Virus-like particles as HIV-1 vaccines

Traditional successful antiviral vaccines have relied mostly on live-attenuated viruses. Live-attenuated HIV vaccine candidates are not ideal as they pose risks of reversion, recombination or mutations. Other current HIV vaccine candidates have difficulties generating broadly effective neutralising antibodies and cytotoxic T cell immune responses to primary HIV isolates. Virus-like-particles (VLPs) have been demonstrated to be safe to administer to animals and human patients as well as being potent and efficient stimulators of cellular and humoral immune responses. Therefore, VLPs are being considered as possible HIV vaccines. Chimeric HIV-1 VLPs constructed with either HIV or SIV capsid protein plus HIV immune epitopes and immuno-stimulatory molecules have further improved on early VLP designs, leading to enhanced immune stimulation. The administration of VLP vaccines via mucosal surfaces has also emerged as a promising strategy with which to elicit mucosal and systemic humoral and cellular immune responses. Additionally, new information on antigen processing and the presentation of particulate antigens by dendritic cells (DCs) has created new strategies for improved VLP vaccine candidates. This paper reviews the field of HIV-1 VLP vaccine development, focusing on recent studies that will likely uncover promising prospects for new HIV vaccines.     

Prophylactic vaccines mimic synthetic CpG oligonucleotides in their ability to modulate immune responses

Synthetic oligonucleotide ligands that bind to toll-like receptors are known to modulate the immune response via the activation of antigen presenting cells, and were therefore proposed as a novel form of vaccine adjuvant. Clinical-grade they are, however, not readily available. Here, we show that commonly used prophylactic vaccines for infectious diseases like measles, mumps and tuberculosis exhibit the same immune modulating behavior as synthetic CpG oligonucleotides in terms of their ability to stimulate IFN-α production and plasmacytoid dendritic cell maturation. Featuring the additional advantages of low-cost and proven safety, these vaccines could therefore be attractive alternatives to CpG oligonucleotides as adjuvants for immunotherapy. This previously undiscovered characteristic of prophylactic vaccines also sheds new light on the mechanisms by which they operate and is extremely interesting for vaccine development. Moreover, the finding that prophylactic vaccines trigger TLRs like synthetic oligonucleotides opens the possibility to predict the immune response of new vaccines.     

An analysis of the role of skin Langerhans cells (LC) in the cytoplasmic processing of HIV-1 peptides after “Peplotion” transepidermal transfer and HLA class I presentation to CD8+ CTLs—An approach to immunization of humans

Skin Langerhans cells (LC) are antigen-presenting cells capable of expressing MHC class I and class II molecules on the plasma membrane. This molecular activity was reviewed to combine the knowledge of peptide presentation by MHC and HLA class I and class II molecules to prime CD8⁺ cytotoxic T cells (CTLs) and CD4⁺ T helper cells, respectively. The possible utilization of the skin dendritic cells for the development of antiviral CTLs and antibodies by synthetic peptides modeled according to the motifs of peptides that naturally interact with the peptide binding grooves of the various HLA haplotypes is discussed and evaluated. It may be possible that the introduction of synthetic viral peptides with motifs to fit the HLA class I haplotypes of a human population to the skin dendritic cells will prime selectively the cellular or the humoral immune responses. This approach may provide a new vaccination technique that applies synthetic virus peptides as vaccines for the immunization of humans. The neuropeptide CGRP interacts with LC and modulates antigen presentation.     

The processing of antigens delivered as DNA vaccines

Summary: The ability of DNA vaccines to provide effective immunological protection against infection and tumors depends on their ability to generate good CD4⁺ and CD8⁺ T‐cell responses. Priming of these responses is a property of dendritic cells (DCs), and so the efficacy of DNA‐encoded vaccines is likely to depend on the way in which the antigens they encode are processed by DCs. This processing could either be via the synthesis of the vaccine‐encoded antigen by the DCs themselves or via its uptake by DCs following its synthesis in bystander cells that are unable to prime T cells. These different sources of antigen are likely to engage different antigen‐processing pathways, which are the subject of this review. Understanding how to access different processing pathways in DCs may ultimately aid the rational development of plasmid‐based vaccines to pathogens and to cancer.     

Efficient antitumor immunity in a murine colorectal cancer model induced by CEA RNA-electroporated B cells

RNA electroporation as a gene delivery method is more feasible and safer as compared with viral vectors. RNA-loaded dendritic cells (DC) have been used to induce T cell responses against tumor rejection antigens and B cells can also act as antigen-presenting cells for cellular vaccines. In this study, we compared B cells and DC, after electroporation with carcinoembryonic antigen (CEA) RNA, for their capacity to generate cytotoxic T lymphocytes and antitumor immunity. Vaccination using these B cells induced levels of IFN-gamma-secreting T cells and cytotoxic T cells comparable to those induced by DC. Intravenous administration was the optimum route for the B cell vaccine, while subcutaneous administration was the optimum route for the DC vaccine. The B cell vaccine predominantly generated CEA-specific CD4(+) T cells, whereas the DC vaccine generated CD8(+) T cells. Moreover, the B cell vaccine induced higher levels of anti-CEA antibodies than the DC vaccine. A heterogeneous prime-boost using B cells and DC failed to show any synergistic effects; however, the B cell vaccine did inhibit tumor growth and prolonged survival to a similar extent as the DC vaccine. Such RNA-electroporated B cells may prove useful as cellular tumor vaccines with potential clinical application.     

Three-day dendritic cells for vaccine development: Antigen uptake,processing and presentation

Background

Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.

Methods

In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.

Results

Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivt RNA.

Conclusions

This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivt RNA as sources of specific antigen.

     

Regulation of the Physiological Functions of Human Dendritic Cells by Recombinant Heat Shock Protein Hsp70

Dendritic cells (DC) are the most important antigen-presenting cells in the body. They are the target of action of various vaccines and dendritic cells have been used as the basis for developing cellular antitumor and antiviral vaccines, i.e., DC vaccines. At the same time, dendritic cells may provide a suitable model for studies of the activity and mechanisms of action of different immunotherapeutic formulations. One aspect of the optimization of the use of dendritic cells for inducing antigen-specific immune responses relates to the use of heat shock proteins (Hsp), particularly Hsp70. This protein can be used to introduce protein antigens into dendritic cells and to control the activity of dendritic cells. Important aspects of achieving these aims include knowledge of dendritic cell physiology and the characteristics of the interaction of Hsp70 and its complexes with antigens with dendritic cells of different levels of differentiation. Human recombinant Hsp70 was found not only to deliver antigens to dendritic cells, but also to regulate the activity of mature dendritic cells and to optimize the induction of antigen-specific cellular immune responses.     

Closely related mycobacterial strains demonstrate contrasting levels of efficacy as antitumor vaccines and are processed for major histocompatibility complex class I presentation by multiple routes in dendritic cells

Mycobacteria expressing recombinant antigens are already being developed as vaccines against both infections and tumors. Little is known about how dendritic cells might process such antigens. Two different mycobacterial species, the fast-growing Mycobacterium smegmatis and the slow-growing M. bovis M. bovis BCG, were engineered to express a model tumor antigen, the K(b)-restricted dominant cytotoxic T-lymphocyte epitope OVA(257-264). Recombinant M. bovis BCG but not recombinant M. smegmatis conferred protection to mice challenged with the B16-OVA tumor cell line. We went on to investigate whether the contrast in antitumor efficacy could be due to differences in how dendritic cells process antigen from the two mycobacterial strains for class I presentation. Both strains of mycobacteria caused phenotypic maturation of dendritic cells, but recombinant M. smegmatis infection led to a greater degree of dendritic cell maturation than recombinant M. bovis BCG infection. Antigen from recombinant M. smegmatis was processed and presented as OVA(257-264) on K(b) molecules by the dendritic cell line DC2.4 but not by bone marrow-derived dendritic cells (BMDC) or splenic dendritic cells. In contrast, antigen from recombinant M. bovis BCG was presented by all three dendritic cell types as long as the mycobacteria were viable. Such presentation was dependent on proteasome function and nascent major histocompatibility complex (MHC) class I molecules in DC2.4 cells but independent of the proteasome and transporter associated with antigen processings (TAP) in BMDC and splenic dendritic cells. These data demonstrate for the first time that antigen vectored by the slow-growing M. bovis BCG but not that vectored by fast-growing, readily destroyed M. smegmatis is processed and presented on MHC class I by in vitro-generated dendritic cells, which has implications for recombinant microbial vaccine development.     

Evaluation of Immunotherapy Efficiency in Mouse CaO-1 Ovarian Carcinoma Treated by Vaccines Based on Dendritic Cells

Injection of dendritic cells, pulsated by tumor lysate or mucin, containing CA 125 antigen, led to a more than 50% inhibition of tumor growth in female CBA mice with transplanted mouse pseudomucinous CaO-1 ovarian carcinoma in comparison with the control. Tumor-associated CA 125 antigen can be used for obtaining dendritic cell vaccines against ovarian malignant tumors. This trend will extend the potentialities of application of antitumor vaccines based on dendritic cells, as clinical use of this technology is limited by the need in patient’s tumor material. Mucin, containing Ca 125 antigen, can be isolated from patient’s serum or obtained by gene engineering technologies as a recombinant peptide. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 147, No. 2, pp. 187-189, February, 2009     

Genetic vaccination for the active immunotherapy of cancer

Molecular biology techniques have given novel impetus to the immunotherapy of cancer because they have catalyzed the identification of several potential tumor antigens, and permitted the generation of vectors for the delivery of genetic material encoding these antigens. Vaccines can be defined "genetic" when the antigen they enclose is present as DNA or RNA. Microrganisms used as vectors can deliver the genetic information, but naked nucleic acids have also been shown to be effective immunogens thanks to built-in adjuvants that activate professional antigen presenting cells. Although gene-based cancer vaccines have been tested in mouse models and selected for pilot clinical trials, enthusiasm has somewhat waned due to an apparently major drawback of cancer vaccination: tumor antigens are weak, and therefore fail to stimulate a sterilizing immune response in tumor-bearing patients. Mouse studies, however, have shown that cancer vaccines are extremely efficacious in establishing a state of active immunosurvellance against tumor growth. This review reconsiders the findings emerging from preclinical studies in the context of our current knowledge of the cellular and molecular bases of the immune responses to vaccines, in an attempt to approach critically the use of genetic vaccination for the treatment of cancer.     

Vaccination of mice with attenuated mutants ofListeria monocytogenes: requirement for induction of macrophage la expression

Isogenic mutant strains ofListeria monocytogenesdemonstrating graded attenuation in mice were used to analyse the correlation between bacterial virulence, ability to induce class II MHC (la) molecules in antigen presenting cells and ability to vaccinate against secondary infection. The mutants used differed only in the amino acid sequence of the thiol–activated hemolysin, Listeriolysin O (LLO). The results indicate thatL. monocytogenesmutants of reduced virulence have the potential to act as vaccines only if they are sufficiently persistant to induce la expression in antigen presenting cells. The findings also suggest that specific mutagenesis of virulence factors, including LLO, could provide an approach for creatingListeria monocytogenesstrains with potential for use as attenuated live vaccines.     

Innovative Cancer Vaccine Strategies Based on the Identification of Tumour-Associated Antigens

The identification of tumour-associated antigens has opened up new approaches to cancer immunotherapy. While past research focused on CD8+ cytotoxic T-cell responses, accumulating evidence suggests that CD4+ T cells also play an important role in orchestrating the host immune response against cancer. In this article, we summarise new strategies for the identification of major histocompatibility complex (MHC) class II-associated tumour antigens and discuss the importance of engaging both CD4+ and CD8+ T cells in cancer immunotherapy. The cloning of MHC class I- or class II-associated antigens has made it possible to develop synthetic and recombinant cancer vaccines that express specific tumour antigens. There are three major types of synthetic and recombinant cancer vaccines: recombinant viral and bacterial vaccines; naked DNA or RNA vaccines; and recombinant protein and peptide vaccines. In this article, we also discuss a new generation of recombinant cancer vaccines, ‘self-replicating’ DNA and RNA vaccines. Studies on the mechanisms of ‘self-replicating’ nucleic acid vaccines revealed that the enhanced immunogenicity was not due to an enhanced antigen expression, suggesting that the quantitative difference may not be as important as the qualitative difference in antigen presentation. The presence of the RNA replicase in the ‘self-replicating’ nucleic acid vaccines mimics alphavirus infection, which triggers the innate antiviral pathways of the host cells. Studies on how viral and cellular modulators of the innate antiviral pathways affect vaccine function should provide molecular insights crucial to future vaccine design.     

Induction of antigen cross-presentation by Toll-like receptors

Cross-presentation is the pathway by which exogenous antigens are routed for presentation on MHC class I for activation of CD8⁺ T cells. This pathway is important for the development of CD8⁺ cytotoxic T lymphocyte responses against tumors and infectious pathogens that do not directly infect APC. We review studies showing that certain Toll-like receptors mediate cross-presentation by dendritic cells, initiating cytosolic processing of antigen after inducing dendritic cell maturation. The implications of these studies for understanding CD8⁺ T cell activation and implementing novel vaccine strategies is considered.     

Gene-based strategies for the immunotherapy of cancer

 T lymphocytes play a crucial role in the host’s immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in preclinical studies at many centers. Gene therapy strategies such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer. Received: 20 January 1997 / Accepted: 17 February 1997     

Alum increases antigen uptake, reduces antigen degradation and sustains antigen presentation by DCs in vitro

Aluminium adjuvants (alum) have been the only widely approved adjuvants for use in human vaccines since the 1920s, however, the mechanism of action of these adjuvants remains elusive. Due to increasing demand for novel adjuvants, a clearer understanding of the mechanisms that allow these important agents to affect adaptive immune responses will make a significant contribution to the rational design of future vaccines. Using a novel approach to tracking antigen and antigen presentation, we demonstrate that alum induces higher antigen accumulation and increased antigen presentation by dendritic cells (DCs) in vitro. Antigen accumulation was 100-fold higher and antigen presentation 10-fold higher following alum treatment when compared with soluble protein alone. We also observed that alum causes an initial reduction in presentation compared with soluble antigen, but eventually increases the magnitude and duration of antigen presentation. This was associated with reduced protein degradation in DCs following alum treatment. These studies demonstrate the dynamic alterations in antigen processing and presentation induced by alum that underlie enhanced DC function in response to this adjuvant.     

Antigen cross-presentation: extending recent laboratory findings to therapeutic intervention

The initiation of adaptive immune responses requires antigen presentation to lymphocytes. In particular, dendritic cells (DCs) are equipped with specialized machinery that promote effective display of peptide/major histocompatibility complexes (MHC), rendering them the most potent stimulators of naive T lymphocytes. Antigen cross-presentation to CD8(+) T cells is an important mechanism for the development of specific cytotoxic T lymphocyte (CTL) responses against tumours and viruses that do not infect antigen-presenting cells. Here, we review recent findings concerning antigen cross-presentation to CD8(+) T lymphocytes. Specific subtypes of DCs in the mouse have been defined as being especially endowed for antigen cross-presentation, and a human homologue of these DCs has recently been described. DC vaccination strategies for the prevention and treatment of human diseases have been under investigation in recent years, but have not generally reached satisfying results. We here provide an overview of new findings in antigen cross-presentation research and how they can be used for development of the next generation of human DC vaccines.     

Two populations of ovine bone marrow-derived dendritic cells can be generated with recombinant GM-CSF and separated on CD11b expression

Whereas studies on dendritic cells in rodents rely largely on bone marrow-derived dendritic cells (BM-DCs), no data are available about BM-DCs in sheep, a species that is largely used for immunology and transplantation studies. We have developed a culture protocol to produce ovine BM-DCs, using 6x(His)-tagged recombinant GM-CSF which was purified from baculovirus-infected insect cells. When ovine bone marrow progenitors were cultured in the presence of recombinant GM-CSF, large numbers of CD11c-positive cells were generated after 6-7 days. The phenotypic appearance of BM-DCs was assessed by flow cytometry and electron microscopy. Two DC subsets were identified that expressed different levels of MHC class II molecules, differed in receptor-mediated endocytosis, and could be separated on CD11b expression. When separated cells were incubated with microbial products, they react differently to those that are considered the TLR2 and TLR4 agonists in other species. Indeed, although CD11b^int/hi cells were partially resistant to maturation induced by lipoteichoic acid or lipopolysaccharide, MHC class II upregulation was observed on CD11b^dull cells. Moreover, these cells had strong stimulatory capacity for CD4 T cells when assayed in allogeneic reactions. This protocol will help analyzing ovine DC interactions with pathogens, and enables future studies on the development of vaccines.     

HLA class I expression on human cancer cells. Implications for effective immunotherapy

Early studies demonstrated the role of cytotoxic T cells as an immune defence mechanism against tumour cells. The demonstration of tumour antigen peptides and their presentation to T cells on major histocompatibility complex class I molecules highlighted the importance of these molecules in effective anti-tumour responses. It is well established that many tumours escape T cell recognition by loss or down regulation of class I molecule expression on the cell surface of tumour cells. Tumours which have lost class I expression are immunoselected and as a result have a propensity for growth and metastatic spread. With the development of cancer vaccine strategies for clinical use, there will be a future role for histocompatibility laboratories in determining class I expression on tumour cells in individual patients. These studies of expression will require not just the demonstration of total class I expression but the demonstration of locus and allele specific class I molecules involved in the relevant tumour peptide presentation. These studies will be pivotal in tailoring individual patient therapies. The identification of appropriate monoclonal antibody reagents for class I expression and techniques used on different kinds of tissue sections will be a component of the forthcoming 13th International Histocompatibility Workshop.     

Dendritic cell elimination as an assay of cytotoxic T lymphocyte activity in vivo

We show in this paper that the survival of antigen-loaded dendritic cells in vivo may be used as a sensitive readout of CTL activity. We have previously shown that dendritic cells labeled with the fluorescent dye CFSE and injected sub-cutaneously into mice migrate spontaneously to the draining lymph node where they persist for several days. In the presence of effector CTL responses, dendritic cells loaded with specific antigen rapidly disappear from the draining lymph node. In this paper we extend the above observations and set up a simple and sensitive method to reveal CTL activity in individual mice in vivo. Dendritic cells were labeled with two different fluorochromes, loaded with antigen or left untreated, and mixed together before injection into mice. We show that only the dendritic cells loaded with specific antigen were cleared from the draining lymph node, while dendritic cells not loaded with antigen remained unaffected. Cytotoxic responses generated by immunization with peptide-loaded dendritic cells, or by infection with influenza virus, could be revealed using this method. Comparison of the differential survival of dendritic cells populations mixed together also allowed us to accurately evaluate the disappearance of dendritic cells, irrespective of variability in the injection site and other parameters. Given the ability of dendritic cells to efficiently take up and present complex antigens, nucleic acids and apoptotic bodies, this method may also allow the evaluation of cytotoxic activity against antigens that are not characterized in terms of peptide epitopes.