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*
Retroviral Immunology Section, Division of Viral Products and
Laboratory of Immunology, Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting T cells. To monitor the contribution of cells at the
site of vaccination to this process, transfected skin was periodically
removed and grafted onto naive recipients. Immediate removal of
vaccinated skin abrogated the development of an immune response.
Low-level IgG production was stimulated when the vaccination site was
left in place for 
5 h, with the strength of this response increasing
the longer the site remained intact (for up to 2 wk). Measurable
primary T cell responses were observed in animals whose vaccination
site remained in place for 1 day. Skin grafts transferred 0 to
24 h postvaccination stimulated a primary immune response in naive
recipients. Memory B and T cells were generated in animals whose site
of vaccination remained intact for 5 to 12 h. Skin transferred
within 12 h of vaccination triggered memory B and T cell
development in graft recipients, while the removal of skin >12 h
postvaccination did not reduce memory in vaccinated mice. These
findings suggest that 1) primary immunity is induced by cells that
migrate rapidly from the site of immunization, 2) nonmigratory cells
influence the magnitude of this primary response, and 3) migratory
cells alone are responsible for the induction of immunologic memory. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When DNA vaccines are administered by gene gun bombardment of the skin or by i.m. injection, a majority of the plasmid is taken up by keratinocytes and muscle cells, respectively (7, 9, 10, 11). Recent evidence suggests that these nonmigratory cells do not contribute to the development of immunity, since primary Ab and CTL responses can be generated despite immediate removal of the site of vaccination (12, 13). Some postulate that primary responses are therefore induced by plasmid or plasmid-containing cells that rapidly leave the site of vaccination. Evidence suggests that bone marrow-derived APC acting far from the site of vaccination contribute to this process (13, 14, 15, 16). Consistent with that hypothesis, dendritic cells in the skin were recently shown to take up DNA-coated beads and migrate within 24 h to the draining lymph nodes (14), where primary immunity develops (17).
Despite ongoing progress in understanding the location and type(s) of
cells that contribute to DNA vaccine-mediated primary immune responses,
the role of cells at the site of injection to the magnitude of the
primary response, and the generation of immunologic memory remains
unclear. We therefore analyzed Ab and cytokine production resulting
from gene gun administration of a plasmid encoding the circumsporozoite
protein (CSP)3 of
malaria to BALB/c mice. In this well-defined system, primary IgG
anti-CSP Ab production arises and can first be detected 2 to 3 wk
postimmunization, while secondary humoral responses achieve
near-maximal levels within 1 wk of boosting (17). The plasmid-encoding
CSP (pCSP) vaccine also activates IFN--secreting T cells with a
memory component that can be detected by restimulation in vitro with Ag
(18). Using this model system, we probed the contribution of gene
gun-vaccinated skin to the development of primary and secondary immune
responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were obtained from Charles River Associates (Wilmington, MA) and maintained in our specific pathogen-free facility. Mice were immunized by gene gun (Bio-Rad, Hercules, CA) administration of 1 µg of pCSP to shaved abdominal skin through an 8-mm hole cut through a Tegaderm mask (3M, St. Paul, MN). Animals were boosted 10 wk later. Serum was collected 1, 3, and 6 wk postimmunization and 1 wk postboost by retro-orbital puncture and stored at -20°C until use. In some experiments, mice were killed by cervical dislocation 4 wk postimmunization and their organs removed aseptically.
Reagents
The CS.1 protein used in ELISA assays is an immunoaffinity-purified fusion protein produced in Escherichia coli, and consists of amino acids 64 to 321 of the intact PyCSP protein fused to 81 amino acids of the nonstructural protein of influenza A (2, 19).
The pCSP DNA vaccine (kind gift of VICAL, Inc., San Diego, CA; company designation VR2507) was constructed by cloning the coding sequence of the Plasmodium yoelii CSP from the nkCMVintPyCSP.1 plasmid (2) into the VR1012 vector, as previously described (20). Expression of pCSP was tested by in vitro transfection of HeLa cells and immunoblot analysis of cell lysates.
DNA-coated gold beads were prepared by combining 100 µg of pCSP and 100 µl of 0.1 M spermidine with 50 mg of gold beads. Plasmid was precipitated onto the beads by slowly adding 200 µl of CaCl2 while vortexing, as previously described (21). Coated beads were centrifuged at 1,000 x g and washed three times in cold 100% ethanol. Beads were resuspended and bound to the inner surface of Gold-Coat Tubing (Bio-Rad) as recommended by the manufacturer. A total of 1 µg of DNA was then delivered to the shaved abdominal skin of adult BALB/c mice using a Helios gene gun (Bio-Rad).
Skin excision/grafting experiments
Donor skin at the vaccination site was removed to the level of the panniculus carnosus by excisional biopsy (10 mm). Subcutaneous tissue was removed from donor skin, which was then grafted heterotopically onto the flanks of prepared recipients. The transplant site was covered with sterile gauze and a pressure bandage for 9 days.
Cytokine ELISA and ELIspot assays
Ninety-six-well Immulon 2 microtiter plates (Dynatech
Laboratories, Alexandria, VA) were coated with 10 µg/ml of
anti-IFN- (clone RA6a2; Lee Biomolecular Research Laboratories,
San Diego, CA) in 0.1 M carbonate buffer (pH 9.6) for 3 h at room
temperature (22). The plates were blocked with PBS-5% BSA for 1 h
and washed with PBS-0.025% Tween-20. Culture supernatants or spleen
cell suspensions prepared in complete medium (RPMI 1640 supplemented
with 10% heat-inactivated FCS, 1.5 mM L-glutamine,
and 100 U/ml of penicillin/streptomycin) starting with 1 x
106 cells/well, were incubated on anticytokine-coated
plates for 8 to 10 h at 37°C in a humidified 5% CO2
incubator (23). Plates were then washed with PBS-Tween and overlaid
with 1 µg/ml of biotinylated anti-IFN- (clone XMG 1.2;
PharMingen, San Diego, CA) at 4°C and then treated with a 1:2000
dilution of avidin-conjugated alkaline phosphatase (Vector
Laboratories, Burlingame, CA) for 2 h at room temperature. After a
final wash, cytokine or ELIspot formation was detected by the addition
of a solution of BCIP/NBT (Kirkegaard and Perry Laboratories,
Gaithersburg, MD).
In some experiments, spleen cells (106 cells/ml) were cultured for 4 days in complete medium plus 10 µg/ml of P16 (the dominant stimulatory T cell epitope on the CSP protein) (24). This in vitro assay selectively stimulated memory T cells (18).
CS.1-specific ELISA assays
Ninety-six-well Immulon 1 microtiter plates were coated with 10 µg/ml of immunoaffinity-purified CS.1 protein in carbonate buffer, pH 9.5 (17). Plates were blocked with PBS-1% BSA, overlaid with serially diluted mouse serum, washed, and reacted with phosphatase-conjugated anti-mouse IgG (Southern Biotechnologies, Birmingham, AL). The concentration of specific Ab was determined by comparison to a standard curve generated using a high-titrated antiserum, as previously described (25).
In all experiments, the GMT of the test group was compared with that of
a control group of similarly vaccinated mice. The % normal IgG
anti-CSP response was calculated by the formula:
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Measurement of primary and secondary immune responses
Previous studies showed that pCSP vaccination induces a primary
IgG anti-CSP Ab response first detectable 2 wk postvaccination and
peaking at 3 wk (17). Memory IgG responses peak at approximately 1 wk
postboost. Primary cytokine responses are characterized by a
significant increase in the number of spleen cells actively secreting
IFN- in vivo 3 to 4 wk postimmunization (18). Memory T cells were
detected by restimulating spleen cells from immunized mice for 4 days
in vitro in the presence of 10 µg/ml of P16.
Statistical analysis
All studies were performed on groups of three mice (studied independently), and were repeated at least once with similar results. Data were analyzed by ANOVA using Fisher’s Exact test as described in the SigmaStat software package.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initial experiments examined the effect of varying the injection pressure and size of vaccine-coated beads on the magnitude of the primary immune response. Gene gun administration of pCSP-coated beads to the abdominal skin of BALB/c mice at pressures ranging from 150 to 450 psi uniformly elicited strong IgG anti-CSP Ab responses. No response was induced when beads were administered at pressures below 100 psi. Optimal IgG anti-CSP Ab production was elicited by immunizing BALB/c mice with 1.6-micron gold beads delivered at 200 psi, and these parameters were used in all subsequent experiments.
Kinetics of the induction of primary and secondary immune responses
Condon et al. recently showed that dendritic cells are triggered to migrate from the skin to the draining lymph nodes following entrance of plasmid-coated beads (14). Whether nonmigratory cells in the epidermis also contribute to the induction/perpetuation of vaccine-induced responses has not been elucidated. To examine this issue, pCSP-vaccinated skin was removed and grafted onto syngeneic recipients at discrete time points following immunization. Donor and recipient BALB/c mice were then studied for the development of humoral (Ab) and T cell-specific (cytokine) responses.
As seen in Figure 1
, neither Ab nor
cytokine production was elicited in mice whose injection site was
removed immediately after immunization. "Boosting" these animals
resulted in an IgG anti-CSP response indistinguishable in magnitude
or kinetics from that of naive mice immunized simultaneously,
suggesting that the immediate removal of vaccinated skin also abrogated
the development of immunologic memory. By comparison, primary IgG
anti-CSP responses were elicited in animals whose vaccination site
was left in place for only 5 h, although this response was 15-fold
lower than that of mice whose vaccination site was never removed.
Progressively stronger humoral responses developed as the vaccination
site was left intact for longer periods, with near maximal levels
achieved at 2 wk (Fig. 1, left
panel).
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, right panel). In contrast, the primary Ab
response generated in these mice was fivefold lower than that of mice
whose vaccination site was never removed.
Primary T cell responses were evaluated by monitoring
cytokine-producing cells in vivo. A significant increase in the number
of spleen cells secreting IFN- was observed in mice whose
vaccination site was left in place for 1 day, but not for 
5 h (Fig. 2). Maximal cytokine production resulted
when the site of immunization was left undisturbed for 1 wk. To
monitor the generation of "memory" T cells, splenocytes from
immunized mice were cultured in vitro with P16, a stimulatory T cell
peptide present on the CSP protein. We previously showed that this
4-day culture activated quiescent Ag-specific T cells (18). Consistent
with results involving memory B cells, the generation of memory T cells
took place quite rapidly, requiring only 5 h (Fig. 2, right panel). Of note, no primary T cell
response was detected at this 5-h time point (Fig. 2, left
panel). Coupled with evidence that transfected
dendritic cells migrate from the skin to the draining lymph nodes (14, 26), these findings suggest that immunologic memory is induced by
dendritic cells that migrate from the skin 5 to 12 h
postvaccination. Alternatively, it might take 5 to 12 h for
nonmigratory cells to transcribe, translate, and secrete the encoded
CSP protein required for the induction of the responses measured
above.
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To differentiate between these alternatives, vaccinated skin was
removed from donor mice and grafted onto naive recipients. Skin
transferred within 5 h of vaccination stimulated IFN-
production by the graft recipient, consistent with the induction of a
primary T cell response (Fig. 2). Yet no primary B cell response was
detected in any skin graft recipient. Given that IgG anti-CSP Ab
production was not induced, and that the magnitude of the primary T
cell response was 40% lower than that of optimally vaccinated donors,
we conclude that the immunogenicity of transfected epidermal cells was
reduced by the process of excision and grafting.
Nevertheless, both B and T cell memory was generated when donor
skin was grafted onto naive recipients within 12 h of vaccination.
As seen in Figure 3, recipients of such
skin grafts mounted an IgG anti-CSP response 1 wk post-pCSP
"boost," in contrast to recipients of skin grafted 1 day
postimmunization, whose anti-CSP response developed 3 wk postboost.
The timing and magnitude of the latter response is indicative of a
primary rather than secondary humoral response. Moreover, memory T
cells were present in recipients of skin vaccinated for 5 h, but not
1 day (Fig. 2). Since it is well documented that cells at the site of
DNA vaccination continue to transcribe, translate, and secrete the
encoded protein for many days postimmunization (27, 28), our results
support the conclusion that migratory cells (rather than secreted
protein) are responsible for the induction of immunologic memory in
both T and B cell subsets, and that such cells are critical for
inducing primary T cell responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Dendritic cells in the epidermis function as specialized APCs, efficiently inducing immune responses against Ags that come into contact with the skin (26, 30). Mor et al. showed that the initial immune response to a DNA vaccine takes place in the draining lymph nodes (17) while Condon et al. showed that dendritic cells transfected by plasmid-coated beads migrate within 24 h from the skin to the draining lymph nodes (14). The precise kinetics of this dendritic cell migration were not delineated, nor was their relative contribution to the generation of primary vs memory responses.
The contribution of Ag-expressing nonmigratory cells to the development of immunity remains controversial. Williams et al. reported that plasmid introduced by particle bombardment directed cells in the epidermis to produce the encoded protein for at least 2 wk (31). Yet since neither muscle cells nor keratinocytes constitutively express the class II MHC or costimulatory molecules required to activate CD4+ Th cells (such as CD40 or B7) (32), their role in this process has been suspect. Yet Ulmer et al. showed that myoblasts transfected in vitro could generate a primary immune response when transplanted into naive recipients (33). Those investigators concluded that Ag shed by the myocytes was taken up and presented by professional APCs, since the induced immune response was restricted by the MHC type of resident APCs rather than transplanted myocytes (34). Further evidence that cells at the vaccination site contribute to the induction of immunity was supplied by Torres et al. (13), who showed that immediate removal of gene gun-vaccinated skin prevented the development of T or B cell responses.
However, this set of findings is inconsistent with two other reports in which normal immune responses were detected despite the immediate removal of muscle and/or skin following DNA vaccination (12, 13). In the latter cases, it is possible that the injected plasmid gained access to the lymphatic or circulatory systems, obviating the need for transfection of cells at the site of immunization. Indeed, plasmid can be detected in serum several hours after i.m. injection of a DNA vaccine (28).
The current studies were undertaken to address these conflicting results and to better define the role of migratory and nonmigratory cells at the site of DNA immunization. Our results indicate that primary immune responses are induced by cells that migrate from the epidermis (presumably dendritic cells) within 24 h of immunization, and that Ag-expressing nonmigratory cells (presumably keratinocytes) influence the magnitude of this response. These conclusions are supported by our finding that: 1) immediate removal of the vaccination site abrogated immunity, 2) primary B and T cell responses were induced when the vaccination site was left intact for 5 to 24 h, 3) transfer of the vaccination site within 5 h of immunization induced a primary T cell response in naive recipients, and 4) the magnitude of this primary response increased the longer the vaccination site was left in place (for up to 2 wk). It should be noted that transfected keratinocytes continue to produce Ag for 2 wk (although maximal protein production occurs within the first 3 days), while transfected dendritic cells emigrate from the epidermis within 24 h (14).
We also found that rapidly migrating cells were solely responsible for
the development of immunologic memory. Leaving the site of vaccination
intact for 5 to 12 h was sufficient for the host to generate
memory B cells that rapidly produced high titrated IgG anti-CSP Abs
when re-exposed to Ag in vivo, and memory T cells that secreted IFN-
when restimulated by Ag in vitro. Similarly, memory T and B cells were
generated when skin vaccinated within 12 h was grafted onto naive
recipients, unlike grafts taken >1 day postvaccination. These data
support the conclusion that T and B cell memory is triggered by cells
that migrate from the epidermis 5 to 12 h postimmunization. These
experiments demonstrate that gene gun vaccination combined with skin
excision/grafting provides a novel method for analyzing the effect of
Ag dose and duration on the induction of primary and memory
responses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dennis Klinman, Bldg. 29A, Rm. 3 D 10, Division of Viral Products, CBER/FDA, Bethesda, MD 20892. E-mail address:
3 Abbreviations used in this paper: CSP, circumsporozoite protein; GMT, geometric mean titer; pCSP, plasmid-encoding CSP.
Received for publication August 22, 1997. Accepted for publication November 5, 1997.
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