**2. A Novel Enhancer Sequence for DNA Vaccine Antigen Expression**

Our group therefore previously investigated the potential of short enhancer sequences derived from a mammalian single-stranded DNA virus—porcine circovirus type I (PCV-1)—for dose-sparing potential and immunogenicity enhancement in a clinically trialed HIV-1 subtype C DNA vaccine [3]. The plasmid vector (pTH) has been well used in preclinical and clinical studies [4–6] and is regarded as being a high-potency vaccine antigen vector for HIV and other agents. It relies on the human cytomegalovirus immediate/early promoter (CMV I/E) enhancer element constituting the promoter Pcmv [7], one of the strongest known promoters in mammalian expression systems, driving in vivo antigen expression with the help of the CMV intron A and the bovine growth hormone polyadenylation signal. It has been used to vector the synthetic HIV-1 subtype C vaccine antigen GrttnC, a polyprotein incorporating Gag, reverse transcriptase (RT), Tat, and Nef sequences, in studies in mice, guinea pigs, monkeys, and humans [8–12].

PCV-1, like all circoviruses, has a compact, genetically dense, bi-directionally transcribed genome of 1759 bp that encodes only a viral capsid protein (*cap* gene) and the replication-associated proteins Rep and Rep- , which derive by alternative splicing from one open reading frame (ORF) (*rep*) (Figure 1). Bidirectional transcription of the two genes originates in the origin of replication (Ori) for *rep*, and in an intron within *rep* for *cap* [13]. In vitro expression studies in human embryonic kidney 293 (HEK293) cells with various constructs derived from the PCV-1 genomes showed that enhancement activity resided in a 70 base pair "core sequence" (C) of the 172 base pair (bp) capsid promoter, Pcap, that includes a putative composite transcription factor binding site comprising CCAAT/enhancer-binding protein beta (C/EBPb), GATA-1, and cAMP response element-binding protein (CREB) sites, as well as a 47 bp conserved late element, or CLE. Inclusion of the 70 bp sequence in the reverse orientation immediately upstream of the Pcmv sequence in pTHgrttnC (yielding pTHCRgrttnC) resulted in 2.4-fold enhancement of polyprotein expression level in vitro following transfection of HEK293 cells, as assessed by Gag p24 ELISA. The cognate sequence from the related PCV-2 was equally effective. The 172 bp Pcap sequence also enhanced luciferase expression in HEK293 cells three-fold when inserted in reverse orientation upstream of the simian virus 40 (SV40) promoter in the commercial pGL vector [3]. Accordingly, we tested the enhancement of immunogenicity in vivo by intramuscular injection of mice with a variety of pTHgrttnC constructs with additives from PCV-1 (Figure 1C): The best enhancement over pTHgrttnC, as assayed by interferon-gamma enzyme-linked immune absorbent spot (IFN-γ ELISPOT) responses to a RT CD8<sup>+</sup> peptide, was obtained using the Pcap (172 bp) insert, after two intramuscular inoculations of 100 μg of pTHPcapRgrttnC DNA (five-fold increase in spot forming units (sfu)/106 splenocytes). Moreover, two inoculations of 10 μg of pTHPcapgrttnC DNA was significantly more immunogenic (3.5-fold) than pTHgrttnC and boosting with 104 plaque forming units (pfu) of modified vaccinia Ankara (MVA) vectoring Grttn showed the same trend (Figure 2). The response to the 10 μg of pTHPcapgrttnC DNA alone was also equivalent to or higher than to 100 μg of pTHgrttnC, indicating that significant dose sparing (10-fold) was possible for the same priming effect for a vaccine-relevant antigen. This proof that a simple enhancement could dramatically improve the functionality of a DNA vaccine vector led to its being employed in subsequent studies in our HIV vaccine research program.

**Figure 1.** Porcine circovirus type-1 (PCV-1) genome arrangement. (**A**) Diagram of the linearized PCV-1 genome, depicted in the orientation cloned into pTHCapgrttnC. The *rep* intron is enlarged and the capsid gene promoter (Pcap) indicated. The core and conserved late elements (CLE) components of Pcap are shown. *rep* = replication associated protein gene, *cap* = capsid protein gene, Prep = *rep* gene promoter, Ori = origin of replication, core = composite host transcription factor binding site. (**B**) DNA sequence of 172 bp PcapR fragment. Putative host transcription factor binding sites are indicated and underlined, CLE motifs are in bold and the minimal PcapR sequence (1252–1238; as identified by Mankertz and Hillenbrand [13]) is highlighted in gray. PCV-1 accession number Y09921. (**C**) Schematic diagrams of plasmids showing assembly of PCV elements. Pcmv = Cytomegalovirus (CMV) promoter, *grttnC* = gene encoding polyprotein of HIV-1 Gag, reverse transcriptase (RT), Tat and Nef, C = 70 bp Pcap core. Figure reproduced from Tanzer et al. [3] under the Creative Commons Attribution (CC-BY) license as specified by BioMed Central.

**Figure 2.** HIV-1 specific IFN-γ ELISPOT responses to pTHgrttnC DNA vaccines containing portions of the PCV-1 genome. Groups of mice were vaccinated intramuscularly with DNA vaccines on days 0 and 28. Two groups of mice were subsequently boosted with 10<sup>4</sup> pfu of modified vaccinia Ankara (MVA) on day 56. A separate group of mice was vaccinated with 10 μg pTH (empty vector) on days 0 and 28 and subsequently boosted with 10<sup>4</sup> pfu of MVA on day 56. \* *p* < 0.001; \*\* *p* < 0.05 Student *t*-test. Figure reproduced from Tanzer et al. [3] under the Creative Commons Attribution (CC-BY) license as specified by BioMed Central.

## **3. Testing the Enhanced DNA Vector with HIV-1 Subtype C pr55Gag**

Strong polyfunctional CD8<sup>+</sup> T cell responses to HIV-1 Gag or Gag-derived antigens have been found to be important for controlling viremia in HIV+ people who are termed "elite controllers." Accordingly, Gag should be and often is included in candidate HIV vaccination regimes, so as to allow early clearance of infected cells at the initial sites of infection, as well as control of spread from these sites and later control of viremia [14]. A subtype C mosaic Gag sequence was chosen to increase the coverage of both CD8<sup>+</sup> and CD4<sup>+</sup> T cell epitopes from that of natural sequences with the hope of reducing the HIV-1 escape pathways [15–18]. Subtype C (HIV-1C) was chosen as it is the most prevalent subtype in the world, accounting for over 50% of all global infections and is the dominant subtype in southern Africa. In a study carried out by our group, the pTHPcapR plasmid backbone [3] was used to construct a DNA vaccine containing an HIV-1 subtype C mosaic *gag* gene, DNA-GagM [19,20].

HEK293T cells transfected with DNA-Gag<sup>M</sup> expressed high levels of Gag (up to 26 ng/mL in the media). The immune responses to the DNA vaccine were evaluated in mice using homologous and heterologous prime boosts with MVA vaccine expressing the matching HIV-1 subtype C mosaic Gag antigen (MVA-GagM). To confirm that the DNA vaccine was immunogenic at a low dose, mice were vaccinated with 10 μg of the DNA vaccine. Mice vaccinated with two doses of DNA-GagM had mean cumulative Gag-specific IFN-γ ELISPOT responses of 882 sfu/10<sup>6</sup> splenocytes (Figure 3). These responses were higher for CD8<sup>+</sup> rather than for CD4<sup>+</sup> Gag peptides (604 and 278 sfu/106, respectively). Mice that received a heterologous prime boost consisting of two doses of DNA-GagM followed by a single dose of MVA-GagM had mean cumulative Gag-specific IFN-γ ELISPOT responses of 2675 sfu/106, that were evenly balanced for both Gag CD4<sup>+</sup> and CD8<sup>+</sup> peptides. Both the homologous and heterologous vaccination regimen elicited a higher proportion of CD8<sup>+</sup> T cells expressing cytokines than CD4<sup>+</sup> T cells. All the cytokine-positive CD8<sup>+</sup> T cells had an effector–memory phenotype. This

study confirmed that the pTHPcapR DNA vector backbone containing the porcine circovirus enhancer elicits high-magnitude, Gag-specific T cell responses in BALB/c mice at a low dose.

**Figure 3.** DNA vaccine elicits high Gag-specific IFN-γ ELISPOT responses both alone and in a heterologous prime boost with MVA. (**A**) Vaccination schedule. DNA-Gag<sup>M</sup> = pTHPcapR containing mosaic *gag*; DNAE = pTHPcapR empty vector; MVA-Gag<sup>M</sup> = MVA containing mosaic *gag*. (**B**) Cumulative IFN-γ ELISPOT CD8<sup>+</sup> and CD4<sup>+</sup> responses of vaccinated mice to HIV-1 Gag peptides. \*\* *p* < 0.01 Student *t*-test of unpaired data.
