Induction of protective CD4+ T cell-mediated immunity by a Leishmania peptide delivered in recombinant influenza viruses

Abstract

The available evidence suggests that protective immunity to Leishmania is achieved by priming the CD4(+) Th1 response. Therefore, we utilised a reverse genetics strategy to generate influenza A viruses to deliver an immunogenic Leishmania peptide. The single, immunodominant Leishmania-specific LACK(158-173) CD4(+) peptide was engineered into the neuraminidase stalk of H1N1 and H3N2 influenza A viruses. These recombinant viruses were used to vaccinate susceptible BALB/c mice to determine whether the resultant LACK(158-173)-specific CD4(+) T cell responses protected against live L. major infection. We show that vaccination with influenza-LACK(158-173) triggers LACK(158-173)-specific Th1-biased CD4(+) T cell responses within an appropriate cytokine milieu (IFN-γ, IL-12), essential for the magnitude and quality of the Th1 response. A single intraperitoneal exposure (non-replicative route of immunisation) to recombinant influenza delivers immunogenic peptides, leading to a marked reduction (2-4 log) in parasite burden, albeit without reduction in lesion size. This correlated with increased numbers of IFN-γ-producing CD4(+) T cells in vaccinated mice compared to controls. Importantly, the subsequent prime-boost approach with a serologically distinct strain of influenza (H1N1->H3N2) expressing LACK(158-173) led to a marked reduction in both lesion size and parasite burdens in vaccination trials. This protection correlated with high levels of IFN-γ producing cells in the spleen, which were maintained for 6 weeks post-challenge indicating the longevity of this protective effector response. Thus, these experiments show that Leishmania-derived peptides delivered in the context of recombinant influenza viruses are immunogenic in vivo, and warrant investigation of similar vaccine strategies to generate parasite-specific immunity.

Figure 1. Cytokine production by LACK_158–173-specific CD4⁺ T cells following influenza-LACK_158–173 priming.

A) Ex vivo IFN-γ production by LACK_158–173-specific CD4⁺ T cells. Cells were obtained from spleens of naïve control mice or PR8-LACK_ins and PR8-LACK_rep mice on day 10 following priming. Cells were cultured in the absence or presence of LACK_158–173 peptide for 12 h and assessed for IFN-γ production by ICS. B) In vitro IFN-γ production by LACK_158–173-specific CD4⁺ T cells. Cells were obtained from spleens of naïve control mice or PR8-LACK_ins and PR8-LACK_rep mice on day 10 following priming. Cells were cultured in the absence or presence of LACK_158–173 peptide for 72 h followed by re-stimulation with PMA and ionomycin, and assessed for IFN-γ production by ICS. Numbers in the upper right quadrant refer to percentage of CD4⁺ T cells producing IFN-γ (raw data). Cells have been gated on CD4⁺ CD44⁺, for statistical analyses no peptide (panel A) and isotype control (panel B) values were subtracted from the raw values. Representative data from one mouse are shown (n = 3, n refers to number of mice, *p = 0.004, **p = 0.0002, ***p = 0.006).

Figure 2. Cytokine profile of LACK_158–175 specific CD4⁺ T cells.

Cells were obtained from spleens of naïve control mice or PR8-LACK_ins and PR8-LACK_rep mice on day 10 following priming. Cells were cultured in the presence of LACK_158–173 peptide for 72 h, and culture supernatants were analysed for cytokine production by capture ELISA. Mean, pooled data ± SD are plotted (n = 3, n refers to number of mice) *p = 0.001, **p = 0.003, ***p = 0.009, ****p = 0.01, ND – below detection level.

Figure 3. Protective efficacy of influenza-LACK_158–173 vaccination.

A) Lesion scores in mice vaccinated with LACK_ins and PR 8 vector controls (pooled data from 2 independent experiments). B) Parasite burdens in mice vaccinated with LACK_ins (n = 5), PR 8 vector control (n = 5), *p = 0.02. C) Cytokine production following challenge infection. Spleens and dNL from vaccinated and control mice were assessed by ex vivo ICS IFN-γ (*p = 0.006). Mean, pooled data from 2 independent experiments ± SEM are plotted (n = 6 for wk 2, n = 5 for wk 6, n refers to number of mice). Cells have been gated on CD4⁺ CD44⁺.

Figure 4. Boosting with serologically distinct recombinant virus increases protective potency of the effector response.

A) Prime-boost and challenge strategy. B) Lesion scores in mice primed with PR8-LACK_ins and boosted with X31-LACK_ins (PR8/X31); and PBS controls. C) Parasite burdens in PR8/X31 vaccinated mice and PBS controls (n = 3 for wk 2 and 6, n = 5 for wk 11), *p = 0.009. D) Cytokine production following challenge. Spleens and dNL from vaccinated and control mice were assessed by ex vivo ICS for IFN-γ production. Mean, pooled data ± SEM are plotted (n = 3 for non-challenged mice, n = 5 for wk 2 and 6); *p = 0.02, **p = 0.03, ***p = 0.005, ****p = 0.001, #p = 0.02, ##p = 0.002, ###p = 0.03. Cells have been gated on CD4⁺ CD44⁺.

Figure 5. Boosting with serologically distinct recombinant virus increases protective potency and longevity of IFN-γ response.

A) Prime-boost with delayed challenge strategy. B) Lesion scores in mice primed with PR8-LACK_ins and boosted with X31-LACK_ins (PR8/X31); and wild type PR8/X31 virus controls. C) Parasite burdens in PR8/X31 vaccinated mice and virus controls (n = 5 each time point, except n = 4 for wk 10 controls), *p = 0.049. D) Cytokine production following challenge. Spleens and dNL from vaccinated and control mice were assessed by ex vivo ICS for IFN-γ production. Mean, pooled data ± SEM are plotted (n = 3 for non-challenged mice, n = 5 for wk 2 and 6; *p = 0.02, **p = 0.01). Cells have been gated on CD4⁺ CD44⁺.

Figure 6. Cytokine production profile of Leishmania specific T cells following long-term prime-boost immunisation.

Cells were obtained from spleens and dLN of immunised and control mice on wk 2 following challenge. Cells were cultured in vitro in the presence of SLA for 72 h, and culture supernatants were analysed for cytokine production. IFN-γ (Th1) levels in splenocytes culture supernatants, and Th2-type cytokine production in dLN on wk 2 post-infection measured by Bio-Plex Pro Mouse Cytokine assay. Mean, pooled data ± SD are plotted (n = 5).