- Open Access
Live attenuated rubella vectors expressing SIV and HIV vaccine antigens replicate and elicit durable immune responses in rhesus macaques
© Virnik et al.; licensee BioMed Central Ltd. 2013
- Received: 8 May 2013
- Accepted: 22 August 2013
- Published: 16 September 2013
Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, however, as well as immunogenicity and antibody persistence, were unknown.
This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells.
Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge.
- Live viral vector
- Rubella vaccine strain RA27/3
- Rhesus macaque
- HIV MPER
- SIV Gag
- Highly immunogenic
- Long lasting
- Memory B cells
Despite the urgent need for a safe and potent vaccine against HIV, efforts to produce a vaccine have been thwarted by antigenic variation, weak immunogenicity of critical epitopes, and short duration of the immune response to HIV vaccine antigens [1–3]. For other viruses, such as measles, mumps and rubella, similar immunogenicity problems were solved by developing live attenuated vaccine strains [4–6]. For HIV and SIV, live attenuated vaccines have been an attractive goal [7, 8], but they may incur risks due to proviral integration and, in some cases, reversion to wild type virus [9, 10]. Instead, live viral vectors with HIV vaccine inserts have been proposed [11–14] to combine the growth and immunogenicity of the vector with the antigenicity of the insert . The live attenuated rubella vaccine strain RA27/3 is a promising viral vector, as its safety and immunogenicity have been established in clinical trials [5, 16]. One dose of rubella vaccine elicits strong humoral and mucosal immunity and protects for life against rubella infection. By presenting HIV and SIV vaccine inserts as rubella antigens, live rubella vectors could enhance the immune response to these antigens.
We have recently identified two insertion sites in the rubella genome, where a foreign gene could be inserted without compromising rubella replication in cell culture [17–19]. The vector constructs were based on the rubella vaccine strain RA27/3 or on wild type rubella [20, 21]. The first insertion site was located in the rubella non-structural region, where a permissive deletion between two Not I restriction sites [18, 22] made room for an insertion at the same site. The insert was expressed as a fusion protein with nonstructural protein P150. This deletion/insertion strategy resulted in the first replicating rubella vectors capable of expressing zGFP, the HIV membrane proximal external region (MPER) determinant, and SIV Gag antigens [17, 18]. However, since each insert was expressed as a fusion protein with P150, preservation of essential P150 functions could limit the size and composition of the insert.
The second insertion site was located in the structural region, between envelope proteins E2 and E1 . This site uncoupled antigen expression from essential viral functions, and it accommodated larger and more complex antigens. At this site, insert expression was controlled by the strong subgenomic promoter, resulting in high-level expression for a longer duration. First generation vectors with a structural site insert retained the Not I deletion. These vectors grew to high titer in cell culture while expressing the insert at a high level . Yet, their replication in vivo was compromised by the Not I deletion. New generation vectors, with structural site inserts and the Not I deletion restored, grew robustly in vivo, while expressing SIV and HIV vaccine antigens at high levels.
The rubella vaccine strain RA27/3 readily infects rhesus macaques . The present study is the first to demonstrate the growth and immunogenicity of rubella vectors in macaques. The new generation rubella vectors infected all animals tested. They elicited a strong immune response to the SIV Gag insert, indicating the potency of vaccine antigens expressed at the structural insertion site. The antibodies were long lasting, and the animals responded strongly to a vector boost, indicating the induction of memory B cells. Rhesus macaques are also susceptible to SIV or Simian-Human immunodeficiency virus (SHIV) infection. This overlapping host range will provide an ideal animal model for immunizing with rubella vectors and testing protection against SIV or SHIV challenge [24, 25].
Vector constructs: replication and expression in cell culture
Three types of vectors were produced, with MPER or Gag sequences inserted into the RA27/3 rubella vaccine background. Type 1 vectors had an insert at the structural site and a compensatory deletion at the Not I site, as described previously . Type 2 vectors had an insertion and deletion at the Not I site in the non-structural region of rubella . The new type 3 vectors have an insertion at the structural site, but they have no deletion at the Not I site. Figure 1A shows the design of a typical type 3 rubella vector. The insertion site is located between envelope glycoproteins E2 and E1. The inserted sequences code for MPER of HIV-1 [18, 19, 26, 27] or for an SIV Gag construct containing four T cell epitopes linked together (called BC-sGag2) [18, 19, 28, 29]. Each antigenic insert (labeled Ag in Figure 1A) is preceded by the transmembrane domain of E2 (E2TM) and the signal peptidase site of E1 (E1SP), and it is followed by another transmembrane domain (TM) and E1SP peptidase site. Signal peptidase cleavage at three sites in the structural polyprotein (red arrows) would release the three rubella structural proteins plus the vaccine insert.
Vector replication in Vero cells was monitored by Western blot with antibodies to the rubella structural proteins C and E1 (Figure 1B, left panel). Rubella vectors expressing HIV MPER or SIV Gag antigen grew as well as the vaccine strain without an insert (left panel). Expression of the MPER-HIVTM insert was detected with anti-MPER monoclonal antibody 2F5 (Figure 1B, middle panel). The MPER insert was strongly expressed as a 10 kDa band (yellow arrowhead), which was absent in the empty rubella control (RA27/3, middle panel), and it was comparable to gp41 in the AT-2 inactivated virus control (SHIV, red arrowhead). Expression of the SIV Gag insert was detected by cross-reaction with HIV immune globulin (Figure 1B, right panel). The BC-sGag2 insert was expressed as a 14 kDa band (green arrowhead), which was absent in the empty rubella control (RA27/3, right panel) and was comparable to the control band for recombinant p55 Gag (lane p55, blue arrowhead). After 5 passages in cell culture, we expanded each vector to create viral stocks expressing MPER or SIV Gag inserts.
Rubella vector stocks for in vivostudies
Titers of rubella vector type 3 stocks
Viral vector (passage)
Estimated titer, PFU/ml
1.5 × 108
1.3 × 107
8.5 × 107
7.7 × 106
Rubella vector replication in vivo
The DNA vaccine consisted of SIV gag and HIV clade B env at the first dose, clade C at the second dose, and both clades for the third dose . Mouth swabs were taken before each dose of live rubella vectors and one and two weeks after the dose to detect viral RNA by RT-PCR. Blood samples were taken before each dose and one, two and six weeks after immunization to analyze the immune response to rubella proteins and to each insert.
Immunogenicity of Gag vector inserts
We compared three vaccinated macaques (two from group 3 and one from group 1) with a panel of five macaques infected with SIVmac251 (Figure 6C). In each case, immunization with Gag vectors elicited anti-Gag antibodies with higher maximum ODs by ELISA and similar endpoint titers as those induced by natural SIV infection. This result indicates the potency of vaccine antigens expressed at the structural insertion site of live rubella vectors.
In contrast, macaques CL6V (Figure 7C) and CL67 (Figure 7D) were initially given two doses of non-replicating type 1 vectors that expressed the same inserts at the same site as the replicating vectors, followed by a third dose with a type 2 vector. Both macaques made a minimal response to the first dose of non-replicating vectors. However, two or three doses of non-replicating vectors could achieve the same antibody titers as a single dose of the same insert in a live vector.
Immunogenicity of MPER vector inserts
We measured anti-Gag antibodies in the same macaques at the same time points (Figure 9B, D, and F). Anti-Gag antibodies peaked 4 to 7 weeks post immunization (week 22 to 25 of the study) and then declined 3-fold or less by 15 weeks. By 38 weeks post immunization (study week 56), they declined another 3-fold or less in two macaques (DCVV and CL49) and 5-fold in the other (CL67). In two macaques (DCVV and CL49), the durability of anti-Gag titers was greater than or equal to anti-rubella titers, and in one case (CL67) anti-Gag titers were less durable over a 9 month period. This suggests that the persistence of anti-Gag antibodies could be comparable to anti-rubella antibodies, which are known to be long-lasting . The durability of anti-Gag antibodies between 7 and 15 weeks after vaccination may be due, in part, to two boosts of DNA vaccine given during this time. However, this would not account for the continued stability observed during weeks 15 to 38 post vaccination, when DNA vaccine was not given.
Response to a rubella vector boost
We waited up to a year for rubella antibodies to decline to a level where we could boost with rubella vectors bearing new antigens. For group 4 macaques, these antibodies declined about 2.5-fold after 6 months, and then remained constant or rose slightly by 1 year. After one year (group 4) or 6 months (group 1), we boosted the macaques with rubella vectors expressing MPER and SIV Gag antigens (week 57 of the study). All five animals showed a prompt rise in antibodies to rubella (data not shown).
Live attenuated rubella vaccine has a proven record of safety and immunogenicity in humans and rhesus macaques. This small RNA virus would be an attractive viral vector, if it could present the major vaccine antigens of other viruses as well as its own. This is the first report showing that live attenuated rubella vectors replicate robustly in vivo while expressing SIV and HIV vaccine antigens. The new vectors infected six out of six rhesus macaques, while eliciting high-titered antibodies to the SIV Gag insert in all of them and to HIV MPER in five out of six. The anti-Gag antibody titers elicited by immunization were greater than or equal to those induced by natural infection with SIV. The antibodies to both inserts have persisted for over 9 months, and they have declined at the same rate as antibodies to rubella, which protect for life. The anti-Gag antibody response was boosted by re-exposure to the vector after six months, indicating the induction of memory B cells. Since rhesus macaques are also susceptible to SIV infection, they will provide an ideal model for testing immunogenicity of novel rubella vectors and protection against SIV or SHIV challenge.
At the start of these studies, rubella vectors faced a series of questions before they could be considered a vaccine candidate. These included: location of insertion sites, size and stability of inserts, adaptation to the live attenuated vaccine strain, replication in vivo, immunogenicity, and concurrent immunization with more than one vector. We previously reported that rubella vectors could grow to high titers in cell culture while expressing SIV and HIV antigens at either of two insertion sites [18, 19]. Inserts at the Not I site in the nonstructural region included zGFP, SIV Gag epitopes, and HIV MPER [17, 18]. These inserts were limited in size and diversity, probably because they were expressed as fusion proteins with rubella nonstructural protein P150, which performs essential viral functions.
However, when the genes were inserted in the structural region, between envelope glycoproteins E2 and E1, insert expression was uncoupled from essential viral functions. This allowed the expression of larger inserts . These inserts were expressed as part of the structural polyprotein under control of the strong subgenomic promoter, leading to high-level antigen expression for a prolonged period. When we switched to the rubella vaccine strain RA27/3, the new vectors were able to express the same inserts as wild type rubella, with little or no loss in vector titer, antigen expression, or insert stability . The vectors with Not I deletion did not replicate well in vivo. This was solved by restoring the deleted sequence, which resulted in replication of type 3 vectors in six out of six macaques. The deleted sequence is part of the “Q” domain in nonstructural protein P150 with unknown function . This region may be important for interferon signaling or suppression. Vectors with the Not I deletion replicate well in Vero cells, which are incapable of interferon production, but they do not replicate in normal WI38 or BSC-1 cells (Virnik et al., manuscript in preparation). Future studies will address this phenomenon.
Live replicating rubella vectors expressing SIV Gag or HIV MPER at the structural insertion site were highly immunogenic in macaques. These vectors could contribute to vaccine potency at each stage of the immune response: by simulating acute infection and triggering innate immunity, they could initiate a stronger immune response to the inserts. Subsequently, exponential growth of the vector would expose the host to increasing doses of antigen each day. Finally, prolonged antigen expression can mimic an ongoing infection [14, 15], leading to germinal center formation, which is needed for immunoglobulin class switching, somatic hypermutation to produce high-affinity antibodies, and maturation of memory B cells . Unlike their non-replicating homologues, live rubella vectors elicited anti-Gag antibodies after a single dose, and the antibody titers continued to rise for four to seven weeks after immunization. These strong stimuli, lasting two weeks or more, explain how live vectors could achieve the same high titers of anti-Gag antibodies as natural SIV infection.
With some other vaccines and vectors, the immune response to SIV and HIV antigens has been short-lived and lacked memory B cells [35, 36]. Transient antibody responses are considered one of the major obstacles to HIV vaccine development . Using live rubella vectors, the anti-Gag and anti-MPER antibodies have persisted for over nine months. They are declining with nearly the same half-life as antibodies to rubella proteins, which protect for life. In general, persistent antibody titers are thought to depend on long-lived plasma cells, while boosting depends on memory B cells, and both are signs of germinal center function during immunization . The primary immune response to these vectors included memory B cells, as shown by boosting 6 months later. The secondary response depended on successful priming, and it could overcome the inhibitory effects of rubella antibodies. Potentially, two or more doses of live rubella vectors, given several years apart, could boost and update immunity to circulating strains of HIV. In addition, the ability to prime and boost memory B cells would allow us to combine rubella vectors with other viral vectors bearing similar HIV vaccine inserts [37, 38], and this will be tested.
Rubella is a small RNA virus that replicates exclusively in the cytoplasm. This location is ideal for eliciting T cell immunity, since it delivers antigens directly into the proteasomal pathway leading to antigen presentation with MHC class I . Priming with DNA vaccine and boosting with the vector gave high levels of Gag specific CD8+ T cells that were comparable to natural infection (Virnik, et al., manuscript in preparation). When two rubella vectors were given simultaneously, they both replicated side by side, as shown by RT-PCR, and they elicited antibodies to both Gag and MPER inserts. Prolonged expression of MPER antigen by rubella vectors may contribute to its immunogenicity and may improve on natural infection, which elicits these antibodies in less than half of the cases [40–43].
The safety and immunogenicity of live attenuated rubella vaccine were demonstrated in rhesus macaques  and in children [5, 6, 16]. Macaques have shown no signs of disease during successful immunization with rubella vectors. The safety of rubella vectors should be comparable to rubella vaccine: if a rubella vector lost its insert, it would revert to the vaccine strain. Given the overlapping host range, rhesus macaques will provide an ideal animal model for testing rubella vectors for immunogenicity and protection against SIV or SHIV challenge. Novel vectors that demonstrate vaccine potency in macaques could be quickly translated into human vaccine design.
We have completed the first successful trial of live rubella vectors in rhesus macaques. Rubella vectors replicated well in all macaques tested and they provide a potent vaccine platform for immunizing with SIV and HIV antigens. These vectors have a number of desirable properties for a vaccine candidate, including: the ability to immunize with more than one antigen at a time. The vectors have shown vaccine potency comparable to natural SIV infection. They elicited long lasting immunity and induced memory B cells that can be boosted months later. They are built on a vaccine platform with known safety and immunogenicity. Due to the overlapping host range of rubella and SIV, rhesus macaques will provide an ideal animal model for testing novel rubella vectors for immunogenicity and protection against SIV or SHIV challenge.
Antigens antibodies and cells
Aldrithiol-2 inactivated SHIV virions with 89.6 envelope and SIV Gag proteins were a kind gift of Drs. Larry Arthur and Jeffrey Lifson at the AIDS Vaccine Program, NCI . Recombinant p55 SIV Gag protein, anti-MPER monoclonal antibody 2F5  and anti-HIV polyclonal antibodies used for BC-sGag2-E2TM detection were provided by the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID. Well-characterized recombinant gp140 SOSIP trimers  were a kind gift of Dr John Moore, Weill Medical College of Cornell University (New York, NY). Monoclonal 2F5 was also provided by Dr. Hermann Katinger, Polymun Scientific (Klosterneuburg, Austria). Polyclonal goat antibodies to rubella structural proteins were purchased from Fitzgerald Industries International, Inc. (Concord, MA). Vero cells were obtained from the American Type Culture Collection (Manassas, VA).
Construction of vectors with inserts in the structural site
The derivation of type 1 and type 2 rubella vectors were described previously [18, 19]. Type 3 vectors were derived from type 1 vectors by cutting out the Not I deleted Hind III-Bgl II fragment from the plasmid DNA encoding type 1 vectors and replacing it with the corresponding intact fragment from the p10RA plasmid coding for the RA27/3 vaccine strain of rubella . For these vectors, the MPER insert was followed by the transmembrane domain of HIV-1 gp41 (MPER-HIVTM vector) and the E1 signal peptide. The BC-sGag2 insert consisted of four T-cell epitopes in tandem (GY9, TE15, CM9 and ME11 of SIV mac239 Gag), followed by the transmembrane domain of E2 (BC-sGag2-E2TM vector), and the E1 signal peptide (Figure 1A). All constructs were verified by sequencing, as described previously . The insert sequences are provided in Figure 2. In addition to previously described inserts, one new insert combined the MPER determinant and the BC-sGag2 determinant in tandem. Another insert consisted of full-length p27 SIV Gag, containing amino acids 135–391 of SIVmac239 Gag, followed by to the E2TM domain.
The protocols for generating infectious rubella RNA from plasmid DNA, transfecting cells, passaging virus in Vero cells, viral stock expansion, sequencing the inserts, determining viral RNA content of rubella vector stocks and their titers were the same as described previously [17, 18]. Briefly, we transcribed plasmid DNA in vitro to produce capped infectious rubella RNA, using RiboMAX Large scale RNA Production System with Ribo m7G(5’)ppp(5’)G cap analog (Promega Corp., Madison, WI). To generate rubella virus, the RNA was transfected into Vero cells, using DMRIEC reagent (Invitrogen Corporation, Carlsbad, CA). After 6–9 days of infection at 37°C, culture supernatant containing virus (passage P0) was collected. Infected cells were transferred onto fresh Vero cells to start a new passage. After several passages, passaging was done with cell-free culture supernatant. To produce virus stock, virus production was scaled up in T75 flasks after 5–6 passages. The supernatant was collected, titered by quantitative RT-PCR and the vector insert was sequenced. Purification of viral RNA from viral supernatants and reverse transcription were performed using QIAamp UltraSens Virus kit (QIAGEN) and High Capacity RNA-to-cDNA Kit (Applied Biosystems), respectively. The cDNA was PCR amplified with illustra PuReTaq Ready-To-Go PCR beads (GE Healthcare) and primers specific for rubella sequences flanking an insertion site, then purified in agarose gel and sequenced using the same primers.
Detection of rubella growth in cell culture and insert expression by Western blot
Rubella structural proteins were detected by Western blot with goat anti-rubella antibodies specific for capsid protein C and envelope protein E1, as described previously [17, 18]. Expression of the MPER-HIVTM and BC-sGag2-E2TM inserts was detected with human monoclonal antibody 2F5 at 1 μg/ml and anti-HIV polyclonal antibodies at 1:2500 dilution, respectively. The second antibody was either horseradish peroxidase-conjugated rabbit anti-goat IgG or goat anti-human IgG at 1:5000 dilution (Santa Cruz Biotechnology, CA). Blots were visualized with enhanced chemiluminescence (GE Healthcare).
Animals and immunizations
Rhesus macaques, between 3 and 16 years of age were obtained from the CBER NHP colony on the NIH campus. All but two were of Indian origin; the two of unknown origin were V200 and V584. The CBER animal research program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All experimental procedures were approved by the CBER Institutional Animal Care and Use Committee and were done in compliance with the current edition of the Guide for the Care and Use of Laboratory Animals. To be selected for the study, all animals passed a screening complete physical exam and a veterinary evaluation of their medical history including serology. Animals were negative for antibodies to rubella, herpes B, STLV-1, SIV, SRV (type D, serology/PCR) 1, 2 and 3. During the study period, the animals were negative for Shigella, Salmonella, Yersinia, Campylobacter and tuberculosis (TB) and free of intestinal pathogenic parasites.
Depending on their size, the macaques were housed in 6–12 sq ft cages and were habituated for more than 3 months. Before entry into the study, all macaques were weighed and tissue typed. They were confirmed seronegative for SIV Gag and rubella antibodies prior to receiving the vectors. All animals received ketamine anesthesia (10 mg/kg/im) for restraint during immunization, bleeding and taking oral swabs.
The 12 macaques were divided into three vaccine groups of three to four each and a control group of two animals (Figure 3). Group 3 was immunized first, with a series of three different rubella vectors, until they showed signs of a vaccine “take”, followed by two doses of DNA vaccine. Group 1 received three doses of DNA vaccine first, followed by one dose of type 3 vectors, the same ones that gave a “take” in group 3. Group 2 received three doses of DNA vaccine and was reserved for future studies. Immunizations were at weeks 0, 5, 11, 18, 25, and 31. Two animals in group 4 received the licensed rubella vaccine, which served as a vector control without an insert, followed by control DNA.
For group 1 macaques, the first two doses of DNA vaccine consisted of four plasmid DNAs: 1 mg DNA coding for SIV gag, 1 mg env DNA (gp140 and gp160), and 200 μg coding for monkey IL-12 . The first dose had env DNA of clade B, while the second dose had clade C env DNA, and the third dose had equal parts of env DNA of both the B and C clades. For the animals in group 3, both DNA boosts contained env DNA of the B and C clades. All DNAs were given by intramuscular injection followed by in vivo electroporation using the Elgen 1000 device (Inovio Pharmaceuticals, Inc., Blue Bell, PA).
Each rubella dose consisted of a pair of rubella vectors of the same type, expressing HIV MPER and SIV Gag determinants. For group 3, the first rubella dose consisted of type 1 vectors, given IM at a dose of 10,000 PFU, followed by a second dose of 30,000 PFU. The next dose was a type 2 vector at 50,000 PFU. Finally, the type 3 vectors were given IM at a dose of 50,000 PFU. Group 1 animals received three doses of DNA vaccine, followed by a single dose of type 3 vectors at 50,000 PFU. This corresponds to about ten times the typical human dose of rubella vaccine.
Blood samples were taken before each vaccine dose and one, two and six weeks after the dose. Mouth swabs were also taken before each dose of rubella vector and one and two weeks later. The blood samples were analyzed by ELISA for antibodies to rubella, SIV Gag and HIV MPER. They were also analyzed by tetramer staining for T cells specific for the CM9 epitope. Mouth swabs were analyzed by RT-PCR using primers specific for the gag and MPER inserts.
Detection of rubella virus in oral fluid specimens from rhesus macaques
PCR primers used in detection of rubella virus in oral fluid specimens
Serum enzyme-linked immunosorbent assay
Monkey antibodies to rubella were detected by ELISA in duplicate wells, as measured on plates coated with rubella antigens (BioCheck, Inc., Foster City, CA) at the recommended serum dilution of 1:40. Macaque antibodies to SIV Gag were detected on plates coated with recombinant p55 Gag protein. For this assay, soft plastic plates were coated with 1 μg/ml recombinant p55 SIV Gag overnight at 4°. They were blocked for 5 minutes with BSA (10 mg/ml). The antibodies were then titered on the plates in PBS with 0.1 mg/ml BSA and 0.05% Triton X100. Macaque antibodies to HIV MPER were detected on plates coated overnight at 4° with recombinant gp140 SOSIP trimers at 1 ug/ml.
Macaque antibodies to MPER peptides were titered on plates coated overnight with two peptides of 14 to 16 amino acids containing the 2F5 and 4E10 epitopes. For each antigen, the plates were incubated with antibodies for 1 hour at 37°, washed twice with the above buffer, and developed with goat anti-monkey IgG conjugated to horseradish peroxidase at a 1:5000 dilution (Santa Cruz Biotechnology, CA). After 30 minutes at 37°, the plates were washed four times and developed with TMB substrate (SureBlue, KPL, Gaithersburg, MD). After ten minutes at room temperature, the reaction was stopped with 1M HCl, and OD450 was determined in an ELISA plate reader (Thermo Scientific).
The authors wish to thank Lewis Shankle of CBER and Dr. Deborah Weiss of Advanced BioScience Laboratories, Inc. for their essential contributions to this work; K. Broderick and N. Sardesai of Inovio Pharmaceuticals, Inc. for the DNA delivery method; Dr. Margherita Rosati of NCI Frederick and Dr. Carol Weiss of CBER for helpful discussions; and Dr. John Moore for providing SOSIP gp140 trimers. This research was supported in part by the NIH Intramural AIDS Targeted Research Program.
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