Daniel O. Villarreal

Alarmin IL-33 acts as an immunoadjuvant to enhance antigen-specific tumor immunity

Studies of interleukin (IL)-33 reveal a number of pleiotropic properties. Here, we report that IL-33 has immunoadjuvant effects in a human papilloma virus (HPV)-associated model for cancer immunotherapy where cell-mediated immunity is critical for protection. Two biologically active isoforms of IL-33 exist that are full-length or mature, but the ability of either isoform to function as a vaccine adjuvant that influences CD4 T helper 1 or CD8 T-cell immune responses is not defined. We showed that both IL-33 isoforms are capable of enhancing potent antigen-specific effector and memory T-cell immunity in vivo in a DNA vaccine setting. In addition, although both IL-33 isoforms drove robust IFN-γ responses, neither elevated secretion of IL-4 or immunoglobulin E levels. Further, both isoforms augmented vaccine-induced antigen-specific polyfunctional CD4(+) and CD8(+) T-cell responses, with a large proportion of CD8(+) T cells undergoing plurifunctional cytolytic degranulation. Therapeutic studies indicated that vaccination with either IL-33 isoform in conjunction with an HPV DNA vaccine caused rapid and complete regressions in vivo. Moreover, IL-33 could expand the magnitude of antigen-specific CD8(+) T-cell responses and elicit effector-memory CD8(+) T cells. Taken together, our results support the development of these IL-33 isoforms as immunoadjuvants in vaccinations against pathogens, including in the context of antitumor immunotherapy.

A synthetic consensus anti–spike protein DNA vaccine induces protective immunity against Middle East respiratory syndrome coronavirus in nonhuman primates

A consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge. Emerging vaccines Public outcry drives vaccine research during outbreaks of emerging infectious disease, but public support for vaccine development dries up when the outbreaks are resolved, frequently leaving promising vaccine candidates sitting on the shelf. DNA vaccines, with their potential for rapid large-scale production, may help overcome this hurdle. Muthumani et al. report the development of a synthetic DNA vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) that induces neutralizing antibodies in mice, macaques, and camels—natural hosts of MERS-CoV. Indeed, macaques vaccinated with this DNA vaccine were protected from viral challenge. These promising results support further development of DNA vaccines for emerging infections. First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

Interleukin 33: a switch-hitting cytokine.

For many years IL-33 has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Interestingly, IL-33 has now emerged as a cytokine with a plethora of pleiotropic properties. Depending on the immune cells targeted by IL-33, it is reported to not only promote Th2 immunity, but also to induce T helper type 1 (Th1) immunity. Furthermore, recent studies have revealed that IL-33 can activate CD8(+) T cells. These new studies provide evidence for its beneficial role in antiviral and antitumor immunity. Here we review the evidence of IL-33 to drive protective T cell immunity plus its potential use as an adjuvant in vaccination and tumor therapy.

Induction of Broad Cytotoxic T Cells by Protective DNA Vaccination Against Marburg and Ebola

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.

Synthetic DNA vaccine strategies against persistent viral infections.

The human body has developed an elaborate defense system against microbial pathogens and foreign antigens. However, particular microbes have evolved sophisticated mechanisms to evade immune surveillance, allowing persistence within the human host. In an effort to combat such infections, intensive research has focused on the development of effective prophylactic and therapeutic countermeasures to suppress or clear persistent viral infections. To date, popular therapeutic strategies have included the use of live-attenuated microbes, viral vectors and dendritic-cell vaccines aiming to help suppress or clear infection. In recent years, improved DNA vaccines have now re-emerged as a promising candidate for therapeutic intervention due to the development of advanced optimization and delivery technologies. For instance, genetic optimization of synthetic plasmid constructs and their encoded antigens, in vivo electroporation-mediated vaccine delivery, as well as codelivery with molecular adjuvants have collectively enhanced both transgene expression and the elicitation of vaccine-induced immunity. In addition, the development of potent heterologous prime–boost regimens has also provided significant contributions to DNA vaccine immunogenicity. Herein, the authors will focus on these recent improvements to this synthetic platform in relation to their application in combating persistent virus infection.

Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG

Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-γ responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.

IL-33 isoforms: their future as vaccine adjuvants?

The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy.

Molecular adjuvant IL-33 enhances the potency of a DNA vaccine in a lethal challenge model.

Identifying new molecular adjuvants that elicit effective vaccine-induced CD8(+) T cell immunity may be critical for the elimination of many challenging diseases including Tuberculosis, HIV and cancer. Here, we report that co-administration of molecular adjuvant IL-33 during vaccination enhanced the magnitude and function of antigen (Ag)-specific CD8(+) T cells against a model Ag, LCMV NP target protein. These enhanced responses were characterized by higher frequencies of Ag-specific, polyfunctional CD8(+) T cells exhibiting cytotoxic characteristics. Importantly, these cells were capable of robust expansion upon Ag-specific restimulation in vivo and conferred remarkable protection against a high dose lethal LCMV challenge. In addition, we demonstrate the ability of IL-33 to amplifying the frequency of Ag-specific KLRG1(+) effector CD8(+) T cells. These data show that IL-33 is a promising immunoadjuvant at improving T cell immunity in a vaccine setting and suggest further development and understanding of this molecular adjuvant for strategies against many obstinate infectious diseases and cancer.

Ubiquitin-like Molecule ISG15 Acts as an Immune Adjuvant to Enhance Antigen-specific CD8 T-cell Tumor Immunity

ISG15 is an ubiquitin-like protein induced by type I interferon associated with antiviral activity. ISG15 is also secreted and known to function as an immunomodulatory molecule. However, ISG15s role in influencing the adaptive CD8 T-cell responses has not been studied. Here, we demonstrate the efficacy of ISG15 as a vaccine adjuvant, inducing human papilloma virus (HPV) E7-specific IFNγ responses as well as the percentage of polyfunctional, cytolytic, and effector CD8 T-cell responses. Vaccination with ISG15 conferred remarkable control and/or regression of established HPV-associated tumor-bearing mice. T-cell depletion coupled with adoptive transfer experiments revealed that ISG15 protective efficacy was CD8 T-cell mediated. Importantly, we demonstrate that ISG15 vaccine-induced responses could be generated independent of ISGylation, suggesting that responses were mostly influenced by free ISG15. Our results provide more insight into the immunomodulatory properties of ISG15 and its potential to serve as an effective immune adjuvant in a therapeutic tumor or infectious disease setting.

Synthetic DNA Approach to Cytomegalovirus Vaccine/Immune Therapy

There is no licensed vaccine or cure for human cytomegalovirus (CMV), a ubiquitous β-herpes virus that infects 60-95 % of adults worldwide. Infection is a major cause of congenital abnormalities in newborns, contributes to development of childhood cerebral palsy and medulloblastoma, can result in severe disease in immunocompromised patients, and is a major impediment during successful organ transplantation. While CMV has been increasingly associated with numerous inflammatory diseases and cancers, only recently has it been correlated with increased risk of heart disease in adults, the number-one killer in the USA. These data, among others, suggest that subclinical CMV infection, or microinfection, in healthy individuals may play more of a causative role than an epiphenomenon in development of CMV-associated pathologies. Due to the myriad of diseases and complications associated with CMV, an efficacious vaccine would be highly valuable in reducing human morbidity and mortality as well as saving billions of dollars in annual health-care costs and disability adjusted life years (DALY) in the developing world. Therefore, the development of a safe efficacious CMV vaccine or immune therapy is paramount to the public health. This review aims to provide a brief overview on aspects of CMV infection and disease and focuses on current vaccine strategies. The use of new synthetic DNA vaccines might offer one such approach to this difficult problem.