Globally almost 39 million people are living with HIV, which causes AIDS by decreasing the number of CD4+ T cells. The current standard of care, Antiretroviral therapy (ART), is an expensive, non-curative treatment that requires patients’ life-long rigorous therapy compliance. As a result, the ART-based treatment costs the Belgian government 240 million euro per year, showing the commercial need for more efficient, cheaper treatment options. Nanobodies (Nbs) have a plethora of interesting properties that are advantageous for the treatment of HIV, such as a wide neutralization breadth and high tissue penetration. Therefore, we aim to generate Nbs against the HIV Env glycoprotein and assess their potential for HIV treatment and imaging applications. Firstly, a DNA immunization protocol will be applied in llamas to produce HIV Env-targeting Nbs, whereafter the most promising candidates will be tested for their ability to image HIV reservoirs in mice. Simultaneously, bispecific T-cell engagers (BiTEs) will be developed which target cytotoxic T cells towards the HIV-infected host cells. These BiTEs will be optimized and undergo extensive in vitro and in vivo testing, combining a variety of in-house assays applied to cellular models, patient-derived samples and HIV infected humanized mouse models. Together, the current research proposal has a high potential to develop a more efficient HIV treatment and establish non-invasive monitoring of HIV reservoirs in patients.