Avian Paramyxovirus 4 Antitumor Activity Leads to Complete Remissions and Long-term Protective Memory in Preclinical Melanoma and Colon Carcinoma Models

Avulaviruses represent a diverse subfamily of nonsegmented negative-strand RNA viruses infecting avian species worldwide. To date, 22 different serotypes have been identified in a variety of avian hosts, including wild and domestic birds. APMV-1 (Avian Paramyxovirus 1), also known as Newcastle disease virus (NDV), is the only avulavirus that has been extensively characterized because of its relevance for the poultry industry and, more recently, its inherent oncolytic activity and potential as a cancer therapeutic. An array of both naturally occurring and recombinant APMV-1 strains has been tested in different preclinical models and clinical trials, highlighting NDV as a promising viral agent for human cancer therapy. To date, the oncolytic potential of other closely related avulaviruses remains unknown. Here, we have examined the in vivo antitumor capability of prototype strains of APMV serotypes -2, -3, -4, -6, -7, -8, and -9 in syngeneic murine colon carcinoma and melanoma tumor models. Our studies have identified APMV-4 Duck/Hong Kong/D3/1975 virus as a novel oncolytic agent with greater therapeutic potential than one of the NDV clinical candidate strains, La Sota. Intratumoral administration of the naturally occurring APMV-4 virus significantly extends survival, promotes complete remission, and confers protection against rechallenge in both murine colon carcinoma and melanoma tumor models. Furthermore, we have designed a plasmid rescue strategy that allows us to develop recombinant APMV-4–based viruses. The infectious clone rAPMV-4 preserves the extraordinary antitumor capacity of its natural counterpart, paving the way to a promising next generation of viral therapeutics. Significance: Discovery of the oncolytic properties of APMV-4 Duck/Hong Kong/D3/1975: a novel cancer therapeutic with natural capacity to exert complete remission and long-term antitumor protection in syngeneic mouse cancer models.


Introduction
The Paramyxoviridae family of viruses includes numerous important pathogens that impact both humans (mumps, measles) and animals (Sendai, cell surface, subsequently triggering the activation of the F protein, promoting fusion of the viral and cell membranes, which allows the entry of the RNPs to the cytoplasm. Genome replication is carried out within the cytoplasm by the viral RNA-dependent RNA polymerase and does not involve any DNA-intermediate stage. After each round of replication, the newly generated viruses are released from the host cell by budding followed by detachment mediated by RBP HN neuraminidase activity (3).
Avian paramyxovirus 1 (APMV-1), commonly known as Newcastle disease virus (NDV), is the most extensively characterized member of the avulaviruses due to the high mortality rate and economic loss caused by virulent strains in the poultry industry (4). Despite the devastating impact of highly pathogenic strains, NDV can be controlled by the prophylactic administration of liveattenuated and/or killed virus vaccines (5). NDV strains are classified as either velogenic (highly virulent), mesogenic (intermediate virulence), or lentogenic (low-virulence or avirulent), in accordance with the severity of the clinical signs displayed by affected chickens (6,7). Regardless of its prevalence and worldwide distribution, NDV strains do not represent a human threat. Occasional human infections are restricted to direct contact with sick birds and resolved with mild flu-like symptoms or conjunctivitis (8). Reported NDV infections in mammals have demonstrated that these avian viruses are neither capable of establishing persistent infection nor counteracting the restriction in host tropism in mammalian cells (9,10). Furthermore, different strains of NDV have been shown to act as strong stimulators of humoral and cellular immune responses at both the local and systemic levels (11)(12)(13). Because of the safety and immunostimulatory properties of NDV in mammals, and because the development of a reverse genetics system (14,15) that allows us to manipulate the genome of negative-strand RNA viruses, several NDV strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens (16)(17)(18).
Over the past three decades, there has been increased interest in the use of NDV as an antineoplastic agent. The inherent antitumor capacity of NDV combines the two major characteristics that define an oncolytic virus (OV): induction of tumor cell death (19), accompanied by the elicitation of antitumor immunity and long-term protection (20,21). From initial reports on its antitumoral potential in the mid-19th century until now, various NDV strains have been utilized in animal models and/or patients with cancer by different routes (intratumoral, locoregional, or systemic), using a multitude of therapeutic approaches, such as viral oncolysates, live cell tumor vaccines, or DC vaccines pulsed with viral oncolysate (22). Nowadays, many research groups, including ours, work toward the development of more efficient NDV-based antitumor strategies (23)(24)(25)(26)(27).
In contrast to what is known about NDV strains, there is limited information on the biology of other avian avulavirus isolates, and there have been no previous studies assessing the oncolytic potential of other closely related APMVs. Here we probed the in vivo oncoytic capacity of other members of the Avulavirinae subfamily. Preclinical syngeneic murine melanoma and colon carcinoma tumor models were challenged intratumorally with prototype viruses from APMV-2, -3, -4, -6, -7, -8, and -9 species so as to compare their survival outcomes with those induced by the clinical candidate NDV LaSota L289A virus (LS-L289A).

Viruses
Modified NDV LaSota-L289A has been described previously (29). APMV viruses isolates were obtained from National Veterinary Services Laboratories,

Phylogenetic Tree of the Attachment Protein of Representative Paramyxoviruses
The amino acid sequence of the attachment protein (RBP, H or G) from the reference viruses of important human and animal paramyxoviruses, as well as those of the 8 APMVs used in this study, were downloaded from Gen-Bank. The sequences were aligned and a maximum likelihood phylogenetic tree was created using MEGA-X v10.0. 5

Amplification and Cloning of a Full-length cDNA of the APMV-4 Genome
Viral RNA was purified from an egg-grown viral stock, following manufacturer instructions from the E.Z.N.A. viral RNA kit (Omega). Next, the viral RNA was used as a template to amplify overlapping fragments of the genome's cDNA using RT-PCR kit SuperScript IV One-Step RT-PCR System (Invitrogen). PCR primers (Supplementary Table S1) were designed to allow cloning by InFusion (Clontech) and to introduce unique restriction sites at nonconserved parts of each intergenic region. Next, the RT-PCR products were cloned into plasmid pUC-18 to obtain intermediate plasmids pUC-APMV4-1, pUC-APMV4-2, and pUC-APMV4-3. Using restriction digestion and ligation, we first combined fragments 1 and 2 into pUC-APMV4-1+2, and then added fragment 3 to obtain pUC-APMV4-1+2+3, which contains the full-length copy of our viral cDNA. Finally, the complete genome sequence was transferred to the rescue plasmid (pAPMV4) under the control of the T7 promoter and a self-cleaving hammerhead ribozyme at 5 , and a self-cleaving hepatitis delta ribozyme and T7 terminator at 3 .

Cloning of the Helper Plasmids pTM1-N, pTM1-P, and pTM1-L
The open reading frames of the viral genes N, P, and L were amplified from the intermediate plasmids pUC-APMV4-1 (N and P genes), and pUC-APMV4-3 (L gene) and cloned by InFusion into the expression plasmid pTM-1 under the control of the T7 promoter.

Rescue of Infectious Recombinant APMV4 by Plasmid Transfection
To rescue the recombinant APMV4, we followed the protocol used in our laboratory to rescue NDV with the APMV-4-specific plasmids described above.

Fluorescence Microscopy
For indirect immunofluorescence staining, the cells were infected for 1 hour at the indicated multiplicity of infection (MOI) in Opti-MEM, after which the inoculum was supplemented with cell-specific complete medium. Cell fixation was performed using 2.5% paraformaldehyde for 15 minutes. Cell-membrane permeabilization was carried out using 0.2% Triton-PBS for 10 minutes and blocked in PBS 1% BSA for 1 hour. The samples were incubated with specific primary antibodies at 1:400 dilution for 1 hour at room temperature. Secondary antibodies (goat anti-chicken Alexa Fluor 568, goat anti-rabbit Alexa Fluor 488; purchased from Invitrogen) were used at a 1:800 dilution for 45 minutes prior to imaging using an EVOS FL cell imagine system (Thermo Fisher Scientific).

In Vitro Cell Viability MTT Assay
Cancer cells were cultured at a confluence of 80% in 24-well plates and infected with our APMV viruses in Opti-MEM at the indicated MOI for 1 hour; cells were then supplemented with complete media. After 24 hours of incubation, the infection media was removed and cells were incubated for 1 hour and 15 minutes with 300 μL of 2.5 mg/mL MTT at 37°C, under light-restricted conditions. Resulting formazan crystals were dissolved with 700 μL of Isopropanol through manual disturbance and a 10-minute, light-restricted incubation on a shaker. The absorbance of each sample was recorded at 570 nm using a BioTek plate reader.

Multistep Replication Kinetics
Cancer cells monolayers in 6-well plates were infected with the specified viruses at a MOI of 0.1 PFU/cell in OptiMEM-I. After 1 hour, the infection media was removed and cells were subsequently incubated with 3 mL of cell specific medium supplemented with 0.3% BSA and 1 μg/mL of TPCK-treated trypsin, to allow for production of fusion-competent viruses. Supernatants were collected at 24, 48, 72, and 96 hours postinfection and titrated by immunofluorescence assay on Vero cells using a polyclonal antiserum specific for each virus serotype.

Rechallenge Studies
A total of 5 × 10 5 cancer cells were engrafted into the contralateral hind leg of mice that shown long lasting complete responses (CR). Age-matching naïve mice were used as control for (i) tumor development and (ii) growth. No viral therapy was used in the postchallenge stage of these studies. Tumor volume was monitored every 48 hours, and mice were humanely euthanized on the day in which the volume exceeded the predefined endpoint (EPP) of 1,000 mm 3 .

Statistical Analysis
Data analysis was performed using GraphPad Prism 9. One-way ANOVA or two-way ANOVA were used to compare multiple groups with one or two independent variables, respectively. Results are expressed as mean value ± SEM or ± SD as indicated. Comparisons of survival curves were performed using the log-rank (Mantel-Cox) test. Survival analysis for each experimental group and study was carried out using the Kaplan-Meier method. P values > 0.05 were considered statistically nonsignificant (ns); *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Data Availability
The data generated in this study are available within the article and its Supplementary Data.

In Vivo Screening of Antitumor Capacity of Selected APMV Viruses in Murine B16-F10 Melanoma and CT26.WT Colon Carcinoma Tumor Models
The preliminary part of our work involved selecting a set of viruses from the 22 species included in the Avulavirinae subfamily. We selected isolates that comply with the biosafety requirements required for BSL-2 laboratory strains, have a confirmed complete genome sequence, lack pathogenicity in chickens and are able to be propagated in embryonated chicken eggs (31)(32)(33)(34)(35)(36)(37). Our final selection included prototype viruses from genus Metaavulavirus (APMV-2, -3, -6, -7, and -8), Orthoavulavirus (APMV-9), and Paraavulavirus (APMV-3 and APMV-4; Fig. 1B; Table 1). The antitumor capacity of selected APMV viruses was tested in vivo in syngeneic B16-F10 melanoma (Fig. 2) and CT26.WT murine colon carcinoma tumor models (Fig. 3).  oncolytic NDV LS-L289A virus was chosen as our reference for antitumoral activity and PBS mock-treated mice were used as our control group for tumor progression and survival.
In B16-F10 melanoma, all selected APMV viruses were able to restrain tumor growth during therapy (day 0 to day 6; Fig. 2B). Following treatment administration, APMV-7, APMV-8, and NDV LS-L289A treated-groups displayed comparable responses leading to a modest improvement on survival. APMV-4 treatment exhibited superior control over tumor growth, thus translating into the most significant benefit in extending survival (Fig. 2D).
In the CT26.WT colon carcinoma model, early suppression of tumor growth was observed in all the experimental groups leading to an overall benefit in survival when compared with the PBS control group (Fig. 3A and B). Sustained regressions that ultimately resolved into complete tumor elimination (CR, or complete response) were achieved by a subset of mice treated with APMV-2, -4, -6, -8, -9, and LS-L289A viruses (Fig. 3C)  further treatment on day 130 of the study. Naïve age-matching BALB/cJ mice were used as a control for tumor growth and survival (Fig. 3D)

Design and Development of a Recombinant APMV-4 Duck/Hong Kong/D3/1975 Virus by Reverse Genetics
APMV-4 demonstrated superior inherent oncolytic capacity in vivo when compared with other selected isolates, including the clinical candidate NDV LS-L289A. This solid response positioned the APMV-4 Duck/Hong Kong/D3/1975 isolate as the strongest candidate for follow-up. Accordingly, we generated an infectious clone of APMV-4 by designing a plasmid-based rescue strategy modeled after the already established system for NDV and other paramyxoviruses (39). Briefly, a pAPMV4 rescue vector containing a full-length antigenomic cDNA of the Duck/Hong Kong/D3/1975 isolate was generated following a multistep cloning strategy using purified viral RNA as template (Fig. 4A).
In parallel, the cloned cDNA was used to generate three helper vectors pTM-APMV4-N, pTM-APMV4-P, and pTM-APMV4-L, encoding the viral proteins N, P, and L, which conform the replication machinery of the virus (Fig. 4B). In both expression constructs, the cDNA is under the transcriptional control of a T7 promoter. To recover the infectious clone rAPMV-4, BSR-T7 cells preinfected with an attenuated MVA-T7 and then transfected with the rescue pAPMV4 and helper plasmids. A total of 24 hours after transfection, the cells and supernatants were inoculated into 8-day-old embryonated chicken eggs. Three days postinoculation, the allantoic fluid was harvested and the presence of infectious rAPMV4 was confirmed by immunofluorescence in cells infected with the harvested allantoic fluid (Fig. 4C).

In Vitro Characterization of APMV-4 Oncolytic Features
To assess the direct effect that APMV-4 has in tumor cells, we examined the replication capacity, cytotoxicity, and proinflammatory responses exerted by both WT and recombinant APMV-4 on melanoma and colon carcinoma cell lines of murine and human origin in vitro (Fig. 5). In these studies, mock-treated and NDV LS-L289A-treated cells were used as comparative references.
To peak titers in all the selected cancer cell lines (Fig. 5A-D). For both viruses, the highest viral titers were reached at 48 hours postinfection in the murine cancer cells lines (Fig. 5A and B) and at 72 hours in the human lines ( Fig. 5C and D). Two-way ANOVA statistical analysis showed significant differences in replicative fitness between APMV-4 and LS-L289A viruses in a time-dependent and cell-specific manner (Fig. 5A-D). At those specific timepoints, AMPV-4 replication outperformed the replication of the recombinant LS-L289A virus.
Virus-mediated cytotoxicity and proinflammatory responses were assessed on cancer cell monolayers infected at a MOI of 1 PFU/cell and incubated for up to 24 hours in absence of TPCK-trypsin, thus limiting the viral replication to a single cycle. Analysis of cell viability by MTT assay at 24 hours postinfection showed no differences in cytolytic activity between APMV-4 and rAPMV-4. Both viruses were able to reduce the viability of B16-F10, CT26.WT, and SK-MEL-2 cell cultures to 60% and to induce 10%-15% viability loss in RKO-E6 cells (Fig. 5E-H). SK-MEL-2 cells showed higher susceptibility to LS-L289A virus, being the only cancer cell line in which significant differences in cytolytic activity were observed between the APMV-4s (40% viability loss) and the LS-L289A virus (60% loss; Fig. 5G).  Fig. S1A and S1B) is indicative that normal cells can more efficiently counteract the progression of viral infection.

Discussion
The main aim of this work was to unveil members within the avian avulaviruses group with inherent antitumor capacity. Considering that the outcome of oncolytic virotherapy depends on the engagement of the host innate and adaptive immune systems (40), we opted for a straightforward in vivo antitumor capacity screening to investigate the therapeutic potential of various APMVs. In our studies, immunocompetent tumor-bearing mice were intratumorally inoculated with our selected isolates following a therapeutic strategy modeled after treatment regimens used by our group and others to study the efficacy of NDV (41)(42)(43). As our reference for anticancer activity, we employed the already characterized recombinant oncolytic NDV LaSota-L289A virus (24,29).
To identify APMVs with similar-or greater oncolytic capacity than the LS-L289A virus, we utilized all our viruses at a dose known to be suboptimal for NDV ( (Table 2). While all the selected APMVs demonstrated the ability to delay tumor growth during treatment administration ( Fig. 2A and B; Fig 3A and B), the analysis of long-term survival and complete remissions underlined the contrasting antineoplastic phenotypes displayed by individual APMVs depending on the type of cancer ( Fig. 2D and Fig. 3C). For example, the antitumor capabilities of the APMV-9 New York isolate-most closely related to NDV-elicited a response similar to PBS in melanoma but was capable of achieving 60% CR and protection against rechallenge in CT26.WT masses. Colon carcinoma tumors showed higher susceptibility to all APMVs (Fig. 3). This differential response to APMV treatment could be due to the contrasting genetic background of these cancers. CT26.WT cells are characterized by an oncogenic version of Kras, MEK (Mapk) amplification and high expression levels Nras (44). Previous works have demonstrated that hyperactivation of Ras/Raf/MEK/ERK signaling sensitizes cells to oncolytic NDV (45)(46)(47). The susceptibility caused by this feature seems to extend to other APMVs, as indicated by the remarkable performance of multiple isolates in the colon carcinoma model. Among the viruses included in these studies, the APMV-4 isolate has shown the greatest antitumor capacity in both cancer models, exceeding the therapeutic potential of all other selected viruses, including the NDV LS-L289A. The advantage of APMV-4 over the LS-L289A virus was further validated in subsequent studies, where experimental conditions were adapted to favor the response of LS-L289A virus by increasing the viral dose to 10 7 PFU (Fig. 6) and, additionally, where the initial tumor volume size was selected to be either more permissive (B16-F10 tumors; Fig. 6B-G) or more challenging (CT26.WT tumors; Fig. 6D-I) for the viruses to achieve complete therapeutic responses.
This isolate has typically been recovered from wild waterfowl worldwide, and AACRJournals.org Cancer Res Commun; 2(7) July 2022 occasionally from domestic ducks, geese, and chickens, although no clinical signs of disease were ever reported in these infected animals (49,50). This avirulent phenotype has been confirmed by experimental inoculations of birds (Table 2) and mammals (51). In our hands, intranasal administration of a high dose of APMV-4 (10 7 PFU) did not compromise the health of inoculated mice ( Supplementary Fig. S2). A complete genome sequence and molecular characterization of the Duck/Hong Kong/D3/1975 strain has been reported previously (32). However, little is known about the molecular biology involved in the virus-host cell interactions. APMV-4 s RBP HN protein has hemagglutinin and neuraminidase activities and is predicted to recognize sialic acids. Its F protein has a monobasic cleavage site (DIPQR↓F) that, although resembling those in avirulent lentogenic NDV strains, has been suggested to capacitate APMV-4 for multicycle replication in certain cell lines in vitro, despite not displaying a canonical furin cleavage site. Up till now, the molecular basis behind this observation remains unknown, and whether this phenomenon is limited to specific cell types or experimental conditions is still unclear. In our replication studies in cancer cells, we were only able to follow multicycle replication with the addition of exogenous TPCK-Trypsin to the infectious media. We did observe, however, that APMV-4 was able to reach higher titers than the LS-L289A virus while exhibiting similar growth kinetics (Fig. 5A-D). Considering all of the above, the distinct dependency of APMV4's F protein on proteolytic activation by either endogenous or secretory proteases could support these differences in viral fitness and, to some extent, be advantageous for the oncolytic activity of the virus in vivo.
In addition, APMV-4 has demonstrated its ability to trigger proinflammatory and death responses in infected cancer cells ( Fig. 5E-P). This oncolytic capability is known to lead to the initiation of a local inflammatory response in the tumor microenvironment necessary for the stimulation of systemic innate and tumor-specific immune responses by the host (38,40). When compared with NDV, we found APMV-4 to be a more potent immune stimulator, leading to an earlier and more robust upregulation of type-I IFN responses. Interestingly, this effect was preserved among the different cancer cells tested ( Fig. 5M-P) and is independent of the levels of viral replication (Fig. 5I-L). Because this distinctive proinflammatory response may be a critical driver of the enhanced therapeutic effect of APMV-4 in vivo, deciphering the molecular biology behind the immunostimulatory capacity of AMPV-4 will be a core aim of our future investigations. To that end, and to answer other questions regarding APMV-4-tumor interactions, we have set up a reverse genetics system for APMV-4 (Fig. 4). The recovered rAPMV-4 infectious clone has shown that it retains the biological behavior (Fig. 5) and inherent antitumor capacity of the natural isolate (Fig. 6).
In summary, our investigation into the antitumor capacity of APMVs has led us to the discovery of APMV-4 Duck/Hong Kong/D3/1975 OV, a novel cancer therapeutic that naturally displays greater antitumor potential than the clinical candidate NDV. From our results, and with technology that enables us to further design and develop improved recombinant therapeutics, the APMV-4 platform has positioned itself as a competitive candidate for translation into the clinic as an anticancer therapeutic for solid tumors. For that matter, rAPMV-4 has the potential to be modified (i) to express transgenes that could allow for the development of cancer-specific therapies, (ii) to express immunotherapeutic molecules, such a cytokines or chemokines, or (iii) to improve already established checkpoint blockade-based therapies. Furthermore, rAPMV-4 could be used to substitute standards of care like radiotherapy in (iv) in situ tumor vaccination approaches, as well as used in (v) combination with small-molecule immunostimulators.