Immunization with HIV protease peptides linked to syngeneic erythrocytes

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 μg) gave no immune responses, while formulating the peptides (50 μg) 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

Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

<p>Abstract</p> <p>Background</p> <p>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 <it>in vitro</it>. 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.</p> <p>Results</p> <p>The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.</p> <p>Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8⁺T cell memory responses, whereas MVA-encoded nef induced CD4⁺T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity.</p> <p>Conclusion</p> <p>This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4⁺or a CD8⁺T cell response depending on the choice of vector.</p

Modulation of tumor sensitivity to effector mechanisms of cytotoxic lymphocytes

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

Intracellular Targeting of CEA Results in Th1-Type Antibody Responses Following Intradermal Genetic Vaccination by a Needle-Free Jet Injection Device

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