Edward Jenner and his contemporaries believed that his variolae vaccinae originated in horses and molecular analyses show that modern vaccinia virus (VACV) strains share common ancestry with horsepox ...virus (HPXV). Given concerns relating to the toxicity of modern VACV vaccines, we asked whether an HPXV-based vaccine might provide a superior alternative. Since HPXV may be extinct and the only specimen of HPXV that has been identified is unavailable for investigation, we explored whether HPXV could be obtained by large-scale gene synthesis. Ten large (10-30 kb) fragments of DNA were synthesized based on the HPXV sequence along with two 157 nt VACV terminal sequences, and were recombined into a live synthetic chimeric HPXV (scHPXV) in cells infected with Shope fibroma virus (SFV). Sequencing of the 212 kbp scHPXV confirmed it encoded a faithful copy of the input DNA. We believe this is the first complete synthesis of a poxvirus using synthetic biology approaches. This scHPXV produced smaller plaques, produced less extracellular virus and exhibited less virulence in mice than VACV, but still provided vaccine protection against a lethal VACV challenge. Collectively, these findings support further development of scHPXV as a novel replication-proficient smallpox vaccine.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
TNX-1800 is a preclinical stage synthetic-derived live attenuated chimeric horsepox virus vaccine engineered to express the SARS-CoV-2 spike (S) gene. The objectives of this study were to assess the ...safety, tolerability, and immunogenicity of TNX-1800 administration in Syrian golden hamsters and New Zealand white rabbits. Animals were vaccinated at three doses via percutaneous inoculation. The data showed that the single percutaneous administration of three TNX-1800 vaccine dose levels was well tolerated in both hamsters and rabbits. At all dose levels, rabbits were more decerning regarding vaccine site reaction than hamsters. Lastly, no TNX-1800 genomes could be detected at the site of vaccination. Post-vaccination, all animals had anti-SARS-CoV-2 spike protein IgG specific antibody responses. These data demonstrate that TNX-1800 infection was limited, asymptomatic, and cleared by the end of this study, and a single dose was able to generate immune responses.
BackgroundPolymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) is pathologically activated immature neutrophil that exerts immunosuppressive functions. PMN-MDSC is short-lived and constantly ...replenished through bone marrow (BM) myelopoiesis. Within the BM, histidine decarboxylase (HDC) expressing immature neutrophil constitute a histaminergic niche that enforces hematopoietic stem cell quiescence and inhibits pathological myelopoiesis.1 Trefoil factor family 2 (TFF2), a partial agonist for CXCR4, inhibits CXCL12-dependent chemotaxis.2 Here we engineered a stabilized TFF2 peptide linked to mouse serum albumin (TFF2-MSA), and investigated its effect on PMN-MDSC and combination with PD1 inhibitor in advanced gastric cancer mouse models.MethodsA syngeneic gastric cancer cell line ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) was grafted subcutaneously into HDC-GFP transgenic mice to trace HDC-GFP+MDSCs. Once tumors reach 200 mm3, mice received TFF2-MSA or/and anti-PD-1 antibody. The CXCR4 antagonist AMD3100 served as comparison to TFF2-MSA. We further established a spontaneous lung metastasis model in which mice bearing ACKP tumors received surgical tumor resection and subsequent TFF2-MSA/anti-PD-1 treatments. In another orthotopic model, ACKP-luciferase cells were implanted to stomach submucosa, and the treatments started after 1 week. At 1 month, flow cytometry and single cell RNA sequencing (scRNA-seq) were performed to study treatment induced immune profile changes.ResultsEither TFF2-MSA or anti-PD-1 monotherapy showed little inhibition of subcutaneous ACKP tumor (15% and 25%, N=10, p>0.05), but their combination dramatically suppressed ACKP growth by 78% in a synergistic manner (p<0.0001). In the lung metastasis model, the combination completely abrogated metastasis in 80% mice (N=10, p<0.0001) and prolonged mice survival (p<0.0001), in contrast to minimal effect of either monotherapy (p>0.05). In the orthotopic model, although either monotherapy only slightly delayed tumor growth, their combination successfully eradicated tumor in 80% mice (N=5, p<0.001). Mechanistically, TFF2-MSA systematically reduced HDC+ PMN-MDSCs in the TME, spleen and blood, and its combination with anti-PD-1 profoundly increased polyfunctional Tim-3-Lag-3-CD8+ T cells in the TME and TCF1+ T cells in the TDLN. Inside BM, TFF2-MSA decreased PMN-MDSC generation from its precursors (MPP3/CMP/GMP/GP) to a level similar to tumor-free mice, and restored hematopoietic stem cell quiescence via histamine feedback loop. Importantly, flow cytometry, scRNA-seq and functional analysis suggested TFF2-MSA treated PMN-MDSCs display a more mature profile with less immunosuppressive property. In contrast, AMD3100 plus anti-PD-1 failed to decrease tumor growth and TME MDSCs possibly due to destruction of BM hematopoietic homeostasis.ConclusionsTFF2-MSA systematically reduced PMN-MDSC, and its immunosuppression and generation in BM, thereby sensitizing advanced gastric cancer to PD-1 inhibitors.ReferencesChen X, Deng H, Churchill MJ, Luchsinger LL, Du X, Chu TH, Friedman RA, Middelhoff M, Ding H, Tailor YH, Wang ALE, Liu H, Niu Z, Wang H, Jiang Z, Renders S, Ho SH, Shah SV, Tishchenko P, Chang W, Swayne TC, Munteanu L, Califano A, Takahashi R, Nagar KK, Renz BW, Worthley DL, Westphalen CB, Hayakawa Y, Asfaha S, Borot F, Lin CS, Snoeck HW, Mukherjee S, Wang TC. Bone Marrow Myeloid Cells Regulate Myeloid-Biased Hematopoietic Stem Cells via a Histamine-Dependent Feedback Loop. Cell Stem Cell. 2017 Dec 7;21(6):747–760.e7.Dubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol Chem. 2009 Feb 6;284(6):3650–62.Ethics ApprovalFor experiments in mouse models, all procedures for mice were approved by the Institutional Animal Care and Use Committee of Columbia University in accordance with institutional and NIH guidelines (protocol number AC-AABV0664).
BackgroundMyeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are potential therapeutic targets in immune checkpoint cancer therapy, particularly for cancers that are unresponsive ...to anti-PD-1 therapy. It has previously been demonstrated that trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can partially suppress MDSC expansion and partially activate tumor immunity through agonism of the CXCR4 receptor.1–3 We investigated whether a novel fusion protein, murine TFF2-murine serum albumin (mTFF2-MSA), designated mTNX-1700, has single agent activity and can improve on the therapeutic effects of anti-PD-1 in the Pan02 syngeneic mouse model of advanced pancreatic cancer.MethodsA syngeneic pancreatic mouse model was developed using the Pan02 pancreatic cell line grafted subcutaneously into C57BL/6 mice. We generated a recombinant fusion protein, designated mTFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing the frequency of dosing. Mice subsequently received mTFF2-MSA, anti-PD-1 antibody (clone RMP1–14) or combination of mTFF2-MSA and anti-PD-1, and tumor volume was measured. At the endpoint, flow cytometry was performed on the tumor, axillary lymph node, blood, and bone marrow, to examine treatment-induced effects on cellular immune profiles.ResultsIn the Pan02 model, on Day 19 of treatment, tumor growth was suppressed (TGI) by mTFF2-MSA alone, anti-PD-1 alone, and by the combination mTFF2-MSA and anti-PD-1 by 23%, 28% and 36% TGI, respectively. In the blood, as measured by flow cytometry, there was a significant increase in total macrophages and M2 macrophages between the control and all treatment groups, and a decrease in monocytes and neutrophils in the groups dosed with anti-PD-1. In the axillary lymph node, there was a significant decrease in VISTA+ cells in both the CD4+ and CD8+ T-cells, and an increase in T regulatory cells in all treatment groups as compared to the control. In addition, an increase in PD-1+ and Lag3+ in both the CD4+ and CD8+ T-cells in the anti-PD-1 and combination treated groups was observed.ConclusionsmTFF2-MSA exhibits single agent activity and is additive to anti-PD-1 antibody checkpoint inhibition in treating the Pan02 syngeneic mouse model of advanced pancreatic cancer.ReferencesDubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol Chem. 2009;284:3650–3662.Dubeykovskaya Z, Si Y, Chen X, Worthley DL, Renz BW, Urbanska AM, Hayakawa Y, Xu T, Westphalen CB, Dubeykovskiy A, Chen D, Friedman RA, Asfaha S, Nagar K, Tailor Y, Muthupalani S, Fox JG, Kitajewski J, Wang TC. Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer. Nat Commun. 2016;7:1–11.Dubeykovskaya Z, Duddempudi PK, Deng H, Valenti G, Cuti, KL, Nagar K, Tailor Y, Guha C, Kitajewski J, Wang TC. Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer. Cancer Gene Ther. 2019;26:48–57.
The ongoing global Monkeypox outbreak that started in the spring of 2022 has reinforced the importance of protecting the population using live virus vaccines based on the vaccinia virus (VACV). ...Smallpox also remains a biothreat and although some U.S. military personnel are immunized with VACV, safety concerns limit its use in other vulnerable groups. Consequently, there is a need for an effective and safer, single dose, live replicating vaccine against both viruses. One potential approach is to use the horsepox virus (HPXV) as a vaccine. Contemporary VACV shares a common ancestor with HPXV, which from the time of Edward Jenner and through the 19th century, was extensively used to vaccinate against smallpox. However, it is unknown if early HPXV-based vaccines exhibited different safety and efficacy profiles compared to modern VACV. A deeper understanding of HPXV as a vaccine platform may allow the construction of safer and more effective vaccines against the poxvirus family. In a proof-of-concept study, we vaccinated cynomolgus macaques with TNX-801, a recombinant chimeric horsepox virus (rcHPXV), and showed that the vaccine elicited protective immune responses against a lethal challenge with monkeypox virus (MPXV), strain Zaire. The vaccine was well tolerated and protected animals from the development of lesions and severe disease. These encouraging data support the further development of TNX-801.
TNX-1800 is a synthetically derived live recombinant chimeric horsepox virus (rcHPXV) vaccine candidate expressing Wuhan SARS-CoV-2 spike (S) protein. The primary objective of this study was to ...evaluate the immunogenicity and efficacy of TNX-1800 in two nonhuman primate species challenged with USA-WA1/2020 SARS-CoV-2. TNX-1800 vaccination was well tolerated with no serious adverse events or significant changes in clinical parameters. A single dose of TNX-1800 generated humoral responses in African Green Monkeys and Cynomolgus Macaques, as measured by the total binding of anti-SARS-CoV-2 S IgG and neutralizing antibody titers against the USA-WA1/2020 strain. In addition, a single dose of TNX-1800 induced an interferon-gamma (IFN-γ)-mediated T-cell response in Cynomolgus Macaques. Following challenge with SARS-CoV-2, African Green and Cynomolgus Macaques exhibited rapid clearance of virus in the upper and lower respiratory tract. Future studies will assess the efficacy of TNX-1800 against newly emerging variants and demonstrate its safety in humans.
BackgroundMyeloid-derived suppressor cells (MDSCs) in the tumor microenvironment are potential therapeutic targets in immune checkpoint cancer therapy, particularly for cancers that are unresponsive ...to anti-PD-1 therapy. It has previously been demonstrated that trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, can partially suppress MDSC expansion and activate tumor immunity through agonism of the CXCR4 receptor.1–3 We investigated whether a novel fusion protein, murine TFF2-murine serum albumin (mTFF2-MSA), has single agent activity and can improve on the therapeutic effects of anti-PD-1 in CT26.wt subcutaneous and CT26-Luciferase (CT26-Luc) orthotopic syngeneic mouse models of advanced colorectal cancer (CRC).MethodsTwo syngeneic colon carcinoma mouse models were developed using the CT26.wt and CT26-Luc CRC cell lines grafted subcutaneously and orthotopically, respectively, into BALB/C mice. We generated a recombinant fusion protein, designated mTFF2-MSA, which contains murine TFF2 fused to murine serum albumin (MSA), for the purpose of increasing half-life and reducing the frequency of dosing. Mice subsequently received mTFF2-MSA, anti-PD-1 antibody (clone 29F.1A12 for subcutaneous study; clone RMP-1–14 for orthotopic study) or combination of mTFF2-MSA and anti-PD-1. Tumor volume, and survival were measured. At the endpoint, flow cytometry was performed on the blood, bone marrow, tumor, and lymph nodes, to examine treatment-induced effects on cellular immune profiles.ResultsIn the CT26.wt model, tumor growth was suppressed by mTFF2-MSA, anti-PD-1 and by the combination of mTFF2-MSA/anti-PD-1 by 16%, 40% and 60%, respectively. Survival in the CT26.wt model on Day 30 treated with vehicle, mTFF2-MSA, anti-PD1 and the combination of mTFF2-MSA and anti-PD-1 was 0%, 40%, 60% and 60%, respectively. In the CT26-Luc model, mTFF2-MSA, anti-PD-1, and the combination of mTFF2-MSA and anti-PD-1 suppressed tumor growth by 42%, 94%, and 94%, respectively. In the CT26-Luc model, neutrophils were significantly reduced in the blood in all treatment groups by flow cytometry. In the bone marrow, a significant reduction in total macrophages, M2 macrophages, and neutrophils was also observed but only in the group treated with anti-PD-1/mTFF2-MSA. In the axillary lymph node, there was a significant reduction in TOX+ cells in both CD4+ and CD8+ T-cells in all treatment groups. In the tumor, there was a significant reduction in total macrophages and M2 macrophages in all treatment groups, while NK cells were also increased, but only in the combination anti-PD-1/mTFF2-MSA treated group.ConclusionsmTFF2-MSA has single agent activity and is additive to anti-PD-1 antibody checkpoint inhibition in treating two syngeneic (subcutaneous and orthotopic) mouse models of advanced colorectal cancer.ReferencesDubeykovskaya Z, Dubeykovskiy A, Solal-Cohen J, Wang TC. Secreted trefoil factor 2 activates the CXCR4 receptor in epithelial and lymphocytic cancer cell lines. J Biol Chem. 2009;284:3650–3662.Dubeykovskaya Z, Si Y, Chen X, Worthley DL, Renz BW, Urbanska AM, Hayakawa Y, Xu T, Westphalen CB, Dubeykovskiy A, Chen D, Friedman RA, Asfaha S, Nagar K, Tailor Y, Muthupalani S, Fox JG, Kitajewski J, Wang TC. Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer. Nat Commun. 2016;7:1–11.Dubeykovskaya Z, Duddempudi PK, Deng H, Valenti G, Cuti, KL, Nagar K, Tailor Y, Guha C, Kitajewski J, Wang TC. Therapeutic potential of adenovirus-mediated TFF2-CTP-Flag peptide for treatment of colorectal cancer. Cancer Gene Ther. 2019;26:48–57.
CD40 is a receptor on the surface of B lymphocytes, the activation of which leads to B cell survival, growth, and differentiation. A yeast two-hybrid screen identified a gene, CRAF1, encoding a ...protein that interacts directly with the CD40 cytoplasmic tail through a region of similarity to the tumor necrosis factor-α (TNF-α) receptor-associated factors. Overexpression of a truncated CRAF1 gene inhibited CD40-mediated up-regulation of CD23. A region of CRAF1 was similar to the TNF-α receptor-associated factors TRAF1 and TRAF2 and so defined a shared TRAF-C domain that was necessary and sufficient for CD40 binding and homodimerization. The CRAF1 sequence also predicted a long amphipathic helix, a pattern of five zinc fingers, and a zinc ring finger. It is likely that other members of the TNF receptor superfamily use CRAF-related proteins in their signal transduction processes.
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Background: Recent studies revealed chemotherapy increases anti-PD1 response of gastric cancer (GC) by reducing myeloid-derived suppressor cells (MDSCs). However, a more specific MDSC-targeted ...treatment is needed to further improve anti-PD1 efficacy in advanced GC. Trefoil factor family 2 (TFF2), a partial agonist of CXCR4 and a secreted anti-inflammatory peptide, can suppress MDSC expansion. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 in syngeneic GC mouse models. Methods: Murine serum albumin (MSA) was fused to murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice then received either TFF2-MSA or anti-PD-1 antibody or both when tumors reached 150 mm
3
. At the endpoint, flow cytometry analysis was performed to examine immune profiles. In parallel, a group of the s.c. tumor bearing mice were followed up for survival differences in response to the treatments. In the orthotopic model, ACKP-luc cells were implanted to stomach submucosa, and recipient mice received the same treatments started after 1 week. In the metastasis model, subcutaneous flank tumors were resected in ACKP-grafted mice once tumors reached 500 mm
3
to permit spontaneous lung metastasis, and mice then received the same treatments. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p>0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p<0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) in a synergistic manner. The combination therapy efficiently reduced tumor HDC-GFP
+
MDSCs by 67%, and profoundly increased tumor-infiltrating cytotoxic CD8
+
T cells by 18 fold. The myeloid/lymphoid progenitor and output ratio in the bone marrow were decreased by the combination, to a similar level of tumor-free mice. While standard chemotherapy (5-fluorouracil and oxaliplatin) showed no efficacy, adding chemotherapy to the combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, in the orthotopic model, the TFF2-MSA/PD-1 antibody combination eradicated GC in 80% mice without recurrence compared to 0% with either monotherapy. Finally, the combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p<0.0001), compared to minimal inhibition with either monotherapy (p>0.05). Conclusions: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of gastric cancer.
•Manufacturing of the smallpox vaccine at the end of the 19th century was the beginning of the vaccine industry in the United States.•There is not information regarding the stocks or seed viruses ...used to manufacture those early vaccines.•Historical information, will allow a better understanding of the origin and evolution of the vaccine used to eradicate smallpox.
For the first 80–90 years after Jenner’s discovery of vaccination in 1796, the main strategy used to disseminate and maintain the smallpox vaccine was arm-to-arm vaccination, also known as Jennerian or humanized vaccination. A major advance occurred after 1860 with the development of what was known as “animal vaccine”, which referred to growing vaccine material from serial propagation in calves before use in humans. The use of “animal vaccine” had several advantages over arm-to-arm vaccination: it would not transmit syphilis or other human diseases, it ensured a supply of vaccine even in the absence of the spontaneous occurrence of cases of cowpox or horsepox, and it allowed the production of large amounts of vaccine. The “animal vaccine” concept was introduced in the United States in 1870 by Henry Austin Martin. Very rapidly a number of “vaccine farms” were established in the U.S. and produced large quantities of “animal vaccine”. These “vaccine farms” were mostly established by medical doctors who saw an opportunity to respond to an increasing demand of smallpox vaccine from individuals and from health authorities, and to make a profit. The “vaccine farms” evolved from producing only smallpox “animal vaccine” to manufacturing several other biologics, including diphtheria- and other antitoxins. Two major incidents of tetanus contamination happened in 1901, which led to the promulgation of the Biologics Control Act of 1902. The US Secretary of the Treasury issued licenses to produce and sell biologicals, mainly vaccines and antitoxins. Through several mergers and acquisitions, the initial biologics licensees eventually evolved into some of the current major American industrial vaccine companies. An important aspect that was never clarified was the source of the vaccine stocks used to manufacture the smallpox “animal vaccines”. Most likely, different smallpox vaccine stocks were repeatedly introduced from Europe, resulting in polyclonal vaccines that are now recognized as “variants” more appropriately than “strains”. Further, clonal analysis of modern “animal vaccines” indicate that they are probably derived from complex recombinational events between different strains of vaccinia and horsepox. Modern sequencing technologies are now been used by us to study old smallpox vaccine specimens in an effort to better understand the origin and evolution of the vaccines that were used to eradicate the smallpox.
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