Diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following H7N9 influenza challenge in mice

Abstract

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8(+) cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K(b)PB(1703, D(b)PA(224), and D(b)NP(366) epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.

Figure 1. Experimental design for analyzing CTL-mediated heterosubtypic immunity against H7N9 virus infection.

8–10 week old female B6 mice were first primed intranasally with 10⁴ TCID₅₀ of an H9N2 virus or 10² TCID₅₀ of an H1N1 virus. The virus-specific primary CTL responses in the bronchoalveolar lavage (BAL) were characterized on day (d) 8 and/or d10 post inoculation (p.i). Blood was collected for Hemagglutination inhibition (HI) assays on d35. The virus-specific memory CTLs in the spleen were characterized on d38. Between 10~12 weeks after the initial priming, the primed mice were intranasally challenged with an H7N9 virus and the H7N9 virus-specific-secondary CTL response in the BAL was characterized on various days between d0 to d14 after challenge infection.

Figure 2. The disease course and primary CTL responses in naive mice after infection with one of two H9N2 or two H1N1 IAVs.

The primary IAV infection was conducted as described in the legends to Fig. 1. (A) Body weight change and (B) virus replication kinetics in mice. (C) The proportion and (D) number of each epitope-specific CTL population, and (E) the total number of the three epitope-specific CTLs in the BAL on d8 or d10. (F) Representative IFN-γ ICS flow cytometry plots from CD8+ T cells in BAL samples. The PB1₇₀₃, PA₂₂₄, and NP₃₆₆ peptide variants specific for each virus were used to stimulate the CTLs to produce IFN-γ for the ICS assay. The data sets represent mean ± SEM; * p<0.05 by Tukey’s test, comparing: (A, B) each virus versus every other virus at that time point, n = 10 per group; (C, D) the indicated epitope versus the other two epitopes in the virus, n = 4–5; (E) the indicated virus versus the other viruses.

Figure 3. The prevalence of epitope-specific CTL memory cells after primary infection with the H9N2 and H1N1 IAVs.

The primary IAV infection was conducted as described in the legends to Fig. 1. (A) The proportion and (B) number of epitope-specific memory CTL populations and (C) the total number of the three epitope-specific memory CTLs in spleen on d38; (D) The epitope-specific memory CTL populations generated by the priming infection that are cross-reactive with non-conserved epitopes in the H7N9 virus; (E) The total number of epitope-specific memory CTLs generated by primary infection targeting both the conserved and nonconserved epitopes in the H7N9 virus. (A-C) The PB1₇₀₃, PA₂₂₄, and NP₃₆₆ peptide variants specific for each virus were used to stimulate memory CTLs to produce IFN-γ, except for (D) where cross-reactive variants were tested. The data sets represent mean ± SEM, n = 4–5 per group.* p<0.05 by Tukey’s test for (A,B, C, E) or by t test for (D), comparing: (A, B) the indicated epitope versus the other two epitopes; (C, E) the indicated virus versus the other three viruses; (D) the nonconserved versus the counterpart conserved epitope.

Figure 4. The disease course and primary CTL responses in naive mice after infection with the H7N9 IAV.

Naïve mice were infected with 1 MLD50 (10^3.5 TCID₅₀) of the H7N9 virus. (A) Body weight change, (B) the proportion and (C, left Y axis) number of each epitope-specific CTL population in the BAL, and (C, right Y axis) the lung virus titer. Data sets represent mean ± SEM, n = 4–5 at each time point. * p<0.05, Tukey’s test, NP versus the other two epitopes. (D) Representative flow cytometry plots for each tetramer-specific CTL response in the BAL. The tetramers used were specific to the K^bPB1₇₀₃, D^bPA₂₂₄, and D^bNP₃₆₆ variants of the H7N9 virus.

Figure 5. The disease course in naive or primed (with H1N1 or H9N2 IAVs 10–12 weeks previously) mice following challenge with the H7N9 virus.

The mice were challenged with 10^4.5 TCID₅₀ H7N9 virus (10 MLD₅₀). (A) The survival ratio and (B) weight loss during the disease course. Data represent mean ± SEM, n = 8–10/group from two independent experiments. (A) * p<0.05, log-rank test, naive versus other four primed groups. (B) * p<0.05, t test, naïve versus the every other four primed groups; # p<0.05, t test, Ck/HK/TP38 versus the every other three primed groups.

Figure 6. Comparing the primary and secondary CTL responses in naïve and Ck/HK/TP38(H9N2)-primed mice challenged with the H7N9 virus.

The mice were challenged with 10^3.5 TCID₅₀ H7N9 virus (1 MLD₅₀). (A) The virus titer in the lung and (B) proportion and (C) number of each epitope-specific CTL population and (D) the combined total number of three epitope-specific CTL populations in the BAL (data represent mean ± SEM, n = 4–5 at each time point). (A, D) * p<0.05, t test, primed versus naïve group. (B, C) * p<0.05, Tukey’s test, the indicated epitope versus the other two epitopes. (E) Representative flow cytometry plots for each tetramer-specific CTL population in the BAL. The tetramers used were specific to the epitope variants of the H7N9 virus.

Figure 7. Comparing the primary and secondary CTL responses in naïve and HK/33892(H9N2)-primed mice challenged with the H7N9 virus.

(A) The virus titer in the lung and (B) proportion and (C) number of each epitope-specific CTL population, and (D) the combined total number for the three epitope-specific CTL populations in the BAL. (E) Representative flow cytometry plots for each tetramer-specific CTL population in the BAL. Details of the data analysis and comparisons are same as shown in the legend to Fig. 6.

Figure 8. Comparing the primary and secondary CTL responses in naïve and PR/8(H1N1)-primed mice challenged with the H7N9 virus.

(A) The virus titer in the lung and (B) proportion and (C) number of each epitope-specific CTL population, and (D) the combined total number for the three epitope-specific CTL populations in the BAL. (E) The proportions of CTL populations specific to AH/1-D^bNP₃₆₆ or PR/8-D^bNP₃₆₆ tetramers in the BAL. (F, G) Representative flow cytometry plots for each tetramer-specific CTL population in the BAL. Details of the data analysis and comparisons are same as shown in the legend to Fig. 6.

Figure 9. Comparing the primary and secondary CTL responses in naïve and CA/4(H1N1)-primed mice challenged with the H7N9 virus.

(A) The virus titer in the lung and (B) proportion and (C) number of each epitope-specific CTL population, and (D) the combined total number for the three epitope-specific CTL populations in the BAL. (E) Representative flow cytometry plots for each tetramer-specific CTL population in the BAL. Details of the data analysis and comparisons are same as shown in the legend to Fig. 6.

Figure 10. Correlation analysis of the protective efficacy and heterosubtypic immunity.

Pearson correlation analysis of the protective efficacy with (A) the magnitude of the H7N9 virus-specific secondary CTL response in the airways on d6 (black line, left Y axis) and d8 (grey line, right Y axis), (B) the total number of priming-virus-specific memory CTL populations generated by the homologous virus (black line); the total number epitope-specific memory CTL populations targeting the conserved and non-conserved cross-reactive epitopes in the H7N9 virus (solid grey line); the number of epitope-specific memory CTL populations which were dominantly recalled during later during challenge infection (dashed grey line). The protective efficacy score was defined by a combination of survival ratio, body weight retention and virus clearance rate; the values of each parameter were normalized to the % maximum and the final score was the average of the three parameters. For (A), because the secondary infections were conducted in different batches, the fold-relationships for a given secondary CTL response in primed mice relative to the primary CTL response in the matched naïve mice were used to enable comparison between experiments. (C) The survival ratio and (D) weight loss of the mice primed with high or low dose of the H9N2 virus after they were challenged with 10^4.5 TCID₅₀ H7N9 viruses. Data represent mean ± SEM, n = 8–10/group, (C) * # p<0.05, log-rank test, * naïve versus other four primed groups, # Ck/HK/TP38 low dose versus high dose and naïve. (D) * p<0.05, t test, HK/33892 high dose versus low dose and naïve.

Figure 11. The disease course in young or aged naïve mice, or primed (with H9N2 or H1N1 virus) aged mice following H7N9 challenge.

The aged females were between 16–18 months at priming and then were challenged about two months later or young (8–10 weeks). Aged matched or young (8–10 weeks) naïve female mice were used for comparisons. (A) The survival ratio and (B) body weight change of the mice after challenge with 10MLD50 of AH/1(H7N9) (data represent mean ± SEM, n = 5–6/group). (C) The body weight change, (D) virus titer in the lung, and (E) the total number of the three-H7N9 virus epitope-specific CTL populations in the BAL samples on d8 after challenge with 1MLD50 of AH/1(H7N9) virus (data represent mean± SEM, n = 3/group). (C, D, E) * p<0.05, t test, indicated group versus the naïve aged group.