Plasmodium vivax apical membrane antigen-1: comparative recognition of different domains by antibodies induced during natural human infection

The Apical Membrane Antigen-1 (AMA-1) of Plasmodium sp. has been suggested as a vaccine candidate against malaria. This protein seems to be involved in merozoite invasion and its extra-cellular portion contains three distinct domains: DI, DII, and DIII. Previously, we described that Plasmodium vivax AMA-1 (PvAMA-1) ectodomain is highly immunogenic in natural human infections. Here, we expressed each domain, separately or in combination (DI-II or DII-III), as bacterial recombinant proteins to map immunodominant epitopes within the PvAMA-1 ectodomain. IgG recognition was assessed by ELISA using sera of P. vivax-infected individuals collected from endemic regions of Brazil or antibodies raised in immunized mice. The frequencies of responders to recombinant proteins containing the DII were higher than the others and similar to the ones observed against the PvAMA-1 ectodomain. Moreover, ELISA inhibition assays using the PvAMA-1 ectodomain as substrate revealed the presence of many common epitopes within DI-II that are recognized by human immune antibodies. Finally, immunization of mice with the PvAMA-1 ectodomain induced high levels of antibodies predominantly to DI-II. Together, our results indicate that DII is particularly immunogenic during natural human infections, thus indicating that this region could be used as part of an experimental sub-unit vaccine to prevent vivax malaria.     

A recombinant measles virus expressing hepatitis B virus surface antigen induces humoral immune responses in genetically modified mice

It has been shown previously that measles virus (MV) can be successfully used to express foreign proteins (M. Singh and M. A. Billeter, J. Gen. Virol. 80:101-106, 1998). To develop an inexpensive MV-based vaccine, we generated recombinant MVs that produce structural proteins of hepatitis B virus (HBV). A recombinant virus that expressed the HBV small surface antigen (HBsAg) was analyzed in terms of its replication characteristics, its genetic stability in cell culture, and its immunogenic potential in genetically modified mice. Although this virus showed a progression of replication slightly slower than that of the parental MV, it appeared to stably maintain the added genetic information; it uniformly expressed the appropriately glycosylated HBsAg after 10 serial passages. Genetically modified mice inoculated with this recombinant MV produced humoral immune responses against both HBsAg and MV proteins.     

The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus

A central goal in vaccinology is the induction of high and sustained Ab responses. Protein-in-adjuvant formulations are commonly used to achieve such responses. However, their clinical development can be limited by the reactogenicity of some of the most potent preclinical adjuvants and the cost and complexity of licensing new adjuvants for human use. Also, few adjuvants induce strong cellular immunity, which is important for protection against many diseases, such as malaria. We compared classical adjuvants such as aluminum hydroxide to new preclinical adjuvants and adjuvants in clinical development, such as Abisco 100, CoVaccine HT, Montanide ISA720, and stable emulsion-glucopyranosyl lipid A, for their ability to induce high and sustained Ab responses and T cell responses. These adjuvants induced a broad range of Ab responses when used in a three-shot protein-in-adjuvant regimen using the model Ag OVA and leading blood-stage malaria vaccine candidate Ags. Surprisingly, this range of Ab immunogenicity was greatly reduced when a protein-in-adjuvant vaccine was used to boost Ab responses primed by a human adenovirus serotype 5 vaccine recombinant for the same Ag. This human adenovirus serotype 5-protein regimen also induced a more cytophilic Ab response and demonstrated improved efficacy of merozoite surface protein-1 protein vaccines against a Plasmodium yoelii blood-stage challenge. This indicates that the differential immunogenicity of protein vaccine adjuvants may be largely overcome by prior immunization with recombinant adenovirus, especially for adjuvants that are traditionally considered poorly immunogenic in the context of subunit vaccination and may circumvent the need for more potent chemical adjuvants.     

Hepatitis B surface antigen vector delivers protective cytotoxic T-lymphocyte responses to disease-relevant foreign epitopes

Although hepatitis B surface antigen (HBsAg) per se is highly immunogenic, its use as a vector for the delivery of foreign cytotoxic T-lymphocyte (CTL) epitopes has met with little success because of constraints on HBsAg stability and secretion imposed by the insertion of foreign sequence into critical hydrophobic/amphipathic regions. Using a strategy entailing deletion of DNA encoding HBsAg-specific CTL epitopes and replacement with DNA encoding foreign CTL epitopes, we have derived chimeric HBsAg DNA immunogens which elicited effector and memory CTL responses in vitro, and pathogen- and tumor-protective responses in vivo, when the chimeric HBsAg DNAs were used to immunize mice. We further show that HBsAg DNA recombinant for both respiratory syncytial virus and human papillomavirus CTL epitopes elicited simultaneous responses to both pathogens. These data demonstrate the efficacy of HBsAg DNA as a vector for the delivery of disease-relevant protective CTL responses. They also suggest the applicability of the approach of deriving chimeric HBsAg DNA immunogens simultaneously encoding protective CTL epitopes for multiple diseases. The DNAs we tested formed chimeric HBsAg virus-like particles (VLPs). Thus, our results have implications for the development of vaccination strategies using either chimeric HBsAg DNA or VLP vaccines. HBsAg is the globally administered vaccine for hepatitis B virus infection, inviting its usage as a vector for the delivery of immunogens from other diseases.     

Development of pilot scale production process and characterization of a recombinant multiepitope malarial vaccine candidate FALVAC-1A expressed in Escherichia coli

Among the four human malarial species, Plasmodium falciparum causes most of the mortality associated with malaria. Several approaches are being pursued to develop a suitable malaria vaccine since it may be the most effective weapon to fight against malaria. A highly immunogenic, synthetic protein consisting of 21 epitopes from pre-erythrocytic and blood stages of P. falciparum (FALVAC-1A) was constructed and expressed in Escherichia coli. This vaccine candidate was highly immunogenic and induced protective antibodies in rabbits when produced through lab-scale processes in milligram quantities. In order to take this vaccine candidate for further clinical trial, we optimized the process for industrial scale production and purification. Here we describe various methods used in pilot scale production and characterization of FALVAC-1A. A fed-batch cultivation process in a bioreactor at 10-L scale was optimized to express the protein in high yields as inclusion bodies in E. coli cells with the recombinant plasmids. Methods to solubilize, capture and purify the target protein from the inclusion bodies were optimized and the resultant protein was >95% pure based on SDS-PAGE and RP-HPLC. This protein was then refolded and nativity was confirmed by Far-UV CD spectroscopy. Final purified protein was characterized to estimate yield, purity, mass and confirmed to be free of host cell proteins, nucleic acids and bacterial endotoxins. This study confirms that industrial scale clinical grade FALVAC-1A can be produced in a cost-effective manner for clinical trials.     

Expression and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.     

Synergism/complementarity of recombinant adenoviral vectors and other vaccination platforms during induction of protective immunity against malaria

The lack of immunogenicity of most malaria antigens and the complex immune responses required for achieving protective immunity against this infectious disease have traditionally hampered the development of an efficient human malaria vaccine. The current boom in development of recombinant viral vectors and their use in prime-boost protocols that result in enhanced immune outcomes have increased the number of malaria vaccine candidates that access pre-clinical and clinical trials. In the frontline, adenoviruses and poxviruses seem to be giving the best immunization results in experimental animals and their mutual combination, or their combination with recombinant proteins (formulated in adjuvants and given in sequence or being given as protein/virus admixtures), has been shown to reach unprecedented levels of anti-malaria immunity that predictably will be somehow reproduced in the human setting. However, all this optimism was previously seen in the malaria vaccine development field without many real applicable results to date. We describe here the current state-of-the-art in the field of recombinant adenovirus research for malaria vaccine development, in particular referring to their use in combination with other immunogens in heterologous prime-boost protocols, while trying to simultaneously show our contributions and point of view on this subject.     

Protein Microarray Analysis of Antibody Responses to Plasmodium falciparum in Western Kenyan Highland Sites with Differing Transmission Levels

Malaria represents a major public health problem in Africa. In the East African highlands, the high-altitude areas were previously considered too cold to support vector population and parasite transmission, rendering the region particularly prone to epidemic malaria due to the lack of protective immunity of the population. Since the 1980’s, frequent malaria epidemics have been reported and these successive outbreaks may have generated some immunity against Plasmodium falciparum amongst the highland residents. Serological studies reveal indirect evidence of human exposure to the parasite, and can reliably assess prevalence of exposure and transmission intensity in an endemic area. However, the vast majority of serological studies of malaria have been, hereto, limited to a small number of the parasite’s antigens. We surveyed and compared the antibody response profiles of age-stratified sera from residents of two endemic areas in the western Kenyan highlands with differing malaria transmission intensities, during two distinct seasons, against 854 polypeptides of P. falciparum using high-throughput proteomic microarray technology. We identified 107 proteins as serum antibody targets, which were then characterized for their gene ontology biological process and cellular component of the parasite, and showed significant enrichment for categories related to immune evasion, pathogenesis and expression on the host’s cell and parasite’s surface. Additionally, we calculated age-fitted annual seroconversion rates for the immunogenic proteins, and contrasted the age-dependent antibody acquisition for those antigens between the two sampling sites. We observed highly immunogenic antigens that produce stable antibody responses from early age in both sites, as well as less immunogenic proteins that require repeated exposure for stable responses to develop and produce different seroconversion rates between sites. We propose that a combination of highly and less immunogenic proteins could be used in serological surveys to detect differences in malaria transmission levels, distinguishing sites of unstable and stable transmission.     

A review of combination adjuvants for malaria vaccines: a promising approach for vaccine development

It is obvious that there is a critical need for an efficient malaria vaccine to accelerate malaria eradication. Currently, recombinant subunit vaccination against malaria using proteins and peptides is gaining attention. However, one of the major drawbacks of this approach is the lack of an efficient and durable immune response. Therefore, subunit vaccines require adjuvants to make the vaccine sufficiently immunogenic. Considering the history of the RTS,S vaccine, it seems likely that no single adjuvant is capable of eliciting all the protective immune responses required in many malarial subunit vaccines and the use of combination adjuvants will be increasingly important as the science of malaria vaccines advances. In light of this, it appears that identifying the most effective mixture of adjuvants with minimal adverse effects offers tremendous opportunities in improving the efficacy of vaccines against malaria. Owing to the importance of a multi-adjuvanted approach in subunit malaria vaccine development, this review paper outlines some of the best known combination adjuvants used in malaria subunit vaccines, focusing on their proposed mechanisms of action, their immunological properties, and their notable results. The aim of the present review is to consolidate these findings to aid the application of these combination adjuvants in experimental malaria vaccines.     

Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence

Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.     

Expression of HBV surface antigen or HIV envelope protein using recombinant adenovirus vectors

Recombinant adenoviruses were constructed that contained either the HBsAg coding sequence or the HIV envelope protein coding sequence. The recombinant adenoviruses can replicate normally in cultured human cells. Cells infected with the adenovirus-HBV recombinant secreted HBsAg into the tissue culture medium. This HBsAg had immunological and physical properties similar to those of the 22-nm particles found in human serum. Expression of HIV envelope protein in cells infected with the adenovirus-HIV recombinant was demonstrated using cytoimmunofluorescence and immunoprecipitation. A hamster model was developed to evaluate the immunogenic properties of adenovirus-HBV recombinants. Hamsters inoculated intranasally with live adenovirus-HBV recombinant produced antibody against both adenovirus and hepatitis B virus surface antigen.     

Protective CD8+ T cell responses against the pre-erythrocytic stages of malaria parasites: an overview

CD8+ T cells have been implicated as critical effector cells in protection against the pre-erythrocytic stage of malaria in mice and humans following irradiated sporozoite immunization. Immunization experiments in animal models by several investigators have suggested different strategies for vaccination against malaria and many of the targets from liver stage malaria antigens have been shown to be immunogenic and to protect mice from the sporozoite challenge. Several prime/boost protocols with replicating vectors, such as vaccinia/influenza, with non-replicating vectors, such as recombinant particles derived from yeast transposon (Ty-particles) and modified vaccinia virus Ankara, and DNA, significantly enhanced CD8+ T cell immunogenicity and also the protective efficacy against the circumsporosoite protein of Plasmodium berghei and P. yeti. Based on these experimental results the development of a CD8+ T cell inducing vaccine has moved forward from epitope identification to planning stages of safety and immunogenicity trials of candidate vaccines.     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax duffy-binding protein

Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.     

Biochemical and immunological properties of a viral hybrid particle expressing the Plasmodium vivax merozoite surface protein 1 C-terminal region

BACKGROUND: Mammalian cells expressing the small hepatitis B virus surface protein (HBs) secrete highly immunogenic 20 nm lipoprotein particles. Previous studies demonstrated that the fusion of foreign sequences into certain regions of HBs leads to chimeric particles carrying epitopes for the foreign peptide, as well as for HBs. The present study investigates immunologic and biochemical properties of the fusion of the C-terminal region of the merozoite surface 1 protein of P. vivax, the most widely distributed human malaria parasite, and HBs (PvMSP1(19)-HBs). MATERIALS AND METHODS: COS7 cells were transfected with a plasmid coding for PvMSP1(19)-HBs. The hybrid products were analyzed by density gradient centrifugation and electron microscopy or detected by metabolic labeling and immunoprecipitation with anti-HBs and patient-derived anti-P. vivax serum. Mice were immunized with the vector and the antibody response was checked by ELISA. RESULTS: The fusion PvMSP1(19)-HBs formed particles of 20-45 nm size, which were secreted from COS7 cells. The particles were immunoprecipitable with anti-HBs and serum of different P. vivax-positive individuals. Immunization of mice with the construct as a genetic vaccine showed that antibodies were raised mostly against the PvMSP1(19) domain and recognized the native protein. CONCLUSION: Due to its biochemical and antigenic properties, the hybrid particle will be useful in future vaccine trials against the asexual blood stages of P. vivax as a genetic and/or a proteic subunit candidate.     

Antibody response of naturally infected individuals to recombinant Plasmodium vivax apical membrane antigen-1

In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43-487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.     

Expression and Characterization of Hepatitis B Surface Antigen in Transgenic Potato Plants

Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.     

Aerosolized adenovirus-vectored vaccine as an alternative vaccine delivery method

Conventional parenteral injection of vaccines is limited in its ability to induce locally-produced immune responses in the respiratory tract, and has logistical disadvantages in widespread vaccine administration. Recent studies suggest that intranasal delivery or vaccination in the respiratory tract with recombinant viral vectors can enhance immunogenicity and protection against respiratory diseases such as influenza and tuberculosis, and can offer more broad-based generalized protection by eliciting durable mucosal immune responses. Controlled aerosolization is a method to minimize vaccine particle size and ensure delivery to the lower respiratory tract. Here, we characterize the dynamics of aerosolization and show the effects of vaccine concentration on particle size, vector viability, and the actual delivered dose of an aerosolized adenoviral vector. In addition, we demonstrate that aerosol delivery of a recombinant adenoviral vaccine encoding H1N1 hemagglutinin is immunogenic and protects ferrets against homologous viral challenge. Overall, aerosol delivery offers comparable protection to intramuscular injection, and represents an attractive vaccine delivery method for broad-based immunization campaigns.     

Immunogenicity of a recombinant protein containing the Plasmodium vivax vaccine candidate MSP1(19) and two human CD4+ T-cell epitopes administered to non-human primates (Callithrix jacchus jacchus)

One of the most promising vaccine candidates against the erythrocytic forms of malaria is the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)). As part of our studies aimed at the development of a Plasmodium vivax malaria vaccine, we characterized the immunogenic properties of a new bacterial recombinant protein containing the P. vivax MSP1(19) and two helper T-cell epitopes, the synthetic universal pan allelic DR epitope (PADRE) and a new internal MSP1 P. vivax epitope (DYDVVYLKPLAGMYK). We found that the recognition of His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE was as good as the recognition of His6MSP1(19) indicating that the presence of the T-cell epitopes PADRE and DYDVVYLKPLAGMYK did not modify the MSP1(19) epitopes recognized by human IgG. The recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE proved to be highly immunogenic in marmosets (Callithrix jacchus jacchus) when administered in incomplete Freund's adjuvant. However, when administered in other adjuvant formulations such as Quil A, CpG ODN 2006 or MPL/TDM, antibody titers to MSP1(19) were significantly lower. Among these three adjuvants, Quil A proved to be the most efficient one generating antibody titers significantly higher than the others. These results indicated that under the circumstances evaluated, adjuvants were key for the immunogenicity of the recombinant protein His6MSP1(19)-DYDVVYLKPLAGMYK-PADRE.     

Removing N-terminal sequences in pre-s1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine

Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.