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Combination of a Sindbis-SARS-CoV-2 spike vaccine and αOX40 antibody elicits protective immunity against SARS-CoV-2 induced disease and potentiates long-term SARS-CoV-2-specific humoral and T-cell immunity

Scaglione, Antonella; Opp, Silvana; Hurtado, Alicia; Lin, Ziyan; Pampeno, Christine; Noval, Maria G; Thannickal, Sara A; Stapleford, Kenneth; Meruelo, Daniel
The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles and metabolic analysis indicate a reprogramming of T-cells in vaccinated mice. Activated T-cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response that can be used as a new candidate to combat SARS-CoV-2. Given the strong T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as, serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.
PMCID:8168399
PMID: 34075383
ISSN: n/a
CID: 4891512

Combination of a Sindbis-SARS-CoV-2 Spike Vaccine and αOX40 Antibody Elicits Protective Immunity Against SARS-CoV-2 Induced Disease and Potentiates Long-Term SARS-CoV-2-Specific Humoral and T-Cell Immunity

Scaglione, Antonella; Opp, Silvana; Hurtado, Alicia; Lin, Ziyan; Pampeno, Christine; Noval, Maria G; Thannickal, Sara A; Stapleford, Kenneth A; Meruelo, Daniel
The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.
PMID: 34394127
ISSN: 1664-3224
CID: 5004522

Sindbis Virus with Anti-OX40 Overcomes the Immunosuppressive Tumor Microenvironment of Low-Immunogenic Tumors

Scherwitzl, Iris; Opp, Silvana; Hurtado, Alicia M; Pampeno, Christine; Loomis, Cynthia; Kannan, Kasthuri; Yu, Minjun; Meruelo, Daniel
Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors.
PMCID:7251545
PMID: 32478167
ISSN: 2372-7705
CID: 4458162

Molecular and metabolic pathways mediating curative treatment of a non-Hodgkin B cell lymphoma by Sindbis viral vectors and anti-4-1BB monoclonal antibody

Yu, Minjun; Scherwitzl, Iris; Opp, Silvana; Tsirigos, Aristotelis; Meruelo, Daniel
BACKGROUND:Limitations to current therapies for treating non-Hodgkin B cell lymphoma include relapse, toxicity and high cost. Thus, there remains a need for novel therapies. Oncolytic viral (OV) therapy has become a promising cancer immunotherapy because of its potential effectiveness, specificity and long-lasting immunity. We describe and characterize a novel cancer immunotherapy combining Sindbis virus (SV) vectors and the agonistic monoclonal antibody (mAb) to the T cell costimulatory receptor, 4-1BB (CD137). METHODS:A20 lymphoma was transfected with luciferase and tumor cells were inoculated to BALB/c mice. Tumor growth was monitored by IVIS imaging. Tumor bearing mice were treated with Sindbis virus, α4-1BB Ab or SV plus α4-1BB Ab. On day 7 after treatment, splenocytes were harvested and surface markers, cytokines, and transcription factors were measured by flow cytometry or Elispot. Splenic T cells were isolated and RNA transcriptome analysis was performed. Tumor cured mice were rechallenged with tumor for testing immunological memory. RESULTS:SV vectors in combination with α4-1BB monoclonal antibody (mAb) completely eradicated a B-cell lymphoma in a preclinical mouse model, a result that could not be achieved with either treatment alone. Tumor elimination involves a synergistic effect of the combination that significantly boosts T cell cytotoxicity, IFNγ production, T cell proliferation, migration, and glycolysis. In addition, all mice that survived after treatment developed long lasting antitumor immunity, as shown by the rejection of A20 tumor rechallenge. We identified the molecular pathways, including upregulated cytokines, chemokines and metabolic pathways in T cells that are triggered by the combined therapy and help to achieve a highly effective anti-tumor response. CONCLUSIONS:Our study provides a novel, alternative method for B cell lymphoma treatment and describes a rationale to help translate SV vectors plus agonistic mAb into clinical applications.
PMID: 31307539
ISSN: 2051-1426
CID: 3977712

Systemically Administered Sindbis Virus in Combination with Immune Checkpoint Blockade Induces Curative Anti-tumor Immunity

Scherwitzl, Iris; Hurtado, Alicia; Pierce, Carolyn M; Vogt, Sandra; Pampeno, Christine; Meruelo, Daniel
Oncolytic viruses represent a promising form of cancer immunotherapy. We investigated the potential of Sindbis virus (SV) for the treatment of solid tumors expressing the human cancer testis antigen NYESO-1. NYESO-1 is an immunogenic antigen frequently expressed in numerous cancers, such as ovarian cancer. We show that SV expressing the tumor-associated antigen NYESO-1 (SV-NYESO1) acts as an immunostimulatory agent, inducing systemic and rapid lymphocyte activation, leading to a pro-inflammatory environment. SV-NYESO1 treatment combined with anti-programmed death 1 (anti-PD-1) markedly augmented the anti-tumor immunity in mice over the course of treatment, resulting in an avid systemic and intratumoral immune response. This response involved reduced presence of granulocytic myeloid-derived suppressor cells in tumors and an increase in the activation of splenic and tumor-infiltrating T cells. Combined therapy also induced enhanced cytotoxic activity of T cells against NYESO-1-expressing tumors. These results were in line with an observed inverse correlation between T cell activation and tumor growth. Finally, we show that combined therapy resulted in complete clearance of NYESO-1-expressing tumors in vivo and led to long-term protection against recurrences. These findings provide a rationale for clinical studies of SV-NYESO1 combined with immune checkpoint blockade anti-PD-1 to be used in the treatment of NYESO-1-expressing tumors.
PMCID:6026467
PMID: 29988525
ISSN: 2372-7705
CID: 3191582

Looking into laminin receptor: critical discussion regarding the non-integrin 37/67-kDa laminin receptor/RPSA protein

DiGiacomo, Vincent; Meruelo, Daniel
The 37/67-kDa laminin receptor (LAMR/RPSA) was originally identified as a 67-kDa binding protein for laminin, an extracellular matrix glycoprotein that provides cellular adhesion to the basement membrane. LAMR has evolutionary origins, however, as a 37-kDa RPS2 family ribosomal component. Expressed in all domains of life, RPS2 proteins have been shown to have remarkably diverse physiological roles that vary across species. Contributing to laminin binding, ribosome biogenesis, cytoskeletal organization, and nuclear functions, this protein governs critical cellular processes including growth, survival, migration, protein synthesis, development, and differentiation. Unsurprisingly given its purview, LAMR has been associated with metastatic cancer, neurodegenerative disease and developmental abnormalities. Functioning in a receptor capacity, this protein also confers susceptibility to bacterial and viral infection. LAMR is clearly a molecule of consequence in human disease, directly mediating pathological events that make it a prime target for therapeutic interventions. Despite decades of research, there are still a large number of open questions regarding the cellular biology of LAMR, the nature of its ability to bind laminin, the function of its intrinsically disordered C-terminal region and its conversion from 37 to 67 kDa. This review attempts to convey an in-depth description of the complexity surrounding this multifaceted protein across functional, structural and pathological aspects.
PMCID:5249262
PMID: 25630983
ISSN: 1464-7931
CID: 1447872

The transition of the 37-kDa laminin receptor (RPSA) to higher molecular weight species: SUMOylation or artifact?

Digiacomo, Vincent; Gando, Ivan A; Venticinque, Lisa; Hurtado, Alicia; Meruelo, Daniel
The 37-kDa laminin receptor (37LRP or RPSA) is a remarkable, multifaceted protein that functions in processes ranging from matrix adhesion to ribosome biogenesis. Its ability to engage extracellular laminin is further thought to contribute to cellular migration and invasion. Most commonly associated with metastatic cancer, RPSA is also increasingly found to be important in other pathologies, including microbial infection, neurodegenerative disease and developmental malformations. Importantly, it is thought to have higher molecular weight forms, including a 67-kDa species (67LR), the expression of which is linked to strong laminin binding and metastatic behavior. The composition of these larger forms has remained elusive and controversial. Homo- and heterodimerization have been proposed as events capable of building the larger species from the monomeric 37-kDa precursor, but solid evidence is lacking. Here, we present data suggesting that higher molecular weight species require SUMOylation to form. We also comment on the difficulty of isolating larger RPSA species for unambiguous identification and demonstrate that cell lines stably expressing tagged RPSA for long periods of time fail to produce tagged higher molecular weight RPSA. It is possible that higher molecular weight species like 67LR are not derived from RPSA.
PMCID:5240582
PMID: 26146125
ISSN: 1689-1392
CID: 1662552

Screening strategies to identify HSP70 modulators to treat Alzheimer's disease

Repalli, Jayanthi; Meruelo, Daniel
Alzheimer's disease, the most common type of dementia, is a progressive brain disease that destroys cognitive function and eventually leads to death. In patients with Alzheimer's disease, beta amyloids and tau proteins form plaques/oligomers and oligomers/tangles that affect the ability of neurons to function properly. Heat shock protein 70 (HSP70) has the ability to prevent aggregation/oligomerization of beta amyloid/tau proteins, making it a potential drug target. To determine this potential, it is essential that we have appropriate in vitro and cell-based assays that help identify specific molecules that affect this aggregation or oligomerization through HSP70. Potential drug candidates could be identified through a series of assays, starting with ATPase assays, followed by aggregation assays with enzymes/proteins and cell-based systems. ATPase assays are effective in identification of ATPase modulators but do not determine the effect of the molecule on beta amyloid and tau proteins. Molecules identified through ATPase assays are validated by thioflavin T aggregation assays in the presence of HSP70. These assays help uncover if a molecule affects beta amyloid and tau through HSP70, but are limited by their in vitro nature. Potential drug candidates are further validated through cell-based assays using mammalian, yeast, or bacterial cultures. However, while these assays are able to determine the effect of a specific molecule on beta amyloid and tau, they fail to determine whether the action is HSP70-dependent. The creation of a novel, direct assay that can demonstrate the antiaggregation effect of a molecule as well as its action through HSP70 would reduce the number of false-positive drug candidates and be more cost-effective and time-effective.
PMCID:4294646
PMID: 25609918
ISSN: 1177-8881
CID: 1440392

TBLR1 as an androgen receptor (AR) coactivator selectively activates AR target genes to inhibit prostate cancer growth

Daniels, Garrett; Li, Yirong; Gellert, Lan Lin; Zhou, Albert; Melamed, Jonathan; Wu, Xinyu; Zhang, Xinming; Zhang, David; Meruelo, Daniel; Logan, Susan K; Basch, Ross; Lee, Peng
Androgen receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. Transducin beta-like-related protein 1 (TBLR1), a core component of the nuclear receptor corepressor complex, shows both corepressor and coactivator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and determined that the activation is dependent on both phosphorylation and 19S proteosome. We showed that TBLR1 physically interacts with AR and directly occupies the androgen-response elements of the affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared with the surrounding benign prostatic glands (P<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen-regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3-1), but not cell proliferation of the prostate cancer. Understanding the molecular switches involved in the transition from AR-dependent growth promotion to AR-dependent growth suppression will lead to more successful treatments for prostate cancer.
PMCID:3947037
PMID: 24243687
ISSN: 1351-0088
CID: 1083962

Sindbis Viral Vectors Transiently Deliver Tumor-associated Antigens to Lymph Nodes and Elicit Diversified Antitumor CD8(+) T-cell Immunity

Granot, Tomer; Yamanashi, Yoshihide; Meruelo, Daniel
Tumors are theoretically capable of eliciting an antitumor immune response, but are often poorly immunogenic. Oncolytic viruses (OVs) have recently emerged as a promising strategy for the immunogenic delivery of tumor-associated antigens (TAAs) to cancer patients. However, safe and effective OV/TAA therapies have not yet been established. We have previously demonstrated that vectors based on Sindbis virus (SV) can inhibit tumor growth and activate the innate immune system in mice. Here, we demonstrate that SV vectors carrying a TAA generate a dramatically enhanced therapeutic effect in mice bearing subcutaneous, intraperitoneal, and lung cancers. Notably, SV/TAA efficacy was not dependent on tumor cell targeting, but was characterized by the transient expression of TAAs in lymph nodes draining the injection site. Early T-cell activation at this site was followed by a robust influx of NKG2D expressing antigen-specific cytotoxic CD8(+) T cells into the tumor site, subsequently leading to the generation of long-lasting memory T cells which conferred protection against rechallenge with TAA-positive as well as TAA-negative tumor cells. By combining in vivo imaging, flow cytometry, cytotoxicity/cytokine assays, and tetramer analysis, we investigated the relationship between these events and propose a model for CD8(+) T-cell activation during SV/TAA therapy.
PMCID:3978799
PMID: 24025748
ISSN: 1525-0016
CID: 781242