Signature for Long-Term Vaccine-Mediated Control o
http://www.100md.com
病菌学杂志 2005年第6期
Emory Vaccine Center and Yerkes National Primate Research Center, Emory University
Emory Vaccine Center and Department of Medicine, Division of Infectious Diseases of Emory University School of Medicine, Atlanta, Georgia
Department of Surgery, Duke University Medical Center, Durham, North Carolina
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
ABSTRACT
In 2001, we reported 20 weeks of control of challenge with the virulent 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) by a Gag-Pol-Env vaccine consisting of DNA priming and modified vaccinia virus Ankara boosting. Here we report that 22 out of 23 of these animals successfully controlled their viremia until their time of euthanasia at 200 weeks postchallenge. At euthanasia, all animals had low to undetectable viral loads and normal CD4 counts. During the long period of viral control, gamma interferon (IFN-)-producing antiviral T cells were present at unexpectedly low breadths and frequencies. Most animals recognized two CD8 and one CD4 epitope and had frequencies of IFN--responding T cells from 0.01 to 0.3% of total CD8 or CD4 T cells. T-cell responses were remarkably stable over time and, unlike responses in most immunodeficiency virus infections, maintained good functional characteristics, as evidenced by coproduction of IFN- and interleukin-2. Overall, high titers of binding and neutralizing antibody persisted throughout the postchallenge period. Encouragingly, long-term control was effective in macaques of diverse histocompatibility types.
INTRODUCTION
Recently, vaccine-elicited cytotoxic T-cell responses have successfully controlled challenges with the 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) (5, 8, 40, 41). These T-cell vaccines do not prevent infection but rather reduce viremia to the very low levels characteristic of humans who are long-term nonprogressors and nontransmitters (31, 39). Most T-cell vaccines have used a heterologous prime-boost regimen in which DNA is used for priming and a live viral vector is used for boosting or serologically distinct live vectors are used for priming and boosting. In these regimens, immunity elicited by the priming vector does not block the boosting vector outside of responses to the common vaccine insert.
Obstacles to the success of T-cell vaccines are the generation of mutant viruses capable of escaping the T-cell response (6, 7), exhaustion of the T-cell response (26, 34, 43), and the dependence of protection on the histocompatibility type of the host (25, 29). Escape mutations can affect the sequence of an epitope or sequences that influence the processing and presentation of an epitope (6, 45). The timing of escape and the localization of escape mutations reflect both the ability of individual cytotoxic T lymphocyte epitopes to control virus replication and the cost of individual mutations to viral fitness (2, 17, 21). A chronic infection also can escape cellular immunity by exhausting responding T cells through constant stimulation by persisting antigen (26, 34, 47). Exhaustion is characterized by a sequential loss of interleukin-2 (IL-2), then tumor necrosis factor alpha, and then gamma interferon (IFN-) production and ultimately apoptosis (44). Exhaustion occurs more rapidly in the absence of CD4 T-cell help (28, 47) and can be a major contributor to the loss of CD8 control for immunodeficiency viruses, which preferentially infect and deplete antiviral CD4 T cells (16, 23). Successful control of human immunodeficiency virus type 1 (HIV-1) in infected humans correlates with the vigor of responding T cells as measured by their ability to coproduce both IL-2 and IFN- (10, 11, 22, 24, 46). IL-2 coproduction is also a characteristic of protective T cells in vaccine-mediated control of immunodeficiency virus challenges in macaques (30).
A host's histocompatibility type determines both the breadth and immunodominance of a CD8 T-cell response and also influences the effectiveness of T-cell vaccines (20). In the macaque model, the presence of the A01 histocompatibility type, which presents the Gag-CM9 (p11c) epitope, can increase the likelihood for the control of viremia (35, 36). This reflects the Gag-CM9 epitope being an immunodominant epitope that requires two mutations for escape due to structural constraints on mutations tolerated in this epitope (19). Macaques with the A01 histocompatibility type frequently show better protection against simian immunodeficiency virus (SIV) infections than macaques without this histocompatibility type (35, 36). For SHIV-89.6P infections, the A01 histocompatibility type has been associated with better protection for two adenovirus-vectored vaccines (25, 29).
Antibody to Env also can contribute to viral control through virus neutralization or antibody-dependent cellular cytotoxicity (38). In the SHIV-89.6P macaque model, Env-binding nonneutralizing antibody appears to contribute to the protection of CD4 T cells and viral control (4). However, the role played by anti-Env antibody is secondary to that of CD8 T cells. In the SHIV-89.6P model, well-contained chronic infections that have generated high titers of neutralizing antibody undergo rapid reemergence if CD8 T cells are removed by depletion (R. R. Amara, C. Ibegbu, F. Villenger, D. C. Montefiori, Y. Xu, P. Nigam, S. Sharma, H. M. McClure, and H. L. Robinson, submitted for publication).
In 2001, we reported the control of a SHIV-89.6P challenge by a vaccine that consisted of priming with DNA and boosting with modified vaccinia virus Ankara (DNA/MVA vaccine) (5). Both immunogens expressed Gag, Pol, and Env of SHIV-89.6. Twenty-four vaccinated animals were challenged with SHIV-89.6P. Only one of these failed to control the infection, and a second animal was lost to trauma during the third year postchallenge. Six controls were challenged. Five of these succumbed to AIDS within the first year postchallenge, whereas the sixth has survived. This study reports temporal viral loads, levels of CD4 T cells, antiviral CD8 and CD4 T-cell responses, and antibody responses up to the time of euthanasia at 200 weeks postchallenge. During this 4-year period, the vaccine successfully protected 11 A01 and 11 non-A01 macaques from viremia and loss of CD4 T cells. This long period of vaccine-mediated control was characterized by a remarkably stable and low-frequency antiviral IFN- T-cell response that consistently maintained the ability to coproduce IL-2.
MATERIALS AND METHODS
Immunogens. The construction and production of immunogens have been previously described (5). The DNA prime (DNA/89.6) expressed SIV Gag, Pol, Vif, Vpx, and Vpr and HIV-1 Env, Tat, and Rev from a single transcript. The rMVA booster (MVA/89.6) expressed SIV Gag and Pol and HIV-1 Env under the control of vaccinia virus early/late promoters (18).
Immunizations and challenge. Young adult rhesus macaques from the Yerkes breeding colony were cared for under guidelines established by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals (5a) with protocols approved by the Emory University Institutional Animal Care and Use Committee. Animal numbers are as follows: 1, RBr-5; 2, RIm-5; 3, RQf-5; 4, RZe-5; 5, ROm-5; 6, RDm-5; 7, RAj-5; 8, RJi-5; 9, RAl-5; 10, RDe-5; 11, RAi-5; 12, RPr-5; 13, RKw-4; 14, RWz-5; 15, RGo-5; 16, RLp-4; 17, RWd-6; 18, RAt-5; 19, RPb-5; 20, RIi-5; 21, RIq-5; 22, RSp-4; 23, RSn-5; 24, RGd-6; 25, RMb-5; 26, RGy-5; 27, RUs-4; 28, RPm-5; 29, RPs-4; and 30, RKj-5. Animals with the A01 allele are indicated with asterisks. Four groups of six rhesus macaques each were primed at 0 and 8 weeks with either 2.5 mg (high dose) or 0.25 mg (low dose) of DNA by intradermal (i.d.) or intramuscular (i.m.) routes with a needleless jet injection device (Bioject, Portland, Oreg.). At 24 weeks, the MVA/89.6 booster immunization (2 x 108 PFU) was injected with a needle both i.d. and i.m. The control group included three mock-immunized animals and three naive animals. At 7 months after the rMVA booster was administered, animals received an intrarectal challenge with SHIV-89.6P, during which approximately 20 intrarectal infectious units (1.2 x 1010 copies of SHIV-89.6P viral RNA) was introduced 15 to 20 cm into the rectum by means of a pediatric feeding tube.
Quantification of SHIV copy number. SHIV copy numbers were determined by using a quantitative real-time PCR as previously described (5). Viral RNA was directly extracted by a MagNa Pure LC robotic workstation (Roche Molecular Biochemicals) with the MagNa Pure LC total nucleic acid isolation kit (Roche), and a reverse transcription reaction was set up in a Perkin-Elmer Applied Biosystems 7700 sequence detection system with the primer sequences 5'-GCAGAGGAGGAAATTACCCAGTAC-3' (forward) and 5'-CAATTTTACCCAGGCATTTAATGTT-3' (reverse) and the probe sequence 6-carboxyfluorescein (FAM)-5'-TGTCCACCTGCCATTAAGCCCGA-3'-6-carboxytetramethylrhodamine (TAMRA) within a conserved portion of the SIV gag gene.
Gag-CM9 tetramer analysis. Tetramer analyses were performed as previously described (5) by surface staining 100 μl of whole blood with antibodies to CD3 conjugated to fluorescein isothiocyanate (FN-18; Biosource International, Camarillo, Calif.), CD8 conjugated to peridinin chlorophyll protein (SK1; Becton Dickinson, San Jose, Calif.), and Gag-CM9 (CTPYDINQM)-Mamu-A01 tetramer conjugated to allophycocyanin. The tetramer-positive cells were represented as a percentage of CD8 T cells.
ICS assays. Intracellular cytokine (ICS) assays were performed as previously described by using four pools of Gag peptides (22-mers overlapping by 11) (5). Peripheral blood mononuclear cells (PBMCs; 106) were stimulated for 2 h at 37°C with 10 μg of peptide in a volume of 200 μl of RPMI with 0.2 μg each of anti-human CD28 and CD49d (Pharmingen, San Diego, Calif.). After 2 h, 800 μl of RPMI containing 10% fetal bovine serum and monensin (10 μg/ml) was added, and the cells were cultured overnight at 37°C. Cells were surface stained with antibodies to CD8 conjugated to peridinin chlorophyll protein (clone SK1; Becton Dickinson), fixed and permeabilized with Cytofix/Cytoperm solution (Pharmingen), and then stained with antibodies to human CD3 (clone FN-18; Biosource International), IFN- (clone B27; Pharmingen), and IL-2 (clone MQ1-17H12; Pharmingen) conjugated to fluorescein isothiocyanate, allophycocyanin, and phycoerythrin, respectively. About 150,000 lymphocytes were acquired on the FACScalibur (BD Biosciences, San Jose, Calif.) and analyzed with FloJo software (Treestar Inc., Ashland, Oreg.). Backgrounds for unstimulated cells were determined for each macaque for each analysis and subtracted from positive results. In general, backgrounds are much less than 0.01%, and macaques with higher backgrounds are excluded from studies. On the basis of our experience, responses with the correct pattern for IFN- responses and frequencies greater than 0.01% of total CD4 or CD8 T cells were considered positive. IL-2 production was scored only for cells coproducing IFN- and IL-2 because unstimulated cells had a high background for IL-2.
Epitope mapping. A two-step strategy was used to map epitopes in SHIV Gag and Env. In the first step, PBMCs from 23 vaccinated animals and the surviving control were screened by enzyme-linked immunospot (ELISPOT) for IFN- production using grids of 125 Gag and 211 Env peptides (15-mers with 11 overlapping amino acids) distributed in 23 Gag and 29 Env peptide pools (27). IFN- ELISPOT analyses were conducted as previously described by using 2 x 105 cells and peptide pools at a final concentration of 5 μg of each peptide/ml (5). Spot-forming units were normalized for 106 cells after subtracting 10 plus two times the average background in two negative control wells. Animals 9, 11, 16, and 19 had high background levels of spot-forming units, and the positive peptide pools could not be determined. In the second step, individual peptides from pools considered positive by ELISPOT were used in intracellular cytokine assays to determine the frequencies of IFN-- and IFN--and-IL-2-producing CD4 and CD8 T cells (see above). Consecutive positive peptides with 11 overlapping amino acids were considered a single epitope.
ELISA for binding antibodies. Enzyme-linked immunosorbent assays (ELISAs) for total antibody to Gag and Env were conducted as previously described (5). SIV Gag p27 produced in Escherichia coli and 89.6 Env produced in transiently transfected 293T cells and captured with sheep antibody against Env (International Enzymes, Fairbrook, Calif.) were used to coat wells. Standard curves were produced by coating with goat anti-monkey immunoglobulin G (IgG) Fc/7S (Accurate Chemical, Westbury, N.Y.) and capturing known amounts of rhesus IgG (Sigma, St. Louis, Mo.). Bound antibody was detected with peroxidase-conjugated goat anti-macaque IgG (Accurate Chemical, Westbury, N.Y.) and tetramethylbenzidine peroxidase substrate (507600; KPL, Gaithersburg, Md.).
RESULTS
Viral load and CD4 counts. All of the DNA/MVA-vaccinated animals became infected by the SHIV-89.6P challenge virus. However, within 9 weeks of the peak of postchallenge viremia, the viral load decreased to near or below 1,000 copies of viral RNA per ml of plasma in most animals (Fig. 1A). In one animal, animal 22 in the low-dose i.m. group, virus rapidly reemerged and the animal progressed to AIDS (Fig. 1C). In the other vaccinated animals, viremia continued to decline, with occasional low-level spikes of replicating virus (42). After the first year, these spikes were scored less frequently. At 200 weeks postchallenge, just prior to euthanasia, 17 animals had levels of viral RNA below our detection limit of 300 copies of viral RNA per ml of plasma (Fig. 1C). Among the remaining five animals, animals 4 and 5 had >1,000 copies of viral RNA per ml of plasma. These two animals, along with the surviving control, were not euthanized so that we could continue to monitor them for possible viral escape. Five of six unvaccinated control animals failed to control their viremia and died within 1 year postchallenge. The surviving unvaccinated animal, animal 30, slowly controlled its viral load, reducing the level of viremia to below 1,000 copies at about 1 year postchallenge. By 171 weeks (3.3 years) postchallenge, its level of viral RNA was undetectable. After challenge, the vaccinated animals maintained overall normal levels of CD4 cells (Fig. 1B). In contrast, all of the unvaccinated controls rapidly lost CD4 cells (Fig. 1D). Among the unvaccinated animals, the one survivor had the least CD4 loss and underwent a slow recovery of its CD4 cells.
CD8 T cells: Gag-CM9 tetramer-specific cells and IFN- response. Antiviral CD8 T-cell counts expanded markedly in response to the viral challenge but then contracted as the infection was controlled (Fig. 2A, B, D, and E). In A01 macaques, tetramer staining revealed 10- to 100-fold expansions in Gag-CM9-specific cells by 2 weeks postchallenge followed by a 5- to 10-fold contraction by 12 weeks postchallenge (Fig. 2A and D). The frequencies of these cells were then stable over the 200 weeks of control in all animals except macaque 20, which showed a slowly rising response. Overall, animal-to-animal differences in the frequencies, which ranged from 0.3 to 5.4% of total CD8 cells, reflected differences in their peak levels of tetramer-positive cells (Fig. 2D). In the unvaccinated A01 monkeys, the peak Gag-CM9 response was 10- to 100-fold lower than in the vaccinated animals (Fig. 2A and D).
Intracellular cytokine assays for CD8 responses to Gag also revealed high responses at 2 weeks postchallenge that then contracted (Fig. 2B and E). In contrast to the frequencies of tetramer-positive cells, which were stable after the initial contraction, the geometric means for the frequencies of IFN--producing CD8 T cells suggested an overall slow decline (Fig. 2A and B). At the peak response, geometric mean titers for anti-Gag CD8 T cells ranged from 1.4 to 6.2% of total CD8 T cells. At euthanasia, 200 weeks postchallenge, this geometric mean had decreased to 0.04 to 0.16% of total CD8 T cells. No significant differences were observed between groups.
CD4 T-cell responses. In contrast to the CD8 T-cell response that peaked at 2 weeks and then contracted, levels of antiviral CD4 T cells were low at 2 weeks but then rose over time (Fig. 2C and F). This result would be consistent with the loss of these cells due to infection during the postchallenge anamnestic response followed by a recovery once the infection had been controlled. At euthanasia, CD4 responses were slightly higher than at 5 weeks postchallenge and had geometric mean titers that ranged from 0.02 to 0.27% of total CD4 T cells. Some animals showed declines and rebounds in their antiviral CD4 responses. These rebounds and declines did not correlate with changes in CD8 T-cell responses or with detectable changes in viral loads. Again, no significant differences were observed between immunization groups.
Epitope mapping. At 140 weeks postchallenge (2.7 years), the IFN- responses of the vaccinated animals and the surviving control were mapped for CD8 and CD4 epitopes (Fig. 3 and Table 1 and 2). These assays revealed average CD8 responses to 1.4 to 2.3 epitopes per group and average CD4 responses to 1.3 to 1.8 epitopes per group. Animals 23 and 5 had the highest numbers of recognized epitopes, with three CD8 and five CD4 for animal 23 and three CD8 and four CD4 for animal 5. Animal 5 was one of the two animals that were spared euthanasia because of the presence of virus at over 1,000 copies per ml of plasma.
In total, the mapping revealed 18 CD8 epitopes and 18 CD4 epitopes residing throughout Gag and Env (Fig. 4). More epitopes were present in Gag than in Env. The Gag epitopes preferentially resided in p27. Sixty percent of the CD8 epitopes and 80% of the CD4 epitopes were present in p27, which represents 43% of Gag. Four of 10 A01 animals recognized peptides in Gag outside of those containing the immunodominant Gag-CM9 epitope (Table 1). Peptide 46 containing the Gag-CM9 epitope also encompassed a CD4 epitope recognized by 8 of 10 A01 monkeys and 1 non-A01 monkey. In Env, the CD8 and CD4 epitopes were distributed evenly across gp120 and gp41, with all CD8 epitopes and all but one CD4 epitope lying outside of the variable regions in Env.
IL-2 response. At the time of epitope mapping, the antiviral CD8 and CD4 responses in the vaccinated animals had good functional characteristics as measured by responding cells that coproduced both IFN- and IL-2 (Table 1 and 2 and Fig. 3). In the vaccinated groups, the average of the cells producing IFN- and IL-2 ranged from 35 to 45% of IFN--producing CD8 cells and from 26 to 49% of IFN--producing CD4 cells. In all, 34 out of 35 CD8 and 25 out of 30 CD4 responses coproduced IL-2. The animal with a CD8 IFN- response that did not coproduce IL-2 had two other CD8 responses, both of which coproduced IL-2 (Table 1, animal 5). The five IFN- CD4 responses that failed to coproduce IL-2 were distributed over five animals (animals 3, 17, 8, 10, and 23), with three of these animals having other CD4 responses that coproduced IL-2 (Table 2). In contrast, all four IFN- responses (two CD8 and two CD4 responses) in the unvaccinated survivor failed to coproduce IL-2.
Stability of the response. The responses to the mapped peptides were remarkably stable over the next year (Table 1 and 2). At 1 year after the initial ICS analyses (week 191), the mapped responses were tested again for IFN- and IL-2 production. Only 5 of the 34 tested CD8 responses and 7 of the 30 tested CD4 responses had declined to undetectable levels. Among the 29 remaining CD8 responses and 23 remaining CD4 responses, only 6 had changed in frequency by more than twofold. For the CD8 cells, the frequencies of IL-2-coproducing cells showed no change over twofold, except for monkey 10, where responses to peptide 46 increased from 0.01 to 0.04%. For the CD4 responses, there were five changes that were greater than twofold, with three of these changes being increases. In the unvaccinated control, the frequencies of three of four IFN- responses increased by more than twofold. These responses, in contrast to those measured at week 140, now showed low frequencies of IL-2-coproducing cells: 12.5% of the IFN--producing CD8 cells and 23% of the IFN--producing CD4 cells coproduced IL-2 (Table 1 and 2).
Comparison of the IFN- responses obtained for the individual peptides with those obtained for Gag pools in March 2004 suggested that there might have been an overall gain in CD4 but not CD8 epitopes. The CD8 responses elicited by the pools were not significantly different from those elicited by the peptides mapped in March 2003 (P = 0.13), whereas the CD4 responses elicited by the pools were significantly higher than those elicited by the mapped peptides (P = 0.005).
Antibody response. After the initial postchallenge anamnestic response, the levels of Gag and Env binding antibody declined by 5- to 10-fold (Fig. 5). In most of the animals, binding antibody then rose slowly over the next 3 years to levels ranging from 10 μg to 1 mg of IgG per ml of serum. The level of Gag or Env binding antibody fell below 10 μg per ml of serum in one animal in the high-dose i.d. group, three animals in the low-dose i.d. group, and two animals in the low-dose i.m. group. These decreases tended to be transient.
In most animals, the titers of neutralizing antibody increased rapidly between 2 and 12 weeks postchallenge from levels that were undetectable at the time of challenge to titers of 103 to 104 for 50% protection against virus-induced killing per ml of serum (Fig. 5C). With time, the levels of neutralizing antibody decreased. At the time of euthanasia, 18 of the 22 animals had titers of neutralizing antibody for SHIV-89.6P between 102 and 103 for 50% protection of MT-2 cells.
DISCUSSION
Here we report on the successful control of viremia in 22 DNA/MVA-vaccinated and SHIV-89.6P-challenged macaques (5). Control was effective up to the time of euthanasia at 200 weeks postchallenge, with all animals effectively maintaining their viremia at low to undetectable levels. CD4 T cells were maintained at normal levels, with no signs of progression to AIDS. Throughout the control period, there were occasional reemergent blips of virus. These blips were most frequent in the first year after challenge, a time when low levels of viral RNA and DNA were still declining (42). They were, however, still occurring at the time of euthanasia and served as a clear indication of the persisting presence of virus.
The signature of the successful control was a low-frequency, low-breadth IFN- T-cell response capable of coproducing IL-2. The importance of this low-level CD8 response to viral control was demonstrated by rapid viral reemergence in a parallel vaccine trial following CD8 T-cell depletion (Amara et al., submitted). A low-level T-cell response also was observed in the one surviving control, albeit this response showed more signs of exhaustion than that in the vaccinated animals. Presumably, the low-level IFN- CD8 T-cell responses reflected the low levels of persisting virus. Similar low levels of IFN- CD8 T cells are also found in successfully drug-treated humans (B. G. Kapogiannis, S. L. Henderson, P. Nigam, S. Sharma, L. Chennareddi, J. G. Herndon, H. L. Robinson, and R. R. Amara, submitted for publication).
The responses that were providing long-term control in the successfully vaccinated animals contrast with those found in HIV progressors and long-term nonprogressors who generally show greater breadths and higher frequencies of IFN--producing cells. A cohort of 23 chronic untreated patients that was mapped for the entire HIV-1 genome recognized a median of 18.5 CD8 epitopes, with a range of 8 to 42 (1). A long-term nonprogressor in this population recognized 42 epitopes. In another study in which ICS was conducted, the height of the IFN- CD8 response ranged from 0.5 to 18.8% of total CD8 T cells for chronic infections and from 5.3 to 8.8% of total CD8 T cells for long-term nonprogressors (32). Our results also contrast with the CD8 T-cell response in a supervised treatment interruption patient, where self-vaccination and then superinfection was associated with the recognition of 25 CD8 epitopes and 27,470 IFN- ELISPOTs per million PBMCs (3).
The long-term cellular response did not undergo the loss of IL-2 production, which is characteristic of infected patients and occurred in the one surviving control (22, 24, 37; Kapogiannis et al., submitted). IL-2 is critical for T-cell proliferation and survival (9), and the loss of IL-2 is the first indication of T-cell exhaustion (43). Coproduction of IL-2 and IFN- by antiviral CD8 T cells has been previously noted as a characteristic of successful vaccine-mediated control in macaques (30). The presence of the T cells that coproduced IFN- and IL-2 at the time of euthanasia is a strong indicator that the vaccine-elicited response was still capable of controlling the reemergent virus.
The long-term relationship between the low levels of residual virus and the IFN- cellular immune response was remarkably stable. However, changes could be detected when individual epitopes were mapped. Between 140 and 191 weeks postchallenge, 5 of 35 CD8 epitopes and 7 of 30 CD4 epitopes declined to frequencies below detection. Whether these changes were due to escape or exhaustion is not known, because viral sequences were below the level that we could recover by PCR.
The frequency of Gag-CM9 tetramer-positive cells was 5- to 10-fold higher than the frequency of peptide-stimulated IFN--producing cells. This was true whether stimulations were with the exact Gag-CM9 peptide or with the 15-mer Gag 46 peptide that contains the Gag-CM9 epitope. Tetramer staining provides a marker for the total population of CD8 T cells recognizing an epitope, whereas our ICS assays reveal only those cells capable of producing IFN- or IFN- and IL-2 in response to peptide stimulation. The small percentage (10 to 20%) of tetramer-positive cells capable of producing IFN- following peptide stimulation in the chronic phase of control contrasted with the peak response, where 50 to 100% of tetramer-positive cells produced IFN- with peptide stimulation. However, in the long chronic period of control, cells coproducing IFN- and IL-2 marked a much larger percentage of IFN--producing cells (20 to 60%) than at peak response (<20%).
Despite low to undetectable levels of viremia and a largely quiescent T-cell response, the antiviral antibody response remained high during the long period of chronic control. The persisting antibody included neutralizing antibody, which undoubtedly contributed to both the control of the infection and the protection of antiviral as well as total CD4 T cells. The 89.6 Env used in our immunizations does not elicit cross-reactive neutralizing antibody for the 89.6P Env in the challenge virus (33). Thus, the protective responses primed by this Env did not depend on the presence of neutralizing antibody at the time of challenge, a phenomenon that has been seen in other SHIV-89.6P vaccine trials using heterologous Envs (4, 15, 29).
Six animals showed declines in Gag and Env antibody. In four of these animals, antibody rebounded, presumably in response to changes in the activity of the persisting virus. These rebounds occurred in the absence of our detecting reemergent virus. Thus, antiviral antibody may provide a more sensitive indicator of the presence of virus than plasma viral RNA, a phenomenon that will likely be true in human trials with well-controlled infections.
Encouragingly, the DNA/MVA vaccine provided excellent protection against a SHIV-89.6P challenge for both A01 and non-A01 macaques. In another study using our Gag-Pol-Env DNA/MVA vaccine, seven additional non-A01 macaques were protected (12). For adeno5-vectored vaccines, protection against SHIV-89.6P has been much more uniform for A01 macaques than for non-A01 macaques (25, 29). Both the MVA and adeno5 vectors elicit high frequencies of responding CD8 T cells during the acute phase of immunization (41). However, the adeno5-based vectors induce higher frequencies of persisting CD8 T cells (13, 14, 41) and may induce a higher ratio of CD8-to-CD4 responses than MVA-vectored immunizations. Thus, the immune responses elicited by the two vectors have different characteristics, which could result in different protective capacities. However, other experimental differences could account for the difference in protective responses (5, 25, 29).
This study in the SHIV model provides continuing hope that a Gag-Pol-Env DNA/MVA vaccine could help control the HIV/AIDS pandemic and suggests that a signature for successful postchallenge control will be a stable, low-frequency, low-breadth, IFN-- plus IL-2-coproducing CD8 and CD4 T-cell response. We suggest that the low levels of the IFN- T-cell response during successful long-term control reflect the relative dormancy of the infection. We also suggest that the presence of IL-2 production in the responding cells provides an indicator that the T-cell response has not been exhausted by persisting antigen and will be capable of controlling reemergent virus.
ACKNOWLEDGMENTS
We are indebted to Research Resources and the veterinary and animal care technicians of the Yerkes National Primate Research Center for their invaluable care and attention to the animals in this trial. We are also indebted to Helen Drake Perrow for expert administrative assistance, to Vanda Bostik, Sunita Sharma, Pragati Nigam, and Lazarus Ofielu for their excellent technical help, to Lakshmi Chennareddi for her help in the statistical analyses, and to Francois Villinger and Bill Kapogiannis for their critiques and suggestions.
This work was supported by National Public Health Service Integrated Preclinical/Clinical AIDS Vaccine Development grant P01-AI 43045, the Emory/Atlanta Center for AIDS Research P30 DA 12121, and the Yerkes National Primate Research Center base grant P51 RR000165.
R.R.A., L.S.W., B.M., and H.L.R. have pending patents for HIV vaccines modeled on the SHIV vaccine reported in this study. H.L.R. also holds a minor equity interest in GeoVax Inc., the company that has licensed the technology.
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Emory Vaccine Center and Department of Medicine, Division of Infectious Diseases of Emory University School of Medicine, Atlanta, Georgia
Department of Surgery, Duke University Medical Center, Durham, North Carolina
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
ABSTRACT
In 2001, we reported 20 weeks of control of challenge with the virulent 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) by a Gag-Pol-Env vaccine consisting of DNA priming and modified vaccinia virus Ankara boosting. Here we report that 22 out of 23 of these animals successfully controlled their viremia until their time of euthanasia at 200 weeks postchallenge. At euthanasia, all animals had low to undetectable viral loads and normal CD4 counts. During the long period of viral control, gamma interferon (IFN-)-producing antiviral T cells were present at unexpectedly low breadths and frequencies. Most animals recognized two CD8 and one CD4 epitope and had frequencies of IFN--responding T cells from 0.01 to 0.3% of total CD8 or CD4 T cells. T-cell responses were remarkably stable over time and, unlike responses in most immunodeficiency virus infections, maintained good functional characteristics, as evidenced by coproduction of IFN- and interleukin-2. Overall, high titers of binding and neutralizing antibody persisted throughout the postchallenge period. Encouragingly, long-term control was effective in macaques of diverse histocompatibility types.
INTRODUCTION
Recently, vaccine-elicited cytotoxic T-cell responses have successfully controlled challenges with the 89.6P chimera of simian and human immunodeficiency viruses (SHIV-89.6P) (5, 8, 40, 41). These T-cell vaccines do not prevent infection but rather reduce viremia to the very low levels characteristic of humans who are long-term nonprogressors and nontransmitters (31, 39). Most T-cell vaccines have used a heterologous prime-boost regimen in which DNA is used for priming and a live viral vector is used for boosting or serologically distinct live vectors are used for priming and boosting. In these regimens, immunity elicited by the priming vector does not block the boosting vector outside of responses to the common vaccine insert.
Obstacles to the success of T-cell vaccines are the generation of mutant viruses capable of escaping the T-cell response (6, 7), exhaustion of the T-cell response (26, 34, 43), and the dependence of protection on the histocompatibility type of the host (25, 29). Escape mutations can affect the sequence of an epitope or sequences that influence the processing and presentation of an epitope (6, 45). The timing of escape and the localization of escape mutations reflect both the ability of individual cytotoxic T lymphocyte epitopes to control virus replication and the cost of individual mutations to viral fitness (2, 17, 21). A chronic infection also can escape cellular immunity by exhausting responding T cells through constant stimulation by persisting antigen (26, 34, 47). Exhaustion is characterized by a sequential loss of interleukin-2 (IL-2), then tumor necrosis factor alpha, and then gamma interferon (IFN-) production and ultimately apoptosis (44). Exhaustion occurs more rapidly in the absence of CD4 T-cell help (28, 47) and can be a major contributor to the loss of CD8 control for immunodeficiency viruses, which preferentially infect and deplete antiviral CD4 T cells (16, 23). Successful control of human immunodeficiency virus type 1 (HIV-1) in infected humans correlates with the vigor of responding T cells as measured by their ability to coproduce both IL-2 and IFN- (10, 11, 22, 24, 46). IL-2 coproduction is also a characteristic of protective T cells in vaccine-mediated control of immunodeficiency virus challenges in macaques (30).
A host's histocompatibility type determines both the breadth and immunodominance of a CD8 T-cell response and also influences the effectiveness of T-cell vaccines (20). In the macaque model, the presence of the A01 histocompatibility type, which presents the Gag-CM9 (p11c) epitope, can increase the likelihood for the control of viremia (35, 36). This reflects the Gag-CM9 epitope being an immunodominant epitope that requires two mutations for escape due to structural constraints on mutations tolerated in this epitope (19). Macaques with the A01 histocompatibility type frequently show better protection against simian immunodeficiency virus (SIV) infections than macaques without this histocompatibility type (35, 36). For SHIV-89.6P infections, the A01 histocompatibility type has been associated with better protection for two adenovirus-vectored vaccines (25, 29).
Antibody to Env also can contribute to viral control through virus neutralization or antibody-dependent cellular cytotoxicity (38). In the SHIV-89.6P macaque model, Env-binding nonneutralizing antibody appears to contribute to the protection of CD4 T cells and viral control (4). However, the role played by anti-Env antibody is secondary to that of CD8 T cells. In the SHIV-89.6P model, well-contained chronic infections that have generated high titers of neutralizing antibody undergo rapid reemergence if CD8 T cells are removed by depletion (R. R. Amara, C. Ibegbu, F. Villenger, D. C. Montefiori, Y. Xu, P. Nigam, S. Sharma, H. M. McClure, and H. L. Robinson, submitted for publication).
In 2001, we reported the control of a SHIV-89.6P challenge by a vaccine that consisted of priming with DNA and boosting with modified vaccinia virus Ankara (DNA/MVA vaccine) (5). Both immunogens expressed Gag, Pol, and Env of SHIV-89.6. Twenty-four vaccinated animals were challenged with SHIV-89.6P. Only one of these failed to control the infection, and a second animal was lost to trauma during the third year postchallenge. Six controls were challenged. Five of these succumbed to AIDS within the first year postchallenge, whereas the sixth has survived. This study reports temporal viral loads, levels of CD4 T cells, antiviral CD8 and CD4 T-cell responses, and antibody responses up to the time of euthanasia at 200 weeks postchallenge. During this 4-year period, the vaccine successfully protected 11 A01 and 11 non-A01 macaques from viremia and loss of CD4 T cells. This long period of vaccine-mediated control was characterized by a remarkably stable and low-frequency antiviral IFN- T-cell response that consistently maintained the ability to coproduce IL-2.
MATERIALS AND METHODS
Immunogens. The construction and production of immunogens have been previously described (5). The DNA prime (DNA/89.6) expressed SIV Gag, Pol, Vif, Vpx, and Vpr and HIV-1 Env, Tat, and Rev from a single transcript. The rMVA booster (MVA/89.6) expressed SIV Gag and Pol and HIV-1 Env under the control of vaccinia virus early/late promoters (18).
Immunizations and challenge. Young adult rhesus macaques from the Yerkes breeding colony were cared for under guidelines established by the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals (5a) with protocols approved by the Emory University Institutional Animal Care and Use Committee. Animal numbers are as follows: 1, RBr-5; 2, RIm-5; 3, RQf-5; 4, RZe-5; 5, ROm-5; 6, RDm-5; 7, RAj-5; 8, RJi-5; 9, RAl-5; 10, RDe-5; 11, RAi-5; 12, RPr-5; 13, RKw-4; 14, RWz-5; 15, RGo-5; 16, RLp-4; 17, RWd-6; 18, RAt-5; 19, RPb-5; 20, RIi-5; 21, RIq-5; 22, RSp-4; 23, RSn-5; 24, RGd-6; 25, RMb-5; 26, RGy-5; 27, RUs-4; 28, RPm-5; 29, RPs-4; and 30, RKj-5. Animals with the A01 allele are indicated with asterisks. Four groups of six rhesus macaques each were primed at 0 and 8 weeks with either 2.5 mg (high dose) or 0.25 mg (low dose) of DNA by intradermal (i.d.) or intramuscular (i.m.) routes with a needleless jet injection device (Bioject, Portland, Oreg.). At 24 weeks, the MVA/89.6 booster immunization (2 x 108 PFU) was injected with a needle both i.d. and i.m. The control group included three mock-immunized animals and three naive animals. At 7 months after the rMVA booster was administered, animals received an intrarectal challenge with SHIV-89.6P, during which approximately 20 intrarectal infectious units (1.2 x 1010 copies of SHIV-89.6P viral RNA) was introduced 15 to 20 cm into the rectum by means of a pediatric feeding tube.
Quantification of SHIV copy number. SHIV copy numbers were determined by using a quantitative real-time PCR as previously described (5). Viral RNA was directly extracted by a MagNa Pure LC robotic workstation (Roche Molecular Biochemicals) with the MagNa Pure LC total nucleic acid isolation kit (Roche), and a reverse transcription reaction was set up in a Perkin-Elmer Applied Biosystems 7700 sequence detection system with the primer sequences 5'-GCAGAGGAGGAAATTACCCAGTAC-3' (forward) and 5'-CAATTTTACCCAGGCATTTAATGTT-3' (reverse) and the probe sequence 6-carboxyfluorescein (FAM)-5'-TGTCCACCTGCCATTAAGCCCGA-3'-6-carboxytetramethylrhodamine (TAMRA) within a conserved portion of the SIV gag gene.
Gag-CM9 tetramer analysis. Tetramer analyses were performed as previously described (5) by surface staining 100 μl of whole blood with antibodies to CD3 conjugated to fluorescein isothiocyanate (FN-18; Biosource International, Camarillo, Calif.), CD8 conjugated to peridinin chlorophyll protein (SK1; Becton Dickinson, San Jose, Calif.), and Gag-CM9 (CTPYDINQM)-Mamu-A01 tetramer conjugated to allophycocyanin. The tetramer-positive cells were represented as a percentage of CD8 T cells.
ICS assays. Intracellular cytokine (ICS) assays were performed as previously described by using four pools of Gag peptides (22-mers overlapping by 11) (5). Peripheral blood mononuclear cells (PBMCs; 106) were stimulated for 2 h at 37°C with 10 μg of peptide in a volume of 200 μl of RPMI with 0.2 μg each of anti-human CD28 and CD49d (Pharmingen, San Diego, Calif.). After 2 h, 800 μl of RPMI containing 10% fetal bovine serum and monensin (10 μg/ml) was added, and the cells were cultured overnight at 37°C. Cells were surface stained with antibodies to CD8 conjugated to peridinin chlorophyll protein (clone SK1; Becton Dickinson), fixed and permeabilized with Cytofix/Cytoperm solution (Pharmingen), and then stained with antibodies to human CD3 (clone FN-18; Biosource International), IFN- (clone B27; Pharmingen), and IL-2 (clone MQ1-17H12; Pharmingen) conjugated to fluorescein isothiocyanate, allophycocyanin, and phycoerythrin, respectively. About 150,000 lymphocytes were acquired on the FACScalibur (BD Biosciences, San Jose, Calif.) and analyzed with FloJo software (Treestar Inc., Ashland, Oreg.). Backgrounds for unstimulated cells were determined for each macaque for each analysis and subtracted from positive results. In general, backgrounds are much less than 0.01%, and macaques with higher backgrounds are excluded from studies. On the basis of our experience, responses with the correct pattern for IFN- responses and frequencies greater than 0.01% of total CD4 or CD8 T cells were considered positive. IL-2 production was scored only for cells coproducing IFN- and IL-2 because unstimulated cells had a high background for IL-2.
Epitope mapping. A two-step strategy was used to map epitopes in SHIV Gag and Env. In the first step, PBMCs from 23 vaccinated animals and the surviving control were screened by enzyme-linked immunospot (ELISPOT) for IFN- production using grids of 125 Gag and 211 Env peptides (15-mers with 11 overlapping amino acids) distributed in 23 Gag and 29 Env peptide pools (27). IFN- ELISPOT analyses were conducted as previously described by using 2 x 105 cells and peptide pools at a final concentration of 5 μg of each peptide/ml (5). Spot-forming units were normalized for 106 cells after subtracting 10 plus two times the average background in two negative control wells. Animals 9, 11, 16, and 19 had high background levels of spot-forming units, and the positive peptide pools could not be determined. In the second step, individual peptides from pools considered positive by ELISPOT were used in intracellular cytokine assays to determine the frequencies of IFN-- and IFN--and-IL-2-producing CD4 and CD8 T cells (see above). Consecutive positive peptides with 11 overlapping amino acids were considered a single epitope.
ELISA for binding antibodies. Enzyme-linked immunosorbent assays (ELISAs) for total antibody to Gag and Env were conducted as previously described (5). SIV Gag p27 produced in Escherichia coli and 89.6 Env produced in transiently transfected 293T cells and captured with sheep antibody against Env (International Enzymes, Fairbrook, Calif.) were used to coat wells. Standard curves were produced by coating with goat anti-monkey immunoglobulin G (IgG) Fc/7S (Accurate Chemical, Westbury, N.Y.) and capturing known amounts of rhesus IgG (Sigma, St. Louis, Mo.). Bound antibody was detected with peroxidase-conjugated goat anti-macaque IgG (Accurate Chemical, Westbury, N.Y.) and tetramethylbenzidine peroxidase substrate (507600; KPL, Gaithersburg, Md.).
RESULTS
Viral load and CD4 counts. All of the DNA/MVA-vaccinated animals became infected by the SHIV-89.6P challenge virus. However, within 9 weeks of the peak of postchallenge viremia, the viral load decreased to near or below 1,000 copies of viral RNA per ml of plasma in most animals (Fig. 1A). In one animal, animal 22 in the low-dose i.m. group, virus rapidly reemerged and the animal progressed to AIDS (Fig. 1C). In the other vaccinated animals, viremia continued to decline, with occasional low-level spikes of replicating virus (42). After the first year, these spikes were scored less frequently. At 200 weeks postchallenge, just prior to euthanasia, 17 animals had levels of viral RNA below our detection limit of 300 copies of viral RNA per ml of plasma (Fig. 1C). Among the remaining five animals, animals 4 and 5 had >1,000 copies of viral RNA per ml of plasma. These two animals, along with the surviving control, were not euthanized so that we could continue to monitor them for possible viral escape. Five of six unvaccinated control animals failed to control their viremia and died within 1 year postchallenge. The surviving unvaccinated animal, animal 30, slowly controlled its viral load, reducing the level of viremia to below 1,000 copies at about 1 year postchallenge. By 171 weeks (3.3 years) postchallenge, its level of viral RNA was undetectable. After challenge, the vaccinated animals maintained overall normal levels of CD4 cells (Fig. 1B). In contrast, all of the unvaccinated controls rapidly lost CD4 cells (Fig. 1D). Among the unvaccinated animals, the one survivor had the least CD4 loss and underwent a slow recovery of its CD4 cells.
CD8 T cells: Gag-CM9 tetramer-specific cells and IFN- response. Antiviral CD8 T-cell counts expanded markedly in response to the viral challenge but then contracted as the infection was controlled (Fig. 2A, B, D, and E). In A01 macaques, tetramer staining revealed 10- to 100-fold expansions in Gag-CM9-specific cells by 2 weeks postchallenge followed by a 5- to 10-fold contraction by 12 weeks postchallenge (Fig. 2A and D). The frequencies of these cells were then stable over the 200 weeks of control in all animals except macaque 20, which showed a slowly rising response. Overall, animal-to-animal differences in the frequencies, which ranged from 0.3 to 5.4% of total CD8 cells, reflected differences in their peak levels of tetramer-positive cells (Fig. 2D). In the unvaccinated A01 monkeys, the peak Gag-CM9 response was 10- to 100-fold lower than in the vaccinated animals (Fig. 2A and D).
Intracellular cytokine assays for CD8 responses to Gag also revealed high responses at 2 weeks postchallenge that then contracted (Fig. 2B and E). In contrast to the frequencies of tetramer-positive cells, which were stable after the initial contraction, the geometric means for the frequencies of IFN--producing CD8 T cells suggested an overall slow decline (Fig. 2A and B). At the peak response, geometric mean titers for anti-Gag CD8 T cells ranged from 1.4 to 6.2% of total CD8 T cells. At euthanasia, 200 weeks postchallenge, this geometric mean had decreased to 0.04 to 0.16% of total CD8 T cells. No significant differences were observed between groups.
CD4 T-cell responses. In contrast to the CD8 T-cell response that peaked at 2 weeks and then contracted, levels of antiviral CD4 T cells were low at 2 weeks but then rose over time (Fig. 2C and F). This result would be consistent with the loss of these cells due to infection during the postchallenge anamnestic response followed by a recovery once the infection had been controlled. At euthanasia, CD4 responses were slightly higher than at 5 weeks postchallenge and had geometric mean titers that ranged from 0.02 to 0.27% of total CD4 T cells. Some animals showed declines and rebounds in their antiviral CD4 responses. These rebounds and declines did not correlate with changes in CD8 T-cell responses or with detectable changes in viral loads. Again, no significant differences were observed between immunization groups.
Epitope mapping. At 140 weeks postchallenge (2.7 years), the IFN- responses of the vaccinated animals and the surviving control were mapped for CD8 and CD4 epitopes (Fig. 3 and Table 1 and 2). These assays revealed average CD8 responses to 1.4 to 2.3 epitopes per group and average CD4 responses to 1.3 to 1.8 epitopes per group. Animals 23 and 5 had the highest numbers of recognized epitopes, with three CD8 and five CD4 for animal 23 and three CD8 and four CD4 for animal 5. Animal 5 was one of the two animals that were spared euthanasia because of the presence of virus at over 1,000 copies per ml of plasma.
In total, the mapping revealed 18 CD8 epitopes and 18 CD4 epitopes residing throughout Gag and Env (Fig. 4). More epitopes were present in Gag than in Env. The Gag epitopes preferentially resided in p27. Sixty percent of the CD8 epitopes and 80% of the CD4 epitopes were present in p27, which represents 43% of Gag. Four of 10 A01 animals recognized peptides in Gag outside of those containing the immunodominant Gag-CM9 epitope (Table 1). Peptide 46 containing the Gag-CM9 epitope also encompassed a CD4 epitope recognized by 8 of 10 A01 monkeys and 1 non-A01 monkey. In Env, the CD8 and CD4 epitopes were distributed evenly across gp120 and gp41, with all CD8 epitopes and all but one CD4 epitope lying outside of the variable regions in Env.
IL-2 response. At the time of epitope mapping, the antiviral CD8 and CD4 responses in the vaccinated animals had good functional characteristics as measured by responding cells that coproduced both IFN- and IL-2 (Table 1 and 2 and Fig. 3). In the vaccinated groups, the average of the cells producing IFN- and IL-2 ranged from 35 to 45% of IFN--producing CD8 cells and from 26 to 49% of IFN--producing CD4 cells. In all, 34 out of 35 CD8 and 25 out of 30 CD4 responses coproduced IL-2. The animal with a CD8 IFN- response that did not coproduce IL-2 had two other CD8 responses, both of which coproduced IL-2 (Table 1, animal 5). The five IFN- CD4 responses that failed to coproduce IL-2 were distributed over five animals (animals 3, 17, 8, 10, and 23), with three of these animals having other CD4 responses that coproduced IL-2 (Table 2). In contrast, all four IFN- responses (two CD8 and two CD4 responses) in the unvaccinated survivor failed to coproduce IL-2.
Stability of the response. The responses to the mapped peptides were remarkably stable over the next year (Table 1 and 2). At 1 year after the initial ICS analyses (week 191), the mapped responses were tested again for IFN- and IL-2 production. Only 5 of the 34 tested CD8 responses and 7 of the 30 tested CD4 responses had declined to undetectable levels. Among the 29 remaining CD8 responses and 23 remaining CD4 responses, only 6 had changed in frequency by more than twofold. For the CD8 cells, the frequencies of IL-2-coproducing cells showed no change over twofold, except for monkey 10, where responses to peptide 46 increased from 0.01 to 0.04%. For the CD4 responses, there were five changes that were greater than twofold, with three of these changes being increases. In the unvaccinated control, the frequencies of three of four IFN- responses increased by more than twofold. These responses, in contrast to those measured at week 140, now showed low frequencies of IL-2-coproducing cells: 12.5% of the IFN--producing CD8 cells and 23% of the IFN--producing CD4 cells coproduced IL-2 (Table 1 and 2).
Comparison of the IFN- responses obtained for the individual peptides with those obtained for Gag pools in March 2004 suggested that there might have been an overall gain in CD4 but not CD8 epitopes. The CD8 responses elicited by the pools were not significantly different from those elicited by the peptides mapped in March 2003 (P = 0.13), whereas the CD4 responses elicited by the pools were significantly higher than those elicited by the mapped peptides (P = 0.005).
Antibody response. After the initial postchallenge anamnestic response, the levels of Gag and Env binding antibody declined by 5- to 10-fold (Fig. 5). In most of the animals, binding antibody then rose slowly over the next 3 years to levels ranging from 10 μg to 1 mg of IgG per ml of serum. The level of Gag or Env binding antibody fell below 10 μg per ml of serum in one animal in the high-dose i.d. group, three animals in the low-dose i.d. group, and two animals in the low-dose i.m. group. These decreases tended to be transient.
In most animals, the titers of neutralizing antibody increased rapidly between 2 and 12 weeks postchallenge from levels that were undetectable at the time of challenge to titers of 103 to 104 for 50% protection against virus-induced killing per ml of serum (Fig. 5C). With time, the levels of neutralizing antibody decreased. At the time of euthanasia, 18 of the 22 animals had titers of neutralizing antibody for SHIV-89.6P between 102 and 103 for 50% protection of MT-2 cells.
DISCUSSION
Here we report on the successful control of viremia in 22 DNA/MVA-vaccinated and SHIV-89.6P-challenged macaques (5). Control was effective up to the time of euthanasia at 200 weeks postchallenge, with all animals effectively maintaining their viremia at low to undetectable levels. CD4 T cells were maintained at normal levels, with no signs of progression to AIDS. Throughout the control period, there were occasional reemergent blips of virus. These blips were most frequent in the first year after challenge, a time when low levels of viral RNA and DNA were still declining (42). They were, however, still occurring at the time of euthanasia and served as a clear indication of the persisting presence of virus.
The signature of the successful control was a low-frequency, low-breadth IFN- T-cell response capable of coproducing IL-2. The importance of this low-level CD8 response to viral control was demonstrated by rapid viral reemergence in a parallel vaccine trial following CD8 T-cell depletion (Amara et al., submitted). A low-level T-cell response also was observed in the one surviving control, albeit this response showed more signs of exhaustion than that in the vaccinated animals. Presumably, the low-level IFN- CD8 T-cell responses reflected the low levels of persisting virus. Similar low levels of IFN- CD8 T cells are also found in successfully drug-treated humans (B. G. Kapogiannis, S. L. Henderson, P. Nigam, S. Sharma, L. Chennareddi, J. G. Herndon, H. L. Robinson, and R. R. Amara, submitted for publication).
The responses that were providing long-term control in the successfully vaccinated animals contrast with those found in HIV progressors and long-term nonprogressors who generally show greater breadths and higher frequencies of IFN--producing cells. A cohort of 23 chronic untreated patients that was mapped for the entire HIV-1 genome recognized a median of 18.5 CD8 epitopes, with a range of 8 to 42 (1). A long-term nonprogressor in this population recognized 42 epitopes. In another study in which ICS was conducted, the height of the IFN- CD8 response ranged from 0.5 to 18.8% of total CD8 T cells for chronic infections and from 5.3 to 8.8% of total CD8 T cells for long-term nonprogressors (32). Our results also contrast with the CD8 T-cell response in a supervised treatment interruption patient, where self-vaccination and then superinfection was associated with the recognition of 25 CD8 epitopes and 27,470 IFN- ELISPOTs per million PBMCs (3).
The long-term cellular response did not undergo the loss of IL-2 production, which is characteristic of infected patients and occurred in the one surviving control (22, 24, 37; Kapogiannis et al., submitted). IL-2 is critical for T-cell proliferation and survival (9), and the loss of IL-2 is the first indication of T-cell exhaustion (43). Coproduction of IL-2 and IFN- by antiviral CD8 T cells has been previously noted as a characteristic of successful vaccine-mediated control in macaques (30). The presence of the T cells that coproduced IFN- and IL-2 at the time of euthanasia is a strong indicator that the vaccine-elicited response was still capable of controlling the reemergent virus.
The long-term relationship between the low levels of residual virus and the IFN- cellular immune response was remarkably stable. However, changes could be detected when individual epitopes were mapped. Between 140 and 191 weeks postchallenge, 5 of 35 CD8 epitopes and 7 of 30 CD4 epitopes declined to frequencies below detection. Whether these changes were due to escape or exhaustion is not known, because viral sequences were below the level that we could recover by PCR.
The frequency of Gag-CM9 tetramer-positive cells was 5- to 10-fold higher than the frequency of peptide-stimulated IFN--producing cells. This was true whether stimulations were with the exact Gag-CM9 peptide or with the 15-mer Gag 46 peptide that contains the Gag-CM9 epitope. Tetramer staining provides a marker for the total population of CD8 T cells recognizing an epitope, whereas our ICS assays reveal only those cells capable of producing IFN- or IFN- and IL-2 in response to peptide stimulation. The small percentage (10 to 20%) of tetramer-positive cells capable of producing IFN- following peptide stimulation in the chronic phase of control contrasted with the peak response, where 50 to 100% of tetramer-positive cells produced IFN- with peptide stimulation. However, in the long chronic period of control, cells coproducing IFN- and IL-2 marked a much larger percentage of IFN--producing cells (20 to 60%) than at peak response (<20%).
Despite low to undetectable levels of viremia and a largely quiescent T-cell response, the antiviral antibody response remained high during the long period of chronic control. The persisting antibody included neutralizing antibody, which undoubtedly contributed to both the control of the infection and the protection of antiviral as well as total CD4 T cells. The 89.6 Env used in our immunizations does not elicit cross-reactive neutralizing antibody for the 89.6P Env in the challenge virus (33). Thus, the protective responses primed by this Env did not depend on the presence of neutralizing antibody at the time of challenge, a phenomenon that has been seen in other SHIV-89.6P vaccine trials using heterologous Envs (4, 15, 29).
Six animals showed declines in Gag and Env antibody. In four of these animals, antibody rebounded, presumably in response to changes in the activity of the persisting virus. These rebounds occurred in the absence of our detecting reemergent virus. Thus, antiviral antibody may provide a more sensitive indicator of the presence of virus than plasma viral RNA, a phenomenon that will likely be true in human trials with well-controlled infections.
Encouragingly, the DNA/MVA vaccine provided excellent protection against a SHIV-89.6P challenge for both A01 and non-A01 macaques. In another study using our Gag-Pol-Env DNA/MVA vaccine, seven additional non-A01 macaques were protected (12). For adeno5-vectored vaccines, protection against SHIV-89.6P has been much more uniform for A01 macaques than for non-A01 macaques (25, 29). Both the MVA and adeno5 vectors elicit high frequencies of responding CD8 T cells during the acute phase of immunization (41). However, the adeno5-based vectors induce higher frequencies of persisting CD8 T cells (13, 14, 41) and may induce a higher ratio of CD8-to-CD4 responses than MVA-vectored immunizations. Thus, the immune responses elicited by the two vectors have different characteristics, which could result in different protective capacities. However, other experimental differences could account for the difference in protective responses (5, 25, 29).
This study in the SHIV model provides continuing hope that a Gag-Pol-Env DNA/MVA vaccine could help control the HIV/AIDS pandemic and suggests that a signature for successful postchallenge control will be a stable, low-frequency, low-breadth, IFN-- plus IL-2-coproducing CD8 and CD4 T-cell response. We suggest that the low levels of the IFN- T-cell response during successful long-term control reflect the relative dormancy of the infection. We also suggest that the presence of IL-2 production in the responding cells provides an indicator that the T-cell response has not been exhausted by persisting antigen and will be capable of controlling reemergent virus.
ACKNOWLEDGMENTS
We are indebted to Research Resources and the veterinary and animal care technicians of the Yerkes National Primate Research Center for their invaluable care and attention to the animals in this trial. We are also indebted to Helen Drake Perrow for expert administrative assistance, to Vanda Bostik, Sunita Sharma, Pragati Nigam, and Lazarus Ofielu for their excellent technical help, to Lakshmi Chennareddi for her help in the statistical analyses, and to Francois Villinger and Bill Kapogiannis for their critiques and suggestions.
This work was supported by National Public Health Service Integrated Preclinical/Clinical AIDS Vaccine Development grant P01-AI 43045, the Emory/Atlanta Center for AIDS Research P30 DA 12121, and the Yerkes National Primate Research Center base grant P51 RR000165.
R.R.A., L.S.W., B.M., and H.L.R. have pending patents for HIV vaccines modeled on the SHIV vaccine reported in this study. H.L.R. also holds a minor equity interest in GeoVax Inc., the company that has licensed the technology.
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