Intranasal monkeypox marmoset model: Prophylactic antibody treatment provides benefit against severe monkeypox virus disease.

Concerns regarding outbreaks of human monkeypox or the potential reintroduction of smallpox into an immunological naïve population have prompted the development of animal models and countermeasures. Here we present a marmoset model of monkeypox and smallpox disease utilizing a relevant poxvirus via a natural exposure route. We found that 1000 plaque forming units (PFU) of Monkeypox virus was sufficient to recapitulate smallpox disease, to include an incubation period of approximately 13 days, followed by the onset of rash, and death between 15 and 17 days. Temporally accurate manifestation of viremia and oral shedding were also features. The number of lesions ranged from no lesions to 299, the most reported in a marmoset exposed to a poxvirus. To both evaluate the efficacy of our antibodies and the applicability of the model system, marmosets were prophylactically treated with two monoclonal antibodies, c7D11 and c8A. Of three marmosets, two were completely free of disease and a single marmoset died 8 days after the mock (n = 1) or PBS control(s) (n = 2). Evaluation of the serum levels of the three animals provided a possible explanation to the animal succumbing to disease. Interestingly, more females had lesions (and a greater number of lesions) and lower viral burden (viremia and oral shedding) than males in our studies, suggesting a possible gender effect

Levels of c8A and c7D11monoclonal antibodies in infected and uninfected marmosets.

<p>Marmosets were subcutaneously injected with a 1:1 mixture of a cocktail containing c8A and c7D1 (20mg/Kg of each antibody per animal). A single animal was not exposed to monkeypox virus (#11). Of the exposed animals, Days 1, 12 and 21 were analyzed for the presence of c7D11 (A) and c8A (B). For infected animals #8, #9 and #10, days are relative to the day of exposure. For animal #11, days are relative to subcutaneous injection of the antibody.</p

Clinical assessment of antibody treated marmosets exposed to monkeypox virus.

<p>Marmosets were sedated every third day at which time body temperature (A), weights (B). Disease score (D) was captured daily. All control animals succumbed to disease, while animals that received targeted antibody treatment survived until at least Day 25 (C). Asterick denotes significance in survival curves between untreated and mock treated versus treated (Log rank, p = 0.0253). Three females (#’s 5, 6 and 8) and three males (#’s 7,8, and 9) were used in this study.</p

Prophylactic treatment with c8A and c7D11 prevents or delays virermia and viral shedding.

<p>EDTA whole blood (WB) and media from oral swabs (OS) were inoculated on to BSC-1 cells, stained, and plaques enumerated five days later. Treated animals (shades of blue); Mock or off-target treated (shades of pink). Limits of detection for whole blood (dashed orange line) and for oral swabs (dashed blue line).</p

Viremia and viral shedding in marmosets exposed to intranasal monkeypox virus.

<p>Whole blood and oral swabs were processed and titrated onto BSC-1 cells. Two of four animals had detectable levels of virus in blood samples on the Day 9. Virus from oral swabs was detectable as early as day 6 (high dosed animal #4). Limits of detection for whole blood (dashed orange line) and for oral swabs (dashed blue line). All animals were positive to varying extents. Notice the onset of oral shedding relative to viremia.</p

Clinical assessment of marmosets exposed to monkeypox virus.

<p>Animal 1–3 were exposed once on Day 0 with an intranasal dose of 100 PFU and subsequently re-exposed with 1000 PFU on day 36. Rectal temperature, (A), and body weight (B), were evaluated every 3 days under sedation. Survival outcome, (C). Animals were evaluated for recumbence, unresponsiveness, and dyspnea and given a disease score, (D). Three males (#1, #2, and #4) and one female (#3) were used in this experiment.</p

Comparison of the clinical course of smallpox and monkeypox in humans and infected nonhuman primates.

<p>To elucidate both the resemblance to human smallpox disease and how our model relates to the “gold standard” of NHP models for poxvirus countermeasures, the figure depicts the human disease (grey outlined box), the IV monkeypox-macaque model (red outlined box) and the IN monkeypox-marmoset model (outlined in green). Arrows are color coded by host and indicate the point of exposure. Days (in the form of a number line) relative to exposure are given for each Human disease manifestations are temporally identical to IN exposed marmosets to include viremia and oral shedding (not shown-see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006581#sec015" target="_blank">discussion</a>), but fever has not yet been detected in marmosets (IV = intravenous; IN = intranasal). This Figure is a modification of Figure 34 from Mucker, 2014 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006581#pntd.0006581.ref036" target="_blank">36</a>].</p

Skin of a mock treated marmoset exhibiting a classical smallpox rash.

<p>Previous to exposure with 1000 PFU of monkeypox virus, animal 5 was treated with non-pox-virus specific antibody. This animal manifested a typical smallpox rash as can be seen on the exposed (hairless) portions of the abdomen and groin (A) a zoomed in portion of the abdomen is shown in B.</p

Rash in marmosets exposed to intranasal monkeypox virus.

<p>A rash was observed in two of the four marmosets, animal 3 (A-G) and animal 4 (H-J). Shown are the head (A), abdomen/groin (B), side of chest and left arm (C), left armpit with enlarged lymph node (D), and urogenital region (F and G) of animal 3. From animal 4, mouth and copious nasal discharge (H), back of skull (I), and lower abdomen (J). Notice that the lesion on animal 4 are in a papular/vesicular stage of rash with few pustules whereas animal 3 has a macular/papular rash that had flattened has areas hemorrhage (petechial in nature).</p

Select hematology from marmosets, some treated, then exposed to intranasally to low dose monkeypox virus.

<p>Prior to challenge, animal 5 was treated with an isotype control antibody, while animals 6 and 7 were treated with PBS. Animals 8, 9, and10 were treated with monkeypox targeted antibody. White blood cells (WBC) (A), lymphocytes (B), monocytes (C) and granulocytes (D) were elevated, and platelets (PLT) (E) decreased in most of the control animals whereas treated animals did not vary much from earlier time points. Data was collected using a Coulter AcT Diff3.</p