Adenovirus expressing ß2-microglobulin recovers HLA class I expression and antitumor immunity by increasing T-cell recognition.

Optimal tumor cell surface expression of human leukocyte antigen (HLA) class I molecules is essential for the presentation of tumor-associated peptides to T-lymphocytes. However, a hallmark of many types of tumor is the loss or downregulation of HLA class I expression associated with ineffective tumor antigen presentation to T cells. Frequently, HLA loss can be caused by structural alterations in genes coding for HLA class I complex, including the light chain of the complex, ß2-microglobulin (ß2m). Its best-characterized function is to interact with HLA heavy chain and stabilize the complex leading to a formation of antigen-binding cleft recognized by T-cell receptor on CD8+ T cells. Our previous study demonstrated that alterations in the ß2m gene are frequently associated with cancer immune escape leading to metastatic progression and resistance to immunotherapy. These types of defects require genetic transfer strategies to recover normal expression of HLA genes. Here we characterize a replication-deficient adenoviral vector carrying human ß2m gene, which is efficient in recovering proper tumor cell surface HLA class I expression in ß2m-negative tumor cells without compromising the antigen presentation machinery. Tumor cells transduced with ß2m induced strong activation of T cells in a peptide-specific HLA-restricted manner. Gene therapy using recombinant adenoviral vectors encoding HLA genes increases tumor antigen presentation and represents a powerful tool for modulation of tumor cell immunogenicity by restoration of missing or altered HLA genes. It should be considered as part of cancer treatment in combination with immunotherapy.

Self-inactivating helper virus for the production of high-capacity adenoviral vectors.

Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >10¹° infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads.

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice.

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS "HLA Peptide Binding Predictions" program, we have identified and further characterized novel H-2 d-restricted CD8+ epitopes within the WHV core (peptides C#12-21, C#18-32, C#19-27, C#61-69) and surface antigens (peptides preS2#10-18, preS2#27-35, S#76-84, S#133-140 and S#257-265), respectively. These peptides bind to H-2 d with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-gamma producing T cells. More importantly, WHV core peptides C#19-27 and C#61-69 and WHV surface peptides S#133-140 and S#257-265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18-32, S#76-84, and S#257-265 resulted in significant production of IFN-gamma. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection.

Antitumoral efficacy of DNA nanoparticles in murine models of lung cancer and pulmonary metastasis.

Polyethylenimine (PEI)-DNA complexes are nanoparticles that are able to efficiently transfer plasmids to the lungs. Interleukin-12 (IL12) gene transfer using PEI may represent an important strategy for lung cancer treatment. In this study, we evaluated the antitumoral efficacy of the administration of PEI-DNA nanoparticles carrying IL12 gene (PEI-IL12) for the treatment of lung cancer and pulmonary metastases in animal models. After inoculation of tumor cells, mice were treated intravenously with a single dose of PEI-IL12, PEI nanoparticles carrying the reporter gene beta-galactosidase (PEI-LacZ) or vehicle. Transgene expression, survival rates and immune response were analyzed in both models. Administration of PEI-LacZ and PEI-IL12 nanoparticles controlled tumor growth and prolonged survival times in both animal models. Although PEI-IL12 and PEI-LacZ administration showed similar antitumoral effects in the lung cancer model, the efficacy of PEI-IL12 was significantly superior in the inhibition of the development of pulmonary metastases. Furthermore, the administration of PEI-DNA nanoparticles results in the production of high levels of proinflammatory cytokines. Our results showed that PEI-DNA nanoparticles are an efficient vector for mediating gene transfer to the lungs, are a potent inducer of the innate immune response and represents an interesting strategy for the treatment of bronchogenic carcinoma and metastatic lung carcinoma.

Efficient recovery of HLA class I expression in human tumor cells after beta2-microglobulin gene transfer using adenoviral vector: implications for cancer immunotherapy.

Here we report a successful use of a non-replicating adenovirus expressing the wild-type human beta2m gene in recovery of normal human leucocyte antigen (HLA) class I expression in beta2m-null cancer cells. Total loss of HLA class I expression in these cell lines is caused by a mutation in beta2m gene and a loss of heterozygosity in chromosome 15 carrying another copy of that gene. Normal HLA class I expression on the tumour cell surface is critical for the successful outcome of cancer immunotherapy as T cells can only recognize tumour-derived peptides in a complex with self-HLA class I molecules. In this report we characterize the newly generated adenoviral vector AdCMVbeta2m and demonstrate an efficient beta2m gene transfer in tumour cell lines of different histological origin, including melanoma, prostate and colorectal carcinoma. The beta2m re-expression lasted for an extended period of time both in vitro and in vivo in human tumour xenograft transplants. We propose that in a subset of cancer patients with structural defect in beta2m gene or chromosome 15, the adenoviral-mediated recovery (or even increase) of HLA class I expression on tumour cells in combination with vaccination or adoptive T-cell therapy can provide a complementary approach to improve the clinical efficacy of cancer immunotherapy.

Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR.

We describe a highly sensitive real-time PCR to detect and measure the development of the liver-stages of malaria parasites in mice infected with sporozoites ranging in number from 25 to more than 164,000, using the same reaction conditions. Furthermore, this assay detects and measures parasite loads in the livers of mice exposed to the bite of a single malaria-infected Anopheles mosquito. This unique method should greatly facilitate studies aimed at evaluating very precisely the efficacy of anti-malarial experimental drug treatments and vaccination regimens in conditions of infection resembling those found in the field.

Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen.

We report that complete protection against malaria and total inhibition of liver stage development and parasitemia was obtained in 100% of BALB/c mice primed with a replication-defective recombinant adenovirus expressing the circumsporozoite (CS) protein of Plasmodium yoelii (AdPyCS), followed by a booster with an attenuated recombinant vaccinia virus, expressing the same malaria antigen, VacPyCS. We found increased levels of activated CS-specific CD8(+) and CD4(+) T cells, higher anti-sporozoite antibody titers, and greater protection in these mice, when the time between priming and boosting with these two viral vectors was extended from 2 to 8 or more weeks. Most importantly, by using this immunization regimen, the protection of the immunized mice was found to be long-lasting, namely complete resistance to infection of all animals 3 1/2 months after priming. These results indicate that immunization with AdPyCS generates highly effective memory T and B cells that can be recalled long after priming by boosting with VacPyCS.

Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36.

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.

alpha -galactosylceramide-activated Valpha 14 natural killer T cells mediate protection against murine malaria.

Natural killer T (NKT) cells are a unique population of lymphocytes that coexpress a semiinvariant T cell and natural killer cell receptors, which are particularly abundant in the liver. To investigate the possible effect of these cells on the development of the liver stages of malaria parasites, a glycolipid, alpha-galactosylceramide (alpha-GalCer), known to selectively activate Valpha14 NKT cells in the context of CD1d molecules, was administered to sporozoite-inoculated mice. The administration of alpha-GalCer resulted in rapid, strong antimalaria activity, inhibiting the development of the intrahepatocytic stages of the rodent malaria parasites Plasmodium yoelii and Plasmodium berghei. The antimalaria activity mediated by alpha-GalCer is stage-specific, since the course of blood-stage-induced infection was not inhibited by administration of this glycolipid. Furthermore, it was determined that IFN-gamma is essential for the antimalaria activity mediated by the glycolipid. Taken together, our results provide the clear evidence that NKT cells can mediate protection against an intracellular microbial infection.

Immunization and infection change the number of recombination activating gene (RAG)-expressing B cells in the periphery by altering immature lymphocyte production.

Recombination activating gene (RAG) expression in peripheral B cells increases after immunization with (4-hydroxy-3-nitrophenyl) acetyl coupled to chicken gamma globulin (NP-CGG) in alum. This increase could result from reinduction of RAG expression or, alternatively, from accumulation of RAG-expressing immature B cells in the periphery. We have used mice that carry a green fluorescent protein (GFP) RAG indicator transgene (RAG2-GFP) to characterize the RAG-expressing B cells in immunized spleens. Most of the RAG2-GFP-expressing B cells in unimmunized spleen are immature B cells. Injection with NP-CGG in alum initially suppresses lymphopoiesis in the bone marrow and decreases the number of immature RAG2-GFP-expressing B cells in the spleen. Recovery of lymphopoiesis in the bone marrow coincides with accumulation of RAG-expressing immature B cells in the spleen. Most of the RAG-expressing cells that accumulate in the spleen after immunization do not proliferate and they are not germinal center cells. Neither the initial suppression of lymphopoiesis nor the subsequent accumulation of RAG-expressing cells in the spleen is antigen dependent, since similar changes are seen with alum alone. Furthermore, such changes in the numbers of developing and circulating immature lymphoid cells are seen after injection with complete Freund's adjuvant or malaria infection. Our experiments suggest that adjuvants and infectious agents cause previously unappreciated alterations in lymphopoiesis resulting in the accumulation of RAG-expressing immature B cells in the spleen.

Differential protein kinase C phosphorylation sites in the L17 ribosomal protein from Leishmania infantum.

Leishmania infantum, the protozoan parasite responsible for leishmaniasis in Europe, is capable of undergoing developmental changes in vitro and provides an excellent model for the study of cell differentiation processes. We have cloned the gene encoding the L17 ribosomal protein. The LiL17 protein family belongs to the macrolide binding site, related to the peptidyl transferase center of the ribosome. Its comparison with other members of the protein family shows several structural differences that may reflect functional variations. The protein kinase C phosphorylation sites display an intermediate pattern involving differences in location and type of residue with respect to all the species considered. Gene-structural analysis suggests the existence of two different encoding genes. The expression of the genes seem to be different with the distinct growth phases of the parasite.

Molecular cloning, cell localization and binding affinity to DNA replication proteins of the p36/LACK protective antigen from Leishmania infantum.

The p36/LACK antigen from Leishmania, an analogue of the receptor for activated protein kinase C (PKC), induces high levels of protection against parasite infection in the BALB/c mouse model. This protection is more than twice as high as that elicited by major parasite antigens such as soluble Leishmania antigen or the main surface protease gp63. We have cloned and purified p36/LACK from Leishmania infantum, the causative agent of visceral leishmaniasis in Europe. This protein belongs to the large family of WD 40 repeat proteins confined to eukaryotes and involved in numerous regulatory functions. Differential solubilization and immunofluorescence experiments indicate that p36/LACK is present close to the kinetoplast disc in the cell cytoplasm, probably bound to multiprotein complexes but not to membrane structures. These complexes probably also include cytoplasm PKC isoforms. The use of a genetically-encoded peptide library indicates that p36/LACK binds sequences present in several proteins involved in DNA replication and RNA synthesis. The recognition and binding sequences present in vacuolar proteins and at the beta-chain of major histocompatability complex (MHC) class II suggest the involvement of this regulatory protein in the early mechanisms triggering the protective immune response of the host against the parasite infection.

Cloning, molecular analysis and differential cell localisation of the p36 RACK analogue antigen from the parasite protozoon Crithidia fasciculata.

The family of the RACK molecules (receptors for activated C kinases) are present in all the species studied so far. In the genus Leishmania, these molecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the parasite trypanosomatid Crithidia fasciculata (CACK). The molecule seems to be encoded by two genes. The sequence analysis of the cloned open reading frame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three of them, shared with the mammalian species, additional to those present in the other protozoa suggesting a certain phylogenetic distance between the protozoon Crithidia fasciculata and the rest of the Trypanosomatidae. The CACK-encoded polypeptide shows an additional sequence of four amino acids at the carboxy-terminal end, which produces a different folding of the fragment with the presence of an alpha-helix instead of the beta-sheet usual in all the other species studied. A similar result is elicited at the amino-terminal end by the change of three amino acid residues. The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainly at the plasma membrane, different from that of the related Leishmania species used as control, that displays a distribution close to the nucleus. Altogether, the data suggest that the existence of the structural differences found may have functional consequences.

Cloning and structural analysis of the gene encoding the ribosomal protein S6 from the parasite Leishmania infantum.

We have cloned the S6 ribosomal protein encoding gene from a Leishmania infantum cDNA library. This parasite protozoon, responsible for leishmaniasis in Europe, is able to undergo developmental changes in vitro and results a good model to study cell differentiation processes. The LiS6 protein sequence indicates its pertinence to the S6 protein family, related to the early mechanisms of cell division, differentiation and activation, and shows an intermediate position between the yeasts and higher eukaryotes. Thus, LiS6 protein has the same amino acid length as that of the higher eukaryotes and certain common features such nucleus entrance sequences and several kinase phosphorylation sites. However, the key functional protein kinase C phosphorylation sites are at different locations and present several threonine instead of the usual serine residues. The gene structural analysis suggest the presence of three different encoding genes that do not present remarkable changes along the different phases of the parasite.

Cloning of the gp63 surface protease of Leishmania infantum. Differential post-translational modifications correlated with different infective forms.

The Leishmania cell surface virulence factor gp63 is a protease family that plays an important role in the survival of the parasite protozoon into the host macrophages. We have cloned and characterised the gp63 gene from L. infantum. The sequence analysis of the gene indicates the existence of a high degree of conservation with the other old world species L. major and L. donovani. The similarity is lower with new world species with the exception of L. chagasi which shows a strikingly high percentage of identity (99-100%). In L. infantum the gp63 gene expresses two polypeptides of 58 and 60 kDa, respectively, which show a similar proteolytic activity. The 60 kDa polypeptide is expressed during the whole life cycle of the promastigote form of the parasite with a moderate increase at the stationary phase of growth while the 58 kDa product, although slightly present in the logarithmic phase, notable increases its expression during the highly infectious stationary phase. RNA analysis showed that the presence in L. chagasi of these two polypeptides correlates with two RNA molecules and with the degree of parasite infectivity, whereas in the case of L. infantum a single 3 kb messenger RNA is detected through the whole promastigote life cycle. Our data indicate that in L. infantum, the differences in gene expression of the gp63 protease family according to parasite phase of growth seem to be due to a differential pattern of glycosilation of the polypeptides which correlates with the different infective forms of the promastigote form of the parasite.