Elicitation of virus-specific humoral immune responses, with their strong CD4+ and CD8+ T-cell immune responses, are critical to good control of HIV-1 [1,2]. Although recent vaccine candidates based on active immunization are intended to stimulate CD4+ and CD8+ T-cell responses, induction of broadly neutralizing antibodies by active immunization has been limited to date [3,4]. In contrast, passive immunization with neutralizing antibody effectively induced sterilizing immunity by preventing the establishment of chronic infection. We and others have reported that chimpanzees can be protected against acute infection with the T-cell line-adapted strain HIV-1IIIB by passive transfer of a mouse–human chimeric anti-HIV-1 V3 mAb . Furthermore, we produced a high-affinity cross-neutralizing humanized mAb, KD-247, by sequential immunization with peptides derived from the V3 region of HIV-1 clade B primary isolates and found that KD-247 yields sterile protection of monkeys against the highly pathogenic simian/human immunodeficiency virus (SHIV) [6,7]. KD-247 is thus considered a promising new immunotherapeutic agent for HIV-1-infected patients .
It was demonstrated that intensive, short-term postinfection therapy with neutralizing immunoglobulin G (IgG) against simian immunodeficiency virus (SIV) can have long-term beneficial effects on disease in a pathogenic primate lentivirus model . Passive transfer of neutralizing antibodies also conferred postinfection prophylaxis against pathogenic SHIVs in macaques [10,11]. Furthermore, passive immunization of pregnant or neonatal monkeys with combinations of mAbs has been reported to completely or partially neutralize SHIV in animal models of mother-to-child transmission of HIV [12,13]. However, whether neutralizing antibody plays a significant role in controlling established HIV infection is unclear. The current aim of antiretroviral therapy remains the maintenance of plasma HIV-1 RNA levels below the limit of detection . In a clinical trial, three passively transferred mAbs, 2G12, 2F5, and 4E10, were shown to delay the rebound of HIV-1 after cessation of antiretroviral therapy; the delay was particularly pronounced in acutely infected individuals . In this study, we evaluated the postinfection effect of KD-247 against CD4+ T-cell loss and increased viral loads in the SHIV model.
Materials and methods
Preparation of KD-247
A high-affinity humanized mAb, KD-247 [Chemical Abstracts Service (CAS) Registry Number: 914257-21-9], was prepared as previously described . Briefly, the mouse mAb C25 was elicited by immunization with six synthetic peptides derived from the V3 region of HIV-1 primary isolates. The complementary-determining regions and partial framework regions of C25 were transferred into the variable region of human IgG. Cells producing the humanized C25, KD-247, were expanded in large-scale culture, and the antibody was purified from the culture supernatants by ion exchange and affinity chromatography.
Pathogenic simian/human immunodeficiency virus challenge to monkeys and postinfection transfer of KD-247
All animals used in this study were mature, male cynomolgus monkeys (Macaca fascicularis) from the Tsukuba Primate Center, the National Institute of Infectious Diseases (NIID) (currently known as the Tsukuba Primate Research Center, National Institute of Biomedical Innovation), Japan. They were housed in accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science, 1987, under the Japanese Law Concerning the Protection and Management of Animals, and were maintained in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of NIID, Japan.
The pathogenic chimeric SHIV-C2/1 is an SHIV-89.6 variant isolated by in-vivo passage in cynomolgus monkeys . Cynomolgus monkeys injected intravenously with SHIV-C2/1 exhibited high levels of viremia and marked CD4+ T-cell depletion within 2 weeks after challenge [16,17]. Six naive monkeys were intravenously inoculated with 20 50% tissue culture infective dose (TCID50) of SHIV-C2/1 and were then given 45 mg/kg weight of KD-247 at 1 h (Cy-1 and Cy-2), 1 day (Cy-3 and Cy-4), or 1 week (Cy-5 and Cy-6) after viral challenge; a single preinfection administration of the mAb at this dosage had exhibited sterile protection against SHIV-C2/1 infection . Two control monkeys (Cy-7 and Cy-8) received 45 mg/kg of purified human normal immunoglobulin (control IgG; Nihon Pharmaceutical, Tokyo, Japan) instead of KD-247 at 1 day after viral challenge. Additional multiple (seven or eight) administrations of the same concentrations of KD-247 or control IgG were given weekly from day 7 for a period of 2.5–3 months. Blood samples were drawn to examine the plasma concentrations of KD-247, SHIV RNA copy numbers, and CD4+ T-cell counts. At approximately 11–13 weeks after viral challenge, necropsies were performed and histological examination and flow cytometric analyses of lymphoid organs were conducted. The schedules of KD-247 administration, blood drawing, and necropsy are shown in Fig. 1(a).
Plasma concentration of KD-247
KD-247 concentrations in macaque plasma were measured by ELISA. Ninety-six-well ELISA plates (MaxiSorp, Nunc A/S, Roskilde, Denmark) were coated with a KD-247-specific antigen, SP13 peptide (GPGRAFGPGRAFGPGRAFC). After blocking and washing, monkey plasma at appropriate dilutions was added and the plates incubated. KD-247 was diluted to concentrations ranging from 2.5 to 40 ng/ml and used as a reference. The wells were washed and then incubated with a detection antibody solution consisting of peroxidase-conjugated antihuman IgG mAb (Kaketsuken, in-house preparation). After final washes, peroxidase substrate was added and the reaction was stopped. The plates were measured for optical density at 450 nm with a precision microplate reader (Emax; Molecular Devices, Menlo Park, California, USA). The concentrations of KD-247 antibody in the plasma were evaluated from a calibration curve drawn with software developed for the reader (SOFTmax; Molecular Devices).
Detection of anti-KD-247 antibodies
Anti-KD-247 antibodies in plasma were detected using 96-well ELISA plates (MaxiSorp) coated with KD-247. After washing and blocking, samples containing test monkey plasma at 1: 400 dilution or a positive control were then added and incubated. A positive control was pooled with rabbit anti-KD-247 plasma at 1: 4000 dilution. The wells were washed, incubated with biotinylated KD-247, and then washed again. Peroxidase-conjugated streptavidin (Sigma Chemical, St. Louis, Missouri, USA) was diluted and added to the wells for reaction.
Real-time reverse transcriptase-PCR quantitation of simian/human immunodeficiency virus RNA in plasma
Plasma viral loads were evaluated by real-time reverse transcriptase PCR (RT-PCR) as described previously [17,18]. Viral RNA in plasma was extracted and purified using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, California, USA). For quantitative analysis of the RNA, the TaqMan system (Applied Biosystems, Foster City, California, USA) was used with primers and probes targeting the SIVmac239 gag region. The viral RNA was amplified using TaqMan EZ RT-PCR Kit (Applied Biosystems) with primers. The RT-PCR product was quantitatively monitored by its fluorescent intensity with ABI7700 (Applied Biosystems). Plasma viral load, which was measured in duplicate, was calculated based on the standard curve of control RNA and RNA recovery rate. The limit of detection was approximately 500 RNA copies/ml.
Flow cytometric evaluation of cell surface antigen expression and absolute cell count
Lymphoid cells for flow cytometric analyses were prepared from intact thymuses, spleens, and lymph nodes. Mouse mAbs conjugated with either fluorescein isothiocyanate (FITC), phycoerythrin, phycoerythrin-Cy5, or peridinin chlorophyll protein (PerCP) were used in flow cytometric analyses to detect cellular expression of monkey CD3 (NF-18; BioSource International, Camarillo, California, USA), human CD4 (SK3; Becton Dickinson, San Jose, California, USA), and CD8 (SK1; Becton Dickinson). To determine absolute cell counts, samples of whole blood were analyzed following the addition of FITC-conjugated anti-CD3 (BioSource), phycoerythrin-conjugated anti-CD4 (Becton Dickinson), and PerCP-conjugated anti-CD8 mAbs (Becton Dickinson), as previously described .
KD-247 concentrations and detection of anti-KD-247 antibodies in monkey plasma
Blood was drawn from monkeys before and after the administration of KD-247 and at necropsy (Fig. 1a). In the monkeys that were given antibody beginning 1 h (Cy-1 and Cy-2) or 1 day (Cy-3 and Cy-4) after challenge with SHIV-C2/1, concentrations of KD-247 peaked at 800–2000 μg/ml at 15 min after injection and were maintained at 200–500 μg/ml until the next administration (as evidenced in blood samples drawn before each administration and within 15 min of the injection) (Fig. 1b i and ii). The plasma concentrations of KD-247 in monkeys treated beginning 1 week after challenge with the virus (Cy-5 and Cy-6) did not remain constant. In particular, the KD-247 maintenance concentrations in Cy-6 after day 22 were below the limit of detection (2.5 μg/ml) of the assay (Fig. 1b iii).
Because KD-247 was repeatedly administered to the monkeys, we also considered the possibility of anti-KD-247 antibody production. Anti-KD-247 antibodies in monkey plasma (1: 400 dilution) were measured using samples collected at necropsy. Binding activity indicated that the number of anti-KD-247 antibodies in Cy-6 was significantly higher than in the other monkeys (Fig. 2a). To clarify the sites of recognition of the anti-KD-247 antibodies in Cy-6, the binding activity of antibodies in Cy-6 plasma to other anti-HIV-1 antibodies was investigated. Rμ5.5 is a reshaped mAb that is equivalent to the entire KD-247 molecule except for antigen-binding sites [6,20], and Cβ1 is a chimeric mAb whose Fc region is equivalent to that of KD-247 . These mAbs were used, as well as KD-247 coated for ELISA. The reaction of these mAbs with the monkey antibodies was detected by biotinylated KD-247 based on a double-antibody capture ELISA. The antibodies bound to KD-247 in Cy-6 plasma reacted with neither Rμ5.5 nor Cβ1 (Fig. 2b). Finally, we examined whether anti-KD-247 antibodies in Cy-6 plasma inhibit the binding of KD-247 to antigen peptides. Two KD-247-specific antigen peptides, SP13 and P20PATH (NNTRRRLSIGPGRAFYARRN), derived from the V3 region of SHIV-C2/1, were coated on ELISA plates and reacted with KD-247 that had been incubated overnight at 4°C with Cy-6 plasma collected on day 0, day 7, or at necropsy. Binding of KD-247 to antigen peptides decreased by approximately 60% after reaction of mAb with Cy-6 plasma collected at necropsy (Fig. 2c). Antibody inhibition of the binding of KD-247 to antigen peptides strongly suggests that the plasma contained an antiidiotype antibody.
Suppression of plasma viral load and of CD4+ T-cell loss in peripheral blood
The kinetics of plasma viral load in monkey plasma is shown in Fig. 3(a). The viral loads were suppressed in monkeys given KD-247 in comparison with those given control IgG. The complete protection previously reported with preadministration of KD-247 was not achieved in these postadministration trials . The CD4+ T-cell counts were maintained at higher levels in monkeys given KD-247 than in the control animals (Fig. 3b). The suppression of viral load and the maintenance effect of KD-247 on CD4+ T cells were similar among the test groups. As each group had only two animals, between-group significant differences were not tested.
Maintenance of CD4+ T cells in various lymphoid tissues
At 11–13 weeks after viral challenge, necropsies of the monkeys given KD-247 were performed and their lymphoid organs were evaluated. All the lymph nodes of the monkeys inoculated with pathogenic SHIV followed by control IgG were atrophied. In contrast, the lymph nodes of all monkeys given KD-247 maintained normal shape. Marked change was observed in the thymus (Fig. 4a); the thymuses of all monkeys given KD-247 were hypertrophic, whereas the thymuses of monkeys inoculated with SHIV alone, or given control IgG, were atrophied. No organ atrophy was observed in any of the groups treated with KD-247. To determine the architecture of the lymph nodes, we examined tissue sections collected at necropsy. Germinal centers were not detected in the lymphoid tissue of monkeys treated with control IgG, but cell architecture was preserved in monkeys given KD-247 (Fig. 4b).
The T-cell subpopulation in the lymphoid tissues of the monkeys was analyzed by flow cytometry (Fig. 5). The CD4+ T cells in the lymph nodes of both the IgG control monkeys (Cy-7 and Cy-8) were nearly depleted. In contrast, a normal level of CD4+CD8− cells was maintained in the lymph nodes of all monkeys given KD-247. The CD4+CD8− T-cell population values in the groups given KD-247 and control IgG were obviously higher and lower, respectively, than the values for the mean − 2 SD in naive control monkeys (n = 15). In the thymus, the absolute cell numbers of the monkeys given control IgG were low and could not be assessed for Cy-7 lymphocytes because of cell depletion. Thymic T-cell subpopulations were composed almost entirely of CD4+CD8+ double-positive cells (Cy-1 = 52%, Cy-2 = 74%, Cy-3 = 75%, Cy-4 = 76%, Cy-5 = 77%, Cy-6 = 75%, and Cy-8 = 71%; naive = 63 ± 15%). In the submandibular and mesenteric lymph nodes and spleen, administration of KD-247 rescued CD4+CD8− cells independently of injection timing; this T-cell subset was not maintained in IgG controls.
Since the development of HAART, the likelihood of progression to AIDS or death has been decreased if CD4+ T-cell counts are properly maintained even when HIV-1 RNA concentrations in peripheral blood are high . This finding suggests importance of maintaining CD4+ T cells in the whole body for the control of HIV/AIDS. In this study, we confirmed that postinfection passive immunization of SHIV-infected monkeys with KD-247 fully rescued CD4+ T-cell loss in various lymphoid tissues and yielded partial protection against increased plasma viral load and loss of CD4+ T cells.
How, then, does postinfection immunization with KD-247 help maintain CD4+ T cells in lymphoid tissues? Immunohistological alterations of the lymph nodes in HIV-infected patients represent a dynamic process, in which an initial florid follicular hyperplasia gives way ultimately to lymphocyte depletion . There are several theories regarding the various direct or indirect mechanisms of CD4+ lymphocyte depletion by HIV . We previously reported that treatment with the humanized neutralizing antibody Rμ5.5 prevented HIV-1-induced atrophic changes in the medulla of engrafted thymic tissue in a thymus/liver-transplanted severe combined immunodeficient murine model . The acute pathogenic SHIV-C2/1-derived clone virus KS661 resulted in increased thymic involution, atrophy, and the depletion of immature T cells, including CD4+CD8+ double-positive cells . Infection with HIV-1, SIV, or SHIV is associated with abnormalities in the number, size, and structure of germinal centers . Progressive depletion of proliferating B cells and disruption of the follicular dendritic cell network in germinal centers within 20 days after SIV challenge have also been reported . Although our study was limited by small group size (two monkeys/group), our data clearly show only minimal differences in CD4+ T-cell levels between groups treated with KD-247 and the IgG control monkeys. The effects of KD-247 on CD4+ T cells were more remarkable in lymph node than in peripheral blood compartments. Accumulation of apoptotic cells has been reported in both lymph nodes and thymus during the second week of highly pathogenic SHIV-C2/1 [17,28] and SHIVDH12R infections . Given the smaller increase in CD4+CD95+ cell populations in peripheral blood mononuclear cells among monkeys that exhibited even partial protection from postchallenge SHIV-C2/1 with a suboptimal dose of KD-247 infusion in previous studies , KD-247 might protect against apoptosis of CD4+ T cells in lymphoid tissues. Thus, in addition to neutralizing antibodies in animals receiving transfusions, passive transfer of KD-247 might help to maintain levels of CD4+ T cells and to preserve the integrity of lymphoid structures, potentially leading to a less pathogenic course of disease progression. The roles played by the antibodies against HIV/AIDS could be clarified by further analyses of immunological function of monkeys treated with KD-247; areas of future research include viral components [30,31], lymphocyte activation [32,33], cytokine spectra , T-cell homeostasis [35,36], dendritic cell functions [37,38], Fc receptor interactions [39,40], and related functions.
Because preinfection experiments have shown that the concentration of KD-247 in plasma is important in protecting monkeys against viral infection , we also measured KD-247 concentrations in plasma samples. The postinfection effect of KD-247 against increased viral load and CD4+ T-cell loss in peripheral blood were evaluated. Monkeys given KD-247 had lower plasma viral loads and less CD4+ T-cell loss than did those treated with control IgG; however, as noted above, each group had only two animals and no statistical analysis was performed. These results in peripheral blood were not very pronounced compared with the phenomena observed in lymphoid organs. Complete protection, which was previously reported with preinfection administration of KD-247 , was not achieved in these postinfection trials. The times and values of viral load peaks were similar in all monkeys, but the increases in viral loads were delayed by administration of KD-247. Interestingly, the ability of KD-247 to suppress viral loads after they peaked did not depend on the timing of administration. In previous preinfection experiments with 45 mg/kg of KD-247, viral challenges were performed 1 day after KD-247 administration, and blood concentrations of KD-247 ranged from 700 to 800 μg/ml immediately before viral challenge in monkeys. Preadministration of these concentrations of KD-247 yielded complete protection against SHIV-C2/1 infection . By contrast, in the current study, the monkeys given KD-247 1 h after challenge with the virus became infected, even though the KD-247 concentrations 15 min after administration ranged from 1000 to 1300 μg/ml (Fig. 1b i). These KD-247 concentrations are considered sufficient to neutralize cell-free viruses that develop after the initial infection and/or are generated one after another following infection in peripheral blood. Therefore, the inability of the antibody administered 1 h after challenge to completely protect against the virus suggests that target cells were infected with the virus within 1 h. The previously reported results of time-dependence studies [10,11,41] of postinfection prophylaxis using SHIV are comparable with those obtained in the present study. The virus might not only infect target cells directly but also evade neutralizing antibody to produce infection in the cells of the peripheral blood and/or the lymphoid tissues [42,43]. Follicular dendritic cells could sustain HIV infection in the presence of neutralizing antibody . Mucosal infection, such as vaginal challenge with SHIV, has been suggested to be a better in-vivo model to evaluate passive immunization [45,46]. The effects of antibodies in the lymph node compartment might be clearly observable using models of mucosal infection, as viruses harbored in lymph nodes after mucosal challenge later appeared in the peripheral blood compartment following systemic spread. Unexpectedly, the maintenance of CD4+ T cells in the lymph nodes in Cy-6 were similar to those in the other monkeys given KD-247, although the mAb was eliminated from the plasma 3 weeks after viral challenge, once anti-KD-247 antibodies were elicited in this monkey. High plasma concentrations of KD-247 seem to be effective in preventing HIV-1 transmission. However, even if high concentrations are not maintained in the blood for a long time, KD-247 could rescue lymphoid CD4+ T cells.
Passive immunization with mAbs has been shown to prevent a variety of diseases, although no mAb products are licensed for use for immunotherapy against HIV/AIDS . In a passive immunization trial with humans, a cocktail of three mAbs was able to delay viral rebound following interruption of antiretroviral therapy . However, differences in the pharmacokinetic profiles of constituent mAbs and cost-related issues of production might affect the development of neutralizing mAb cocktail drugs [47,48]. In contrast, KD-247 itself neutralizes primary isolates including chemokine (C–C motif) receptor 5 (CCR5)-tropic viruses with a matching narrow-neutralization sequence motif [6,7]. KD-247 is expected to be useful as a novel reagent for immune protection against HIV/AIDS, because the mAb might not only directly neutralize the virus but also maintain CD4+ T cells in lymphoid tissues.
This work was supported by the ‘Panel on AIDS’ of the United States–Japan Cooperative Medical Science Program and the Health Science Foundation, Japan.
T. Murakami planned experiments, wrote the manuscript, and analyzed the laboratory data; M. Honda conducted the study, planned the experiments, and wrote the manuscript; Y. Eda planned and conducted the experiments; T. Nakasone, K. Someya, N. Yoshino, M. Kaizu, and Y. Izumi performed the animal experiments and analyzed the laboratory data; Y. Ami and H. Matsui performed the animal experiments and the pathological analyses; K. Shinohara managed the animal experiments; N. Yamamoto supervised the study.
Part of the information was presented at the 16th Annual Meeting of the Japanese Society for AIDS Research, 28–30 November 2002, Nagoya, Japan (abstract 249).
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