HIV infection results in a complex pattern of impairments that involves all components of the immune system.1-3 Antiretroviral therapy (highly active antiretroviral treatment [HAART]) is associated with a certain degree of immune reconstitution; nevertheless, functional defects persist even in patients who have successfully undergone HAART for long periods.4-9 Thus, residual functional defects can be seen in T helper (Th) cells, cytotoxic T lymphocytes (CTLs), and B lymphocytes, even in patients in whom HAART results in near-complete suppression of circulating HIV viremia and in restoration of CD4 T-cell counts to physiologic numbers.4-9 It is important to underline that because the thymus is still present in children, the likelihood for success of HAART-associated immune restoration should be higher in children than in adults. As a matter of fact, a number of studies have shown that successful therapy in prepubertal patients results in dramatic increases of the thymic mass and, consequently, in satisfying restoration of CD4 T-cell counts and function.10-19
A question that has not yet found an unequivocal answer is whether immune responses elicited by vaccination are fully restored in HIV-infected and HAART-treated individuals. In particular, it is still not clear whether the persistence of vaccine-induced immune responses is comparable in HIV-infected and -uninfected subjects. Contrasting data are reported. Therefore, whereas immunization with pneumococcal, measles, and rubella vaccines were shown to elicit optimal responses,20-23 tetanus immunization resulted in antigen-specific responses of limited duration.24 The results obtained with influenza vaccination are even more controversial, because contrasting results have shown that influenza vaccination elicits immune functions comparable to those observed in healthy controls and that a compromised response to vaccination is seen in HIV-infected patients.25-33
Annual routine vaccination against influenza is recommended in children, in whom the health burden caused by this infectious disease is even greater than in the elderly in terms of incidence and hospitalization.34,35 Because influenza can cause severe complications in HIV-infected patients, influenza vaccination is particularly useful in such patients. Recently, a virosomal-adjuvanted subunit influenza vaccine was shown to be well tolerated and endowed with improved immunogenicity.36-40 To evaluate the amplitude and duration of the cell-mediated and humoral immune responses elicited by such vaccine preparation in pediatric HIV infection, we designed a study protocol enrolling HIV-infected children in whom long-term HAART resulted in an optimal immunovirologic response to therapy.
The success of a vaccination protocol is determined by the ability of the vaccine to elicit increases in antibody (Ab) titers that ensure protection against influenza disease. These criteria have a strong epidemiologic/public health value but are not designed to verify finer immunologic parameters. We thus performed in-depth immunologic analyses to verify the effect of vaccination on a single-patient level. The primary outcome of this study was to evaluate vaccine-induced cell-mediated and humoral immune responses at 1 month after vaccination and the incidence of local and systemic vaccine-related adverse events (AEs); the secondary outcome was to investigate the persistence of such responses 6 months after the vaccination.
MATERIALS AND METHODS
Study Design and Population
This was an observational, prospective, open-label study enrolling HIV-infected children (N = 24) and age- and gender-matched healthy controls (N = 14) undergoing influenza vaccination in the Department of Pediatrics University of Milan, Luigi Sacco Hospital (Milan, Italy) during the period spanning October 15 to November 15, 2005. Inclusion criteria for the HIV-infected children were undetectable viral load, >6 months of HAART, and having never received influenza vaccination before. Exclusion criteria were a contraindication for the influenza vaccine, sensitivity to any component of the vaccine, acute respiratory and/or allergic illness, history of egg allergy, severe atopia, fever >38°C (axillary), treatment with immunosuppressive drugs (including systemic corticosteroids), and having received blood transfusions or immunoglobulins during the previous 3 months. HIV infection was diagnosed and classified in accordance with the Centers for Disease Control and Prevention (CDC) criteria for immunologic (classes 1, 2, and 3) and clinical (classes N, A, B, and C) disease stages.41 The parents of eligible children or their legal guardians received a careful explanation about the aim of the study; if they agreed, they signed a written consent form. The Ethics Committee of the Luigi Sacco Hospital approved the study protocol.
HIV-infected children (8 in CDC class A and 16 in CDC class B+C, 11 in CDC class 1 and 13 in CDC class 2+3) were clinically stable and had long-lasting control of viral replication and immune recovery (Table 1). All showed an undetectable plasma viral load and had a long duration of HAART exposure (mean = 85 months; range: 21 to 116 months). The median nadir CD4 T-cell count was 244 cells/μL (see Table 1). CD4 T- lymphocyte and B-lymphocyte counts and percentages and CD8 T-lymphocyte counts were comparable in HIV-infected children and in healthy controls, whereas CD8 T-cell percentages were increased in HIV-infected children (see Table 1).
All children received 1 dose (0.5 mL) of a trivalent virosome-adjuvanted influenza vaccine (Inflexal V; Berna Biotech AG, Berne, Switzerland), licensed in Italy for the 2005 to 2006 season. This vaccine contains 15 μg each of A/New Caledonia/20/99 (H1N1)-like, A/California/7/2004 (H3N2)-like, and B/Shanghai/361/2002-like purified influenza surface antigens hemagglutinin, and neuraminidase, integrated into the lipid membrane of the virosome with solvent added to 0.5 mL.42,43 The vaccine was injected intramuscularly in the deltoid muscle or in the lateral upper thigh. Immunogenicity assessments were made before vaccination (baseline) and 1 month and 6 months after vaccination.
Safety assessments of local and systemic reactions were made by the investigators at baseline and at the 1-month follow-up visit and by the parents/legal guardians for 4 days after vaccination. Children were examined for the presence of local AEs (pain/tenderness, redness and swelling/induration) and questioned for systemic AEs (body temperature >38°C, shivering, and malaise/irritability).
Detection of Hemagglutination-Inhibiting Antibodies
Serum samples were collected immediately before and 1 and 6 months after the immunization. The samples were simultaneously examined for hemagglutination-inhibiting (HI) Abs using standard microtiter assays for each of the influenza strains contained in the vaccine.44 The HI Ab titer was expressed as the reciprocal of the highest dilution inhibiting agglutination. Minimum response was defined as seroconversion or as a 4-fold or greater increase in Ab titer in the postimmunization samples. Ab titers ≥1:40 were considered protective against influenza infection. To allow the calculation of the HI geometric mean titers (GMTs), a titer of 1:5 was assigned arbitrarily to nonresponder vaccinees. The HI Abs were evaluated according to criteria described in the guideline of the European Agency for the Evaluation of Medical Products (EMEA).45 Because there are no EMEA-defined criteria for the pediatric population, immunogenicity was assessed on the basis of the criteria for adults aged 18 to 60 years, which require at least 1 of the following criteria to be met for each strain to confirm immunogenicity: (1) seroconversion (≥4-fold increase in HI Ab titer, with a titer of ≥1:40 being reached in >40% of the subjects), (2) seroprotection (HI Ab titer of ≥1:40 in >70% of the subjects), and (3) GMT (>2.5-fold increase in the HI Ab GMT).
Blood Sample Collection and Separation of Peripheral Blood Mononuclear Cells
Whole blood was collected by venipuncture in Vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA; Becton Dickinson, Rutherford, NJ). Peripheral blood mononuclear cells (PBMCs) were separated on lymphocyte separation medium (Organon Teknica, Durham, NC) and washed twice in phosphate-buffered saline (Organon Teknica). The number of viable leukocytes was determined by trypan blue exclusion. All analyses were performed on freshly collected cells.
Measurement of Influenza-Specific IgG1 and IgG3
A final concentration of 3 μg/mL of Inflexal V vaccine (Berna Biotech AG, Berne, Switzerland) (New Caledonia 20/99, 591 μg hemagglutinin (HA)/mL; New York 55/2004, 553 μg HA/mL; and Jiangsu 10/2003, 544 μg HA/mL) was used to coat the wells overnight at 4°C. A blocking solution (3% bovine serum albumin [BSA] in Tris-buffered saline [TBS]) was added for 2 hours at 37°C before incubation with plasma. Six serial dilutions (1:1, 1:10, 1:100, 1:400, 1:1600, and 1:6400) of patient plasma were evaluated to define the best condition. After washing, secondary Abs to IgG1 (100 ng/mL; mouse antihuman IgG1 fucose [Fc] fragment horseradish peroxidase [HRP] conjugate; Chemicon Int., Temecula, CA) or IgG3 (300 ng/mL; mouse antihuman IgG3 HRP conjugate; Serotec Ltd, Oxford, UK) were added for 1 hour at 37°C. The reaction was developed with O-phenylenediamine (0.4 mg/mL; Sigma, St. Louis, MO) in 0.05 M of phosphate-citrate buffer containing 0.03% sodium perborate (pH 5.0, Sigma) and stopped after 30 minutes by adding 0.4 N of H2SO4. Optical density was measured at 450 nm (620-nm reference filter).
Enzyme-Linked Immunospot Assays
Ninety-six-well nitrocellulose plates were precoated with a first layer of monoclonal Abs to interferon-γ (IFNγ) or interleukin (IL)-2 (MABTECH, Nacka, Sweden). PBMCs were added in duplicate wells at a rate of 2 × 205 cells/well and were unstimulated or stimulated with the Inflexal V vaccine (1:2000 final concentration) in the presence or the absence of neutralizing anti-CD4 monoclonal Ab. IFNγ production by CD4 T lymphocytes was blocked by preincubating PBMCs with 100 ng/mL of neutralizing recombinant human monoclonal Ab to CD4. IL-2 production by CD8 T cells was prevented by preincubating PBMCs with 100 ng/mL of neutralizing recombinant human monoclonal Ab to CD8 (R&D Systems, Minneapolis, MN). Plates were incubated overnight at 37°C in 7% CO2; the cells were then discarded and the plates incubated at room temperature for 3 hours. A second biotinylated anti-IFNγ or anti-IL-2 monoclonal Ab (7-B6 to 1 biotin; MABTECH), followed by streptavidin-conjugated alkaline phosphatase (MABTECH) for 2 hours, was subsequently used. Cytokine-producing cells were detected using an alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories, Hercules, CA). Finally, plates were dried and spots were counted using an automated enzyme-linked immunospot (ELISPOT) reader (EliScan Aelvis; Aelvis GmbH, Hannover, Germany). Influenza-specific responses were reported as the number of spot-forming units (SFUs) per 106 mononuclear cells after subtraction of background cytokine secretion.
Seroconversion and seroprotection rates in the 2 groups were compared using the Fisher exact test or the χ2 test (χ2). The Student t test was used to compare GMTs of HI Abs, lymphocyte subset counts, and age. A P value <0.05 was considered significant for all the statistical tests.
Response to Vaccination: Adverse Effects
No serious systemic or local vaccine-related AEs were observed after vaccination at clinical examination. A total of 9 solicited AEs were reported by 4 HIV-infected children (16.6%) and by 2 healthy controls (14.3%). Local reactions (pain/tenderness) occurred in 3 HIV-infected patients and in 2 healthy controls. Systemic events (body temperature >38°C axillary) were observed in 2 children per group.
Response to Vaccination: Seroconversion and Seroprotection Rates
Pre- and postvaccination seroconversion and seroprotection rates for each of the 3 vaccine antigens were tested in paired serum samples. Tables 2 and 3 show the HI Ab responses to the vaccine. The EMEA criteria for seroconversion and seroprotection 1 month after immunization were fulfilled for all 3 influenza strains in HIV-infected children and healthy controls. Nevertheless, HIV-infected children showed lower seroconversion and seroprotection rates as compared with those observed in healthy controls (SC: A/H3N2: 54% vs. 71%, A/H1N1: 71% vs. 93%, B: 71% vs. 86%). These differences, however, did not reach statistical significance. Finally, stratification of children according to age revealed no difference in seroconversion rates in children younger or older than 9 years in HIV-infected children or healthy controls.
Before immunization, a significantly lower percentage of HIV-infected children showed the presence of protective A/H1N1-specific Ab titers compared to healthy controls (≥1:40; P = 0.014), whereas the percentage of individuals with protective A/H3N2- and B-specific Ab titers was similar in both groups. One month after immunization, an increase in the percentage of subjects with a protective Ab titer against all 3 influenza strains was seen in both groups, although, again, the increase was lower in HIV-infected children (A/H3N2 and A/H1N1: 79% vs. 93%, B: 75% vs. 93%). This difference was retained 6 months after immunization (A/H3N2: 54% vs. 71%, A/H1N1: 75% vs. 93%, B: 62% vs. 93%) and became statistically significant (P = 0.04) for B-specific Ab titers. Similar to what was observed for seroconversion, seroprotection rates were similar at both time points after vaccination when younger (<9 years) and older (>9 years) HIV-infected and -uninfected children were analyzed separately.
Response to Vaccination: Geometric Mean Antibody Titers
GMTs against all 3 influenza strains were significantly increased at 1 month and 6 months after immunization in HIV-infected children and in healthy controls. At both time points, however, A/H3N2- and A/H1N1-specific GMTs were significantly lower (P > 0.01) in HIV-infected children compared with healthy controls. These data are shown in Table 3.
The time course of GMTs against all 3 influenza strains according to the preexistence of Ab for any specific strains at baseline are shown in Table 4. The numbers of HIV-infected and -uninfected children without preexisting Abs (ie, HI Ab <10) were as follows: A/H3N2, 14 of 24 HIV-infected children versus 7 of 14 healthy controls; A/H1N1, 10 of 24 HIV-infected children versus 2 of 14 healthy controls; and B, 7 of 24 HIV-infected children versus 5 of 14 healthy controls. GMTs for any specific strain were comparable in HIV-infected children and healthy controls without preexisting Abs at 1 month and 6 months after immunization. Conversely, lower GMTs for A/H3N2 and A/H1N1 strains were observed at both time points in HIV-infected children with preexisting Abs (A/H3N2: P < 0.02 at 1 and 6 months, A/H1N1: P < 0.0006 at 1 month, and P < 0.004 at 6 months).
Response to Vaccination: Influenza-Specific IgG3 Antibodies
Influenza -specific IgG1 and IgG3 titers were measured in plasma of all patients. Whereas a comparable augmentation of influenza-specific IgG1 Ab was seen in both groups of children (data not shown), results presented in Figure 1 indicate that a more than 2-fold increase in influenza-specific IgG3 was seen 1 month after vaccination in healthy controls alone. This difference was statistically significant (P = 0.01).
Response to Vaccination: Influenza-Specific CD4 and CD8 T Lymphocytes
Influenza-specific CD8 IFNγ-secreting T lymphocytes and CD4 IL-2-secreting T lymphocytes were analyzed in all individuals using an ELISPOT assay. Results showed that on vaccination, IFNγ-producing influenza-specific CD8 T lymphocytes were significantly increased 1 month after immunization in HIV-infected patients (P > 0.001) and healthy controls (P = 0.02). This difference was still significant 6 months after vaccination in healthy controls (P > 0. 01) but not in HIV-infected children. At both time points considered, the number of IFNγ-producing influenza-specific CD8 T lymphocytes was statistically higher in healthy controls compared with HIV-infected children (P > 0.01 in both cases).
Vaccination also increased the number of IL-2-producing influenza-specific CD4 T lymphocytes. The increase was statistically significant compared with baseline in healthy controls alone (P > 0.001 at months 1 and 6). Finally, a significantly larger number of IL-2-producing influenza-specific CD4 T lymphocytes were induced by immunization in healthy controls compared with HIV-infected children (P = 0.01 at both time points). These results are shown in Figure 2.
The question of whether immune responses to vaccines are impaired in HIV-infected individuals, and whether the functionality of the immune system can be drastically improved by antiviral therapy, is still not fully elucidated. This question is even more relevant in the pediatric setting, where the presence of an active thymus suggests the possibility that an effective therapy could elicit nearly complete immune restoration.10-19 The immunogenicity of vaccines in HIV-infected children has been analyzed by a number of investigators, showing that an immune response against Pneumococcus, rubella, measles, and tetanus, among others, can be elicited in these children.20-33 Conversely, in the few studies that have performed in-depth analyses to dissect the immune response stimulated by such vaccines, it was shown that the magnitude and duration of Ab response was lower in HIV-infected individuals as compared with controls.46-49
To our knowledge, no study has been performed in children with a full immunovirologic response to HAART, and no study has evaluated immunogenic persistence at 6 months after influenza vaccination. To shed light on this question, we studied the response to influenza vaccine in a group of HIV-infected children with an optimal response to HAART. The vaccine used was a virosomal influenza vaccine. Although, the US Advisory Committee on Immunization Practices recommends 2 doses of influenza vaccine for children younger than 9 years of age,50 protocols based on a single dose were shown to be as effective. In particular, Salleras et al40 administered a single dose of virosomal influenza vaccine in 966 children (94% unprimed; L. Salleras, personal communication, 2006) ranging in age from 3 to 14 years, observing an effectiveness of 88.4% in preventing laboratory-confirmed cases of influenza. Virosomes represent a novel vaccine formulation; they closely mimic the native virus and have the capacity to elicit broad immune responses, activating the humoral and cellular arms of the adaptive immune system.42,43 In particular, the vaccine used, Inflexal V, is an adjuvanted influenza vaccine characterized by exceptional purity, because it is completely viral RNA- and thimerosal-free. This vaccine has been shown to be highly immunogenic, effective, and safe in healthy children and in children with cystic fibrosis.36-40 Our results indicate that a single dose of virosomal influenza vaccine elicits effective immune responses in HIV-infected children and in healthy controls. The results also show that even if EMEA criteria for registration of influenza vaccines are fulfilled up to 6 months post vaccination, in the presence of undetectable HIV plasma viremia and CD4 T-cell counts comparable to those of healthy uninfected controls, the response to vaccination is lower in HIV-infected children. Immune impairment may thus persists even in the context of an apparent perfect response to therapy. Finally, the results of the present study indicate that virosomal-adjuvanted vaccine is safe and well tolerated in HIV-infected children.
From a public health point of view, it is interesting to observe that the EMEA criteria for seroconversion and seroprotection45 were satisfied by the vaccine protocol in both groups of children. Despite this favorable overall result, more in-depth analyses in HIV-infected children as compared with healthy children indicate that (1) the percentage reaching the criteria for seroconversion and seroprotection is lower, (2) the duration of response is shorter, and (3) GMT Ab titers are reduced. These observations suggest that, although useful at analyzing the efficacy of a vaccine at the population level, the EMEA criteria might be inadequate in particular settings, such as HIV infection.
CD4 T-lymphocyte counts and percentages reached by HAART-treated children were optimal for their age and fully comparable to those seen in healthy controls of the same age. This observation notwithstanding, data showed that T lymphocytes were functionally impaired in HIV-infected children. The discrepancy between CD4 T-cell numbers and functions has been described early in HIV research.51,52 Therefore, it may be concluded that the presence of a conspicuous number of CD4 T lymphocytes does not warrant their functionality. As a matter of fact, it is known that a complex pattern of functional impairment is observed during the progression of infection in HIV-infected patients.53 Conversely, whereas it was convincingly shown that HAART can restore CD4 T-cell counts to an almost optimal degree, functional immune defects are much less susceptible to being fully reverted by therapy.4-9,54,55 The response to a protein antigen, such as that used in this vaccine, needs full collaboration between T and B lymphocytes56; we observed the presence of defects in both of these cell types. Thus, influenza-stimulated IL-2 production by CD4 T lymphocytes, IFNγ production by CD8 T lymphocytes, and influenza-specific Ab production by B lymphocytes were fully defective in HIV-infected children compared with healthy controls. These data raise the possibility that an immune defect in antigen processing and/or presentation could be involved in the impairment of the immune response observed in HIV-infected children. Antigen presentation was shown to be defective only in the final stages of disease;55 preliminary data obtained in the children enrolled in the study showed that the expression of major histocompatibility complex (MHC) class I and class II molecules was not reduced on the immune cells of HIV-infected patients, suggesting that antigen presentation is not impaired in this setting.
An isotype switch occurs after B cells have been stimulated by antigens and is finely regulated by cytokines produced by the T lymphocytes present in the cellular milieu.57 In particular, IFNγ and IL-12 stimulate IgG3 production, whereas IL-4 favors the generation of IgG1.58-60 Antigen-specific Ab subclasses can thus be used as a biologic indicator to characterize antigen-specific immune responses functionally. IFNγ production by CD8 T cells was reduced in HIV-infected children; as a result, influenza vaccination was associated with a significant increase of antigen-specific IgG3 in healthy controls alone. These findings indicate that defects in Th1-like cytokine production that cannot be reversed by antiviral therapy persist even in the face of a complete immune restoration.
In conclusion, whereas a single injection of virosomal vaccine elicits strong immune responses in healthy children, the effect of a single dose administration is reduced in HIV-infected children, even in the context of an apparently totally satisfactory immunovirologic response to HAART. The persistence of immune defects in HIV-infected children prevents the possibility of mounting fully efficient immune responses to vaccination. The efficacy of different, more frequent, and/or more intense vaccine schedules should be considered in these children.
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