A polyvalent RNA vaccine reduces the immune imprinting phenotype in mice and induces neutralizing antibodies against omicron SARS-CoV-2.

Immune imprinting is now evident in COVID-19 vaccinated people. This phenomenon may impair the development of effective neutralizing antibodies against variants of concern (VoCs), mainly Omicron and its subvariants. Consequently, the boost doses with bivalent vaccines have not shown a significant gain of function regarding the neutralization of Omicron. The approach to design COVID-19 vaccines must be revised to improve the effectiveness against VoCs. Here, we took advantage of the self-amplifying characteristic of RepRNA and developed a polyvalent formulation composed of mRNA from five VoCs. LION/RepRNA Polyvalent induced neutralizing antibodies in mice previously immunized with LION/RepRNA D614G and reduced the imprinted phenotype associated with low neutralization capacity of Omicron B.1.1.529 pseudoviruses. The polyvalent vaccine can be a strategy to handle the low neutralization of Omicron VoC, despite booster doses with either monovalent or bivalent vaccines.

Psychiatric complications in Graves' disease.

Background: Mood disorders are common in Graves' disease despite treatment. The pathogenic mechanisms involved are unknown and so is whether previous psychiatric disease influences these symptoms. Methods: This is a longitudinal study conducted in Sweden on 65 women with newly diagnosed Graves' disease and 65 matched controls. Participants were examined during hyperthyroidism and after 15 months of treatment. Examinations included blood sampling, and psychiatric testing with the Comprehensive Psychopathological Rating Scale for Affective Syndromes and the Structured Clinical Interview for DSM-IV - Axis I Disorders. We also performed two analyses of a national population-based registry to determine previous psychiatric diagnoses and previous prescriptions of psychoactive drugs in (i) all patients we asked to participate and (ii) all Swedish women given a diagnosis of hyperthyroidism during 2013-2018, comparing them to matched controls. Results: There was no increased previous psychiatric comorbidity in Graves' patients compared to controls. There was no higher prevalence of psychiatric diagnoses and prescriptions of psychoactive drugs between (i) included GD patients compared to those who declined participation and (ii) women with a hyperthyroidism diagnosis in 5 years prior to their diagnosis, compared to matched controls. Depression scores and anxiety scores were higher in patients compared to controls both during hyperthyroidism (depression (median (IQR): 7.5 (5.0-9.5) vs 1.0 (0.5-2.5) P < 0.001), anxiety: 7.7 (5.0-11) vs 2.5 (1.0-4.0) P < 0.001) and after treatment (depression: 2.5 (1.5-5.0) vs 1.5 (0.5-3.5) P < 0.05), anxiety: 4.0 (2.5-7.5) vs 3.0 (1.5-5.0) P < 0.05). Patients with a previous psychiatric condition, mild eye symptoms, and a younger age had more anxiety at 15 months compared to patients without these symptoms and a higher age (all p<0.05). Conclusion: Graves' disease affects patients' mood despite treatment. A previous psychiatric condition, mild eye symptoms, and a younger age increase the vulnerability for long-lasting symptoms and require specific attention.

Toll-Like Receptor 4 Agonist Injection With Concurrent Radiotherapy in Patients With Metastatic Soft Tissue Sarcoma: A Phase 1 Nonrandomized Controlled Trial.

Importance: Metastatic soft tissue sarcomas (STSs) have limited systemic therapy options, and immunomodulation has not yet meaningfully improved outcomes. Intratumoral (IT) injection of the toll-like receptor 4 (TLR4) agonist glycopyranosyl lipid A in stable-emulsion formulation (GLA-SE) has been studied as immunotherapy in other contexts. Objective: To evaluate the safety, efficacy, and immunomodulatory effects of IT GLA-SE with concurrent radiotherapy in patients with metastatic STS with injectable lesions. Design, Setting, and Participants: This phase 1 nonrandomized controlled trial of patients with STS was performed at a single academic sarcoma specialty center from November 17, 2014, to March 16, 2016. Data analysis was performed from August 2016 to September 2022. Interventions: Two doses of IT GLA-SE (5 µg and 10 µg for 8 weekly doses) were tested for safety in combination with concurrent radiotherapy of the injected lesion. Main Outcomes and Measures: Primary end points were safety and tolerability. Secondary and exploratory end points included local response rates as well as measurement of antitumor immunity with immunohistochemistry and T-cell receptor (TCR) sequencing of tumor-infiltrating and circulating lymphocytes. Results: Twelve patients (median [range] age, 65 [34-78] years; 8 [67%] female) were treated across the 2 dose cohorts. Intratumoral GLA-SE was well tolerated, with only 1 patient (8%) experiencing a grade 2 adverse event. All patients achieved local control of the injected lesion after 8 doses, with 1 patient having complete regression (mean regression, -25%; range, -100% to 4%). In patients with durable local response, there were detectable increases in tumor-infiltrating lymphocytes. In 1 patient (target lesion -39% at 259 days of follow-up), TCR sequencing revealed expansion of preexisting and de novo clonotypes, with convergence of numerous rearrangements coding for the same binding sequence (suggestive of clonal convergence to antitumor targets). Single-cell sequencing identified these same expanded TCR clones in peripheral blood after treatment; these T cells had markedly enhanced Tbet expression, suggesting TH1 phenotype. Conclusions and Relevance: In this nonrandomized controlled trial, IT GLA-SE with concurrent radiotherapy was well tolerated and provided more durable local control than radiotherapy alone. Patients with durable local response demonstrated enhanced IT T-cell clonal expansion, with matched expansion of these clonotypes in the circulation. Additional studies evaluating synergism of IT GLA-SE and radiotherapy with systemic immune modulation are warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT02180698.

A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation.

RNA vaccines possess significant clinical promise in counteracting human diseases caused by infectious or cancerous threats. Self-amplifying replicon RNA (repRNA) has been thought to offer the potential for enhanced potency and dose sparing. However, repRNA is a potent trigger of innate immune responses in vivo, which can cause reduced transgene expression and dose-limiting reactogenicity, as highlighted by recent clinical trials. Here, we report that multivalent repRNA vaccination, necessitating higher doses of total RNA, could be safely achieved in mice by delivering multiple repRNAs with a localizing cationic nanocarrier formulation (LION). Intramuscular delivery of multivalent repRNA by LION resulted in localized biodistribution accompanied by significantly upregulated local innate immune responses and the induction of antigen-specific adaptive immune responses in the absence of systemic inflammatory responses. In contrast, repRNA delivered by lipid nanoparticles (LNPs) showed generalized biodistribution, a systemic inflammatory state, an increased body weight loss, and failed to induce neutralizing antibody responses in a multivalent composition. These findings suggest that in vivo delivery of repRNA by LION is a platform technology for safe and effective multivalent vaccination through mechanisms distinct from LNP-formulated repRNA vaccines.

Clinical trials of self-replicating RNA-based cancer vaccines.

Therapeutic cancer vaccines, designed to activate immune effectors against tumor antigens, utilize a number of different platforms for antigen delivery. Among these are messenger RNAs (mRNA), successfully deployed in some prophylactic SARS-CoV2 vaccines. To enhance the immunogenicity of mRNA-delivered epitopes, self-replicating RNAs (srRNA) that markedly increase epitope expression have been developed. These vectors are derived from positive-strand RNA viruses in which the structural protein genes have been replaced with heterologous genes of interest, and the structural proteins are provided in trans to create single cycle viral replicon particles (VRPs). Clinical stage srRNA vectors have been derived from alphaviruses, including Venezuelan Equine Encephalitis (VEE), Sindbis, and Semliki Forest virus (SFV) and have encoded the tumor antigens carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), prostate specific membrane antigen (PSMA), and human papilloma virus (HPV) antigens E6 and E7. Adverse events have mainly been grade 1 toxicities and minimal injection site reactions. We review here the clinical experience with these vaccines and our recent safety data from a study combining a VRP encoding HER2 plus an anti-PD1 monoclonal antibody (pembrolizumab). This experience with VRP-based srRNA supports recent development of fully synthetic srRNA technologies, where the viral structural proteins are replaced with protective lipid nanoparticles (LNP), cationic nanoemulsions or polymers.

Nonclinical safety evaluation of two vaccine candidates for herpes simplex virus type 2 to support combined administration in humans.

Herpes simplex virus type 2 (HSV-2) is the most common cause of genital disease worldwide. The development of an effective HSV-2 vaccine would significantly impact global health based on the psychological distress caused by genital herpes for some individuals, the risk transmitting the infection from mother to infant, and the elevated risk of acquiring HIV-1. Five nonclinical safety studies were conducted with the replication defective HSV529 vaccine, alone or adjuvanted with GLA-SE, and the G103 subunit vaccine containing GLA-SE. A biodistribution study was conducted in guinea pigs to evaluate distribution, persistence, and shedding of HSV529. A preliminary immunogenicity study was conducted in rabbits to demonstrate HSV529-specific humoral response and its enhancement by GLA-SE. Three repeated-dose toxicity studies, one in guinea pigs and two in rabbits, were conducted to assess systemic toxicity and local tolerance of HSV529, alone or adjuvanted with GLA-SE, or G103 containing GLA-SE. Data from these studies show that both vaccines are safe and well tolerated and support the ongoing HSV-2 clinical trial in which the two vaccine candidates will be given either sequentially or concomitantly to explore their potential synergistic and incremental effects.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine.

BACKGROUND: In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. METHODS: We rapidly developed a replicating RNA vaccine expressing the B.1.1.529 spike and evaluated immunogenicity in mice and hamsters. We also challenged hamsters with B.1.1.529 and evaluated whether vaccination could protect against viral shedding and replication within respiratory tissue. FINDINGS: We found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. INTERPRETATION: Our data suggest that B.1.1.529-targeted vaccines may provide superior protection against B.1.1.529 but pre-existing immunity and timing of boosting may need to be considered for optimum protection. FUNDING: This research was supported in part by the Intramural Research Program, NIAID/NIH, Washington Research Foundation and by grants 27220140006C (JHE), AI100625, AI151698, and AI145296 (MG).

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease following challenge with heterologous variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late 2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoCs) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second-generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform can be updated to target emergent VoCs, elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Since 2019, the SARS-CoV-2 virus has spread worldwide and caused hundreds of millions of cases of COVID-19. Vaccines were rapidly developed to protect people from becoming severely ill from the virus and decrease the risk of death. However, new variants ­ such as Alpha, Beta and Omicron ­ have emerged that the vaccines do not work as well against, contributing to the ongoing spread of the virus. One way to overcome this is to create a vaccine that can be quickly and easily updated to target new variants, like the vaccine against influenza. Many of the vaccines made against COVID-19 use a new technology to introduce the RNA sequence of the spike protein on the surface of SARS-CoV-2 into our cells. Once injected, our cells use their own machinery to build the protein, or 'antigen', so the immune system can learn how to recognize and destroy the virus. Here, Hawman et al. have renovated an RNA vaccine they made in 2020 which provides immunity against the original strain of SARS-CoV-2 in monkeys and mice. In the newer versions of the vaccine, the RNA was updated with a sequence that matches the spike protein on the Beta or Alpha variant of the virus. Both the original and updated vaccines were then administered to mice and hamsters to see how well they worked against SARS-CoV-2 infections. The experiment showed that all three vaccines caused the animals to produce antibodies that can neutralize the original, Alpha and Beta strains of the virus. Vaccinated hamsters were then infected with one of the three variants ­ either matched or mismatched to their vaccination ­ to see how much protection each vaccine provided. All the vaccines reduced the amount of virus in the animals after infection and mitigated damage in their lungs. But animals that received a vaccine which corresponded to the SARS-CoV-2 strain they were infected with had slightly better protection. These findings suggest that these vaccines work best when their RNA sequence matches the strain responsible for the infection; however, even non-matched vaccines still provide a decent degree of protection. Furthermore, the data demonstrate that the vaccine platform created by Hawman et al. can be easily updated to target new strains of SARS-CoV-2 that may emerge in the future. Recently, the Beta variant of the vaccine entered clinical trials in the United States (led by HDT Bio) to evaluate whether it can be used as a booster in previously vaccinated individuals as well as unvaccinated participants.

SARS-CoV2 variant-specific replicating RNA vaccines protect from disease and pathology and reduce viral shedding following challenge with heterologous SARS-CoV2 variants of concern.

Despite mass public health efforts, the SARS-CoV2 pandemic continues as of late-2021 with resurgent case numbers in many parts of the world. The emergence of SARS-CoV2 variants of concern (VoC) and evidence that existing vaccines that were designed to protect from the original strains of SARS-CoV-2 may have reduced potency for protection from infection against these VoC is driving continued development of second generation vaccines that can protect against multiple VoC. In this report, we evaluated an alphavirus-based replicating RNA vaccine expressing Spike proteins from the original SARS-CoV-2 Alpha strain and recent VoCs delivered in vivo via a lipid inorganic nanoparticle. Vaccination of both mice and Syrian Golden hamsters showed that vaccination induced potent neutralizing titers against each homologous VoC but reduced neutralization against heterologous challenges. Vaccinated hamsters challenged with homologous SARS-CoV2 variants exhibited complete protection from infection. In addition, vaccinated hamsters challenged with heterologous SARS-CoV-2 variants exhibited significantly reduced shedding of infectious virus. Our data demonstrate that this vaccine platform elicits significant protective immunity against SARS-CoV2 variants and supports continued development of this platform.

Intratumoral expression of IL-12 from lentiviral or RNA vectors acts synergistically with TLR4 agonist (GLA) to generate anti-tumor immunological memory.

Systemic interleukin-12 (IL12) anti-tumor therapy is highly potent but has had limited utility in the clinic due to severe toxicity. Here, we present two IL12-expressing vector platforms, both of which can overcome the deficiencies of previous systemic IL12 therapies: 1) an integrating lentiviral vector, and 2) a self-replicating messenger RNA formulated with polyethyleneimine. Intratumoral administration of either IL12 vector platform resulted in recruitment of immune cells, including effector T cells and dendritic cells, and the complete remission of established tumors in multiple murine models. Furthermore, concurrent intratumoral administration of the synthetic TLR4 agonist glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE) induced systemic memory T cell responses that mediated complete protection against tumor rechallenge in all survivor mice (8/8 rechallenged mice), whereas only 2/6 total rechallenged mice treated with intratrumoral IL12 monotherapy rejected the rechallenge. Taken together, expression of vectorized IL12 in combination with a TLR4 agonist represents a varied approach to broaden the applicability of intratumoral immune therapies of solid tumors.

The Importance of RNA-Based Vaccines in the Fight against COVID-19: An Overview.

In recent years, vaccine development using ribonucleic acid (RNA) has become the most promising and studied approach to produce safe and effective new vaccines, not only for prophylaxis but also as a treatment. The use of messenger RNA (mRNA) as an immunogenic has several advantages to vaccine development compared to other platforms, such as lower coast, the absence of cell cultures, and the possibility to combine different targets. During the COVID-19 pandemic, the use of mRNA as a vaccine became more relevant; two out of the four most widely applied vaccines against COVID-19 in the world are based on this platform. However, even though it presents advantages for vaccine application, mRNA technology faces several pivotal challenges to improve mRNA stability, delivery, and the potential to generate the related protein needed to induce a humoral- and T-cell-mediated immune response. The application of mRNA to vaccine development emerged as a powerful tool to fight against cancer and non-infectious and infectious diseases, for example, and represents a relevant research field for future decades. Based on these advantages, this review emphasizes mRNA and self-amplifying RNA (saRNA) for vaccine development, mainly to fight against COVID-19, together with the challenges related to this approach.

Long-term survival of patients with stage III colon cancer treated with VRP-CEA(6D), an alphavirus vector that increases the CD8+ effector memory T cell to Treg ratio.

BACKGROUND: There remains a significant need to eliminate the risk of recurrence of resected cancers. Cancer vaccines are well tolerated and activate tumor-specific immune effectors and lead to long-term survival in some patients. We hypothesized that vaccination with alphaviral replicon particles encoding tumor associated antigens would generate clinically significant antitumor immunity to enable prolonged overall survival (OS) in patients with both metastatic and resected cancer. METHODS: OS was monitored for patients with stage IV cancer treated in a phase I study of virus-like replicon particle (VRP)-carcinoembryonic antigen (CEA), an alphaviral replicon particle encoding a modified CEA. An expansion cohort of patients (n=12) with resected stage III colorectal cancer who had completed their standard postoperative adjuvant chemotherapy was administered VRP-CEA every 3 weeks for a total of 4 immunizations. OS and relapse-free survival (RFS) were determined, as well as preimmunization and postimmunization cellular and humoral immunity. RESULTS: Among the patients with stage IV cancer, median follow-up was 10.9 years and 5-year survival was 17%, (95% CI 6% to 33%). Among the patients with stage III cancer, the 5-year RFS was 75%, (95%CI 40% to 91%); no deaths were observed. At a median follow-up of 5.8 years (range: 3.9-7.0 years) all patients were still alive. All patients demonstrated CEA-specific humoral immunity. Patients with stage III cancer had an increase in CD8 +TEM (in 10/12) and decrease in FOXP3 +Tregs (in 10/12) following vaccination. Further, CEA-specific, IFNγ-producing CD8+granzyme B+TCM cells were increased. CONCLUSIONS: VRP-CEA induces antigen-specific effector T cells while decreasing Tregs, suggesting favorable immune modulation. Long-term survivors were identified in both cohorts, suggesting the OS may be prolonged.

ZVex™, a dendritic-cell-tropic lentivector, primes protective antitumor T cell responses that are significantly boosted using heterologous vaccine modalities.

Therapeutic cancer vaccines must induce high levels of tumor-specific cytotoxic CD8 T cells to be effective. We show here that tumor-antigen specific effector and memory T cell responses primed with a non-integrating, dendritic-cell targeted lentiviral vector (ZVex™) could be boosted significantly by either adjuvanted recombinant protein, adenoviral vectors, or self-replicating RNA. These heterologous prime-boost regimens also provided significantly better protection in murine tumor models. In contrast, homologous prime-boost regimens, or using the lentiviral vector as a boost, resulted in lower T cell responses with limited therapeutic efficacy. Heterologous prime-boost regimens that utilize ZVex as the prime may be attractive modalities for therapeutic cancer vaccines.

An Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates.

The coronavirus disease 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale up. Here, we developed an Alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with lipid inorganic nanoparticles (LIONs) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in the lung and spleen. Prime-only immunization of aged (17 months old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. In nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in five intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.

Single-dose replicating RNA vaccine induces neutralizing antibodies against SARS-CoV-2 in nonhuman primates.

The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.

Intratumoral immune activation with TLR4 agonist synergizes with effector T cells to eradicate established murine tumors.

Effective T cell-based immunotherapy of solid malignancies requires intratumoral activity of cytotoxic T cells and induction of protective immune memory. A major obstacle to intratumoral trafficking and activation of vaccine-primed or adoptively transferred tumor-specific T cells is the immunosuppressive tumor microenvironment (TME), which currently limits the efficacy of both anti-tumor vaccines and adoptive cell therapy (ACT). Combination treatments to overcome TME-mediated immunosuppression are therefore urgently needed. We combined intratumoral administration of the synthetic toll-like receptor 4 agonist glucopyranosyl lipid A (oil-in-water formulation, G100) with either active vaccination or adoptive transfer of tumor-specific CD8 T cells to mice bearing established melanomas or orthotopically inoculated glioblastomas. In combination with cancer vaccines or ACT, G100 significantly increased expression of innate immune genes, infiltration and expansion of activated effector T cells, antigen spreading, and durable immune responses. Complete tumor regression of both injected and non-injected tumors was observed only in mice receiving combination immunotherapy. TLR4-based intratumoral immune activation may be a viable approach to enhance the efficacy of therapeutic cancer vaccines and ACT in patients.

Therapeutic efficacy of PD1/PDL1 blockade in B16 melanoma is greatly enhanced by immunization with dendritic cell-targeting lentiviral vector and protein vaccine.

While immune checkpoint inhibition is rapidly becoming standard of care in many solid tumors, immune checkpoint inhibitors (ICIs) fail to induce clinical responses in many patients, presumably due to insufficient numbers of tumor-specific T cells in the tumor milieu. To this end, immunization protocols using viral vectors expressing tumor-associated antigens are being explored to induce T cell responses that synergize with ICIs. However, the optimal combination of vaccine and immune checkpoint regimen remains undefined. Here, a dendritic cell-targeting lentiviral vector (ZVex®) expressing the endogenous murine tyrosinase-related protein 1 (mTRP1), or the human tumor antigen NY-ESO-1, was explored as monotherapy or heterologous prime-boost (HPB) vaccine regimen together with recombinant tumor antigen in the murine B16 melanoma model. PD1/PDL1 blockade significantly enhanced ZVex/mTRP1, but not ZVex/NY-ESO-1, induced immune responses in mice, whereas the opposite effect was observed with anti-CTLA4 antibody. Anti-tumor efficacy of anti-PD1, but not anti-PDL1 or anti-CTLA4, was significantly enhanced by ZVex/mTRP1 and HPB vaccination. These results suggest mechanistic differences in the effect of checkpoint blockade on vaccine-induced immune and anti-tumor responses against self versus non-self tumor antigens, possibly due to tolerance and state of exhaustion of anti-tumor T cells.

Structural brain changes in hyperthyroid Graves' disease: protocol for an ongoing longitudinal, case-controlled study in Göteborg, Sweden-the CogThy project.

INTRODUCTION: Cognitive impairment and reduced well-being are common manifestations of Graves' disease (GD). These symptoms are not only prevalent during the active phase of the disease but also often prevail for a long time after hyperthyroidism is considered cured. The pathogenic mechanisms involved in these brain-derived symptoms are currently unknown. The overall aim of the CogThy study is to identify the mechanism behind cognitive impairment to be able to recognise GD patients at risk. METHODS AND ANALYSIS: The study is a longitudinal, single-centre, case-controlled study conducted in Göteborg, Sweden on premenopausal women with newly diagnosed GD. The subjects are examined: at referral, at inclusion and then every 3.25 months until 15 months. Examinations include: laboratory measurements; eye evaluation; neuropsychiatric and neuropsychological testing; structural MRI of the whole brain, orbits and medial temporal lobe structures; functional near-infrared spectroscopy of the cerebral prefrontal cortex and self-assessed quality of life questionnaires. The primary outcome measure is the change in medial temporal lobe structure volume. Secondary outcome measures include neuropsychological, neuropsychiatric, hormonal and autoantibody variables. The study opened for inclusion in September 2012 and close for inclusion in October 2019. It will provide novel information on the effect of GD on medial temporal lobe structures and cerebral cortex functionality as well as whether these changes are associated with cognitive and affective impairment, hormonal levels and/or autoantibody levels. It should lead to a broader understanding of the underlying pathogenesis and future treatment perspectives. ETHICS AND DISSEMINATION: The study has been reviewed and approved by the Regional Ethical Review Board in Göteborg, Sweden. The results will be actively disseminated through peer-reviewed journals, national and international conference presentations and among patient organisations after an appropriate embargo time. TRIAL REGISTRATION NUMBER: 44321 at the public project database for research and development in Västra Götaland County, Sweden (https://www.researchweb.org/is/vgr/project/44321).

Reduced DNA methylation and psychopathology following endogenous hypercortisolism - a genome-wide study.

Patients with Cushing's Syndrome (CS) in remission were used as a model to test the hypothesis that long-standing excessive cortisol exposure induces changes in DNA methylation that are associated with persisting neuropsychological consequences. Genome-wide DNA methylation was assessed in 48 women with CS in long-term remission (cases) and 16 controls matched for age, gender and education. The Fatigue impact scale and the comprehensive psychopathological rating scale were used to evaluate fatigue, depression and anxiety. Cases had lower average global DNA methylation than controls (81.2% vs 82.7%; p = 0.002). Four hundred and sixty-one differentially methylated regions, containing 3,246 probes mapping to 337 genes were identified. After adjustment for age and smoking, 731 probes in 236 genes were associated with psychopathology (fatigue, depression and/or anxiety). Twenty-four gene ontology terms were associated with psychopathology; terms related to retinoic acid receptor signalling were the most common (adjusted p = 0.0007). One gene in particular, COL11A2, was associated with fatigue following a false discovery rate correction. Our findings indicate that hypomethylation of FKBP5 and retinoic acid receptor related genes serve a potential mechanistic explanation for long-lasting GC-induced psychopathology.