Filorab1’s immunogenicity and lack of side-effects in captive chimpanzees bode well for its use to protect wild chimpanzees and gorillas endangered by EBOV. Filorab1 produced immune responses comparable to those observed in the only previous EBOV vaccine trial on captive chimpanzees using a virus-like particle (VLP) vaccine22 but did so with only one dose rather than the three doses given in the VLP study. Both robust immune response to a single dose and oral delivery are massive advantages for the field vaccination of wild apes that are difficult to locate in dense forest and fear human approach.

We are already taking further steps towards realizing the potential of oral vaccination as an ape conservation tool, including ongoing tests of both oral bait prototypes on wild apes and methods for quantifying and controlling rates of vaccine bait uptake by both apes and non-target species. Important future steps include heat stabilization of vaccine for longer viability under hot forest conditions, non-invasive assays for vaccine safety and immunogenicity, and field trials on wild apes. Because of the special position that wild apes hold in the public imagination, this work could provide a particularly powerful proof of the more general principle that oral vaccination is a safe and efficient way to protect endangered species against a large and growing pathogen threat. EBOV challenge experiments on macaques might also be used to establish the protective effect of oral delivery of filorab1. However, conservation funds are scarce and the cost of such trials conducting such trials in a BSL4 facility would likely equal or exceed that of all other activities combined.

It seems likely that further safety and immunogenicity trials on captive chimpanzees will not be part of the development program. In principle, research that benefits wild chimpanzee conservation is exempt under the new ESA regulations banning medical research on chimpanzees. In practice, all of the biomedical facilities that held chimpanzees have or are in the process of “retiring” their populations to sanctuaries: sanctuaries which are philosophically opposed to invasive biomedical research. The Pan African Sanctuary Alliance also recently voted to oppose biomedical research on chimpanzees at its member institutions. Biomedical research facilities in developed countries other than the United States no longer hold chimpanzees. And extensive informal outreach suggests that, although zoos have the facilities to conduct safe and rigorous trials and are sympathetic to the conservation objectives, they are unwilling to risk the public backlash that hosting vaccine trials might evoke. This really may be the final vaccine trial on captive chimpanzees: a serious setback for efforts to protect our closest relatives from the pathogens that push them ever closer to extinction in the wild.

This study also has some useful implications for future vaccine trials involving other captive species. First, although our relatively small sample size necessitates caution, the results imply that the psychological stress levels of captive study animals may modulate the antibody response to vaccination. This is not surprising given both published results on the substantial effects of stress on human immune responses and the extensive literature on stress responses of captive animals to experimental procedures and housing conditions. It is, however, surprising that so little attention seems to have been paid to this phenomenon in the design and analysis of vaccine trials involving captive animals. Stress induced variation in immune response is a problem both because it reduces inferential power (by introducing variance unexplained by experimental treatments) and because eventual recipients of the vaccine (often humans) will often not be subject to equally high or persistent stress levels. Thus, more effort may need to be put into mitigating and controlling for animal stress: both experimentally and statistically. A good start would be vaccine trials with larger samples in which sources of stress were experimentally manipulated (e.g. oral vs hypodermic immobilization, vaccination without immobilization). The ban on chimpanzee research effectively precludes such trials as a part of our ape conservation program.

The second implication of our study is that the key to successfully mitigating and controlling for stress is to carefully discriminate between acute and chronic stressors. Much opposition to the use of chimpanzees in biomedical research has rested on the assertion that confinement of chimpanzees in small experimental cages during trials subjects chimpanzees to psychological stress of a severity comparable to that induced by persistent torture11. However, the relatively rapid attenuation of stress responses in our study suggests that chimpanzees did not suffer severely from severe, chronic stress due to either confinement in small cages or social isolation. For instance, chimpanzees were no longer losing weight by Day 7 and were gaining weight by Day 28 (Fig. 2A). Similarly, WBC peaked at Day 0 and serum glucose at Day 14. Furthermore, the longer VLP vaccine trial conducted earlier at New Iberia using identical housing and handling protocols showed chimpanzees serum glucose decaying to baseline levels by Day 56. In fact, the combined data from the two chimpanzee studies show a pattern of serum glucose rise and fall very similar to that seen in baboons acclimating to captivity19 but with a substantially lower peak implying less severe stress (Fig. 3B). Chimpanzee titers for alkaline phosphatase, an enzyme whose serum concentration responds very quickly to acute stress, peaked at Day -8 and decayed approximately exponentially to near background level by day 28 (Fig. 3D): a pattern also very similar to that seen in the baboon study.

The relatively quick rise and fall seen in the values of stress correlates suggest that homeostatic mechanisms successfully down-regulated the chimpanzee stress responses to experimental conditions. Of particular interest is the observation that chimpanzees that did not always voluntarily present for sedation showed mean glucose and WBC values respectively 11% and 15% higher than chimpanzees that always voluntarily presented (t test glucose p = 0.023, WBC p = 0.033), with glucose and WBC peaks above the lowest daily mean value for non-voluntary presenters that were 69% and 46% higher than voluntary presenters. That voluntary presenters showed an earlier serum glucose peak (Fig. 3B) could be interpreted as more rapid down-regulation of the stress response to sedation. Results from the earlier VLP vaccine trial show that chimpanzee glucose did not rise (Fig. 3B) or weight drop (Fig. 3A) in response to closely-spaced sedations at the end of the study (Days 70, 77, and 84), again consistent with down-regulated stress responses to sedation.

These results suggest that contrary to the claims of animal welfare advocates, housing conditions, per se, may not be a major source of severe stress in trials on captive primates. Apparently, husbandry improvements (e.g. paired cages that allow grooming and other social contact) can successfully minimize social isolation and confinement-related stress of the kind responsible for the behavioral and physiological pathologies that originally galvanized animal welfare advocates to oppose biomedical testing on non-human primates. Rather, the most effective focus of efforts to mitigate stress may be on improved protocols for sedation. For example, sedation stress might be reduced by training of study animals to present voluntarily15, distraction tactics23, oral sedatives24, or newly developed non-invasive assays of antibodies25 in secreted and excreted body fluids (e.g. urine, saliva, feces). Data on the behavioural response to sedation as well as hematological and physiological correlates of stress should also be used to statistically control for immune response.