Protein expression from unintegrated HIV-1 DNA introduces bias in primary in vitro post-integration latency models

To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency

Vaccinia Virus Expressing Interferon Regulatory Factor 3 Induces Higher Protective Immune Responses against Lethal Poxvirus Challenge in Atopic Organism

Vaccinia virus (VACV) is an enveloped DNA virus from the Orthopoxvirus family, various strains of which were used in the successful eradication campaign against smallpox. Both original and newer VACV-based replicating vaccines reveal a risk of serious complications in atopic individuals. VACV encodes various factors interfering with host immune responses at multiple levels. In atopic skin, the production of type I interferon is compromised, while VACV specifically inhibits the phosphorylation of the Interferon Regulatory Factor 3 (IRF-3) and expression of interferons. To overcome this block, we generated a recombinant VACV-expressing murine IRF-3 (WR-IRF3) and characterized its effects on virus growth, cytokine expression and apoptosis in tissue cultures and in spontaneously atopic Nc/Nga and control Balb/c mice. Further, we explored the induction of protective immune responses against a lethal dose of wild-type WR, the surrogate of smallpox. We demonstrate that the overexpression of IRF-3 by WR-IRF3 increases the expression of type I interferon, modulates the expression of several cytokines and induces superior protective immune responses against a lethal poxvirus challenge in both Nc/Nga and Balb/c mice. Additionally, the results may be informative for design of other virus-based vaccines or for therapy of different viral infections

Development of Eczema Vaccinatum in Atopic Mouse Models and Efficacy of MVA Vaccination against Lethal Poxviral Infection

<div><p>Smallpox vaccine based on live, replicating vaccinia virus (VACV) is associated with several potentially serious and deadly complications. Consequently, a new generation of vaccine based on non-replicating Modified vaccinia virus Ankara (MVA) has been under clinical development. MVA seems to induce good immune responses in blood tests, but it is impossible to test its efficacy in vivo in human. One of the serious complications of the replicating vaccine is eczema vaccinatum (EV) occurring in individuals with atopic dermatitis (AD), thus excluding them from all preventive vaccination schemes. In this study, we first characterized and compared development of eczema vaccinatum in different mouse strains. Nc/Nga, Balb/c and C57Bl/6J mice were epicutaneously sensitized with ovalbumin (OVA) or saline control to induce signs of atopic dermatitis and subsequently trans-dermally (t.d.) immunized with VACV strain Western Reserve (WR). Large primary lesions occurred in both mock- and OVA-sensitized Nc/Nga mice, while they remained small in Balb/c and C57Bl/6J mice. Satellite lesions developed in both mock- and OVA-sensitized Nc/Nga and in OVA-sensitized Balb/c mice with the rate 40–50%. Presence of mastocytes and eosinophils was the highest in Nc/Nga mice. Consequently, we have chosen Nc/Nga mice as a model of AD/EV and tested efficacy of MVA and Dryvax vaccinations against a lethal intra-nasal (i.n.) challenge with WR, the surrogate of smallpox. Inoculation of MVA intra-muscularly (i.m.) or t.d. resulted in no lesions, while inoculation of Dryvax t.d. yielded large primary and many satellite lesions similar to WR. Eighty three and 92% of mice vaccinated with a single dose of MVA i.m. or t.d., respectively, survived a lethal i.n. challenge with WR without any serious illness, while all Dryvax-vaccinated animals survived. This is the first formal prove of protective immunity against a lethal poxvirus challenge induced by vaccination with MVA in an atopic organism.</p></div

Percentage (%) of Nc/Nga, Balb/c and C57Bl/6 mice with 1 or more satellite lesions 7 days p.i. with WR.

<p>n =  number of mice in each group.</p><p>Percentage (%) of Nc/Nga, Balb/c and C57Bl/6 mice with 1 or more satellite lesions 7 days p.i. with WR.</p

Expression of TIM-3 on Plasmacytoid Dendritic Cells as a Predictive Biomarker of Decline in HIV-1 RNA Level during ART

Depletion and functional impairment of circulating plasmacytoid dendritic cells (pDCs) are characteristic attributes of HIV-1-infection. The mechanism of dysfunction of pDCs is unclear. Here, we studied the development of phenotype of pDCs in a cohort of HIV-1-infected individuals monitored before the initiation and during a 9-month follow up with antiretroviral therapy (ART). Using polychromatic flow cytometry, we detected significantly higher pDC-surface expression of the HIV-1 receptor CD4, regulatory receptor BDCA-2, Fc&gamma; receptor CD32, pDC dysfunction marker TIM-3, and the marker of killer pDC, TRAIL, in treatment-na&iuml;ve HIV-1-infected individuals before initiation of ART when compared to healthy donors. After 9 months of ART, all of these markers approached but did not reach the expression levels observed in healthy donors. We found that the rate of decline in HIV-1 RNA level over the first 3 months of ART negatively correlated with the expression of TIM-3 on pDCs. We conclude that immunogenic phenotype of pDCs is not significantly restored after sustained suppression of HIV-1 RNA level in ART-treated patients and that the level of the TIM-3 expressed on pDCs in treatment na&iuml;ve patients could be a predictive marker of the rate of decline in the HIV-1 RNA level during ART

Immune responses of atopic Nc/Nga mice immunized with MVA and Dryvax.

<p>Mock-sensitized Nc/Nga mice were inoculated using a vaccination stamp trans-dermally (t.d.) or intra-muscularly (i.m.) with 10⁸ PFU of a purified stock of MVA or t.d. with 10⁷ PFU of a purified stock of Dryvax, and sacrificed 3 weeks later. (A) Levels of total serum IgG₁ and IgG_2a antibodies specific to VACV were determined by ELISA (dilution 1∶1,000); median values are indicated. Results of 2 independent experiments. (B) Neutralization capacity of mice sera expressed as number of plaques detected after a 12-h incubation of the input virus with the individual sera; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114374#s2" target="_blank">Materials and Methods</a> section 6 for experimental details. Median values of the resulting numbers of plaques are indicated. Results of 1–2 independent experiments. *P<0.05, **P<0.01.</p

Histological features of the skin of Nc/Nga, Balb/c and C57Bl/6 mice 7 days after mock or WR inoculation by vaccination stamp.

<p>Mock- (A, C) and OVA-sensitized (B, D) mice were mock-inoculated with OVA in PBS (A, B) or inoculated with 10⁷ PFU of a purified stock of WR with OVA in PBS (C, D). Hematoxylin-eosin stained skin sections. Original magnification: (A, B) upper panel 100x, lower panel 400x; (C, D) upper panel 100x, lower panel 200x with inserts 400x. (A) Nc/Nga - Mild mixed predominantly mononuclear dermal inflammatory infiltrate. Thickening of epidermis, increased thickness of dermis due to proliferation of the connective tissue. Balb/c - Mild fibrosis and no or minimal mixed inflammation in the dermis. C57Bl/6 - Minimal mixed dermal inflammatory infiltrate in the skin, focal thickening of epidermis. (B) Nc/Nga – Very mild mixed dermal inflammatory infiltrate. Thickening of epidermis and parakeratosis (↓), increased thickness of dermis. Balb/c - Thickening of epidermis, mild fibrosis and mild mixed dermal inflammation. C57Bl/6 - Focal thickening of epidermis, very mild mixed dermal inflammatory infiltrate. (C, D) WR-inoculated mice. In all strains - mixed dermal inflammatory infiltrate with an epidermal erosion/ulcer, covered by a crust. Florid necrotizing folliculitis with high incidence of chromatin debris →, eosinophils found in Nc/Nga, Balb/c and C57Bl/6 mice in about 5, 1 and 3%, respectively – shown in the inserts. Representative results of skin sections of 3-5 animals.</p

Primary and secondary lesions in the skin of Nc/Nga, Balb/c and C57Bl/6 mice after WR inoculation by vaccination stamp.

<p>Mock- and OVA-sensitized mice were inoculated with 10⁷ PFU of a purified stock of WR with OVA in PBS, sacrificed at indicated days p.i, and skin biopsies were taken. (A) Size of primary lesions 7 days p.i. Data represent mean +/− S.E.M. *P<0.05, **P<0.01. Results of 2 experiments with n = 6–9 mice in each group. (B) WR titers (PFU/ml) in the site of WR inoculation 7 days p.i.; median values are indicated. No statistically significant differences. Results of 1–2 independent experiments. (C) Time course of WR growth. Data represent mean +/− S.E.M.; n = 3–12 mice in each group. No statistically significant differences. Results of 3 and 2 independent experiments in Nc/Nga mice and Balb/c mice, respectively. (D) Site of primary inoculation (upper panels) and a satellite lesion (lower panels) in mock-sensitized, WR-inoculated Nc/Nga mice 7 days p.i. Immunohistochemical detection of WR (right), control hematoxylin-eosin staining (left); original magnification 200x. Positive IHC signal (brown) overlaps with the sites of increased apoptosis (→).Representative results of 3 skin sections.</p

Quantification of mast cells in Toluidine blue-stained skin sections from mock- and OVA-sensitized mice at the day of inoculation and 7 days after mock- or WR-inoculations.

<p>Data represent mean +/− S.E.M.; n = 2–5 mice in each group. ND, not determined. Statistically significant decrease in the number of mast cells compared to analogously sensitized Nc/Nga mice,</p>x<p>P<0.1,</p><p>*P<0.05,</p><p>**P<0.01;</p><p>***P<0.001. Statistically significant decrease in the number of mast cells in OVA-sensitized Balb/c mice compared to mock-sensitized Nc/Nga mice.</p>a<p>P<0.05.</p><p>Quantification of mast cells in Toluidine blue-stained skin sections from mock- and OVA-sensitized mice at the day of inoculation and 7 days after mock- or WR-inoculations.</p

Survival of atopic Nc/Nga mice immunized with MVA or Dryvax after a lethal intranasal challenge with VACV strain WR.

<p>(A) Body weight changes of mock-immunized (PBS), MVA- or Dryvax-immunized Nc/Nga mice after lethal i.n. challenge with WR. Results are expressed as mean +/− S.E.M. At day 10, all mock-immunized mice were spontaneously dead. Results of 2 independent experiments performed for each virus strain together with the PBS controls. (B) Levels of total serum IgG₁ and IgG_2a antibodies specific to VACV determined 14 days after a lethal challenge with VACV strain WR determined by ELISA in sera dilutions 1∶10,000–1∶20,000 and 1∶100,000–1∶200,000 for IgG₁ and IgG_2a, respectively, and expressed as relative values; median values are indicated. Results of 2 independent experiments.</p