Molecular genetic relationship of the 3' untranslated region among Thai dengue-3 virus, Bangkok isolates, during 1973-2000.

Dengue virus serotype 3 (DENV-3) was associated with severe dengue epidemics in Thailand during 1973-1999. We studied Thai DENV-3 viruses isolated from hospitalized children in Bangkok with differing disease severity during that period. Viruses were sequenced at their 5' and 3' untranslated regions (UTRs), which are regions that play a pivotal role in viral replication. Our results indicated that the primary sequences as well as the secondary structures at both ends of Thai DENV-3 viruses were highly conserved over almost three decades. We found nucleotide insertions and deletions at the variable region (VR) that is located just downstream of the nonstructural protein 5 (NS5) stop codon among these viruses. The phylogenetic tree derived from the size heterogeneity of VR in the 3' UTR divided DENV-3 into four genotypes, and Thai DENV-3 viruses in this study belonged to genotype II. The replication efficiency of the candidate viruses with different lengths at the VR were assessed in the mosquito (C6/36) and human (HepG2) cell lines. Our results show that the viruses with nucleotide insertions at VR replicated better than the virus that contained deletions. Our findings indicate that Thai DENV-3 demonstrated a remarkable conservation of nucleotides over 28 years. Correlation with disease severity suggests that both primary sequences and secondary structures of the 3' UTR do not appear correlated with disease severity in humans.

Development of a novel DNA SynCon tetravalent dengue vaccine that elicits immune responses against four serotypes.

The increased transmission and geographic spread of dengue fever (DF) and its most severe presentations, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), make it one of the most important mosquito-borne viral disease of humans. Four distinct serotypes of dengue viruses are transmitted to humans through the bites of the mosquitoes. Currently there is no vaccine or antiviral drug against DV infections. Cross-protection between dengue virus serotypes is limited and antibody dependent enhancement (ADE) contributes significantly to the severity of the disease. The major challenge is to induce a broad durable immune response against all four serotypes of dengue virus simultaneously while avoiding the possible exacerbation of risk of developing the severe forms of disease through incomplete or modified responses. In order to address this worldwide concern, we present a synthetic consensus (SynCon) human codon optimized DNA vaccine that elicits immunity against all four dengue serotypes. We cloned consensus DIII domain of E protein from all serotypes and expressed them as a single open reading frame in a mammalian expression vector, called pDV-U-DIII (dengue-vaccine universal). In mice, this dengue-universal construct elicits significant level of anti-DIII antibody that neutralizes all four dengue subtypes and prevents cell death induced by dengue infection. This is the first SynCon DNA vaccine that provides tetravalent immunity against all four serotypes of dengue virus.

Coimmunization with an optimized IL15 plasmid adjuvant enhances humoral immunity via stimulating B cells induced by genetically engineered DNA vaccines expressing consensus JEV and WNV E DIII.

The Japanese encephalitis virus (JEV) and West Nile virus (WNV) are responsible for a large proportion of viral encephalitis in humans. Currently, there is no FDA approved specific treatment for either, though there are attempts to develop vaccines against both viruses. In this study, we proposed novel genetically engineered DNA vaccines against these two neurotrophic flaviviruses. The structural domain III (DIII) of E protein from these viruses is reported to carry dominant epitopes that induce neutralizing antibodies. Therefore we created consensus sequence of DIII domain across numerous strains of JEV and WNV. Based on the consensus amino acid sequence, synthetic codon and RNA optimized DIII-expressing DNA vaccine constructs with an efficient leader sequence were synthesized for immunization studies. In addition, we also constructed a genetically engineered IL15 DNA vaccine molecular adjuvant for co-stimulating the immune response against DIII clones. Vaccine constructs were delivered into BALB/C mice intramuscularly followed by electroporation using the CELLECTRA in vivo electroporator. We have observed that the combined delivery of both WNV DIII and IL15-ECRO DNA vaccine constructs resulted in not only the highest level of antibody against DIII, but also enhanced cross reactivity with two other antigens tested. Also, coimmunization with IL15 plasmid further increased the immune response by four- to five-fold. Importantly, we have shown that IL15 coimmunization adjuvanted humoral responses against DIII antigens by elevating the level of antibody secreting B cells. Such a DNA vaccine approach may better help to control potential travel related infectious agents such as JEV.

Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding an E6/E7 fusion antigen.

Human papillomavirus type 16 (HPV-16) infection is associated with a majority of cervical cancers and a significant proportion of head and neck cancers. Here, we describe a novel-engineered DNA vaccine that encodes a HPV-16 consensus E6/E7 fusion gene (pConE6E7) with the goal of increasing its antitumor cellular immunity. Compared to an early stage HPV-16 E7 DNA vaccine (pE7), this construct was up to five times more potent in driving E7-specific cellular immune responses. Prophylactic administration of this vaccine resulted in 100% protection against HPV E6 and E7-expressing tumors. Therapeutic studies indicated that vaccination with pConE6E7 prevented or delayed the growth of tumors. Moreover, immunization with pConE6E7 could also partially overcome immune tolerance in E6/E7 transgenic mice. Such DNA immunogens are interesting candidates for further study to investigate mechanisms of tumor immune rejection in vivo.

Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine.

An important goal for human immunodeficiency virus (HIV) vaccines is to develop immunogens that induce broader and more potent cellular immune responses. In this study of DNA vaccine potency, we constructed a novel subtype B env gene (EY2E1-B) with the goal of increasing vaccine antigen immune potency. The vaccine cassette was designed based on subtype B-specific consensus sequence with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The V1 and V2 loops were shortened and the cytoplasmic tail was truncated to prevent envelope recycling. Three different strains of mice (BALB/c, C57BL/6, and HLA-A2 transgenic mice) were immunized three times with pEY2E1-B or the primary DNA immunogen pEK2P-B alone. The analysis of specific antibody responses suggested that EY2E1-B could induce a moderate subtype B-specific antibody response. Moreover, this construct was up to four times more potent at driving cellular immune responses. Epitope mapping results indicated that there is an increase in the breadth and magnitude of cross-reactive cellular responses induced by the EY2E1-B immunogen. These properties suggest that such a synthetic immunogen deserves further examination for its potential to serve as a component antigen in an HIV vaccine cocktail.

Immunogenicity testing of a novel engineered HIV-1 envelope gp140 DNA vaccine construct.

DNA vaccines expressing the envelope (env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating strong immune responses. In this study, we described the development of a recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-gamma ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4+, CD8+, and CCR+ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity.

Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway.

The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV.

Expression and evolutionary analysis of West Nile virus (Merion strain).

The authors report a new strain of West Nile virus (WNV) with the expression analysis of its individual open reading frames. Since its sudden appearance in the summer of 1999 in New York City, the virus has spread rapidly across the continental United States into Canada and Mexico. Besides, its rapid transmission by various vectors, the spread of this virus through organ transplantation, blood transfusion, and mother-child transmission through breast milk is of concern. In order to understand molecular variations of WNV in North America and to generate new tools for understanding WNV biology, a complete clone of WNV has been constructed. Investigations so far have focused only on half of its genes products and a detailed molecular and cell biological aspects on all of WNV gene have yet to be clearly established. The open reading frames of WNV were recovered through an reverse transcriptase-polymerase chain reaction (RT-PCR)-PCR using brain tissue from a dead crow collected in Merion, PA, and cloned into a mammalian expression vector. The deduced amino acid sequences of individual open reading frames were analyzed to determine various structural motifs and functional domains. Expression analysis shows that in neuronal cells, C, NS1, and NS5 proteins are nuclear localized whereas the rest of the antigens are confined to the cytoplasm when they are expressed in the absence of other viral antigens. This is the first report that provides an expression analysis as well as intracellular distribution pattern for all of WNV gene products, cloned from an infected bird. Evolutionary analysis of Merion strain sequences indicates that this strain is distinct phylogenetically from the previously reported WNV strains.

Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help.

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8(+) T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8(+) T cell proliferation and IFN-gamma secretion, and strong induction of long-lived CD8(+) T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8(+) T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8(+) T cell function, we show that in the partial, but not total, absence of CD4(+) T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8(+) T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.

Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway.

West Nile virus (WNV) is a member of the Flaviviridae family of vector-borne pathogens. Clinical signs of WNV infection include neurologic symptoms, limb weakness, and encephalitis, which can result in paralysis or death. We report that the WNV-capsid by itself induces rapid nuclear condensation and cell death in tissue culture. Apoptosis is induced through the mitochondrial pathway resulting in caspase-9 activation and downstream caspase-3 activation. Capsid gene delivery into the striatum of mouse brain or interskeletal muscle resulted in cell death and inflammation, likely through capsid-induced apoptosis in vivo. These studies demonstrate that the capsid protein of WNV may be responsible for aspects of viral pathogenesis through induction of the apoptotic cascade.

Carboxyl terminus of hVIP/mov34 is critical for HIV-1-Vpr interaction and glucocorticoid-mediated signaling.

Human immunodeficiency virus, type 1 (HIV-1) vpr is a highly conserved gene among lentiviruses. The diverse functions of Vpr support interactions of this HIV accessory protein with host cell partners of important pathways. hVIP/mov34 (human Vpr Interacting Protein) is one of these identified Vpr ligands. hVIP is a 34-kDa member of the eIF3 family that is vital for early embryonic development in transgenic mice and important in cell cycle regulation. Its interaction with Vpr, however, is not yet clearly defined. Therefore, we constructed a panel of deletion mutants of this cytoplasmic cellular ligand to map the protein domain that mediates its interaction with Vpr. We observed that the carboxyl-terminal region of hVIP is critical for its interaction with Vpr. In the absence of Vpr or HIV infection, full-length hVIP is expressed in the cytoplasm. The cytoplasmic localization pattern of full-length hVIP protein, however, is shifted to a clear nuclear localization pattern in cells expressing both hVIP and Vpr. In contrast, Vpr did not alter the localization pattern of hVIP mutants, which have their carboxyl-terminal domain deleted. The movement of hVIP supported prior work that suggested that Vpr triggers activation of the GR receptor complex. In fact, we also observed that dexamethasone moves hVIP into the nucleus and that glucocorticoid antagonists inhibit this effect. Interestingly, the expression of an hVIP carboxyl-terminal mutant, which is not responsive to Vpr, is also not responsive to dexamethasone. These data illustrate that the carboxyl-terminal domain of hVIP is critical for mediating hVIP-Vpr interaction as well as for its glucocorticoid response. These results support the view that hVIP is a member of the complex array of nucleocytoplasmic shuttling proteins that are regulated by HIV infection and glucocorticoids.