Vaccine 22 (2004) 2586–2598 Codon optimization and ubiquitin conjugation of human immunodeficiency virus-1 Tat lead to enhanced cell-mediated immune responses Lakshmi Ramakrishna a , Krishnamurthy Kumar Anand a , Marthandan Mahalingam a , Kumarasamypet M. Mohankumar a , Shilpa Ramani b , Nagadenahalli B. Siddappa a , Udaykumar Ranga a,∗ a Molecular Virology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur (PO), Bangalore 560064, India b Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India Received 5 August 2003; received in revised form 13 November 2003; accepted 11 December 2003 Available online 20 January 2004 Abstract The transactivator protein, Tat, is a potential candidate for developing a vaccine against human immunodeficiency virus (HIV-1). Since Tat is not immunodominant, especially when delivered as a genetic vaccine, we expressed codon-optimized subtype-C Tat as a molecular conjugate of ubiquitin, to elicit antigen-specific cell-mediated immune responses. Immunization of mice with different ubiquitin-Tat constructs elicited a strong cellular, but not a humoral, immune response. The combination of codon-optimization and ubiquitin-mediated processing of Tat induced a Th-1 type cellular immune response that was detectable without in vitro stimulation, suggesting its potential utility for destruction of virus-infected cells via CTL-mediated lysis. Preliminary attempts at characterizing the immunodominant regions identified a novel T-helper epitope within the core domain of Tat. © 2004 Published by Elsevier Ltd. Keywords: HIV-1 subtype-C Tat; Molecular adjuvant; DNA vaccine 1. Introduction A wide variety of attempts at developing a vaccine for hu- man immunodeficiency virus type 1 (HIV-1) focused mainly on the envelope (env) gene of the virus. Although env vac- cines conferred protection against autologous viral strains, antigenic variation is a challenge for vaccine design [1]. A need for developing multi-component vaccines is being increasingly realized, to induce broader immune responses against the viral infection, by incorporating multiple viral antigens [2]. Extensive work from various laboratories has identified the viral structural proteins, gag and pol, and vi- ral regulatory proteins Nef, transactivator protein (Tat) and Rev, as potential candidates for vaccine development [3–5]. The regulatory genes of the virus that are expressed early during the viral life cycle have the advantage of stimu- lating immune responses with faster kinetics. Additionally, ∗ Corresponding author. Tel.: +91-80-2362-2750x2241; fax: +91-80-2362-2766. E-mail address: udaykumar@jncasr.ac.in (U. Ranga). their sequences are also conserved to a greater extent. The viral transactivator protein has been delivered as a candi- date antigen in several vaccination formats including as a peptide [6,7], a protein [8], a toxoid [6,9], a DNA vac- cine [10–13], and a recombinant virus [13,14]. Although the results of these studies have been conflicting, the im- portance of anti-Tat immune responses as correlates of pro- tection has been established. Cellular immune responses to the regulatory proteins of HIV-1, especially Tat, are con- sidered important for protection against disease progression [15,16]. Strategies employing Tat as a vaccine must, however, overcome the problem of poor immunogenicity of this viral antigen. Immune responses to Tat usually are absent in the majority of the seropositive subjects and when present are of low magnitude [17]. Especially when Tat is delivered in the form of a DNA vaccine, strategies to enhance the host im- mune response are essential, as this strategy of immunization is not efficient. Vaccination using DNA induces weak hu- moral and cell-mediated responses even when administered in multiple doses and requires adjuvants or boosting with 0264-410X/$ – see front matter © 2004 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2003.12.007