A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

www.nature.com/scientificreports OPEN received: 10 February 2016 accepted: 15 June 2016 Published: 30 June 2016 A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice Khamis Tomusange1, Danushka Wijesundara1, Jason Gummow1, Tamsin Garrod2, Yanrui Li1, Lachlan Gray3,4, Melissa Churchill3, Branka Grubor-Bauk1 & Eric J. Gowans1 DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAXsTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. The HIV Tat protein is required for efficient virus replication1,2 and is released into the extracellular milieu by infected cells where it can be taken up by other cells to increase transcription from the HIV long terminal repeat (LTR)1. The tat gene is more genetically stable than the env gene and immunogenic Tat epitopes are conserved across HIV-1 subtypes in group M3. Thus, a Tat vaccine may provide cross protection across all group M viruses, which account for a majority of HIV infections globally4. These attributes make Tat a potential component of an HIV vaccine. As it has been difficult to generate canonical Env-specific neutralising antibodies (NAb)5, the production of high titre anti-Tat NAb might be a feasible alternative to control HIV replication and delay disease onset. Tat-based HIV vaccines are safe and generate high titre Tat-specific humoral and cell mediated immunity (CMI) that correlate with asymptomatic infection or slower disease progression in humans6,7 and animals. However, native Tat is poorly immunogenic, easily oxidised and degraded by proteolysis1. Thus, protecting Tat from these detrimental processes improves the immunogenicity of Tat-based vaccines8,9. We now report a novel strategy that incorporates Tat into heptamers by fusing Tat with the oligomerisation domain of the C4 binding protein (C4b-p), an inhibitor of the classical and lectin pathways of complement10. Previously the most effective form, termed IMX313, a hybrid derived from the oligomerisation domains of human and murine C4b-p α-chains11–13 induced oligomerisation of P. falciparum or M. tuberculosis antigens and enhanced the immunogenicity and protective efficacy of the resultant vaccines11–14. The IMX313 amino acid sequence is <20% identical to the murine or human C4b-p oligomerisation domains, thus IMX313 does not induce antibodies12, and has recently been shown to be safe for use in humans15 making it a practical molecular adjuvant. However, as the adjuvanticity of IMX313 requires efficient secretion of the oligomerised protein14, a TPA leader sequence was introduced upstream of the 1 Virology Laboratory, Basil Hetzel Institute, Discipline of Surgery, University of Adelaide, Adelaide, South Australia, Australia. 2Royal Australasian College of Surgeons, Adelaide, South Australia, Australia. 3Centre for Biomedical Research, Burnet Institute, Melbourne VIC, Australia. 4Department of Infectious Diseases, Monash University, Melbourne VIC, Australia. Correspondence and requests for materials should be addressed to E.J.G. (email: eric. ) Scientific Reports | 6:29131 | DOI: 10.1038/srep29131 1 www.nature.com/scientificreports/ Figure 1. Vaccine constructs and Tat expression. (A) Schematic representation of the vaccine constructs; (B) reducing Western blot analysis of Tat in cell lysates from HEK293T cells transfected with plasmid DNA encoding the different forms of Tat (tracks 2–5) and (C) non-reducing Western blot analysis of Tat in supernatant fluids of HEK293T cells transfected with pVAX-Tat-IMX313 (track 2) or pVAX-sTat-IMX313 (track 3) DNA. The 42 kDa β-actin protein was used as loading control for the Western blot. Blots in Fig. 1B,C are cropped for clarity and conciseness; full length blots of these figures are presented in supplementary figure S1A,B. sequences encoding Tat and IMX313 to drive expression of the oligomerised protein into the secretory pathway. Indeed, DNA and recombinant viral vaccines encoding IMX313 are in advanced clinical testing16. DNA vaccines are stable, readily manufactured, induce humoral and CMI, and are licensed for veterinary use17, emphasising their potential in developing human vaccines. To improve the immunogenicity of Tat-based DNA vaccines, we fused Tat to IMX313, and compared the immunogenicity and protective efficacy of this vaccine with that of the corresponding vaccines lacking IMX313 with a major thrust to elicit robust humoral responses. Results Tat oligomerisation. Four plasmids, each encoding a different form of HIV Tat were constructed (Fig. 1A). In this study, the DNA vaccine encoding the native form of Tat protein was used as the benchmark against which the efficacy of plasmids encoding secreted and/or oligomerised forms of Tat were compared. Native Tat antigen, was not used as a control , as others have shown that its immunogenicity is affected due to its susceptibility to oxidation and proteolysis1. To confirm expression of Tat, HEK293T cells were transfected with plasmid DNA, and Western blot analysis performed on all four cell lysates (Fig. 1B) and supernatant fluids from pVAX-Tat-IMX313 or pVAX-sTat-IMX313-transfected cells (Fig. 1C). Oligomers of protein-IMX313 fusions can only be detected in non-reducing conditions12,14 and consequently in a reducing Western blot, 11, 14, 18 and 20 kDa bands corresponding to proteins expressed from pVAX-Tat, pVAX-sTat, pVAX-Tat-IMX313 and pVAX-sTat-IMX313, respectively, were detected (Fig. 1B) and supplementary figure S1A (for full size blot).The assay was repeated in the absence of β-Me and bands of ~18 kDa from pVAX-Tat-IMX313-and ~140 kDa from pVAX-sTat-IMX313transfected cells detected (Fig. 1C) and supplementary figure S1B (for full l (...truncated)