In order to address the potential issue of vaccine/therapeutic interference we employed a uniformly lethal rhesus macaque model of EBOV infection9. In brief, sixteen animals were divided into three experimental groups (n = 5/group) and one control animal. Animals in one group were given the rVSV-ZEBOV vaccine on day –1 and then received the anti-EBOV GP mAb therapeutic MIL77 on days 3, 6, and 9 at 20 mg/kg/dose after EBOV exposure. The MIL77 immunotherapeutic was selected based on availability and previous results in EBOV-challenged NHPs5. We reduced the dose of MIL77 from a therapeutically proven dose of 50 mg/kg to 20 mg/kg5 to deliver a dose on the margin of protection and accentuate any potential interference from the rVSV-ZEBOV vaccine. Animals in the second experimental group only received the rVSV-ZEBOV vaccine on day –1 and animals in the third experimental group were only treated with MIL77 on days 3, 6, and 9 post-infection (dpi) (20 mg/kg/dose). The single EBOV challenge control animal was not vaccinated or given mAb therapy and succumbed to disease 9 days postexposure. Importantly, 12 historical control rhesus macaques challenged via the same route with the same EBOV seed stock and target dose all succumbed 6 to 9 days after challenge (Figure 1a)9,14.
The unvaccinated/untreated control animal developed clinical symptoms of EBOV disease (EVD) beginning at 5 dpi (Figure 1b, Supplementary Table 1),, and succumbed to disease at 9 dpi (Figure 1a).. Animals that were either vaccinated day –1 with rVSV-ZEBOV or treated day 3, 6, and 9 dpi with MIL77 all developed clinical illness with 2/5 and 4/5 animals in each group surviving, respectively (Figure 1c,d).. Notably, all five animals that were vaccinated day –1 with rVSV-ZEBOV and subsequently treated on days 3, 6, and 9 with MIL77 survived to the study endpoint (28 dpi) without developing any clinical signs of EVD. There was a significant difference in survival between the rVSV-ZEBOV + MIL77 treated group and the control animal (p = 0.0253, Mantel-Cox log-rank test), and between the rVSV-ZEBOV + MIL77 and rVSV-ZEBOV treatment groups (p = 0.0486, Mantel-Cox log rank test) (Figure 1a).. No significant difference was detected between the experimental control animal in this study and historical control (HC) rhesus macaques (N = 12). However, differences were detected when comparing the experimental treatment groups with the HC NHPs (p = 0.0003 for rVSV-ZEBOV + MIL77 vs. HC; p = 0.0072 for rVSV-ZEBOV vs. HC; p = 0.0009 for MIL77 vs. HC).
There were notable differences in clinical pathology and the course of EVD between the experimental treatment groups. A single animal (#180206) in the dual-treatment group developed fever 1 dpi, which was most likely vaccine associated, whereas the control animal and 4/5 animals each in the vaccination only and mAb treatment only groups developed fever 6–9 dpi, which coincided with the appearance of other signs of EVD (Figure 1b-d, Supplementary Tables 2,3).. Post-mortem pathological findings in the control animal and vaccinated or treated animals that succumbed was consistent with previous reports of EVD in macaques (Figure 3d, h, l, p, t, and x)15,16. Animals surviving challenge, including all animals in the rVSV-ZEBOV + MIL77 treatment group, exhibited no significant gross or histopathological findings (Figure 3a-c, e-g, i-k, m-o, q-s, u-w).
Infectious rVSV-ZEBOV was detected by plaque assay up to 2 days post-vaccination in all vaccinated animals except one (#180295) which had low level (1.4 log10 pfu/ml) viremia on day 4 post-vaccination. Detection of circulating EBOV genomic RNA (vRNA) and infectious virus was performed by RT-qPCR and plaque assay titration, respectively. Consistent with historical controls challenged with the same EBOV seed stock, the experimental control animal had 2 log10 pfu/ml of infectious EBOV by 3 dpi and 8.46 log10 GEq of vRNA by 6 dpi, which then peaked on 9 dpi at euthanasia for both detection methods (Figure 2a, Supplementary Figure 1). The vaccine only group had detectable EBOV vRNA by 6 dpi in 4/5 animals and by 9 dpi in the remaining animal. Infectious EBOV was detected in the same group by 6 dpi in 2/5 animals with peak viral titers comparable to both the experimental and HC animals (Supplementary Figure 1).. In the MIL77 only group, vRNA was detected by 3 dpi in 3/5 animals and in 5/5 by 9 dpi, but none had detectable circulating infectious EBOV. In stark contrast, none of the rVSV-EBOV + MIL77 animals had detectable EBOV vRNA or detectable circulating infectious EBOV at any point postexposure (Figure 2a, c, and d),, consistent with the total lack of clinical scoring in this group.
We used ELISA-based detection to estimate total host derived anti-VP40 IgM and IgG as well as anti-GP IgM. Notably, the rVSV-ZEBOV + MIL77 (3/5) group and the rVSV-ZEBOV animals that survived had detectable IgM to VP40 and GP by 6 dpi, yet the MIL77 IgM responses were at or below the limit of detection for the assays. All animals from the rVSV-ZEBOV + MIL77, and any surviving animals from the rVSV-ZEBOV or MIL77 groups, had clear evidence of circulating IgG antibodies against VP40 at 9 dpi through the end of the study. Interestingly, significantly higher levels of circulating MIL77 were detected in the rVSV-ZEBOV + MIL77 animals on 9 dpi where the trend continued out to 14 dpi, suggesting that consumption of the therapeutic occurred at a higher rate in the MIL77 animals and vaccinated animals were supplemented with host derived humoral responses thereby dampening the clearance of the therapeutic. In all groups, no MIL77 was detected at the study endpoint (28 dpi). The 2013–16 West African and current EBOV epidemic in the DRC, both of previously unprecedented proportion, have demonstrated the critical need for efficacious medical countermeasures. However, the potential for deleterious interference between different modes of treatment presents a possible barrier to the development and approval of protocols utilizing a combinatorial approach. Studies in NHPs investigating protection by the rVSV-ZEBOV vaccine when administered as a postexposure intervention have demonstrated only partial protection suggesting that additional postexposure countermeasures may be necessary17,18. Indeed, seroconversion offering protective immunity does not occur before 3 days post-vaccination in NHPs19, and the same is likely true in humans20. Given that all current candidate postexposure mAb therapeutics in clinical trials target the EBOV GP, which is also the antigenic immunogen displayed by the rVSV-ZEBOV vaccine vector, significant concern exists regarding the potential for interference between these types of products. Accordingly, we performed a narrowly focused study utilizing rhesus monkeys to model a scenario likely occurring during the current outbreak in DRC; namely, high-risk exposure to EBOV in individuals recently vaccinated with rVSV-ZEBOV. To assess the potential contraindication of subsequent mAb treatment, we treated a cohort of vaccinated animals with the MIL77 mAb cocktail at days 3, 6, and 9 dpi. Surprisingly, instead of interference, we observed clear therapeutic benefit, where animals vaccinated before EBOV challenge and then subsequently treated postexposure were afforded complete protection without any observable clinical disease. In contrast, animals that received vaccination only or mAb treatment only displayed significant signs of clinical EVD, and in the case of the vaccine only group, limited protection.
It has recently been demonstrated that induction of potent innate immune effector mechanisms occurs in the context of rVSV-ZEBOV vaccination21. Indeed, others have shown modest widening of the therapeutic window upon administration of exogenous interferon-alpha modalities, although neither approach was enough to induce protection 22,23. While the precise mechanism is unclear, the scenario presented here suggests reduction of circulating infectious EBOV complements the induction of vaccine-induced EBOV immunity, ultimately reducing morbidity and likely contributing to survival. Of note, a precedent for a tandem approach of vaccination and mAb treatment for postexposure treatment is the standard protocol for rabies virus exposure, which recommends both vaccination with the inactivated rabies virus vaccine and treatment with human rabies immunoglobulin24. Our study suggests that a similar approach to treatment may be appropriate for high-risk EBOV exposure and that mAb therapy post-vaccination may improve clinical outcome in recently vaccinated individuals.