Jérémy Béguin
email: jeremy.beguin@vet-alfort.fr

Virginie Nourtier

Murielle Gantzer

Sandrine Cochin

Johann Foloppe

Jean-Marc Balloul

Eve Laloy

Dominique Tierny

Bernard Klonjkowski

Eric Quemeneur

Christelle Maurey

Philippe Erbs

Safety studies and viral shedding of intramuscular administration of oncolytic vaccinia virus TG6002 in healthy beagle dogs

Keywords: Oncolytic virotherapy, Vaccinia virus, TG6002, FCU1, Cancer, Safety, Canine, Translational research

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background

Cancer is a leading cause of mortality throughout the world for both humans and dogs. The incidence of cancer ranges from 1 to 2% in the canine population and accounts for about half of the deaths in dogs older than 10 years [START_REF] Merlo | Cancer incidence in pet dogs: findings of the animal tumor registry of Genoa, Italy[END_REF][START_REF] Kelsey | Epidemiologic studies of risk factors for Cancer in pet dogs[END_REF]. Despite progress in the diagnosis and treatment of advanced canine cancers, complete longlasting remissions are still infrequent. Therefore, new therapeutic approaches are needed.

The use of oncolytic viruses to treat cancer is an emerging field in cancer research and therapy. Oncolytic virotherapy is a promising therapeutic option based on the ability of engineered viral vectors to selectively replicate in cancer cells leading to their lysis and to deliver genes encoding therapeutic proteins into cancer cells [START_REF] Miest | New viruses for cancer therapy: meeting clinical needs[END_REF]. Many viruses have been studied in preclinical and clinical studies, including adenovirus, vaccinia virus, herpes virus, parvovirus, picornavirus and reovirus in both human and veterinary medicine [START_REF] Harrington | Optimizing oncolytic virotherapy in cancer treatment[END_REF][START_REF] Patil | Oncolytic virotherapy in veterinary medicine: current status and future prospects for canine patients[END_REF][START_REF] Sánchez | Oncolytic viruses for canine Cancer treatment[END_REF]. Vaccinia virus (VACV) is a large, double stranded DNA virus, and member of the poxviridae family. Its ability to efficiently replicate, lyse host cells, and evade immune responses make VACV an attractive candidate for human and veterinary oncolytic virotherapy [START_REF] Haddad | Genetically engineered Vaccinia viruses as agents for Cancer treatment, imaging, and transgene delivery[END_REF]. VACV has been shown to replicate and lyse tumor cells within 72 h of infection [START_REF] Kirn | Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer[END_REF]. Furthermore, VACV replicates in cell cytoplasm, preventing the integration of viral DNA into host chromosomes [START_REF] Shen | Fighting cancer with vaccinia virus: teaching new tricks to an old dog[END_REF]. VACV has also been shown to exhibit broad tumor tropism [START_REF] Kirn | Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer[END_REF]. In human medicine, Pexa-Vec (pexastimogene devacirepvec, JX-594, SillaJen Biotherapeutics, Seoul, South Korea) is the most advanced VACV oncolytic product. It is derived from a VACV strain engineered to express GM-CSF and has successfully entered Phase III clinical trials [START_REF] Breitbach | Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans[END_REF]. An oncolytic VACV, designated as TG6002, has been developed with deletion of the thymidine kinase (TK) and the ribonucleotide reductase (RR) loci in its genome resulting in attenuated virulence and enhanced tumor-specific targeting [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. To enhance therapeutic efficacy, the chimeric gene FCU1 was inserted in the TG6002 genome. FCU1 encodes a bifunctional fusion protein combining cytosine deaminase and uracil phosphoribosyltransferase activities. FCU1 converts the non-toxic prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic compound 5-fluorouracil (5-FU), and further into 5-fluorouracil-monophosphate, which inhibits DNA and protein synthesis [START_REF] Erbs | In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene[END_REF]. In murine xenograft mice treated by TG6002 followed by per os 5-FC administration, high levels of 5-FU were detected in tumors [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. In this model, TG6002 in combination with 5-FC has significant antitumor efficacy against a large range of human tumors [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. Another study has also showed relevant oncolytic features of TG6002 as an oncolytic therapy on canine cancer cell lines, mouse xenografts and canine mammary tumor explants [START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF]. Intratumoral injections of TG6002 in canine mammary tumor cells grafted onto mice lead to a significant decrease in tumor size [START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF]. Administration of 5-FC to those mice significantly improved the antitumor activity of TG6002. Finally, canine mammary tumor explants cultured with TG6002 and 5-FC, allowed the assessment of tumor necrosis, and conversion of 5-FC into 5-FU [START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF].

Evaluation of TG6002 in cancer-bearing dogs could be beneficial for both humans and dogs. As spontaneous canine tumors are relevant models for translational research in oncology, they can provide useful preclinical data for human medicine [START_REF] Rowell | Dog models of naturally occurring cancer[END_REF][START_REF] Regan | Clinical, pathological, and ethical considerations for the conduct of clinical trials in dogs with naturally occurring Cancer: a comparative approach to accelerate translational drug development[END_REF][START_REF] Paoloni | Translation of new cancer treatments from pet dogs to humans[END_REF]. Moreover, new therapeutic strategies are needed to improve therapeutic options in veterinary medicine. Biosafety is a major issue with oncolytic viruses for both patients and the environment. VACV infection is generally associated with cutaneous pock lesions which participate in the shedding of the virus [START_REF] Buller | Poxvirus pathogenesis[END_REF]. Clinical trials using attenuated oncolytic VACV in patients diagnosed with cancer have reported the development of mucocutaneous pustules after treatment [START_REF] Breitbach | Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans[END_REF][START_REF] Park | Use of a targeted oncolytic poxvirus, JX-594, in patients with refractory primary or metastatic liver cancer: a phase I trial[END_REF][START_REF] Heo | Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer[END_REF][START_REF] Downs-Canner | Phase 1 study of intravenous Oncolytic poxvirus (vvDD) in patients with advanced solid cancers[END_REF][START_REF] Mell | Phase I trial of intravenous Oncolytic Vaccinia virus (GL-ONC1) with Cisplatin and radiotherapy in patients with Locoregionally advanced head and neck carcinoma[END_REF][START_REF] Kung | Isolation of vaccinia JX594 from pustules following therapy for hepatocellular carcinoma[END_REF][START_REF] Biondo | POSTER phase I clinical trial of a genetically modified Oncolytic Vaccinia virus GL-ONC1 with green fluorescent protein imaging[END_REF]. Similar lesions have been described in dogs receiving a TK-deleted VACV [START_REF] Appel | Immune response to vaccinia virus and recombinant virus products in dogs[END_REF]. VACV is known to remain detectable in urine and feces for a long time [START_REF] Lederman | Eczema vaccinatum resulting from the transmission of vaccinia virus from a smallpox vaccinee: an investigation of potential fomites in the home environment[END_REF][START_REF] Abrahão | Long-lasting stability of Vaccinia virus strains in murine feces: implications for virus circulation and environmental maintenance[END_REF][START_REF] Essbauer | Long-lasting stability of vaccinia virus (orthopoxvirus) in food and environmental samples[END_REF]. Thus, before the use of TG6002 in pet dogs, evaluations of safety and viral shedding are needed. Considering the promising results obtained by the intratumoral route on xenograft models, intramuscular injections were chosen to mimic this route of administration in healthy dogs.

The present study aims at assessing the tolerability and viral shedding following intramuscular injections of escalating doses of TG6002 in healthy dogs. This study is a prerequisite for a phase 1 trial of intratumoral delivery of TG6002 in pet dogs suffering from incurable cancers.

Results

Single intramuscular injection of TG6002 was well tolerated in four healthy dogs

During the single injection phase, only a decrease in body weight scored as grade 1 was recorded for all dogs (Fig. 1a). The median percentage value of maximal weight loss was 6.05%. The decrease in body weight did not seem related to the viral titer. Only one dog (Dog 1 treated with 1 × 10 6 PFU/kg of TG6002) had a transient increase in temperature (39.4 °C, grade 1) 7 days after TG6002 administration (Fig. 1b). Hyperthermia was not seen in the other dogs. Furthermore, no mucocutaneous or skin lesions at the injection site, nor any other clinical abnormalities were found. In addition, hematological and biochemical analyses performed on day 14 did not reveal toxicity (Fig. 2) [see Additional file 1]. For Dog 2, treated with 5 × 10 6 PFU/kg of TG6002, total proteins were stable but in the lower part of the reference interval at days 0 and 14. For Dog 3, treated with 1 × 10 7 PFU/kg of TG6002, a moderate decrease of total proteins was noticed between day 0 and 14 [see Additional file 1]. As maximal tolerated dose (MTD) was not reached, the highest tested dose of 5 × 10 7 PFU/kg was selected for administration in the second phase of the study.

Repeated intramuscular injections of 5 × 10 7 PFU/kg of TG6002 with oral 5-FC administration were well tolerated in three healthy dogs During the repeated injections phase, only a decrease in body weight scored as grade 1 was observed in two dogs (Fig. 3a). The median percentage value of maximal weight loss was 7.89%. No hyperthermia was observed (Fig. 3b). Neither mucocutaneous nor skin lesions at the injection site, nor other clinical abnormalities were found. For all dogs, an increase in the white blood cell count was noticed at days 9 and 21 (Fig. 4). On day sixteen even with values within the reference interval, a decrease in white blood cell count was noticed in all dogs. (Fig. 4). No significant abnormalities were seen in the biochemical analytes reference intervals for all dogs [see Additional file 2]. Dog 7 showed lethargy (grade 1), anorexia (grade 3), vomiting (grade 2), diarrhea (grade 2), transient melena (grade 2) and decreased body weight (grade 2) 1 day after the third injection. Analysis of serum biochemistry values revealed an increase in blood urea nitrogen (BUN) (12.70 mmol/L; reference interval: 3.30 to 10.00 mmol/L) (grade 2) with normal creatinemia (51.00 μmol/L; reference interval: 36.00 to 106.00 μmol/L) and a slight increase in alkaline phosphatase (158 UI/L; reference interval: 29 to 153 UI/L) [see Additional file 2]. No hematological abnormalities were observed (Fig. 4). Abdominal ultrasound examination was normal. Supportive care consisting of gastrointestinal protectants, a histamine type-2 receptor antagonist and supportive diet was initiated for the digestive disorders. Oral administration of kaolin and pectin (Kaopectate, Zoetis, Malakoff, France) 5 ml twice daily and sucralfate (Ulcar, Sanofi-Aventis, Gentilly, France) 1 g three times a day, with intravenous injections of ranitidine (Azantac, Laboratoire Glaxosmithkline, Marly-le-Roi, France) 1 mg/kg twice daily were dispensed. As melena was seen, an associated risk for bacterial translocation was determined, enrofloxacin (Xeden, CEVA santé Animale, Libourne, France) 5 mg/kg once daily per os was administered. Four days later, a complete resolution of clinical signs was seen and treatments were stopped at day 22. A progressive weight recovery was also observed until the thirty-fifth day. To evaluate histopathological abnormalities and viral shedding, euthanasia under general anesthesia was elected for Dog 7. Anesthesia was performed using an initial intravenous administration of 0.2 mg/kg of butorphanol (Torbugesic, Zoetis, Malakoff, France), 3 mg/kg of ketamine (Ketamine 1000, Virbac, Carros, France) and 15 μg/kg of medetomidine (Domitor, Orion Corporation, Espoo, Finland). After the dog was sedated, an intravenous injection of 180 mg/kg of sodium pentobarbital solution (Dolethal, Vetoquinol, Magny Vernois, France) was given. Postmortem examination revealed no relevant pathological changes, especially in the digestive tract, liver and kidneys. Histological analyses only revealed a slight degenerative renal tubulopathy with intra-epithelial pigment, a generalized vacuolar hepatopathy and a slight diffuse necrotic hepatitis. Immunohistochemical analyses of kidney and liver samples using VACV antibody were negative.

Discussion

Oncolytic viruses are gaining ground as an alternative therapy in veterinary oncology. Several studies involving adenovirus, myxoma virus, Sendai virus, reovirus and vesicular stomatitis virus have been conducted in dogs and cats [START_REF] Westberg | Treatment efficacy and immune stimulation by AdCD40L gene therapy of spontaneous canine malignant melanoma[END_REF][START_REF] Von Euler | Efficient adenovector CD40 ligand immunotherapy of canine malignant melanoma[END_REF][START_REF] Siddiqui | A phase I trial of hyperthermia-induced interleukin-12 gene therapy in spontaneously arising feline soft tissue sarcomas[END_REF][START_REF] Jourdier | Local immunotherapy of spontaneous feline fibrosarcomas using recombinant poxviruses expressing interleukin 2 (IL2)[END_REF][START_REF] Macneill | Safety of an Oncolytic Myxoma virus in dogs with soft tissue sarcoma[END_REF][START_REF] Hwang | Oncolytic reovirus therapy: pilot study in dogs with spontaneously occurring tumours[END_REF][START_REF] Ilyinskaya | Oncolytic Sendai virus therapy of canine mast cell tumors (a pilot study)[END_REF][START_REF] Naik | Comparative oncology evaluation of intravenous recombinant Oncolytic vesicular stomatitis virus therapy in spontaneous canine Cancer[END_REF][START_REF] Cejalvo | Remission of spontaneous canine tumors after systemic cellular Viroimmunotherapy[END_REF]. Promising results have been reported by intratumoral or intravenous routes on dogs and cats with cancer. No study has yet evaluated an oncolytic VACV allowing intratumoral production of a chemotherapy drug. Our study describes the dose, clinical toxicities and viral shedding after intramuscular administration of TG6002 with oral 5-FC in seven healthy immune-competent dogs. As TG6002 is engineered from VACV, evaluating both the safety and the shedding profiles of this attenuated virus remains essential. Thus, to provide more reliable information on tolerance, seven healthy dogs with a competent immune system were selected for this study. Due to ethical concerns, safety studies evaluating oncolytic viruses only involve small numbers of healthy animals [START_REF] Autio | Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory beagles[END_REF][START_REF] Autio | Attenuated Semliki Forest virus for cancer treatment in dogs: safety assessment in two laboratory beagles[END_REF][START_REF] Leblanc | Safety studies on intravenous administration of oncolytic recombinant vesicular stomatitis virus in purpose-bred beagle dogs[END_REF][START_REF] Smith | Administration of a Conditionally Replicative Oncolytic Canine Adenovirus in Normal dogs[END_REF].

Considering the low number of dogs in our study, it is important to remain cautious about the absence of major toxicities.

Data collected in this preclinical study on seven dogs indicate that administration of TG6002 was well tolerated in all dogs. MTD was not reached even at the highest tested dose of 5 × 10 7 PFU/kg. Only transient weight loss for all dogs and digestive disorders for one dog out of seven were observed. One transient history of hyperthermia was noticed for one dog out of seven receiving TG6002 at the lowest dosage. During the second part of the study, a decrease in the white blood cell count was observed on day 16 for all dogs. Hematological changes were transient and values remained within the reference intervals. For all dogs (n = 3) receiving three injections of TG6002, increases of neutrophil, lymphocyte and monocyte counts were noticed after the second injection. An inflammatory response secondary to VACV injection was suspected. After the third injection, decreases of neutrophil, lymphocyte, monocyte, eosinophil and basophil counts were observed. Even if a stress response could not be excluded, hematological changes were suspected to be induced by viral injection. Similar variations have been described in laboratory beagles receiving intravenous administrations of an oncolytic VACV encoding CD40 ligand [START_REF] Autio | Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory beagles[END_REF]. Leukopenia has also been reported in human trials with intratumoral oncolytic VACV. In an intratumoral dose escalation clinical trial using a Western Reserve Strain Oncolytic VACV only a grade 1or 2 leukopenia was reported in one human patient out of 16 (6.3%) treated with 1 × 10 9 PFU [START_REF] Zeh | First-in-man study of western reserve strain oncolytic vaccinia virus: safety, systemic spread, and antitumor activity[END_REF]. In pediatric cancer patients, only a grade 3 lymphopenia was reported in one patient out of seven receiving intratumoral injection of Pexa-Vec at 1 × 10 7 PFU/kg [START_REF] Cripe | Phase 1 study of intratumoral Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients[END_REF]. However, as 5-FU can induce bone marrow depression, it would have been interesting to evaluate serum concentrations of 5-FU in our study.

Other adverse events reported in laboratory beagles, receiving intravenous administration of an oncolytic VACV encoding CD40 ligand, included transient grade 1 fever (n = 1/2) and grade 3 seizure (n = 1/2) [START_REF] Autio | Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory beagles[END_REF]. Additionally, only a grade 1 increase in alkaline phosphatase (n = 1/2) and a mild decrease in albumin concentration (grade 1) (n = 1/2) were noticed without hematological or urine abnormalities [START_REF] Autio | Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory beagles[END_REF]. In human trials adverse events secondary to intratumoral or intravenous administration of oncolytic VACV included fever, rigors, abdominal pain, nausea, vomiting, tiredness and headache [START_REF] Heo | Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer[END_REF][START_REF] Downs-Canner | Phase 1 study of intravenous Oncolytic poxvirus (vvDD) in patients with advanced solid cancers[END_REF][START_REF] Cripe | Phase 1 study of intratumoral Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients[END_REF].

Infection with VACV is generally characterized by the development of cutaneous and mucocutaneous pock lesions [START_REF] Buller | Poxvirus pathogenesis[END_REF]. Human cancer patients receiving attenuated oncolytic VACV have been reported to develop these lesions [START_REF] Breitbach | Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans[END_REF][START_REF] Heo | Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer[END_REF][START_REF] Mell | Phase I trial of intravenous Oncolytic Vaccinia virus (GL-ONC1) with Cisplatin and radiotherapy in patients with Locoregionally advanced head and neck carcinoma[END_REF][START_REF] Kung | Isolation of vaccinia JX594 from pustules following therapy for hepatocellular carcinoma[END_REF]. Safety of a TK-deleted VACV (VTK-79) administered by intradermal, subcutaneous or intranasal routes has been reported in laboratory beagles [START_REF] Appel | Immune response to vaccinia virus and recombinant virus products in dogs[END_REF]. Only intradermal injections of 10 7 PFU of VTK-79 have been reported to induce the development of small nodules at the site of inoculation [START_REF] Appel | Immune response to vaccinia virus and recombinant virus products in dogs[END_REF]. However, no pock lesions were observed throughout our study even if dosages were greater than 10 7 PFU. Deletions of both TK and RR genes have been shown to improve the safety profile of VACV, due to its high attenuation in normal tissues, compared to a single deleted oncolytic vector [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. As VACV has a tropism for cutaneous and mucocutaneous tissues, the intramuscular route of administration could also explain the lack of pock lesions.

TG6002 has a tumor-selective viral replication induced by the double TK-RR deletion of the TG6002 genome and does not display pathogenic effects in normal tissue [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. No pock lesions nor any other major clinical abnormalities were observed in our study. The lack of clinical adverse events confirms the safety profile of TG6002. However, the use of healthy dogs in our study could have limited the assessment of adverse events. The dogs in this study were cancer free, but in dogs with tumors the viral load could be higher and allow viral amplification. Thus, further studies of tolerance and viral shedding must be performed in dogs with cancer.

In our study, the transient mild weight loss seen in all dogs could have been induced by virus administration. Weight loss was observed for all dogs receiving TG6002 alone (n = 4) or with 5-FC (n = 3). One dog (Dog 7) developed gastrointestinal signs which are likely related to 5-FC or TG6002. 5-FC is an antifungal agent, mainly used against strains of Cryptococcus and Candida. 5-FC penetrates fungal cells where it is deaminated by cytosine deaminase to 5-FU. It acts as an antimetabolite by competing with uracil, thereby interfering with pyrimidine metabolism and eventually RNA and protein synthesis. It is thought that 5-FU is converted into 5-fluoro-2′-deoxyuridylate which inhibits thymidylate synthesis and ultimately DNA synthesis. In humans, 5-FC is rapidly absorbed with a bioavailability of 76-89% [START_REF] Cutler | Flucytosine kinetics in subjects with normal and impaired renal function[END_REF]. It penetrates well into most body sites and is mostly eliminated by glomerular filtration with a 3 to 4 h half-life [START_REF] Block | Pharmacological studies with 5-fluorocytosine[END_REF][START_REF] Block | Flucytosine and amphotericin B: hemodialysis effects on the plasma concentration and clearance. Studies in man[END_REF]. A dose-dependent bone marrow depression (anemia, leukopenia, thrombocytopenia) and an increase in hepatic enzymes are reported [START_REF] Vermes | Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions[END_REF]. Adverse events reported in dogs also include toxic epidermal necrolysis of the scrotum, nasal planum, lips and eyelids [START_REF] Malik | Suspected drug eruption in seven dogs during administration of flucytosine[END_REF][START_REF] Panciera | Management of cryptococcosis and toxic epidermal necrolysis in a dog[END_REF]. These cutaneous adverse events resolve with discontinuation of 5-FC [START_REF] Malik | Suspected drug eruption in seven dogs during administration of flucytosine[END_REF]. No cutaneous lesions nor hematological disorders induced by 5-FC were observed in our study. In human medicine, blood concentration should not exceed 100 mg/L within 72 h of administration of 5-FC [START_REF] Ashbee | Therapeutic drug monitoring (TDM) of antifungal agents: guidelines from the British Society for Medical Mycology[END_REF]. Thus, it would have been interesting to evaluate serum concentrations of 5-FC and 5-FU in our dogs. Studies suggest that 5-FC can be converted into cytotoxic 5-FU by intestinal microorganisms leading to intestinal disorders [START_REF] Harris | Conversion of 5fluorocytosine to 5-fluorouracil by human intestinal microflora[END_REF][START_REF] Vermes | An in vitro study on the active conversion of flucytosine to fluorouracil by microorganisms in the human intestinal microflora[END_REF]. Due to the potential intestinal conversion of 5-FC into 5-FU, the dosage of 5-FU in the feces and blood would have been interesting. In a phase 1 human trial, grade 1 or 2 nausea (n = 6/16, 37.5%), grade 1 or 2 vomiting (n = 2/12; 12.5%), grade 1 or 2 diarrhea (n = 2/16, 12.5%) and grade 3 gastrointestinal hemorrhage (n = 1/16; 6.3%) were reported after intratumoral injections of oncolytic VACV [START_REF] Zeh | First-in-man study of western reserve strain oncolytic vaccinia virus: safety, systemic spread, and antitumor activity[END_REF]. For Dog 7, only an increase of BUN was noticed on biochemical analysis which was suggestive of dehydration or gastrointestinal bleeding. Post mortem examination did not reveal bowel lesions causing gastrointestinal bleeding. Even if no macroscopic intestinal lesions were observed during the necropsy, qPCR assay as well as histological and immunohistochemical analyses, would have been interesting to identify the causes of the gastrointestinal disorders.

Viral shedding detection is also important in the environmental risk assessment for this novel therapy. Viral shedding has been evaluated in dogs after intradermal or subcutaneous or intranasal administration of the TK-deleted VACV (VTK-79) [START_REF] Appel | Immune response to vaccinia virus and recombinant virus products in dogs[END_REF]. Even if dogs with induced pock lesions were in close contact with sentinel dogs, none of these control animals developed VACV antibodies, which suggested the absence of viral shedding [START_REF] Appel | Immune response to vaccinia virus and recombinant virus products in dogs[END_REF]. In a study with intravenous oncolytic VACV administrations in healthy dogs, the viral load, detected by qPCR, declined quickly in blood samples during the 4 h after infusion and viral DNA was not detected in feces, saliva and urine samples collected at 1, 2, or 4 days after virus administration [START_REF] Autio | Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory beagles[END_REF]. In human clinical trials, viral genome has also been detected in patients' blood after intratumoral administration of oncolytic VACV [START_REF] Heo | Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer[END_REF][START_REF] Hwang | A mechanistic proof-of-concept clinical trial with JX-594, a targeted multimechanistic oncolytic poxvirus, in patients with metastatic melanoma[END_REF].

In the present study, neither infectious virus nor viral genome copies were detectable in blood, urine, saliva and feces. The absence of viral shedding can be explained by the characteristics of the viral vector and the route of administration. Due to the deletion of TK and RR genes, the virus can only replicate in dividing cells [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. Intramuscular administration of viral vector in healthy dogs was not expected to be associated with replication of the vector. As TG6002 was designed for intratumoral injection, this administration route might promote vector replication in tumors. Thus, viral burden would likely be higher in dogs with tumors that allow viral amplification. Viral replication leads to the production of FCU1 protein and the conversion of 5-FC to 5-FU. Expression of exogenous proteins without replication of the virus is considered negligible. Thus, 5-FU production was not expected after intramuscular injections in healthy dogs. In this context, tolerance and viral shedding must be evaluated further on dogs with cancer.

Previous studies evaluating oncolytic potency of TG6002 in cell lines and xenograft models have shown promising results [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF][START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF]. However, murine xenograft models have limitations including an impaired immune system, tumor size and the non-spontaneous origin of the tumor. Highly relevant animal models are needed to assess the efficacy of treatment before human trials. In contrast to rodents, cancers arising in dogs have several commonalities with human cancers. Dogs share the same environment as their owners, their immune system is intact, and cancer progression is spontaneous resulting in similar complexity, clonality, and immune suppression as seen in man [START_REF] Vail | Spontaneously occurring tumors of companion animals as models for human Cancer[END_REF]. In addition, the same cancerassociated genes and histological features have been found in both species for several cancers such as urothelial carcinoma or mammary carcinoma [START_REF] Uva | Comparative expression pathway analysis of human and canine mammary tumors[END_REF][START_REF] Knapp | Naturally-occurring canine transitional cell carcinoma of the urinary bladder a relevant model of human invasive bladder cancer[END_REF]. It is now well recognized that dogs with spontaneous cancer serve as a good model for several human cancers [START_REF] Knapp | Naturally occurring cancer in pet dogs: important models for developing improved cancer therapy for humans[END_REF]. One study has already established the oncolytic potency of TG6002 with 5-FC in canine mammary tumor explants [START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF]. Histological analyses of ex vivo canine mammary adenocarcinoma explants cultured with TG6002 and 5-FC, allowed assessment of tumor necrosis and conversion of 5-FC into 5-FU [START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF]. Considering dogs as a relevant model in oncology, and the promising in vitro results combined with the safety profile of TG6002 observed in the seven dogs in this study, intratumoral injections of 5 × 10 7 pfu/kg of TG6002 can be considered for clinical trial in dogs with spontaneous tumors. Individual dogs will benefit from effective treatment and the results are expected to help other dogs. Furthermore, the findings are expected to help in human oncology treatments.

Conclusion

Intramuscular injections of TG6002 at 5 × 10 7 PFU/kg with concurrent administrations of 5-FC was well tolerated in seven healthy dogs. The evaluation of viral shedding did not reveal TG6002 excretion in the environment. These results support the future evaluation of TG6002 in pet dogs with spontaneous tumors. A veterinary study will also provide critical data for the clinical development of TG6002 as a human cancer therapy. This translational approach fits well with the "One Health -One Medicine" concept and may contribute to the development of new therapies for animal and human cancers.

Methods

Viral vector

TG6002 was derived from the Copenhagen strain of VACV with targeted deletions of the thymidine kinase (J2R) and the large subunit of ribonucleotide reductase (I4L) genes [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. TG6002 expressing the fusion gene FCU1 (ΔI4LΔJ2R/FCU1 VACV) under the control of the p11K7.5 promoter was constructed and as previously described [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF]. TG6002 was produced according to the Good Manufacturing Production on CEF, and virus stock was titrated on CEF by plaque assay.

Laboratory dogs

Seven adult healthy male Beagle dogs (Harlan Laboratories, Gannat, France) were used. The mean weight was 10.4 ± 1.1 kg (mean ± standard deviation). All dogs were acclimatized for 7 days before the start of the experiment and had regular inspections to detect any sanitary, or behavioral events. All dogs were under the care of a licensed veterinarian. The dogs were housed individually in inox-steel bar boxes with a resin soil substrate and a softwood chips litter. The room temperature was 19 °C (+/-2 °C) with a humidity greater than 35%, and the day/night cycle was 12:12 h. Dogs were fed twice daily with a certified commercial canine diet and given potable water ad libitum. Dogs were not euthanized at the end of the study, but instead returned to the facility colony except in the case of grade 3 or grade 4 clinical, hematological or biochemical adverse events [START_REF]Common terminology criteria for adverse events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.1[END_REF].

Study design

This study was conducted in accordance with European legislation and French regulations on the protection of animals used for scientific purposes (Directive 2010/63/EU, 2010; Code rural, 2018; Décret no. 2013-118, 2013) and complied to the recommendations of the "Charte nationale portant sur l'éthique en expérimentation animale" established by the "Comité National de Réflexion Ethique sur l'Expérimentation Animale" (CNREEA-Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation-Ministère de l'Agriculture et de l'Alimentation). The study protocol (n°1431_v2) was approved by the VetAgro Sup Ethical Committee (C2EA No. 18) and the Ministry of National Education, Higher Education and Research. Informed written consent to participate was required from the owners.

The first part of the study consisted of determining the MTD in four dogs, with each dog receiving a single different dose of TG6002 by intramuscular injection. The second part of the study assessed tolerability of several injections of TG6002 at the MTD, identified in the first part of the study, in combination with 5-FC administration. Intramuscular injections were chosen to mimic intratumoral route of administration in healthy dogs. The overall schedule of the study is shown in Fig. 5.

In the first part of the study, four dogs were randomized for single intramuscular injection of TG6002. The doses chosen in this study were similar to the doses administered in a clinical trial using TG6002 in human patients with advanced gastrointestinal tumors (NCT03724071) [START_REF]Study of TG6002 (VV TK-RR-FCU1) in combination with 5-FC in patients with advanced gastrointestinal tumors[END_REF]. Dog 1 received 1 × 10 6 PFU/kg, Dog 2 received 5 × 10 6 PFU/kg, Dog 3 received 1 × 10 7 PFU/kg and Dog 4 received 5 × 10 7 PFU/kg, diluted with NaCl 0.9%. A single intramuscular (quadriceps or lumbar muscle) injection with a maximal volume of 0.5 ml/kg per site was performed in each dog. Multiple site injections were performed if the volume was over 0.5 ml/kg. Injections were performed in a dedicated room of the laboratory animal facility and outside the room housing the dogs. To detect any side effects, injections were performed early in the morning to allow for observations and administrations between dogs were spaced by 7 days.

Dogs were evaluated once-a-day by a physical examination over 14 days, after the injection of TG6002. Complete blood counts were performed using a Procyte Hematology analyzer (IDEXX Laboratory Inc., Westbrook, Maine, United States) before and 14 days after the injection of TG6002. Biochemistry analyses were performed Fig. 5 Study chart and sample collection for safety evaluation of TG6002 in dogs. The first part of the study was conducted using four dogs to determine the maximal tolerated dose of TG6002. The second part of the study included three new dogs that were injected with TG6002 at the maximal dose determined in the first part of the study. The dogs were given three intramuscular injections of TG6002 with one-week intervals between injections. Four days after each injection, 5-FC was orally administered for three days. Samples collections for virus detection, complete blood count and blood chemistry are indicated before and 14 days after the injection of TG6002 using a Catalyst Biochemistry analyzer (IDEXX Laboratory Inc., Westbrook, Maine, United States).

Body fluid samples (blood, saliva, urine and feces) were collected at days 0 (before virus injection), 1 and 2 and analysed to assess viral shedding. The MTD was defined as the highest dose of TG6002 that did not cause major side effects.

Three new dogs (Dog 5, Dog 6, Dog 7) were randomized for evaluating the tolerability to multiple injections combined with 5-FC. Dogs received three intramuscular injections of TG6002 at day 0, 7 and 14 at the defined MTD. Four days after each injection, 5-FC (Toronto Research Chemicals, North York, ON, Canada) was orally administered at a dose of 100 mg/kg twice a day for 3 days. Injections were performed in a dedicated room of the laboratory animal facility and outside the room housing the dogs. To detect any side effects, injections were performed early in the morning to allow for observations and administrations between dogs were spaced by 7 days. Dogs were evaluated once-a-day by physical examination until 2 weeks after the last administration of 5-FC. Complete blood counts were performed at day 0, 9, 16, 21 and 35 using a Procyte Hematology analyzer (IDEXX Laboratory Inc., Westbrook, Maine, United States). Biochemistry analyses were performed at days 0, 16 and 35 using a Catalyst Biochemistry analyzer (IDEXX Laboratory Inc., Westbrook, Maine, United States).

Bodily fluid samples (blood, saliva, urine and feces) were collected at day 0 (before virus injection), 1, 2, 7 (before second virus injection), 8, 9, 14 (before third virus injection), 15, 16, 21, and 35 and analysed to assess viral shedding.

Adverse events

Assessment of adverse events was performed according to the Veterinary Cooperative Oncology Group Common Terminology Criteria for Adverse Events v1.1 guidelines [START_REF]Common terminology criteria for adverse events (VCOG-CTCAE) following chemotherapy or biological antineoplastic therapy in dogs and cats v1.1[END_REF]. Adverse events were monitored throughout the study by daily physical examination, complete blood count and biochemistry analyses.

Viral shedding

Viral shedding was evaluated for both part of the study, on blood, saliva, urine and feces collected before and during the 48 h after each injection of TG6002. Additional evaluations were performed at day 21 and day 35 during the second part of the study (Fig. 5). Five milliliters of blood were collected in an EDTA tube, saliva samples were taken with buccal swabbing (Universal viral transport kit, Becton Dickinson, Franklin Lakes, New Jersey, United States), 5 ml of urine were collected by ultrasonography guided vesical puncture and placed in a sterile Falcon tube, and one gram of feces was transferred to a sterile Falcon tube. All samples were stored at -80 °C until analysis. Viral shedding was evaluated by viral titration by plaque assay on CEF (for blood, saliva and urine) and by qPCR assay (for blood, saliva, urine and feces).

Viral titration by plaque assay on CEF

Viral titration by plaque assay was performed on CEF, each sample (blood, saliva, urine) was tested after dilution of 100 μl of sample in phosphate buffered saline supplemented with 1% fetal calf serum (FCS, Life Technologies, Carlsbad, California, United States) and 1% cations (magnesium acetate 100 μg/mL, calcium chloride 100 μg/mL, Merck, Darmstadt, Germany) up to 1 ml. Diluted samples were sonicated three times for 5 min at room temperature and were titrated in triplicate on CEF by plaque assay as previously described [START_REF] Foloppe | The enhanced tumor specificity of TG6002, an armed Oncolytic Vaccinia virus deleted in two genes involved in nucleotide metabolism[END_REF].

Viral quantification by qPCR assay

DNA was extracted from 50 μl of the whole blood, saliva, and urine samples using an automatic MagMax96 Deep Well (Life Technologies, Carlsbad, California, United States). Eight hundred milligrams of feces sample were diluted in 20 ml of phosphate buffered saline, sonicated and centrifuged for 4 min at 1400 rpm. DNA extraction was performed on 50 μl of sample using the MagMax 96 Viral RNA Isolation kit (Life Technologies, Carlsbad, California, United States) and the automatic MagMax96 Deep Well (Life Technologies, Carlsbad, California, United States). qPCR amplification was performed with the Multiplex Quantitect kit (Qiagen, Hilden, Germany) and was based on primers designed on VACV ITR region sequence (forward primer: CGATGA TGGAGTAATAAGTGGTAGGA, reverse primer: CACCGACCGATGATAAGATTTG, probe: ACTGAT TCCACCTCGGG). A standard curve was generated for absolute quantification by using the plasmid pTG15212 which contains the cloned VACV ITR sequence at both ends of the virus and located between position 6352 to 8180 (relative to Gene Bank sequence U94848) in the 5'region and position 169,909 to 171,737 in the 3′ region of the viral genome. The ratio of plasmid/virus was two copies of pTG15212 for one copy of TG6002. Absolute quantification was performed by using a standard curve of pTG15212 plasmid. Positive controls were included with spiked samples of 10 μl of the plasmid pTG15212 (10,000 copies) solution. All samples were run in triplicate. The limit of detection for analysis was 150 copies/ ml for whole blood, 300 copies/ml for urine and 36 copies/mg for feces. It was not possible to define detection limits for saliva, since the amount of collected saliva was exceedingly low.

Histological and immunohistochemical analyses

Histological analyses were performed on Hematoxylin-Eosin-Saffron stained tissue sections. Immunohistochemical analyses were performed on kidney and liver samples using a rabbit anti-VACV polyclonal antibody (dilution 1/500) (B65101R, Meridian Life Science, Memphis, Tennessee). Immunohistochemical analyses were performed using a DXT automat (Ventana Medical Systems, Roche Diagnostics, Basel, Switzerland) with the streptavidin-biotin-peroxidase complex method with 3, 3′-Diaminobenzidine as a substrate and hematoxylin counterstaining Formalin-fixed. As previously described, a canine mammary adenocarcinoma explant infected with TG6002 was used as positive control [START_REF] Béguin | Abstract 1446: characterization, evaluation and safety studies of the oncolytic Vaccinia virus TG6002 for canine cancer therapy[END_REF].

Fig. 1

 1 Fig. 1 Weight (a) and temperature (b) of dogs after a single intramuscular injection of TG6002. No significant change in the weight and temperature of dogs was noticed after a single intramuscular injection of TG6002. Arrows indicate TG6002 administrations. Dotted lines represent the reference interval

Fig. 2

 2 Fig. 2 White blood cell counts of dogs after a single intramuscular injection of TG6002TG6002 did not induce significant changes in any white blood cells after a single injection. a: leukocytes, b: neutrophils, c: lymphocytes, d: monocytes, e: eosinophils, f: basophils. Arrows indicate TG6002 administrations. Dotted lines represent the reference interval.

Fig. 3

 3 Fig. 3 Weight (a) and temperature (b) of dogs after three intramuscular injections of TG6002. No significant change in the weight and temperature of dogs was noticed after repeated intramuscular injections of TG6002 at 5 × 10 7 PFU/kg. Arrows indicate TG6002 administrations. Dotted lines represent the reference interval

Fig. 4

 4 Fig. 4 White blood cell counts of dogs after three intramuscular injections of TG6002. TG6002 did not induce significant changes in any white blood cells after repeated injections of TG6002 at 5 × 10 7 PFU/kg. a: leukocytes, b: neutrophils, c: lymphocytes, d: monocytes, e: eosinophils, f: basophils. Arrows indicate TG6002 administrations. Dotted lines represent the reference interval

Acknowledgments

We would like to thank Caroline Schenkels and Thomas Donnelly for editorial assistance.

Funding

No funding was received for this study.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Supplementary information

Supplementary information accompanies this paper at https://doi.org/10. 1186/s12917-020-02524-y.

Additional file 1. Biochemical analyses of dogs after single intramuscular injection of TG6002. No significant anomalies were noted. Bold numbers refer to values outside the reference interval.

Additional file 2. Biochemical analyses of dogs after three intramuscular injections of TG6002. No significant anomalies were noted. Bold numbers refer to values outside the reference interval.

Additional file 3. Detection of virus shedding by plaque assay after single intramuscular injection of TG6002. Infectious VACV was not detected in blood, urine and saliva samples by plaque assay method from dogs treated with escalating doses of TG6002. All samples were run in triplicate.

Additional file 4. Detection of virus shedding by plaque assay after three intramuscular injections of TG6002. Infectious VACV was not detected in blood, urine and saliva samples by plaque assay from dogs treated with three successive injections of TG6002 at 5 × 10 7 PFU/kg. All samples were Additional file 5. Detection of virus shedding by qPCR assay after a single intramuscular injection of TG6002. VACV DNA was not detected in blood, urine and saliva samples by qPCR assay from dogs treated with escalating doses of TG6002. All samples were run in triplicate.

Additional file 6. Detection of virus shedding by qPCR assay after three intramuscular injections of TG6002. VACV DNA was not detected in blood, urine and saliva samples by qPCR assay from dogs treated with three successive injections of TG6002 at 5 × 10 7 PFU/kg. All samples were run in triplicate.

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FARHAD RE ´ZAI Seyedmohammad Tabaie

Bertrand C D Flipo

Jonathan Cormier

Mohammad Jahazi
email: mohammad.jahazi@etsmtl.ca

Farhad Re

Post-Weld Heat Treatment of Additively Manufactured Inconel 718 Welded to Forged Ni-Based Superalloy AD730 by Linear Friction Welding

A new post-weld heat treatment (PWHT) cycle was designed for novel dissimilar linear friction welding (LFW) of selective laser melted (SLM) Inconel 718 (IN718) to AD730 forged nickel-based superalloy. The microstructure and hardness of the joints after the PWHT are investigated and compared with those of as-linear friction welded samples. The precipitation of c¢ + c¢¢ is determined as the main mechanism to increase the mechanical properties of SLM IN718 alloy. These particles coarsened during heat treatment at 1253 K and double aging. The results show that the thermomechanical history of linear friction welded joints can affect the microstructure of IN718 alloy such as the morphology of d phase after solution treatment (ST) from the platelike in the weld zone (WZ) to the needlelike in the base material (BM). It was found that in AD730, nanometric size c¢ particles reprecipitated close to the weld line during rapid cooling after welding. The presence of ultrafine c¢ particles and coarsening of the remaining particles in the microstructure of the alloy, during PWHT, can enhance the strength and hardness. The developed PWHT resulted in uniform hardness across the new dissimilar joint.

I. INTRODUCTION

LINEAR friction welding (LFW) is a solid-state welding technique that overcomes defects induced by fusion welding methods. This technique is very fast and flexible and has recently been applied for the manufacturing and repair of blade integrated disks (blisks) in the aerospace industry as well as in other turbine applications. [1][2][3][4] Alloys used for such applications are made of precipitation-hardened nickel (Ni)-based superalloys. Due to the large volume fraction of the second-phase particles in these alloys, they are susceptible to crack formation. The latter could occur in both the weld zone (WZ) and the heat-affected zone (HAZ) during welding or post-weld heat treatment (PWHT). [1,5,6] Formation of intergranular liquation cracking, microshrinkage, strain-age cracking associated with melting and solidification, and PWHT are the main reasons why newly designed Ni-based superalloys (c¢ volume fraction of more than 40 pct) are categorized as ''unweldable'' alloys by conventional fusion welding techniques. [1,7] Selective laser melting (or laser powder bed fusion) is one of the main additive manufacturing (AM) techniques. AM presents significant advantages compared to conventional methods, such as forging, by reducing the number of fabrication steps and the manufacturing of parts with complex geometry. [8][9][10] The microstructure of fabricated parts contains inhomogeneity in the fraction of precipitates arising from the high thermal gradient of the parts during fabrication. Any inhomogeneity can be controlled by applying different post-heat-treating cycles. However, such PWHT may not be enough to erase tailored microstructure buildup. [9,[11][12][13][14][15][16] As reported in Reference 17 developing a hybrid technique (SLM + LFW) involves some difficulties such as controlling and tailoring the microstructure of the alloy after SLM and after joining. A number of research studies [7,[18][19][20][21][22] have reported that changes in the microstructure of the as-linear friction welded alloy take place that can affect the chemical composition, grain sizes, and strengthening phases, and can induce undesirable eutectic compounds and liquation. The preceding changes could result in different mechanical properties such as hardness in different regions. Hence, to enhance the microstructure stability and to tackle undesirable variation in hardness and residual stresses, a PWHT cycle is required. [1,[23][24][25][26][27] The high-temperature exposure and high heating (400 K/s to 500 K/s) and cooling (150 K/s to 250 K/s) rates present in the LFW process led to smaller average particle sizes and volume fraction of secondary phases, resulting in hardness reduction in the WZ in comparison to the base material (BM). [28,29] Qin et al. [30] simulated the heat generated during LFW of superalloys and its impact on the temperature and plastic deformation behavior of the alloy. Their simulated results were validated by microstructure observation, which showed the influence of plastic deformation heat on phase transformation. In the case of the friction welding of dissimilar superalloys, [7,22] the significant differences existing between the microstructures, particularly in terms of the type and the volume fraction of the strengthening phase (c¢/c¢¢) and grain size, could make it particularly difficult to attain a sound dissimilar joint. Roder et al. [START_REF] Roder | Proceedings of the Sixth International Special Emphasis Symposium on Superalloy[END_REF] reported on tensile tests on IN718 alloy inertia friction welded to INCOLOY* 909 at different temperatures. In these tests, failure occurred in the WZ of IN718 alloy due to the absence of c¢¢ strengthening precipitates. However, regarding the LFW of AD730,** Masoumi et al. [18,[START_REF] Masoumi | [END_REF] revealed that failure and crack initiated in the BM and a final fracture occurred in the zone adjacent to the thermomechanical affected zone (TMAZ) during tensile testing at room temperature. They reported that the observed higher strength of the WZ as compared to that of the TMAZ resulted from finer grain microstructure and reprecipitation of fine secondary phase in the WZ. The c¢ particles (30 to 200 nm) reprecipitated in the WZ [33,[START_REF] Panella | Superalloys[END_REF] at a higher proportion than did those of the TMAZ and the BM during cooling after LFW. They noted that the PWHT cycle led to c¢ reprecipitation in the TMAZ and restored the tensile properties of the linear friction welded samples. [18] Smith et al. [START_REF] Smith | [END_REF]36] reported that the PWHT of linear friction welded IN718 samples improved the hardness and tensile properties (at room temperature) of the weldment, likely partially due to an increase in the volume fraction of c¢¢ phase resulting from complete d phase dissolution. It was observed that the PWHT resolutionized the d phase uniformly even when the starting materials varied significantly in terms of d phase volume fraction. Therefore, applying the PWHT on linear friction welded samples was compatible. Chaturvedi [37] and Chaturvedi and Han [38] reported that the precipitate strengthening mechanisms in IN718 varied (changing between precipitate shearing and coherency strain strengthening mechanisms) based on the size, morphology, and volume fraction of the primary strengthening precipitates (i.e., spheroidal c¢ and ellipsoidal c¢¢), which were highly influenced by the aging duration and heat treatment peak temperature. In many studies, [26,[39][40][41] in the case of cast and wrought or AM IN718, the dissolution of strengthening precipitates and their coarsening and the formation of d phase are microstructure evolutions that have been shown to play crucial roles in the decline of mechanical properties such as fatigue life, stress-rupture life, creep resistance, and tensile strength. [42] Damodaram et al. [29] reported that c'' particles dissolved in the WZ and the HAZ. Due to coarser grains in the HAZ, this zone was weaker than the WZ; therefore, failure occurred in the HAZ. However, post-weld heat treated samples (solution-treated (ST) and ST + aged (STA)) failed in the BM. The STA sample after welding exhibited the best stress-rupture properties. The authors explained that this could be attributed to the moderate coarsening of the grains at the WZ induced by homogenization.

Selective laser melting can be designed and combined with the LFW process as a developing welding process to create and to accelerate the manufacture or repair of blisks with dissimilar alloys. The main objective of the present study was to develop new and specific PWHT cycles for novel linear friction welded dissimilar joints of polycrystalline Ni-based superalloy AD730 to as-SLM IN718 and to homogenized SLM IN718 (H-SLM IN718). Therefore, a comparative study between as-linear friction welded and PWHT states was carried out to investigate the relationship of heat treatment, microstructural characteristics, and hardness. The obtained hardness measurements are compared with other reported results in the literature.

II. EXPERIMENTAL PROCEDURES

A. Post-Weld Heat Treatment

The as-received forged AD730 alloy, provided by Aubert & Duval, had an average grain size of 41.36 ± 9.14 lm and a volume fraction of c¢ of 40 pct (for all c¢ classes). SLM-125HL AM equipment, at IMT-Mines Albi in France, was used to fabricate IN718 superalloy. Samples were fabricated in the Z-direction, which is perpendicular to the fabrication platform for LFW conditions. Two rectangular AD730 samples (37-mm length 9 26-mm width 9 13-mm height) were linear friction welded to an as-SLM IN718 and an H-SLM IN718 (homogenized at 1373 K for 1 hour and water quenched) with the same dimensions. Their chemical compositions are provided in Table I.

The FW34-E20 LFW machine at TWI Ltd was used for the LFW process in this investigation. The following process parameters were used to produce welded specimens: friction pressure of 228 MPa, oscillation frequency of 40 Hz, amplitude of 3 mm, and forge pressure of 340 MPa as the last step of the LFW. The oscillation displacement occurred perpendicular to the SLM building direction and on the top part of the SLM samples. Therefore, the contact surface in the AD730 was in touch with the top of the SLM fabricated simples. Three welds were post-weld heat treated in this study: two as-SLM IN718-AD730 (W1) and one as H-SLM IN718-AD730 (W2).

Figure 1 shows the PWHT cycle used in the present investigation. Depending on the microstructure of the SLM IN718 and the precipitation hardening treatment for the IN718 and AD730 alloys, one of the samples of W1 was ST at 1253 K for 1 hour and water quenched. It should be noted that the solution treatment heat treatment is for IN718 alloy. The samples were subsequently aged at 1003 K for 12 hours, cooled in the furnace (55 K/h), and held at 893 K for 10 hours, followed by air cooling (W1-STA). The second sample of W1 and the W2 sample were directly aged and are identified as W1-DA and W2-DA, respectively. Regarding the industrial aging treatment of AD730 alloy at 1003 K to 1033 K for 8 to 10 hours and the first aging step of SLM IN718 alloy at 993 K to 1033 K for 8 to 10 hours, in this study, the temperature and time of the first aging step (for dissimilar welds) are modified (i.e., temperature 1003 K and time change from 8 to 12 hours). It should be noted that the second aging step (893 K for 10 hours) cannot affect the precipitation hardening in AD730. However, the time was increased from 8 hours (the usual aging time for IN718 alloy) to 10 hours to compare the results with the literature.

B. Microstructure Analysis

The as-linear friction welded samples were cross sectioned by electrical discharge machining (EDM) from the center of the welded samples along the longitudinal axis and perpendicular to the welding direction. The waterless Kalling and Marble solutions were used to reveal the microstructural features, which were examined using a tabletop scanning electron microscope (SEM) (Hitachi TM3000) and a field emission-SEM (FESEM) (Hitachi SU-8230) (Japanese Multinational Engineering and the Electronics Conglomerate Company, Tokyo, Japan). Furthermore, a Quad energy-dispersive X-ray spectroscopy (EDS) detector and a Bruker electron backscatter diffraction (EBSD) detector were also used in the analysis.

For EBSD investigation, samples were polished using a Buehler VIBROMET (Illinois, USA) with colloidal silica suspension (0.05 lm) and then an ion beam milling system (IM 400Plus, Hitachi) for optimum surface preparation and removal of any subsurface damage during the polishing stages. For regions close to the weld lines (or WZ), where very fine recrystallized grains could be present, a fine step size of 90 nm was used. The nonrecrystallized regions were examined by a coarser step size of 0.47 lm. At least 300 grains were characterized for each region.

C. Microhardness Test

The Vickers microhardness (HV) profile was measured on cross-sectioned post-weld heat treated samples throughout the weldment by using an automatic Future-Tech Vickers machine. The obtained results were compared with as-linear friction welded samples (W1 and W2). A load of 200 g (HV0.2) and a dwell time of 15 seconds were employed for all measurements from the weld interface toward the BM (a distance of ~3 mm). The hardness examination was conducted on both linear friction welded and post-weld heat treated samples. The measurements were repeated between three and five times for different distances. The average values are reported with errors between 0.4 pct (in the WZ and the BM) and 2.1 pct (in the TMAZ). W1-STA) specimen (Figures 2(a) and(c)). In the W2-DA (Figure 2(d)), the as-fabricated microstructure, the columnar grains, and the cellular structures were partially removed after homogenization (before LFW) and replaced with equiaxial grains; however, a highly oriented structure could be observed inside the grains. Ni et al. [13] reported that partial recrystallization is beneficial in decreasing the anisotropy for mechanical properties. However, on AD730, no grain size change was observed since 1253 K was too low a temperature to induce any grain growth or recrystallization. [43,44] As shown in Figures 2 and3, in addition to grain size, different types of secondary phases precipitated after PWHT in different regions. Figure 3 shows the evolution of the shape, location, and volume fraction (V f ) of the d phase in IN718 alloy (W1-STA) from the BM zone to the WZ. In the BM, two d-phase morphologies were observed, i.e., intragranular needlelike and platelike d phases at grain boundaries (intergranular) in the vicinity of granular and long-striped Laves particles (Nb: 21.58, Mo: 1.88, Ti: 1.77 (at. pct), on average). The presence of d phase particles (Nb: 9.92, Mo: 1.74, Ti: 1.75 (at. pct), on average) of varied sizes can be observed within the grains (larger) and at grain boundaries (smaller). Furthermore, the dimensions (e.g., length and width) of the d particles increased from the BM to the WZ. This evolution was also observed in the microstructures of W1-DA and W2-DA on the IN718 side, where the fraction of the d phase was less than that of W1-STA (Figure 4). As shown in Figure 4, d particles are larger and platelike or semispherical in the WZ and in the TMAZ (from the weld interface to ~1700 lm).

On the IN718 side, due to the heat treating at 1253 K and aging on W1-STA, the microstructure contains more d phase within the grain and at the grain boundaries. Moreover, in the BM, this phase experienced greater d precipitation than in the TMAZ and the WZ (Figures 3 and4).

The d phase formed in the segregated regions (interdendritic) where the Laves phase (enriched Nb) precipitated after fabrication and solidification, but not so in the core of dendrites having a low content of Nb. This indicates that the formation of this phase is a function of Nb local concentration. [45] Since the amount and size of the d phase depends on the thermomechanical history of the alloy, [30] the dissolution and precipitation of platelike and spherical d phase particles both occurred during the deformation [46] and underwent a transformation during the thermomechanical process such as the one in LFW (Figure 4). The level of deformation during the LFW decreases from the WZ to the BM. [47,48] As shown in Figure 4(a), the volume fraction of d phase increased from the WZ to the BM in all samples. As a result of deformation and dissolution (due to the higher temperature close to the weld line), the platelike d phase that had been formed during the SLM process (and before STA) in grain boundaries was transformed, as precursor, dissolved/transformed into short granular rods or semispherical d phase particles (Figures 4(b) and(c)). The other platelike particles could be formed during the ST. [49] As also reported previously, [17,50] there is an evolution in size, shape, and volume fraction of precipitates, from the WZ to the BM, on both sides. Figures 5(a) and(b) show the microstructure in the WZ in the W1 and W1-DA samples, on the AD730 side, before and after the PWHT, respectively.

In the WZ, on the AD730 side, the c¢ phase was completely dissolved during LFW. Figure 5(a) shows no secondary phase precipitates close to the weld line after welding. However, the ultrafine c¢, which could have reprecipitated due to the high cooling rate after LFW, were coarsened (primary c¢) or newly precipitated (secondary + tertiary c¢) (44 ± 12.2 nm) within grains (as well as W2-DA sample) (Figure 5(c) and(d)) after the PWHT. In W1-STA, c¢ particles are larger (189 nm ± 29) due to the heat treatment (or ST) before aging (Figure 6). The heat treatment temperature (1253 K) used for W1-STA is less than the solvus temperature of the c¢ phase (~1378 to 1383 K) and of secondary c¢ particles (1323 to 1353 K) in a fine grain AD730. [33,51] Therefore, on the AD730 side, fine secondary c¢ dissolved during the heat treatment at 1253 K, while undissolved or large particles coarsened, potentially affecting the volume fraction of c¢ in the microstructure (from ~160 nm in W1-DA to ~250 nm W1-STA at the BM). The variation of the volume fraction and the size of c¢ in AD730 is provided in Figure 7 for W1-DA and W1-STA. It should be noted that primary c¢ was not observed in the WZ of the samples (Figure 5(a) and (b) and Figure 6(a)), and the V f is reported for all categories.

Due to the temperature gradient between the WZ and the BM during LFW, almost all of the c¢ dissolved in the WZ, where the temperature had reached 1473 K to 1523 K, and c¢ primary and secondary were partially dissolved in the TMAZ, where the temperature had reached 1173 K to 1373 K. After PWHT (DA), ultrafine c¢ (2 to 10 nm [18,33] ) remained in the WZ of as-linear friction welded samples (in AD730), coarsened slowly during the first step of DA at 1003 K (for 12 hours) in W1-DA and W2-DA samples. It must be noted that these particles could be reprecipitated and further coarsened (by 3 times close to the WZ and by 1.6 times in the BM) during the heat treatment at 1253 K and during double aging (W1-STA). However, the ST time and temperature are not long enough for the precipitation of primary c¢, although comparing W1-STA with W1-DA shows that it was coarsened, as shown in Figure 7(b). According to the Lifshitz-Slyozov-Wagner (LSW) equation, [52] which describes the growth kinetics of the c''/c¢ precipitate, these precipitates coarsen gradually with aging time. [53,54] As also reported in References 5, 55 and 56 on the AD730 side, the shape of the c¢ precipitate could change from spherical to quasi-spherical and cubic (more rounded edges) as a result of coarsening that occurs during heat treatment at 1253 K or aging (Figures 6(c) and(d)).

In the IN718 alloy, in the as-linear friction welded conditions, secondary particles were dissolved (except for the Laves phase and MC carbides) close to the WZ due to the LFW temperature (1473 to 1523 K), which was higher than the c¢ and c'' dissolution temperatures (c¢ = 1243 K and c'' = 1203 K). On the SLM and H-SLM IN718 sides, small particles of the Laves phase dissolved and their constitutional elements (Nb and Mo) diffused to the c matrix due to high temperatures (1473 to 1523 K). For their part, the large Laves particles were partially dissolved and transformed to granular and finer particles since the cooling rate was high and, therefore, the time for the evolution was short. [12,17,57] After the PWHT (both DA and STA samples), very fine c¢ and c¢¢ precipitated and were observed in IN718 (in both SLM and H-SLM conditions) in all zones. Generally, the same fraction of c¢/c'' is expected close to the WZ of W1-DA and W2-DA. However, as we go from the WZ toward the BM, the volume fraction of the c'' phase in W2-DA could be significantly higher than in W1-STA, but slightly lower than in W1-DA due to some possible c¢¢ phase remnants after the SLM process in the microstructure of W1 (in the BM and almost in the TMAZ). It should be noted that both the Laves phase (Ni, Cr, Fe) 2 (Nb, Mo, Ti) (Nb-rich) and the d phase (in W1-STA) Ni 3 Nb (Nb and Ti-rich), in W1-STA and W1-DA, can slow the precipitation of the c'' and c¢ strengthening phases. [13,14] As reported in References 15, 58 and 59 carefully adjusting Nb, Ti, and Al is one the most effective ways of controlling the thermal stability and precipitation of c'' and c¢. The d particles were recognized by their morphology and their Nb and Ti contents, which were, respectively, lower than those of the Laves particles (Table II). From the EDS results, it can be seen that the blocky particles close to the weld line are Laves phase due to their lower amounts of Nb and Ti than MC carbides. [60][START_REF] Mitchell | Superalloys[END_REF][START_REF] Mitchell | [END_REF] The Nb concentration in the d and in the Laves phases in the TMAZ was higher than in the WZ and lower than in the BM due to the thermomechanical gradient from the WZ to the BM during LFW, which changed the rate and diffusion distance of the elements according to References 12 and.17

As shown in Figures 8(a) through (i), the Laves phase fraction increased from the weld line toward the BM in all three samples. For example, Figure 8(a) shows that the Laves phase fraction in the BM of the W1-AD sample (on the IN718 side) was more than in the others. In W1-STA (Figures 8(d

B. Microhardness

Figure 9 shows the evolution of the hardness in the post-weld heat treated samples (W1 fi W1-DA, W1-STA, and W2 fi W2-DA) and its variation in different regions. Applying the proposed PWHT helped the back diffusion of segregated elements in the Laves particles into the matrix solid solution and precipitation of c¢, c¢¢, and d phases with the desired morphology in the IN718 alloy. It also induced reprecipitation of c¢ in AD730, thereby allowing for the restoration of the hardness in the weldment, as discussed in detail subsequently. It must be noted that the hardness data for as-linear friction welded conditions (W1 and W2) are reported from a recent publication by the present authors. [17] After PWHT, the hardness increased as compared to that of the as-linear friction welded samples (W1 and W2) in all zones. Table III provides the microhardness comparison values between the PWHT and as-linear friction welded conditions for both alloys in all zones. In order to better illustrate the variations, the data are presented in terms of percentage difference (i.e., ((PWHT-AW)/AW) 9 100) in each zone of both alloys. Furthermore, in the last two columns of Table III, the hardness differences of post-weld heat treated samples in different locations or between the two alloys are reported.

An overall analysis of the data reported in Table III shows that the highest hardness variation occurred in the WZ of IN718 (90 pct for W2-DA) and in the TMAZ of AD730 (33.5 pct for W1-DA). The minimum change was seen in the WZ of AD730, the BM of IN718 in W1-DA and W1-STA samples, and the TMAZ of IN718 in the W2-DA sample. However, the relative increase in the hardness in the BM zone of IN718 in the W2-DA sample was higher than in all three samples. After PWHT, for each alloy, the maximum difference in the hardness was measured between the WZ and the BM, as reported in column 7 of Table III. These changes, ranging between 17 are 25 HV, are not necessarily related to variations in grain size, as will be discussed subsequently. In the last column of Table III, the maximum hardness difference in the WZ of IN718 and AD730 after PWHT is reported. These data will be used to discuss the influence of microstructural changes during PWHT on hardness evolution in each alloy in the following sections.

On the IN718 side, the increase in the hardness is mainly attributable to the precipitation of c¢ + c¢¢ as strengthening phases, as well as to the coarsening of precursor c¢ and c¢¢ particles remaining from the SLM process in W1-DA in the TMAZ and in the BM. In the WZ, the reprecipitation of these phases with a higher volume fraction is generally expected to increase the hardness more than in other zones. Due to the dissolved Nb-rich Laves phase in the WZ, the amount of Nb (as c¢¢ forming element) increases in the matrix and enhances the precipitation of c¢¢ during PWHT. As a result, the volume fraction of c¢ + c¢¢ could increase in the IN718 alloy; thus, an 83 to 90 pct change in hardness is observed in this zone (Figure 9 and Table III). However, in W1-STA, due to the precipitation of d phase during the ST, the Nb content necessary for precipitation of c¢¢ decreases in the matrix; hence, a lower fraction of c¢¢ is expected in W1-STA than in the other two samples, and the change in hardness is less than in them.

Furthermore, as reported in References 7, 40 and 63 the coherent strain strengthening induced by the precipitation of c¢¢ and c¢ phases is the main strengthening mechanism in the IN718 alloy. Furthermore, it is well known that the mismatch induced by the c¢¢ phase in the matrix results in a stress field. Dislocation movement also induces the stress in the matrix. Therefore, interactions between these fields can block the dislocation movements and increase the strength of the material. [7,40,64] Therefore, the precipitation of mainly c¢¢ and c¢ after PWHT and grain refinement [17] and solid-solution strengthening [12,16,17,65,66] could enhance the hardness of the IN718 alloy. Although grain refinement is a mechanism used to increase the hardness in as-linear friction welded conditions (W1 and W2), the grain sizes did not change after PWHT. Therefore, the influence of secondary phases on the mechanical properties of the SLM IN718 should be considered in the present situation.

It is notable that the DA sample (W1-DA) shows the highest hardness (the blue line in Figure 9), although the Laves phase is still observed due to the low aging temperature. In W2-DA, due to the homogenization before LFW, the Laves particles could partially be eliminated in the microstructure. However, in W1-STA, the formation of d phase (needlelike) was observed close to the Laves phase after heat treatment at 1253 K. This indicates that the d phase had precipitated out from the Laves phase dissolution, as also reported in Reference 45. The direct-aged W1-DA sample is expected to have higher ultimate tensile stress but lower elongation than both W1-STA and W2-DA samples. Sui et al. [15] reported that the presence of Laves phases (as in W1-DA) could have a strengthening effect, even if not coherent with the matrix. Sui et al. [15] reported that the formation and presence of the Laves phase is accompanied by notable niobium microsegregation, which causes an inhomogeneous distribution and coarsening of c¢¢ phase. After homogenization on SLM IN718 (i.e., H-SLM in W2), as well as a significant reduction in the volume fraction of the Laves phase, [12,17] long-striped Laves phases are almost eliminated and transformed into spherical particles, the c'' phase distribution becomes uniform, and the residual stress accumulation could decrease. [15,16] Huang et al. [14] and Marchese et al. [67] reported that due to repeated rapid heating and cooling during the SLM process, dislocation density increases. This leads to a gradual increase in the level of residual stresses, which can act as a strain hardening source. Therefore, the dislocation density can remain at a relatively high level after the DA due to low-temperature treatment.

It should be noted that on the IN718 side in W1 (Figure 9, black line), the Laves phase volume fractions were ~18.5 pct and 12 to 15 pct in the BM and the TMAZ, respectively. Prior to the PWHT, the average hardness of this sample in the BM was ~60 HV higher than in the TMAZ. However, after PWHT (Figure 9, blue line), the difference in hardness between the WZ and the TMAZ decreased. This indicates that the hardness in the TMAZ was even higher (~15 HV) than the WZ due to the precipitation of c¢ + c''. The reduction of Nb content in the Laves phase makes the Laves crystal structure unstable. [57] On the contrary, a high concentration of Nb will diffuse again into the c matrix and will contribute to the precipitation of c'' and d phases (Ni 3 Nb).

On the AD730 side (Figure 9 and Table III), as mentioned previously, the PWHT restored the hardness in the weld regions. The hardness increased in the WZ of all samples due to the coarsening of the remaining ultrafine c¢ during PWHT. However, this increase in the hardness was substantial in the TMAZ (~23 to 33.5 pct in Table III), although the grain size was smaller than in the WZ (~5 lm in the WZ vs ~25 lm in the TMAZ). In the TMAZ, a simultaneous coarsening of the remaining c¢ particles (primary + secondary + tertiary) and c¢ reprecipitation could increase the hardness more than in other zones. Chamanfar [START_REF] Chamanfar | Mining and Materials Engineering[END_REF] reported that primary c¢ particles remain at the grain boundaries of Waspaloy during thermomechanical processing and inhibit grain growth. In contrast, Huang et al. [22] reported that the presence of the intragranular secondary and tertiary c¢¢ particles are the main reasons for the higher strength of the 720Li alloy as compared to IN718. The hardness increases to a maximum value with increasing particle size and then it declines. This maximum value was referred to as the ''strongest size,'' which depends on the c¢ volume fraction. [START_REF] Chamanfar | Mining and Materials Engineering[END_REF][START_REF] Jackson | [END_REF] The preceding behavior was also observed in the BM as compared to the WZ, as also reported in Reference 18 in the case of an LFW of AD730.

Wrought superalloys, such as Waspaloy and AD730, are prone to reheat cracking during PWHT. The higher the c¢ content or the concentration of c¢ forming elements, the higher will be the susceptibility to reheat cracking. [1,24] However, the HAZ was not observed in the as-linear friction welded samples. Besides, no depletion of the matrix from c¢ forming elements related to c¢ precipitation during PWHT was observed due to the application of subsolvus heat treatment.

As reported in the last column in Table III, the difference in hardness between the two alloys after PWHT was around 19 HV for W1-DA and W2-DA in the WZ. This difference was higher (27 HV) in W1-STA due to the stress relief in the WZ and the dissolution of Laves phase by Laves + c (in W1) fi Laves + d + c during the ST. The preceding results could be compared with those reported on dissimilar friction welding of IFW IN718-U720Li, [22] linear friction welded IN718-IN713LC, [7] and IFW IN718-INCOLOY 909; [START_REF] Roder | Proceedings of the Sixth International Special Emphasis Symposium on Superalloy[END_REF] the hardness differences between the two alloys were 65 HV (WZ and BM), 12 HV (WZ and BM), and 28 HV (for WZ) and 60 HV(for BM), respectively. It must be noted that the preceding results were obtained after applying one-step aging for PWHT. However, interestingly, hardness measurements in all zones across the weldments, after the proposed two-step aging, show that the maximum homogeneity occurred on both alloys of the W1-DA sample (Table III) (average of 17 HV for AD730 and 15 HV for SLM IN718 between the WZ and the BM). The hardness differences between the WZ and BM were compared with those published in the literature and are summarized in the following (Figure 10).

(1) IN718-U720Li: [22] ~48 HV for IN718 and ~45

HV for U720Li. (2) IN718-IN713LC: [7] ~70 HV for IN718 and ~1 20 HV for IN713LC. (3) IN718-INCOLOY 909: [START_REF] Roder | Proceedings of the Sixth International Special Emphasis Symposium on Superalloy[END_REF] sample was smaller (30 to 40 HV) than in other weldments, the hardness was more homogenous on the side of IN718 in W1-DA.

Therefore, applying two-step aging on the LFWs of dissimilar superalloys could provide more homogeneity to the hardness of each alloy and to the weldment. A more homogenous microstructure after the PWHT could potentially provide more isotropic mechanical properties in the joint. In addition to homogeneity, the presence of d phase within grain boundaries of IN718 alloy in W1-STA and a larger grain size in W2 and W1-STA (in both alloys) can improve creep resistance. Masoumi et al. [START_REF] Masoumi | [END_REF] reported that a specific PWHT (solutionizing at 1173 K for 2 hours and aging at 1003 K for 8 hours) on a linear friction welded fine grain AD73 alloy resulted in a creep strength that was superior to that of as-linear friction welded joints.

IV. CONCLUSIONS

In this study, PWHT cycles were carried out on linear friction welded dissimilar Ni-based superalloys fabricated by two different methods. The phase transformation and evolution of secondary phases across the weld line were studied following PWHT. The impact of the microstructure evolution on the microhardness variation was also evaluated. The main findings can be summarized as follows.

1. The presence of ultrafine c¢ particles in the WZ of AD730 alloy in as-linear friction welded samples was revealed, and the particles coarsened during PWHT. 2. The precipitation of the c'' phase in post-weld heat treated SLM and H-SLM IN718 (W1-DA and W2-DA) is the main mechanism used to increase the hardness on the side of IN718 alloy. 3. A comparison of hardness measurements in W1-DA with W1-STA and their microstructure evolution from the WZ to the BM shows that stress relief can be induced by the PWHT cycle and likely reduce the amount of the Laves phase in the microstructure of SLM IN718. However, the hardness in W2-DA (on IN718 side) was greater (~14 HV) than in the W1-STA in the TMAZ, which can be attributed to the participation of Nb in the d phase formation after ST (i.e., constitutional elements of Laves phase diffused into the c matrix during homogenization in W2). Hence, less Nb was available for c'' precipitation during aging. 4. A double aging cycle based on the precipitation temperatures of the secondary phases in both alloys caused a more homogenous hardness in the joint as compared to the one-step aging previously reported in the literature.

Fig. 1 -

 1 Fig. 1-Schematic of the PWHT cycle for all linear friction welded samples.

Fig. 2 -

 2 Fig. 2-SEM micrograph in the BM zone of IN718 alloy after LFW and PWHT for (a) W1, (b) W2, (c) W1-DA, (d) W2-DA, and (e) W1-STA.

Fig. 3 -

 3 Fig. 3-SEM images from sample W1-STA on the side of the SLM IN718 alloy in the (a) BM, (b) intergranular and intragranular d phase at the BM, (c) TMAZ, and (d) WZ.

Fig. 4 -

 4 Fig. 4-Size and volume fraction (V f ) evolution of the d phase from the WZ toward the BM, on the SLM IN718 side, in different samples after PWHT: (a) volume fraction, (b) length, and (c) width of d particles.

Fig. 5 -

 5 Fig. 5-Precipitation of secondary phases in the weld interface of W1 sample after PWHT: (a) as-linear friction welded, (b) W1 direct aged, (c) on the DA-SLM IN718 side in the WZ, and (d) very fine secondary and probably tertiary c¢ in DA-AD730 in the WZ.

  ) through (f)), the remaining Laves particles of as-linear friction welded W1 were transformed into d and c¢¢ due to the ST and aging treatment, respectively. The W2-DA sample (Figures 8(g) through (i)) contained the lowest amount of Laves particles due to the homogenization treatment that took place before LFW. The evolution of the Laves phase in the W1 and W2 samples at different locations from the weld line to the BM region has been reported in Reference 17. Spherical (or semispherical) Laves particles within the grains in the WZ were transformed to long-striped shapes in the interdendritic regions in the HAZ and BM. Therefore, variations of secondary phases, such as c¢, c¢/c¢¢, d, carbides, and Laves, in IN718 (SLM and H-SLM) and AD730 alloys could provide different mechanical properties for the dissimilar joint.

Fig. 6 -

 6 Fig. 6-SEM images of c¢ precipitates in sample W1-STA: (a) and (c) in the WZ and (b) and (d) in the BM of AD730 side.

Fig. 7 -

 7 Fig. 7-Variation of (a) the volume fraction and (b) the size of primary and secondary (and tertiary) c¢ on the AD730 side in W1-DA and W1-STA samples from the weld line to the BM.

Fig. 8 -

 8 Fig. 8-Precipitation of secondary phases in the microstructure after the PWHT: (a) through (c) W1-DA, (d) through (f) W1-STA, and (g) through (i) W2-DA on the IN718 (SLM and H-SLM) sides (a), (d), and (g) at the BM, (b), (e), and (h) at the TMAZ, and (c), (f), and (i) at the WZ.

Fig. 9 -

 9 Fig. 9-Microhardness profiles as a function of distance from the weld line of two linear friction welded samples. The hardness profiles of W1 and W2 samples are captioned from Ref.17.

Fig. 10 -

 10 Fig. 10-Hardness distribution across the weld interface in three other dissimilar joints after PWHT.

Table I .

 I Chemical Compositions of the Bulk of Alloys (Weight Percent)

								Elements
	Alloy	Ni	Fe	Cr	Co	Mo	W	Al	Ti	Nb	B	C	Zr	Si
	Forged AD730	bal	4.00	15.70	8.50	3.10	2.70	2.25	3.40	1.10	0.010	0.015	0.030	-
	SLM IN718	bal	15.70	20.54	0.10	3.13	-	0.34	1.17	5.10	0.002	0.040	0.018	0.01

Table II .

 II EDS Chemical Composition Analysis of Laves and d Phases in the Post-Weld Heat Treated Samples in the TMAZ

			Main Constituent Elements (At. Pct)
	Phase Sample	Ni	Nb	Mo	Ti	Fe	Cr
	Laves W1-DA	bal 34.87 1.74 7.76	9.74 18.09
		W1-STA		20.17 1.45 2.63	5.06	8.90
		W2-DA		17.46 1.34 3.84 13.07 16.01
	d	W1-DA	bal	9.09 2.42 8.62 15.59	9.63
		W1-STA		11.10 1.14 3.97	6.52	7.19
		W2-DA		8.15 2.17 6.13 14.58 12.45

Table III .

 III Microhardness Comparisons between Post-Weld Heat Treated and As-Linear Friction Welded Samples in Different Zones

	PWHT/		WZ	TMAZ	HAZ	BM	Max. Difference at Post-Weld	Max. Difference in Hardness between
	AW	Alloy	(Pct)	(Pct)	(Pct)	(Pct)	Heat Treated Zones (HV)	Post-Weld Heat Treated Alloys (HV)
	W1-DA/	IN718	88.2	78	42	46.5	17 (WZ-BM)	19 (WZ)
	W1	AD730 13.5	33.5	-	26.1	15 (WZ-BM)
	W1-STA/	IN718	83	65	-	41.6	10 (WZ-BM)	27 (WZ)
	W1	AD730 12.6	31.4	-	22.7	24 (WZ-BM)
	W2-DA/	IN718	90	70	-	73	20 (WZ-BM)	18 (WZ)
	W2	AD730 17	23	-	25.3	25 (WZ-BM)

ACKNOWLEDGMENTS

The authors gratefully acknowledge the support from the Natural Sciences and Engineering Research Council of Canada (NSERC) 2018-03889 through a discovery grant. Thanks to Professor Mamoun Medraj and Dr. Eslam Fayed, Concordia University, for their involvement with the heat treatment of the welded samples. The authors also express their gratitude to Aubert & Duval Co. and Dr. Alexandre Devaux for providing forged AD730 samples. Technical support provided by Dr. Mohammad Saadati for microscopic experiments is gratefully acknowledged. The authors extend special thanks to TWI Ltd for carrying out the LFW of the samples.