Replication-defective Adenoviral Vector Research Articles

The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo.

Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.

A Novel Vaccine Strategy to Prevent Cytauxzoonosis in Domestic Cats.

Cytauxzoonosis is caused by Cytauxzoon felis (C. felis), a tick-borne parasite that causes severe disease in domestic cats in the United States. Currently, there is no vaccine to prevent this fatal disease, as traditional vaccine development strategies have been limited by the inability to culture this parasite in vitro. Here, we used a replication-defective human adenoviral vector (AdHu5) to deliver C. felis-specific immunogenic antigens and induce a cell-mediated and humoral immune response in cats. Cats (n = 6 per group) received either the vaccine or placebo in two doses, 4 weeks apart, followed by experimental challenge with C. felis at 5 weeks post-second dose. While the vaccine induced significant cell-mediated and humoral immune responses in immunized cats, it did not ultimately prevent infection with C. felis. However, immunization significantly delayed the onset of clinical signs and reduced febrility during C. felis infection. This AdHu5 vaccine platform shows promising results as a vaccination strategy against cytauxzoonosis.

Adenovirus vector-mediated single chain variable fragments target the nucleocapsid protein of porcine epidemic diarrhea virus and protect against viral infection in piglets.

Porcine epidemic diarrhea virus (PEDV) mainly infects the intestinal epithelial cells of pigs, causing porcine epidemic diarrhea (PED). In particular, the virus causes severe diarrhea, dehydration, and death in neonatal piglets. Maternal immunity effectively protects neonatal piglets from PEDV infection; however, maternal antibodies can only prevent PEDV attachment and entry into target cells, but have no effects on intracellular viruses. Intracellular antibodies targeting virus-encoded proteins are effective in preventing viral infection. We previously identified four single chain variable fragments (scFvs), ZW1-16, ZW3-21, ZW1-41, and ZW4-16, which specifically targeted the PEDV N protein and significantly inhibited PEDV replication and up-regulated interferon-λ1 (IFN-λ1) expression in host cells. In our current study, the four scFvs were subcloned into replication-defective adenovirus vectors to generate recombinant adenoviruses rAdV-ZW1-16, rAdV-ZW3-21, rAdV-ZW1-41, and rAdV-ZW4-16. ScFvs were successfully expressed in Human Embryonic Kidney 293 (HEK293) cells and intestinal porcine epithelial cell line J2 (IPEC-J2) and were biosafe for piglets as indicated by body temperature and weight, scFv excretion in feces, IFN-γ and interleukin-4 (IL-4) expression in jejunum, and pathological changes in porcine tissue after oral administration. Western blotting, immunofluorescence, and immunohistochemical analyses showed that scFvs were expressed in porcine jejunum. The prophylactic effects of rAdV-ZW, a cocktail of the four rAdV-scFvs, on piglet diarrhea caused by PEDV was investigated. Clinical symptoms in piglets orally challenged with PEDV, following a two-time treatment with rAdV-ZW, were significantly reduced when compared with PEDV-infected piglets treated with phosphate buffered saline (PBS) or rAdV-wild-type. Also, no death and jejunal lesions were observed. ScFv co-localization with the PEDV N protein in vivo was also observed. Next, the expression of pro-inflammatory serum cytokines such as tumor necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, and IFN-λ was assessed by enzyme-linked immunosorbent assay (ELISA), which showed that scFvs significantly suppressed PEDV-induced pro-inflammatory cytokine expression and restored PEDV-inhibited IFN-λ expression. Therefore, our study supported a promising role for intracellular scFvs targeting the PEDV N protein to prevent and treat diarrhea in PEDV-infected piglets.

Heterologous chimpanzee adenovirus vector immunizations for SARS-CoV-2 spike and nucleocapsid protect hamsters against COVID-19

Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines.

Role of Adenoviruses in Cancer Therapy.

Cancer is one of the leading causes of death in the world, which is the second after heart diseases. Adenoviruses (Ads) have become the promise of new therapeutic strategy for cancer treatment. The objective of this review is to discuss current advances in the applications of adenoviral vectors in cancer therapy. Adenoviral vectors can be engineered in different ways so as to change the tumor microenvironment from cold tumor to hot tumor, including; 1. by modifying Ads to deliver transgenes that codes for tumor suppressor gene (p53) and other proteins whose expression result in cell cycle arrest 2. Ads can also be modified to express tumor specific antigens, cytokines, and other immune-modulatory molecules. The other strategy to use Ads in cancer therapy is to use oncolytic adenoviruses, which directly kills tumor cells. Gendicine and Advexin are replication-defective recombinant human p53 adenoviral vectors that have been shown to be effective against several types of cancer. Gendicine was approved for treatment of squamous cell carcinoma of the head and neck by the Chinese Food and Drug Administration (FDA) agency in 2003 as a first-ever gene therapy product. Oncorine and ONYX-015 are oncolytic adenoviral vectors that have been shown to be effective against some types of cancer. The Chiness FDA agency has also approved Oncorin for the treatment of head and neck cancer. Ads that were engineered to express immune-stimulatory cytokines and other immune-modulatory molecules such as TNF-α, IL-2, BiTE, CD40L, 4-1BBL, GM-CSF, and IFN have shown promising outcome in treatment of cancer. Ads can also improve therapeutic efficacy of immune checkpoint inhibitors and adoptive cell therapy (Chimeric Antigen Receptor T Cells). In addition, different replication-deficient adenoviral vectors (Ad5-CEA, Ad5-PSA, Ad-E6E7, ChAdOx1–MVA and Ad-transduced Dendritic cells) that were tested as anticancer vaccines have been demonstrated to induce strong antitumor immune response. However, the use of adenoviral vectors in gene therapy is limited by several factors such as pre-existing immunity to adenoviral vectors and high immunogenicity of the viruses. Thus, innovative strategies must be continually developed so as to overcome the obstacles of using adenoviral vectors in gene therapy.

Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy.

Gene therapy is currently in the public spotlight. Several gene therapy products, including oncolytic virus (OV), which predominantly replicates in and kills cancer cells, and COVID-19 vaccines have recently been commercialized. Recombinant adenoviruses, including replication-defective adenoviral vector and conditionally replicating adenovirus (CRA; oncolytic adenovirus), have been extensively studied and used in clinical trials for cancer and vaccines. Here, we review the biology of wild-type adenoviruses, the methodological principle for constructing recombinant adenoviruses, therapeutic applications of recombinant adenoviruses, and new technologies in pluripotent stem cell (PSC)-based regenerative medicine. Moreover, this article describes the technology platform for efficient construction of diverse “CRAs that can specifically target tumors with multiple factors” (m-CRAs). This technology allows for modification of four parts in the adenoviral E1 region and the subsequent insertion of a therapeutic gene and promoter to enhance cancer-specific viral replication (i.e., safety) as well as therapeutic effects. The screening study using the m-CRA technology successfully identified survivin-responsive m-CRA (Surv.m-CRA) as among the best m-CRAs, and clinical trials of Surv.m-CRA are underway for patients with cancer. This article also describes new recombinant adenovirus-based technologies for solving issues in PSC-based regenerative medicine.

Relapsed Refractory Acquired Thrombotic Thrombocytopenic Purpura (aTTP) Following COVID-19 Vaccination

Introduction:Acquired thrombotic thrombocytopenic purpura (aTTP) due to an acquired deficiency in the enzyme ADAMTS13 leads to ultra-large von Willebrand multimers, thrombocytopenia and microangiopathic hemolytic anemia. Complications include microvascular and macrovascular thrombosis. We present an unusual case of a patient with a history of refractory aTTP who experienced relapsed aTTP following COVID-19 vaccine.Case Description:A 57-year-old African-American male with a history of refractory aTTP experienced a relapse following 3 years of remission after receiving COVID-19 vaccination. The patient was initially diagnosed with aTTP in 2016, after presenting with symptoms of dark urine, mild headaches and transient episodes of aphasia and paresthesia. Due to symptoms and persistently low ADAMTS13 levels, he required prolonged and extensive treatment including over 5 weeks of daily therapeutic plasma exchange (TPE), followed by gradual reduction in frequency of TPE sessions, as well as trials of rituximab, eculizumab, steroids, mycophenolate mofetil and bortezomib. Ultimately, he achieved remission after 9 months of intermittent TPE, 3 months of weekly bortezomib 1 mg/m 2, mycophenolate mofetil up-titrated to 1,750 mg twice daily, and then slowly tapered off over a 2-year period. The patient was doing well for 3 years without manifestations of aTTP (2 years off all therapeutics), until he developed a petechial rash 7 weeks after receiving the second dose of the Moderna COVID-19 vaccine. He was found to have acute thrombocytopenia with platelets of 38 x 10 9/L (normal range 135-317 x 109/L), from a baseline of 200-300 x 10 9/L. He was referred to the emergency department, where additional labs were notable for mildly elevated LDH of 508 U/L (normal range 122-222 U/L), hemoglobin of 12.4 g/dL (normal range 13.2-16.6 g/dL), creatinine at baseline, and peripheral blood smear showing 1-3 schistocytes per high-powered field. ADAMTS13 activity level was t <5% (normal >/= 70%), with positive ADAMTS13 inhibitor screen and titer of 1.5 (normal <0.4), consistent with relapsed aTTP.The patient was admitted to the hospital, and initiated on daily TPE, with steroids and diphenhydramine prior to each TPE session. He quickly improved with TPE alone , but given his history of refractory aTTP, he was discharged on weekly rituximab for 4 weeks and caplacizumab 11 mg daily for 30 days. His platelets remained stable within the upper limit of normal during his 30 day course of caplacizumab. However, 3 weeks after completion of caplacizumab, he had an acute drop in his platelets to 23 x 10 9/L. His ADAMTS13 level was again found to be <5%, and inhibitor level was the highest that it had ever been at 11.4. He was again hospitalized and underwent 8 sessions of daily TPE, as well as re-initiation of caplacizumab, mycophenolate mofetil 500 mg bid (with increasing taper), and a prednisone taper. Intravenous Cyclophosphamide 750 mg/m 2 was also added every 3 weeks. With this regimen, patient's platelet count normalized and remain stable, and his ADAMTS13 activity level has reached 52-59%.Discussion:Cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) have been described as a complication following vaccination with formulations containing replication-defective adenoviral vectors (AstraZeneca-Oxford and Johnson&Johnson COVID-19 vaccines)(Arepally and Ortel 2021, Simpson, Shi et al. 2021). VITT and aTTP are both immune-mediated, however, VITT is distinct and pathogenically linked to autoimmune heparin-induced thrombocytopenia (HIT), given the presence of anti-platelet factor 4 antibodies in these patients, whereas aTTP is due to reduction in ADAMTS13 level, secondary to an antibody inhibitor of ADAMTS13 (Arepally and Ortel 2021). Recently, cases have been reported of de novo aTTP developing shortly after COVID-19 vaccination with all available vaccines, except the Moderna (mRNA-1273) vaccine (Al-Ahmad, Al-Rasheed et al. 2021, de Bruijn, Maes et al. 2021, Maayan, Kirgner et al. 2021, Ruhe, Schnetzke et al. 2021, Waqar, Khan et al. 2021, Yocum and Simon 2021). Additionally, cases of relapsed aTTP have been described following only the BNT162B2 (Pfizer-BioNTech) vaccine (Maayan, Kirgner et al. 2021, Sissa, Al-Khaffaf et al. 2021). This is the first case, to our knowledge, reported in the literature of aTTP following vaccination with Moderna's mRNA-1273 vaccine. DisclosuresNo relevant conflicts of interest to declare.

Abstract PS13-28: Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy

Abstract Introduction: Amongst all breast cancers, Triple Negative Breast Cancer (TNBC) account for 15-20% of all the cases. TNBC affect younger patients, and is more prevalent in African-American women. The poor prognosis for this very aggressive tumor subtype is exacerbated by the lack of specific targeted therapy against the disease. Although TNBC initially respond very well to chemotherapy, paradoxically the disease-free survival is very short. It has been showed that TNBC have higher rates of CD8+ T-cells infiltration, and express high level of PD-L1. Together, these data provide a strong rationale for the combination of chemotherapy and immunotherapy to treat TNBC patients. In this study, we investigated the response to pre-priming the tumor with one round of docetaxel and IL-12, followed by anti-PD1 maintenance in mouse E0771 and 4T1 TNBC syngeneic models. We hypothesized that docetaxel will promote the release of neo-antigens, while IL-12 will activate immunity specific to these antigens and anti-PD1 therapy prevent the exhaustion of those T-cells. Materials/Methods: Mouse TNBC E0771 and 4T1 cell lines were injected in the mammary fat pad of C57BL/6, and Balb/c mice respectively. On day 1, the mice received a single dose (20mg/kg) of docetaxel and one intratumoral injection (1.25x109) of mAdv.IL-12, a replication defective adenoviral vector containing mIL-12 (mouse) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (provided by Dr. Chen, HMRI). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week (2 cycles) starting 5 days post docetaxel and Il-12 treatment. At the end of the study, IFN-gamma levels were measured from blood and tumor samples; tumor sizes were compared between treatment groups (Control/mAdv.IL-12/anti-PD1/and various Combination), as well as survival curves. The metastatic burden to the lungs (H&amp;E), as well as the apoptosis in the tumor (TUNNEL) were assessed by IHC. Results: In both 4T1 and E0771 tumor models, triple combination of docetaxel + IL-12 followed by anti-PD1 significantly reduced tumor size compared to both single agents, and double combination. In the Triple Combo group, 1 mice had lung metastasis vs all of them in the other treatment groups. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination groups compared to single agents. There was more apoptosis in the triple combo group as indicated by TUNNEL. Conclusion:Our preliminary data strongly supports that treating TNBC models with docetaxel and mAdv.IL-12 followed by anti-PD1 significantly slows down tumor growth, and decrease lung metastasis incidence. We are actively investigating the mechanism through which the response is achieved. Citation Format: Ann Cassany Anselme, Wei Qian, Jianying Zhou, Roberto R. Rosato, Jenny C. Chang. Pre-treating TNBC with docetaxel and il-12 enhances anti-PD1 efficacy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-28.

Increasing Efficacy of Enveloped Whole-Virus Vaccines by In situ Immune-Complexing with the Natural Anti-Gal Antibody.

The appearance of variants of mutated virus in course of the Covid-19 pandemic raises concerns regarding the risk of possible formation of variants that can evade the protective immune response elicited by the single antigen S-protein gene-based vaccines. This risk may be avoided by inclusion of several antigens in vaccines, so that a variant that evades the immune response to the S-protein of SARS-CoV-2 virus will be destroyed by the protective immune response against other viral antigens. A simple way for preparing multi-antigenic enveloped-virus vaccines is using the inactivated whole-virus as vaccine. However, immunogenicity of such vaccines may be suboptimal because of poor uptake of the vaccine by antigen-presenting-cells (APC) due to electrostatic repulsion by the negative charges of sialic-acid on both the glycan-shield of the vaccinating virus and on the carbohydrate-chains (glycans) of APC. In addition, glycan-shield can mask many antigenic peptides. These effects of the glycan-shield can be reduced and immunogenicity of the vaccinating virus markedly increased by glycoengineering viral glycans for replacing sialic-acid units on glycans with α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R). Vaccination of humans with inactivated whole-virus presenting α-gal epitopes (virusα-gal) results in formation of immune-complexes with the abundant natural anti-Gal antibody that binds to viral α-gal epitopes at the vaccination site. These immune-complexes are targeted to APC for rigorous uptake due to binding of the Fc portion of immunecomplexed anti-Gal to Fcγ receptors on APC. The APC further transport the large amounts of internalized vaccinating virus to regional lymph nodes, process and present the virus antigenic peptides for the activation of many clones of virus specific helper and cytotoxic T-cells. This elicits a protective cellular and humoral immune response against multiple viral antigens and an effective immunological memory. The immune response to virusα-gal vaccine was studied in mice producing anti-Gal and immunized with inactivated influenza-virusα-gal. These mice demonstrated 100-fold increase in titer of the antibodies produced, a marked increase in T-cell response, and a near complete protection against challenge with a lethal dose of live influenza-virus, in comparison to a similar vaccine lacking α-gal epitopes. This glycoengineering can be achieved in vitro by enzymatic reaction with neuraminidase removing sialic-acid and with recombinant α1,3galactosyltransferase (α1,3GT) synthesizing α-gal epitopes, by engineering host-cells to contain several copies of the α1,3GT gene (GGTA1), or by transduction of this gene in a replication-defective adenovirus vector into host-cells. Theoretically, these methods for increased immunogenicity may be applicable to all enveloped viruses with N-glycans on their envelope.

A next generation vaccine against human rabies based on a single dose of a chimpanzee adenovirus vector serotype C.

Rabies, caused by RNA viruses in the Genus Lyssavirus, is the most fatal of all infectious diseases. This neglected zoonosis remains a major public health problem in developing countries, causing the death of an estimated 25,000–159,000 people each year, with more than half of them in children. The high incidence of human rabies in spite of effective vaccines is mainly linked to the lack of compliance with the complicated administration schedule, inadequacies of the community public health system for local administration by the parenteral route and the overall costs of the vaccine. The goal of our work was the development of a simple, affordable and effective vaccine strategy to prevent human rabies virus infection. This next generation vaccine is based on a replication-defective chimpanzee adenovirus vector belonging to group C, ChAd155-RG, which encodes the rabies glycoprotein (G). We demonstrate here that a single dose of this vaccine induces protective efficacy in a murine model of rabies challenge and elicits strong and durable neutralizing antibody responses in vaccinated non-human primates. Importantly, we demonstrate that one dose of a commercial rabies vaccine effectively boosts the neutralizing antibody responses induced by ChAd155-RG in vaccinated monkeys, showing the compatibility of the novel vectored vaccine with the current post-exposure prophylaxis in the event of rabies virus exposure. Finally, we demonstrate that antibodies induced by ChAd155-RG can also neutralize European bat lyssaviruses 1 and 2 (EBLV-1 and EBLV-2) found in bat reservoirs.

Vaccination of Macaques with DNA Followed by Adenoviral Vectors Encoding Simian Immunodeficiency Virus (SIV) Gag Alone Delays Infection by Repeated Mucosal Challenge with SIV.

Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication-defective adenovirus (rAd) vaccine vectors are at the forefront of this work and have been tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV Gag or other nonenvelope proteins induces T cell responses that control virus load but disappointingly is unsuccessful so far in preventing infection, and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomolgus macaques (MCM), Macaca fascicularis, vaccinated with unmodified SIV gag alone in a DNA prime followed by an rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low-dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection, a significant reduction in peak virus load was observed in all vaccinated animals, including those vaccinated with modified gag These findings suggest that a nonpersistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV type 1 (HIV-1) infection. The mechanisms are probably distinct from those of antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus-infected cells and may be mediated in part by CD4 T cells.IMPORTANCE The simian immunodeficiency virus (SIV) macaque model represents the best animal model for testing new human immunodeficiency virus type 1 (HIV-1) vaccines. Previous studies employing replication-defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with an rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to the envelope and neutralize the virus or mediate antibody-dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established has also shown some success. In contrast, the vaccine described here mediates resistance by a different mechanism from that described above, which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.

Expression and Characterization of Human Vascular Endothelial Growth Factor Produced in SiHa Cells Transduced with Adenoviral Vector

The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.

Abstract 2261: Understanding the mechanism underlying resistance to immunotherapy in TNBC

Abstract Triple Negative Breast Cancer (TNBC) accounts for 15-20% of all breast cancer cases. TNBC patients have the worst outcome amongst all breast cancers; and do not respond well to conventional therapies. Although TNBC patients have a high amount of tumor infiltrating lymphocytes, and express higher level of PD-L1 compared to other breast cancer subtypes, they do not all respond to PD-1 blockade therapy. IL-12 is a cytokine produced by dendritic cells (DCs), and other APCs, known to induce the activation of natural killer cells, cytotoxic T-cells, and induce a Th1 phenotype. Although the immune activating potential of Il-12 are known, its systemic uses have been limited due to high toxicity. Using three different tumor models (PDXs: BCM 4913, MC1 in humanized mice and E0771 syngeneic model C57/Bl), we are investigating the response of combining intratumoral injection of Adenovirus IL-12 (ad.Il-12) and anti-PD1 therapy. Materials/Methods: E0771, a mice TNBC cell line was injected in the mammary fat pad of C57BL/6 mice. The same procedure was followed using MC1 and BCM 4913, in humanized NSG-MGM3 mice. The mice received weekly intratumoral injection of ad.IL-12; a replication defective adenoviral vector containing mIL-12 (mouse) and hIL-12 (PDX) cDNA under the transcriptional control of Rous sarcoma virus long terminal repeat (received from Dr. Chen). Anti-PD1 (InVivoMab anti-mouse PD-1 CD279) was administered 3 times a week, Pembrolizumab (once a week). Post treatment, INF-gamma level were measured from blood and tumor samples, in addition, tumor sizes were compared between treatment groups (Control/ad.IL-12/anti-PD1/ad.IL-12 + anti-PD1), as well as survival curves. Immunohistochemistry was used to assess levels on TILs in the tumors. Results: Ad.Il-12 and PD-1 blockade therapy combination was more effective at reducing tumor growth in BCM4913, while in E00771 and MC1 combination treatment showed no difference compared to single agent. In all the models, levels of INF-gamma in the tumor were significantly upregulated in the combination group compared to control and single agents, while no difference was found between treatment groups from blood samples. Tumor growth in the combination group and the treated group were slower compared to the controls in BCM 4913. IHC data indicate a higher level of TILs in the treatment groups, with a statistically significant difference in the combination group compared to the others. Conclusion: Our preliminary data suggest that intratumoral injection of IL-12 confine the release of INF-gamma in the tumor microenvironment, and mitigate the toxicity associated with systemic admission of ad.IL-12. Treatment response is not achieved in every model, suggesting an underlying mechanism which confers resistance to the treatment. We are currently investigating different models in the hope to identify markers of responders versus non responders. Citation Format: Ann C. Anselme, Wei Qian, Anthony Kozielski, Roberto R. Rosato, Jenny C. Chang. Understanding the mechanism underlying resistance to immunotherapy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2261.

Administration of granulocyte-colony stimulating factor (G-CSF) to pigs results in a longer mean survival time after exposure to Streptococcus suis

The use of immunomodulators is a promising alternative to the use of antibiotics for therapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease. Previously we demonstrated a replication-defective adenovirus vector that expresses porcine granulocyte colony-stimulating factor (G-CSF) elicited a sustained neutrophilia, lasting nearly 3 weeks, which may be beneficial to prevent bacterial diseases during times of peak incidence. In a pilot study using the vectored G-CSF with a Caesarian-derived, colostrum-deprived (CDCD) pig model of Streptococcus suis disease, only 1 of 4 pigs given G-CSF developed disease, while 3 of 4 non-treated pigs developed Streptococcal disease. In a subsequent study using a larger number of pigs, although there was no difference in overall survival, there was a longer mean survival time in G-CSF treated pigs. S. suis infection is more severe in CDCD pigs than conventionally raised pigs, consequently results in the field may be superior to the ones reported in this study. Although there were positive effects from the use of G-CSF in this study, further research is needed to determine if improved clinical outcomes could be achieved under field conditions and whether the use of G-CSF in pigs to induce a sustained increase in circulating neutrophil numbers may be useful as an adjunct to antibiotics to diminish the severity of Streptococcal disease, especially during times of stress and pathogen exposure such as post-weaning.

Safety and immunogenicity of a potential checkpoint blockade vaccine for canine melanoma.

Human immunotherapy with checkpoint blockades has achieved significant breakthroughs in recent years. In this study, a checkpoint blockade vaccine for canine melanoma was tested for safety and immunogenicity. Five healthy adult dogs received a mixture of three replication-defective chimpanzee-derived adenoviral vectors, one expressing mouse fibroblast-associated protein (mFAP) and the others expressing canine melanoma-associated antigens Trp-1 or Trp-2 fused into Herpes Simplex-1 glycoprotein D, a checkpoint inhibitor of herpes virus entry mediator (HVEM) pathways. The vaccine mixture was shown to be well tolerated and increased frequencies of canineTrp-1-specific activated CD8+ and CD4+ T cells secreting interferon-(IFN)-γ, tumor necrosis factor (TNF)-α, or interleukin (IL)-2 alone or in combinations in four and five out of five dogs, respectively. To avoid excessive bleeds, responses to cTrp-2 were not analyzed. All dogs responded with increased frequencies of mFAP-specific activated CD8+ and CD4+ T cells. The results of this safety/immunogenicity trial invite further testing of this checkpoint blockade vaccine combination in dogs with melanoma.Electronic supplementary materialThe online version of this article (10.1007/s00262-018-2201-5) contains supplementary material, which is available to authorized users.

A Replicating Single-Cycle Adenovirus Vaccine Against Ebola Virus.

Recent West African Ebola virus (EBOV) epidemics have led to testing different anti-EBOV vaccines, including a replication-defective adenovirus (RD-Ad) vector (ChAd3-EBOV) and an infectious, replication-competent recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (rVSV-EBOV; also known as rVSV-ZEBOV). While RD-Ads elicit protection, when scaled up to human trials, the level of protection may be much lower than that of vaccines containing viruses that can replicate. Although a replication-competent Ad (RC-Ad) vaccine might generate a level of protection approximating that of rVSV, this infectious vector would also risk causing adenovirus disease. We recently described a "single-cycle" adenovirus (SC-Ad) vector that amplifies antigen genes like RC-Ad, but that avoids the risk of adenovirus infection. Here we have tested an SC-Ad6 vector expressing the glycoprotein (GP) from a 2014 EBOV strain in mice, hamsters, and rhesus macaques. We show that SC-Ad6-EBOV GP induces a high level of serum antibodies in all species and mediates significant protection against pseudo-challenge with rVSV-EBOV expressing luciferase in mice and hamsters. These data suggest that SC-Ad6-EBOV GP may be useful during future EBOV outbreaks.

Prime-boost vaccination with recombinant protein and adenovirus-vector expressing Plasmodium vivax circumsporozoite protein (CSP) partially protects mice against Pb/Pv sporozoite challenge

Vaccine development against Plasmodium vivax malaria lags behind that for Plasmodium falciparum. To narrow this gap, we administered recombinant antigens based on P. vivax circumsporozoite protein (CSP) to mice. We expressed in Pichia pastoris two chimeric proteins by merging the three central repeat regions of different CSP alleles (VK210, VK247, and P. vivax-like). The first construct (yPvCSP-AllFL) contained the fused repeat regions flanked by N- and C-terminal regions. The second construct (yPvCSP-AllCT) contained the fused repeat regions and the C-terminal domain, plus RI region. Mice were vaccinated with three doses of yPvCSP in adjuvants Poly (I:C) or Montanide ISA720. We also used replication-defective adenovirus vectors expressing CSP of human serotype 5 (AdHu5) and chimpanzee serotype 68 (AdC68) for priming mice which were subsequently boosted twice with yPvCSP proteins in Poly (I:C) adjuvant. Regardless of the regime used, immunized mice generated high IgG titres specific to all CSP alleles. After challenge with P. berghei ANKA transgenic parasites expressing Pb/PvVK210 or Pb/PvVK247 sporozoites, significant time delays for parasitemia were observed in all vaccinated mice. These vaccine formulations should be clinically tried for their potential as protective universal vaccine against P. vivax malaria.

Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine

Type I interferons, such as interferon alpha (IFN-α), contribute to innate antiviral immunity by promoting production of antiviral mediators and are also involved in promoting an adaptive immune response. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating and costly viruses to the swine industry world-wide and has been shown to induce a meager IFN-α response. Previously we administered porcine IFN-α using a replication-defective adenovirus vector (Ad5-IFN-α) at the time of challenge with virulent PRRSV and demonstrated an increase in the number of virus-specific IFNγ secreting cells, indicating that the presence of IFN-α at the time of infection can alter the adaptive immune responses to PRRSV. In the current experiment, we explored the use of IFN-α as an adjuvant administered with live-attenuated PRRSV vaccine as a method to enhance immune response to the vaccine. Unlike the previous studies with fully virulent virus, one injection of the Ad5-IFN-α abolished replication of the vaccine virus and as a result there was no detectible adaptive immune response. Although IFN-α did not have the desired adjuvant effect, the results further highlight the use of IFN-α as a treatment for PRRSV infection.

The immunoprotection of transplant mice allograft by suppressor of cytokine signaling 1 gene-modified dendritic cells induces T cell anergy

Objective To study the regulation of high expression of suppressor of cytokine signaling 1 (SOCS1) molecule in dendritic cells (DC), and to verify the effect of DC-SOCS1 on T cell function and allografts immune protective effects in vitro and in vivo. Methods DC were modified with high affinity replication-defective adenoviral vector Ad5F35 over-expressing SOCS1. The phenotype of transduced mature DC and the function were analyzed, and the mechanism of inducing donor-specific CD4+ T cells hyporesponsiveness was explored. The mouse allogenic islet transplantation model was established, and 24 h prior to transplantation each recipient mouse was given DC cells, DC-SOCS1 cells or equal amount of phosphate buffer (PBS), named DC group, DC-SOCS1 group and control group respectively. The survival of islet grafts of transplanted recipients was observed, and on the day 7 and the rejection day post transplantation, the islet graft glucose tolerance, pathological changes, the percentage of T cell subsets in the spleen and draining lymph nodes as well as related cytokines were observed. The possible associated mechanisms of SOCS1 gene-modified mature DC mitigating immune rejection and inducing transplant tolerance in vivo were explored. Results Genetic modified Ad5F35 dramatically increased the transduction efficiency of DC compared to regular Ad. The experimental data demonstrated there was a tendency that DC overexpressing SOCS1 can induce DC low down the express of MHC and costimulatory molecules, potentiate the ability of DC-SOCS1 to induce donor specific T cell anergy in mixed lymphocytes reaction (MLR) assay. DC-SOCS1 significantly prolonged the survival time of the islet allograft with a median survival time of (37.00±2.19) days, which is significantly longer than control [(20.00±0.55) d, P=0.008] and DC group [(10.00±0.45) d, P=0.001]. Flow cytometric analysis revealed that the DC-SOCS1 dramatically reduced the population of Th1 and Tc1 [(9.92±1.57)%, P=0.027], CD4 [(17.77±2.80)%, P=0.007] and CD8 [(14.92±2.18)%, P=0.004] in spleen lymphocytes compared to control group and DC group on day 7, and a reduction of CD4 [(26.00±2.02)%, P=0.034] was observed in draining lymph nodes compared to DC group. Conclusion SOCS1 genetic modification efficiently confers mDC with low express of MHC and costimulatory molecules, and allows them to induce T cell hyporesponsiveness in vitro. The DC-SOCS1 pretreated recipients exhibit immune tolerance and prolong islet allograft survival in vivo. Key words: Dendritic cells; Gene transduction; Suppressor of cytokine signaling 1; Islet transplantation; Immune tolerance

Transgene Expression and Host Cell Responses to Replication-Defective, Single-Cycle, and Replication-Competent Adenovirus Vectors.

Most adenovirus (Ad) vectors are E1 gene deleted replication defective (RD-Ad) vectors that deliver one transgene to the cell and all expression is based on that one gene. In contrast, E1-intact replication-competent Ad (RC-Ad) vectors replicate their DNA and their transgenes up to 10,000-fold, amplifying transgene expression markedly higher than RD-Ad vectors. While RC-Ad are more potent, they run the real risk of causing adenovirus infections in vector recipients and those that administer them. To gain the benefits of transgene amplification, but avoid the risk of Ad infections, we developed “single cycle” Ad (SC-Ad) vectors. SC-Ads amplify transgene expression and generated markedly stronger and more persistent immune responses than RD-Ad as expected. However, they also unexpectedly generated stronger immune responses than RC-Ad vectors. To explore the basis of this potency here, we compared gene expression and the cellular responses to infection to these vectors in vitro and in vivo. In vitro, in primary human lung epithelial cells, SC- and RC-Ad amplified their genomes more than 400-fold relative to RD-Ad with higher replication by SC-Ad. This replication translated into higher green fluorescent protein (GFP) expression for 48 h by SC- and RC-Ad than by RD-Ad. In vitro, in the absence of an immune system, RD-Ad expression became higher by 72 h coincident with cell death mediated by SC- and RC-Ad and release of transgene product from the dying cells. When the vectors were compared in human THP-1 Lucia- interferon-stimulated gene (ISG) cells, which are a human monocyte cell line that have been modified to quantify ISG activity, RC-Ad6 provoked significantly stronger ISG responses than RD- or SC-Ad. In mice, intravenous or intranasal injection produced up to 100-fold genome replication. Under these in vivo conditions in the presence of the immune system, luciferase expression by RC and SC-Ad was markedly higher than that by RD-Ad. In immunodeficient mice, SC-Ad drove stronger luciferase expression than RC- or RD-Ad. These data demonstrate better transgene expression by SC- and RC-Ad in vitro and in vivo than RD-Ad. This higher expression by the replicating vectors results in a peak of expression within 1 to 2 days followed by cell death of infected cells and release of transgene products. While SC- and RC-Ad expression were similar in mice and in Syrian hamsters, RC-Ad provoked much stronger ISG induction which may explain in part SC-Ad′s ability to generate stronger and more persistent immune responses than RC-Ad in Ad permissive hamsters.