Selective pre-priming of HA-specific CD4 T cells restores immunological reactivity to HA on heterosubtypic influenza infection

Abstract

A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.

Fig 1. The suppression of novel responses demonstrated following a secondary influenza infection is associated with a marked decline in antigenic load.

A: Mice were infected with 300,000 EID₅₀ of X-31 (H3N2) influenza and then were rested for 8 weeks. These mice, together with a cohort of naïve mice, were then infected with 50,000 EID₅₀ of x139 (H1N1) influenza. Mice only infected with X-31 8 weeks prior served as a control for waning immunity. CD4 T cell responses in splenocytes derived from 3 to 4 individual mice per group were quantified at day 8 post x139 infection by IL-2 Elispot assay, with data presented as the average spot count per million CD4 T cells after subtracting background. Error bars represent the standard error of the mean. B: Sera from these same 3 to 4 individual mice per group were pooled and the titer of HA-specific antibodies in each group was quantified using an HA ELISA assay, with data presented as the average of duplicate wells. C: Viral load in the lungs of 3–4 individual mice per group was determined by quantitative PCR using primers and probe derived from the M1 protein at days 2, 4, and 8 following x139 infection. Samples were run in quadruplicate, with the average qC for each sample determined and plotted on a standard curve. Data are presented as the average number of copies per mL, with error bars representing the standard error of the mean.

Fig 2. Reactivity following HA peptide priming is not influenced by previous X-31 infection.

A cohort of B10.S mice was infected with 300,000 EID₅₀ of X-31 (H3N2) influenza and rested for 4 weeks. These previously infected mice, together with a cohort of naïve mice, were then immunized intraperitoneally with a pool of 5 H1 HA peptides in alum as described. Spleens were harvested at day 10 post immunization and CD4 T cells were isolated by MACS cell purification, with reactivity to a panel of selected epitopes determined by Elispot assay. A: Reactivity to selected peptide-epitopes as measured by IL-2 Elispot assay; B: Reactivity to peptide-epitopes quantified by IFNγ Elispot. Data represent the results obtained from 3–6 individual mice per experiment group, with the spot count normalized to 10⁶ CD4 T cells after subtracting background and then averaged.

Fig 3. Experimental design to examine the effect of establishing HA-specific memory by peptide immunization prior to secondary infection.

B10.S mice were infected with 300,000 EID₅₀ of X-31 (H3N2) influenza virus and were rested for four weeks. Groups 1 and 3 were then immunized with a pool of 5 HA peptides in alum IP as described while Group 2 and 4 were only immunized with alum in PBS (sham). After 4 weeks, Groups 1, 2 and 4 were infected with 50,000 EID₅₀ of x139 (H1N1) influenza virus. Spleen, mediastinal lymph nodes, and serum were harvested at days 8 or 24 following secondary infection (Group 4 only had serum harvested).

Fig 4. Immunization with a pool of HA peptides restores CD4 T cell reactivity to the selected HA epitopes following a secondary infection.

Mice were infected and immunized as depicted in Fig 3. At day 8 following the secondary infection, individual spleens and mediastinal lymph nodes were harvested. CD4 T cells were isolated by MACS cell purification and reactivity to a panel of peptide-epitopes was measured by IL-2 Elispot assay. A: CD4 T cell reactivity in the spleen. Data represent results from 9–13 individual mice averaged in each experimental group. B: CD4 T cell reactivity within the mediastinal lymph node. Data presented represent the average results from 6–13 individual mice in each experimental group. All data are presented as the spot count normalized to 10⁶ CD4 T cells after subtracting background, with error bars depicting the standard error of the mean. * = p<0.05; ** = p<0.01; *** = p<0.001 by Kruskal-Wallis one-way ANOVA test.

Fig 5. Establishing H1-specific CD4 T cell memory prior to secondary challenge partially restores the H1-specific antibody response.

A: Mice were infected and immunized as depicted in Fig 3 and serum antibody responses were measured by H1 HA ELISA assay at day 8 following secondary infection. Data demonstrate the average OD from serum obtained from between 8–10 individual mice in the X31-HA, X31-HA-x139, and X31-sham-x139 groups and 3 individual mice in the naïve and sham-x139 groups, with error bars depicting the standard error of the mean. ** = p<0.01 using a linear mixed effect model for pairwise group comparisons. B: Mice were infected and immunized as depicted in Fig 3 and serum neutralizing antibody responses were determined by microneutralization assay at day 24 following secondary infection. Squares represent the neutralizing antibody titer in individual mice, with the geometric mean of the microneutralization titer shown by a line and error bars depicting the 95% confidence interval. * = p<0.05 by Wilcoxon rank-sum test.

Fig 6. H3-specific antibody responses are not boosted following secondary infection with the x139 H1N1 virus.

To evaluate for evidence of original antigenic sin, serum from mice in each group was tested for antibody against the A/Brisbane/10/07 H3 HA protein by ELISA assay at 24 days following x139 viral challenge. Data represent the average OD obtained at a given serum dilution from 5–6 individual mice per experimental group, with the exception of the sham-x139 group where only 2 mice were examined. There were no statistically significant differences between H3 antibody levels in the sham-immunized (X31-sham-x139) or HA-immunized (X31-HA-x139) mice when compared to antibody in the mice initially primed with the X31 virus and immunized with HA peptides without a subsequent H1 viral infection (X31-HA).