DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life

The relative deficiency of T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) responses in early life is associated with an increased susceptibility to infections by intracellular microorganisms. This is likely to reflect a preferential polarization of immature CD4 T cells toward a Th2 rather than a Th1 pattern upon immunization with conventional vaccines. In this report, it is shown that a single immunization within the first week of life with DNA plasmids encoding viral (measles virus hemagglutinin, Sendai virus nucleoprotein) or bacterial (C fragment of tetanus toxin) vaccine antigens can induce adult-like Th1 or mixed Th1/Th2 responses indicated by production of IgG2a vaccine-specific antibodies and preferential secretion of interferon-γ (IFN-γ) compared with interleukin (IL)-5 by antigen-specific T cells, as well as significant CTL responses. However, in spite of this potent Th1-driving capacity, subsequent DNA immunization was not capable of reverting the Th2-biased responses induced after early priming with a recombinant measles canarypox vector. Thus, DNA vaccination represents a novel strategy capable of inducing Th1 or mixed Th1/Th2 and CTL responses in neonates and early life, providing it is performed prior to exposure to Th2-driving conventional vaccine antigens.

Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. One area of interest is the development of new immune adjuvants. Immunostimulatory oligodeoxynucleotides containing the CpG motif (CpG ODN) can induce production of a wide variety of cytokines and activate B cells, monocytes, dendritic cells, and NK cells. Using the 38C13 B cell lymphoma model, we assessed whether CpG ODN can function as immune adjuvants in tumor antigen immunization. The idiotype served as the tumor antigen. Select CpG ODN were as effective as complete Freund’s adjuvant at inducing an antigen-specific antibody response but were associated with less toxicity. These CpG ODN induced a higher titer of antigen-specific IgG2a than did complete Freund’s adjuvant, suggesting an enhanced TH1 response. Mice immunized with CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to that seen in mice immunized with complete Freund’s adjuvant. We conclude that CpG ODN are effective as immune adjuvants and are attractive as part of a tumor immunization strategy.

Immunization with live attenuated simian immunodeficiency virus induces strong type 1 T helper responses and β-chemokine production

Immunization with live attenuated simian immunodeficiency virus (SIV) strains has proved to be one of the most effective strategies to induce protective immunity in the SIV/macaque model. To better understand the role that CD4+ T helper responses may play in mediating protection in this model, we characterized SIV-specific proliferative and cytokine responses in macaques immunized with live attenuated SIV strains. Macaques chronically infected with live attenuated SIV had strong proliferative responses to SIV proteins, with stimulation indices of up to 74. The magnitude of the proliferative response to SIV Gag varied inversely with the degree of attenuation; Gag-specific but not envelope-specific responses were lower in animals infected with more highly attenuated SIV strains. SIV-specific stimulation of lymphocytes from vaccinated macaques resulted in secretion of interferon-γ, IL-2, regulated-upon-activation, normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β but not IL-4 or IL-10. Intracellular flow cytometric analysis documented that, in macaques vaccinated with SIVmac239Δnef, up to 2% of all CD4+T cells were specific for SIV p55. The ability of live attenuated SIV to induce a strong, sustained type 1 T helper response may play a role in the success of this vaccination approach to generate protection against challenge with wild-type SIV.

DNA immunization: Induction of higher avidity antibody and effect of route on T cell cytotoxicity

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon γ production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.

Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis

H-2Kb-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181–188 (TRP-2) and connexin 37 residues 52–59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Iab+ cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.

Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: A phase I trial

The carbohydrate antigen globo H commonly found on breast cancer cells is a potential target for vaccine therapy. The objectives of this trial were to determine the toxicity and immunogenicity of three synthetic globo H-keyhole limpet hemocyanin conjugates plus the immunologic adjuvant QS-21. Twenty-seven metastatic breast cancer patients received five vaccinations each. The vaccine was well tolerated, and no definite differences were observed among the three formulations. Serologic analyses demonstrated the generation of IgM antibody titers in most patients, with minimal IgG antibody stimulation. There was significant binding of IgM antibodies to MCF-7 tumor cells in 16 patients, whereas IgG antibody reactivity was observed in a few patients. There was evidence of complement-dependent cytotoxicity in several patients. Affinity column purification supported the specificity of IgM antibodies for globo H. On the basis of these data, globo H will constitute one component of a polyvalent vaccine for evaluation in high-risk breast cancer patients.

Intralymphatic immunization enhances DNA vaccination

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8+ cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.

Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.

Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule.

Graves disease is an autoimmune thyroid disease characterized by the presence of antibodies against the thyrotropin receptor (TSHR), which stimulate the thyroid to cause hyperthyroidism and/or goiter. By immunizing mice with fibroblasts transfected with both the human TSHR and a major histocompatibility complex class II molecule, but not by either alone, we have induced immune hyperthyroidism that has the major humoral and histological features of Graves disease: stimulating TSHR antibodies, thyrotropin binding inhibiting immunoglobulins, which are different from the stimulating TSHR antibodies, increased thyroid hormone levels, thyroid enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen. The results suggest that the aberrant expression of major histocompatibility complex class II molecules on cells that express a native form of the TSHR can result in the induction of functional anti-TSHR antibodies that stimulate the thyroid. They additionally suggest that the acquisition of antigen-presenting ability on a target cell containing the TSHR can activate T and B cells normally present in an animal and induce a disease with the major features of autoimmune Graves.

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.