Potent effect of KISS1-54 DNA vaccine compared with KISS1-10 DNA vaccine in inhibiting the fertility of female rats

BackgroundMost studies on immunocastration currently focused on male animals. However, immunization of male animals does not completely inhibit sexual behavior and fertility. This study aimed to compare the immunocastration effect of KISS1 DNA vaccines encoding different lengths of kisspeptins in female rats for effective castration effects on both male and female rats.MethodsFifteen female rats were randomly divided into three groups. The rats in T1 group or T2 group was orally given respectively KISS1-54 or KISS1-10 DNA vaccines with fused tPA signal peptide, and the control group (Group C) was orally administered with empty vector vaccine, at a dose of 5 × 10⁹ CFU/rat at weeks 0, 3 and 6 of the study. Blood samples were collected by retroorbital bleeding before primary immunization and at weeks 3 and 9 after primary immunization.ResultsBoth KISS1-54 and KISS1-10 DNA vaccines induced the body’s humoral immune response, and the anti-kisspeptin antibody titres in the T1 group were significantly higher than that in T2 and C groups (p < 0.05). The rats in T1 group has lower serum kisspeptin and estradiol levels than those in T2 and C groups and smaller litter size of rats than those in the control group after mating (p < 0.05). No significant difference was observed between T2 and C groups. The levels of KISS1 and GPR54 mRNA in the hypothalamus and ovaries of the T1 group were significantly lower than that in control group. However, the levels of KISS1 mRNA in the T2 group were significantly lower than that in the control group only in ovaries (p < 0.05).ConclusionThe oral KISS1-54 DNA vaccine with fused tPA signal peptide was more effective than that KISS1-10 DNA vaccine in suppressing fertility of female rats.     

Design,synthesis and evaluation of a gonadotropin releasing hormone-based subunit vaccine in rams (Ovis aries)

Immunocastration using gonadotropin-releasing hormone (GnRH)-based vaccines has been investigated in rams to reduce aggressive and sexual behaviour and to control meat quality. Despite considerable efforts, a practical GnRH vaccine has yet to be developed for rams. In the present study, a A GnRH-lipopeptide vaccine (GnRH-LP) including two copies of GnRH, 2-amino-d,l-hexadecanoic acid (C16), and a unique T helper epitope, was examined in rams. Rams received a primary and secondary vaccination of GnRH-LP without additional adjuvant (Group 1) or with the adjuvant AdjuVac™ (Group 2). In both Group 1 and 2 anti-GnRH antibody titres increased after secondary vaccination, however, the antibody titres were higher (p < 0.01) for rams in Group 2. The latter rams showed a marked decrease in testicular size. The marked and sustained reduction in testicular size in rams treated with GnRH-LP + AdjuVac™ provides the basis for an effective immunocastration vaccine in rams.     

An optimized SIV DNA vaccine can serve as a boost for Ad5 and provide partial protection from a high-dose SIVmac251 challenge

One limitation in the development of an improved cellular response needed for an effective HIV-vaccine is the inability to induce robust effector T-cells capable of suppressing a heterologous challenge. To improve cellular immune responses, we examined the ability of an optimized DNA vaccine to boost the cellular immune responses induced by a highly immunogenic Ad5 prime. Five Chinese rhesus macaques received pVax encoding consensus (con) gag/pol/env intramuscularly (IM) with electroporation followed by the Merck Ad5 gag/pol/nef vaccine. A second group of five animals were vaccinated with Merck Ad5 gag/pol/nef followed by pVax gag/pol/env. One year following vaccination, Ad5-prime DNA-boosted monkeys and four unvaccinated controls received an intrarectal challenge with 1000 ID50 SIV(mac)251. The quality and magnitude of the T-cell response was analyzed by ELISpot and polyfunctional flow cytometry. We observed that an Ad5-prime DNA-boost resulted in significantly elevated SIV-specific T-cell responses even compared with animals receiving a DNA-prime Ad5-boost. Ad5 prime DNA boosted animals were capable of suppressing a pathogenic SIV(mac)251 challenge. Peak control correlated with the expansion of HLA-DR(+) CD8(+) T-cells two weeks post-infection. These data illustrate that high optimization of a DNA vaccine can drive of immune responses primed by a robust vector system. This previously unachievable feature of these newly optimized DNAs warrants future studies of this strategy that may circumvent issues of serology associated with viral vector prime-boost systems.     

Co-administration of certain DNA vaccine combinations expressing different H5N1 influenza virus antigens can be beneficial or detrimental to immune protection

Achieving broad-spectrum immunity against emerging zoonotic viruses such as avian influenza H5N1 and other possible pandemic viruses will require generation of cross-protective immune responses. Strong antibody responses generated against the H5HA protein are protective, however, antigenic variation between diverging isolates can interfere with virus neutralization. The current study investigates co-administration of an H5 HA DNA vaccine with other variable and conserved influenza antigens (NA, NP, and M2). All antigens were derived from the A/Hanoi/30408/2005 (H5N1) virus and the contribution towards overall protection and immune activation was assessed against lethal homologous and heterologous challenges. An (HA + NA) combination afforded the best protection against homologous challenge and (HA + NP) was comparable to HA alone against heterologous A/Hong Kong/483/1997 challenge. Interestingly, combining all four H5 antigens at a single site did not improve protection against matched challenge and unexpectedly reduced survival by 30% against a heterologous challenge. Survival was also significantly decreased against heterologous challenge following combination of (HA + NP) with an unrelated antigen. Although there were no significant changes in antibody titres, significantly lower T-cell responses were detected against all antigens except HA in each combination. Co-administration of the vaccines at different injection sites restored T-cell responses but did not improve overall protection. Similar observations were also recorded following combination of HA and NP antigens using two different adenovirus-based backbones. Overall, the data suggest that co-administering certain H5N1 antigens offer better or comparable protection to HA alone, however, combining extra antigens may be unnecessary and lead to unfavourable immune responses.     

Immunization of cattle with a BHV1 vector vaccine or a DNA vaccine both coding for the G protein of BRSV

A gE-negative bovine herpesvirus 1 (BHV1) vector vaccine carrying a gene coding for the G protein of bovine respiratory syncytial virus (BRSV) BHV1/BRSV-G) induced the same high degree of protection in calves against BRSV infection and BHV1 infection as a multivalent commercial vaccine. A DNA plasmid vaccine, carrying the same gene as the BHV1/BRSV-G vaccine, significantly reduced BRSV shedding after BRSV infection compared with that in control calves, but less well than the BHV1/BRSV-G vaccine. Flow cytometric analysis showed a significant relative increase of γ/δ⁺ T cells in peripheral blood after BRSV challenge-infection of the calves of the control group but not in the vaccinated groups. These results indicate that the G protein of BRSV can induce significant protection against BRSV infection in cattle, and that the BHV1/BRSV-G vaccine protects effectively against a subsequent BRSV and BHV1 infection.     

Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (>1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination.     

一种CpG佐剂增强HIV-1DNA疫苗免疫原性的研究

目的以一种CpG寡聚核苷酸为HIV-1DNA疫苗候选佐剂,研究该CpG佐剂增强DNA疫苗免疫原性,体外促进DC细胞成熟等特点。方法在Balb/c小鼠模型上连续3次联合免疫HIV-1DNA疫苗及CpG佐剂,通过IFN-γ、IL-2ELISPOT及ELISA检测HIV特异性细胞免疫反应及体液免疫应答强度;体外制备小鼠骨髓来源的树突状细胞,通过FACS技术、高通量细胞因子检测等方法评价CpG佐剂刺激活化DC的能力。结果 CpG能够增强HIV-1DNA疫苗诱导的特异性细胞免疫反应水平,降低DNA疫苗使用剂量;CpG体外刺激原代小鼠骨髓来源的树突状细胞(BMDC),能显著上调CD40、CD80、CD86等BMDC表面共刺激分子的表达,活化BMDC并分泌各型细胞因子IL-5、IL-12p70,促炎症因子IL-1α、IL-1β、IL-6、IL-10、MIP-2、KC、MIG、Eotaxin、GM-CSF等以发挥佐剂效应。结论综合体内体外实验数据,证实该型CpG能够充分活化BMDC,显著提高HIV-1DNA疫苗免疫原性,降低疫苗使用剂量,可成为HIV-1DNA疫苗临床试验用候选佐剂。     

Immunogenicity of novel nanoparticle-coated MSP-1 C-terminus malaria DNA vaccine using different routes of administration

An important aspect in optimizing DNA vaccination is antigen delivery to the site of action. In this way, any alternative delivery system having higher transfection efficiency and eventual superior antibody production needs to be further explored. The novel nanoparticle, pDNA/PEI/γ-PGA complex, is one of a promising delivery system, which is taken up by cells and is shown to have high transfection efficiency. The immunostimulatory effect of this novel nanoparticle (NP) coated plasmid encoding Plasmodium yoelii MSP1-C-terminus was examined. Groups of C57BL/6 mice were immunized either with NP-coated MSP-1 plasmid, naked plasmid or NP-coated blank plasmid, by three different routes of administration; intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c). Mice were primed and boosted twice at 3-week intervals, then challenged 2 weeks after; and 100%, 100% and 50% mean of survival was observed in immunized mice with coated DNA vaccine by i.p., i.v. and s.c., respectively. Coated DNA vaccine showed significant immunogenicity and elicited protective levels of antigen specific IgG and its subclass antibody, an increased proportion of CD4⁺ and CD8⁺ T cells and INF-γ and IL-12 levels in the serum and cultured splenocyte supernatant, as well as INF-γ producing cells in the spleen. We demonstrate that, NP-coated MSP-1 DNA-based vaccine confers protection against lethal P. yoelii challenge in murine model across the various route of administration and may therefore, be considered a promising delivery system for vaccination.     

Development of a DNA vaccine for chicken infectious anemia and its immunogenicity studies using high mobility group box 1 protein as a novel immunoadjuvant indicated induction of promising protective immune responses

Chicken infectious anaemia (CIA) is an economically important and emerging poultry disease reported worldwide. Current CIA vaccines have limitations like, the inability of the virus to grow to high titres in embryos/cell cultures, possession of residual pathogenicity and a risk of reversion to virulence. In the present study, a DNA vaccine, encoding chicken infectious anaemia virus (CIAV) VP1 and VP2 genes, was developed and co-administered with truncated chicken high mobility group box 1 (HMGB1ΔC) protein in young chicks for the evaluation of vaccine immune response. CIAV VP1 and VP2 genes were cloned in pTARGET while HMGB1ΔC in PET32b vector. In vitro expression of these gene constructs was evaluated by Western blotting. Further, recombinant HMGB1ΔC was evaluated for its biological activity. The CIAV DNA vaccine administration in specific pathogen free chicks resulted in moderately protective ELISA antibody titres in the range of 4322.87 ± 359.72 to 8288.19 ± 136.38, increased CD8⁺ cells, and a higher titre was observed by co-administration of novel adjuvant (HMGB1ΔC) and booster immunizations. The use of vaccine with adjuvant showed achieving antibody titres nearly 8500, titre considered as highly protective, which indicates that co-immunization of HMGB1ΔC may have a strong adjuvant activity on CIAV DNA vaccine induced immune responses. The able potential of HMGB1 protein holding strong adjuvant activity could be exploited further with trials with vaccines for other important pathogens for achieving the required protective immune responses.     

The antitumor immunopreventive effects of a DNA vaccine against CYP26a1 on mouse breast carcinoma

Background

CYP26a1, which functioned mainly as a retinoic acid (RA) catabolic enzyme, has been shown to be oncogenic and to support cell survival in many breast carcinoma cells.

Objectives

The purpose of the study was to investigate the antitumor effect of a DNA vaccine targeting CYP26a1 on breast tumors development in mice which highly express CYP26a1 and to further clarify its potential mechanism.

Methods

After three times immunization of the DNA vaccine, the BALB/c mice were inoculated with the engineered 4T1 breast cancer cells expressing CYP26a1. Primary tumors were measured every 4 days after tumor cell inoculation. The primary tumors were surgically removed and weighted after 30 days of inoculation. The anti-CYP26a1 antibody titer of the antiserum was measured by an enzyme-linked immunosorbent assay (ELISA). The effect of the vaccine on apoptosis of the primary tumor was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Apoptosis-related proteins in primary tumor were detected by Western blotting. The expression of the Th1 and Th2 type cytokines was detected by RT-PCR.

Results

The vaccine could elicit the production of anti-CYP26a1 antibody and significantly inhibit the growth of the primary tumor compared to the control groups (p < 0.05). The vaccine induced the apoptosis of the primary tumor with the increase in expression of apoptosis-related proteins p53, Caspase3 and Fas. Furthermore, the vaccine increased the expression of the Th1 cytokine, but not the expression of Th2 cytokine.

Conclusion

Our study shows that the vaccine targeting CYP26a1 significantly inhibits the primary tumor growth and progression by activating the apoptosis pathway and by eliciting both humoral and cellular immune responses.     

A DNA vaccine encoding the E protein of West Nile virus is protective and can be boosted by recombinant domain DIII

West Nile Virus (WNV) is an emerging pathogenic flavivirus with increasing distribution worldwide. Birds are the natural host of the virus, but also mammals, including humans, can be infected. In some cases, a WNV infection can be associated with severe neurological symptoms. All currently available WNV vaccines are in the veterinary sector, and there is a need to develop safe and effective immunization technologies, which can also be used in humans. An alternative to current vaccination methods is DNA immunization. Most current DNA vaccine candidates against flaviviruses simultaneously express the viral envelope (E) and membrane (prM) proteins, which leads to the formation of virus-like particles. Here we generated a DNA plasmid, which expresses only the E-protein ectodomain. Vaccination of mice stimulated anti-WNV T-cell responses and neutralizing antibodies that were higher than those obtained after immunizing with a recombinant protein previously shown to be a protective WNV vaccine. A single dose of the plasmid was sufficient to protect animals from a lethal challenge with the virus. Moreover, immunogenicity could be boosted when DNA injection was followed by immunization with recombinant domain DIII of the E-protein. This resulted in significantly enhanced neutralizing antibody titers and a more prominent cellular immune response. The results suggest that the WNV E-protein is sufficient as a protective antigen in DNA vaccines and that protection can be significantly improved by adding a recombinant protein boost to the DNA prime.     

Nucleic acid (DNA) immunization as a platform for dengue vaccine development

Since the early 1990s, DNA immunization has been used as a platform for developing a tetravalent dengue vaccine in response to the high priority need for protecting military personnel deployed to dengue endemic regions of the world. Several approaches have been explored ranging from naked DNA immunization to the use of live virus vectors to deliver the targeted genes for expression. Pre-clinical animal studies were largely successful in generating anti-dengue cellular and humoral immune responses that were protective either completely or partially against challenge with live dengue virus. However, Phase 1 clinical evaluation of a prototype monovalent dengue 1 DNA vaccine expressing prM and E genes revealed anti-dengue T cell IFNγ responses, but poor neutralizing antibody responses. These less than optimal results are thought to be due to poor uptake and expression of the DNA vaccine plasmids. Because DNA immunization as a vaccine platform has the advantages of ease of manufacture, flexible genetic manipulation and enhanced stability, efforts continue to improve the immunogenicity of these vaccines using a variety of methods.     

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

Background

DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion.

Methodology and principal findings

Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T_H1 (IgG2a) but not T_H2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses.

Conclusions

We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.     

Protective immune response against Toxoplasma gondii elicited by a recombinant DNA vaccine with a novel genetic adjuvant

Previous immunological studies from our laboratory have demonstrated the potential role of Toxoplasma gondii antigens SAG1 and GRA2 as vaccine candidates. To further evaluate the vaccine's effects, a series of recombinant DNA vaccines pVAX1-SAG1, pVAX1-GRA2 and pVAX1-SAG1-GRA2, termed pSAG1, pGRA2 and pSAG1-GRA2, respectively, were constructed. A plasmid pVAX1-S/PreS2, termed pSPreS2 encoding hepatitis B virus (HBV) surface antigen (HBsAg) S and PreS2 as a novel genetic adjuvant, was also constructed. The expression abilities of those DNA plasmids were examined in HFF cells by Western blotting. Then BALB/c mice were intramuscularly immunized with DNA plasmids and followed by challenging with the highly virulent T. gondii RH strain. The results demonstrated that the recombinant DNA vaccine pSAG1-GRA2 was capable of eliciting high levels of antibodies, a Th1 type of immune response with significant production of IFN-γ and low levels of IL-4 or IL-10 in BALB/c mice, and partial protection against the acute phase of toxoplasmosis as compared to pSAG1, pGRA2 and controls. In addition, the adjuvant pSPreS2 formulated with DNA vaccine induced a Th1 type of immune response and therefore might be a novel genetic adjuvant to DNA vaccine for further investigation.     

Effects of a novel recombinant Gonadotropin-Releasing Hormone-1 vaccine on the reproductive function of mixed-breed dogs (Canis familiaris) in Taiwan

Immunocastration is an effective alternative to surgical castration for controlling the population of animals. As gonadotropin-releasing hormone (GnRH) regulates the reproductive endocrine system in mammals, it is a target antigen for vaccine formulation. Through this study, we evaluated the effectiveness of a recombinant subunit GnRH-1 vaccine for the immunocastration of the reproductive function of 16 mixed-breed dogs (Canis familiaris) provided voluntarily by different households. All the dogs were deemed clinically healthy prior to and during the experiment. A specific anti-GnRH immune response was detected at Week 4, which was maintained for at least 24 weeks after vaccination. Moreover, decreased levels of sexual hormones (testosterone as well as progesterone and estrogen, respectively) were observed in both male and female dogs. Estrous suppression was apparent in female dogs, and testicular atrophy and poor semen quality (concentration, abnormality, and viability) were observed in male dogs. In conclusion, the recombinant subunit GnRH-1 vaccine could successfully suppress fertility and delay the estrous cycle in canines. These results support the efficacy of the recombinant subunit GnRH-1 vaccine; thus, it is a suitable candidate for fertility control in dogs.     

A novel DNA vaccine for reduction of PRRSV-induced negative immunomodulatory effects: A proof of concept

BackgroundViral-induced interleukin (IL)-10 and regulatory T lymphocytes (Tregs) are believed to play a major role in shaping the immunological and clinical outcomes following Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infection. Recently, it has been shown that PRRSV nucleocapsid (N) protein can induce IL-10 production which is essential for induction of PRRSV-specific Tregs. We hypothesized that immunity to N protein should reduce PRRSV-induced negative immunomodulatory effects which will be essential for establishing proper anti-PRRSV immunity in infected pigs.ObjectivesTo investigate the immunomodulatory effects of DNA vaccine encoding a linearized, truncated form of PRRSV-N protein (pORF7t) which was designed to preferentially induce cell-mediated immunity against PRRSV N protein.MethodImmunomodulatory effects of the novel DNA vaccine were investigated in an experimental vaccinated-challenged model. In addition, long-term immunomodulatory effects of the DNA vaccine were investigated in vaccinated pigs kept at the PRRSV-positive environment until the end of the fattening period. Pigs were vaccinated either prior to or following natural PRRSV infection.ResultThe results indicated that pORF7t could modulate the anti-PRRSV immune responses and promote the control of viral replication in the vaccinated-challenged pigs. Immunized pigs exhibited increased numbers of PRRSV-specific activated CD4⁺CD25⁺ lymphocytes, reduced numbers of PRRSV-specific Tregs, and rapid viral clearance following infection. In a long-term study, regardless of the time of vaccination, DNA vaccine could modulate the host immune responses, resulted in enhanced PRRSV-specific IFN-γ producing cells, and reduced numbers of PRRSV-specific Tregs, without evidence of enhanced antibody responses. No vaccine adverse reaction was observed throughout the study.ConclusionThis study revealed the novel concept that PRRSV-specific immunity can be modulated by induction of cell-mediated immunity against the nucleocapsid protein. This concept could potentially benefit the development of PRRSV management and control strategies.     

Comparative performance of a licensed anthrax vaccine versus electroporation based delivery of a PA encoding DNA vaccine in rhesus macaques

DNA vaccination is a promising immunization strategy that could be applied in the development of vaccines for a variety of prophylactic and therapeutic indications. Utilizing anthrax protective antigen as a model antigen, we demonstrate that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection. Two administrations of a DNA vaccine with electroporation elicited anthrax toxin neutralizing antibody responses in 100% of rhesus macaques. Toxin neutralizing antibodies were sustained for the nearly 1-year study duration and were correlated with protection against subsequent lethal Bacillus anthracis spore challenge. Collectively, electroporation mediated DNA vaccination conferred protection comparable to that observed following vaccination with an FDA approved anthrax vaccine.     

Immunogenicity, safety, biodistribution and persistence of ADVAX, a prophylactic DNA vaccine for HIV-1, delivered by in vivo electroporation

ADVAX is a DNA-based candidate HIV vaccine that was safe but weakly immunogenic when delivered intramuscularly (IM) in humans. Studies were performed in animal models to determine whether an alternative delivery method, in vivo electroporation (EP), could improve the immunogenicity of ADVAX while maintaining an acceptable safety profile. Immunization of mice with ADVAX with or without EP at weeks 0, 3, and 6, revealed significantly higher gamma interferon ELISpot responses to all antigens in the EP groups. Antigen-specific CD4+ and CD8+ T cell responses, as quantified by intracellular cytokine staining, both improved significantly with EP. Evaluation of repeat-dose toxicity of ADVAX-EP in rabbits did not reveal any safety concerns. Biodistribution studies of ADVAX delivered IM and with EP in rats indicated that the vaccine was localized predominantly to the administration site in both groups. PCR-based quantitation of residual plasmid at Day 60 indicated that the potential for integration events into the host genome was low for both IM and EP delivery. Taken together, these data supported the clinical development of ADVAX delivered with EP in human volunteers.     

HSV-1 amplicon vectors that direct the in situ production of foot-and-mouth disease virus antigens in mammalian cells can be used for genetic immunization

HSV-1 amplicon vectors encoding heterologous antigens were capable to mediate in situ generation of protein synthesis and to generate a specific immune response to the corresponding antigens. In this study, foot-and-mouth disease (FMD) virus antigens were used to generate a genetic vaccine prototype. The amplicons were designed to provide a high safety profile as they do not express any HSV-1 genes when packaged using a helper virus-free system, and they are able to encapsidate several copies of the transgene or allow the simultaneous expression of different genes. Virus-like particles were produced after cell processing of the delivered DNA. Inoculation of mice with 5 × 10⁵ transducing units of amplicon vectors resulted in FMDV-specific humoral responses in the absence of adjuvants, which were dependent on the in situ de novo production of the vector-encoded antigens. Challenge of mice vaccinated with these amplicons with a high dose of live virus, resulted in partial protection, with a significant reduction of viremia. This work highlights the potential use of a HSV-1 amplicon vector platform for generation of safe genetic vaccines.     

Development of a DNA vaccine expressing a secreted HIV-1 gp41 ectodomain that includes the membrane-proximal external region

A limited number of sites on the HIV-1 Envelope protein are vulnerable to broadly neutralizing antibodies (bnAbs). One of these sites, the membrane proximal external region (MPER), is located at the C-terminus of the gp41 ectodomain (gp41_ecto). This highly conserved sequence is bound by several well-characterized bnAbs. Efforts to produce a gp41 immunogen are in part hampered by the MPER’s hydrophobicity and propensity to induce aggregation. We sought to produce a DNA vaccine expressing a gp41_ecto that is both secreted from mammalian cells and maintains binding by bnAbs to the MPER. Through in silico analysis, we predicted regions of gp41_ecto that could induce aggregation and possibly hinder secretion. We generated deletion mutants of gp41_ecto and tested their ability to be secreted by mammalian cells. Upon deletion of regions in either the fusion peptide (FP) or MPER, secretion of the gp41_ecto increased. In an effort to both augment secretion and maintain binding by bnAbs, we developed constructs with the FP deletion and introduced point mutations in the MPER. Two constructs (gp41 ΔFP and gp41 ΔFP+I682E) maintained binding by gp41 MPER-specific bnAbs (4E10, Z13e1 and 10E8). These were evaluated as DNA vaccines in a mouse model. Both vaccines proved to be immunogenic and appeared to elicit some MPER-specific antibodies that bound gp41 ectodomain-derived proteins but not short peptides spanning the MPER. No neutralizing capacity was detected against a clade C virus containing a homologous MPER.