Review Article Replacing, Reducing and Refining the Use of Animals in Tuberculosis Vaccine Research Rachel Tanner and Helen McShane The Jenner Institute, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK Summary Tuberculosis (TB) remains a serious global health threat and an improved vaccine is urgently needed. New candidate TB vaccines are tested using preclinical animal models such as mice, guinea pigs, cattle and non-human primates. Animals are routinely infected with virulent Mycobacterium tuberculosis (Mtb) in challenge experiments to evaluate protective efficacy, raising ethical issues regarding the procedure of infection itself, symptoms of disease and humane end-points. We summarize the importance and limitations of animal models in TB vaccine research and review current alternatives and modifications in the context of the NC3Rs framework for replacing, reducing and refining the use of animals for scientific purposes. Keywords: tuberculosis, vaccine, animal models, Mtb challenge, 3Rs 1 Introduction human-like pulmonary and extrapulmonary lesions observed (Basaraba, 2008; McMurray et al., 1996). Despite the utility of Tuberculosis (TB) is the world’s most deadly infectious disease, small animals in early screens, larger animal models such as with an estimated 9.6 million new cases and 1.5 million deaths cattle and NHPs are considered more relevant to human TB. annually (WHO, 2016). Incidence of infection in endemic coun- NHPs are naturally susceptible to infection with Mtb, and de- tries remains very high despite good coverage with BCG (Ba- velop the most human-like disease with latency and reactivation cillus Calmette-Guérin) vaccine, the only currently available (Flynn et al., 2015). BCG confers some level of protection in vaccine (Mahomed et al., 2006; Moyo et al., 2010). There is a NHPs, which can be quantified through a variety of clinical and desperate need for a more efficacious vaccine. New candidate nonclinical parameters (Sharpe et al., 2010). In the absence of TB vaccines are currently tested for safety, immunogenicity and a surrogate marker of protection from TB disease, animal Mtb efficacy using preclinical animal models such as mice, guinea infection models remain an essential pre-requisite for novel pigs, cattle and non-human primates (NHPs). Mice are the most vaccine candidates progressing to clinical trials. widely used species due to the potential for screening a high To evaluate the protective efficacy of a candidate TB vaccine, number of candidates at low cost and the availability of gene animals must be infected with virulent Mtb in a challenge exper- knockout strains to characterize the immune response (Apt and iment following vaccination. While M. bovis challenge studies Kramnik, 2009). However, research in species other than mice in cattle are classified as “mild” in severity by the UK Home is becoming more commonplace with increasing availability Office due to a lack of clinical symptoms, Mtb challenge ex- of immunological reagents (McShane and Williams, 2014). periments in mice, guinea pigs and NHPs are generally defined Guinea pigs have emerged as a useful model, replicating many as “moderate”. Moderate severity indicates that the animals aspects of Mtb infection in humans such as granuloma forma- are likely to experience “short term moderate pain, suffering tion, dissemination and caseating necrosis (Clark et al., 2015). or distress or long-lasting mild pain, suffering or distress… or They are also considered a more stringent model in discrimi- moderate impairment of the well-being or general condition”1. nating the efficacy of different vaccines due to the variety of As TB disease progresses, animals may experience loss of body 1 https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/276014/NotesActualSeverityReporting.pdf Received July 28, 2016; This is an Open Access article distributed under the terms of the Creative Accepted September 22, 2016; Commons Attribution 4.0 International license (http://creativecommons.org/ Epub September 26, 2016; licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in https://doi.org/10.14573/altex.1607281 any medium, provided the original work is appropriately cited. ALTEX 34(1), 2017 157 TANNER AND MCSHANE weight, fever and respiratory distress and if left untreated will This framework, now formalized in national and international eventually die of pulmonary insufficiency (Gupta and Katoch, legislation, provides the basis of our discussion on the use of 2009). As this is unethical, humane euthanasia at predefined animals in TB vaccine research. clinical endpoints, which will be discussed later in this report, is now enforced by UK Home Office legislation. In addition to welfare concerns, the many differences be- 2 Replacement tween animal models and human TB bring into question the predictive value of such studies. “Protection” in animal The National Centre for the 3Rs (NC3Rs) in the UK describes models, as determined by the outcome of Mtb challenge ex- replacement as “methods that avoid or replace the use of animals periments, is on a continuous spectrum and usually defined in defined as ‘protected’ under the Animals (Scientific Procedures) terms of a relative improvement in a disease-related readout Act 1986, amended 2012 (ASPA) in an experiment where they such as bacterial load, pathology score or long-term survival. would have otherwise been used”2. Protected animals in the UK A vaccine is considered to provide protection even if there is a refer to all living vertebrates except humans. Alternatives in- measurable bacterial load or pathology in the organs or if some clude using humans, in vitro/cell culture models, computational/ animals do not survive (Elias et al. 2005; McShane and Wil- mathematical modelling, or less sentient animals. All of these liams, 2014; Vordermeier et al., 2009). In humans, however, have been reported in the context of TB vaccine development. efficacy is binary and defined as the prevention of TB disease using clinical endpoints; any individual developing disease, 2.1 Use of humans however minimal, is not protected (McShane and Williams, Given the differences between human and animal manifes- 2014). Clearly, an artificial aerosol challenge is very differ- tations of TB disease, one may argue that a more appropriate ent from natural transmission in humans, and the laboratory focus would be the target species. Controlled human challenge strains of Mtb commonly used (such as H37Rv) are genetical- models have been successfully implemented for other patho- ly dissimilar to clinical isolates (Niemann and Supply, 2014), gens, including those responsible for malaria and typhoid with much higher challenge doses employed (McShane and (Marwick, 1998; Sauerwein et al., 2011), and are a valuable tool Williams, 2014). This issue has recently been addressed with for assessing vaccine efficacy. However, the safety and ethical new advances in ultra-low dose challenge, as discussed be- barriers to challenging humans with live virulent mycobacteria low. In addition to these fundamental differences in the mod- have thus far limited the development of an in vivo challenge el itself, animals are genetically distinct from humans, with model for TB. several discrepancies in both innate and adaptive immunity Already licensed for use in humans, BCG represents a po- between mice and humans (Mestas and Hughes, 2004). The tential surrogate for Mtb challenge, and is a safe replicating widely used Balb/c and C57BL/6 mouse strains do not exhibit mycobacterium that causes a contained, short-term infection caseating granuloma formation following Mtb infection (Orme in immunocompetent individuals. A BCG challenge model has and Basaraba, 2014) and manifest a chronic phase of disease recently been described in which participants were challenged unlike latent Mtb infection in humans (Rhoades et al., 1997). with intradermal (ID) BCG. Skin biopsies of the challenge site Furthermore, responses in genetically diverse humans will be were taken 2 weeks later. BCG load was quantified by culture considerably more variable than in an inbred laboratory animal and quantitative polymerase chain reaction (qPCR) (Harris et strain. Although outbred mice give a diversified picture of TB, al., 2014; Minassian et al., 2012). The model demonstrated the which may be more representative of human disease, larger ability to detect differences in anti-mycobacterial immunity in- group sizes are required to offset the increase in variability duced by BCG and MVA85A (modified vaccinia Ankara 85A, a (Niazi et al., 2015). new-generation vaccine against tuberculosis) vaccination, with The predictive value of animal challenge models in deter- a significant inverse correlation between immune signatures, mining TB vaccine efficacy in humans is uncertain, and will particularly IFN-γ and IL-17 pathways, and BCG load detect- remain unclear until a successful vaccine is developed. Fur- ed by qPCR (Harris et al., 2014). A dose escalation study and thermore, there is evidence from studies of other diseases that comparison of BCG SSI and BCG TICE has also been reported animal models can fail to reliably predict safety in humans (Minhinnick et al., 2016). (Suntharalingam et al., 2006; McKenzie et al., 1995). Other One criticism of intradermal challenge is that it does not mim- difficulties include the large numbers of animals required, and ic the natural route of infection, and to that end a clinical trial the nature and slow