Chih-Hsiang Leng

A novel dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity

A novel dengue vaccine candidate comprised of a consensus dengue virus envelope protein domain III (cED III) was developed to fight against dengue virus infection. The amino acid sequence of this novel cED III was obtained by alignment of amino acid sequences from different isolates of the four serotypes of dengue viruses. A proof-of-concept study demonstrated that BALB/c mice immunized with the recombinant cED III developed neutralizing antibodies against all serotypes of dengue virus. Moreover, formulation of recombinant cED III with aluminum phosphate could induce long-lasting antibody responses and anamnestic neutralizing antibody responses following challenge with dengue virus at week 28 after priming. These results demonstrate the possibility of developing a single tetravalent vaccine against dengue viral infections.

An improved SUMO fusion protein system for effective production of native proteins

Expression of recombinant proteins as fusions with SUMO (small ubiquitin‐related modifier) protein has significantly increased the yield of difficult‐to‐express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky‐end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 5′‐end and XhoI at the 3′‐end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His6‐Smt3‐X) in which the protein of interest (X) is fused to a hexahistidine (His6)‐tagged Smt3. Smt3 is the yeast SUMO protein. His6‐Smt3‐X was purified by Ni2+ resin. Removal of His6‐Smt3 was performed on the Ni2+ resin by an engineered SUMO protease, His6‐Ulp1403–621‐His6. Because of its dual His6 tags, His6‐Ulp1403–621‐His6 exhibits a high affinity for Ni2 resin and associates with Ni2+ resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni2+‐resin column. The eluant contains only the native target protein. Such a one‐column protocol is useful in developing a better high‐throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult‐to‐produce authentic proteins.

A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design

We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.

Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus

The neutralization titer of a hemagglutinin (HA)-specific neutralizing antibody against new isolates reflect both the antigenic drift and the conformation status of HA protein in these new influenza viruses. Since most antigenic sites are in the HA1 domain of HA, using HA1 domain of influenza virus as antigen is of great importance in vaccine development. In this study, we investigate different purification processes for optimizing the immunological properties of an Escherichia coli-expressed HA1 domain (rH5HA1) of influenza H5N1 virus. rH5HA1 was expressed as inclusion bodies and extracted with 6M guanidine hydrochloride (GnHCl)/PBS buffer. The best condition for generating HA1-specific neutralization determinants is on-column oxidative refolding procedures with GSH/GSSG and l-arginine buffer. Others refolding procedures such as using high-pH buffer and/or different detergent solubilizations were found to be ineffective producing neutralization epitope recognized by a HA1-specific neutralizing monoclonal antibody that was raised against H5N1 virus.

Emulsified Nanoparticles Containing Inactivated Influenza Virus and CpG Oligodeoxynucleotides Critically Influences the Host Immune Responses in Mice

Background Antigen sparing and cross-protective immunity are regarded as crucial in pandemic influenza vaccine development. Both targets can be achieved by adjuvantation strategy to elicit a robust and broadened immune response. We assessed the immunogenicity of an inactivated H5N1 whole-virion vaccine (A/Vietnam/1194/2004 NIBRG-14, clade 1) formulated with emulsified nanoparticles and investigated whether it can induce cross-clade protecting immunity. Methodology/Principal Findings After formulation with PELC, a proprietary water-in-oil-in-water nanoemulsion comprising of bioresorbable polymer/Span®85/squalene, inactivated virus was intramuscularly administered to mice in either one-dose or two-dose schedule. We found that the antigen-specific serum antibody responses elicited after two doses of non-adjuvanted vaccine were lower than those observed after a single dose of adjuvanted vaccine, PELC and the conventional alum adjuvant as well. Moreover, 5 µg HA of PELC-formulated inactivated virus were capable of inducing higher antibodies than those obtained from alum-adjuvanted vaccine. In single-dose study, we found that encapsulating inactivated virus into emulsified PELC nanoparticles could induce better antibody responses than those formulated with PELC-adsorbed vaccine. However, the potency was rather reduced when the inactivated virus and CpG (an immunostimulatory oligodeoxynucleotide containing unmethylated cytosine-guanosine motifs) were co-encapsulated within the emulsion. Finally, the mice who received PELC/CpG(adsorption)-vaccine could easily and quickly reach 100% of seroprotection against a homologous virus strain and effective cross-protection against a heterologous virus strain (A/Whooper swan/Mongolia/244/2005, clade 2.2). Conclusions/Significance Encapsulating inactivated H5N1 influenza virus and CpG into emulsified nanoparticles critically influences the humoral responses against pandemic influenza. These results demonstrated that the use of PELC could be as antigen-sparing in preparation for a potential shortage of prophylactic vaccines against local infectious diseases, in particular pandemic influenza. Moreover, the cross-clade neutralizing antibody responses data verify the potential of such adjuvanted H5N1 candidate vaccine as an effective tool in pre-pandemic preparedness.

A Novel Single-Dose Dengue Subunit Vaccine Induces Memory Immune Responses

To protect against dengue viral infection, a novel lipidated dengue subunit vaccine was rationally designed to contain the consensus amino acid sequences derived from four serotypes of dengue viruses. We found that the lipidated consensus dengue virus envelope protein domain III (LcED III) is capable of activating antigen-presenting cells and enhancing cellular and humoral immune responses. A single-dose of LcED III immunization in mice without extra adjuvant formulation is sufficient to elicit neutralizing antibodies against all four serotypes of dengue viruses. In addition, strong memory responses were elicited in mice immunized with a single-dose of LcED III. Quick, anamnestic neutralizing antibody responses to a live dengue virus challenge were elicited at week 28 post-immunization. These results demonstrate the promising possibility of a future successful tetravalent vaccine against dengue viral infections that utilizes one-dose vaccination with LcED III.

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates.

Abstract The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6M urea, then purified by Ni2+-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166–180, 356–375, and 396–410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156–175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156–175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1–30, 86–100, 306–320, and 351–365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2Kd CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-γ secretion cell assay and analysis by flow cytometry, peptides containing residues 81–95 were found to be capable of stimulating both CD4+ and CD8+ cell proliferation in vitro. We also only observed that peptides corresponding to residues 336–350 were capable of stimulating IFN-γ production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

A consensus envelope protein domain III can induce neutralizing antibody responses against serotype 2 of dengue virus in non-human primates

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.

Recombinant Lipidated HPV E7 Induces a Th-1-Biased Immune Response and Protective Immunity against Cervical Cancer in a Mouse Model

The E7 oncoprotein of human papillomavirus (HPV) is an ideal target for developing immunotherapeutic strategies against HPV-associated tumors. However, because protein-based immunogens alone are poor elicitors of the cytotoxic T-lymphocyte (CTL) responses, they have been difficult to exploit for therapeutic purposes. In this study, we report that a recombinant lipoprotein consisting of inactive E7 (E7m) biologically linked to a bacterial lipid moiety (rlipo-E7m) induces the maturation of mouse bone marrow-derived dendritic cells through toll-like receptor 2 (TLR2), skews the immune responses toward the Th1 responses and induces E7-specific CTL responses. We further studied the ability of rlipo-E7m to provide protection against a TC-1 tumor cell challenge in an animal model. Mice prophylactically immunized with two 10-µg doses of rlipo-E7m were found to be free of TC-1 tumor growth. Experiments in a therapeutic immunization model showed that the tumor volume in mice receiving a single dose of rlipo-E7m was less than 0.01 cm3 on day 40, whereas the tumor volume in mice treated with rE7m was 2.28±1.21 cm3. The tumor volume of the entire control group was over 3 cm3. In addition, we demonstrated that the CD8+ T cells play a major role in anti-tumor immunity when administration of rlipo-E7m. These results demonstrate that rlipo-E7m could be a promising candidate for treating HPV-associated tumors.

Dengue-1 Envelope Protein Domain III along with PELC and CpG Oligodeoxynucleotides Synergistically Enhances Immune Responses

The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus.