Levels Of Antibody Production Research Articles

Effect of chemical adjuvants on DNA vaccination

DNA vaccination is useful for generating immune responses, particularly the cell-mediated immune response, in a wide variety of species. However, DNA vaccination generally induces only relatively weak responses; hence, various approaches have been developed recently in order to improve its efficacy or immunopotency. The use of a chemical adjuvant is one of them. Previously we have shown that Bupivacaine or Marcaine can modulate immune responses induced by DNA vaccines [Proc. Natl. Acad. Sci. 90 (1993) 4156]. Following that lead, we have recently tested several additional chemicals for their usefulness as adjuvants in DNA inoculation. Of a total of five chemicals tested, levamisole exhibited strongest Th1 stimulatory activity whereas Tween 80 showed weakest Th1 activity, as determined by IgG2a production, and saline formulation induced weak T cell proliferation and DTH, in animals inoculated with a DNA construct expressing the foot–mouth disease viral capsule protein VP1. Furthermore, co-inoculation of levamisole increased the production of IFN-γ by more than 100-fold as compared to that by DNA inoculation formulated in saline. In contrast, a previously reported chemical adjuvant, bupivacaine, stimulated only modest levels of overall antibody production, with relatively low level of Ig2a. These results demonstrate the usefulness of various chemicals, particularly levamisole, for modulating the outcome of DNA vaccination, in both the intensity of the immune response and the polarity of such response (toward Th1).

Immune deviation and ocular infections with varicella zoster virus

Since experimental, herpes simplex virus-induced acute retinal necrosis (ARN) develops in mice only if the mice fail to acquire virus-specific delayed hypersensitivity (DH) and despite their production of anti-viral antibodies (i.e. ACAID), I investigated whether a similar situation exists for patients with either varicella zoster virus (VZV)-induced ARN or anterior uveitis caused by VZV. Patients with either acute VZV-induced ARN, anterior uveitis with dermatitis (herpes zoster ophthalmicus, ZO-AU), or anterior uveitis without dermatitis (zoster sine herpete, ZSH-AU) were skin-tested with VZV to evaluate DH. The formal diagnoses of ARN associated with VZV, ZO-AU, and ZSH-AU were established by PCR analysis of the ocular samples and/or by the Goldmann-Witmer coefficient to determine levels of local antibody production. ARN, ZO-AU, and ZSH-AU activity were assessed clinically, and DH skin tests were repeated three months after onset when ocular recovery had taken place. All patients with VZV-induced skin disease alone (control group) displayed intense DH when tested with VZV antigen. In contrast, subsets of patients with ARN or ZO-AU displayed loss of VZV-specific DH. Patients with the most severe ARN or ZO-AU had the lowest DH responses to VZV antigens. Serum anti-VZV antibody titers were higher in ARN patients than in normal controls, and the anti-viral titer correlated inversely with the intensity of anti-VZV DH responses. VZV-specific DH responses were restored in patients who recovered from ARN. Patients with ZSH-AU also failed to display VZV-specific DH. The absence of DH reactivity to VZV antigens (i.e. immune deviation) appears to be a concomitant feature of VZV uveitis of high intensity, implying that virus-specific DH may interfere with the emergence of VZV-induced ARN or anterior uveitis.

Angiotensin-converting enzyme genotype is a predictive factor in the peak panel-reactive antibody response

The presence of a high panel-reactive antibody (PRA) level represents an independent risk factor for early graft failure and chronic allograft dysfunction. It has also been reported that patients with the ACE-DD and AGT-AA genotypes display poorer chronic allograft function. We investigated the effects of gene polymorphisms of the renin angiotensin system (RAS) on anti-HLA antibody production among renal transplant candidates. Genotyping was performed on 133 dialysis patients for the ACE (I/D) and AGT (M235T) as well as the type 1 (A1166C) and type 2 (A1223G) angiotensin II receptor genes. Patients with a peak PRA ≥ 30% were considered to be positive for anti-HLA antibody (40.6% of 133 patients). Genetic polymorphisms of the RAS were not associated with anti-HLA antibody production at this PRA level. Another analysis comparing the 29 patients with a peak PRA ≥50% with the 104 patients with a peak PRA <50% showed that previous transplants, the presence of ACE-DD genotype, history of blood transfusions, and dialysis duration were all associated with the high levels of antibody production by univariant analysis. A multivariate analysis using a logistic regression model revealed previous transplants, the presence of ACE-DD genotype, and history of blood transfusions to be predictors of anti-HLA antibody production. The ACE-DD genotype is an important risk factor for higher PRA levels. This study suggests that genetic control of RAS activity correlates with production of anti-HLA antibodies, possibly explaining the relationship to chronic allograft outcome.

IL-10 Augments Antibody Production inin VitroImmunized Lymphocytes by Inducing a Th2-Type Response and B Cell Maturation

An in vitro immunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4(+) and CD8(+) T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4(+) and CD8(+) T cells, as well as in CD19(+) B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages.

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.

Impact of conventional chemotherapy on levels of antibodies against vaccine-preventable diseases in children treated for cancer.

Intensive chemotherapy in children with malignancies causes partial immune deficiency, including long-term impairment of humoral immunity. We investigated the levels of antibodies against measles, mumps, polio, rubella, diphtheria, tetanus, and Haemophilus type b (Hib) in 139 children at the time of diagnosis of the malignant disease, during chemotherapy, after cessation of intensive treatment, and after re-vaccination. In general, cytostatic therapy resulted in a significant lowering of antibody levels. A decline of antibodies below the protective level as a consequence of cytostatic treatment was observed in 6% of the children for measles and mumps, in 18%, 12%, and 25% for polio types 1, 2, and 3, and in 21% for diphtheria. By contrast, rubella and tetanus antibodies remained within the protective range in all cases of this study. Re-vaccination 3 to 5 months after cessation of chemotherapy produced antibody levels about as high as those measured prior to therapy. Only 6 out of 83 children with previously positive antigen titres did not respond to re-vaccination. Vaccination or re-vaccination failed in 5 of 13 non-responders for more than 1 antigen, indicating a decreased reactability to vaccinations in some patients.

Mouse myeloma cell line Sp2/0 multidrug-resistant variant as parental cell line for hybridoma construction.

The use of multidrug-resistant variant of Sp2/0 mouse myeloma cell line SpEBr-5 as a partner for making mouse hybridoma producing monoclonal antibodies is described here. The resulting hybridoma cell line 1F7 was characterized with a high level of monoclonal antibody production and karyotype containing all normal mouse chromosomes. 1F7 cells were separately selected for resistance to ethidium bromide (EBr) and adriamycin (ADR) and different mechanisms of drug resistance were found in these cell variants. The resistance in ADR-selected 1F7 cells was due to amplification and overexpression of mdr genes. In EBr-resistant 1F7 cells, mdr genes were overexpressed without amplification. Substantially decreased level of Topo II activity in both cell lines also suggests the existence of additional mechanisms for MDR phenotype of hybridoma cells. Finally, adriamycin-resistant 1F7 hybridoma cell variant was found to produce higher level of specific immunoglobulins due to the increased level of Iggamma(2b) heavy chain mRNA.

New animal model of emotional stress: Behavioral, neuroendocrine and immunological consequences

This report describes a new model of emotional stress, which was induced by randomly giving an empty water bottle to rats during watering periods per day for 14 consecutive days. The behavioral, endocrinological and immunological consequences were investigated. The data showed that the emotional stress activated both the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, leading to the increased blood levels of corticosterone and catecholamine. It also elicited attacking and exploring behavior, suppressed the immune function of the rats, including leukocyte counts, weight of the spleen, and the level of specific anti-ovalbumin IgG antibody production. Presenting no water and no empty bottle to rats only evoked the exploring behavior, increased the corticosterone level and decreased the leukocyte counts. These findings demonstrate a role of psychological factors on behavioral, endocrinological and immunological functioning. The animal model described in the present study may serve as an analogue mimicking emotional stress experienced in humans (e.g. anger and/or anxiety), and may be useful for further studying the complex relationships among emotional stress, behavior, and immune function.

Peculiarities of immunocorrective effects of the bone marrow regulatory peptides (myelopeptides)

Myelopeptides (MPs) are low-molecular-weight immunoregulatory peptides of bone marrow origin. The peculiarities of their immunoregulatory effects are demonstrated with two of the six synthesized MPs, MP-1 (Phe-Leu-Gly-Phe-Pro-Thr) and MP-2 (Leu-Val-Val-Tyr-Pro-Trp). It is shown that MP action is directed to the damaged links of immunity. MP-1 enhances a decreased level of antibody production in cyclophosphamide (Cy)-treated mice, but does not influence the antibody formation in normal animals. MP-2 inhibits the tumor growth more in a tumor-bearing organism as the tumor size gets larger, insofar as MP-2 antitumor effect is concerned, by its ability to recover functional activity of T lymphocytes suppressed by tumor products. Selective immunocorrective effects of MPs are based on ligand–receptor interactions. Using FITC-labeled MP-1 and [ 3H]-labeled MP-2, specific binding of these peptides with appropriate cell populations is shown. The cytofluorimetric analysis revealed a target cell for MP-1—CD4 + T lymphocyte (T helper). The data obtained suggest that MPs are endogenic immunoregulators which participate in the maintenance of immune homeostasis.

Starburst Conjugates of Proteins with Carbon-Chain Polymers Containing Low Molecular Biologically Active Compounds: Synthesis and Immunogenicity

The synthesis of starburst polymer–protein conjugates on the basis of bovine serum albumin and horseradish peroxidase was performed with the aim to study the possibilities of regulation of the immune response against the components of the conjugates. These polymers had one-point binding between the protein and the modifying carbon-chain polymer that contained 1-vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (a salsolinol analogue) or bradykinin (peptide hormone) residues in its carbon chain. Changes in the chemical nature of the carbon-chain part of the polymer–protein conjugate were shown to increase or decrease the level of antibody production both against the low-molecular compounds attached to the polymeric fragments and against the protein carrier.

Influenza hemagglutinin peptides fused to interferon gamma and encapsulated in liposomes protects mice against influenza infection

The immunogenicity of a peptide vaccine may be improved by fusing antigen to a cytokine and administering this chimeric protein in a particulate delivery system. We have investigated this using a vaccine comprising an immunodominant T cell epitope and a B cell epitope from influenza haemagglutinin (HATB) fused to interferon gamma and encapsulated in liposomes (HATB/IFN-γ/lipo). Controls comprised groups receiving HATB/IFN-γ mixed with liposomes, HATB incorporated in liposomes or heat inactivated PR8 influenza virus (HI PR8). IFN-γ production in mice treated with HATB/IFN-γ/lipo was significantly higher than in mice inoculated with either HATB/IFN-γ mixed with liposomes or HATB incorporated in liposomes but less than HI PR8. Lung viral titres were significantly lower in mice treated with HATB/IFN-γ/lipo compared with those treated with HATB/IFN-γ mixed with liposomes. HI PR8-treated mice recorded a nil viral titre. There was no correlation between the level of antibody production and clearance of virus from the lungs. These data suggest that particulate delivery systems may be useful adjuncts to improve immune responses to chimeric proteins and to induce protection against disease.

Construction of engineered CHO strains for high-level production of recombinant proteins.

We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a plasmid carrying a loxP-green fluorescent protein (GFP) fusion gene and a DHFR gene, we screened colonies by fluorescent intensity. We selected 16 clones that expressed high levels of GFP and carried one copy of the plasmid in their chromosomes and treated them with methotrexate (MTX) to examine their ability for DHFR-mediated gene amplification. Two clones, MK1 and MK2, showed increased GFP expression upon gene amplification. In those clones, the loxP-GFP gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a loxP-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal loxP by Cre recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. In human monoclonal antibody production, after gene-targeting of loxP in MK2 and gene amplification with MTX, the MTX-resistant colonies showed high levels of antibody production. The most productive clone was able to produce 160 mg/l in 7 days in a low-protein medium in a spinner-flask.

Maternal and newborn immunization with a human immunodeficiency virus-1 immunogen in a rodent model.

We examined immunization with an inactivated, gp120-depleted human immunodeficiency virus (HIV) antigen in incomplete Freund's adjuvant (IFA), also containing a sequence of immunostimulatory (ISS) DNA, during the last trimester of pregnancy and neonatally in a rat model. Pregnant rats were immunized in the third trimester and their litters were immunized during the newborn period. In addition, litters of rats from non-immunized mothers were immunized during the neonatal period. As another control, pregnant rats were immunized and their litters analysed. Supernants from peripheral blood mononuclear cells (PBMCs) were assayed from newborns at 4 weeks of age for HIV-specific interferon-gamma (IFN-gamma), HIV-specific regulated on activation, normal, T-cell expressed, and secreted (RANTES), and serum for p24 antigen-specific immunoglobulin G (IgG) production. In the animals whose pregnant mothers were immunized and were also immunized during the neonatal period, we observed HIV-specific IFN-gamma production and HIV-specific RANTES production, but weak p24 IgG antibody production. Animals immunized only during the neonatal period developed the highest levels of HIV-specific IFN-gamma production, but somewhat lower levels of HIV-specific RANTES and p24 IgG antibody production. The group of animals whose mothers had received immunizations during the last trimester of pregnancy, but were not immunized during the neonatal period, developed the strongest p24 IgG antibody levels, but little or undetectable HIV-specific IFN-gamma or RANTES production. Neonatal immunization resulted primarily in cell-mediated immune responses, while animals born to mothers who were immunized during the last trimester had primarily an antibody-mediated immune response. Immunization of pregnant animals followed by neonatal immunization resulted in a mixed cell-mediated/antibody type profile in the neonatal animal. Future studies should provide insights into neonatal immunity and potential vaccine approaches to prevent neonatal infection and perinatal transmission.

Cholesterol inclusion in liposomes affects induction of antigen-specific IgG and IgE antibody production in mice by a surface-linked liposomal antigen.

In the previous study, we investigated the induction of ovalbumin (OVA)-specific antibody production in mice by OVA-liposome conjugates made using four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids. All of the OVA-liposome conjugates tested induced IgE-selective unresponsiveness. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes with the highest membrane fluidity, suggesting that the membrane fluidity of liposomes affects their adjuvant effect. In this study, liposomes with five different cholesterol inclusions, ranging from 0% to 43% of the total lipid, were made, and the induction of OVA-specific antibody production by OVA-liposome conjugates was compared among these liposome preparations. In contrast to the results in the previous study, liposomes that contained no cholesterol and possessed the lowest membrane fluidity demonstrated the highest adjuvant effect for the induction of IgG antibody production. In addition, when the liposomes with four different lipid compositions were used, OVA-liposome conjugates made using liposomes that did not contain cholesterol induced significantly higher levels of anti-OVA IgG antibody production than did those made using liposomes that contained cholesterol and, further, induced significant production of anti-OVA IgE. These results suggest that cholesterol inclusion in liposomes affects both adjuvanticity for IgG production and regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes.

Intranasal immunization with SIV virus-like particles (VLPs) elicits systemic and mucosal immunity

By using a baculovirus expression system, we have successfully produced simian immunodeficiency virus (SIV)-like particles (VLPs) with high levels of biologically active SIV envelope (Env) incorporated on their surfaces. To study whether SIV VLPs represent effective mucosal immunogens, we immunized groups of mice with VLPs alone or VLPs plus the mucosal adjuvant cholera toxin (CT) by the intranasal (i.n.) route. High levels of serum IgG antibody production were achieved in mice immunized intranasally with SIV VLPs, and the antibody response was found to be antigen dose-dependent. The IgG1 and IgG2a ratio indicates that immune responses induced by SIV VLPs are Th1 oriented. Mice immunized with VLPs plus CT were found to exhibit higher serum antibody responses than those immunized with VLPs alone ( P<0.001). Furthermore, IgA antibody production was detected in both saliva and vaginal fluid from mice mucosally immunized with SIV VLPs. Higher levels of IgA were found in vaginal fluid than in saliva in animals immunized with SIV VLPs plus CT ( P<0.05). Higher neutralizing activity to SIV 1A11 was also found in serum of animals immunized with SIV VLPs plus CT. Moreover, increased numbers of MHC I-restricted peptide-specific IFN-γ and IL-4 producing T cells were detected in both splenocytes and lymph nodes by intranasal immunization of SIV VLP plus CT. These results suggest that VLPs are effective mucosal antigens that can induce both humoral and cellular immune responses at systemic and mucosal sites.

Immunostimulatory Actions of Lactobacilli: Mitogenic Induction of Antibody Production and Spleen Cell Proliferation by Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus

We investigated the ability of heat-treated Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus to act as direct inducers of antibody production and cellular proliferation. Yogurt starter-derived strains of L. delbrueckii subsp. bulgaricus and L. acidophilus induced high levels of antibody production by murine splenocytes in vitro . Both IgM and IgG isotypes were produced. In contrast, two different strains, L. delbrueckii subsp. bulgaricus (ATCC 11977) and L. acidophilus (ATCC 521) did not induce antibody production by murine splenocytes. While all four strains were able to induce some degree of splenocyte proliferation, the yogurt starter-derived strain of L. acidophilus was the most potent inducer. These results indicate a mitogenic effect of certain strains of lactobacilli on murine splenocytes, resulting in polyclonal antibody production. The ability of heat-treated lactic acid bacteria to induce antibody production and proliferation suggests the involvement of a heat-resistant structural component in non-specific activation of the immune system by these strains of lactic acid bacteria.

BmTI antigens induce a bovine protective immune response against Boophilus microplus tick

Boophilus microplus trypsin inhibitors (BmTIs) present in larvae were preliminarily characterized as active proteins, approximately 10–18 kDa, by SDS-PAGE. BmTIs showed trypsin inhibitory activity on reverse zymography containing gelatin (0.03%) and also inhibited others serine proteinases (human neutrophil elastase and human plasma kallikrein). Bos indicus, Nelore breed calves, previously sensitized with BmTIs and challenged with tick larvae (20,000 larvae/animal), showed 72.8% efficacy to interfere in tick development with 69.7% and 71.3% reduction of both tick number and egg weight, respectively. Cattle BmTIs antiserum titer was approximately 1:8000. The maximum level of BmTIs antibody production was detected 40 days after the first immunization by ELISA. Our preliminary results suggest that B. microplus serine proteinase inhibitors may play a role in the tick larvae fixation and feeding processes. Therefore, the development of antibodies against BmTIs might impair the normal parasitism.

Immune responses of interferon gamma (IFN-gamma) knock out mice to repeated Haemaphysalis longicornis (Acari: Ixodidae) nymph infestations.

To investigate the immunological mechanisms of acquired resistance to tick infestation, interferon gamma (IFN-gamma) deficient mice (IFN-gamma mice) were used to assess interleukin-4 (IL-4) and antibody production levels against tick salivary gland antigen on three successive infestations with Haemoaphysalis longicornis Neumann nymphs. The engorged body weight of the ticks decreased during the second and third infestations. Similar observations were noted in IFN-gamma+/+ mice. However, the engorged body weight of the ticks from IFN-gamma +/+ mice were considerably lower than those from IFN-gamma-/- mice. A marked increase in antibody production during the second and third infestations was observed indicating that IFN-gamma-/- mice could acquire immunological resistance against H. longicornis nymphs. Moreover, IL-4 levels were higher during the first and third infestations but decreased during the second infestation. IL-4 levels were significantly higher in IFN-gamma-/- mice than in IFN-gamma+/+ mice. We have shown here that the statistically significant high IL-4 levels observed in IFN-gamma-/- mice may be a result of type 2 helper cell (Th2) polarization. However, the apparently higher IL-4 levels during the first and third infestations and the notable decline during the second infestation suggest that other cytokines or factors in the host immune system may play a part in regulating IL-4 levels.

Use of Taguchi's methods as a basis to optimize hybridoma cell line growth and antibody production in a spinner flask.

Taguchi's methods were used for the design of an experimental strategy aimed at optimizing cell density and monoclonal antibody (mAb) production from a spinner flask hybridoma culture. 23G11 is an antibody to the human leukocyte adhesion molecule, CR3 or beta 2 integrin (CD11b/CD18). It recognizes specifically the A-domain of the alpha subunit CD11b. Anti beta 2 integrin monoclonal antibodies hold a great potential for preventing inflammation mediated tissue injuries. An L8 orthogonal experimental design was used to investigate four different culture components: stirring speed, nature of serum, concentration of serum and nature of media (RPMI 1640 or RPMI 1640 supplemented with glucose and glutamine). The experiments were conducted using two levels for each factor studied and a direct ELISA test was used to estimate the level of antibody production. Statistical analysis of the collected data pointed to the stirring speed and serum concentration, and the interaction between these parameters, as the components that affected cell growth. Antibody production was affected by these factors and by the nature of medium but also by the following two interactions: stirring speed/nature of serum and stirring speed/concentration of serum. This study emphasizes the value of using Taguchi's methods as a basis for optimization of mAb production from a hybridoma culture, in cost effective and significantly less labor intensive ways.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.