Long-term seroprotective response of trivalent seasonal influenza vaccine in HIV-infected children,regardless of immunogenicity before immunisation

Abstract

Influenza vaccination can reduce disease in HIV-infected children. The durability of the antibody response after trivalent influenza vaccine is important for management. The aim of this prospective study was to assess the durability of seroprotection for trivalent influenza vaccine strains and the factors effecting seroprotective response regardless of immunogenicity before trivalent influenza vaccine at one and six months after immunisation. Hemagglutination inhibition assay was done at one and six months. Seventy-five HIV-infected children were enrolled after vaccination. Four children were lost to follow-up. None of the children had confirmed influenza infection between immunisation and hemagglutination inhibition at six months after influenza vaccination. Seventy-one children were included in the final analysis and immunogenicity of trivalent influenza vaccine strains at one and six months. Of these, 27 (38%) had complete seroprotection (Group A) and 44 (62%) had non-complete seroprotection (Group B). Sex, age and the body mass index of both groups were not different from each other (p > 0.05). There was a higher mean CD4 level and more children with RNA ≤40 copies/mL among Group A compared with Group B (p < 0.05). Other factors did not differ significantly. The durability of the seroprotective response after trivalent influenza vaccine was associated with a high CD4 level and virological suppression before vaccination.

Keywords

Immunogenicity trivalent influenza vaccine HIV-infected children

Introduction

Seasonal influenza is a serious public health problem that causes severe illness and death in high-risk populations.¹ Influenza is a contagious respiratory illness that is mainly benign and usually self-limiting.² However, more severe influenza disease was reported in HIV-infected patients with poor vaccine immunogenicity.³ At present, influenza vaccination is the most appropriate strategy to reduce the disease burden and transmission.⁴ Generally, influenza vaccine is the best protection against influenza among young healthy adults and older children, and it also reduced the risk of influenza-related paediatric intensive care unit admissions by 74% during the flu seasons from 2010 to 2012.^5,6

The seasonal influenza vaccine has conventionally been trivalent, including two influenza A strains and one influenza B strain. The antigen type to be included in the vaccine is reviewed by the World Health Organization (WHO) annually. After vaccination, influenza antibody level peaks in two to four weeks and subsequently declines over time.⁷ The effectiveness of the influenza vaccine depends on at least two factors: the characteristics of the vaccinated person along with the match between viruses used to make the vaccine and the circulating virus.⁸

The immune protection of influenza vaccine among HIV-infected patients is controversial because the magnitude of antibody response can significantly vary and does not reach the minimum protective level in some cases.⁹ Previous studies have demonstrated that HIV-infected persons generate a lower initial antibody level after influenza vaccination compared to immunocompetent hosts.^10–12 Furthermore, study data regarding HIV-infected adults demonstrated that 54% had generated a seroprotective response at day 28, and 28% had a durable response at six months post-vaccination.¹³ In contrast, HIV-infected children had a high proportion of seroprotection rates after prime and booster doses of influenza vaccine (75% and 71%, respectively).¹⁴ One study demonstrated that 79% of HIV-infected children had generated a seroprotection response after influenza vaccination.¹⁵

The data of durability of the antibody response after influenza vaccination are important for management. To date, data regarding durability of immunogenicity after influenza immunisation in HIV-infected children have been limited. Therefore, we evaluated immunogenicity at one month and six months after influenza vaccination in HIV-infected children regardless of immunogenicity before influenza vaccination at Bamrasnaradura Infectious Diseases Institute, Thailand.

Materials and methods

Study design

This was a prospective study to assess the seroprotective response in HIV-infected children to trivalent influenza vaccine (TIV), regardless of immunogenicity before receipt of TIV. The primary objective was to identity the factors associated with complete seroprotection for all three TIV strains (Group A) and non-complete seroprotection (Group B). Group A was defined as complete seroprotection for all three TIV strains both at one month (30 ± 2 days) and six months. Group B was defined as non-complete seroprotection for all three TIV strains at one month or six months. The secondary objective was to compare the immunogenicity of those who had received TIV at both one month and six months between Group A and Group B. Seroprotection was defined as a titer of ≥40 post-vaccination for A/California/7/2009 (H1N1)-derived strain using NYMC X-179A, A/Victoria/361/2011 (H3N2)-like strain using IVR-65 and B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei Wujiagang/158/2009 strains. The study was performed at Bamrasnaradura Infectious Diseases Institute, Thailand during the 2012 to 2013 season. This study was reviewed and approved by the Ethical Committee for Research in Human Subjects, Department of Diseases Control, Ministry of Public Health, Thailand, and by the institutional review board. Written informed consent was obtained from parents or legal guardians before children were included in this study.

Study population and sample size calculation

After vaccination of an appropriate influenza vaccine, children were enrolled between 24 April 2013 and 19 December 2013. Children included in this study had the following: six months to ≤18 years of age, HIV infection, influenza vaccine, antiretroviral therapy and a CD4 of more than 15%. All children were diagnosed with HIV infection by HIV antibody or PCR and perinatal infection. All children were contacted for information of influenza infection. Adherence to antiretroviral therapy was defined as children who took ≥95% of their antiretroviral pills, which is key to success in HIV-infected treatment among children.¹⁶ In this study site, the majority of HIV-infected children were adolescents with high CD4 levels. Therefore, the sample size was calculated by a proportion formula assuming the seroprotection rate at six months after influenza vaccine among healthy adults was 82%,¹⁷ and the minimum sample size was calculated to be 57.

Vaccine formulation and administration

TIV for the 2012 to 2013 season contained A/California/7/2009 (H1N1)-derived strain using NYMC X-179A, A/Victoria/361/2011 (H3N2)-like strain using IVR-65 and B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei-Wujiagang/158/2009 strains. All TIV were provided by Department of Diseases Control, Ministry of Public Health, Thailand. Doses were provided in pre-filled 0.25 mL or 0.5 mL, single-dose syringes or in 0.5 mL, single-dose vials. Vaccine potency was assessed periodically by a routine stability monitoring program. Children were immunised with the appropriate dose of vaccine based on age at the time of enrolment; 0.25 mL for children six to <36 months of age and 0.5 mL for children three to <9 years of age as recommended by the Advisory Committee on Immunization Practices for the 2012–2013 season.⁴ Children received one or two doses of the study vaccine four weeks apart based on their influenza vaccine histories. All immunisations were administered by intramuscular injection into the anterolateral thigh or deltoid region.

Hemagglutination inhibition assay

Blood samples were collected at one month and six months after the final vaccination. Hemagglutination inhibition (HAI) antibody titers against TIV strains were assessed at the Siriraj Influenza Cooperative Research Center, Department of Microbiology, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand. HAI assay for detection of influenza antibody was performed as previously described.¹⁸ The replicating viruses including A/California/07/2009(H1N1) virus, A/Victoria/361/2011(H3N2)- like virus and B/Wisconsin/2010-like virus were used as the test antigens. Fifty microlitres of the test serum were treated with 150 µl of receptor destroying enzyme (Denka Seiken, Tokyo, Japan) overnight at 37℃ for removal of non-specific inhibitor and followed by heat inactivation at 56℃ for 30 min to destroy the enzyme activity and the heat-labile inhibitors. At the last step of treatment, the serum was absorbed with goose red blood cells for 1 h at 4℃ for removal of non-specific agglutinators. The treated serum at the initial dilution of 1:10 was serially two-fold diluted in phosphate-buffered saline in duplicate wells of the microtiter V-shaped plate. The diluted serum at a final volume of 25 µl was mixed with a 25 µl volume of each test antigen at the concentration of four hemagglutination units. The reaction plate was incubated for 30 min at room temperature and then added with 50 μl of 0.5% goose erythrocyte suspension per well and incubated at 4℃ for 30 min. The HAI antibody titer was determined based on the last serum dilution that completely inhibited hemagglutination reaction.

Post-vaccination influenza active surveillance

Post-vaccination influenza active surveillance was performed between April 2013 and June 2014. The study team telephoned the parents/legal guardians of the study children every one month and there were the self-reports of illnesses in study children from parents/legal guardians. Children or their parents/legal guardians were instructed to contact the study team in the event of influenza-like illness (ILI) that is defined as a sudden onset of symptoms and at least one of four systemic symptoms (fever/feverishness, malaise, headache or myalgia) and at least one of three respiratory symptoms (cough, sore throat or shortness of breath).¹⁹ Children with a diagnosis of ILI were instructed to return to the clinic for nasal swab for rapid influenza test. Each child was followed-up six months after TIV.

Statistical analysis

All continuous data such as age, BMI, baseline and nadir CD4 cell counts, HIV RNA level, Hct were compared with Student’s t-test. Categorical data such as sex, previous influenza vaccine, a proportion of adherence ≥95% to antiretrovirals (adherence ≥95%), a proportion of RNA ≤ 40 copies/mL and receipt of highly active antiretroviral therapy (HAART) before TIV were compared with the Chi square test. A p value < 0.05 was considered statistically significant. All hypothesis tests were two-sided.

Results

Study population

Figure 1 shows a flow chart of the enrolment and follow-up of the study children. Seventy-five children were enrolled after receiving an appropriate influenza vaccine. Four children (5%) were excluded because of loss to follow-up. Seven ILI-infected children had negative rapid influenza test results. None of the children had confirmed influenza infection between immunisation and HAI at six months after influenza vaccination. Seventy-one children without confirmed influenza infection were included in the final analysis. Of these, 27 (38%) had complete seroprotection (Group A), and 44 (62%) had non-complete seroprotection (Group B).

Figure 1.

Flow diagram enrolment and follow-up of study children.

Table 1 displays the demographics and characteristics of the study children. The majority were male in both groups. The mean ages of Group A and Group B were 13.35 and 11.44 years, respectively. The mean BMI of Group A and Group B were 17.42 kg/m² and 18.72 kg/m², respectively. The demographics and characteristics of both Groups A and B were not significantly different from each other (p < 0.05).

Table 1.

Demographics and characteristics of study children.

Factor	Group A seroprotective for all 3 strains at 1 and 6 mo. (N = 27)	Group B non-seroprotective for all 3 strains at 1 or 6 mo. (N = 44)	p
Sex, n
Male	14	24	1.00
Female	13	20
Age (years), mean ± SD	13.35 ± 3.90	11.44 ± 5.20	0.104
BMI (kg/m²), mean ± SD	17.42 ± 3.30	18.72 ± 4.09	0.165

BMI: body mass index.

Risk factors of study children

The risk factors of the study children are shown in Table 2. The mean nadir CD4 levels before influenza vaccination in Group A and Group B were 958 and 809 cells/µL, respectively. The CD4 levels among Group A were higher than those of Group B (p < 0.05).

Table 2.

Risk factors of study children.

Factor	Group A seroprotective for all three strains at one mo. and six mo. (N = 27)	Group B non-seroprotective for all three strains at one mo. or six mo. (N = 44)	p
History of receiving influenza vaccine within the previous year	21	39	0.220
Adherence ≥ 95%	21 (78%)	33 (75%)	1.00
Nadir CD4 cell count, mean (cells/µL) before influenza vaccine	958 ± 328	809 ± 386	0.048
Peak log₁₀ plasma HIV RNA before influenza vaccine, median (range)	1.3 (1.3–4.4)	1.3 (1.3–147.7)	0.441
RNA ≤ 40 copies/mL	26 (96%)	34 (77%)	0.043
Hct (g/dL)	38.41 ± 3.53	34.89 ± 3.67	0.558
HAART			0.456
PI	14 (52%)	28 (64%)
Non PI	13 (48%)	16 (36%)

HAART: highly active antiretroviral therapy; PI: protease inhibitor.

The proportion of RNA ≤ 40 copies/mL in Group A (96%) was also higher than that of Group B (77%) (p < 0.05). History of receiving influenza vaccine within the previous year, a proportion of adherence ≥95%, Hct (g/dL), and intake of protease inhibitor (PI)-based HAART were not significantly different between Groups A and B (p > 0.05).

Seroprotection for TIV strains

Table 3 shows the seroprotection rates for the TIV strains used in this study. At one month after influenza vaccine immunisation, the seroprotection rates for A/California/7/2009 (H1N1)-derived strain using NYMC X-179 A, A/Victoria/361/2011 (H3N2)-like strain using IVR-65 and B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei-Wujiagang/158/2009 strains were 83%, 73% and 75%, respectively. At six months, seroprotection was 68%, 72% and 66%, respectively. Seroprotection for A/California/7/2009 (H1N1)-derived strain using NYMC X-179 A had trend to be more than the other strains at one month. At six months, seroprotection for B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei-Wujiagang/158/2009 had trend to be less than that of the other strains. At six months, seroprotective response for each strain had a declining trend, and they did not differ significantly between one month and six months.

Table 3.

Seroprotection for A/California/7/2009 (H1N1)-derived strain using NYMC X-179A, A/Victoria/361/2011 (H3N2)-like strain using IVR-65 and B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei-Wujiagang/158/2009 strains.

Strains in TIV	Total (n = 71)	HAI antibody titer ≥ 40		p
		Month after TIV
		1 mo.	6 mo.
A/California/7/2009 (H1N1)-derived strain using NYMC X-179A	71	59 (83%)	48 (68%)	0.051
A/Victoria/361/2011 (H3N2)-like strain using IVR-65	71	52 (73%)	51 (72%)	1.000
B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei-Wujiagang/158/2009	71	53 (75%)	47 (66%)	0.358

TIV: trivalent influenza vaccine; HAI: hemagglutination inhibition.

Type of seroprotection for TIV strains

Table 4 shows type of seroprotection for TIV strains. At one month, the numbers of study children with seroprotection for all three TIV strains, two TIV strains and one TIV strain were 36/71 (51%), 23/71 (32%) and 10/71 (14%), respectively. Of the 36 children in the seroprotection for three TIV strains group at one month, the numbers declined at six months to 27/36 (75%) with seroprotection for three TIV strains, 8/36 (22%) with seroprotection for two TIV strains and 1/36 (3%) with seroprotection for one TIV strain.

Table 4.

Type of seroprotection for TIV strains.

At 1 month	N	Types of strain	At six months	N	Types of strain
HAI antibody titer ≥ 40			HAI antibody titer ≥ 40
Three strains	36	All strains	3 Strains	27	All strains
			2 Strains	8	3 H1N1/H3N2 3 H1N1/B 2 H3N2/B
			1 Strain	1	1 H3N2
			No strain	0
Two strains	23	9 H1N1/H3N2 11 H1N1/B 3 H3N2/B	3 Strains	0	–
			2 Strains	19	8 H1N1/H3N2 8 H1N1/B 3 H3N2/B
			1 Strain	4	1 H3N2, 3 B
			No strain	0	–
One strain	10	3 H1N1, 3 H3N2,4 B	3 Strains	0
			2 Strains	0
			1 Strain	8	1 H1N1, 4 H3N2, 3 B
			No strain	2	–
HAI antibody titer < 40	2	–	HAI antibody titer < 40	2	–

H1N1: A/California/7/2009 (H1N1)-derived strain using NYMC X-179A strain H3N2: A/Victoria/361/2011 (H3N2)-like strain using IVR-65 strain B: B/Wisconsin/1/2010-like strain using NYMC BX-39 derived from B/Hubei-Wujiagang/158/2009 strain.

TIV: trivalent influenza vaccine; HAI: hemagglutination inhibition.

Twenty-three children with seroprotection for two TIV strains at one month consisted of 9/23 (39%) H1N1/H3N2, 11/23 (48%) H1N1/B and 3/23 (13%) H3N2/B. The numbers in this group declined to 19/23 (83%) with seroprotection for two TIV strains, 4/23 (17%) with seroprotection for one TIV strain and none of children were seroprotection for three TIV strains and non-seroprotection at six months.

Ten children in the seroprotection for one TIV strain group at one month consisted of 3/10 (30%) H1N1, 3/10 (30%) H3N2 and 4/10 (40%) B. The numbers in this group declined to 8/10 (80%) with seroprotection for one TIV strain, 2/10 (20%) with no seroprotection and none of children were seroprotection for two and three TIV strains at six months. Two children with non-seroprotective responses to three strains at one month were non-seroprotection at six months.

Discussion

The durability of seroprotection for influenza vaccine often spans an estimated six months.⁴ However, data on the durability of seroprotective response after TIV among HIV-infected children are limited. In this study, we found that HIV-infected children are less likely to maintain a seroprotective response for all three strains in TIV for at least six months after immunisation. Only 27/71 (38%) of children had a complete seroprotective response at both one month and six months. This study demonstrated that complete seroprotective response was associated with a high CD4 level and RNA ≤ 40 copies/mL. Because a high level of CD4 leads to improved cellular immunity and humoral immunity, a B cell-stimulated antibody response to influenza vaccine and a higher HIV viral load related to reduced post-vaccination IL-10 levels may affect vaccine response.²⁰ The findings of this study are compatible with other studies that have reported higher ratios of CD4 to CD8 and virological suppression at the time of immunisation associated seroprotective response after influenza vaccine.^14,21 In addition, an association between poor responses to influenza vaccines and higher viral loads and/or lower current or nadir CD4 counts has been reported.^22–25 Other risk factors such as history of receiving influenza vaccine within the previous year, a proportion of adherence ≥95%, Hct (g/dL), and intake of PI were not associated with a complete seroprotection response. In contrast to this study, a previous study showed that HIV-infected patients who had received antiretroviral therapy often affected vaccine responses.²⁶ Interestingly, the previous annual influenza vaccine did not affect seroprotection in this study because all of the children had received annual influenza vaccines 10 to 12 months apart from each other. This lack of effect was caused by waning immunity in those 10 to 12 months and the antigenic variation of virus every year.^4,8

This study demonstrated a low proportion of complete seroprotection for all vaccine strains after receipt of TIV. However, the seroprotection rates after TIV for each strain were high at one month (83%, 73% and 75%, respectively). This was similar to another study in which HIV-infected children had a high seroprotection rate of 75% after inoculation with the inactivated split-virion AS03-adjuvanted pandemic H1N1(2009) vaccine,¹⁴ while another previous study showed that the seroprotection rates of the inactivated influenza A (H1N1) pdm 2009 vaccine in healthy children were 78 to 98% at 21 days.²⁷ A previous study in response to TIV reported that 73% of healthy children exhibited protective responses.²⁸ In contrast, a similar previous study showed the proportion of seroconversion for H1N1, H3N2 and influenza B strains in HIV-infected children were 47.5, 50.0 and 40.0%, respectively.²⁹

Although all seroprotective responses of each strain had a declining trend at six months, the proportions of seroprotection did not differ significantly between one month and six months. This was similar to a previous study that reported HIV patients with seroprotection at day 28 were associated with a durable antibody response.¹³ None of the children with seroprotection for TIV strains at six months were non-seroprotective response at one month because the seroprotection or HAI antibody titer ≥40 at one month represented a durable antibody response.¹³

Post-vaccination influenza active surveillance was performed. There were low levels of influenza activity during this study that may have led to decreased incidence of influenza-infected cases. None of the children had confirmed influenza infection in this study. It was similar to another study, in a positive way, as the lack of influenza illnesses likely reduced confounding regarding the potential effects of intercurrent influenza infection on antibody levels.¹³

This study had some limitations. It did not evaluate immunogenicity before immunisation because HAI prior to immunisation was not done. However, the main objective was to assess the durability of the immunogenicity for TIV strains, regardless of immunogenicity before immunisation. Also, the sample size was small and children with high CD4 levels had less influenza illness and severe complications. Another limitation was that although we contacted children for information of illnesses by visits, telephone and the self-reports to exclude children with confirmed influenza infections between immunisation and HAI at six months from the final analysis, it would have been difficult to distinguish between immunogenicity generated by the vaccine strains and asymptomatic or mild influenza infection.

In conclusion, children with a high CD4 level and virological suppression before vaccination were more likely to sustain a seroprotective response for the vaccine strains, whereas children with a lower CD4 level and detectable HIV RNA demonstrated a lower durability of seroprotective response. These findings may support influenza vaccination during a high CD4 level and virological suppression to increase the efficacy and immunogenicity of the vaccine.

Footnotes

Acknowledgements

The manuscript was written with assistance. The authors thank all the study participants and staff of Bamrasnaradura Infectious Diseases Institute, Ministry of Public Health, Nonthaburi, Thailand.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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