Boosting of Cross-Reactive and Protection-Associated T Cells in Children After Live Attenuated Influenza Vaccination

MATERIAL AND METHODS

RESULTS

In the current study, we examined the ability of LAIV to induce cross-reactive antibodies and T cells in children. To evaluate the immune responses to the heterologous H1N1 strain, we chose the historical Solomon Islands/06(H1N1) strain, which circulated 7 years before our study, and before 10 (71%) of the children were born. To evaluate the heterologous H3N2, we chose the Switzerland/13(H3N2) strain, which was antigenically distinct from the Victoria/12-like(H3N2)/Texas/12(H3N2) vaccine strain. This circulated in 2014–2015, the year after our study, so none of the children could have been exposed. There is a larger genetic distance between the H1N1 (0.22 in evolutionary distance) than the H3N2 strains (0.025 in evolutionary distance; Figure 1).

DISCUSSION

Natural infection provides the basis for cross-reactive T cells, and historical studies provide evidence for heterovariant protection after influenza infection [1, 6, 18]. Current research toward the desired universal influenza vaccine includes focus on T cells and their potential for cross-protection across different IAVs. As opposed to inactivated influenza vaccines, the LAIV mimics natural infection, inducing broad T-cell responses [8, 9]. We hypothesized that these T cells could cross-react to heterovariant influenza strains. Our study found that up to 70% of children had preexisting cross-reactive, influenza-specific T cells in response to heterosubtypic IAVs in the absence of preexisting antibodies (70% to H1N1, 60% to H3N2). Furthermore, we found that the LAIV boosted T-cell responses to genetically diverse, wild-type IAVs, to which the children could not have been previously exposed and to which LAIV did not generally induce antibody responses.

Cross-reactive T cells are considered important in limiting disease severity and death in the absence of strain-specific antibodies, such as during a pandemic [2, 6]. It is noteworthy that the overall levels of preexisting heterovariant influenza-specific IFN-γ T cells were high, with most children having levels of >100 SFUs/10⁶ PBMCs before vaccination, which were boosted after receipt of LAIV. These findings indicate a considerable number of preexisting influenza-specific T cells with cross- reactive potential in our pediatric population, confirming findings at a population level (>20 SFUs/10⁶ PBMCs) [7].

These preexisting T cells may explain the diversity of clinical symptoms after infection, from asymptomatic to fulminant pneumonia [19]. Remarkably, in children without detectable HI antibodies to divergent strains, we found high levels of cross-reactive T cells in all but 2 children before vaccination. Importantly, our study found that LAIV boosted cross-reactive T-cell responses to historical and drifted wild-type IAVs not included in the vaccine. This important finding could imply a considerable protective effect at the population level if LAIV is administered to children. Indeed, signs of herd immunity, despite circulation of drifted influenza strains, as well as a reduction in child hospitalization rates, have been observed in the United Kingdom, where widespread LAIV vaccination of school children was conducted, [20, 21].

A previous study by Mohn et al [9], the longest reported follow-up of cellular responses in LAIV vaccinated children, showed increased levels of T cells for up to 1 year. The results from the current study provide proof of concept that LAIV boosts cross-reactive and protection-associated CD8⁺ T cells. This important finding may help explain the clinical observation that LAIV provides protection from culture- confirmed illness in the absence of a boost in HI antibodies [22]. This is also supported by the correlation in CD4⁺ and CD8⁺ T-cell responses, but no correlation between the cellular and HI responses [8].

Similarly, we found no correlation between HI titer and CD8⁺ T-cell responses, and children with high HI titers showed no increase in CD8⁺ T-cells, while children with low HI titers showed and increase, indicating a response in the cellular or humoral immunecompartement, but not both (Supplementary Figures 3 and 4). One child (15 years old) responded in both the humoral and cellular compartments, and 1 (3 years old) did not respond in either compartment, highlighting the individual variation in human immune responses. However, because LAIV is dependent on viral replication to elicit immune responses, we cannot exclude the possibility that LAIV viruses are neutralized by preexisting antibodies. In agreement with our pediatric findings, a Russian LAIV study among young adults found cellular immune responses in all subjects without an increase in HI antibody titers [23]. In the Russian study, baseline CD4⁺ and CD8⁺ responses were predictive of post-LAIV cellular immune responses. Although the Russian LAIV is considered more immunogenic, it induced HI antibodies in ≤50% of the subjects, suggesting that the lack of HI response we found is more likely linked to the LAIV itself, rather than to the Russian or US/European LAIV.

Our study is the first to our knowledge to show long-term, cross-reactive CD8⁺ T-cell responses after LAIV vaccination, with the youngest children having the largest increases. Generally, the T-cell response after infection is thought to be rapid, and the peak was earlier than day 7 in adults during the 2009 pandemic [24]. Although we found significant increases 28 days after LAIV, larger increases may have been detected if we had sampled at an earlier time point.

Cross-reactive T-cell responses are directed primarily to conserved internal viral proteins (M1, NP, PB1) [1, 2]. CD8⁺ T cells have been found essential in limiting disease, and providing protection on heterosubtypic challenge in animal studies [12, 25]. Moreover, human studies have shown that preexisting CD8⁺ and CD4⁺ T cells, specific for these conserved proteins, have been associated with lower viral shedding and less severe influenza disease [1, 2, 7]. Interestingly, lower levels of CD8⁺ T cells were observed in adults hospitalized with pandemic influenza disease, perhaps leaving them vulnerable to severe disease [26, 27]. Whether it is the magnitude or the functionality of the CD8⁺T-cell response that has a protective effect remains to be elucidated [28]. We found a significant increase in the CD8⁺ IFN-γ-response after LAIV, with the greatest response toward the NP protein, supporting the importance of including NP in broadly protective T-cell–inducing vaccines [7].

Our study is limited by its few subjects, and larger studies will therefore be needed to confirm our findings. Furthermore, we do not know the duration of the CD8⁺ T cells beyond 1 year, hence the need for repeated annual vaccination. However, if these CD8⁺ T cells provide protection from severe illness, LAIV could prove to be an important public health countermeasure in a situation with mismatch of epidemic and seasonal vaccine strains, or in the face of a novel pandemic virus, before a strain-specific vaccine is available. The finding that 50% of the children had increases in CD8⁺ T cells may be explained by the HLA genetic diversity, with the peptides perhaps not covering all HLA types.

It is debated whether LAIV is effective in older age groups. Children’s developing immune systems, combined with the impact of previous exposures to influenza, may influence the age-related response to LAIV. Our results show age-dependent differences in the response, supporting immunization of the youngest children with 2 doses, although most respond after the first dose. Importantly, our findings suggest that if a child has a CD8⁺ T-cell response, this will be cross-reactive. The single child that did not respond with CD8⁺ T cells after vaccination did not respond to either cross-reactive strain.

In contrast to cross-reactive T cells, only strain-specific antibodies neutralize the virus. However, a study in LAIV vaccinated children found an induction of cross-reactive HI antibodies to a drifted H3N2 strain, and the children who were vaccinated but nevertheless infected showed milder symptoms than infected controls [29]. Interestingly, although LAIV protected against a closely related H3N2 strain, recent epidemiological studies showed no sterilizing immunity against more antigenically divergent strains [19]. However, cross-reacting neutralizing antibodies have been found for closely related strains [30–32].

Overall, we did not find cross-reactive antibodies to the Solomon Islands/06(H1N1) strain, except in 2 older children, indicating earlier infection. However, 3 children responded to the drifted Switzerland/13(H3N2) strain, which has a closer genetic relationship to the Texas/12(H3N2) vaccine strain (Figure 1). The general lack of antibody response may also explain the failure of LAIV to provide sterilizing immunity in the 2013–2014 season, when the Switzerland/13(H3N2) variant emerged. However, we found durable vaccine, strain-specific antibodies 1 year after vaccination, with lower responses to the H1N1 than to the H3N2 strain, similar to findings during the 2012–2013 influenza season [9, 13]. This strain variability could be a result of the current H1N1 strain being less immunogenic than during earlier seasons, an issue being addressed by the vaccine manufacturer [33, 34] Currently, there is disparity between LAIV effectiveness data in the United States and in Europe. In the United States, the lack of effectiveness caused concern and withdrawal of the prior LAIV recommendation. In Europe, LAIV has been found to provide protection and is recommended. The reason for these differences is currently unknown, and investigation is ongoing, but it could be related to the vaccine, the viruses, or the population’s exposure history [20, 35].

Efficacy studies measure viral replication in the upper respiratory tract in patients with influenza like illness; cross-reactive T cells cannot provide sterilizing immunity but could limit severe disease. Perhaps an improvement in LAIV efficacy studies would be to divide vaccinated subjects who become infected into mild and severe infection groups, potentially illuminating a clinical protective effect. Indeed, a study in the United Kingdom found LAIV to reduce hospitalizations in children [20]. Norway does not have effectiveness data, but surveillance data for the study year indicated that approximately 60% and 30% of influenza infections were due to H1N1 and H3N2 strains, respectively [36].

In conclusion, our unique trial is the first to show long-term cross-reactive T cells elicited by LAIV in children. Importantly, our study found that these preexisting and protection- associated CD8⁺ T cells were detectable in young children in the absence of antibodies, which is proof of concept that the LAIV boosts CD8⁺ T-cell responses to conserved antigens. The responses were durable, indicating that a cellular immune response could possibly last through a whole influenza season. Although our findings were limited by small numbers, they support our hypothesis that the LAIV has the potential to provide cross-protective immunity to drifted and potentially heterovariant strains. Hence, it could possibly be a step toward the desired universal influenza vaccine.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Supplementary Material

Notes

Acknowledgments.  We thank the children and parents who altruistically participated in this study. We also thank the staff at the pediatric clinical trial unit and Ear Nose and Throat Department at Haukeland University Hospital and Sarah Lartey and staff at the Influenza Centre for their help with conducting the study. We especially thank Dr Othmar Engelhardt at the National Institute for Biological Standards and Control, South Mimms, United Kingdom, for providing the heterologous Switzerland/12(H3N2) strain and for valuable input to the study. We thank Astra Zeneca for recommendations and assisting with vaccine purchase and import.

The following reagent was obtained through the National Institutes of Health Biodefense and Emerging Infections Research Resources Repository, National Institute of Allergy and Infectious Diseases: peptide arrays, peptides for expected conserved major histocompatibility complex class I epitopes of influenza virus A proteins, NR-2667.

Financial support.  The study was funded intramurally by the Influenza Centre at the University of Bergen. The Influenza Centre is funded by the Ministry of Health and Care Services, Norway, the Norwegian Research Council Globvac program (grant 220670/H10), the European Union (UNIVAX 601738), and Innovative Medicines Initiative (IMI) (IMI115672 FLUCOP), Helse Vest, and the K. G. Jebsen Centre for Influenza Vaccines.

Potential conflicts of interest.  All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.