Short Communication: Immunogenicity of an Inactivated Influenza Vaccine and Postvaccination Influenza Surveillance in HIV-Infected and Noninfected Children and Adolescents

Abstract

Individuals infected with HIV are at higher risk for severe cases of seasonal influenza infection and should receive annual doses of vaccine. Our objectives were to evaluate the immunogenicity of an influenza vaccine in 37 HIV-infected patients (HIV group) compared to 29 uninfected individuals (control group) and to carry out a clinical and virological surveillance of influenza during a 6-month follow-up. Both groups received the vaccine recommended for the southern hemisphere in 2008. Antibody responses to antigens H1N1, H3N2, and B were measured in blood samples at vaccination (T0) and after 1 month (T1). Influenza surveillance was performed by weekly telephone calls for a follow-up period of 6 months. Nasal washes were taken from subjects with respiratory symptoms. The direct immunofluorescence assay in house polymerase chain reaction (PCR) and real-time PCR were used for the detection of different respiratory viruses. The median age of the participants was 13.3 years (sd = 2.2) and 12.1 years (sd = 1.3) for the HIV group and control group, respectively. One month after vaccination (T1), both groups showed significant increases in the antibody geometric mean titers (GMTs) for all antigens. However, healthy controls showed higher values for antigens A/H1N1 and A/H3N2 (p = 0.002 and 0.001, respectively). There was a higher increase in the percentage of HIV-uninfected subjects with protective A/H1N1 antibodies (96.6%) compared to HIV-infected vaccinees (67.6%) at T1 (p = 0.004). Rhinovirus (27.7%) and coronavirus (22.5%) were the most prevalent agents identified in HIV-infected individuals. In the control group, the viruses most frequently found were rhinovirus (24.2%) and adenovirus (21.2%). The seroprotection rate for the H1N1 antigen was higher in the control group, which also showed a greater increase in GMTs for H1N1 and H3N2 antigens after immunization. Viral agents were identified in 39/60 (65%) episodes of respiratory infections from the HIV-infected group and in 17/32 episodes (53.1%) from the control group (p = 0.273).

P atients with immunosuppressive diseases such as acquired immunodeficiency syndrome caused by infection with human immunodeficiency virus (HIV) are among the most vulnerable to severe forms of influenza virus infection. Those who are affected may have prolonged viral replication, an extended duration of influenza symptoms, and a high rate of influenza-related mortality.¹ Since the early 1990s, immunization guidelines have recommended yearly vaccination against influenza virus in HIV-infected patients as a priority precaution.² However, the capacity of individuals to respond to vaccines with protective titers of the requisite antibody depends on the degree of immune impairment at the time of immunization.^3,4

Thus, people infected with HIV are potentially susceptible to influenza viruses even if properly vaccinated. Previous studies focused on the immunogenicity of influenza vaccines rather than on the close postvaccination influenza surveillance during seasonal epidemics.⁵

Taking into account the scarcity of data in the literature, this study followed up a cohort of HIV-infected children and adolescents who had been vaccinated with a single dose of influenza vaccine during the 2008 influenza season in São Paulo, Brazil. The aim was to evaluate the immunogenicity of the vaccine and to carry out clinical and virological surveillance of influenza in this population.

The open-label prospective study involved a cohort of 37 perinatally HIV-infected children and adolescents (mean age: 13.2 ± 2.2 years) who were followed-up at the outpatient clinic of the Division of Pediatric Infectious Diseases, Federal University of São Paulo, Brazil. HIV infection was classified in accordance with the United States Centers for Disease Controls and Prevention (CDC) disease stage criteria for clinical Categories N, A, B, and C and immunological Categories 1, 2, and 3.⁶ A control group was comprised of 29 HIV-uninfected subjects from a public school (mean age: 12.1 years ± 1.3 years) who received influenza vaccination as the primary immunization in May 2008. All HIV-infected subjects had been vaccinated against influenza in the previous season.

The protocol was approved by the Ethics Committee of Federal University of São Paulo and all parents or legal guardians gave written informed consent prior to enrollment in the study.

The children received one dose (0.5 ml, intramuscularly in the deltoid muscle) of inactivated influenza vaccine (Sanofi Pasteur) containing three strains of influenza viruses (A/Solomon Islands/3/2006-H1N1, A/Brisbane/12/2007-H3N2, and B/Florida/4/2006 hemagglutinin) that had been purified and fragmented following the recommendations of the World Health Organization (WHO) for the 2008 season in the southern hemisphere.

The study population was contacted weekly until the end of the influenza season to identify symptomatic respiratory infections and to collect nasal washes for virus identification. Symptomatic respiratory infections were defined by the presence of two or more signs and symptoms such as cough, runny nose, nasal obstruction, shortness of breath, and wheezing with or without fever (T ≥37.5°C).

Response to vaccination (seroconversion) was defined as an increase of antibody titers ≥4 times the baseline value. Antibody titers ≥1:40 were considered protective against influenza infection (seroprotection).

A baseline blood sample (5 ml) was collected from both groups immediately before (T0), and 1 month after immunization (T1). Serum samples (1 ml) were separated from whole blood by centrifugation, placed in microtubes, and stored at −20°C until required. A hemagglutination inhibition (HI) test was conducted according to standard procedure.^7,8 The HI antibody titer was expressed as the reciprocal of the highest dilution that inhibited agglutination.

Nasal washes were taken from study participants with symptomatic respiratory infections. The direct immunofluorescence assay (DFA) was used for rapid detection of influenza A virus (INF A) and B (INF B) and for the differential diagnosis of respiratory syncytial virus (RSV) and parainfluenza virus (PIV) types 1, 2, and 3 using a commercial kit (Dako). In-house polymerase chain reaction (PCR) and real-time PCR were used for detection of adenovirus (ADV), coronavirus (hCoV), rhinovirus (HRV), metapneumovirus (hPMV), and bocavirus (BocaV). Both DFA and PCR were used for detection of INF A and INF B. Viral RNA was obtained from nasopharyngeal samples with a QIAmp Viral RNA kit (Qiagen) and the cDNA synthesis was performed using the High Capacity cDNA kit (Applied Biosystems), according to the manufacturer's instructions. The amplification of target sequences was performed using specific protocols for each type of virus.

The CD4⁺ T cell counts and viral load of HIV-infected patients were assessed 1–2 months pre- and postvaccination. Demographic characteristics of patients and CDC 1994 classification of HIV infection were obtained from medical records.

Chi-square test or Fisher's exact test was used for between-group comparisons of seroconversion and seroprotection rates. The Student's t test or the nonparametric Mann–Whitney test was used to compare geometric mean titers (GMTs) of HI antibodies, lymphocyte subset counts, and age. Correlations between GMTs and number of previous influenza vaccine doses, CD4 values, and HIV viral load were evaluated by Pearson or Spearman correlation tests, when appropriate. The McNemar test was used for comparing seroprotection rates of the same group at different time points. For all statistical analyses, differences were considered significant when p < 0.05.

The groups did not differ in regard to gender (p = 0.804), but the HIV-infected patients were older than the control group (p = 0.003) (Table 1). The mean ± standard deviation age (years) at enrollment was 13.3 ± 2.2 and 12.1 ± 1.3 for the HIV-infected and control groups, respectively. Most of the 37 HIV-infected subjects (78.4%) had symptoms listed for clinical category B or C and 51.4% were considered as immunologic category 3 (CDC, 1994) (Table 1). Fifty percent of the participants had an HIV viral load below the limit of detection (50 copies/ml) before immunization (T0).

Table 1.

Demographic Characteristics of the Study Population

	HIV
	Positive	Negative	p
Sex			0.804
Female	18 (48.7)	15 (51.7)
Male	19 (51.3)	14 (48.3)
Age			0.030^a
Mean (dp)	13.3 (2.2)	12.1 (1.3)
Median (min–max)	12.7 (9.9–18.0)	12.0 (10.3–14.6)

Clinical category	n	%
N	3	8.1
A	5	13.5
B	16	43.2
C	13	35.2

Immunological category	n	%
1	4	10.8
2	14	37.8
3	19	51.4

Mann–Whitney test.

At T0, there were no significant differences in the antibody GMTs against all vaccine antigens between the two groups. One month after vaccination (T1), both groups showed significant increases in the antibody GMTs for all antigens. However, healthy controls showed higher values for antigens A/H1N1 and A/H3N2 (p = 0.002 and 0.001, respectively; Table 2). At baseline, both groups had no significant differences in antibody protective titers for the antigens A/H1N1 (p = 0.460), A/H3N2 (p = 0.565), and B (p = 0.140). The same occurred between groups for A/H3N2 and B antigens 1 month (T1) after vaccination (p = 0.095 and p = 0.764, respectively). However, there was a higher increase in the percentage of HIV-uninfected subjects with protective A/H1N1 antibodies (96.6%) compared to HIV-infected vaccinees (67.6%) at the same time point (p = 0.004) (Table 3). Also, the percentage of subjects from the HIV-uninfected group with a 4-fold or greater increase of A/H1N1 and A/H3N2 antibody titers was higher than that found in the HIV group (p = 0.03 and p = 0.01, respectively). There were no significant differences in GMTs for all antigens according to the number of vaccine doses received in previous influenza seasons. Pre- and postimmunization assessments showed no significant differences in CD4⁺ counts [mean CD4 pre = 748.5 (sd = 343.4); mean CD4 post = 746.3 (sd = 394.3); p = 0.966] or HIV viral load [mean VL pre = 3.38 log (sd = 0.92); mean VL post = 3.45 (sd = 0.91); p = 0.544] in the HIV-infected group.

Table 2.

Values of Geometric Mean Titers for Vaccine Antigens A/H1N1, A/H3N2, and B, and Time Points in HIV-Infected and Uninfected Subjects

		HIV
Antigens	Time point ^a	Infected	Uninfected	p values
A/H1N1	Baseline	18.6 [12.6–27.3]^b	14.0 [9.0–21.7]	0.235^c
	1 mo-post	46.5 [29.4–73.3]	132.2 [92.3–189.2]	0.002^c
A/H3N2	Baseline	17.9 [12.7–25.1]	16.1 [9.8–26.4]	0.719^d
	1 mo-post	57.1 [34.1–95.6]	198.4 [119.3–329.9]	0.001^d
B	Baseline	59.3 [39.1–89.9]	40.0 [23.2–69.0]	0.239^d
	1 mo-post	77.1 [49.7–119.5]	64.5 [37.7–110.4]	0.600^d

Prevaccination (baseline) and 1 month (mo) postvaccination.

95% confidence intervals are reported in brackets.

Mann–Whitney test.

Student's t test.

Table 3.

Protection Rates for Influenza Antigens A/H1N1, A/H3N2, and B Before and 1 Month After Vaccination of HIV-Infected and Uninfected Subjects

	HIV
Antigen	Infected Soroprotection rate [IC 95%]	Uninfected Soroprotection rate [IC 95%]	p^a
Prevaccination
AgH1	32.4 [18.0–49.8]	24.1 [10.3–43.5]	0.586
AgH3	37.8 [22.5–55.2]	31.0 [15.3–50.8]	0.611
AgB	75.7 [58.8–88.2]	58.6 [38.9–76.5]	0.185
Postvaccination (1 month)
AgH1	67.6 [50.2–82.0]	96.6 [82.2–99.9]	0.004
AgH3	75.7 [58.8–88.2]	93.1 [77.2–99.2]	0.095
AgB	75.7 [58.8–88.2]	72.4 [52.8–87.3]	0.491

Fisher's exact test.

Postvaccination surveillance detected 92 episodes of respiratory infections in 29 HIV-infected children and in 19 uninfected controls. Viral agents were identified in 39/60 (65%) episodes from HIV-infected group and in 17/32 episodes (53.1%) from the control group (p = 0.273). The viruses diagnosed in the HIV group and control group were, respectively: ADV (n = 8 and 6), hPMV (n = 1 and 2), HRV (n = 16 and 8), hCoV (n = 14 and 0), and INF B (n = 0 and 1). There was one case of coinfection hMPV/ADV in an HIV-uninfected child and no cases of INF A, parainfluenza, RSV, and BocaV in both groups during the follow-up.

Although several investigators have already analyzed the immunogenicity of different vaccines in HIV-infected children,^9

–12 incorporation of a follow-up period with virological surveillance has rarely been considered.¹³ Antibody responses vary according to the type of vaccine antigens, age at vaccination, and levels of immune impairment, and tend to be lower than in uninfected individuals.¹⁴ Contrasting data have been reported regarding the durability of immune responses elicited by vaccination in HIV-infected subjects. Immunization with measles, rubella, hepatitis A, and pneumococcal vaccines elicits optimal responses,^11,12,15,16 unlike immunization against tetanus, which produces an antibody response of limited duration.¹⁷ Influenza vaccination has also produced contradictory results.⁵

Presently, no significant differences between the groups in terms of GTMs against all three influenza strains were evident prior to immunization. All the healthy controls were not previously vaccinated against influenza, in contrast to the HIV-infected children who had already received several doses of influenza vaccine. Even in the context of an apparently satisfactory immunological response to highly active antiretroviral therapy (HAART) as seen in our patients, the effect of several doses of vaccine in previous influenza seasons could be reduced in HIV-infected children. One study reported that higher prevaccination antibody titers in the control group and annually repeated vaccination of HIV-infected individuals did not lead to higher postvaccination antibody titers.¹⁸

In the current study, the antibody protective rates were also similar in both groups before immunization. In contrast, a significantly lower percentage of protective A/H1N1-specific antibody titers in HIV-infected children compared to healthy controls preimmunization was reported.¹⁹ Presently, 1 month after immunization GMTs were significantly increased in both groups for all antigens, although the control group had GMT values significantly higher for the antigens A/HIN1 and A/H3N2. The seroprotection and seroconversion rates also varied according to the vaccine antigens. Whereas the postvaccination seroprotection for the antigen A/H1N1 was significantly higher in HIV-uninfected children (96.5%) compared to HIV-infected subjects (67.6%), this could not be shown for the other vaccine antigens. Seroconversion rates were similar in both groups for antigen B, but it was higher in healthy controls for the antigens A/H1N1 and A/H3N2. Another study found no differences regarding the antibody protective rates 1 month after vaccination between HIV-infected and uninfected children and the seroprotection conferred for all vaccine antigens persisted through an entire influenza season.¹³

Influenza virus vaccine can decrease chemokine receptor CCR5 expression on CD4 T lymphocytes, although this immunomodulatory effect does not seem to affect HIV replication in children receiving HAART.²⁰ We found no significant differences in HIV viral load and in CD4⁺ counts postimmunization. Similar results were previously reported, with no significant difference in the levels of HIV viral load between the moment of application of the influenza vaccine and 14–20 days and 60–90 days after its administration.²¹

Several viruses could be identified during vaccination follow-up, highlighting that sensitive techniques are crucial to confirm influenza infection, considering the clinical similarities with other respiratory infections. In addition, our study also demonstrated that respiratory symptoms following influenza vaccination are generally caused by other respiratory viruses and are not necessarily indicative of vaccine failure.

A single case of INF B was detected in the control group 1 day after immunization, suggesting that the patient was already infected at the time of vaccination.

Although the present study had a limited sample size, the observation of a lack of influenza infection among the HIV subjects during follow-up offers supporting evidence for the substantial benefit provided by influenza vaccination among HIV-infected children and adolescents.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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