Shainn Wei Wang

Control of Simian/Human Immunodeficiency Virus Viremia and Disease Progression after IL-2-Augmented DNA-Modified Vaccinia Virus Ankara Nasal Vaccination in Nonhuman Primates

A successful HIV vaccine may need to stimulate antiviral immunity in mucosal and systemic immune compartments, because HIV transmission occurs predominantly at mucosal sites. We report here the results of a combined DNA-modified vaccinia virus Ankara (MVA) vaccine approach that stimulated simian/human immunodeficiency virus (SHIV)-specific immune responses by vaccination at the nasal mucosa. Fifteen male rhesus macaques, divided into three groups, received three nasal vaccinations on day 1, wk 9, and wk 25 with a SHIV DNA plasmid producing noninfectious viral particles (group 1), or SHIV DNA plus IL-2/Ig DNA (group 2), or SHIV DNA plus IL-12 DNA (group 3). On wk 33, all macaques were boosted with rMVA expressing SIV Gag-Pol and HIV Env 89.6P, administered nasally. Humoral responses were evaluated by measuring SHIV-specific IgG and neutralizing Abs in plasma, and SHIV-specific IgA in rectal secretions. Cellular responses were monitored by evaluating blood-derived virus-specific IFN-γ-secreting cells and TNF-α-expressing CD8+ T cells, and blood- and rectally derived p11C tetramer-positive T cells. Many of the vaccinated animals developed both mucosal and systemic humoral and cell-mediated anti-SHIV immune responses, although the responses were not homogenous among animals in the different groups. After rectal challenge of vaccinated and naive animals with SHIV89.6P, all animals became infected. However a subset, including all group 2 animals, were protected from CD4+ T cell loss and AIDS development. Taken together, these data indicate that nasal vaccination with SHIV-DNA plus IL-2/Ig DNA and rMVA can provide significant protection from disease progression.

Enterovirus 71 infection of human dendritic cells.

Enterovirus 71 (EV71) causes death and long-term neurologic sequelae in hundreds of thousands of young children, but its pathogenesis remains elusive. Dendritic cells (DCs) play a crucial role in antiviral immunity by functioning as professional antigen-presenting cells to prime T cells and by secreting cytokines to modulate immune responses. Here, we show that EV71 productively infected human immature DCs and expressed viral antigen in DCs. EV71 entry into DCs was partially mediated by DC-SIGN. Further analyses revealed that EV71 increased the viability, activation, release of cytokines, interleukin-6, interleukin-12, and tumor necrosis factor-α in DCs. Moreover, EV71 enabled DCs to stimulate T-cell proliferation. Collectively, these findings suggest that EV71 infection of human DCs in vivo is very likely to elicit protective immunity, because in infected mice, both T cells and IL-6 function to reduce mortality.

Effective Induction of Simian Immunodeficiency Virus-Specific Systemic and Mucosal Immune Responses in Primates by Vaccination with Proviral DNA Producing Intact but Noninfectious Virions

ABSTRACT We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

Galectin-1 Binds to Influenza Virus and Ameliorates Influenza Virus Pathogenesis

ABSTRACT Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant (Kd ) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.

RNA Incorporation Is Critical for Retroviral Particle Integrity after Cell Membrane Assembly of Gag Complexes

ABSTRACT The nucleocapsid (NC) domain of retroviruses plays a critical role in specific viral RNA packaging and virus assembly. RNA is thought to facilitate viral particle assembly, but the results described here with NC mutants indicate that it also plays a critical role in particle integrity. We investigated the assembly and integrity of particles produced by the human immunodeficiency virus type 1 M1-2/BR mutant virus, in which 10 of the 13 positive residues of NC have been replaced with alanines and incorporation of viral genomic RNA is virtually abolished. We found that the mutations in the basic residues of NC did not disrupt Gag assembly at the cell membrane. The mutant Gag protein can assemble efficiently at the cell membrane, and viral proteins are detected outside the cell as efficiently as they are for the wild type. However, only ∼10% of the Gag molecules present in the supernatant of this mutant sediment at the correct density for a retroviral particle. The reduction of positive charge in the NC basic domain of the M1-2/BR virus adversely affects both the specific and nonspecific RNA binding properties of NC, and thus the assembled Gag polyprotein does not bind significant amounts of viral or cellular RNA. We found a direct correlation between the percentage of Gag associated with sedimented particles and the amount of incorporated RNA. We conclude that RNA binding by Gag, whether the RNA is viral or not, is critical to retroviral particle integrity after cell membrane assembly and is less important for Gag-Gag interactions during particle assembly and release.

Andrographolide exerts anti-hepatitis C virus activity by up-regulating haeme oxygenase-1 via the p38 MAPK/Nrf2 pathway in human hepatoma cells.

This study aimed to evaluate the anti‐hepatitis C virus (HCV) activity of andrographolide, a diterpenoid lactone extracted from Andrographis paniculata, and to identify the signalling pathway involved in its antiviral action.

An SHIV DNA/MVA rectal vaccination in macaques provides systemic and mucosal virus-specific responses and protection against AIDS

We explored the use of a simian-human immunodeficiency virus (SHIV) DNA vaccine as an effective mucosal priming agent to stimulate a protective immune response for AIDS prevention. Rhesus macaques were vaccinated rectally with a DNA construct producing replication-defective SHIV particles, and boosted with either the same DNA construct or recombinant modified vaccinia virus Ankara (MVA) expressing SIV Gag, SIV Pol, and HIV Env (MVA-SHIV). Virus-specific mucosal and systemic humoral and cell-mediated immune responses could be stimulated by this approach but were present inconsistently among the vaccinated animals. Rectal vaccination with either SHIV DNA alone or SHIV DNA followed by MVA-SHIV induced SIV Gag/Pol- or HIV gp120-specific IgA in rectal secretions of four of seven animals. However, the gp120-specific rectal IgA antibody responses were not durable and had become undetectable in all but one animal shortly before rectal challenge with pathogenic SHIV 89.6P. Only the macaques primed with SHIV DNA and boosted with MVA-SHIV demonstrated SHIV-specific IgG in plasma. In addition, these animals developed more consistent antiviral cell-mediated responses and had better preservation of CD4 T cells following challenge with SHIV 89.6P. Our study demonstrates the utility of a rectal DNA/MVA vaccination protocol for the induction of diverse responses in different immunological compartments. In addition, the immunity achieved with this mucosal vaccination regimen is sufficient to delay progression to AIDS.

Nasal DNA-MVA SIV vaccination provides more significant protection from progression to AIDS than a similar intramuscular vaccination.

Preventive human immunodeficiency virus (HIV) vaccination may require induction of virus-specific immune responses at mucosal sites to contain viral infection locally after exposure, as most HIV infections occur through mucosal surfaces. We compared the efficacy of an intranasal or intramuscular Simian immunodeficiency virus (SIV)+ interleukin (IL)-2+IL-15 DNA/SIV–MVA (modified vaccinia virus Ankara) vaccination in preventing disease progression in SIVmac251 intrarectally challenged rhesus macaques. SIV-specific rectal IgA responses were more significantly persistent in nasally vaccinated than in intramuscularly vaccinated animals. No significant differences were observed in the magnitude of systemic T-cell responses between the two groups, although the nasal immunization induced more significant anti-SIV T-cell responses in the colorectal mucosa. After challenge, CD4+ central memory (CM) T-cell preservation and significant disease-delay were observed in both vaccination groups. However, nasally vaccinated animals had more significant early preservation of circulating and colorectal CD4+ CM T cells, of circulating CD4+/α4β7+ effector memory (EM) T cells, and a longer disease-free interval when compared with the intramuscularly vaccinated or control groups. Regardless of vaccination status, long-term viremia control and preservation of CD4+ CM T cells was detected in animals with significantly higher systemic CD8+/tumor necrosis factor (TNF)-α+ and CD8+/interferon (IFN)-γ+ T-cell responses and higher SIV-specific CD4+/IL-2+ responses in colorectal T cells.

A single nucleotide in stem loop II of 5'-untranslated region contributes to virulence of enterovirus 71 in mice.

Background Enterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored. Principal Findings In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5′-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5′-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5′-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. Conclusions These results presented the first reported virulence determinant in EV71 5′-UTR and first position discovered from unadapted isolates.

Cell Confluence-induced Activation of Signal Transducer and Activator of Transcription-3 (Stat3) Triggers Epithelial Dome Formation via Augmentation of Sodium Hydrogen Exchanger-3 (NHE3) Expression

Cell confluence induces the activation of signal transducer and activator of transcription-3 (Stat3) in various cancer and epithelial cells, yet the biological implications and the associated regulatory mechanisms remain unclear. Because confluent polarized epithelia demonstrate dome formation and sodium influx that mimic the onset of differentiation, we sought to elucidate the role of Stat3 in association with the regulation of selective epithelial transporters in this biological phenomenon. This study established the correlation between Stat3 activation and cell confluence-induced dome formation in Madin-Darby canine kidney cells (MDCK) by following Stat3 activation events in dome-forming cells. Epifluorescent and confocal microscopy provided evidence showing specific localization of phosphorylated Stat3 Tyr705 in the nuclei of dome-forming cells at initial stages. The relationship was further elucidated by the establishment of tetracycline-inducible expression of constitutive Stat3 mutant (Stat3-C) in MDCK cells or expression of dominant negative Stat3 (Stat3-D) stable cell lines (MDCK and NMuMG). Dome formation was promoted by the expression of Stat3-C but inhibited by Stat3-D. Two trans-epithelial transporters, NHE3 and ENaC α-subunit, were found to be increased during cell confluence. Interestingly, NHE3 expression could be specifically up-regulated by Stat3-C but inhibited by Stat3-D through promoter regulation, whereas NHE1 and ENaC α-subunit were not affected by Stat3 expression. Application of NHE3 shRNA, NHE3 inhibitors (EIPA and S3226) suppressed confluence-induced dome formation in MDCK or NMuMG cells. These results demonstrate a cell confluence-induced Stat3 signaling pathway in epithelial cells in triggering dome formation through NHE3 augmentation.