Protein Boosting Research Articles

Abstract 472: Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors.

Abstract Cancer vaccines continue to be of global importance. In the HIV/SIV non-human primate models replicating adenovirus has shown promise as a vaccine vector. Priming with replicating recombinant host-range mutant adenovirus (Ad5hr) followed by envelope (Env) protein boosting, elicits strong cellular, humoral and mucosal immunity, and protection in non-human primates. Based on the results using Ad5hr in the HIV/SIV non-human primate models replicating Ad4 vaccines are being developed for HIV/AIDS, and Influenza. These vectors may also be of value for cancer. A limitation of replicating Ad vectors is the transgene carrying capacity which is about 3000 base pairs (bp). Using an Ad5hr encoding full-length single-chain HIVBal gp120 linked to rhesus CD4 D1D2 domains (rhFLSC; DeVico et al., PNAS 2007) we systematically deleted the genes encoding E4-orf1 to E4-orf4. The latter virus with a 1100 bp deletion expands the total carrying capacity to ∼4000 bp. Using plaque assays we were unable to determine any differences in viral progeny production that could be attributed to the deletions. We assessed the virus-host interactions by evaluating the cell cycle profile of infected cells over time. Here too no differences could be attributed to the deletions. The Ad replication cycle is divided into early and late phases with regard to viral DNA synthesis. Western blots confirmed that the parental and deleted viruses expressed similar transgene levels in a temporally regulated manner similar to Ad late proteins. Splenocytes and sera from mice vaccinated with the parental, the E4-deleted variants, or with an Ad5hr empty vector at 0 and 4 weeks were collected at week 6 and used to evaluate whether the deletions altered transgene or vector immunogenicity. Levels of Env-specific cytokines responses by T-cells were negligible for mice immunized with the empty vector yet positive for those immunized with the parental and E4-deleted viruses. Ad responses were similar in all 6 groups. Interestingly, while the 5 immunization groups yielded similar binding antibody titers against rhFLSC and gp120 the sera showed differential binding patterns for Ad antigens. Thus while little to no host-cell or cellular immunogenicity differences distinguish the E4 deleted viruses from the parent, the differential binding patterns for Ad antigens opens the possibility that the E4 gene products may influence Ad antigen recognition. These differences may prove useful in the design of replicating Ad vaccine vectors. Citation Format: Michael A. Thomas, Rui Song, Marjorie Robert-Guroff. Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus vaccine vectors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 472. doi:10.1158/1538-7445.AM2013-472

DETERMINATION OF THE CHROMIUM CONTENT IN SOME SELECTED VEGETABLES IN SOUTH‐WEST ZONE, NIGERIA

Chromium is necessary for insulin function and aids nutrient uptake by cells, regulation of blood sugar, moderation of serum cholesterol levels, contribution to leans muscles mass, enhancement of protein synthesis and boosting of immune system amongst many others.Vegetables constitute the cheapest source of essential trace element for the majority of people living in developing countries. The main objective of this work is to determine the chromium content in twelve selected common vegetables in Southwestern NigeriaChromium concentration was determined in the fresh vegetables and the vegetable extracts. Result indicated that in the fresh samples, the level of chromium range from 2.3750×10−4 ± 3.0923×10−5 mg/L to 5.8×10−4 ± 4.6904×10−5 mg/L while the chromium content in vegetable extracts range from 1.2868×10−5 ± 9.704×10−6 mg/L to 3.650×10−5 ± 1.2868×10−6mg/L respectively. A notable difference was observed in the concentration of chromium, as the fresh samples were significantly higher in chromium content when compared with the extract (juice) vegetable samples. The level of chromium was highest in Vernonia amygdalina juice extracts while Curdoscololus aconilofolius fresh samples contained the highest level of chromium among the fresh samples. The recommended dietary daily allowance of chromium according to the United Nation is 50–200ug which shows that the vegetables studied are all good sources. Eating these vegetables will therefore be beneficial most especially to those with insulin resistance, stroke, cardiovascular and cancer disease.

Priming with DNA encoding functional trimers and boosting with soluble trimeric protein elicited tier 2 neutralizing antibodies in non-human primates

Background All known HIV-1 gp120-directed broadly neutralizing antibodies efficiently recognize fully cleaved JR-FL spikes, however, the non-neutralizing gp120-directed antibodies only recognize the uncleaved JR-FL spikes. Therefore, as an immunogen, cleaved, functional spikes may selectively present neutralizing epitopes to B cells, perhaps more efficiently eliciting neutralizing antibodies. However, attempts to make soluble versions of Env that fully mimic the viral spike are yet to be successful. In vitro, this plasmid encodes for fully cleaved, cell-surface, trimeric JR-FL Env spikes, but only when expressed from a mini-LTR in a non-codon optimized state. In vivo, the LTR-driven, non-codon-optimized Env plasmid DNA, likely requires tat co-transfection in trans, and rev expression in cis, for efficient Env expression. Methods To present functional, cleaved spikes to the immune system, we inoculated non-human primates (NHPs) with non-codon optimized JR-FL Env expressing plasmid DNA along with plasmid expressing Tat protein by electroporation. DNA priming was followed by boosts with soluble JR-FL gp140 trimers in adjuvant. As controls codon-optimized and CMV-driven Env plasmids were used to prime and three animals received protein in adjuvant only. Results We report that the DNA priming elicited reasonable ELISA binding titers in NHPs and that the elicitation of neutralizing antibodies against Tier 1 viruses. Following trimer protein boosting of the DNA-primed animals, reasonable titers of Tier 2 neutralizing antibodies in the A3R5 assay were elicited. For the Tier 2 isolates tested to date, much of the neutralizing activity in sera maps to the CD4 binding site. Conclusion

Broadly neutralizing immune responses against hepatitis C virus induced by vectored measles viruses and a recombinant envelope protein booster.

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.

Motif-optimized subtype A HIV envelope-based DNA vaccines rapidly elicit neutralizing antibodies when delivered sequentially

HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants.

455. Enhanced DNA Vaccine with Protein Boost Delivered by EP Expands B and T Cell Responses and Neutralizing Phenotype In Vivo

455. Enhanced DNA Vaccine with Protein Boost Delivered by EP Expands B and T Cell Responses and Neutralizing Phenotype In Vivo

662. Broad Neutralizing Immune Responses Against Hepatitis C Virus Induced by Vectored Measles Virus and a Recombinant Envelope Protein Boost

662. Broad Neutralizing Immune Responses Against Hepatitis C Virus Induced by Vectored Measles Virus and a Recombinant Envelope Protein Boost

Immune Responses Induced by Heterologous Boosting of Recombinant Bacillus Calmette-Guerin with Ag85B-ESAT6 Fusion Protein in Levamisole-Based Adjuvant

In the present study, we evaluated the effectiveness of a levamisole-based adjuvant (ADL) to enhance the ability of the Ag85B-ESAT6 fusion protein to boost immune responses after primary vaccination with recombinant bacillus Calmette-Guerin (rBCG) in Balb/c mice. The results were compared with that of the control adjuvant formulation of dimethyl dioctadecylammonium bromide (DDA) and monophosphoryl lipid A (MPL), which has previously been shown to induce T-helper type 1 (Th1)-biased responses. Enzyme-linked immunospot (ELISPOT) assay with Ag85B and ESAT6 derived peptides corresponding to CD4+ and CD8+ T cell restricted epitopes and cell surface immunostaining indicated that Ag85B-ESAT6/ADL predominantly triggered activation of CD4+ T cells. Functional CD8+ T cells with interferon (IFN)-γ production or cytotoxicity were undetectable all vaccinated mice. The ADL adjuvant modified T-helper (Th) subtypes by up-regulating multiple signature cytokines. Furthermore, profiles of the immunoglobulin G (IgG) subtypes indicated ADL enhanced the secretion of Th1-associated IgG2a antibodies and decreased the yield of Th2-associated IgG1 subtype. These observations suggest that the ADL adjuvant formulated with a protein booster may induce Th1-biased cellular and humoral immune responses to primary vaccination with a live attenuated bacterial TB vaccine.

Replicating Adenovirus-Simian Immunodeficiency Virus (SIV) Recombinant Priming and Envelope Protein Boosting Elicits Localized, Mucosal IgA Immunity in Rhesus Macaques Correlated with Delayed Acquisition following a Repeated Low-Dose Rectal SIV mac251 Challenge

We have shown that sequential replicating adenovirus type 5 host range mutant human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) recombinant priming delivered first intranasally (i.n.) plus orally and then intratracheally (i.t.), followed by envelope protein boosting, elicits broad cellular immunity and functional, envelope-specific serum and mucosal antibodies that correlate with protection from high-dose SIV and simian/human immunodeficiency virus (SHIV) challenges in rhesus macaques. Here we extended these studies to compare the standard i.n./i.t. regimen with additional mucosal administration routes, including sublingual, rectal, and vaginal routes. Similar systemic cellular and humoral immunity was elicited by all immunization routes. Central and effector memory T cell responses were also elicited by the four immunization routes in bronchoalveolar lavage fluid and jejunal, rectal, and vaginal tissue samples. Cellular responses in vaginal tissue were more compartmentalized, being induced primarily by intravaginal administration. In contrast, all immunization routes elicited secretory IgA (sIgA) responses at multiple mucosal sites. Following a repeated low-dose intrarectal (i.r.) challenge with SIV(mac251) at a dose transmitting one or two variants, protection against acquisition was not achieved except in one macaque in the i.r. immunized group. All immunized macaques exhibited reduced peak viremia compared to that of controls, correlated inversely with prechallenge serum antienvelope avidity, antibody-dependent cellular cytotoxicity (ADCC) titers, and percent antibody-dependent cell-mediated viral inhibition. Both antibody avidity and ADCC titers were correlated with the number of exposures required for infection. Notably, we show for the first time a significant correlation of vaccine-induced sIgA titers in rectal secretions with delayed acquisition. Further investigation of the characteristics and properties of the sIgA should elucidate the mechanism leading to this protective effect.

Immune Responses in Wild-type Mice Against Prion Proteins Induced Using a DNA Prime-Protein Boost Strategy

Objective To break immune tolerance to prion (PrP) proteins using DNA vaccines. Methods Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests. Results Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response. Conclusion Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.

Strong viremia control in vaccinated macaques does not prevent gradual Th17 cell loss from central memory

It has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributed to Th17 and Tc17 cells. Th17 cells can be infected with HIV/SIV, however whether effective vaccination leads to their maintenance following viral challenge has not been addressed. Here we retrospectively investigated if a vaccine regimen that potently reduced viremia post-challenge preserved Th17 and Tc17 cells, thus adding benefit in the absence of sterilizing protection. Rhesus macaques were previously vaccinated with replication-competent Adenovirus recombinants expressing HIV tat and HIV env followed by Tat and gp140 protein boosting. Upon SHIV 89.6P challenge, the vaccines exhibited a significant 4 log reduction in chronic viremia compared to sham vaccinated controls which rapidly progressed to AIDS [39]. Plasma and cryopreserved PBMC samples were examined pre-challenge and during acute and chronic infection. Control macaques exhibited a rapid loss of CD4 + cells, including Th17 cells. Tc17 cells tended to decline over the course of infection although significance was not reached. Immune activation, assessed by Ki-67 expression, was associated with elevated chronic viremia of the controls. Significantly increased plasma IFN-γ levels were also observed. No increase in plasma LPS levels were observed suggesting a lack of microbial translocation. In contrast, vaccinated macaques had no evidence of immune activation within the chronic phase and preserved both CD4 + T-cells and Tc17 cells in PBMC. Nevertheless, they exhibited a gradual, significant loss of Th17 cells which concomitantly displayed significantly higher CCR6 expression over time. The gradual Th17 cell decline may reflect mucosal homing to inflammatory sites and/or slow depletion due to ongoing low levels of SHIV replication. Our results suggest that potent viremia reduction during chronic SHIV infection will delay but not prevent the loss of Th17 cells.

Vaccination with DNA vector expressing chlamydial low calcium response protein E (LcrE) against Chlamydophila pneumoniae infection

Chlamydophila pneumoniae is an obligate intracellular human pathogen, which causes acute respiratory tract infections and can also cause chronic infections. C. pneumoniae possess type III secretion system (TTSS), which allows them to secrete effector molecules into the inclusion membrane and the host cell cytosol. Low calcium response protein E (LcrE) is a part of TTSS. The gene of LcrE in a 6His-tagged form was cloned from C. pneumoniae CWL029, expressed and purified from Escherichia coli using the HIS-select TALON CellThru Resin, this gene was also cloned into a eukaryotic expression vector (pΔRC). One group of BALB/c mice received an intramuscular pΔRC inoculation then the mice were immunized with purified LcrE protein; the second group of mice was immunized two times with the recombinant plasmid (pΔRCLcrE), and the third group was primed with pΔRCLcrE inoculation then boosted with LcrE protein. LcrE-specific antibody response was induced by DNA immunization with a shift towards Th1 isotype pattern compared to protein-immunization, this shifting pattern was observed in plasmid primed then protein-boosted animals. DNA immunization given as a priming and followed by a protein booster significantly reduced the number of viable bacteria in the lungs after challenge with C. pneumoniae. These results confirm that immunization with pΔRCLcrE can be an effective part of a vaccination schedule against C. pneumoniae.

A Trimeric, V2-Deleted HIV-1 Envelope Glycoprotein Vaccine Elicits Potent Neutralizing Antibodies but Limited Breadth of Neutralization in Human Volunteers

A key missing element in the development of a successful human immunodeficiency virus (HIV) vaccine is an immunogen that can generate broadly cross-neutralizing antibodies against primary isolates of the virus. This phase 1 clinical trial employed a DNA prime and subunit envelope protein boost in an attempt to generate cellular and humoral immune responses that might be desirable in a protective HIV vaccine. Priming was performed via intramuscular injection with gag and env DNA adsorbed to polylactide coglycolide microspheres, followed by boosting with a recombinant trimeric envelope (Env) glycoprotein delivered in MF59 adjuvant. The DNA prime and protein boost were generally safe and well-tolerated. Env-specific CD4(+) cellular responses were generated that were predominantly detected after Env protein boosting. Neutralizing antibody responses against the homologous SF162 viral isolate were remarkably strong and were present in the majority of vaccine recipients, including a strong response against CD4-induced epitopes on gp120. Despite the promising potency of this vaccine approach, neutralization breadth against heterologous tier 2 strains of HIV-1 was minimal. Potent neutralization against neutralization-sensitive strains of HIV is achievable in humans through a DNA prime, recombinant oligomeric Env protein boost regimen. Eliciting substantial breadth of neutralization remains an elusive goal. NCT00073216.

Induction of mucosal and systemic antibody and T-cell responses following prime-boost immunization with novel adjuvanted human immunodeficiency virus-1-vaccine formulations

As sexual transmission of human immunodeficiency virus-1 (HIV-1) occurs via the mucosa, an ideal HIV-1 vaccine should induce both mucosal and systemic immunity. We therefore sought to evaluate the induction of mucosal responses using a DNA env prime–gp120 protein boost approach in which sequential nasal and parenteral protein administration was performed with two novel carbohydrate-based adjuvants. These adjuvants, Advax-M and Advax-P, were specifically designed for mucosal and systemic immune enhancement, respectively. Murine intranasal immunization with gp120/Advax-M adjuvant elicited gp120-specific IgA in serum and mucosal secretions that was markedly enhanced by DNA priming. Boosting of DNA-primed mice with gp120/Advax-M and gp120/Advax-P by sequential intranasal and intramuscular immunization, or vice versa, elicited persistent mucosal gp120-specific IgA, systemic IgG and memory T- and B-cell responses. Induction of homologous, but not heterologous, neutralizing activity was noted in the sera of all immunized groups. While confirmation of efficacy is required in challenge studies using non-human primates, these results suggest that the combination of DNA priming with sequential nasal and parenteral protein boosting, with appropriate mucosal and systemic adjuvants, could generate strong mucosal and systemic immunity and may block HIV-1 mucosal transmission and infection.

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

P04-24. Frequencies of HIV Env-specific B cells and neutralizing antibodies during DNA-prime/protein-boost immunization with three different Env immunogens*

gp140, 0.44% (0.35–0.52%) for the gp120, and 0.35% (0.13–0.56%) for the D368R gp140 immunized animals. However, no significant correlations can be drawn between the titer of Env specific antibodies and frequency of Env specific B cells in any group. The wt-gp140 was a more effective immunogen than m-gp120 in eliciting weak cross-NAbs. Interestingly, the wt-gp140 was also more effective in eliciting cross-NAbs than the D368Rgp140; potentially suggesting that the development of cross-NAbs by wt-gp140 depends on the structural integrity of the CD4-binding site. Conclusion Protein boosting is required for the development of high frequencies of anti-HIV Env antibodies, and ability of trimeric gp140 to elicit cross-NAbs will depend on the structural integrity of the CD4-binding site.

Immune responses to polyethylenimine-mannose-delivered plasmid DNA encoding aFasciola giganticafatty acid binding protein in mice

Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an immune response in mice, by delivering the gene coding for this protein with mannosylated-polyethylenimine (PEI) to peritoneal cells. Mice were immunized with 50 microg recombinant plasmid DNA (Group I) or DNA-PEI-mannose (a 22 kDa linear cationic polymer with mannose ligand) (Group II) via the intraperitoneal route. Antibody studies showed no significant humoral immune response evoked to this DNA immunization with either PEI-mannose-delivered or naked DNA. However, on protein boosting of these DNA-primed mice there was a significant enhancement of antibody titre. Flow cytometric bead array was used to measure quantities of interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) cytokines. Overexpression of T-helper 1 (Th1) cytokines such as IFN-gamma and TNF, with a lower but significant expression of the T-helper 2 (Th2) cytokine IL-5 was detected. Gene delivery using polyethylenimine-mannose ligand showed significant expression of IFN-gamma and TNF (P < 0.05), but no significant difference in IL-2, IL-4 and IL-5 (P>0.05) cytokine expression was observed between naked-DNA- and mannosylated PEI-DNA-delivered mice. Naked- or PEI-delivered-DNA immunization produced insignificant levels of IL-2 and IL-4 (P>0.05) cytokines in both groups of mice.

Improved efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by introduction of a novel adjuvant epitope

DNA vaccine represents an attractive approach for cancer treatment by inducing active immune-deprivation of gastrin-releasing peptide (GRP) from tumor cells, the growth of which is dependent on the stimulation of GRP. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen of six copies of the B cell epitope GRP 18–27 (GRP6). In order to increase the potency of this DNA vaccine, multiple strategies have been applied including DNA-prime protein-boost immunization and introduction of a foreign T-helper epitope into DNA vaccine. Mice vaccinated DNA vaccine boosting with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies as well as inhibition effect on the growth of murine prostate carcinoma, superior to the treatment using DNA alone or BCG priming HSP65-GRP6 protein boosting. Furthermore, the introduction of a novel foreign T-helper epitope into the GRP DNA vaccine showed a markedly stronger humoral immune response against GRP and tumor rejection even than the DNA-prime protein-boost strategy. No further stronger immunogenicity of this foreign T-helper epitope modified DNA vaccine was observed even using the strategy of modified DNA vaccine-priming and HSP65-GRP6 boosting method. The data presented demonstrate that improvement of potency of anti-GRP DNA vaccine with the above two feasible approaches should offer useful methods in the development of new DNA vaccine against growth factors for cancer immunotherapy.

Improved immunogenicity and protective efficacy of a replicon DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A by electric pulses and protein boosting

Vaccination by intramuscular injection of naked DNA is very efficient in mouse model, but immunogenicity of DNA vaccines needs to be improved in human use. Thus, we wanted to determine whether suitable electric pulses-mediated DNA delivery technology and DNA prime-protein boost regimen could improve the immunogenicity and protective efficacy of the replicon DNA vaccine pSCARSHc in mouse model. In this study, the immunogenicity and protective efficacy of the replicon DNA vaccine pSCARSHc following electric pulses were dramatically improved in Balb/c mice. Also, priming of the immune response by DNA vaccination followed by a single booster with AHc protein immunogen resulted in very high levels of ELISA and neutralization antibodies and afforded more efficient protection against botulinum neurotoxin serotype A. Therefore, these methods described here potentially provide suitable strategies in developing an efficacious vaccine against Clostridium botulinum neurotoxin serotype A.

Mucosal Adjuvanticity of a Shigella Invasin Complex with DNA-Based Vaccines

Protection against many infectious diseases may require the induction of cell-mediated and mucosal immunity. Immunization with plasmid DNA-based vaccines has successfully induced cell-mediated immune responses in small animals but is less potent in humans. Therefore, several methods are under investigation to augment DNA vaccine immunogenicity. In the current study, a mucosal adjuvant consisting of an invasin protein-lipopolysaccharide complex (Invaplex) isolated from Shigella spp. was evaluated as an adjuvant for DNA-based vaccines. Coadministration of plasmid DNA encoding the Orientia tsutsugamushi r56Karp protein with Invaplex resulted in enhanced cellular and humoral responses in intranasally immunized mice compared to immunization with DNA without adjuvant. Mucosal immunoglobulin A, directed to plasmid-encoded antigen, was detected in lung and intestinal compartments after Invaplex-DNA immunization followed by a protein booster. Moreover, immunization with Invaplex elicited Shigella-specific immune responses, highlighting its potential use in a combination vaccine strategy. The capacity of Invaplex to enhance the immunogenicity of plasmid-encoded genes suggested that Invaplex promoted the uptake and expression of the delivered genes. To better understand the native biological activities of Invaplex related to its adjuvanticity, interactions between Invaplex and mammalian cells were characterized. Invaplex rapidly bound to and was internalized by nonphagocytic, eukaryotic cells in an endocytic process dependent on actin polymerization and independent of microtubule formation. Invaplex also mediated transfection with several plasmid DNA constructs, which could be inhibited with monoclonal antibodies specific for IpaB and IpaC or Invaplex-specific polyclonal sera. The cellular binding and transport capabilities of Invaplex likely contribute to the adjuvanticity and immunogenicity of Invaplex.