Persistent Zika Virus Clinical Susceptibility despite Reduced Viral Burden in Mice with Expanded Virus-Specific CD8+ T Cells Primed by Recombinant Listeria monocytogenes

Abstract

Vaccines against Zika virus (ZIKV) infection that target CD8⁺ T cells are of considerable interest because Abs may enhance infection susceptibility. However, whether CD8⁺ T cells are protective or promote susceptibility to clinical infection symptoms remains uncertain. To more precisely investigate ZIKV-specific CD8⁺ T cells in isolation, we engineered a Listeria monocytogenes-based vector to express a single MHC class I-restricted immune dominant peptide, E294-302, from ZIKV envelope protein. We show accumulation of activated ZIKV-specific CD8⁺ T cells primed by recombinant L. monocytogenes is associated with reductions in circulating virus levels after ZIKV challenge in type I IFN receptor-deficient mice and wildtype mice administered neutralizing Abs against type I IFN receptor. Interestingly, susceptibility to ZIKV clinical infection including weight loss and mortality each persists and is neither significantly improved nor worsened compared with isogenic L. monocytogenes-primed control mice. These data demonstrating persistent ZIKV clinical susceptibility despite reduced viral burden in mice with expanded virus-specific CD8⁺ T cells highlights the need for targeting other adaptive immune components in developing vaccines against ZIKV infection.

FIGURE 1.

Recombinant Lm-ZIKV primes accumulation of virus-specific memory CD8⁺ T cells poised for re-expansion in response to ZIKV challenge. (A) Accumulation of splenic CD8⁺ T cells with ZIKV H-2D^b:E_294–302 specificity day 7 after Lm-ZIKV or Lm-HSV inoculation. (B) Numbers of H-2D^b:E_294–302-specific CD8⁺ T cells in the spleen at each time point after Lm-ZIKV inoculation. (C) Relative expression of CD44 and CD62L by E_294–302-specific compared with bulk CD8⁺ splenocytes of mice day 35 after Lm-ZIKV infection. (D) Cytokine production by H-2D^b:E_294–302-specific (line histogram) compared with bulk (gray shaded histogram) CD8⁺ T cells after PMA/ionomycin stimulation of splenocytes from mice 35 days after Lm-ZIKV or Lm-HSV infection. (E) Percent H-2D^b:E_294–302-specificity among CD8+ T cells in each tissue 5 days after ZIKV challenge of mice infected 35 days prior with Lm-ZIKV compared with Lm-HSV. (F) Number and fold-expansion of H-2D^b:E_294–302-specific CD8⁺ splenocytes prior to compared with 5 days after ZIKV challenge for the mice described in panel E. Each point represents the data from an individual mouse, combined from at least two experiments each with similar results. Bar, mean ± SEM. * p<0.05; ** p<0.01; **** p<0.001.

FIGURE 2.

Clinical susceptibility persists in Lm-ZIKV primed mice. (A) Progression of clinical symptoms, weight loss, and survival after MR766 ZIKV challenge of IFNAR^−/− mice infected 35 days prior with Lm-ZIKV compared with Lm-HSV. (B) ZIKV genomic copies in the serum (per mL), liver, and brain 5 days after MR766 ZIKV challenge for the mice described in panel A. (C) Progression of clinical symptoms, weight loss, and survival after MR766 ZIKV challenge of IFNAR+/+ mice infected 35 days prior with Lm-ZIKV compared with Lm-HSV, and administered anti-IFNAR antibody beginning one day prior to ZIKV challenge (0.4 mg day-1; 0.2 mg, day +1; 0.2 mg day +3; 0.2 mg, day +5). (D) ZIKV genomic copies in the serum (per mL), liver, and brain 5 days after MR766 ZIKV challenge for the mice described in panel C. The number of mice in each group for clinical disease progression, weight loss and mortality is shown. Each point represents the results from an individual mouse, combined from at least two experiments each with similar results. Bar, mean ± SEM. * p<0.05; ** p<0.01

FIGURE 3.

Boosting expansion of ZIKV-specific CD8⁺ T cells does not enhance clinical or virological susceptibility. (A) Schematic for administering one compared with two Lm-ZIKV prior to ZIKV challenge. (B) Numbers of ZIKV H-2D^b:E_294–302 CD8⁺ splenocytes amongst IFNAR^−/− mice primed 21 days prior with either a single or two Lm-ZIKV doses. (C) Progression of clinical symptoms, weight loss, and survival after MR766 ZIKV infection for mice described in panel A. (D) ZIKV genomic copies in the serum (per mL), liver, and brain 5 days after MR766 ZIKV challenge for the mice described in panel A. The number of mice in each group for clinical disease progression, weight loss and mortality is shown. Each point represents the results from an individual mouse, combined from at least two experiments each with similar results. Bar, mean ± SEM. ** p<0.01.

FIGURE 4.

Attenuated ZIKV PRVABC59 infection dissociates virologic and clinical susceptibility primed by recombinant Lm. (A) Progression of clinical symptoms and weight loss after PRVABC59 ZIKV challenge amongst IFNAR^−/− mice infected 35 days prior with Lm-ZIKV compared with Lm-HSV. (B) ZIKV levels in the serum (per mL), liver, and brain, 5 days after PRVABC59 ZIKV challenge amongst mice described in panel A. (C) Percent fetal resorption 3 days after PRVABC59 ZIKV challenge in mice mid-gestation during allogeneic pregnancy, previously infected with Lm-ZIKV compared with Lm-HSV 35 days prior to mating. (D) ZIKV genomic copies in fetal tissue (fetus and placenta) for the concepti of mice described in panel C. The number of mice in each group for clinical disease progression, weight loss and mortality is shown. Each point represents the results from an individual mouse (concepti or pregnant female as indicated), combined from at least two experiments each with similar results. Bar, mean ± SEM. * p<0.05; **** p<0.0001.