Could the cytokine upend animal-based vaccine testing and development? A perspective
Two primates in a laboratory cage. USDA/ Public Domain
Lakshman Varanasi, PhD, Science Associate October 3, 2020
Animal models in biomedical research constitute a perpetual ethical challenge. They trouble our conscience but we need them for what they enable, viz therapeutic and vaccine development. In an effort to address animals’ rights without stymying research, Russell and Burch proposed the 3Rs concept in 1959, in their book “The Principles of Humane Experimental Research”. The 3 Rs stand for Replacement, Reduction, and Refinement, and together, the three elements of the concept comprise the paradigm for current EU regulations on animal testing. The first of these, Replacement, is defined as “the substitution of insentient material for conscious living higher animals”, and is, in essence, a nod to ex vivo, or in vitro procedures, that dispense with the higher animal model. Since then, the vaccine industry has made some headway in this direction. Implementation of the 3 Rs requires consideration of the various aspects of vaccine development and how these can be individually modelledin vitro. It will improve as our understanding of the biology of infections and immune responses (to antigens) evolves and matures.
Alas, the pathogenesis/ pathophysiology of a live-attenuated viral vaccine has multiple determinants, and can be holistically modelled (for the purpose of understanding its safety, virulence, or toxicity/cytoxicity) in intact organ systems in a living organism, preferably one that is evolutionarily close to the Homo sapiens. This precludes the in vitro model and ensures the animal model’s continued relevance. The neurovirulence test (NVT) highlights the problem of abandoning the monkey model completely. Neurovirulence is the capacity of a pathogen to cause disease of the nervous system. It is exhibited by the various neurotropic viruses, such as, the polio, mumps, Japanese Encephalitis, rabies, herpes simplex, and West Nile viruses. Viruses are fastidious in that they attach a specific tissue or tissue type. Tropism refers to this peculiar affinity. Non-animal NVT methods are currently unavailable, although some partial alternatives for individual viruses are in development; for instance, the PCR-based in vitro test (MAPREC) and the TgPVR21 transgenic mouse model for testing neurovirulence and potency for the oral polio vaccine; the latter was adopted after it was shown to be comparable to the Monkey Neurovirulence Test (MNVT). For the mumps virus, “preliminary studies have demonstrated a very good correlation between virus growth kinetics in vitro and neurovirulence potential in vivo”, where the in vitro assay is directed at measuring the degree of viral neuroattenuation.
Few non-animal in-vitro alternatives for testing of vaccine potency and safety are available, although again, they are not complete replacements of the animal models; they are screening tools at best. The complex interaction between the various immune system components cannot yet be modelled in an in vitro system.
Cytokines may provide a way out though. Antigenic challenge, naturally or through a vaccine, effects a cytokine response as part of the larger immune/ inflammatory response. The cytokines are chemicals that quickly amplify the response by recruiting other immune components. The pertinent questions then are: Can the cytokine response to a vaccine (antigen) be a measure of its potency (or another property)? Is the cytokine response proportional to vaccine (antigen) dose? Current knowledge indicates that it is likely. This can be deduced from the cytokine profile post antigenic stimulation of immune cells in vitro. The type and intensity of the immune response is dictated by cytokine production by T-helper (Th) cells/lymphocytes. Differences in the relative levels of individual cytokines have been observed in supernatants from murine spleen cultures after stimulation with tetanus vaccine from different batches of variable quality. Importantly, higher cytokine release has also been observed to correspond to a higher (neutralising) antibody titre. The concept certainly has precedent, and is now a need.
T-lymphocyte secreted cytokines can now be routinely assayed using commercially available in vitro kits. The cytokine response (from a mere trickle to a storm) is a measurable immunological phenotype, peculiar to the subject, that can be leveraged for vaccine testing. To put it differently, Cytokines, possibly from human progenitor cell-culture supernatants, can be used as a surrogate measure for the immunogenicity and potency of a vaccine, both qualitatively and quantitatively. The human-specific readout from such assays can reduce the uncertainty implicit in the animal modelsystem.
A new test platform needs to be made available to, and then accepted by, the industry. Vaccine manufacturers may be loath to move to a system that is new (and requires training and operational changes) and likely to require of them, more than any regulatory hurdle for review and approval, a change of mindset. The validated cytokine-based assay will quickly find takers, provided its benefits over the current standard are made evident, we daresay. It will enable high-throughput screening/ testing and greater integration with routine vaccine development procedures. Such in vitro human surrogate safety-related testing platforms would be a step into the future, one that would doubtless makeMessrs. Russell and Burch happy.