Original Article | Published:

Intravenously injected Newcastle disease virus in non-human primates is safe to use for oncolytic virotherapy

Cancer Gene Therapy volume 21, pages 463471 (2014) | Download Citation

Abstract

Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.

Introduction

Newcastle disease virus (NDV) is a single-stranded negative-sense RNA virus belonging to the genus Avulavirus within the family of Paramyxoviridae.1 NDV infections were first recognized early in the twentieth century as a cause of high mortality and economic loss in the poultry industry.2 Consequently, prophylactic vaccination of poultry is currently applied on a large scale, and (suspected) outbreaks are rigorously acted upon.3 NDV strains vary widely in pathogenicity, which can be attributed mostly to the (multi)basic cleavage site of the fusion (F) protein.4,5 Strains can be classified into three pathotypes: lentogenic (low virulent, no mortality in susceptible hosts), mesogenic (intermediate virulent, <10% mortality) or velogenic (highly virulent viscerotropic or neurotropic, 10–100% mortality).6,7

Human infections with NDV have been observed after exposure to virus while handling infected birds or cadavers and lyophilized or aerosolized NDV vaccine. The associated symptoms have been described as mild: acute conjunctivitis and laryngitis, occasionally accompanied by low-grade fever and chills, with a rapid and spontaneous resolution.8, 9, 10, 11, 12, 13, 14 Human-to-human transmission has never been reported.

During the last decade, NDV has become a renewed focus of research in the field of oncolytic viruses.15,16 The tumor-specific replication and ability to induce a robust antiviral and antitumor immune response make the virus a good candidate for further development for virotherapy in cancer patients. Early clinical trials employing wild-type NDV strains for (intravenous) oncolytic therapy have shown NDV to be generally safe.17, 18, 19, 20, 21, 22, 23, 24 Several studies have demonstrated that increasing the virulence of recombinant NDV (rNDV) by editing the F protein cleavage site results in better oncolytic activity, but also in higher pathogenicity in chickens.25, 26, 27 Surprisingly, detailed information on preclinical safety testing of oncolytic NDVs is scarce, and a comparison of different (non-)virulent strains in non-human primate models has never been described.

In our efforts to further translate oncolytic NDV to the clinic, we evaluated the toxicity, biodistribution and shedding of oncolytic NDV after injection of non-human primates with three different strains. Two recombinant strains (rNDV), one lentogenic and one mesogenic, were compared with a commercially available clonal vaccine strain, because this vaccine strain has a long history of safe usage in the poultry industry. Similar to clinical trials that employed wild-type NDV strains for intravenous oncolytic therapy, we generated the viruses in embryonated chicken eggs.17, 18, 19, 20, 21, 22, 23, 24 In the midst of our experiments, it was reported that naive human serum is able to neutralize NDV generated in embryonated chicken eggs, in contrast to virus generated in human cells.28 This was explained by the fact that egg-generated viruses do not express human regulators of complement activity (CD46 and CD55) on their viral envelope and are therefore susceptible to neutralization by complement when incubated with non-homologous (for example, human or primate) serum. In the current study, we confirm that non-heat inactivated (non-HI) primate and human sera neutralized NDVs generated in embryonated eggs in vitro and we discuss the implications of these results for our study and the use of NDV for virotherapy.

Materials and methods

Cell lines and culture conditions

BSR-T7, Vero clone 118 (Vero-118) and MRC-5 cells were cultured as described before.29,30 All media and supplements were purchased from GIBCO (Life Technologies, Bleiswijk, the Netherlands).

Virus preparation

A full-length cDNA clone of lentogenic NDV strain La Sota (pNDV-F0) and expression plasmids pCIneo-NP, pCIneo-P and pCIneo-L were kindly provided by B Peeters from the Central Veterinary Institute of Wageningen UR, the Netherlands.6 To create a full-length NDV with multibasic cleavage site in the fusion protein, the amino-acid sequence of the protease cleavage site was changed from 112GRQGR↓L117 (lentogenic) to 112RRQRR↓F117 (mesogenic; pNDV-F3aa) by means of site-directed mutagenesis as described earlier.25 Recombinant viruses (rNDV-F0 and rNDV-F3aa) were rescued using a method adapted from the original method described previously.6 Briefly, BSR-T7 cells were transfected with 5 μg of pNDV-F0 or pNDV-F3aa, 2.5 μg of pCIneo-NP, 1.25 μg of pCIneo-P and 1.25 μg of pCIneo-L using 10 μl lipofectamine (Life Technologies). Three days later, 200 μl of BSR-T7 supernatant was injected into the allantoic cavity of 10-day-old specified pathogen-free embryonated chicken eggs. After incubation in a humidified egg incubator at 37 °C for 2 or 3 days (rNDV-F3aa or rNDV-F0, respectively), allantoic fluid was harvested and presence of virus demonstrated by hemagglutination assay, as described before.31 Samples displaying hemagglutination were passaged once more in eggs to increase virus titer, and allantoic fluid was harvested after 2 or 3 days. Fresh allantoic fluid was purified and concentrated by ultracentrifugation at 27 000 r.p.m. for 2 h at 4 °C using a 30%/60% sucrose gradient. Aliquots of purified rNDV were stored at −80 °C. Recombinant virus stocks were sequenced to confirm the sequence of the protease cleavage site of the fusion protein (F0/F3aa). Stocks were titrated by end-point dilution assay in Vero-118 cells, as described before.30

Additional rNDV-F0 and rNDV-F3aa stocks were grown in Vero-118 and MRC-5 cells. To this end, cells were infected with MOI 0.1 in the presence of 20 μg ml−1 trypsin (Lonza, Breda, the Netherlands) and virus was harvested after 3 days.

Non-recombinant plaque-purified (egg-generated) clonal vaccine strain AviPro ND C131 was obtained from Lohmann Animal Health (Cuxhaven, Germany). Vials containing 1 × 109 EID50 were reconstituted and diluted (if needed) in cold phosphate-buffered saline (PBS) immediately before use, and vials were used only once.

Ethics statement

All experiments involving animals were conducted strictly according to the European guidelines (EU directive on animal testing 86/609/EEC) and the Dutch legislation (Experiments on Animals Act, 1997). The experimental protocol was reviewed and approved by an independent animal experimentation ethical review committee, not affiliated with Erasmus MC (DEC consult number EMC2921).

Animals and experimental design

Nine juvenile (average age 5 years), male NDV-seronegative cynomolgus macaques (Macaca fascicularis) were used. All animal handling was performed under light ketamine/medetomidine anesthesia, and atipamezole was administered after handling to reverse the effect of medetomidine. Three weeks before the start of the experiment, a Data Storage Tag centi-Temperature probe (Star-Oddi, Brussels, Belgium) was implanted intraperitoneally, set to register temperature every 10 min. Figure 1 displays a detailed experimental timeline. Animals were housed in groups of three per isolator (A: AviPro ND C131; B: rNDV-F0; C: rNDV-F3aa). Two animals (inject 1 and 2) per group were injected i.v. into the posterior tibial vein with escalating viral doses (day 0: 1 × 107; day 1: 1 × 108; day 2: 1 × 109 EID50 or TCID50), the third animal (control) served as contact animal. Animal wellbeing was observed daily throughout the experiment, and the animals were weighed on days 0–4, 7, 14 and 21. Samples (see below) were collected just before and 2 h after injection on days 0, 1 and 2, and also once on days 3, 4, 7, 14 and 21. The two injected animals per group were killed on day 4 and the contact animal on day 21, and all animals underwent full necropsy.

Figure 1
Figure 1

Timeline of experimental setup. Time is depicted in days (d) and hours (h). Inject 1 and 2, inoculated animals 1 and 2; control, contact animal; S, sample time point; A, Avipro ND C131; B, rNDV-F0; C, rNDV-F3aa; †, euthanasia.

Samples

Eye, nose, throat and rectum swabs were collected in virus transport medium.32 After vortexing, 200 μl medium was used for RNA isolation and residual medium was stored at −80 °C for virus isolation later on.

Small volume blood samples were taken from an inguinal vein and collected into Vacuette Z Serum Sep Clot Activator and K3EDTA tubes (both from Greiner Bio One, Alphen aan de Rijn, the Netherlands). Clotted blood samples were centrifuged and 100 μl separated serum was assayed with Piccolo BioChemistry Panel Plus Reagent Discs (Abaxis, Darmstadt, Germany), which were processed using a Piccolo Xpress chemistry analyzer (Abaxis) following the manufacturer’s instructions. Measurements were obtained for glucose, blood urea nitrogen, creatinine, calcium, albumin, total protein, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyltransferase, amylase and C-reactive protein. Reference values (if indicated) were obtained from the supplemental data of a publication by Xie et al.33

Hundred microliters of EDTA blood (AviPro ND C131 and rNDV-F0 groups only) was tested using a pocH-100iV automated counter (Sysmex, Etten-Leur, the Netherlands). Owing to biosafety issues, these data could not be obtained for animals in the rNDV-F3aa group. Five hundred microliters of EDTA blood was incubated with 10 ml Red Blood Cell Lysis Buffer (Roche, Woerden, the Netherlands) for 10 min, centrifuged, washed with PBS and centrifuged again. The cellular pellet, representing mostly white blood cells, was resuspended in TRIzol (Life Technologies) and stored at −80 °C. Remaining EDTA blood was centrifuged to separate plasma, of which 200 μl was used for RNA isolation.

Necropsy

Animals were killed by exsanguination under deep anesthesia. All collected organ samples were transferred to tubes containing either 10% neutral-buffered formalin or RNAlater (Life Technologies), or were frozen without additives at −80 °C. Broncho-alveolar lavage was performed by direct infusion of PBS into the right main bronchus. Recovered broncho-alveolar lavage fluid was centrifuged and the cellular pellet resuspended in TRIzol. Organ samples in RNAlater were stored at −80 °C, thawed later and transferred to tubes containing a quarter inch ceramic sphere in virus transport medium. After homogenization using a FastPrep 24 tissue homogenizer (MP Biomedicals, Eindhoven, the Netherlands), samples were centrifuged and cleared supernatant was used for RNA isolation.

RNA isolation and quantitative reverse transcription-PCR

Samples stored in TRIzol were processed according to the manufacturer’s instructions to isolate RNA. Two hundred microliters of other samples (swabs, plasma and organ homogenates) were combined with 300 μl lysis buffer of the Total Nucleic Acid Isolation kit (Roche) and RNA was isolated in a volume of 50 μl using a MagNA Pure LC machine (Roche) following the manufacturer’s instructions.

NDV-specific quantitative reverse transcription-PCR was performed using 5 μl (TRIzol samples) or 19.5 μl (MagNA Pure samples) RNA in an ABI PRISM 7000 Sequence Detection System using TaqMan Fast Virus 1-Step Master Mix (both from Life Technologies) in a total volume of 30 μl. The NDV-specific primers used were described by Wise et al.34 The reverse transcriptase step was 5 min at 50 °C, followed by 95 °C for 20 s. Cycling consisted of 45 cycles of 3 s denaturation at 95 °C, 5 s annealing at 54 °C and 31 s extension at 60 °C.

Virus isolation

A volume of 200 μl medium from collected swabs or 200 μl supernatant of homogenized dry frozen organs was injected in duplicate into 10-day-old specified pathogen-free embryonated chicken eggs. After 2 or 3 days (samples containing rNDV-F3aa or Avipro ND C131 and rNDV-F0, respectively), allantoic fluid was harvested and tested for the presence of virus by hemagglutination assay.

NDV serology

Sera were tested for NDV-specific antibodies by hemagglutination inhibition assay using specified pathogen-free turkey erythrocytes.31 To neutralize neuraminidase activity, serum samples were pretreated with cholera filtrate for 16 h at 37 °C, followed by inactivation for 1 h at 56 °C. Diluted chicken polyclonal anti-NDV antibody was used as positive control.

Histopathology

Samples for histological examination were stored in 10% neutral-buffered formalin (lungs after inflation with formalin), embedded in paraffin, sectioned at 4 mm and stained with hematoxylin and eosin for examination by light microscopy. The following tissues were examined by light microscopy for the presence of histopathological changes: lungs, primary bronchus, trachea, liver, spleen, kidney, nasal septum, nasal concha, eye and conjunctiva.

In addition, separate sections were stained for NDV using an immunoperoxidase method. Tissue sections were mounted on coated slides (KliniPath, Duiven, the Netherlands), deparaffinized, rehydrated and endogenous peroxidase was blocked by incubating slides in 3% H2O2 in bidest for 10 min. Antigen was retrieved by boiling slides in Tris buffer (pH 9) for 15 min and sections were subsequently washed in PBS containing 0.05% Tween-20. Slides were incubated in PBS with 0.1% BSA (Aurion, Wageningen, the Netherlands) for 10 min at room temperature. After this, slides were incubated with monoclonal mouse anti-NDV antibody (HyTest, Turku, Finland) in PBS with 0.1% BSA for 1 h at room temperature. Mouse IgG2a (R&D Systems, Abingdon, UK) was used as isotype control. After washing, slides were incubated with goat anti-mouse antibody (Southern Biotech, Birmingham, AL, USA) labeled with horseradish peroxidase for 30 min at room temperature. horseradish peroxidase activity was revealed by incubating slides in 3-amino-9-ethylcarbazole in N,N-dimethylformamide for 10 min. Slides were imbedded in Imsolmount and overlayed with Pertex (both from KliniPath). Brain tissue from a cormorant (Phalacrorax auritus) known to be infected with virulent NDV was used as positive control.

Virus neutralization assay

Virus neutralization assays were performed as described previously.35 Briefly, three human sera and three naive primate sera were left untreated (non-HI) or heat inactivated (HI) for 30 min at 56 °C. Twofold serial dilutions of the sera starting at a 1:10 dilution were mixed 1:1 with 100 TCID50 of rNDV. After incubation at 37 °C for 1 h, the serum–rNDV mixture was transferred to 96-well plates containing Vero-118 cells. Plates were incubated for 1 h at 37 °C, and inoculum was replaced by 200 μl fresh medium. After 6 days, end-point dilutions were read by scoring cytopathic effect.

Results

To compare the safety of three different oncolytic NDVs, two macaques per group were injected i.v. with lentogenic Avipro ND C131, lentogenic rNDV-F0 or mesogenic rNDV-F3aa (Figure 1). A dose-escalation design was chosen to observe potential toxicity at different doses. To study transmission, naive animals were co-housed with the injected animals.

Clinical and vital signs

During the experiments, none of the injected animals displayed behavioral changes or overt signs of illness. Specifically, no conjunctivitis or ocular, oral or nasal discharge was observed. Animals were lively and displayed normal appetite similar to the period before the start of the experiment. No change in stool consistency or color was observed. All injected animals maintained their body weight (Figure 2a).

Figure 2
Figure 2

Clinical parameters recorded throughout the experiment. (a) Animals were weighed at indicated time points. (b) Body temperature was recorded using an intraperitoneally implanted probe (Star-Oddi). (c) Detailed view of body temperature just after third Newcastle disease virus injection. Blue circles and green squares: inoculated animals 1 and 2; red triangles: contact animal. d.p.i., days post first inoculation; h.p.i., hours post first inoculation. Black box in b: area of detailed view in c.

Animals injected with Avipro ND C131 had a short-term fever peak higher than 40 °C after injection with the highest dose (1 × 109 50% egg infectious dose (EID50)) on day 2, which decreased rapidly the same day (Figures 2b and c). Animals injected with the medium (1 × 108 50% tissue culture infectious dose (TCID50)) and highest dose (1 × 109 TCID50) of rNDV-F3aa also had a short-term fever peak after injection on days 1 and 2 with a maximum around 39.5 °C, which also decreased rapidly (Figures 2b and c). Sharp decreases in registered temperature during the first 5 days can be attributed to the general anesthesia animals underwent for sampling and injection.

Similar to the injected animals, none of the contact animals displayed signs of illness. All contact animals maintained their body weight up to 3 weeks after the start of the experiment (Figure 2a). In contrast to the injected animals, none of the contact animals had an abnormal increase of body temperature during the experiment (Figures 2b and c; data after day 4 not shown).

Hematology and serum chemistry

Hemoglobin concentration in all injected animals dropped from an average of 10.0 mmol l−1 at the start of the experiment, to an average of 7.1 mmol l−1 at day 4, which was still within normal limits (Figure 3a; reference value 6.7–9.7 mmol l−1).33 This decrease was probably due to the blood sampling during the first few days. Leukocyte counts were on the low side of normal and slightly lower than normal in one of the animals injected with rNDV-F0, however, no overt leukocytopenia or leukocytosis was observed (Figure 3b). Platelet counts did not show any abnormalities (Figure 3c).

Figure 3
Figure 3

Hematological parameters for animals injected with lentogenic viruses and their co-housed contact animals. Owing to biosafety issues, these parameters could not be obtained for animals injected with rNDV-F3aa. EDTA blood samples were analyzed using an automated counter (Sysmex pocH-100iV) to obtain a basic blood profile. Blue circles and green squares: inoculated animals 1 and 2; red triangles: contact animal. d.p.i., days post first inoculation. Reference values33 are indicated by the gray area between the dotted lines.

In all injected animals, kidney function, as measured by blood urea nitrogen and creatinine concentration, did not deteriorate during the experiment (Figures 4a and b). Animals injected with Avipro ND C131 had slight increases of liver enzyme concentrations, with a maximum alanine aminotransferase of 94 U l−1 (reference value: 11–78 U l−1)33 and aspartate aminotransferase of 184 U l−1 (reference value: 23–71 U l−1)33, which seemed to decrease spontaneously on day 4 (Figures 4c and d). Animals injected with rNDV-F0 or rNDV-F3aa also had slight increases in liver enzyme concentrations, but much less than animals injected with Avipro ND C131. Gamma glutamyltransferase values did not increase during the experiment, although they were generally higher than the reference values (Figure 4e; reference value: 18–65 U l−1).33 A peak in serum amylase concentration was detected in one animal injected with Avipro ND C131, whereas other injected animals had no abnormalities in their serum amylase concentrations (Figure 4f). C-reactive protein was detected (detection limit >5 mg l−1) in multiple serum samples of injected animals, but no peaks in concentration were observed (Figure 4g). As there are no reference values for amylase or C-reactive protein measurements in cynomolgus macaques, it is difficult to evaluate the measurements obtained. Other serum chemistry markers (glucose, calcium, albumin, total protein and alkaline phosphatase) were not found to be abnormal during the course of the experiment (data not shown).

Figure 4
Figure 4

(ag) Serum chemistry parameters in samples taken just before injection. Samples taken at indicated time points were assayed using an automated serum chemistry analyzer (Piccolo Xpress) to obtain a basic serum chemistry profile. Blue circles and green squares: inoculated animals 1 and 2; red triangles: contact animal. ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; BUN, blood urea nitrogen; CRP, c-reactive protein; d.p.i., days post first inoculation; GGT, gamma glutamyltransferase. Reference values33 are indicated by the gray area between the dotted lines.

Hematologic parameters obtained for contact animals showed the same pattern as obtained from the injected animals: slight decreases in hemoglobin concentration over time and normal leukocyte and platelet counts (Figure 3). Biochemistry values obtained for the contact animals revealed that blood urea nitrogen and creatinine, did not increase during the experiment (Figures 4a and b). In contrast with animals injected with Avipro ND C131, the contact animal did not show elevated liver enzymes (Figures 4c and d). The contact animal in the rNDV-F3aa group showed slightly elevated liver enzymes, whereas injected animals did not (Figures 4c and d). Gamma glutamyltransferase and amylase measurements did not show abnormalities (Figures 4e and f). Finally, there was a peak observed in C-reactive protein concentration in the contact animal in the rNDV-F3aa group of 86 mg l−1 (Figure 4g).

Virological data

All injected animals had detectable NDV-RNA in their plasma and white blood cells immediately after injection of the lowest dose until euthanasia on day 4 (Figure 5a). Animals injected with either virus showed shedding of viral RNA from eyes, nose and throat, but not from their rectum, starting around the time of injection of the medium dose, with a peak after injection of the highest dose (Figure 5b). A low number of quantitative reverse transcription-PCR-positive swab samples (almost exclusively those taken on day 2) were also found positive in virus culture, without a correlation between Ct value and culture results.

Figure 5
Figure 5

Virological parameters from samples taken from animals just before and 2 h after injection. Blood (a) and swab (b) samples were collected at indicated time points. Total RNA was isolated and Newcastle disease virus (NDV)-specific quantitative reverse transcription-PCR (qRT-PCR) was performed on all samples (positive: +, negative: −). qRT-PCR-positive swab samples were cultured in specified pathogen-free (SPF) embryonated chicken eggs (positive = green, negative = red, not cultured = blank). A, Avipro ND C131; B, rNDV-F0; C, rNDV-F3aa; c, contact animal; d, days post first inoculation; h, hours; i1 and i2, inoculated animals; WBC, white blood cell.

A few samples collected from the eyes, nose and throat of the contact animals were found to be positive for NDV-RNA (Figure 5b). However, viable virus was isolated only once from a throat swab (rNDV-F0, day 2+2 h). Sporadically, the contact animals also had detectable NDV-RNA in plasma or white blood cell samples (Figure 5a).

Hemagglutination inhibition assay conducted on sera collected from the contact animals 21 days after exposure revealed that contact animals had not seroconverted (data not shown).

Necropsy and histopathology

Samples of organs collected upon necropsy were tested for the presence of NDV-RNA with quantitative reverse transcription-PCR. I.v. injection with all three strains of NDV resulted in a systemic distribution of viral RNA mainly to the (upper and lower) respiratory tract, spleen, liver and kidney (Figure 6). No differences in distribution were observed between the three groups (Avipro ND C131 vs rNDV-F0 vs rNDV-F3aa). Virus was not isolated from the RNA-positive samples.

Figure 6
Figure 6

Virological parameters at necropsy. Organs were collected on day 4 (inoculated animals) or day 21 (contact animal) post first inoculation. Tissue samples were homogenized and supernatant was used to isolate total RNA. Newcastle disease virus (NDV)-specific quantitative reverse transcription-PCR (qRT-PCR) was performed on all samples (positive: +, negative: −). qRT-PCR-positive swab samples were cultured in specified pathogen-free (SPF) embryonated chicken eggs (negative=red, not cultured=blank). A, Avipro ND C131; B, rNDV-F0; BAL. bronchi-alveolar lavage; C, rNDV-F3aa; c, contact animal; i1 and i2, inoculated animals; LN, lymph node; RLL, right lower lobe; RUL, right upper lobe; TB, tracheo-bronchiolar.

No gross pathological changes were observed at necropsy of injected animals. The following incidental lesions were detected upon light microscopy: mild acute focal suppurative bronchoadenitis in one animal injected with Avipro ND C131 and mild acute focal suppurative rhinitis with hemorrhage in one animal injected with rNDV-F3aa. Otherwise, no microscopic lesions were detected in any of the evaluated tissues of any of the injected macaques. Immunohistochemical staining for NDV revealed no infected cells in tissues that were positive for NDV-RNA. None of the rare incidental lesions detected were considered to be related to the treatment.

One incidental lesion was detected in a contact animal by light microscopy: mild acute focal alveolar hemorrhage in the animal in the rNDV-F0 group, considered not to be related to exposure to virus. Immunohistochemical staining for NDV again revealed no infected cells in evaluated tissues. None of the organs collected from contact animals on day 21 tested positive for NDV-RNA (Figure 6).

Virus neutralization

Virus neutralization assays were conducted with virus stocks generated in embryonated chicken eggs and in human or primate cells. These viruses were tested against a panel of human and primate sera (Table 1). This experiment revealed that indeed non-HI naive human and primate sera, but not HI sera, neutralized rNDVs generated in embryonated chicken eggs. This was in contrast with rNDVs generated in primate (Vero-118) or human (MRC-5) cells, which managed to escape neutralization by non-HI naive human and primate sera.

Table 1: Virus neutralization assay using rNDVs generated in eggs, MRC-5 or Vero-118 cells.

Discussion

Limited information is available on preclinical biosafety regarding administration of oncolytic NDV in a human-like animal model. Therefore, we carried out a safety study comparing the toxicity, biodistribution and shedding of NDV strains differing in virulence in non-human primates.

A non-recombinant, commercially available vaccine strain was included, as this virus has a long history of safe usage in vaccinating poultry. Two other (recombinant) strains were tested as they are candidates for future virotherapy with oncolytic NDV, and are suitable for further development using reverse genetics. These two rNDVs differed in virulence as defined by their F protein cleavage site. Considering virulent NDV strains as potential oncolytic viruses, it is also important to know whether the virus is capable of spreading to the environment, as this could lead to accidental infection of susceptible hosts, and could thus also pose a serious threat to the poultry industry or domestic bird species.

We found that i.v. injection of a high dose of all three strains was safe for the animals. No clinical signs of illness were observed in the vaccine or recombinant virus-injected animals, except short-term fever in animal injected with the highest dose Avipro ND C131 and rNDV-F3aa. Hematologic and serum chemistry parameters were mostly within normal range, with the exception of slightly elevated liver enzymes upon Avipro ND C131 injection. This could be related to the presence of preservatives and lyophilization products in vials of this vaccine strain, specifically peptone, magnesium sulphate, sucrose or gelatin. We hypothesize that other incidental findings of abnormalities in measured biochemistry parameters are most likely unrelated to the experiment. Owing to biosafety issues, we were not able to measure hematologic parameters for animals injected with rNDV-F3aa. However, it is unlikely that these parameters would differ much from the measurements obtained from animals injected with non-virulent strains, as there was no indication for increased pathogenicity (see below) of the virulent strain in this non-human primate model.

Analysis of the distribution of NDV showed that i.v. injection resulted in systemic spread, with most of the viral RNA ending up in the respiratory tract, spleen and liver. Histological examination, however, did not reveal infected cells in any of the examined organs, indicating that substantial virus replication in these organs did not occur.

We observed shedding of virus from injected animals, irrespective of the strain used and we cannot exclude transmission of virus. However, productive infection of the contact animals did not occur, as no seroconversion was detected. Earlier studies of human infection with NDV reported mostly on workers in the poultry industry having contact with live virus either when handling infected or dead poultry or lyophilized or aerosolized vaccine strains.8, 9, 10, 11, 12, 13, 14 The exposure of these human cases was probably to a higher dose than the contact animals in the present study, as they developed symptoms within a couple of days and seroconverted 1–2 weeks after exposure. We therefore argue that contact animals in the present study were not exposed to high concentrations of shed NDV, as they did not develop any symptoms nor did they seroconvert after 3 weeks of observation. Nonetheless, appropriate biosafety measures preventing environmental spread have to be considered when administrating high-dose oncolytic NDV.

Preclinical studies employing NDV as a vaccine vector using subcutaneous, intranasal and/or intratracheal administration showed that NDV as vaccine vector is highly attenuated in non-human primates.36, 37, 38, 39, 40, 41 However, it is difficult to compare these studies with the one we present here, as these animals were not i.v. inoculated and the viral doses administered in these vaccine studies were relatively low.

Several clinical phase I/II trials have been described previously, in which high-dose wild-type oncolytic NDV was injected i.v. into patients with advanced and/or metastatic solid cancer.17, 18, 19, 20, 21, 22, 23, 24 These trials employed naturally occurring, in vitro selected egg-grown oncolytic strains of NDV. Viral doses used in these clinical studies were comparable to the dose administered in the present study: up to 34–120 × 109 EID50 m−2 (body surface) as compared with an equivalent dose of 4 × 109 TCID50 m−2 recombinant virus in our study, noting that egg titration is generally about 10-fold more sensitive than tissue culture titration.20,21,42 A study recruiting 14 patients suffering from glioblastoma multiforme showed that i.v. injection of a high dose of the lentogenic oncolytic strain NDV-HUJ was safe, with grade I-II fever as only adverse event.20 Infectious virus was recovered from blood, saliva and urine samples up to 96 h after injection. Two other phase I/II studies with a total of 95 patients showed that i.v. injection of a virulent NDV strain (PV701) was safe in patients with advanced or metastatic solid tumors.18,21 Common side effects included fever, diarrhea and slight transient elevation of liver transaminases (but only in patients with hepatic metastases). Shedding of injected virus was also observed up to 3 weeks after injection. Measures to prevent environmental spread of this virulent NDV strain were not described.18,21 Overall, the results of our present preclinical safety study with (recombinant) NDV strains corresponds well with the results previously reported on phase I/II clinical trials.

During the execution of our animal experiments, it was reported that NDV generated in embryonated chicken eggs or non-human cells is susceptible to neutralization by human serum through complement binding and activation.28 This was clearly an important finding related to our experiment. Upon testing, we confirmed that non-HI primate and human sera neutralized egg-generated NDV, in contrast to NDV generated in primate or human cells. All clinical trials thus far have used egg-generated virus stocks and still reported positive effects for NDV virotherapy as well as shedding of infectious virus, indicating that egg-generated NDV is still capable of replication.17, 18, 19, 20, 21, 22, 23, 24 The efficacy of NDV virotherapy might increase when using virus stocks generated in human or primate cells. However, neutralization by complement might also be important for safe administration of high doses of NDV, and abolishing this could result in more toxicity and a higher risk of shedding to the environment. More information should therefore be obtained on the safety and oncolytic efficacy of virotherapy with NDV stocks generated in human or primate cells.

Our data in a non-human primate model using recombinant avirulent and virulent NDV strains corroborate with the data from clinical trials with wild-type strains. Based on the lack of virus replication in organs, the absence of NDV-related lesions and hematologic or biochemistry abnormalities, we conclude that the i.v. administration of oncolytic NDV generated in eggs is safe, even when using high doses of a virulent strain, when taking appropriate biosafety measures to prevent environmental spread. Future research has to elucidate whether NDV generated in human or primate cells has a similar safety profile and oncolytic efficacy.

References

  1. 1.

    . A summary of taxonomic changes recently approved by ICTV. Arch Virol 2002; 147: 1655–1663.

  2. 2.

    . Over een in Ned.-Indië heerschende ziekte onder het pluimvee. Nederlands-Indische bladen voor Diergeneeskunde 1926; 38: 448–450.

  3. 3.

    Council of the European Communities. Council Directive 92/66/EEC of 14 July 1992 introducing Community measures for the control of Newcastle disease. In. , 1992.

  4. 4.

    , , . Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity. Arch Virol 1993; 128: 363–370.

  5. 5.

    , , , . Virulence of newcastle disease virus: what is known so far? Vet Res 2011; 42: 122.

  6. 6.

    , , , . Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol 1999; 73: 5001–5009.

  7. 7.

    OIE: World Organisation for Animal Health Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. In. , 2012.

  8. 8.

    . Conjunctival haemorrhage due to an infection of Newcastle virus of fowls in man; laboratory and contact infection. Br J Ophthalmol 1946; 30: 260–264.

  9. 9.

    , . Human infection with the Newcastle virus of fowls. AMA Arch Ophthalmol 1950; 44: 573–580.

  10. 10.

    , , , , . An outbreak of conjunctivitis due to Newcastle disease virus (NDV) occurring in poultry workers. Am J Public Health Nations Health 1952; 42: 672–678.

  11. 11.

    , , , . Newcastle disease virus in man; results of studies in five cases. J Lab Clin Med 1952; 40: 736–743.

  12. 12.

    . Pathogenicity and immunology of Newcastle disease virus (NVD) in man. Am J Public Health Nations Health 1955; 45: 742–745.

  13. 13.

    , . Outbreaks of conjunctivitis due to the Newcastle disease virus among workers in chicken-broiler factories. Br Med J 1965; 2: 1514–1517.

  14. 14.

    , . Newcastle disease conjunctivitis with subepithelial infiltrates. The Br J Ophthalmol 1973; 57: 694–697.

  15. 15.

    . Oncolytic viruses: an approved product on the horizon? Mol Ther 2010; 18: 233–234.

  16. 16.

    , . Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions. Future Microbiol 2012; 7: 347–367.

  17. 17.

    , . Use of Newcastle disease virus vaccine (MTH-68/H) in a patient with high-grade glioblastoma. JAMA 1999; 281: 1588–1589.

  18. 18.

    , , , , , et al. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J Clin Oncol 2002; 20: 2251–2266.

  19. 19.

    , , , , , et al. MTH-68/H oncolytic viral treatment in human high-grade gliomas. J Neurooncol 2004; 67: 83–93.

  20. 20.

    , , , , , et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol Ther 2006; 13: 221–228.

  21. 21.

    , , , , , et al. A phase 1 clinical study of intravenous administration of PV701, an oncolytic virus, using two-step desensitization. Clin Cancer Res 2006; 12: 2555–2562.

  22. 22.

    , , , , , et al. Combined treatment of pediatric high-grade glioma with the oncolytic viral strain MTH-68/H and oral valproic acid. APMIS 2006; 114: 731–743.

  23. 23.

    , , , , , et al. An optimized clinical regimen for the oncolytic virus PV701. Clin Cancer Res 2007; 13: 977–985.

  24. 24.

    , , , , , et al. Phase 1 clinical experience using intravenous administration of PV701, an oncolytic Newcastle disease virus. Curr Cancer Drug Targets 2007; 7: 157–167.

  25. 25.

    , , , , , et al. Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus. Cancer Res 2007; 67: 8285–8292.

  26. 26.

    , , , . Engineered newcastle disease virus as an improved oncolytic agent against hepatocellular carcinoma. Mol Ther 2010; 18: 275–284.

  27. 27.

    , , , , , et al. Velogenic Newcastle disease virus as an oncolytic virotherapeutics: in vitro characterization. Appl Biochem Biotechnol 2012; 167: 2005–2022.

  28. 28.

    , , , , . Incorporation of host complement regulatory proteins into Newcastle disease virus enhances complement evasion. J Virol 2012; 86: 12708–12716.

  29. 29.

    , , , , , et al. Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA. J Virol 2004; 78: 8264–8270.

  30. 30.

    , , , , . Different responses of human pancreatic adenocarcinoma cell lines to oncolytic Newcastle disease virus infection. Cancer Gene Ther 2014; 21: 24–30.

  31. 31.

    . The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J Exp Med 1942; 75: 49–64.

  32. 32.

    , , , , , et al. Practical considerations for high-throughput influenza A virus surveillance studies of wild birds by use of molecular diagnostic tests. J Clin Microbiol 2009; 47: 666–673.

  33. 33.

    , , , , , et al. Age- and sex-based hematological and biochemical parameters for Macaca fascicularis. PLoS One 2013; 8: e64892.

  34. 34.

    , , , , , et al. Development of a real-time reverse-transcription PCR for detection of newcastle disease virus RNA in clinical samples. J Clin Microbiol 2004; 42: 329–338.

  35. 35.

    , , , , , et al. Influenza virus subtype cross-reactivities of haemagglutination inhibiting and virus neutralising serum antibodies induced by infection or vaccination with an ISCOM-based vaccine. Vaccine 1999; 17: 2512–2516.

  36. 36.

    , , , , , et al. Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates. J Virol 2005; 79: 13275–13284.

  37. 37.

    , , , , , et al. Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens. Proc Natl Acad Sci USA 2007; 104: 9788–9793.

  38. 38.

    , , , , , et al. Immunization of primates with a Newcastle disease virus-vectored vaccine via the respiratory tract induces a high titer of serum neutralizing antibodies against highly pathogenic avian influenza virus. J Virol 2007; 81: 11560–11568.

  39. 39.

    , , , , , et al. Delivery to the lower respiratory tract is required for effective immunization with Newcastle disease virus-vectored vaccines intended for humans. Vaccine 2009; 27: 1530–1539.

  40. 40.

    , , , , , . Respiratory tract immunization of non-human primates with a Newcastle disease virus-vectored vaccine candidate against Ebola virus elicits a neutralizing antibody response. Vaccine 2010; 29: 17–25.

  41. 41.

    , , , , , et al. Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian influenza virus protect against virus challenge in monkeys. J Virol 2010; 84: 1489–1503.

  42. 42.

    , , , , . Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey, and man. Cancer Chemother Rep 1966; 50: 219–244.

Download references

Acknowledgements

This study was supported by the Virgo consortium, funded by the Dutch government project number FES0908, and by the Netherlands Genomics Initiative (NGI) project number 050-060-452. We thank B Peeters (Central Veterinary Institute of Wageningen UR, Lelystad, the Netherlands) for providing plasmids for recombinant NDV. We thank T Leighton (Canadian Cooperative Wildlife Health Centre, Saskatoon, Canada) for providing a positive control NDV-infected tissue. We thank V Vaes and D de Meulder (EDC Erasmus MC, Rotterdam, the Netherlands) for assisting with animal housing and experiments.

Author information

Affiliations

  1. Department of Surgery, Erasmus MC, Rotterdam, The Netherlands

    • P R A Buijs
    •  & C H J van Eijck
  2. Viroclinics Biosciences, BV, Rotterdam, The Netherlands

    • G van Amerongen
  3. Department of Viroscience, Erasmus MC, Rotterdam, The Netherlands

    • S van Nieuwkoop
    • , T M Bestebroer
    • , P R W A van Run
    • , T Kuiken
    • , R A M Fouchier
    •  & B G van den Hoogen

Authors

  1. Search for P R A Buijs in:

  2. Search for G van Amerongen in:

  3. Search for S van Nieuwkoop in:

  4. Search for T M Bestebroer in:

  5. Search for P R W A van Run in:

  6. Search for T Kuiken in:

  7. Search for R A M Fouchier in:

  8. Search for C H J van Eijck in:

  9. Search for B G van den Hoogen in:

Competing interests

The authors declare no conflict of interest.

Corresponding author

Correspondence to B G van den Hoogen.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/cgt.2014.51

Further reading