Production of an antigenic C4(V3)6 multiepitopic HIV protein in bacterial and plant systems

A C4(V3)6 multiepitopic protein was designed in an effort to pursue broad immunization against the human immunodeficiency virus (HIV). This C4(V3)6 chimeric protein is based on sequences of gp120, including epitopes from the fourth conserved domain (C4) and six tandem repeats of the third variable domain (V3), which represent different HIV isolates. The histidine-tagged C4(V3)6 was subsequently over-expressed in a recombinant Escherichia coli strain, and purified by immobilized metal ion affinity chromatography. Expression of the C4(V3)6 in both tobacco and lettuce plants was also achieved with no toxic effects on plant growth as transgenic plants were phenotypically normal. Moreover, the functional C4(V3)6 protein showed HIV antigenic determinants. The implications of these findings on the development of a new low-cost HIV vaccine are discussed.     

Impact of epitope permutations in the antibody response of mice to a multi-epitope polypeptide of the V3 loop of human immunodeficiency virus type 1

Our group have produced in Escherichia coli and evaluated the immunogenicity of different multi-epitope polypeptides (MEPs) bearing one copy of V3 loop sequential B cell epitopes from several isolates of human immunodeficiency virus type 1 (HIV-1) gp120. One of these MEPs called TAB9 comprises the 15 central amino acids of the V3 loop from isolates LR150, JY1, RF, MN, BRVA and IIIB in this order. Antibodies against all V3 regions were elicited after immunization of rabbits, macaques and humans with TAB9. In contrast, mice immunized with this protein only developed antibodies against epitopes JY1, LR150 and MN in that order (JY1>LR150>MN>RF, BRVA, IIIB) resembling an immunodominant gradient from the N-terminus to the C-terminal portion of this construction. To assess what role the location of the V3 epitopes in TAB9 could play, we constructed the protein TAB16, by altering the position of V3 epitopes in TAB9 primary structure and compared the pattern of antibodies elicited by both MEPs in H-2(d) Balb/c mice. The MEP TAB16 elicited antibody titers comparable to that of the sera from mice immunized with TAB9. There were no statistical differences in antibody titers between both groups (P>0.05). JY1, LR150 and MN V3 epitopes were again immunodominant in mice immunized with TAB16 fusion protein. The highest antibody titers detected in both groups among V3 epitopes corresponded to JY1, now located at the C-terminus of the permuted chimera. Antibodies against V3 epitopes RF, BRVA and IIIB were again not detected. Additionally, the MN V3 epitope showed to be significantly more immunogenic in its new orientation in TAB16, possibly as a result of a higher degree of accessibility in the surface of the protein. The results of the present investigation strongly suggest that the sequential order or the intramolecular position of V3 epitopes inside the primary structure of TAB9 and TAB16 MEPs does not interfere with the global immunogenicity or with the hierarchy of immunodominance of these regions.     

Chloroplast expression of an HIV envelop-derived multiepitope protein: towards a multivalent plant-based vaccine

The development of a vaccine is still a priority in the fight against human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). Since conventional vaccine strategies have failed to provide a highly immunoprotective effect, approaches based on the rational design of vaccines composed of multiple HIV neutralizing epitopes have been proposed as potential vaccines. The aim of this study is to design a multiepitopic protein (Multi-HIV) carrying several neutralizing epitopes from both gp120 and gp41 as an effort to develop a new broad immunization scheme against HIV. This Multi-HIV was initially produced in a recombinant Escherichia coli strain either as a single protein or fused to glutathione-S-transferase. These proteins were purified by immobilized metal ion affinity chromatography and shown to be antigenic by positive reactivity in Western blot analyses using sera from HIV-positive patients for labeling. Since global immunization strategies are often limited by costs, platforms that require minimal processing are the priority in this field. Therefore, we explored the possibility of using transplastomic tobacco plants as an experimental model of a low cost plant-based vaccine against HIV. Transplastomic tobacco plants carrying the multi-HIV gene were developed and verified by PCR analyses. The expected Multi-HIV recombinant protein was localized in the chloroplast as proven first by confocal microscopy and subsequently by Western blot analysis. Tobacco-derived Multi-HIV protein was clearly able to evoke humoral responses in mice when orally administered without adjuvants. This report constitutes an effort to explore a new low-cost candidate that could have future implications on the development of affordable HIV vaccines.     

Pre-Clinical Development of a Recombinant,Replication-Competent Adenovirus Serotype 4 Vector Vaccine Expressing HIV-1 Envelope 1086 Clade C

Background

There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV-1 infection or limits in vivo viral replication. The objective of these studies is to develop a replication-competent, vaccine vector based on the adenovirus serotype 4 (Ad4) virus expressing HIV-1 envelope (Env) 1086 clade C glycoprotein. Ad4 recombinant vectors expressing Env gp160 (Ad4Env160), Env gp140 (Ad4Env140), and Env gp120 (Ad4Env120) were evaluated.

Methods

The recombinant Ad4 vectors were generated with a full deletion of the E3 region of Ad4 to accommodate the env gene sequences. The vaccine candidates were assessed in vitro following infection of A549 cells for Env-specific protein expression and for posttranslational transport to the cell surface as monitored by the binding of broadly neutralizing antibodies (bNAbs). The capacity of the Ad4Env vaccines to induce humoral immunity was evaluated in rabbits for Env gp140 and V1V2-specific binding antibodies, and HIV-1 pseudovirus neutralization. Mice immunized with the Ad4Env160 vaccine were assessed for IFNγ T cell responses specific for overlapping Env peptide sets.

Results

Robust Env protein expression was confirmed by western blot analysis and recognition of cell surface Env gp160 by multiple bNAbs. Ad4Env vaccines induced humoral immune responses in rabbits that recognized Env 1086 gp140 and V1V2 polypeptide sequences derived from 1086 clade C, A244 clade AE, and gp70 V1V2 CASE A2 clade B fusion protein. The immune sera efficiently neutralized tier 1 clade C pseudovirus MW965.26 and neutralized the homologous and heterologous tier 2 pseudoviruses to a lesser extent. Env-specific T cell responses were also induced in mice following Ad4Env160 vector immunization.

Conclusions

The Ad4Env vaccine vectors express high levels of Env glycoprotein and induce both Env-specific humoral and cellular immunity thus supporting further development of this new Ad4 HIV-1 Env vaccine platform in Phase 1 clinical trials.     

Infection-induced antibodies against the major outer membrane protein of Campylobacter jejuni mainly recognize conformational epitopes

The major outer membrane protein (MOMP) of Campylobacter jejuni is an abundant surface protein with a pore-forming function and may be a potential candidate for vaccine development. Despite the fact that MOMP is immunogenic and the recombinant MOMP (rMOMP) can be readily produced in Escherichia coli, the nature of the antibody response to MOMP during in vivo infection is not well understood. In this study, various methods involving detergent replacement and liposome reconstitution were used to refold rMOMP, and antibody responses to MOMP elicited in Campylobacter-colonized chickens were evaluated using sera from chickens either naturally or experimentally infected by C. jejuni. The results demonstrated that proteoliposomes restored the reactivity of rMOMP to rabbit antibodies elicited by native MOMP, indicating the recovery of native MOMP conformation by this refolding method. Importantly, sera from naturally or experimentally infected chickens reacted weakly with denatured rMOMP, but strongly with rMOMP reconstituted in proteoliposome, suggesting that the chicken antibody response to MOMP is predominantly directed against conformational epitopes. These observations provide direct evidence for conformation-dependent humoral responses to MOMP induced by Campylobacter infection, demonstrate that C. jejuni MOMP is immunogenic in its natural host and suggest that proteoliposomes may be potentially used for the evaluation of rMOMP-based vaccines.     

Glycoconjugate Vaccine Containing Escherichia coli O157:H7 O-Antigen Linked with Maltose-Binding Protein Elicits Humoral and Cellular Responses

Glycoconjugate is one of the most efficacious and safest vaccines against bacterial pathogens. Previous studies of glycoconjugates against pathogen E. coli O157:H7 focused more on the humoral responses they elicited. However, little was known about their cellular responses. In this study, we exploited a novel approach based on bacterial protein N-linked glycosylation system to produce glycoconjugate containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein and examined its humoral and cellular responses in BALB/c mice. The transfer of E. coli O157:H7 O-antigen to MBP was confirmed by western blot and MALDI-TOF MS. Mice injected with glycoconjugate O-Ag-MBP elicited serum bactericidal antibodies including anti-E. coli O157:H7 O-antigen IgG and IgM. Interestingly, O-Ag-MBP also stimulated the secretion of anti-E. coli O157:H7 O-antigen IgA in intestine. In addition, O-Ag-MBP stimulated cellular responses by recruiting Th1-biased CD4⁺ T cells, CD8⁺ T cells. Meanwhile, O-Ag-MBP induced the upregulation of Th1-related IFN-γ and downregulation of Th2-related IL-4, and the upregulation of IFN-γ was stimulated by MBP in a dose-dependent manner. MBP showed TLR4 agonist-like properties to activate Th1 cells as carrier protein of O-Ag-MBP. Thus, glycoconjugate vaccine E. coli O157:H7-specific O-Ag-MBP produced by bacterial protein N-linked glycosylation system was able to elicit both humoral and Th1-biased cellular responses.     

Changes in the immunogenic properties of soluble gp140 human immunodeficiency virus envelope constructs upon partial deletion of the second hypervariable region

Immunization of macaques with the soluble oligomeric gp140 form of the SF162 envelope (SF162gp140) or with an SF162gp140-derived construct lacking the central region of the V2 loop (DeltaV2gp140) results in the generation of high titers of antibodies capable of neutralizing the homologous human immunodeficiency virus type 1 (HIV-1), SF162 virus (Barnett et al. J. Virol. 75:5526-5540, 2001). However, the DeltaV2gp140 immunogen is more effective than the SF162gp140 immunogen in eliciting the generation of antibodies capable of neutralizing heterologous HIV-1 isolates. This indicates that deletion of the V2 loop alters the immunogenicity of the SF162gp140 protein. The present studies were aimed at identifying the envelope regions whose immunogenicity is altered following V2 loop deletion. We report that the antibodies elicited by the SF162gp140 immunogen recognize elements of the V1, V2, and V3 loops, the CD4-binding site, and the C1 and C2 regions on the homologous SF162 gp120. With the exception of the V1 and V2 loops, the same regions are recognized on heterologous gp120 proteins. Surprisingly, although a minority of the SF162gp140-elicited antibodies target the V3 loop on the homologous gp120, the majority of the antibodies elicited by this immunogen that are capable of binding to the heterologous gp120s tested recognize their V3 loops. Deletion of the V2 loop has two effects. First, it alters the immunogenicity of the V3 and V1 loops, and second, it renders the C5 region immunogenic. Although deletion of the V2 loop does not result in an increase in the immunogenicity of the CD4-binding site per se, the relative ratio of anti-CD4-binding site to anti-V3 loop antibodies that bind to the heterologous gp120s tested is higher in sera collected from the DeltaV2gp140-immunized animals than in the SF162gp140-immunized animals. Overall, our studies indicate that it is possible to alter the immunogenic structure of the HIV envelope by introducing specific modifications.     

Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain

Human immunodeficiency virus type 1 (HIV-1) subtype C infections are on the rise in Sub-Saharan Africa and Asia. Therefore, there is a need to develop an HIV vaccine capable of eliciting broadly reactive immune responses against members of this subtype. We show here that modified HIV envelope (env) DNA vaccines derived from the South African subtype C TV1 strain are able to prime for humoral responses in rabbits and rhesus macaques. Priming rabbits with DNA plasmids encoding V2-deleted TV1 gp140 (gp140TV1DeltaV2), followed by boosting with oligomeric protein (o-gp140TV1DeltaV2) in MF59 adjuvant, elicited higher titers of env-binding and autologous neutralizing antibodies than priming with DNA vaccines encoding the full-length TV1 env (gp160) or the intact TV1 gp140. Immunization with V2-deleted subtype B SF162 env and V2-deleted TV1 env together using a multivalent vaccine approach induced high titers of oligomeric env-binding antibodies and autologous neutralizing antibodies against both the subtypes B and C vaccine strains, HIV-1 SF162 and TV1, respectively. Low-level neutralizing activity against the heterologous South African subtype C TV2 strain, as well as a small subset of viruses in a panel of 13 heterologous primary isolates, was observed in some rabbits immunized with the V2-deleted vaccines. Immunization of rhesus macaques with the V2-deleted TV1 DNA prime/protein boost also elicited high titers of env-binding antibodies and moderate titers of autologous TV1 neutralizing antibodies. The pilot-scale production of the various TV1 DNA vaccine constructs and env proteins described here should provide an initial platform upon which to improve the immunogenicity of these subtype C HIV envelope vaccines.     

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein(NP) and extracellular domain of matrix protein 2(M2e) genes from the influenza A virus A/Beijing/30/95(H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34(PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP-and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD_(50) of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.     

融合表达小鼠IgG2b-Fc可增强人类免疫缺陷病毒1型Gag DNA疫苗的免疫原性

为阐明小鼠IgG2b-Fc对DNA疫苗免疫原性的增强作用,首先构建表达人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)CN54Gag基因的DNA疫苗及表达Gag与小鼠IgG2b-Fc融合基因的DNA疫苗,限制性酶切和DNA测序结果表明这两个疫苗均构建成功,蛋白免疫印迹结果也显示其正确表达。然后,利用上述DNA疫苗接种C57BL/6小鼠,比较两个DNA疫苗所诱导的特异性体液免疫反应和特异性细胞免疫反应。结果显示,融合表达小鼠IgG2b-Fc对特异性细胞免疫和体液免疫反应均有增强作用,但只有对特异性体液免疫反应的增强作用有统计学意义。     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements

Background

It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed.

Methods and Findings

Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge.

Conclusion

CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.     

Oral immunization with a lettuce-derived <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile toxin B subunit induces neutralizing antibodies in mice

Transgenic plants serve as attractive systems for the production and delivery of subunit vaccines, thus expression of an enterotoxigenic Escherichia coli (ETEC) antigen in an edible plant may lead to the development of a viable oral vaccine against cholera and ETEC diarrhea. In this study, expression of the heat labile toxin B subunit (LTB) from ETEC was performed in lettuce, and its immunological characterization was investigated. A total of 27 independent transgenic lines were established following Agrobacterium-mediated transformation. Selected lettuce lines were subjected to GM1-ELISA to confirm the proper quaternary structure of the LTB protein. Levels of accumulation of the pentameric LTB reached up to 0.05% of the total soluble protein (TSP) in T1 and T2 progenies of these lines. Oral immunization of Balb/c mice was conducted using three weekly doses of lettuce-derived LTB. This elicited specific and significant antibody responses in both serum and intestinal tissues. Moreover, mice immunized with lettuce-derived LTB showed diminished intestinal fluid accumulation following challenge with the cholera toxin. This study demonstrated that this plant-based vaccine may contribute to immunization practices against diarrheal diseases.     

A chemically defined synthetic vaccine model for HIV-1.

Multiple Ag peptide (MAP) system without the use of a protein carrier was used as a vaccine model in three species of animals. Synthetic peptides from the V3 region of the gp120 of IIIB, RF and MN HIV-1 isolates were used as the Ag. MAP consisting of various chain lengths, from 11 to 24 residues, were prepared in a monoepitope configuration containing four repeats of each individual peptide. In parallel, they were synthesized in a diepitope configuration adding at the carboxyl-terminus of the V3 peptides a conserved sequence, known to be a Th cell epitope of gp120. The antibody response elicited by the monoepitope constructs was species-dependent. Rabbits produced immunity against all nine peptides, whereas mice were strongly reactive mainly to the longest sequence of the IIIB isolate. The immune response of guinea pigs was intermediate to those of rabbits and mice. Diepitope MAPs were immunogenic in all three species and elicited significantly higher titers than those raised by the immunization with the monoepitope MAPs. The response was type specific; the high-titered antibodies were reactive mostly against the isolate from which the peptides were derived, with a small cross-reactivity in ELISA between IIIB and RF strains. The dominant antigenic site of the B cell epitope, IIIB sequence, was located at the amino and central part of the MAP and a sequence overlapping the putative V3 reverse-turn was particularly reactive with the raised antibodies. Moreover, sera from the immunized animals inhibited virus-dependent cell fusion. These results show that MAP, with a chemically defined structure and without the use of a protein carrier, can be potentially useful for the design of synthetic HIV-1 vaccine candidates.     

Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.     

TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4x. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines.     

Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs) specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6) by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein) and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90%) of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.     

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.     

Immunogenicity comparison of a multi-antigenic peptide bearing V3 sequences of the human immunodeficiency virus type 1 with TAB9 protein in mice.

The multiple antigenic peptide system (MAP) has been proposed as a novel and valuable approach for eliciting antibodies for peptides and developing synthetic vaccines. Multi-epitope polypeptides (MEP) have also been developed as an alternative to the recombinant approach for vaccines. The V3 loop from the HIV type 1 (HIV-1) external glycoprotein (gp120) contains the principal neutralization domain (PND). Antibodies against this region neutralize HIV-1 in vitro and in vivo. In this work, a novel presentation of di-epitope MAP was synthesized. A monomeric MAP carrying two identical JY1 V3 sequences as B-cell epitopes and the 830-843 region of tetanus toxoid as a T-helper cell epitope was synthesized. This basic structure was covalently linked to produce a four-JY1-branched homodimer (JY1-MAP4). Additionally, six different monomeric MAPs, bearing four copies of V3 from isolates LR150, JY1, RF, MN, BRVA and IIIB, were synthesized. These monomers were conveniently linked among themselves to produce homodimeric and heterodimeric MAPs of eight V3 branches (V3-MAP8). JY1-MAP8 elicited higher antibody titers in Balb/c mice than JY1-MAP4. The immunogenicity of two different, hexavalent V3-MAP8 mixtures and the MEP TAB9, which tandems the same six V3 sequences in a single molecule, were compared. The antibody response against the mixtures of the heterodimeric MAP showed a wider recognition pattern of the V3 region, while the homodimeric cocktail showed an intermediate pattern. Antibodies elicited by TAB9 recognized only the JY1, LR150 peptides. These results emphasize the influence of V3 epitope presentation upon the characteristics of the antibody response generated.     

In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.