Genital herpes simplex virus type 2 infection in humanized HIV-transgenic mice triggers HIV shedding and is associated with greater neurological disease.

BACKGROUND: Epidemiological studies consistently demonstrate synergy between herpes simplex virus type 2 (HSV-2) and human immunodeficiency virus type 1 (HIV-1). Higher HIV-1 loads are observed in coinfected individuals, and conversely, HIV-1 is associated with more-severe herpetic disease. A small animal model of coinfection would facilitate identification of the biological mechanisms underlying this synergy and provide the opportunity to evaluate interventions. METHODS: Mice transgenic for HIV-1 provirus and human cyclin T1 under the control of a CD4 promoter (JR-CSF/hu-cycT1) were intravaginally infected with HSV-2 and evaluated for disease progression, HIV shedding, and mucosal immune responses. RESULTS: HSV-2 infection resulted in higher vaginal HIV loads and genital tissue expression of HIV RNA, compared with HSV-uninfected JR-CSF/hu-cycT1 mice. There was an increase in genital tract inflammatory cells, cytokines, chemokines, and interferons in response to HSV-2, although the kinetics of the response were delayed in HIV-transgenic, compared with control mice. Moreover, the JR-CSF/hu-cycT1 mice exhibited earlier and more-severe neurological disease. The latter was associated with downregulation of secretory leukocyte protease inhibitor expression in neuronal tissue, a molecule with antiinflammatory, antiviral, and neuroprotective properties. CONCLUSIONS: JR-CSF/hu-cycT1 mice provide a valuable model to study HIV/HSV-2 coinfection and identify potential mechanisms by which HSV-2 facilitates HIV-1 transmission and HIV modulates HSV-2-mediated disease.

HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression.

HSV triggers intracellular calcium release to promote viral entry. We hypothesized that Akt signaling induces the calcium responses and contributes to HSV entry. Exposure of human cervical and primary genital tract epithelial, neuronal, or keratinocyte cells to HSV serotype 2 resulted in rapid phosphorylation of Akt. Silencing of Akt with small interfering RNA prevented the calcium responses, blocked viral entry, and inhibited plaque formation by 90% compared to control siRNA. Susceptibility to infection was partially restored if Akt was reintroduced into silenced cells with an Akt-expressing plasmid. HSV-2 variants deleted in glycoproteins B or D failed to induce Akt phosphorylation, and coimmunoprecipitation studies indicated that Akt interacts with glycoprotein B. Cell-surface expression of Akt was rapidly induced in response to HSV exposure. Miltefosine (50 µM), a licensed drug that blocks Akt phosphorylation, inhibited HSV-induced calcium release, viral entry, and plaque formation following infection with acyclovir-sensitive and resistant clinical isolates. Miltefosine blocked amplification of HSV from explanted ganglia to epithelial cells; viral yields were significantly less in miltefosine compared to control-treated cocultures (P<0.01). Together, these findings identify a novel role for Akt in viral entry, link Akt and calcium signaling, and suggest a new target for HSV treatment and suppression.

Griffithsin protects mice from genital herpes by preventing cell-to-cell spread.

Griffithsin, which binds N-linked glycans on gp120 to prevent HIV entry, has the most potent HIV-1 inhibitory activity described for any antiviral lectin and is being developed for topical preexposure prophylaxis. The current studies were designed to further assess its potential by exploring its activity against herpes simplex virus 2 (HSV-2), a cofactor for HIV acquisition, in vitro and in a murine model. Safety was evaluated by examining its impact on epithelial barrier integrity in polarized cultures and testing whether repeated intravaginal dosing potentiates the susceptibility of mice to genital herpes. Griffithsin displayed modest inhibitory activity against HSV-2 if present during viral entry but completely blocked plaque formation if present postentry, reduced plaque size, and prevented cell-to-cell spread. These in vitro findings translated to significant protection against genital herpes in mice treated with 0.1% griffithsin gel. Griffithsin, but not placebo gel, prevented viral spread (visualized with a luciferase-expressing virus), significantly reduced disease scores, and resulted in greater survival (P < 0.05, log rank test). Protection persisted when HSV-2 was introduced in seminal plasma. Although griffithsin triggered a small decline in transepithelial electrical resistance in polarized cultures, this did not translate to any significant increase in the ability of HIV to migrate from the apical to the basolateral chamber nor to an increase in susceptibility to HSV-2 in mice treated with griffithsin gel for 7 days. These findings demonstrate that griffithsin inhibits HSV-2 by a unique mechanism of blocking cell-to-cell spread and support its further development for HIV and HSV-2 prevention.

Herpes simplex virus 2 (HSV-2) prevents dendritic cell maturation, induces apoptosis, and triggers release of proinflammatory cytokines: potential links to HSV-HIV synergy.

Herpes simplex virus 2 (HSV-2) may cause frequent recurrences, highlighting its ability to evade host defense. This study tested the hypothesis that HSV-2 interferes with dendritic cell (DC) function as an escape mechanism, which may contribute to enhanced HIV replication in coinfected populations. Immature monocyte-derived human DCs were exposed to live or UV-inactivated HSV-2 or lipopolysaccharide. Little or no increase in the maturation marker CD83 was observed in response to HSV-2 and HSV-2 exposed DCs were impaired in their ability to present antigen (influenza) to T cells. Exposure to UV-inactivated virus stimulated a modest, but significant increase in CD83, suggesting that viral gene expression contributes to the block in DC maturation. The functional impairment of HSV-2-exposed DCs could be partially attributed to the induction of apoptosis. Live and inactivated HSV-2 triggered an increase in the number of early and late apoptotic cells in both the infected and bystander cell populations; apoptosis was associated with a decrease in cellular FLICE-inhibitory protein (c-FLIP). Paradoxically, HSV-2 induced Akt phosphorylation, which typically promotes DC maturation and survival. Despite these aberrant responses, live and inactivated HSV-2 induced the release of cytokines into culture supernatants, which were sufficient to activate HIV-1 replication in latently infected U1 cells. Together, these findings suggest that in the presence of overt or subclinical HSV-2, the function of mucosal DCs would be impaired. These responses may allow HSV to escape immune surveillance but may also promote HIV infection and contribute to the epidemiological link between HIV and HSV.

MiniCD4 microbicide prevents HIV infection of human mucosal explants and vaginal transmission of SHIV(162P3) in cynomolgus macaques.

In complement to an effective vaccine, development of potent anti-HIV microbicides remains an important priority. We have previously shown that the miniCD4 M48U1, a functional mimetic of sCD4 presented on a 27 amino-acid stable scaffold, inhibits a broad range of HIV-1 isolates at sub-nanomolar concentrations in cellular models. Here, we report that M48U1 inhibits efficiently HIV-1(Ba-L) in human mucosal explants of cervical and colorectal tissues. In vivo efficacy of M48U1 was evaluated in nonhuman primate (NHP) model of mucosal challenge with SHIV(162P3) after assessing pharmacokinetics and pharmacodynamics of a miniCD4 gel formulation in sexually matured female cynomolgus macaques. Among 12 females, half were treated with hydroxyethylcellulose-based gel (control), the other half received the same gel containing 3 mg/g of M48U1, one hour before vaginal route challenge with 10 AID(50) of SHIV(162P3). All control animals were infected with a peak plasma viral load of 10(5)-10(6) viral RNA (vRNA) copies per mL. In animals treated with miniCD4, 5 out of 6 were fully protected from acquisition of infection, as assessed by qRT-PCR for vRNA detection in plasma, qPCR for viral DNA detection in PBMC and lymph node cells. The only infected animal in this group had a delayed peak of viremia of one week. These results demonstrate that M48U1 miniCD4 acts in vivo as a potent entry inhibitor, which may be considered in microbicide developments.

Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention.

The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

Dynamics of cell-mediated immune responses to cytomegalovirus in pediatric transplantation recipients.

CMI responses, combined with quantification of CMV DNA (DNAemia), may identify transplantation recipients at risk for invasive disease. PBMC were collected in pediatric transplantation candidates at one, three, and six months post-transplant in 10 subjects (six renal, three cardiac, one stem cell) and at single time points in eight HC and 14 children greater than one yr post-transplant (LTTx). Cells were stimulated with anti-CD3mAb or CMV pp65 peptide pools and responses assessed by IFNG enzyme-linked immunosorbent spot assay and cytokine secretion. IFNG responses to anti-CD3mAb were significantly lower pretransplant relative to HC and were further decreased at one and three months post-transplant, but recovered to levels comparable to HC by six months. Responses to pp65 among CMV-seropositive recipients followed a similar pattern but recovered by three months. CMV-seropositive LTTx and HC showed a Th1 cytokine response to pp65 stimulation. Three LTTx subjects developed CMV DNAemia; two demonstrated decreased responses to anti-CD3mAB (and pp65 in the CMV seropositive subject) at the onset of DNAemia, which recovered as DNAemia resolved. Monitoring CMI in children is feasible and may provide an adjunct biomarker to predict CMV progression and recovery.

Alternative luciferase for monitoring bacterial cells under adverse conditions.

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.