For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269 -277]. However, the decreased ability of the immune system to mount a robust immune response to selfantigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO 2Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-␣ (mTNF-␣) independently to pNO2Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-␣ IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-␣ and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO 2Phe 43 mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.retinol-binding protein ͉ tumor necrosis factor ͉ vaccination ͉ p-nitrophenylalanine ͉ genetic code