Plasmid Vaccines and Plasmid Cytokine Adjuvant
Product VRC-3900 (HIV-1)
This precursor plasmid vaccine construct (6438 bp) encodes the gag gene of the HXB2 strain of HIV-1 (). Briefly, it is built on the pVR1012x/s plasmid backbone contaexining a kanamycin resistance gene, the human cytomegalovirus (CMV) promoter/enhancer (containing the immediate early 5′ untranslated region and intron), and the bovine growth hormone (BGH) polyadenylation sequence. This backbone differs from a related backbone (Hartikka et al., 1996
) used in products described below by removal of an Xmn
I site in the region following the BGH poly A sequence and replacement with an Sfi
I site. This was done to ensure that at least one Sfi
I site was present in each plasmid construct to permit use of Sfi
I in the integration studies. When the gene insert contained an Sfi
I site, the pVR1012 backbone was used. When it did not, the pVR1012x/s backbone, with the introduced Sfi
I site, was used. A synthetic version of the gag gene using codons optimized for expression in human cells was inserted into this plasmid backbone (pVR1012x/s). The nucleotide sequence of the synthetic gag gene shows little homology to the HXB2 gene, but the protein encoded is the same. This candidate vaccine did not enter clinical trials because development of multigenic constructs was pursued instead.
Materials and Methods—Products Tested
Product 1 (HIV-1): VRC-4302
This first-generation plasmid vaccine candidate (9170 bp) was generated by inserting into the pVR1012x/s plasmid backbone a synthetic human codon–optimized gag gene from the HXB2 strain of HIV-1 ligated in frame with sequences encoding a synthetic (human codon optimized) pol polyprotein from the NL4−3 strain of HIV-1. To allow for the natural translational frameshifting, the synthetic coding region of the last four amino acids of the nucleocapsid protein through the rest of gag plus an additional three amino acids from pol were replaced with the authentic viral sequences from NL4−3. This introduces the site required for frameshifting and restores the ability to express all the gag proteins. Mutations were introduced into the protease-, RT-, and integrase-encoding sequences to destroy the biological activities of these viral proteins for safety while retaining the maximal number of potential epitopes for immunogenicity when inoculated into humans as a vaccine candidate (Huang et al., 2001
). A phase I trial of this product was completed, and this product also served as a comparator plasmid for later products tested in biodistribution studies.
Product 2 (HIV-1)
The product is a combination of two plasmids, VRC-2805 and VRC-4306. VRC-2805 (6869 bp) consists of a HIV-1 clade B env gene inserted into the pVR1012x/s backbone, described above. The env gene contains the following modifications: a human codon–optimized sequence from HXB2 into which sequences from BaL were inserted to change the tropism from the CCR4 receptor to the CXCR5 receptor. The chimeric env gene was truncated to include the entire SU protein–coding region and a portion of TM, including the fusion domain, the transmembrane domain, and regions important for oligomerization. A portion of the fusion/cleavage domain has been deleted, as has been the interspace between heptads 1 and 2. This version of env is referred to as gp145Δcfi (Chakrabarti et al., 2002
). VRC-4306 (9790 bp) encodes human codon–optimized gag, pol, and nef genes expressed as a fusion protein. Deletions have been introduced into the protease-, RT-, and integrase-coding regions to destroy the biological activities of these viral proteins for safety reasons. In addition, the authentic viral sequences were included in the region to allow for frameshifting (as described above for Product 1 [HIV-1]). The gag portion of the fusion protein is from the HXB2 strain, and the pol and nef portions are from the NL4−3 strain (the entire nef including the initial ATG is included in the construct). This was inserted into the pVR1012 backbone. This HIV-1 clade B combination product was not tested in clinical trials on its own because a multiclade approach was chosen for clinical development. However, the two components of this vaccine candidate are included among the components of a product that proceeded into clinical trials (see description of Product 6 [HIV-1] below for more details).
Product 3 (HIV-1)
This product is a combination of two plasmids. VRC-2805 is a component of Product 2 (HIV-1) described above. VRC-4302 is Product 1 (HIV-1) described above. This HIV-1 clade B Product 3 also did not proceed to clinical trials because a multiclade approach was chosen for clinical development.
Product 4 (cytokine adjuvant plasmid)
VRC-7000 (6086 bp) encodes a fusion protein human IL-2/Ig inserted into the pVR1012x/s backbone. The entire coding region of the human IL-2 cDNA is included (native sequence without mutations), as is the Fc portion of a human IgG2 (Barouch et al., 2004
Product 6 (HIV-1)
This product is a combination of six plasmids. VRC-2805 (clade B env gene) and VRC-4306 (clade B gag-pol-nef) are described above in Product 2 (HIV-1). VRC-4311 (9786 bp) consists of a human codon–optimized gene encoding a gag-pol-nef fusion protein (modified as described for VRC-4306) from a clade C sequence identified as GenBank accession number U52953. The stop codon TAG was removed from the gag-pol gene, and the nef gene from the same virus was ligated in. The fusion sequence was inserted into the pVR1012 backbone. VRC-5309 (6829 bp) consists of a human codon–optimized gene encoding the env protein (modified as described for VRC-2805 to gp145Δcfi) from HIV-1 clade C 97ZA012 strain inserted into the pVR1012x/s backbone. VRC-4313 (9783 bp) consists of a human codon–optimized gene encoding a gag-pol-nef fusion protein (modified as described for VRC-4306) from a HIV-1 clade A strain identified as GenBank accession number AF004885 (gag-pol) and AF069670 (nef) inserted in the pVR1012 backbone. VRC-5305 (6836 bp) consists of a human codon–optimized gene encoding the env protein (modified as described for VRC-2805 to gp145Δcfi) from HIV-1 clade A 92rw020 strain inserted into the pVR1012x/s backbone. This product did not proceed into clinical trials although a product consisting of four of the six plasmids (VRC-2805, VRC-4306, VRC-5309, and VRC-5305) contained in this product was tested in a completed phase I trial and is being tested in other ongoing phase I trials. The basis for deciding to clinically develop this four-plasmid product instead of Product 6 (HIV-1) was as a result of murine (Kong et al., 2003
) and nonhuman primate (NHP, unpublished data) immunogenicity studies. Further NHP studies led to the development of another six-plasmid product containing the CMV/R promoter (see below in Product 12 [Ebola] for description) and separating the gag, pol, and nef onto separate plasmids (Barouch et al., 2005
). The preclinical studies described herein and in the companion article for Product 6 (HIV-1), as well as those for Product 12 (Ebola), along with phase I clinical data, supported the development of this newer generation six-plasmid product which has also entered phase I testing (Martin et al., 2005
) and is proceeding into advanced clinical development as a prime for an adenovirus type 5-vectored boost HIV vaccine regimen.
Product 7 (Ebola)
This first-generation Ebola product (Sullivan et al., 2000
; Xu et al., 1998
) consists of a combination of four plasmids. VRC-6401 (7329 bp) consists of a synthetic human codon–optimized gene expressing the nucleoprotein (NP) of the Zaire strain of Ebola virus inserted into the pVR1012x/s backbone. VRC-6201 (7087 bp) consists of a synthetic human codon–optimized gene expressing the full-length glycoprotein (GP) of the Sudan strain of Ebola virus inserted into the pVR1012x/s backbone. VRC-6301 (7036 bp) consists of a synthetic human codon–optimized gene expressing the full-length GP protein of the Ivory Coast strain of Ebola virus inserted into the pVR1012x/s backbone. VRC-6001 (7188 bp) consists of a synthetic human codon–optimized gene expressing the full-length GP protein of the Zaire strain of Ebola virus inserted into the pVR1012x/s backbone. However, this product did not proceed to clinical trial because of observed in vitro
cytotoxicity with the full-length GP constructs (Sullivan et al., 2005
) and the theoretical safety concerns this phenomenon raised; modified GP vectors were developed.
Product 12 (Ebola)
This second-generation Ebola product consists of a combination of three plasmids. The backbone, referred to as CMV/R, used for each of these plasmids differs from the pVR1012x/s and pVR1012 plasmid backbones in that it substitutes the Human T-Lymphotrophic Virus (HTLV-1) Long Terminal Repeat (LTR) R-U5 region for the CMV immediate early region 1 enhancer to improve gene expression (Barouch et al., 2005
). VRC-6402 (6625 bp) consists of a synthetic human codon–optimized gene expressing the NP protein of the Zaire strain of Ebola virus. VRC-6605 (6337 bp) consists of a synthetic human codon–optimized gene expressing a transmembrane-deleted GP protein of the Zaire strain of Ebola virus. VRC-6204 (6327 bp) consists of a synthetic human codon–optimized gene expressing the transmembrane-deleted GP protein of the Sudan/Gulu strain of Ebola virus. A phase I trial of this product has been completed and represents the first clinical trial of an Ebola virus vaccine candidate in the world.
Product 15 (SARS)
VRC-8318 (8164 bp) encodes a human codon–optimized gene expressing the S glycoprotein from the Urbani strain of SARS in the CMV/R backbone described above. The transmembrane region and a portion of the cytoplasmic domain were included (Yang et al., 2004
). This first-generation SARS product is being tested in an ongoing clinical trial and represents the first SARS vaccine candidate to be tested in clinical trial in the United States.
Product 17 (WNV)
VRC-8109 (6982 bp) encodes human codon–optimized WNV preM and E protein genes from the NY99 strain in the pVR1012 backbone. In addition, a leader sequence, which has also been human codon optimized, from the SA-14 isolate of Japanese encephalitis virus has been included. This first-generation WNV is being tested in an ongoing human clinical trial.
The earlier studies involved comparison to a DNA plasmid vaccine from the U.S. Navy's malaria program, which had been previously tested preclinically and clinically. This product is briefly described in Epstein et al. (2002
. Later studies involved comparison to Product 1 (HIV-1, VRC-4302) described above.
Many of the studies were performed with the Biojector 2000 (referred hereafter as Biojector) device, a needleless injection system cleared by FDA for i.m. and s.c. injections of liquid medications, including vaccines. The liquid vaccine is forced through a tiny orifice held against the skin creating a very fine, high-pressure stream that penetrates the skin, depositing the vaccine in the tissue beneath. The system has three components: the reusable device, a sterile single-use disposable needle-free syringe, and a CO2 cartridge. Earlier studies in mice were performed with needle and syringe because the precise device to be used in our clinical trials was not suitable for the size of the species without modification.
Biodistribution Study Designs
Different groups of animals were inoculated for biodistribution studies than for repeated-dose toxicology studies for the following reasons. In the biodistribution analyses, the animals were only inoculated once to determine where the inoculum subsequently biodistributed, whereas in the repeated-dose toxicology studies, the animals were repeatedly dosed. The timing of terminations in the biodistribution studies (generally 1 week and 1 and 2 months post-inoculation) was markedly different from the repeated-dose inoculations and timing of terminations (generally 2 days and 2 weeks after last inoculation) in the repeated-dose toxicology studies.
Studies A, B, and C were performed by BioReliance (Rockville, MD) and involved inoculation of 6- to 7-week-old male and nonpregnant female Hsd:ICR (CD-1) mice with 100 mcg (on the order of 1011 molecules) of DNA plasmid vaccine by either the i.m. or i.v. (data not shown) routes. These were compared to inoculation of phosphate-buffered saline (PBS) by each route. Animals were inoculated at one time point, and five animals per gender per inoculation route (test article; one animal per gender per inoculation route control) were terminated at study days 8 and 50. The following organs were collected for biodistribution analyses: gonads, brain, kidneys, mesenteric lymph nodes, lung, liver, right thigh muscle (injection site in case of i.m. inoculation), bone marrow (left femur), heart, and blood. Tissues were snap frozen in liquid nitrogen after being placed in sterile vials. In addition to tissues being collected for biodistribution, animals were monitored for morbidity and mortality twice daily and for clinical signs of toxicity (body position; locomotor activity; secretions from eyes, nose, or mouth; coat condition; respiration; excretions; muscle tone; body tremors) daily (data not shown). Body weights were measured at days 1, 8, and 50 (prior to termination; data not shown). Gross pathology was observed at termination (data not shown). These studies were performed in compliance with Good Laboratory Practices (GLP) regulations (21 CFR 58).
Studies D, E, F, and G were performed by GeneLogic (formerly Therimmune) and involved inoculation of 12- to 13-week-old New Zealand white rabbits with 2 mg (moderate human dose, on the order of 1014 molecules) of DNA plasmid delivered i.m. by the Biojector needleless injection device, which is the method also used for injection of the candidate vaccines in clinical studies. In each study, comparison was made to PBS and to a comparator plasmid (VCL-2510 [malaria] for Study D, VRC-4302 [HIV-1] for Studies E, F, and G) for which there was prior biodistribution, toxicology, and integration data, as well as clinical safety data. Animals were inoculated at one time point and terminated by sodium thiopental injection (Study D), Nembutal sodium injection (Studies F and G), or sodium pentobarbital injection (Study E) and exsanguination at days 8 or 9, 30 or 31, and 60 or 61. The variance in study days for termination between studies was based on pragmatic scheduling considerations at the animal facility, and the time points were approximatly 1 week and 1 and 2 months post-inoculation on study day 1. The designs of these studies were consistent with FDA recommendations current at the time of study initiation, as well as the refinement of protocols as experience was gained by the VRC. The following organs were collected for biodistribution analyses: blood, gonads, liver, thymus (in Studies F and G only), heart, lung, adrenal glands (in Studies F and G only), kidney, spleen, mesenteric lymph nodes (Study D), right and left popliteal lymph nodes (collected separately in Studies F and G) or contralateral popliteal lymph nodes (in Studies D and E), subcutis at injection site, thigh muscle at injection site, bone marrow (from left femur in Studies F and G), and brain. Paired organs were processed together. Tissues and bone marrow cells were snap frozen in liquid nitrogen after being placed in sterile vials and stored at 70 C. In addition, animals were monitored for morbidity (tremors, convulsions, salivation, diarrhea, lethargy, coma, and atypical behavior) and mortality twice daily and for clinical signs of toxicity (evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, automonic and central nervous systems, and somatomotor and behavior patterns) prior to dosing, weekly, and at termination (data not shown). Body weights were taken prior to dosing, weekly, and at termination (fasted), and food consumption was measured daily (data not shown). These studies were performed in compliance with GLP regulations.
Integration Study Design
Studies H and I were performed at the University of Michigan (in-life portion) and by Althea Technologies, Inc. (San Diego, CA) (DNA extraction and PCR analysis). They involved inoculation of 6- to 7-week-old female CD-1 mice (n = 9 in Study H, n = 10 in Study I) with 100 mcg (split into two sites per animal) i.m. into the right and left quadriceps muscles. At 28 days postinoculation, injected muscle tissue was harvested. Specimens were pooled (all right muscles together, all left muscles together), minced, placed into extraction buffer (0.5 mg/ml proteinase K, 50mM Tris [pH 8.0], 100mM EDTA, 100mM NaCl, 0.5% Tween 20) and incubated overnight at 56°C. DNase-free RNAse A (0.3 mg/ml) was added and incubated at 37°C for 30 min. Each specimen was extracted three times with equal volumes of buffered phenol, then chloroform. DNA was precipitated with two volumes of ethanol at room temperature. DNA was pelleted, rinsed in 70% ethanol, and air-dried at room temperature. Pellets were resuspended in 2.4 ml 10mM Tris (pH 8.0), and concentrations were determined by UV spectrophotometer. Thirty mcg of right or left quadriceps muscle DNA were loaded into each of 24 agarose tube gels, for a total of 720 mcg. The gels were run at 100 V using field-inversion gel electrophoresis for 3 h and using bacteriophage lambda cut with HindIII and KpnI as size markers. These pooled, purified specimens were used for PCR analysis. Additionally, the purified DNA was digested with 10 units SfiI/mcg DNA, overnight at 50°C. After digestion, the DNA was precipitated and subjected to a second round of purification (~ 50 mcg total DNA for each pooled specimen). This was again extracted from the gel and used for PCR analysis. These studies were not conducted in compliance with GLP, but as research studies.
Tissue Processing and DNA Extraction for Studies A, B, and C
Genomic DNA (gDNA) was isolated from tissues using the Promega Wizard Genomic DNA Purification Kit. DNA was diluted with sterile nuclease-free water to a final concentration of 0.1 mcg/mcL for use. Ten mcL of sample was tested per PCR reaction.
Tissue Processing and DNA Extraction for Studies D, E, F, G, H, and I
Generally, 200 mg of tissue were processed for DNA extraction. Tissues were physically homogenized and subjected to digestion with proteinase K. DNA was extracted with the BioRobot M48 Workstation (Qiagen, Valencia, CA) using reagents and protocols recommended by the manufacturer. A naïve tissue sample was included with each run of the BioRobot to serve as a sentinel control for contamination. The concentration of the eluted DNA was determined by UV spectrophotometry and adjusted to a final concentration suitable for quantitative PCR (qPCR).
qPCR Analysis for Studies A, B, and C
A real-time quantitative modification of the TaqMan PCR technique in which the amplicon is detected by a sequence-specific fluorogenic probe was used. Primers and probes were selected using the Primer Express Primer Design Software (PE Applied Biosystems, Foster City, CA). The forward primer used was 5′-TGGGATCTCCACGCGAAT-3′, the reverse primer was 5′-GGAAGCTCCGCCGCTACC-3′, and the probe was 5′-CCATGTCCGGAACACGTACCCGA-3′ labeled with the 6-Carboxyfluorescein (6-FAM) reporter dye (PE Applied Biosystems, Foster City, CA). Amplification of the target sequence was performed in the ABI PRISM 7700 Sequence Detector System using 1 × Universal TaqMan buffer containing AmpErase UNG and AmpliTaq Gold DNA Polymerase with primers at a final concentration of 0.4lμM and the probe at 80nM. Tissue was separately spiked with 100 copies of the plasmids of interest to monitor qPCR inhibition. Controls in which there was no DNA, a negative control (mouse gDNA), the spiked controls, and a standard curve (two series of 10-fold template dilutions of the plasmids of interest from 100 to 105 copies) were also run. Assay performance characteristics were established as follows. Values of less than one copy were considered not biologically relevant and were scored as negative. The limit of detection (LOD) of the assay was considered to be 1 copy, and the limit of quantitation (LOQ, bound of linear range) of the assay was considered to be > 10 copies. Such specimens were scored as positive. Therefore, values between one and nine copies were regarded as nonquantifiable. The qPCR in Studies A, B, and C was performed by BioReliance.
qPCR Analysis for Studies D, E, F, G, H, and I
A TaqMan qPCR assay was designed to target the bovine growth hormone polyadenylation signal sequence, an element common to the plasmid backbone of all constructs. Primers and probe were designed using Primer Express software (Applied Biosystems, Inc., Foster City, CA). The forward primer used was 5′-TGAAGAATTGACCCGGTTCCT-3′, the reverse primer was 5′-GTACTTTAGCGGGTGGGATTGA-3′, and the probe was 5′-FAMTTCTCTGTGACACACCCTGTCCACGC-TAMRA-3′. TaqMan reactions were performed in a 96-well plate using the ABI PRISM 7700 instrument. Amplification of the target sequence was performed in duplicate reactions each containing up to 1 μg gDNA from tissue, and 2× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) with primers and probe at final concentrations of 600nM and 200nM, respectively. A third replicate reaction was performed spiked with 100 copies of the target sequence to monitor qPCR inhibition. Cycling conditions were 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 30 s and 63°C for 1 min. Quantification of the target sequence in each specimen was determined using a standard curve of plasmid DNA diluted in a background of gDNA isolated from a naïve animal. Standards, controls containing no template, sentinel extraction controls, and background gDNA controls were all run in duplicate reactions. Assay performance characteristics were established, and the assay was qualified for use with each transgene insert and animal model. The LOD of this assay was 10 copies of target sequence of gDNA extracted from tissues. The LOQ of this assay was 50 copies of target sequence/mcg gDNA extracted from tissues. qPCR analyses in Studies D, E, F, G, H, I, and J were performed by Althea Technologies, Inc.
Statistical Analyses on the PCR Results
Wilcoxon rank-sum tests were used to compare the distributions at the first and last time points. Two-sided p values from this procedure are reported for each test article, using both the thigh muscle and the subcutis data, where available. For readings that were reported as below the LOD or LOQ, a set value just below the limit was inserted; the use of nonparametric, rank-based tests allowed these discrete observations to be included in the comparison of the distributions. To adjust for the two comparison sites, a threshold of 0.025 was used as a cutoff for statistical significance. Larger p values are simply reported as “Not Significant.” For graphical displays, data were transformed using logarithms due to the strongly skewed distributions and non-constant variance.