Why did my mouse or rabbit die after peptide antigen injection?

Usually the injection of a peptide conjugated to an adjuvant such as Keyhole Limpet Protein (KLH) or bovine serum albumin (BSA) results in the production of polyclonal antibodies very useful to biological research. However, some peptides can cause problems when used as antigens during antibody production in mice and rabbits and possibly in other animals as well.

The origin of horror autotoxicus: Jules Bordet reported in 1898 of the phenomenon of immune hemolysis which led Paul Ehrlich to assign his assistant Julius Morgenroth to perform more detailed studies. In the following years they described the hemolytic antibodies that result when animals are injected with the blood of unrelated species. This led Ehrlich to postulate the existence of what he termed horror autotoxicus. This is the unwillingness of the organism to endanger itself by the formation of toxic autoantibodies (Silverstein 2001). Even thought the concept of immunity from disease dates back to Greece in the 5^th century BC, the study of the immune system has only began in earnest in the 19 century. Much progress has been made, during the last century, on our understanding of immunoregulation and tolerance induction. However, we still don’t understand how exactly the immune system works. Furthermore, the scientific community does not agree on all models developed in the past to explain all interactions of the immune system.

Wack et al. in 1996 could show that antigen induced thymocyte apoptosis can occur after injecting peptides NP68 (ASNENMDAM) from the nucleoprotein of influenza virus A/NT/60/68, and peptide NP34 (ASNENMETM), from the nucleoprotein of influenza virus A/PR/8/34, into F5 and F5/NP mice. The authors report that during thymic maturation, a large number of thymocytes undergo apoptosis, and only a small fraction are selected. Eventually they leave the thymus as mature T cells. Thymocyte death in situ was postulated to explain the imbalance between high thymocyte turnover rate and low output rate of mature T cells from the thymus. Their data showed that the injected peptides can induce cell death (apoptosis) in the thymus cortex in the mice studied.

Pedotti et al. in 2001 encountered animals that died very rapidly after the administration of the self-antigen myelin proteolipid protein (PLP) amino acids 139 through 151, or PLPp(139-151). The researchers where attempting to develop a peptide-based antigen-specific therapy to treat experimental autoimmune encephalomyelitis (EAE). They report that hypersensitivity occurred within a few minutes of injection and the death of the mice occurred within 30 minutes in 43% of the mice studied. The nature and time-course of the observed symptoms were reported as anaphylactic shock. EAE can refer either to experimental autoimmune encephalomyelitis or experimental allergic encephalomyelitis. Although EAE is classically a prototypic T helper 1 (TH1) cell–mediated autoimmune disease, it can also be induced by TH2 cells. Anaphylaxis is associated with exposure to a foreign antigen that is often derived from medication, insect venom or food. The authors report in the cited paper that, after self-tolerance to myelin is destroyed, anaphylaxis may be triggered by a self-antigen, in this case a myelin peptide. They state that - "Horror autotoxicus", which was initially described by Ehrlich, may not only include autoimmunity to self, but may also encompass immediate hypersensitivity to self, which can lead to shock and rapid death.

Liu et al. in 2006 report that the immunization of acutely infected mice causes lethal shock. BALB/c mice that had been infected with Lymphocytic Choriomeningitis Virus (LCMV) were inoculated with the synthetic peptide representing the LCMV epitope NP118 (RPQASGVYM) or saline alone. The mice injected with saline displayed no clinical signs. However, all of the peptide-injected mice died within 24 hours after the injection. The authors state that the same effector function that can protect the host can also be harmful if they are not tightly regulated. Furthermore, they report that the administration of a peptide to virus-immune recipient mice can lead to the synchronous activation of preexisting virus-specific CD8+ T cells with serious or even lethal consequences.

Ridwan et al. in 2010 performed studies to optimize the in vivo activity of a proteolipid protein-bifunctional peptide inhibitor molecule (PLP-BPI) to suppress EAE in mice and to evaluate pharmokinetic profiles of PLP-BPI. One part of their study included peptides that minimize the risk for an anaphylactic response during treatment. The significance of studies like these is to find synthetic therapeutic peptides that show inhibitory activity of the target and have a low risk to induce anaphylactic shock.

Taken together, these examples show that there is a small risk that some peptides used for antibody production can have a lethal effect on the immunized animals.

References:

1. Fei Liu, Ralph Feuer, Daniel E. Hassett, and J. Lindsay Whitton. Peptide vaccination of mice immune to LCMV or vaccinia virus causes serious CD8+ T cell–mediated, TNF-dependent immunopathology. The Journal of Clinical Investigation 116:465-475 (2006).
2. Rosetta Pedotti, Dennis Mitchell, Jochen Wedemeyer, Marcela Karpuj, Dorothée Chabas, Eyas M. Hattab, Mindy Tsai, Stephen J. Galli & Lawrence Steinman. An unexpected version of horror autotoxicus: anaphylactic shock to a self-peptide. Nature Immunology 2, 216 - 222 (2001).
3. Rahmawati Ridwan, Paul Kiptoo, Naoki Kobayashi, Scott Weir, Michael Hughes, Todd Williams, Rondang Soegianto, and Teruna J. Siahaan; Antigen-Specific Suppression of Experimental Autoimmune Encephalomyelitis by a Novel Bifunctional Peptide Inhibitor: Structure Optimization and Pharmacokinetics. JPET 332:1136–1145, 2010.
4. Arthur M. Silverstein; Autoimmunity versus horror autotoxicus: The struggle for recognition. Nature Immunology 2001, pp279 – 281.
5. Erwan Ventre, Lilia Brinza1, Stephane Schicklin1, Julien Mafille, Charles-Antoine Coupet, Antoine Marçais, Sophia Djebali, Virginie Jubin, Thierry Walzer and Jacqueline Marvel. Negative Regulation of NKG2D Expression by IL-4 in Memory CD8 T Cells. The Journal of Immunology October 1, 2012 vol. 189 no. 7 3480-3489.
6. Andreas Wack, Heather M. Ladyman1, Owen Williams, Kathleen Roderick, Mary A. Ritter1 and Dimitris Kioussis Direct visualization of thymocyte apoptosis in neglect, acute and steady-state negative selection International Immunology, Vol 8, No. 10, pp 1537-1548.

Definition of Horror autotoxicus: Means literally, the horror of self-toxicity. A term coined by the great German bacteriologist and immunologist Paul Ehrlich (1854-1915) to describe the body's innate aversion to immunological self-destruction. As we now know the immune system can occasionally attack itself and does so in autoimmune disorders.