The natural history of HIV-1 infection is characterized by progressive decline in CD4 T-cell number and function that places infected persons at risk for opportunistic infections and neoplasms [1–5]. In recent years significant immune reconstitution has been achieved by highly active antiretroviral therapy (HAART) resulting in a dramatic decline in HIV-1 related morbidity and mortality [6–9]. While in the short term, restoration of immune function seems to largely protect persons from the major opportunistic complications of advanced disease, functional immune restoration is often incomplete [10–18] and the long-term clinical significance of this ‘subclinical’ immune deficiency is unclear.
At the same time, there is uncertainty regarding the optimal timing for the initiation of HAART [19–22]. While some delay in therapy initiation may not increase short-term risks for opportunistic infections, the effects of sustained HIV replication on restoration of functional immunologic competence have been incompletely explored.
The ability to respond to immunization can serve as a model for host defense against microbial challenge. Microbial challenge and vaccine antigen challenge can test both afferent and efferent limbs of immune recognition. In addition, quantification of responses to immunization can provide a detailed characterization of immunologic competence that reflects the ability to mount a response to a new challenge. Importantly, circulating CD4 T-cell counts alone may be incomplete predictors of immunologic competence. Our earlier studies have demonstrated that both viral replication and immune activation predict poorer responses to immunization in HIV infected persons on HAART, while higher level expression of the co-receptor for T-cell activation – CD28 – predicts better functional responses . Moreover, a near doubling of CD4 T-cell numbers after interleukin-2 administration in HAART treated persons with moderately advanced disease does not improve responses to immunization .
For these reasons, we immunized HIV-infected patients who had normalized their CD4 T-cell counts on suppressive antiretroviral therapies in order to learn if delaying the initiation of HAART administration affected the functional immunologic reserve irrespective of virologic control and current CD4 T-cell levels. We found that pretreatment CD4 T-cell nadirs and the expression of CD28 on CD4 T cells predicted the ability to respond to immunization while CD4 T-cell numbers at the time of immunization did not.
HIV-1 infected patients followed at the John T. Carey Special Immunology Unit of University Hospitals of Cleveland and the Infectious Diseases outpatient clinic at MetroHealth Medical Center in Cleveland, Ohio, USA with most recent CD4 T-cell counts > 450 × 106/l, and plasma HIV RNA levels consistently < 400 copies/ml during the previous 12 months while treated with at least three antiretroviral drugs were enrolled between January and June 2001. All eligible patients (n = 32) identified in this consecutive sample volunteered to participate. Ten HIV-1 seronegative subjects participated as a control group.
Individuals with an acute febrile illness, a history of adverse events after immunizations, a history of cytokine therapy or antineoplastic chemotherapy and pregnant or nursing women were excluded. Informed consent was obtained from all patients and controls in accordance with US Department of Health and Human Services guidelines. This study was approved by the institutional review boards of University Hospitals of Cleveland and the MetroHealth Medical Center.
Immunizations and immunologic evaluations
Immunization with diphtheria/tetanus toxoid (0.5 ml, Diphtheria/Tetanus Toxoid Fluid, Aventis Pasteur, Swiftwater, Pennsylvania, USA) was by intramuscular injection; keyhole limpet hemocyanin (KLH; 0.1 mg/0.1 ml, KLH-ImmuneActivator, Intracel Corp., Rockville, Maryland, USA) was administered by intradermal injection.
Subjects were evaluated at baseline and immunized on days 3 and 31. Lymphocyte proliferation and serologic responses were measured twice before immunization and on days 17, 31 and 59. For baseline values, pre-immunization data from days 1 and 3 were combined.
Immunologic and virologic evaluations
Lymphocyte subsets were enumerated in freshly obtained whole blood using directly labeled murine monoclonal antibodies against CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62L, CD95, HLA-DR and CD38 (PharMingen, San Diego, California, USA) by three-color flow cytometry using ACTG consensus methods . Absolute lymphocyte counts were derived from complete blood counts and leukocyte differential counts.
Peripheral blood mononuclear cells were prepared by Ficoll-Hypaque density sedimentation, and were used for assays of lymphocyte proliferation (LP) in response to antigens of Candida albicans (CASTA; 20 μg/ml; Greer Laboratories, Lenoir, North Carolina, USA), Cytomegalovirus (CMV; 1 : 40 dilution; BioWhittaker, Walkersville, Maryland, USA), diphtheria toxoid (DT; 2 limiting flocculation units (LFU)/ml; Wyeth-Ayerst, Marieta, Pennsylvania, USA), HIV p24 (3.35 μg/ml, Protein Sciences Corporation, Meriden, Connecticut, USA), KLH (10 μg/ml) Mycobacterium avium complex (MAC; 5 μg/ml; kindly provided by R. Wallis, UMDNJ, Newark, New Jersey, USA); tetanus toxoid (TT; 2 LFU/ml; Wyeth-Ayerst), streptokinase (SK; 200 μg/ml; Sigma, St. Louis, Missouri, USA), and pokeweed mitogen (PWM; 5 μg/ml; Sigma). Proliferation was also measured in response to control culture supernatants from baculovirus cell lines for HIV-1 p24 (Protein Sciences Corporation) and uninfected control cell lysates for CMV antigen (BioWhittaker). Results are expressed as the stimulation index (SI), defined as the ratio of the median counts per minute (c.p.m.) of quadruplicate cultures with antigen to the median c.p.m. in culture medium without antigen. SI values < 1 were imputed a value of 1.
Delayed-type hypersensitivity (DTH) responses to antigens of Candida albicans (1 : 5000/0.1 ml; Candin Skin Test Antigen for Cellular Hypersensitivity, Allermed Laboratories, San Diego, California, USA), TT (0.08 LFU/0.1 ml; Tetanus Toxoid USP, Aventis Pasteur), and KLH (0.05 mg/0.1 ml, KLH-ImmuneActivator) were measured 48–72 h after intradermal administration using the ball-pen technique .
Anti-tetanus, anti-diphtheria and anti-KLH IgG antibodies were measured by enzyme immunoassays as described elsewhere .
Plasma HIV-1 RNA was measured using quantitative HIV-1 RNA PCR assays (Amplicor HIV-1 MONITOR; Roche Molecular Systems, Branchburg, New Jersey, USA) with a lower limit of detection of 400 copies/ml.
Definition of responses to immunization
A stimulation index of 10 or more and a 0.67 log10 SI increase from baseline was chosen to define a vaccination response because only 11% of subjects had an SI to the presumed neoantigen KLH > 10 prior to immunization and the standard deviation (SD) in SI to the control antigen Candida was 0.67 log10 in the absence of intervention.
A 10-mm induration was considered a stringent criterion for a positive skin test response . Because with time and without intervention, the induration response to Candida changed a mean ± SD of 2 ± 6 mm, we also required that the postimmunization induration represented at least a 6-mm increase from baseline.
Antibody levels > 0.1 U/ml that also represented a fourfold increase in antibody concentration following vaccination were considered a response [27,28].
To summarize functional immune responses, three cumulative immune scores were developed. The first score (score A) included results of all evaluations (antibody levels, LP and DTH) after immunization. A response as defined received one point and a non-response zero points. All points were added to derive the score. Since many patients had significant baseline immune responses to tetanus that did not increase after immunization, inclusion of responses to this antigen could dilute real differences in responses. We therefore generated a second score that excluded tetanus evaluations (score B). Finally, since excluding only tetanus responses could be considered arbitrary, we generated a third score—the score fraction (score C). For this analysis, all positive baseline responses (SI > 10, DTH > 10, antibody > 0.1U/ml) were excluded from consideration and a fractional response was generated. The denominator represented the total possible number of responses a given participant could have developed and the numerator represented the total number of responses that the participant actually developed after immunization.
Continuous variables were compared using non- parametric testing (Mann–Whitney or Kruskal–Wallis) and categorical variables were compared using the χ2 test. Correlations were performed using the Spearman rank correlation and variables were tested for normality using the Kolmogorov–Smirnoff test. P values were considered significant if < 0.05; no corrections were made for multiple comparisons. Variables found to be significantly associated with the immunization score (P < 0.1) were entered into a linear regression model to determine the relationship between nadir CD4 T-cell counts and immunization scores. Models were selected using the R-squared method. The dependent (immunization score) and independent variables were tested for normality. Independent variables were also tested for colinearity using variance inflation factor and no significant multicolinearity problems were found. No heteroscedasticity was found when analyzing the variance of residuals. Statistical analyses were computed using SPSS software (version 10.0.7, SPSS corp., Chicago, Illinois, USA).
Thirty-two HIV-1 infected patients and 10 HIV-1 seronegative age-matched controls were enrolled. Three patients who could not adhere to the protocol schedule withdrew. One female control subject discontinued participation when fever and chills occurred after the second immunization with DT/TT and KLH on day 31. Data from these four subjects are not included in the analysis.
Twenty-nine HIV-1 infected patients (27 male, two female; 21 Caucasian, six African–American, two Hispanic) and nine controls (two male, seven female, eight Caucasian, one African–American) received all skin tests and immunizations and were included in the final analysis. To explore whether there tended to be a difference in functional immune responses according to HIV status and nadir CD4 T-lymphocyte count, we divided the cohort of HIV-infected patients in two groups according to the median nadir CD4 T-cell count. Baseline characteristics of the study population thus divided are shown in Table 1. Nine of 15 patients with nadir CD4 T-cell counts < 250 × 106/l had a history of AIDS defining illnesses (Pneumocystis carinii pneumonia, four; Kaposi′s sarcoma, three; disseminated MAC infection, two; HIV-associated wasting, two; HIV-associated dementia, two; Candida esophagitis, two; disseminated histoplasmosis, one; pulmonary tuberculosis, one). Some patients had more than one AIDS-defining illness.
Accurate information on prestudy immunization with DT/TT was not available for three patients in group I, two patients in group II, and one HIV-1 seronegative control subject (11%). In all other patients, the median interval between the last DT/TT immunization and study entry was 42 months (range, 13–129 months) for group I, 37 months (range, 14–95 months) for group II and 40 months (range, 13–120 months) for controls. These intervals were not significantly different.
Lymphocyte phenotypes at time of first immunization are presented in Table 2. Lymphocyte phenotypes were comparable in the patient groups.
HIV-1 infected patients had fewer naive CD4 T cells (CD45RA+CD62L+) (group A versus C, P < 0.005; group B versus C, P < 0.01), fewer CD4 T cells expressing the co-stimulatory molecule CD28 (P < 0.05), and more total CD8 T cells (group A versus C, P < 0.005; group B versus C, P < 0.05) and activated CD8 T cells (HLA-DR+ CD38+; P < 0.001) than the controls.
Antibody levels, lymphoproliferation and DTH skin reactions
Median functional immune responses to immunization are shown in Fig. 1. All patients and controls had protective anti-toxin levels against tetanus and diphtheria (> 0.01 IU/ml ) at study entry whereas none of the patients or controls had KLH-specific antibodies prior to immunization. On the last evaluation following immunization (day 59) patients had significantly lower antibody concentrations against DT (P < 0.005) and KLH (P < 0.001) than controls.
The median lymphocyte proliferation responses to KLH (P < 0.05; P < 0.005), TT (P < 0.05; P < 0.005) and DT (P < 0.05; P < 0.005) were also lower in HIV-infected patients at baseline and after immunization when compared to those of controls, while SI in response to either MAC, SK, CMV, C. albicans, HIVp24 and PWM were comparable pre- and postimmunization in patients and controls. The magnitudes of DTH skin test indurations in response to the recall antigen TT were smaller in patients than in controls prior to immunization (P < 0.01), but were comparable among the groups after immunization on day 59. After immunization, DTH responses to KLH were smaller in patients than in controls (P < 0.005).
Immune response scores
When the data were analyzed by univariate regression analysis the CD4 T-cell nadir significantly correlated with and predicted the magnitude of responses to immunization regardless of the immune response scores used (Spearman′s ρ = 0.5–0.6; P < 0.005 to P < 0.001) (Table 3, Fig. 2). In addition, the percentage (score B, ρ = 0.5; P < 0.005) or number (score C, ρ = 0.04; P < 0.05) of CD4 T cells that co-expressed CD28 at the time of immunization also were correlated with immune response scores in HIV-1 infected patients (Fig. 3). The expression of CD28 on CD4 T cells and the nadir CD4 T-cell count were both independent predictors of the immune response scores (IRS) and did not correlate with each other. Of note, the relationship between the CD4 T-cell nadir and the IRS values was linear without an apparent threshold. Importantly, there was no correlation between the IRS and CD4 T-cell counts at the time of immunization (ρ = 0.2; P = 0.42).
In multivariate models using the variables correlated with the immune response scores in univariate analyses, the nadir CD4 T-cell count (score A, B and C) and the absolute (score C) or percentage (scores A and B) of CD4 T cells that co-expressed CD28 were the only independent variables included in the final model (Table 3). The model using CD4+CD28+ T cells and nadir CD4 T-cell counts explained approximately half of the variability seen in the patients′ immune response scores.
Using a model of in vivo immune response to immunization challenge, we analyzed functional and phenotypic indicators of immune recovery in patients with chronic HIV-1 infection who initiated suppressive antiretroviral therapies at a broad range of CD4 T-cell count nadirs and who experienced ‘normalization’ of circulating CD4 T-cell numbers thereafter.
In contrast to previous investigations that compared the pretreatment CD4 T-cell nadir and immune-phenotype and -function in the response to HAART [10,15,16,25,30–32] we here performed a comprehensive examination of in vivo immune function, including measurement of antibody concentrations, LP and DTH, in response to vaccination with recall antigens and a presumed neo-antigen. We found that analyses of responses to immunization can discriminate functional differences in immune status even in persons with comparable circulating CD4 T-cell counts. HAART-treated patients with normal CD4 T-lymphocyte counts exhibited a broad range of responses to immunization. These responses to immunization could be predicted by the number or percentage of CD4 T lymphocytes co-expressing CD28 at the time of immunization and by the nadir pretreatment CD4 T-cell count. Our results suggest that even when CD4 T-cell counts have ‘normalized’ prior immune decline determines current immune competence. Changes in the immune phenotype persist in HIV-1 infected patients even after years of suppressive antiviral therapy [10,15].
It should be noted that we studied a highly selected group of chronically HIV-1 infected patients, each of whom had a very favorable and durable response to antiretroviral treatment and an associated normalization of CD4 T-cell counts. Thus in an unselected patient population who initiate HAART, even more profound effects of CD4 T-cell nadir on functional restoration might be expected. Whether longer duration of suppressive therapy will result in greater enhancement of functional immune reserve remains to be determined.
While CD4 T-cell counts provide an indicator of immune competence in HIV disease, reasonably predicting short-term risk for opportunistic infection , the relationship between CD4 T-cell nadir and functional immune competence is incompletely understood. Several cross-sectional studies [19–22] and a recently reported collaborative analysis of 13 prospective cohort studies show that delaying antiretroviral therapy is associated with a greater risk of opportunistic infection and death . In these studies CD4 T-cell counts at the time of infection or deaths have not been reported.
Our results are in agreement with earlier studies that failed to show normalization of antibody and cellular responses to immunization [23,35,36] in HIV-1 infected patients with moderately advanced disease. We show here a linear relationship between the immune response scores and the nadir CD4 T-cell count suggesting that functional immune restoration is attenuated by prior depletion of the CD4 T-cell pool without an apparent threshold for complete immune reconstitution. Whether such a threshold exists should be determined in prospective studies enrolling larger numbers of patients.
Complete normalization of lymphocyte phenotoypes was not achieved in these successfully treated patients as numbers of circulating CD4+CD45RA+CD62L+ naive T-cells and CD4 T-cells expressing the CD28 co-receptor for activation were lower and numbers of circulating CD8 T-cells and CD8 HLA-DR+, CD38+ activated T-cells were elevated in patients when compared to HIV-1 seronegative persons. Ongoing activation of CD8 T-cells and lower numbers of naive CD4 T-cells were observed in HIV infected individuals regardless of the CD4 T-cell nadir and there was no relationship among the numbers of HLA-DR+, CD38+CD8+ T-cells and CD4+CD45RA+ CD62L+ T-cells or the IRS. Since naive T-cell restoration may be delayed yet persistent [37–42], the on-treatment period in this study may have been insufficient to permit ‘complete’ naive T-cell reconstitution. Longer duration of follow-up in these patients will be needed to learn if subclinical immune deficiency persists and predicts long-term morbidities or if it progressively normalizes.
CD28 expression on CD4 T-cells is diminished in untreated HIV-1 infection [43,44] and failure of co-stimulation through this receptor may contribute to decreased lymphocyte proliferation  and anergy  after T-cell receptor engagement. Cellular proliferation is a critical factor in immunization responses. We have demonstrated earlier a profound defect in proliferation after T-cell receptor engagement among CD4 and CD8 T-cells in HIV infection [47,48] that is also predicted by CD4 T-cell nadir  and is at least in part related to diminished CD28 expression (S.F. Sieg and M.M. Lederman, unpublished data).
Sustained suppression of viral replication results in increases in the frequency of CD28 expression [49,50]. Here we confirmed a previous finding of our group that CD28 expression on CD4 T-cells is an important predictor of the ability to respond to immunization in HIV-1 infection . We suspect, therefore, that proliferation failure of T-cells in HIV disease, in part related to diminished expression of CD28, is an important determinant of suboptimal responses to immunization. Our results suggest that quantification of CD28 expression on CD4 T-cells may be superior to the enumeration of absolute circulating CD4 T-cells alone as a surrogate marker of the functional immune status in HIV-1 infected patients.
It remains unclear why the numbers of activated CD8 T-cells expressing HLA-DR and CD38 do not return to normal levels despite longstanding suppression of viral replication [15,49,51]. Whether this activation state is sustained by low level viral replication or whether the activation and survival of these cells persists without antigen exposure needs to be further explored .
We here show that even after normalization of CD4 T-lymphocyte numbers, in vivo immune function as measured by responses to immunization is significantly impaired in persons who delayed therapy initiation. Although this study was not designed primarily to evaluate the optimal timing of treatment initiation in chronic HIV-1 infection, our data suggest that delaying treatment significantly increases the risk of a persistent functional immune defect. Our data demonstrate that even in those persons who normalize CD4 T-cell numbers and are largely protected from opportunistic infection, immune deficits persist.
Interpretation of our results is limited in several ways. Detailed pretreatment phenotypic and functional analyses were not available. Only patients with longstanding suppression of viral replication and ‘normalization’ of CD4 T-cell counts (> 450 × 106/l) on HAART were included in the study and only a fraction of the patients with low CD4 T-cell nadirs were likely to reach a CD4 T-cell count > 450 × 106/l permitting inclusion in this study. In this regard our findings probably underestimate the magnitude of the persistent immune defect attributable to delayed treatment initiation. Since persons with CD4 T-cell restoration on HAART have to date been protected from major AIDS-defining complications, the long-term clinical relevance of a persistent partial immune deficiency remains to be determined. The clinical significance of our findings regarding decisions for the timing of treatment initiation is therefore unclear.
In summary, we found that even in persons with longstanding suppression of viral replication who achieve normalization of circulating CD4 T-cell counts, functional immune-reconstitution is incomplete in patients who start HAART at more advanced stages of HIV-1 infection. Complete functional immune reconstitution is progressively attenuated by CD4 T-cell depletion prior to the initiation of HAART and is also predicted by the expression of the CD28 co-receptor for T-cell activation on CD4 T-lymphocytes. Thus, delay in initiation of therapy results in impaired functional immune restoration. The long-term consequences of persistent subclinical immune deficiency in this setting are not known and should be monitored prospectively.
The authors thank all patients and volunteers for their participation in the study, the physicians and staff of the John T. Carey Special Immunology Unit at University Hospitals of Cleveland for their assistance and for their dedicated patient care, H. Lederman and A. Swift (Johns Hopkins University) for measuring antibody levels, and D. Dorazio, R. Mohner and M. Banchy for technical assistance.
Sponsorship: Supported by grants AI36219, AI50501, and AI38858 from the National Institutes of Health, grant awards from the Foundation for AIDS and Immune Research and Agouron Pharmaceuticals, and generous donations to our AIDS research fund.
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