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Scientists have recognized human genes that fight against the SARS-CoV-2 viral infection. The information about genes and their function can help to control infection and aids the understanding of crucial factors that causes severe infection. These novel genes are related to interferons, the frontline fighter in our body’s defense system and provide options for therapeutic strategies.
Sumit K. Chanda, Ph.D., professor and director of the Immunity and Pathogenesis Program at Sanford Burnham Prebys reported in the article that they focused on better understanding of the cellular response and downstream mechanism in cells to SARS-CoV-2, including the factors which causes strong or weak response to viral infection. He is the lead author of the study and explained that in this study they have gained new insights into how the human cells are exploited by invading virus and are still working towards finding any weak point of virus to develop new antivirals against SARS-CoV-2.
With the surge of pandemic, researchers and scientists found that in severe cases of COVID-19, the response of interferons to SARS-CoV-2 viral infection is low. This information led Chanda and other collaborators to search for interferon-stimulated genes (ISGs), are genes in human which are triggered by interferons and play important role in confining COVID-19 infection by controlling their viral replication in host.
The investigators have developed laboratory experiments to identify ISGs based on the previous knowledge gathered by the outbreak of SARS-CoV-1 from 2002-2004 which was similar to COVID-19 pandemic caused by SARS-CoV-2 virus.
The article reports that Chanda mentioned “we found that 65 ISGs controlled SAR-CoV-2 infection, including some that inhibited the virus’ ability to enter cells, some that suppressed manufacture of the RNA that is the virus’s lifeblood, and a cluster of genes that inhibited assembly of the virus.” They also found an interesting fact about ISGs that some of these genes revealed control over unrelated viruses, such as HIV, West Nile and seasonal flu.
Laura Martin-Sancho, Ph.D., a senior postdoctoral associate in the Chanda lab and first author of the study reported in the article that they identified 8 different ISGs that blocked the replication of both SARS-CoV-1 and CoV-2 in the subcellular compartments responsible for packaging of proteins, which provide option to exploit these vulnerable sites to restrict infection. They are further investigating whether the genetic variability within the ISGs is associated with COVID-19 severity.
The next step for researchers will be investigating and observing the biology of variants of SARS-CoV-2 that are evolving and affecting vaccine efficacy. Martin-Sancho mentioned that their lab has already started gathering all the possible variants for further investigation.
“It’s vitally important that we don’t take our foot off the pedal of basic research efforts now that vaccines are helping control the pandemic,” reported in the article by Chanda.
“We’ve come so far so fast because of investment in fundamental research at Sanford Burnham Prebys and elsewhere, and our continued efforts will be especially important when, not if, another viral outbreak occurs,” concluded Chanda.
AstraZeneca’s potential coronavirus vaccine is likely to provide protection against contracting Covid-19 for about a year, the company’s chief executive told a Belgian radio station on Tuesday.
The British drugmaker has already begun human trials of the vaccine developed by the University of Oxford, with a phase I trial in Britain due to end soon and a phase III trial already begun, Pascal Soriot told broadcaster Bel RTL.
“We think that it will protect for about a year,” Soriot said.
AstraZeneca said on Saturday that it had signed contracts with France, Germany, Italy and the Netherlands to supply the European Union with up to 400 million doses of the potential vaccine.
It has also agreed deals with Britain and the United States.
“If all goes well, we will have the results of the clinical trials in August/September. We are manufacturing in parallel. We will be ready to deliver from October if all goes well,” Soriot said.
This piece at Forbes by Bill Haseltine has set off a lot of comment – it’s a look at the Oxford group’s vaccine candidate as compared to the SinoVac candidate, and you may recall (background here) that these are the two teams that have separately reported that their vaccines appear to protect rhesus monkeys from infection after exposure to the coronavirus. Haseltine has some criticisms of the Oxford data, and as you will see from that link to his name, his opinions deserve to be taken seriously. So what’s going on? Update: here’s the take on this at BioCentury.
Looking at the preprint on the Oxford results, Haseltine has a problem with the claim that the monkeys were protected from infection by a dose of ChAdOx1 nCoV-19. The key data are in the preprint’s Figure 3. The Oxford team checked for viral RNA several different ways. One was using bronchoaveolar lavage (BAL fluid), a sampling technique that involves running a bronchoscope down into the lungs and washing out aveolar spaces – a pretty darn invasive assay, which is why you don’t hear about it all that much compared to the still-not-so-nonivasive nose swabs. BAL fluid of the virus-exposed unvaccinated animals showed coronavirus genomic RNA throughout the study, and viral subgenomic RNA (more indicative of active replication) at days 3 and 5 after exposure. Meanwhile, the vaccinated animals showed the genomic RNA in only two monkeys, and no subgenomic RNA at all.
So far, so good. But both vaccinated and unvaccinated monkeys showed the same amount of viral genomic RNA from nose swab samples (Figure 3c). That’s the test that’s used out in the human population, and that means that the vaccinated animals would still be declared as positive for the coronavirus after being exposed to it. And the other thing that Haseltine notes is that the amount (the “titer”, in the lingo) of neutralizing antibodies in the blood of the vaccinated animals does not appear to be that high. You’d like to be able to dilute the blood antibody samples down by hundreds of times or even a thousandfold and still see antiviral activity in an in vitro assay, but in the Oxford case the activity started disappearing at about fortyfold dilution (Figure 2b).
On the positive side, 2/3 of the unvaccinated animals showed clear evidence of viral pneumonia at autopsy, but none of the vaccinated ones did. The conclusion is that the vaccinated animals were indeed infected – the vaccine did not protect against that – but that the disease was definitely less severe. But these results mean that the virus might well still be transmissible from people who had been so vaccinated, even if the disease course itself was not as deadly. You’d want to do better than that, if you can. Haseltine’s take is “Time will tell if this is the best approach. I wouldn’t bet on it.”
Haseltine compares these results to the SinoVac inactivated virus vaccine, and finds that that one looks better – at its highest dose, no viral RNA was recovered from the tissues of the vaccinated animals, for example. This sort of “sterilizing immunity” is what you’d want to aim for – it gives the virus nowhere to go in the human population if you can vaccinate enough people. But it’s worth noting that the SinoVac results were from three doses of their vaccine (versus one of the Oxford candidate), and the viral exposure challenge was about half as strong (total viral particles) as what the Oxford paper used. The Oxford group also inoculated their monkeys in both the upper and lower respiratory tract, while the SinoVac team used a single inoculation in the trachea. So I agree with that tweet linked from AndyBiotech; I don’t think that a head-to-head comparison is fair. But Haseltine’s point stands, that the results as we have them from the ChAdOx1 nCoV-19 vaccine did not actually protect monkeys from infection.
New Coronavirus Passive Vaccine Developed by Israeli Researchers
Reporter: Irina Robu, PhD
Researchers at Bar-Ilan University have identified short amino acid sequences that could help the development of a vaccine against COVID-19 virus. Of the 25 epitopes that were discovered to be 100% identical to SARS, seven are theoretically efficient vaccine candidates. Their research indicate that they could cover as much as 87% of the world population
Their study has identified a set of immunodominant epitopes from the SARS-CoV-2 proteome, which are capable of generating antibody and cell mediated immune responses. The epitopes, known as antigenic determinants, are the part of the antigen that binds to a specific antigen receptor on the surface of B cells or T cells and are able to provoke an immune response.
It is known that immune response occurs within an organism for the purpose of defending against foreign invaders such as viruses, bacteria, parasites and fungi. The immune responses that are based on specific immunodominant epitopes contain the generation of both antibody- and cell-mediated immunity against pathogens. Such immunity can facilitate fast and effective elimination of the pathogen. The end result is a passive vaccine capable of capable of activating both cellular and humoral immune responses in humans.
According to the team at Bar-Ilan University, the mapped coronavirus epitopes with those of the influenza virus. And they found that 85% of the sequence identity with experimentally detected epitopes of Severe Acute Respiratory Syndrome-related coronavirus (SARS-CoV).
Additional analysis indicated that the epitopes are non-allergic and non-toxic to humans and have very low risk for generating autoimmune responses. The team is looking to partner with companies to build vaccine constructs and test them in-vitro and on animal trials before starting any clinical trials.
JP Morgan Healthcare Conference Update: Sage, Mersana, Shutdown Woes and Babies
Published: Jan 10, 2019By Alex Keown
With the J.P. Morgan Healthcare Conference winding down, companies remain busy striking deals and informing investors about pipeline advances. BioSpace snagged some of the interesting news bits to come out of the conference from Wednesday.
SAGE Therapeutics – Following a positive Phase III report that its postpartum depression treatment candidate SAGE-217 hit the mark in its late-stage clinical trial, Sage Therapeutics is eying the potential to have multiple treatment options available for patients. At the start of J.P. Morgan, Sage said that patients treated with SAGE-217 had a statistically significant improvement of 17.8 points in the Hamilton Rating Scale for Depression, compared to 13.6 for placebo. The company plans to seek approval for SAGE-2017, but before that, the FDA is expected to make a decision on Zulresso in March. Zulresso already passed muster from advisory committees in November, and if approved, would be the first drug specifically for postpartum depression. In an interview with the Business Journal, Chief Business Officer Mike Cloonan said the company believes there is room in the market for both medications, particularly since the medications address different patient populations.
Mersana Therapeutics – After a breakup with Takeda Pharmaceutical and the shelving of its lead product, Cambridge, Mass.-based Mersana is making a new path. Even though a partial clinical hold was lifted following the death of a patient the company opted to shelve development of XMT-1522. During a presentation at JPM, CEO Anna Protopapas noted that many other companies are developing therapies that target the HER2 protein, which led to the decision, according to the Boston Business Journal. Protopapas said the HER2 space is highly competitive and now the company will focus on its other asset, XMT-1536, an ADC targeting NaPi2b, an antigen highly expressed in the majority of non-squamous NSCLC and epithelial ovarian cancer. XMT-1536 is currently in Phase 1 clinical trials for NaPi2b-expressing cancers, including ovarian cancer, non-small cell lung cancer and other cancers. Data on XMT-1536 is expected in the first half of 2019.
Novavax – During a JPM presentation, Stan Erck, CEO of Novavax, pointed to the company’s RSV vaccine, which is in late-stage development. The vaccine is being developed for the mother, in order to protect an infant. The mother transfers the antibodies to the infant, which will provide the baby with protection from RSV in its first six months. Erck called the program historic. He said the Phase III program is in its fourth year and the company has vaccinated 4,636 women. He said they are tracking the women and the babies. Researchers call the mothers every week through the first six months of the baby’s life to acquire data. Erck said the company anticipates announcing trial data this quarter. If approved, Erck said the market for the vaccine could be a significant revenue driver.
“You have 3.9 million birth cohorts and we expect 80 percent to 90 percent of those mothers to be vaccinated as a pediatric vaccine and in the U.S. the market rate is somewhere between $750 million and a $1 billion and then double that for worldwide market. So it’s a large market and we will be first to market in this,” Erck said, according to a transcript of the presentation.
Denali Therapeutics – Denali forged a collaboration with Germany-based SIRION Biotech to develop gene therapies for central nervous disorders. The two companies plan to develop adeno-associated virus (AAV) vectors to enable therapeutics to cross the blood-brain barrier for clinical applications in neurodegenerative diseases including Parkinson’s, Alzheimer’s disease, ALS and certain other diseases of the CNS.
AstraZeneca – Pharma giant AstraZeneca reported that in 2019 net prices on average across the portfolio will decrease versus 2018. With a backdrop of intense public and government scrutiny over pricing, Market Access head Rick Suarez said the company is increasing its pricing transparency. Additionally, he said the company is looking at new ways to price drugs, such as value-based reimbursement agreements with payers, Pink Sheet reported.
Amarin Corporation – As the company eyes a potential label expansion approval for its cardiovascular disease treatment Vascepa, Amarin Corporation has been proactively hiring hundreds of sales reps. In the fourth quarter, the company hired 265 new sales reps, giving the company a sales team of more than 400, CEO John Thero said. Thero noted that is a label expansion is granted by the FDA, “revenues will increase at least 50 percent over what we did in the prior year, which would give us revenues of approximate $350 million in 2019.”
Government Woes – As the partial government shutdown in the United States continues into its third week, biotech leaders at JPM raised concern as the FDA’s carryover funds are dwindling. With no new funding coming in, reviews of New Drug Applications won’t be able to continue past February, Pink Sheet said. While reviews are currently ongoing, no New Drug Applications are being accepted by the FDA at this time. With the halt of NDA applications, that has also caused some companies to delay plans for an initial public offering. It’s hard to raise potential investor excitement without the regulatory support of a potential drug approval. During a panel discussion, Jonathan Leff, a partner at Deerfield Management, noted that the ongoing government shutdown is a reminder of how “overwhelmingly dependent the whole industry of biotech and drug development is on government,” Pink Sheet said.
Other posts on the JP Morgan 2019 Healthcare Conference on this Open Access Journal include:
NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic α-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.
Cancer Res. 2013 Jul 1;73(13):3842-51. doi: 10.1158/0008-5472.CAN-12-1974. Epub 2013 May 23.
Avirulent Toxoplasma gondii generates therapeutic antitumor immunity by reversing immunosuppression in the ovarian cancer microenvironment.
Reversing tumor-associated immunosuppression seems necessary to stimulate effective therapeutic immunity against lethal epithelial tumors. Here, we show this goal can be addressed using cps, an avirulent, nonreplicating uracil auxotroph strain of the parasite Toxoplasma gondii (T. gondii), which preferentially invades immunosuppressive CD11c(+) antigen-presenting cells in the ovarian carcinoma microenvironment. Tumor-associated CD11c(+) cells invaded by cps were converted to immunostimulatory phenotypes, which expressed increased levels of the T-cell receptor costimulatory molecules CD80 and CD86. In response to cps treatment of the immunosuppressive ovarian tumor environment, CD11c(+) cellsregained the ability to efficiently cross-present antigen and prime CD8(+) T-cell responses. Correspondingly, cps treatment markedly increased tumor antigen-specific responses by CD8(+) T cells. Adoptive transfer experiments showed that these antitumor T-cell responses were effective in suppressing solid tumor development. Indeed, intraperitoneal cps treatment triggered rejection of established ID8-VegfA tumors, an aggressive xenograft model of ovarian carcinoma, also conferring a survival benefit in a related aggressive model (ID8-Defb29/Vegf-A). The therapeutic benefit of cps treatment relied on expression of IL-12, but it was unexpectedly independent of MyD88 signaling as well as immune experience with T. gondii. Taken together, our results establish that cps preferentially invades tumor-associated antigen-presenting cells and restores their ability to trigger potent antitumor CD8(+) T-cell responses. Immunochemotherapeutic applications of cps might be broadly useful to reawaken natural immunity in the highly immunosuppressive microenvironment of most solid tumors.
Toll-like receptor 3 (TLR3) agonists have been extensively used as adjuvants for anticancer vaccines. However, their immunostimulatory effects and precise mechanisms of action in the presence of antineoplastic monoclonal antibodies (mAbs) have not yet been evaluated. We investigated the effect of TLR3 agonists on cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against head and neck cancer (HNC) cells, as well as on dendritic cell (DC) maturation and cross-priming of epidermal growth factor receptor (EGFR)-specific CD8+ T cells. The cytotoxic activity of peripheral blood mononuclear cells (PBMCs) or isolated natural killer (NK) cells expressing polymorphic variants (at codon 158) of the Fcγ receptor IIIa (FcγIIIa) was determined in 51Cr release assays upon incubation with the TLR3 agonist poly-ICLC. NK cell stimulation was measured based on activation and degranulation markers, while DC maturation in the presence of poly-ICLC was assessed using flow cytometry. The DC-mediated cross priming of EGFR-specific CD8+ T cells was monitored upon in vitro stimulation with tetramer-based flow cytometry. TLR3-stimulated, unfractionated PBMCs from HNC patients mediated robust cetuximab-dependent ADCC, which was abrogated by NK-cell depletion. The cytolytic activity of TLR3-stimulated NK cells differed among cells expressing different polymorphic variants of FcγRIIIa, and NK cells exposed to both poly-ICLC and cetuximab expressed higher levels of CD107a and granzyme B than their counterparts exposed to either stimulus alone. Poly-ICLC plus cetuximab also induced a robust upregulation of CD80, CD83 and CD86 on the surface of DCs, a process that was partially NK-cell dependent. Furthermore, DCs matured in these conditions exhibited improved cross-priming abilities, resulting in higher numbers of EGFR-specific CD8+ T cells. These findings suggest that TLR3 agonists may provide a convenient means to improve the efficacy of mAb-based anticancer regimens.
Evidence has been accumulating since the middle of the last century, first from animal models and later from studies in cancer patients, that the immune system can recognise and reject tumours. The goal of tumour immunology has been to understand the components of the immune system that are important for tumour immunosurveillance and tumour rejection to understand how, when, and why they fail in cases of clinical disease. Immunotherapy, which involves strengthening the cancer patient’s immune system by improving its ability to recognise the tumour or providing a missing immune effector function, is one treatment approach that holds promise of a life-long cure [4].
Studies of cancer–immune system interactions have revealed that every known innate and adaptive immune effector mechanism participates in tumour recognition and control [5]. The first few transformed cells are detected by NK cells through their encounter with specific ligands on tumour cells. This leads to the destruction of some transformed cells and the uptake and processing of their fragments by macrophages and dendritic cells. In turn, these macrophages and dendritic cells are activated to secrete many inflammatory cytokines and present tumour cell-derived molecules to T- and B cells. Activation of T- and B cells leads to the production of additional cytokines that further promote activation of innate immunity and support the expansion and production of tumour-specific T cells and antibodies, respectively. The full power of the adaptive immune system leads to the elimination of remaining tumour cells and, importantly, to the generation of immune memory to specific tumour components that will serve to prevent tumour recurrence.
Effectors of adaptive immunity, such as CD4+ helper T cells, CD8+ cytotoxic T cells, and antibodies, specifically target tumour antigens; i.e. molecules expressed in tumour cells, but not in normal cells. Tumour antigens are normal cellular proteins that are abnormally expressed as a result of genetic mutations, quantitative differences in expression, or differences in posttranslational modifications [5]. In tumour types that have a well-documented viral origin, such as cervical cancer, caused by the human papillomavirus [5], or hepatocellular carcinoma caused by the hepatitis B virus [6], viral proteins can also serve as tumour antigens and targets for antitumour immune response [7].
The first indication that tumours carried molecules distinct from those on the normal cell of origin was derived from immunising mice with human tumours and selecting antibodies that recognised human tumour cells but not their normal counterparts. The major question was whether some, or all, of these molecules would also be recognised by the human immune system. 2011 was an important anniversary for human tumour immunology, marking 20 years since the publication by van der Bruggen et al. [8] that described the cloning of MAGE-1, a gene that encodes a human melanoma antigen recognised by patient’s antitumour T cells. This was not a mutant protein; its recognition by the immune system was due to the fact that it was only expressed by transformed, malignant cells and, with the exception of testicular germ cells, was not expressed in normal adult tissue. Many similar discoveries followed, with each new molecule providing a better understanding of what might be good targets for different forms of cancer immunotherapy. Tumour antigens have been tested as vaccines, as targets for monoclonal antibodies, and as targets for adoptively transferred cytotoxic T cells. There is a wealth of publications from preclinical studies targeting these antigens and results from phase I/II clinical trials. Recently, these studies were critically reviewed and a list of tumour antigens with the largest body of available data compiled [9]. The goal was to encourage faster progress in the design, testing, and approval of immunotherapeutic reagents that incorporate or target the most promising antigens.
As highlighted in the article two scenarios which present problems emerged:
In the past, immunotherapy was referred to as ‘passive’ (e.g. the infusion of preformed immune effectors, such as antibodies, cytokines, or activated T cells, NK cells, or lymphokine-activated killer cells), presumably acting directly on the tumour and independent of the immune system or ‘active’ (e.g. vaccines), designed to activate and therefore be dependent on the patient’s immune system. it has since become clear that both passive and active immunotherapies depend on the patient’s immune system for long-term tumour control or complete tumour elimination. anticancer monoclonal antibodies are a well-established class of immunotherapeutic agent. HOWEVER, The potential of these antibodies is drastically undermined by their administration relatively late in the disease course, when the patient’s immune system is largely compromised. Under more optimal conditions, antibody treatment might result not only in the direct cytostatic or cytotoxic effect on the tumour cell, but also in the loading of antibody-bound tumour antigens onto antigen presenting cells (APC) in the tumour microenvironment. The resultant cross-presentation to antitumour T- and B cells could result in additional antibodies to these antigens being produced, and propagation of the immune response at the tumour site would maintain tumour elimination long after the infused monoclonal antibody is gone.
The same scenario could be predicted for adoptively transferred T cells. Unlike antibodies, transferred T cells persist longer and may provide a memory response [14]; however, as long as the memory response is restricted to one clone, or a limited number of clones, then antigen-negative tumours will be able to escape. In addition, cancer vaccines encounter large numbers of immunosuppressive Tregand MDSC in circulation, as well as immunosuppressive cell-derived soluble products that flood the lymph nodes, preventing maturation of APCs and activation of T cells. Even when vaccines are delivered in the context ofex vivomatured and activated dendritic cells, their ability to activate T cells is compromised by the high-level expression of various molecules on T cells that block this process.
The scenarios proposed above present a rather bleak picture of the potential of immunotherapy to achieve the cure for cancer that has eluded standard therapy [15]. Interestingly, failures of some standard therapies are beginning to be ascribed to their inability to activate the patient’s immune system [16]. However, rather than seeing the picture as a deterrent, it should be considered as a road map, providing at least two major directions for new developments in immunotherapy.
The first direction is to continue using the old classes of immunotherapy that target the cancer directly, but to use them in combination with therapies that target the immune system in the tumour microenvironment, such as cytokines, suppressors of Treg or MDSC activity, or antibodies that modulate T-cell activity. The recently approved antibody, ipilimumab, which acts to sustain cytotoxic T-cell activity by augmenting T-cell activation and proliferation, is one example of such an immunomodulatory agent [17].
The other direction is to use immunotherapies, both old and new, for preventing cancer in individuals at high risk [18]. Studies of the tumour microenvironment are providing information about immunosurveillance of tumours from early premalignant lesions to more advanced dysplastic lesions to cancer. At each step, tumour-derived and immune system-derived components have a unique composition that will have distinct effects on immunotherapy. Because these premalignant microenvironments are less developed and immunosuppression is less entrenched, it should be easier to modulate towards the elimination of abnormal cells.
The combination of viral vaccination with intratumoral (IT) administration of CpG ODNs is yet to be investigated as an immunotherapeutic treatment for solid tumors. Here, we show that such a treatment regime can benefit survival of tumor-challenged mice. C57BL/6 mice bearing ovalbumin (OVA)-expressing EG.7 thymoma tumors were therapeutically vaccinated with adenovirus type 5 encoding OVA (Ad5-OVA), and the tumors subsequently injected with the immunostimulatory TLR9 agonist, CpG-B ODN 1826 (CpG), 4, 7, 10, and 13 days later. This therapeutic combination resulted in enhanced mean survival times that were more than 3.5× longer than naïve mice, and greater than 40% of mice were cured and capable of resisting subsequent tumor challenge. This suggests that an adaptive immune response was generated. Both Ad5-OVA and Ad5-OVA + CpG IT treatments led to significantly increased levels of H-2 K(b)-OVA-specific CD8+ lymphocytes in the peripheral blood and intratumorally. Lymphocyte depletion studies performed in vivo implicated both NK cells and CD8+ lymphocytes as co-contributors to the therapeutic effect. Analysis of tumor infiltrating lymphocytes (TILs) on day 12 post-tumor challenge revealed that mice treated with Ad5-OVA + CpG IT possessed a significantly reduced percentage of regulatory T lymphocytes (Tregs) within the CD4+ lymphocyte population, compared with TILs isolated from mice treated with Ad5-OVA only. In addition, the proportion of CD8+ TILs that were OVA-specific was reproducibly higher in the mice treated with Ad5-OVA + CpG IT compared with other treatment groups. These findings highlight the therapeutic potential of combining intratumoral CpG and vaccination with virus encoding tumor antigen.
Adv Drug Deliv Rev. 2009 Mar 28;61(3):268-74. doi: 10.1016/j.addr.2008.12.005. Epub 2009 Jan 7.
The use of an adenovirus transduced to express a prostate cancer antigen (PSA) as a vaccine for the treatment of prostate cancer has been shown to be active in the destruction of antigen-expressing prostate tumor cells in a pre-clinical model, using Balb/C or PSA transgenic mice. The destruction of PSA-secreting mouse prostate tumors was observed in Ad/PSA immunized mice in a prophylaxis study with 70% of the mice surviving long term tumor free. This successful immunotherapy was not observed in therapeutic studies in which tumors were established before vaccination and the development of anti-PSA immune response was not as easily generated in PSA transgenic mice. Immunization of conventional and transgenic animals was enhanced by incorporating a collagen matrix into the immunizing injection. Therefore the need to strengthen anti-PSA and anti-prostate cancer immunity was an obvious next step in developing a successful prostate cancer immunotherapy. Because the use ofimmunostimulatory CpG motifs was shown to enhance immune responses to a wide variety of antigens, our studies incorporated CpG into the Ad/PSA vaccine experimental plans. The results of the subsequent studies demonstrated a dichotomy where Ad/PSA plus CpG enhanced the in vivo destruction of PSA-secreting tumors and the survival of experimental animals, but revealed that the number and in vitro activities of antigen specific CD8+ T cells was decreased as compared to the values observed when the vaccine alone was used for immunization. The dichotomous observations were confirmed using another antigen system, OVA also incorporated into a replication defective adenovirus. Despite the reduction in antigen-specific CD8+ cells after vaccine plus CpG immunization the enhanced destruction of sc and systemic tumors was shown to be mediated entirely by CD8+ T cells. Finally, the reduction of the CD8+ T cells was the result of an observed decrease in the proliferation of the antigen specific cell population.
DNA vaccines can induce impressive specific cellular immune response (IR) when taking advantage of their recognition as pathogen-associated molecular patterns (PAMP) through Toll-like receptors (TLR) expressed on/in cells of the innate immune system. Among the many types of PAMP,immunostimulatory DNA, so-called CpG motifs, was shown to interact specifically with TLR9, which is expressed in plasmacytoid dendritic cells(pDC), a key regulatory cell for the activation of innate and adaptive IR. We now report that CpG motifs, when introduced into the backbone, are a useful adjuvant for plasmid-based DNA (pDNA) vaccines to induce melanoma antigen-specific protective T cell responses in the Cloudman M3/DBA/2 model. The CpG-enriched pDNA vaccine induced protection against subsequent challenge with melanoma cells at significantly higher levels than its parental unmodified vector. Preferential induction of an antigen-specific, protective T cell response could be demonstrated by (i) induction of antigen-dependent tumor cell protection, (ii) complete loss of protection by in vivo CD4+/CD8+T cell- but not NK cell-depletion, and (iii) the detection of antigen-specific T cell responses but not of relevant NK cell activity in vitro. These results demonstrate that employing PAMP in pDNA vaccines improves the induction of protective, antigen-specific, T cell-mediated IR.
J Biomed Sci. 2016 Jan 25;23(1):16. doi: 10.1186/s12929-016-0238-3.
DNA vaccines have emerged as an attractive approach for the generation of cytotoxic T lymphocytes (CTL). In our previous study, we found That Toll like receptor (TLR) ligands are promising candidates for the development of novel adjuvants for DNA vaccine. To improve the efficacy of DNA vaccine directed against human papillomavirus (HPV) tumors, we evaluated whether co-administration of a TLR4 ligand, monophosphoryl lipid A (MPL), and Natural Killer T Cell Ligand α-Galactosylceramide(α-GalCer) adjuvants with DNA vaccine would influence the anti-tumor efficacy of DNA vaccinations.
METHODS:
We investigated the effectiveness of α-GalCer and MPL combination as an adjuvant with an HPV-16 E7 DNA vaccine to enhance antitumor immune responses.
RESULTS:
By using adjuvant combination for a DNA vaccine, we found that the levels of lymphocyte proliferation, CTL activity, IFN- γ, IL-4 and IL-12 responses, and tumor protection against TC-1 cells were significantly increased compared to the DNA vaccine with individual adjuvants. In addition, inhibition of IL-18 signaling during vaccination decreased IFN-γ responses and tumor protection, and that this inhibition suggested stimulatory role of IL-18 in adjuvant effects of α-GalCer and MPL combination.
CONCLUSION:
The strong adjuvanticity associated with α-GalCer/MPL combination may to be an important tool in the development of novel and strong cancer immunotherapy.
Cancer Sci. 2015 Dec;106(12):1659-68. doi: 10.1111/cas.12824. Epub 2015 Nov 18.
Immune-enhancing adjuvants usually targets antigen (Ag)-presenting cells to tune up cellular and humoral immunity. CD141(+) dendritic cells (DC) represent the professional Ag-presenting cells in humans. In response to microbial pattern molecules, these DCs upgrade the maturation stage sufficient to improve cross-presentation of exogenous Ag, and upregulation of MHC and costimulators, allowing CD4/CD8 T cells to proliferate and liberating cytokines/chemokines that support lymphocyte attraction and survival. These DCs also facilitate natural killer-mediated cell damage. Toll-like receptors (TLRs) and their signaling pathways in DCs play a pivotal role in DC maturation. Therefore, providing adjuvants in addition to Ag is indispensable for successful vaccine immunotherapy for cancer, which has been approved in comparison with antimicrobial vaccines. Mouse CD8α(+) DCs express TLR7 and TLR9 in addition to the TLR2 family (TLR1, 2, and 6) and TLR3, whereas human CD141(+) DCs exclusively express the TLR2 family and TLR3. Although human and mouse plasmacytoid DCs commonly express TLR7/9 to respond to their agonists, the results on mouse adjuvant studies using TLR7/9 agonists cannot be simply extrapolated to human adjuvant immunotherapy. In contrast, TLR2 and TLR3 are similarly expressed in both human and mouse Ag-presenting DCs. Bacillus Calmette-Guerin peptidoglycan and polyinosinic-polycytidylic acid are representative agonists for TLR2 and TLR3, respectively, although they additionally stimulate cytoplasmic sensors: their functional specificities may not be limited to the relevant TLRs. These adjuvants have been posted up to a certain achievement in immunotherapy in some cancers. We herein summarize the history and perspectives of TLR2 and TLR3 agonists in vaccine-adjuvant immunotherapy for cancer.
Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells have traditionally been classified as a component of the innate immune system, they have recently been shown in mice and humans to exhibit certain features of immunological memory, including an ability to undergo a clonal-like expansion during virus infection, generate long-lived progeny (i.e. memory cells), and mediate recall responses against previously encountered pathogens–all characteristics previously ascribed only to adaptive immune responses by B and T cells in mammals. To date, the molecular events that govern the generation of NK cell memory are not completely understood. Using a mouse model of cytomegalovirus infection, we demonstrate that individual pro-inflammatory IL-12, IL-18, and type I-IFN signaling pathways are indispensible and play non-redundant roles in the generation of virus-specific NK cell memory. Furthermore, we discovered that antigen-specific proliferation and protection by NK cells is mediated by the transcription factor Zbtb32, which is induced by pro-inflammatory cytokines and promotes a cell cycle program in activated NK cells. A greater understanding of the molecular mechanisms controllingNKcell responses will provide novel strategies for tailoring vaccinesto target infectious disease.
Cambridge Healthtech Institute’s inaugural Cancer Immunotherapy and Combinations meeting will convene immuno-oncology researchers, cancer immunotherapy developers, and technology providers to discuss next-generation approaches and combinations, including small molecule development, to enhance the efficacy of checkpoint inhibitors.
BISPECIFIC ANTIBODIES – DUAL TARGETING
FEATURED PRESENTATION: ANTI-PD1 OR CD137 ENHANCES NK-CELL CYTOTOXICITY TOWARDS CD30+ HODGKIN LYMPHOMA INDUCED BY CD30/CD16A TANDAB AFM13 Martin Treder, Ph.D., CSO, R&D, Affimed
In vivo Efficacy of Bispecific Antibodies Targeting Two Immune-Modulatory Receptors Jacqueline Doody, Ph.D., Vice President, Immunology, F-star Biotechnology, Ltd
Dual-Targeting Bispecific Antibodies for Selective Neutralization of CD47 on Cancer Cells Krzysztof Masternak, Ph.D., Head, Biology, Therapeutic Antibody Discovery, Novimmune
Update on MCLA-134: A Biclonics® Binding Two Immunomodulatory Targets Expressed by T Cells Mark Throsby, Ph.D., CSO, Merus
The ImmTAC Technology: A Cutting-Edge Immunotherapy for Cancer Treatment Samir Hassan, Ph.D., Director, Translational Research & Development, Immunocore Ltd.
RADIOTHERAPY AND CHEMOTHERAPY – PD-1 COMBINATIONS
Rational Development of Combinations of Antiangiogenic Therapy with Immune Checkpoint Blockers Using Mouse Models of HCC and Cirrhosis Dan Duda, D.M.D., Ph.D., Associate Professor, Steele Laboratories for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School
Harnessing the Immune Microenvironment of Gastrointestinal Cancers Using Combined Modalities Osama Rahma, M.D., Assistant Professor, Internal Medicine/Oncology, University of Virginia
AGONIST – PD-1 AND CTLA-4 COMBINATIONS
The Role of the Target in the Disposition and Immunogenicity of an Anti-GITR Agonist Antibody Enrique Escandón, Ph.D., Senior Principal Scientist, DMPK and Disposition, Merck
Combination of 4-1BB Agonist and PD-1 Antagonist Promotes Antitumor Effector/Memory CD8 T Cells Changyu Wang, Ph.D., Director, Cancer Immunology, Pfizer
Combination Immunotherapy with Checkpoint Blockade, Agonist Anti-OX40 mAb, and Vaccination Rescues Anergic CD8 T Cells William Redmond, Ph.D., Associate Member, Laboratory of Cancer Immunotherapy, Earle A. Chiles Research Institute, Providence Portland Medical Center
Interactive Breakout Discussion Groups with Continental Breakfast
This session features various discussion groups that are led by a moderator/s who ensures focused conversations around the key issues listed. Attendees choose to join a specific group and the small, informal setting facilitates sharing of ideas and active networking. Continental breakfast is available for all participants.
Topic: Small Molecule Targeting of IDO1 and TDO for Cancer Immunotherapy
Moderator: Rogier Buijsman, Ph.D., Head, Chemistry, Netherlands Translational Research Center B.V. (NTRC)
Overcoming challenges of current IDO1 inhibitors lacking selectivity over TDO and having suboptimal drug-like properties
Advances in IDO1 and TDO inhibitor screening
Is selective IDO1 or TDO inhibitors is required, or a dual IDO1/TDO inhibitor is preferred to obtain optimal efficacy and safety in the clinic?
Topic: Strategies for Developing Bispecific Antibodies for Cancer Immunotherapy
Moderator: Krzysztof Masternak, Ph.D., Head, Biology, Therapeutic Antibody Discovery, Novimmune
Considerations for efficacy in vitro and in vivo: selectivity for tumor cells, ADCP, ADCC, in vivo efficacy (xenograft models)
Insights into mechanisms of action
Safety considerations: binding selectivity, PK and tox studies
Topic: Combining Standard Antiangiogenic Therapy with Immune Checkpoint Inhibitors
Moderator: Dan Duda, D.M.D., Ph.D., Associate Professor, Steele Laboratories for Tumor Biology, Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical School
Will checkpoint combination with chemotherapy or other targeted agents prove to have too many toxicity issues?
How do we minimize overlapping toxic effects of radiation and immunotherapy?
How to optimize the sequencing of these two treatment modalities?
SMALL MOLECULE INHIBITORS AS SINGLE AND CHECKPOINT COMBINATION AGENTS
Selective Small Molecule Inhibitors of IDO1 and TDO for Cancer Immunotherapy Rogier Buijsman, Ph.D., Head, Chemistry, Netherlands Translational Research Center B.V. (NTRC)
Potent and Selective Small Molecule USP7 Inhibitors for Cancer Immunotherapy Suresh Kumar, Ph.D., Director, R&D, Progenra, Inc.
Epigenetic Agents for Combination with Cancer Immunotherapy Svetlana Hamm, Ph.D., Head, Biology, Translational Pharmacology, 4SC Group
VACCINES AND CHECKPOINT BLOCKADE IMMUNOTHERAPY
Immunotherapy for Mesothelioma with an in vivo DC Vaccine and PD-1/PD-L1 Blockade Huabiao Chen, M.D., Ph.D., Principal Investigator, Vaccine and Immunotherapy Center, Massachusetts General Hospital
Bringing Together Checkpoint Inhibition with Vaccines Using Customizing Capsids Willie Quinn, Ph.D., President & CEO, Bullet Bio
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Researchers trying to develop an anti-malaria vaccine in pregnant women have stumbled upon an unexpected discovery that could potentially transform cancer therapy. They revealed that the carbohydrate that the malaria parasite attaches itself to – an oncofetal chondroitin sulfate – which is expressed in the placenta is the same as the one displayed on proteoglycans in cancerous cells.
By conjugating diphtheria toxin to recombinant malaria protein VAR2CSA (rVAR2), the researchers were able to inhibit in vivo tumor growth and metastasis. The study demonstrates how a parasite-derived protein can be exploited to target a common but complex, cancer-associated modification. The researchers have already tested thousands of samples from brain tumors to leukemia and found that the malaria protein is able to inhibit more than 90% of all types of tumors. While this approach seems very promising a major drawback is that this treatment cannot be applied to pregnant women. Since the rVAR2-conjugated toxin cannot distinguish placenta from tumor, it would inhibit placenta growth as well. The research team wants to conduct human testing but the earliest possible scenario they predict would be in 4 years.
Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein.
Cancer Cell. 2015 Oct 12;28(4):500-14. http://dx.doi.org:/ 10.1016/j.ccell.2015.09.003.
Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.
Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA.
Malar J. 2008 Jun 6;7:104. doi: 10.1186/1475-2875-7-104.
BACKGROUND:
Pregnancy malaria is caused by Plasmodium falciparum-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA in vitro; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.
METHODS:
To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire var2csa coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.
RESULTS:
The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.
CONCLUSION:
Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.
AM (Pregnancy Associated Malaria) is a major health problem in malaria-endemic areas and on a world basis it affects millions of pregnant women and their offspring. The presence of parasites in the placenta of pregnant women can have serious consequences for both mother and child including: maternal anaemia, premature delivery, low birth weight and increased infant mortality [1]. In malaria endemic areas, children acquire clinical immunity after multiple infections, and adults are in general protected against malaria. Women who have acquired immunity against malaria during childhood become susceptible to malaria during pregnancy due to novel parasite phenotypes expressing unique antigens not encountered during childhood infections [2,3]. In areas of high parasite transmission PAM mainly affects primigravidae since immunity is acquired as a function of gravidity [1]. Protective antibodies target proteins expressed on the surface of infected erythrocytes (IE), which mediate binding to syncytiotrophoblasts. By this process, the parasite is not filtered through the spleen and thus avoids exposure to effector mechanisms, which clear erythrocytes infected with late blood stage parasite from circulation [4]. The best characterized surface protein is thePlasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) [5,6], which is encoded by the polymorphic var gene family containing 50–60 copies per parasite genome [7]. The PfEMP1 family constitutes high-molecular proteins of 200–400 kDa, which are highly polymorphic. Different PfEMP1 molecules have different receptor specificities, therefore switching between expression of various var genes in a mutually exclusive manner allows the parasite to modify its adhesion properties (reviewed in [8]). PfEMP1 proteins include three to seven Duffy-binding-like (DBL) domains, which belong to a parasite adhesion-domain super-family present in erythrocyte invasion ligands called: erythrocyte-binding ligands (EBL). Antibodies against PfEMP1 can interfere with parasite binding and the successive acquisition of a broad range of PfEMP1 antibodies is important for the acquisition of immunity during childhood [9–13]. Several molecules such as ICAM-1 [14], VCAM-1 [15], thrombospondin [16], CD36 [17], and chondroitin sulfate A (CSA) [18,19] have been identified as host receptors for PfEMP1. In the placenta IE exclusively bind to the glycosaminoglycan CSA [19,20]. The parasite protein mediating IE adhesion to CSA in the placenta is a distinct member of the PfEMP1 protein family, named VAR2CSA [21]. High levels of anti-VAR2CSA antibodies are correlated with favourable birth outcome and they are acquired as a function of parity [22]. Disruption of the var2csa gene causes the loss of the IE’s ability to bind CSA [23]. VAR2CSA is a large IE surface-expressed antigen consisting of six DBL domains with a total estimated molecular mass of 350 kDa. The ultimate aim of PAM vaccine development is to define a VAR2CSA construct capable of eliciting antibodies that inhibit binding of IEs to CSA. However, several of the VAR2CSA domains have in vitro affinity to CSA [24–26] and this complicates vaccine design. It is thus of high priority to define the minimal epitopes within each domain and inter-domains that have affinity to CSA.
Phage display is a strong and widely used tool for mapping protein ligand interactions and has in several studies been used to define adhesive parts of proteins present on the surface of different organisms causing infectious diseases (reviewed in [27]). Phage display has also been extensively used in malaria research. For vaccine development Casey and others [28], used phage display to isolate a phage-derived peptide that mimic an important epitope of AMA-1 and had the ability to induce functionally protective antibodies. Lanzillotti and others [29], used a phage display library to search for P. falciparum encoded motifs involved in erythrocyte invasion, and identified regions in EBA-175 and Ebl-1 like proteins binding to receptors on the human erythrocyte. EBA and Ebl belong to the same super family of Duffy-binding-like proteins as the DBL domains from VAR2CSA. We were thus encouraged to use this technique to search for CSA-binding motifs in VAR2CSA. In this study a phage display library was constructed based on the exon1 coding region of VAR2CSA. The library was biopanned on different sources of glycosaminoglycans (GAG) including: immobilized bovine CSA; immobilized proteoglycans purified from placentas – CSPG; CSA-expressing CHO cells, and BeWo cells derived from the human placental syncytiotrophoblasts. Five regions of VAR2CSA potentially involved in in vivo parasite sequestration were identified and are thus potential candidate components of a multivalent PAM vaccine.
A DBL2 peptide binds to CSA and not to CSC. (A) Peptide binding assay to CSA: P2b (red) binds to CSA (■) in a peptide concentration-specific manner and not to the plate (▲). The control peptide (blue) does not bind to neither the plate (×) nor CSA (◆). (B) Peptide binding assay to CSC: P2b (red) does not bind to either CSC (■) or to the plate (▲). The control peptide (blue) does not bind to neither the plate (×) nor CSC (◆).
Multiple linear VAR2CSA regions have affinity for proteoglycans
The exon1 of var2csa has 9171 bp and was PCR amplified and cloned into the T7select 415-1b phage vector. The plasmid was propagated and the insert containing the whole var2csa exon1 was cut out and digested with DNase1 to generate 200 bp fragments. The var2csa fragments were used to create a T7 Phage display library as described in the material and methods section. This vector is described to display 415 copies of peptides on the surface of the T7 capsid [31].
To ensure that all parts of VAR2CSA exon1 were present in this constructed library a number of clones were sequenced before biopanning. No sequences were overrepresented and sequences belonging to all six DBL domains were present in the library (Figure (Figure1A,1A, black). The var2csa phage display library was biopanned four rounds on: CHO cells (two independent assays); human placental choriocarcinoma cell line BeWo (four independent assays); bovine CSA (two independent assays); biotinylated CSA (two independent assays) and chondroitin sulfate proteoglycans of human placenta (CSPG) (two independent assays). Control biopannings were done on ELISA plates coated with BSA (Figure (Figure1A,1A, Blocking Bf, blue). From each assay 15 clones were sequenced. Figure Figure11 shows the frequency by which different phages expressing particular VAR2CSA regions were identified after the different types of biopanning. Biopanning on CSA resulted in enrichment of a single region (20% of all phages) corresponding to a peptide sequence in DBL3 (Figure (Figure1A,1A, green). CSA coated directly on ELISA plates might not be very efficient; therefore the biopanning was subsequently repeated using biotinylated CSA (bCSA). This resulted in enrichment of phages representing sequences present in DBL1, DBL2 and DBL4 (Figure (Figure1A,1A, red). In addition, biopanning on human placental CSPG resulted in enrichment of phages representing DBL4 and DBL5 sequences (Figure (Figure1A,1A, yellow). However, the DBL4 sequence was also present in 18% of the control BSA biopannings (Figure (Figure1A,1A, blue). To further identify proteoglycan-binding regions, the VAR2CSA phage display library was biopanned on BeWo and CHO cells expressing CSPG and CSA, respectively (Figure (Figure1B).1B). Enrichment of phages expressing peptides from DBL2, DBL3, DBL4 and DBL5 was seen. Again the DBL4 sequence was also detected in the phages from the control biopanning. DBL2 and DBL3 phages binding to the GAG expressing cells contained VAR2CSA sequences that overlapped with the sequences identified by the CSA biopanning. The DBL5 sequence overlapped with the clone identified by CSPG biopanning. In summary, five relatively short stretches of VAR2CSA appeared to bind soluble GAGs as well as GAGs expressed on cells. No none-DBL regions (i.e. NTS, ID1 or ID2) were identified as GAG binding.
Frequency of identifed phages sorted according to the identity of the VAR2CSA region and the method of biopanning. The var2csaphage display library was biopanned four rounds on each of the following: biotinylated CSA (A, red); Chondroitin sulfate proteoglycans …
Mapping the phage display selected regions on models of VAR2CSA DBL domains and comparing with the previous described surface-expressed epitopes
Structural models of VAR2CSA 3D7 DBL domains have previously been produced using the solved DBL structures in EBA-175 and Pkα-DBL as templates [36]. As part of previous work we identified regions on native VAR2CSA, which were accessible to antibodies [26,36]. In the current study, the CSA-binding regions defined by the phage display screening were mapped onto the models and compared to the previous findings (Figure (Figure2).2). Interestingly, in DBL2, DBL3 and DBL5 there was a high degree of overlap between the CSA-binding regions and the surface-exposed regions (Figure (Figure2,2, green). Peptides residing in DBL1 and DBL4 did not map to the predicted surface exposed regions. All mapped regions, except the DBL1 region, mapped to the S2 subdomain of the DBL domains. The CSA-binding regions mapped in DBL2 and DBL5 are in close vicinity to the chemokine-binding site region of Pkα-DBL Duffy [37].
Structural models of VAR2CSA DBL domains showing surface-exposed regions and GAG binding regions. The surface-exposed epitopes previously determined by depleting female IgG plasma on parasites expressing VAR2CSA are shown in blue. The GAG binding regions identified by phage display assays are shown in yellow. The overlap of surface-exposed regions and GAG binding regions is shown in green.
……..
Malaria parasites causing PAM have been shown to bind to glycosaminoglycans in the intevillous space of the placenta. These parasites also bind specifically to bovine CSA [18], CSPG [38] as well as BeWo [39] and CHO cells having CSPG on the surface [18]. The binding between the parasite and the placental CSPG is most likely mediated through the parasite expressed protein, VAR2CSA. VAR2CSA is a large multidomain protein and for vaccine development it is important to define which regions of VAR2CSA are responsible for the interaction with CSPG. In the present study a var2csa phage display library was biopanned on five different CSA containing targets (bovine CSA, bovine bCSA, human placental CSPG, BeWo and CHO cells) in 12 independent experiments. Five regions of VAR2CSA repeatedly showed affinity for the different CSA preparations. The CSA-binding peptides identified with the phage display approach were based on the linear sequence of VAR2CSA. However, the CSA binding region might be conformational and involve peptides from several domains. It is preferable that results obtained by phage display assays are confirmed by showing that peptides corresponding to the identified regions also possess binding capacity. The var2csa phage display library used in this study was constructed from DNA fragments of 100–150 bp, and the corresponding peptides are thus 34–75 aa. These long peptides were difficult to synthetize and were unstable in solution and we thus had to divide some of the phage regions into several synthetic peptides. Furthermore, the structure of peptides in solution might be very different from peptides bound to a phage. These factors could explain why only one out of seven synthetic peptides could have its binding to CSA confirmed.
DBL2, DBL3 and DBL5 domains of VAR2CSA have previously been shown to bind to CSA [24–26] and the surface-exposed regions within these domains have been mapped [26,36]. Three of the five peptides are located on surface-exposed parts of the previously described CSA-binding domains and two of these peptides map to regions on the DBL domains, which are in close proximity to the ligand-binding region of Pk-alfa-DBL [37]. These findings show an agreement between two independent approaches, which strengthens the present results. No CSA-binding epitopes were found in the highly polymorphic DBL6 domain, which previously has been shown to bind CSA indicating the presence of conformational CSA binding regions in this domain [25].
Phage display was used to identify GAG binding linear regions of VAR2CSA. Five regions located in five different domains were found to have affinity for both immobilized CSA and CSA expressed on the surface of cells. The most frequently observed GAG binding phages mapped to DBL2, 3, 4 and DBL5, and except DBL4 all these domains have been shown to bind CSA in vitro. These results are supported by data published by Andersen and others [36], demonstrating that the phage display defined CSA-binding regions in DBL2, 3, 5 all locate to areas of VAR2CSA that appear to be exposed on the native molecule. The DBL2 CSA binding peptide showed specific binding to CSA and affinity-purified antibodies against the same phage display identified region reacted with the surface of infected erythrocytes. This work is the first step in defining small regions of VAR2CSA, which can be used in an adhesion blocking sub-unit vaccine protecting pregnant women against PAM.
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Ricke CH, Staalsoe T, Koram K, Akanmori BD, Riley EM, Theander TG, Hviid L. Plasma antibodies from malaria-exposed pregnant women recognize variant surface antigens on Plasmodium falciparum-infected erythrocytes in a parity-dependent manner and block parasite adhesion to chondroitin sulfate A. J Immunol. 2000;165:3309–3316. [PubMed]
David PH, Hommel M, Miller LH, Udeinya IJ, Oligino LD. Parasite sequestration in Plasmodium falciparum malaria: spleen and antibody modulation of cytoadherence of infected erythrocytes. Proc Natl Acad Sci USA. 1983;80:5075–5079. doi: 10.1073/pnas.80.16.5075. [PMC free article][PubMed][Cross Ref]
Smith JD, Chitnis CE, Craig AG, Roberts DJ, Hudson-Taylor DE, Peterson DS, Pinches R, Newbold CI, Miller LH. Switches in expression of Plasmodium falciparum var genes correlate with changes in antigenic and cytoadherent phenotypes of infected erythrocytes. Cell. 1995;82:101–110. doi: 10.1016/0092-8674(95)90056-X. [PMC free article][PubMed][Cross Ref]
NEW YORK (GenomeWeb) – A malaria vaccine may be more effective against parasites whose genotype matches that of the vaccine itself, according to researchers from Harvard University and the Fred Hutchinson Cancer Research Center.
Reporting this week in the New England Journal of Medicine, researchers evaluated malarial genotypes of individuals enrolled in a phase III trial of GlaxoSmithKline’s vaccine, RTS,S/ASO1.
The vaccine was previously evaluated in a large phase III trial in Africa in more than 15,000 children and was found to confer “moderate protective efficacy against clinical disease and severe malaria that wanes over time,” according to the study authors.
The mechanism by which the vaccine confers protection is incompletely understood, although it is known to target a specific protein produced by thePlasmodium falciparum malaria parasite called circumsporozoite protein. However, the circumsporozoite protein contains regions where polymorphisms can occur, including a conserved tandem repeat with a length polymorphism between 37 and 44 repeat unit, and numerous polymorphisms within the C-terminal region of protein.
Researchers hypothesized the vaccine might be less effective against malaria parasites with polymorphisms in those regions.
To test this theory, they used PCR and next-generation sequencing on both Illumina’s MiSeq instrument and Pacific Biosciences’ RS II. The researchers targeted and sequenced the circumsporozoite protein C-terminal and as well as a control region with the MiSeq from children enrolled in the clinical trial who had become infected with malaria. They used the PacBio system to sequence the longer repeat region.
Over 4,000 samples were sequenced on the MiSeq and over 3,000 on the PacBio. Samples included patients at multiple time points after they received the vaccine.
Genetic data of the malaria parasite was evaluated from 1,181 kids between the ages of five and 17 months who received the RTS,S vaccine and 909 who received a control vaccine, all of whom had developed clinically confirmed malaria.
Over two-thirds of patents had “complex infections,” defined as being founded by two or more distinct parasite lineages, the authors reported. Patients that received the RTS,S vaccine were more likely to have complex infections — 71 percent had complex infections compared to 61 percent of patients who received the control vaccine.
Looking at the relationship between polymorphisms to the C-terminal region and vaccine efficacy, the researchers found that one-year post vaccination, the C-terminal region in the malaria parasite matched that of the vaccine in 139 individuals, but was a mismatch in 1,951 individuals. Thus, cumulative vaccine efficacy against malaria with a perfect genotype match at the C-terminal site was 50.3 percent. For those without a perfect match, efficacy was 33.4 percent.
In addition, efficacy was higher immediately after receiving the vaccine. Through six months post vaccination, efficacy was 70.2 percent in individuals with a matched genotype and 56.3 percent in those with mismatched genotypes.
Looking at the relationship between the number of repeats and vaccine efficacy, the researchers found a non-significant effect with increasing repeats and vaccine efficacy.
The results suggest that among children between the ages of five and 17 months the RTS,S vaccine “has greater activity against malaria parasites with matched circumsporozoite protein allele than against mismatched malaria,” the authors concluded, and overall vaccine efficacy will depend on the genotype of the local parasite population.
In addition, the authors noted, “Genetic surveillance of circumsporozoite protein sequences in parasite populations could inform the development of future vaccine candidates targeting polymorphic malaria proteins.”
In the name of Translation from a food born pathogen to a friendly vaccine: Listeria monocytogenes
Curator: Demet Sag, PhD, CRA, GCP
Is it a far fetch? Friend or Foe?
Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen bacterium. It is used as a prototypes for an experimental model to understand the fundamental processes of adaptive immunity and virulence. 10 species of L. monocytogenes is identified in both humans and animals, L. ivanovii mainly infects ungulates (eg. sheep and cattle), while other species (L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, L. rocourtiae, L. fleischmannii and L. weihenstephanensis) are essentially saprophytes. Within the species of L. monocytogenes, several serovars (e.g., 4b, 1/2a, 1/2b and 1/2c) are highly pathogenic and account for a majority of clinical isolations.
Gram-negative bacteria has inner and outer membranes and they are most studied; yet mechanics of protein secretion across the single cell membrane of Gram-positive is not. The protein secretion in gram positive bacteria is complex not only it requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface but also folding specifically. As a result, protein folding mechanism and stability investigated for the role of PrsA2 and PrsA-like so that optimizing the virulence and protein secretion become possible.
Pathogen: Listeriosis
Listeria monocytogenes is a food-borne pathogen determined in 1980s causing an opportunistic disease called listeriosis which is widespread in nature being part of the faecal flora of many mammals. In addition to contaminated food resources (1-10%), may occur sporadically or in outbreaks. It can be difficult to control and may cause severe clinical outcomes, especially in pregnant women, children and the elderly. The mechanism of pathogenity based on simply altering the actin cytoskeleton structure. Infection causes a spectrum of illness, ranging from febrile gastroenteritis to invasive disease, including bacteraemia, sepsis, and meningoencephalitis.
This organisms copes well with bile acids and acidic environment such as glutamate decarboxylase and arginine deiminase systems to survive in competitive microbiome of GI.
This information may benefit developing effective vaccines, designing pharmabiotics; even including probiotics, prebiotics, or phages.
Nutrition:
Altering dietary habit assumed to control a disease. The effects of various fatty acids on bacterial clearance and disease outcome through suppression or activation of immune responses can’t be simplified down to one or two kinds of fatty acids in foodborne pathogens. Commonly they have a specialized carbohydrate metabolism so they can utilize fatty acids of host and the host may use the end products for an energy resource. The compared food-borne pathogens include Salmonella sp., Campylobacter sp.,Shiga toxin-producing Escherichia coli, Shigella sp., Listeria monocytogenes, and Staphylococcus aureus.
Genetics:
This bacterium has a complex transcriptional machinery to adept, invade several types of cells, and survive. It happens through RNA-based regulation in bacteria in cell biology at the chromatin level during bacterial infection. This includes clathrin, atypical mitochondrial fragmentation, and several hundred non-coding RNAs (ncRNAs) in the Listeria genome. Patho-epigenetics becoming an attractive field. Improved bioinformatics may help to classify these changes under specific regulatory mechanisms and networks to determine their function and use.
The Toxin, Vaccine and Immunotheraphy
The virulence of Listeria monocytogenes mainly depends on a listeriolysin O (LLO) which is a thiol-activated, cholesterol-dependent, pore-forming toxin, and highly immunogenic. In addition, biochemically, LLO, a toxin that belongs to the family of cholesterol-dependent cytolysins (CDCs), exhibits potent cell type-non-specific toxicity and is a source of dominant CD4(+) and CD8(+) T cell epitopes. Hence, it is the major target for innate and adaptive immune responses in different animal models and humans.
As a result, during infection bacteria escape from phagocytosis, allow bacteria to infest the cells and multiply. Thus, due to it’s naturally immunomodulation role this mechanisms is under investigation so that it can be used for cancer immunotherapies for developing immune tolerance. Since it has effective cytotoxicity. Thus, co-administration of this toxin or using as an adjuvant with vaccine vectors are also under research. LLO has diverse biological activities such as cytotoxicity, apoptosis induction, endoplasmic reticulum stress response, modulation of gene expression,
Since FDA approved Sipuleucel-T (Provenge, Dendreon, Seattle, WA), which consists of antigen-loaded dendritic cells (DCs), there is a boom in immunotherapy applications. Yet, there is a shortcoming of this application because of its limited scope in immune response. However, Listeria monocytogenes (Lm) naturally targets DCs in vivo and stimulates both innate and adaptive cellular immunity. Lm-based vaccines engineered to express cancer antigens have demonstrated striking efficacy applications.
Meningitis
On the other hand, there is a caution to be taken in clinics since L. monocytogenes most often presents as acute bacterial meningitis, particularly in weaken immune system of patients such as elderly, already sick patients as secondary infection/opportunistic, and those with already immune fragile state. L. monocytogenes CNS the infections may present as acute bacterial meningitis, meningoencephalitis, or acute encephalitis.
References and Further readings:
PMCID: PMC3574585 PMID: 22595054
Le DT(1), Dubenksy TW Jr, Brockstedt DG. “Clinical development of Listeria monocytogenes-based immunotherapies”. 20. Semin Oncol. 2012 Jun;39(3):311-22. doi: 10.1053/j.seminoncol.2012.02.008.
PMCID: PMC3987759 PMID: 24826075
Liu D(1).“Molecular approaches to the identification of pathogenic and nonpathogenic Listeriae”. 16. Microbiol Insights. 2013 Jul 22;6:59-69. doi: 10.4137/MBI.S10880. eCollection 2013.
PMCID: PMC4385656 PMID: 25874208
Hernández-Flores KG(1), Vivanco-Cid H(2). Biological effects of listeriolysin O: implications for vaccination. Biomed Res Int. 2015;2015:360741. doi: 10.1155/2015/360741. Epub 2015 Mar 22.
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TABLE 4: Current Interventions______________________________________________________________________________________________________________
ABSTRACT:
Overall purpose is to find a method to manipulate IDO for clinical applications, mainly the focus of this review is is cancer prevention and treatment. The first study proving the connection between IDO and immune response came from, a very natural event, a protection of pregnancy in human. This led to discover that high IDO expression is a common factor in cancer tumors. Thus, attention promoted investigations on IDO’s role in various disease states, immune disorders, transplantation, inflammation, women health, mood disorders.
Many approaches, vaccines and adjuvants are underway to find new immunotherapies by combining the power of DCs in immune response regulation and specific direction of siRNA. As a result, with this unique qualities of IDO, DCs and siRNA, we orchestrated a novel intervention for immunomodulation of IDO by inhibiting with small interference RNA, called siRNA-IDO-DCvax. Proven that our DCvax created a delay and regression of tumor growth without changing the natural structure and characterization of DCs in melanoma and breast cancers in vivo. (** The shRNA IDO- DCvax is developed by Regen BioPhrama, San Diego, CA , Thomas Ichim, Ph.D, CSO. and David Koos, CEO)
Double-Edged Sword of IDO: The Good and The Bad for Clinical intervention and Developments
IDO almost has a dual role. There is a positive side of high expression of IDO during pregnancy(29; 28; 114), transplants (115; 116; 117; 118; 119), infectious diseases(96)and but this tolerance is negative during autoimmune-disorders (120; 121; 122), tumors of cancer (123; 124; 117; 121; 125; 126; 127) (127), and mood disorders (46). The increased IDO expression has a double-edged sword in human physiology provides a positive role during protection of fetus and grafts after transplantations but becomes a negative factor during autoimmune disorders, cancer, sepsis and mood disorders.
Prevention of allogeneic fetal rejection is possible by tryptophan metabolism (26) rejecting with lack of IDO but allocating if IDO present (29; 28; 114). These studies lead to find “the natural regulation mechanism” for protecting the transplants from graft versus host disease GVHD (128) and getting rid of tumors.
The plasticity of mammary and uterus during reproduction may hold some more answers to prevent GVHD and tumors of cancer with good understanding of IDO and tryptophan mechanism (129; 130). After allogeneic bone marrow transplants the risk of solid tumor development increased about 80% among 19,229 patients even with a greater risk among patients under 18 years old (117). The adaptation of tolerance against host mechanism is connected to the IDO expression (131). During implantation and early pregnancy IDO has a role by making CD4+CD25+Foxp3+ regulatory T cells (Tregs) and expressing in DCs and MQs (114; 132; 133).
Clonal deletion mechanism prevents mother to react with paternal products since female mice accepted the paternal MHC antigen-expressing tumor graft during pregnancy and rejected three weeks after delivery (134). CTLA-4Ig gene therapy alleviates abortion through regulation of apoptosis and inhibition of spleen lymphocytes (135).
Immune System and IDO DCs are the orchestrator of the immune response (56; 57; 58) with list of functions in uptake, processing, and presentation of antigens; activation of effector cells, such as T-cells and NK-cells; and secretion of cytokines and other immune-modulating molecules to direct the immune response. The differential regulation of IDO in distinct DC subsets is widely studied to delineate and correct immune homeostasis during autoimmunity, infection and cancer and the associated immunological outcomes. Genesis of antigen presenting cells (APCs), eventually the immune system, require migration of monocytes (MOs), which is originated in bone marrow. Then, these MOs move from bloodstream to other tissues to become macrophages and DCs (59; 60).
Initiation of immune response requires APCs to link resting helper T-cell with the matching antigen to protect body. DCs are superior to MQs and MOs in their immune action model. When DCs are first described (61) and classified, their role is determined as a highly potent antigen-presenting cell (APC) subset with 100 to 1000-times more effective than macrophages and B-cells in priming T-cells. Both MQs and monocytes phagocytize the pathogen, and their cell structure contains very large nucleus and many internal vesicles. However, there is a nuance between MQ and DCs, since DCs has a wider capacity of stimulation, because MQs activates only memory T cells, yet DCs can activate both naïve and memory T cells.
DCs are potent activators of T cells and they also have well controlled regulatory roles. DC properties determine the regulation regardless of their origin or the subset of the DCs. DCs reacts after identification of the signals or influencers for their inhibitory, stimulatory or regulatory roles, before they express a complex repertoire of positive and negative cytokines, transmembrane proteins and other molecules. Thus, “two signal theory” gains support with a defined rule. The combination of two signals, their interaction with types of cells and time are critical.
In short, specificity and time are matter for a proper response. When IDO mRNA expression is activated with CTL40 ligand and IFNgamma, IDO results inhibition of T cell production (4). However, if DCs are inhibited by 1MT, an inhibitor of IDO, the response stop but IgG has no affect (10). In addition, if the stimulation is started by a tryptophan metabolite, which is downstream of IDO, such as 3-hydroxyantranilic or quinolinic acids, it only inhibits Th1 but not Th2 subset of T cells (62).
Furthermore, inclusion of signal molecules, such as Fas Ligand, cytochrome c, and pathways also differ in the T cell differentiation mechanisms due to combination, time and specificity of two-signals. The co-culture experiments are great tool to identify specific stimuli in disease specific microenvironment (63; 12; 64) for discovering the mechanism and interactions between molecules in gene regulation, biochemical mechanism and physiological function during cell differentiation.
As a result, the simplest differential cell development from the early development of DCs impact the outcome of the data. For example, collection of MOs from peripheral blood mononuclear cells (PBMCs) with IL4 and GM-CSF leads to immature DCs (iDCs). On next step, treatment of iDCs with tumor necrosis factor (TNF) or other plausible cytokines (TGFb1, IFNgamma, IFNalpha, IFNbeta, IL6 etc.) based on the desired outcome differentiate iDCs into mature DCs (mDCs). DCs live only up to a week but MOs and generated MQs can live up to a month in the given tissue. B cells inhibit T cell dependent immune responses in tumors (65).
AutoImmune Disorders:
The balance of IDO expression becomes necessary to prevent overactive immune response self-destruction, so modulation in tryptophan and NDA metabolisms maybe essential. When splenic IDO-expressing CD11b (+) DCs from tolerized animals applied, they suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen (136).
The role of Nicotinamide prevention on type 1 diabetes and ameliorates multiple sclerosis in animal model presented with activities of NDAs stimulating GPCR109a to produce prostaglandins to induce IDO expression, then these PGEs and PGDs converted to the anti-inflammatory prostaglandin, 15d-PGJ(2) (137; 138; 139). Thus, these events promotes endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma. (137).
Modulating the immune response at non-canonical at canonocal pathway while keeping the non-canonical Nf-KB intact may help to mend immune disorders. As a result, the targeted blocking in canonical at associated kinase IKKβ and leaving non-canonocal Nf-kB pathway intact, DCs tips the balance towards immune supression. Hence, noncanonical NF-κB pathway for regulatory functions in DCs required effective IDO induction, directly or indirectly by endogenous ligand Kyn and negative regulation of proinflammatory cytokine production. As a result, this may help to treat autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, inflammatory bowel disease, and multiple sclerosis, or allergy or transplant rejection.
While the opposite action needs to be taken during prevention of tumors, that is inhibition of non-canonical pathway. Inflammation induces not only relaxation of veins and lowering blood pressure but also stimulate coagulopathies that worsen the microenvironment and decrease survival rate of patients after radio or chemotherapies.Cancer Generating tumor vaccines and using adjuvants underway (140).
Clinical correlation and genetic responses also compared in several studies to diagnose and target the system for cancer therapies (127; 141; 131). The recent surveys on IDO expression and human cancers showed that IDO targeting is a candidate for cancer therapy since IDO expression recruiting Tregs, downregulates MHC class I and creating negative immune microenvironment for protection of development of tumors (125; 27; 142). Inhibition of IDO expression can make advances in immunotherapy and chemotherapy fields (143; 125; 131; 144).
IDO has a great importance on prevention of cancer development (126). There are many approaches to create the homeostasis of immune response by Immunotherapy. However, given the complexity of immune regulations, immunomodulation is a better approach to correct and relieve the system from the disease. Some of the current IDO targeted immunotherapy or immmunomodulations with RNA technology for cancer prevention (145; 146; 147; 148; 149; 150) or applied on human or animals (75; 151; 12; 115; 152; 9; 125) or chemical, (153; 154) or radiological (155). The targeted cell type in immune system generally DCs, monocytes (94)T cells (110; 156)and neutrophils (146; 157). On this paper, we will concentrate on DCvax on cancer treatments.
T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)
IDO and the downstream enzymes in tryptophan pathway produce a series of immunosuppressive tryptophan metabolites that may lead into Tregs proliferation or increase in T cell apoptosis (62; 16; 27; 158), and some can affect NK cell function (159).
The interesting part of the mechanism is even without presence of IDO itself, downstream enzymes of IDO in the kynurenine tryptophan degradation still show immunosuppressive outcome (160; 73) due to not only Kyn but also TGFbeta stimulated long term responses. DC vaccination with IDO plausible (161) due to its power in immune response changes and longevity in the bloodstream for reversing the system for Th17 production (162).
Clinical Interventions are taking advantage of the DC’s central role and combining with enhancing molecules for induction of immunity may overcome tolerogenic DCs in tumors of cancers (163; 164).
The first successful application of DC vaccine used against advanced melanoma after loading DCs with tumor peptides or autologous cell lysate in presence of adjuvants keyhole limpet hematocyanin (KLH) (165). Previous animal and clinical studies show use of DCs against tumors created success (165; 166; 167) as well as some problems due to heterogeneity of DC populations in one study supporting tumor growth rather than diminishing (168).
DC vaccination applied onto over four thousand clinical trial but none of them used siRNA-IDO DC vaccination method. Clinical trials evaluating DCs loaded ex vivo with purified TAAs as an anticancer immunotherapeutic interventions also did not include IDO (Table from (169). This table presented the data from 30 clinical trials, 3 of which discontinued, evaluating DCs loaded ex vivo with TAAs as an anticancer immunotherapy for 12 types of cancer [(AML(1), Breast cancer (4), glioblastoma (1), glioma (2), hepatocellular carcinoma (1), hematological malignancies (1), melanoma (6), neuroblastoma sarcoma (2), NSCLC (1), ovarian cancer (3), pancreatic cancer (3), prostate cancer (10)] at phase I, II or I/II.
Tipping the balance between Treg and Th17 ratio has a therapeutic advantage for restoring the health that is also shown in ovarian cancer by DC vaccination with adjuvants (161). This rebalancing of the immune system towards immunogenicity may restore Treg/Th17 ratio (162; 170) but it is complicated. The stimulation of IL10 and IL12 induce Treg produce less Th17 and inhibiting CTL activation and its function (76; 171; 172) while animals treated with anti-TGFb before vaccination increase the plasma levels of IL-15 for tumor specific T cell survival in vivo (173; 174) ovarian cancer studies after human papilloma virus infection present an increase of IL12 (175).
Opposing signal mechanism downregulates the TGFb to activate CTL and Th1 population with IL12 and IL15 expression (162; 173). The effects of IL17 on antitumor properties observed by unique subset of CD4+ T cells (176) called also CD8+ T cells secrete even more IL17 (177).
Using cytokines as adjuvants during vaccination may improve the efficacy of vaccination since cancer vaccines unlike infections vaccines applied after the infection or disease started against the established adoptive immune response. Adjuvants are used to improve the responses of the given therapies commonly in immunotherapy applications as a combination therapy (178).
Enhancing cancer vaccine efficacy via modulation of the microenvironment is a plausible solution if only know who are the players. Several molecules can be used to initiate and lengthen the activity of intervention to stimulate IDO expression without compromising the mechanism (179). The system is complicated so generally induction is completed ex-vivo stimulation of DCs in cell lysates, whole tumor lysates, to create the microenvironment and natural stimulatory agents. Introduction of molecules as an adjuvants on genetic regulation on modulation of DCs are critical, because order and time of the signals, specific location/ tissue, and heterogeneity of personal needs (174; 138; 180). These studies demonstrated that IL15 with low TGFb stimulates CTL and Th1, whereas elevated TGFb with IL10 increases Th17 and Tregs in cancer microenvironments.
For example Ret-peptide antitumor vaccine contains an extracellular fragment of Ret protein and Th1 polarized immunoregulator CpG oligonucleotide (1826), with 1MT, a potent inhibitor of IDO, brought a powerful as well as specific cellular and humoral immune responses in mice (152).
The main idea of choosing Ret to produce vaccine in ret related carcinomas fall in two criterion, first choosing patients self-antigens for cancer therapy with a non-mutated gene, second, there is no evidence of genetic mutations in Ret amino acids 64-269. Demonstration of proliferating hemangiomas, benign endothelial tumors and often referred as hemangiomas of infancy appearing at head or neck, express IDO and slowly regressed as a result of immune mediated process.
After large scale of genomic analysis show insulin like growth factor 2 as the key regulator of hematoma growth (Ritter et al. 2003). We set out to develop new technology with our previous expertise in immunotherapy and immunomodulation (181; 182; 183; 184), correcting Th17/Th1 ratio (185), and siRNA technology (186; 187). We developed siRNA-IDO-DCvax. Patented two technologies “Immunomodulation using Altered DCs (Patent No: US2006/0165665 A1) and Method of Cancer Treatments using siRNA Silencing (Patent No: US2009/0220582 A1).
In melanoma cancer DCs were preconditioned with whole tumor lysate but in breast cancer model pretreatment completed with tumor cell lysate before siRNA-IDO-DCvax applied. Both of these studies was a success without modifying the autanticity of DCs but decreasing the IDO expression to restore immunegenity by delaying tumor growth in breast cancer (147) and in melanoma (188). Thus, our DCvax specifically interfere with Ido without disturbing natural structure and content of the DCs in vivo showed that it is possible to carry on this technology to clinical applications.
Furthermore, our method of intervention is more sophisticated since it has a direct interaction mechanism with ex-vivo DC modulation without creating long term metabolism imbalance in Trp/Kyn metabolite mechanisms since the action is corrective and non-invasive.
First, prevention of tumor development studies targeting non-enzymatic pathway initiated by pDCs conditioned with TGFbeta is specific to IDO1 (189).
Second, IDO upregulation in antigen presenting cells allowing metastasis show that most human tumors express IDO at high levels (123; 124).
Third, tolerogenic DCs secretes several molecules some of them are transforming growth factor beta (TGFb), interleukin IL10), human leukocyte antigen G (HLA-G), and leukemia inhibitory factor (LIF), and non-secreted program cell death ligand 1 (PD-1 L) and IDO, indolamine 2.3-dioxygenase, which promote tumor tolerance. Thus, we took advantage of DCs properties and Ido specificity to prevent the tolerogenicity with siRNA-IDO DC vaccine in both melanoma and breast cancer.
Fourth, IDO expression in DCs make them even more potent against tumor antigens and create more T cells against tumors. IDOs are expressed at different levels by both in broad range of tumor cells and many subtypes of DCs including monocyte-derived DCs (10), plasmacytoid DCs (142), CD8a+ DCs (190), IDO compotent DCs (17), IFNgamma-activated DCs used in DC vaccination. These DCs suppress immune responses through several mechanisms for induction of apoptosis towards activated T cells (156) to mediate antigen-specific T cell anergy in vivo (142) and for enhancement of Treg cells production at sites of vaccination with IDO-positive DCs+ in human patients (142; 191; 192; 168; 193; 194). If DCs are preconditioned with tumor lysate with 1MT vaccination they increase DCvax effectiveness unlike DCs originated from “normal”, healthy lysate with 1MT in pancreatic cancer (195). As a result, we concluded that the immunesupressive effect of IDO can be reversed by siRNA because Treg cells enhances DC vaccine-mediated anti-tumor-immunity in cancer patients.
Gene silencing is a promising technology regardless of advantages simplicity for finding gene interaction mechanisms in vitro and disadvantages of the technology is utilizing the system with specificity in vivo (186; 196). siRNA technology is one of the newest solution for the treatment of diseases as human genomics is only producing about 25,000 genes by representing 1% of its genome. Thus, utilizing the RNA open the doors for more comprehensive and less invasive effects on interventions. Thus this technology is still improving and using adjuvants. Silencing of K-Ras inhibit the growth of tumors in human pancreatic cancers (197), silencing of beta-catenin in colon cancers causes tumor regression in mouse models (198), silencing of vascular endothelial growth factor (VGEF) decreased angiogenesis and inhibit tumor growth (199).
Combining siRNA IDO and DCvax from adult stem cell is a novel technology for regression of tumors in melanoma and breast cancers in vivo. Our data showed that IDO-siRNA reduced tumor derived T cell apoptosis and tumor derived inhibition of T cell proliferation. In addition, silencing IDO made DCs more potent against tumors since treated or pretreated animals showed a delay or decreased the tumor growth (188; 147)
Clinical Trials:
First FDA approved DC-based cancer therapies for treatment of hormone-refractory prostate cancer as autologous cellular immunotherapy (163; 164). However, there are many probabilities to iron out for a predictive outcome in patients.
Table 2 demonstrates the current summary of clinical trials report. This table shows 38 total studies specifically Ido related function on cancer (16), eye (3), surgery (2), women health (4), obesity (1), Cardiovascular (2), brain (1), kidney (1), bladder (1), sepsis shock (1), transplant (1), nervous system and behavioral studies (4), HIV (1) (Table 4). Among these only 22 of which active, recruiting or not yet started to recruit, and 17 completed and one terminated.
Most of these studies concentrated on cancer by the industry, Teva GTC ( Phase I traumatic brain injury) Astra Zeneca (Phase IV on efficacy of CRESTOR 5mg for cardiovascular health concern), Incyte corporation (Phase II ovarian cancer) NewLink Genetics Corporation Phase I breast/lung/melanoma/pancreatic solid tumors that is terminated; Phase II malignant melanoma recruiting, Phase II active, not recruiting metastatic breast cancer, Phase I/II metastatic melanoma, Phase I advanced malignancies) , HIV (Phase IV enrolling by invitation supported by Salix Corp-UC, San Francisco and HIV/AIDS Research Programs).
Many studies based on chemotherapy but there are few that use biological methods completed study with IDO vaccine peptide vaccination for Stage III-IV non-small-cell lung cancer patients (NCT01219348), observational study on effect of biological therapy on biomarkers in patients with untreated hepatitis C, metastasis melanoma, or Crohn disease by IFNalpha and chemical (ribavirin, ticilimumab (NCT00897312), polymorphisms of patients after 1MT drug application in treating patients with metastatic or unmovable refractory solid tumors by surgery (NCT00758537), IDO expression analysis on MSCs (NCT01668576), and not yet recruiting intervention with adenovirus-p53 transduced dendric cell vaccine , 1MT , radiation, Carbon C 11 aplha-methyltryptophan- (NCT01302821).
Among the registered clinical trials some of them are not interventional but observational and evaluation studies on Trp/Kyn ratio (NCT01042847), Kyn/Trp ratio (NCT01219348), Kyn levels (NCT00897312, NCT00573300), RT-PCR analysis for Kyn metabolism (NCT00573300, NCT00684736, NCT00758537), and intrinsic IDO expression of mesenchymal stem cells in lung transplant with percent inhibition of CD4+ and CD8+ T cell proliferation toward donor cells (NCT01668576), determining polymorphisms (NCT00426894). These clinical trials/studies are immensely valuable to understand the mechanism and route of intervention development with the data collected from human populations
Future Actions for Molecular Dx and Targeted Therapies:
Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors. (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)
Current survival or response rate is around 40 to 50 % range. By using specific cell type, selected inhibition/activation sequence based on patient’s genomic profile may improve the efficacy of clinical interventions on cancer treatments. Targeted therapies for specific gene regulation through signal transduction is necessary but there are few studies with genomics based approach.
On the other hand, there are surveys, observational or evaluations (listed in clinical trials section) registered with www.clinicaltrials.gov that will provide a valuable short-list of molecules. Preventing stimulation of Ido1 as well as Tgfb-1gene expression by modulating receptor mediated phosphorylation between TGFb/SMAD either at Mad-Homology 1 (MH1) or Mad-Homology 1 (MH2) domains maybe possible (79; 82; 80). Within Smads are the conserved Mad-Homology 1 (MH1) domain, which is a DNA binding module contains tightly bound Zinc atom.
Smad MH2 domain is well conserved and one the most diverse protein-signal interacting molecule during signal transduction due to two important Serine residues located extreme distal C-termini at Ser-Val-Ser in Smad 2 or at pSer-X-PSer in RSmads (80). Kyn activated orphan G protein–coupled receptor, GPR35 with unknown function with a distinct expression pattern that collides with IDO sites since its expression at high levels of the immune system and the gut (63) (200; 63).
The first study to connect IDO with cancer shows that group (75). The directly targeting to regulate IDO expression is another method through modulating ISREs in its promoter with RNA-peptide combination technology. Indirectly, IDO can be regulated through Bin1 gene expression control over IDO since Bin1 is a negative regulator of IDO and prevents IDO expression. IDO is under negative genetic control of Bin1, BAR adapter–encoding gene Bin1 (also known as Amphiphysin2). Bin1 functions in cancer suppression since attenuation of Bin1 observed in many human malignancies (141; 201; 202; 203; 204; 205; 206) . Null Bin-/- mice showed that when there is lack of Bin1, upregulation of IDO through STAT1- and NF-kB-dependent expression of IDO makes tumor cells to escape from T cell–dependent antitumor immunity.
This pathway lies in non-enzymatic signal transducer function of IDO after stimulation of DCs by TGFb1. The detail study on Bin1 gene by alternative spicing also provided that Bin1 is a tumor suppressor. Its activities also depends on these spliced outcome, such as Exon 10, in muscle, in turn Exon 13 in mice has importance in role for regulating growth when Bin1 is deleted or mutated C2C12 myoblasts interrupted due to its missing Myc, cyclinD1, or growth factor inhibiting genes like p21WAF1 (207; 208).
On the other hand alternative spliced Exon12A contributing brain cell differentiation (209; 210). Myc as a target at the junction between IDO gene interaction and Trp metabolism. Bin1 interacts with Myc either early-dependent on Myc or late-independent on Myc, when Myc is not present. This gene regulation also interfered by the long term signaling mechanism related to Kynurenine (Kyn) acting as an endogenous ligand to AHR in Trp metabolite and TGFb1 and/or IFNalpha and IFNbeta up regulation of DCs to induce IDO in noncanonical pathway for NF-kB and myc gene activations (73; 74). Hence, Trp/Kyn, Kyn/Trp, Th1/Th17 ratios are important to be observed in patients peripheral blood. These direct and indirect gene interactions place Bin1 to function in cell differentiation (211; 212; 205).
Table 3 contains the microarray analysis for Kyn affect showed that there are 25 genes affected by Kyn, two of which are upregulated and 23 of them downregulated (100). This list of genes and additional knowledge based on studies creating the diagnostics panel with these genes as a biomarker may help to analyze the outcomes of given interventions and therapies. Some of these molecules are great candidate to seek as an adjuvant or co-stimulation agents. These are myc, NfKB at IKKA, C2CD2, CREB3L2, GPR115, IL2, IL8, IL6, and IL1B, mir-376 RNA, NFKB3, TGFb, RelA, and SH3RF1. In addition, Lip, Fox3P, CTLA-4, Bin1, and IMPACT should be monitored.
In addition, Table 4 presents the other possible mechanisms. The highlights of possible target/biomarkers are specific TLRs, conserved sequences of IDO across its homologous structures, CCR6, CCR5, RORgammat, ISREs of IDO, Jak, STAT, IRFs, MH1 and MH2 domains of Smads. Endothelial cell coagulation activation mechanism and pDC maturation or immigration from lymph nodes to bloodstream should marry to control not only IDO expression but also genesis of preferred DC subsets. Stromal mesenchymal cells are also activated by these modulation at vascular system and interferes with metastasis of cancer. First, thrombin (human factor II) is a well regulated protein in coagulation hemostasis has a role in cell differentiation and angiogenesis.
Protein kinase activated receptors (PARs), type of GPCRs, moderate the actions. Second, during hematopoietic response endothelial cells produce hematopoietic growth factors (213; 214). Third, components of bone marrow stroma cells include monocytes, adipocytes, and mesenchymal stem cells (215). As a result, addressing this issue will prevent occurrence of coagulapathologies, namely DIC, bleeding, thrombosis, so that patients may also improve response rate towards therapies. Personal genomic profiles are powerful tool to improve efficacy in immunotherapies since there is an influence of age (young vs. adult), state of immune system (innate vs. adopted or acquired immunity). Table 5 includes some of the current studies directly with IDO and indirectly effecting its mechanisms via gene therapy, DNA vaccine, gene silencing and adjuvant applications as an intervention method to prevent various cancer types.
CONCLUSION
IDO has a confined function in immune system through complex interactions to maintain hemostasis of immune responses. The genesis of IDO stem from duplication of bacterial IDO-like genes. Inhibition of microbial infection and invasion by depleting tryptophan limits and kills the invader but during starvation of trp the host may pass the twilight zone since trp required by host’s T cells. Thus, the host cells in these small pockets adopt to new microenvironment with depleted trp and oxygen poor conditions. Hence, the cell metabolism differentiate to generate new cellular structure like nodules and tumors under the protection of constitutively expressed IDO in tumors, DCs and inhibited T cell proliferation.
On the other hand, having a dichotomy in IDO function can be a potential limiting factor that means is that IDOs impact on biological system could be variable based on several issues such as target cells, IDO’s capacity, pathologic state of the disease and conditions of the microenvironment. Thus, close monitoring is necessary to analyze the outcome to prevent conspiracies since previous studies generated paradoxical results.
Current therapies through chemotherapies, radiotherapies are costly and effectiveness shown that the clinical interventions require immunotherapies as well as coagulation and vascular biology manipulations for a higher efficacy and survival rate in cancer patients. Our siRNA and DC technologies based on stem cell modulation will provide at least prevention of cancer development and hopefully prevention in cancer.
11. References
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