Technology transfer hub for pandemic influenza vaccine.

Increase of influenza vaccine production capacity in developing countries has been identified as an important element of global pandemic preparedness. Nevertheless, technology transfer for influenza vaccine production to developing country vaccine manufacturers has proven difficult because of lack of interested technology providers. As an alternative to an individual provider-recipient relationship, a technology and training platform (a "hub") for a generic non-proprietary process was established at a public sector European manufacturer's site. The conditions for setting up such a platform and the potential applicability of this model to other biologicals are discussed.

Hepatitis C virus structural proteins do not prevent human dendritic cell maturation.

AIM: Infection with hepatitis C virus (HCV) results in chronic hepatitis in more than 70% of cases. Alterations in the maturation of dendritic cells (DC) might play a role in the immune system's inability to eliminate the virus, although viral factors that could be involved have not been identified. This study in vitro investigated whether HCV structural proteins affect maturation of monocyte-derived DC. METHODS: HCV proteins (core, E1, E2) were expressed by transduction with recombinant adenoviruses of immature DC. The ability of these transduced DC to respond to a maturation stimulus was evaluated by measuring cell surface markers, allogenic lymphocyte stimulation and interleukin (IL)-12 production. RESULTS: Expression of HCV structural proteins did not modify DC maturation in the presence of lipopolysaccharide, as determined by their phenotype and stimulatory functioning. IL-12 secretion was not affected by HCV protein expression in mature DC. CONCLUSION: Our results suggest that HCV structural proteins do not affect maturation of monocyte-derived DC by lipopolysaccharide. These findings are important for further studies to clarify the pathogenesis of chronic HCV infection and towards the rational design of cellular vaccine approaches for immunotherapy against hepatitis C.

Ethical considerations related to the provision of care and treatment in vaccine trials.

Ethical principles of beneficence and justice combined with international human rights norms and standards create certain obligations on researchers, sponsors and public health authorities. These include treatment provision for participants enrolled in clinical trials of vaccines, drugs and other new preventive and curative technologies and methods. However, these obligations are poorly defined in practical terms, inconsistently understood or inadequately applied. Vaccine clinical trial designs normally define standards of prevention applicable to the population where the trial is to take place. The present document addresses specifically the setting of standards applicable to care and treatment in vaccine trials. The lack of clear guidance on how to achieve the optimal synergy between the development of new health technologies, on the one hand, and the promotion and protection of ethical and human rights principles, on the other, is a barrier to the progress of health research and therefore to the advancement of public health. The World Health Organization and UNAIDS have engaged in a series of consultations in Africa, the Americas, Asia and Europe to reflect on how this aim could best be achieved. This document highlights the outcome of these consultations. It proposes a structured approach to consensual decision making in the context of the clinical trial of vaccines against such public health challenges as HIV and newly emerging or threatening epidemics. A structured approach involving investigators and sponsors in a consultative process with trial communities and other stakeholders in research will ensure that the needs and legitimate expectations of trial participants are appropriately met, obligations towards them are delivered and, as a result, ethical research is facilitated in the interest of public health.

Malaria vaccine development: progress and challenges.

A safe and effective malaria vaccine would contribute greatly to the control and prevention of the disease. Although a review of global activity in malaria vaccine development does reflect significant activity, progress has remained slow. This article discusses the current vaccine candidates, with emphasis on those in the clinic, and explains the numerous challenges to making and evaluating malaria vaccines, which have resulted in only a few approaches being adopted and repeatedly evaluated. Against a parasite with more than 5200 genes, the lack of definitive knowledge regarding the nature of protective immunity and absence of reliable surrogates of protection are among the key challenges to a rational evaluation and prioritization of candidate vaccines. Pursuing the current R&D strategies may not result in the availability of a vaccine with characteristics suitable to impact significantly on disease morbidity in developing countries. Therefore, it is critical that the main challenges to malaria vaccine development be unambiguously identified and collectively addressed.

Review on flavivirus vaccine development. Proceedings of a meeting jointly organised by the World Health Organization and the Thai Ministry of Public Health, 26-27 April 2004, Bangkok, Thailand.

In light of the continuous spread of human pathogenic flaviviruses, in particular the mosquito-transmitted species, vaccine development remains a high priority on the public health agenda. On 26-27 April 2004, a conference was held in Bangkok, Thailand, to review current status of flavivirus vaccine development and related issues, focussing on dengue (DEN) and Japanese encephalitis (JE). This event, co-sponsored by the World Health Organization (WHO) and the Thai Ministry of Public Health, reviewed the progress made with vaccine development, sero-epidemiological studies and other accompanying activities critical for vaccine development and vaccination. The considerable interest in and awareness of the flavivirus diseases and their prevention by public health decision makers, as well as the establishment of two dedicated programmes for dengue and Japanese encephalitis vaccine development raise hopes that new or improved vaccines will become available in the coming years.

HCV core, NS3, NS5A and NS5B proteins modulate cell proliferation independently from p53 expression in hepatocarcinoma cell lines.

Several reports have shown that activity and/or expression of p53 can be modulated by Hepatitis C virus (HCV) proteins and may interfere with normal regulation of cell growth. In order to understand the relationship between p53 function and HCV proteins expression, we have investigated potential effects of the core, NS3, NS5A and NS5B proteins on Huh-7 (p53 +/+) and Hep3B (p53 -/-) cell proliferation. The effect of HCV proteins transiently expressed after recombinant-adenoviral infection was analyzed by Western blot, crystal violet and propidium iodide staining. Expression of the core, NS3, NS5A or NS5B proteins inhibited cell proliferation and blocked both cell lines in the G2/M phase of the cell cycle. c-myc and p53 expression were respectively induced and increased in Huh-7 cells only following expression of the Core protein. No expression of p21(waf1/cip1) could be detected and expression of cyclin A, cdk2 and p27(Kip1) were independent of HCV protein's expression. Our results show that the effect of core, NS3, NS5A and NS5B on cell proliferation is independent of p53 expression and that only the Core protein, induces the expression of both c-myc and p53.

Recombinant vaccinia viruses expressing immunoglobulin variable regions efficiently and selectively protect mice against tumoral B-cell growth.

The variable regions of the immunoglobulin (Ig) expressed on the surface of a malignant B cell can be considered tumor-specific antigens and, as such, could be targets for immunotherapeutic approaches. However, because until now the immunization procedures have been complex and have given only partial protection, it was necessary to find new methods of immunotherapy. Here, we present a successful method of vaccination against B-cell tumors in a murine model. We produced recombinant vaccinia viruses (rVV) expressing the heavy and the light chain of surface Ig of a patient's malignant B cells and we tested the ability of these rVV to protect immunized mice against tumor growth of transfectomas producing the same human Ig. The protection of the mice was complete and specific to the variable region of the immunizing heavy chain although specific lymphoproliferative and cytotoxic responses were not detectable in vitro. The protection was strictly dependent on the presence of CD4 T cells and asialo GM1+ cells. Furthermore, tumor protection clearly required gamma-interferon and was partially inhibited by blocking the Fas-Fas ligand interaction. We also show, in a murine syngeneic model, that rVV expressing a poorly mutated Ig protects against the growth of Ig-producing tumor.

Persistence of pathogenic challenge virus in macaques protected by simian immunodeficiency virus SIVmacDeltanef.

Live attenuated simian immunodeficiency virus (SIV) is the most efficient vaccine yet developed in monkey models of human immunodeficiency virus infection. In all successful vaccine trials, attenuation was achieved by inactivating at least the nef gene. We investigated some virological and immunological characteristics of five rhesus macaques immunized with a nef-inactivated SIVmac251 molecular clone (SIVmac251Deltanef) and challenged 15 months later with the pathogenic SIVmac251 isolate. Three animals were killed 2 weeks postchallenge (p.c.) to search for the challenge virus and to assess immunological changes in various organs. The other two animals have been monitored up for 7 years p.c., with clinical and nef gene changes being noted. The animals killed showed no increase in viral load and no sign of a secondary immune response, although the challenged virus was occasionally detected by PCR. In one of the monkeys being monitored, the vaccine virus persisted and an additional deletion occurred in nef. In the other monkey that was monitored, the challenge and the vaccine (Deltanef) viruses were both detected by PCR until a virus with a hybrid nef allele was isolated 48 months p.c. This nef hybrid encodes a 245-amino-acid protein. Thus, our results show (i) that monkeys were not totally protected against homologous virus challenge but controlled the challenge very efficiently in the absence of a secondary immune response, and (ii) that the challenge and vaccine viruses may persist in a replication-competent form for long periods after the challenge, possibly resulting in recombination between the two viruses.

Vaccines for parasitic diseases.

Significant effort and progress has occurred over the last several years in the development of vaccines against three main tropical parasitic diseases (malaria, leishmaniases and schistosomiasis). However, an effective vaccine is not yet available. The difficulties in developing a vaccine against parasitic disease are complicated not only by the necessity to identify (and produce) appropriate, protective antigens but also a lack of complete understanding of the types of immune responses needed for protection. Despite these hurdles, several candidate vaccines are under development for each disease; at least one promising vaccine candidate exists that is in late stage clinical testing.

Recombinant vaccinia virus encoding human MUC1 and IL2 as immunotherapy in patients with breast cancer.

Polymorphic epithelial mucin, encoded by the MUC1 gene, is present at the apical surface of glandular epithelial cells. It is over-expressed and aberrantly glycosylated in most breast tumors, resulting in an antigenically distinct molecule and a potential target for immunotherapy. This transmembrane protein, when produced by tumor cells, is often cleaved into the circulation, where it is detectable as a tumor marker (CA 15.3) by various antibodies, allowing for early detection of recurrences and evaluation of treatment efficacy. The objective of the current study was to examine the clinical and environmental safety and immunogenicity of a live recombinant vaccinia virus expressing the human MUC1 and IL2 genes (VV TG5058), referred to here as TG1031. The study was an open-label phase 1 and 2 trial in nine patients with advanced inoperable breast cancer recurrences to the chest wall. The patients were vaccinated intramuscularly with a single dose of TG1031; three patients were treated at each of three progressive dose levels ranging from 5x10(5) to 5x10(7) plaque-forming units. A boost injection at their original dose level was administered in patients responding immunologically, clinically, or both. Vaccination resulted in no significant clinical adverse effects, and there was no environmental contamination by live TG1031. All patients had been vaccinated as children, and patients treated at the highest dose level mounted a significant anti-vaccinia antibody response. None of the nine patients had a significant increase in MUC1-specific antibody titers after one single injection, whereas five patients had a detectable increase in vaccinia virus antibody titers. Peripheral blood mononuclear cells of one patient at the intermediate dose level showed a proliferative response to in vitro culture with vaccinia virus, with a stimulation index of 6. A second patient treated at the intermediate dose level had a stimulation index of 7 to MUC1 peptide and of 14 after a boost injection. This patient had a concomitant decrease in carcinoembryonic antigen serum levels and remained clinically stable for 10 weeks. Evidence of MUC1-specific cytotoxic T lymphocytes was detected in two patients. Immunohistochemical analysis revealed an increase in T memory cells (CD45RO) in tumor biopsies after vaccination. The absence of serious adverse events, together with the documentation of immune stimulations in vivo, warrant the further use of TG1031 in immunotherapy trials of breast cancer.

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the env precursor interferes with membrane fusion at a post-CD4 binding step.

CLIV is a multiple antigen peptide ([PTKAKRRVVQREKR](4)-K(2)-K-betaA) that encompasses the cleavage region of the human immunodeficiency virus type 1 (HIV-1) envelope precursor. It displays an antiviral activity against HIV-1 and HIV-2 and inhibits HIV-1 Env-mediated cell-to-cell fusion. This effect has previously been attributed to interference with Env processing, resulting in the expression of a nonfusogenic envelope [Virology (1998) 247, 137]. However, we show here that CLIV does not alter the status of Env cleavage at steady state. Using various aggregation/syncytium assays that allow us to discriminate between gp120/CD4 binding and binding followed by gp41-mediated fusion, we demonstrate that CLIV inhibits a step of the cell-to-cell fusion process after CD4 binding. We demonstrate also that CLIV binds at 37 degrees C to a single class of protein present at the CD4(+) cell surface (Scatchard analysis: K(d) = 8 nM; B(max) = 10(4) sites/cell) and that the fusion inhibition activity seems to correlate with binding to this proteic component. In contrast, CLIV interacts with neither membrane-inserted nor CD4-associated Env. We therefore propose that CLIV interferes after Env/CD4 binding with a step of the membrane fusion process that may involve the C-terminal domain of gp120.

Redirected cellular cytotoxicity by infection of effector cells with a recombinant vaccinia virus encoding a tumor-specific monoclonal antibody.

Cytotoxicity is an important function of the immune system that results in the destruction of cellular targets by humoral and/or cellular mechanisms. We wanted to assess the possibility of targeting the lytic function of immune cells toward cancer cells, which express the gene coding for a known tumor antigen (Ag) (GA733-2/epithelial cell adhesion molecule), using a viral vector encoding a monoclonal antibody (mAb) specific for said tumor Ag (CO17-1A). To this end, we have constructed recombinant vaccinia viruses expressing the sequences corresponding to mAb CO17-1A, which recognizes a specific Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. The recombinant vectors encoding either a secreted or membrane-anchored form of CO17-1A mAb were used to infect effector cells, which were subsequently assessed for their cytotoxic activity. The recombinant viruses were able to infect both granulocyte-macrophage colony-stimulating factor-activated human macrophages and Ag-stimulated murine cytotoxic T lymphocytes. Infected granulocyte-macrophage colony-stimulating factor-activated macrophages were found to be able to kill GA733-2-expressing tumor cells. Likewise, infected cytotoxic T lymphocytes, although conserving their original alloreactivity, gained the capability of killing GA733-2-expressing cancer cells.

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines.

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen.

Mucosal immunity and tolerance: relevance to vaccine development.

The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen-presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S-IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected antigens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue-damaging inflammatory reactions triggered by persistent microorganisms.

Lack of evidence for an immunosuppressive role for MUC1.

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1-and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells.

Properties of HIV envelope expressed in the presence of SPC3, an Env-derived peptide drug under phase II clinical trials.

A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4+ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4+ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.

Production of cholera toxin B subunit in Lactobacillus.

The intracellular expression of the B subunit of cholera toxin (CTB) was first achieved in Lactobacillus paracasei LbTGS1.4 with an expression cassette including the P25 promoter of Streptococcus thermophilus combined with the translation initiation region from the strongly expressed L. pentosus D-lactate dehydrogenase gene (ldhD). Secretion of CTB was next attempted in L. paracasei LbTGS1.4 and L. plantarum NCIMB8826 with four different signal sequences from exported proteins of lactic acid bacteria (Lactococcus lactis Usp45 and PrtP, Enterococcus faecalis unknown protein and S. pyogenes M6 protein). Host-dependent secretion of CTB was clearly observed: whereas none of the secretion cassettes led to detectable CTB in the extracellular fraction of L. paracasei LbTGS1.4, secretion of CTB molecules was clearly achieved with three of the selected signal sequences in L. plantarum NCIMB8826.