Varicella is an acute, highly infectious disease caused by varicella zoster virus (VZV). Children generally develop mild disease. After the initial infection, VZV establishes latency in the dorsal route ganglia. Reactivation, resulting in zoster, is thought to occur when alterations in the balance between the virus and host factors allow local replication of the virus in the ganglion and axonal transport to the skin [1,2]. Complications of varicella are bacterial superinfection, cerebellitis and encephalitis. VZV is very common in temperate climates, where approximately 95% of the young adult population has serological evidence of previous infection . In tropical areas VZV is less common . After migration to colder areas individuals are at risk of contracting varicella at an older age, which increases the risk of developing complications .
Both virus-specific IgG and T cells can be detected after varicella infection  as well as after VZV vaccination of healthy individuals . Virus-specific IgG probably neutralizes free viral particles, and therefore helps to counter reinfection upon repeated exposure. This is supported by the observation that the early administration of varicella zoster hyperimmune immunoglobulin protects against VZV infection after close contact with a contagious individual with active varicella. Both CD4 and CD8 cytotoxic T cells are thought to suppress reactivation from viral latency. Exposure to an individual with chickenpox gives an increase in VZV-specific T cells and reduces the risk of zoster later in life [8,9].
Individuals with untreated HIV-1 infection show continuous viral replication and subsequent loss of CD4 T cells, which eventually causes an immune deficiency and opportunistic infections as a consequence. VZV infection in these patients can cause severe chickenpox, with major morbidity and mortality [10,11]. Even during treatment with HAART, HIV-1-infected adults were found to have an increased risk of reactivation of VZV, causing shingles . In our cohort of HIV-1-infected children, we recently observed that during treatment with HAART serological protection against VZV after natural infection fades over time despite immune reconstitution . HAART may thus not normalize the risks of recurrent or reactivating VZV infection in HIV-1-infected patients.
Since 1974 a live attenuated vaccine against VZV has been available . Routine vaccination of all children without a history of varicella is recommended in the United States . However, vaccination of children with any known immune deficiency is not recommended, because of the potential of disseminated viral infection. To date, VZV immunization of HIV-1-infected children has been restricted to children with stable disease [stage N1 or A1 according to Centers for Disease Control and Prevention (CDC) classification , with CD4 T-lymphocyte percentages greater than or equal to 25%] , based on a study that excluded severely immunocompromised children . These HIV-1-infected patients would benefit most from an effective vaccination, in order to prevent severe forms of primary wild-type VZV infection.
A high plasma viral load is correlated with a rapid CD4 T-cell decline and disease progression [18–21]. HIV enters its target cell by binding to CD4 and one of the chemokine receptors CCR5 and CXCR4 as a co-receptor , upregulated during immune activation during acute viral infections or vaccination . It was hypothesized that vaccination of HIV-1-infected individuals may make T cells more susceptible to HIV-1 entry.
The aim of the study was to evaluate the safety and efficacy of VZV vaccination of HIV-1-infected children irrespective of CDC category or treatment. To this end, children were followed intensively for the development of clinical symptoms, and both cellular and humoral responses were measured.
Patients and methods
From June 2002 to June 2005 all patients in the Paediatric Amsterdam Cohort on HIV (n = 78) were evaluated for inclusion in the study. Inclusion criteria were: HIV-1 seropositive, VZV seronegative, under the age of 18 years, lymphocyte count above 700 cells/μl, and without any active disease. Of 22 eligible HIV-1-infected children in our paediatric cohort, 15 consented to participate. During vaccination two children were not being treated with antiretroviral drugs, others had been on HAART for a median of 289 weeks (Table 1).
All HIV-1-negative household members of the patients included were also tested for VZV serology. If tested VZV seronegative, all HIV-1-negative household members of the patients included were also offered VZV vaccination. All six eligible HIV-negative sibs consented to participate.
HIV-1-infected children in our cohort manifesting clinical VZV symptoms during the same 3-year period of study were included for comparison. In our cohort, eight children had clinically evident chickenpox (of whom four were VZV IgG positive before chickenpox), and four VZV-seropositive children had clinical herpes zoster during treatment with HAART (Table 2). At the first symptoms of chickenpox the children had been on HAART for a median of 132 weeks. The children who developed herpes zoster had been on HAART for a median of 14 weeks.
Written informed consent was obtained from all patients, controls and caregivers. The medical ethical committee of the Academic Medical Centre approved the research protocol.
All VZV-seronegative patients received the VZV vaccine. Study visits and blood samples were planned at 2, 3, 4 and 6 weeks after primary immunization. All patients received a second immunization 3–6 months after the primary immunization, with an evaluation of the serological and cellular responses after 6 weeks.
Varicella vaccine (Varilrix; GlaxoSmithKline UK, Uxbridge, UK) containing the live-attenuated VZV Oka strain was administered subcutaneously as per the manufacturer's recommendations for immunocompromised children.
Lymphocytes, T-cell subsets
Blood samples were collected and the numbers of B cells (CD19+), T cells (CD3+) and T-cell subsets (CD3+CD4+, CD3+CD8+) were determined by standard flow cytometry procedures, as described before in detail . In short, 100 μl ethylenediammine tetraacetic acid anticoagulated whole blood was incubated with fluorescent label conjugated monoclonal antibodies (concentrations according to manufacturer's instructions). CD4 T-cell and CD8 T-cell subsets were additionally defined by monoclonal antibodies specific for CCR5-FITC (CD195; BD Biosciences, San Jose, California, USA) and CXCR4-PE (CD184; BD Biosciences). The number of activated CD4 and CD8 T cells were determined using monoclonal antibodies against HLA-DR-PE (BD Biosciences) and CD38-FITC (Beckman Coulter, Miami. Florida). Analysis of cells was performed using a FACScan flowcytometer and CellQuest software (BD Biosciences). Peripheral blood mononuclear cells (PBMC) were isolated from blood samples using standard density gradient centrifugation techniques, after separation of the plasma. Both plasma and PBMC were stored until further analyses.
Antigen-specific T-cell proliferation assay
In-vitro T-cell proliferation to VZV antigen was measured using 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, Oregon, USA) dye dilution assay according to the manufacturer's protocol. Briefly, after thawing, PBMC were resuspended in phosphate-buffered saline at a final concentration of 5–10 × 106 cells/ml and labeled with 0.5 μmol (final concentration) of CFSE for 8 min at room temperature. Labeling was stopped using human pool serum (Sanquin, Amsterdam, the Netherlands). Cells were washed and subsequently resuspended in RPMI (BioWhittaker, Verviers, Belgium), supplemented with 10% human pool serum and antibiotics (penicillin/streptomycin).
CFSE-labeled PBMC were cultured for 6 days at 37°C with VZV antigen in a final concentration of 20 μl/ml (Microbix Biosystems, Toronto, Canada), previously defined as the optimal effective dose for lymphocyte stimulation . Cells were stimulated otherwise with CD3 and CD28 monoclonal antibodies to define T-cell reactivity under optimal lymphocyte proliferation induction at final concentrations of 0.3 and 2 μg/ml, respectively (Sanquin Research, Amsterdam, the Netherlands). At day 6, cells were stained extracellularly with fluorochrome-conjugated monoclonal antibody against CD4 and CD8 (BD Biosciences). Cells were fixed in Cellfix (BD Biosciences), and 60 000–300 000 events were acquired using FACScalibur flow cytometer (BD Biosciences). The stimulation index (SI) was calculated by dividing the percentage of proliferating (CFSE low) CD4 or CD8 T cells after stimulation with VZV by the percentage of proliferation after stimulation without VZV antigen (control).
Plasma HIV-1-RNA determination
The plasma HIV-1-RNA concentration (plasma viral load) was determined using Versant HIV-1 RNA 3.0 (Bayer, Tarrytown, New York, USA). All tests were performed according to the instructions of the manufacturers.
Varicella zoster virus serology
Specific IgG to VZV was determined by Vidas immunoassay (Biomerieux, Lyon, France) following the instructions of the manufacturers. The test values of this assay were converted to IU/ml as determined by van der Zwet et al. . An antibody level greater than 0.14 IU/ml was regarded as positive. Seropositivity was defined by the presence of specific IgG after the age of 18 months to exclude any confounding contribution of maternal antibodies in the very young. Serological tests within 3 months after the administration of blood products were excluded from the analyses.
Varicella zoster virus culture
VZV culture on human embryo lung cells and VERO cells was performed, using standard procedures.
Statistical analyses were performed using SPSS for Windows version 11.5 (SPSS, Chicago, Illinois, USA). All P values were two-tailed. P values smaller than 0.05 were considered statistically significant. Continuous data were analysed using a Mann–Whitney U test. Categorical data were compared with a Fisher's exact test. Correlation was tested using the Spearman's correlation test. SI was modeled using a mixed model incorporating repeated measurements. This model handles missing data adequately by estimating the outcome using a ‘first order autoregressive’ structure. Differences in these estimates between different levels of the variable were tested for significance using t-statistics. Age correction for CD4 and CD8 T cells was done by dividing the counts by the mean of an age-matched healthy control group .
Baseline characteristics of study population
Twelve out of 13 patients on HAART (92%) included in this study had an undetectable plasma viral load at first vaccination. The median nadir percentage of CD4 T lymphocytes was 19%, 12 out of 15 vaccinated children had a nadir percentage below 25% and six even below 15%, having by definition a severe immune deficiency . CD4 and CD8 T-cell counts were comparable between HIV-1-infected and HIV-negative children (P = 1.0 and P = 0.3, respectively; Table 1).
Safety of the vaccine
After vaccination none of the 15 HIV-1-positive children experienced a serious adverse event related to primary or secondary vaccination. The plasma viral load did not change within the 6 weeks after the first or second immunization, irrespective of treatment or detectable plasma viral load at the time of immunization. VZV DNA was only detectable once in the whole blood of a single patient at 2 weeks after the first immunization (100 copies/ml). The patient had no clinical complaints or signs of varicella infection upon physical examination. All cultures for VZV from throat swabs remained negative.
No change in immune activation markers on T cells
Vaccination could induce immune activation of T cells and the upregulation of HIV co-receptors on CD4 T cells, which would lead to more target cells for HIV, potentially resulting in an increase in the plasma viral load. During the weeks after vaccination we measured the CD4 and CD8 T-cell counts, CCR5 and CXCR4 receptors, and immune activation as assessed by HLA-DR and CD38. At baseline the median percentages of CCR5 and CXCR4-positive CD4 and CD8 T cells were comparable between HIV-1-infected and HIV-negative children (P = 0.5, P = 0.07, P = 0.8, P = 0.9, respectively; Table 1). CD4 and CD8 T-cell counts did not change over time after immunization (Fig. 1a and b). The number of activated T cells as assessed by HLA-DR and CD38 expression remained unchanged (data not shown). The expression of both chemokine receptors CCR5 and CXCR4 did not show any change in the weeks after primary and secondary immunization (Fig. 1c–f).
Low humoral response rate of HIV-1-infected children
At 6 weeks after each immunization the VZV-specific serological response was assessed. After primary vaccination, five out of 15 HIV-1-infected children seroconverted for VZV. The median VZV IgG was 0.12 IU/ml. After booster vaccination, nine of the 15 children (60%) became VZV IgG positive. In the HIV-negative siblings, the response rate was three out of six (50%) after primary vaccination and six out of six (100%) after boosting. In addition, the anti-VZV IgG titres in HIV-1-infected children after two vaccinations against VZV were significantly lower than those of the HIV-negative siblings (median 0.2 versus 4.6 IU/ml, P = 0.002; Fig. 2). Of the nine children who seroconverted, seven were retested at 24 weeks after the second immunization. VZV-specific IgG was no longer detectable in two of the children. Of the five VZV-seronegative children who remained seronegative after two immunizations, two received a third vaccination. Only one of these two children seroconverted.
Low varicella zoster virus IgG titres after wild-type varicella zoster virus in HIV-1-infected children
To investigate antibody responses against VZV in natural VZV infection in HIV-infected children, we studied eight children with clinically evident chickenpox (of whom 50% had been VZV seropositive in the past), and an additional four children with clinical herpes zoster during treatment with HAART (Table 2). The median nadir percentages of CD4 T lymphocytes of the children who had wild-type VZV or herpes zoster were 16 and 7%, respectively. Only six of these eight children with wild-type varicella were VZV IgG positive after chickenpox. The titres of anti-VZV IgG after chickenpox in HIV-1-infected children were significantly lower than in HIV-negative siblings after vaccination (median 0.8 versus 4.6 IU/ml, P = 0.04). The titre after chickenpox was no different from the titre after vaccination of HIV-1-infected children (median 0.8 versus 0.2 IU/ml, P = 0.3).
The four children with herpes zoster had previously tested positive for VZV IgG. However, one child lost VZV antibodies during HAART, before herpes zoster. All four children were VZV seropositive after herpes zoster. The titre after wild-type zoster in HIV-1-infected children (0.4 IU/ml) was also lower than the titre after the immunization of HIV-negative siblings (P = 0.02), but was comparable with the immunization of HIV-1-infected children (P = 0.4).
Varicella zoster virus-specific T-cell responses after vaccination
The cellular immune response upon vaccination was measured after stimulation with VZV antigen using CFSE dye dilution assay and was expressed as SI. We measured separately the VZV-specific CD4 and CD8 T-cell proliferative responses after 6-day stimulation. The occurrence of VZV-specific CD4 and CD8 T-cell proliferation after primary and secondary vaccination in an HIV-1-infected child and an HIV-negative sibling are shown with the corresponding CD4 and CD8 SI (Fig. 3a and b and Table 3).
All children showed a peak in SI at approximately 4 weeks after immunization. The median peak CD4 SI of HIV-1-infected children was 6.9 [interquartile range (IQR) 3.0–18.1] and of HIV-negative siblings 3.3 (IQR 2.0–8.2, P = 0.2). The median peak CD8 SI of HIV-1-infected children was 0.7 (IQR 0.4–1.8) and of HIV-negative siblings 1.0 (IQR 0.7–1.4, P = 0.5; Fig. 3c). Also after the second immunization an increase in cellular reactivity was seen. The median CD4 SI at 6 weeks after the second vaccination was 4.2 in HIV-1-infected children and 3.9 in HIV-negative children. The median CD8 SI reached 1.5 in HIV-1-infected children and 1.6 in HIV-negative children (Fig. 3c). No difference over time was seen in either CD4 or CD8 proliferation between HIV-1-infected children and HIV-negative siblings [interaction term (SI*time) P = 0.5 and P = 0.7, respectively].
Age at baseline was negatively correlated with the anti-VZV IgG titre at 6 weeks after the second vaccination (r = −0.7, P = 0.007), but not with the peak in VZV-specific T-cell response (r = −0.15, P = 0.6) or the T-cell response 6 weeks after the second vaccination (r = −0.27, P = 0.4). The nadir age-adjusted CD4 T-cell ratio was neither correlated with the T-cell response nor with the serological response or peak of the CD4 T-cell response. We also found no correlation with any of the immunological outcomes and baseline age-adjusted CD4 T-cell ratio. The T-cell response at 6 weeks after the second vaccination was positively correlated with the antibody titre at the same timepoint (r = 0.6, P = 0.03). However, none of the three children above 13 years of age seroconverted, whereas nine of 12 children younger than 13 years of age demonstrated seroconversion upon immunization.
This study describes the results of VZV vaccination in a single-centre study in VZV-seronegative HIV-1-infected children. The live-attenuated VZV vaccine was well tolerated by both previously immunocompromised HIV-1-infected children treated with HAART despite previous immunodeficiency, as well as by stable, non-progressing HIV-1-infected children.
VZV vaccination of 15 HIV-1-infected children in our cohort and six healthy siblings was undertaken under strict surveillance by weekly clinical checks for fever, vesicular lesions and other clinical symptoms that could be associated with VZV infection by the Oka vaccine strain. VZV vaccination of HIV-1-infected children did not result in any adverse reaction upon vaccination. Specific T-cell responses against VZV were induced in all, but seroconversion occurred in only 60% of the children after two vaccinations. Three documented VZV contacts did not result in breakthrough infection in any of these VZV-immunized HIV-infected children, even in the presence of low VZV-specific titres. Again VZV titres did not rise afterwards (data not shown).
We show that the number of CCR5 or CXCR4-positive cells was stable in the weeks after vaccination, and plasma viral loads also did not increase upon VZV vaccination in any of the children, either with or without HAART. These data suggest that VZV vaccination has no effect on HIV-1 infection. Therefore, VZV vaccination can be considered safe in HIV-1-infected children during HAART despite previous immunodeficiency.
Current guidelines recommend vaccination of HIV-1-infected children with a well-maintained immune system . However, most children are tested HIV-1 positive at a later stage of the disease. Effectiveness in our study was similar to the seroconversion rate of 60% after two doses, as reported in 41 HIV-1-infected children with N1 or A1 disease . In this study severely immunocompromised patients were excluded.
Early trials of VZV vaccination of healthy immunocompetent children and adults showed high seroconversion rates (up to 100%) after primary vaccination. Serological and cellular responses persisted 3–4 years postimmunization in approximately 95% [6,25]. Clinical reactions were generally mild, being limited to 5–10% of recipients in the form of a mild generalized rash with less than 50 papules and some vesicles. These results are in line with our findings in HIV-negative siblings who showed 100% seroconversion after two vaccinations.
Children on chemotherapy or radiotherapy for malignant disease showed cellular and humoral immune responses after vaccination; however, at a lower level compared with otherwise healthy individuals . Lymphocyte counts below 700 cells/μl resulted in more frequent and severe side effects. This observation underlines the need for a re-established immunity before the vaccine can be administered safely. In our study, immune reconstitution was established because HAART was started months to years before immunization, and the plasma viral load was undetectable in 12 out of 15 vaccinated children. Seroconversion rates in immunocompromised patients are lower than in healthy children. Also in HIV-1-infected children only 60% had detectable VZV-specific IgG.
Our study provides new evidence that HIV-1-infected children remain immunodeficient, even when properly treated with HAART. First, serological responses to vaccination were significantly less than those observed in HIV-negative siblings or previously described in the literature [6,27]. Second, a relatively high number of wild-type VZV reinfections were observed in VZV-seropositive HIV-infected children, and once reinfected, titres did not rise steeply. Whether life-long immunity upon re-exposure [8,28] is maintained in HIV-infected individuals remains to be shown upon further follow-up.
The lack of any clinical symptom after vaccination and only one positive VZV polymerase chain reaction result in blood indicates that the immune system of children during HAART is able to mount an adequate primary cellular immune response upon vaccination. However, in the presence of comparable T-cell reactivity between HIV-1-positive and HIV-negative children, the serological response was much lower in HIV-1-infected individuals. Although the short-term restoration of immune function seems to protect individuals from vaccine-related varicella, functional immune restoration and immunological fine tuning is still incomplete, resulting in absent and low VZV-specific IgG. Perturbations in B-cell responsiveness were found not only to be caused by impaired CD4 T-cell help, but were also intrinsic to the detrimental changes in the B-cell compartment itself [29,30]. In our study we show that even during successful treatment with HAART, B-cell function remained disturbed. The absence of serological memory induction may also explain the increased rate of herpes zoster during HAART .
In conclusion, VZV vaccination of HIV-1-infected children is safe during HAART, even in children with previous CDC stage C events. Repeated vaccination of VZV-seronegative HIV-positive children seems mandatory, because primary and secondary vaccine failures frequently occur. Additional research with more children and longer follow-up is needed to determine further the effectiveness of vaccination against chickenpox by breakthrough wild-type infection or shingles by reactivated VZV.
The authors are indebted to the patients, their parents and caretakers for their participation, to the HIV nurses Atie van der Plas and Eugenie le Poole for their enthusiasm and care of the paediatric cohort, and to Mrs H. Schuitemaker and Mrs D. Pajkrt for critically reading and commenting on the manuscript.
Sponsorship: This study was financially supported by a grant from the Dutch AIDS foundation (grant 2002 7006).
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