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Active Anti-Interferon-α Immunization: A European-Israeli, Randomized, Double-Blind, Placebo-Controlled Clinical Trial in 242 HIV-1-Infected Patients (the EURIS Study)

Gringeri, A.*; Musicco, M.; Hermans, P.; Bentwich, Z.§; Cusini, M.; Bergamasco, A.; Santagostino, E.*; Burny, A.#; Bizzini, B.**; Zagury, D.#; the EURIS Study Group

Journal of Acquired Immune Deficiency Syndromes & Human Retrovirology: 1 April 1999 - Volume 20 - Issue 4 - pp 358-370

This randomized, double-blind, placebo-controlled, phase II/III study was designed to evaluate safety, immunogenicity, and efficacy of an active anti-interferon-α (anti-IFN-α) vaccine in asymptomatic HIV-1-infected patients. The active immunization was aimed at inducing anti-IFN-α antibodies to counteract IFN-α overproduction. In all, 242 patients, recruited between December 1995 and July 1996 in eight centers in Europe and Israel, with CD4+ counts from 100 to 634 cells/mm3 who were receiving or not receiving antiretroviral therapy (including protease inhibitors) were randomized to receive either anti-IFN-α vaccine or placebo. The anti-IFN-α immunization regimen consisted of three priming injections delivered intramuscularly at 1-month intervals in a water-in-oil emulsion of inactivated recombinant IFN-α-2b (i-IFN-α) followed by intramuscular booster injections of i-IFN-α adsorbed onto calcium phosphate every 3 months. Immunogenicity to vaccine was defined as an increase of anti-IFN-α antibody level of more than twofold the preimmunization value. Clinical progression, changes in antiretroviral treatment, and decrease of CD4+ counts to <200 cells/mm3 were considered endpoints for efficacy evaluation. Contrary to our previous experience, in which six to seven oil priming injections induced a >90% response rate, the three oil-adjuvanted injections in this trial were suboptimal because only 40 of 122 vaccinees (33%) had raised anti-IFN-α antibody following immunization. In vaccinees, both antibody responders (AbRV) and nonresponders (AbNRV), the tolerance to the vaccine was good and was without evidence of significant safety concerns. During the course of the trial, 62% of vaccine responders, 64% of nonresponders, and 63% of placebo patients elected to add protease inhibitor-containing regimens as new treatment guidelines were established, resulting in a marked decrease in clinical and laboratory progression such that the expected endpoints of the study could not be achieved and further follow-up was halted. Despite the unexpectedly low immunogenicity and fewer than expected endpoints, anti-IFN-α vaccine recipients, in comparison with placebo recipients, showed a lower rate of disease progression, nonelective treatment changes, and/or CD4+ count decrease to <200 cells/mm3, but the difference was not statistically significant. Nevertheless, the subgroup of patients immunized to IFN-α who experienced a rise in anti-IFN-α antibodies had a significantly lower rate of occurrence of HIV-1-related events and of any combination of the endpoints compared with those of either placebo patients or vaccinees who failed to develop anti-IFN-α antibodies, the latter two groups behaving similarly. Further studies of this approach are warranted because these data suggest a beneficial effect of this adjuvant approach.

*A. Bianchi Bonomi Hemophilia and Thrombosis Center, Istituto Ricovero e Cura a Carattere Scientifico Maggiore Hospital and University of Milan; †Istituto Technologie Biomediche Avanzate, National Research Council, Milan, Italy; ‡Division of Infectious Diseases, CHU St. Pierre, Brussels, Belgium; §R. Ben Ari Institute of Clinical Immunology and AIDS Center, Kaplan Medical Center, Rehovot, Israel; Sexually Transmitted Diseases Center, Dermatology Department, Istituto Ricovero e Cura a Carattere Scientifico Maggiore Hospital and University of Milan, Milan; ¶Clinica di Malattie Infettive e Tropicali, University of Brescia and Spedali Civili di Brescia, Brescia, Italy; #Department of Molecular Biology and J. Bordet Institute, Free University of Brussels, Brussels, Belgium; and **Laboratoire de Physiologie Cellulaire, Pierre and Marie Curie University, Paris, France

Address correspondence and reprint requests to Alessandro Gringeri, Institute of Internal Medicine, IRCCS Maggiore Hospital, Via Pace, 9, 20122, Milan, Italy.

Manuscript received June 26, 1998; accepted December 7, 1998.

Progressive T-cell immunosuppression and constitutive T-cell loss characterize HIV-1 infection. These phenomena are due to lysis of infected cells following immune activation, with the subsequent release of virions (1), and to HIV-1-induced immune disorders associated with cytokine dysregulation (2). In particular, the impairment of cytokine network homeostasis is characterized by decrease of interleukin-2 (IL-2) and IL-12 secretion and overproduction of interferon-α (IFN-α) (3-6).

Overproduction of IFN-α (5,6), which is known to inhibit cell proliferation and to induce apoptosis of activated T cells (7), was detected in tissues of patients with early stages of HIV-1 infection (8). High levels of IFN-α above the critical level of 30 IU/ml, which predicts progression (9) are present in sera of AIDS/AIDS-related complex (ARC) patients and of macaque monkeys (Macaca mulatta) with AIDS-like disease, but are absent in sera of asymptomatic HIV-1-infected chimpanzees and asymptomatic simian immunodeficiency virus (SIV)-infected macaques (10). IFN-α overproduction results from IFN-α-induced activation of antigen-presenting cells, including macrophages and dendritic cells (11). Some authors have also shown that HIV-1 envelope gp160/gp120 glycoproteins induce endogenous production of IFN-α and IFN-γ (12). Moreover, anti-IFN-α antibodies could partly restore in vitro the reduced IL-2 production by peripheral blood mononuclear cells (PBMCs) originating from ARC/AIDS patients.

Current antiviral therapy for HIV-1 infection, based on reducing HIV-1 replication rates, especially those using combination of reverse transcriptase and protease inhibitors have shown efficacy (13), but residual reservoirs of replicative competent viruses persist and may account for emergence of multidrug resistance and treatment failures (14,15). New biologic approaches under investigation, including hydroxyurea, anti-gag proteins vaccination, and cytokine-modulating therapy, such as IL-2 and IL-12 administration (16-18) and anti-IFN-α immunization (19-23), may enhance the capacity to clear or down-modulate these latent persistent populations of virus.

Several reports indicate that a vaccine with inactivated IFN-α is both safe and feasible (19-26). In particular, a 9-month, randomized, placebo-controlled study in 22 HIV-1-infected Italian patients (21) showed that active immunization with inactivated IFN-α adjuvanted with mineral oil (IFA) in water-in-oil emulsion for six priming injections and with Ca(OH)PO4 in water suspension for boosting injections was safe and highly immunogenic, whereas a study with IFN-α absorbed into Ca(OH)PO4 for priming and boosting injections failed to elicit an immune response (26).

Long-term (>30 months) follow-up of patients on clinical trials showed that anti-IFN-α immunization consisting of repeated injections of water-in-oil preparations for priming (21,23), increased specific antibodies and decreased circulating IFN-α levels (23), even in severely immunocompromised patients who were receiving antiretroviral therapy. Moreover, these clinical trials provided additional evidence on the role of IFN-α in AIDS immunopathogenesis, in that the occurrence rate of AIDS-related clinical manifestations was found to increase in patients with high circulating IFN-α levels.

These data prompted us to carry out a multicenter, double-blind, placebo-controlled phase II/III trial with three objectives: to confirm the safety, tolerance, and compliance of IFN-α, vaccine preparation; to determine whether the use of three injections of inactivated IFN-α in incomplete Freund's adjuvant (IFA) followed by boosters in calcium phosphate is optimal to induce IFN-α antibodies; and to evaluate whether, in association to antiretroviral therapy, those HIV-1-infected patients who were immunized to IFN-α (i.e., antibody responder vaccinees) show clinical improvement in comparison to nonimmunized patients (i.e., antibody nonresponders and placebo recipients).

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This trial was designed to evaluate safety, immunogenicity, and efficacy of an active anti-IFN-α immunization in HIV-1-infected patients. Eligibility criteria were asymptomatic HIV-1 disease, CD4+ cell counts ranging from 100 to 650 cells/mm3 and signed informed consent to participate in the trial. Concurrent treatment with immunosuppressive drugs or active IFN-α was considered as an exclusion criterion, but patients could be treated with antiretroviral drugs providing that no change in the treatment schedule had been introduced in the previous 3 months. Concomitant chronic hepatitis B and C virus (HCV) infections were not exclusion criteria (21-23). Patients were randomly assigned to receive in a double-blind fashion either an anti-IFN-α or a placebo preparation. Separate randomization lists were drawn for patients with CD4+ counts ≤350 cells/mm3 (group A) and >350 cells/mm3 (group B) and for each recruiting center.

CD4+ cell count and viral load are accepted surrogate markers of disease progression. Our patients, at inclusion and at any time during follow-up, were allowed to be treated with antiretrovirals that are known to influence these surrogate markers. For this reason, efficacy evaluation was based on three clinical endpoints: death; occurrence of clinical signs related to HIV-1 disease progression (U.S. Centers for Disease Control and Prevention [CDC] stage B or C) and changes or initiation of antiretroviral therapy for disease progression, evidence of treatment failure, or both; decrease of CD4+ count to <200 cells/mm3 was added as a secondary endpoint for those patients with a starting value above the threshold. A change or initiation of antiretroviral therapy was considered to result from clinical and/or biologic deterioration of HIV-1 disease status in case of occurrence of HIV-1-related symptoms or signs (CDC group B and C), increase in plasma viremia, CD4+ counts falling to <200 cells/mm3 or CD4+ cell counts decreasing >30% compared with pretreatment values. Any change of treatment due to side effects, commercial availability of new drugs, patients' request, and/or adhesion to national or international guidelines for treatment of HIV-1 disease was not considered an endpoint. Among our patients, 63% of placebo and vaccine recipients elected to change treatment regimens when protease inhibitors become widely available. Investigators were required by the study protocol to specify the reason of any change of antiretroviral treatment.

The study was planned to provide follow-up observation of all patients for 24 months on an intention-to-treat basis. However, the Steering Committee terminated the trial in February 1998 because the elective addition of antiviral protease inhibition had dramatically reduced trial endpoints (discussed later). Patients remained in the trial even if they had demonstrated one or more of the study endpoints. Assessment of the endpoints and laboratory testing were performed at 3-month intervals or whenever needed. Determinations of CD4+ and CD8+ lymphocyte subsets were performed by fluorescent activated cell flow cytometry at each trial Center. HIV-1 p24 antigenemia was centrally measured by sandwich enzyme-linked immunoassay (Abbott Laboratories, North Chicago, IL, U.S.A.). Plasma HIV-1 viremia was centrally evaluated in most patients (85.5%) by multitarget DNA-RNA PCR, according to the previously reported quantitative method (Virionquant, Paris, France), with a detection threshold of 10 HIV-1 RNA equivalent copies/ml (27). The remaining 14.5% were assayed by Amplicor HIV-1 Monitor (Roche Diagnostic Systems, Nutley, NJ, U.S.A.), with a threshold of 400 HIV-1 RNA copies/ml. HIV-1 viremia was expressed as HIV-1 RNA equivalent copies × 104/ml. Routine standard laboratory examinations, including liver function tests, were also performed.

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Vaccine Preparation and Immunization Regimen

The immunogen was an albumin-free preparation of recombinant human IFN-α-2b (Biosidus, Buenos Aires, Argentina), 2.5 × 108 IU/mg of protein, inactivated by chemical treatment (25). The treatment resulted in a completely inactivated IFN-α (i-IFN-α) preparation, as assayed by conventional biologic test based on inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) on Madin-Darby bovine kidney (MDBK) cells, but with preservation of its immunogenicity.

The immunogen was injected intramuscularly three times at 1-month intervals at a dose of 250 μg, as a water-in-oil emulsion (IFA, mineral oil ISA-050 by Seppic, Paris, France). Priming injections were followed by booster injections administered every 3 months, with the immunogen adsorbed onto a calcium phosphate gel (Superfos, Copenhagen, Denmark) in water. This immunizing regimen using a reduced number (three) of oil (i.e., IFA) injections for priming was chosen to reduce the risk of local discomfort and pain observed in previous studies (21-23). Indeed, CaOHPO4 injections are less painful than oil injections. In addition, vaccine preparations adjuvanted with CaOHPO4 in water are more stable at room temperature and easier to handle at mass level than oil emulsions that are stable in the longer term under more strictly controlled conditions. Furthermore, CaOHPO4, which did not prove to be appropriate for priming in the ANRS Paris Trial (26), could represent an effective adjuvant for booster inoculations, as shown in a previous trial (23).

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Sera of enrolled patients collected every 3 months starting from the first injection were blindly evaluated by enzyme-linked immunosorbent assay (ELISA) for anti-IFN-α antibodies and the results expressed as optical density (OD). Because of the blind evaluation, it was not until the code was broken that the lack of adequate immunogenicity for the three injections priming regimen was appreciated. According to study protocol, the variation of anti-IFN-α antibody levels was used for differentiating two groups of immunized subjects: immunized patients, who developed increased anti-IFN-α antibody levels (antibody responder vaccinees [AbRV]) and nonimmunized patients, who did not, including vaccine recipients (antibody nonresponder vaccinees [AbNRV]). The placebo (Pla) group received the adjuvant without the immunogen. The antibody responders had to demonstrate in any determinations following immunization an anti-IFN-α antibody level >0.5 OD together with a twofold increase over preimmunization values. These criteria rely on preliminary observations in >600 HIV-1-infected patients, in the sera of whom spontaneous increases of anti-IFN-α antibody levels have never exceeded 1.5-fold (unpublished data). Rise of ELISA titers of IFN-α antibody was assayed by comparing serum samples collected before and after immunization at random from 80 patients. Elevated ELISA titers in postimmunization sera were determined starting from a serum dilution 1:250 up to 1:32,000 and expressed as the highest dilution at which OD was increased over twofold dilution compared with those of preimmunization sera. Rise of ELISA titers in samples collected at different times from any placebo individual (control) was <1:250. (data not shown).

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Circulating IFN-α

IFN-α levels in sera were measured according to the standard biologic IFN assay, using the inhibition of the cytopathic effect (CPE) on MDBK cells induced by VSV in presence of increasing twofold dilutions of tested sera. Titers were expressed as IU. As previously reported (9), titers ≥30 IU were considered significantly high.

Moreover, the IFN-α neutralization capacity (INC) was evaluated by IFN neutralization assay according to the procedures of Fall et al. (9). The test was performed in the 150 patients for whom the cohort of serum samples was available. INC of tested sera was expressed as the ratio of the highest dilution of exogenous IFN-α in presence of tested serum (at a 1:20 dilution) resulting in 50% CPE to the highest dilution of exogenous IFN-α in presence of a seronegative standard serum (at a 1:20 dilution) resulting in 50% CPE. INC changes (increase or decrease) were taken into account when over 2 log dilution differences were found in sera collected during at least two periods of the follow-up compared with the serum taken at enrollment.

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Power of the Study and Statistical Analysis

Assuming a frequency of clinical disease progression, reduction of CD4+ counts to <200 cells/mm3, and antiretroviral treatment change or initiation of 15% in the placebo group, 200 patients would have provided a power of 80% to detect a statistically significant decrease of these endpoints to 5% in the vaccine group. Interim blind analyses were planned every 12 months from the time the first patient received the first injection to check for occurrence of unexpected adverse events, to monitor the frequency of events, and to evaluate consistent differences between vaccine and placebo recipients.

On February 1998, after a median follow-up of 478 days (range, 9-717 days), a last interim analysis was carried out. This blind analysis showed a slight but not significant difference between the two study groups (Fig. 1; Table 1). Because this difference was very unlikely to become significant over the remaining 6-month follow-up, taking into account the role of protease inhibitors, which had dramatically reduced the occurrence of endpoints, including clinical events and CD4+ cell counts, the Steering Committee decided to stop the trial.

Kaplan-Meier survival analysis was performed for each study outcome and for any combination of them, comparing placebo recipients with vaccine recipients on an intention-to-treat basis. Crude relative risks of vaccine recipients were calculated, taking placebo recipients as the reference population. To adjust estimates for potential prognostic variables (CD4+ cell counts and HIV-1 plasma viremia at enrollment), Cox's model was used. Using the standard error of the regression coefficients, 95% confidence intervals (CI) of relative risks (RR) were calculated. A preintended analysis was planned in the subgroup of patients immunized to IFN-α, that is, those who responded to the vaccine by raising their anti-IFN-α antibodies (antibody responders). This group was compared with vaccinees who did not develop antibodies or placebo recipients. None of the analyses combined these groups. Fisher's exact test was used to evaluate associations between parameters and clinical outcome.

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Safety, Tolerability, and Compliance

From December 1995 to July 1996, 242 asymptomatic HIV-1-infected patients were enrolled by eight clinical centers in three countries (Belgium, Israel, and Italy). The characteristics of enrolled patients, according to randomly assigned treatment, are shown in Table 2. Randomization succeeded in balancing the prognostic variables both in placebo and vaccine recipients. The two groups were similar for age, gender, risk factor, number of years from the first HIV-1-seropositive test, number of HCV-coinfected patients, HIV-1 viremia, and CD4+ and CD8+ cell counts at enrollment and for the percentage of patients receiving an antiretroviral treatment either before or electively (63%) during the course of the trial. In all, 4 patients, 2 in the placebo group and 2 in the vaccine group, were lost to follow-up within 1 month from the first injection (9-28 days) and they were accordingly excluded from the analysis.

Vaccination was well tolerated. The most frequently reported side effect was local discomfort at the site of injection and fever. In addition, 68 patients reported local pain (25%), and 39 of those had fever >38°C lasting not longer than 2 days and the therapy for which was then reduced to common analgesics. No patients, whether placebo or vaccine recipients, had to discontinue the immunization program as a result of related complications. The most adverse reaction was the formation of a cold abscess at the site of injection; it was observed in 2 patients in response to the first priming injection of water-in-oil emulsion. Subsequently, these 2 patients were only given injections of i-IFN-α adsorbed onto Ca(OH)PO4 in water; these patients have been included in the analysis. No other complications were found following >700 oil emulsion injections as well as Ca(OH)PO4 water injections performed in this trial. Biochemical and hematologic tests did not reveal any significant variation from preimmunization values, except in 2 patients who showed a marked increase of transaminase levels (>5 times the normal upper limit). These two study subjects were chronically HCV-infected patients and treatment was discontinued, the code was broken, and they were found to be receiving placebos. None of the patients showed an increase of HIV-1 viral load after priming or boosting injections (data not shown).

None of the enrolled patients died during the 18-month follow-up period. Clinical progression of HIV-1 infection from asymptomatic status (CDC stage A) to symptomatic status (CDC stage B or C) was observed in 37 of the 242 patients (15.3%); HIV-1-related signs and symptoms included multidermatomeric herpes zoster (20 cases), oral or systemic candidiasis (7 cases), oral hairy leukoplakia (4 cases), Kaposi's sarcoma (2 cases), Mycobacterium tuberculosis infection (1 case), cytomegalovirus-related enteritis (1 case), aggressive intraepithelial uterine carcinoma (1 case), chronic ulcerative anal herpes simplex infection (1 case), blastocystosis (1 case), atypical mycobacteriosis (1 case), and giant disseminated molluscum contagiosum (1 case).

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IFN-α Antibody Response to the Vaccine

Forty patients responded to vaccination with increased anti-IFN-α antibody titers. These antibody responder vaccinees (AbRV) represented 33% of vaccine recipients (Table 3). In most of these study subjects (>90%), the circulating IFN-α antibodies increased more than twofold to 10-fold, following the third injection. These antibody responders were all vaccine recipients and none were placebo recipients, which confirmed the immunogenicity of the vaccine preparation. In the antibody responders, postimmunization serum titers were in the range of 1:2,000 to 1:32,000 whereas these titers were <1:500 in tested antibody nonresponder vaccinees (AbNRV) as well as in placebo (Pla; data not shown).

Some 85% of AbRV (i.e., 30 of 35 patients assayed) exhibited an increase of INC of their sera, as detected by the standard IFN-α neutralization assay. In 75% of these study subjects (22 patients), the serum INC levels highly increased over 3 log2 dilutions compared with levels found at enrollment. By contrast, in AbNRV and Pla groups, serum INC remained unchanged in most patients but declined in a consistent number of study subjects.

At enrollment, circulating IFN-α levels were similar in AbRV, AbNRVs, and Pla recipients. At the end of follow-up, mean values of serum IFN-α levels, even though below critical levels for disease progression (i.e., <30 IU/ml) in the three groups of patients, were significantly lower in AbRVs (8.6 IU/ml) compared with AbNRVs and Pla recipients (mean values, 14.1 and 10.9 IU/ml, respectively) by analysis of variance for repeated measures (F = 3.15; p < .05). Further individual analysis showed that at enrollment, a similar percentage of patients with significantly high levels of serum IFN-α (30 IU/ml) in AbRVs (15%) and nonimmunized patients (AbNRVs and Pla recipients, 20%), and at the end of follow-up, 15% of nonimmunized patients maintained or increased IFN-α levels >30 IU/ml, compared with <3% of antibody responder vaccinees. The difference was statistically significant (p < .05).

Characteristics of AbRVs are reported in Table 4: age, gender, risk factors, and HIV-1 plasma viremia were similar to those of AbNRVs and Pla recipients. Furthermore, circulating antibody levels of various specificities, including tetanus toxoid, HIV-1 gp160, p24, p17, and nef and tat peptides, were at enrollment and following immunization of comparable magnitude in both AbRVs and AbNRVs as ascertained by Student's t-test for paired samples. Finally, no difference in level and increase of anti-IFN-α antibodies was found between patients receiving or not antiretroviral therapy, including protease inhibitors, during the follow-up period (data not shown).

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Clinical Endpoints

Kaplan-Meier survival analysis was carried out on occurrence of HIV-1-related signs, treatment changes, or initiation due to disease progression and decrease of CD4+ counts to <200 cells/mm3; the latter was analyzed in patients with CD4+ counts at baseline >200 cells/mm3. Differences between the two groups were observed, vaccine recipients having slightly better event-free survival than placebo recipients (Fig. 1; Table 1); however, these differences were not statistically significant. The surveillance committee determined that the rate of clinical and laboratory endpoints was reduced in the two groups, because of the introduction of protease inhibitors, to a level that the likelihood of achieving the study aims was not feasible, resulting in the trial follow-up being terminated. Overall, 154 of 242 enrolled patients were given protease inhibitors during the study period (Table 2), of whom 79 (51%), added drugs because of clinical or laboratory progression.

A Kaplan-Meier survival analysis, comparing patients who were AbRVs with AbNRVs and Pla recipients separately (Fig. 2; Table 4), showed a significantly lower rate of all study endpoints in antibody responder vaccinees. More numerous patients from group B with CD4+ counts >350 cells/mm3 (25 individuals) experienced a rise of anti-IFN-α antibodies than patients from group A with CD4+ counts ≤350 cells/mm3 (15 individuals; Table 5). This result, which confirms previous findings (22), was expected, inasmuch as CD4+ cell counts reflect the immune system's status, and thus confirmed previous findings. As a consequence, CD4+ cell counts of AbRVs were significantly higher than those of AbNRVs, but not those of the Pla group (p = .16) Furthermore, AbRVs at enrollment exhibited significantly lower viral load than AbNRVs (Table 5). Important to note, median CD4+ cell counts and plasma viremia of AbRVs within each stratum were absolutely similar to those of AbNRVs and Pla groups (Table 5). Since during HIV-1 infection CD4+ cell counts as well as plasma viremia are known to be biologic markers of disease progression (28), it was necessary to control the Kaplan-Meier analysis for CD4+ cell count and HIV-1 plasma viremia at baseline, to ascertain whether the rise of IFN-α antibody obtained through active immunization per se accounted for reduction of AIDS progression in AbRVs (Table 4). The statistical adjustment for these parameters at enrollment did not alter the direction, magnitude, or statistical significance of the crude estimates of risk of AbRVs compared with those of AbNRVs and Pla recipients (Table 4). In particular, the RR of occurrence of HIV-1-related clinical signs was reduced in AbRVs by 75% compared with AbNRVs or Pla recipients. Similarly, the RR of occurrence of any of the study outcomes was reduced by 50% in AbRVs. Moreover, the rate of occurrence of all the study endpoints was always lower in AbRVs compared with those of AbNRVs and Pla recipients, as shown by stratification for CD4+ cell counts and plasma viremia in Table 5. Interestingly, the Kaplan-Meier clinical survival analysis comparing the AbNRV subgroup to Pla group showed no significant differences between these two groups even controlled for CD4+ cell counts, which were somewhat lower in AbNRVs compared with findings in Pla recipients (median, 297 and 348, respectively; p = .08; Table 4).

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Rise of IFN-α Antibodies Is Inversely Associated to AIDS-Related Clinical Signs Rate

Because previous reports showed that the level of circulating IFN-α was highly correlated to clinical manifestations (23) and to fast-progressor HIV-1-infected patients (29), we questioned whether the rise of IFN-α antibodies, enhancing the capacity to neutralize circulating IFN-α in AbRVs could significantly contribute to the reduction of AIDS occurrence observed in this group (Tables 4 and 5). To eliminate any bias due to differences of CD4+ cell counts and plasma viremia at enrollment, the adjustment for these parameters, as mentioned previously, did not alter the statistical differences of risk in AbRVs versus AbNRVs and Pla recipients (Table 4).

Furthermore, the results of the Fisher's exact test showed the following:

1. As expected, in vaccinees, a significant correlation was found between high CD4+ cell count and low plasma viremia and anti-IFN-α response (p = .03 and p = .01, respectively). Indeed, patients of vaccine group B (CD4+ counts >350 cells/mm3) had an antibody response rate better than those of the vaccine group A (CD4+ counts < 350 cells/mm3).

2. As anticipated, a positive correlation of lower occurrence of clinical manifestations with higher CD4+ cell counts (RR = 0.55) and lower plasma viremia (RR = 0.91) at enrollment was found (even though not statistically significant in these series of patients: p = .16 and p = .85, respectively).

3. Importantly, the risk of occurrence of AIDS-related symptoms was significantly reduced in patients with rise of anti-IFN-α antibody (RR = 0.25, p = .05).

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This international, multicenter, randomized, double-blind, placebo-controlled trial was designed to evaluate safety, immunogenicity, and efficacy of an active immunization against IFN-α. The purpose of anti-IFN-α immunization was to counteract IFN-α overproduction, which is characteristic of HIV-1 infection (5,6) and plays a central role in HIV-1-induced immunosuppression (8). The safety of the immunization against this cytokine, reported by previous studies (21-23), was confirmed by this study, during which only 2 cold abscesses occurred following >700 priming (water-in-oil) injections and none after booster (water) injections. In previous studies on anti-IFN-α, immunization of patients untreated with antiretroviral therapy, 6 to 7 monthly repeated injections of i-IFN-α elicited an anti-IFN-α antibody response rate of >80% of patients (21-23). In the current trial, in which only three oil injections were given for priming, only 33% of patients exhibited an anti-IFN-α antibody response, showing that the immunization scheme applied was not optimal. Even though CaOHPO4 water suspension for priming injections, as used in the ANRS trial, did not allow expression of the immunogenicity (22), previous trials showed that CaOHPO4 adjuvant was effective after priming for booster injection (23). Because in this trial only a low percentage of patients (33%) developed IFN-α antibodies after the three oil injections used for priming, we concluded that optimization of IFN-α immunization regimen was not achieved in this trial, and an optimal immunization schedule using six to seven oil priming injections should increase the percentage of AbRVs to >80% (21). Such an optimal immunizing regimen should enhance the study's power to show an effect.

As previously reported (22,23), in AbRVs, increased serum INC and lower serum IFN-α levels were also observed following immunization, by contrast to AbNRVs and Pla recipient groups.

The recent availability of protease inhibitors for HIV-1 disease treatment and their use by enrolled patients according to those patients' request and treatment guidelines was an important confounder of trial analysis, because they lowered the rate of endpoints that would have been achieved based on our power calculation. Although no significant difference in clinical endpoints could be detected in vaccine and placebo recipients, on subgroup analysis those who had a positive antibody response to active immunization against IFN-α (i.e., AbRVs) had a statistically significant lower risk of occurrence of clinical and/or biologic signs of progression (Fig. 2) compared with AbNRVs and Pla recipients (Table 4).

The lower risk of disease progression in AbRVs versus AbNRVs and Pla recipients could be ascribed either to the effect of the anti-IFN-α immunization, to a selective bias at enrollment, or to both. The AbRV group exhibited higher CD4+ cell count and lower plasma viremia at enrollment, as anticipated by our previous study showing lower antibody response when CD4+ counts were <350 cells/mm3 (22). Because CD4+ cell count and plasma viremia are known to be important predictors of the risk to disease progression and to evaluate whether the rise of IFNa antibodies was correlated to reduction of AIDS progression, it was necessary to control for these major markers of AIDS progression, that is, CD4+ cell count and plasma viremia in the Kaplan-Meier statistical analysis. Adjustment for CD4+ cell count and viremia did not alter the statistical significance of reduced risk of progression in AbRVs compared with that of AbNRVs and Pla recipients. This statement is further confirmed by the results of Fisher's exact test. This test showed that low levels of CD4+ cell counts at enrollment influence, but not significantly, AIDS progression, whereas the rise of IFN-α antibodies reduces significantly AIDS-related symptoms (p = .05). This result is consistent with previous clinical reports that demonstrated that a high circulating IFN-α level is strongly correlated to AIDS evolution (23) and fast progression (28). Furthermore, recent experimental data stressing that IFN-α (the well-known immunosuppressive cytokine [29] overproduced by infected patients [6,8]) mediates AIDS immunosuppression (30), and thus may provide an explanation of why the rise of IFNa antibodies is inversely correlated to AIDS progression. These data further showed that the cellular immune response of PBMCs inhibited in vitro by HIV-1 can be restored in presence of anti IFN-α antibodies. Therefore, the rise of specific antibodies neutralizing the overproduction of IFN-α, found at early stages in infected lymphoid foci and at later stages in sera of HIV-1-infected patients (6), should block the spontaneous evolution of HIV-1 replication toward immune collapse and AIDS.

Nevertheless, we recognize it remains possible that more subtle yet unidentified differences between the subgroups at enrollment may have contributed to the better behavior of AbRV patients. In any event, statistical analysis of the results of this trial supports our assumption that reduction of AIDS-related symptoms in AbRVs may be secondary to the rise of IFN-α antibody in this subpopulation of patients. It supports our decision to continue to evaluate the clinical efficacy of this approach in a larger efficacy trial.

In conclusion, although the power of this trial has been reduced by two unforeseen confounders, that is, introduction of triple therapy delaying/modifying the kinetics of endpoints and suboptimal immunizing regimen reducing the number of AbRVs, this double-blind, placebo-controlled clinical trial carried out on 242 patients divided into groups receiving or not receiving antiretroviral therapy supports the safety of the IFN-α vaccine and suggests those with a rise in anti-IFN-α antibodies, have improved prognosis.

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The EURIS Study Group is represented by: F. Adorni (Coordinating Center, Milan, Italy), J-M. Andrieu (Paris, France), A.G. Angius (Milan, Italy), A. Barath (Brussels, Belgium), G.P. Cadeo (Brescia, Italy), M. Carcagno (Buenos Aires, Argentina), G.P. Carosi (Brescia, Italy), C. Chiaro (Coordinating Center, Milan, Italy), C.M. Farber (Brussels, Belgium), M. Feldman (Rehovot, Israel), K. Kabeya (Brussels, Belgium), A. Lachgar (Paris, France), H. Le Buanec (Paris, France), W. Lu (Paris, France), P.M. Mannucci (Coordinating Center, Milan, Italy), M. Morfini (Florence, Italy), M. Muça-Perja (Coordinating Center, Milan, Italy), R. Naaman (Rehovot, Israel), E. O Doherty (Brussels, Belgium), L. Palvarini (Brescia, Italy), A. Rocino (Naples, Italy), S. Sant (Coordinating Center, Milan, Italy), M. Schiavoni (Bari, Italy), S. Sprecher (Brussels, Belgium), M. Stein (Rehovot, Israel), A. Turano (Brescia, Italy), J.F. Zagury (Paris, France), R. Zerboni (Milan, Italy).

Acknowledgments: This work has been supported scientifically and financially by Neovacs (Paris, France). We acknowledge Biosidus (Buenos Aires, Argentina) and Seppic (Paris, France) for generously supplying the immunizing and adjuvant reagents, the patients for their great collaborative participation, and Robert C. Gallo for his continued advice, scientific discussions, and encouragement.

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AIDS clinical trial; Anti-interferon-α immunization; Anti-cytokine vaccination

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