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Journal of Virology, December 2005, p. 14887-14898, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14887-14898.2005

CD8+ and CD20+ Lymphocytes Cooperate To Control Acute Simian Immunodeficiency Virus/Human Immunodeficiency Virus Chimeric Virus Infections in Rhesus Monkeys: Modulation by Major Histocompatibility Complex Genotype

Hanwen Mao, Bernard A. P. Lafont, Tatsuhiko Igarashi, Yoshiaki Nishimura, Charlie Brown, Vanessa Hirsch, Alicia Buckler-White, Reza Sadjadpour, and Malcolm A. Martin*

Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

Received 21 July 2005/ Accepted 5 September 2005


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ABSTRACT
 
We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIVDH12R-Clone 7, like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+ T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIVDH12R-Clone 8 experience only partial and transient losses of CD4+ T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+ and CD20+ lymphocytes in suppressing the replication of the attenuated SHIVDH12R-Clone 8 and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+ or CD20+ cells during the SHIVDH12R-Clone 8 acute infection resulted in the rapid, complete, and irreversible loss of CD4+ T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+ cells; SHIVDH12R-Clone 8 was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+ and CD20+ B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.


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INTRODUCTION
 
Recent reports have described the massive infection and systemic depletion of CD4+ memory T lymphocytes in rhesus macaques during the initial weeks of acute simian immunodeficiency virus (SIV) infections (21, 26). A similar rapid loss of CD4+ T cells from the gut mucosa has been observed during acute infections of recently human immunodeficiency virus type 1 (HIV-1)-exposed individuals (4). Despite this severe insult to the immune system, potent virus-specific CD8+ cytotoxic T lymphocyte (CTL) responses are detected contemporaneously with the control of plasma viremia during both HIV-1 and SIV infections (3, 19, 20). Because virus-specific neutralizing antibodies (NAbs) first become demonstrable following the suppression of viremia and the titers measured are quite low (29, 38), B lymphocytes are not thought to play a major role during the early stages of HIV-1 infection.

It is now appreciated that prompt and rigorous control of acute primate lentivirus infections is key for durably controlling virus replication and preventing the subsequent development of disease. For example, when potent antiretroviral therapy is initiated in rhesus monkeys within 24 h of SIV inoculation, plasma viremia is markedly suppressed during or following cessation of treatment (22). A similar 28-day treatment regimen, begun on day 5 postinoculation in SIV/HIV chimeric virus (SHIV)-infected animals, resulted in durable suppression of virus replication in three of four treated macaques and a 4-year disease-free clinical course (14). Passive transfer of high-titer monoclonal or polyclonal neutralizing antibodies prior to SHIV challenge can also successfully abort the primary virus infection and in several instances resulted in sterilizing protection (24, 34, 37, 46). In addition, genetic determinants affecting major histocompatibility complex (MHC) class I alleles (17, 28, 30, 31, 35), chemokines (11, 50), and chemokine receptors (7, 23, 41, 47) have been shown to alter the balance between susceptibility/resistance to both HIV-1 and other primate lentiviruses. The dose dependency of the full-blown SHIV-induced immunodeficiency syndrome (rapid and complete depletion of CD4+ T lymphocytes within weeks of virus inoculation was observed with large, but not small [<625 50% tissue culture infective doses {TCID50}], virus inocula [9]) is yet another illustration of the race between vigorous SHIV replication/systemic dissemination and containment by effective host responses (9, 14).

The recent development and use of humanized monoclonal antibodies (MAbs) to deplete specific immune cell populations has provided an in vivo approach to study the contributions of individual lymphocyte subsets in controlling lentiviral infections in nonhuman primates (15, 16, 25, 43, 44). In this study, MAbs were used to deplete CD8+ or CD20+ cells to assess their role in controlling the acute infection of an attenuated molecularly cloned SHIV, designated SHIVDH12R-Clone 8. Unlike the isogenic and highly pathogenic SHIVDH12R-Clone 7, which causes a rapid, systemic, and irreversible depletion of CD4+ T cells and immunodeficiency requiring euthanasia within 13 to 30 weeks of virus inoculation, SHIVDH12R-Clone 8 induces a transient loss of CD4+ T cells, low to undetectable levels of postpeak plasma viremia, and a benign clinical course even when large amounts of virus (≥5,000 TCID50) are inoculated (40). Not unexpectedly, MAb-mediated ablation of CD8+ cells at the time of SHIVDH12R-Clone 8 inoculation of Mamu-A*01-negative animals resulted in high sustained levels of postpeak plasma viremia, the rapid and complete loss of CD4+ T cells, and the induction of immunodeficiency. Surprisingly, administration of the anti-CD20 MAb also induced the full-blown fatal clinical syndrome typical of SHIVDH12R-Clone 7. In contrast to these results, the depletion of CD20+ cells in Mamu-A*01-positive rhesus monkeys at the time of their infection with SHIVDH12R-Clone 8 did not lead to the rapid and complete loss of CD4+ T lymphocytes or the development of disease, whereas the administration of the anti-CD8 MAb induced a rapidly downhill clinical course requiring euthanasia. Together, these results indicate that depending on the MHC genetic background, CD8+ and CD20+ lymphocytes play various and cooperative roles in controlling acute primate lentivirus infections.


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MATERIALS AND METHODS
 
Animal experiments. Rhesus macaques (Macaca mulatta) were maintained in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals (5) and were housed in a biosafety level 2 facility; biosafety level 3 practices were followed. Animals were anesthetized with intramuscular injections of ketamine hydrochloride (Ketaject; Phoenix Pharmaceutical, Inc., St. Joseph, Mo.) and acepromazine maleate (Fermenta Animal Health Co., Kansas City, Mo.) during phlebotomies, administration of MAbs, and virus inoculations. Monkeys were euthanized as previously described (13).

Virus. The construction and characterization of the SHIVDH12R-Clone 7 and SHIVDH12R-Clone 8 molecular clones and their use to generate virus stocks have been described previously (40). In the experiments reported, unless specifically stated in the figure legends, 5,000 TCID50 of the same SHIVDH12R-Clone 7 or SHIVDH12R-Clone 8 stocks was inoculated into the saphenous veins of anesthetized animals.

Treatment of monkeys with MAbs. CD8+ cells were depleted by administering three doses of a mouse-human chimeric anti-CD8 antibody (cM-T807; Centocor, MA) over a 7-day period (on days 0, 3, and 7) as previously described (43). The first dose was given subcutaneously (10 mg/kg), followed by two intravenous doses (5 mg/kg). The course of cM-T807 treatment was initiated 1 day following virus inoculation. B cells were depleted by three weekly intravenous injections (20 mg/kg) of the monoclonal mouse-human chimeric anti-human CD20 antibody Rituxan (IDEC Pharmaceutical Corp., San Diego, CA). The first Rituxan injection was administered 4 days prior to virus inoculation. Two control animals were treated with an isotype-matched control MAb (Synagis; MedImmune, Gaithersburg, MD), following the routes and schedules used for anti-CD8 or anti-CD20 MAbs, respectively. The depletion of both lymphocyte subsets in the peripheral blood and lymph nodes was monitored by flow cytometry. The loss of CD20+ lymphocytes in lymph node specimens was also monitored immunohistochemically. Tissue sections were deparaffinized, subjected to high-temperature, high-pressure unmasking, and then blocked with 3% normal horse serum and 1% nonfat milk in phosphate-buffered saline (PBS) and incubated with mouse anti-human CD20 (DAKO, M0755), goat anti-mouse immunoglobulin G (IgG) biotinylated secondary antibody (Vector Labs), ABC-peroxidase reagent (Vector Labs), and then diaminobenzidine. All stained sections were examined on a Zeiss Axiophot microscope.

Plasma viral RNA measurements. Plasma viral RNA levels were determined by real-time PCR (ABI Prism 7700 sequence detection system; Applied Biosystems, Foster City, Calif.) utilizing reverse-transcribed viral RNA from macaque plasma samples as previously described (9).

Lymphocyte immunophenotyping and tetramer staining. EDTA-treated blood samples were stained with fluorochrome-conjugated MAbs CD3{varepsilon}-fluorescein isothiocyanate (FITC), CD4-allophycocyanin (APC), CD8-peridinin chlorophyll protein (PerCP), and CD20-phycoerythrin (PE) (BD Biosciences, San Diego, CA) and analyzed by flow cytometry (FACSCalibur; BD Biosciences). In depletion experiments, blood samples and lymph node suspensions from antibody-treated animals were stained with CD3{varepsilon}-PerCP, CD4-APC (BD Biosciences), CD8-PE (clone DK25; DakoCytomation, Carpinteria, CA), and CD20-FITC (clone B9E9; Immunotech, Beckman Coulter, Marseille, France). Preliminary experiments demonstrated that the staining anti-CD8 (clone DK25) and anti-CD20 (clone B9E9) MAbs did not compete with the depleting anti-CD8 (cM-T807) and anti-CD20 (Rituxan, C2B8) MAbs, respectively. For tetramer analyses, blood samples were surface stained with CD3{varepsilon}-FITC, CD8-PerCP, CD28-PE (Becton Dickinson, San Jose, CA), and SIV Gag iTAG (CM9[CTPYDINQM])-MamuA*01 tetramer-APC (Beckman Coulter, Immunomics Operations, San Diego, CA) as previously described (2).

Intracellular cytokine detection. Suspensions (2.0 x 106 cells/ml) of freshly isolated peripheral blood mononuclear cells (PBMC) from rhesus macaque whole blood were incubated for 1 hour at 37°C with 1 µg/ml of anti-CD28 and anti-CD49d monoclonal antibodies (BD Biosciences, San Jose, CA) and stimulated with CM9 peptide (0.5 µM) or mock treated. Brefeldin A (Sigma, St. Louis, MO) was added (10 µg/ml) to block cytokine secretion, and the cells were incubated for an additional 5 h at 37°C. After being washed, the cells were stained for 30 min at 4°C with CD3{varepsilon}-FITC, CD8-PerCP, and APC-conjugated SIV Gag iTAG (CM9)-Mamu-A*01 tetramer. Following treatment with fluorescence-activated cell sorter lysing solution (BD Biosciences) and washing, the cells were permeabilized using fluorescence-activated cell sorter permeabilizing solution 2 (BD Biosciences) and then stained with gamma interferon (IFN-{gamma})-PE (BD Biosciences PharMingen). Acquisition was performed using a FACScalibur flow cytometer (BD Biosciences), collecting at least 50,000 CD3+ CD8+ lymphocytes in list mode. Data were analyzed with CellQuest Pro (BD Biosciences).

Proliferation of Mamu-A*01-positive CD8+ T lymphocytes by CFSE dilution assay. Rhesus macaque B-lymphocyte cell lines (B-LCL) were established by infecting freshly isolated PBMC with Herpesvirus papio present in the supernatant of the 594S cell line (39). The stable B-cell lines were maintained in RPMI 1640 medium (Biowhittaker, Walkersville, MD) supplemented with 20% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 U/ml streptomycin (R20 medium). Stable B-LCL and freshly purified PBMC, collected from animals at 2 weeks postinfection, were washed in 37°C prewarmed PBS plus 0.5% FBS and then resuspended in the same buffer at 10 x 106 cells/ml. Cells were stained with 5 -and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) at 10 µM for 10 min at 37°C and washed once with 10 volumes of PBS plus 0.5% FBS and twice with culture medium. Two million B-LCL cells were pulsed with the CM9 peptide at 0.2 µM or mock pulsed for 1 h at 37°C. After incubation, the cells were irradiated (3,000 rads) and washed twice with culture medium, and 0.5 x 106 pulsed and CSFE-stained autologous B cells were mixed with 2.0 x 106 CFSE-stained PBMC in 1 ml medium and incubated at 37°C for 6 days. Following cultivation, the cells were collected, washed twice with PBS plus 0.5% FBS, and surface stained with CD8-PerCP and APC-conjugated SIV Gag iTAG (CM9)-Mamu-A*01 tetramer. Acquisition and data analysis were performed as described above.

Antiviral IgG/IgM enzyme-linked immunosorbent assay (ELISA) and virus neutralization assays. Anti-SHIV IgG in the plasma of SHIVDH12R-Clone 8-infected macaques was measured using Vironostika HIV-1 Microelisa System (Organon Teknika, Durham, NC) kits as specified by the manufacturer with the following modifications. Antigen-coated microwell plates were first blocked with 5% nonfat milk in PBS (350 µl per well). Samples of macaque plasma were diluted 1:6 in 5% PBS-nonfat milk and incubated for 1 h at 37°C, followed by incubation overnight at 4°C. Horseradish peroxidase-conjugated polyclonal goat anti-monkey IgG (Nordic Immunological Laboratories, The Netherlands) (1:2,000 dilution in 5% PBS-nonfat milk) was used as the secondary antibody to measure levels of antiviral IgG. The same procedure was used to detect HIV-1 specific IgM, except that horseradish peroxidase-conjugated polyclonal goat antiserum to monkey IgM (Fc specific) diluted 1:1,000 (Nordic Immunologic Laboratories) was used.

Anti-SHIV NAbs in the plasma of infected monkeys were evaluated in a 2-week assay that measures 100% neutralization, as previously described (46). Individual plasma samples were diluted threefold, using plasma from uninfected rhesus monkeys as diluent. A 5-µl aliquot of each plasma dilution was incubated with 5 TCID50 (in 5 µl) of SHIVDH12R-Clone 8 at room temperature for 1 h and was then used to infect MT-4 cells in quadruplicate. Infected cultures were maintained for 2 weeks, and virus replication was monitored by 32P-reverse transcriptase assays (49).


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RESULTS
 
Highly pathogenic and attenuated molecular SHIV clones induce distinct clinical outcomes in infected rhesus monkeys. We previously reported the construction of the SHIVDH12R-Clone 7 and SHIVDH12R-Clone 8 molecular clones from PBMC DNA prepared from a rhesus monkey infected with the highly pathogenic SHIVDH12R derivative SHIVDH12R-PS1 (40). Monkeys infected with SHIVDH12R-Clone 7 typically exhibit an extremely rapid, complete, systemic, and irreversible depletion of their CD4+ T cells and high sustained levels of plasma viral RNA (Fig. 1A and B). These animals were euthanatized between weeks 13 and 30 postinoculation because of symptoms of immunodeficiency including severe anorexia, intractable diarrhea, marked weight loss, and opportunistic infections. In contrast, macaques inoculated with SHIVDH12R-Clone 8 experience a significant but transient loss of CD4+ T cells during the first 3 weeks of infection, which gradually return to near preinfection values by week 40 post infection (Fig. 1A). These monkeys develop a peak viremia (mean of 3.2 x 107 [ranging from 0.39 x 107 to 5.4 x 107] RNA copies/ml plasma) that is approximately 4 times lower than that measured in animals infected with the highly pathogenic SHIVDH12R-Clone 7 (mean of 12.6 x 107 [ranging from 4.9 x 107 to 16 x 107] RNA copies/ml plasma) (Fig. 1B). The high levels of plasma viral RNA in SHIVDH12R-Clone 8-infected macaques rapidly decline and remain low or undetectable for extended periods of time. With the exception of a single animal (BH30), which experienced a sudden sharp depletion of CD4+ T cells beginning at week 145 that was accompanied by a marked elevation of its plasma viremia (from <1.0 x 102 copies/ml at week 137 to 1 x 106 copies/ml at week 145), the SHIVDH12R-Clone 8-infected monkeys have shown no signs of disease during 3 to 5 years of observation (Fig. 1C). This experiment clearly shows that SHIVDH12R-Clone 8 is attenuated compared to SHIVDH12R-Clone 7; the prolonged uneventful clinical course induced by SHIVDH12R-Clone 8 very likely reflects the presence of an intact and functional immune system that is able to effectively control the acute virus infection and prevent the development of immunodeficiency for extended periods of time.



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  		FIG. 1. SHIVDH12R-Clone 7 and SHIVDH12R-Clone 8 induce distinct clinical outcomes in infected rhesus monkeys. Rhesus monkeys were inoculated intravenously with the indicated amounts (TCID50) of SHIVDH12R-Clone 7 or SHIVDH12R-Clone 8. The levels of CD4+ T cells (A and C) and plasma viral RNA (B) in the peripheral blood were monitored over the first 40 weeks (A and B) or over a 220-week period (C).

Treatment of individual rhesus macaques with an anti-CD8 MAb during the acute SHIVDH12R-Clone 8 infection is associated with the rapid induction of immunodeficiency. The capacity of an anti-CD8 MAb to eliminate CD8+ lymphocytes in vivo was initially assessed by administering three doses of cM-T807 to uninfected rhesus monkeys. As reported by Schmitz etal. (43), we observed a rapid decline of circulating CD8+ cells to background levels within 24 h of cM-T807 treatment and preservation of CD4+ T and B lymphocytes in naive animals (data not shown). In these uninfected monkeys, CD8+ lymphocytes in the blood returned to preinjection levels within 2 to 4 weeks of anti-CD8 MAb treatment. The contribution of CD8+ lymphocytes in controlling the SHIVDH12R-Clone 8 infection was then evaluated by administering the anti-CD8 MAb on days +1, +4, and +8 relative to the intravenous inoculation of two monkeys (CE96 and CK3A) with virus (Fig. 2A). In these infected animals, cM-T807 also caused an immediate and complete loss of CD8+ cells, which lasted 3 to 4 weeks. The treatment of a third SHIVDH12R-Clone 8-infected macaque (CK1K) with an isotype-matched control MAb was associated with a transient reduction in the number of circulating CD8+ lymphocytes, a depletion typically seen at week 2 in SHIV-infected animals (13).



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  		FIG. 2. Treatment of individual rhesus macaques with an anti-CD8 MAb during acute SHIVDH12R-Clone 8 infection. Animals (CE96 and CK3A) were injected intravenously with the anti-CD8 MAb beginning 1day following virus inoculation. Peripheral blood CD8+ lymphocyte (A), CD4+ lymphocyte numbers (B), and plasma viral RNA levels (C) were measured at the indicated times. Animal CK1K received an isotype-matched control MAb. The arrows indicate the times of antibody treatment on days +1, +4, and +8 relative to SHIVDH12R-Clone 8 inoculation.

In contrast to the untreated SHIVDH12R-Clone 8-infected monkeys, which experienced prompt control of viremia and a benign clinical course (Fig. 1), the two infected animals receiving cM-T807 sustained a rapid, complete, and irreversible loss of CD4+ lymphocytes (Fig. 2B), similar to that observed for macaques infected with SHIVDH12R-Clone 7 (Fig. 1A). Both of these SHIVDH12R-Clone 8-infected monkeys also developed high and sustained levels of plasma viral RNA (Fig. 2C), no detectable SHIV-specific IgG antibody responses, and a rapid downhill clinical course requiring euthanasia at weeks 11 and 13 postinfection, respectively, because of marked anorexia and weight loss. A SHIVDH12R-Clone 8-infected animal, CK1K, which received an isotype-matched control antibody experienced the characteristic partial and transient loss of CD4+ T cells and readily controlled its postpeak plasma viremia (Fig. 2B and C). These results show that CD8+ lymphocyte responses in SHIVDH12R-Clone 8-inoculated animals are required to suppress virus replication and prevent the development of disease.

Treatment of individual rhesus macaques with anti-CD20 MAbs during acute SHIVDH12R-Clone 8 infection also results in uncontrolled plasma viremia and the development of disease. The role of B cells in durably controlling the acute SHIVDH12R-Clone 8 infection in rhesus monkeys was examined by administering three weekly injections of the depleting anti-CD20 MAb Rituxan. In a preliminary experiment, Rituxan treatment of an uninfected animal (CE43) resulted in the rapid depletion of CD20+ lymphocytes in the peripheral blood (Fig. 3A) and lymph nodes (data not shown). This marked loss of B lymphocytes lasted 4 weeks, at which time this subset gradually returned to pretreatment values (Fig. 3A). This effect was similar to that previously reported following Rituxan administration to uninfected rhesus monkeys (44) and was accompanied by some reduction of total circulating CD8+ lymphocytes in macaque CE43.



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  		FIG. 3. Treatment of individual rhesus macaques with an anti-CD20 MAb during the acute SHIVDH12R-Clone 8 infection. An uninfected monkey (CE43) was injected intravenously with three doses of the anti-CD20 MAb Rituxan, and the numbers of CD4+, CD8+, and CD20+ lymphocytes in its peripheral blood were determined (A). Intravenous administration of Rituxan to animals CJ55 and CJ61 was started 4 days prior to SHIVDH12R-Clone 8 inoculation. The number of peripheral blood CD20+ lymphocytes (B), number of CD4+ lymphocytes (C), plasma viral RNA levels (D), and CD8+ lymphocyte numbers (E) were measured at the indicated times. Animal CJ6F received an isotype-matched control MAb. The arrows indicate the times of the weekly antibody treatments.

Two additional rhesus monkeys (CJ55 and CJ61) were similarly treated with the anti-CD20 MAb and, on day 4 following the initiation of Rituxan treatment, were inoculated intravenously with SHIVDH12R-Clone 8. The administration of the anti-CD20 antibody also resulted in the rapid elimination of CD20+ lymphocytes in these infected animals; at the time of virus inoculation, circulating B cells were not detectable (Fig. 3B). Somewhat unexpectedly, the two Rituxan-treated SHIVDH12R-Clone 8-infected macaques experienced a rapid irreversible depletion of their CD4+ T lymphocytes, no detectable virus-specific antibody responses, and persistently high levels of plasma viremia (Fig. 3C and D), requiring euthanasia at weeks 9 and 14, respectively, due to severe weight loss, anorexia, and diarrhea. As shown in Fig. 3E, this surprising change in the pathogenic phenotype associated with SHIVDH12R-Clone 8 infection of rhesus monkeys was not due to any significant difference in the CD8+ lymphocyte levels induced by Rituxan treatment. The transient reduction of circulating CD8+ cells usually observed between weeks 1 and 2 in SHIV-infected animals also occurred in these macaques. It is also worth noting that in contrast to the case for the uninfected anti-CD20 MAb-treated macaque, the loss of CD20+ cells in the peripheral blood and lymph nodes in both SHIVDH12R-Clone 8-infected monkeys was irreversible. As a control, another SHIVDH12R-Clone 8-inoculated monkey (CJ6F), receiving an isotype-matched irrelevant MAb, experienced the transient loss of CD4+ T lymphocytes and low to undetectable postpeak plasma viremia (Fig. 3C and D), as typically observed in infected but untreated animals (Fig. 1). Taken together, the experiments shown in Fig. 2 and 3 indicate that both CD8+ and CD20+ lymphocytes are required to control the nonpathogenic SHIVDH12R-Clone 8 infections in vivo.

SHIV-specific antibodies do not become detectable until 2 to 3 weeks after virus inoculation. The principal role of B cells in controlling the acute SHIVDH12R-Clone 8 infection is commonly thought to be their capacity to generate virus-specific antibodies. To assess the temporal appearance of such antibodies, we initially examined sequential plasma samples collected from SHIVDH12R-Clone 8-inoculated monkeys that either had not been treated with the anti-CD20 MAb (macaques 429 and BH30) or had received the isotype control antibody (macaque CK1K). At a plasma dilution of 1:6, SHIV-specific IgG antibodies first became demonstrable by ELISA between days 17 and 21 in the three animals (Fig. 4A).



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  		FIG. 4. Production of binding and neutralizing antibodies in untreated monkeys inoculated with SHIVDH12R-Clone 8. Virus-specific IgG (A) and IgM (B) binding antibodies were measured by ELISA using plasma samples, diluted 1:6, collected at the indicated times. OD, optical density. Neutralization of SHIVDH12R-Clone 8 was determined from the 32P-reverse transcriptase activity released into the medium on day 14 from quadruplicate cultures (C). Autoradiograms of 32P-reverse transcriptase assays, using plasma samples diluted 1:3 and collected at the indicated times, are shown.

IgM production and secretion occur earlier in the B-cell developmental pathway than those of IgG. We therefore measured the levels of SHIV-specific IgM in the plasma of the infected monkeys treated with the anti-CD8 or anti-CD20 MAbs. As shown in Fig. 4B, low and transient levels of IgM were detected in the two recipients of the anti-CD8 MAb (CK3A and CE96) but not in the animals (CJ61 and CJ55) treated with the anti-CD20 MAb. These data demonstrate that the depletion of B cells in anti-CD20 MAb-treated macaques abrogated any virus-specific antibody production, whereas the administration of the anti-CD8 MAb had little effect on the IgM responses to SHIVDH12R-Clone 8.

Since binding assays are not necessarily a good measure of virus NAbs, we next examined the capacity of plasma samples from three untreated SHIVDH12R-Clone 8-infected macaques to block spreading virus infections in cultured MT4 cells, using 32P-reverse transcriptase activity released into the medium on day 14 as the readout (46). In this assay, nonneutralized supernatants from replicate cultures appear as black dots on the autoradiograms; the absence of dots indicates the complete neutralization of virus infectivity. In order to detect any residual neutralization activity, relatively high concentrations (1:3 dilutions) of plasma, collected at multiple times from three untreated SHIVDH12R-Clone 8-infected monkeys, were tested against small amounts (5 TCID50) of SHIVDH12R-Clone 8. As shown in the autoradiograms in Fig. 4C, no neutralizing activity was demonstrable in any of the animals during the first 2 weeks of infection. NAbs did become detectable in all three monkeys between weeks 3 and 4 after virus inoculation. These results show that antiviral antibodies are generated during the course of SHIVDH12R-Clone 8 infections, but they emerge relatively late during the acute infection, suggesting that other B-cell functions may be involved in suppressing virus replication and preventing disease development.

Administration of anti-CD8 but not anti-CD20 MAbs to Mamu-A*01-positive monkeys inoculated with the nonpathogenic SHIVDH12R-Clone 8 results in uncontrolled virus replication and disease development. Expression of the Mamu-A*01 MHC class I allele in Indian-origin rhesus monkeys has been associated with slower disease progression in SIV-challenged animals (30, 31, 35, 36). This has been shown to be a consequence of the Mamu-A*01 allele binding to SIV epitopes recognized by dominant CTL responses during the acute viral infection (35). Site-specific PCR (18) was therefore used to assess the Mamu-A*01 status of the previously studied, randomly selected rhesus monkeys that had been treated with the depleting anti-CD8 and anti-CD20 MAbs and then inoculated with SHIVDH12R-Clone 8. All of these animals proved to be Mamu-A*01 negative (data not shown).

Having found that both CD8+ and B cells are needed for the durable suppression of virus replication in Mamu-A*01-negative macaques, we wished to assess the role of B cells in controlling the acute SHIVDH12R-Clone 8 infection in Mamu-A*01-positive animals. In an initial experiment, two Mamu-A*01-positive monkeys (CE4D and CE6F) were inoculated with SHIVDH12R-Clone 8 and experienced a partial and transient depletion of CD4+ T cells and durable low levels of postpeak viremia (data not shown), a response similar to that we previously observed in untreated SHIVDH12R-Clone 8-infected Mamu-A*01-negative animals (Fig. 1). Treatment of two other Mamu-A*01-positive monkeys (H680 and CF6T) with the anti-CD8 MAb at the time of the primary SHIVDH12R-Clone 8-infection resulted in the rapid and complete elimination of their CD8+ cells (Fig. 5A). Unlike the case for the cM-T807-treated SHIVDH12R-Clone 8-infected Mamu-A*01-negative macaques, the CD8+ lymphocytes never reappeared following anti-CD8 treatment in either of the infected Mamu-A*01-positive animals (compare Fig. 2A and 5A). Both of these virus-infected animals experienced a rapid and complete depletion of their CD4+ T lymphocytes (Fig. 5B), high levels of postpeak plasma viral RNA (Fig. 5C), and the onset of symptomatic disease requiring euthanasia at weeks 7 and 10.5 postinoculation, respectively, an outcome similar to that seen in the Mamu-A*01-negative macaques (compare to Fig. 2B and C).



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  		FIG. 5. Administration of anti-CD8 MAbs is associated with a rapid and complete depletion of CD4+ cells in Mamu-A*01-positive monkeys inoculated with the nonpathogenic SHIVDH12R-Clone 8. Animals (CF6T and H680) received the anti-CD8 antibody beginning 1 day following infection. The numbers of peripheral blood CD8+ lymphocytes (A), numbers of CD4+ lymphocytes (B), and plasma viral RNA levels (C) were measured at the indicated times. The arrows indicate the times of antibody treatment.

Two additional Mamu-A*01-positive monkeys were treated with the anti-CD20 MAb at the time of SHIVDH12R-Clone 8 inoculation, and both experienced an immediate loss of circulating B lymphocytes (Fig. 6A). As shown in Fig. 6B, these two animals sustained significant but incomplete depletions of their circulating CD4+ T cells. Macaque CK48 promptly controlled its postpeak viremia, whereas monkey H678 has continued to generate moderate amounts of plasma viral RNA (Fig. 6C). The reduced capacity of animal H678 to control SHIVDH12R-Clone 8 replication was associated with the prolonged suppression of its circulating CD20+ lymphocytes, which remained below a level of 5 cells/µl throughout the initial 18 weeks of infection (Fig. 6A). Although the CD4+ T-cell levels have remained depressed in this macaque, both of the Rituxan-treated, SHIVDH12R-Clone 8-infected Mamu-A*01-positive animals have remained healthy and free of disease for more than 11 months. Thus, in contrast to the case for Mamu-A*01-negative animals, in which the Rituxan-induced elimination of B cells at the time of an acute SHIVDH12R-Clone 8 infection resulted in the complete and irreversible loss of CD4+ T lymphocytes and the rapid onset of immunodeficiency (Fig. 3), similar anti-CD20 treatment of two infected Mamu-A*01-positive monkeys was associated with an asymptomatic clinical outcome.



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  		FIG. 6. Administration of anti-CD20 MAb to Mamu-A*01-positive monkeys inoculated with SHIVDH12R-Clone 8 is not associated with a rapid and complete loss of CD4+ T cells. Rituxan was administered to animals H678 and CK48 4 days prior to intravenous inoculation with 5,000 TCID50 of SHIVDH12R-Clone 8. The numbers of peripheral blood CD20+ lymphocytes (A), numbers of CD4+ lymphocytes (B), and plasma viral RNA levels (C) were measured at the indicated times.

The absence of CD20+ lymphocytes during acute SHIVDH12R-Clone 8 infection does not affect the appearance of virus-specific CTLs. Studies of B-cell-deficient mice have shown that in addition to antibody production, B cells contribute to the activation of both CD4+ and CD8+ T cells via their antigen presentation functions (12). To ascertain whether Rituxan-mediated depletion of B lymphocytes possibly altered antiviral CTL responses during viral infection, CM9 tetramer-staining assays were performed on samples of whole blood collected from four Mamu-A*01-positive rhesus monkeys at various times following SHIVDH12R-Clone 8 inoculation. Two of the macaques (CK48 and H678) had been treated with the depleting anti-CD20 MAb, and two (CE4D and CE6F) were untreated. As shown in Fig. 7, tetramer-positive CD8+ T cells first became detectable in all four animals on day 11 postinfection, reaching a peak of 10 to 19 positive CD3+ CD8+ cells/µl (3.2% of the CD3+ CD8+ cells) in both recipients of the anti-CD20 MAb on day 14. The levels of tetramer-binding CD8+ T cells in the untreated monkeys stabilized during weeks 2 to 6 postinoculation and then gradually fell to lower values by week 15.



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  		FIG. 7. Gag peptide CM9-specific CD8+ T cells in infected Mamu-A*01-positive macaques. Sequential tetramer-positive CD8+ T-lymphocyte levels in anti-CD20 MAb-treated or untreated Mamu-A*01-positive animals during the SHIVDH12R-Clone 8 infection are shown.

In addition to monitoring of their absolute levels in the blood, the functional properties of tetramer-positive CD8+ T lymphocytes were examined in two different assays. IFN-{gamma} production by CM9 tetramer-positive CD8+ T cells, following in vitro stimulation with the CM9 Gag peptide, was not affected by the depletion of CD20+ lymphocytes (Table 1). In fact, the frequency of IFN-{gamma}-producing CD8+ T cells was somewhat higher than that observed in the two untreated animals. Similarly, the capacity of tetramer-positive CD8+ T cells to proliferate following 6 days of CM9 peptide stimulation in the presence of exogenously provided autologous B cells, as measured in a CFSE dilution assay, was not significantly different in the anti-CD20 MAb-treated monkeys and the untreated animals (Table 2). Taken together, these results indicate that elimination of CD20+ lymphocytes during the acute SHIVDH12R-Clone 8 infection did not significantly affect the generation and maintenance of antiviral CTLs.


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  		TABLE 1. Percentages of CM9-stimulated tetramer-positive CD8+ T cells secreting IFN-{gamma} at week 2 postinfection


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  		TABLE 2. Proliferation of tetramer-positive CD8+ T cells following 6 days of CM9 peptide stimulation


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DISCUSSION
 
Despite numerous reports suggesting that CD20+ lymphocytes and the humoral responses they generate play a relatively minor role in controlling primate lentiviral infections, our results clearly show that the loss of either CD8+ or CD20+ lymphocytes at the time of the primary SHIVDH12R-Clone 8 infection drastically altered the uneventful and asymptomatic clinical course typically observed in Mamu-A*01-negative monkeys. Macaques not expressing the Mamu-A*01 allele and treated with either a CD8+ or a CD20+ lymphocyte-depleting MAb experienced a rapid and complete loss of CD4+ T cells and sustained high levels of postpeak plasma viremia and developed immunodeficiency within 4 months of inoculation. In contrast, Mamu-A*01-positive animals receiving the same anti-CD20 MAb, but not the anti-CD8 MAb regimen, were able to control the SHIVDH12R-Clone 8 infection and have remained clinically asymptomatic.

These results are consistent with studies of Friend murine leukemia virus (MLV), which showed that the adoptive transfer of splenocytes or an admixture of CD4+, CD8+, and CD19+ cells from vaccinated animals conferred sterilizing immunity against a subsequent virus challenge (8). This finding indicated that lymphocyte subsets mediating both cellular and humoral responses were needed for complete protection against this acute murine retrovirus infection. Combinations of any two lymphocyte subsets failed to prevent MLV infection but did mediate regression of the virus-induced splenomegaly. In the mouse model, the protective effects of B cells were attributable to the antibodies they generated, since B-cell-deficient mice were poorly protected by a vaccination regimen that elicited only cellular immune responses whereas the passive transfer of virus-neutralizing antibodies, in combination with the latter vaccination regimen, provided significantly greater protection than either the antibodies or immune T cells alone (27). Our results are also consistent with a recent report examining the role of CD8+ lymphocytes in controlling a SIVmac251 challenge in recipients of a live attenuated SIV vaccine (42). That study showed that the depletion of CD8+ cells at the time of virus challenge of vaccinated animals resulted in only a partial loss of control of the ensuing plasma viremia (compared to untreated vaccinated monkeys) and suggested that both humoral and cellular immune responses were contributing to the protection observed.

MAb-mediated ablation of CD20+ lymphocytes has been previously used to assess the role of B cells during acute SIV infections of rhesus monkeys, but the effects observed were quite modest. In one study, the administration of the MAb to animals at the time of SIVmac251 inoculation resulted in the delayed appearance and lower titers of neutralizing antibodies, but the levels of peak viremia and the subsequent decline of plasma viral RNA over a 28-day observation period were similar to those observed in monkeys treated with a control antibody (44). In a second study, Rituxan was administered to three macaques inoculated with SIV-M5, an attenuated SIVmac239 derivative lacking five N-linked glycosylation sites mapping to the gp120 envelope glycoprotein (16). In untreated SIV-M5-infected animals, plasma viremia was rapidly controlled to barely detectable levels by week 20 postinoculation, whereas MAb administration resulted in high set-point levels of viral RNA in two of three SIV-M5-infected monkeys. Neither of these SIV studies reported any effect of the CD20+ lymphocyte depletion on disease induction.

We have previously reported that the immunodeficiency induced by the highly pathogenic SHIVs is the result of the direct infection and killing of an extraordinarily large fraction of quiescent naive and memory CD4+ T cells during the acute infection (32, 33). In the SHIV/macaque model, if sufficient numbers of naive CD4+ T cells are eliminated within the first 7 to 10 days of infection, there will be inadequate total residual helper T-cell activity and the infected animal is destined to rapidly succumb to immunodeficiency. If, however, an animal is infected with an attenuated SHIV strain (e.g., SHIVDH12R-Clone 8) or inoculated with relatively small amounts (e.g., <500 TCID50) of a highly pathogenic SHIV (e.g., SHIVDH12R-Clone 7), the CD4+ T-cell depletion is usually partial and transient and the battle between the virus and the immune system is shifted in favor of the immune system (9, 40). The results obtained in this study imply that B cells in combination with CD8+ lymphocytes provide some important function(s) during the critical first 7 to 10 days of the acute infection that effectively suppresses SHIVDH12R-Clone 8 replication and its capacity to induce disease. The production of NAbs, capable of contributing to the control of a primary SHIVDH12R-Clone 8 infection, is certainly the B-cell function that comes immediately to mind. However, there is a large body of published literature reporting that virus-specific NAbs arise subsequent to the suppression of HIV-1, SIV, and SHIV viremia; our own results showing that such NAbs first become detectable during the third week of SHIVDH12R-Clone 8 infections (Fig. 4) would also suggest that NAbs might not be critical in controlling virus replication during the primary infection. However, as shown in Fig. 4, low and transient levels of virus-specific IgM were detectable between days 5 and 22 in these animals, which could contribute to the virus control observed.

Numerous studies have reported that HIV-1-induced nonneutralizing antibodies, detectable within the initial weeks of the acute HIV-1 infection, may contribute to the control of virus replication in exposed individuals via opsonization, antibody-dependent cellular cytotoxicity, or complement-mediated inactivation mechanisms. In the case of antibody-dependent cellular cytotoxicity, antibodies able to inhibit primary HIV-1 strains can be measured contemporaneously with the appearance of antiviral CTLs (6). These nonneutralizing antibodies have been shown to block the replication of both autologous and heterologous virus strains in the presence of natural killer effector cells as measured in ex vivo assays (10). Antibodies present in HIV-1-infected persons at or shortly following peak plasma viremia have also been reported to mediate complement-mediated inactivation of both autologous and heterologous HIV-1 isolates by direct lysis of virions (1, 48).

In addition to giving rise to antibody-producing cells, B lymphocytes have also been reported to contribute to the activation and maintenance of CD4+ and CD8+ T cells. In the context of lymphocytic choriomeningitis virus-infected mice, adoptive transfer experiments have shown that B-cell-deficient animals exhibit a profound CD4 helper T-cell deficit and reduced interleukin-2 and IFN-{gamma} secretion by CD8+ T lymphocytes (12). However, experiments assessing vaccine-mediated control of Friend MLV found that T cells from immunized B-cell-deficient mice were equivalent to T cells from wild-type animals in contributing to the resistance against a subsequent virus challenge (27). This observation is also consistent with the results obtained in our study, which show that virus-specific CTLs emerge, as measured by tetramer binding, even when B cells are undetectable during the acute SHIV infection (Fig. 7).

Previous studies have shown that Mamu-A*01-positive rhesus monkeys control their postpeak SIV plasma viremia more effectively and, in some instances survive longer, than Mamu-A*01-negative animals (30, 31, 35, 36). This resistance to the pathogenic effects of SIV was markedly enhanced in previously vaccinated macaques when the Mamu-A*01 allele was present in combination with the Mamu-B*17 and Mamu-B*29 MHC class 1 alleles (35). The reported impact of Mamu-A*01 expression in SHIV-infected rhesus monkeys is less clear. In unvaccinated animals, the peak and set-point plasma viral RNA levels were not statistically different in Mamu-A*01-positive and -negative macaques, although one study reported that Mamu-A*01-positive monkeys had longer survival times and greater preservation of lymph node architecture (36, 45, 51). In contrast to these experiments, which have assessed the role of Mamu-A*01 in the control of known pathogenic primate lentiviruses, our study examined a more subtle parameter: disease development in Mamu-A*01-positive and -negative monkeys induced by the attenuated SHIVDH12R-Clone 8 in the absence of CD8+ or CD20+ lymphocytes. Potent Mamu-A*01-positive restricted CTL responses effectively controlled viral replication, even in the absence of B cells in SHIVDH12R-Clone 8-infected monkeys, and prevented the development of immunodeficiency.

The results described in this report clearly show that prompt and effective immunologic responses directed against primate lentiviruses are required to moderate the acute infection and prevent the induction of disease. All components of the immune system play critical roles in this process. In Mamu-A*01-negative rhesus monkeys, both B and T lymphocytes were needed at the time of SHIVDH12R-Clone 8 infection to ensure a benign clinical outcome. Our results also show that a balance exists between different arms of the immune system in controlling SHIV infections. Host genetic factors, exemplified by different MHC haplotypes, can shift the balance of the composite response in either direction. In Mamu-A*01-positive macaques, CTLs were clearly dominant and still able to suppress SHIVDH12R-Clone 8 replication in the absence of B lymphocytes. Nonetheless, the potency of this response was demonstrably different in the two Rituxan-treated Mamu-A*01-positive animals as monitored by postpeak plasma viremia and CD4+ T-cell levels (Fig. 6). The variable control of virus replication in these two monkeys very likely reflects the expression of other MHC alleles in each animal, which in combination with Mamu-A*01 could further augment or diminish the antiviral cellular response, as has been previously described (35).


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ACKNOWLEDGMENTS
 
We are indebted to Keith Reimann and Centocor for kindly providing the anti-human CD8 MAb (cM-T807) and to Genetech for anti-human CD20 antibody (Rituxan). We also thank Joel Beren, Liza Murray, Jennifer Post, Liz Scanlon, Shelley Lower, Frances Banks, and Wes Thornton for their assistance in the care and maintenance of our animals; Christopher Erb and Ronald Plishka for determining viral RNA levels; Michael Eckhaus for help in interpreting histopathological specimens; and Charles Buckler for arranging and scheduling animal experiments.

This research was supported by the Intramural Research Program of NIAID (NIH).


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FOOTNOTES
 
* Corresponding author. Mailing address: NIAID, NIH, Bldg. 4, Room 315, 4 Center Drive, MSC 0460, Bethesda, MD 20892-0460. Phone: (301) 496-4012. Fax: (301) 402-0226. E-mail: malm{at}nih.gov. Back


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Journal of Virology, December 2005, p. 14887-14898, Vol. 79, No. 23
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.23.14887-14898.2005




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