1 II Department of Infectious Diseases, L. Sacco Hospital, Via G.B. Grassi 74, 20157, Milan; 2 Preclinic Department of Immunology, L. Sacco Hospital, Milan University, Milan, Italy
Received 11 February 2003; returned 12 March 2003; revised 29 April 2003; accepted 29 April 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: immune reconstitution, lymphocyte proliferation, Pneumocystis carinii, highly active antiretroviral therapy, protease inhibitors
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Observational studies suggested that P. carinii primary and secondary prophylaxis can be safely discontinued when CD4 > 200 cells/mm3. However, sporadic PCP has been reported above that threshold.1115 In our ward, we observed three cases of PCP at CD4 > 250 cells/mm3 in patients receiving non-nucleoside reverse transcriptase inhibitor (nNRTI)-based antiretroviral therapy. We studied the response to non-specific and P. carinii-specific antigens (PcAg) in index cases and control groups by lymphocyte proliferation (LP), since it could be clinically useful to have functional tests for specific immunity to opportunistic agents for HIV-1-related clinical decisions.16 HIV-1 infection induces changes in CD4 T cell phenotype and repertoire depletion which are not immediately restored by antiretroviral or immuno-based therapies.17,18 It has been demonstrated that HIV-1 infection of human macrophages modulates cytokine responses to P. carinii so that a virological dissociation (high viral load with relatively high CD4 cell count under therapy) could provide clinically relevant information not provided by a CD4 count.19
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A) 28 HIV-1-infected patients naive to HAART with documented PCP and CD4 count < 200 cells/mm3.
B) 22 asymptomatic HIV-1 positive patients naive to HAART with three subsequent determinations of CD4 > 200 cells/mm3.
C) 44 HIV-1-infected patients immune-reconstituted by HAART (starting antiretroviral therapy with CD4 < 200 cells/mm3 but with CD4 > 200 cells/mm3 for at least three subsequent determinations when tested).
D) 14 healthy HIV-1-uninfected controls.
HAART was defined as a combination of two NRTIs and one protease inhibitor (PI) or nNRTI.
LP assay
After blood collection in EDTA Vacutainer tubes, peripheral blood mononuclear cell (PBMC) separation (FICOLL-Hypaque) was carried out within 4 h of withdrawal. For the LP assay, we used previously published protocols with rat-derived P. carinii antigens and human samples.2023
LP was carried out by measuring [3H]thymidine incorporation after 7 days of culture at 37°C in a 5% CO2 incubator; 105 PBMCs/well were incubated in medium alone (MED) or together with PcAg, FLU, ENV, PHA as antigens in four replicates for each antigen. PcAg was added in a 10:1 ratio of trophozoite/PBMC. On day 6, the wells were pulsed with radioactive thymidine (Amersham) and harvested 18 h later, and the incorporated activity measured as cpm.
P. carinii antigen preparation
Trophozoites were obtained from P. carinii-free SpragueDawley rats infected with P. carinii by transtracheal inoculation and expanded onto human embryonic lung (HEL 299 ATCC CCL137) cells sheeted on microcarrier beads in spinner flasks as previously reported.24,25 Pelleted microorganisms were washed twice in PBS, counted on Giemsa-stained calibrated drops and used fresh or thawed after cryopreservation, at a 10:1 ratio of trophozoites/PBMC. To monitor the purity of P. carinii antigens, aliquots were microscopically checked for morphology, and cultured for bacteria and fungi; lipopolysaccharide (LPS) contamination was excluded by Limulus assay (E-toxate; Sigma, Italy). European guidelines for animal experimentation were followed in the animal study.
Other antigens
MED: culture medium, no stimulator, negative control, background values; PHA: phytohaemagglutinin, positive control for mitogenic activity (2.5 µg/mL); ENV: pool of five antigenic peptides from envelope of HIV-1 (25 mM);26 FLU: influenza virus vaccine prepared with a mixture of A/Taiwan, A/Shangai and B/Victoria (24 mg/mL).
LP assay was carried out twice on each patient, once with fresh PBMC and then, from the same blood sample, with frozen PBMC.
Assessment of clinical and laboratory parameters
The following data were analysed for each HIV-1-infected patient: demographic features, sex, age, risk factor, viral load, absolute counts of CD4, CD8, natural killer (NK) and B cells, and occurrence and type of antiretroviral therapy. For patients with PCP, microscopically diagnosed by bronchoalveolar lavage (BAL) after standard bronchoscopy, data were collected during the overt disease. CD4 count nadir and duration of HAART were also recorded in immune-reconstituted patients.
Informed consent was obtained from all patients and healthy controls involved in the study, according to European guidelines for good clinical practice.
Evaluation of results and statistical analysis
After verification of Gaussian distribution, statistics (two-tailed t-test with P < 0.05) were carried out both on LP data (crude cpm to each antigen minus noise background) and stimulation index (SI), defined as the ratio of median counts per minute of quadruplicate culture with antigen to the median counts per minute in culture medium alone without antigen for PHA, ENV, FLU and PcAg in groups A, B, C and D. A two-tailed t-test with P < 0.05 was also carried out after stratification of immune-reconstituted patients (group C) into two groups (receiving PI-based or nNRTI-based HAART) in order to assess differences in age, CD4 nadir count, therapy duration, actual flow cytometry subsets, viral load, occurrence of opportunistic events, and results of LP to different antigens.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Mean baseline CD4 count before starting HAART (nadir) in immune-reconstituted patients was 73.8 (S.D. 64.7) with an increase in CD4 count of 389.7 (S.D. 151.1) cells after 23.9 months of therapy, without statistically relevant differences between PI-based versus PI-sparing antiretroviral regimens. Table 2 shows the characteristics and lymphocyte proliferation responses of 43 immune-reconstituted patients treated with PI-based or nNRTI-based antiretroviral therapy. One immune-reconstituted patient receiving both nevirapine and indinavir was not included in the statistical analysis. After stratification of immune-reconstituted patients according to therapy (PI versus nNRTI regimens) no differences were seen for CD4 nadir, HAART duration, or actual CD4, CD8, NK or B cell counts.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two-thirds of cases of PCP with relatively high CD4 number under nNRTI-based HAART may have reflected this modulation as suggested by high viral load, but in one case the HIV-1 RNA was <50 copies/mL. We previously detected antipneumocystic activity of various PIs in vitro whereas other authors did not confirm this observation in an animal model using a different scoring system.42,43 However, a partial, aspecific but clinically protective effect against P. carinii could be hypothesized for immune-reconstituted patients receiving PIs in their HAART regimens, since no patient with abnormally low Pc-specific LP receiving PIs developed PCP, although prophylaxis was stopped. In addition to the possible effect on opportunistic microorganisms, interesting studies demonstrated a non-virological possible modulation of host cell proteasome activity by ritonavir. Recently published studies suggest that subtle differences in immune restoration may exist after PI-based versus PI-sparing HAART, despite comparable degrees of viral suppression.44,45 Clinical studies have already analysed patterns of T cell repopulation, virus load reduction and restoration of T cell function in HIV-1-infected persons during therapy with different antiretroviral agents.4648 In our study, three cases of PCP in patients with CD4 > 250 cells/mm3 occurred in nNRTI-containing HAART regimens. Among 44 immune-reconstituted patients, after stratification according to therapy, we found a similar LP without statistically relevant differences in PI-containing versus nNRTI-containing associations.
A specific PcAg LP could be considered for doubtful situations (i.e. patients starting HAART with low CD4 nadir), for a safer clinical decision on discontinuing prophylaxis. In our study, the 25th percentile of the median value of LP to P. carinii of asymptomatic HIV-1 patients naive to HAART is proposed as a cut-off value to discriminate among immune-reconstituted patients those who, showing lower values, could be at risk of developing PCP although having a CD4 count above the threshold, since all PCP patients with CD4 < 200 cells/mm3 had an LP value below this value and all healthy controls, and 70% of group C had an LP value above the same value. This value is also consistent with the observation that 10% of PCP are seen in HIV-1 patients with CD4 > 200 cells/mm3 naive to antiretroviral therapy.
Our in vitro data confirm the clinical observation that certain HIV-1-infected patients retain a relative immunodeficiency that is not reflected in the CD4 cell counts, and are at risk of developing opportunistic events.27,45 This observation reinforces the idea that CD4 count and T helper (TH) cell function are independent variables. Thus, TH dysfunction may be observed in HIV-1-seropositive asymptomatic individuals even before a critical decline in CD4 count.49 These data also confirm that restoration of the ability to produce IL-2 upon antigen stimulation can be observed independently of dramatic changes in CD4 counts in HIV-1-seropositive individuals undergoing therapy.26,47
Such a refined measure of specific immunity, as LP to P. carinii antigen, is not hereby proposed as a routine test since it requires further prospective validation on a wider cohort. However, if validated, it could be clinically useful when focused on patients starting HAART with a very low CD4 nadir or experiencing a sudden decrease in CD4 count, for a safer decision on discontinuing or restarting therapy.50 A further application would be the discrimination of differences in immune reconstitution among patients receiving different HAART, i.e. PI- versus nNRTI-containing regimens, in addition to clinical follow up for opportunistic events.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Autran, B., Careelain, G., Li, T. S. et al. (1997). Positive effects of combined antiretroviral therapy on CD4+ T cells homeostasis and function in advanced HIV-1 disease. Science 227, 1126.[CrossRef]
3 . Sepkowitz, K. A. (1998). Effect of HAART on natural history of AIDS-related opportunistic disorders. Lancet 351, 22830.[ISI][Medline]
4 . Simonds, R. J., Hughes, W. T., Feinberg, J. et al. (1995). Preventing Pneumocystis carinii pneumonia in persons infected with human immunodeficiency virus. Clinical Infectious Diseases 21, S448.[ISI][Medline]
5 . Cassone, A., De Bernardis, F., Torosantucci, A. et al. (1999). In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors. Journal of Infectious Diseases 180, 44853.[CrossRef][ISI][Medline]
6 . Cassone, A., Tacconelli, E., De Bernardis, F. et al. (2002). Antiretroviral therapy with protease inhibitors has an early, immune reconstitution-independent beneficial effect on Candida virulence and oral candidiasis in human immunodeficiency virus-infected subjects. Journal of Infectious Diseases 185, 18895.[CrossRef][ISI][Medline]
7
.
Korting, H. C., Schaller, M., Eder, G. et al. (1999). Effects of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on in vitro activities of secreted aspartyl proteinases of Candida albicans isolates from HIV-infected patients. Antimicrobial Agents and Chemotherapy 43, 203842.
8 . Atzori, C., Angeli, E., Mainini, A. et al. (2000). In vitro activity of HIV-1 protease inhibitors against P. carinii. Journal of Infectious Diseases 181, 162934.[CrossRef][ISI][Medline]
9
.
Derouin, F. & Santillana-Hayat, M. (2000). Anti-Toxoplasma activities of antiretroviral drugs and interactions with pyrimethamine and sulfadiazine in vitro. Antimicrobial Agents and Chemotherapy 44, 25757.
10 . Sgadari, C., Barillari, G., Toschi, E. et al. (2002). HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma. Nature Medicine 8, 22532.[CrossRef][ISI][Medline]
11 . Schneider, M. M., Borleffs, J. C., Stolk, R. P. et al. (1999). Discontinuation of prophylaxis for Pneumocystis carinii pneumonia in HIV-1-infected patients treated with highly active antiretroviral therapy. Lancet 353, 2013.[CrossRef][ISI][Medline]
12
.
Ledergerber, B., Mocroft, A., Reiss, P. et al. (2001). Discontinuation of secondary prophylaxis against Pneumocystis carinii pneumonia in patients with HIV infection who have a response to antiretroviral therapy. New England Journal of Medicine 344, 16874.
13 . Bender, M. & Sax, P. E. (2001). Discontinuing prophylaxis against Pneumocystis carinii pneumonia. New England Journal of Medicine 344, 1639.
14
.
Le Moal, G., Breux, J. P. & Roblot, F. (2001). Discontinuing prophylaxis against Pneumocystis carinii pneumonia. New England Journal of Medicine 344, 163940.
15 . Lim, L., Street, A. C. & Lewin, S. R. (2001). Pneumocystis carinii pneumonia after cessation of secondary prophylaxis in a patient on highly active antiretroviral therapy with a CD4 cell count greater than 200/mm3. AIDS 15, 19123.[CrossRef][ISI][Medline]
16
.
Girard, P. M. (2001). Discontinuing Pneumocystis carinii prophylaxis. New England Journal of Medicine 344, 2223.
17 . Connors, M., Kovacs, J. A., Krevat, S. et al. (1997). HIV infection induces changes in CD4+ T-cell phenotype and depletions within the CD4+ T-cell repertoire that are not immediately restored by antiviral or immune-based therapies. Nature Medicine 3, 53340.[ISI][Medline]
18 . Lange, C. G., Lederman, M. M., Madero, J. S. et al. (2002). Impact of suppression of viral replication by highly active antiretroviral therapy on immune function and phenotype in chronic HIV-1 infection. Journal of Acquired Immune Deficiency Syndromes 30, 3340.[ISI][Medline]
19 . Kandil, O., Fishman, J. A., Koziel, H. et al. (1994). Human deficiency virus type 1 infection of human macrophages modulates the cytokine response to Pneumocystis carinii. Infection and Immunity 62, 64450.[Abstract]
20 . Chatterton, J. M., Joss, A. W., Pennington, T. H. et al. (1999). Usefulness of rat-derived antigen in the serodiagnosis of Pneumocystis carinii infection. Journal of Medical Microbiology 48, 6817.[Abstract]
21 . Hagler, D. N., Deepe, G. S., Pogue, C. L. et al. (1998). Blastogenic responses to Pneumocystis carinii among patients with human immunodeficiency (HIV) infection. Clinical and Experimental Immunology 74, 713.
22 . Forte, M., Maartens, G., Campbell, F. et al. (1992). T-lymphocyte responses to Pneumocystis carinii in healthy and HIV-positive individuals. Journal of Acquired Immune Deficiency Syndromes 5, 40916.[Medline]
23 . Theus, S. A., Sawhney, N., Smulian, A. G. et al. (1998). Proliferative and cytokine responses of human T lymphocytes isolated from human immunodeficiency virus-infected patients to the major surface glycoprotein of Pneumocystis carinii. Journal of Infectious Diseases 177, 23841.[ISI][Medline]
24 . Bartlett, M. S., Fishman, J. A., Queener, S. F. et al. (1998). New rat model of Pneumocystis carinii infection. Journal of Clinical Microbiology 26, 11002.
25 . Lee, C. H., Bauer, N. L., Shaw, M. M. et al. (1993). Proliferation of rat Pneumocystis carinii on cells sheeted on micro-carrier beads in spin- ner flasks. Journal of Clinical Microbiology 31, 165962.[Abstract]
26 . Clerici, M., Stocks, N. I., Zajac, R. A. et al. (1989). Interleukin-2 production used to detect antigenic peptide recognition by T-helper lymphocytes from asymptomatic, HIV-seropositive individuals. Nature 339, 3835.[CrossRef][ISI][Medline]
27 . Connick, E., Lederman, M. M., Kotzin, B. L. et al. (2000). Immune reconstitution in the first year of potent antiretroviral therapy and its relationship to virologic response. Journal of Infectious Diseases 181, 35863.[CrossRef][ISI][Medline]
28 . Li, T. S., Tubiana, R., Katlama, C. et al. (1998). Long-lasting recovery in CD4 T-cell function and viral-load reduction after highly active antiretroviral activity in advanced HIV-1 disease. Lancet 351, 16826.[CrossRef][ISI][Medline]
29 . Komanduri, K. V., Viswnathan, M. N., Wieder, E. D. et al. (1998). Restoration of cytomegalovirus-specific CD4+ T-lymphocyte responses after ganciclovir and highly active antiretroviral therapy in individuals infected with HIV-1. Nature Medicine 4, 9536.[ISI][Medline]
30 . Fournier, S., Rabian, C., Alberti, C. et al. (2001). Immune recovery under highly active antiretroviral therapy is associated with restoration of lymphocyte proliferation and interferon gamma production in the presence of Toxoplasma gondii antigens. Journal of Infectious Diseases 183, 158691.[CrossRef][ISI][Medline]
31 . Dworkin, M. S., Hanson, D. L., Kaplan, J. E. et al. (2000). Risk for preventable opportunistic infections in persons with AIDS after antiretroviral therapy increases CD4+ T lymphocyte counts above prophylaxis thresholds. Journal of Infectious Diseases 182, 6115.[CrossRef][ISI][Medline]
32
.
Herrod, H. G., Valenski, W. R., Woods, D. R. et al. (1981). The in vitro response of human lymphocytes to Pneumocystis carinii antigen. Journal of Immunology 126, 5961.
33 . Kovacs, J. A., Halpern, J. L., Lundgren, B. et al. (1989). Monoclonal antibodies to Pneumocystis carinii: identification of specific antigens and characterization of antigenic differences between rat and human isolates. Journal of Infectious Diseases 159, 6070.[ISI][Medline]
34
.
Walzer, P. D. & Linke, M. J. (1987). A comparison of the antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting. Journal of Immunology 138, 225765.
35 . Smulian, A. G., Theus, S. A., Denko, N. et al. (1993). A 55 kDa antigen of Pneumocystis carinii: analysis of the cellular immune response and characterization of the gene. Molecular Microbiology 7, 74553.[ISI][Medline]
36 . Harmsen, A. & Stankiewicz, M. (1990). Requirement for CD4+ cells in resistance to Pneumocystis carinii pneumonia in mice. Journal of Experimental Medicine 172, 93745.[Abstract]
37 . Roths, J. B. & Sidman, C. L. (1992). Both immunity and hyperresponsiveness to Pneumocystis carinii result from transfer of CD4+ but not CD8+ T cells into severe combined immunodeficiency mice. Journal of Clinical Investigation 90, 6738.[ISI][Medline]
38 . Garvy, B. A., Ezekowitz, R. A. & Harmsen, A. G. (1997). Role of gamma interferon in the host immune and inflammatory responses to Pneumocystis carinii infection. Infection and Immunity 65, 3739.[Abstract]
39 . Garvy, B. A., Wiley, J. A., Gigliotti, F. et al. (1997). Protection against Pneumocystis carinii pneumonia by antibodies generated from either T helper 1 or T helper 2 responses. Infection and Immunity 65, 50526.[Abstract]
40
.
Hanano, R., Reifenberg, K. & Kaufmann, S. H. (1998). Activated pulmonary macrophages are insufficient for resistance to Pneumocystis carinii. Infection and Immunity 66, 30514.
41 . Chen, W., Havell, E. & Harmsen, A. G. (1992). Importance of endogenous tumor necrosis alpha and gamma interferon in host resistance against Pneumocystis carinii infection. Infection and Immunity 60, 127984.[Abstract]
42 . Atzori, C. & Cargnel, A. (2002). Human immunodeficiency virus protease inhibitors and Pneumocystis carinii. Journal of Infectious Diseases 185, 16923.
43 . Walzer, P. & Cussion, M. (2002). Reply to: human immunodeficiency virus protease inhibitors and Pneumocystis carinii. Journal of Infectious Diseases 185, 16934.[CrossRef][ISI]
44
.
André, P., Groettrup, M., Klenerman, P. et al. (1998). An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proceedings of the National Academy of Sciences, USA 95, 131204.
45 . Smith, K. Y., Steffens, C. M., Truckenbrod, A. et al. (2002). Immune reconstitution after successful treatment with protease inhibitor-based and protease inhibitor-sparing antiretroviral regimens. Journal of Acquired Immune Deficiency Syndromes 29, 5445.[ISI][Medline]
46 . Clerici, M., Roilides, E., Butler, K. M. et al. (1992). Changes in T-helper cell function in human immunodeficiency virus-infected children during didanosine therapy as a measure of antiretroviral activity. Blood 80, 2196202.[Abstract]
47 . Kelleher, A. D., Carr, A., Zaunders, J. et al. (1996). Alterations in the immune response of human immunodeficiency virus (HIV)-infected subjects treated with an HIV-specific protease inhibitor, ritonavir. Journal of Infectious Diseases 173, 3219.[ISI][Medline]
48 . Pakker, N. G., Roos, M. T., van Leeuwen, R. et al. (1997). Patterns of T-cell repopulation, virus load reduction, and restoration of T-cell function in HIV-infected persons during therapy with different antiretroviral agents. Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology 16, 31826.[ISI][Medline]
49 . Clerici, M., Stocks, N. I., Zajac, R. A. et al. (1989). Detection of three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging. Journal of Clinical Investigation 84, 18929.[ISI][Medline]
50 . Miller, V., Phillips, A. N., Clotet, B. et al. (2002). Association of virus load, CD4 cell count, and treatment with clinical progression in human immunodeficiency virus-infected patients with very low CD4 cell counts. Journal of Infectious Diseases 186, 18997.[CrossRef][ISI][Medline]
|