Modulation of immune responses using adjuvants to facilitate therapeutic vaccination.

Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies.

Prevention and treatment of COVID-19 disease by controlled modulation of innate immunity.

The recent outbreak of coronavirus disease 2019 (COVID-19), triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an enormous threat to global public health and economies. Human coronaviruses normally cause no or mild respiratory disease but in the past two decades, potentially fatal coronavirus infections have emerged, causing respiratory tract illnesses such as pneumonia and bronchitis. These include severe acute respiratory syndrome coronavirus (SARS-CoV), followed by the Middle East respiratory syndrome coronavirus (MERS-CoV), and recently the SARS-CoV-2 coronavirus outbreak that emerged in Wuhan, China, in December 2019. Currently, most COVID-19 patients receive traditional supportive care including breathing assistance. To halt the ongoing spread of the pandemic SARS-CoV-2 coronavirus and rescue individual patients, established drugs and new therapies are under evaluation. Since it will be some time until a safe and effective vaccine will be available, the immediate priority is to harness innate immunity to accelerate early antiviral immune responses. Second, since excessive inflammation is a major cause of pathology, targeted anti-inflammatory responses are being evaluated to reduce inflammation-induced damage to the respiratory tract and cytokine storms. Here, we highlight prominent immunotherapies at various stages of development that aim for augmented anti-coronavirus immunity and reduction of pathological inflammation.

Therapeutic Immunization against Glioblastoma.

Glioblastoma is the most common form of brain cancer in adults that produces severe damage to the brain leading to a very poor survival prognosis. The standard of care for glioblastoma is usually surgery, as well as radiotherapy followed by systemic temozolomide chemotherapy, resulting in a median survival time of about 12 to 15 months. Despite these therapeutic efforts, the tumor returns in the vast majority of patients. When relapsing, statistics suggest an imminent death dependent on the size of the tumor, the Karnofsky Performance Status, and the tumor localization. Following the standard of care, the administration of Bevacizumab, inhibiting the growth of the tumor vasculature, is an approved medicinal treatment option approved in the United States, but not in the European Union, as well as the recently approved alternating electric fields (AEFs) generator NovoTTF/Optune. However, it is clear that regardless of the current treatment regimens, glioma patients continue to have dismal prognosis and novel treatments are urgently needed. Here, we describe different approaches of recently developed therapeutic glioma brain cancer vaccines, which stimulate the patient's immune system to recognize tumor-associated antigens (TAA) on cancer cells, aiming to instruct the immune system to eventually attack and destroy the brain tumor cells, with minimal bystander damage to normal brain cells. These distinct immunotherapies may target particular glioma TAAs which are molecularly defined, but they may also target broad patient-derived tumor antigen preparations intentionally evoking a very broad polyclonal antitumor immune stimulation.

Immune adjuvants as critical guides directing immunity triggered by therapeutic cancer vaccines.

Tumor growth is controlled by natural antitumor immune responses alone or by augmented immune reactivity resulting from different forms of immunotherapy, which has demonstrated clinical benefit in numerous studies, although the overall percentage of patients with durable clinical responses remains limited. This is attributed to the heterogeneity of the disease, the inclusion of late-stage patients with no other treatment options and advanced tumor-associated immunosuppression, which may be consolidated by certain types of chemotherapy. Despite variable responsiveness to distinct types of immunotherapy, therapeutic cancer vaccination has shown meaningful efficacy for a variety of cancers. A key step during cancer vaccination involves the appropriate modeling of the functional state of dendritic cells (DCs) capable of co-delivering four critical signals for proper instruction of tumor antigen-specific T cells. However, the education of DCs, either directly in situ, or ex vivo by various complex procedures, lacks standardization. Also, it is questioned whether ex vivo-prepared DC vaccines are superior to in situ-administered adjuvant-guided vaccines, although both approaches have shown success. Evaluation of these variables is further complicated by a lack of consensus in evaluating vaccination clinical study end points. We discuss the role of signals needed for the preparation of classic in situ and modern ex vivo DC vaccines capable of proper reprogramming of antitumor immune responses in patients with cancer.

Therapeutic dendritic cell-based cancer vaccines: the state of the art.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells, capable of initiating proper adaptive immune responses. Although tumor-infiltrating DCs are able to recognize cancer cells and uptake tumor antigens, they often have impaired functions because of the immunosuppressive tumor milieu. Therefore, DCs are targeted by therapeutic means either in vivo or ex vivo to facilitate tumor antigen presentation to T cells and induce or promote efficient antitumor immune responses in cancer patients. This immunotherapeutical approach is defined as specific active tumor immunotherapy or therapeutic cancer vaccination. In this review we briefly discuss general aspects of DC biology, followed by a thorough description of the current knowledge and optimization trends of DC vaccine production ex vivo, including various approaches for the induction of proper DC maturation and efficient loading with tumor antigens. We also discuss critical clinical aspects of DC vaccine application in cancer patients, including protocols of administration (routes and regimens), individualization of tumor immunotherapy, prediction and proper evaluation of immune and clinical responses to immunotherapy, and the critical role of combining tumor immunotherapy with other cancer treatment strategies to achieve maximal therapeutic effects.

Outer membrane vesicle-based intranasal vaccines.

Delivery of vaccines via the mucosal route is regarded as the most effective mode of immunization to counteract infectious diseases that enter via mucosal tissues, including oral, nasal, pulmonary, intestinal, and urogenital surfaces. Mucosal vaccines not only induce local immune effector elements, such as secretory Immunoglobulin A (IgA) reaching the luminal site of the mucosa, but also systemic immunity. Moreover, mucosal vaccines may trigger immunity in distant mucosal tissues because of the homing of primed antigen-specific immune cells toward local and distant mucosal tissue via the common mucosal immune system. While most licensed intramuscular vaccines induce only systemic immunity, next-generation mucosal vaccines may outperform parenteral vaccination strategies by also eliciting protective mucosal immune responses that block infection and/or transmission. Especially the nasal route of vaccination, targeting the nasal-associated lymphoid tissue, is attractive for local and distant mucosal immunization. In numerous studies, bacterial outer membrane vesicles (OMVs) have proved attractive as vaccine platform for homologous bacterial strains, but also as antigen delivery platform for heterologous antigens of nonbacterial diseases, including viruses, parasites, and cancer. Their application has also been extended to mucosal delivery. Here, we will summarize the characteristics and clinical potential of (engineered) OMVs as vaccine platform for mucosal, especially intranasal delivery.

A Prospective, Cohort Study of SITOIGANAP to Treat Glioblastoma When Given in Combination With Granulocyte-Macrophage Colony-Stimulating Factor/Cyclophosphamide/Bevacizumab/Nivolumab or Granulocyte-Macrophage Colony-Stimulating Factor/Cyclophosphamide/Bevacizumab/Pembrolizumab in Patients Who Failed Prior Treatment With Surgical Resection, Radiation, and Temozolomide.

Background: Glioblastoma (GBM) is the most common primary, malignant brain tumor in adults and has a poor prognosis. The median progression-free survival (mPFS) of newly diagnosed GBM is approximately 6 months. The recurrence rate approaches 100%, and the case-fatality ratio approaches one. Half the patients die within 8 months of recurrence, and 5-year survival is less than 10%. Advances in treatment options are urgently needed. We report on the efficacy and safety of a therapeutic vaccine (SITOIGANAP: Epitopoietic Research Corporation) administered to 21 patients with recurrent GBM (rGBM) under a Right-to-Try/Expanded Access program. SITOIGANAP is composed of both autologous and allogeneic tumor cells and lysates. Methods: Twenty-one patients with rGBM received SITOIGANAP on 28-day cycles in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), cyclophosphamide, bevacizumab, and an anti-programmed cell death protein-1 (anti-PD-1) monoclonal antibody (either nivolumab or pembrolizumab). Results: The mPFS was 9.14 months, and the median overall survival (mOS) was 19.63 months from protocol entry. Currently, 14 patients (67%) are at least 6 months past their first SITOIGANAP cycle; 10 patients (48%) have received at least six cycles and have a mOS of 30.64 months and 1-year survival of 90%. The enrollment and end-of-study CD3+/CD4+ T-lymphocyte counts strongly correlate with OS. Conclusions: The addition of SITOIGANAP/GM-CSF/cyclophosphamide to bevacizumab and an anti-PD-1 monoclonal antibody resulted in a significant survival benefit compared to historic control values in rGBM with minimal toxicity compared to current therapy.

Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation.

Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases.

Phase II study of ERC1671 plus bevacizumab versus bevacizumab plus placebo in recurrent glioblastoma: interim results and correlations with CD4+ T-lymphocyte counts.

AIM: ERC1671 is an allogeneic/autologous therapeutic glioblastoma (GBM) vaccine - composed of whole, inactivated tumor cells mixed with tumor cell lysates derived from the patient and three GBM donors. METHODS: In this double-blinded, randomized, Phase II study bevacizumab-naive patients with recurrent GBM were randomized to receive either ERC1671 in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine® or sargramostim) and cyclophosphamide plus bevacizumab, or placebo plus bevacizumab. Interim results: Median overall survival (OS) of patients treated with ERC1671 plus bevacizumab was 12 months. In the placebo plus bevacizumab group, median OS was 7.5 months. The maximal CD4+ T-lymphocyte count correlated with OS in the ERC1671 but not in the placebo group. CONCLUSION: The addition of ERC1671/GM-CSF/cyclophosphamide to bevacizumab resulted in a clinically meaningful survival benefit with minimal additional toxicity.

Development of a chicken 5 K microarray targeted towards immune function.

BACKGROUND: The development of microarray resources for the chicken is an important step in being able to profile gene expression changes occurring in birds in response to different challenges and stimuli. The creation of an immune-related array is highly valuable in determining the host immune response in relation to infection with a wide variety of bacterial and viral diseases. RESULTS: Here we report the development of chicken immune-related cDNA libraries and the subsequent construction of a microarray containing 5190 elements (in duplicate). Clones on the array originate from tissues known to contain high levels of cells related to the immune system, namely Bursa, Peyers patch, thymus and spleen. Represented on the array are genes that are known to cluster with existing chicken ESTs as well as genes that are unique to our libraries. Some of these genes have no known homologies and represent novel genes in the chicken collection. A series of reference genes (ie. genes of known immune function) are also present on the array. Functional annotation data is also provided for as many of the genes on the array as is possible. CONCLUSION: Six new chicken immune cDNA libraries have been created and nearly 10,000 sequences submitted to GenBank [GenBank: AM063043-AM071350; AM071520-AM072286; AM075249-AM075607]. A 5 K immune-related array has been developed from these libraries. Individual clones and arrays are available from the ARK-Genomics resource centre.

Th1/Th2 polarization by viral and helminth infection in birds.

Mammals developed an immune system able to functionally polarize into so-called type 1 or type 2 immune pathways, to resolve infections with intracellular and extracellular pathogens, respectively. In the well-studied avian immune system of the chicken, however, no evidence for polarized immunity could be found, as yet. To investigate whether these two major arms of mammalian immunity, regulated by a T helper (Th)1/Th2 cytokine balance, evolved similarly in birds, chickens were exposed to a prevalent intracellular (viral) or extracellular (helminth) infection. By using semi-quantitative RT-PCR analysis we provide evidence that polarization of Th1/Th2 type immunity extends beyond mammalian species, and, therefore, has been evolutionary conserved for more than 300 million years, when the lineages of mammalian and avian vertebrates are assumed to have segregated.

Choice and Design of Adjuvants for Parenteral and Mucosal Vaccines.

The existence of pathogens that escape recognition by specific vaccines, the need to improve existing vaccines and the increased availability of therapeutic (non-infectious disease) vaccines necessitate the rational development of novel vaccine concepts based on the induction of protective cell-mediated immune responses. For naive T-cell activation, several signals resulting from innate and adaptive interactions need to be integrated, and adjuvants may interfere with some or all of these signals. Adjuvants, for example, are used to promote the immunogenicity of antigens in vaccines, by inducing a pro-inflammatory environment that enables the recruitment and promotion of the infiltration of phagocytic cells, particularly antigen-presenting cells (APC), to the injection site. Adjuvants can enhance antigen presentation, induce cytokine expression, activate APC and modulate more downstream adaptive immune reactions (vaccine delivery systems, facilitating immune Signal 1). In addition, adjuvants can act as immunopotentiators (facilitating Signals 2 and 3) exhibiting immune stimulatory effects during antigen presentation by inducing the expression of co-stimulatory molecules on APC. Together, these signals determine the strength of activation of specific T-cells, thereby also influencing the quality of the downstream T helper cytokine profiles and the differentiation of antigen-specific T helper populations (Signal 3). New adjuvants should also target specific (innate) immune cells in order to facilitate proper activation of downstream adaptive immune responses and homing (Signal 4). It is desirable that these adjuvants should be able to exert such responses in the context of mucosal administered vaccines. This review focuses on the understanding of the potential working mechanisms of the most well-known classes of adjuvants to be used effectively in vaccines.

Use of ERC-1671 Vaccine in a Patient with Recurrent Glioblastoma Multiforme after Progression during Bevacizumab Therapy: First Published Report.

Glioblastoma multiforme is a highy aggressive tumor that recurs despite resection, focal beam radiation, and temozolamide chemotherapy. ERC-1671 is an experimental treatment strategy that uses the patient's own immune system to attack the tumor cells. The authors report preliminary data on the first human administration of ERC-1671 vaccination under a single-patient, compassionate-use protocol. The patient survived for ten months after the vaccine administration without any other adjuvant therapy and died of complications related to his previous chemotherapies.

First clinical results of a personalized immunotherapeutic vaccine against recurrent, incompletely resected, treatment-resistant glioblastoma multiforme (GBM) tumors, based on combined allo- and auto-immune tumor reactivity.

Glioblastoma multiforme (GBM) patients have a poor prognosis. After tumor recurrence statistics suggest an imminent death within 1-4.5 months. Supportive preclinical data, from a rat model, provided the rational for a prototype clinical vaccine preparation, named Gliovac (or ERC 1671) composed of autologous antigens, derived from the patient's surgically removed tumor tissue, which is administered together with allogeneic antigens from glioma tissue resected from other GBM patients. We now report the first results of the Gliovac treatment for treatment-resistant GBM patients. Nine (9) recurrent GBM patients, after standard of care treatment, including surgery radio- and chemotherapy temozolomide, and for US patients, also bevacizumab (Avastin™), were treated under a compassionate use/hospital exemption protocol. Gliovac was given intradermally, together with human GM-CSF (Leukine(®)), and preceded by a regimen of regulatory T cell-depleting, low-dose cyclophosphamide. Gliovac administration in patients that have failed standard of care therapies showed minimal toxicity and enhanced overall survival (OS). Six-month (26 weeks) survival for the nine Gliovac patients was 100% versus 33% in control group. At week 40, the published overall survival was 10% if recurrent, reoperated patients were not treated. In the Gliovac treated group, the survival at 40 weeks was 77%. Our data suggest that Gliovac has low toxicity and a promising efficacy. A phase II trial has recently been initiated in recurrent, bevacizumab naïve GBM patients (NCT01903330).

Characterization of the first nonmammalian T2 cytokine gene cluster: the cluster contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 that appears to be a pseudogene, and a gene encoding another cytokinelike transcript, KK34.

A genomics approach based on the conservation of synteny was used to develop a bacterial artificial chromosome (BAC) contig across the chicken T2 cytokine gene cluster. Sequencing of representative BACs showed that the chicken genome encodes genes for the homologs of mammalian interleukin-3 (IL-3), IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). These sequences represent the first T2 cytokines found outside of mammals, and their location demonstrates that the T2 cluster is ancient (at least 300 million years old). Four of these genes (IL-3, IL-4, IL-13, and GM-CSF) are expressed at the mRNA level and can be expressed as recombinant protein. In contrast to the other four genes, the chicken IL-5 (ChIL-5) gene we sequenced lacks a recognizable promoter and regulatory sequences in the predicted 3'-untranslated region (3'-UTR). Further, there is no evidence for its expression at the mRNA level. We, therefore, hypothesize that it is a pseudogene. Genomic analysis revealed that a recently characterized cytokinelike transcript, KK34, not identified in our initial analysis of the BAC sequence, is also encoded in this cluster. This gene may represent a duplication of an ancestral IL-5 gene and may encode the functional homolog of IL-5 in the chicken.

Vaccine adjuvant technology: from mechanistic concepts to practical applications.

Distinct types of immune responses are required for efficient elimination of different pathogens. Programming of the desired type of immune response by safe nonreplicating vaccines requires suitable vaccine adjuvants. Adjuvants largely determine the magnitude and quality of immune responses specific for the coadministered antigen. Unfortunately, rational vaccine design requiring a rational choice of vaccine adjuvant, is hampered by a lack of knowledge about the mechanism(s) of vaccine adjuvant activity. The current review addresses different critical immunological processes possibly explaining adjuvant functions. In addition, we discuss traditional vaccine adjuvant formulations and their possible mode of action. Finally, we reflect on the latest technologies for the identification of novel adjuvants using molecular analysis of immune activation and functional genomics.

Antigen delivery systems and immunostimulation.

The immune system evolved to free the host from invading noxious pathogens. Vaccines are inoculated as a prophylactic measure in order to program the immune system for accelerated recognition and elimination of specific pathogens. During vaccination the immune system is exposed to attenuated or inactivated microorganisms, or their fragments. The immune response to these structures, in contrast to virulent pathogens, is often inadequate for the generation of memory cells or immune effector elements such as antibodies, perforines, granzymes or cytokines. Vaccine adjuvants help to overcome these limited responses. They provide instructive signals for the host immune system by mimicking the conditions associated with virulent infection. Hence, they either enhance and prolong expression of antigen components to reactive T cells in lymph nodes (signal 1) or they increase expression of membrane-bound or soluble costimulatory molecules (signal 2). The enhancement of both signals by vaccine adjuvants is not mutually exclusive. Moreover, adjuvants may encode a third signal instructing the type of immune reaction to be generated. Supported by animations this presentation addresses putative immunological concepts of vaccine adjuvant activity, a phenomenon long been known as "the immunologist's dirty little secret". Insight in the mechanisms that underlie adjuvant-induced immunostimulation and generation of memory cells will facilitate rational vaccine design.

Oil-based emulsion vaccine adjuvants.

Vaccine adjuvants are critical components in experimental and licensed vaccines used in human and veterinary medicine. When aiming to evoke an immune response to a purified antigen, the administration of antigen alone is often insufficient, unless the antigen contains microbial structures or has a natural particulate structure. In most cases, the rationale to use an adjuvant is obvious to the experimental immunologist or the professional vaccinologist, who is familiar with the nature of the antigen, and the aim of the vaccine to elicit a specific antibody response and/or a specific type of T cell response. In this unit, we describe protocols to formulate antigens with oil-based emulsions. Such emulsions represent a major prototype adjuvant category that is frequently used in experimental preclinical vaccines, as well as veterinary and human vaccines.

Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: to use or not to use?

Accumulation of firm evidence that clinically apparent cancer develops only when malignant cells manage to escape immunosurveillance led to the introduction of tumor immunotherapy strategies aiming to reprogramm the cancer-dysbalanced antitumor immunity and restore its capacity to control tumor growth. There are several immunotherapeutical strategies, among which specific active immunotherapy or therapeutic cancer vaccination is one of the most promising. It targets dendritic cells (DCs) which have a unique ability of inducing naive and central memory T cell-mediated immune response in the most efficient manner. DCs can be therapeutically targeted either in vivo/in situ or by ex vivo manipulations followed by their re-injection back into the same patient. The majority of current DC targeting strategies are based on autologous or allogeneic tumor-associated antigens (TAAs) which possess various degrees of inherent tolerogenic potential. Therefore still limited efficacy of various tumor immunotherapy approaches may be attributed, among various other mechanisms, to the insufficient immunogenicity of self-protein-derived TAAs. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species was suggested to overcome the natural immune tolerance to self TAAs. Xenoantigens are supposed to differ sufficiently from self antigens to a degree that renders them immunogenic, but at the same time preserves an optimal homology range with self proteins still allowing xenoantigens to induce cross-reactive T cells. Here we discuss the concept of xenogeneic vaccination, describe the cons and pros of autologous/allogeneic versus xenogeneic therapeutic cancer vaccines, present the results of various pre-clinical and several clinical studies and highlight the future perspectives of integrating xenovaccination into rapidly developing tumor immunotherapy regimens.

Dendritic cells and their role in tumor immunosurveillance.

Dendritic cells (DCs) comprise a heterogeneous population of cells that play a key role in initiating, directing and regulating adaptive immune responses, including those critically involved in tumor immunosurveillance. As a riposte to the central role of DCs in the generation of antitumor immune responses, tumors have developed various mechanisms which impair the immunostimulatory functions of DCs or even instruct them to actively contribute to tumor growth and progression. In the first part of this review we discuss general aspects of DC biology, including their origin, subtypes, immature and mature states, and functional plasticity which ensures a delicate balance between active immune response and immune tolerance. In the second part of the review we discuss the complex interactions between DCs and the tumor microenvironment, and point out the challenges faced by DCs during the recognition of tumor Ags. We also discuss the role of DCs in tumor angiogenesis and vasculogenesis.