Immunogenicity of trivalent influenza vaccine
The immunogenicity per-protocol analysis was limited to 83 (86.5%) of 96 children (Fig. 1). Postvaccination, seroconversion was observed in 47.5, 50.0, and 40.0% of TIV recipients for A/Brisbane(H1N1), A/Uruguay(H3N2), and B/Florida strains, respectively (P < 0.0001 for all strains compared to placebo recipients; Table 2). Among TIV recipients, less than 40% of younger children seroconverted to any vaccine strain, and this was significantly lower for A/Uruguay(H3N2) compared to older age group (Table 2).
The proportion of TIV recipients with postvaccination HAI at least 1:40 was lower in the younger compared to older age group for A/Uruguay(H3N2) (52.2 vs. 94.1%; P = 0.005) and B/Florida (43.5 vs. 88.2%; P = 0.004; Table 3). Similarly, fewer younger TIV recipients (31.0%) had HAI at least 1:110 to A/Uruguay(H3N2) compared to older TIV recipients (52.9%; P = 0.013; Table 2). The proportion of TIV recipients with HAI at least 1:215 was less than 30% to A/Brisbane(H1N1), less than 24% to A/Uruguay(H3N2), and less than 12% to B/Florida in both age groups (Table 2). Postvaccination, GMTs were lower in the younger compared to older age group of TIV recipients for A/Uruguay(H3N2) and B/Florida (Table 2).
Efficacy of trivalent influenza vaccine against seasonal influenza illness
Investigation for influenza virus occurred in 123 children who were investigated on 144 illness occasions, including 74 (51.4%) which fulfilled ILI criteria (Table 3). Multiple illness visits spaced at least 7 days apart were undertaken in 20 children, including one with three illness visits. The median age of children was 18.8 months (range 6–54.6) for ILI and 20.9 months (range 6–54.6) for confirmed influenza illness. There were 19 episodes of confirmed influenza illness among TIV recipients and 21 in placebo recipients. The dominant strain of influenza virus identified was H3N2 (37 of 40 cases). Two TIV recipients, aged 13.5 and 21.7 months with CD4+% of 27.6 and 36.8 and HIV viral load of less than 40 and more than 750 000 copies/ml at enrollment, respectively, experienced two episodes of confirmed H3N2 illness spaced 13 and 38 days apart.
The overall incidence (per 100 child-weeks) was 2.6 (95% CI 2.2–3.1) for medically attended respiratory illness, including 1.3 (95% CI 1.1–1.7) for ILI and 0.7 (95% CI 0.5–1.0) for confirmed influenza illness (Table 3). The incidence rates of ILI or confirmed influenza illness did not differ between study arms in the intent-to-treat or per-protocol analysis (Table 3). There were three TIV recipients in the immunogenicity subset with confirmed H3N2 influenza illness, all of whom received both TIV doses and were on ART. These children were 21.4, 25.0, and 54.6 months of age and had CD4+% of 24.3, 31.3, and 31.4, respectively. Two children less than 36 months failed to mount an immune response to any vaccine strain, although postvaccination HAI titers were 1:40 for each vaccine strain in the older child.
There were one TIV and two placebo recipients hospitalized for pneumonia during the influenza season, none of whom tested positive for influenza virus. One death occurred in a placebo recipient outside of a health facility during the seasonal influenza period, which was attributed to pneumonia but not tested for influenza virus.
Phylogenetic analysis on 21 randomly selected wild-type H3N2 viruses from study participants formed two clusters with a mean protein distance of 1.2 and 2.6%, respectively, to the vaccine strain (Supplemental digital material, http://links.lww.com/QAD/A267). Site-by-site amino acid analysis of the H3N2 viruses (Fig. 2) showed evidence of drift in reference to the A/Uruguay(H3N2) vaccine strain, as indicated by mismatched amino acid changes mapped to the immunodominant epitopes A (S138A), B (K158N; N189K; P194L), and D (K173Q; T212A).
Safety of trivalent influenza vaccine
There were no grade 3 or above solicited adverse events following either the first or second dose of study vaccine. The only solicited adverse events documented among TIV recipients within 3 days of either dose of vaccine were pain at site of injection (n = 1; 2.3%), fever at least 37.5oC (n = 2; 4.2%), induration at injection site (n = 1; 2.3%), and weakness (n = 2; 4.5%).
To our knowledge, this is the first randomized-controlled study which evaluated the efficacy of TIV in HIV-infected children. In our study of children mainly 6–35 months of age, who are at heightened risk of severe influenza illness independent of HIV infection , TIV vaccination did not prevent confirmed seasonal influenza illness or ILI. The lack of efficacy was in part corroborated by the poor immunogenicity of TIV against the dominant wild-type circulating strain, particularly in the younger age group (32.0% seroconversion).
Possible reasons for the lack of efficacy in our study include those which are inherent to any study evaluating TIV vaccine efficacy. These include the unpredictability of the severity of the influenza virus which emerges and possible vaccine strain mismatch to the wild-type strain. Genotypic analysis of H3N2 isolates from our study revealed only a 1.2% change in mean protein distance compared to A/Uruguay-like virus. These amino acid changes, however, affected key positions in three hemagglutinin epitopes (A, B, and D). The changes in epitopes A (S138A) and B (P194L) corresponded to positions that form part of the receptor-binding site . Immunodominant epitopes A and B induce high efficiency neutralizing antibodies. Whereas single epitope changes for A or B are associated with minor antigenic drift, mutations resulting in two or more changed epitopes as in our study are considered as major antigenic drift which necessitates selection of a new vaccine strain . HAI analysis of the wild-type H3N2 strains was not possible due to the phenotypic characteristic of recent H3 strains which do not hemagglutinate red blood cells from various species . A reference serum to A/Uruguay(H3N2)(2007), however, was negative in HAI assays against A/Perth/16/2009, suggesting a similar result could be expected for wild-type strains with the same mutations at receptor-binding site positions 138 and 194 .
The lack of efficacy may also be attributed to the poor immunogenicity of TIV, particularly in younger HIV-infected children. Although we did not enroll a control group of HIV-uninfected children, the seroconversion rates in the younger age group were lower compared to the 77.8% (H3N2) to 86.1% (Influenza/B) among healthy Canadian children aged 6–35 months , in whom an identical TIV formulation and dosing concentration was evaluated. The seroconversion rates in the Canadian study were also generally higher compared to our study's older age group. The impaired immune responses to TIV in our study may be due to HIV-induced impaired B-lymphocyte function, required for immune responses to TIV, which persist even following immune reconstitution with ART [17–19]. Impaired memory B-cell response to TIV in HIV-infected individuals has been attributed to deficiencies in serum and B-cell activating factor and APRIL (a proliferating-inducing ligand) and alteration in their receptors .
Traditionally HAI titers at least 1:40 have been used as a putative measure of relative protection against influenza illness in adults [20,21]. The relevance of this threshold in children is unclear [22,23]. Additionally, a HAI titer of 1:40 is associated with only 50% protection against influenza illness in adults [20,23], despite the likely presence of underlying cell-mediated immunity to influenza induced by previous exposure to influenza antigens from vaccination or from circulating wild-type influenza-virus. This underlying cell-mediated immunity is associated with cross-protection against different influenza strains , and boosting thereof by vaccination may contribute towards enhancing the efficacy of TIV in adults, which may also in-part mitigate the effect of any mismatch between the vaccine and circulating wild-type influenza strains. Although cell-mediated immune responses are induced in young children immunized with live-attenuated influenza vaccine (LAIV), such responses are less evident with TIV in the absence of previous exposure to related vaccine strains . In addition, young children are more likely to be influenza-naive and consequently may require higher HAI titers, although vaccine strain mismatch may be more relevant in them compared to that in adults. Black et al.  modeled that in children, HAI titers of at least 1:110 and at least 1:215 were predictive of 50 and 70% protection, respectively. In our study, an exploratory analysis identified only 13% of younger and 52.9% of older TIV recipients achieved HAI titers at least 110 to A/Uruguay (H3N2), and less than 30% in both age groups had titers at least 1:215 for any vaccine strain. The one vaccine failure due to H3N2 involved a 54.6-month-old child in our study, who had postvaccination H3N2-HAI titer of 1:40.
Most other immunogenicity studies on influenza vaccine in HIV-infected children have involved older HIV-infected children and adolescents [25–30]. Some of these studies preceded management of HIV-infected children with combination, triple ART [25,26,28], and more recently have focused on children/adolescents on ART who were immunologically reconstituted and virologically suppressed [29–31]. Among HIV-infected children on ART, immune responses to TIV correlated with immunological status prior to vaccination, with moderate-to-severe immunocompromised children benefiting from a second dose of vaccine . In addition, the magnitude of immune response and proportion of children with presumed seroprotective levels were lower than in healthy controls for at least two vaccine strains .
The lack of efficacy in our study was surprising, considering the 75% efficacy reported in the same setting among HIV-infected adults despite only modest seroconversion rates to TIV (47.4–60.8%) . In addition to the slightly lower seroconversion rates to TIV in this study (40–50%), other reasons for the difference in TIV efficacy between HIV-infected adults and children may include that the adults were possibly more likely to have established underlying cell-mediated immunity to influenza virus because of more frequent exposure to wild-type virus over their life time. This would have possibly enhanced their protection against influenza illness because of the role of cell-mediated immunity in clearing influenza infection as well as conferring some protection against influenza strains which are heterologous to the vaccine strain .
Recent studies have evaluated alternate influenza vaccine formulations, such as virosomal adjuvant vaccines [33,34], MF59-adjuvant and ASO3-adjuvant influenza vaccine, and LAIV [35–40], for safety and immunogenicity in HIV-infected children. Palma et al.  reported immunogenicity of MF59-adjuvanted monovalent H1N1 influenza vaccine in HIV-infected children following a single dose of vaccine, in whom seroconversion rate was 60%, but less than that in healthy controls (82%). In healthy children aged 6–72 months, the MF59-adjuvanted influenza vaccine was 81% more efficacious than unadjuvanted TIV. This included 79% efficacy against confirmed influenza illness in children aged 6–35 months and 92% efficacy in children aged 36–72 months, which was approximately double the efficacy observed for nonadjuvanted TIV .
Limitations of our study include being a single-center study because of it primarily been investigator-funded. Furthermore, the evaluable sample size (n = 403) only had 40% power to detect a 50% reduction in confirmed influenza based on the 10% attack rate observed among placebo recipients. The sample size was, however, sufficient to detect at least 73% vaccine efficacy at 80% power based on the observed attack rate. Although the study did not complete enrollment of the targeted 550 participants prior to the peak of the influenza season, this was offset by only 4.1% (n = 17) of enrollees, rather than the projected 20%, being lost to follow-up or withdrawn. A further limitation of our study is that it was conducted over a single influenza season. The study is, therefore, unable to definitively conclude the relative roles of poor TIV immunogenicity in younger children or antigenic drift of the circulating wild-type H3N2 away from the vaccine strain as being responsible for the lack of efficacy observed against confirmed influenza illness. Also, very few (<18%) of the children in our study were breastfed at any stage. It is possible that predominantly breastfed children may have mounted better immune responses, as breastfeeding contributes to boosting of the infant adaptive and innate immune system .
The results from our study do not support current recommendations for immunization of young HIV-infected children with nonadjuvanted TIV, even if on ART. We propose that further multicentered controls studies, which include a placebo arm and which are powered to investigate efficacy against confirmed influenza illness and not only immunogenicity, are required. Such studies should consider evaluating higher strength doses of TIV in young children, as well as the possible roles of LAIV and adjuvanted TIV in protecting HIV-infected children against influenza illness.
The authors would like to thank all the children and their parents for their participation in this study. In addition, the support of the nursing, pharmacy, and general staff at Perinatal HIV Research Unit and data clerks at the Respiratory and Meningeal Pathogens Research is acknowledged.
S.A.M. and A.V. contributed to the study conceptualization and design. First draft of the article was cowritten by S.A.M. and A.V. S.D., H.C., E.L., and T.T. were involved in participant follow-up and management. M.V. and F.T. were responsible for laboratory testing of influenza virus. A.W. was responsible for doing the hemagglutinin inhibition tests. C.L.C. was involved in protocol development and study logistics. M.V. and F.T. made input on the article sections on genotypic characteristic of the wild-type influenza virus, and A.W. on the immunogenicity data. All the authors critically reviewed the article and contributed to the final draft.
The study was funded in part through an unrestricted grant from Secure the Future Fund. The rest of the study, including vaccine procurement, was investigator-funded. Secure the Future did not have any input into the study design, data analysis, or write-up of the article.
Conflicts of interest
There are no conflicts of interest.
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children; efficacy; HIV; immunogenicity; influenza vaccine; pneumonia; safety
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