CpG, suggesting that the DNA vaccine may not have had a priming effect.

Challenge with HBV following Vaccination As expected, all animals that had developed a protec-

tive level of anti-HBs ( 6 10 mIU/ml) prior to challenge were protected ( table 1 ). Also as expected, chimpanzee 5872 (positive infection control) developed a productive HBV infection ( fi g. 2 ).


Neither of the two DNA-vaccinated chimpanzees had developed detectable anti-HBs before challenge. One of these animals (1573) appeared to be protected despite the lack of detectable prechallenge anti-HBs. This chimpan-zee developed protective levels of antibodies during the incubation period of hepatitis B much more rapidly than occurs in a naïve animal ( fi g. 2 ), suggesting that the DNA

vaccine had primed a humoral response, albeit undetect-able; it is further possible that the animal actually had a subclinical infection that boosted any priming that had occurred; however, this is unlikely since no HBV DNA was detectable ( fi g. 3 ) and there was no evidence that it had seroconverted to anti-HBc with weekly testing over 6 months, and was still negative even 2.5 years after chal-

- --

Fig. 1. Anti-HBs levels, in mIU/ml, generated by different vaccination strategies. The animals received injections at 0 and 4 weeks with either Engerix-B, Engerix-B + CpG ODN, HBs expressing DNA vaccine, or the combina-tion of the DNA vaccine at 0 weeks and Engerix-B + CpG ODN at 4 weeks. Anti-HBs levels were determined on plasma samples collected biweekly.

Vaccines Animals Weeks after challenge

0–5 6 7–11 12–14 15–18 19–24

Engerix-B + CpG 5960 –/–1 –/– –/– –/– –/– –/–Prime and boost 1581 –/– –/– –/– –/– –/– –/–

Engerix-B 5855 –/– –/– –/– –/– –/– –/–Prime and boost 1580 –/– –/– –/– –/– –/– –/–

DNA vaccine 1573 –/– –/– –/– –/– –/– –/–Prime and boost 1574 –/– +/– +/+ +/+ +/+ +/–

DNA vaccine prime 1578 –/– –/– –/– –/– –/– –/–Engerix-B + CpG boost 1579 –/– –/– –/– –/– –/– –/–

Infection control 5872 –/– +/– +/+ +/– –/– –/–

1 HBsAg/HBeAg (see text for details).

Table 1. HBsAg and HBeAg following challenge of vaccinated chimpanzees with hepatitis B virus


Intervirology 2006;49:144–151 148

Fig. 2. Response of chimpanzee 5872 (un-vaccinated) and chimpanzees 1574 and 1573 (DNA vaccine) to challenge with HBV. Serology is expressed as ratios of sam-ple OD to cutoff OD. Values of 6 1 are pos-itive.


lenge when last tested. Furthermore, it is unlikely that there was a failure of the viral challenge to establish infec-tion considering the high dose of 10 3.5 CID 50 of the infec-tious virus [16] , the consistent infectivity of this challenge pool in many previous studies [12] , and the fact that two other animals challenged at the same time with the same inoculum did develop productive infections ( fi g. 3 ). The other DNA-vaccinated chimpanzee (1574) became in-fected and appeared to have a more prolonged and severe course of infection than the saline treated control.

Discussion

Despite the clear effi cacy of current HBV vaccines, it would be highly advantageous for use in both developed and less developed areas of the world to have a two-dose regimen that could induce seroprotective responses in a high proportion of individuals within weeks rather than months. We have evaluated this possibility in chimpan-zees using the novel adjuvant CpG ODN combined with a commercial HBV vaccine and/or a CpG-optimized DNA vaccine expressing HBsAg.

Early mouse studies on DNA vaccines consistently found that they were superior to protein-based vaccines for both kinetics and magnitude of response, including for HBsAg [11] , and this suggested much promise for their ability to rapidly induce protective immunity in hu-mans, and possibly even allow the development of a sin-

gle-dose vaccine for use in developing areas of the world. Unfortunately DNA vaccines have not proven to be as effective in larger species, and early results in human clin-ical trials have been uniformly disappointing. In a previ-ous study in chimpanzees we demonstrated the induction of a strong anti-HBs response following IM administra-tion of a fairly large dose (2 mg) of a DNA vaccine encod-ing HBsAg [12] . We had also shown in mice the possibil-ity to make DNA vaccines more immunogenic through inserting additional optimized CpG motifs to supple-ment those naturally present in the plasmid backbone, and to remove, through site-directed mutagenesis, most neutralizing DNA sequences that can counteract the im-mune stimulatory effects [10] . Somewhat surprisingly, considering earlier success in chimpanzees by ourselves [12] and others [17] , the DNA vaccine in this study did not generate any detectable anti-HBs, despite the CpG optimization that did make them more immunogenic in mice. The best that can be said is that one of two chim-panzees that received two doses of DNA vaccine ap-peared to have been primed for a humoral response since it developed protective anti-HBs shortly after infectious challenge and was thus protected from developing hepa-titis. This chimpanzee (1573) did not have detectable CTL measured with a chromium-release assay with re-stimulated peripheral blood mononuclear cells recovered 2 weeks after boost (data not shown); however, this does not necessarily rule out a role of T cells since this assay is technically diffi cult and not highly sensitive, and we did

Fig. 3. Viremia in chimpanzees following HBV challenge. Levels of HBV DNA in se-rum recovered weekly for 24 weeks follow-ing challenge were measured by real-time PCR. Values are shown as the log 10 of val-ues in IU/ml with levels higher than 2.0 be-ing considered positive.


Intervirology 2006;49:144–151 150

not test for other markers of the host’s adaptive immune response. The observation of priming for an antibody re-sponse without detectable antibody is consistent with those of postexposure vaccination studies of hepatitis B in which the development of anti-HBs during the incuba-tion period can prevent productive infection [1] . Neither of the two chimpanzees that received one dose of DNA vaccine followed by a dose of recombinant protein vac-cine appeared to have immune priming by the DNA vac-cine.

The differences in results between the earlier and pres-ent studies may be due to different dosing schedules (0, 8, 16 and 27 weeks and 0 and 4 weeks, respectively) and/or the larger animals in the present study that resulted in a lower weight proportional dose (0.07 mg/kg vs. 0.3 mg/kg). The differences in results are unlikely to be due to use of different plasmid vectors since the CpG-optimized vector was found to be superior to the nonoptimized vec-tor in mice [10] .

Nevertheless, these results demonstrate that unless im-proved delivery systems are developed for DNA vaccines, they will not be viable for use in humans as these doses of DNA are considerably higher than what would be af-fordable and practical for prophylactic human use. Al-though much effort has been put towards optimizing DNA vaccines, including new formulations and different modes and routes of delivery, highly successful DNA vac-cination is still elusive in primates [18–20] .

The observation that one DNA-vaccinated chimpan-zee (1574) had a more prolonged and more severe infec-tion than the unvaccinated control (5872) may be due to chance, and indeed, we could not demonstrate CTL in peripheral PBMCs (data not shown), but vaccine-induced enhancement of certain infections is a well-recognized phenomenon and should be kept in mind when develop-ing vaccines designed to stimulate cell-mediated immu-nity. Indeed, more severe hepatitis B, characterized by higher and more prolonged ALT elevations and higher and more prolonged viremia, has been observed in chim-panzees whose CD4 cellular or CD8 cellular response was compromised when compared to a control chimpanzee [21] ; the markers of hepatitis B in those chimpanzees re-sembled the response in chimpanzees 1574 and 5872, respectively.

In contrast to the disappointing results with the DNA vaccine, use of CpG ODN as an adjuvant to a commercial recombinant protein HBV vaccine greatly enhanced vac-cine immunogenicity. Preliminary results for 8-week anti-HBs levels were previously reported [22] , and we now additionally show improved kinetics and higher

anti-HBs at all time points compared to control vaccine. Of particular interest was the early protective response. Engerix-B alone induced a marginally protective humor-al response by 2 weeks after the fi rst vaccine dose. In con-trast, Engerix-B + CpG ODN was capable of inducing anti-HBs levels that were 10-fold greater than the mini-mum protective levels (10 mIU/ml) 2 weeks following the fi rst vaccine dose. The primary role of an adjuvant is the reduction in the threshold of immune activation, and these observations directly support, in chimpanzees, pre-vious observations in mice [10] and orangutans [6] that CpG ODN is a potent adjuvant when used in combina-tion with alum. CpG ODN reduces the threshold of im-mune activation through its infl uence on the maturation of B cells and antigen-presenting cells [23, 24] .

In summary, this study in chimpanzees fails to dem-onstrate utility of naked plasmid DNA vaccines for pri-mates. In contrast, it provides clear evidence that addi-tion of CpG to recombinant protein HBV vaccines can greatly enhance immunogenicity, resulting in earlier and stronger responses. If this translates to humans, it may be possible to develop a two-dose vaccine that would even be superior to the current three-dose vaccine. Such a vac-cine would be benefi cial for vaccination of at-risk popula-tions such as health care workers and those living in en-demic areas. Furthermore, a CpG-containing HBV vac-cine might also reduce the incidence of hypo- and nonresponsiveness to HBsAg, as has been demonstrated in genetically hyporesponsive orangutans [6] . Indeed such possibilities have been supported by results from fi rst clinical studies in man where addition of CPG 7909 (an ODN of the same sequence as used in this study) to Engerix-B has shown 100% of healthy volunteers devel-oping protective titers after only two doses [25] and im-proved response rates and higher anti-HBs titers in HIV-infected patients who failed previous vaccination [26] . Enhanced immunogenicity of HBsAg with a CpG ODN (1018 ISS) but without alum was also recently reported in healthy volunteers [27] .


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