Methionine tumor starvation by erythrocyte-encapsulated methionine gamma-lyase activity controlled with per os vitamin B6.

Erymet is a new therapy resulting from the encapsulation of a methionine gamma-lyase (MGL; EC number 4.4.1.11) in red blood cells (RBC). The aim of this study was to evaluate erymet potential efficacy in methionine (Met)-dependent cancers. We produced a highly purified MGL using a cGMP process, determined the pharmacokinetics/pharmacodynamics (PK/PD) properties of erymet in mice, and assessed its efficacy on tumor growth prevention. Cytotoxicity of purified MGL was tested in six cancer cell lines. CD1 mice were injected with single erymet product supplemented or not with vitamin B6 vitamer pyridoxine (PN; a precursor of PLP cofactor). NMRI nude mice were xenografted in the flank with U-87 MG-luc2 glioblastoma cells for tumor growth study following five intravenous (IV) injections of erymet with daily PN oral administration. Endpoints included efficacy and event-free survival (EFS). Finally, a repeated dose toxicity study of erymet combined with PN cofactor was conducted in CD1 mice. Recombinant MGL was cytotoxic on 4/6 cell lines tested. MGL half-life was increased from <24 h to 9-12 days when encapsulated in RBC. Conversion of PN into PLP by RBC was demonstrated. Combined erymet + PN treatment led to a sustained Met depletion in plasma for several days with a 85% reduction of tumor volume after 45 days following cells implantation, and a significant EFS prolongation for treated mice. Repeated injections in mice exhibited a very good tolerability with only minor impact on clinical state (piloerection, lean aspect) and a slight decrease in hemoglobin and triglyceride concentrations. This study demonstrated that encapsulation of methioninase inside erythrocyte greatly enhanced pharmacokinetics properties of the enzyme and is efficacy against tumor growth. The perspective on these results is the clinical evaluation of the erymet product in patients with Met starvation-sensitive tumors.

Asparagine Synthetase Expression and Phase I Study With L-Asparaginase Encapsulated in Red Blood Cells in Patients With Pancreatic Adenocarcinoma.

OBJECTIVES: Asparaginase encapsulated in erythrocytes (ERY-ASP) is a potentially effective drug in patients with pancreatic adenocarcinoma (PAC) with null/low asparagine synthetase (ASNS) expression. Our aims were to assess ASNS expression in PAC from a large cohort and its prognostic and/or predictive value and to conduct a phase I trial with ERY-ASP in patients with metastatic PAC. METHODS: Asparagine synthetase expression was evaluated using immunohistochemistry in resected PAC (471 patients) and in pairs of primary tumor and metastases (55 patients). Twelve patients were included in the phase I trial and received a single administration of ERY-ASP (25-150 IU/kg). RESULTS: Null/low ASNS expression was found in 79.4% of the resected PAC with a high concordance between primary tumor and metastases. Asparagine synthetase expression was significantly correlated with sex and CXCR4 expression. In the phase I trial, ERY-ASP was well tolerated by patients with metastatic PAC. No patient had DLTs, and 6 patients had at least 1 ERY-ASP causally related adverse event out of the 12 adverse events reported. CONCLUSIONS: Given the high rate of PAC with null/low ASNS expression and the good tolerability profile of ERY-ASP, ERY-ASP should be evaluated in further clinical studies in metastatic PAC.

Innovative approach in Pompe disease therapy: Induction of immune tolerance by antigen-encapsulated red blood cells.

Pompe disease is a glycogen storage disease caused by acid α-glucosidase enzyme deficiency. Currently, the unique treatment is lifelong enzyme replacement therapy ERT with frequent intravenous administration of the recombinant analog alglucosidase-α (AGA), which ultimately generates a sustained humoral response resulting in treatment discontinuation. Our aim is to use the tolerogenic properties of antigen-encapsulated red blood cells (RBCs) to abolish the humoral response against AGA and to restore tolerance to replacement therapy. To demonstrate that our approach could prevent the AGA-induced immune response, mice were intravenously injected three times with AGA encapsulated into RBCs before being sensitized to AGA with several adjuvant molecules. Control animals received injections of free AGA instead of the encapsulated molecule. One-week after treatment with AGA-loaded RBCs, a strong decrease in specific humoral response was observed despite three stimulations with AGA and adjuvant molecules. Furthermore, this specific immunomodulation was maintained for at least two months without affecting the overall immune response. AGA-loaded RBCs represent a promising strategy to induce or restore tolerance in Pompe disease patients who develop hypersensitivity reactions following repeated AGA administrations.

A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague.

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

Construction and tropism characterisation of recombinant viruses exhibiting HIV-1 env gene from seminal strains.

Genetic differences between blood and mucosal-derived HIV-1 strains have been widely reported. As amplification of HIV-1 strains from mucosal samples including semen or saliva by co-culture has low sensitivity, we developed the construction of chimeric viruses expressing wild-type seminal HIV-1 envelope protein. Chimeric viruses were produced by co-transfection of a V1-V3 deleted pNL 43 vector and PCR fragments spanning the deleted region, amplified from HIV-1 RNA positive seminal plasma samples. After an initial testing of co-receptor usage by a tropism recombinant test, replication capacity and amplification of these recombinant viruses were assessed using PBMC. Four chimeric replicative strains, all using CXCR4 as coreceptor, were produced. The interaction between cell-free viral particles and reporter cell lines was assessed by confocal microscopy. These replicative chimeras exhibiting HIV-1 env from seminal strains represent useful tools for the in vitro study of the heterosexual transmission of HIV-1 and testing of microbicide activity.

Opposite immune reactivity of serum IgG and secretory IgA to conformational recombinant proteins mimicking V1/V2 domains of three different HIV type 1 subtypes depending on glycosylation.

The V1/V2 domain of the HIV-1 gp120 envelope protein has been shown to contribute to viral cell tropism during infection and also to viral recognition by neutralizing monoclonal antibodies. However, this domain has been poorly investigated. Carbohydrates have been demonstrated to dramatically influence immune reactivity of antisera to viral glycoprotein antigens. In this study, DNA sequences coding for V1/V2 domains from HIV-1 primary isolates of three subtypes (A, B, and C) were subcloned into a secretion vector and used to transfect CHO cells that are able to achieve the glycosylation of proteins. The structure of purified recombinant V1/V2 proteins was tested using two anti-V1/V2 monoclonal antibodies directed against either a linear or a conformational and glycosylation-dependent epitope (8.22.2 and 697-D). Serum or saliva of 14/82 seropositive patients with anti-V1/V2 reactivity demonstrated good recognition of the recombinant proteins. Deglycosylation of the recombinant proteins was found to increase the reactivity of the serum IgG to the clade A and C but not to clade B V1/V2 domain demonstrating that the recognition of glycosylation sites by serum IgG is clade dependent. When considering SIgA from parotid saliva, deglycosylation of all recombinant proteins tested decreased the reactivity, suggesting that glycosylation plays an important role in the recognition of V1/V2 domain target epitopes by this class of antibodies. In conclusion, these results suggest the influence of carbohydrate moieties on the specificity of the antibodies to the V1/V2 domain produced during HIV infection and the potential importance of viral glycans in vaccine responses after mucosal administration.

Amount of seminal IL-1beta positively correlates to HIV-1 load in the semen of infected patients.

BACKGROUND: Cytokines present in the sperm could influence the heterosexual transmission of HIV by modulating viral titers and influencing the early immune response in the vaginal mucosa. OBJECTIVES: To assess the relation between cytokine concentrations and HIV status in the seminal plasma. STUDY DESIGN: Twenty-five HIV positive subjects were tested for cytokine content and HIV-1 load in seminal and blood plasma through a cross-sectional study. RESULTS: HIV positive subjects exhibited a significantly higher amount of seminal IL-1beta as compared to a group of 33 HIV negative controls. In HIV positive subjects, amounts of IL-1beta and HIV-1 RNA in semen were significantly correlated and a trend for correlation was found between seminal IL-1beta and blood HIV-1 RNA. Amount of seminal IL-1beta was significantly lower in patients under HAART, according to the decrease of their viral loads in blood and semen. CONCLUSIONS: Considering that IL-1beta is known to enhance viral replication and to promote immune response, its dosage in semen could represent an interesting marker for identifying patients at high risk for HIV heterosexual transmission.

Selective sequestration of X4 isolates by human genital epithelial cells: Implication for virus tropism selection process during sexual transmission of HIV.

X4 and R5 HIV strains are present in the semen of men infected with HIV but R5 isolates are transmitted preferentially. The role of human epithelial cells in this selection is addressed. Three human cervical cell lines-CaSki, SiHa, and HEC1A-and normal human vaginal cells from HIV-negative donors were characterized for HIV receptor expression and incubated with X4 and R5 laboratory-adapted strains or primary isolates. The infection was assessed by detection of intracellular HIV DNA. The three cell lines were shown to express on their surface the CXCR4 and GalCer molecules, but not the CD4 and CCR5 ones. The three cell lines and normal human vaginal cells were found to be selectively permissive to X4 HIV entry; the preincubation of the cell lines with rhSDF-1 inhibited this infection. The detection of the intracellular proviral DNA in the cell lines and in normal human vaginal cells demonstrated a selective integration of X4 strains. Additional experiments showed that no extracellular RNA was detected in the supernatants of HEC1A cells infected by X4 isolates either after 18 days of culture or after incubation with PHA-stimulated PBMCs and that no transmission occurred after co-culture between infected HEC1A cells and PHA-stimulated PBMCs. These results suggest specific sequestration of X4 strains by genital epithelial cells, which could explain, at least in part, the HIV tropism selection process during sexual intercourse.

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction.

Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1beta through the nuclear factor-kappaB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

HIV-1 infection of Langerhans cells in a reconstructed vaginal mucosa.

We have developed an in vitro reconstructed vaginal mucosa integrating Langerhans cells (LCs), obtained by differentiation of umbilical cord blood CD34(+) hematopoietic progenitor cells, and, in this model, we have investigated the infection of LCs by human immunodeficiency virus type 1 (HIV-1). Proviral DNA of X4 (LAI and NL4-3) and R5 (Ba-L) HIV-1 strains were detected in LCs integrated in the reconstituted mucosa. Infection of LCs could be specifically inhibited by the chemokines stromal cell-derived factor 1 (SDF-1) and RANTES (regulated on activation, normally T cell-expressed and -secreted), confirming the presence of functional coreceptors on LCs generated in vitro. A complete inhibition of LCs, by use of azidothymidine, a reverse-transcriptase inhibitor, was also observed. Moreover, HIV-1-infected LCs of the reconstructed mucosa were able to transmit R5 or X4 HIV-1 strains to activated peripheral blood mononuclear cells. Such a model could be a useful tool to study the mechanisms involved in transmission of HIV in the female genital tract.

Detection of enterovirus in human skeletal muscle from patients with chronic inflammatory muscle disease or fibromyalgia and healthy subjects.

Enterovirus RNA has been found previously in specimens of muscle biopsy from patients with idiopathic dilated cardiomyopathy, chronic inflammatory muscle diseases, and fibromyalgia or chronic fatigue syndrome (fibromyalgia/chronic fatigue syndrome). These results suggest that skeletal muscle may host enteroviral persistent infection. To test this hypothesis, we investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay the presence of enterovirus in skeletal muscle of patients with chronic inflammatory muscle diseases or fibromyalgia/chronic fatigue syndrome, and also of healthy subjects. Three of 15 (20%) patients with chronic inflammatory muscle diseases, 4 of 30 (13%) patients with fibromyalgia/chronic fatigue syndrome, and none of 29 healthy subjects was found positive. The presence of VP-1 enteroviral capsid protein was assessed by an immunostaining technique using the 5-D8/1 monoclonal antibody; no biopsy muscle from any patient or healthy subject was found positive. The presence of viral RNA in some muscle biopsies from patients exhibiting muscle disease, together with the absence of VP-1 protein, is in favor of a persistent infection involving defective viral replication.