A Combination DNA and Attenuated Simian Immunodeficiency Virus Vaccine Strategy Provides Enhanced Protection from Simian/Human Immunodeficiency Virus-Induced Disease
ABSTRACT
Among the most effective vaccine candidates tested in the simian immunodeficiency virus (SIV)/macaque system, live attenuated viruses have been shown to provide the best protection from challenge. To investigate if preimmunization would increase the level of protection afforded by live attenuated SIVmac239Δnef (Δnef), macaques were given two priming immunizations of DNA encoding SIV Gag and Pol proteins, with control macaques receiving vector DNA immunizations. In macaques receiving the SIV DNA inoculation, SIV-specific cellular but not humoral responses were readily detectable 2 weeks after the second DNA inoculation. Following boosting with live attenuated virus, control of Δnef replication was superior in SIV-DNA-primed macaques versus vector-DNA-primed macaques and was correlated with higher levels of CD8+/gamma-interferon-positive and/or interleukin-2-positive cells. Challenge with an intravenous inoculation of simian/human immunodeficiency virus (SHIV) strain SHIV89.6p resulted in infection of all animals. However, macaques receiving SIV DNA as the priming immunizations had statistically lower viral loads than control animals and did not develop signs of disease, whereas three of seven macaques receiving vector DNA showed severe CD4+ T-cell decline, with development of AIDS in one of these animals. No correlation of immune responses to protection from disease could be derived from our analyses. These results demonstrate that addition of a DNA prime to a live attenuated virus provided better protection from disease following challenge than live attenuated virus alone.
Human immunodeficiency virus (HIV) infection and AIDS continue to be a growing problem for the world's population. Current estimates by UNAIDS suggest that 40 million people are currently infected with HIV throughout the world (44). While antiretroviral therapy in many cases has been able to slow disease progression and most likely virus transmission rates, treatment availability is severely limited, especially in developing countries, where the majority of HIV infections still occur (44). The current challenge facing the AIDS pandemic is the development of a safe, effective vaccine. Roadblocks to such a successful vaccine include the presence of multiple subtypes in many regions of the world, the further development of recombinant subtypes, the issue of sterilizing immunity, and the ability of the virus to escape from immune surveillance.
Over the years, the simian immunodeficiency virus (SIV)-infected macaque has provided a valuable model system for investigations into HIV pathogenesis, for development of new vaccine strategies for HIV, and for retooling older vaccine strategies for HIV. Many vaccine approaches have been tested using the SIV/macaque model, including purified/recombinant protein, killed virus, live attenuated virus, prime boost, recombinant viral and bacterial vectors, and most recently, DNA-based (either alone or with a protein or recombinant vector-based boost) approaches (4, 5, 8, 9, 13, 15-22, 24, 25, 27, 30, 33, 35, 36, 45). While a number of these strategies have provided some level of protective effect, live attenuated viruses have been shown to provide the highest levels of protection (11, 14, 15, 26, 28, 31-33, 39, 43, 47, 48). Attenuated SIVs have been shown to induce broad cellular and humoral immune responses and have provided protective effects from a number of different challenge systems, including both homologous and heterologous viruses (11, 28, 29, 31, 37, 41-43). Still, despite these encouraging results, precise correlates of immunity have not been elucidated.
However, the development of attenuated virus vaccines by using the SIV system has not been without problems. Issues that have arisen as the result of live attenuated virus vaccination in the SIV/macaque model system include the time-dependent evolution of maximum protective effects, the inverse relationship between level of attenuation and level of protection, the lack of immune correlates of protection, and the development of AIDS disease in vaccinated animals, most notably neonates (2, 6, 7, 12, 28, 41, 42, 47, 48). Understanding the basis of the mechanisms behind these issues as well as possibly resolving them is a major focus of research in the live attenuated virus arena. To address the issue of increased protection using live attenuated viruses, we have investigated the inclusion of a DNA immunization prior to vaccination with live attenuated virus.
MATERIALS AND METHODS
Animals, DNA, viruses, and cells.
Juvenile rhesus macaques were utilized for these studies. A total of 18 macaques were randomly divided into two groups of 7 macaques each and one group of 4 macaques (controls). Animals were housed at the Yerkes National Primate Research Center and handled under the strict guidelines of AAALAS and the Emory University IACUC. The Gag-Pol DNA vaccine was constructed by the introduction of a stop codon and a unique EcoRI restriction site at the junction of reverse transcriptase and integrase in the Gag-Pol-Env DNA vaccine (5) and has been described previously (4). The truncated Gag-Pol sequences were recombined at an EcoRI restriction endonuclease site 86 bp upstream of the Tat start site with an HIV type 1 (HIV-1) subclone which included vpu, tat, rev, and the HIV-1 ADA env with an internal BglII deletion. The Gag-Pol insert was cloned into the pGA1 expression vector (GenBank accession no. AF425297 ). Expression of HIV-1 Env by this DNA was not directly tested. However, previous results (4) and results presented here show that no immune responses to HIV-1 Env are generated using this DNA.
A titered stock of SIVmac239Δnef (Δnef) was kindly provided by R. Desrosiers of the New England National Primate Center. A titered stock of simian/human immunodeficiency virus (SHIV) strain SHIV89.6p was kindly provided by K. Reimann of the Beth Israel Deaconess Medical Center. Viruses were stored under liquid nitrogen until use.
Peripheral blood mononuclear cells (PBMC) were isolated from macaque whole-blood samples by centrifugation over lymphocyte separation media. Cells were then used immediately for immunological assays.
DNA and virus inoculations.
For immunization, 1.5 mg of either SIV or vector DNA was injected intradermally into macaques using a Bioject device. Injections were distributed over 10 sites along the arms and legs of the macaques. Week 0 denotes the first DNA injection. The second immunization was given in an identical manner at week 8. The SIVmac239Δnef virus stock was used directly for inoculation without dilution. Animals were inoculated intravenously with 5 ng of p27 equivalents. For challenge, a 1:300 dilution of SHIV89.6p was prepared in RPMI medium containing 10% heat-inactivated fetal bovine serum (FBS), and macaques received 1 ml of this stock intravenously.
Immunological assays.
PBMC proliferation assays were done according to previously published methods (5). Briefly, for proliferation assays, purified PBMC from macaques were stimulated in triplicate for 5 days with the indicated antigen in 200 ml of RPMI at 37°C under 5% CO2. Supernatants from 293T cells transfected with DNA expressing SIV Gag and Pol or HIV-1 Env were used directly as antigens (final concentration of ∼0.5 μg of p27 Gag per milliliter). Supernatants from mock DNA (vector alone)-transfected cells served as negative controls. On day 6, cells were pulsed with 1 μCi of [3H]thymidine per well for 16 to 20 h. Cells were harvested with an automated cell harvester (TOMTEC Harvester 96, model 1010; Hamden, CT) and counted with a Wallac 1450 MICROBETA scintillation counter (Gaithersburg, MD). Stimulation indices are the counts of [3H]thymidine incorporated in PBMC stimulated with SIV antigens divided by the counts of [3H]thymidine incorporated by the same PBMC stimulated with mock antigen.
Intracellular cytokine staining (ICS) assays were performed as described previously (3, 5, 40). Briefly, approximately 1 × 106 PBMC were stimulated in 5-ml polypropylene tubes in RPMI containing 10% heat-inactivated FBS, anti-human CD28, anti-human CD49d (1 μg per ml each; Pharmingen, Inc., San Diego, CA) in 1 ml. Peptide pools (15mer overlapping by 11; 25 peptides per pool) specific for SIV Gag were used for stimulations at a final concentration of 5 μg/ml. After 2 h of incubation at 37°C, 10 μl of RPMI containing 10% FBS and monensin (10 μg/ml final concentration) was added and cells were cultured for an additional 4 h at 37°C at an angle of 5°. Cells were surface stained with fluorochrome-conjugated antibodies to CD8 (clone SK1; Becton Dickinson) at 8 to 10°C for 30 min, washed once with cold phosphate-buffered saline (PBS) containing 2% FBS, and fixed and permeabilized with Cytofix/Cytoperm solution (Pharmingen, Inc.). Cells were then incubated with fluorochrome-conjugated antibodies to macaque CD3 (clone FN18; Biosource), gamma interferon (IFN-γ) (clone B27; Pharmingen), and interleukin-2 (IL-2) (clone MQ1-17H12; Pharmingen) in Perm wash solution (Pharmingen) for 30 min at 4°C. Cells were washed twice with Perm wash and once with plain PBS and resuspended in 1% formalin in PBS. Approximately 200,000 lymphocytes were acquired on a FACSCaliber and analyzed using FloJo software (Treestar, Inc., San Carlos, CA). Lymphocytes were identified based on their scatter patterns; CD3+ CD8− cells were considered CD4+ T cells, and CD3+ CD8+ cells were considered CD8+ T cells. Using this assay, we could detect antigen-specific CD4+ and CD8+ T cells at frequencies as low as 0.01% of the respective total cells. In unvaccinated controls and prebleeds of vaccinated macaques, the frequencies of Gag-specific CD4+ and CD8+ T cells were below 0.01%.
SIV-specific antibodies.
A commercially available whole-virus HIV-2 enzyme-linked immunosorbent assay (ELISA) kit (Bio-Rad, Hercules, CA) was used to detect antibodies to SIV present in the plasma of immunized and challenged macaques. Levels of SIV-specific antibodies were quantitated by titration with serial twofold dilutions of plasma. SIV seropositive status of macaques was confirmed by Western blot analysis using commercially available strips (Zeptometrix, Buffalo, NY).
Neutralizing antibodies to SHIV89.6p.
To detect antibodies neutralizing the challenge virus, SHIV89.6p, a recently described assay was utilized (34). Briefly, neutralization was measured as a function of reduction in luciferase reporter gene expression after a single round of virus infection in TZM-bl cells as described previously (34). TZM-bl cells were obtained from the NIH AIDS Research and Reference Reagent Program, as contributed by John Kappes and Xiaoyun Wu. These cells are engineered to express CD4 and CCR5 (38) and contain integrated reporter genes for firefly luciferase and Escherichia coli β-galactosidase under control of an HIV-1 long terminal repeat (46). Briefly, cell-free virus (50% tissue culture infective dose of 200) was incubated with serial dilutions of test samples in triplicate in a total volume of 150 μl for 1 h at 37°C in 96-well flat-bottomed culture plates. Freshly trypsinized cells (10,000 cells in 100 μl of growth medium containing 75 μg/ml DEAE dextran and 2.5 μM indinavir) were added to each well. One set of control wells received cells plus virus (virus control), and another set received cells only (background control). After a 48-h incubation, 100 μl of cells was transferred to 96-well black solid plates (Costar, Corning, NY) for measurements of luminescence using Bright Glo substrate solution as described by the supplier (Promega, Madison, WI). Neutralization titers are the dilutions at which relative luminescence units were reduced by 50% compared to levels for virus control wells after subtraction of background relative luminescence units.
Plasma viral load determinations.
SIV and SHIV89.6p plasma viral loads in macaques were quantitated using a modification of a real-time PCR methodology as previously described (23). Briefly, for the determination of SHIV/SIVmac239 copy number, viral RNA from 0.2 ml of EDTA-anticoagulated cell-free plasma was directly extracted by a MagNa Pure LC robotic workstation (Roche Molecular Biochemicals) with a MagNa Pure LC total nucleic acid isolation kit (Roche). RNA was eluted with 60 ml of elution buffer and frozen at −80°C until analysis. A one-step reverse transcriptase PCR (RT-PCR) method using the TaqMan EZ RT-PCR kit was performed. Fifteen microliters of purified plasma RNA was reverse transcribed in a final 50-μl volume containing 10 μl of 5× EZ TaqMan buffer; 2 mM Mn(CH3OO)2; 0.3 mM dATP, dCTP, and dGTP; 0.6 mM dUTP; 200 nM of each primer; a 125-nM concentration of the 6-carboxyfluorescein (FAM)-labeled probe; 5 U of recombinant Tth DNA polymerase; and 1 U of AmpErase uracil N-glycosylase. (All reagents are from Applied Biosystems, Foster City, CA). The primer sequences within a conserved portion of the SIV gag gene were 5′-GCAGAGGAGGAAATTACCCAGTAC-3′ (forward; 24 nucleotides [nt]), 5′-CAATTTTACCCAGGCATTTAATGTT-3′ (reverse; 25 nt), and FAM-5′-TGTCCACCTGCCATTAAGCCCGA-3′-TAMRA, where TAMRA is 6-carboxytetramethylrhodamine (probe sequence; 23 nt). An Applied Biosystems 7700 sequence detection system with the PCR profile of 50°C for 2 min, 60°C for 30 min, and 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min, was used.
Absolute quantification of SHIV/SIVmac239 RNA copy number was determined by comparison with an external standard curve consisting of prepared RNA standard transcripts quantified from the initial stock concentration and subsequently confirmed by the SIV B-DNA method (Bayer Diagnostics, Emeryville, CA). All specimens were extracted and amplified in duplicate, with the mean results reported. With a 0.15-ml plasma input, the assay has a sensitivity of 80 RNA copies per milliliter of plasma and a linear dynamic range of 102 to 108 RNA copies/ml (R = 0.995). The efficiencies of the RT-PCR varied from 100% to 90%, with corresponding slopes of −3.6 to −3.3. The intra-assay coefficient of variation was 20% for samples containing 104 SHIV RNA copies per milliliter and 25% for samples containing 103 to 102 SHIV RNA copies per milliliter. This assay does not distinguish between SIV and SHIV.
Statistical analyses.
The Wilcoxon rank sum test was used for two-sample comparisons. For comparisons involving plasma viral loads over time, the area under the curve (AUC) of viral load versus time was calculated for each macaque and used as the dependent measure (postvaccination, weeks 28 to 61; postchallenge, weeks 71 to 101). The Freeman-Halton test and Fisher's exact test were used, respectively, for 3-by-2 and 2-by-2 contingency tables involving comparisons of the proportion of animals in each group that showed depletion of CD4 cells. Spearman's rank correlation test was used to assess the relationships between AUCs and immune responses. Between-group comparisons involving control and vaccine groups were based upon the AUCs between weeks 71 and 85, because three of four controls had died after week 85. The Bonferroni method was used to adjust P values for multiple comparisons.
RESULTS
Immunization strategy and protocol.
This study was designed to address the hypothesis that priming macaques with DNA might increase the level of immunogenicity and protection afforded by live attenuated virus vaccination. Figure 1 shows a schematic of the immunization and challenge schedule for these experiments. Based upon the previous work of Amara et al. (4, 5), it was decided that two DNA inoculations would be administered, at weeks 0 and 8. For each DNA immunization, a total of 1.5 mg was inoculated into each animal. Seven rhesus macaques served as controls and received 1.5 mg of pGA2 (vector DNA) distributed over 10 sites on the arms and legs (group 1: animal codes RNs6, RVs6, RPm6, RPi6, RMk6, RWq6, and RUs6). Similarly, seven rhesus macaques received 1.5 mg of pGA2/M2Gag-Pol (SIV DNA) in the same manner (group 2: animal codes RKb6, RJk6, RDl6, RQi6, RVj6, RIo6, and RQp6).
Immune responses following the DNA immunizations were examined using SIV-protein-induced proliferation of lymphocytes and ICS. At 2 weeks following the second DNA inoculation (week 10), proliferative responses of lymphocytes from macaques in both groups were determined following stimulation with an SIV Gag-Pol protein. The data in Fig. 2 show that PBMC derived from macaques immunized with SIV DNA proliferated in response to stimulation with SIV Gag-Pol protein. Cells from these animals showed various levels of proliferative potential, with stimulation indices (SIs) ranging from 5 to 57. One animal, RDl6, was not tested because a sufficient number of cells were not available. In contrast, PBMC derived from macaques immunized with vector DNA did not show proliferative responses following in vitro stimulation with SIV Gag-Pol protein. Proliferative responses were also tested against HIV-1 Env. None of the PBMC samples obtained from either the SIV-DNA-immunized macaques or the vector-DNA-immunized macaques responded to the HIV-1 Env protein (data not shown).
ICS assays did not yield any measurable levels of immune response (data not shown). Additionally, ELISAs (whole virus) for SIV-specific antibody in serum were negative at both 10 and 20 weeks post-DNA immunization (data not shown). These results demonstrate that DNA immunization was able to induce very low levels of T-cell responses to SIV proteins that were measurable only with an in vitro proliferation assay.
Booster immunization with live attenuated SIV.
As a booster to the two DNA priming immunizations, macaques received an intravenous inoculation of SIVmac239Δnef at week 25 (17 weeks following the second DNA immunization). Following this inoculation, evaluations of immunological reactivity continued and macaques were also monitored for plasma viral loads and levels of circulating CD4+ T cells. Figure 3 shows the circulating CD4+ T-cell counts and plasma viral loads for all animals. In vector-DNA-immunized animals, a slight, transient drop in CD4+ T-cell levels was evident in the 2 weeks immediately following Δnef administration, but levels recovered to preinoculation levels and did not show additional variances prior to challenge (Fig. 3C). In SIV-DNA-immunized animals, circulating CD4+ T cells remained at fairly constant levels at all times following Δnef inoculation (Fig. 3D).
Plasma viral loads were measured for all animals beginning 2 weeks post-Δnef inoculation (Fig. 3A, B, and E). Peak viral loads were detected for all animals at the 2-week time point. Interestingly, at this time, plasma virus levels were, in general, higher in SIV-DNA-immunized animals than in the vector control animals, as demonstrated both with individual animals (Fig. 3A and B) and by calculation of geometric means within the two groups (Fig. 3E). However, the difference in the means between the two groups was not statistically significant (P = 0.200; Wilcoxon rank sum test). Following peak viremia, plasma viral loads quickly declined to undetectable levels in all animals just 1 week later. In most of the vector-DNA-immunized animals (Fig. 3A), plasma viral loads rebounded to various levels. Only two animals (RPi6 and RVs6) had plasma viral loads consistently below the level of detection beyond 7 weeks postinoculation. Viral loads in two animals, RUs6 and RMk6, showed increasing levels from 16 weeks postinoculation onward. In contrast, SIV-DNA-immunized animals (Fig. 3B) controlled viremia very well. Only one animal, RDl6, showed a rebound of plasma viral loads following the 3-week time point. This animal proved to be an outlier in the group. An additional animal, RVj6, showed undetectable plasma virus loads (following the 2-week time point), except for low-level detection at the 8-week time point. If the geometric means are followed through the period before challenge, the SIV-DNA-immunized macaques have consistently lower viral loads than the vector-DNA-immunized macaques (Fig. 3E). Additionally, if weeks 28 to 61 are examined and the AUCs are compared for each group, the SIV-DNA-immunized macaques have statistically significantly lower viral loads during this period (P = 0.037; Wilcoxon rank sum test).
Higher frequencies of Gag-specific CD8 cells following ΔNef infection.
To begin an examination of the immune response following Δnef vaccination, SIV-specific T-cell functions were analyzed. The frequencies of Gag-specific IFN-γ- and IL-2-producing CD8+ and CD4+ T cells were assessed using an ICS assay as described in Materials and Methods. Following Δnef administration, IFN-γ-producing CD8 responses were readily detectable in both groups (Fig. 4A). At 2 weeks following Δnef, the frequencies of Gag-specific IFN-γ-producing + T cells (Fig. 4A) were much higher in the SIV-DNA-primed group (ranging from 0.3 to 7.4%) than in the vector-DNA-primed group (ranging from below detection limit [0.01%] to 0.3%). At this time, the median frequency of Gag-specific IFN-γ-producing CD8+ T cells in the SIV-DNA-primed group (1.3%) was statistically significantly higher than that of the vector-DNA-primed group (0.05%) (P = 0.005; Wilcoxon rank sum test). By 25 weeks following SIVΔnef administration, the median frequency of Gag-specific IFN-γ-producing CD8 T cells in the SIV-DNA-primed group contracted about 26-fold. However, the vector-DNA-primed group demonstrated a threefold increase in Gag-specific IFN-γ-producing CD8 T cells. Thus, at this time (week 49 of the study), the frequency of IFN-γ-producing CD8+ T cells was very similar for both groups and differences in median values were not statistically significant.
A similar pattern was also observed for the IL-2-producing CD8+ T cells (Fig. 4B). At 2 weeks following Δnef vaccination, the median frequency of IL-2-producing CD8+ T cells in the SIV-DNA-primed macaques was 16-fold lower than the median frequency of IFN-γ-producing CD8+ T cells (Fig. 4, compare panels A and B). However, as with the IFN-γ-producing CD8+ T cells, the frequencies of IL-2-producing CD8+ T cells were much higher in the SIV-DNA-primed group (ranging from 0.01 to 0.2%) than in the vector-DNA-primed group (0.01%). In the latter group, the frequency of IL-2-producing CD8+ T cells was below our detection limit (0.01%) for all except one animal, RVs6. Comparing the median frequencies of IL-2+/CD8+ T cells, the SIV-DNA-primed macaques had a significantly higher level than the vector-DNA-primed macaques (0.08% versus 0.01%, respectively; P = 0.008; Wilcoxon rank sum). By 24 weeks following Δnef, the vector-DNA-primed group demonstrated low but detectable levels of IL-2-producing CD8+ T cells for all animals (Fig. 4B). At this time, both the SIV-DNA-primed and the vector-DNA-primed groups had similar levels of IL-2-producing CD8+ T cells. The difference was not statistically significant.
Lower frequencies of Gag-specific CD4 cells following ΔNef infection in the DNA-primed group.
Following ΔNef administration, the IFN-γ-producing CD4+ T-cell responses were readily detectable for both groups (Fig. 4C). At 2 weeks following Δnef, the frequency of IFN-γ-producing CD4+ T cells was much lower in the SIV-DNA-primed group (ranging from below the detection limit [0.01%] to 0.12%) than in the vector-DNA-primed group (ranging from 0.02% to 0.35%). At this time, the mean frequency of IFN-γ-producing CD4+ T cells in the SIV-DNA-primed group (0.02%) was 3.5-fold lower than that of the vector-DNA-primed group (0.07%). However, this difference was not statistically significant (P = 0.23; Wilcoxon rank sum test of medians). By 24 weeks following SIVΔnef, the mean frequency of Gag-specific IFN-γ-producing CD4+ T cells increased about threefold for the SIV-DNA-primed group and about twofold for the vector-DNA-primed group. At this time, the mean frequencies of IFN-γ-producing CD4+ T cells were not statistically significant between the two groups (0.14% versus 0.06%; P = 0.13; Wilcoxon rank sum test).
At 2 weeks following Δnef infection, the frequencies of IL-2-producing CD4+ T cells were very low for both groups and were below our detection limit in the majority of animals. By 24 weeks following SIVΔnef, however, these cells were present in all of the vaccinated macaques except one macaque from the SIV-DNA-primed group. At this time, both groups had similar mean frequencies of IL-2-producing CD4+ T cells (0.05% versus 0.03%). Again, the difference in frequencies was not statistically significant (P = 0.26; Wilcoxon rank sum test).
SIV-specific antibodies.
As an additional analysis of immune function, we investigated SIV-specific antibody levels of the immunized macaques. As stated above, following the two DNA immunizations, no anti-SIV antibodies were detected in any animals. Following Δnef vaccination, SIV-specific antibodies could be detected in both groups of macaques. Table 1 shows the levels of antibodies reactive to whole virus at three separate time points following Δnef administration. As can be seen in this table, all animals had significant ELISA titers to SIV by 6 weeks post-Δnef administration. The titers are somewhat variable over time, dependent upon the macaque, but in general increase with time after Δnef infection. The medians of both groups for each time point are also presented in the table. No statistical difference is evident between the vector-DNA-primed and SIV-DNA-primed groups (P = 0.1; Wilcoxon rank sum test).
Summary of prechallenge parameters.
Table 2 shows a comparative analysis of the major parameters examined following immunization with DNA and attenuated virus in these macaques. As indicated above, the major differences between SIV-DNA-primed and vector-DNA-primed macaques occurred in the setpoint viral loads and in the CD8+ T-cell responses. No statistical differences were evident elsewhere. If we examine correlations, we find that only two correlations can be derived. The first is the correlation between proliferation before Δnef vaccination and the viral load setpoint following Δnef vaccination. The ability of PBMC to proliferate in response to Gag-Pol antigen negatively correlates with the viral load setpoint (AUC; P = 0.03 [Table 2]). Next, both CD8+/IFN-γ+ and CD8+/IL-2+ responses at 2 weeks following Δnef vaccination negatively correlate with the viral load setpoint following Δnef vaccination. These results show that an enhanced immune response can favorably affect viral loads in Δnef-vaccinated macaques.
Viral loads and CD4 cell levels following SHIV challenge.
At week 65, all macaques were challenged intravenously with 1 ml of a 1:300 dilution of SHIV89.6p (kindly provided by K. Reimann). Additionally, four naïve macaques were also challenged with the same dose to act as controls. Following challenge, viral loads and immune parameters were examined. Figure 5 shows the results of viral load and peripheral CD4+ T-cell analysis.
At 2 weeks following challenge, most vaccinated macaques showed a transient peak viremia (Fig. 5A and B). All animals became infected with SHIV89.6p, as demonstrated by differential PCR amplification of the SIV nef gene (data not shown). In the four naïve macaques, viral loads also peaked at 2 weeks post-SHIV challenge (Fig. 5C). Median plasma virus levels in this naïve group of macaques were significantly higher than those in the two immunized groups at week 67 (2 weeks postchallenge) (P = 0.018; Wilcoxon rank sum test).
Following peak viremia, three of seven macaques in the vector-DNA-primed group showed subsequently increasing levels of plasma viral loads (RMk6, RUs6, and RVs6). These viral loads maximized at between 106 and 108 SIV RNA copies/ml at week 85 (20 weeks postchallenge), higher than the initial viral peak following SHIV89.6p challenge. The other four macaques appeared to control the initial peak of viral replication. Still, despite initial control, three of these four macaques showed variable increases in viral loads at later times, with two of these animals having increasing viral loads past week 100. In contrast, macaques in the SIV-DNA-primed group showed good control of virus replication, with a secondary peak at week 85 postinfection in five of the seven macaques, albeit at lower levels than the initial peak (Fig. 5B). Three of the four naïve macaques showed increasing viral loads following the initial partial control observed at 3 weeks postchallenge (Fig. 5C). If weeks 71 to 85 are examined by comparing the AUCs of the viral loads (Fig. 5G), the SIV-DNA-primed group has significantly lower viral loads than does the naïve control group (P = 0.036; Wilcoxon rank sum test), but the vector-DNA-primed group does not (P = 0.200; Wilcoxon rank sum test).
Examination of the peripheral CD4+ T-cell levels of the immunized groups showed an initial drop in levels for most macaques at 2 weeks following SHIV challenge (Fig. 5D and E). In all immunized animals, this initial drop recovered to normal levels. Subsequently, three of the seven vector-DNA-primed macaques showed dramatic decreases in CD4 cell counts (Fig. 5D). The first was RMk6, which showed a dramatic decrease in peripheral CD4+ T-cell levels beginning by week 72 (7 weeks postchallenge). This macaque eventually developed AIDS and was sacrificed at week 94. The second macaque to show CD4+ T-cell decline was RUs6, which showed a less dramatic decrease beginning by week 78 (13 weeks postchallenge). The third animal was RPm6, with peripheral CD4+ T-cell levels beginning to decline by week 85 (20 weeks postchallenge). By the end of the study, all three of these macaques had fewer than 100 CD4+ T cells/μl of blood. With the SIV-DNA-primed macaques, no incidence of T-cell decline was detected following the initial postchallenge drop described above (Fig. 5E). All animals had rebounding CD4+ T-cell counts and remained at normal levels for the length of the study (113 weeks total). In contrast, three of four unimmunized macaques showed dramatic, irreversible CD4+ T-cell decline following SHIV89.6p inoculation (Fig. 5F). The fourth macaque showed an initial drop to low levels, which rebounded slightly but was consistently below 500 cells/μl. If comparative analyses are performed between each of the immunized macaque groups and the unimmunized control group, there is a significant difference in the ability of the SIV DNA/attenuated virus vaccine to protect against CD4+ T-cell decline (P = 0.006; Fisher's exact test), but not so with the vector DNA/attenuated virus vaccine strategy (P = 0.194; Fisher's exact test). This suggests that the addition of the DNA priming significantly enhanced the protective effects of attenuated virus vaccination.
Immune responses following challenge.
As with the analyses post-Δnef inoculation, we examined both the CD4+ and the CD8+ T-cell compartments for immune responses following challenge with SHIV89.6p. For CD4+ T cells, most animals in both immunized groups showed little measurable activity to SIV Gag peptides on the day of challenge, as determined by ICS for IFN-γ (Fig. 6A, B, and C). At 2 weeks postchallenge, most animals in both groups showed increased levels of SIV-specific T-cell activity to Gag. The exceptions were RUs6 in the vector-DNA-primed group and RQi6 and RKb6 in the SIV-DNA-primed group. By 20 weeks postchallenge, CD4+ T-cell responses to SIV Gag were still detectable in all immunized animals. Of the vector-DNA-primed macaques, five of the seven animals showed equal or greater reactivity in the CD4+ T-cell compartment at 20 weeks postchallenge versus week 2 postchallenge (Fig. 6A). However, of the SIV-DNA-primed macaques, six of seven animals showed equal or less reactivity at 20 weeks postchallenge versus 2 weeks postchallenge (Fig. 6B). Geometric mean titers of CD4+/IFN-γ+ T cells were not much different between groups (Fig. 6C).
As with the CD4+ T-cell compartment, macaques in both immunized groups had similar levels of SIV-Gag-reactive CD8+/IFN-γ+ T cells on the day of challenge. Following challenge, increases in SIV-Gag-specific IFN-γ+ CD8 cells were observed with all animals in both groups by 2 weeks postinfection (Fig. 6D and E). At 20 weeks postinfection, levels of SIV-Gag-specific CD8+/IFN-γ+ cells were still increasing in five of the seven vector-DNA-primed macaques, whereas they were decreasing in five of the seven SIV-DNA-primed macaques. Geometric mean titers of SIV-Gag-specific CD8+/IFN-γ+ T cells were higher at 2 weeks postchallenge in the SIV-DNA-primed group but were higher at 20 weeks postchallenge in the vector-DNA-primed group (Fig. 6F). The differences of the medians at this point were statistically significant (P = 0.01; Wilcoxon rank sum test). IL-2 responses for both groups were similar across both the CD4+ and the CD8+ T-cell compartments (data not shown).
Finally, we analyzed the development of antibody to see if this contributed in any way to the protective effects. We hypothesized that it would not because of the totally heterologous challenge (SIV Env prime versus HIV89.6 Env challenge). Binding SIV-specific antibody titers were comparable in the SIV-DNA-primed and vector-DNA-primed groups (Table 3), similar to results seen after Δnef inoculation. No statistical differences were evident either prior to or after challenge with SHIV89.6p (Table 4). Neutralization assays using plasma samples from both groups were also performed. As seen in Table 4, neutralizing activity to SHIV89.6p was undetectable at 3 and 6 weeks postchallenge. By 20 weeks postchallenge, a number of immunized macaques developed low to moderate levels of neutralizing antibody. However, there were no statistically significant differences in neutralizing activity between the vector-DNA-primed macaques and the SIV-DNA-primed macaques.
As a result of the challenge, three of six macaques in the vector DNA group developed extremely low CD4+ T-cell counts, with one succumbing to AIDS. In contrast, none of the seven macaques in the SIV DNA group showed these CD4+ T-cell declines and none developed AIDS. All four of the challenge animals showed severely depleted CD4+ T cells, and three of these macaques developed AIDS and were sacrificed. At the end of 115 weeks, all animals remaining were sacrificed due to the end of the study.
Summary of analyzed parameters.
Table 5 presents a comparative analysis of parameters examined following challenge of macaques with SHIV89.6p. As can be seen, statistically significant differences were observed mainly between the SIV-DNA-primed group of macaques and the control, naïve macaques. This was evident for both viral loads (peak and setpoint) and protection from disease (CD4+ T-cell decline). Unlike the results following immunization, no correlations could be found between immune response and viral load or prevention of disease in this study. However, these results strongly suggest an enhancement of protection by combining a DNA prime with live attenuated virus vaccination.
DISCUSSION
Despite a number of vaccine candidates being tested using the SIV/macaque model system over a number of years, live attenuated viruses still have shown the best protective effects from numerous challenge viruses (11, 14, 15, 26, 28, 31-33, 39, 47, 48). However, the immunity elicited by attenuated viruses is time dependent and is not fully protective in some cases (12, 28, 31, 47, 48). The current study was an effort to improve upon the immune responses and protective effects induced by vaccination with a live attenuated virus, by inclusion of a DNA-based priming immunization prior to SIVmac239Δnef infection. The DNA immunogen utilized was an SIV Gag-Pol expression vector and was administered intradermally in two separate immunizations at 0 and 8 weeks to a group of seven rhesus macaques. A control group of macaques received an empty DNA expression vector.
Similar to results from previous studies (4, 5, 8, 17, 24), DNA immunization induced a very low level of T-cell response to SIV Gag antigens in the absence of a detectable humoral immune response. Subsequent boosting was accomplished at week 25 with live attenuated SIVmac239Δnef administered as an intravenous inoculation. This attenuated virus was chosen over other, more attenuated viruses to ensure that the DNA priming would not prevent infection.
Following Δnef administration, plasma viral loads in both groups of macaques peaked by 2 weeks after infection (week 27 of the study). Interestingly, the peak viral loads were consistently more elevated in the macaques receiving the SIV DNA priming immunization, although the mean levels were not statistically higher than those of the vector DNA macaques. The basis for the increased viral replication in the SIV-DNA-primed group could represent an increased presence of activated (or activatable) CCR5+/CD4+ memory T cells available as targets for SIV infection as a consequence of priming. Previous studies utilizing DNA as a priming method have not utilized a live SIV as a follow-up boost. However, upon challenge in these studies, higher viral loads in DNA-immunized macaques were not observed (4, 5, 8, 17, 21, 24). This is likely due to extensive boosting with other immunogens prior to challenge. The mechanism of the phenomenon of elevated viral replication in our immunized macaques will require additional investigation.
Despite having higher peak viral loads, the SIV-DNA-primed macaques controlled viral replication more effectively than did vector-DNA-primed macaques. By 3 weeks after Δnef administration, viral loads were undetectable in five of seven animals in the vector DNA group and in seven of seven animals in the SIV DNA group. Subsequent rebounds of viral load were observed with most macaques in the vector-DNA-primed group but with only one macaque of the SIV-DNA-primed group. Studies utilizing SIVmac239Δnef as a vaccine strategy have shown various results concerning detectable plasma viral loads following the initial peak. Reports from some investigators have indicated that most macaques control virus replication well following infection, with plasma virus loads becoming undetectable (1, 12). However, similar to the results found in our study, other reports have shown that plasma virus loads can indeed rebound to detectable levels for long periods of time (10, 31, 43).
The differences between macaques able to control Δnef replication and those unable to control replication have not been previously investigated. In the study presented here, control of Δnef viral replication inversely correlated with statistically higher levels of pre-Δnef vaccination proliferative responses and peak postvaccination IFN-γ- and IL-2-secreting CD8+ T cells. No significant differences were observed in the induction of IFN-γ- or IL-2-producing CD4+ T cells. Additionally, no significant differences in levels of anti-SIV binding antibodies were seen between the two groups following Δnef immunization. The ability to control replication of live attenuated viruses in these types of studies may depend on the levels of IFN-γ- and IL-2-secreting T cells.
In this study, a highly rigorous, heterologous challenge was utilized: intravenous inoculation of SHIV89.6p. It was expected that all macaques would become infected with this challenge, and indeed this did occur. Plasma viral loads increased in most animals from both immunized groups, and no statistical differences were observed for mean peak viral loads. However, both immunized groups had statistically lower peak viral loads than the control, unimmunized group that received SHIV89.6p. Also indicative of SHIV infection in both immunized groups was the brief decline in circulating CD4+ T-cell count. These results are similar to previously published results by Lewis et al., where all macaques immunized with Δnef became infected with SHIV (31).
Differences between the immunized groups became evident following the rebound of CD4+ T-cell levels. Control of virus replication was more pronounced in the SIV-DNA-primed group, with six of seven macaques showing a decrease to undetectable levels (versus three of seven in the vector DNA group). Progression to disease was subsequently observed only with the vector-DNA-primed group, with three of seven macaques developing CD4+ T-cell deficiencies and one macaque succumbing to AIDS. No indicators of disease were observed with any of the macaques in the SIV-DNA-primed group. Because of the heterologous nature of the challenge virus, antibodies were not expected to play a role in protection from SHIV challenge. This was confirmed by neutralizing antibody studies, which revealed no presence of anti-SHIV89.6p neutralizing antibodies until much later after challenge.
Despite numerous studies illustrating the efficacious nature of immunization with live attenuated SIVmac239Δnef, issues remain concerning the safety, immunogenicity, and overall protective effects. Deletion of additional portions of the SIVmac239 genome have resulted in further attenuation but also have resulted in viruses which do not offer as great of a protective effect. In the study presented here, a priming immunization was shown to achieve better control of a live attenuated virus immunization and was also shown to positively aid in protecting macaques from disease development due to SHIV infection. No correlation was found when comparing levels of immune response (either humoral or cellular) to either viral loads or progression to disease following challenge. These results demonstrate that improvements upon live attenuated virus can be made. Still, before live attenuated viruses could feasibly be utilized for immunization against HIV, safety issues would need to be resolved. Added priming prior to live attenuated virus vaccination could improve upon safety, especially with a more attenuated virus, but this would need to be tested with the macaque model system. Additionally, a priming immunization could circumvent the problem of time needed for full protective effects of live attenuated virus vaccination.
FIG. 1.
FIG. 2.
FIG. 3.
FIG. 4.
FIG. 5.
FIG. 6.
TABLE 1.
| Immunization group and animal | Antibody titera at wk post-Δnef administration | ||||
|---|---|---|---|---|---|
| 6 | 19 | 27 | |||
| Vector DNA | |||||
| RUs6 | 12,800 | 6,400 | 12,800 | ||
| RPi6 | 3,200 | 6,400 | 6,400 | ||
| RPm6 | 6,400 | 12,800 | 6,400 | ||
| RMk6 | 6,400 | 12,800 | 25,600 | ||
| RWq6 | 3,200 | 12,800 | 12,800 | ||
| RNs6 | 6,400 | 6,400 | 6,400 | ||
| RVs6 | 6,400 | 6,400 | 6,400 | ||
| SIV DNA | |||||
| RVj6 | 12,800 | 6,400 | 6,400 | ||
| RIo6 | 3,200 | 3,200 | 3,200 | ||
| RQp6 | 12,800 | 12,800 | 6,400 | ||
| RJk6 | 3,200 | 3,200 | 3,200 | ||
| RDl6 | 6,400 | 25,600 | 51,200 | ||
| RQi6 | 1,600 | 1,600 | 3,200 | ||
| RKb6 | 800 | 800 | 1,600 | ||
| Medianb (vector DNA / SIV DNA) | 6,400 / 5,829 | 9,143 / 7,657 | 10,971 / 10,743 | ||
a
Tested with a whole-virus commercial ELISA kit. Endpoint titers were determined by serial twofold dilutions of plasma.
b
Arithmetic medians of all values per group.
TABLE 2.
| Parameter | Value for group | P valuei | R | ||
|---|---|---|---|---|---|
| SIV DNA | Vector DNA | ||||
| Peak VLa | 1.85E+05 | 3.94E+04 | 0.2 | ||
| VL setpoints | |||||
| Wk 44b | 6.90E+01 | 2.12E+02 | 0.09 | ||
| AUC (wk 28-61)c | 1,200 | 11,118 | 0.037* | ||
| Anti-SIV antibody titerd | 5,829 | 6,400 | 0.1 | ||
| Responses of cell types | |||||
| CD8+/IFN-γ+e | 1.31 | 0.05 | 0.005* | ||
| CD8+/IL-2+e | 0.08 | 0.01 | 0.008* | ||
| CD4+/IFN-γ+e | 0.02 | 0.07 | 0.23 | ||
| CD4+/IL-2+e | 0.01 | 0.01 | 0.93 | ||
| CD8+/IFN-γ+f | 0.05 | 0.14 | 0.2 | ||
| CD8+/IL-2+f | 0.03 | 0.06 | 0.46 | ||
| CD4+/IFN-γ+f | 0.06 | 0.14 | 0.13 | ||
| CD4+/IL-2+f | 0.03 | 0.05 | 0.26 | ||
| Correlationsg | |||||
| Wk 10 proliferation vs VL AUC (wk 28-61)h | 0.03* | −0.62 | |||
| Wk 27 CD8+/IFN+ cells vs VL AUC | 0.02* | −0.69 | |||
| Wk 27 CD8+/IL-2+ cells vs VL AUC | 0.01* | −0.72 | |||
| Wk 27 CD4+/IFN+ cells vs VL AUC | 0.56 | 0.17 | |||
| Wk 27 CD4+/IL-2+ cells vs VL AUC | 0.95 | −0.01 | |||
a
Peak viral loads (VL) (SIV RNA copies/ml) from week 27. Geometric means of each group are reported.
b
VL setpoints were determined using geometric means for week 44.
c
AUC was calculated for each macaque, and the median for each group is reported. Significance was determined using the Wilcoxon rank sum test.
d
Comparison of median anti-SIV antibody titers for SIV DNA and vector DNA groups at week 31.
e
Measured as ICS responses (median frequency) to SIV Gag on week 27.
f
Measured as ICS responses (median frequency) to SIV Gag on week 49.
g
Correlations were tested using Spearman's rank correlation test.
h
Proliferation to Gag-Pol protein; VL are setpoints for weeks 28 to 61 in all correlations.
i
P values are based on the Wilcoxon rank sum test between groups. Asterisks denote statistically significant differences.
TABLE 3.
| Immunization group and animal | Whole-virus anti-SIV antibody titera at: | ||||
|---|---|---|---|---|---|
| 4 Wk prechallenge | 6 Wk postchallenge | 20 Wk postchallenge | |||
| Vector DNA | |||||
| RUs6 | 51,200 | 102,400 | 51,200 | ||
| RPi6 | 25,600 | 51,200 | 25,600 | ||
| RPm6 | 25,600 | 51,200 | 51,200 | ||
| RMk6 | 51,200 | 102,400 | 25,600 | ||
| RWq6 | 25,600 | 51,200 | 25,600 | ||
| RNs6 | 25,600 | 51,200 | 51,200 | ||
| RVs6 | 25,600 | 102,400 | 25,600 | ||
| SIV DNA | |||||
| RVj6 | 12,800 | 102,400 | 25,600 | ||
| RIo6 | 25,600 | 51,200 | 12,800 | ||
| RQp6 | 25,600 | 102,400 | 25,600 | ||
| RJk6 | 3,200 | 51,200 | 6,400 | ||
| RDl6 | 102,400 | 204,800 | 102,400 | ||
| RQi6 | 6,400 | 25,600 | 12,800 | ||
| RKb6 | 3,200 | 12,800 | 12,800 | ||
| Median (vector DNA / SIV DNA) | 32,914 / 25,600 | 73,143 / 78,629 | 36,571 / 28,343 | ||
a
Tested with a whole-virus commercial ELISA kit.
TABLE 4.
| Immunization group and animal | Antibody neutralization titer to SHIV89.6pa at wk postchallenge | ||||
|---|---|---|---|---|---|
| 3 | 6 | 20 | |||
| Vector DNA | |||||
| RUs6 | <20 | <20 | 41 | ||
| RPi6 | <20 | <20 | 1,017 | ||
| RPm6 | <20 | 29 | 468 | ||
| RMk6 | <20 | <20 | 68 | ||
| RWq6 | <20 | <20 | 29 | ||
| RNs6 | <20 | <20 | <20 | ||
| RVs6 | <20 | <20 | 655 | ||
| SIV DNA | |||||
| RVj6 | <20 | <20 | 65 | ||
| RIo6 | <20 | <20 | 1,086 | ||
| RGp6 | <20 | <20 | 605 | ||
| RJk6 | <20 | <20 | 65 | ||
| RDl6 | <20 | <20 | <20 | ||
| RQi6 | <20 | <20 | 121 | ||
| RKb6 | <20 | <20 | <20 | ||
a
In vitro neutralization assay using TZM-b1 cells and SHIV89.6p virus.
TABLE 5.
| Parameter | Post-SHIV89.6p challenge result for group | P valueh | ||||
|---|---|---|---|---|---|---|
| SIV DNA | Vector DNA | Controls | ||||
| Peak VLa | ||||||
| SIV DNA vs vector DNA | 1.31E+04 | 7.22E+03 | 1 | |||
| SIV DNA vs controls | 1.31E+04 | 7.02E+07 | 0.018* | |||
| Vector DNA vs controls | 7.22E+03 | 7.02E+07 | 0.018* | |||
| SIV DNA and vector DNA vs controls | 1.31E+04 | 7.22E+03 | 7.02E+07 | 0.001* | ||
| CD4+ T-cell declineb | ||||||
| SIV DNA and vector DNA vs controls | 0 of 7 | 3 of 7 | 4 of 4 | 0.004* | ||
| SIV DNA vs controls | 0 of 7 | 4 of 4 | 0.006* | |||
| Vector DNA vs controls | 3 of 7 | 4 of 4 | 0.388 | |||
| VL setpoints | ||||||
| Wk 78c | 1.40E+02 | 1.56E+03 | 1.70E+05 | 0.047*† | ||
| AUC (wk 71-85)d | 2.67E+06 | 3.11E+08 | 4.66E+08 | 0.036*† | ||
| Anti-SIV antibody titere | 78,629 | 73,143 | 0.79 | |||
| Responses of cell types | ||||||
| CD8+/IFN-γ+f | 0.39 | 0.14 | 0.15 | |||
| CD8+/IL-2+f | 0.14 | 0.03 | 0.09 | |||
| CD4+/IFN-γ+f | 0.19 | 0.14 | 0.93 | |||
| CD4+/IL-2+f | 0.02 | 0.03 | 1 | |||
| CD8+/IFN-γ+g | 0.05 | 0.44 | 0.02* | |||
| CD8+/IL-2+g | 0.01 | 0.02 | 0.35 | |||
| CD4+/IFN-γ+g | 0.10 | 0.11 | 0.44 | |||
| CD4+/IL-2+g | 0.02 | 0.02 | 1 | |||
a
Peak viral loads (VL) from week 67. Geometric means of each group are reported.
b
CD4+ T-cell decline was determined as consistent CD4+ T-cell counts of <500. Three-way comparisons were tested using the Freeman and Halton method. Two-way comparisons were tested using Fisher's exact test.
c
VL setpoints were determined using geometric means for week 78.
d
CAUC was calculated for each macaque, and the median for each group is reported. Significance was determined using the Wilcoxon rank sum test.
e
Comparison of median anti-SIV antibody titers for SIV DNA and vector DNA groups at week 85.
f
Measured as ICS responses (median frequency) to SIV Gag on week 67.
g
Measured as ICS responses (median frequency) to SIV Gag on week 85.
h
P values are based on the Wilcoxon rank sum test between groups. Asterisks denote statistically significant differences, and daggers (†) denote statistically significant differences between SIV DNA and control groups only.
Acknowledgments
We thank the Yerkes National Primate Research Center animal care staff and Research Resources personnel for their excellent care and handling of the animals in this study. Additionally, we thank David Lee and Barbara Sokolik-Wolak for technical assistance.
This work was supported by grants AI-49789 (to F.J.N.) and RR-00165 (to the Yerkes National Primate Research Center).
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© 2005 American Society for Microbiology.
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Received: 22 April 2005
Accepted: 15 September 2005
Published online: 15 December 2005
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2005. A Combination DNA and Attenuated Simian Immunodeficiency Virus Vaccine Strategy Provides Enhanced Protection from Simian/Human Immunodeficiency Virus-Induced Disease. J Virol 79:.
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