Biodistribution and toxicology evaluation of a recombinant measles Schwarz‐based Lassa vaccine in cynomolgus macaques

MV‐LASV is an investigational measles Schwarz‐based vaccine for the prevention of Lassa fever. A repeated‐dose toxicity study in cynomolgus macaques was performed to assess the biodistribution and local and systemic toxicological effects. Monkeys received three immunizations of MV‐LASV or saline intramuscularly with a 2‐week interval. An increase in anti‐measles antibodies confirmed the reaction of the immune system to the vaccine backbone. Clinical observations, body weight, body temperature, local tolerance, electrocardiogram parameters, various clinical pathology parameters (hematology, coagulation urinalysis, serum chemistry, and C‐reactive protein) were monitored. Gross pathology and histopathology of various tissues were evaluated. MV‐LASV induced a mild increase in fibrinogen and C‐reactive protein concentrations. This coincided with microscopic inflammation at the injection sites which partially or fully resolved following a 3‐week recovery period. Viral RNA was found in secondary lymphoid organs and injection sites and gall bladder. No viral shedding to the environment was observed. Overall, the vaccine was locally and systemically well tolerated, supporting a first‐in‐human study.


| INTRODUCTION
Lassa fever (LF) is a viral hemorrhagic illness caused by a negative stranded RNA virus of the family Arenaviridae, the Lassa virus (LASV) (Buchmeier et al., 2013). The pathogen represents a major threat in countries of Western Africa including Nigeria, Guinea, Liberia, Sierra Leone, Togo, and Benin where it is endemic (McCormick et al., 1987;Yadouleton et al., 2020). Usually, humans become infected through contact with a peridomestic rodent of the genus Mastomys, which acts as the reservoir host (McCormick et al., 1987;Woyessa et al., 2019). In addition, human-to-human transmission has been observed in nosocomial settings through contact with body fluids of infected patients (Edington & White, 1972). The severity of LF ranges from asymptomatic infection to fatal hemorrhagic fever, with severe sequelae such as hearing deficits reported in up to one third of survivors (Cummins et al., 1990). The virus causes between 100,000 and 300,000 estimated cases and 5000 infections each year (Gunther & Lenz, 2004). Although a vaccination would be most effective in controlling the spread of LF, there are currently no licensed vaccines on the market. The only treatment available is based on ribavirin. However, this molecule is not readily available in the countries in which LF is endemic, and its efficacy is controversial (Merson et al., 2021;Murphy & Ly, 2021). To accelerate vaccine development, LF was included in the WHO's Research and Development (R&D) blueprint list of priority pathogens. In addition, the Coalition of Epidemic Preparedness Innovations (CEPI) prioritized the need for the development of vaccines against LF/LASV and supported various preventive Lassa vaccine projects including the candidate investigated here.
Several LASV vaccine candidates, almost exclusively based on the prototypic LASV Josiah strain (Clade IV), are developed using different approaches, such as inactivated or live attenuated viruses, deoxyribonucleic acid (DNA) vaccines, various vectors, recombinant proteins, or nanocarriers bearing recombinant envelope glycoprotein (Salami et al., 2019). As the glycoprotein GPC is the only protein exposed on the virion surface and thus the primary target of humoral immunity, most development efforts have focused on this antigen. However, in seropositive individuals from endemic regions, LASV-specific CD4 + Tcells recognizing epitopes in both GPC and the nucleoprotein NP were identified (Meulen et al., 2004;ter Meulen et al., 2000). Consequently, selected vaccine candidates have included both GPC and NP antigens. While GPC is required for receptor binding and viral fusion with membranes (Pennington & Lee, 2022), NP encapsulates the viral genome, is involved in viral replication and transcription (Pinschewer et al., 2003), and acts as an immune suppressor by blocking interferon (IFN) signaling (Hastie et al., 2011).
MV-LASV was the first LASV vaccine candidate awarded CEPI funding for clinical development. It is a live attenuated recombinant measles Schwarz vectored vaccine virus that was originally constructed by Institut Pasteur Lyon (Mateo et al., 2019). The measles genome was modified to encode two heterologous antigens derived from the LASV strain Josiah-GPC and a modified NP. The NP contains two amino acid changes in order to block its exonuclease activity which interferes with IFN I induction in antigen presenting cells (Martinez-Sobrido et al., 2007). Of note, a vaccine candidate for the prevention of Chikungunya fever based on the same backbone was previously evaluated in Phase 1 and 2 clinical studies, showing a favorable safety and immunogenicity profile (Ramsauer et al., 2015;Reisinger et al., 2018).
The immunogenicity and efficacy of MV-LASV was assessed in a series of nonhuman primate (NHP) studies, demonstrating the candidate protects cynomolgus macaques from lethal challenge with homologous and heterologous LASV strains even when the vaccine candidate was administered up to a year before challenge (Mateo et al., 2019(Mateo et al., , 2021. Prior to enter clinic, we evaluated the toxicity and biodistribution of MV-LASV and conducted a study in cynomolgus macaques under Good Laboratory Practices (GLP) conditions, the results of which are discussed here. MV-LASV administered intramuscularly (IM) induced no local or systemic toxicity and showed tissue distribution comparable with the parental measles Schwarz vaccine, supporting the initiation of a first-in-human study.

| Study design
The repeated-dose toxicity study and the biodistribution assessment  , 2015;Reisinger et al., 2018) and dose levels previously tested in a NHP efficacy study (Mateo et al., 2019). A three-dose schedule was used to represent the number of doses plus one to be used in the human clinical trial. Details on respective experimental procedure, examination and analysis can be found in the sections below.

| Test item: MV-LASV
MV-LASV is a measles-based prophylactic vaccine candidate for the prevention of LF. The measles virus (MV) vector pTM-MVSchwarz which was used the generate MV-LASV has been described previously (Combredet et al., 2003).

| Control item
The control article was 0.9% sodium chloride for Injection (saline).

| Animals
A total of 10 male and 10 female experimentally naïve cynomolgus monkeys approximately 25 to 33 months at transfer were transferred from the stock colony. The animals were originally received from Worldwide Primates Inc. (USA). All animals were prescreened by the vendor prior study allocation and were tested measles negative. The country of origin of all monkeys was China. Animals were quarantined upon arrival. During the 16-day acclimation period, the animals were observed daily with respect to general health and any signs of disease.

| Administration
The test article was provided ready to use. A daily qualitative assessment of food intake/appetite was performed for all animals as part of the cage-side observations. Quantitative food consumption measurements were not conducted.
Electrocardiographic examinations were performed on all animals pretreatment (2-week period prior first immunization), on Day 10, within 3 days prior to the terminal necropsy, and within 3 days prior to the recovery necropsy. Standard electrocardiograms (ECGs) were recorded at 50 mm/s. Using an appropriate lead the RR, PR, and QT intervals, and QRS duration were measured, and heart rate was determined. Corrected QT (QTc) interval was calculated using a procedure based on the method described by Bazett (1920).

| Specimen collections and evaluations
Hematology, coagulation, and clinical chemistry evaluations were conducted on all surviving animals twice pretreatment (Days À14 and À9) and prior to the terminal (Day 31) and recovery (Day 50) necropsies. Additionally, evaluation of C-reactive protein (CRP) and fibrinogen were conducted on all animals pretreatment (Day À14), 2 days (±30 min), and 7 days post dose 1 and prior to the terminal (Day 31) and recovery (Day 50) necropsies. A summary of all analyzed parameters is given in Table S1.
Blood samples were collected into tubes containing K2EDTA for evaluation of hematology parameters and sodium citrate for evaluation of coagulation parameters. Plasma samples for additional fibrinogen were analyzed on the day of collection. Blood samples were also collected into serum separator tubes without anticoagulant, allowed to clot at controlled room temperature and processed to serum for evaluation of clinical chemistry parameters. Blood samples were collected into nonadditive, barrier free tubes and centrifuged at controlled room temperature. The resulting serum was stored frozen at À60 C to À90 C until analyzed for CRP. Urine samples were collected using steel pans placed under the cages for at least 16 h, and urine parameters were evaluated. Hematology analysis was assessed with ADvia 2120i, Coagulation with STA Compact Max, Clinical Chemistry with ATU5800, and Urinalysis with Clinitek Atlas v7.11.
Bone marrow smears were collected and preserved.
For the immunogenicity, evaluation blood samples (approximately 1 ml) were collected via the femoral vein once at the pretreatment period, predose on Day 29, and prior to recovery necropsy on Day 50. Blood samples were collected in tubes without anticoagulant, allowed to clot at controlled room temperature, and centrifuged at controlled room temperature within 30 min of collection.

| Antimeasles IgG ELISA
Immune response against the vaccine vector was determined by antimeasles IgG ELISA. Testing was performed by VRL Laboratories, San Antonio (USA). The assay was qualified showing >90% sensitivity and specificity. Antigen plates were coated with measles antigen (MVinfected cell lysate) and cell line control antigen (noninfected cell lysate). Test sera were diluted 1:50 using 5% Milk Diluent, and 100 μl of the 1:50 diluted test sera were added to the appropriate wells, and the plates were incubated at 37 C for 1 h. Plates were washed, and bound antibodies were detected by a secondary antibody linked to horse-radish peroxidase (HRP). The enzymatic activity of bound HRP was detected by addition of 100 μl/well ABTS HRP substrate (KPL).
The optical density (OD) (absorbance values) was read spectrophotometrically at 405 nm using a Molecular Devices vMax plate reader.
Any value above or equal to cut-off 0.127 was reported as positive, values below cut-off as negative.

| Gross pathology, organ weights, and histopathology
At necropsy, animals were fasted, sedated, and euthanized. Before exsanguination, animals were also weighed. The animals were examined carefully for any external abnormalities. The abdominal, thoracic, and cranial cavities were examined for abnormalities. The organs were removed, examined, and placed in fixative. All designated tissues (see Table S2, microscopic examination) were fixed in neutral buffered formalin (NBF) except the eyes and testes which were placed in modified Davidson's fixative and then transferred to 70% ethanol for up to 3 days prior final placement in NBF. Organs were weighed and the appropriate organ weight ratios (relative to body and brain weights) were calculated. All tissues required for microscopic examination were embedded in paraffin wax, sectioned, and stained with hematoxylin-eosin.

| Sample processing for biodistribution and shedding analysis
Samples (whole blood, urine, nasal, and oral swabs) were collected pretreatment, on Days 15 (predose), 31 (terminal necropsy animals only), and 50 from all recovery necropsy animals. The animals were fasted prior collection. Samples were collected from animals sequentially for the pretreatment collection and in group order to reduce the potential for cross contamination.
Whole blood was collected via the femoral artery/vein. Samples were placed in Qiagen ® RNA Protect Animal Blood Tubes, inverted eight to 10 times prior placing on wet ice. Whole blood samples were stored on wet ice or refrigerated (2 C to 8 C) for no more than 1 week prior to RNA isolation. Following RNA isolation, both samples were stored frozen (À60 C to À90 C) until analyzed for viral shedding.
Urine was collected from steel pans under the cages. Urine (approximately 1.5 ml/sample) was collected, centrifuged (2 C to 8 C) at 800 g for 10 ± 1 min. As MV is a cell-associated virus, urine pellets were resuspended in 0.5-ml phosphate-buffered saline (PBS). The resulting urine pellets were divided into two approximately equal aliquots. The remaining urine supernatant was retained after centrifugation. Urine samples (pellet in PBS) were individually contained in Nonstick, RNase-free microfuge tubes and were stored frozen at 60 C to 90 C until analyzed for viral shedding.
Nasal and oral swabs were collected into FLOQSwabs ® , processed and stored frozen (À60 C to À90 C) until RNA isolation. For infectivity assessment, nasal and oral swabs were collected into RNAse-free tubes (Sigma-Virocult ® swabs) containing Virocult medium and snapped at the break point. Once the cap was screwed on tightly, the swab was deemed to be "captured" securely. The samples were stored frozen (À60 C to À90 C) at the Testing Facility and maintained for possible future analysis.
Tissue samples of selected organs (see Table S2) were collected (up to 150 mg per organ), put into 2-ml microfuge tubes prefilled with 1.5-ml RNALater. Samples were stored refrigerated (2 C to 8 C) for up to 1 week until processed to remove RNALater and subsequently stored frozen (À60 C to À90 C) until analyzed.

| RT-qPCR
Total RNA was extracted from cynomolgus tissues and purified using  the tissue RNA samples were tested at 1000 ng per reaction, the blood RNA samples were tested at 100 ng per reaction, and the urine and swab (nasal and oral) samples were tested at 5 μl per well. If it was not possible to load the amounts specified above for a specific sample (because the RNA concentration was too low or the sample volume was limiting), a smaller amount of sample RNA was analyzed.
All sample reactions were run in triplicate, and the third reaction was spiked with 200 copies of in vitro transcribed RNA template to evaluate potential RT-qPCR inhibition. Concentration limits were set as following: LOD/LLOQ: 25 copies of measles RNA per reaction. Samples below the LOD/LLOQ are identified as BLOD.

| Infectivity assay
Five hundred microliter PBS with Pen-Strep and Amphotericin B was added to each swab vial and mixed thoroughly. Next, 250 μl was used for virus isolation on Vero cells, and the remaining aliquot was stored in Trizol LS below À60 C for potential future RNA isolation. Swab samples in PBS were then subjected to serial dilutions-neat, 1:2, 1:4, and 1:8. All generated samples were transferred onto seeded Vero cells and incubated for several days. The cells were observed for cytopathic effects.

| Statistical analysis
The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared with the control group using the following analysis:

| RESULTS
To support a Phase 1 clinical trial of MV-LASV, the local and systemic toxicity of MV-LASV was assessed in cynomolgus macaques, because these animals are highly susceptible to MV infection. Monkeys were injected IM with MV-LASV with a dose of 2.1 Â 10 6 TCID50 on Days 1, 15, and 29. The dose volume was 1.0 ml/animal/injection, and one injection site was used for each administration. Two days after the last injection (i.e., on Day 31), six animals per group were euthanized. The remaining four animals per group were kept for a 2-week treatmentfree period to analyze the reversibility of any findings and were euthanized on Day 50. Table 1 shows the study design.

| Clinical monitoring
All animals survived to the scheduled terminal and recovery necropsies. No mortality or morbidity was observed in any of the groups ( There were no MV-LASV-related changes in group mean body weights as shown in Figure 1A

| Clinical pathology
There were no test article-related effects on hematology, clinical chemistry, and urinalysis endpoints (see Tables S3-S6). All fluctuations were considered sporadic, consistent with biologic variation and/or negligible in magnitude, and not related to test article administration. Analysis of the coagulation parameters revealed an increase in fibrinogen concentration in both sexes following MV-LASV administration (Table 3)

| Gross pathology, organ weights, and histopathology
Necropsy performed after three immunizations revealed no test article-related macroscopic findings or organ weight changes (see Figure 2). All macroscopic observations or organ weight differences like lower mean thymus weight in MV-LASV treated females at necropsy Day 50 were considered incidental, commonly observed in this species and, therefore, toxicologically irrelevant.
Microscopic findings attributable to test article administration were seen at the injection sites and consisted of increased severity and incidence of minimal to mild acute and chronic muscle inflammation and minimal mixed inflammatory infiltration of the subcutis in animals with necropsy on Day 31, with resolution over the recovery phase in animals with necropsy Day 50 (Table 5). Inflammation was characterized by a mixture of lymphocytes, plasma cells, and macrophages (chronic inflammation), with occasional accompanying neutrophils (acute inflammation) infiltrating through the muscle fibers.
Rarely, this was seen with other accompanying findings such as myofiber regeneration, myofiber degeneration, myofiber mineralization, or hemorrhage, although these were seen at low incidence and often identified in the control groups at similar incidence and severity. Additionally, there was test article-related increased incidence of subcutaneous infiltration/inflammation of mixed leukocytes, consisting of infiltrates of lymphocytes, plasma cells, neutrophils, and rare macrophages within the superficial and deep subcutis overlying the injection site. These are considered to be secondary to the injection and represent leakage of the test article into the subcutis during the administration procedure. To evaluate now the immune response against the vaccine vector backbone, antimeasles IgG antibody titers were measured in sera at baseline and after dosing ( Figure 3). All animals were tested negative for anti-MV specific IgG at baseline (sample collected during pretreatment phase), with the exception of one Group 2 male animal which was weakly positive. The antibody titer of this one Group 2 animal may have dropped below the cut-off limit during the initial screening phase or our assay was more sensitive. Animals in the control group remained negative throughout the study (i.e., no specific measles antibodies could be detected). All animals in Group 2 were clearly seropositive on Day 29 (predose third immunization). The sera of Group 2 animal number 2001 had an at least fourfold higher OD value on Day 29 (predose), showing that the immunization induced a specific immune response against the vaccine vector backbone. There was a further increase in antimeasles immune response from Day 29 to 50 for animals in Group 2 which were part of the recovery group.

| Viral shedding in body fluids
To evaluate the potential risk of the vaccine virus MV-LASV shedding to the environment, the presence of measles viral RNA was assessed in different body fluids (including whole blood, urine, nasal and oral swabs) by measles genome specific RT-qPCR. All samples in which viral RNA was detected by RT-qPCR were subsequently analyzed by infectivity assay.
Of the 4 time points at which shedding was analyzed, only one nasal swab sample collected from one Group 2 animal on Day 31 contained detectable viral RNA, with 1409 copies of MV-LASV viral genome per swab. The infectivity analysis of this sample however revealed it to be noninfectious. All remaining nasal swabs and all samples of blood, urine, and oral swabs from all dosed animals were negative.

| Biodistribution in organs and tissues
The GPC protein of LASV is a membrane-bound protein and functions as receptor for LASV triggering entry and fusion. As the insertion of Tissues containing detectable levels of viral RNA are summarized in Table 6. By Day 31, MV-LASV viral RNA was detected in spleen, Day 50 with all injection site, cervical lymph node, and popliteal lymph node tissues turning negative. One exception was the mesenteric lymph node, due to a higher copy number at 519 copies per microgram of RNA from one animal. Importantly, no viral RNA was detected in brain, heart, kidneys, intestine, larynx, liver, lung, spinal cord, ovaries, or testes.
The biodistribution samples collected from the vehicle control animals (Group 1) were negative except for one injection site sample from one animal. Investigation revealed that this result was due to a contamination event.

| DISCUSSION
The measles vectored platform technology is built upon one of the safest and most efficacious vaccines available, the live attenuated measles vaccine. Measles vaccination has been used for more than 50 years in over one billion children. It has been demonstrated to be safe (WHO, 2009) and to induce life-long immunity (Vandermeulen et al., 2007). Different MV vaccine strains exist (e.g., Edmonston Zagreb, Schwarz, AIK-C, Moraten, Attenuvax, and Rubeovax), developed from wild-type virus by passaging multiple times in different cell cultures under different conditions (Bankamp et al., 2011). This passaging strategy has caused a reduction in virulence while retaining the T A B L E 3 Coagulation parameters of MV-LASV and control-treated monkeys  (Griffin & Oldstone, 2009). Furthermore, the virus forms a helical nucleocapsid and pleomorphic particles allowing the incorporation of long foreign genes into the viral genome. In addition, it delivers the antigen directly to dendritic cells and macrophages (the most effective antigen presenting cells), a major advantage for vaccine development (Allen et al., 2018). Taking advantage of all these characteristics, different groups have developed methods to genetically manipulate this negative strand RNA virus into a versatile chimeric or recombinant vector (Muhlebach, 2017). MV-LASV is based on such a measles vector technology.
To assess the toxicity and biodistribution profile of measles-based vaccine vectors, the animal species should be permissive for MV replication. In addition, the animals should express the relevant receptors in a comparable manner to humans. Wild-type MV can infect immune cells via CD150 interaction and epithelial cells via Nectin-4 (Muhlebach et al., 2011;Noyce & Richardson, 2012); the attenuated MV strains can also use the ubiquitous receptor CD46 present an all nucleated cells (Dhiman et al., 2004). MVs are generally noninfectious in rodent species, except for cotton rats which show limited replication in certain tissues (Niewiesk, 2009) (Kobune et al., 1996). Moreover, cynomolgus macaques are susceptible to LASV infection, and the protective efficacy of MV-LASV has been demonstrated in this animal model (Mateo et al., 2019(Mateo et al., , 2021. Thus, the cynomolgus macaque was selected specifically for use in the repeated-dose toxicity and biodistribution study.  (Baldrick, 2016;Green, 2015). In addition, these findings also correlated with microscopic inflammation at injection sites (acute and chronic inflammation of the muscle and mixed inflammation/infiltration of the subcutis), and partially or fully resolved following a 3-week recovery period. Interestingly, also the saline control group showed signs of inflammation at the injection site, albeit to a lower degree, indicating that these are common findings associated with intramuscular injection as documented in historical control data and need not be considered adverse.
As already mentioned further above MV-LASV is based on an attenuated measles vaccine strain which should have a wider tissue tropism than the wild-type MV. Moreover, MV-LASV also expresses the membrane-bound LASV GPC which could act as a receptor and could therefore also modify the biodistribution of the virus. To assess this, tissue samples were collected 2 days and 3 weeks after the third RNA could be detected in urine up to 2 weeks post immunization (Rota et al., 1995) and in few studies also in secretions of throat and nasopharynx (Morfin et al., 2002;Tramuto et al., 2015). In a Phase 1 clinical trial with oncolytic recombinant Edmonston-based virus MV-CEA and MV-NIS given intraperitoneally, however, no detection in urine or saliva was reported (Galanis et al., 2015(Galanis et al., , 2010. Immunization of monkeys with the parental measles Schwarz virus resulted in Note: The differences in incidence and/or severity had resolved by the recovery necropsy, on Day 50. F I G U R E 3 Antimeasles IgG ELISA of MV-LASV and control-treated monkeys. Blood was collected during the pretreatment phase (pretest), D29 prior dosing, and D50 (only recovery animals). Cut-off limit is set at 0.127 nm. All values above are considered positive.
Overall, the toxicity, biodistribution, and vector shedding profile of MV-LASV was comparable with the parental measles Schwarz vaccine Rouvax and another measles vectored HIV vaccine candidate MV1-F4 as published by Lorin et al., 2012. Thus, MV-LASV was advanced to clinical testing. The first-in-human study was recently completed (ClinicalTrials.gov NCT04055454) and has shown an acceptable safety and tolerability profile (manuscript accepted).

ACKNOWLEDGMENTS
We thank Sylvain Baize and Mathieu Mateo for providing the MV-LASV vaccine virus. We thank Stephene Rose (formerly CRL) and Tyler Plachta for conducting the monkey study at CRL and for statistical analysis. We thank Haiyan Ma (formerly CRL) for setting up the validated RT-qPCR assay. We would like to thank CEPI for funding of the study and the CEPI project team for their continuous support and helpful discussions. The single sample that was tested positive in the vehicle control group resulted from a contamination event. b Includes cecum, colon, and rectum. c Includes duodenum, ileum, and jejunum.