Here, an Ang II peptide vaccine effectively reduced BP and prevented cardiovascular complications such as cardiac fibrosis by stimulating anti-Ang II antibody production without severe adverse effects. Our working hypothesis about Ang II vaccine therapy was evaluated in each step of the study (see ). As shown in , mice are immunized with Ang II-KLH and adjuvants to circumvent T cell tolerance. As an immunization phase, antigen-presenting cells (APCs) phagocytose Ang II-KLH and present a T cell epitope of Ang II-KLH to T cells through the major histocompatibility complex (MHC), and T cells recognize it through the T cell epitope and become activated (i.e., differentiate to effector T cells) (step 1). B cells, specific to Ang II phagocytose Ang II-KLH and present the T cell epitope of Ang II-KLH to T cells through MHC. Then, B cells differentiate to plasmacytes and produce antibodies with the help of activated T cells (effector T cells) (step 2).
Conceptual schematic of the experiment.
In this study, we confirmed that the vaccine with Ang II-KLH and adjuvants efficiently induced an antibody titer of Ang II and induced T cell activation. Generally, antigens must contain both B-cell and T-cell epitopes. We and others have confirmed that antibodies against Ang II have been produced successfully
, confirming the existence of a B-cell epitope in Ang II. As for T-cell epitopes, we perform a T cell proliferation assay and ELISPOT assay. The results show that Ang II-KLH and KLH induced T cell activation, but Ang II did not (–), which may suggest that only KLH contains a T-cell epitope. Therefore, eight amino acids of Ang II could activate B cells but not T cells in our animal model. This situation is reflected in the relationship between hapten and its carrier, in which hapten has only a B-cell epitope and the carrier possesses a T-cell epitope.
Based on this finding, we used Ang II and KLH as hapten and its carrier to successfully produce an antibody against Ang II which was assisted by helper T-cell activation.
The immune response against pathogens comprises factors that activate both T cells and B cells. It lasts until the invaders are removed. However, the “natural boost” for most autoimmune diseases is generated by a self-antigen, and leads to the maintenance of chronically high antibody titers because the factors activating T cells and B cells are inherent to the body and cannot be cleared.
Therefore, we also investigated the response to endogenous Ang II after immunization, as shown in . In this case, APCs do not present T cell epitope of Ang II to T cells. Hence, endogenous Ang II does not activate T cells (i.e., cause them to differentiate to effector T cells) (step 1). Because B cells do not present the T cell epitope of Ang II to T cells, B cells are not stimulated because of the lack of the help of the effector T cells. Although B cells are sometimes stimulated by an antigen without the help of T cells, Ang II does not directly stimulate B cells (step 2). Similar to a previous report
_ENREF_14_ENREF_14_ENREF_14, our study showed that the Ang II vaccine did not maintain a high titer after the last boost (). Furthermore, a large amount of Ang II administration did not induce antibody production in immunized mice (). This result implies that endogenous Ang II does not induce the activation of T cells or the production of anti-Ang II antibodies, improving its safety for clinical use.
The use of vaccines targeting self-antigens has recently been reported for cancer 
, rheumatoid arthritis 
, Alzheimer’s disease 
, hypertension 
, and dyslipidemia.
Because safe and effective drug therapies have already been established for several of these conditions, adverse vaccine effects should be carefully considered, especially in hypertension and dyslipidemia. The key goals of this approach are avoiding T-cell activation in response to self and the reversible inhibition of the target molecule. The Alzheimer’s disease clinical trial was halted because the participants developed aseptic meningoencephalitis due to an autoimmune response.
Because amyloid beta-induced T-cell activation was considered responsible for the adverse event, we examined whether Ang II had the ability to trigger a T-cell-mediated immune response using T cell proliferation and ELISPOT assays. Splenocytes from the immunized mice responded to Ang II-KLH and KLH, but not to Ang II (), which showed that Ang II does not induce T cell activation. In another case, vaccine may have some possibilities to induce the production of immune complex by the antigen-antibody reaction. For example, in the case of renin vaccine, the autoimmune nephritis was induced by the observation of immune complex depositions same as Lupus nephritis or IgA nephropathy. 
In this study, immunized mice did not show any findings even after Ang II infusion, which suggest that Ang II vaccine did not induce autoimmune nephritis unlike renin vaccine in mice. Therefore, we suggest that this vaccine does not induce an autoimmune disease because KLH is a completely “non-self” antigen in the human body and Ang II-KLH only exists during immunization.
In conclusion, we propose that this Ang II vaccine is safe, does not carry the risk of an anti-Ang II T cell-mediated autoimmune response, and effectively induces the production of anti-Ang II antibody. In this study we employed Ang II infusion in mice after the antibody titer peaked, which corresponds to preventive therapy. As a therapeutic model, the Ang II vaccine was also effective at reducing blood pressure in SHR
. In this study, we successfully showed that Ang II vaccine was also effective at reducing blood pressure in relatively old SHR. Although further studies are necessary to develop the Ang II vaccine into a real treatment for hypertensive patients, future modifications supporting the clinical application of this vaccine may identify a novel immunotherapy for hypertension.