Correlations of integrin-expressing cells between tissues.

<p>Graphs display Spearman’s correlation (r_s), and both unadjusted and adjusted p values (n = 10). P values adjusted for multiple comparisons are marked with asterisks (p*).</p

Integrin-expressing CD4^negT cells isolated from blood, cervix and rectum and their co-expression with CD69.

<p>(A) Representative flow cytometry plots for the identification of α4^-β7^-, αE⁺β7^hi, α4⁺β7^hi, α4^intβ7^int, α4⁺β1⁺, and CD69⁺ on CD4^negT cell populations in blood, cervix and rectum; (B) αE⁺β7^hi (blue), α4⁺β7^hi (red), α4^intβ7^int (green) and α4⁺β1⁺ (gray) expression on CD4^negT cells isolated from blood, cervix and rectum. (C) Frequency of CD69-expressing cells on α4^-β7^-CD4^negT cells (black), αE⁺β7^hiCD4^negT cells (blue), α4⁺β7^hiCD4^negT cells (red), α4^intβ7^intCD4^negT cells (green) and α4⁺β1⁺CD4^negT cells (gray). Data from 45 female subjects presented as median (IQR). *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P</i> < 0.001 ****<i>P</i> < 0.0001, as calculated by Friedman Test, followed by Wilcoxon signed rank-test, and adjusted for multiple comparisons using step-down procedure.</p

Anti-α4 and anti- β7 co-staining as means to the identification of αE⁺β7^hi T cell population.

<p>(A) Representative flow cytometry plots for the identification of α4^-/negβ7^hi, α4⁺β7^hi, α4^intβ7^int and α4⁺β7^-T cell populations in blood, cervix and rectum; (B) Frequency of α4^-/lowβ7^hi, α4^intβ7^int and α4⁺β7^hi on CD4⁺ and CD4^negT cells expressing αE. Data from 10 female subjects presented as median and interquartile range (IQR).</p

Integrin-expressing CD4⁺ and CD4^negT cell densities in the blood, cervix and rectum.

<p>(A) CD4^neg:CD4⁺ ratio of all CD3⁺cells expressing α4^-β7^-, αE⁺β7^hi, α4⁺β7^hi, α4^intβ7^int or α4⁺β1⁺ in blood, cervix and rectum. (B) The densities of αE⁺β7^hi, α4⁺β7^hi and α4⁺β1⁺T cells in this pie charts were drawn based on integrin-expressing T cells in each tissue and does not account for the total density of T cells in each site. Data from 45 female subjects presented as median (IQR). *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P</i> < 0.001 ****<i>P</i> < 0.0001, as calculated by Friedman Test, followed by Wilcoxon signed rank-test, and adjusted for multiple comparisons using step-down procedure.</p

Integrin-expressing CD4⁺T cells isolated from blood, cervix and rectum and their co-expression with CCR5 and CD69.

<p>(A) Representative flow cytometry plots for the identification of α4^-β7^-, αE⁺β7^hi, α4⁺β7^hi, α4^intβ7^int α4⁺β1⁺, CCR5⁺ and CD69⁺ on CD4⁺T cell populations in blood, cervix and rectum; (B) α4^-β7^-CD4⁺T cells (black), αE⁺β7^hi (blue), α4⁺β7^hi (red), α4^intβ7^int (green) and α4⁺β1⁺ (gray) expression on CD4⁺T cells isolated from blood, cervix and rectum. (C) Frequency of CCR5-expressing cells on α4^-β7^-CD4⁺T cells (black), αE⁺β7^hiCD4⁺T cells (blue), α4⁺β7^hiCD4⁺T cells (red), α4^intβ7^int CD4⁺T cells (green) and α4⁺β1^hiCD4⁺T cells (gray). (D) Frequency of CD69-expressing cells on α4^-β7^-CD4⁺T cells (black), αE⁺β7^hiCD4⁺T cells (blue), α4⁺β7^hiCD4⁺T cells (red), α4^intβ7^int CD4⁺T cells (green) and α4⁺β1⁺CD4⁺T cells (gray). Data from 45 female subjects presented as median (IQR). *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P</i> < 0.001 ****<i>P</i> < 0.0001, as calculated by Friedman Test, followed by Wilcoxon signed rank-test, and adjusted for multiple comparisons using step-down procedure.</p

Examining the Species-Specificity of Rhesus Macaque Cytomegalovirus (RhCMV) in Cynomolgus Macaques

<div><p>Cytomegalovirus (CMV) is a highly species-specific virus that has co-evolved with its host over millions of years and thus restricting cross-species infection. To examine the extent to which host restriction may prevent cross-species research between closely related non-human primates, we evaluated experimental infection of cynomolgus macaques with a recombinant rhesus macaque-derived CMV (RhCMV-eGFP). Twelve cynomolgus macaques were randomly allocated to three groups: one experimental group (RhCMV-eGFP) and two control groups (UV-inactivated RhCMV-eGFP or media alone). The animals were given two subcutaneous inoculations at week 0 and week 8, and a subset of animals received an intravenous inoculation at week 23. No overt clinical or haematological changes were observed and PBMCs isolated from RhCMV-eGFP inoculated animals had comparable eGFP- and IE-1-specific cellular responses to the control animals. Following inoculation with RhCMV-eGFP, we were unable to detect evidence of infection in any blood or tissue samples up to 4 years post-inoculation, using sensitive viral co-culture, qPCR, and Western blot assays. Co-culture of urine and saliva samples demonstrated the presence of endogenous cynomolgus CMV (CyCMV) cytopathic effect, however no concomitant eGFP expression was observed. The absence of detectable RhCMV-eGFP suggests that the CyCMV-seropositive cynomolgus macaques were not productively infected with RhCMV-eGFP under these inoculation conditions. In a continued effort to develop CMV as a viral vector for an HIV/SIV vaccine, these studies demonstrate that CMV is highly restricted to its host species and can be highly affected by laboratory cell culture. Consideration of the differences between lab-adapted and primary viruses with respect to species range and cell tropism should be a priority in evaluating CMV as vaccine vector for HIV or other pathogens at the preclinical development stage.</p></div

Inoculation and sampling schedule.

<p>Twelve cynomolgus macaques were randomly assigned into three groups, RhCMV-eGFP (N = 6), UV-inactivated RhCMV-eGFP control (N = 2), and media control (N = 4). The animals received one subcutaneous inoculation at week 0 with 7x10⁷ PFU of RhCMV-eGFP or UV-inactivated RhCMV-eGFP, or media alone. A subset of animals was subcutaneously boosted at week 8 with 2x10⁷ PFU of RhCMV-eGFP or UV-inactivated RhCMV-eGFP, while the remaining animals received media alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121339#pone.0121339.t001" target="_blank">Table 1</a>). At week 23, one UV-inactivated RhCMV-eGFP control animal (4M) and three media control animals (1M, 2M, 6M) received an intravenous inoculation with RhCMV-eGFP (7x10⁷ PFU). Sample collection schedule is described.</p

Anti-eGFP antibody responses present in RhCMV-eGFP and UV-inactivated RhCMV-eGFP control animals.

<p>Anti-eGFP antibody responses were determined by ELISA assay, values represented by optical density (OD). The mean of the replicate wells for each animal (A) or the mean of the animals in each group (B) are shown with error bars denoting the standard deviation. The dashed vertical lines represent subcutaneous inoculations and the solid vertical line represents intravenous RhCMV-eGFP inoculation of four control animals (1M, 2M, 4M, 6M). B) The mean from the animals in each group that were omitted from the second subcutaneous boost at week 8 (RhCMV-eGFP group) or the intravenous inoculation at week 23 (UV-inactivated group) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121339#pone.0121339.t001" target="_blank">Table 1</a>) are plotted with an inverted arrow (◉).</p

Intracellular cytokine staining of eGFP and IE-1 stimulated PBMCs.

<p>^aThe values represent the percentage of total CD8⁺ and CD4⁺ T cell responses (background subtracted). Following background subtraction, the negative or zero values were annotated with a dash (-) and the responses that were greater than two times the background are highlighted in bold.</p><p>Intracellular cytokine staining of eGFP and IE-1 stimulated PBMCs.</p

eGFP protein expression is not detected in RhCMV-eGFP inoculated cynomolgus macaques.

<p>Total protein was isolated from urine co-cultures in Telo-RF infected cells and probed by Western blot analyses with antibodies specific for eGFP (27 kDa), IE (72 kDa), and β-actin (42 kDa) as a lysate control. A) Analyses were performed following the two subcutaneous inoculations (week 16). Animals are listed by animal number as XX. B) In vitro infected Telo-RF cells were used for the eGFP controls with RhCMV-eGFP-infected cells as a positive control (+) and CyCMV_Ott-infected cells as a negative control (-). Western blots from different experiments were combined for analyses.</p