Correlational analyses between PD1⁺ICOS⁺ Tfh-like subsets and memory B cells.

<p>Fold changes in different Tfh-like subsets (indicated as Tfh1, Tfh2, and Tfh17) at D7 over the pre-vaccinated time points were compared to the fold changes in the memory B cells at the indicated days (over the pre-vaccinated time points).</p><p>^<i>a</i><i>r</i> indicates correlation coefficient from Spearman rank correlation analyses; <i>p</i> indicates <i>p</i> value from the Spearman correlational rank analyses.</p><p>^<i>b</i> NS, not significant.</p><p>^<i>c</i> The frequencies of Tfh17 subsets in some subjects were 0% at the baseline; therefore, we could not analyze some of the subjects. For the Tfh1 and 2 subsets, 10 subjects for Gardasil were compared, and it was 8 for Cervarix at D7, 14, and 30. For the other 3 time points, the numbers ranged 5–8 in both groups</p><p>Correlational analyses between PD1⁺ICOS⁺ Tfh-like subsets and memory B cells.</p

Frequency distributions of different subsets of circulating Tfh-like cells.

<p>(A) Percentages of circulating Tfh1-, Tfh2-, and Tfh17-like subsets from the Gardasil and Cervarix groups are shown. (B) Frequencies of PD1/ICOS double positive cells, (C) frequencies of PD1⁺ ICOS^- cells, and (D) frequencies of double negative cells within each subset were plotted over time. N = 10–11 (pre-vac to D30) and N = 6–8 (M6 to M7) for Gardasil. N = 8 (pre-vac to D30) and N = 5–6 (M6 to M7) for Cervarix. Paired, one-tailed Wilcoxon rank sum analyses were performed between the pre-vac time point and each of the post-vaccination time point for the first vaccination. For the third vaccination (M6 to M7), M6 was used as the baseline for the analyses. Black horizontal bars indicate the medians. For the comparison of the two vaccine groups described in the “HPV vaccines induced circulating PD1⁺ICOS⁺ Tfh1-like cells” section, paired, two-tailed Wilcoxon rank sum analyses were performed.</p

Circulating CXCR5⁺CD4⁺ T Follicular-Like Helper Cell and Memory B Cell Responses to Human Papillomavirus Vaccines

<div><p>Through the interaction of T follicular helper (Tfh) cells and B cells, efficacious vaccines can generate high-affinity, pathogen-neutralizing antibodies, and memory B cells. Using CXCR5, CXCR3, CCR6, CCR7, PD1, and ICOS as markers, Tfh-like cells can be identified in the circulation and be classified into three functionally distinct subsets that are PD1⁺ICOS⁺, PD1⁺ ICOS^-, or PD1^-ICOS^-. We used these markers to identify different subsets of CXCR5⁺CD4⁺ Tfh-like cells in response to highly immunogenic and efficacious vaccines for human papillomaviruses (HPV): Cervarix and Gardasil. In this small study, we used PBMC samples from 11 Gardasil recipients, and 8 Cervarix recipients from the Vaccine Research Center 902 Study to examine the induction of circulating Tfh-like cells and IgD^-CD38^HiCD27⁺ memory B cells by flow cytometry. PD1⁺ICOS⁺ CXCR3⁺CCR6^-CXCR5⁺CD4⁺ (Tfh1-like) cells were induced and peaked on Day (D) 7 post-first vaccination, but not as much on D7 post-third vaccination. We also observed a trend toward increase in PD1⁺ICOS⁺ CXCR3^-CCR6^-CXCR5⁺CD4⁺ (Tfh2-like) cells for both vaccines, and PD1⁺ICOS⁺ CXCR3^-CCR6⁺CXCR5⁺CD4⁺ (Tfh17-like) subset was induced by Cervarix post-first vaccination. There were also minimal changes in the other cellular subsets. In addition, Cervarix recipients had more memory B cells post-first vaccination than did Gardasil recipients at D14 and D30. We found frequencies of memory B cells at D30 correlated with anti-HPV16 and 18 antibody titers from D30, and the induction levels of memory B cells at D30 and PD1⁺ICOS⁺Tfh1-like cells at D7 post-first vaccination correlated for Cervarix. Our study showed that induction of circulating CXCR5⁺CD4⁺ Tfh-like subsets can be detected following immunization with HPV vaccines, and potentially be useful as a marker of immunogenicity of vaccines. However, further investigations should be extended to different cohorts with larger sample size to better understand the functions of these T cells, as well as their relationship with B cells and antibodies.</p></div

Longitudinal analysis of circulating Tfh1-, Tfh2-, Tfh17-like subsets and their PD1 and ICOS expression.

<p>One of the HPV vaccine samples (Gardasil) is shown. The contour plots on the left most column show the expression of CXCR3 and CCR6 on CXCR5⁺ CM cells in samples collected from the indicated time points that are shown on the right. These two markers were used to identify Tfh1-like (blue), Tfh2-like (red), and Tfh17-like (purple) subsets. The percentages of each of these subsets are shown inside the plots. The second, third, and fourth columns of plots show the expression of PD1 and ICOS in each of the Tfh-like subset. In the upper right quadrants, the percentages of PD1⁺ ICOS⁺ Tfh-like cells are shown. The percentages of PD1⁺ ICOS^- and double negative cells are indicated in the lower right and lower left quadrants, respectively.</p

Classification of circulating Tfh-like cell subsets.

<p>This classification is based on the model proposed by Schmitt <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137195#pone.0137195.ref019" target="_blank">19</a>].</p><p>Classification of circulating Tfh-like cell subsets.</p

Generation of IgD^-CD38^HiCD27⁺ memory B cells after vaccination.

<p>(A) Gating strategy used to identify IgD^-CD38^HiCD27⁺ memory B cells is shown. (B) Percentages of memory B cells in Gardasil or Cervarix immunized recipients were plotted over time. Statistical analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137195#pone.0137195.g003" target="_blank">Fig 3</a>. Black bars indicate the medians. For the comparison of the two vaccine groups described in the “A higher frequency of IgD^-CD38^HiCD27⁺ memory B cells was present in Cervarix recipients at D30 post-first vaccination” section, paired, two-tailed Wilcoxon rank sum analyses were performed. N = 10–11 (pre-vac to D30) and N = 6–8 (M6 to M7) for Gardasil. N = 8 (pre-vac to D30) and N = 5–6 (M6 to M7) for Cervarix. (C and D) ELISA were performed to determine the titers of anti-HPV16 (C) and-HPV18 (D) IgG. Geometric mean antibody titers (EU/ mL) ± 95% confidence intervals in log₁₀ scale were plotted as a function of time. The plots on the left show the titers post-first vaccination, and the plots on the right show the titers after the third vaccination. Paired, two-tailed Wilcoxon rank sum analyses were performed. N = 12–15 Gardasil, and N = 8–12 for Cervarix.</p

Memory B cell frequencies at D30 correlate with the titers of anti-HPV16 and-HPV18 antibodies.

<p>Spearman rank correlational analyses were performed on the percentages of IgD^-CD38^HiCD27⁺ B cells at D30 post-first vaccination with the antibody titers of anti-HPV16 and-HPV18 antibodies from D30. The data were plotted in log₁₀ scale for both axes.</p

Correlational analyses between PD1⁺ICOS⁺ Tfh-like subsets and antibody titers at D30 and M7.

<p>Fold changes in different Tfh-like subsets (indicated as Tfh1, Tfh2, and Tfh17) at D7 over the pre-vaccinated time point were compared to the fold changes in the antibody titers at the indicated time points (over the pre-vaccinated time points).</p><p>^a<i>r</i> indicates correlation coefficient from Spearman correlation rank analyses; <i>p</i> indicates <i>p</i> value from the Spearman correlational rank analyses.</p><p>^<i>b</i> NS, not significant.</p><p>^<i>c</i> The frequencies of Tfh17 subsets in some subjects were 0% at the base line; therefore, we could not analyze some of the subjects. For the Tfh1- and 2 subsets, 10 subjects for Gardasil were compared, and it was 8 for Cervarix for D30. For M7, the numbers ranged 5–8 in both groups.</p><p>Correlational analyses between PD1⁺ICOS⁺ Tfh-like subsets and antibody titers at D30 and M7.</p

DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial

<div><p>Background</p><p>DNA vaccine immunogenicity has been limited by inefficient delivery. Needle-free delivery of DNA using a CO₂-powered Biojector® device was compared to delivery by needle and syringe and evaluated for safety and immunogenicity.</p><p>Methods</p><p>Forty adults, 18–50 years, were randomly assigned to intramuscular (IM) vaccinations with DNA vaccine, VRC-HIVDNA016-00-VP, (weeks 0, 4, 8) by Biojector® 2000™ or needle and syringe (N/S) and boosted IM at week 24 with VRC-HIVADV014-00-VP (rAd5) with N/S at 10¹⁰ or 10¹¹ particle units (PU). Equal numbers per assigned schedule had low (≤500) or high (>500) reciprocal titers of preexisting Ad5 neutralizing antibody.</p><p>Results</p><p>120 DNA and 39 rAd5 injections were given; 36 subjects completed follow-up research sample collections. IFN-γ ELISpot response rates were 17/19 (89%) for Biojector® and 13/17 (76%) for N/S delivery at Week 28 (4 weeks post rAd5 boost). The magnitude of ELISpot response was about 3-fold higher in Biojector® compared to N/S groups. Similar effects on response rates and magnitude were observed for CD8+, but not CD4+ T-cell responses by ICS. Env-specific antibody responses were about 10-fold higher in Biojector-primed subjects.</p><p>Conclusions</p><p>DNA vaccination by Biojector® was well-tolerated and compared to needle injection, primed for greater IFN-γ ELISpot, CD8+ T-cell, and antibody responses after rAd5 boosting.</p><p>Trial Registration</p><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/show/NCT00109629" target="_blank">NCT00109629</a></p></div

VRC 011 Disposition Flow Diagram of Screening, Randomization and Vaccination Completion.

<p>VRC 011 Disposition Flow Diagram of Screening, Randomization and Vaccination Completion.</p