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Why infection research in India must become relevant to humans

What works in mice may not work in humans, especially in infectious diseases, argue Surat Parvatam* and Karishma Kaushik**.

doi:10.1038/nindia.2020.30 Published online 15 February 2020

Drugs developed using animal models may not work in human clinical trials.

© Pixabay

Those working in the field of tuberculosis (TB) research will know that mice samples infected with Mycobacterium tuberculosis, the bacteria that causes TB, are widely used to study how the disease spreads in their lungs.

Mouse is the animal of choice to study TB, constituting almost 61% of all models globally1. In India, the preference for mice models is higher – at 84% (Fig.1). However, these rodents are not the natural hosts of Mycobacterium tuberculosis, and do not show lung cavitation, a key feature in human TB where lung cells cave-in and die. Therefore, anti-TB drugs developed using animal models may not work in human clinical trials2.

Fig. 1. Experimental animal models used for TB research in India.
This is true for many other infections such as cystic fibrosis, a genetic disease that makes the lungs susceptible to life-threatening bacterial pneumonias3. Genetically modified mice with cystic fibrosis do not develop lung infections, unlike humans4. Piglets, on the other hand, recapitulate most human manifestations of such lung infections, but are difficult to work with because of their size, associated costs and ethics.

Similarly, results of skin wound infection studies on mice may not be replicable in humans. The way wounds heal in mice is fundamentally different from that in humans. In mice, skin defects heal by contraction. In humans, new tissues regrow and proliferate in the wound bed. Infections of the wound bed typically occur in this proliferative stage, and understanding this is important to study wound infections.

Besides these fundamental differences, animal studies are also plagued with issues of reproducibility5, costs, and translation.

According to an analysis, only 37 per cent of animal studies were replicable in human clinical trials6. Eighty five per cent of animal studies fail during early clinical trials, and only half of the studies that make it to Phase III, are finally approved. Major ethical concerns with animal research have led to mandates, including the 4Rs – replacement, reduction, refinement and rehabilitation – limiting the scope for such experimental studies.

Why we need to replace animals with human-relevant models

Globally, scientists are increasingly advocating for biologically-relevant laboratory models that are not only reproducible, cost-effective, and ethical but also closely mimic human biology. In September 2019, the U.S. Environmental Protection Agency (EPA) announced that it would stop funding studies on mammals by 2035, making it the first federal agency to set a firm timeline to phase out animal research.

In India, this discussion has gained substantial traction, with a recent publication outlining a road-map to develop non-animal technologies. This is a critical need because of the multiple issues surrounding animal research brought to fore by a scathing 2003 report by India government’s Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The report pointed out the deplorable animal housing conditions, unacceptable procedural standards, and poor regulatory supervision across 400 animal-care facilities in the country.

Given these major deviations, there are concerns around pre-existing infections or diseases confounding results of the experimental infection. Researchers will also have to be vigilant about uncontrolled transmission of pathogens to other animals in the facility, and accidental escape of infected animals into the community.

Alternatives to animals

Organoids, or three-dimensional miniature organ-like structures are emerging as an exciting area of research (Fig. 2a). They overcome the drawback of classical laboratory cultures, which flatten cells into two dimensions, leading to loss of tissue structure and form.

Fig. 2a. Strategies for creating 3D tissues or organoids.
To make organoids, embryonic cells or stem cells created from adult human samples (called induced pluripotent stem cells – iPSCs), or even primary human cells, are grown in the laboratory. These cells are given specific growth factors that coax them to form organ-like micro-structures. Several organoids have been created, including the brain, stomach, gastrointestinal, kidney, liver, lung and skin, by scientists around the world. These organoids resemble human tissues in terms of structure and gene expression profile7, 8.

In 2016, a study reported that the growth of brain organoids exposed to the zika virus was reduced by 40% compared to unexposed organoids. This resembled the real life malady of microcephaly, or small head (brain) size, a birth defect that cripples zika-infected infants.

The other alternative to animal models is organ-on-a-chip platforms. These are memory stick-sized clear polymers with two parallel hollow channels lined with different types of human cells, and separated by a porous membrane to enable cell communication (Fig 2b). For instance, a gut-on-a-chip device consists of intestinal epithelial and capillary cells, mimicking the intestine-blood vessel interface9. In this platform, intestinal cells could differentiate into villi, and be stably cultured with an assortment of gut microbes, enabling exploration of the role of gut microbes in various diseases.

Fig. 2b: Organ-on-a-chip: (Left panel) Inside architecture of a gut-on-a-chip; (right panel) tissue-on-a-chip for various organs.
This organ-on-a-chip approach has led to miniaturized versions of lungs, bones, liver, placenta and the like. The U. S. National Institute of Health is currently funding a 142 million dollar Tissue Chip Program to expand this technology and develop 3D tissue chips that can model human diseases.

The logical extension of an organ-on-a-chip is an infection-on-a-chip platform that uses 3D tissue models to replicate human infections. In 2018, a liver-on-chip was infected with the Hepatitis B virus (HBV) and maintained for at least 40 days. The system could recapitulate all stages of the virus life cycle, and also elicit an immune response much like the human liver. This is important, given that the pathogen-immunity interactions are critical to infection states.

While hugely promising, these model systems require additional components and further refinements. Most organoids lack relevant microenvironmental factors such as blood vessels and immune cells. Recent studies have tried to overcome this by co-culturing different types of cells, including immune cells, and by expressing proteins that aid vascular development10, 11.

Human-relevant infection research in India

While regulations governing cosmetic and pesticide testing in India recognise human-cell based alternatives, the field of human-relevant disease and infection biology is still nascent.

The Indian Council of Medical Research (ICMR) has recommended capacity building amongst various stakeholders, fostering Centres of Excellence to validate non-animal methods.

In 2019, the ICMR announced plans to establish a Centre of Excellence in Human-Pathway-Based Biomedicine and Risk Assessment in Hyderabad which will aim to develop alternatives to animal testing. A science policy think-tank, the Centre for Predictive Human Model Systems, also promotes human-relevant methodologies. The centre is a collaborative effort between the Atal Incubation Centre-Centre for Cellular and Molecular Biology (AIC-CCMB) at Hyderabad and the Humane Society International-India.

In the last four years, human-relevant technologies received around 0.2 per cent of India’s Department of Biotechnology's total funding. Increasing funding will enable the regulatory acceptance for these new methodologies. Besides, improving awareness, training, and engaging regulators during the early stages of development will also help. 

Building on human-relevant models to mimic infection states could provide unprecedented insights into infection pathophysiology and accelerate the development of novel therapeutics. Leading research in this area that promises to have a major impact on the future of medicine could be an opportunity for India. It also underscores India’s commitment to animal welfare and ethical science, as a responsible international player.

[*Senior Research Associate, Centre for Predictive Human Model Systems (Atal Incubation Centre-CCMB) and **Assistant Professor/Ramalingaswami Fellow, Institute of Bioinformatics and Biotechnology (University of Pune)]


References

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2. Singh, A. K. et al. Animal models of tuberculosis: Lesson learnt. Indian J.Med Res. 147, 456–463 (2018) doi: 10.4103/ijmr.IJMR_554_18

3. Billerbeck, E. et al. Animal models for hepatitis C. Springer book chapter (2013)

4. Dickson, I. A mouse model of hepatitis A virus infection. Nat. Rev. Gastroenterol. Hepatol.(2016) doi: 10.1038/nrgastro.2016.172

5. Mogil, J. S. Laboratory environmental factors and pain behavior: The relevance of unknown unknowns to reproducibility and translation. Lab Animal 46, 136–141 (2017) doi: 10.1038/laban.1223

6. van der Worp, H. B. et al. Can animal models of disease reliably inform human studies? PLoS Med (2010)

7. Kim, S. et al. Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids. PNAS 116, 10824–10833 (2019)

8. Vlachogiannis, G. et al. Patient-derived organoids model treatment response of metastatic gastrointestinal cancers. Science 359, 920–926 (2018)

9. Huh, D. et al. Microfabrication of human organs-on-chips. Nat. Protocols. 8, 2135-57 (2013) doi: 10.1038/nprot.2013.137

10. Cakir, B. et al. Engineering of human brain organoids with a functional vascular-like system. Nat. Methods. 16, 1169–1175 (2019) doi: 10.1038/s41592-019-0586-5

11. Neal, J. T. et al. Organoid Modeling of the tumor immune microenvironment. Cell 175, 1972-1988. e16 (2018)