Article
Reference
Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies
WEERATNA, RD, et al.
Abstract
Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system.
Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1-7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg-expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN [...]
WEERATNA, RD, et al . Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies. FEMS Immunology and Medical Microbiology , 2001, vol. 30, no. 3, p. 241-7
DOI : 10.1111/j.1574-695X.2001.tb01577.x PMID : 11335145
Available at:
http://archive-ouverte.unige.ch/unige:56145
Disclaimer: layout of this document may differ from the published version.
Priming of immune responses to hepatitis B surface antigen in young mice immunized in the presence of maternally derived antibodies
Risini D. Weeratna
a;*, Cynthia L. Brazolot Millan
b, Michael J. McCluskie
a, Claire-Anne Siegrist
c, Heather L. Davis
a;b;d;ea Coley Pharmaceutical Canada, 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9
b Loeb Health Research Institute at the Ottawa Hospital, Ottawa, ON, Canada
c WHO Collaborating Center for Neonatal Vaccinology, Department of Pathology and Pediatrics, University of Geneva, Geneva, Switzerland
d School of Rehabilitation Sciences, Faculty of Health Sciences, Ottawa, ON, Canada
e Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada Received 4 December 2000; received in revised form 16 February 2001; accepted 21 February 2001
Abstract
Early vaccination is necessary to protect infants from various infectious diseases. However, this is often unsuccessful largely due to the immaturity of the neonatal immune system. Furthermore, maternally derived antibodies can interfere with active immunization. We have previously shown in young mice that immune responses against several different antigens can be improved by the addition of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN). In this study we have evaluated immunization of newborn (1^7-day-old) BALB/c mice against hepatitis B surface antigen (HBsAg), with alum and/or CpG ODN, in the presence of high levels of maternal antibody against HBsAg (anti-HBs). Seroconversion rates and anti-HBs titers were compared to those induced by a HBsAg- expressing plasmid, since other studies had suggested DNA vaccines to be superior to protein vaccines in young mice with maternal antibody. HBsAg/alum/CpG ODN was superior to DNA vaccine in inducing HBsAg-specific CTL responses in young mice in the presence of maternally transferred anti-HBs antibodies. However, B cell responses to both HBsAg/alum/CpG ODN and DNA vaccines remained weak in the presence of maternally transferred anti-HBs antibodies. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords: Immunization; CpG ODN; DNA vaccine; Maternally transferred antibody
1. Introduction
One of the greatest achievements in modern medical science has been the development of e¡ective vaccines re- sulting in a remarkable reduction of many infectious dis- eases, including the complete eradication of smallpox [1].
Although most live attenuated, whole killed or subunit vaccines o¡er good protective immunity to adults, their success in immunizing infants is limited, probably due to the immature state of their immune system. Successful vaccination of newborns can be further hampered by the presence of maternally derived antibodies [2]. Although antibodies provided by the mother in utero or in colos- trum/breast milk can protect against numerous viral and bacterial pathogens, such passive immunity only lasts for a
short time, after which infants become vulnerable to in- fection. Therefore, it is important to develop vaccination strategies that are capable of inducing active immune re- sponses in the presence of maternally derived antibodies.
Several studies in animal models have shown that DNA vaccines can successfully prime immune responses in mice in the presence of maternal antibodies [3^5]. However, several other studies showed contrasting results [6,7]. Fur- thermore, results in human clinical trials with DNA vac- cines have been disappointing with most failing to induce strong antibody responses [8^10]. A number of safety con- cerns must also be addressed before use of DNA vaccines in healthy infants.
Numerous studies have shown that oligodeoxynucleoti- des containing immunostimulatory CpG motifs (CpG ODN) have potent Th1-like adjuvant properties when used with a wide variety of antigens [11^19]. We have previously shown that hepatitis B surface antigen (HBsAg) administered with the adjuvant combination of alum and
* Corresponding author. Tel.: +1 (613) 798-5555 ext. 16152;
Fax: +1 (613) 761-5354; E-mail: rweeratna@coleycanada.com
www.fems-microbiology.org
CpG ODN can prime strong humoral and cell-mediated immunity in mice injected 3 days after birth [17]. Herein, we examine the potential of this vaccine to induce B cell and CTL priming in young mice in the presence of mater- nally derived antibodies, and compare it to a HBsAg-ex- pressing DNA vaccine.
2. Materials and methods 2.1. Animals
Adult BALB/c mice were purchased from Charles River Laboratories (Montreal, QC, Canada) and housed at the animal care facility at Loeb Health Research Institute at Ottawa Hospital-Civic Site. Naive female mice or female mice with high antibody titers against HBsAg (anti-HBs) following immunization with HBsAg/alum/CpG [19] were bred onsite and checked daily for births. Immunization was carried out on groups of 5^12 pups aged 1, 3 or 7 days, with the date of birth being the day that the pups were ¢rst found. Pups were weaned and sexes sepa- rated at 3 weeks of age. Some pups (n= 25) were not immunized but were bled at weekly intervals for 12 weeks to determine the kinetics of decline of maternally derived anti-HBs.
2.2. Immunization of young mice
Mice were immunized with HBsAg at 1, 3 or 7 days of age using either the protein/adjuvant or DNA vaccine, both of which were administered by intramuscular (IM) injection. The total dose (see below) was administered in two equal injections of 10 Wl each, given bilaterally into the posterior thigh muscles.
Subunit vaccines contained recombinant HBsAg (0.1Wg Wl31, subtype ay, yeast-derived; Genzyme Diagnostics, San Carlos, CA, USA) at a ¢nal concentration of 0.05 mg ml31. The HBsAg was combined with alum (25 Wg Al3 Wg31 protein), CpG ODN (10 Wg CpG ODN Wg31 protein, using CpG ODN #1826 comprised of sequence 5P-TCCATGACGTTCCTGACGTT-3P, made with a nu- clease resistant phosphorothioate backbone, Coley Phar- maceutical Group, Wellesley, MA, USA) or alum plus CpG ODN [19].
The DNA vaccine encoded the major hepatitis B virus envelope protein (S) of subtypeayunder the control of the early immediate cytomegaloviral promoter and had a CpG-optimized backbone, as previously described (pMCG50-S) [20]. Plasmid DNA was puri¢ed on Qiagen DNA puri¢cation columns (Qiagen GmbH, Hilden, Ger- many), redissolved in 0.15 M NaCl and used at 5.0 mg ml31.
All mice were bled 4, 6, 8, 10 and 12 weeks post-immu- nization by retro-orbital puncture and plasma was recov- ered for enzyme-linked immunosorbent assay (ELISA). At
12 weeks post-immunization, the animals were injected IM with 1Wg HBsAg without adjuvant (`challenge'), and then were bled 3 and 7 days later to evaluate anti-HBs re- sponses. At 7 days post-challenge, mice were killed and spleens were removed. CTL activity was measured as de- scribed previously [21]. Brie£y, splenocytes were re-stimu- lated for 5 days using mouse mastocytoma cells expressing HBsAg (p815-S). CTL activity was measured by 51Cr re- lease assay.51Cr-labeled p815-S cells were used as targets.
A group of mice (n= 25) with maternally derived anti- HBs antibodies was left unimmunized and bled at bi- weekly intervals up to 12 weeks of age to determine the kinetics of decline of maternally transferred antibodies.
Subsequently, they were injected with 1Wg HBsAg without adjuvant and anti-HBs and CTL responses were evaluated 7 days later (`challenge control').
2.3. Evaluation of immune responses
Anti-HBs titers were determined by end-point dilution ELISA as previously described [19]. Titers for total IgG, IgG1 and IgG2a isotypes were de¢ned as the highest di- lution that resulted in an absorbance (OD450) value two times greater than that of the pre-immune plasma, with a cut-o¡ value of 0.05. Data were reported as group geo- metric mean titers þ the standard errors of the mean (GMT þ S.E.M.) of values of individual samples, which themselves were the average of triplicate assays. Serocon- version was de¢ned as a titerv100.
Antigen-speci¢c CTL responses of individual animals were measured using splenocytes recovered from immu- nized animals at 7 days post-`challenge', as described pre- viously [21].
2.4. Statistical analysis
Statistical analyses were performed using the InStat pro- gram (Graph PAD Software, San Diego, CA, USA). The statistical di¡erences between groups were determined by Student'st-test (for two groups) or by one-factor ANOVA followed by Tukey's test (for three or more groups) on raw data (for CTL) or on transformed data (log10, for antibody titers).
3. Results
3.1. Persistence of maternal antibodies
Twenty ¢ve pups born to four di¡erent mothers (with post-partum anti-HBs titers between 2.1 and 3.3U103) were used to evaluate the rate of decline of maternally transferred anti-HBs antibodies. The pups had very high anti-HBs titers early in life; at 2 weeks of age these were 4^6 times higher than those in their mothers. The levels of maternally derived antibody declined rapidly over the ¢rst
R.D. Weeratna et al. / FEMS Immunology and Medical Microbiology 30 (2001) 241^247 242
4 weeks of life and then more slowly. They were almost undetectable by 11 weeks of age (Fig. 1).
3.2. CTL responses in young mice immunized with protein or DNA vaccines
HBsAg-speci¢c CTL responses were assessed using sple- nocytes obtained from animals immunized in the presence of maternally derived anti-HBs antibodies (Fig. 2). All vaccine formulations tested were capable of inducing strong CTL responses in 7-day-old animals in spite of the presence of maternally transferred anti-HBs antibodies at the time of immunization. However, HBsAg/alum/CpG was capable of inducing CTL in animals injected 1 day after birth, and CTL activity in animals immunized at all ages (1, 3 or 7 days) was signi¢cantly higher than in con- trol animals (P60.05) at all e¡ector:target (E:T) ratios
(100:1, 50:1 or 10:1, data not shown for 100:1 and 10:1 ratio). Animals immunized with DNA 3 and 7 days after birth, but not at day 1, had signi¢cantly higher CTL ac- tivity compared to controls at 100:1 and 50:1 E:T ratios (P60.05). Animals immunized with HBsAg/alum 3 days after birth also had signi¢cantly higher CTL activity com- pared to challenge controls at 100:1 and 50:1 E:T ratios.
In contrast, animals immunized at 1, 3 or 7 days after birth with HBsAg/CpG did not have signi¢cant CTL re- sponses.
3.3. Anti-HBs antibody responses in young mice immunized with protein or DNA vaccines
None of the mice immunized in the presence of mater- nal antibodies had a detectable anti-HBs response (titer v100) at 12 weeks of age (a time at which the maternal antibodies had essentially disappeared) irrespective of the age at which they were vaccinated and the vaccine formu- lation used, including DNA vaccine. At this time, mice were `challenged' with 1 Wg HBsAg without adjuvant to determine whether there was an anamnestic response which would indicate priming by the early life immuniza- tion (Fig. 3, upper panel). According to our previous work, adult mice immunized with HBsAg alone require at least 4 weeks to seroconvert to titersv100 [19]. There- fore, high titers seen in other groups at 7 days post-chal- lenge indicate priming by the antigen formulation given early in life. None of the `challenge control' mice that were ¢rst immunized at 12 weeks of age with 1Wg HBsAg developed a detectable anti-HBs response by 7 days.
Among mice immunized with DNA vaccine and HBsAg/alum/CpG alum 7 days after birth in the presence of maternally derived anti-HBs, seroconversion (anti-HBs titers s100 upon challenge) reached 57% and 43%, re- spectively (Table 1). However, antibody titers remained
Fig. 1. Decline of maternally derived anti-HBs antibodies over time. Fe- male BALB/c mice were immunized with 1 Wg HBsAg. Animals with high anti-HBs titers were mated with naive BALB/c males. The anti- HBs levels of the litters born to these vaccinated females (n= 25) were measured over time by end-point ELISA.
Fig. 2. HBsAg-speci¢c CTL responses generated in the presence of maternal antibodies. BALB/c mice were immunized with 10Wg DNA vaccine or 1Wg HBsAg using di¡erent adjuvants as indicated in theXaxis at 1, 3 or 7 days after birth and challenged with 1Wg HBsAg at 12 weeks post-immuniza- tion. At 7 days post-challenge, spleens were removed and splenocytes were used for measuring CTL activity by51Cr release assay. CTL activity is indi- cated as % speci¢c lysis at an E:T ratio of 50:1. Each bar represents the mean % speci¢c lysis of all animals in a group (n= 5^12) þ S.E.M. The groups that show signi¢cantly higher (P60.05) CTL activity compared to the control group have been indicated by asterisks.
low (Fig. 3, upper panel) compared to immunization with the same vaccine formulations in the absence of anti-HBs antibodies of maternal origin, which led to 100% serocon- version and much higher overall anti-HBs titers (Fig. 3, lower panel).
In groups immunized in the presence of maternally de- rived anti-HBs antibody, plasma samples obtained 7 days post-challenge were assayed for IgG1 and IgG2a isotypes to determine the Th polarization of the response, a high IgG2a antibody titer being recognized as indicating a pref- erential Th1-type response in BALB/c mice. Isotypes were not determined for samples taken 1 or 3 days post-chal-
lenge or following immunization with HBsAg/CpG or HBsAg/alum at any age, since antibody titers were too low to give meaningful results. In control animals immu- nized at day 7 in the absence of maternally transferred anti-HBs antibodies, the antibody responses were either mixed (DNA immunization) or Th2-biased (HBsAg/
alum, HBsAg/alum/CpG) (Fig. 4A). Interestingly, immu- nization in the presence of maternal antibodies led to a di¡erent pattern of anti-HBsAg isotypes. Indeed, IgG1 responses elicited by DNA or HBsAg/alum/CpG were in- hibited to a much larger extent than IgG2a antibodies (Fig. 4B). This resulted in IgG2a/IgG1 ratios of 88
Fig. 3. Anti-HBs antibody responses generated in the presence and absence of maternal antibodies. BALB/c mice were immunized with either 10Wg of DNA vaccine or 1Wg HBsAg using di¡erent adjuvants as indicated in theXaxis in the presence (upper panel) or absence (lower panel) of maternally transferred anti-HBs antibodies and challenged with 1Wg HBsAg at 12 weeks post-immunization. The age of mice at immunization is indicated above each graph. Each bar represents the GMT of total IgG for the group (n= 5^12) at 7 days post-challenge as determined in triplicate by end-point dilu- tion ELISA assay. End-point titers were de¢ned as the highest plasma dilution that resulted in an absorbance value (OD450) two times greater than that of control non-immune plasma, with a cut-o¡ value of 0.05.
Table 1
Number of responders and % seroconversion at 12 weeks post-immunization of BALB/c mice with maternally derived anti-HBs antibodies Age at vaccination No. responders/total number of animals (% seroconversion)
DNA vaccine HBsAg+
Alum CpG ODN Alum+CpG ODN
Day 1 4/12 (33) 2/9 (22) 0/5 (0) 2/8 (25)
Day 3 0/5 (0) 0/5 (0) 1/6 (17) 4/6 (67)
Day 7 4/7 (57) 1/10 (10) 1/7 (14) 3/7 (43)
Mice were immunized at days 1, 3 and 7 of age with di¡erent vaccine formulations.
R.D. Weeratna et al. / FEMS Immunology and Medical Microbiology 30 (2001) 241^247 244
(HBsAg/alum/CpG) or 7 (DNA vaccine), compared to ra- tios of 0.03 or 0.81 in mice respectively immunized with HBsAg/alum/CpG or DNA in the absence of maternal antibodies. Thus, maternal antibodies exerted signi¢cantly more inhibition of IgG1 than of IgG2a B cell priming.
4. Discussion
Although the inhibitory e¡ect is reversible with the eventual decline of maternally derived antibodies, the pe- riod of reduced vaccine e¤cacy can last well beyond the period when maternally derived antibodies are at a su¤- cient level to protect the infants [2]. Immunization with live attenuated pathogens, DNA vaccines, or recombinant viral vectors can occasionally induce immune responses despite the presence of maternally derived antibodies [3^
5,22^24]. This was postulated to result from the in vivo antigen synthesis in host cells that may result in direct cell- to-cell antigen presentation without interference by circu- lating antibody. However, other studies showed failure to induce good immunity using DNA vaccines, live attenu- ated vaccines or recombinant viral vectors when adminis- tered in the presence of maternally transferred antibodies.
This inconsistency in results may be due to the presence of high levels of maternal antibodies in proportion to the amount of vaccine antigen used, age of the animal at time of immunization and di¡erences in the speci¢city of antibodies of maternal origin and those induced by vac- cines administered neonatally [25].
CpG ODN has been shown to have potent adjuvant e¡ects with numerous antigens in adult mice [19], and with HBsAg in non-human primates [26] and humans [27]. Furthermore, CpG ODN has been shown to over- come hyporesponsiveness to HBsAg in young non-human primates [28]. CpG ODN has strong synergy with other adjuvants, including alum [29]. In very young mice, a HBsAg/alum/CpG vaccine induces stronger anti-HBs and
CTL responses than a DNA vaccine expressing HBsAg [17], and alum/CpG was e¡ective in inducing adult-like Th1 responses to a panel of vaccine antigens [30].
In the present study, we demonstrate that HBsAg/alum/
CpG was as e¡ective as a DNA vaccine in immunizing newborn mice in the presence of maternally derived anti- bodies against HBsAg. However, the strength of the anti- body responses remained much weaker compared to mice immunized at the same age that had no maternally trans- ferred anti-HBs antibodies. Therefore, the presence of ma- ternal antibodies reduced the induction of B cell responses regardless of the vaccine formulation. This study also demonstrated that even a HBsAg-encoding DNA vaccine could not escape interference from maternal antibodies, as indicated by poor B cell responses. This is in accordance with the current understanding that in vivo synthesized antigens have to be released from host cells produce the antigen to become available for binding by infant B cells, a process leading to their simultaneous exposure to maternal antibodies. It will be interesting to con¢rm that this inhi- bition is due to the unavailability of antigen synthesized in vivo by DNA vaccines to the B cells, as a result of the formation of antigen^antibody complexes, rather than to B cell anergy. In addition, it is interesting to note that some vaccine formulations (HBsAg/alum/CpG and DNA) although not very strong, do allow a certain degree of B cell priming. Therefore, it is likely that this response could be further enhanced by optimizing the antigen dose, adjuvant dose and the age of immunization (to allow ma- ternal antibodies to deplete to a lower titer).
Even though the overall titers were low, and no statis- tical signi¢cance was achieved between groups due to high inter-group variation in antibody titers (likely due to dif- ferences in maternally transferred antibody levels amongst animals), the humoral response in mice immunized with HBsAg/alum/CpG was much more strongly Th1-biased (high proportion of IgG2a antibodies) than with the DNA vaccine. Unexpectedly, priming with HBsAg/alum/
Fig. 4. Di¡erence in IgG isotypes generated by the use of di¡erent vaccination strategies. BALB/c mice were immunized at 7 days after birth as de- scribed for Fig. 3 in the absence (A) or presence (B) of maternally transferred anti-HBs antibodies and challenged with 1Wg HBsAg at 12 weeks post- immunization. Their plasma was assayed for IgG isotypes at 7 days post-challenge. Gray bars indicate IgG1 titers and black bars indicate IgG2a titers.
CpG or DNA in the presence of maternal antibodies led to much better preserved IgG2a than IgG1 antibodies, compared to control 7-day-old mice immunized with no maternally transferred anti-HBs antibodies. In fact, there was no signi¢cant inhibition of post-challenge IgG2a anti- bodies following HBsAg/alum/CpG priming, compared to a s3 log10 inhibition of IgG1 antibodies. This di¡erence in the nature of the immune response could be due to the di¡erence in antigen uptake by professional antigen pre- senting cells, i.e. distinct receptor-mediated uptake of anti- gen via Fc receptors of HBsAg/Ab IgG1 or IgG2a com- plexes in animals with maternally transferred anti-HBs antibodies vs. uptake of exogeneous antigen in animals without maternally transferred anti-HBs antibodies. An- other hypothesis is that the lower antigen dose available to the immune system in animals immunized in the pres- ence of maternally derived anti-HBs antibodies would fa- vor priming of IgG2a antibodies. Antigen dose has indeed been shown to play an important role in determining the nature of the immune response [31,32]. Last, this di¡er- ential inhibition of IgG1 compared to IgG2a antibodies could re£ect preferential binding of HBsAg epitopes by maternal IgG1 rather than IgG2a antibodies, resulting in preferential inhibition of IgG1 versus IgG2a B cell prim- ing. Future experiments will try to address this interesting issue.
In contrast to inhibition of B cell responses by maternal antibodies, there was strong induction of CTL responses by HBsAg/alum/CpG vaccine from a very early age (1- day-old) despite the presence of high maternally derived antibody titers. DNA vaccines were also capable of induc- ing strong CTL responses, albeit only in older animals (3- day-old). These results are consistent with previous studies which showed that high titers of maternally transferred antibodies which inhibited B cell responses did not a¡ect T cell responses induced by four di¡erent measles vaccine formulations [33], or tetanus toxoid vaccine [2]. Further- more, the fact that maternal antibodies do not inhibit T cell responses may explain the e¤cacy of the current vac- cination strategy, which involves administering HBV vac- cine together with high concentrations of anti-HBs immu- noglobulins to infants born to HBV-infected mothers.
Under these circumstances, cell-mediated immunity con- sisting of CTL and, as shown more recently, Th1-type cytokines are needed to protect the infants rather than antibody responses [34].
In conclusion, this study shows that maternal antibodies hinder the induction of e¡ective B cell responses but not T cell responses to both DNA and protein vaccines against HBsAg. However, some B cell priming was observed with HBsAg/alum/CpG and DNA vaccines. Further studies are needed to optimize the conditions (i.e. antigen dose, adju- vant dose, and maternal antibody titers at time of immu- nization) that would help overcome the interference of maternal antibodies.
Acknowledgements
We wish to thank Amanda Boyd and Lu Zhang for their excellent technical assistance. This work was sup- ported by Coley Pharmaceutical Group Inc., and an oper- ating grant from the Medical Research Council (Canada) to H.L.D., who is also the recipient of an Ontario Minis- try of Health Career Scientist Award.
References
[1] Arita, I. (1999) Standardization of smallpox vaccines and the erad- ication programme ^ a WHO perspective. Dev. Biol. Stand. 100, 31^
37.
[2] Siegrist, C.A., Cordova, M., Brandt, C., Barrios, C., Berney, M., Tougne, C., Kovarik, J. and Lambert, P.H. (1998) Determinants of infant responses to vaccines in presence of maternal antibodies. Vac- cine 16, 1409^1414.
[3] Manickan, E., Yu, Z. and Rouse, B.T. (1997) DNA immunization of neonates induces immunity despite the presence of maternal anti- body. J. Clin. Invest. 100, 2371^2375.
[4] Hassett, D.E., Zhang, J. and Whitton, J.L. (1997) Neonatal DNA immunization with a plasmid encoding an internal viral protein is e¡ective in the presence of maternal antibodies and protects against subsequent viral challenge. J. Virol. 71, 7881^7888.
[5] Van Drunen Littel-van den Hurk, S., Braun, R.P., Lewis, P.J., Kar- vonen, B.C., Babiuk, L.A. and Griebel, P.J. (1999) Immunization of neonates with DNA encoding a bovine herpesvirus glycoprotein is e¡ective in the presence of maternal antibodies. Viral Immunol. 12, 67^77.
[6] Monteil, M., Le Potier, M.F., Guillotin, J., Cariolet, R., Houdayer, C. and Eloit, M. (1996) Genetic immunization of seronegative one- day-old piglets against pseudorabies induces neutralizing antibodies but not protection and is ine¡ective in piglets from immune dams.
Vet. Res. 27, 443^452.
[7] Wang, Y., Xiang, Z., Pasquini, S. and Ertl, H.C. (1998) E¡ect of passive immunization or maternally transferred immunity on the antibody response to a genetic vaccine to rabies virus. J. Virol. 72, 1790^1796.
[8] MacGregor, R.R., Boyer, J.D., Ugen, K.E., Lacy, K.E., Gluckman, S.J., Bagarazzi, M.L., Chattergoon, M.A., Baine, Y., Higgins, T.J., Ciccarelli, R.B., Coney, L.R., Ginsberg, R.S. and Weiner, D.B.
(1998) First human trial of a DNA-based vaccine for treatment of human immunode¢ciency virus type 1 infection: safety and host re- sponse. J. Infect. Dis. 178, 92^100.
[9] Calarota, S., Bratt, G., Nordlund, S., Hinkula, J., Leandersson, A.C., Sandstrom, E. and Wahren, B. (1998) Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 351, 1320^1325.
[10] Wang, R., Doolan, D.L., Le, T.P., Hedstrom, R.C., Coonan, K.M., Charoenvit, Y., Jones, T.R., Hobart, P., Margalith, M., Ng, J., Weiss, W.R., Sedegah, M., de Taisne, C., Norman, J.A. and Ho¡- man, S.L. (1998) Induction of antigen-speci¢c cytotoxic T lympho- cytes in humans by a malaria DNA vaccine. Science 282, 476^480.
[11] Freidag, B.L., Melton, G.B., Collins, F., Klinman, D.M., Cheever, A., Stobie, L., Suen, W. and Seder, R.A. (2000) CpG oligodeoxynu- cleotides and interleukin-12 improve the e¤cacy ofMycobacterium bovisBCG vaccination in mice challenged with M. tuberculosis. In- fect. Immun. 68, 2948^2953.
[12] von Hunolstein, C., Teloni, R., Mariotti, S., Recchia, S., Ore¢ci, G.
and Nisini, R. (2000) Synthetic oligodeoxynucleotide containing CpG motif induces an anti-polysaccharide type 1-like immune response R.D. Weeratna et al. / FEMS Immunology and Medical Microbiology 30 (2001) 241^247
246
after immunization of mice withHaemophilus in£uenzaetype b con- jugate vaccine. Int. Immunol. 12, 295^303.
[13] McCluskie, M.J. and Davis, H.L. (1999) CpG DNA as mucosal adjuvant. Vaccine 18, 231^237.
[14] Stacey, K.J. and Blackwell, J.M. (1999) Immunostimulatory DNA as an adjuvant in vaccination againstLeishmania major. Infect. Immun.
67, 3719^3726.
[15] Walker, P.S., Scharton-Kersten, T., Krieg, A.M., Love-Homan, L., Rowton, E.D., Udey, M.C. and Vogel, J.C. (1999) Immunostimula- tory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-Q-dependent mechanisms. Proc. Natl. Acad. Sci. USA 96, 6970^6975.
[16] Deml, L., Schirmbeck, R., Reimann, J., Wolf, H. and Wagner, R.
(1999) Immunostimulatory CpG motifs trigger a T helper-1 immune response to human immunode¢ciency virus type-1 (HIV-1) gp 160 envelope proteins. Clin. Chem. Lab. Med. 37, 199^204.
[17] Brazolot Millan, C.L., Weeratna, R., Krieg, A.M., Siegrist, C.A. and Davis, H.L. (1998) CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proc. Natl. Acad. Sci. USA 95, 15553^15558.
[18] Moldoveanu, Z., Love-Homan, L., Huang, W.Q. and Krieg, A.M.
(1998) CpG DNA: a novel adjuvant for systemic and mucosal im- munization with in£uenza virus. Vaccine 16, 1216^1224.
[19] Davis, H.L., Weeratna, R., Waldschmidt, T.J., Tygrett, L., Schorr, J.
and Krieg, A.M. (1998) CpG DNA is a potent enhancer of speci¢c immunity in mice immunized with recombinant hepatitis B surface antigen. J. Immunol. 160, 870^876.
[20] Krieg, A.M., Wu, T., Weeratna, R., E£er, S.M., Love-Homan, L., Yang, L., Yi, A.K., Short, D. and Davis, H.L. (1998) Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc. Natl. Acad. Sci. USA 95, 12631^12636.
[21] McCluskie, M.J. and Davis, H.L. (1998) CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice. J. Immunol.
161, 4463^4466.
[22] Monteil, M., Le Potier, M.F., Cariolet, R., Houdayer, C. and Eloit, M. (1997) E¡ective priming of neonates born to immune dams against the immunogenic pseudorabies virus glycoprotein gD by rep- lication-incompetent adenovirus-mediated gene transfer at birth.
J. Gen. Virol. 78, 3303^3310.
[23] Periwal, S.B., Speaker, T.J. and Cebra, J.J. (1997) Orally adminis- tered microencapsulated reovirus can bypass suckled, neutralizing maternal antibody that inhibits active immunization of neonates.
J. Virol. 71, 2844^2850.
[24] Osterhaus, A., van Amerongen, G. and van Binnendijk, R. (1998) Vaccine strategies to overcome maternal antibody mediated inhibi- tion of measles vaccine. Vaccine 16, 1479^1481.
[25] Siegrist, C.A. (2000) Vaccination in the neonatal period and early infancy. Int. Rev. Immunol. 19, 195^219.
[26] Hartmann, G., Weeratna, R.D., Ballas, Z.K., Payette, P., Blackwell, S., Suparto, I., Rasmussen, W.L., Waldschmidt, M., Sajuthi, D., Purcell, R.H., Davis, H.L. and Krieg, A.M. (2000) Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164, 1617^
1624.
[27] Davis, H.L., Morris, M.L., E£er, S.M., Cameron, D.W. and Heath- cote, J. (2000) CpG ODN is safe and highly e¡ective in humans as adjuvant to HBV vaccine: Preliminary results of Phase I trial with CpG ODN 7909. In: The Third Annual Conference on Vaccine Re- search, S25, p. 47.
[28] Davis, H.L., Suparto, I.I., Weeratna, R.R., Jumintarto, Iskandriati, D.D., Chamzah, S.S., Ma'ruf, A.A., Nente, C.C., Pawitri, D.D., Krieg, A.M., Heriyanto, Smits, W. and Sajuthi, D.D. (2000) CpG DNA overcomes hyporesponsiveness to hepatitis B vaccine in orang- utans. Vaccine 18, 1920^1924.
[29] Weeratna, R.D., McCluskie, M.J., Xu, Y. and Davis, H.L. (2000) CpG DNA induces stronger immune responses with less toxicity than other adjuvants. Vaccine 18, 1755^1762.
[30] Kovarik, J., Bozzotti, P., Love-Homan, L., Pihlgren, M., Davis, H.L., Lambert, P.H., Krieg, A.M. and Siegrist, C.A. (1999) CpG oligodeoxynucleotides can circumvent the Th2 polarization of neo- natal responses to vaccines but may fail to fully redirect Th2 re- sponses established by neonatal priming. J. Immunol. 162, 1611^
1617.
[31] Sarzotti, M., Robbins, D.S. and Ho¡man, P.M. (1996) Induction of protective CTL responses in newborn mice by a murine retrovirus.
Science 271, 1726^1728.
[32] Constant, S.L. and Bottomly, K. (1997) Induction of Th1 and Th2 CD4T cell responses: the alternative approaches. Annu. Rev. Im- munol. 15, 297^322.
[33] Siegrist, C.A., Barrios, C., Martinez, X., Brandt, C., Berney, M., Cordova, M., Kovarik, J. and Lambert, P.H. (1998) In£uence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime-boost strategies in mice. Eur. J. Immunol. 28, 4138^4148.
[34] Guidotti, L.G., Rochford, R., Chung, J., Shapiro, M., Purcell, R.
and Chisari, F.V. (1999) Viral clearance without destruction of in- fected cells during acute HBV infection. Science 284, 825^829.
