Ontogeny of the B- and T-cell response in a primary Zika virus infection of a dengue-naïve individual during the 2016 outbreak in Miami, FL

<div><p>Zika virus (ZIKV) is a mosquito-borne flavivirus of significant public health concern. In the summer of 2016, ZIKV was first detected in the contiguous United States. Here we present one of the first cases of a locally acquired ZIKV infection in a dengue-naïve individual. We collected blood from a female with a maculopapular rash at day (D) 5 and D7 post onset of symptoms (POS) and we continued weekly blood draws out to D148 POS. To establish the ontogeny of the immune response against ZIKV, lymphocytes and plasma were analyzed in a longitudinal fashion. The plasmablast response peaked at D7 POS (19.6% of CD19⁺ B-cells) and was undetectable by D15 POS. ZIKV-specific IgM was present at D5 POS, peaked between D15 and D21 POS, and subsequently decreased. The ZIKV-specific IgG response, however, was not detected until D15 POS and continued to increase after that. Interestingly, even though the patient had never been infected with dengue virus (DENV), cross-reactive IgM and IgG binding against each of the four DENV serotypes could be detected. The highest plasma neutralization activity against ZIKV peaked between D15 and D21 POS, and even though DENV binding antibodies were present in the plasma of the patient, there was neither neutralization nor antibody dependent enhancement (ADE) of DENV. Interestingly, ADE against ZIKV arose at D48 POS and continued until the end of the study. CD4⁺ and CD8⁺ T-cells recognized ZIKV-NS2A and ZIKV-E, respectively. The tetramer positive CD8⁺ T-cell response peaked at D21 POS with elevated levels persisting for months. In summary, this is the first study to establish the timing of the ontogeny of the immune response against ZIKV.</p></div

ZIKV induced maculopapular rash.

<p>Upon admission into the study photos of the patient’s rash were taken D3 POS. This rash was neither itchy, nor sensitive to the touch. (A) A close-up of the initiation point of the rash on the front of the torso near the midline. (B) The rash then spread from the torso to the neck and head.</p

Phylogenetic tree of ZIKV isolated from Hu0015 compared to previously sequenced ZIKV genomes.

<p>A detailed maximum likelihood phylogenetic analysis of published ZIKV genomes from the Pacific and Americas (Asian genotype, from 2013–2016). The pink branches represent isolates from the 2016 ZIKV outbreak in Florida. Hu0015 is one of the first ZIKV sequences from autochthonous transmission in the contiguous US and clades with one of four ZIKV lineages detected during the outbreak in Florida. The scale of 0.005 represents nucleotide substitutions per site in the viral genome.</p

Ontogeny of the plasmablast response of a primary ZIKV infection in a flavivirus-naïve individual.

<p>The blue box in each flow graph represents the frequency of Pbs from isolated PBMCs as a percentage of total CD19⁺ B-Cells. The Pb frequency was significantly elevated at D5 POS when compared to a naïve uninfected individual as well as to convalescent phase PBMCs from Hu0015 (D148 POS).</p

Neutralization titers against ZIKV and DENV.

<p>(A) Neutralization titers were performed by flow cytometry at several time points against ZIKV-Paraiba/2015 and the NEUT₅₀ was calculated based on a non-linear regression. Peak NEUT₅₀ occurred at D15 POS. Hu0002 was used as a flavivirus-naïve control and Hu0004 was a DENV- and ZIKV-exposed control. (B) Plaque reduction neutralization tests (PRNTs) were also performed against ZIKV and all four DENV serotypes. PRNT₅₀ was calculated as 50% neutralization of plaques based on control virus wells and reported as a dilution of patient plasma. (N/A = samples not run).</p

Ontogeny of the T-cell response.

<p>(A) Using PBMCs from D106 POS and mega-pools consisting of overlapping 15-mer peptides from each protein of ZIKV in ICS assays, we observed that the CD4⁺ T-cells responded to ZIKV-NS2A. (B) The CD8⁺ T-cells responded to ZIKV-E. (C) To further analyze the CD8⁺ T-cell response, the peptide pool that was responsible for the ZIKV-E response was fine mapped in ICS assays into ten individual 15-mer peptides. The response was directed against only one of the 15-mer peptides. (D) Using this peptide, epitope predictions for the patient’s MHC Class I alleles were made for all possible 8-, 9-, 10-, and 12-mer peptides. These peptides were synthesized and tested in and IFN-γ ELISPOT dilution assay. (E) We compared an alignment of the 9-mer minimal optimal peptide to ZIKV-E and to DENV-E. The amino acids, indicated in red, are amino acid differences from the reference ZIKV sequence. (F) The minimal optimal peptide was then used to make a tetramer, and this was used to track the ontogeny of ZIKV-specific CD8⁺ T-cell in this patient. A tetramer response was present at D5 POS, with a peak at D21 POS, and then remained level from D48 to D148 POS.</p