Viral spread of wt VACV in infected animals.

<p>Groups of 3 C57BL/6 mice were infected intranasally with 10⁶ pfu of wt VACV. Animals remained untreated (A) or were treated one day post-infection with 10⁷ pfu of v50ΔB13RMγ (B). Replication of v50ΔB13RMγ in the tissues from the treated group was examined by X-gal staining (C). Error bars indicate the standard error of the mean. Data represent a pool of two independent experiments using 3 mice per group. </p

Measurement of IFN-γ in the serum following treatment.

<p>Groups of 5 C57BL/6 mice were mock-infected or infected IN with 10⁶ pfu (~100 LD_50s) of wt VACV. One day post infection mice were either mock-treated or treated with 10⁷ pfu of v50ΔB13R or v50ΔB13RMγ. After 24 hours, serum was harvested and an ELISA was performed to measure IFN-γ. The horizontal line indicates the average and error bars indicate the standard error of the mean. Comparisons were done using the Mann-Whitney test. One asterisk (*) represents p<0.008 in comparison to the wt/v50ΔB13RMγ group. Two asterisks (**) represents p<0.016 in comparison to the wt/v50ΔB13RMγ group. </p

Protection of wt VACV infected animals by post-exposure vaccination with v50ΔB13RMγ at one, two and three days post-infection.

<p>Groups of 5 to 10 C57BL/6 mice were infected intranasally with 10⁶ pfu (~100 LD_50s) of wt VACV. Animals were treated with 10⁷ pfu of v50ΔB13RMγ at one (▲), two (●) or three (♦) days post-infection. One group was infected with 10⁷ pfu (~1000 LD_50s) of wt VACV and treated with 10⁷ pfu of v50ΔB13RMγ at one day post-infection (∆). Controls animals were infected with wt VACV and then mock-treated (■), or animals were mock-infected and mock-treated (◊). (A) Comparison of survival curves was done using the log-rank test. (B) Recovery of wt VACV infected animals by post-exposure vaccination with v50ΔB13RMγ at one, two and three days post-infection. The graph indicates the relative sickness of each group of animals during the course of the infection. Lines ending prematurely indicate death of all the animals from the group. A value of 0 indicates that all the animals from that group were healthy.</p

Post-exposure protection of animals after intranasal infection with a lethal dose of wt VACV and subsequent treatment with different VACV mutants.

<p>(A) Weight loss. Groups of 5 to 10 four-week-old C57BL/6 mice were infected intranasally with 10⁶ pfu (~100 LD_50s) of wt VACV and treated 1 dpi with 10⁷ pfu of VACVE3LΔ7C (○), VACVE3LΔ26C (●), VACVΔE3L (♦), VACVΔE3L::ATVeIF2α (□), v50ΔB13RMγ (▲) and v50ΔB13RMIL-18 (∆). There were two groups of control animals, one group was infected with wt VACV alone and was mock-treated (■), the second group of animals was mock-infected and mock-treated (◊). Each mouse was weighed at the indicated times. Average percentage of initial weight of the animals infected with each virus is plotted versus time (days post-infection). Lines end at the death of one animal. Error bars indicate the standard error of the mean. (B) Survival curve. The data represent a pool of two independent experiments using group sizes of 5 mice. (C) v50ΔB13RMγ dose response. Groups of 10 four-week-old C57BL/6 mice were infected intranasally with 10⁶ pfu of wt VACV (~100 LD_50s) and treated one day post-infection with doses of 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ and 5 x 10⁸ pfu of v50ΔB13RMγ (▲).</p

Histopathologic comparison and infection progression in the nasal cavity section.

<p>Mice were infected IN with 10⁶ pfu (~100 LD_50s) of wt VACV and treated one day post-infection with 10⁷ pfu of v50ΔB13RMγ. Mice were sacrificed at 7-8 days post-infection. All representative sections were stained with polyclonal antibodies against VACV. S=septum, T=maxilloturbinate, M=meatus (air passage), i=incisor. (A) Maxilloturbinate section, 2 mm depth, 100X magnification (B) Whole section, 5-6 mm depth. 10X magnification. Lines indicate 200 μm length.</p

Post-exposure protection of wt VACV infected animals treated with v50ΔB13RMγ using different routes of treatment.

<p>Groups of 12 to 15 C57BL/6 mice were infected intranasally with 10⁶ pfu of wt VACV. Animals were treated one day post-infection with 10⁷ pfu of v50ΔB13RMγ intranasally, IN (▲), intranasally using the other nostril, INON (∆), intramuscularly, IM (○) or via scarification, SCA (<b>●</b>). One group of animals was infected with wt VACV and then mock-treated (■), another group was mock-infected and mock-treated (◊). Comparison of survival curves was done using the log-rank test.</p

Protection of wt ECTV infected animals by post-exposure vaccination with v50ΔB13RMγ at one day post-infection.

<p>Groups of 30 C57BL/6 mice were infected intranasally with 5 x 10³ pfu (~50 LD_50s) of wt ECTV. Animals were treated by FP route with saline (■), 3 x 10⁷ pfu of v50ΔB13RMγ (▲) or v50ΔB13R (●) at one day post-infection. One group of animals was mock infected and mock-treated (◊). The data were pooled from two independent experiments using group sizes of 10 and 20 mice. <i>P</i> values (log-rank test) show the significance of difference with respect to ECTV/Mock. The boxed <i>P</i> value shows the significance of the difference between v50ΔB13RMγ and v50ΔB13R.</p

Figure 5

<p>Augmented CD4⁺ HIV-1 immune responses to Gag peptide pools in exposed uninfected neonatal cord blood upon the removal of CD4⁺CD25⁺ Treg cells. A) IL-2 production by undepleted whole cord blood and peripheral blood mononuclear cells (MNCs) derived CD4⁺ T-cells (open white symbols) and CD25-depleted MNCs derived CD4⁺ T-cells (closed black symbols) is depicted. B) Flow cytometry plots from an exposed uninfected neonate (Patient 63) representing HIV-1 Gag induced IL-2 production in undepleted whole CBMC derived CD4⁺ T-cells and CD25-depleted CBMC derived CD4⁺ T-cells.</p

Figure 1

<p>CD4⁺ and CD8⁺ T cell immune responses were measured by cytokine flow cytometry. A) Gating strategy for the identification of polyfunctional IFN-gamma/TNF-alpha CD8⁺ T cell responses. B) Shown are representative data for the unstimulated and HIV-gag-specific response from subject PB-INF-4 after an 18 h <i>in vitro</i> stimulation.</p

Figure 3

<p>Polyfunctional CD8⁺ T cell immune responses to the HIV-1 Gag peptide pool were detected by cytokine flow cytometry. Responses were measured in the cord blood of unexposed neonates (CB-UNEX; n = 4), HIV-1-exposed uninfected neonates (CB-EU; n = 6), and in the peripheral blood of HIV-1-exposed-uninfected infants (PB-EU 7 mo; n = 9) and young children (PB-EU 20 mo; n = 7), and in HIV-1-infected infants (PB-INF 7 mo; n = 5) and young children (PB-INF 25 mo; n = 5). Each group is represented by a different symbol.</p