Strong HIV-1-Specific T Cell Responses in HIV-1-Exposed Uninfected Infants and Neonates Revealed after Regulatory T Cell Removal

BACKGROUND: In utero transmission of HIV-1 occurs on average in only 3%–15% of HIV-1-exposed neonates born to mothers not on antiretroviral drug therapy. Thus, despite potential exposure, the majority of infants remain uninfected. Weak HIV-1-specific T-cell responses have been detected in children exposed to HIV-1, and potentially contribute to protection against infection. We, and others, have recently shown that the removal of CD4(+)CD25(+) T-regulatory (Treg) cells can reveal strong HIV-1 specific T-cell responses in some HIV-1 infected adults. Here, we hypothesized that Treg cells could suppress HIV-1-specific immune responses in young children. METHODOLOGY/PRINCIPAL FINDINGS: We studied two cohorts of children. The first group included HIV-1-exposed-uninfected (EU) as well as unexposed (UNEX) neonates. The second group comprised HIV-1-infected and HIV-1-EU children. We quantified the frequency of Treg cells, T-cell activation, and cell-mediated immune responses. We detected high levels of CD4(+)CD25(+)CD127(−) Treg cells and low levels of CD4(+) and CD8(+) T cell activation in the cord blood of the EU neonates. We observed HIV-1-specific T cell immune responses in all of the children exposed to the virus. These T-cell responses were not seen in the cord blood of control HIV-1 unexposed neonates. Moreover, the depletion of CD4(+)CD25(+) Treg cells from the cord blood of EU newborns strikingly augmented both CD4(+) and CD8(+) HIV-1-specific immune responses. CONCLUSIONS/SIGNIFICANCE: This study provides new evidence that EU infants can mount strong HIV-1-specific T cell responses, and that in utero CD4(+)CD25(+) T-regulatory cells may be contributing to the lack of vertical transmission by reducing T cell activation

Induction of Potent Humoral and Cell-Mediated Immune Responses by Attenuated Vaccinia Virus Vectors with Deleted Serpin Genes

Vaccinia virus (VV) has been effectively utilized as a live vaccine against smallpox as well as a vector for vaccine development and immunotherapy. Increasingly there is a need for a new generation of highly attenuated and efficacious VV vaccines, especially in light of the AIDS pandemic and the threat of global bioterrorism. We therefore developed recombinant VV (rVV) vaccines that are significantly attenuated and yet elicit potent humoral and cell-mediated immune responses. B13R (SPI-2) and B22R (SPI-1) are two VV immunomodulating genes with sequence homology to serine protease inhibitors (serpins) that possess antiapoptotic and anti-inflammatory properties. We constructed and characterized rVVs that have the B13R or B22R gene insertionally inactivated (vΔB13R and vΔB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50ΔB13R and v50ΔB22R). Virulence studies with immunocompromised BALB/cBy nude mice indicated that B13R or B22R gene deletion decreases viral replication and significantly extends time of survival. Viral pathogenesis studies in immunocompetent CB6F(1) mice further demonstrated that B13R or B22R gene inactivation diminishes VV virulence, as measured by decreased levels of weight loss and limited viral spread. Finally, rVVs with B13R and B22R deleted elicited potent humoral, T-helper, and cytotoxic T-cell immune responses, revealing that the observed attenuation did not reduce immunogenicity. Therefore, inactivation of immunomodulating genes such as B13R or B22R represents a general method for enhancing the safety of rVV vaccines while maintaining a high level of immunogenicity. Such rVVs could serve as effective vectors for vaccine development and immunotherapy

Use of a recombinant vaccinia virus expressing interferon gamma for post-exposure protection against vaccinia and ectromelia viruses.

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses

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

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 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

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

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

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