Introduction
Recently, a small reservoir of latently infected, resting memory CD4 T cells harbouring replication competent, integrated provirus was demonstrated in patients despite prolonged antiretroviral combination therapy with suppression of plasma HIV-1 RNA levels below 50-200 copies/ml[1,2]. The number of these cells decreased only very slowly with increasing time on therapy[3]. This reservoir might be a major barrier for the eradication of HIV by antiretroviral agents alone.
Treating patients for many years with antiretroviral combination therapy is undesirable given the side-effects of such therapy[4,5]. Therefore, it is necessary to develop strategies to increase the turnover rate of the reservoir of latently infected T cells. One strategy might be the activation of the resting T cells, in the continued presence of antiretroviral therapy[6,7]. Activation of these cells may result in apoptosis of these cells[8], or in virus replication within these cells, resulting in their lysis, with antiretroviral drugs preventing new infections[6]. In vitro, resting T cells of HIV-1 patients could be activated by CD3 monoclonal antibody or by the combination of the cytokines TNF-agr;, IL-2 and IL-6, resulting in HIV-1 replication[6].
In the present study, we investigated the concept of in-vivo stimulation with OKT3 and IL-2. OKT3 is an IgG2a murine monoclonal antibody directed against the CD3 molecule[9,10]. Because IL-2 plays a critical role in OKT3-driven cell proliferation[11], the effects exerted by OKT3 treatment might be enhanced by the administration of recombinant IL-2[9-11].
Materials and methods
Patients and treatment
Three patients (008, 010 and 002) were studied. Antiretroviral therapy had been started 9-15 months earlier with zidovudine, lamivudine, abacavir, nevirapine, indinavir and ritonavir[12]. Patient 010 stopped abacavir and nevirapine within 10 days because of hypersensitivity reactions and instead received hydroxyurea until 10 weeks before the OKT3/IL-2 treatment.
When the plasma HIV-1 RNA load had been below 5 copies/ml for at least 26 weeks (Fig. 1), the patients were treated with OKT3 and recombinant human IL-2. On five consecutive days 5 mg of OKT3 (Janssen-Cilag BV, Tilburg, the Netherlands) were given as a 2 h continuous infusion[13], but on the first day 2.5 mg was given for safety reasons. From days 2-6, rhIL-2 (Chiron, Amsterdam, the Netherlands) was added at a rate of 4.5 MIU twice a day subcutaneously. Antiretroviral therapy was continued. Because of the side-effects experienced by the first patient (008), the IL-2 dosage was decreased to 2 MIU twice a day in patients 010 and 002. The OKT3/IL-2 cycle was to be repeated after 2 weeks. The study was approved by the Medical Ethics Committee of our hospital and informed consent was obtained from all three patients.
;)
Fig. 1.:
Time course for changes of HIV-1 RNA in plasma (copies/ml), and changes in HIV-1 DNA copy number per 106 peripheral blood CD4 T cells, after the initiation of antiretroviral therapy, which was started on day 0. *When retested less than 5 copies/ml.
Follow-up
Lymph node biopsies were obtained by surgical excision 4-6 weeks before the OKT3 treatment and on the fourth day of the first (patient 002) or the second (patient 010) OKT3/IL-2 cycle, fixed in Parafix™, and embedded in paraffin.
OKT3 concentrations and OKT3 antibody levels in serum were determined using enzyme-linked immunosorbent assay[14]. To determine whether anti-idiotype or anti-isotype antibodies were produced, an irrelevant mouse IgG2a antibody was used[14].
The quantification of HIV-1 RNA in plasma was performed using the NucliSens HIV-1 QT assay (Organon Teknika, Boxtel, the Netherlands). When RNA levels decreased to below 50 copies/ml[12], an initial input volume in the assay of 2 ml plasma was used combined with the ultrasensitive protocol adaptation, resulting in a lower quantification limit of 5 copies/ml.
HIV-1 RNA in tissue sections was quantitated by first performing in-situ hybridization with 35S radiolabelled antisense HIV-1 RNA probes, with sense probes used as a control[15]. This was followed by measurements in the phosphor storage imager[16]. One pCi/mm2 represents 555 viral genome equivalents. HIV-1 expressing mononuclear cells in the tissue sections were counted in a darkfield microscope. Positive cells were considered to have more than 20 silver grains per 200 μm2.
HIV-1 DNA from at least 1 × 106 mononuclear cells was isolated using the TRIzol reagent (Gibco BRL, Life Technologies Inc., Grand Island, MD, USA). The cellular DNA was recovered, aliquoted and a mutant plasmid was added (400, 80, 16, and 3 copies). A competitive nested polymerase chain reaction was performed on the HIV-1 pol region[17]. The DNA copy number was expressed as copy number per 106 CD4 T cells present in peripheral blood mononuclear cells (PBMC).
Culture of resting CD4 T cells
HLA-DR-CD4 T cells were isolated from 50 ml freshly obtained blood[18,19]. Virus was isolated from limiting-diluted cells[20].
Results
Clinical side-effects
The side-effects consisted of spiking fever, headache, nausea, vomiting, diarrhea and anemia. These side-effects started within several hours after administration and persisted undiminished during the entire OKT3/IL-2 cycle. Because of the side-effects patients 008 and 002 declined a second cycle. Although no prolonged periods of hypotension were recorded, patient 008 developed acute renal failure caused by acute tubular necrosis after 6 days. Temporary hemodialysis was required, after which his renal function recovered completely. In the following patients the dosage of IL-2 was decreased, intravenous fluid administration was increased and dopamine 3 μg/kg/min was added as a continuous infusion[21-23]. Furthermore, patient 008 developed seizures on the 19th day after the start of treatment. A magnetic resonance imaging scan showed white-matter abnormalities[24]. Within a few weeks all neurological signs and symptoms disappeared and anti-epileptic medication was withdrawn. Finally, patient 010 developed a short period of subclinical hypothyroidism[25].
A profound lymphocytopenia persisted during the entire treatment with OKT3[14]. Likewise, CD4 T cell counts dropped from 320-690 × 106/l to below 10 × 106/l as early as one hour after the start of treatment. Lymphocyte and T cell counts started to recover after day 8.
Serum levels of OKT3 and OKT3 antibodies
Serum concentrations of OKT3 at the end of the infusions were 132-225 ng/ml during the first day and 566-1148 ng/ml during days 2-5, and trough concentrations ranged from 11 to 44 ng/ml after the first dose and from 53.7 to 133 ng/ml after subsequent dosages.
None of our patients developed precipitating antibodies against OKT3. However, all patients developed both anti-idiotype and anti-isotype antibodies against OKT3, as early as 11 days after the start of treatment. On day 15, patient 010 started a second OKT3/IL-2 cycle. This time he developed only minor clinical signs and symptoms, the degree of lymphocytopenia in the peripheral blood (23%) was much less than during the first cycle (1%), and the concentration of OKT3 measured at the end of the first infusion was only 5.3 ng/ml, as opposed to 132 ng/ml during the first cycle. We concluded that the anti-OKT3 antibodies significantly reduced the effects of OKT3. Therefore, OKT3 infusions were stopped after 3 days, and the IL-2 dosage increased to 4.5 MIU twice a day.
Plasma HIV-1 RNA
After the start of antiretroviral therapy, plasma HIV-1 RNA levels declined within 1-9 weeks to levels below 5 copies/ml (Fig. 1).
Patient 008, who had a plasma HIV-1 RNA load below 5 copies/ml for 37 weeks before the start of the OKT3/IL-2 treatment, had a plasma HIV-RNA level of 110 copies/ml immediately before the first dose of OKT3. His HIV-1 RNA plasma levels peaked at 1500 copies/ml at day 5 of the OKT3/IL-2 protocol, and dropped again to below 5 copies at day 16 (Fig. 1). Plasma HIV-1 RNA levels of the other two patients remained below 5 copies/ml during the entire treatment and follow-up period, up to 6 weeks after the first day of OKT3.
HIV-1 RNA in lymph nodes
During OKT3/IL-2 the number of viral RNA genome equivalents in the lymph nodes increased from 555 to 1532/mm2 in patient 010 and from 683 to 1332/mm2 in patient 002 (Fig. 2), but no increase in the number of productively infected cells (≤ 1/106 paracortical cells; approximately 106 paracortical cells counted) was seen.
Fig. 2.:
HIV-1 RNA in-situ hybridization on lymph nodes taken from patients 010 (a,b) and 002 (c,d) 4-6 weeks before (a,c) and on day 4 of the OKT3/IL-2 cycle (b,d).
HIV-1 DNA in peripheral blood mononuclear cells
During OKT3/IL-2, HIV-1 DNA in the PBMC dropped to undetectable levels in patient 010 and 002 in parallel to the disappearance of CD4 T cells from the peripheral blood. In patient 008, CD4 T cells did not completely disappear from the circulation and HIV-1 DNA could be measured in all samples obtained during the OKT3/IL-2 treatment. Upon the reappearance of the CD4 T cells, HIV-1 DNA returned to pre-OKT3/IL-2 levels.
Culture of resting CD4 T cells
In patient 008, the number of resting CD4 lymphocytes in peripheral blood harbouring replication-competent HIV before OKT3/IL-2 treatment (10-19/per 106 resting CD4 cells) was in the same order of magnitude as the number thereafter (13-19/106). In the other two patients the frequency of these cells was one or less per 106 resting CD4 T cells before and after OKT3/IL-2 treatment, and it is therefore not possible to assess whether the treatment influenced the size of this HIV reservoir.
Discussion
With regard to the most important endpoints, the three patients showed very consistent results, despite variations in the OKT3/IL-2 dosages administered.
The OKT3 serum levels in our patients were above the concentrations needed to induce the activation and proliferation of lymphocytes in vitro[10,26]. OKT3/IL-2 treatment indeed resulted in T cell activation and proliferation[27]. This was evidenced by early TNF-agr; and IL-2 peaks[27-29], which were in the range of the concentrations that have been demonstrated to induce HIV-1 replication in latently infected, resting CD4 cells in vitro[6]. Furthermore, the percentage of CD4 and CD8 T cells expressing CD38 increased to almost 100%, and the number of Ki67+ cells in lymph nodes increased 10-fold[27].
The goal of this T cell activation was to induce HIV-1 replication in latently infected cells. In one patient an increase in plasma HIV-1 RNA was seen, whereas in the other two patients only lymph node HIV-1 RNA increased. In the lymph nodes no productively infected cells could be demonstrated, and it was therefore not possible to identify the cellular source of HIV replication, because in lymph nodes both lymphocytes and macrophages carrying integrated HIV-DNA have been demonstrated[18]. The temporary increase in HIV-1 RNA did not result in ‚viral escape‚, because plasma HIV-1 RNA levels returned to (008) or remained at (010 and 002) levels below 5 copies/ml.
The fact that immunostimulation did not result in a significant decrease of total HIV-1 DNA is not surprising, because only a minute fraction of HIV-1 DNA in PBMC is considered to represent replication-competent HIV[18]. Therefore, even if the reservoir of replication-competent HIV was eliminated, this would probably not notably affect the total HIV-1 DNA concentration in PBMC as assessed by polymerase chain reaction.
The side-effects of the therapy were serious. During the first days the side-effects could be attributed to OKT3-induced cytokine release[28], and during days 2-6 also to rhIL-2[25,30]. The patients rapidly developed antibodies against the mouse monoclonal OKT3, which significantly reduced its immunostimulatory effects. In up to 46% of transplant patients, the administration of OKT3 results in the (often temporary) presence of antibodies directed against OKT3[31]. The formation of antibodies can be delayed and reduced in incidence and intensity using corticosteroids[14]. We deliberately did not administer corticosteroids because of the restraining effect that corticosteroids might have on T cell activation and viral replication[6].
The early development of anti-OKT3 antibodies causes difficulties in treating patients with consecutive courses. More than one T cell activating cycle is probably required to ‚flush out‚ the entire latent reservoir[7]. Therefore, additional strategies to activate these latently infected cells in vivo must be considered.
Acknowledgements
The authors would like to thank the following manufacturers: Glaxo-Wellcome for providing abacavir; Boehringer-Ingelheim for providing nevirapine; Janssen-Cilag, Tilburg, the Netherlands, for providing OKT3; and Chiron, Amsterdam, the Netherlands, for providing rhIL-2. They also thank K. de Blok and R.Th. Krediet, nephrologists, and P. Portegies, neurologist, for clinical support, and S. Jansen, study nurse, and the nursing staff of F5N for excellent patient care.
References
1. Wong JK, Hezareh M, Gunthard HF,
et al.
Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.Science 1997,
278:1291-1295.
2. Finzi D, Hermankova M, Pierson T,
et al.
Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.Science 1997,
278:1295-1300.
3. Finzi D, Blankson J, Siliciano JD,
et al.
Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999,
5:512-517.
4. Brinkman K, ter Hofstede HJM, Burger DM, Smeitink JAM, Koopmans PP.
Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway.AIDS 1998,
12:1735-1744.
5. Carr A, Samaras K, Burton S,
et al.
A syndrome of peripheral lipodystrophy, hyperlipidaemia and insulin resistance in patients receiving HIV protease inhibitors.AIDS 1998,
12:F51-58.
6. Chun TW, Engel D, Mizell SB, Ehler LA, Fauci AS.
Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines.J Exp Med 1998,
188:83-91.
7. Ho DD.
Towards HIV eradication or remission: the tasks ahead.Science 1998,
280:1866-1867.
8. Green DR, Scott DW.
Activation-induced apoptosis in lymphocytes.Curr Opin Immunol 1994,
6:476-487.
9. Ellenhorn JDI, Hirsch R, Schreiber H, Bluestone JA.
In vivo administration of anti-CD3 prevents malignant progressor tumor growth.Science 1988,
242:569-571.
10. Ellenhorn JDI, Woodle ES, Ghobreal I, Thistlethwaite JR, Bluestone JA.
Activation of human T cells in vivo following treatment of transplant recipients with OKT3.Transplantation 1990,
50:608-612.
11. Meuer SC, Hussey RE, Cantrell DA,
et al.
Triggering of the T3-Ti antigen-receptor complex results in clonal T-cell proliferation through an interleukin 2-dependent autocrine pathway.Proc Natl Acad Sci U S A 1984,
81:1509-1513.
12. Weverling GJ, Lange JMA, Jurriaans S,
et al.
Alternative multidrug regimen provides improved suppression of HIV-1 replication over triple therapy.AIDS 1998,
12:F117-122.
13. Buysmann S, Hack CE, van Diepen FNJ, Surachno J, ten Berge IJM.
Administration of OKT3 as a two-hour infusion attenuates first-dose side effects.Transplantation 1997,
64:1620-1623.
14. Chatenoud L, Baudrihaye MF, Chkoff N, Kreis H, Goldstein G, Bach JF.
Restriction of the human in vivo immune response against the mouse monoclonal antibody OKT3.J Immunol 1986,
137:830-838.
15. Fox CH, Cottler-Fox M.
In situ hybridization for the detection of HIV RNA in cells and tissues. In:
Current protocols in immunology. Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober W (editors). New York: Wiley; 1993.
16. Fox CH, Hoover S, Currall VR, Bahre HJ, Cottler-Fox M.
HIV in infected lymph nodes.Nature 1994,
370:256.
17. Bruisten SM, Frissen PH, van Swieten P,
et al.
Prospective longitudinal analysis of viral load and surrogate markers in relation to clinical progression in HIV type-1 infected persons.AIDS Res Hum Retroviruses 1997,
13:327-335.
18. Chun TW, Carruth L, Finzi D,
et al.
Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection.Nature 1997,
387:183-188.
19. Chun TW, Finzi D, Margolick J, Chadwick K, Schwartz D, Siliciano RF.
In vivo fate of HIV-1-infected T cells: quantitative analysis of the transition to stable latency.Nat Med 1995,
1:1284-1290.
20. Koot M, van ‚t Wout AB, Kootstra NA, de Goede REY, Tersmette M, Schuitemaker H.
Relation between changes in cellular load, evolution of viral phenotype, and the clonal composition of virus populations in the course of human immunodeficiency virus type 1 infection.J Infect Dis 1996,
173:349-354.
21. .Mercatello A, Hadj-Aïssa A, Négrier S,
et al.
Acute renal failure with preserved renal plasma flow induced by cancer immunotherapy.Kidney Int 1991,
40:309-314.
22. Shalmi CL, Dutcher JP, Feinfeld DA,
et al.Acute renal dysfunction during interleukin-2 treatment: suggestion of an intrinsic renal lesion.J Clin Oncol 1990,
8:1839-1846.
23. Memoli B, De Nicola L, Libetta C,
et al.
Interleukin-2-induced renal dysfunction in cancer patients is reversed by low-dose dopamine infusion. Am J Kidney Dis 1995,
26:27-33.
24. van Laar JM, van Buchem MA, Weyl N, Cleton FJ.
Reversible neurotoxicity during interleukin-2 therapy for metastatic renal cell carcinoma.Eur J Cancer 1995,
31A:1895-1897.
25. Kovacs JA, Vogel S, Albert JM,
et al.
Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency virus.N Engl J Med 1996,
335:1350-1356.
26. Suthanthiran M, Wiebe ME, Stenzel KH.
Effect of immunosuppressants on OKT3 associated T cell activation: clinical implications.Kidney Int 1987,
32:362-367.
27. Van Praag M, Prins J, Ten Berge I, Schellekens P, Lange J.
Cytokine and chemokine levels in HIV-1 positive patients treated with anti-CD3 monoclonal antibody (OKT3) and recombinant IL-2.6th Conference on Retroviruses and Opportunistic Infections. Chicago, February 1999 [abstract 29].
28. Chatenoud L, Ferran C, Legendre C,
et al.
In vivo cell activation following OKT3 administration.Transplantation 1990,
49:697-1202.
29. Goumy L, Ferran C, Merite S, Bach JF, Chatenoud L.
In vivo anti-CD3-driven cell activation. Cellular source of induced tumor necrosis factor, interleukin-1β, and interleukin-6.Transplantation 1996,
61:83-87.
30. Davey RT Jr, Chaitt DG, Piscitelli SC,
et al.
Subcutaneous administration of interleukin-2 in human immunodeficiency virus type 1-infected persons.J Infect Dis 1997,
175:781-1289.
31. Thistlethwaite JR Jr, Stuart JK, Mayes JT,
et al.
Complications and monitoring of OKT3 therapy.Am J Kidney Dis 1988,
11:112-119.
