The relative contribution of neutralizing antibodies (nAbs) and MHC-restricted cytotoxic T lymphocyte (CTL) responses to overall immune protection against HIV-1 infection is not clear at this point. However, it is generally considered from evidence of HIV-1-infected, long-term non-progressors or uninfected individuals, that generating both would be most beneficial [1–6]. As models for vaccine development against AIDS, the challenge of macaques with simian immunodeficiency virus (SIV) or chimeric SHIV constructs approximates HIV-1 infection in humans. Pathogenic strains of SHIV cause loss of CD4+ T cells, albeit more rapidly than HIV-1-induced loss in humans, and viral infection is dependent on HIV-1 envelope glycoproteins [7,8]. Direct evidence for the role of CD8+ T cells in protection was recently shown by anti-CD8 antibody treatment of pathogenic SIV-infected macaques. Depletion of CD8+ T cells during primary or chronic infection resulted in increased viral loads and more rapid disease progression [9–11].
For this study, we used rhesus macaques that previously were enrolled in a pilot study to assess the safety and immunogenicity of a DNA prime/gp160 boost vaccination protocol in neonates . These vaccinees were then sequentially challenged with non-pathogenic and pathogenic strains of SHIV. A summary of the vaccinations and virus challenges for the five animals enrolled in this study is shown in Table 1 and Figure 1. The first two virus challenges were with non-pathogenic SHIV-vpu+, a chimeric virus containing tat, rev, vpu and env of the HIV-IIIB laboratory isolate within a SIVmac239 backbone [13,14]. All five animals had evidence of viral containment and only two animals, RDt-5 and RMw-5, seroconverted and developed anti-Gag antibodies as detected by Western blot analysis. However, sterilizing immunity was not observed in any animal. The five animals were then challenged with pathogenic, heterologous SHIV89.6P [7,8]. Two animals (RDw-5 and RDt-5) had a peak of low-level SHIV-vpu+ viremia, but no evidence of SHIV89.6P infection. Two other animals, RFw-5 and RMw-5, became virus positive for both SHIV-vpu+ and SHIV89.6P. All four animals were protected from the dramatic CD4+ T-cell loss seen in unvaccinated animals. Animal RGt-5 also became positive for both SHIV strains and had a drop in CD4+ T cells that slowly recovered. By 8 weeks after the SHIV89.6P challenge, all animals had anti-Gag antibodies, indicating that viral infection was strong enough and of sufficient duration to cause seroconversion. In conclusion, the vaccinated animals were partially protected from high peak viral loads and completely protected from severe CD4+ T-cell loss.
To directly test the role of CD8+ T lymphocytes in the observed protection from high virus loads and disease, we treated these monkeys with anti-CD8 mAb. A rapid drop of peripheral blood CD8+ T-cell counts was accompanied by a sharp rise in plasma viremia and cell-associated virus. Additional data suggest a secondary protective mechanism acted to contain both SHIV89.6P proliferation from reaching pathogenic levels in three animals and high level viremia in the other two monkeys. The possibility of nAbs contributing to this secondary mechanism of protection is discussed.
Materials and methods
Rhesus monkeys (Macaca mulatta) were housed at the Yerkes Regional Primate Research Center (YRPRC), Atlanta GA, a facility fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All procedures were approved by the Animal Care and Use Committees of Emory University and the Dana-Farber Cancer Institute.
Anti-CD8 antibody administration
cM-T807, a mouse-human chimeric anti-CD8 mAb, was administered according to an optimal schedule and dosage determined previously [11,15]. Monoclonal antibody adminstration was initiated 5 months after SHIV89.6P challenge for all animals except RGt-5 which was given mAb 9 months post-SHIV89.6P challenge. Monkeys were given cM-T807 at 5 mg/kg body weight on day 0 subcutaneously. The mAb was administered intravenously on days 3 and 7 at the same dose.
Enumeration of T-cell subsets
CD8+ T-cell levels in whole blood were measured by four-color flow cytometry as described previously  with CD8-PE (DK25; Dako, Carpinteria, California, USA), CD4-FITC (OKT4; Ortho Diagnostics Systems, Raritan, New Jersey, USA), CD20-ECD (B1; Beckman Coulter, Miami, Florida, USA) and CD3-APC (FN18). DK25 is able to bind to CD8 in the presence of cM-T807. Absolute CD4+ and CD4+4B4+ T-cell values in peripheral blood were measured separately as described .
Measurement of viral load
Blood samples were analyzed by quantitative TaqManEZ reverse-transcriptase-polymerase chain reaction (RT-PCR), peripheral blood mononuclear cell (PBMC) cocultivation assays, DNA PCR, and RT-PCR as described [12,17,18]. In brief, for TaqManEZ RT-PCR analysis of plasma, RNA was extracted from sodium citrate-anticoagulated plasma with QIAamp Viral RNA Mini kits (Qiagen, Valencia, California, USA). RT-PCR was performed as a one-tube, one-enzyme assay with the TaqMan EZ RT-PCR kit (PE Biosystems, Foster City, California, USA) and run on an ABI Prism 7700 Sequence Detection System (PE Biosystems). The primers and probe used amplified a 92 bp fragment in the SIV gag region. The lower limit of detection of the assay was 100 RNA copies per ml plasma.
For DNA PCR, high molecular weight genomic DNA was isolated from frozen monkey PBMC using DNAzol (Molecular Research Center, Cincinnati, Ohio, USA) under the manufacturer's recommended conditions. To distinguish between SHIV-vpu+ and SHIV89.6P proviral DNA, specific oligonucleotides spanning the 140-bp deletion in the env gene of SHIV89.6P [7,8] were employed. One microgram of genomic PBMC DNA was amplified with primers 8329 to 8353 (5′-GGCAAGTTTGTGGAATTGG TTTGA-3′) and 8903 to 8926 (5′-CCTTGTCT AATCCTCCTGGGGATT-3′) (GeneBank; Accession number: U89134). Sensitivity of the assay was 1 proviral copy/1 μg of genomic DNA. The PCR product for the env sequences of SHIV-IIIB (homologous to SHIV-vpu+) and for SHIV89.6P are 498 and 358 bp, respectively. In order to detect the SHIV89.6P provirus in monkey PBMC DNA samples containing high numbers of SHIV-vpu+ proviral copies, a SHIV89.6P-specific DNA PCR assay was employed. Genomic PBMC DNA (1.0 μg) was amplified with primers 8164 to 8195 (5′-ATGTTAGTTGGAGTAA TAAATCTGTGGATGAT-3′) and 8918 to 8948 (5′-TCACAAGAGAGTGAGCTCAAGCCCTTGTCT-3′). Then, 1 μl of amplicons was amplified by second-round (nested) PCR with primers 8546 – 8575 (5′-AGAGAGAGACAGAGACAGATCCGGTCCATC-3′) and 8875 to 8904 (5′-TTGGCAGTATCCATCTTC CACCTCTGCTAA-3′). The specificity of this PCR for a unique SHIV89.6P env sequence was demonstrated previously using PBMC from SHIV-vpu+ and SHIV89.6P-inoculated control animals . The sensitivity of both DNA PCR assays was 1 proviral copy/1 μg of genomic DNA (approximately equivalent to 150 000 cells).
RT-PCR was used to confirm the differential SHIV identification in cM-T807-treated animals. RNA was extracted using TRIZOL LS reagent (Life Technologies, Gaithersberg, Maryland, USA) and reverse transcription was performed using a kit from Promega (Madison, Wisconsin, USA). The cDNA synthesis reaction product was diluted as noted in the corresponding figure legend and the PCR reactions were performed as described above for both differential SHIV-vpu+/SHIV89.6P identification and for SHIV89.6P-specific identification.
For co-cultivation analysis, PBMC from each animal were serially diluted and cultured in duplicate in the presence of CEMx174/GFP cells, a B-cell/T-cell hybrid cell line expressing green fluorescent protein under transcriptional control of the HIV-2 terminal repeat element. After 21 days, plates were scored for fluorescent cell-positive wells. The p27 Gag antigen in culture supernatants was also determined using a commercial ELISA kit (Coulter, Miami, Florida, USA) to confirm results.
Neutralizing antibody measurements
Antibody-mediated neutralization of SHIV-HXBc2 (homologous to SHIV-vpu+) and SHIV89.6P was assessed in an MT-2 cell killing assay as described [19,20]. Briefly, 50 μl of cell-free virus containing 500 TCID50 (dose giving 50% infectivity in tissue culture) were added to multiple dilutions of test serum in 100 μl of growth medium (RPMI-1640 containing 12% fetal bovine serum and 50 μg gentamicin/ml) in triplicate in 96-well culture plates. The mixtures were incubated for 1 h at 37°C followed by the addition of MT-2 cells (5 × 104 cells in 100 μl) to each well. Infection led to extensive syncytium formation and virus-induced cell killing in approximately 4–6 days in the absence of antibodies. Neutralization was measured by staining viable cells with Finter's neutral red in poly-l-lysine-coated plates. The percentage protection was determined by calculating the difference in absorption (A540) between test wells (cells + serum sample + virus) and virus control wells (cells + virus), dividing this result by the difference in absorption between cell control wells (cells only) and virus control wells, and multiplying by 100. Neutralization was measured at a time when virus-induced cell killing in virus control wells was greater than 70% but less than 100%. Neutralization titers are given as the reciprocal dilution required to protect 50% of cells from virus-induced killing. Cell-free stocks of SHIV-HXBc2 and SHIV89.6P were prepared in H9 cells and human PBMC, respectively.
SIVmac-specific CTL were measured in MHC Class I Mamu-A*01+ animals, RDt-5 and RDw-5, by tetramer staining as described [11,21,22]. Fluorochrome-labeled Mamu-A*01/SIV Gag peptide (p11C, C-M) tetramers were used to stain PBMC at indicated time points, and the results show the percentage of CD8αβ+ (2ST8-5H7; Beckman Coulter) T lymphocytes that bound tetramer.
Anti-CD8 treatment induces a loss of CD8+ T cells and a rise in viremia
After anti-CD8 mAb treatment, CD8+ T-cell levels in peripheral blood dropped from a pretreatment range of 23–47% to < 1% for four of the five animals by day 7 and for all five animals by day 14 (Fig. 2). CD8+ T cells reappeared after day 14 and returned to near normal levels. The degree and rapidity of the CD8+ T-cell decline and recovery were comparable with previous findings using the cM-T807 mAb at identical dose-schedules, in which a near total depletion of CD8+ T cells in peripheral lymph nodes also occurred [11,15]. The animals served as their own controls; in previous CD8+ T-cell depletion studies, administration of an unrelated control mAb had no effect on CD8+ T cell or virus levels in SIVmac251-infected monkeys . Coincident with the decline in CD8+ T cells was an increase in plasma viremia and infectious PBMC within all five animals (Fig. 2, Table 2). Peak viremia was observed at day 14 post cM-T807 treatment in monkeys RFw-5 and RMw-5. More frequent viral load measurements were precluded by an emphasis on frequent CD8+ T-cell measurements and day-to-day fluctuations in viremia may have occurred. However, over the time points measured, viremia rose no higher after day 7 in monkey RDw-5, and in RDt-5 and RGt-5, viremia was highest on day 7 and started to decline despite no observable increase in CD8+ T-cell levels in peripheral blood. In addition, cell-associated virus levels in all animals were at least 10–500 fold lower than the lowest estimated average levels in separate groups of naive virus-challenged control monkeys (Table 2). This large difference in cytoviremia between the cM-T807-treated vaccinees and naive, SHIV89.6P-challenged controls was also reflected in large differences in viral RNA levels [107, 1.5 × 106 and 7.8 × 104 copies/ml for animals RFw-5, RMw-5 and RGt-5, respectively (Fig. 2b), versus 2.3 × 107– 8.4 × 107 copies/ml for four naive controls (data not shown)].
Only single SHIV isolates are detected in cM-T807-treated animals
As mentioned, the cM-T807 mAb-treated monkeys were previously exposed to two heterologous strains of SHIV through sequential challenges. It was, therefore, important to determine which one of the two virus strains predominated in each animal during the peak of viremia following anti-CD8 mAb treatment. The viral RNA levels (Fig. 2) during the time of CD8+ T-cell depletion were measured by an RT-PCR assay that does not discriminate between the two SHIV strains. Therefore, a differential DNA PCR assay was used to measure proviral DNA of each SHIV strain in monkey PBMC. Animals RDw-5 and RDt-5 had no evidence of SHIV89.6P proviral DNA following anti-CD8 mAb treatment, as only SHIV-vpu+ proviral DNA was detected (Fig. 3a, b). In contrast, only SHIV89.6P provirus was detected in monkeys RFw-5, RMw-5, and RGt-5. Although an increase in SHIV-vpu+ replication with anti-CD8 mAb treatment in these three monkeys cannot be completely ruled out, based on the sensitivity of the DNA PCR assays, SHIV-vpu+ levels were at least 10-fold lower than SHIV89.6P levels (data not shown). These differential diagnostic DNA PCR data were confirmed by endpoint titration of RT-PCR products from plasma samples at the time of peak virus titers (Fig. 3c, d).
cM-T807-treated macaques show loss of SIV Gag-specific CTL
Coincidentally, the two antibody-treated monkeys that showed no evidence of SHIV89.6P infection expressed the rhesus major histocompatibility complex (MHC) class I allele Mamu A*01. SIV-specific CTL in these monkeys were enumerated using Mamu A*01/viral peptide tetramers [11,21,22]. In both monkeys, SIV-mac-specific CTL were present at low, yet significant levels prior to cM-T807 treatment (Table 3). Upon reappearance of CD8+ T cells and coincident with the drop in viral RNA loads, the percentage of SIVmac-specific CTL rose to levels six to twelve times higher than at the start of cM-T807 treatment. These results suggest that CD8+ CTL activity contributed to the subsequent drop of viremia associated with the reappearance of CD8+ cells in cM-T807-treated monkeys.
Animals show no loss of CD4+ T cells despite a rise of pathogenic SHIV levels
SHIV89.6P infection of naive macaques causes a rapid loss of CD4+ T cells from peripheral blood within 2 weeks following exposure (see Fig. 4b, naive controls and [7,8]). However, despite previous exposure to SHIV89.6P and the increase of virus titers following anti-CD8 mAb treatment, no animal in this study exhibited an appreciable decrease in CD4+ T cells (Fig. 4). This finding was of particular interest for animal RGt-5. This monkey had a pronounced drop in CD4+ T-cell counts when first challenged with SHIV89.6P (Fig 5; due to non-specific staining for CD4 alone at a single time point after SHIV89.6P challenge, only CD4+4B4+ memory T-cell numbers are shown). CD4+ T lymphocytes slowly recovered to levels of about 500/μl of blood. Despite cM-T807 treatment and a rapid drop of CD8+ T-cell numbers, RGt-5 maintained absolute numbers of CD4+ T cells between 180 and 465/μl of blood.
High levels of neutralizing antibodies coincide with signs of supplemental control of viremia
Three observations suggested that the experimental animals utilized a protective mechanism distinct from CD8+ T-cell-mediated control over virus replication during the time of CD8+ T lymphocyte depletion: (1) the cell-associated virus levels were much lower in all animals compared with levels in virus-challenged, naive control monkeys; (2) the decline in viremia after day 7 and before the reappearance of CD8+ T cells in animals RDw-5, RDt-5, and RGt-5; and (3) the finding that there was no drop in CD4+ T cells in monkeys RFw-5, RMw-5, and RGt-5 despite a clear rise in levels of pathogenic SHIV89.6P RNA. To determine whether nAb might have played a role in the control of viremia in the absence of CD8+ T-cell immunity, titers of nAb in all five monkeys were measured against both SHIV-IIIB (homologous to SHIV-vpu+) and SHIV89.6P (Table 3). RDw-5 and RDt-5 had strong nAb responses to SHIV-IIIB at the start of cM-T807 treatment. Neutralizing antibody titers quickly rose 12 to 15 times during the time of increasing viremia and CD8+ T-cell depletion. In accordance with the inability to detect SHIV89.6P in RDw-5 and RDt-5 by PCR analysis, no nAb activity to SHIV89.6P was detected in the sera of these monkeys. The two monkeys that were SHIV89.6P-positive yet maintained normal CD4+ T-cell counts, RFw-5 and RMw-5, had significant anti-SHIV89.6P nAb titers at the start of cM-T807 administration. Animal RFw-5 had very high titers that slightly diminished during the rise and subsequent fall of viremia. Animal RMw-5 showed an anamnestic 15-fold rise in nAb titers to SHIV89.6P in the 2 weeks immediately after the start of anti-CD8 mAb treatment. Neither monkey exhibited any change in nAb responses to SHIV-vpu+, in agreement with our inability to detect recurrent SHIV-vpu+ viremia in these animals that would have provided a possible anamnestic boost to antibody titers. The third SHIV89.6P positive monkey, RGt-5, had a similar anamnestic nAb response to SHIV89.6P that peaked 2 weeks following the start of cM-T807 treatment. Neutralizing antibodies to SHIV-IIIB, present at the start of anti-CD8 mAb treatment, became undetectable in this animal.
The results of this study clearly show the importance of CD8+ T cells as a primary mechanism for the control of viremia in SHIV-infected macaques. The transient rise in viral RNA loads that coincided with CD8+ T-cell depletion resembled the pattern reported previously in SIVmac251-infected monkeys [10,11]. Thus, there was a sharp drop in CD8+ T cells upon the administration of anti-CD8 mAb and a corresponding rise in plasma viral RNA levels. With the reappearance of CD8+ T cells, there was a coincident drop in viral RNA loads. However, in contrast to studies using chronically SIV-infected animals, the monkeys in this study were previously shown to have contained virus infection, at least partially, following a series of homologous and heterologous SHIV challenges subsequent to DNA prime/protein boost vaccination. Here, we also provide evidence suggesting the role of a secondary control mechanism that limited the spread of infectious, pathogenic SHIV89.6P in the absence of CD8+ T-cell protection. The presence of high titer nAbs coincided with this apparent secondary means of immune protection.
Until now, the relative importance of CD8+ T lymphocyte immunity and nAb titers to protection from lentiviral infection and pathogenicity has not been clear. For this study, we enrolled a set of animals that were positive for pre-existing nAb. By effectively removing the virus-specific CD8+ T-cell immunity through mAb cM-T807 administration, the possible contribution of neutralizing antibodies to the control of viremia could be assessed as an isolated parameter. Neutralizing antibodies in the animals were first detected following either the first or second gp160 inoculation of the pilot vaccination protocol described; priming inoculation with DNA alone failed to elicit nAb . The initial nAb reactivity in all five was directed solely against homologous SHIVIIIB Env. SHIV89.6P-reactive nAb was detected only after the heterologous SHIV89.6P rechallenge and was seen by week 8 post-rechallenge in the animals that became SHIV89.6P positive. As such, upon CD8+ T-cell depletion, a likely contribution of nAbs as an additional control mechanism became apparent in several animals. Monkeys RDt-5 and RDw-5 were both SHIV89.6P negative but had rises in SHIV-vpu+ viral RNA and developed strong anamnestic nAb responses to the virus upon CD8+ T-cell depletion. The rise in nAb titers coincided with a leveling off (RDw-5) or an actual decline in viral RNA measurements (RDt-5), despite undetectable CD8+ T-cell levels on day 7 and day 14. Moreover, cell-associated virus levels in these animals remained low relative to the observed levels of cell-associated virus in naïve animals challenged with the same virus stocks (Table 2).
Among the three vaccinated monkeys with recurrent SHIV89.6P viremia during CD8+ T-cell ablation, one (RGt-5) also showed an early drop of viral RNA levels after day 7, even in the absence of CD8+ T-cell recovery. The possible protective role of nAbs was especially notable in this animal, in which the susceptibility to SHIV89.6P pathogenesis and CD4+ T-cell loss was observed when the animal was first challenged with heterologous SHIV89.6P previously. When CD4+ T-cell levels were at their nadir after this first SHIV89.6P challenge, RGt-5 had a low nAb titer to SHIV89.6P of only 29, identical to the measured titer at the start of cM-T807 treatment. This nAb level was apparently too low to offer protection against CD4+ T-cell loss after this first challenge. Following cM-T807 treatment, however, nAb levels quickly rose to high levels in an anamnestic response. As a consequence, the recurrent SHIV89.6P viremia did not cause CD4+ T-cell loss. These results suggest that humoral immunity kept SHIV89.6P loads below pathogenic thresholds in animal RGt-5 .
In contrast to the pattern observed for RGt-5, plasma viral RNA levels in the other two animals with recurrent SHIV89.6P viremia (RFw-5 and RMw-5) continued to rise after day 7 of cM-T807 treatment. However, in neither animal did viremia reach a pathogenic threshold level . Thus, CD4+ T-cell counts remained normal and again, there was only a limited rise in the numbers of infectious PBMC (Table 2). Neutralizing antibody titers to SHIV89.6P were high in both monkeys. Although it is unclear in these cases why the strong nAb responses in each animal did not halt the rise in viral RNA levels, it is possible that the observed increase is due to nAb escape variants of SHIV89.6P . Sufficient time had elapsed (6–9 months) from the first challenge with SHIV89.6P for the virus in those animals to escape antibodies that initially neutralized the parental challenge virus. Neutralizing antibody assays using virus isolated at the time of CD8+ T-cell depletion would provide clearer evidence of a more definitive role of nAb as a secondary mechanism for the containment of virus replication. Unfortunately, multiple attempts by two separate laboratories were unsuccessful at isolating virus from serum or plasma samples during this period.
In all animals, the rise in the number of infectious PBMC upon CD8+ T-cell depletion was severely limited relative to the numbers seen in naive animals challenged with identical virus stocks. However, we did not see as strong a containment of plasma viral RNA levels. One possibility for this difference is that the measured viral RNA levels represent both infectious and non-infectious viral particles. Thus, in the absence of immune surveillance by antiviral CD8+ T cells, existing cells harboring proviral DNA can produce virus but nAb can cover the particles and prevent widespread infection of new target cells. Our unsuccessful attempts to isolate infectious virus from serum or plasma at times of peak viral RNA support this idea, and the low levels of infectious PBMC we observed are consistent with this role of nAb to contain the spread of infection.
In summary, our study suggests that both CD8+ T cells and nAb are important in the control of viremia and disease in chronic lentiviral infection. We postulate that immune surveillance by specific antiviral CD8+ T cells eliminates virus-producing cells and thus is primarily responsible for the low levels of viral RNA observed during the chronic phase of infection in our animals. Here we provide evidence to suggest that virus-specific nAb form a second line of defense to keep infectious virus levels from reaching pathogenic thresholds in the absence of protective CD8+ T-cell surveillance.
We thank S. Sharp and C. Gallegos for the preparation of this manuscript and Centocor for permitting the use of cM-T807 in these studies.
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Keywords:© 2002 Lippincott Williams & Wilkins, Inc.
primate; neutralization; antibodies; CD8+ T cells; cellular immunity; viral infections