A Human T-Cell Leukemia Virus Type 1 Regulatory Element Enhances the Immunogenicity of Human Immunodeficiency Virus Type 1 DNA Vaccines in Mice and Nonhuman Primates

Abstract

Plasmid DNA vaccines elicit potent and protective immune responses in numerous small-animal models of infectious diseases. However, their immunogenicity in primates appears less potent. Here we investigate a novel approach that optimizes regulatory elements in the plasmid backbone to improve the immunogenicity of DNA vaccines. Among various regions analyzed, we found that the addition of a regulatory sequence from the R region of the long terminal repeat from human T-cell leukemia virus type 1 (HTLV-1) to the cytomegalovirus (CMV) enhancer/promoter increased transgene expression 5-to 10-fold and improved cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens. In cynomolgus monkeys, DNA vaccines containing the CMV enhancer/promoter with the HTLV-1 R region (CMV/R) induced markedly higher cellular immune responses to HIV-1 Env from clades A, B, and C and to HIV-1 Gag-Pol-Nef compared with the parental DNA vaccines. These data demonstrate that optimization of specific regulatory elements can substantially improve the immunogenicity of DNA vaccines encoding multiple antigens in small animals and in nonhuman primates. This strategy could therefore be explored as a potential method to enhance DNA vaccine immunogenicity in humans.Plasmid DNA vaccines have shown promise as a novel vaccination modality based on their simplicity and versatility (31,32,36). In particular, DNA vaccines can elicit potent and protective cellular and humoral immune responses in a variety of small-animal models (10). However, they have proven substantially less immunogenic in nonhuman primate studies and in clinical trials to date (8,19,33).Several approaches have been explored to improve the immunogenicity of DNA vaccines. Our laboratories and others have demonstrated that the addition of plasmids expressing cytokines and immunomodulatory molecules can substantially augment DNA vaccine-elicited immune responses in both mice and nonhuman primates (3,4,15,16,21,34,37). However, the practical requirements of manufacturing and establishing the safety of the plasmid cytokines prior to the initiation of clinical trials may prove a limitation of this strategy (7, 26). Other approaches involve the addition of polymer adjuvants (29) and the use of in vivo electroporation techniques (24, 35). These strategies have similarly proven effective in animal models, but their practical utility in clinical trials has yet to be demonstrated.In this study, we investigate a novel strategy involving optimization of regulatory elements in the backbone of the plasmid DNA vaccine. DNA vaccines often utilize a cytomegalovirus (CMV) enhancer, promoter, and intron to drive high-level expression of a transgene in mammalian cells (32,38). Here, we explore the effects of adding the regulatory R region from the 5Ј long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1), which acts as a transcriptional and posttranscriptional enhancer (30). We find that these CMV/R DNA vaccines elicit substantially higher human immunodeficiency viru...

Effect of promoter strength on protein expression and immunogenicity of an HSV-1 amplicon vector encoding HIV-1 Gag

“…These promoters included: two retroviral promoters (the Rous sarcoma virus [RSV] and the myeloproliferative sarcoma virus [MPSV] long terminal repeat [LTR]) as well as the major human cytomegalovirus immediate-early promoter [CMV], and several hybrid promoters derived from this element; these included a composite CMV/ chicken ß-actin promoter [CAG] [19], a modified CMV promoter containing regulatory sequences from the human T-cell leukemia virus type-1 LTR [CMV-R] [20], and a CMV promoter linked to the CMV Intron A sequence [CMV-I] [21,22].…”

“…Promoters chosen for this study included the ubiquitously active human cytomegalovirus immediate early (CMV) promoter, as well as three hybrid regulatory elements derived from this promoter. These hybrid promoters contained combinations of the CMV promoter/enhancer together with (i) its associated Intron A element (CMV-I) [21,22,[32][33][34], (ii) the regulatory R region from the long terminal repeat (LTR) of the human T-cell leukemia virus type 1 (CMV-R) [20], and (iii) the chicken β-actin promoter and the woodchuck hepatitis virus regulatory element (CAG) [19]. Each of these composite CMV-based promoters has been reported to mediate improved levels of gene expression and/or enhanced immune responses to encoded antigens, when compared to the basic CMV promoter [35,36].…”

“…The basic CMV promoter/ enhancer was used from a modified pVax-based amplicon plasmid and the gag insert cloned in with HindIII and XbaI. The CMV promoter/enhancer and the associated Intron A sequence were PCR amplified from plasmid VRC5309 (provided by the NIH VRC) using forward (5′-ACCATATGCACGCGTGTGTGAAATACCGCA-3′) and reverse oligonucleotide primers (5′-TGGCGATATCTCTAGAGCGGCCGCGATATC-3′) [20,23] (VRC5309 was generously provided by Dr. Gary Nabel of the NIH Vaccine Research Center, VRC). The 1.6 kb PCR product was digested with MluI and XbaI and cloned into the amplicon backbone to create CMV-I.…”

HSV-1 amplicon vectors elicit polyfunctional T cell responses to HIV-1 Env, and strongly boost responses to an adenovirus prime

“…Three different HSVgp120 amplicon vectors and an E1-deleted, replication defective type 5 adenovirus (rAd5) vector were constructed. The three HSVgp120 amplicon vectors all contained the same HIV-1 gp120 insert, placed under the transcriptional control of three different promoters: the major human cytomegalovirus immediate early promoter (cmv), a modified cmv promoter containing regulatory sequences from the human T-cell leukemia virus type 1 LTR (cmvr) [39], and the herpes simplex virus immediate early 4/5 promoter (hsv). HIV-1 gp120 expression in the rAd5 vector was transcriptionally controlled by the cmv promoter.…”

“…Animals were inoculated with one of three HSV-1 amplicon vectors which encoded HIV-1 gp120 under the transcriptional control of three different promoters: (1) the major human cytomegalovirus (CMV) immediate early (cmv) promoter, (2) a modified cmv promoter containing regulatory sequences from the human T-cell leukemia virus type 1 LTR (cmvr) [39], and (3) the herpes simplex virus immediate early 4/5 promoter (hsv). Additional groups of animals received a recombinant adenovirus type-5 encoding the same HIV-1 gp120 gene (rAd5gp120 (cmv)), under the control of the CMV promoter (cmv) or a mixed inoculum containing the rAd5gp120 (cmv) vector and the HSVgp120 (hsv) vector.…”