Introduction
The aim of this function is to demonstrate the ability of the vaccine to prevent infection, reduce infection burden or pathology, or protect Mtb infected animals from progressing to TB disease (POD). Different animal species, modes of infection and disease endpoints can be used. Although there is no single standardised animal model of TB, there are several recognised animal models/study designs that can be used to compare different vaccine candidates. The most appropriate model or study design will vary according to the type ofvaccine, and also the target population. It is generally recommended to perform studies in mouse challenge models initially. When selecting the mouse model(s) it is important to keep in mind that host genetics, vaccine type, vaccination route, and the strain and duration of Mtb challenge influence the protection levels achievable with TB vaccine candidates in mice (Pozo-Ramos and Kupz, 2025). Moreover, an important consideration in all pre-clinical animal efficacy studies is the challenge dose of Mtb, as different mechanisms of vaccine-induced protective immunity may be operating at different challenge doses. This is described in Challenge dose considerations for mice.
It is recommended, particularly from Stage B onwards, to use vaccine doses produced under controlled circumstances (GLP-like) and perform protection experiments using standardised methodology. Such studies can include blinded analyses to ensure un-biased interpretation of the results. As mentioned under pre-clinical immunogenicity, there is no validated animal model that is accepted as being predictive for TB vaccine efficacy in humans. Therefore, a pragmatic approach is recommended. For neonate/infant TB vaccine candidates, BCG is a suitable benchmark. However, without such direct comparators available for adult vaccines the developer has to weigh added value of efficacy studies in higher models versus the cost in time and resources, to make pivotal decisions.
In this TB vaccine development pathway, animal studies are referred to as pre-clinical because of an emphasis on providing guidance for animal testing performed before entering the clinical stages i.e. up to Stage C. References are made to formal, published documents some of which use the terminology of non-clinical testing because they describe both pre- and post-clinical animal studies.
Vaccine technology specific considerations and target specific considerations
The objectives and the related preclinical protection/efficacy described in this guidance remain the same regardless of the vaccine type.
- Demonstrate protection (usually) in a small animal Mycobacterium tuberculosis (Mtb) infection model
- Compare to benchmark, if available
- Protection in a small animal Mtb infection model demonstrated
- Protection statistically better than a relevant benchmark, if available
The ability of a candidate to reduce or limit the progression of experimental infection/diseasewith Mtb must be demonstrated in anappropriate animal model. Different animal species, modes of infection and read-outs of disease are used. These are described in reviews such as Cardona and Williams 2017, Singh and Gupta 2018, Williams and Orme 2016. Typically, efficacy is first demonstrated in mice or guinea pigs. There are many exampleso f these species being used for testing the efficacy of TB vaccines which are illustrated in the following references; Stylianou et al 2018, Doherty et al., 2004, Reed and Lobet 2005, Williams et al.,2005, Brandt et al., 2004, Clark et al., 2016.
The strain of Mtb used for challenge should be well-characterised and from a source that allows reproducibility between experiments [see also “Challenge strains in preclinical models”]. The design of the vaccination schedule should be informed by previously generated immunogenicity data. In the case of protection studies in mice, the strain of mice used should also be consistent with the strain used to demonstrate immunogenicity. [see also “Mouse strains”] In the absence of a correlate of protection, evaluating multiple vaccine dose levels in early-stage challenge studies should be considered. The outcome measures used to demonstrate protection typically include a reduction in bacterial burden in lungs and other relevant organs (mostly spleen), survival, or quantifiable changes in pathological features of TB disease, for high dose challenge studies. In experiments with a challenge dose of 1-3 CFU, prevention of infection and prevention of dissemination to the contralateral lung or spleen can also be measured. A statistically significant change compared to a relevant control group must be demonstrated. Statistical power calculations based upon a pre-defined target level of protection should be used to design experiments with appropriate group sizes. Of note, group sizes needed are much larger when using (ultra) low dose challenge. The control group against which efficacy is compared must be justifiable and appropriate to the nature and intended TPP of the candidate. For example, for a vector vaccine, empty vectors could be included and a novel live-attenuated whole cell vaccine should show an improvement over BCG unless other substantial benefits would justify an equivalent level of protection to BCG. If a candidate is a part of a heterologous prime-boost regimen, the combined regimen must be significantly more protective than the individual components. If the prime-boost regimen has BCG as a critical component e.g. boosting BCG in infants, there is an expectation that protection greater than BCG must be demonstrated.
The selection of the most suitable model for specific vaccine candidates is complex and organisations such as TBVI or the Collaboration for Tuberculosis Vaccine Discovery (CTVD) will provide access to advice via, for example, product development teams (PAX – TBVI) or the research communities established by CTVD, in particular the NHP community.
- Confirm robust protection e.g. in a small animal Mycobcterium tuberculosis (Mtb) infection model in an independent lab or second animal model/ species
- Review immunogenicity and protection data in small animals
- Decide on use NHP study. If yes, design study and prepare read-outs to evaluate protection
- Protection statistically better than a relevant benchmark, if available, reproduced independently in same species or confirmed in a second animal model
- Immunogenicity and protection data support proposed mode of protection, and support the NHP (or another advanced model) study design
- Read-outs for NHP, or other model, ready
At Stage B, the emphasis is on confirming the protective efficacy of the candidate. It is helpful if efficacy can be confirmed in a second animal model to demonstrate that the effect is biologically robust. This second model might be a guinea pig model or a mouse model which is more stringent e.g. involves a more virulent Mtb challenge strain (e.g. HN878), or uses a mouse strain which is more susceptible to disease such as CBA, DBA/2, C3H and 129/SvJ mice (Medina and North, 1998), or in outbred mice such as Collaborative Cross (CC) or Diversity Outbred (DO) (Hackett etal., 2022) or if a higher dose Mtb challenge was used in Stage A, to use a more physiologic infectious challenge dose (e.g., 1-3 CFU). Regardless, it is imperative that the data are robust and that the protective effect can be reproduced. Studies aimed at elucidating the mechanism of action should be considered (e.g., T-cell depletion or passive antibody transfer). Head-to-head evaluation of vaccine candidates in well-characterized mouse and guinea pig models using standardized study designs by independent laboratories could also be helpful. This can be available as a service, please check NIH (NIAID/NIH pre-clinical models) and TBVI for current availability. Immunogenicity and efficacy studies conducted in Stages A and B should be used to support the design and execution of experiments in the advanced model(s) in Stage C, including the identification of a primary endpoint upon which Stage C studies will be powered.
- Confirm protection or Proof of Concept (PoC) (Note: the animal models for evaluation should be justified based on candidate’s proposed mechanism of action)
- Protection from Mtb challenge statistically better than BCG and/or relevant benchmark using primary endpoint in 2 (animal) models, as demonstrated by a read-out with high statistical power for the group size.
Though also the NHP model is not validated, it could be a valuable model for efficacy evaluation in stage C (see also “Animal Model Considerations"), as a wide range of pathology, bacteriology, imaging and immunity analyses can be deployed for efficacy readout in fixed endpoint designs. However, time-to-humane endpoint strategies are feasible but poised with some ethical concern and increased cost and standard measures of clinical chemistry and haematology for monitoring, for example systemic inflammation and loss of protein or infection-associated anaemia, appear more robust only when a higher dose challenge is applied, which has become an uncommon approach lately. Typically, single low dose challenge with Mtb strain Erdman K01, available as standardized stocks from BEI Resources, is used for infectious challenge of NHP, either by aerosol inhalation or endobronchial instillation. Also, a repeated limiting dose challenge model has been defined with infection occurring more as a stochastic event, to further support longitudinal readoutsof infection and immunity. In concordance with what has been raised under functions 5 and 6 on safety and immunogenicity already, also for efficacy evaluation in NHP mycobacterial pre-exposure conditions (e.g. prior BCG vaccination, NTM exposure or 'latent' Mtb infection) should be considered. Various experimental strategies, based on alternative dosing and/or delivery of BCG, have yielded prevention of infection (POI) and prevention of disease (POD) signals, which set an important mark on the dynamic range of NHPTB infection modelling. Regarding the time response dynamic, various imaging approaches have been used in NHP vaccine studies. Beyond conventional chestX-ray, many laboratories monitor TB over time by computed tomography (CT) either or not in combination with positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) or more experimental tracers to track the host response after infectious challenge. Post infection sampling and immune response measurements should be considered for extended study designs, sinc ethey can support (diagnostic) biomarker studies or the investigation of protective immunity in the face of the intruding pathogen.