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Factors Associated with the Development of Neonatal Tolerance After the Administration of a Plasmid DNA Vaccine¹

Motohide Ichino^*, Gil Mor^*, Jackie Conover^*, Walter R. Weiss, Mitsuhiro Takeno^*, Ken J. Ishii^* and Dennis M. Klinman^2,*

^* Section of Retroviral Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; and Malaria Program, Naval Medical Research Institute, Bethesda, MD 20889

	Abstract

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A plasmid DNA vaccine encoding the circumsporozoite protein of malaria (pCSP) induces tolerance rather than immunity when administered to newborn mice. We find that this tolerance persists for >1 yr after neonatal pCSP administration and interferes with the induction of protective immunity in animals challenged with live sporozoites. Susceptibility to tolerance induction wanes rapidly with age, disappearing within 1 wk of birth. Higher doses of plasmid are more tolerogenic, and susceptibility to tolerance is not MHC-restricted. CD8⁺ T cells from tolerant mice suppress the in vitro Ag-specific immune response of cells from adult mice immunized with pCSP. Similarly, CD8⁺ T cells from tolerant mice transfer nonresponsiveness to naive syngeneic recipients. These findings clarify the cellular basis and factors contributing to the development of DNA vaccine-induced neonatal tolerance.

	Introduction

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Vaccine development is being revolutionized by the ability of Ag-encoding DNA plasmids to induce cellular and humoral immune responses against pathogenic viruses, parasites, and bacteria (1, 2, 3, 4, 5). DNA vaccines have successfully prevented infection in a variety of animal models and are currently undergoing phase I clinical trials in humans. These vaccines are composed of Ag-encoding genes whose expression is regulated by a strong mammalian promoter expressed on a plasmid backbone of bacterial DNA (1, 6). Cells transfected by DNA vaccines transcribe, translate, and express the encoded proteins in the context of self MHC (1, 6, 7).

Many DNA vaccines induce strong protective immune responses in adult animals (1, 2, 3, 8, 9). For example, the plasmid encoding the circumsporozoite protein of the Plasmodium yoelii malaria parasite (pCSP)³ stimulates a protective immune response in adult BALB/c mice (3); this response is characterized by the production of Th1-type cytokines, IgG anti-CSP Abs, and CTLs (3, 10). A similar vaccine (directed against the CSP of P. falciparum) was reported recently to stimulate a strong CTL response in adult human volunteers (11). However, most vaccines intended for human use are administered to infants and children. Due to the immaturity of their immune system, newborns exposed to foreign Ags are at risk of developing tolerance rather than immunity (12). A number of factors influence the development of neonatal tolerance, including the nature, concentration, and mode of Ag presentation to the immune system, and the age of the host (13, 14, 15). Because the protein encoded by a DNA vaccine is produced endogenously and expressed in the context of self MHC, the potential exits for the neonatal immune system to recognize it as "self", resulting in tolerance rather than immunity.

Consistent with such a possibility, we showed that pCSP induced tolerance rather than immunity when administered to newborn BALB/c mice (16). Not all DNA vaccines are tolerogenic: several investigators have elicited immunity by administering DNA vaccines to newborn mice and non-human primates (15, 17, 18, 19, 20, 21, 22). However, we reproducibly observed that neonatal animals treated with pCSP were unable to generate T or B cell responses when challenged with pCSP as adults (16). The current work examines the influence of Ag dose, recipient age, and MHC haplotype on the development of tolerance, as well as the role of T cells in this process.

	Materials and Methods

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Reagents

The pCSP DNA vaccine (a kind gift of Vical, Inc., San Diego, CA; manufacturer’s designation VR2507) was constructed by cloning the DraI-EcoRV fragment of the PyCSP gene into the HincII site of pBluescript II SK⁺ (Stratagene, La Jolla, CA) and then transferring it into the SalI/Klenow-filled and BamHI sites of a kCMVinBL vector (a modified pUC18-based plasmid pCMVintBl) (7), where the ampicillin-resistance gene was replaced with a kanamycin resistance gene using the pBluescript XhoI/Klenow-filled and BamHI restriction endonuclease sites located 5' and 3', respectively, to the PyCSP coding sequence, as described previously (3). Expression of PyCSP was tested by in vitro transfection of BHK cells and by immunoblot analysis of cell lysates.

Plasmid DNA was purified using an EndoFree plasmid maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Briefly, lysates from bacteria transformed with VR2507 were passed over a Qiagen column and then sterilized by ethanol precipitation. These lysates were shown to be endotoxin-free before being dissolved in sterile PBS for injection.

CS.1 is an immunoaffinity-purified fusion protein that is produced in Escherichia coli and consists of amino acids (aa) 64–321 of the intact PyCSP protein fused to 81 aa of the nonstructural protein of influenza A (3, 23). A synthetic peptide corresponding to PyCSP 280–295 aa, SYVPSEQILEFVKQI (designated P16), was used to induce IFN- production from pCSP-immunized T cells in vitro (16, 24).

Animals

Female BALB/c, C57BL/6, C3H/HeJ, and A/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in the specific pathogen animal colony at the Center for Biologics Evaluation and Research. Mice were immunized between 1 day and 7 wk of age and boosted from 6 wk to 1 yr of age. Adults were immunized in the quadriceps muscle with 40 µg of plasmid DNA, whereas mice <1 wk of age were injected with 1–100 µg of pCSP in the gluteus maximus. A total of 50 µg of CS.1 protein emulsified in CFA was injected i.p. into adult mice, whereas 20 µg of soluble CS.1 was injected i.p. into newborns (25).

At 3–6 wk postimmunization, animals were bled by retroorbital puncture and killed by cervical dislocation; their spleens were removed aseptically. Serum was stored at -70°C and assayed for Ab by ELISA, whereas freshly isolated cells were examined for cytokine production.

CSP-specific Ab assay

We coated 96-well Immulon 1 microtiter plates (Dynatech, Alexandria, VA) with 10 µg/ml of immunoaffinity-purified CS.1 protein in 0.1 M carbonate buffer, pH 9.5 (10). Plates were blocked with PBS/1% BSA, overlaid with serially diluted mouse serum, washed, and reacted with phosphatase-conjugated anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL). The concentration of specific Ab was determined by comparison with a standard curve that had been generated using a high-titered anti-serum.

IFN--specific ELISA assay

Spleen cell suspensions (2.5 x 10⁵ cells/ml) were prepared in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FCS, 1.5 mM L-glutamine, and 100 U/ml of penicillin/streptomycin). A total of 100 µl of the cell suspensions from naive, immunized, or tolerized mice (in some cases depleted of CD4⁺ or CD8⁺ T cells) was mixed and stimulated for 3 days in U-bottom microtiter plates with 10 µg/ml P16 peptide in a 5% CO₂ in air, 37°C incubator. The culture supernatants from these wells were collected and stored at -70° until assayed, as described previously (26).

We coated 96-well Immulon 2 microtiter plates with 10 µg/ml of anti-IFN- (clone RMMG-1, Biosource International, Camarillo, CA) in PBS for 5 h at room temperature (27). The plates were blocked with PBS/5% BSA for 1 h and washed with PBS/0.025% Tween 20. Culture supernatants diluted in PBS/1% BSA were added to the anti-IFN--coated plates for 2 h, washed with PBS-Tween, and overlaid with 1 µg/ml of biotinylated anti-IFN- (clone R4-6A2, PharMingen, San Diego, CA) followed by a 1/5000 dilution of streptavidin-alkaline phosphatase (PharMingen) for 2 h at room temperature. After a final wash, the concentration of IFN- was determined colorimetrically by comparison with a standard curve that had been generated using known concentrations of cytokine.

Cell purification

Spleen cells (10⁶/ml) were incubated with biotinylated anti-CD4 or anti-CD8 Abs (Becton Dickinson, San Jose, CA) for 15 min at 4°C. Cells were washed and reacted with avidin-conjugated magnetic cell sorting (MACS) microbeads (Miltenyi Biotec, Sunnyvale, CA) for 15 min at 4°C. Phenotype-positive cells were then deleted using the MACS magnetic purification system (Miltenyi Biotec) (28). The remaining cells were <0.4% phenotype-positive as determined by staining sorted populations with fluorescein-labeled phenotype-specific mAbs (PharMingen).

Adoptive transfer

A single-cell suspension was prepared from the spleens of three to five 10-wk-old naive or pCSP-tolerized donors. Next, 5 x 10⁷ cells were injected i.v. into 6-wk-old syngeneic recipients. After 4 days, recipients were immunized i.m. with 40 µg of pCSP; their Ab response was monitored 3 wk later.

Sporozoite challenge

At 3 wk after the final pCSP treatment, mice were challenged by an i.v. injection of 100 sporozoites from the nonlethal 17 x NL strain of P. yoelii (>10 ID₅₀). These sporozoites were raised in Anopheles stephensi mosquitoes and were obtained by salivary gland dissection. To document malaria infection, Giemsa-stained blood films were examined on days 6, 10, and 14 postchallenge. The presence of 1/1000 infected RBCs at any time was taken as evidence of active infection.

Data analysis

All results represent the average of more than four individually tested mice per group. Statistical significance was established using the Student t test.

	Results

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Factors influencing the induction of neonatal tolerance

The pCSP DNA vaccine is composed of the gene encoding the circumsporozoite protein (CSP) of P. yoelii malaria inserted into the 1020 plasmid vector (3, 7). Our lab showed previously that i.m. injections of pCSP into adult BALB/c mice induced a strong, protective, immune response characterized by the production of IgG anti-CSP Abs and the induction of Ag-specific CTLs (10). In contrast, the same plasmid induced a profound state of immune tolerance when administered to 1- to 2-day-old mice (Table I and 16). Tolerance was characterized by the lack of a cellular or humoral immune response to treatment with pCSP at 6–7 wk of age.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Neonatal mice (n 5/group) were treated with pCSP and then reexposed to plasmid at 6–7 wk of age. Mice were deemed tolerant if their serum IgG anti-CSP titer at 3 wk after the second pCSP injection was within two SDs of the mean titer of unvaccinated littermate controls (15 ± 9). As seen in Table I, mice that responded to vaccination generated much higher IgG anti-CSP Ab responses (400 ± 108). A, Age effect: BALB/c mice were injected i.m. with 10 µg/mouse of pCSP at the indicated time after birth. B, Dose effect: 1- to 2-day-old BALB/c mice were injected i.m. with 1–100 µg/mouse of pCSP. C, MHC effect: 1- to 2-day-old mice were injected i.m. with 10 µg/mouse of pCSP.

The duration of tolerance was examined by repeatedly rechallenging neonatally tolerized animals with pCSP at 6–10 wk intervals. These studies used only those mice found to be tolerant when challenged with pCSP at 6 wk of age. As seen in Table II, these mice remained tolerant for the duration of the experiment (>1 yr). To determine whether repeated exposure to pCSP contributed to this persistence of tolerance, neonatally tolerized mice were "rested" for 6–12 mo and then reexposed to pCSP. All animals remained tolerant for 6 mo, and most mice remained tolerant for one full year in the absence of pCSP reexposure (Table II). The site of pCSP injection was examined by PCR and RT-PCR for the persistence of plasmid or CSP mRNA. Neither could be detected longer than 3 mo after pCSP administration (data not shown). Of interest, the magnitude of the serum IgG anti-CSP response of mice that broke tolerance after 1 yr resembled that of a conventional primary (rather than memory) immune response.

Naive BALB/c mice are susceptible to infection by P. yoelii sporozoites, whereas adult BALB/c mice repeatedly vaccinated with pCSP are generally protected (Fig. 2 and 16). We revaccinated normal and neonatally tolerized mice with pCSP and then challenged them with 100 live sporozoites (>10 ID₅₀). As seen in Fig. 2, repeated vaccination protected normal adult mice but not tolerized mice from malaria. These results suggest that the tolerance induced by neonatal DNA vaccination could potentially prevent the host from developing a protective pathogen-specific immune response.

View larger version (8K): [in this window] [in a new window]   FIGURE 2. BALB/c mice were treated with pCSP at the times shown (10 µg/mouse as newborns, 40 µg/mouse as adults). Mice were challenged by an i.v. injection of 100 live sporozoites at 3 wk after the final treatment. The presence of 1 infected cell per 1000 RBCs on Giemsa-stained blood films at 6–14 days postinjection was interpreted as active infection. n = 10 mice/group.

To gain insight into the cellular basis of this tolerant state, 5 x 10⁷ spleen cells from naive or neonatally tolerized mice were adoptively transferred into normal syngeneic recipients. Recipients were immunized 4 days later with 40 µg of pCSP. As seen in Table III, spleen cells from tolerized donors inhibited the development of a primary IgG anti-CSP response in recipient mice. In contrast, a normal immune response developed in the recipients of bone marrow cells from tolerized donors or of spleen cells from naive donors. To examine the phenotype of the cells responsible for this suppression, splenocytes from tolerized mice were depleted of CD4⁺ or CD8 T⁺ cells before transfer. Recipients of CD8- but not CD4-depleted splenocytes responded normally to pCSP immunization, suggesting that CD8⁺ T cells were required for tolerance induction (Table IV).

View this table: [in this window] [in a new window]   Table V. Cellular basis of neonatal tolerance: in vitro analysis of Ag-induced IFN- production1

	Discussion

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Whereas adult BALB/c mice repeatedly vaccinated with pCSP mount strong protective Ab and CTL responses when challenged with live sporozoites (Fig. 2 and 3), the administration of this vaccine to newborn mice frequently results in neonatal tolerance. Tolerized BALB/c mice are unable to generate Ab, cytokine, or cytotoxic T cell responses against pCSP-encoded Ags (16). We find that this induction of tolerance is critically dependent upon the timing of vaccine administration. Although 2-day-old mice were extremely susceptible to tolerance induction, pCSP was never tolerogenic when administered to mice that were >7 days of age.

Among 1- to 2-day-old mice, increasing the dose of pCSP increased the likelihood of tolerance developing, with 100% of mice being tolerized by 100 µg of pCSP. Presumably, an early expression of pCSP-encoded Ags presented in the context of self MHC can result in tolerance. In animals that were not tolerized, pCSP had no discernible effect. Thus, mice treated with pCSP as neonates and reexposed to the same plasmid 6 wk later either mounted no response (evidence of tolerance) or developed a low-titered IgG anti-CSP response (indistinguishable from a primary response). Once tolerance was established, it persisted long-term (1 yr) without the need for additional pCSP. It is not clear whether the CSP Ag produced by transfected cells persisted for this period (potentially taken up and stored by dendritic cells); however, the tolerogenic plasmid itself disappeared within 3 mo.

Of particular interest, CD8⁺ T cells from tolerant mice transferred nonresponsiveness to naive recipients. This observation is reminiscent of classical "infectious tolerance" as described by Gershon and Kondo (31) (see review by Schwartz (32)). Other investigators have shown that suppressor cells are present in neonatal mice (33, 34). However, many mechanisms might account for the development of tolerance, and many of these involve cells that are not of the CD8⁺ phenotype (32). Indeed, in our own studies, active suppression was not always observed (see legend to Fig. 1). Given the complexity of these issues, further study will be needed to clarify the contribution of CD8⁺ T cells to pCSP-induced tolerance.

Animal studies and preliminary results from phase I clinical trials indicate that DNA vaccines can be safely administered to adults. However, our findings indicate that a CSP-encoding DNA vaccine has the potential to induce tolerance rather than immunity in newborns. This finding is in contrast to the activity of plasmids encoding a variety of other malaria proteins (such as PySSP2 and PyHEP17 (35)) examined in our lab and to those encoding Ags from flu, rabies, and hepatitis B that have been studied by other investigators (18, 19, 20, 21, 22). In those cases, neonatal plasmid administration primed recipients to mount Ag-specific Ab and/or CTL responses, rather than inducing tolerance.

Our studies of DNA vaccines against multiple malaria Ags used the same 1012/1020 vectors. Because tolerance was observed with pCSP but not with these other plasmids, we conclude that it is the nature of the encoded Ag rather than the vector that determines whether a plasmid will be tolerogenic. In this context, soluble CSP protein is not immunogenic in neonatal mice, suggesting that poorly immunogenic Ags may be more likely to induce neonatal tolerance. Given the importance of vaccines in preventing childhood diseases, our observations support the need for thorough safety and efficacy testing of each DNA vaccine targeted for use in children or newborns. This may be accomplished by studying the effect of these vaccines in young animals whose immune system reflects the functional activity and maturational age of the targeted human population.

	Acknowledgments

 
We thank Vical, Inc. for kindly providing the plasmid DNA used in these experiments.

	Footnotes

 
¹ This work was supported in part by a grant from the National Vaccine Program. W.R.W. was supported by Naval Medical Research and Development Command Work Units 611102A.S13.00101-BFX.1431 and 612787A.870.00101.EFX.1432.

² Address correspondence and reprint requests to Dr. Dennis M. Klinman, Building 29A, Room 3D10, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892. E-mail address:

³ Abbreviations used in this paper: pCSP, plasmid encoding the circumsporozoite protein of the Plasmodium yoelii malaria parasite; MACS, magnetic cell sorting.

Received for publication September 25, 1998. Accepted for publication December 21, 1998.

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