Kristian B Haller

Abstract: HIV-1 protease is an important target for anti-HIV therapy but has not received much attention as a vaccine antigen. To investigate the immunogenic properties of HIV-1 protease, we designed DNA plasmids encoding variants of the protease gene. Mutations resulting in enzymatic inactivation (D25N) and resistance to standard antiretroviral drugs (V82F/I84V) were introduced in order to examine the impact of the enzymatic activity on immunogenicity and the possibility to induce immune responses against drug resistant protease, respectively. The enzymatic inactivation of protease resulted in significantly increased in vitro expression as well as in vivo immunogenicity. The inactivated protease was highly immunogenic in both BALB/c and HLA-A0201 transgenic C57Bl/6 mice, and the immunogenicity was retained when the gene was delivered as a part of a multigene HIV-1 DNA vaccine. The drug resistance mutations hampered both the cellular and humoral immune responses, as the mutations also affect both CD4 and CD8 T cell epitopes. Taken together, our data demonstrates the possibility to drastically increase the immunogenicity of HIV-1 protease.

Abstract: In vivo electroporation (EP) has proven to significantly increase plasmid transfection efficiency and to augment immune responses after immunization with plasmids. In this study, we attempted to establish an immunization protocol using intradermal (i.d.) EP. BALB/c mice were immunized with a plasmid encoding HIV-1 p37Gag, either i.d. with the Derma Vax EP device, intramuscularly (i.m.) without EP, or with combinations of both. A novel FluoroSpot assay was used to evaluate the vaccine-specific cellular immune responses. The study showed that i.d. EP immunizations induced stronger immune responses than i.m. immunizations using a larger amount of DNA and that repeated i.d. EP immunizations induced stronger immune responses than i.m. priming followed by i.d. EP boosting. Two and three i.d. EP immunizations induced immune responses of similar magnitude, and a short interval between immunizations was superior to a longer interval in terms of the magnitude of cellular immune responses. The FluoroSpot assay allowed for the quantification of vaccine-specific cells secreting either gamma interferon (IFN-γ), interleukin-2 (IL-2), or both, and the sensitivity of the assay was confirmed with IFN-γ and IL-2 enzyme-linked immunosorbent spot (ELISpot) assays. The data obtained in this study can aid in the design of vaccine protocols using i.d. EP, and the results emphasize the advantages of the FluoroSpot assay over traditional ELISpot assay and intracellular staining for the detection and quantification of bifunctional vaccine-specific immune responses.

Abstract: A plasmid DNA vaccine, encoding a truncated form of human CEA fused to a T-helper epitope (CEA66 DNA) was delivered three times intradermally at 2 mg or intramuscularly at 8 mg by Biojector® to patients with colorectal cancer. Prior to the first vaccination, all patients received cyclophosphamide (300 mg/m²) intravenously. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously with each vaccination. All patients completed the vaccine schedule. There were no grade 3 or 4 adverse events (AE). The most frequently reported AE grades 1 and 2 were injection site reactions, fatigue, headache, arthralgia, chest tightness and myalgia. Vaccination with CEA66 DNA in combination with GM-CSF was well tolerated and no signs of autoimmunity have been detected.

Abstract: Immunotherapy in patients with HIV-1 infection aims to restore and broaden immunological competence, reduce viral load and thereby permit longer periods without combined antiretroviral treatment (cART). Twelve HIV-1-infected patients on cART were immunized on the skin with DNA plasmids containing genes of several HIV-1 subtypes with or without the addition of hydroxyurea (HU), or with placebo. The mean net gain of HIV-specific CD8+ T cell responses were higher and broader in the HIV DNA vaccine groups compared to non-vaccinated individuals (p<0.05). The vaccine-induced immune responses per se had no direct effect on viral replication. In all patients combined, including placebo, the viral set point after a final structured therapy interruption (STI) was lower than prior to initiation of cART (p=0.003). Nadir CD4 levels appeared to strongly influence the post-STI viral titers. After the sixth immunization or placebo, patients could stay off cART for a median time of 15 months. The study shows that HIV DNA immunization induces broader and higher magnitudes of HIV-specific immune responses compared to structured therapy interruptions alone. Although compromised by small numbers of patients, the study also demonstrates that well-monitored STI may safely function as an immunological read out of HIV vaccine efficacy.

Abstract: The Notch ligand delta-like ligand 4 (DLL4) is an essential component expressed by endothelial tip cells during angiogenic sprouting. We have described a conceptually novel therapeutic strategy for targeting tumor angiogenesis and endothelial tip cells based on DNA vaccination against DLL4. Immunization with DLL4-encoding plasmid DNA by in vivo electroporation severely retarded the growth of orthotopically implanted mammary carcinomas in mice by induction of a nonproductive angiogenic response. Mechanistically, vaccination brought about a break in tolerance against the self-antigen, DLL4, as evidenced by the production of inhibitory and inherently therapeutic antibodies against mouse DLL4. Importantly, no evidence for a delayed wound healing response, or for toxicity associated with pharmacological blockade of DLL4 signaling, was noted in mice immunized with the DLL4 vaccine. We have thus developed a well-tolerated DNA vaccination strategy targeting the endothelial tip cells and the antigen DLL4 with proven therapeutic efficacy in mouse models of mammary carcinoma; a disease that has been reported to dramatically induce the expression of DLL4. Conceivably, induction of immunity toward principal mediators of pathological angiogenesis could provide protection against recurrent malignant disease in the adjuvant setting.

Abstract: It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vaxâ„¢ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.

Abstract: Heterologous boost immunisation is considered the most efficient way to enhance DNA-primed immune responses. We have previously shown that administration of recombinant carcinoembryonic antigen (CEA) efficiently boosts humoral responses in mice primed with CEA DNA. However, clinical grade recombinant proteins are far more intriguing to produce than plasmid DNA. Therefore, the possibility to use plasmid DNA for both priming and boosting would be beneficial. With the prospect of future use in a clinical trial, we investigated if electroporation-mediated delivery of DNA could be used to boost DNA-primed immune responses to CEA. The Biojector was used to prime BALB/c mice intradermally three times with CEA66 DNA, encoding an intracellular modified form of CEA. Twelve weeks after the last prime, the animals received either one injection of recombinant CEA or one intradermal injection of twtCEA DNA, encoding the wild type CEA fused to a tetanus T helper epitope, in combination with electroporation. Boosting with rCEA protein did not enhance T cell responses to CEA but induced CEA-specific IgG in 4 of 8 mice. In contrast, intradermal delivery of twtCEA DNA by electroporation led to a tenfold increase in IFN-gamma-producing CD8+ T cells, compared to the levels obtained after the third priming immunisation. The DNA boost also induced high CEA-specific IgG titers in all immunised animals (8/8). The data suggests that a late DNA boost, in combination with enhanced DNA delivery by electroporation, could be used to enhance the efficiency of DNA vaccination and substitute for a heterologous protein boost vaccination.

Abstract: Cancer is one of the leading causes of death in the Western world. Therapeutic vaccination to target minimal residual disease or prevent tumor recurrence represents an interesting and novel alternative for treatment of tumor diseases. T-cell peptide epitopes are commonly used as vaccine candidates for the induction of antitumor immune responses. By modifying the amino-acid sequence of the peptide at certain, so-called anchor positions, the binding affinity to MHC class I and the immunogenicity of the peptide can be improved. Vaccination with the modified peptide analogue can then be used to induce an immune response to the wild-type epitope.

Abstract: IFN-gamma, a pleiotropic immune regulator, is implicated in both tumor immune surveillance and selection of tumor variants resistant to immune control, i.e., immunoediting. In uveal melanoma patients, elevated serum levels of IFN-gamma correlate with the spread of metastasis and represent a negative prognostic marker. Treatment with IFN-gamma boosted the MHC class I presentation machinery in uveal melanoma cells but suppressed their MHC class I-restricted CTL lysis. Tumor cells exposed to IFN-gamma efficiently activated specific CTL but were less susceptible to permeabilization by perforin and exhibited a decreased capacity to bind and incorporate granzyme B. These results define a novel mechanism of resistance to granule-mediated CTL lysis in human tumors. Furthermore, the data suggest that immunoediting is not limited to genetic or epigenetic changes resulting in stable cellular phenotypes but also involves an inducible modulation of tumor cells in response to a microenvironment associated with immune activation.

Abstract: The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens. Furthermore, we show that the DNA priming increases the number of T-cell epitopes recognized after the MVA boost. In the prime/boost-immunized animals, a significant proportion of CD8(+) T cells were stained positive for both interferon-gamma (IFN-gamma) and interleukin-2 (IL-2), a feature that has been associated with control of HIV-1 infection in long-term non-progressors. The HIV-1-specific antibody levels were moderate after the plasmid DNA immunizations but increased dramatically after the MVA boost. Although the initial injection of MVA induced significant levels of vaccinia-neutralizing antibodies, the HIV-specific responses were still significantly boosted by the second MVA immunization. The results from this study demonstrate the potency of this combination of DNA plasmids and MVA construct to induce broad and high levels of immune responses against several HIV-1 proteins of different subtypes.

Abstract: New potent vaccine adjuvants are desirable for increasing the efficacy of novel vaccine modalities such as DNA and peptides. We therefore tested if syngeneic erythrocytes could serve as delivery vectors for selected HIV peptides and compared the potency of these constructs to immunization with peptides in phosphate buffered saline or in incomplete Freunds adjuvant. Immunization of mice with peptides in a low dose (5 ng) coupled to erythrocytes induced a weak immune response in mice. These peptides alone (5 microg) gave no immune responses, while formulating the peptides (50 microg) in IFA induced strong homologous immunity as well as prominent cross reactivity to a related mutant epitope. Thus, vaccine delivery using syngeneic erythrocytes, although attractive for clinical use, might be of limited value due to the low amount of antigen that can be loaded per erythrocyte.

Abstract: In preparation for a clinical trial in patients diagnosed with colorectal cancer, a vaccination strategy targeting the carcinoembryonic antigen (CEA) was evaluated in mice using a GMP-produced plasmid DNA vaccine, CEA66, encoding a truncated form of the tumour-associated antigen, CEA. The GMP-produced CEA DNA vaccine was also evaluated for toxicity. Repeated intradermal administration of the GMP-produced vaccine using a novel needle-free jet injection device (Biojector) induced robust CD4 and CD8 T-cell responses in mice, and did not result in any vaccine-related toxicity. In a heterologous DNA prime/protein boost setting, cellular immune responses were of higher magnitude in animals primed with CEA66 DNA than in animals receiving repeated doses of recombinant CEA protein. These responses were further enhanced if recombinant murine granulocyte-macrophage colony-stimulating factor was given as an adjuvant prior to vaccination. In contrast to repeated administration of recombinant CEA protein as a single modality vaccine, the heterologous CEA66 DNA prime/rCEA boost vaccination strategy resulted in a qualitatively broader immune response, and supports clinical testing of this vaccination regimen in humans.

Abstract: The route and method of immunization, as well as the cellular localization of the antigen, can influence the generation of an immune response. In general, intramuscular immunization results in Th1 responses, whereas intradermal delivery of DNA by gene gun immunization often results in more Th2 responses. Here we investigate how altering the cellular localization of the tumor antigen CEA (carcinoembryonic antigen) affects the quality and amplitude of DNA vaccine-induced antibody responses in mice following intradermal delivery of DNA by a needle-free jet injection device (Biojector). CEA was expressed either in a membrane-bound form (wild-type CEA) or in two truncated forms (CEA6 and CEA66) with cytoplasmic localization, where CEA66 was fused to a promiscuous T-helper epitope from tetanus toxin. Repeated intradermal immunization of BALB/c mice with DNA encoding wild-type CEA produced high antibody titers of a mixed IgG1/IgG2a ratio. In contrast, utilizing the DNA construct that resulted in intracellular targeting of CEA led to a reduced capacity to induce CEA-specific antibodies, but instead induced a Th1-biased immune response.

Abstract: The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types in vitro. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed.

Abstract: Mechanisms responsible for resistance of tumors to death receptor-mediated damage by cytotoxic lymphocytes are not well understood. Uveal melanoma cells expressed Fas but were insensitive to Fas triggering induced by bystander cytotoxic T lymphocytes or a Fas-specific agonistic antibody; this could not be ascribed to tumor counterattack against T cells or general resistance of the tumors to apoptosis. Treatment with inhibitors of metalloproteases rendered uveal melanomas sensitive to Fas-mediated cytotoxicity. Metalloprotease inhibitors did not affect the expression of Fas but increased the surface expression of Fas ligand (FasL), which correlated with the disappearance of soluble FasL from culture supernatants of tumor cells. FasL eluted from the surface of uveal melanomas specifically inhibited cytotoxic T lymphocyte lysis of tumor cells pretreated with an inhibitor of metalloproteases. In addition to uveal melanomas, a number of other tumor cell lines of various cellular origins were sensitized to Fas-mediated cytotoxicity by metalloprotease inhibitors. Our results show that autocrine secretion of FasL shields tumor cells from Fas-mediated killing by cytotoxic lymphocytes. This defines a novel mechanism of tumor escape from immune surveillance.

Abstract: It is demonstrated that similar to interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) induces coordinated changes at different steps of the major histocompatibility complex (MHC) class I processing and presentation pathway in nonprofessional antigen-presenting cells (APCs). TNF-alpha up-regulates the expression of 3 catalytic immunoproteasome subunits--LMP2, LMP7, and MECL-1--the immunomodulatory proteasome activator PA28 alpha, the TAP1/TAP2 heterodimer, and the total pool of MHC class I heavy chain. It was also found that in TNF-alpha--treated cells, MHC class I molecules reconstitute more rapidly and have an increased average half-life at the cell surface. Biochemical changes induced by TNF-alpha in the MHC class I pathway were translated into increased sensitivity of TNF-alpha--treated targets to lysis by CD8(+) cytotoxic T cells, demonstrating improved presentation of at least certain endogenously processed MHC class I--restricted peptide epitopes. Significantly, it was demonstrated that the effects of TNF-alpha observed in this experimental system were not mediated through the induction of IFN-gamma. It appears to be likely that TNF-alpha--mediated effects on MHC class I processing and presentation do not involve any intermediate messengers. Collectively, these data demonstrate the existence of yet another biologic activity exerted by TNF-alpha, namely its capacity to act as a coordinated multi-step modulator of the MHC class I pathway of antigen processing and presentation. These results suggest that TNF-alpha may be useful when a concerted up-regulation of the MHC class I presentation machinery is required but cannot be achieved by IFN-gamma. (Blood. 2001;98:1108-1115)

Abstract: Today, ample evidence demonstrates a clear role for the immune system in the battle against cancer. However, the relatively high rate of mutation and proliferation of tumor cells, in combination with the selective pressure exerted by the immune system, can potentially lead to the generation of genetically altered tumor cells, which are able to evade recognition by the immune system and continue to grow and form tumors. Increased knowledge of the mechanisms allowing tumors to escape from the immune system is of great importance in facilitating the design of effective immunotherapeutic regimens against cancer. The work described in this thesis was aimed at identifying new mechanisms of tumor escape as well as possible ways to counteract them. We have identified TNF-alpha as a potent modulator of MHC class I antigen presentation in tumors. TNF-alpha-treatment led to enhanced expression of several molecules in the MHC class I antigen processing and presentation pathway, including the IFN-inducible subunits of the proteasome, LMP2, LMP7 and MECL-1, the transporters associated with antigen presentation (TAP) and MHC class I heavy chain. These changes resulted in increased stability of surface MHC class I complexes, presumably due to an increased supply of peptides suitable for binding to MHC class I molecules, and enhanced susceptibility of TNF-alpha-treated tumors to antigen-specific lysis by cytotoxic T-lymphocytes (CTLs). Our results suggest a role for TNF-alpha as a potent immunomodulator in IFN-gamma unresponsive tumors. Investigating the possible effects of cytokines on the sensitivity of tumor cells to different CTL effector mechanisms, we found that IFN-gamma protects uveal melanoma cells from CTL-mediated lysis. We also demonstrated that despite potent upregulation of antigen presentation in uveal melanoma cells, IFN-gamma-treated tumor cells were less sensitive to lysis by CTL. Granzyme B is an apoptosis-inducing effector molecule released by CTLs upon triggering of the T-cell receptor. IFNgamma-treated uveal melanoma cells bound less granzyme B than their untreated, or TNF-alpha-treated, counterparts. Cleavage of the granzyme B substrate Bid was reduced in uveal melanoma cells following treatment with IFN-gamma. This correlated with a reduced expression of the cationindependent mannose-6-phosphate receptor (CI-MPR), a receptor for granzyme B, and decreased CTL-lysis of IFN-treated uveal melanoma cells. In another study, we examined the regulatory role of IFN-gamma on the sensitivity of uveal melanoma cells to the lytic activity of perforin, another major constituent of cytolytic granules. We demonstrated that IFN-gamma induces resistance of uveal melanoma cells to plasma membrane lysis by perforin. This was not a result of proteolytic inactivation of perform by either cathepsin B, known to protect CTL from perforin-mediated suicide, or other proteases. Protection from perforin lysis correlated with IFN-gamma-induced growth arrest in the G1-phase of the cell cycle, and reduced binding of perform to IFN-gamma-treated OCM1 cells. In light of the current data, we propose a mechanism were IFN-gamma-induced growth arrest leading to structural changes in the plasma membrane results in decreased perforin binding capacity of the tumor cell and protection from perforin. Our results demonstrate that, in response to IFN-gamma, tumors can escape the immune system through the active acquisition of a CTL-resistant phenotype, characterized by impaired sensitivity to granule-mediated killing. The second major effector mechanism employed by CTL is the engagement of death receptors expressed on target cells. The production of soluble Fas ligand (sFasL) completely protected uveal melanoma cells from killing via Fas. Inhibition of metalloproteases on the surface of tumor cells prevented shedding of Fast, and rendered uveal melanoma cells sensitive to Fasmeditated lysis by CTL. The protective effect of Fast, was not due to tumor counter-attack or reduced lytic potential of CTL, but transfer of sFasL-containing culture supernatant protected normally Fas-sensitive cells from killing induced both by FasLexpressing lymphocytes and a agonistic antibody to Fas. We speculated that soluble Fast, bind to Fas receptors expressed on tumor cells, thereby preventing their activation by Fas-inducing effector molecules. Our findings demonstrate the existence of a novel mechanism of tumor escape from death receptor-mediated killing by cytotoxic lymphocytes, and point to a new rationale for the use of metalloprotease inhibitors as cancer therapeutic agents.

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