Distribution and absence of generalized lesions in mice following single dose intramuscular inoculation of the vaccine candidate MVA-MERS-S.

Modified Vaccinia Virus Ankara (MVA) is a highly attenuated and replication-deficient virus serving as vaccine against infectious diseases. Here, we assessed the in vivo distribution of a recombinant MVA candidate vaccine against the Middle Eastern Respiratory Syndrome (MVA-MERS-S) in mice. Intramuscularly inoculated mice were necropsied at different time points and examined by histology, immunohistochemistry and real-time PCR. We detected inflammation and myonecrosis at the parenteral site and hyperplasia of the draining lymph nodes. MVA-MERS-S did not result in detectable lesions in tissues peripheral to the parenteral site and draining lymph nodes. Real-time PCR analysis of >240 tissue samples detected MVA-DNA predominantly at the injection site and in the draining lymph nodes, and suggested continuous clearance of the candidate vaccine during the observation period. Levels of parenteral site inflammation and hyperplasia of draining lymph nodes were considered in line with immunological responses to vaccine inoculation.

Introduction

Modified Vaccinia Virus Ankara (MVA) is a highly attenuated vaccinia virus serving as a well-established viral vector system used for developing vaccines against infectious diseases [1,2]. MVA is largely replication-deficient in mammalian cells, but grows well in chicken embryo fibroblasts [3,4]. Non-replicating MVA vaccines have an excellent safety profile in preclinical models using irradiated rabbits, immunosuppressed macaques, SCID and immunocompetent mice, and in clinical testing in humans using different inoculation routes [[5], [6], [7], [8], [9]].

A recombinant MVA vaccine expressing the full length spike protein of Middle Eastern Respiratory Syndrome Coronavirus (MVA-MERS-S) proved to be suitable for production at an industrial scale, immunogenic, and protective against MERS-CoV infections in mice and dromedaries [10,11]. Here we wished to generate additional preclinical data on the in vivo distribution of MVA-MERS-S in order to prepare for the evaluation of the MVA-MERS-S vector vaccine in a first-in-man phase I clinical trial. This ‘biodistribution’ of a candidate vaccine is important to estimate risks potentially associated with an in vivo application of the virus. Relevant aspects include monitoring for signs of virus replication and for potential side effects of the vaccination in the selected animal model. For instance, a dispersion of virus from the parenteral site of inoculation to excretory organs could be indicative of possible viral shedding and spillover into the general environment. Although MVA has been investigated as a safe vector vaccine candidate for years, there are not many studies regarding distribution in sites peripheral to the administration site.

In this study, we assessed the distribution of MVA-MERS-S after intramuscular application in the mouse model using histology, immunohistochemistry, and PCR to detect virus-associated lesions, viral and recombinant antigen in situ, and viral DNA.

Materials & methods

Animals

C57BL/6N mice (6–10 weeks old) were maintained under specified pathogen-free conditions. Animals were allowed to adjust to the facilities (one week) before vaccination experiments were performed and had free access to food and water. Experiments were in compliance with the German regulations for animal experimentation (Animal Welfare Acts).

Mice were inoculated by intramuscular injection with 107 (PCR-study) or 108 plaque-forming units (pfu, histology-study) of MVA-MERS-S or a control recombinant MVA-GFP-mCherry [12] or PBS into the thigh. Mice were monitored daily for signs of disease and were euthanized and necropsied at different time points (Table 1 ).

Table 1

Distribution studies of MVA-MERS-S using qPCR and histology.

A C57BL/6N mice
Group	Treatment	Animals		Necropsy, animals per time point post inoculation								Organs
	Single i.m. inoculation	♀	♂	16 h	24 h	2 d	3 d	4 d	6 d	7 d	21 d
qPCR	MVA-MERS-S	10	17	–	6	–	6	–	–	6	9	See Part B
	PBS	10	3	–	4	–	3	–	–	3	3
Histology	MVA-MERS-S	1	5	1	1	1	1	1	1	–	–	See Fig. 1
	MVA-GFP-mCherry	1	5	1	1	1	1	1	1	–	–
	PBS	4	–	–	1	–	1	–	2	–	–

Cycle threshold (Ct) > 36.

Necropsy, qPCR, histology and immunohistochemistry

A full necropsy was performed and organ samples for PCR and histology/immunohistochemistry were taken. An orthopoxvirus-specific TaqMan-PCR-assay was used to detect viral-DNA [13]. Samples (Table 1A) were frozen at −80 °C until DNA was isolated using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions. For real-time PCR (qPCR) the QuantiTect probe PCR kit (Qiagen) was used. A quantification cycle cut-off ≤38 was regarded specific.

For histology, tissues (Fig. 1 ) were paraformaldehyde-fixed, paraffin-embedded, cut and sections were stained with hemalum-eosin. Skull, rectum and urinary bladder were taken from 48 h on. Skull and whole thigh were decalcified.

Fig. 1

Organ lesions and antigen distribution: Depicted are cumulative results from histological analysis of sampled organs in sections stained with hemalum-eosin and immunohistochemistry. Total number of organs/tissues sampled (white bars), organs/tissues with lesions (grey bars), lesions with VACV-antigen detection (black bars). A MVA-MERS-S inoculated mice. B MVA-GFP-mCherry inoculated mice.

Anti-VACV-immunohistochemistry (IHC) was performed on all tissues as previously described [14] with heat-induced epitope-retrieval. Anti-MERS-S-IHC was performed using a polyclonal rabbit serum (1:1000; Sino Biological, 100208 RP-02) without epitope-retrieval. IHC positive and negative controls were included.

Results

Histological analyses

MVA-MERS-S inoculated animals

Macroscopic examination revealed swelling of the left thigh with muscular pallor, mild edema and hemorrhage, and swelling of draining lymph nodes. Macroscopic lesions in other organs attributable to MVA inoculation were not observed.

Histologically, at all time points, there was absence of lesions attributable to MVA inoculation in any tissue other than the parenteral site and draining lymph nodes (Fig. 1). At the early time points, there was separation of muscle fibers through inoculum and edema, muscle fiber degeneration and necrosis at the parenteral site. Interstitial areas, adjacent adipose tissue and inoculum were predominantly infiltrated by many neutrophils. At the late time points there was evidence of muscular regeneration, characterized by myoblast tubes and internalized nuclei and many predominantly mononuclear infiltrates in the interstitia and surrounding the inoculum. The draining lymph nodes revealed increased paracortical lymphocytic cellularity (hyperplasia).

VACV antigen was detected only at the parenteral site. Other tissues including draining lymph nodes revealed no VACV antigen. VACV antigen was located in the cytoplasm of spindle cells in the interstitium (Fig. 2 ) and phagocytic cells (macrophages and neutrophils) in the interstitium or surrounding inoculum. MERS-S antigen was detected in MVA-MERS-S inoculated animals only at the parenteral site in the cytoplasm of similar spindle cells with a comparable distribution pattern (Fig. 2). Draining lymph nodes displayed no specific MERS-S antigen-staining.

Fig. 2

A PBS inoculated mouse (72 h), left thigh, VACV-immunohistochemistry, no signal. B MVA-MERS-S inoculated mouse (16 h), left thigh, VACV-antigen in spindle cells (center, brown color) and inoculum (bottom right). C MVA-GFP-mCherry inoculated mouse (48 h), left thigh, VACV-antigen in similar spindle cells. D MVA-MERS-S inoculated mouse (24 h), left thigh, cytoplasmic MERS-S-antigen in interstitial spindle cell. Bars = 50 μm.

MVA-GFP-mCherry inoculated animals

The distribution and severity of lesions were remarkably similar to the MVA-MERS-S inoculated animals. Macroscopic and histologic examinations revealed similar findings in the parenteral site and draining lymph nodes. Acute inflammation, characterized by edema and many infiltrating neutrophils with myofiber degeneration and necrosis were prevalent at the early time points. At later time points, the infiltrate became predominantly mononuclear and there was evidence of muscle regeneration. Macroscopic and microscopic lesions in other organs attributable to MVA inoculation were not observed at any time point. VACV-antigen was only detected at the parenteral site within the cytoplasm of phagocytic and spindle cells (Fig. 2). Other tissues including draining lymph nodes revealed no VACV antigen.

Saline inoculated animals

Macroscopic examination revealed no lesions. Histological analysis displayed mild injection-associated lesions at the parenteral site consisting of band-like degeneration of single muscle fibers, regeneration at later time points and focal scarce interstitial leukocytic infiltrates. The subiliac lymph node of one animal revealed mildly increased paracortical cellularity. Histological changes attributable to inoculation were not detected in any other tissue in any animal. A specific reaction product was not observed in either of the two IHCs.

PCR analyses

MVA-DNA was detected only in MVA-MERS-S-inoculated mice at the parenteral site (Ct-range: 19–36) and draining lymph nodes (Ct-range: 33–35) at days 1, 3 and 7 days post inoculation. Only in three animals, single organs were positive (Table 1B).

Discussion

This study was done to contribute to the development of the vaccine candidate MVA-MERS-S towards clinical phase I testing. Here, we report data supporting the biodistribution assessment of MVA-MERS-S after intramuscular application in the mouse model. With regard to systemic spread, potential excretion and side effects we analyzed tissue samples by histology, immunohistochemistry and qPCR.

As determined by histology, the candidate vaccine did not produce generalized lesions or organ lesions peripheral to the parenteral site. MERS-S- and VACV-antigen remained restricted to the parenteral site and were not observed in draining lymph nodes. Anti-VACV-IHC did not reveal antigen in any of the analyzed peripheral tissues underlining the incompetence of MVA to result in generalized spreading. Inoculation of mice with the control virus MVA-GFP-mCherry produced a reaction pattern indistinguishable from MVA-MERS-S underlining the maintenance of a well confined vector virus infection. Moreover, the limited distribution of MVA-MERS-S was further confirmed by the results of the qPCR analysis detecting relevant amounts of MVA-DNA only at the injection site and in draining lymph nodes. In three animals, high Ct-values of MVA-DNA were obtained in lung and liver. These PCR signals can probably be attributed to marginal quantities of vector DNA, which was dislodged from the parenteral site. Similar studies also detected minimal amounts of vector DNA in single organs, however, attempts of MVA re-isolation were negative [6,8]. Therefore, it seems unlikely that these signals represent viable virions.

Importantly, MVA-DNA was not found in excretory organs (kidneys, rectum including feces and urinary bladder including urine). Thus, the risk of MVA excretion and spillover of the genetically modified organism into the general environment is regarded negligible.

Other studies using recombinant MVA candidate vaccines also report locally confined lesions even in severely immunosuppressed animals. While MVA-DNA can remain detectable by PCR for several weeks after inoculation, attempts to isolate infectious virus proved to be negative. Histology in either case did not reveal peripheral lesions associated with MVA inoculation [5,8].

In this study, the observed local inflammation infiltrating the inoculum and adjacent tissue is not regarded as undesired reaction but part of the physiologic immune reaction against the vaccine virus. The detection of VACV antigen in phagocytic cells supports an efficient in vivo targeting of antigen presenting cells following the inoculation of MVA vector vaccines. The degree and extent of inflammation and myonecrosis in this study is in accordance with the ratio of inoculum volume in relation to parenteral site (mouse muscle) volume, which is expected to be considerably smaller in human vaccinees. The hyperplasia observed in the draining lymph nodes again reflects the immune competence of the host and is characteristic for an early response to inflammation at the draining site [15].

We conclude that a single intramuscular injection of equivalents of a human dose of MVA-MERS-S in C57BL/6 mice is without lesions in tissues other than the parenteral site. Further, viral-DNA was not found in other peripheral organs, with the exception of very low copy numbers detected in single samples. Thus, this distribution study in mice provides relevant preclinical data further supporting the biological safety of the MVA-MERS-S candidate vaccine.

Conflicts of interest

The authors declare they have no conflict of interest.