SIV gp120/140 reactivity at week 20 post rRRV-SIVcmv-nfl inoculation.

<p><b>A)</b> Sera obtained at week 20 following rRRV-SIVcmv-nfl inoculation were serially diluted and tested for the reactivity to gp120 via an ELISA using recombinant SIVmac239 gp120 to coat the ELISA plates. Subsequently, reactive antibodies were detected with a horseradish-peroxidase-conjugated anti-rhesus IgG antibody. Furthermore, sera of two SIVmac239Δ<i>nef</i>-infected animals (week 18 post infection) and sera obtained from two monkeys that had received rRRV expressing a codon-modified version of SIVmac239 gp160 (week 19 post inoculation) were tested [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007143#ppat.1007143.ref024" target="_blank">24</a>]. <b>B)</b> Similarly, the monkey sera were serially diluted and tested for reactivity to gp140 by ELISA, using recombinant SIVmac239 gp140 to coat the ELISA plates. Reactive antibodies were detected with a horseradish-peroxidase-conjugated anti-rhesus IgG antibody.</p

Characteristics of research animals.

<p>Characteristics of research animals.</p

Serum neutralization of SIVmac316 at week 20 post rRRV-SIVcmv-nfl vaccination^a.

<p>Serum neutralization of SIVmac316 at week 20 post rRRV-SIVcmv-nfl vaccination<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007143#t002fn001" target="_blank">^a</a>.</p

Memory phenotype of vaccine-induced CD8+ T-cell responses in <i>Mamu-A*01+</i> rRRV-SIVcmv-nfl vaccinees.

<p>The frequencies of Mamu-A*01/Gag CM9 and Mamu-A*01/Tat SL8 tetramer+ CD8+ T-cells in PBMC at week 10 post rRRV-SIVcmv-nfl inoculation are shown in the left panels for r11089 (A) and r11099 (B). The middle panels show the delineation of memory subsets within the tetramer+ gate based on the differential expression of CD28 and CCR7. Three subsets were identified: central memory (T_CM; CD28+CCR7+), transitional memory (T_EM1; CD28+CCR7-), and effector memory (T_EM2; CD28-CCR7-). The histograms in the right panels show the levels of granzyme B expressed by tetramer+ CD8+ T-cells (blue lines). The red lines correspond to tetramer+ CD8+ T-cells stained with an isotype-matched control monoclonal antibody.</p

SIVmac316 neutralization titers at week 20 post rRRV-SIVcmv-nfl inoculation in rhesus monkeys.

<p>Sera from the rRRV-SIVcmv-nfl-inoculated monkeys taken at week 20 post inoculation were serially diluted and subjected to neutralization assays against SIVmac316 utilizing TZM-bl cells. A pool of sera from SIVmac239-infected monkeys served as a positive control and pooled sera from specific pathogen free (SPF) monkeys as a negative control. The dashed line indicates 50% relative light units representing 50% neutralization activity.</p

Humoral immune responses following infection of rhesus monkeys with rRRV-SIVcmv-nfl.

<p>Sera obtained at the indicated time points following rRRV-SIVcmv-nfl inoculation were diluted 1:20 and tested for the presence of <b>A)</b> anti-RRV responses via an ELISA utilizing ELISA plates coated with purified wild type RRV lysate. Reactive antibodies were detected with a horseradish-peroxidase-conjugated anti-rhesus IgG antibody. <b>B)</b> Similarly, the sera were tested for the presence of anti-gp140 antibodies via an ELISA using recombinant SIVmac239 gp140 to coat the ELISA plates. Reactive antibodies were detected with a horseradish-peroxidase-conjugated anti-rhesus IgG antibody. <b>C)</b> The obtained sera were tested for the presence of anti-gp120 antibodies via an ELISA using recombinant SIVmac239 gp120 to coat the ELISA plates. Reactive antibodies were detected with a horseradish-peroxidase-conjugated anti-rhesus IgG antibody.</p

Position of the SIVcmv-nfl expression cassette in recombinant RRV.

<p>A near full-length genome (nfl) sequence of SIVmac239 with a 520 bp deletion (red X) in the 5’ Long Terminal Repeat (LTR) region, a deletion in the <i>pol</i> region spanning 306 bp, corresponding to the active site of the reverse transcriptase, and a 414 bp deletion in the 3’ LTR region, was generated via Gibson cloning. Numbering system corresponds to that of Regier and Desrosiers [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007143#ppat.1007143.ref016" target="_blank">16</a>]. Subsequently, utilizing overlaps containing PmeI restriction sites, the cytomegalovirus immediate-early enhancer and promoter (pCMV), the SIV-nfl sequence containing a C-terminal V5-tagged <i>nef</i> open reading frame (ORF), and the bovine growth hormone (BGH) polyA signal were inserted between the left terminal repeats (TR) and the first ORF R1 of RRV via Gibson cloning. Full length recombinant RRV was obtained from subsequent transfection of overlapping cosmid clones [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007143#ppat.1007143.ref021" target="_blank">21</a>].</p

rRRV-SIVcmv-nfl vaccination elicits Gag CM9- and Tat SL8-specific CD8+ T-cell responses in <i>Mamu-A*01+</i> macaques.

<p>Fluorescently-labeled Mamu-A*01 tetramers folded with peptides corresponding to the Gag CM9 (A) or Tat SL8 (B) epitopes were used to monitor the ontogeny of vaccine-induced CD8+ T-cell responses in the two <i>Mamu-A*01+</i> rRRV-SIVcmv-nfl-inoculated monkeys (r11089 and r11099).</p

Production of replication-defective SIV particles by rRRV-SIV-nfl.

<p><b>A)</b> Early passage rhesus fibroblast (RF) cells were grown in 6-well culture plates and subsequently infected with 3.5x10⁹ genome copies of the indicated recombinant SIV-nfl. The RF cell culture supernatants were collected at the indicated time points post infection and subjected to an antigen capture assay to measure the levels of SIV Gag p27. Supernatant of uninfected RF cells served as a negative control. <b>B)</b> Cell culture supernatant was harvested from early passage rhesus fibroblast (RF) cells infected with the respective recombinant RRV-SIV-nfl strains. Subsequently, CEMx174 cells were infected with these supernatants containing 20ng of p27 as measured by an antigen capture assay. An equal amount (20ng of p27) of SIVmac239 wild-type virus produced in HEK239T-cells served as a positive control. The supernatants from the CEMx174 cell cultures were collected at the indicated days post infection and were used in an antigen capture assays measuring p27 levels. Supernatant of uninfected RF cells served as a negative control. <b>C)</b> Early passage rhesus fibroblast (RF) cells grown in T75 culture flasks were infected with rRRV-SIVcmv-nfl and harvested at days 4–6 post infection. Subsequently, the cells were fixed with 2.5% glutaraldehyde in sodium cacodylate buffer. Transmission electron microscopic (TEM) images were taken at The Core Electron Microscopy Facility, University of Massachusetts. Shown are SIV particles budding from cell membrane (filled arrowheads). The bar in the lower left corner represents 500 nm <b>D</b>) TEM image showing free SIV particles including mature SIV particles (filled arrowheads) containing cylindrical, rod-shaped nucleoid as well as immature particles lacking any nucleoid structures (empty arrowheads). The bar in the lower left corner represents 200 nm. In the inset on the upper right, a TEM image of recombinant RRV derived from a separate image is shown.</p

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